AN ABSTRACT OF THE THESIS OF
Timothy McLagan Hazzard for the degree of Doctor of Philosophy in Animal Science
presented on August 26. 1997. Title: Mechanisms of Estradiol -1713- Induced Down-
Regulation of Ovine Uterine Oxytocin Receptors During the Estrous Cycle.
Abstract Approved:
Redacted for Privacy
Fredrick Stormshak
To determine whether chronic treatment with estradio1-17f3 (E2) maintained the
corpus luteum (CL), 10 mature ewes were injected s.c. for 20 days beginning on Day 4 of
the cycle with either 500 lig E2 in corn oil or vehicle. Laparotomy of E2-treated ewes on
Day 24 revealed the presence of previously marked original CL. Treatment increased the
mean interestrous interval for 4 of 5 animals (p < 0.001). Therefore, to determine whether
the increase in the interestrous interval was due to an effect on the function and (or)
concentration of uterine oxytocin receptors (OTR), 20 ewes were injected with E2 or
vehicle as above from Days 4-14 of the cycle. On Day 15, ewes were injected i.v. with
oxytocin (OT) or saline, and blood samples were collected for 13,14-dihydro-15-ketoprostaglandin F2, (PGFM) analysis at frequent intervals for 60 min and progesterone (P4)
analysis at time 0. Laparotomy was performed after blood collection to remove uterine
endometrium from saline-treated ewes only for OTR analysis. Estradiol treatment
maintained mean serum concentrations of P4 (p < 0.05), and reduced uterine OTR
concentrations (p = 0.002). Treatment with E2 reduced OT-induced concentrations of
PGFM (p < 0.01). These data suggest that chronic E2 treatment can prolong the
interestrous interval by reducing uterine concentrations of OTR and hence OT-induced
secretion of prostaglandin Flo. (PGF2.).
To examine the effects of E2 and P4 on uterine OTR, OTR mRNA and P4 receptors
(PR), 15 ewes were injected with either 250 lig E2 or vehicle as above or 10 mg P4 on
Days 11-14 following vehicle treatment on Days 4-10. Endometrium and CL were
removed on Day 15 of the cycle. Mean luteal weights were greater in E2- and P4-treated
ewes (p < 0.05). Endometrial OTR and OTR mRNA were greater in control than in E2
and P4-treated ewes. In a subsequent experiment it was determined that endometrial PR
did not differ between P4-treated and control ewes. To ascertain whether chronic E2
treatment of ewes during the cycle would alter PGF2.-induced secretion of luteal OT, 20
ewes were injected with 250 pg E2 or vehicle as above. On Day 15, ewes were injected
i.v. with PGF2c, or saline and blood samples were collected for OT analysis at frequent
intervals for 30 min. Prostaglandin Fu-induced secretion of OT was similar between
treatment groups (p > 0.05), suggesting that chronic E2 treatment of ewes during the cycle
does not act directly on the ovary to alter production of OT. Collectively, these data
suggest that in cycling ewes exposure to increased concentration of E2 or P4 causes down-
regulation of the OTR as a consequence of genomic suppression of the OTR gene.
©Copyright by Timothy McLagan Hazzard
August 26, 1997
All Rights Reserved
Mechanisms of Estradio1-1713 -Induced Down-Regulation of Ovine Uterine Oxytocin
Receptors During the Estrous Cycle
by
Timothy McLagan Hazzard
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed August 26, 1997
Commencement June 1998
Doctor of Philosophy thesis of Timothy McLagan Hazzard presented on August 26, 1997
APPROVED:
Redacted for Privacy
Major Professor, representing Animal Science
Redacted for Privacy
Head of Depa ment o Animal Sciences
Redacted for Privacy
Dean of Graduat
chool
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Redacted for Privacy
Timoth McLagan Hazz
,
Author
ACKNOWLEDGEMENTS
I would like to take this time to thank a number of friends and supporters that have
helped me to survive the last few years. First, I would like to thank my mentor Dr.
Fredrick "Stormy" Stormshak for his invaluable guidance and fatherly advice, humorous
anecdotes, and many "swift kicks" that he has freely offered (sometimes too freely!). I
would like to thank by fellow graduate students for their help and support through the
long days we have shared in the laboratory. First, Jennifer Bertrand who, although often
quite a pessimist, was always willing to spend a little extra time helping me figure out
problems both scientific and personal. Diana Whitmore-Takahashi and Joan Burke for
quickly breaking me in to the "do's and don'ts" of the Stormshak lab. Shelby Filley for
her philosophy on "working hard to get somewhere". Ursula Bechert for hours of
entertaining e-mail, animal stories and always making me feel like I know everything,
"where is the...., How do you do....". Sara "Superchick" Supancic (who got while the
gettin's good) for her smiles and extremely positive attitude about everything. Kelly
Pinckard (Hazzard) my student, labworker, labmate and finally fiancee who I will mention
later. I must also thank many of my graduate peers. Derek "the sperm man" McLean for
his quick wit and scientific inspiration, Susan "llama lady" Powell for making me take
lunch breaks, Jennifer "loud mouth" Duncan for her frank yet sincere comments, and Tina
"major of the week" Clark for her ability to become the sister I never had.
I would like to thank those faculty members who served on my graduate
committee, especially Dr. Fred Menino for his humor (I think!), and athletic prowess (Go
Squirrels!) and Dr. Robert Mason for our many runs and his ever enlightening but always
profane chats. I would like to express my sincere gratitude to the many student workers
that helped "check the animals", particularly Clint Abbe who not only gave me a
wonderful yet browbeaten view of married life (Yes dear!), but for opening up his home
for me during the last month of my stay.
I would like to acknowledge Dr. Dave Thomas of the Department of Statistics for
his assistance in the statistical analyses, and Drs. Mark Mirando and OV Slayden for their
advice in performing the radioreceptor assays. Additionally, I would like to thank Dr.
Gordon Niswender for the progesterone antiserum, Dr. Dieter Schams for the oxytocin
antiserum, Dr. William Thatcher for the PGFM antiserum, and Dr. Tadashi Kimura for the
oxytocin receptor cDNA, for without these supplies there would be no project.
Most importantly I want to express my appreciation and love to my family and
future family. My parents have always been a source of never ending support for any
endeavor I have chosen to undertake. They have always been proud of me and have never
hesitated to express this. They should be credited for the educational status I have
attained. I also want to thank my "unbenounced" cousin Laura Hulburd (Hazzard) for
offering her wisdom, support and home to me over the past few years, she has quickly
become family to me. I would like to thank "mom and dad" Pinckard for including me in
their family times, and many home cooked meals. Finally, I want to thank my fiancee Kelly
Pinckard and her "our" daughter Alexa for hours of down-time and fun. "My student"
Kelly has become an important part of my life since the time I asked her in the 4°C walk-in
"so..are you married?", to the time that I proposed. She has been supportive and my
partner in crime both in the lab and especially when the "cat" is away.
TABLE OF CONTENTS
REVIEW OF THE LITERATURE
CHARACTERISTICS OF THE MAMMALIAN ESTROUS CYCLE:
A BRIEF OVERVIEW
Page
1
1
HYPOTHALAMO-PITUITARY-OVARIAN MECHANISMS
Hypothalamic and Pituitary Hormones
The Ovary
Feedback Mechanisms
3
FOLLICULAR PHASE
Stages of the Follicle
Recruitment
Selection and Dominance
Follicular Atresia
Hormones of the Ovarian Follicle
Two Cell Theory of Estradiol Production
Oxytocin
Ovulation
Morphology and Physical Changes
Inflammatory-like Reaction
9
9
Steroid ogenesis
LUTEAL PHASE
Luteinization
The Corpus Luteum
Morphology
Progesterone synthesis
Luteotropins
Prolactin
Maternal Recognition of Pregnancy
Luteolysis
Prostaglandin F2a
Mechanisms of action for PGF
Pulsatile Requirement of PGF2.
Oxytocin
Oxytocin-PGF2. Feedback Loop
3
7
8
11
12
14
15
15
16
18
18
20
21
23
23
25
25
27
32
36
37
45
48
49
52
53
55
TABLE OF CONTENTS (CONTINUED)
Steroid Regulation
Progesterone
Estrogen
Receptor Interaction
Page
56
57
59
62
STATEMENT OF THE PROBLEM
65
DOWN-REGULATION OF OXYTOCIN RECEPTORS AND
SECRETION OF PROSTAGLANDIN F2, AFTER CHRONIC
TREATMENT OF EWES WITH ESTRADIOL-17p
67
INTRODUCTION
67
MATERIALS AND METHODS
Animals
Treatments
Experiment 1
Experiment 2
Steroid Radioimmunoassays
Progesterone Assay
Estrogen Assay
Prostaglandin Metabolite Assay
Oxytocin Receptor Analysis
Estrogen Receptor Exchange Assay
Statistical Analysis
68
68
69
69
69
70
70
72
72
74
76
77
RESULTS
Experiment 1
Experiment 2
78
78
78
DISCUSSION
82
IMPACT OF CHRONIC TREATMENT OF EWES WITH
ESTRADIOL -17[3 OR PROGESTERONE ON OXYTOCIN
RECEPTOR GENE EXPRESSION AND OVARIAN OXYTOCIN
SECRETION
INTRODUCTION
87
87
TABLE OF CONTENTS (CONTINUED)
MATERIAL AND METHODS
Animals
Treatments
Experiment 1
Experiment 2
Experiment 3
Oxytocin Receptor Analysis
Progesterone Receptor Exchange Assay
Steroid Radioimmunoassays
Progesterone Assay
Oxytocin Assay
RNA Extraction and Northern Blotting
Statistical Analysis
Page
89
89
90
90
91
91
92
92
93
93
93
94
96
RESULTS
97
DISCUSSION
101
GENERAL CONCLUSIONS
105
BIBLIOGRAPHY
109
LIST OF FIGURES
a
Figure
1A.
1B.
2.
3.
4.
Saturation analysis of OTR in ovine endometrium. Duplicate
aliquots of membrane protein from ewes (n=5) were incubated
with each concentration of [311] -0T alone (total binding) or with
a 200-fold excess of unlabeled OT (nonspecific binding). Specific
binding was determined as the difference between total and
nonspecific binding.
79
Regression of bound OT in endometrial membranes as determined
using a single saturating concentration of [3H] OT
(Y = 0.52x - 4.51, R2 = 0.99).
80
Plasma concentrations of PGFM in control (CO) and E2-treated
ewes on Day 15 of the cycle after i.v. injection of saline (S) or
20 IU USP OT. Because of different mean basal concentrations
of PGFM among groups, each point represents the adjusted mean
for the covariate (-5 and 0 time points) for each ewe. Standard
errors are given for each mean in the results.
81
Northern Blot of OTR mRNA from Day 15 ovine endometrium.
Each lane represents one ewe. Lane 1: ovine nonlactating mammary
gland tissue (negative control). Lanes 2-4: animals received 10 mg
progesterone s.c. Days 10-14. Lanes 5-7: animals received 250 lig
E2 s.c. Days 4-14. Lanes 8-10: animals received 2 ml CO s.c. Days
4-14 (positive controls). The mRNA for OTR is represented by four
differentially polyadenylated variants (1.65-6.20 kb). 18S rRNA was
probed to determine equality of loading.
98
Plasma concentrations of oxytocin in control (CO) and E2-treated e
wes on Day 15 of the cycle after iv. injection of saline (S) or 125 pg
PGF2a (Estrumate ®) at time 0. The common estimate of the standard
error was 15.18. Each point represents the mean concentration of OT
of five animals/group.
99
MECHANISMS OF ESTRADIOL-17P-INDUCED DOWNREGULATION OF OVINE UTERINE OXYTOCIN RECEPTORS
DURING THE ESTROUS CYCLE
REVIEW OF THE LITERATURE
CHARACTERISTICS OF THE MAMMALIAN ESTROUS CYCLE:
A BRIEF OVERVIEW
The mammalian estrous cycle is characterized by four distinct stages: proestrus,
estrus, metestrus and diestrus. The hormonal and utero-ovarian morphological events that
occur during each stage control the behavior of the animal and prepare the reproductive
tract for the possibility of a pregnancy, respectively.
Estrus, or the period of the estrous cycle when mating occurs, usually coincides
with ovulation in domestic mammals. Primates have a menstrual cycle with ovulation
occurring at the end of an extended follicular phase, which does not coincide with
behavioral signs of male receptivity in most species. Estrus is characterized by high
systemic concentrations of estrogen secreted by preovulatory Graafian follicles. High
serum concentrations of estrogen stimulate the pulsatile release of gonadotropin-releasing
hormone (GnRH) from the hypothalamus and a subsequent release of luteinizing hormone
(LH) from the anterior pituitary. Preovulatory serum concentrations of LH rapidly
increase in response to the estrogen-induced release of GnRH, and initiate mechanisms in
the Graafian follicle that result in ovulation. Follicular rupture is then followed by the LH-
2
induced transformation of the granulosa cells into lutein cells, which become the only
major source of progesterone for most mammalian species (Hafez, 1993).
The transition period between female receptivity (estrus) and the rise in
concentration of progesterone is termed metestrus. At this time, the corpus luteum (CL)
is developing from the collapsed follicle into a transient endocrine organ that will maintain
the uterus in a state capable of maintaining a pregnancy.
During diestrus, the comparatively longest stage of the estrous cycle, the CL
matures and continues to produce progesterone. Luteal progesterone acts to suppress
further release of LH and thus inhibits ovulation. Serum concentrations of progesterone
continue to increase over the subsequent 6 to 10 days before leveling off and eventually
declining if the ovulated ovum was not fertilized to initiate development of a viable
embryo.
Proestrus is marked by the decline in progesterone concentrations and the
development of a dominant follicle(s) that is destined to ovulate in response to the LH
surge. Follicular growth occurs in response to increased basal concentrations of LH
whose secretion from the pituitary is provoked by the increasing levels of estrogen being
secreted by the developing follicle. Concomitantly, structural regression of the CL occurs
with the consequent reduction in systemic concentrations of progesterone. This sequence
of events promotes behavioral estrus with ovulation.
The duration of each stage varies among different mammalian species, and thus, so
do the lengths of the estrous cycles. Importantly, some animals (cow, sow) cycle
throughout the year and only become acyclic due to pregnancy or environmental stressors
3
such as temperature. Others experience a period of anestrus, which is linked to the annual
photoperiod. In species such as sheep and horses, reproduction of females commences
due to the decreasing or increasing of daylight hours, and are termed "short-day breeders"
and "long-day breeders", respectively (Hafez, 1993).
HYPOTHALAMO-PITUITARY-OVARIAN MECHANISMS
Hypothalamic and Pituitary Hormones
Hormonal regulation of bodily processes such as metabolism, growth, lactation
and reproduction all begin in a region of the brain, the hypothalamus, that differentiates
from the ventral portion of the diencephalon during brain development. The hypothalamus
consists of multiple neurosecretory nuclei producing a number of neurohormones affecting
the pituitary gland. Many of these hormones are secreted into a highly vascularized region
called the median eminence, and are absorbed into a portal system that supplies the
anterior pituitary gland. Thus, these neurohormones are known as releasing or inhibiting
hormones because they exert a direct effect on the pituitary to either release or suppress
specific hormones. For example, GnRH from the hypothalamus induces a release of
LH/follicle-stimulating hormone (FSH) from the anterior pituitary. Although many of the
nuclei in the hypothalamus more commonly secrete one neurohormone only, a great deal
of overlap in production of these hormones exists. For example, the supraoptic nuclei and
the paraventricular nuclei both produce oxytocin and vasopressin (Hadley, 1992).
4
However, it is more commonly thought that the source of oxytocin is the paraventricular
nuclei.
The pituitary gland is located beneath the third ventricle of the brain, and is
composed of the neurohypophysis (posterior pituitary) and the adenohypophysis (anterior
pituitary). The neurohypophysis originates from an evagination of the diencephalon while
the anterior pituitary develops from an outpocketing of the oral cavity known as Rathke's
pouch. Although the fusion of these developmentally different areas forms the pituitary
gland, clearly each area maintains a different function (Hadley, 1992).
The adenohypophysis is a glandular structure with no continuous neural
innervation from the hypothalamus. This portion of the pituitary is composed of multiple
cell types that respond to the hypothalamic releasing or inhibiting hormones secreted into
the median eminence and carried by the hypothalamo-hypophyseal portal system. There
are three categories of tropic hormones produced and secreted by the adenohypophysis,
separated by similarity of chemical structure. The glycoproteins all consist of two peptide
subunits, a and p, and an assortment of carbohydrate moieties. The a subunits are the
same for LH, FSH and thyroid-stimulating hormone (TSH), however the 13 subunits are
different and denote the specificity of each hormone (Boothby et al, 1981). The
gonadotropins, LH and FSH, provoke a response in the ovaries or testes. Luteinizing
hormone causes the synthesis of testicular androgens in males and follicular androgens in
preovulatory (proestrus) females as well as gamete release (ovulation in females). After
ovulation, LH is also responsible for the morphological change of granulosa cells to the
lutein cells that hypertrophy to form the corpus luteum. Follicle-stimulating hormone is
5
involved with gamete preparation (follicular development in the mammalian female), as
well as the conversion of androgens to estrogens by follicular granulosa cells. The third
glycoprotein secreted by the adenohypophysis is TSH. As the name suggests, this
hormone stimulates the thyroid gland to release triiodothyronine (T3) and thyroxine (T4),
which influence metabolic homeostasis (Norris, 1997).
The second category of tropic pituitary hormones consists of growth hormone
(GH) and prolactin (PRL). These are both large polypeptide hormones that are very
similar in structure, and overlap in function. Some common effects of these two hormones
involve osmoregulation, selective tissue growth and lactation. However, PRL has weaker
effects on body growth and metabolism than GH. Although GH causes growth in many
tissues, it does so indirectly through production of insulin-like growth factors (IGF's).
Therefore, the main effect of GH is to stimulate the release of IGF's from the liver, which
are carried systemically to target tissues, or induce a release of IGF's at a more local level
(Norris, 1997).
The third category of adenohypophyseal tropic factors consists of several
hormones derived from a prohormone known as proopiomelanocortin (POMC).
Hormones such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating
hormone (MSH), lipotropin (LPH), and [3-endorphin are all variations in posttranslational
processing of POMC. Corticotropic cells in the adenohypophysis produce ACTH and pLPH as cleavage products of POMC. However, enzymes in melanotrope cells of the pars
intermedia (fused area between the adenohypophysis and neurohypophysis), differentially
cleave ACTH to a-MSH; and cleaves 13-LPH to I3-endorphin. Adrenocorticotropic
6
hormone stimulates release of corticosteroids from the adrenal cortex, which are involved
in intermediary metabolism and other bodily functions. Alpha-MSH induces the release of
melanin from skin cells, which give the skin or hair a darker color (Norris, 1997).
Unlike the adenohypophysis, the neurohypophysis is an extension of the
neurosecretory neurons originating in the hypothalamus. Therefore, the hormones
secreted from the neurohypophysis are actually produced in the nuclei of the
hypothalamus. Oxytocin (OT) and vasopressin are both nonapeptides originating in the
hypothalamus and are stored, pending proper neuro-stimulus for secretion, in the
neurohypophysis. Nonapeptides are synthesized as a larger propeptide, which is
hydrolyzed to yield the appropriate nonapeptide and neurophysin (approximately 92 amino
acid peptide). Neurophysins are thought to play some carrier function in transporting the
nonapeptide to the axonal storage site in the neurohypophysis. Vasopressin acts
differentially based on the concentration in the blood. In high doses, vasopressin increases
blood pressure by affecting the vascular smooth muscle, which causes diuresis through an
increase in the glomerular filtration rate in the kidneys. However, in low doses vasopressin
acts as an antidiuretic thus preventing diuresis. Because of this effect, vasopressin is also
termed antidiuretic hormone (ADH). Oxytocin stimulates smooth muscle contraction in
the reproductive tract of both males (vas deferens) and females (myometrium) as well as
contraction of myoepithelial cells around the alveoli and ducts of the mammary gland in
females. Muscular contractions in the reproductive tract serve the purpose of both
propelling sperm during ejaculation, and movement of sperm in the female to enhance
fertilization. Oxytocin has also been used medically during pregnancy to successfully
7
induce labor. Release of OT involved in milk letdown is part of a neuroendocrine reflex
arc. Stimulation of the teat by the newborn sends neural impulses to the brain, which
reach the hypothalamus and initiate an action potential in the neurosecretory neurons
storing oxytocin in the neurohypophysis, resulting in secretion of the hormone (Hadley,
1992; Norris, 1997).
The Ovary
The ovary is the female gonad that produces the gametes necessary for
reproduction. However, the ovary is also a major endocrine organ responsible for the
production of sex steroid hormones (estrogen and progesterone), prostaglandins and
multiple peptide hormones (relaxin, inhibin, activin, follistatin and oocyte maturation
inhibitor). These compounds are produced and secreted by two distinct cell populations in
the follicles, and in some species, by the supporting stroma (interstitial cells; Hansel et al.,
1973). Follicular development occurs in waves throughout the estrous cycle of domestic
animals and ultimately the dominant follicle(s) of a wave ovulate to form corpora lutea.
Those follicles not ovulating undergo a form of degeneration referred to as atresia (Norris,
1997). Follicular thecal cells are the primary source of androgens while granulosa cells are
the primary source of estrogens in the ovary (follicular development will be discussed in
further detail in subsequent sections). Importantly, many of these compounds (estrogen,
inhibin and activin) are produced and secreted due to stimulation by pituitary
gonadotropes, FSH and LH. Thus, regulation of follicular development and ovulation
8
depends on a cascade of hormonal release originating in the brain and continuing in the
ovary, forming what is termed the hypothalamic-pituitary-ovarian axis.
Feedback Mechanisms
Feedback mechanisms are common throughout biochemical pathways and are
usually characterized as being negative or positive. As stated above, hypothalamic
hormones act in a positive manner to induce the release of pituitary hormones, which in
turn stimulate ovarian secretion of steroids. However, the possibility of retrograde flow
from the hypothalamo-hypophysial-portal system (Page and Bergland, 1977), and the
accumulation of pituitary hormones in the circulatory system, provide a means by which
these hormones can feedback to inhibit secretion of their corresponding hypothalamic
releasing hormones. Synthesis, storage and release of hypothalamic hormones are
regulated by three types of feedback mechanisms: "ultra-short loop", "short loop" and
"long loop" feedback (Hafez, 1993). An example of "ultra-short loop" feedback would be
the regulation of hypothalamic hormone production and release by the same hormone,
thus it is actually a form of self-regulation. Modification of hypothalamic hormone
secretion by pituitary hormones would be "short loop" feedback, while stimulated
production of gonadal hormones traveling through the circulatory system to affect
hypothalamic or pituitary output would be an example of "long loop" feedback. All three
types of feedback mechanisms can have either a positive or negative effect on the
9
secretory activity of the stimulating hormones, and examples of both will be mentioned
throughout this thesis.
FOLLICULAR PHASE
Stages of the Follicle
As previously mentioned, the ovary is not only the site of gametogenesis, but also
a major endocrine organ. However, the established connections between gametogenesis
and endocrine factors cannot be explained without first detailing the physical structure and
components of the ovary: the follicle. At any time during the reproductive years of a
female, there are constant morphological changes occurring in the ovary, representing the
development of the primordial follicles to mature follicles that will either ovulate or
undergo atresia. Unlike males, who produce sperm throughout their lives, the female
produces all of the primordial follicles of her reproductive life during fetal development
(primates, ruminants) or early in neonatal life (rodents, rabbits; Fortune, 1994). At this
point, the oocyte located within the follicle, is arrested in the diplotene stage of meiosis I
of development (referred to as dictyate stage), and awaits the appropriate signals
necessary to initiate resumption of meiotic division. Stages of follicular development are
classified by changes in oocyte morphology and size, as well as the organization of the
granulosa (cumulus oophorus) cells surrounding the oocyte (Gore-Langton and
Armstrong, 1994).
10
Follicular development begins with the primordial follicles, which constitute a
nongrowing follicle "reserve" in the ovarian cortex. Depletion of this "reserve" occurs
throughout the reproductive life of the female, and is characterized by the growth of the
primordial follicle. During the onset of follicular growth, the oocyte begins to grow and
the flattened pre-granulosa cells of the primordial follicle become cuboidal and begin to
proliferate (Hirshfield, 1991). The factors that initiate follicle development remain
unknown (Fortune, 1994). However, both primordial and primary follicular development
are independent of gonadotropins (Richards and Midgley, 1976), and recently Wandji et
al. (1996), suggested that the ovarian stroma exerts inhibitory control over the initiation of
primordial follicle growth. Secondary follicles are characterized by the presence of the
zona pellucida, a glycoprotein matrix immediately surrounding the oocyte, as well as the
proliferation of granulosa cells into two or more continuous layers. Granulosa cells are
surrounded by the basement lamina, which separates the granulosa from the thecal cells.
Tertiary (antrum, Graafian) follicles contain an antrum filled with follicular fluid,
composed of secretions from the surrounding cells (Edwards, 1974), as well as a
continually proliferating granulosa cell layer (Gore-Langton and Armstrong, 1994). The
hypothesis for recruitment of the tertiary follicle for ovulation or atresia is discussed
below.
11
Recruitment
Although the female has a reserve of nongrowing follicles in the ovary, the patterns
of development of large follicles in mammals do not appear to be random. Instead,
development occurs during particular times of the reproductive cycle. Follicular
development in rats, humans and pigs, occurs only during the follicular phase, and is
otherwise suppressed (Fortune, 1994). Pache et al. (1990) demonstrated in humans that a
cohort of follicles begin to develop during the early follicular phase, from which only one
follicle continues to grow in the late follicular phase while others regress. Importantly, no
ovulatory-size follicles develop during the luteal phase in humans, rats and pigs. In
contrast, follicular development involving ovulatory-size follicles throughout the cycle
occurs in cattle, sheep and horses. In heifers, ultrasonography revealed that two or three
times during the cycle a wave of 3 to 6 follicles begin to grow larger than 4 to 5 mm in
diameter (Sirois and Fortune, 1988). After several days of growth, one follicle continues
to grow and the others regress. Similarly, in ewes both laparoscopy and ultrasonography
revealed that peaks of follicular activity above a constant basal level occurs two or three
times during the cycle (Noel et al., 1993; Schrick et al., 1993). However, these peaks of
follicular activity result in multiple follicles that do not regress to leave a single dominant
follicle. Thus, ewes are considered to display a continuous entry, growth and atresia of
follicles that result in the presence of several ovulatory-size follicles (Schrick et al., 1993).
In fact, the number of large follicles was demonstrated to be 2 to 3 times the ovulation
rate in ewes (Noel et al., 1993). These data suggest a lack of follicular dominance in this
12
polyovular species. Nevertheless, two follicular wave patterns may occur during the
estrous cycle of ewes while in heifers two or three waves have been detected. However,
only the wave immediately preceding luteal regression (second or third wave), results in
ovulation of the largest follicle(s) (Ginther et al., 1989; Noel et al., 1993). Thus, a signal
is responsible for the initiation of growth or recruitment of a group of follicles in a non-
random pattern. Experiments measuring plasma concentrations of FSH throughout the
bovine estrous cycle have demonstrated slightly higher concentrations before the first,
second and third follicular waves (Walters and Schallenberger, 1984; Adams et al., 1992).
Gong et al. (1996) demonstrated in heifers, that FSH was required for antral follicles
mm to continue growth up to 9 mm in diameter. In primates, plasma FSH is slightly
elevated during the early follicular phase as compared with the late follicular and luteal
phases (Abraham et al., 1972). Thus, slight elevations in plasma FSH concentrations are
the required signal that stimulates follicular recruitment in mammals (Fortune, 1994).
Selection and Dominance
Finally, recruitment ends with the selection of one follicle to continue to grow at
the expense of the other follicles in its cohort. These follicles are called "dominant"
follicles because not only do they continue to grow, but they differentiate functionally
from the subordinate follicles in ways that prepare them for ovulation. For instance, one
prominent characteristic of the dominant follicle is the ability to produce tremendous
13
amounts of estradiol- 1713 (Ireland et al., 1983). Increased estradiol production appears to
arise from increases in the ability of granulosa cells to aromatize androgens, which will be
discussed below. More recently, researchers have examined the mRNA expression of
enzymes responsible for steroid synthesis in an effort to determine molecular markers
characteristic of dominant follicles (Tian et al., 1995; Xu et al., 1995, Bao et al., 1997a,b).
Activity of steroidogenic enzymes such as cytochrome P450 side-chain cleavage
(P450scc), 3P-hydroxysteroid dehydrogenase (313-HSD), and cytochrome P450 17ahydroxylase all increased in thecal cells within 24 hours after prostaglandin Fla (PGF2.)-
induced luteolysis in heifers (Tian et al., 1995). However, the expression of 3P-HSD was
higher in healthy follicles as compared with atretic follicles, and dominant follicles
expressed 3P-HSD in granulosa cells (Bao et al., 1997b). Therefore, expression of 3PHSD mRNA in granulosa cells may be associated with the mechanism of dominant follicle
selection during the follicular wave. Alternatively, the expression of mRNA for FSH
receptor (FSHr) and LH receptor (LHr) was detected in granulosa and thecal cells,
respectively (Xu et al., 1995). However, these researchers further demonstrated the
expression of LHr in granulosa cells of healthy >9 mm follicles, suggesting the importance
of LHr expression in selection of follicular dominance. Finally, Bao et al. (1997a)
suggested that follicular recruitment in cattle was associated with granulosa expression of
P450scc and cytochrome P450 aromatase (aromatase), while the process of follicular
selection was associated with initiation of LHr mRNA in granulosa cells. In any case,
hormonal steroidogenic expression, feedback mechanisms, and receptor expression are all
14
cues temporally spaced to bring about the final selection that establishes dominance among
a cohort of follicles.
Follicular Atresia
Although the penultimate reason for folliculogenesis is ovulation, many follicles
develop before the appropriate signals are present to cause ovulation. Follicular
development is initiated, but, at a certain point these follicles undergo a form of
degeneration termed atresia. Atresia is associated with several characteristics such as
degeneration of the oocyte, hypertrophy of theca cells (Erickson et al., 1985), and the
appearance of pyknotic granulosa cells (Byskov, 1978). When and why atretic follicles
appear remains to be a contested point. Richards (1980) suggested that most follicles
undergo atresia at an early stage of growth in the absence of appropriate hormonal
assistance (FSH and LH). Conversely, Hirshfield (1991) implied that most follicles become
atretic near the end of development. Uniquely, this latter study described follicular
development in the rat as a series of follicular cell generations, with atresia most
commonly occurring during the 8th or 9th granulosa cell generation. Similarly, the highest
rates of atresia occur near the end of follicular development in cattle (generations 10 to
13; Lussier et al., 1987). Only those follicles that are exposed to specific signals at the
time they reach this stopping point (8th or 9th granulosa cell generation) will continue to
develop subsequent granulosa cell generations and ovulate. Although FSH has been
previously described to be necessary in recruitment of follicles, a variety of hormonal
15
triggers in addition to FSH have been hypothesized to cause atresia. Circulating
concentrations of gonadotropins, androgens and estrogens in excess (Harman et al., 1975;
Clark et al., 1981) or insufficient quantities of gonadotropins and estrogens (McNatty et
al., 1979; Richards, 1980), have all been associated with atresia. In fact one of the
primary characteristics of the impending atresia is a high follicular fluid concentration of
androgens due to decreased aromatase activity (Maxson et al., 1985). Further, metabolic
stress involved with increasing cell number has also been implicated with atresia
(Hirshfield, 1991). However, a definitive cause for why follicles do not continue to grow
past a certain point without the proper signals remains to be clarified.
Hormones of the Ovarian Follicle
Two Cell Theory of Estradiol Production
As mentioned previously, estradiol is produced by follicles. This steroid hormone
is reproductively important because it is responsible for estrous behavior in some species,
and is involved in a feedback mechanism with the hypothalamus, regulating the LH surge
and ovulation. Interestingly, in many species the mechanisms by which estradiol is
synthesized involves the coordinated effort of both follicular cell types; theca and
granulosa cells. The two-cell theory of estrogen production states that LH acts on the
theca interna to stimulate production of androgens from cholesterol derived progestins
(Falck et al., 1962; Richards, 1980). While granulosa cells lack the enzyme (17ahydroxylase) necessary for androgen production, androstenedione more so than
16
testosterone (Fortune and Hansel, 1985), does diffuse across the basement membrane to
the granulosa cells. These cells contain the FSH-induced enzyme aromatase (Berndtson et
al., 1995), which converts thecal derived androgens to estrogens. Although both cell
types produce progestins (Fortune and Hansel, 1979a), incubations of follicle wall
(granulosa and theca interna cells) yielded greater production of androstenedione than
theca cells alone (Fortune, 1986), suggesting that granulosa cells supply the necessary
progestins for androgen biosynthesis by the theca cells. Thus, the production of steroids
from theca interna and granulosa cells are dependent on the specific precursors from each
cell type as well as regulation by the gonadotropins.
Oxytocin
Oxytocin is another important hormone produced by the preovulatory follicles of
cows (Wathes et al., 1984; Schams et al., 1985) and ewes (Wathes et al., 1986).
However, the production of follicular OT seems to be species specific as Stock et al.
(1995), demonstrated that preovulatory follicles of mares are not a source of OT. Bovine
granulosa cells from preovulatory follicles have been identified as the source of OT in
vitro (Schams et al., 1985), and the location of OT mRNA synthesis (Holtorf et al., 1989).
This led to a series of investigations into the role of OT in regulating preovulatory
follicular steroidogenesis. Aladin-Chandrasekher and Fortune (1990), demonstrated that
OT stimulated progesterone production by granulosa cells in vitro, and this occurred just
prior to the LH/FSH surge (Voss and Fortune, 1991a). Thus, OT was hypothesized to
have a role in the steroidogenic shift from estradiol production to progesterone
17
production. A subsequent study was conducted to determine the developmental pattern of
OT secretion and the response to gonadotropins by the granulosa and thecal cells during
the follicular phase (Voss and Fortune, 1991b). This study demonstrated that granulosa
cells are the primary source of OT throughout the follicular phase, as OT secretion was
only produced in theca cell cultures contaminated by granulosa cells. Further, more
oxytocin was secreted during coculture of early and midfollicular phase theca and
granulosa cells than by granulosa cells alone, and this response was induced by LH and
FSH. Results of this study suggest that the LH/FSH surge stimulates the secretion of
oxytocin. Increased secretion of oxytocin after the LH/FSH surge is attributable to the
accumulation of oxytocin/neurophysin I mRNA in bovine granulosa cells (Voss and
Fortune, 1992). Voss and Fortune (1993) examined the effect of estradiol on oxytocin
secretion by bovine granulosa cells, and demonstrated that estradiol in low doses
stimulated oxytocin secretion while high doses inhibited secretion. These data suggest
that the elevated concentrations of estradiol in the follicular fluid of early and midfollicular phase follicles (Fortune and Hansel, 1985) may inhibit OT production, while the
decline in estradiol levels after the LH surge may allow stimulation of oxytocin synthesis
by gonadotropins. In addition, progesterone stimulated OT secretion by cultures of
bovine granulosa cells (Voss and Fortune, 1993). Thus, both OT and progesterone may
amplify the ability of gonadotropins to stimulate the production of the other hormone.
However, whether progesterone regulates OT mRNA expression is unclear. In any case, a
role for steroids in modulating follicular oxytocin secretion exists, suggesting that the
18
presence of OT during the periovulatory period may involve regulation of luteinization
and/or ovulatory events.
Ovulation
Mammalian ovulation is a unique event that involves the rupture of healthy tissue
at the surface of the ovary (Espey, 1994). Ovulation is initiated at the moment a surge of
gonadotropins stimulates the dominant follicle(s) (Espey and Lipner, 1994). The pituitary
surge of gonadotropins consists of both LH and FSH, and although an LH surge can lead
to as much as a 100-fold increase in circulating concentrations of this hormone
(Scaramuzzi et al., 1972), FSH has also been demonstrated to induce ovulation by itself in
monkeys (Schenken et al., 1984). In fact, many researchers have suggested that a
combination of LH and FSH are needed to ensure an optimal ovulatory stimulus
(Nalbandov et al., 1973; Galway et al., 1990). In either case, the surge of gonadotropins
that initiates ovulation bind to receptors in the follicular cells activating signal transduction
pathways that result in phosphorylation processes and protein synthesis (Espey and
Lipner, 1994).
Morphology and Physical Changes
Although the pituitary surge of gonadotropins appears to initiate ovulatory events,
many morphological changes must occur in the dominant follicle to cause rupture.
Rupture occurs at the apex of the follicle (where the stigma forms) and consists of
19
multiple tissue layers (Espey and Lipner, 1994). The outermost layer is a single-celled
layer of cuboidal epithelial cells that surrounds the entire ovary. The second layer is the
tunica albuginea, a fibroblast and collagen sheath, which surrounds the ovary and provides
external structural integrity. The third layer is the theca externa cells of the follicle, and
also contains fibroblasts within this collagen bound matrix of cells. The fourth layer
consists of the steroid secreting theca interna cells that line the basement membrane of the
follicle, and the fifth and final layer is the granulosa cell layer surrounding the antrum.
Thus, the release of the oocyte from the follicle depends on the dissociation of multiple
cell layers, as well as the degradation of the collagen matrix that maintains ovarian
integrity (Espey, 1994). With the activation of phosphorylation events and protein
synthesis within follicular cells, it is not too surprising that researchers have studied
proteolysis as a means for follicular rupture. Plasminogen activator is a protease
associated with the preovulatory degradation of the follicle wall (Galway et al., 1990).
Plasminogen activator cleaves both serum and follicular fluid plasminogen to the active
enzyme plasmin, which activates collagenase to degrade the collagen matrix supporting
the follicle wall and results in ovulation (Strickland and Beers, 1976; Beers and Strickland,
1978). Galway et al. (1990) demonstrated that recombinant FSH injected into immature
rats stimulated tissue plasminogen activator (tPA) enzyme activity and mRNA expression
to reach peak levels 12 hours after injection, providing evidence for the role of tPA in the
FSH induction of ovulation. However, FSH is not the only gonadotropin capable of
stimulating PA. Reich et al. (1985) demonstrated that LH was as potent a stimulator of
PA activity as FSH. More importantly, this latter study illustrated the importance of
20
follicular steroidogenesis in ovulation by demonstrating the ability of exogenous estradiol
to enhance the LH induction of PA.
Inflammatory-like Reaction
Ovulation has been likened to an inflammatory reaction (Espey, 1994).
Inflammation generally occurs as a result of a disturbance to the plasma membrane, such
as occurs when epithelial cells of the skin are perturbed by abrasion. With any disturbance
of the plasma membrane, prostaglandins are produced from a release of arachidonic acid
from the membrane phospholipids (Espey and Lipner, 1994), initiating a cascade of events
leading to the activation of prostaglandin synthetase and lipoxygenase. Importantly, part
of the inflammatory process includes vasodilation, hyperemia, edema and collagenolysis,
suggesting a synergistic role with PA and proteolysis. The first implication of an
association of prostanoids with the ovulatory process was presented in a report by
Labhsetwar (1971), who found that prostaglandin Fla (PGF2a) could induce both luteolysis
and ovulation in hamsters. Evidence of a role of prostanoids at the follicular level was
further documented by findings that intrafollicular concentrations of PGF2a and
prostaglandin E2 (PGE2) increased in the ovulatory follicle just prior to ovulation
(Ainsworth et al., 1975; Armstrong and Zamecnik, 1975; Bauminger and Linder, 1975).
The action of at least PGE2 has been demonstrated to mimic the action of FSH on
granulosa cells and LH on theca cells (reviewed by Armstrong, 1981). Thus, PGE2 may
be able to substitute for or enhance the function of gonadotropins at certain stages of
follicular development. Inhibition of prostaglandin synthesis by drugs does not interfere
21
with ovum maturation, activation of adenylyl cyclase, steroidogenesis, nor luteinization
(Tsafriri and Dekel, 1994). In fact, an inhibitor of prostaglandin synthesis, indomethacin,
which causes a reduction in PGE2 and PGF2 within minutes of an intravenous (i.v.)
injection in rabbits (Espey et al., 1986), was found to inhibit only follicular rupture in rats,
rabbits, gilts and ewes (Armstrong and Grinwich, 1972; O'Grady et al., 1972; Tsafriri et
al., 1972; Ainsworth et al., 1979; Murdoch and McCormick, 1991), without the inhibition
of luteinization and progesterone synthesis (Espey and Lipner, 1994). Another factor
involved with the inflammatory response is tumor necrosis factor (TNF), which is present
in the ovary (Brannstrom et al., 1994; Terranova, 1997). Tumor necrosis factor has been
demonstrated to induce ovulation in rats, but not as successfully as LH. However, when
TNF and LH were added simultaneously to ovaries in vitro, the number of ovulations
increased fourfold over LH alone (Brannstrom et al., 1995). Thus, TNF is involved with
ovulatory events, but the mechanism by which TNF mediates ovulation remains unknown.
Steroidogenesis
Changes in steroidogenesis occur in the preovulatory follicle during the late
follicular phase primarily consisting of a transition from the production of androgens and
estrogens to that of progestins (Fortune and Hansel, 1979b). The change in
steroidogenesis has been associated with the gonadotropin surge and ovulation (Espey and
Lipner, 1994). At the beginning of ovulation, estradiol synthesis is high while
progesterone synthesis is very low. However, during the first half of the ovulatory
process estradiol production drops to negligible quantities while progesterone production
22
increases substantially. Thus, it is logical to assume that estradiol is not needed for
ovulation, and that luteinization of follicular tissue begins to occur before actual ovulation
(Espey and Lipner, 1994). This brings into consideration the importance of progesterone
in ovulation, as ovulation cannot occur unless a follicle begins to luteinize and produce
progesterone (Espey, 1994). Snyder et al. (1984) demonstrated the necessity of
progesterone in the ovulatory process by using an inhibitor of 3 P-HSD, epostane. In this
study, rats were used to demonstrate that the inhibition of follicular steroidogenesis and
ovulation induced by epostane, could be overcome by administration of exogenous
progesterone. A further study with ewes treated with isoxazol, an inhibitor of
progesterone synthesis, resulted in the inhibition of ovulation in this species (Murdoch et
al., 1986). Thus, the presence of progesterone at the time of ovulation is somehow
involved in regulating ovulation. One possible effect of progesterone was demonstrated
by Tanaka et al. (1992) who measured ovarian PA and kallikrein (a serine protease)
activity in rats. Epostane was used to block ovulation, and was also found to inhibit PA
and kallikrein activities in a dose-dependent manner. The inhibitory action of epostane
was reversible by the treatment of animals with progesterone, suggesting the increase of
follicular progesterone at the time of ovulation regulates ovarian kallikrein and PA
activities.
23
LUTEAL PHASE
Luteinization
Luteinization and the resulting formation of the corpus luteum are a direct result of
the preovulatory gonadotropin surge. Theca interna and granulosa cells of the
preovulatory follicle undergo morphological and biochemical changes that transform these
follicular cells into the lutein cells of the corpus luteum. Following the ovulatory stimulus,
the basement membrane breaks down, and blood vessels from the theca interna invade the
ruptured antral cavity (Corner, 1919). Growth of new capillaries, angiogenesis, begins
chronologically at the time of basement membrane breakdown (Tsukada et al., 1996),
forming a complex network of vessels in the previously avascular granulosa cell layers
(Bassett, 1943). Angiogenesis plays an important role in the development and
nourishment of the corpus luteum, thus opening investigation into the role of angiogenic
factors in corpus luteum development. Gospodarowicz et al. (1985) purified a diffusible
substance responsible for triggering the early vascular changes that occur in the
developing corpus luteum, and found it to be related to fibroblast growth factor (FGF).
Previous research suggested FGF to be mitogenic for cultured granulosa cells in rats, pigs
and humans (Gospodarowicz and Bialecki, 1979). However, FGF lacks a hydrophobic
signal peptide thought to be necessary for extracellular transport (Redmer et al., 1985).
Thus, although FGF has mitogenic activity, other growth factors with diffusible properties
were thought to be responsible for neovascularization in the corpus luteum. Leung et al.
24
(1989) identified a potent mitogen for vascular endothelial cells called vascular endothelial
growth factor (VEGF). Importantly, VEGF is a secreted protein with a potential to be
involved in the regulation of vascular endothelial growth and angiogenesis. Vascular
endothelial growth factor expression has been demonstrated in the corpus luteum of rats
(Phillips et al., 1990), ewes (Redmer et al., 1996), and primates (Ravindranath et al.,
1992). Further, VEGF expression is upregulated during early luteal development
(Ravindranath et al., 1992; Redmer et al., 1996), when vascular growth is at its peak.
Finally, Koos (1995) demonstrated that human chorionic gonadotropin (hCG) induces
VEGF expression in female rats, suggesting a role of this peptide in the ovulatory process
as well as in luteal vascular growth. Therefore, angiogenic activity in the early
development of the corpus luteum is of fundamental importance in promoting the early
development of this ephemeral endocrine gland.
Further morphological and functional changes occur to the thecal and granulosa
cells during luteinization. Thecal cells undergo significant hypertrophy and hyperplasia
(O'Shea et al., 1980). Within 24 hours after the LH surge, these cells begin to migrate
into the ruptured follicular cavity (O'Shea et al., 1979), and become dispersed among the
luteinizing granulosa cells. Granulosa cells, meanwhile, have intracellular morphological
changes, such as the accumulation of smooth endoplasmic reticulum, and the development
of tubular cristae in the mitochondria (Niswender and Nett, 1994). These changes are
characteristic of steroid-secreting cells. As previously mentioned, steroidogenesis changes
from androgen/ estrogen production to progestin production at the time of ovulation.
Enzymatically, the activity of cytochrome P450scc increases following the gonadotropin
25
surge, while 17a-hydroxylase activity decreases (Rodgers et al., 1986, 1987; Lauber et al.,
1991), thus enhancing the conversion of cholesterol to progestins and preventing further
conversion of progesterone to androgens.
Morphological changes also occur at the cell surface. Following ovulation, FSH
receptors in plasma membranes of granulosa cells are internalized and gene expression for
these receptors is reduced (Nakamura, et al., 1991); although FSH receptors have been
detected in corpora lutea of hamsters (Oxyberry and Greenwald, 1982) and cows (Matins
et al., 1984). Receptors for LH in granulosa cells are also down-regulated following the
LH surge (Segaloff et al., 1990; Lapolt et al., 1990; Nakamura et al., 1991), but
expression of these receptors reoccurs approximately 48 hours after the gonadotropin
surge (Braden et al., 1994). The importance of these surface receptor changes will be
discussed below.
The Corpus Luteum
Morphology
The direct result of luteinization in normally cycling animals, is the formation of a
transient endocrine organ called the corpus luteum. Morphologically, the corpus luteum
consists of at least four types of luteal cells: two types of steroid-secreting cells, vascular
endothelial cells, and fibroblasts (Wiltbank, 1994). Steroidogenic luteal cells appear to be
derived from the two distinct follicular steroid-secreting cells; thecal and granulosa cells.
Therefore, it is of no surprise that the steroidogenic cells comprising the corpus luteum,
26
can be differentiated into two distinct cell types. Cell size, among other differences, is the
most obvious characteristic that distinguishes these cells, and has led to their designation
as small and large luteal cells (Koos and Hansel, 1981). Small and large luteal cells
contain numerous mitochondria, and an abundance of smooth endoplasmic reticulum,
which is common in steroid-secreting cells. However, unlike small luteal cells, large luteal
cells also contain a rough endoplasmic reticulum and secretory granules indicative of
protein secreting activity (Niswender and Nett, 1994). Secretory granules contain a
number of different components including oxytocin (Rodgers et al., 1983; Wathes et al.,
1983; Sawyer et al., 1986), neurophysin (Fields and Fields, 1986; Sawyer et al., 1986),
and relaxin (Fields and Fields, 1980; Fields, 1984), which have been demonstrated to be
released by exocytosis (Sawyer et al., 1979). Furthermore, the number of secretory
granules present in the large luteal cells changes during the estrous cycle of the cow, with
the highest percentage of these cells containing secretory granules during the mid-luteal
phase (Fields et al., 1992).
Small and large luteal cells also differ morphologically in ways that affect their
response to hormones. Variations in cell response are mainly due to differences in
receptor expression in the cell membrane. Fitz et al. (1982) demonstrated that small luteal
cells from superovulated ewes possess substantially more receptors for LH, while large
luteal cells contain the majority of receptors for prostaglandins. However, other
researchers have demonstrated that large and small luteal cells from normally cycling ewes
(Harrison et al., 1987), cows (Chegini et al., 1991), and rats (Nelson et al., 1992), contain
similar numbers of LH receptors. Chegini et al. (1991) also suggested that small luteal
27
cells contain a significant number of prostaglandin receptors, but this is probably due to
the presence of high-affinity binding sites for PGF2. on large luteal cells and low-affinity
binding sites on small luteal cells (Balapure et al., 1989). Whatever the case, both
steroidogenic luteal cell types have cell surface receptors that bind ligands and initiate
some form of intracellular activity.
The processes of ovulation and luteinization includes inflammatory-like events and
neovascularization. Therefore, it is no surprise that the other luteal cell types are vascular
endothelial cells and fibroblasts. Vascular endothelial cells, which line the lumen of blood
vessels in the corpus luteum, make up the largest percentage of luteal cells (approximately
50%), but are only a small percentage of the total corpus luteum volume (10%; Wiltbank,
1994). However, the role of vascular formation is extremely important in establishing the
luteal blood flow necessary to provide steroid precursors and to transport secreted
hormones to the general circulation. Fibroblasts infiltrate the corpus luteum at the time of
basement membrane breakdown, and have been demonstrated to induce luteal cell division
in vitro (Gospodarowicz and Birdwell, 1977). Further, FGF infused into the ovarian
artery for 12 hours suppressed the secretion of estradiol and androstenedione, suggesting
a paracrine effect on luteal function (Scaramuzzi and Downing, 1995).
Progesterone synthesis
Progesterone is the main steroid hormone produced and secreted by the corpus
luteum. This steroid is the ovarian "hormone of pregnancy" (Corner and Allen, 1929;
Allen and Corner, 1930), and is responsible for preparing the reproductive tract for
28
embryo implantation, as well as the subsequent maintenance of the pregnancy. Incredibly,
corpus luteum production of progesterone reaches approximately 1016 molecules of
progesterone . min"' . g"' of luteal tissue during the midluteal phase of the cycle in ewes
(Stormshak et al., 1963). These secretion rates are dependent on the activity and
efficiency of both steroidogenic luteal cells, and the rapid transport of secreted
progesterone to the general circulation. The apparent production of progesterone is
reliant on the presence of precursors necessary for continuous metabolic synthesis, and the
factors that regulate such a system.
The synthesis of progesterone requires a source of cholesterol, which can come
from either de novo synthesis or plasma lipoproteins. Luteal cells have been demonstrated
to produce cholesterol from acetate as first demonstrated by Savard and Casey (1964), but
appear to prefer the delivery of cholesterol by way of lipoproteins (Gwynne and Strauss,
1982). Cholesterol bound to lipoproteins is delivered either as low-density lipoproteins
(LDL) or high-density lipoproteins (HDL) depending on the species. Progesterone
secretion has been demonstrated to increase in cattle, after luteal cells were incubated with
LDL or HDL (Pate and Condon, 1982; Carroll et al., 1992). In sheep, both LDL and
HDL stimulated progesterone secretion by large luteal cells and LH-stimulated small luteal
cells (Wiltbank et al., 1990); however, HDL-induced release of progesterone was greater.
These researchers also demonstrated that small luteal cells utilize the endogenous lipid
droplets, within the cytoplasm, to produce progesterone. These cholesterol stores were
eventually depleted during long cultures or after LH stimulation. Ovine large luteal cells
29
do not contain lipid droplets (Niswender and Nett, 1994), and appear to need lipoproteins
for progesterone production (Wiltbank et al., 1990).
Low-density and high-density lipoproteins bind to specific receptors on the surface
of the steroidogenic luteal cells, which facilitate the transport of the lipoprotein from the
outside to the inside of the cell. The uptake of cholesterol carried by LDL occurs by way
of receptor-mediated endocytosis (Brown and Goldstein, 1986). Once the LDL is
internalized it is combined with lysosomes and degraded to liberate cholesterol. Free
cholesterol is then utilized for steroid biosynthesis, or esterified and stored as lipid droplets
(Flint and Armstrong, 1973). Cholesterol uptake by luteal cells is mainly related to
concentrations of lipoprotein receptors. In the human, LDL receptors increased in
granulosa cells treated with hCG or 8-bromo-cAMP (Golos and Strauss, 1987). Rat luteal
tissue treated with hCG or LH increased binding of HDL (Strauss et al., 1982).
Importantly, progesterone production is enhanced when bovine luteal cells were incubated
in the presence of either LDL or HDL (Pate and Condon, 1982; 0' Shaughnessy and
Wathes, 1985). However Carroll et al. (1992) demonstrated that lysine-modified LDL
(which does not bind to the LDL receptor) stimulated progesterone secretion by bovine
luteal cells, suggesting the delivery of LDL cholesterol may not require receptor binding.
Further, a mechanism other than receptor-mediated endocytosis may be responsible for
cholesterol transfer from HDL, because Paavola et al. (1985) demonstrated that delivery
of cholesterol from HDL may not require internalization. Whatever the type of lipoprotein
or the mechanism for cholesterol delivery, the source of cholesterol for steroid
biosynthesis is lipoproteins (Wiltbank, 1994).
30
Cholesterol is converted to pregnenolone in the mitochondria and this steroid
eventually gives rise to progesterone in the endoplasmic reticulum. Thus, cholesterol
transport across the mitochondrial membrane in necessary for steroid synthesis to occur.
Ghosh et al. (1987) suggested that cholesterol transport from the outer to the inner
mitochondrial membrane is the rate-limiting step in steroid synthesis. However, until
recently the transport process was an enigma. Clark et al. (1994) demonstrated that a LHinduced regulatory protein was responsible for cholesterol transport into the mitochondria,
and proposed to call this protein the "steroidogenic acute regulatory protein" (StAR).
Further investigations suggested that mRNA expression of StAR was closely associated
with steroid production in mice (Clark et al., 1995), thus, considerably strengthening the
evidence that StAR is a key mediator of steroidogenesis. Presence of mRNA for StAR
protein declined in hypophysectomized ewes; however, treatment with LH rescued StAR
protein expression to concentrations not different from those of control ewes (Juengel et
al., 1995). Finally, the human ovary expresses mRNA for StAR, which was induced by 8bromo-cAMP, a cyclic nucleotide analogue, suggesting a signal transduction link to LH
(Kiriakidou et al., 1996). Cholesterol transported by StAR reaches the inner mitochondrial
membrane, the site of P4508. This enzyme converts cholesterol to pregnenolone, which
is then changed in the smooth endoplasmic reticulum to progesterone by 313-HSD. The
resulting final product is then secreted, because the capacity of the luteal cell to store
progesterone is limited (Niswender and Nett, 1994).
The basal synthesis and secretion of progesterone, regardless of species, appears to
be the result of LH stimulation. In small luteal cells, progesterone secretion increases
31
greatly when stimulated by LH in sheep and rats (Fitz et al., 1982; Nelson et al., 1992),
and ovine luteal LH receptor mRNA is highest during the midluteal phase of the cycle
(Smith et al., 1996). Luteinizing hormone binds to a G-protein coupled receptor in the
plasma membrane, and activates adenylyl cyclase. Activation of adenylyl cyclase increases
the intracellular second messenger, cAMP, which stimulates protein kinase A (PKA).
Marsh (1975) suggested that cAMP is the intracellular signal by which LH activates the
small luteal cell. The activated catalytic subunit of PKA then uses Mg*ATP to
phosphorylate various regulatory proteins (enzymes) within the cell.
Luteinizing hormone may stimulate progesterone secretion through changes in
PKA activation, cholesterol transport, or uptake of cholesterol from lipoproteins.
Cholesterol esterase, which releases cholesterol from pools of esterified fatty acids, is
activated by cAMP-dependent protein kinase (Caffrey et al., 1979). Although cholesterol
esterase appears to be a target for LH-induced regulation of steroidogenesis, other
steroidogenic enzymes (P450scc and 3r3-HSD) are apparently not regulated by LH
(Wiltbank et al., 1993).
Although LH induces progesterone secretion from small luteal cells, LH has very
little effect on large luteal cell secretion of this steroid in ewes (Fitz et al., 1982; Nelson et
al., 1992). Furthermore, incubation of ovine large luteal cells with LH does not increase
intracellular cAMP concentrations or secretion of progesterone (Hoyer et al., 1984).
However, large luteal cells secrete the majority of luteal phase progesterone in ewes
(Niswender et al., 1985), and tend to secrete progesterone at a higher rate than small
luteal cells under basal conditions (Fitz et al., 1982; Alila et al., 1988; Nelson et al., 1992).
32
Mechanisms responsible for the LH-independent secretion of progesterone by large luteal
cells are not well understood, but possible explanations have been proposed. Lucy et al.
(1993) demonstrated that growth hormone receptors (GHr) were present on bovine large
luteal cells, suggesting that GH may enhance steroidogenesis in these cells. However, this
appears to be species specific, as highly specific expression of GHr was not detected in
porcine cells (Yuan and Lucy, 1996). As previously mentioned, large luteal cells contain
prostaglandin receptors (Fitz et al., 1982; Feng and Almond, 1996), suggesting a role for
prostaglandin regulation of large luteal cells. Richards et al. (1994) demonstrated that
prostaglandin E2 (PGE2) stimulates progesterone production from porcine large luteal cells
but not small luteal cells. Interestingly, the LH surge stimulates PGF2a receptor mRNA
expression in bovine corpora lutea (Tsai et al., 1996), however, mRNA expression was
not defined to luteal cell type. Differential regulation of progesterone production by small
and large luteal cells may occur to ensure that systemic concentrations of progesterone are
more than adequate for establishment and maintenance of pregnancy.
Luteotropins
The common definition of a "tropic" hormone is defined as "influencing the
activity of a specified gland". Thus, in the case of the corpus luteum, the primary
"luteotropic" hormone in most species is LH. As previously discussed, LH is important in
ovulation and luteinization. However, the role of LH in regulating luteal function has been
an active area of research for many years. In what follows an attempt will be made to
describe data of studies demonstrating a pituitary origin for luteotropins in various species.
33
In the cow, LH was first suggested to be luteotropic by Simmons and Hansel
(1964). In these early studies, the estrous cycle was prematurely shortened by injections
of oxytocin on days 2 to 6 after estrus, so the effect of gonadotropin preparations could be
observed on luteal weights and progesterone contents. The results suggested that LH or
LH-containing pituitary preparations were capable of overcoming the inhibitory effects of
oxytocin on the corpus luteum. A subsequent study, demonstrated that pure bovine LH
injected into cows on day 16 extended the cycle from 20 days in control animals to 36
days in LH-treated animals, and this effect was lost when LH was heated with 6 M urea to
denature this compound (Donaldson and Hansel, 1965). Finally, anti-bovine LH
administered on days 2 to 6 of the cycle to remove endogenous plasma LH, reduced luteal
weights and progesterone contents in intact heifers (Snook et al., 1969). Thus, without
doubt, many studies demonstrated the necessity of LH for maintenance of the corpus
luteum in the bovine.
In ewes, hypophysectomy on the day of ovulation prevented the formation of the
corpus luteum, while hypophysectomy on day 5 resulted in regression of the partially
formed corpus luteum (Kaltenbach et al., 1968a). Similarly, hypophysectomy of pregnant
ewes on days 20 to 23 of gestation resulted in corpus luteum regression and reabsorption
of the embryos and membranes 7 days later. However, treatment of hypophysectomized
ewes with a mixture of LH-FSH maintained pregnancy until days 27 to 29 of gestation
(end of the study) in these animals (Kaltenbach et al., 1968b). Hypophysectomy on day 5
resulted in fewer small luteal cells than in controls, but no change in the number of large
luteal cells was observed (Farin et al., 1990). Apparently, an LH-deprived corpus luteum
34
is incapable of a normal pattern of growth and development, but continues to function for
a week in the absence of this gonadotropin. Conversely, intact ewes continuously infused
with ovine LH beginning 10 to 12 days after estrus, prolonged the life span of the corpus
luteum until infusion was terminated on day 30 after ovulation (Karsch et al., 1971b).
Considerably less information is available about the role of LH as a luteotropin in
the sow and mare. Spies et al. (1967) treated pregnant gilts on days 25 to 29 of gestation
with anti-ovine LH and demonstrated the complete loss of embryos as well as corpus
luteum regression due to treatment. However, these researchers were unable to cause
luteal regression in non-pregnant gilts after treatment with the same antiserum on days 7
to 11 of the estrous cycle. Furthermore, corpora lutea developed after hypophysectomy
(du Mesnil du Buisson and Leglise, 1963) or hypophysial stalk transection (Anderson et
al., 1967) at estrus in gilts, but were smaller than those of controls at day 12 postestrus.
Interestingly, exogenous estradiol -1713 has been demonstrated to maintain the corpora
lutea of cyclic gilts (Gardner et al., 1963; Garbers and First, 1969). Thus, it seems likely
that follicular estradiol may act locally or in combination with LH as the luteotropic
hormones in the sow. In the mare, the corpus luteum regresses after treatment with
antiserum against a gonadotropin fraction of equine pituitary extracts (Pineda et al., 1972),
suggesting a role for LH in the maintenance of the corpus luteum for horses.
In the rat and mouse it appears that luteal function during the estrous cycle is
minimally dependent upon hypophyseal gonadotropins. Hypophysectomy of the rat
shortly after ovulation does not alter normal life span of the corpora lutea during the cycle
(Smith, 1930). However, the anterior pituitary hormone PRL is luteotropic in
35
hypophysectomized rats when administered within 56 hours after an ovulatory injection of
LH (Malven, 1969). Interestingly, this study further demonstrated that PRL was luteolytic
if it was not administered within 80 hours after ovulation. Therefore, the effects of PRL
appear to depend on the age of the corpora lutea. In the case of the hamster, a
"luteotropic complex" consisting of PRL, LH and FSH are required for normal
progesterone secretion (Grady and Greenwald, 1968).
The rabbit, having no cycle, is an induced-ovulator. Thus, the ovulatory surge of
LH occurs as a result of cervical stimulation. Uniquely, in this species it appears that LH
functions to cause ovulation only, as 50 lig of LH administered on day 9 of
pseudopregnancy caused luteal regression (Stormshak and Casida, 1964). However, the
presence of follicular estradiol is essential for maintenance of luteal function (Hilliard,
1973). Removal of follicular estradiol by way of x-irradiation (Keyes and Armstrong,
1968), or luteinization in response to exogenous LH (Kelly and Stormshak, 1969), results
in corpus luteum regression. The rabbit corpus luteum is richly endowed with estrogen
receptors suggesting that the effect of estrogen is mediated via a genomic pathway (Lee et
al., 1971).
Finally, pituitary luteotropins in the bitch have been investigated. Luteinizing
hormone was demonstrated to be involved with ovulation and progesterone synthesis in
this species (Wildt et al., 1979). Concannon (1980) examined the importance of LH as a
luteotropin in the dog. In this study, bitches hypophysectomized 10, 35, 40, 45 and 50
days postestrus had declining systemic progesterone concentrations within 3 to 17 days of
surgery. Importantly, plasma progesterone concentrations were increased within 3 hours
36
after subsequent treatment with LH. More recently, however, the role of PRL as a
luteotropin in the bitch was investigated by Onclin and Verstegen (1997). In this study,
cabergoline, a dopamine agonist inhibitory to PRL release was injected into pregnant and
non-pregnant bitches 30 days after the LH surge. Mean plasma PRL, and subsequently
progesterone concentrations, were decreased for 4 to 5 days following treatment,
suggesting the luteotropic importance of PRL in dogs. However, exogenous PRL given
twice daily on days 30 or 40 of gestation did not induce progesterone secretion in this
species. Thus, the bitch has two luteotropins, LH and PRL, which appear to work in
concert to maintain the corpus luteum and stimulate progesterone production.
Prolactin
As previously mentioned, PRL has been demonstrated to be luteotropic in rats and
dogs (Wildt et al., 1979; Concannon, 1980; Niswender et al., 1985; Onclin and Verstegen,
1997). However, very little evidence exists for the role of PRL as a luteotropin in most
domestic animals. In fact, plasma PRL concentrations rise slightly at the time of estrus,
but no marked changes have been correlated with corpus luteum function in heifers
(Swanson and Hafs, 1971). McCracken et al. (1971) infused prolactin into the ovarian
artery of luteal phase ewes and were unable to stimulate progesterone secretion.
Furthermore, infusion of intact ewes with PRL failed to prolong the life span of the corpus
luteum (Karsch et al., 1971a). Interestingly, PRL maintained progesterone secretion by
aged corpora lutea in hysterectomized gilts treated from days 110 to 120 postestrus
(Felder et al., 1988). A subsequent study by Li et al. (1989) demonstrated that PRL
37
maintains progesterone secretion as well as morphology of aging corpora lutea for at least
10 days after hypophysectomy in hysterectomized gilts. Thus, PRL appears to have a
luteotropic role in pigs.
Maternal Recognition of Pregnancy
In the event of a pregnancy, a luteotropic signal is necessary to inhibit the demise
of the corpus luteum, thus ensuring a continuous production of progesterone to maintain
the proper uterine environment for the developing embryo. However, unlike during the
cycle, the luteotropins of pregnancy are from an embryological source. The phrase
"maternal recognition of pregnancy" has been used to describe how the mother responds
to the presence of the conceptus within her, and importantly how the conceptus acts to
maintain corpus luteum function. There are species differences in the methods utilized for
maintaining a functional corpus luteum, therefore a species by species approach is taken to
define these differences.
The primate and rodent have the most invasive placentas. When the blastocyst
hatches from the zona pellucida, it immediately attaches to the uterine wall. Thus,
implantation can occur and placental membranes develop connecting the maternal and
fetal blood supply. In the primate, the corpus luteum is maintained due to the trophoblast
secretion of chorionic gonadotropin (CG), which is very similar to LH and also classified
in the same group of glycoprotein hormones. Secretion of CG occurs by days 7 to 9 of
pregnancy, at the time of implantation (Hearn et al., 1991), and is detectable in the
maternal circulation by days 8 to 11 (Lenton et al., 1982). The human placenta is capable
38
of producing enough progesterone to support the pregnancy after the first month of
gestation (Auletta and Flint, 1988). Thus initially, CG appears to be essential to sustain
luteal progesterone concentrations (Hodgen and Itskovitz, 1988).
The rat and primate both have a hemochorial discoid placenta, however, the
luteotropins necessary for pregnancy maintenance of the corpus luteum are different.
Gestation length in the rat is 22 days, however, an infertile mating will result in a
pseudopregnancy of about 12 to 13 days. Thus, the corpus luteum of pseudopregnancy
regresses well before parturition would normally occur in a pregnant rat, suggesting the
role of a luteotropin in both early pregnancy and the second half of gestation. Prolactin is
the luteotropin of early pregnancy (Morishige and Rothchild, 1974), rescuing the corpus
luteum of the estrous cycle from regression. However, these researchers demonstrated
that the corpus luteum is dependent on PRL for only the first 7 days of early pregnancy.
The second half of pregnancy in rats is dependent on a few placentally derived hormones.
Placental luteotropin increased uterine weight on day 12 in pseudopregnant
hypophysectomized rats (Linkie and Niswender, 1972), which coincides with the time of
regression in the pseudopregnant animal. Rat placental lactogen (rPL), isolated from
placental tissue and uterine decidua, displays prolactin-like activity (Kelly et al., 1975;
Robertson and Friesen, 1975), and was demonstrated to be detectable in serum on days 8
thru 15 (Kelly et al., 1975; Robertson et al., 1982). The rat placenta also secretes a
luteotropin with LH-like activity, rat chorionic gonadotropin (rCG), which is not secreted
by the decidua (Jayatilak et al., 1984). Finally, intraluteal estrogen has been demonstrated
to be important for elevated serum concentrations of progesterone during the second half
39
of pregnancy (Gibori and Keyes, 1978). However, the rat corpus luteum only produces
large quantities of estrogens in the presence of androgens (Gibori and Keyes, 1978), which
are elevated during days 12 to 22 of gestation (Gibori et al., 1979). Gibori and Sridaran
(1981) hypophysectomized and hysterectomized pregnant rats to determine the source of
late pregnancy androgens. These data demonstrated the placenta to be the source of
androgens converted by the ovaries and fetuses to estrogens during the second half of
pregnancy. Thus, for the rat a series of placental substances replace pituitary PRL of early
pregnancy (and pseudopregnancy) at about mid-gestation, providing the necessary signals
needed in maternal recognition of pregnancy (Roberts et al., 1996).
Corpora lutea of the sow are the only source of progesterone for maintenance of
pregnancy, and progesterone replacement in ovariectomized sows is sufficient for
pregnancy to continue (Ellicott and Dzuik, 1973). Thus, to prolong the life span of the
corpus luteum during pregnancy, a luteotropin is necessary. Estrogen released by the
trophoblast as it elongates is probably the signal for maternal recognition of pregnancy in
this species. As previously mentioned, estrogen is luteotropic during the estrous cycle of
the sow. However, exogenous estrogens have been demonstrated to extend luteal
function for several months in non-pregnant sows after only 5 days of treatment (days 11
to 15; Gardner et al., 1963; Frank et al., 1977). Relative to luteal maintenance, treatment
with estrogens is similar to the effects of estrogen synthesized by the conceptus as it
begins to elongate. In the sow, the conceptus synthesizes and secretes estrogens by day
12 of pregnancy (Gadsby et al., 1980; Heap et al., 1981). Thus, the corpus luteum of
40
early pregnancy appears to be maintained, at least initially, by a conceptus source of
estrogens.
Maternal recognition of pregnancy in the mare also involves the conceptus. As the
conceptus grows, it remains spherical, and the trophoblast does not elongate like in the
sow (Roberts et al., 1996). However, the conceptus must come in contact with the entire
endometrium to prevent luteal regression (Niswender and Nett, 1994). Thus, during the
critical period for recognition of pregnancy (days 12 to 14), the embryo migrates
throughout the uterus to establish contact with the endometrium (Ginther, 1993). The
embryo traverses the entire uterus about every 2 hours during the recognition period
(Leith and Ginther, 1984). Although embryo migration involves the secretion of some
factor that inhibits luteal regression, the factor(s) remains unknown (Roberts et al., 1996).
Goff et al. (1993) have suggested that 17a-hydroxyprogesterone, the major steroid
synthesized by the equine blastocyst, may have a role in pregnancy recognition because it
is a prominent metabolite during this critical period. The horse conceptus does secrete
chorionic gonadotropin (eCG), which appears in the maternal serum at about day 35 of
gestation (Squires and Ginther, 1975). Equine CG induces premature ovulation of
developing follicles, producing secondary corpora lutea, which aid in progestational
support of the pregnancy. Thus, eCG is luteotropic but obviously not responsible for
recognition of pregnancy in the mare.
The conceptuses of the ewe and cow are well known to secrete an antiluteolytic
trophoblast protein, responsible for pregnancy recognition. Through molecular cloning
techniques, bovine trophoblast protein-1 (bTP-1) was found to have 89.7% homology
41
with ovine trophoblast protein-1 (oTP-1), and an amino acid sequence homology of
80.4% (Imakawa et al., 1989). Thus, because bTP-1 and oTP-1 are similar trophoblast
interferons, Roberts et al. (1992) recommended the common nomenclature for ruminant
trophoblast interferon, as interferon tau (INFT). Thus, for ease of explanation the
identification and characterization of INFT for both species will be discussed together.
In ewes, early investigations established a window of early pregnancy whereby the
embryos presence was necessary to extend the life span of the corpus luteum (Moor and
Rowson, 1966a,b). In these studies, removal of the embryo before day 12 resulted in
luteal regression. However, embryos removed after day 13 did not initiate immediate
luteal regression, which did ultimately occur. Thus, maternal recognition of pregnancy
occurs during days 12 to 13 in the ewe. Similarly, the critical period in cows was
determined to be days 16 to 17 (Betteridge et al., 1980; Northey and French, 1980). The
importance of the embryo was further demonstrated when homogenates of day 14 to 16
ovine conceptuses (Ellinwood et al., 1979a; Martal et al., 1979), or 17 to 18 day-old
bovine conceptuses ( Northey and French, 1980), were injected into the uterine lumen of
these animals. These studies demonstrated that conceptus homogenates prolonged luteal
function when injected into the uterine lumen. Martal et al. (1979) suggested that the
substance secreted by the conceptus was a protein, because this component was no longer
functional after heating or proteolytic digestion.
In 1982, Godkin et al. described the purification of a low molecular weight protein
released into culture medium by day 13 to 21 ovine conceptuses. This secretory protein
had a molecular weight of about 17,000, and was soon designated ovine trophoblast
42
protein-1 (Godkin et al., 1984a). Ovine trophoblast protein-1 (oTP-1) extended the life
span of the corpus luteum when infused into the uteri of non-pregnant ewes (Godkin et
al., 1984b; Vallet et al., 1988). Vallet et al. (1988) also demonstrated that when this
protein was removed from a pool of conceptus secretory products by immunoadsorption,
infusion of the resulting products failed to increase the interestrous interval. Thus, oTP-1
and the discovery of a similar protein in cows (bTP-1; Bartol et al., 1985), were
established as antiluteolysins.
Unlike CG of primates, these TP's are secreted into the lumen of the uterus and do
not enter the maternal circulation nor do they have a direct luteotropic effect (Godkin et
al., 1984a). These authors also demonstrated that incubation of oTP-1 with endometrial
explants from day 12 non-pregnant ewes, resulted in increased protein release into the
incubation medium. Thus, these proteins appear to have a local effect on the uterus
(Bazer et al., 1986). The luteolysin PGF2a is secreted by the uterus and enters the
circulatory system through the uterine vein. Inskeep et al. (1975) suggested that a factor
from the conceptus was capable of overcoming the luteolytic effect of PGF2a when mated
ewes did not return to estrus after injection of this luteolysin. Progesterone concentrations
in pregnant ewes treated with PGF2a between days 10 and 30 were different depending on
the day of treatment (Silvia and Niswender, 1986). Specifically, progesterone
concentrations in pregnant ewes on days 13 and 16 initially declined within 24 hours
following PGF2a treatment, however, serum progesterone returned to pre-treatment
concentrations 36 hours later. Conversely, pregnant ewes treated with PGF2c, on other
days (10, 19, 22, 26 and 30) did not return to pre-treatment concentrations, suggesting
43
that the embryo provides a temporary resistance to luteal regression. Interestingly, the
concentrations of PGF2a are higher in pregnant ewes than non-pregnant ewes (Peterson et
al., 1976). Zarco et al. (1988a) investigated the mode of PGF2a secretion at the time of
luteolysis or maintenance of pregnancy in the ewe. In this study, pulse frequencies and
serum concentrations of prostaglandin F24 metabolite (PGFM) were measured in pregnant
and non-pregnant ewes from days 12 to 17 post-estrus. Basal concentrations were higher
in pregnant ewes on days 13 to 17; however, the pulse frequencies of PGF2c, were lower in
these ewes. Thus, modification of pulsatile release of PGF2c, appeared to be an important
difference that may be linked to luteal maintenance during pregnancy. Fincher et al.
(1986) demonstrated that day 12 to 14 uterine infusions of conceptus secretory proteins
suppressed the estradiol-induced rise in PGFM concentrations in non-pregnant ewes, and
this suppression was similar to that of estradiol-treated pregnant ewes. In any case, the
secretion of oTP-1 by the conceptus seems likely to be responsible for the altered pattern
of PGF2c, secretion in pregnant ewes (Godkin et al., 1982; Godkin et al., 1984b; Fincher et
al., 1986; Vallet et al., 1988).
The ovine and bovine conceptus also secretes PGF,c, and PGE2 as well as IFM
(LaCroix and Kahn, 1982; Findlay et al., 1983). LaCroix and Kann (1982) determined
that PGE2 synthesis from incubated endometrium was twice as great in pregnant than non-
pregnant ewes. These data established the conceptus as another source of this eicosanoid,
which has been observed to be elevated during the critical period for maternal recognition
of pregnancy, and suggested it to be the factor responsible for prolonging the life span of
the corpus luteum (Ellinwood et al., 1979b). In support of this possibility, PGE2 has been
44
demonstrated to be antiluteolytic when infused into the uterine lumen of non-pregnant
ewes (Pratt et al., 1977; Magness et al., 1981; Reynolds et al., 1981).
The mechanisms by which IFNt alters the secretion of PGF2a is still under
investigation. Mirando et al. (1990) demonstrated that ovine conceptus proteins blocked
oxytocin stimulation of inositol triphosphate (1P3) production, the second-messenger for
oxytocin-stimulated secretion of PGF2, in ovine endometrium. These data suggested a
role for IFNT in the inhibition of oxytocin receptor formation, revealing a need for further
investigation into the oxytocin receptor changes associated with pregnancy. Ovine
conceptus secretory proteins injected twice daily from days 11 to 15 post-estrus, reduced
endometrial receptor concentrations for estrogen, progesterone and oxytocin in cyclic
ewes (Mirando et al., 1993b). Spencer and Bazer (1995) investigated the tissue-specific
expression of these steroid hormone receptors in both cyclic and pregnant ewes. Results
of this research illustrated a pattern of receptor expression that varied between pregnant
and non-pregnant ewes. Specifically, endometrial estrogen receptor (ER) mRNA and
progesterone receptor (PR) mRNA in cyclic ewes increased between days 11 to 15 and 13
to 15, respectively. Endometrial PR mRNA similarly increased between days 11 to 17 in
pregnant ewes. However, endometrial ER mRNA expression decreased between days 11
to 15 in pregnant ewes. Therefore, endometrial expression of ER mRNA is specifically
suppressed during the early stages of pregnancy, while PR mRNA displays a similar
pattern in both cyclic and pregnant ewes. Spencer et al. (1995a) demonstrated that IFNt
infused into ovariectomized ewes treated with steroids did not stabilize PR mRNA or
protein expression in the endometrium. However, IFNt did suppress endometrial ER
45
mRNA expression and oxytocin receptor formation. Because estradiol treatment
increased oxytocin receptor formation in ovariectomized control ewes (no IFNt), Spencer
et al. (1995b) infused cyclic ewes with IFNt beginning on day 10 post-estrus and
subsequently injected estradiol benzoate on day 12. Results of this study demonstrated
that ovine IFNt inhibited the estradiol-induced increase in ER mRNA and oxytocin
receptor formation, suggesting that IFNt prevents luteolysis in pregnant ewes by down-
regulating these receptors. Finally, IFNt has been suggested to suppress ER transcription
through a negative-acting transcriptional mechanism which prevents the estradiol-induced
increases in endometrial oxytocin receptor expression (Spencer and Bazer, 1996). Thus,
exposure to IFNt differentially inhibits endometrial receptor expression, apparently
altering the uterine response to endocrine signals, and ultimately resulting in maternal
recognition of pregnancy.
Luteolysis
A cycle is defined as, "an interval of time during which a sequence of a recurring
succession of events or phenomena is completed." If an animal does not become
pregnant, the reproductive cycle must come to an end so that a nonpregnant animal can
return to a potentially fertile state. Because the corpus luteum is the transient endocrine
organ that appears and disappears in each cycle, the regulation of this organ dictates the
cycle length. Previously, I have discussed the luteotropins that maintain the corpus
46
luteum. Obviously in the absence of an embryo there must be luteolysins, which cause the
demise of the corpus luteum, and terminate the cycle.
A uterine factor has been demonstrated to be involved in luteolysis in cattle, sheep
(Wiltbank and Casida, 1956), pigs (Spies et al., 1960), horses (Stabenfeldt et al., 1974)
and pseudopregnant rats (Butcher et al., 1969). In these studies, animals were
hysterectomized to determine the effect of the uterus on the life span of the corpora lutea.
In all cases, hysterectomy prolonged the life span of the corpus luteum. However,
hysterectomy does not extend the life span of corpora lutea in primates (Castracane et al.,
1979) and dogs (Hadley, 1975). These data suggest that the uterus does not play an
active role in luteolysis of all species.
Although hysterectomy suggested that the uterus produced a luteolysin of general
systemic nature, many studies have demonstrated that the effects of the uterus on luteal
life span are exerted locally. Inskeep and Butcher (1966) demonstrated that unilaterally
hysterectomized ewes did not display the same pattern of corpora lutea regression as
observed in the completely hysterectomized ewe. Instead, these authors discovered that
the presence of one uterine horn was sufficient to cause luteal regression in the adjacent
ovary, while luteal regression did not occur in the ovary adjacent to the hysterectomized
horn. These effects of hysterectomy were subsequently confirmed in cattle (Ginther et al.,
1967). However, no effect of unilateral hysterectomy has been demonstrated in rabbits
(Hunter and Casida, 1967) and horses (Ginther and First, 1971). In these latter studies,
there was no significant evidence of differences in local action of the uterine horn on
corpora lutea maintenance or regression. Results of these investigations suggest that in
47
some species with bipartite or bicornuate uteri a factor produced within the uterine horn
must act locally to regulate luteal life span.
A mechanism for transport of a uterine derived luteolysin to the ovary has been
established for the ewe and cow. Although no portal system exists between the uterus and
ovary, blood vessels supplying the anterior portion of the uterine horn were ligated above
and beside the ovary in ewes (Inskeep and Butcher, 1966), and cows (Hixon and Hansel,
1974), resulting in maintenance of the corpus luteum. Ligation of the uterine vein draining
the intact horn and reconnection to the remaining portion of the uterine vein of the
hysterectomized horn resulted in luteal regression of those corpora lutea in the ovary
adjacent to the removed horn in ewes (Ginther et al., 1973), and cows (Mapletoft et al.,
1976). Similarly, this investigation demonstrated that the ovarian artery adjacent to the
uterine intact side could be ligated and reconnected to the ovarian artery on the
hysterectomized side to cause luteal regression. Thus, although no direct vascular
connections exist between the ovary and uterus, the luteolytic factor appears to pass
directly from the uterine vein to the ovarian artery (Ginther, 1974). Anatomically, the
ovarian artery of sheep and cattle is convoluted and in close apposition to the uterine vein.
Thus, a local counter-current transfer of the uterine luteolysin, (found in high
concentrations in the uterine vein) can cross into the ovarian artery (area of low
concentration) to affect the corpus luteum (Stabenfeldt, 1992). Interestingly, the rabbit
and horse do not have extensive contact between the uterine vein and ovarian artery
(Ginther, 1974). This anatomical difference provides an explanation for the previously
48
mentioned exception that in these two species there is no effect of unilateral hysterectomy
on corpus luteum function (Hunter and Casida, 1967; Ginther and First, 1971).
Prostaglandin F2,
Phariss and Wyngarden (1969) were first to demonstrate that the abundant uterine
prostaglandin, PGF2a, could be administered in high doses (1 mg kg-1
day') to rats and
cause a significant shortening of pseudopregnancy by luteolysis. This breakthrough in
research led to subsequent studies, the results of which demonstrated that PGF2a was
luteolytic in sheep (McCracken et al., 1970; Thorburn and Nicol, 1971), cattle (Rowson et
al., 1972), horses (Allen and Rowson, 1973), pseudopregnant mice (Bartke et al., 1972),
hamsters (Harris and Murphy, 1981), rabbits (Scott and Rennie, 1970), and to a limited
extent in swine (Gleeson, 1974). In these studies, PGF2a was demonstrated to be
luteolytic when infused into the uterine vein, uterine body or ovarian artery, supporting the
idea of a counter-current mechanism of transfer. However, luteolysis only occurs after
prolonged treatment with high concentrations of PGF2a in primates (Auletta et al., 1984)
and dogs (Concannon and Hansel, 1977). The uterus has no luteolytic effect in these
species, suggesting that if PGF2a is luteolytic in these animals it must arise elsewhere in the
body. Prostaglandin Fla was further confirmed to be the luteolytic factor when the
prostaglandin inhibitor, indomethacin, was demonstrated to block spontaneous luteolysis
in cattle and sheep (Lewis and Warren, 1977). These data document the importance of
synthesis and secretion of PGF2a in luteolysis. Finally, active and passive immunization
against PGF2a was demonstrated to prevent luteal regression in sheep (Scaramuzzi and
49
Baird, 1976) and cattle (Fairclough et al., 1981). Together, these studies support a role
for PGF2a as the luteolysin for large domestic species and rodents.
Mechanisms of action for PGF2a
Progesterone concentrations decrease during luteolysis and this decline has been
correlated with increasing concentrations of PGF2a (McCracken et al., 1972). Thus, the
effect of PGF2a on the luteal cells has been investigated to elucidate the mechanism by
which decreased steroidogenesis occurs. As previously mentioned, large luteal cells
possess most of the receptors for PGF2a (Fitz et al., 1982). In these cells, PGF2a binds its
receptor and activates the signal transduction pathway involving protein kinase C (PKC;
Leung et al., 1986), which phosphorylates further proteins including an actin-binding
protein called myristoylated alanine-rich C kinase substrate (MARCKS; Orwig and
Stormshak, 1994). Protein kinase C can be activated by the phorbol ester, phorbol 12myristate 13-acetate (PMA), which has been demonstrated to inhibit the production of
progesterone in ovine large and LH-stimulated small luteal cells (Wiltbank et al., 1989b).
McGuire et al. (1992) examined the in vivo effect of PMA by treating ewes with the
phorbol ester on day 10 postestrus and found a rapid reduction in serum concentrations of
progesterone. Thus, the effects of phorbol esters in vitro and in vivo appear to be similar.
Wiltbank et al. (1989a) demonstrated that in vitro treatment with PGF2a increased free
calcium concentrations in ovine large but not small luteal cells. The increase in
intracellular calcium concentrations was dose- and time-dependent, suggesting calcium is a
second messenger mediating the luteolytic action of PGF2a. Prostaglandin Fa, has also
50
been suggested to have a direct cytotoxic effect on luteal cells (Fitz et al., 1984), which
appears to be mediated by intracellular calcium concentrations (Sawyer et al., 1990).
However, intracellular signaling may effect steroid biosynthesis. Pate and Condon (1989)
treated bovine luteal cells with PGF2a and demonstrated a decrease in de novo cholesterol
synthesis. Hawkins et al. (1993) treated ewes with PGF2a on day 10 postestrus to
determine how steady state levels of mRNA encoding 3P-HSD changed in the corpus
luteum. These researchers found that corpora lutea collected 1 or 8 hours after PGF2c,
injection had decreased (>80% reduction) expression of 3P-HSD compared with controls.
Murdoch et al. (1996) proposed a possible mechanism by which steroidogenic enzymes,
ultimately responsible for progesterone synthesis and luteal function, are inactivated by
PGF2a. These investigators measured quantities of mRNA for ubiquitin, an early marker
protein of cell death that binds other cellular proteins targeting them for degradation, in
corpora lutea collected from ewes 2 or 16 hours following treatment with PGF2.. Results
of this study demonstrated that prostaglandin Fla induced ubiquitin expression in large
luteal cells before the precipitous decline of progesterone characteristic of luteolysis.
These data suggest that PGF2c, disrupts progesterone biosynthesis by the ubiquitination of
steroidogenic regulatory enzymes. Because LH induces progesterone secretion from small
luteal cells, PGF2c, regulation of LH receptor (LHr) and LHr mRNA expression has been
investigated. The number of LHr binding sites present in the ovine corpora lutea decrease
during luteolysis (Diekman et al., 1978a). In a subsequent study, these authors
demonstrated that the reduction in LHr binding sites at luteolysis is due to PGF2e, secretion
(Diekman et al., 1978b). Smith et al. (1996) demonstrated that LHr mRNA content in day
51
10 ovine corpora lutea collected after PGF2a-induced luteolysis, decreased by 6 hours after
treatment and were approximately 60% of control concentrations within 24 hours. In any
case, the cellular and molecular actions of PGF2a ultimately decrease the synthesis and
secretion of progesterone that occurs during luteolysis.
Prostaglandin Fla may stimulate factors from endothelial cells that effect
steroidogenesis. As previously mentioned, angiogenesis occurs at the time of corpora
lutea formation. Thus, endothelial cells, the most abundant cell type in the corpora lutea,
are present throughout the life span of the corpus luteum, possibly mediating the effects of
hormones on the large and small luteal cells. Endothelial cells have been demonstrated to
produce a family of peptides called endothelins (Levin, 1996). Girsch et al. (1995)
examined the luteolytic effect of PGF2a on bovine large luteal cells cultured alone or with
endothelial cells in the presence of forskolin, an activator of adenylyl cyclase.
Prostaglandin Fa, inhibited forskolin activity only when incubated in co-cultures,
suggesting an endothelial cell involvement in paracrine regulation of steroidogenic
function. A subsequent study demonstrated that PGF2a inhibited progesterone production
from bovine luteal cells and concomitantly stimulated the production of an endothelial cell
factor, endothelin-1 (ET-1; Girsh et al., 1996a). Prostaglandin F2, inhibition of
progesterone only occurred in cultures with both luteal and endothelial cells. Girsh et al.
(1996b) also examined the regulation of ET-1 in the bovine corpus luteum by PGF2a.
Expression of ET-1 mRNA was demonstrated to be highest during luteolysis, which was
further confirmed when intact cows were treated with PGF2a on day 10 postestrus.
Collectively, these studies suggest a role for ET-1 in PGF2a- induced luteolysis.
Although
52
the mechanism of ET-1 action remains unclear, these latter researchers suggested that
PGFacinduced ET-1 production causes hypoxic conditions that lead to luteal regression
by causing constriction of the arterioles supplying the corpora lutea. Further, it has been
hypothesized that binding of ET-1 to luteal cells may mediate PGF2einduced changes in
steroidogenesis that decrease progesterone production around luteolysis.
Pulsatile Requirement of PGF2c,
Interestingly, PGF2a is released from the uteri of ewes (Barcikowski et al., 1974),
and cows (Nancarrow et al., 1973) as a series of discrete pulses that first appear
immediately prior to the onset of luteal regression (Thorburn et al., 1972). Pulse duration
and magnitude are different among these species, but they typically occur at 6 to 8 hour
intervals. In sheep, pulses of uterine PGF2a release occur every 6 hours with the duration
of the pulse lasting about 1 hour (McCracken, 1984). The number of episodic pulses of
PGF2a in a 24 hour period appears to be critical in causing luteolysis. Schramm et al.
(1983) demonstrated that four 1 hour pulses of PGF22. infused into the ovarian artery every
6 hours over a period of 19 hours on day 12 resulted in regression of corpora lutea in only
one out of four ewes. However, the addition of a fifth pulse within a 25 hour period
resulted in luteolysis in four out of four animals. Further, a single 1 hour pulse of PGF2a
was ineffective in causing luteal regression, and resulted in a temporary decrease in serum
progesterone concentrations. Thus, for physiological regression of the corpus luteum, a
relatively short pulse frequency of PGF2c, (every 6 hours) over the period of a day is
required.
53
Oxytocin
Secretion of PGF2a from the uterus during luteolysis, is stimulated by oxytocin
(Roberts and McCracken, 1976; Fairclough et al., 1980, 1984). Pulses of oxytocin or
neurophysin VII have been demonstrated to coincide with pulses of PGF2a during
luteolysis in sheep (Fairclough et al., 1980; Flint and Sheldrick, 1983), and cattle (Vighio
and Liptrap, 1986). Wathes and Swann (1982) discovered the presence of oxytocin in the
corpora lutea of ewes. This ovarian source of oxytocin contributes to the serum
concentrations of oxytocin during the luteal phase of the estrous cycle (Sheldrick and
Flint, 1981; Flint and Sheldrick, 1982). Specifically, Rodgers et al. (1983) concluded that
the ovine large luteal cells almost exclusively store oxytocin over small luteal cells, and
these cells are capable of oxytocin synthesis in vitro. Further, the use of
immunohistochemistry and electron microscopy localized oxytocin-neurophysin peptide to
the secretory granules of ovine and bovine large luteal cells (Fields and Fields, 1986),
suggesting that this luteal peptide is secreted by exocytosis.
Ivell and Richter (1984) sequenced the bovine oxytocin gene products of luteal
and hypothalamic origin, and found both sources of the gene produced identical transcripts
except for a difference in polyadenylation. Expression of the luteal oxytocin gene has
been demonstrated to be highest early in the estrous cycle in cows and ewes (Ivell et al.,
1985; Fehr et al., 1987; Jones and Flint, 1988). Northern blot analysis revealed that luteal
oxytocin-neurophysin mRNA in the ewe increases during luteinization, reaches maximum
concentrations by day 3 and declines to low levels by day 9 of the estrous cycle (Jones and
Flint, 1988). Interestingly, this study measured maximal luteal oxytocin concentrations on
54
day 7. Thus in the ewe and cow (Fehr et al., 1987), a lag between peak mRNA and
oxytocin concentrations is present, and may be attributable to the time taken for posttranslational processing of the oxytocin-neurophysin prohormone.
Although oxytocin was known to induce PGF2c, secretion from the uterus, the
source of oxytocin was initially thought to be the posterior pituitary. Thus, with the
discovery of an alternative source of oxytocin (ovarian), experiments were conducted to
examine the regulation of luteal oxytocin secretion. Flint and Sheldrick (1983)
demonstrated through frequent plasma sampling that two-thirds of detected surges of
uterine secretion of PGF2a were accompanied by increased serum concentrations of
oxytocin. Further, ewes injected with cloprostenol (PGF2a analogue) on day 8 postestrus
had decreased serum concentrations of progesterone, followed 24 hours later with a
decrease in jugular oxytocin concentrations. Importantly, the corpus luteum was
considered to be a significant source of oxytocin when 98% of the available oxytocin was
secreted within 45 minutes of cloprostenol treatment.
Regulation of oxytocin production may be through estrogen as estrogen response
elements (ERE) and estrogen responsiveness have been demonstrated for the 5' flanking
region of the human and rat oxytocin gene (Richard and Zingg, 1990; Adan et al., 1991).
However, estrogen responsiveness appears to be species specific as the imperfect ERE in
the upstream promoter sequence of the bovine oxytocin gene does not respond to
estrogen (Adan et al., 1991). Recently, the oxytocin gene has been demonstrated to be
expressed in the uterine epithelium of the pregnant rat (Lefebvre et al., 1992). This
implies a paracrine or autocrine control of oxytocin on uterine oxytocin receptors. A
55
subsequent study investigated the role of ovarian steroids in the regulation of the oxytocin
gene (Lefebvre et al., 1994). This study demonstrated that exogenous estradiol increased
uterine oxytocin mRNA after 2 days of exposure in ovariectomized rats. Administration
of progesterone alone does not change the mRNA concentration, however, this steroid
increased oxytocin mRNA by sevenfold when given concomitantly with estradiol. These
data suggest that the rat oxytocin gene is regulated by steroids in vivo, and further that
progesterone and estradiol act synergistically to attain maximal expression of this gene. In
any case, with multiple sources of oxytocin available, the potential roles of uterine PGF2c,
and oxytocin in luteolysis appeared to involve a positive feedback loop.
Oxytocin-PGF2c, Feedback Loop
As with any positive feedback loop, activation must start at one site or the other.
Considering the functional relationship between the uterus and ovary, the question "what
comes first the pulse of PGF2a or oxytocin" was asked. Moore et al (1986) demonstrated
that concentrations of PGF2,, in utero-ovarian venous plasma temporally increased before
concentrations of oxytocin were detectable, suggesting that the initiation of the positive
feedback loop occurs in the uterus. However, oxytocin was only suggested to be the
signal that stimulates PGF2a secretion. Hooper et al. (1986) illustrated the importance of
pituitary oxytocin by collecting carotid artery and jugular venous plasma for analysis of
this hormone. In this study, pituitary and luteal releases of oxytocin occurred
simultaneously, and were frequently associated with pulses of PGF2a. Further, more
oxytocin pulses were detected in the jugular vein than the carotid artery or vena cava of
56
ewes on day 13 postestrus (Walker et al., 1997). Neurohypophyseal oxytocin is regularly
released, during the follicular phase of intact ewes and in ovariectomized ewes receiving
estrogen replacement, in episodic bursts lasting a few minutes and occurring at a
frequency of 3 pulses/hour (Silvia et al., 1991). These observations suggest a role for
estrogen in stimulating pituitary oxytocin release. Treatment of ovariectomized ewes with
estradiol provoked high frequency pulses of oxytocin release that occurred every 3 hours
beginning 24 hours after treatment, which led McCracken et al. (1991) to propose that
alteration in pulses of pituitary oxytocin is the signal that stimulates pulsatile secretion of
uterine PGF2, initiating the positive feedback loop.
Steroid Regulation
It is clear that the positive feedback loop is important for maintaining regular
estrous cycles in ruminants. However, this utero-ovarian feedback loop is not functional
for most of the estrous cycle. In fact, oxytocin stimulated release of uterine PGF2c, occurs
during only a short portion of the estrous cycle (Roberts and McCracken, 1976; Roberts
et al., 1976; Fairclough et al., 1984; Silvia and Taylor, 1989), and the uterine response to
oxytocin develops late in the estrous cycle as well. Thus, it is of no surprise that many
studies have investigated the regulation of this mechanism by steroids, peptides, embryos,
as well as many other factors.
57
Progesterone
Progesterone has been demonstrated to be important for the proper timing of
luteolysis (Ginther, 1968; Ottobre et al., 1980). Progesterone administered 8 and 32
hours postestrus shortened the estrous cycle to 11 days in treated ewes (Ottobre et al.,
1980) and this response was caused by premature secretion of PGF2,. This is supported
by the data of Scaramuzzi et al. (1977) who showed that treatment of ovariectomized
ewes with progesterone for 7 to 14 days restored normal serum concentrations of PGF2.
from low basal levels. Additionally, ovariectomized ewes treated with progesterone twice
daily to mimic the increasing concentrations of luteal phase progesterone, only responded
with an oxytocin-induced release of PGF2a after 10 days of treatment (Homanics and
Silvia, 1988). Apparently, the uterus has to be under a long period of progestational
influence (10 to 14 days) for spontaneous secretion of PGF2. to occur.
The effect of progesterone on the uterus may be to regulate the concentrations of
oxytocin receptors, which have been demonstrated to be present in the endometrium of
ewes during proestrus and estrus (Sheldrick and Flint, 1985). Vallet et al. (1990)
increased concentrations of endometrial oxytocin receptors in ovariectomized ewes after
progesterone treatment for 10 to 12 days. In ovariectomized cows treated with
exogenous hormones to mimic the pattern observed during the estrous cycle, prolonged
treatment with progesterone increased endometrial oxytocin receptor concentrations by
day 16 (Lamming and Mann, 1995). This rise in uterine oxytocin receptors was similar to
that observed during the luteal phase of intact cyclic cows. Further, Raw and Silvia
(1991) found that prolonged exposure of ovine endometrial tissue to progesterone in vivo
58
increased oxytocin-induced PKC activity, which stimulates prostaglandin secretions.
Thus, the presence of progesterone appears to be necessary to induce luteolysis. Leavitt
et al.
(1985)
infused ovariectomized ewes with progesterone for 5 days during continuous
infusion with estradiol -17p to examine changes in endometrial receptor concentrations. In
this study, the withdrawal of progesterone after 5 days of treatment increased endometrial
concentrations of estrogen, progesterone, and oxytocin receptors compared with controls
(continued progesterone infusion). Similarly, oxytocin receptor concentrations increased
2
days after progesterone withdrawal in ovariectomized ewes continuously treated for
days with this steroid (Zhang et al.,
1992).
9
Thus, progesterone appears to be suppressing
endometrial oxytocin receptor activity in ewes. Such an effect of progesterone is
consistent with an early proposal by McCracken et al.
(1984)
that this steroid regulated
estrogen and oxytocin receptors.
Results of research conducted by McCracken et al.
(1984)
suggested that
progesterone inhibits synthesis of receptors for estradiol, and that synthesis of oxytocin
receptors is an estrogen-dependent process. Accordingly, as long as progesterone
suppressed estrogen receptor concentrations, estradiol could not induce oxytocin receptor
synthesis. In general, the effects of progesterone appear to be either stimulatory or
inhibitory, but most likely progesterone acts congruently with other factors to regulate the
life span of the corpus luteum.
59
Estrogen
Endogenous estradiol also appears to affect the timing of luteolysis. However, the
response of various species to exogenous estradiol has varied depending on dose of
steroid administered, time of the estrous cycle administered and duration of treatment.
Supraphysiological doses of estradiol -17(3 injected into intact ewes beginning on day 4
postestrus and continued for up to 18 days (day 22 postestrus) blocked luteolysis in intact
ewes (Piper and Foote, 1968,1970). Corpora lutea in estradiol-treated ewes did not
regress by the end of treatment as determined by similar luteal weights between treated
and control ewes. However, estradiol was demonstrated to be necessary for luteolysis
when ovarian follicles were destroyed by irradiation (Karsch et al., 1970; Zhang et al.,
1991). Further, estradiol administered during the midluteal phase of the estrous cycle will
induce premature luteal regression in ewes and cows (Wiltbank et al, 1961; Stormshak et
al., 1969; Hawk and Bolt, 1970; Warren et al., 1973; Hixon and Flint, 1987), suggesting
that estradiol has a paradoxical effect on luteal life span depending on when it is
administered during the estrous cycle.
The apparent paradoxical affect of estradiol on luteolysis seems likely to depend
upon previous exposure of the uterus to progesterone. Ovariectomized ewes treated with
progesterone for 14 days, and subsequently infused with estradiol -1713 48 hours later, had
increased plasma concentrations of PGF2c, (Scaramuzzi et al., 1977). Estradiol
administration for as little as 6 hours increased oxytocin-induced uterine secretion of
PGF2. after 10 days of progesterone treatment in ovariectomized ewes, but had no effect
on days 2 or 6 of progesterone treatment (McCracken, 1980). However, estradiol
60
injected into seasonally anestrous ewes whose uteri were not exposed to progesterone
increased plasma PGF2e, concentrations, which were further increased by exogenous
oxytocin (Sharma and Fitzpatrick, 1974). In contrast, chronic exposure of uteri of
ovariectomized ewes to physiological concentrations of estradiol (15 days) did not result
in any oxytocin-induced release of PGF2a on days 5, 10 or 15 of treatment (Homanics and
Silvia, 1988). Nevertheless, this study demonstrated that exogenous estradiol enhanced
the ability of progesterone to stimulate uterine responsiveness to oxytocin, which occurs in
intact ewes as well (Hixon and Flint, 1987).
Systemic concentrations of estradiol also appears to have an effect on uterine
responsiveness to oxytocin. Quantitative effects of estradiol and progesterone
concentrations on oxytocin-induced PGF2a release were investigated by Beard et al.
(1994). In this study, ovariectomized ewes were treated with either low to high or high to
low ratio of estradiol and progesterone, succeeded by an injection of oxytocin 12 days
after treatment commenced. Results suggested that concentrations of high estradiol and
low progesterone stimulated oxytocin-induced PGFM response, while high progesterone
and low estradiol concentrations inhibited this response. Mann and Lamming (1995b)
demonstrated a reduction in the oxytocin-induced secretion of PGF2c, in steroid-treated
ovariectomized cows infused with subphysiological concentrations of estradiol. Thus,
these studies suggest that systemic concentrations of estradiol appear to have an effect on
the strength of the luteolytic signal.
Uterine changes in oxytocin receptor concentrations are influenced by the
spatiotemporal presence of estrogen and progesterone. Estradiol treatment alone
61
suppresses oxytocin receptor concentrations by day 7 in ovariectomized ewes (Val let et
al., 1990). However, estradiol treatment preceded by 10 days of progesterone treatment
increased concentrations of oxytocin receptors in ovariectomized (Vallet et al., 1990) and
intact ewes (Hixon and Flint, 1987). Progesterone withdrawal after concomitant
treatment of ovariectomized ewes with silastic implants containing estradiol increased
concentrations of oxytocin receptors (Zhang et al., 1992). Onapristone, a progesterone
receptor antagonist, injected on day 13 following injections of estradiol on days 11 and 12
increased uterine concentrations of oxytocin receptors (Walker et al., 1997). Thus, the
ability of estradiol to increase uterine concentrations of oxytocin receptors appears to be
dependent on a progesterone primed uterus.
Although regulation of the oxytocin receptor by the estrogen receptor is still not
clear, evidence exists for the regulation of the oxytocin receptor gene by estradiol.
Estradiol has been demonstrated to up-regulate oxytocin receptor mRNA levels in the
uterus, anterior pituitary and central nervous system (ventromedial hypothalamus,
amygdala, and hippocampus) of the ovariectomized rat (Quinones -Jenab et al., 1997).
The rat oxytocin receptor gene and 5' flanking region have been characterized (Rozen et
al., 1995). Analysis of the 5' flanking region of the oxytocin receptor gene failed to reveal
the presence of a classical palindromic estrogen response element (ERE), but six widely
spaced half-palindromes of the ERE were detected. Theoretically, estrogen could mediate
activation of the gene through these sites. More recently, Bale and Dorsa (1997) used an
alternative technique to sequence the 5' flanking region of the rat oxytocin receptor gene
and identified an internal segment of this area that was additional to the originally
62
published sequence. Upon examination, this segment contained the sequence representing
a palindromic ERE with a difference in only one base. Importantly, estradiol was
demonstrated to induce oxytocin receptor gene expression after binding to the newly
characterized ERE. Thus, in the rat, estradiol does directly induce oxytocin receptor
expression.
Receptor Interaction
The significance of uterine receptors for estrogen, progesterone, and oxytocin have
been mentioned throughout this section. Development of oxytocin receptors in the
luminal epithelium of the uterine endometrium is a critical step in the initiation of luteolysis
in the ewe (Wathes and Lamming, 1995). As previously suggested, luteolysis, and thus
the timing of oxytocin receptor development is determined by the concentration (Mann
and Lamming, 1995a,b) and most importantly the secretory pattern (Beard et al., 1994) of
estradiol and progesterone. To further understand the role of these receptors in luteolysis
and maternal recognition of pregnancy, a temporal view of receptor development and
steroid regulation will be discussed.
McCracken et al. (1984) proposed a model to explain the hormone mediated
events controlling the secretion of PGF2c, from the uterine endometrium. According to
this hypothesis, the uterus eventually becomes refractory to luteal phase concentrations of
progesterone probably due to down-regulation of the progesterone receptor. The
declining action of progesterone on the uterus eventually enables serum concentrations of
estradiol to up-regulate estrogen receptor concentrations. Increased concentrations of
63
estrogen receptor stimulates the expression of the oxytocin receptor gene, with a
consequent increase in endometrial oxytocin receptors. At this point, systemic
concentrations of oxytocin can interact with the newly synthesized oxytocin receptors to
stimulate the release of uterine PGF2., initiating the utero-ovarian feedback mechanism
and luteolysis. This hypothesis suggests that interrelationships between different receptor
types must exist to coordinate the events surrounding luteolysis or maternal recognition of
pregnancy.
Wathes and Hamon (1993), demonstrated that oxytocin receptors first develop in
the luminal epithelium on day 14 postestrus in the ewe. However, these authors observed
that the rise in oxytocin receptor concentrations precedes estrogen and progesterone
receptor development in the luminal epithelium. A subsequent study examined the timing
and additive effects of estrogen, progesterone and oxytocin in ovariectomized ewes on the
expression of uterine receptors for these hormones (Wathes et al., 1996). In this study,
ewes were treated with hormones to mimic the natural pattern and concentrations found
during the estrous cycle. As expected, progesterone treatment (overlapping low doses of
estradiol), immediately decreased estrogen receptor, oxytocin receptor, and its own
receptor over a period of time. Estradiol treatment alone increased estrogen and
progesterone receptors, but decreased oxytocin receptors and sustained this decrease until
day 14. Progesterone-induced inhibition of oxytocin receptors only appeared to be in the
luminal epithelium (the required site for oxytocin receptor expression to induce luteolysis)
and declined by day 14. However, no progesterone receptors were present in luminal
epithelial cells between days 8 and 14. Therefore, the inhibitory effect of progesterone on
64
oxytocin receptor concentrations is unlikely to be the direct effect of this steroid. Further,
the rise in estrogen receptors that occurred in progesterone-treated ewes by day 10
preceded the rise in oxytocin receptors by 2 days, suggesting that estrogen may not be
essential in the initiation of oxytocin receptor synthesis. In conclusion, it appears at this
time that the presence of the oxytocin receptors in the luminal epithelium is necessary for
luteolysis. However, the role of estrogen and progesterone in regulating the entire
process of luteolysis seems unlikely. It is likely that factors other than these steroid
hormones are contributing to the timing of oxytocin receptor development.
65
STATEMENT OF THE PROBLEM
Use of commercially available PGF2 analogues to induce luteolysis and
synchronize estrus in domestic species is well established. However, PGF2c, is not widely
used in some animal production industries because of the variability in time to estrus by
individual animals (Zarco et al., 1988b), which precludes maximizing the use of artificial
insemination. Thus, the study of factors regulating the estrous cycle, especially those that
induce luteolysis, are of significance from the standpoint of improving the technology for
animal production.
The current dogma with respect to luteolysis in sheep and cattle is based upon the
proper initiation of a utero-ovarian feedback mechanism involving secretion of the
oxytocin-induced uterine luteolytic factor, PGF2, upon oxytocin binding to its
endometrial receptor (McCracken et al., 1972; Roberts and McCracken, 1976;
McCracken et al., 1984). Over the last three decades, research has been conducted to
examine the various components of the luteolytic mechanism. Some of these studies have
focused on measuring changes in ovarian secretion of hormones and uterine receptors for
progesterone and estrogen, or measuring changes in concentrations of oxytocin receptors
(OTR) during the various stages of the cycle or in hormone-treated ovariectomized
ruminants. During pregnancy in sheep and cattle, the embryo interferes with the luteolytic
mechanism (Bazer, 1992, Ott et al., 1993; Meyer et al., 1995). Thus, the study of
pregnancy maintenance and "maternal recognition of pregnancy" has provided valuable
information about the role of the embryo in suppressing luteolysis. In these species the
66
embryo secretes a protein that inhibits luteolysis by regulating uterine concentrations of
endometrial estrogen and oxytocin receptors (Mirando et al., 1993b; Spencer et al.,
1995c), which are integral components of the luteolytic mechanism. These studies have
clearly shown the importance of suppressing estrogen and oxytocin receptors to prevent
luteolysis. The need to develop improved contraceptives to control our ever-increasing
world population and to have commercially available more effective agents to synchronize
estrus in livestock serves as the impetus for the present research to further explore and
define the utero-ovarian factors involved in regulating the life span of the corpus luteum.
It has long been known that exogenous estradiol can alter the duration of the
cycle. However, the role of estrogens in regulating the life span of the corpus luteum is a
paradox. Administration of estradiol at midcycle in the ewe causes premature luteal
regression (Stormshak et al, 1969; Warren et al., 1972) through an increase in uterine
oxytocin receptors (Hixon and Flint, 1987). Conversely, Piper and Foote (1968)
demonstrated that chronic treatment of ewes with estradiol at the beginning of the cycle
prolonged luteal life span and increased the interestrous interval. Thus, investigating
mechanisms by which estrogen treatment regulates the life span of the corpus luteum
provides a useful model in which to further study the interrelationships among factors that
govern luteolysis. It is anticipated that defining these factors and their interrelationships
may lead to more precise and defined methods of regulating the estrous cycle and thus
promote development of better products and methods of synchronizing estrus in domestic
animals.
67
DOWN-REGULATION OF OXYTOCIN RECEPTORS AND
SECRETION OF PROSTAGLANDIN F2, AFTER CHRONIC
TREATMENT OF EWES WITH ESTRADIOL-1713
INTRODUCTION
Administration of estradio1-17r3 (E2) to ewes during the mid-luteal phase of the
estrous cycle causes premature luteal regression (Stormshak et al., 1969; Warren et al.,
1973). However, daily injection of E2 initiated early during the cycle prolongs the life span
of the corpus luteum (Piper and Foote, 1968). Thus, exogenous E2 appears to have a
paradoxical effect on the life span of the corpus luteum (CL); accordingly it can either
promote luteal maintenance or regression in the ewe.
Prostaglandin F2, (PGF2a) secreted by the uterine endometrium is considered to
be the luteolytic hormone in the ewe (McCracken et al., 1972; Roberts et al., 1976). This
eicosanoid has been demonstrated to cause the secretion of luteal oxytocin (Wathes and
Swann, 1982) at the end of the cycle in ewes (Flint and Sheldrick, 1983), which binds to
high affinity binding sites for oxytocin in the endometrium during proestrus and estrus
(Sheldrick and Flint, 1985) to stimulate the pulsatile secretion of PGF2a. Consequently, a
functional interrelationship exists between the CL and the uterus through a positive
feedback mechanism (McCracken et al., 1984). Uterine responsiveness to oxytocin (OT)
in the ewe develops prior to luteal regression (Fairclough et al., 1980; Silvia et al., 1992)
and as systemic concentrations of progesterone (P4) wane, ovarian secretion of E2
promotes increased concentration of endometrial OT receptors (Sheldrick and Flint,
68
1985). Results of research with ovariectomized ewes injected with E2 and P4, to mimic
the hormonal pattern in estrous ewes, indicate that an increase in uterine oxytocin binding
sites occurs in response to a treatment regimen of P4 followed by E2 (Vallet et al., 1990;
Beard et al., 1994). However, Vallet et al. (1990) also demonstrated that injections of E2
alone or preceding P4 treatment reduced the number of OT receptors (OTR) in the uterus
by Day 7 after treatment. The ability of exogenous E2 to either shorten or prolong luteal
life span is presumably related to its capacity to alter uterine concentrations of OTR and
secretion of PGF2.
The present study consisted of two experiments; the objective of the first being to
determine if chronic exposure of ewes to E2 initiated early in the estrous cycle would
prolong the life of the CL. Because CL were maintained by this chronic treatment
regimen, the objectives of the succeeding experiment were to determine whether chronic
E2 treatment of ewes during the cycle would alter uterine concentrations of OT and
estrogen receptors and, if so, test the ability of the uterus to secrete PGF2a in response to
exogenous OT.
MATERIAL AND METHODS
Animals
Mature ewes were checked for behavioral estrus twice daily with a vasectomized
ram. After at least one estrous cycle of normal duration (15.5-18.0 days), ewes were
69
assigned randomly to treatments. All experimental procedures and surgeries were
performed in accordance with the Institutional Animal Care and Use Committee.
Treatments
Experiment 1
Ten mature ewes were assigned randomly in equal numbers to one of two groups
receiving: 1) corn oil (CO) or 2) 500 pg of E2 (Sigma Chemical Co., St. Louis, MO)
dissolved in 2 ml of CO. It is recognized that this daily dosage of E2 exceeds physiological
levels but was considerably less than the maximal dosage used by Piper and Foote (1968).
Ewes in both groups were injected s.c. daily from Day 4 of the cycle (estrus = Day 0) until
detected behavioral estrus or until Day 24 post-estrus, whichever occurred first. All ewes
were subjected to midventral laparotomies on Day 7 of the cycle to mark the existing CL
with India ink. Animals were anesthetized by i.v. injection of 5% sodium pentothal and
anesthesia was maintained by halothane-oxygen inhalation. All surgical procedures were
performed under aseptic conditions. Subsequent laparotomies of Eftreated ewes were
performed on Day 24 to determine if the marked CL were maintained and whether
secondary CL formation occurred as a result of treatment.
Experiment 2
Twenty mature ewes were assigned randomly in equal numbers to four groups in a
2x2 factorial arrangement. Treatment consisted of two concentrations of E2 dissolved in
70
2 ml of CO (0 and 500 gg) and two concentrations of OT (0 and 20 IU USP). Injections
(s.c.) of estradio1-1713 were initiated on Day 4 of the cycle and continued daily until Day
14. Jugular blood was collected by use of vacutainer tubes on Day 15, approximately 24 h
after the last E2 injection, to measure serum concentrations of E2 and P4. On Day 15, ewes
received either 1 ml of physiological saline or 1 ml of saline containing OT (20 IU USP) i.v.
with jugular blood collected into heparinized vacutainer tubes at -5 and 0 min prior to
injection and then 2, 5, 10, 20, 30, 40, 50, and 60 min post-injection. Tubes were placed
on ice for transport to the laboratory where blood was immediately centrifuged at 4°C for
10 min at 2450 x g and the plasma separated and stored at -20°C until assayed for 13,14dihydro-15-keto prostaglandin P2a, the PGF2a metabolite (PGFM). Laparotomies were
performed to expose the uterus only in those ewes receiving saline injection on Day 15.
Approximately 3 g of caruncular and intercaruncular endometrium were collected from the
horn adjacent to the ovary bearing the CL. If CL were present in both ovaries,
endometrium was collected from both uterine horns. Tissue was immediately frozen in
liquid N2 and stored at -80°C until assayed for OTR.
Steroid Radioimmunoassays
Progesterone Assay
Progesterone was extracted from 100 111 of plasma in duplicate with
benzene:hexane (1:2) by vortexing for 30 sec. Samples were incubated at -20°C overnight
before the organic phase was decanted into new tubes, dried down and used in the assay.
71
A third tube for each sample containing 100 Ill plasma plus 4000 cpm [3H] progesterone
(92 Ci/mmol, Dupont NEN, Boston, MA) was extracted to determine the extraction
efficiency. Mean extraction efficiency measured for all samples was 95 f 0.6%.
Radioimmunoassay was performed using P4 standards (5 pg\ tube to 800 pg\ tube)
in ethanol. Ethanol standards (100 111, in triplicate) or extracted samples (100 gl in
duplicate) were pipetted into 12 x 75 mm borosilicate glass tubes and evaporated under
air. When tubes were dry, 100 ill of phosphate buffered saline with gelatin (GPBS; 0.01
M NaPO4 pH 7.0, 0.14 M NaCI, 1:10000 thimerosal, 0.1% gelatin) were added to the
total count and non-specific binding tubes. Standards and sample tubes received 100 IA of
GPBS with anti-progesterone-11-BSA (Dr. Gordon Niswender, Colorado State
University, Fort Collins, CO) in a 1:2400 dilution. All tubes were vortexed and incubated
at room temperature for 30 min before adding 20000 cpm [3H] progesterone in 100 ill
GPBS. Tubes were vortexed and incubated overnight at 4°C. The following day, 1 ml of
cold GPBS was added to the total count tubes, and 1 ml dextran-coated charcoal [2.5 g/1
washed neutral writ charcoal (Sigma), 0.25 g/lDextran T-70 (Pharmacia, Uppsala,
Sweden)] in GPBS was added rapidly to all other tubes. Tubes were vortexed and
incubated at 4°C for 15 min, then centrifuged at 2450 x g for 10 min. The supernatant
from each tube was decanted into a 20 ml glass scintillation vial containing 6 ml Ecolume
scintillation cocktail (ICN Pharmaceutical Inc., Irvine, CA). Vials were counted in a
Beckman LS-6000 liquid scintillation counter.
Standard curves were plotted and unknown concentrations determined using the
RIA AID computer program (Robert Maciel Association, Inc, Arlington, MA). Analysis
72
of 18 replicates of a pooled plasma sample (70 ±2 pg/tube) resulted in an intraassay
coefficient of variation of 6.93%. Sensitivity of the assay was 0.05 ng/tube.
Estrogen Assay
Estradiol was analyzed by use of RIA in a single assay (David Hess, Oregon
Regional Primate Research Center) as described by Goodman (1978). Analysis resulted in
an intraassay coefficient of variation of 7.4%. Sensitivity of the assay was 0.5 pg/tube.
Prostaglandin Metabolite Assay
Plasma samples were analyzed for PGFM using a modified procedure described
by Guilbault et al. (1984). Prostaglandin free plasma was obtained from two anestrous
ewes by administering two 4 ml injections of a cylcooxygenase inhibitor (flunixin
meglumine; Banamine, Butler Company, Dublin, OH; 50 mg/ml) i.m. at 16 h intervals.
Blood was collected into evacuated 100 ml bottles containing 1430 USP units of heparin-
sodium salt 4 h after the second injection. Blood was immediately centrifuged at 4°C for
20 min at 2450 x g and the plasma was separated and stored at -20°C. Standards of
PGFM were prepared by serial dilution of a stock solution (1 mg/ml in ethanol, Cayman
Chemical Co., Ann Arbor, MI). Final standard concentrations were 0, 2.5, 3.75, 5, 10,
25, 50, 100, and 200 pg/100 IA in duplicate. Each tube contained 200 jAl prostaglandin
free plasma and 100 pi of appropriate PGFM standard. Experimental samples (200iil
plasma) were added to 100 til buffer. All tubes received 100 p1 of bovine y globulin (5
g/100 ml), were briefly vortexed and incubated for 15 min. A 100 p1 aliquot of 1:25000
73
rabbit antiserum for PGFM (gift from W.W. Thatcher, University of Florida) was added to
all tubes except the non-specific binding tubes, which received 100 'al of buffer. Tubes
were briefly vortexed and incubated for 30 min. Finally, 100 ill of 18000 dpm 13,14dihydro-15-keto[5,6,8,9,11,12,14(n)-311] prostaglandin Fa, (178 Ci/mmol; Amersham
Corp., Arlington Heights, IL) were added to make a final volume of 600 gl/tube. All
tubes were briefly vortexed and incubated for 1 h followed by overnight incubation at
4°C. A 750 IA aliquot of cold 40% solution of polyethylene glycol-8000 in water (40%
PEG) was added to precipitate the bound PGFM. Tubes were vortexed for 1 min
followed by centrifugation at 1620 x g for 30 min at 4°C to separate bound from free
hormone. Tubes were then inverted to drain the supernatant for 10 min followed by the
addition of 750 i_il of buffer and 750 i_il 40% PEG. All tubes were vortexed for 1 min after
each addition followed by a final centrifugation for 30 min. Tubes were again inverted to
drain the supernatant and the pellets were resuspended in 1 ml of buffer by vortexing for 2
min. Tube contents were poured into a scintillation vial containing 6 ml Ecolume cocktail
(ICN Pharmaceutical Inc.). Vials were vortexed and allowed to stand overnight before
counting.
The sensitivity of the assay was established at 25 pg/ml, thus all values measured at
less than the level of sensitivity were given this value. Six repeated measurements of a
single pooled plasma sample (206 ±5 pg/ml) among five assays resulted in intra- and
interassay coefficients of variation of 5.3 and 6.2%, respectively.
74
Oxytocin Receptor Analysis
Endometrium was analyzed for oxytocin receptors using a modified procedure
described by Mirando et al. (1993a). Approximately 1 g of tissue was rapidly minced
while frozen with a single-edge razor blade and placed into a 50 ml conical tube containing
20 ml of ice-cold wash buffer A (0.9% NaCI, 1 mM EDTA). The tube was briefly
vortexed and centrifuged at 2000 x g for 10 min at 4°C. Supernatant was discarded and
the pellet was rinsed twice with 20 ml buffer A. Final resuspension was in 15 ml of
homogenization buffer B (25 mM Tris-HC1, 0.25 M sucrose). Tissue was homogenized
six times with a Tekmar (Cincinnati, OH) tissumizer at speed 60 (medium-high) for 5 sec
and then allowed to cool in an ice bath for 15 sec between each homogenization. The
homogenate was transferred to a Duall glass tissue grinder and further homogenized
before centrifuging at 2000 x g for 10 min (4°C). The supernatant was transferred to a 25
ml tube and centrifuged at 4°C for 90 min at 80000 x g. The supernatant was discarded
and the pellet gently rinsed three times with 5 ml membrane diluting buffer C (25 mM
Tris -HCI, 0.01% NaN3) with final resuspension in 3 ml. Prior to storing the pellet
suspension at -80°C, an aliquot was analyzed for protein concentration using the Bio-Rad
Protein Assay (Bio-Rad Laboratories, Hercules, CA).
Specific binding of OT to endometrial membrane preparations (110 gg protein/100
Ill) from each ewe was determined in duplicate samples by adding increasing
concentrations of [tyrosy1-2,6-314]-0T (0.125 - 1 pM) alone or in combination with 200 -
fold excess of unlabeled OT for a total volume of 200 IA Labeled OT (48.5 Ci/mmol,
75
Dupont NEN) was diluted to appropriate concentrations in buffer D consisting of 25 mM
Tris-HC1, 0.01% NaN3, 0.2% BSA and 20 mM MnC12. Sample tubes were vortexed and
incubated for 60 min at 25°C. Thereafter, incubation tubes were placed on ice and to
each was added 750 IA of cold buffer D and 1 ml of membrane pelleting buffer E (40%
polyethylene glycol, 25 mM Tris-HC1, 0.01% NaN3). Tubes were then vortexed and
centrifuged at 2000 x g for 15 min (4°C). The supernatant was decanted and the tubes
inverted for 10 min (4°C) before resuspension by addition of 1 ml each of buffers D and
E, followed by centrifugation as described above. After decanting the supernatant and
allowing the inverted tubes to drain for 10 min the pellet was resuspended in 500 µl of
buffer C and decanted into scintillation vials containing 5 ml of Cytoscint ES+ (ICN
Pharmaceutical Inc., Irvine, CA). Vials were vortexed and allowed to stand overnight at
room temperature before counting. Oxytocin specifically bound to membrane receptors
was determined by difference between total binding (samples incubated with labeled OT)
and nonspecifically bound hormone (samples incubated with labeled OT and a 200-fold
excess of unlabeled OT). Dissociation constants (Kd) for each treatment group were then
determined by Scatchard analysis using LIGAND (Munson and Rodbard, 1980).
Binding of [311]-0T to ovine endometrial tissue preparations was saturable (Fig.
1A). Assay validity was determined by measuring [3H] -OT bound to increasing protein
concentrations prepared from endometrial tissue collected on Day 15 post-estrus (Fig.
1B). A regression of fmol OT bound/µg membrane protein assayed (Y = 0.52x - 4.51, R2
=0.99) was linear. Intra- and interassay coefficients of variation, determined from ovine
76
endometrium preparation assayed at 2.5 nM [3H] -OT in each assay, were 2.6 and 4.2%,
respectively.
Estrogen Receptor Exchange Assay
Uterine tissue was analyzed for estrogen receptors (ER) using a modified version
of the progesterone receptor exchange assay described by Slayden et al. (1994). Briefly,
400 mg frozen endometrium were homogenized in 10 vol of TEDGM [10 mM Tris -HCI,
1.5 mM EDTA, 1 mM dithiothreitol (DTT), 10% glycerol, 25 mM sodium molybdate] on
ice with a Duall glass tissue grinder. The homogenate was centrifuged at 2000 x g for 10
min at 4°C to separate the cytosolic fraction (supernatant) from the nuclear fraction. The
nuclear pellet was washed three times by gentle resuspension in 1 ml of TEDGM before
recentrifuging as above. After washing, the pellet was resuspended in 4 ml TEDK (10
mM Tris-HC1, 1.5 mM EDTA, 1 mM DTT, 0.8 M KC1) and extracted on ice for 1 h. A
200-p1 aliquot of the extract was frozen for DNA analysis using the procedure described
by Schneider (1957). The nuclear and crude cytosolic fractions were then centrifuged at
100000 x g for 1 h. The resulting supernatants were stored on ice until required.
Radioligand [6,7-3H(N)] E2 (50 Ci/ mmol, Dupont NEN Boston, MA) was diluted
to a concentration of 120 nM (333000 dpm/ 25 pl) in both TEDGM and TEDK. Nuclear
and cytosolic fractions (100 pl) were each assayed in duplicate by addition of increasing
concentrations of labeled E2 (0.94 - 60 nM) or labeled E2 plus a 200-fold excess of
unlabeled E2 for a total volume of 150 pl. Nuclear fractions were incubated at 37°C for
77
30 min and then both nuclear and cytosolic fractions were incubated overnight (18 h) at
4°C.
Bound receptors were separated from the free ligand using Sephadex (Pharmacia
and Upjohn, Kalamazoo, MI) LH-20 mini columns at 4°C. Briefly, columns were rinsed
three times with 300 ill TEDGM or TEDK before the samples were loaded onto the top
of the columns. After 30 min the columns were eluted into scintillation vials with two 300-
µl aliquots of respective buffer. The eluate was then diluted with 5 ml of Ecolume
cocktail, vortexed, and counted.
Binding of [3H] -E2 to ovine endometrial tissue preparations was saturable at a
concentration of 4 nM [3H] -E2. Specifically bound hormone was determined by
subtracting nonspecifically bound steroid from total bound steroid. The Kds were then
determined by Scatchard analysis using LIGAND (Munson and Rodbard, 1980).
Statistical Analysis
Effects of E2 treatment on the duration of the interestrous interval, the serum
concentrations of E2 and P4 on Day 15, and the uterine concentrations of OTR and ER
protein were all tested for significance by t-test using Statgraphics (STSC, Inc., Rockville,
MD). Data on PGFM concentrations were log transformed because of heterogeneity of
variance and subjected to repeated measures analysis of covariance using the general linear
model procedure of SAS (1993). Covariance analysis was used because of differing
baseline serum concentrations of PGFM among groups prior to injection of OT or saline
78
(p<0.05). The covariate consisted of the average of the -5 and 0 h concentration of
PGFM for each ewe, which was used to adjust subsequent mean concentrations of PGFM.
Data presented in graphic form are of non-log transformed values (Fig. 2).
RESULTS
Experiment 1
Administration of E2 (500 gg/day) from Days 4 to 24 post-estrus increased the
mean interestrous interval compared with that of controls ( mean ± SE; treated, 51.8 ± 1.7
vs. controls, 17.6 ± 0.4 days, p < 0.001). Marked CL were maintained in 4 of 5 treated
animals by Day 24 post-estrus with no newly induced CL present. A single ewe detected
in estrus prior to the end of treatment (Day 19) was excluded from the statistical analysis
for mean interestrous interval.
Experiment 2
Administration of E2 from Days 4 to 15 maintained the function of CL in treated
ewes as determined from an increased concentration of P4 on Day 15 (treated, 2.20 ± 0.40
vs. controls, 0.97 ± 0.30 ng/ml; p < 0.05). Serum concentrations of E2 in treated ewes on
Day 15 of the cycle were increased compared with concentrations in control ewes (14.3 ±
3.5 vs. 6.5 ± 0.8 pg/ ml; p < 0.05). Concentrations of OTR in the endometrium from
79
0
2
4
6
8
10
[31-1]-0XYTOCIN (nM)
Figure 1A. Saturation analysis of OTR in ovine endometrium. Duplicate aliquots of
membrane protein from ewes (n=5) were incubated with each concentration of [3H] -OT
alone (total binding) or with a 200-fold excess of unlabeled OT (nonspecific binding).
Specific binding was determined as the difference between total and nonspecific binding.
80
120
100
80
60
40
20
00
I
I
I
I
50
100
150
200
250
MEMBRANE PROTEIN (jig)
Figure 1B. Regression of bound OT in endometrial membranes as determined using a
single saturating concentration of [3H] -OT (Y = 0.52x - 4.51, R2 = 0.99).
81
TIME (min)
Figure 2. Plasma concentrations of PGFM in control (CO) and E2-treated ewes on Day 15
of the cycle after i.v. injection of saline (S) or 20 IU USP OT. Because of different mean
basal concentrations of PGFM among groups, each point represents the adjusted mean for
the covariate (-5 and 0 time points) for each ewe. Standard errors are given for each
mean in the results.
82
E2-treated ewes were lower than in controls (26 ± 11 vs. 252 ± 58 fmol/mg protein, Kd
=1.54 nM, p=0.002). Because of an OTR concentration of 988 frnol/mg protein, one
control animal was considered an outlier and was not included in the statistical analysis for
OTR concentration. Chronic E2 treatment had no effect on total nuclear or cytoplasmic
endometrial ER concentration compared to controls (nuclear ER: treated, 0.61 ± 0.23 vs.
controls, 0.44 ± 0.16 finol/pg DNA; Kd = 0.55 nM, p = 0.55; cytosolic ER; treated, 2.33 ±
0.48 vs. controls, 4.21 ± 1.09 fmol/pg DNA; Kd = 0.38 nM, p = 0.15). Response of ewes
to injection of OT on Day 15 of the cycle, as determined by measurement of plasma
concentrations of PGFM, differed markedly between control and E2-treated ewes
(estradiol x oxytocin x time interaction, p < 0.01; Fig. 2). Plasma concentrations of PGFM
in E2-treated and control ewes injected with saline on Day 15 did not differ. However,
injection of OT into control ewes on Day 15 caused a rapid increase in plasma PGFM that
attained maximal levels by 10 min after injection and remained elevated for the ensuing 50
min sampling period. In contrast, injection of OT into E2-treated ewes provoked a slight
increase in plasma concentration of PGFM that returned to basal level by 20 min.
DISCUSSION
Results of our research indicate that chronic administration of E2 to ewes beginning
on Day 4 of the cycle prolongs the life span of the CL. These data confirm those of Piper
and Foote (1968) who first reported that chronic E2 treatment of ewes prolonged luteal life
83
span. Serum concentrations of P4 in E2-treated ewes on Day 15 of the cycle were
significantly greater than those of control ewes, and were comparable to those of ewes in
the mid-luteal phase of the cycle as previously measured in our laboratory (Slayden and
Stormshak, 1990). Corpora lutea in E2-treated ewes that were marked with India ink on
Day 7 of the cycle were still present on Day 24 post-estrus. It is not known how long
these corpora lutea remained functional but these ewes did not exhibit behavioral estrus
until approximately another 27 days had elapsed. The possibility exists that chronic E2
treatment may have stimulated secretion of luteinizing hormone in quantities that were
luteotropic in the ewe (Kaltenbach et al., 1968b; Karsch et al., 1971b). However, based
upon available data, it is more likely that the effect of E2 on luteal maintenance was due to
altered uterine function.
McCracken (1984) demonstrated that E2 acts to up-regulate OTR at the end of the
normal cycle ( Day 15), and subsequently proposed (McCracken et al., 1984) that
luteolysis in the ewe involves a positive feedback loop in which luteal OT binding to
uterine OTR stimulates secretion of PGF2c, and vice versa. If one accepts this hypothesis,
it is logical to assume that failure of uterine OTR to be upregulated at the appropriate time
would result in luteal maintenance. Indeed, maintenance of CL in the pregnant ewe is due
to the ability of a trophoblast protein, interferon t, to reduce the concentrations of uterine
estrogen and oxytocin receptors (Spencer et al., 1995a). In our study, E2 treatment
resulted in a 10-fold reduction in the concentration of uterine OTR on Day 15. These data
are similar to those of Vallet et al. (1990) who reported that E2 injections into
ovariectomized ewes decreased uterine OTR concentrations when quantified 7 days after
84
initiating treatment. Thus, the response of ewes to chronic treatment with
pharmacological levels of E2 during the luteal phase is not comparable with endogenous
E2-induced upregulation of OTR dictated by the McCracken model.
It is unlikely that chronic E2 stimulated secretion of posterior pituitary OT in
sufficient quantities to cause OTR down-regulation. Although i.v. infusion of E2 (2-4
µg/h) into ovariectomized ewes for 12-18 h decreased the interval between pulsatile bursts
of OT secretion, the quantity of hormone released from the neurohypophysis was
approximately only one-tenth the amount normally released from the CL (McCracken et
al., 1995). Rat uterine epithelium is a source of OT (Lefebvre et al., 1992), suggesting
that in this species uterine OT may contribute to systemic concentrations of the hormone
as well as act locally to down-regulate the concentration of uterine OTR. Administration
of E2 to ovariectomized rats resulted in a sevenfold increase in uterine OT mRNA
(Lefebvre et al., 1994), and this response was further increased in the presence of P4
suggesting a synergistic effect of these steroids. However, preliminary data from our
laboratory fail to provide evidence that chronic treatment of ewes with E2 results in any
increase in systemic concentrations of OT of pituitary or uterine origin (Hazzard et al.,
1996).
The observed reduction in uterine concentration of OTR in E2-treated ewes cannot
be attributed to a reduction in uterine estrogen receptors. Uterine concentrations of
estrogen receptor in treated ewes were similar to those of control ewes in which luteal
regression was underway. Similarly, Spencer et al. (1995b) reported up-regulation of
uterine OTR without a change in ER in ovariectomized ewes treated with P4 and E2
85
compared with animals receiving P4 alone. In our study, E2 treatment markedly increased
serum concentration of this steroid which apparently was able to stimulate uterine
production of ER even though systemic concentrations of P4 were increased. Ability of a
comparable dosage of estradiol to induce uterine ER in the face of high systemic
concentrations of P4 in the ewe has been reported (Zelinski et al., 1980). Others have also
reported that exogenous estrogen, presumably acting via its receptor, can up-regulate
ovine endometrial OTR formation when systemic concentrations of P4 are increased and
hence result in luteal regression (Hixon and Flint, 1987; Beard and Lamming, 1994). The
possibility that E2-induced down-regulation of uterine OTR is mediated indirectly by
sustained luteal production of P4 cannot be excluded. In ewes continuously infused with
OT to promote down-regulation of uterine OTR and luteal maintenance, a PGF2a-induced
reduction in serum concentrations of P4 resulted in uterine OTR up-regulation (Flint and
Sheldrick, 1985; Sheldrick, 1992).
Estradiol administered to ewes on a daily basis apparently acts directly on the
uterus to suppress concentrations of OTR. Estradiol has been demonstrated to induce
uterine OTR mRNA in rats (Larcher et al., 1995), but not through binding of ER to the
classical estrogen response elements in the 5' flanking region of the OTR gene (Zingg et
al., 1995). It is conceivable that the markedly increased concentration of E2 in treated
ewes suppresses synthesis of uterine OTR via regulation of other transcriptional factors
(Webb et al., 1993). Chronic E2 treatment of immature rats drastically suppresses uterine
metabolism and protein synthesis (Stormshak et al., 1976).
86
Although the exact mechanism to explain the reduction in OTR concentration is
unclear at this time, exogenous E2 may affect the ability of the uterus to synthesize and
secrete PGF2a. In our study, E2 reduced the basal concentration of PGFM compared to
that of control ewes, but did not impair OTR function as reflected by the OT-induced
secretion of PGF2a on Day 15. However, the magnitude of the response to OT was
significantly less in E2-treated than OT-treated control ewes. These data are similar to
those of Sharma and Fitzpatrick (1974), who also demonstrated an OT-induced release of
PGF2a in E2-treated anestrous ewes. Vallet et al. (1990) demonstrated that by Day 7 after
initiating treatment, exogenous E2 had reduced uterine OTR concentrations in
ovariectomized ewes. However, these receptors were not functional as determined by
lack of OT-induced increases in serum concentrations of PGF2a metabolite (PGFM).
In conclusion, the results of this study can be compared to the regulatory events
that occur in the pregnant ewe. The conceptus of the pregnant ewe produces interferon ti
which has been demonstrated to inhibit endometrial ER and OTR formation (Spencer et
al., 1995a), leading to the suppression of the OT-induced release of PGF2c, and hence
luteolysis. In cyclic ewes, the present study suggests chronic E2 treatment similarly downregulates the concentration of OTR and reduces the OT-induced release of PGF2a.
However, there was no measurable change in the concentration of endometrial ER in the
E2-treated ewe. Therefore, chronic treatment of ewes with E2 has the same effect in terms
of luteal maintenance as the presence of a conceptus, but appears to act via an entirely
different mechanism(s) to down-regulate the OTR.
87
IMPACT OF CHRONIC TREATMENT OF EWES WITH
ESTRADIOL-1713 OR PROGESTERONE ON OXYTOCIN
RECEPTOR GENE EXPRESSION AND OVARIAN OXYTOCIN
SECRETION
INTRODUCTION
During the past two decades it has become central dogma that luteolysis during the
estrous cycle of ruminants occurs as a consequence of a positive feedback system between
luteal oxytocin (OT), which stimulates secretion of the uterine luteolysin, prostaglandin
Fa, (PGF2a), and vice versa (Flint and Sheldrick, 1982; Flint and Sheldrick, 1983;
McCracken et al., 1984). An integral aspect of this feedback system is the coordinated
actions of estrogen and progesterone in regulating uterine concentrations of receptor to
which OT binds to initiate the pulsatile release of PGF2a.
We have previously demonstrated that chronic treatment of ewes with estradiol1713 (E2) beginning on Day 4 of the estrous cycle prevented an increase in endometrial
concentrations of oxytocin receptors (OTR) at the expected time of luteolysis, prolonged
luteal life span and maintained normal systemic concentrations of progesterone (Hazzard
and Stormshak, 1997). However, the acquired data failed to provide a clue as to whether
the suppression in uterine concentrations of OTR was due to a direct effect of E2 or
indirectly via the sustained production of progesterone. Progesterone suppresses uterine
concentrations of estrogen receptor (ER) in E2 treated ovariectomized ewes (Koligian and
Stormshak, 1977) and uterine concentrations of OTR in intact ewes (Roberts et al., 1976;
88
Sheldrick and Flint, 1985) and ovariectomized ewes (Lau et al., 1992a; Zhang et al.,
1992). Thus, the sustained production of progesterone in ewes subjected to chronic E2
treatment may account for the observed reduction in uterine OTR. Regardless of which
steroid in chronic E2-treated ewes causes the change in uterine OTR, it is not known
whether the effect is genomic or nongenomic. There is at least some circumspect basis for
assuming that E2 may act directly on the genome to alter uterine production of OTR. In
rats, an estrogen response element (ERE) has recently been reported to exist in the 5'
flanking region of the OTR gene (Bale and Dorsa, 1997). It is conceivable that the
promoter region of the ovine OTR gene also contains an ERE, thus providing for E2
regulation of gene expression. Presently, there is no evidence for the existence of a
progesterone response element (PRE) present in the promoter region of the OTR gene
(Rozen et al., 1995; Bale and Dorsa, 1997). On the other hand, there is some evidence
that progesterone may be acting via a nongenomic mechanism to regulate uterine
production of OT in the rat (Zingg et al., 1995). Whether the sustained concentrations of
progesterone present in the chronic E2-treated ewe alters uterine concentration of
progesterone receptor (PR) during the otherwise normal period of luteolysis is of
associated relevance in ascertaining if its mode of action is genomic.
Ovine large luteal cells that are the source of OT (Rodgers et al., 1983) have been
reported to contain estrogen receptors (Glass et al., 1984) and the gene for OT has been
shown to contain an ERE upstream of the transcription start site (Richard and Zingg,
1990; Zingg et al., 1995). Because chronic exposure of the ewe to E2 results in a
pharmacological state, it is conceivable that this treatment regimen may alter synthesis and
89
(or) storage of luteal OT. Such an effect of this treatment could be ascertained by
challenging the chronic E2-treated ewe with PGF2a to induce the secretion of luteal OT
(Flint and Sheldricic, 1982). A reduction in luteal OT production may in part contribute to
absence of a luteolytic mechanism in the chronic E2-treated ewe.
The present study was conducted to: 1) examine the effects of chronic E2 and
progesterone treatment on uterine concentrations of OTR and associated changes in OTR
mRNA and progesterone receptors during the late luteal phase of the cycle, and 2)
ascertain whether chronic E2 treatment of ewes during the cycle would alter luteal OT
production.
MATERIALS AND METHODS
Animals
Mature ewes were checked for behavioral estrus twice daily with a vasectomized
ram. After at least one estrous cycle of normal duration (15.5-18.0 days), ewes were
assigned randomly to treatment groups. All experimental procedures and surgeries were
performed in accordance with the NIH guide for the Care and Use of Laboratory Animals
at Oregon State University.
90
Treatments
Experiment 1
Fifteen mature ewes were assigned randomly in equal numbers to three treatment
groups to compare the effects of exogenous E2 and progesterone on uterine
concentrations of OTR and OTR mRNA. Daily dosage of E2 administered to ewes in this
experiment (and Exp. 3 below) was one-half of that recently used in a similiar series of
experiments in our laboratory (Hazzard and Stormshak, 1997) but, nevertheless, was
considered to be sufficiently pharmacological to alter uterine function as based upon the
observations of Hawk and Bolt (1970). Treatments consisted of a control group and two
groups receiving either 250 iig of E2 or 10 mg of progesterone in 2 ml of corn oil
(CO) /day. Injections of E2 (s.c.) were initiated on Day 4 of the cycle and continued for a
total of 11 days; progesterone-treated ewes received 2 ml CO s.c. on Days 4-10 and
progesterone on Days 11-14. Ewes were anesthetized and laparotomized on Day 15 as
previously described (Hazzard and Stormshak, 1997). All CL were counted, enucleated,
and weights were recorded. An incision was made through the antimesometrial uterine
tissue parallel to the long axis of the horn to expose the lumen. Approximately 3 g of
caruncular and intercaruncular endometrium from the uterine horn adjacent to the ovary
bearing the CL were collected, immediately frozen in liquid N2, and stored at -80°C until
assayed for OTR and OTR mRNA.
91
Experiment 2
This experiment was conducted to determine whether progesterone treatment of
ewes, as described for Exp. 1 above, altered uterine concentrations of PR. Ten mature
ewes were assigned randomly in equal numbers to two treatment groups. Treatments
consisted of two concentrations of progesterone (0 or 10 mg) dissolved in 2 ml of CO.
Injections (s.c.) were initiated on Day 11 of the cycle and continued for a total of 4 days.
All ewes were laparotomized on Day 15, and endometrial tissue was removed from the
uterine horn adjacent to the ovary bearing the CL, maintained at 4°C and immediately
assayed for PR.
Experiment 3
The primary objective of this experiment was to examine the effect of chronic E2
treatment of ewes on luteal secretion of OT in response to a PGF2c, challenge. Twenty
mature ewes were assigned randomly in equal numbers to four treatment groups in a 2x2
factorial arrangement. Treatment consisted of two doses of E2 (0 or 250 lig) dissolved in
2 ml of CO and two doses of PGF2a analogue (Estrumate ®, Mobay Corp., Shawnee, KS; 0
or 125 gg). Control and E2-treated ewes were injected s.c. for 11 days as described for
Exp. 1. Jugular blood samples collected by use of vacutainer tubes on Day 15 were
allowed to clot overnight at 4°C before centrifuging at the same temperature for 10 min at
2,450 x g. Sera were removed and stored at -20°C until assayed for progesterone (n=9
each for control and E2-treated groups). After collection of blood samples for
progesterone analysis, all ewes received either 1 ml physiological saline or 1 ml of PGF2.
92
analogue (125 gg) i.v. on Day 15, with jugular blood collected in heparinized vacutainer
tubes at 0 min before injection and then 2, 5, 10, 20, and 30 min after injection. Upon
collection, 10 I.LI of 5 mg/ml 1,10-phenanthroline (Sigma Chemical Company, St. Louis,
MO) in ethanol and 20 gl 0.5M EDTA were added to each tube to suppress oxytocinase
activity. All tubes were mixed by inversion and placed on ice for transport to the
laboratory where blood was immediately centrifuged as described above. Plasma was
removed and stored at -20°C until assayed for OT.
Oxytocin Receptor Analysis
Endometrium was analyzed for OTR with a procedure described by Hazzard and
Stormshak (1997) by methods adapted from Mirando et al. (1993a). Single-point
saturation analysis of ovine endometrium preparation assayed at 3.0 nM [3H] -OT resulted
in intraassay and interassay coefficients of variation of 4.3 and 14.0%, respectively.
Progesterone Receptor Exchange Assay
Endometrial PR concentrations were analyzed using a procedure described by
Slayden et al. (1994), and modified as follows. Radioligand promegestone, [17a-methyl311]-R5020 (86 Ci/mmol; Dupont NEN, Boston, MA) was diluted to a concentration of
120 nM (573000 dpm/25 gl) in the appropriate buffers. Nuclear and cytosolic fractions
were each assayed in duplicate (100 gl/ tube) by addition of 25 gl of labeled R5020 (3-120
93
nM) or labeled R5020 plus 25 gl of 200-fold excess unlabeled R5020 for a total volume of
150 IA To determine affinity of R5020 for glucocorticoid receptors, dexamethasone (10
nM) was added to additional samples, and assayed in duplicate at 8 nM concentration of
[31-1]-R5020. There was no indication of binding of [311] -R5020 with the glucocorticoid
receptor (p > 0.05, Student's t-test). Specifically bound hormone was determined by
substracting nonspecifically bound steroid (3.7 ± 0.7%) from total bound steroid. Binding
of [31-1]-R5020 to ovine endometrium was saturable at a concentration of 8 nM of labeled
ligand.
Steroid Radioimmunoassays
Progesterone Assay
Progesterone was extracted from 100 gl of serum with benzene:hexane (1:2) and
subsequently quantified by use of RIA as described by Koligian and Stormshak (1976).
Analysis of a quality-control sample of progesterone (75 pg/tube) resulted in an intraassay
coefficient of variation of 7.8%. Sensitivity of the assay was 0.05 ng/tube.
Oxytocin Assay
Oxytocin was extracted from 1 ml of plasma and assayed as described by Bertrand
and Stormshak (1996) using methods adapted from Abdelgadir et al. (1987) and Schams
(1983). For plasma extraction, a Waters vacuum manifold was utilized with Sep-Pak Plus
C-18 cartridges (Waters Chromatography Division, Millipore Corp., Milford, MA). Mean
94
extraction efficiency was 61 ± 0.5%. Oxytocin antibody was generously provided by Dr.
Dieter Schams, Technical University of Munich, Freising-Weihenstephan, Germany.
Analysis of a stock plasma sample of OT (3.5 pg/tube) resulted in intraassay and
interassay coefficients of variation of 5.9 and 15.6%, respectively. Sensitivity of the assay
was 0.125 pg/tube.
RNA Extraction and Northern Blotting
Endometrial tissues (approximately 300 mg) from each of three control, chronic
E2-treated and progesterone-treated ewes and an equivalent quantity of nonlactating ovine
mammary gland tissue (negative control) were pulverized with a mortar and pestle over a
dry-ice/ethanol bath and then placed in 10 vol (3 ml) TRI REAGENT (Molecular
Research Center, Inc., Cincinnati, OH), a single-step total RNA isolation reagent
consisting of a mono-phase solution of phenol and guanidine thiocyanate (Chomczynski,
1993). Extraction of total RNA was performed according to the manufacturer's protocol,
which included procedures for homogenization of tissue, precipitation of RNA with
isopropanol, and centrifugation. Final resuspension of recovered RNA was in 50 ill
diethylpyrocarbonate-treated (DEPC) water, which was incubated at 55-60°C for 10 min.
Recovered RNA was quantitated by A260 spectrophotometric measurement and assessed
for purity by A260/280 ratio.
Samples were denatured at 65°C for 10 min with 4 X Sample Buffer (1 part 10 X
Northern Buffer [0.2 M HEPES, 10 mM EDTA, pH 7.8], 1.6 parts 37% formaldehyde,
95
and 5 parts deionized formamide) in a ratio of 1:3. Denatured samples (30 pg total RNA)
plus 2 pi of 10% Bromophenol Blue were loaded onto a 1.25% formaldehyde agarose gel
and electrophoresed in 1 X Northern Buffer for 2 h at 70 V. The gel was then rinsed for
40 min in DEPC-treated water before equilibrating in 10 X SSC buffer (1 X SSC = 0.15
M NaC1, 15 mM sodium citrate, pH 7.0) for 20 min. Transfer of RNA onto MagnaGraph,
Nylon membrane (Microns Separations Inc., Westboro, MA) was performed by capillary
transfer. After 12-18 h, the membrane was rinsed for 10 min in 6 X SSC, allowed to air
dry on filter paper (10-15 min) and the RNA UV-crosslinked to the membrane for 30 sec.
The membrane was remoistened in 1 X SSPE (3 M NaC1, 0.2 M NaH2PO4H20, 20 mM
EDTA, pH 7.4) before staining for 3 min (0.02% Methylene Blue, 0.3 M sodium acetate,
pH 5.5), and destaining for 15 min in 1 X SSPE to visualize the 18S and 28S rRNA bands.
Following visualization and destaining, the membrane was baked for 1 h at 80°C in a
vacuum oven before removing the stain from the membrane with a strip buffer (0.1 X
SSPE/0.1% SDS) for 15 min.
Probes for OTR and 18S RNA were made as follows: The template for the OTR
probe was a 0.8 kb BamBI/Pst I fragment excised from the human OTR gene plasmid
insert (a gift from Dr. T. Kimura, Osaka University Medical School, Osaka, Japan). The
probe was made from the cDNA template by random hexanucleotide priming with
EasytideTM a-32P-dCTP (Dupont NEN) as the radioactive label (Prime-a-Gene® Labeling
System, Promega, Madison, WI). Unincorporated label was removed by passage through
a Sephadex® 0-50 column (Quick SpinTM columns, Boehringer Mannheim, Indianapolis,
IN). The 18S probe was made from eukaryotic rRNA (1406R; Olsen et al., 1986) and
96
end-labeled with y-32P-dATP (Dupont NEN) using T4 polynucleotide kinase (Promega).
Unincorporated label was removed using a Sephadex® G-25 spin column (Boehringer
Mannheim).
Hybridization buffer (5 X SSPE, 5 X Denhardt's solution [0.1% each BSA, Ficoll
and polyvinylpyrrolidone; Sigma], 1% SDS, 100 mg/ml salmon testes DNA for
hybridization [Sigma], and 50% deionized formamide) was incubated with the membrane
at 42°C for at least 4 h by automated rotation in a hybridization oven (Robbins Scientific,
Sunnyvale, CA). Following incubation, the OTR cDNA probe was added to the
hybridization solution to obtain approximately 4 x 106 cpm/ml, and incubated overnight at
42°C. Membranes were washed at room temperature for 15 min three times in 1 X
SSPE/0.1%SDS, followed by one wash for 15 min at 50°C in strip buffer. Blots were
then wrapped in plastic wrap and exposed to a storage phosphor screen (Molecular
Dynamics, Sunnyvale, CA) for 4-5 days. Screens were scanned by a Phosphor Imager SI
and visualized with Image QuaNT software (Molecular Dynamics). Before using the 18S
rRNA probe, blots were incubated at 70°C for 30 min in strip buffer to remove any OTR
probe. Thereafter, the blots were prehybridized and then rehybridized overnight at room
temperature and visualized as mentioned above.
Statistical Analysis
Effect of E2 treatment on the uterine concentrations of OTR were tested for
significance by ANOVA using Statgraphics (STSC, Inc., Rockville, MD). Because
97
uterine concentrations of OTR in progesterone-treated ewes were below the detection
limits of the assay, data on these animals were not included in the statistical analysis.
Effects of treatments on luteal weights were tested by analysis of covariance using number
of CL as the independent variable among groups. Differences among group means were
further tested for significance by using orthogonal contrasts. Data on serum
concentrations of progesterone and effects of progesterone treatment on the uterine
concentrations of PR were tested for significance by t-test using Statgraphics (STSC,
Inc.). Plasma concentrations of OT were subjected to repeated measures analysis using
the general linear model procedure of SAS (1993). Data were considered significantly
different at (p < 0.05).
RESULTS
Chronic treatment of ewes with E2 from Days 4-14 of the estrous cycle or daily
injection of progesterone during Days 11-14 of the cycle reduced endometrial
concentrations of OTR compared with that of controls (E2, 14 ± 11, progesterone < 1 vs.
controls, 348 ± 140 frnol/mg protein; p < 0.05). These data are supported by analysis of
endometrial OTR mRNA, which demonstrated that both E2 and progesterone treatments
may inhibit expression of the OTR gene (Fig. 3). All four differentially polyadenylated
variants of mRNA (1.65, 2.5, 3.9 and 6.2 kb) for the ovine OTR gene (Flint et al., 1995)
were visible in Day 15 endometrium of control ewes (Fig. 3; lanes 8-10) but none were
98
1
2
3
4
5
6
7
8
9
10
- 0. 6.2
3.9
4. 2.5
1.65
18S
Figure 3. Northern Blot of OTR mRNA from Day 15 ovine endometrium. Each lane
represents one ewe. Lane 1: ovine nonlactating mammary gland tissue (negative control).
Lanes 2-4: animals received 10 mg progesterone s.c. Days 10-14. Lanes 5-7: animals
received 250 pig E2 s.c. Days 4-14. Lanes 8-10: animals received 2 ml CO s.c. Days 4-14
(positive controls). The mRNA for OTR is represented by four differentially
polyadenylated variants (1.65-6.20 kb). 18S rRNA was probed to determine equality of
loading.
99
-- 200
E
7:3)
a.
E 180
0
0 160
TIME (min)
Figure 4. Plasma concentrations of oxytocin in control (CO) and E2-treated ewes on Day
15 of the cycle after i.v. injection of saline (S) or 125 gig PGF2a (Estrumate ®) at time 0.
The common estimate of the standard error was 15.18. Each point represents the mean
concentration of OT of five animals/group.
100
evident in endometria of steroid-treated ewes. Absence of OTR mRNA in endometria of
treated ewes was not due to unequal loading of total RNA because no differences in
quantities of 18S rRNA among samples were detected. Additionally, the absence of
detectable OTR mRNA in nonlactating mammary gland tissue is consistent with the data
reported by Flint et al. (1995) and indicates the specificity of the probe for the OTR
mRNA.
Treatment of ewes with progesterone from Days 11-14 (Exp. 2) did not alter
uterine concentrations of cytoplasmic PR compared with that of control ewes
(progesterone-treated, 200 ± 40 vs. controls, 180 ± 40 fmol/ mg DNA; p > 0.05).
Unfortunately, uterine concentrations of nuclear PR could not be quantitated because
concentrations were below the level of sensitivity of the assay. Chronic treatment of ewes
with 250 pg of E2 daily during the cycle or administration of progesterone during the late
luteal phase of the cycle resulted in CL on Day 15 that were heavier than those of controls
(E2-treated, 394 ± 23, progesterone-treated, 492 ± 67 vs. controls, 307 ± 43 mg; p < 0.05
for each treatment compared with controls). Consistent with these weights of CL, mean
serum concentrations of progesterone in chronic E2-treated ewes on Day 15 (Exp. 3) were
greater than in controls (1.33 vs. 0.92 ng/ ml), but the difference was not significant
statistically. However, the mean of control ewes is based upon eight animals because one
ewe in this group had a serum concentration of 4.36 ng/ ml, fivefold greater than the mean
serum concentration of progesterone in the other animals. Because of this unusually
increased serum concentration of progesterone, this ewe was considered to be an outlier
and the datum of this animal was not included in the statistical analysis.
101
Injection of PGF2a analogue on Day 15 of the cycle did not alter induced secretion
of OT in chronic E2-treated and control ewes (p > 0.05; Fig. 4). However, compared with
saline-injected ewes, injection of PGF2a into both chronic E2-treated and vehicle-injected
ewes markedly increased plasma concentrations of OT (p < 0.05).
DISCUSSION
Results of these research endeavors indicate that chronic treatment of ewes with E2
from early to late in the estrous cycle suppresses endometrial OTR gene expression as
reflected by the absence of measurable quantities of OTR mRNA on Day 15. In view of
this observation, it is not surprising that analysis of the same endometrial tissue for OTR
also revealed a significant reduction in quantities of this receptor in E2-treated ewes. This
latter finding is consistent with our recent data demonstrating that chronic E2-treatment of
ewes during the cycle caused down-regulation of uterine OTR without affecting uterine
concentrations of ER (Hazzard and Stormshak, 1997). The dosage of E2 used in our
current study, although only one-half of that used in our previous experiments, was
apparently sufficient to provoke similar changes in uterine concentrations of OTR.
Similarly, the effectiveness of this dosage of E2 in altering the luteolytic mechanism is
supported in part by the greater weight of CL and serum concentrations of progesterone in
treated compared with those of control ewes on Day 15. Thus, there is some indication
that daily administration of 250 tig of E2, like the dosage of 500 lig of E2 used previously
102
(Hazzard and Stormshak, 1997), did sustain luteal function but no prediction can be
advanced as to how long these CL would persist and continue to function once treatment
ceased. Ewes treated with 500 lig of E2 from Days 4-24 postestrus exhibited a mean
interestrous interval of over 50 days (Hazzard and Stormshak, 1997).
Because our previous studies demonstrated that chronic treatment of ewes with E2
during the cycle prolonged luteal function, it was hypothesized that the sustained
production of progesterone actually mediated the effect of chronic E2 in down-regulating
uterine OTR. This hypothesis was tested in Exp. 1 by administering progesterone to ewes
during the late luteal phase of the cycle (Days 11-14). The effect of progesterone on
endometrial concentrations of OTR and OTR mRNA was similar to that evoked by
chronic E2; i.e., endometria contained no OTR mRNA and corresponding concentrations
of OTR were too low to measure with accuracy. On this basis one might conclude that
the effect of chronic E2 treatment of ewes on OTR gene expression is indeed mediated by
progesterone. The ability of this steroid to suppress OTR gene expression is consistent
with the report of Stewart et al. (1993) who found an absence of endometrial OTR mRNA
in the ewe during the progesterone-dominated luteal phase of the cycle. The mechanism
by which progesterone acts to suppress expression of the OTR gene is not known.
Regardless of the specific mechanism by which progesterone acted to inhibit OTR gene
expression this steroid must have exerted its effect when bound to its receptor. However,
there was no significant difference in the endometrial concentration of cytoplasmic PR
between control and progesterone-treated ewes. Cytoplasmic concentrations of PR were
lower than those reported to be present during the midluteal phase of the cycle of the ewe
103
(Zelinski et al., 1980) and nuclear concentrations of PR were below the detection limits of
the assay. Based upon the data of Wathes et al. (1996) it is likely that the PR measured on
Day 15 in progesterone-treated ewes were localized predominantly in the caruncular
stroma. On Day 14 of the cycle OTR are found primarily in the uterine lumenal epithelium
(Wathes and Hamon, 1993; Wathes et al., 1996). If one accepts this apparent difference
in tissue distribution of OTR and PR then it is logical to propose that progesterone acts on
stromal cells to stimulate the production of some factor capable of suppressing OTR gene
expression in the lumenal epithelial cells. The fact that OTR apparently increase after
prolonged treatment of ovariectomized ewes with progesterone (Vallet et al., 1990) may
be due to eventual accumulation of E2 resulting from the peripheral conversion of the
progesterone to this estrogen (Beard et al., 1994).
In view of the above possible mode of action of progesterone, it is conceivable that
the suppressive effects of chronic E2 and progesterone on OTR gene expression likely
involve independent mechanisms. For example, in intact ewes (Sheldrick and Flint, 1985)
and cows (Jenner et al., 1991) endometrial concentrations of OTR are maximal at estrus
as systemic concentrations of E2 are increasing and then decline by Days 3-5 of the cycle.
The reduction in uterine OTR cannot be attributed to inhibition by progesterone because
luteal production of this steroid is minimal at these times. Further, a similar response to E2
has been observed to occur in ovariectomized cows after pretreatment with progesterone
to mimic the natural luteal phase of the cycle (Lamming and Mann, 1995) and in
ovariectomized ewes given no previous progesterone treatment (Vallet et al., 1990).
These data suggest that in the intact ewe and cow, follicular secretion of E2 at estrus
104
causes down-regulation of uterine OTR, perhaps by the same mechanism as chronically
administered E2 in the ewe.
Flint and Sheldrick (1982) were the first to demonstrate that an exogenous
analogue of PGF2a induced the secretion of luteal OT in ewes during the late stages of the
estrous cycle. Because ovine large luteal cells are the source of OT and also contain ER
(Glass et al., 1984) it was hypothesized that chronic treatment of ewes with E2 may alter
luteal production/secretion of this nonapeptide. However, results of the present study
indicate that chronic treatment of ewes with E2 did not affect PGF2a- induced secretion of
OT on Day 15. These data suggest that although chronic E2 treatment interferes with
luteolysis by down-regulating OTR, the PGF2c, inducible component of the positive
feedback system in these animals is fully functional.
In conclusion, it appears that chronic treatment of ewes with E2 during the entire
cycle or treatment of ewes with progesterone only during the late luteal phase of the cycle,
suppresses OTR gene expression with a concomitant reduction in endometrial
concentration of the receptor. Data from this study suggest that the suppressive effects of
each steroid on endometrial OTR are the consequence of each hormone acting through a
distinctly different mechanism. Further, at this time, there is no evidence that chronic E2
treatment of ewes during the cycle has any impact on the ability of the CL to secrete OT.
105
GENERAL CONCLUSIONS
Research was conducted to establish that daily administration of E2 could extend
the interestrous interval in ewes when treatment was initiated on Day 4 postestrus.
Chronic E2-treated ewes failed to exhibit behavioral estrus within the normal 17 day
estrous cycle characteristic of sheep. While these data support those of Piper and Foote
(1968), the mechanism by which luteolysis was inhibited remained to be clarified. In a
subsequent study, examination of ovine uterine tissue on Day 15 demonstrated that
chronic E2 treatment of ewes down-regulated endometrial OTR concentrations.
McCracken et al. (1984) proposed that follicular phase E2 increases production of uterine
ER, which are requisite for stimulating OTR gene expression. However, in our study the
concentration of endometrial ER were not altered by E2 treatment. Thus, the reduction in
OTR observed in the E2-treated ewe could not be attributable to a reduction in
endometrial concentrations of ER. Similarly, up-regulation of uterine OTR without a
change in ER has been reported in ovariectomized ewes treated with progesterone and E2
as compared with animals receiving progesterone alone (Spencer et al., 1995b). Thus,
changes in uterine OTR concentrations in steroid-treated ewes do not always heed to the
hypothesis of McCracken (1984).
Uterine oxytocin receptors are a necessary component of the luteolytic mechanism,
therefore the decreased concentration of these receptors could prevent the necessary
secretion of the luteolytic hormone, PGF2a. In actuality, measurable PGF2a secretion
(PGFM) in response to exogenous oxytocin was reduced in chronic E2-treated animals.
106
The attenuation of oxytocin-induced secretion of PGFM in E2-treated ewes suggests that
receptor function was not impaired by treatment, but was characteristic of decreased
concentrations of OTR. Similarly, research in ovariectomized ewes demonstrated that
oxytocin-induced secretion of PGFM did not occur in ewes without significant uterine
OTR concentrations (Vallet et al., 1990). In contrast, chronic E2 treatment of ewes for 11
days does not appear to alter Day 15 PGF2-induced secretion of oxytocin. Although
chronic E2 treatment down-regulates OTR, altering the luteolytic mechanism, the PGF2a-
induced component of the luteolytic mechanism remains intact in the face of
supraphysiological concentrations of E2. These data suggest that the effect of chronic E2
treatment appears to specifically affect the uterine component of the luteolytic mechanism.
Chronic treatment of ewes with E2 maintained the life span of corpora lutea
beyond Day 15 postestrus. Serum concentrations of progesterone in the first set of
experiments were also maintained at midluteal phase concentrations in treated ewes.
These data suggest that the functionality of corpora lutea in E2-treated ewes is extended,
and thus elevated concentrations of progesterone on Day 15 may play a role in regulating
the luteolytic mechanism. Lau et al. (1992a) showed that progesterone treatment
decreased concentrations of OTR in ovariectomized ewes. Further, a subsequent study
demonstrated that although progesterone reduced endometrial concentrations of OTR in
ovariectomized ewes, upon withdrawl of exogenous progesterone, OTR concentrations
increased to pre-treatment levels (Lau et al., 1992b). Therefore, in our study the midluteal
phase serum concentrations of progesterone measured on Day 15 suggest that chronic E2
treatment may reduce endometrial concentrations of OTR indirectly through the sustained
107
production of progesterone. Consequently, subsequent experiments were conducted to
determine if exogenous progesterone could also induce a reduction in endometrial
concentrations of OTR similar to that observed after chronic treatment with E2. Results of
our research indicated that progesterone treatment from Days 11 to 14 of the estrous
cycle inhibited the increase in endometrial concentrations of OTR associated with
luteolysis. Analysis of mRNA for the OTR gene in Day 15 endometria demonstrated that
the effect of chronic E2 or progesterone treatment was to suppress expression of the OTR
gene. These data support the hypothesis that the effect of chronic E2 treatment in the ewe
may be mediated by the sustained luteal phase concentrations of progesterone, and
suggests that E2 and progesterone may act genomically to block the increase in
endometrial OTR. However, the genomic mechanisms of action of chronic E2 and
progesterone appear to be different. Although an estrogen response element (ERE) has
been demonstrated in the 5' flanking region of the rat OTR gene, to date no response
element for progesterone has been detected in this region (Rozen et al., 1995; Bale and
Dorsa, 1997). However, the promoter region of the ovine OTR gene has not been
characterized, thus the possibility of E2 and progesterone response elements in this region
cannot be excluded. Interestingly, although progesterone has been demonstrated to inhibit
OTR formation (Zhang et al., 1992), through endometrial progesterone receptors (PR;
McCracken et al., 1984), recent data on localization of endometrial PR and OTR suggests
that on Days 14 and 15 of the cycle, the two receptor types are not colocalized within the
same endometrial cell. Wathes et al. (1996) demonstrated that OTR are found principally
in the luminal epithelium on Day 14 in steroid-treated ovariectomized ewes. Conversely,
108
progesterone receptors are found predominantly in the caruncular stroma, and are not
detectable in the luminal epithelium after Day 8 of the cycle. However, progesterone must
act through a receptor mediated response. In our study progesterone treatment of ewes
did not induce a change in endometrial PR concentrations, suggesting that progesterone
was acting through the basal concentrations of PR present in the stroma. These data
suggest that in the chronic E2- or progesterone-treated ewe, progesterone may act
genomically to stimulate the production of some factor(s) from the endometrial stromal
cells that suppress OTR gene expression in luminal epithelial cells with a consequent
down-regulation of endometrial concentrations of OTR.
It has long been known that exogenous E2 can regulate the estrous cycle. The
treatment of ewes with chronic estradiol has provided us with a model to examine the
mechanism by which steroids can regulate luteolysis. Future research is needed to
sequence the 5' flanking region of the ovine OTR gene. The promoter sequence of this
gene would prove invaluable for further contemplation of factors that regulate the OTR,
as well as furnish evidence for steroid involvement. Differential tissue expression of OTR
and PR could be examined using a nuclear transcription runoff assay to measure specific
steroid-induced gene transcription in endometrial stromal and epithelial cells. This
procedure would hopefully elucidate possible factors, stimulated by progesterone, that
may regulate OTR gene expression in luminal epithelial cells. Isolation of a factor(s) from
these cells could then be tested in a promoter construct system, to determine if the element
could induce expression of the OTR gene. These experiments would, without doubt,
provide insight into the molecular control of luteolysis.
109
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