AN ABSTRACT OF THE THESIS OF
Diana L. Whitmore for degree of Master of Science in Animal Sciences presented on
March 13, 1995. Title: Corpus Luteum Function in Hysterectomized and Unilaterally
Hysterectomized Ewes Treated with Gonadotropin-Releasing Hormone.
Abstract approved:
Fredrick Stormshak
Two experiments were conducted to examine the effects of exogenous GnRH on
luteal function in hysterectomized (HYST) and unilaterally hysterectomized (UHYST)
ewes. In each experiment, crossbred ewes were assigned randomly in equal numbers into
four groups in a 22 factorial arrangement. Treatments consisted of two levels of GnRH (0
and 100 gg/day) and two levels of hysterectomy (Exp. 1, none and UHYST; Exp. 2, none
and HYST). On day 12 of an estrous cycle, all ewes in Exp. 1 (n = 16) were unilaterally
ovariectomized and in eight ewes the uterine horn adjacent to the remaining ovary was
removed, while in Exp. 2 (n = 20) the entire uterus was removed from one-half of the
ewes. In each experiment CL in the remaining ovary or ovaries were enucleated. After
subsequent estrus, one-half of the control and UHYST or HYST ewes were injected i.v.
with GnRH on days 2 and 3 (Exp. 1) or day 2 only (Exp. 2) while the remaining ewes
were injected similarly with saline. Jugular blood was collected for 60 min after injection
for analysis of serum LH (Exp. 1) and periodically thereafter for analysis of serum
progesterone (P4) (Exp. 1 and 2), and plasma oxytocin (OT) (Exp. 1). In Exp. 1,
catheterization of the caudal vena cava was performed on all ewes on day 4 and periodic
plasma samples were collected for OT and prostaglandin Fla (PGF2a) analysis. In Exp. 1
injection of GnRH increased serum concentrations of LH within 60 minutes compared
with those of saline-treated ewes (P = .01). The GnRH-induced secretion of LH in intact
and UHYST ewes on day 2 was greater than on day 3 (P = .05). Treatment with GnRH
did not alter jugular or vena cava concentrations of OT in intact or UHYST ewes on d 5
to 10 after estrus. However, mean jugular plasma concentrations of OT on days 12 and
14 were greater in all intact vs all UHYST ewes (P = .006), with levels of OT in saline-
treated intact ewes being significantly greater than those of the other groups. Overall,
vena cava levels of PGF2a did not differ significantly among treatment groups; however,
on days 10 to 14 those intact ewes receiving GnRH had higher concentrations of PGFat
compared to those ewes of other groups (P = .07). Treatment with GnRH was without
effect on serum concentrations of P4 but levels of this steroid were greater in all UHYST
ewes compared with those in intact ewes (P = .09). In Exp. 2, HYST ewes that were
injected with GnRH had lower serum levels of P4 on days 4 to 12 (P = .04) than GnRH­
treated intact or saline-treated intact and HYST ewes. Therefore, while luteal P4
production by intact and UHYST or HYST ewes was not consistently altered by
exogenous GnRH, levels of this hormone were affected by the presence or absence of the
uterus.
Corpus Luteum Function in Hysterectomized and Unilaterally Hysterectomized Ewes
Treated with Gonadotropin-Releasing Hormone
by
Diana L. Whitmore
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed March 13, 1995
Commencement June 1995
Master of Science thesis of Diana L. Whitmore presented on March 13, 1995
APPROVED:
Major Professor, representing Animal Sciences
Head
meniV. f Ani
.1. ciences
Redacted for privacy
Dean of Graduate
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
Diana L. Whitmore, Author
ACKNOWLEDGMENTS
So many people have been instrumental in helping me to achieve my goals in
academia, that I really don't know where to start! I would like to begin by thanking the
entire Department of Animal Sciences. Everyone has been so friendly and giving, that you
can't help but feel at home in this department. Particularly, I would like to recognize Dr.
Fredrick Stormshak, my major professor. If he hadn't hired me as an employee back in
1991, I most certainly would have never continued on to graduate school. Through his
encouragement, I threw my B.S. in Zoology (emphasis in marine biology) in the back seat
and continued on to get a M.S. in Animal Sciences. What a change! Thanks for believing
in me Stormy!
I would like to thank those who have helped me with my research; Beth Olenchek,
Pam Thineson, Mike Lutz and Ursula Bechert. Thanks to all those faculty and students
who participated in intramural volleyball and softball; participating in those teams are
some of my fondest memories here. Those fellow graduate students, past and present,
who have supported and believed in me. Kyle "Kansas" Orwig, who was kind enough to
let me catch a ride to work my first year but he made me pay for it by continuously kicking
my butt in one-on-one basketball; I think I still owe him "Big Gulp". Joan Burke, whose
drive and determination made me feel so guilty for not working as hard as her, that I
eventually did. Jennifer Bertrand, with whom I've shared an office since Joan's departure.
Jennifer never ceases to amaze me with her fantastic brain power; this woman can spew
information from classes she took in high school as well as spot a typo from across a
crowded room. Tim "Captain Planet" Hazzard who makes me feel guilty for throwing
away a gum wrapper (I'm still waiting for those "corn starch" cookies Tim). Shelby Filley,
who decided to come back to college for her Ph.D. after ten years off from school, juggles
a family and still manages to have a cheerful attitude! Maybe its all that basketball? I can't
forget Bill Schutzer, up in the Great Holtini's laboratory, who had to put up with me in
almost every one of his classes our first year in graduate school (thanks for making
biochemistry a little more bearable). My final thank you to my pals at OSU goes to Mike,
my canine friend. Without our occasional noontime walks and talks, graduate school
would have been alot more stressful. Thanks for the advice Mike; you can have the
whipped cream off my hot chocolate anytime!
I would like to extend special thanks to my family. My parents, Donald and
Louise Whitmore, who have always encouraged me to pursue my dreams and to continue
learning; even after it blew up in their faces! You see, my dad is one of those people who
likes to have an answer for every question. So in those few occasions that he didn't know
the answer, he would...well...make one up. However, I never knew he was making up
answers until I came to college. At this fine institution, I finally found out that the really
pretty flower in the shape of a star wasn't really named the "star flower", and that black
bird with the red patch on its wings wasn't really named the "midnight red" bird but rather
the red-winged black bird (I liked dad's name better though C)).
I'd also like to thank my two older sisters, Donna Helman and Debbie Uyeda, who
despite my pet names for them as "my evil step sisters" have given me endless support and
confidence. My fiancee, Dean Takahashi, who has been tremendously patient during these
last few months with me coming home late (and grouchy). Without his love and support
through the years, I never would have made it! We will be married on March 25, 1995, in
Pearl City, Hawaii.
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
2
THE ESTROUS CYCLE: AN OVERVIEW
2
SEASONAL ANESTRUS
The Gonadotropin-Releasing Hormone Pulse Generator
Effect of Photoperiod on Anestrus
The Pineal Gland and Melatonin
5
10
FOLLICULOGENESIS
12
OVULATION OF THE DOMINANT FOLLICLE
14
CHARACTERISTICS OF THE CORPUS LUTEUM IN THE NONPREGNANT ANIMAL
Morphology and Biochemistry of the Corpus Luteum
Hormones Synthesized by the Corpus Luteum
Steroid Hormones
Progesterone
Estrogen
Peptide Hormones: A Focus on Oxytocin
15
HYPOTHALAMIC-HYPOPHYSEAL HORMONES INFLUENCING
THE CORPUS LUTEUM DURING THE ESTROUS CYCLE
Hypothalamic Hormones: An Overview of GonadotropinReleasing Hormone
Pituitary Hormones
Luteinizing Hormone
Follicle Stimulating Hormone
Oxytocin
Prolactin
34
46
ROLE OF THE UTERUS IN REGULATING LUTEAL LIFE SPAN
Effects of Hysterectomy
Utero-ovarian Functional Interrelationships and Luteolysis
Action of Prostaglandins
49
49
52
54
5
9
16
19
19
20
23
27
34
38
40
43
45
TABLE OF CONTENTS (Continued)
Role of Oxytocin
Luteotropic Mechanism
56
57
STATEMENT OF THE PROBLEM
60
EXPERIMENTS 1 AND 2: CORPUS LUTEUM FUNCTION
IN HYSTERECTOMIZED AND UNILATERALLY
HYSTERECTOMIZED EWES TREATED WITH
GONADOTROPIN-RELEASING HORMONE
62
INTRODUCTION
MATERIALS AND METHODS
Experiment 1
Experiment 2
Radioimmunoassays
Enzyme Immunoassay
Statistical Analysis
RESULTS
DISCUSSION
SUMMARY
BIBLIOGRAPHY
62
63
63
65
66
67
68
68
70
72
80
LIST OF FIGURES
Page
Figure
1.
Jugular serum concentrations
of LH in
intact or unilaterally
74
hysterectomized (UHYST) ewes for 60 min after i.v. treatment with
either saline or GnRH (100 µg/d) on d 2 (A) and 3 (B) of the cycle.
Time 0 is just prior to injection.
2.
Jugular plasma concentrations
of OT in
intact or unilaterally
75
hysterectomized (UHYST) ewes treated with either saline or GnRH
(100 ug on d 2 and 3) over d 5 to 14 of the cycle.
3.
Vena cava plasma concentrations of OT in intact or unilaterally
76
hysterectomized (UHYST) ewes treated with either saline or GnRH
(100 lig on d 2 and 3) over d 5 to 14 of the cycle.
4.
Vena cava plasma concentrations of PGF2a in intact or unilaterally
hysterectomized (UHYST) ewes treated with either saline or GnRH
77
(100 ug on d 2 and 3) over d 5 to 14 of the cycle.
5.
Jugular
serum
concentrations
of P4
in
intact or
unilaterally
78
hysterectomized (UHYST) ewes treated with either saline or GnRH
(100 ug on d 2 and 3) over d 5 to 14 of the cycle.
6.
Jugular serum concentrations of P4 in intact or hysterectomized
(HYST) ewes treated with either saline or GnRH (100 ug on d 2) over
d 4 to 12 of the cycle.
79
CORPUS LUTEUM FUNCTION IN HYSTERECTOMIZED AND
UNILATERALLY HYSTERECTOMIZED EWES TREATED WITH
GONADOTROPIN-RELEASING HORMONE
INTRODUCTION
The corpus luteum is a transient endocrine gland that is required for the
maintenance of pregnancy in mammals (Niswender and Nett, 1988; Niswender et al.,
1994). The primary function of this ovarian structure is to produce progesterone, which
prepares the uterine endometrium for the implantation of the fertilized ovum and maintains
early pregnancy. In domestic animals, fertilization rates are typically high and
approximately 70% of ovulations give rise to live births (Roberts et al., 1990). The loss of
embryos typically occurs within the first three weeks of pregnancy and at least some of
this loss appears to be due to inadequate corpus luteum function (Niswender and Nett,
1988). Reproductive failure has been implicated as one of the most costly and limiting
factors in the livestock industry with the overall economic impact well into the billions of
dollars (Gerrits et al., 1979). Therefore, efforts to understand the regulation of the life
span and function of the corpus luteum is of the utmost importance for improving
reproductive efficiency in domestic animals. The following is an attempt to review the
literature pertaining to the factors regulating the life span of the corpus luteum of several
mammalian species and more specifically the effects of injecting gonadotropin-releasing
hormone on the luteal function of the ewe.
2
LITERATURE REVIEW
THE ESTROUS CYCLE: AN OVERVIEW
Most sexually mature mammalian females are subject to cyclic changes in
reproductive activity. These changes may be coordinated with alterations in the seasonal
environment so that young are born at a time when both climate and food availability are
optimal. Seasonal breeding occurs in most wild animals, whereas only some domesticated
animals, such as sheep, goats and horses, exhibit breeding seasons while cattle and swine
do not (Hafez, 1987). Ewes and goats are "short-day" breeders, meaning they become
sexually active in the fall when daylight hours are decreasing, thus parturition occurs in
early spring when temperatures are warm and forages are plentiful. The mare, in contrast,
is a "long day" breeder, becoming sexually active in the spring, but due to a year long
gestation period, birth also occurs in the spring. In addition to the external factors
mentioned, the presence of a male can also influence onset of estrus in several species
(Hafez, 1987).
In numerous mammals, sexual behavior is closely linked to ovulation and is
dependent on ovarian production of estradiol to stimulate estrous behavior (Asdell et al.,
1945). It is only during this phase of the estrous cycle that the female is sexually receptive
to the male. In contrast, most species of non-human primates do not limit sexual activity
to any one phase of the menstrual cycle, although peaks in sexual activity are still observed
midcycle, around the time of ovulation (Hadley, 1992a).
Duration of the estrous cycle varies among species as well as individual animals.
The estrous cycle of the ewe is approximately 16-18 days, the cow, sow, and doe 20-21
days, and that of the mare is 20-24 days. Internal factors regulate the character of the
estrous cycle, including hormones emanating from the hypothalamus, pituitary, ovary and
uterus.
3
The estrous cycle is divided into four stages: estrus, metestrus, diestrus and
proestrus. Based upon ovarian structures present, the estrous cycle can also be divided
into two phases, luteal and follicular; both phases are somewhat overlapping in
domesticated animals. The luteal phase encompasses the period of maximal functional
activity of the corpus luteum (CL) and lasts 14-15 days in the ewe and 16-17 days in the
cow and sow. The follicular phase coincides with proestrus and lasts 2-3 days in the ewe
and doe and 3-6 days in the cow and sow (Hafez, 1987).
Estrus is characterized by behavioral changes, the most obvious being the female
standing for the male to mount. The period of behavioral estrus lasts approximately 24-36
hours in ewes, 32-40 hours in does, 18-19 hours in cows, 48-72 hours in sow and 4-8
days in mare. Behavioral estrus is closely coordinated with ovulation and is the only time
in which the female is sexually receptive to the male. Ovulation of the ovum occurs
primarily as a result of a peak release of luteinizing hormone (LH) and follicle stimulating
hormone (FSH) from the anterior pituitary, which are under the control of gonadotropinreleasing hormone (GnRH) from the hypothalamus.
During metestrus, the granulosa and theca cells lining the walls of the recently
ovulated follicle undergo a luteinization process to become the luteal cells. As the corpus
luteum continues to develop, two distinct cell types emerge that are termed small and
large luteal cells based on their different sizes. In the ewe, progesterone secretion begins
to rise to significant levels by 30 hours post-ovulation due to the formation of the
functional CL (Caldwell et al., 1972). The increase in luteal progesterone is thought to be
under the control of LH (Niswender and Nett, 1988).
Diestrus is characterized by maximal luteal secretion of progesterone. The CL
formed from the granulosa and theca cells is now fully functional and capable of secreting
increased levels of progesterone. Estradiol of ovarian origin is capable of increasing the
sensitivity of the hypothalamic GnRH pulse generator to progesterone negative feedback,
which indirectly suppresses the release of LH and prevents ovulation (Karsch, 1987).
Progesterone is also capable of directly inhibiting the secretion of LH from the pituitary in
an estradiol-dependent manner as demonstrated in pituitary stalk-sectioned ewes subjected
to periodic boluses of GnRH (Girmus and Wise, 1992).
Proestrus, characterized by declining serum concentrations of progesterone, occurs
around days 13-14 of the ovine estrous cycle due to the regression of the existing CL
(Caldwell et al., 1972). Identification of the uterine hormone prostaglandin Fla (PGF2a)
as the substance that brings about the demise of the CL was first demonstrated in
pseudopregnant rats (Pharriss and Wyngarden, 1969), but soon after was shown to occur
in other species such as cattle, sheep, goats, horses and guinea pigs (Chaichareon et al.,
1974; Douglas and Ginther, 1972; Lauderdale, 1972; McCracken et al., 1970b; Ott et al.,
1980). The manner in which PGF2a exerts its effect on the CL varies between species. In
ruminants such as cattle and sheep, the luteolytic effect of PGF2a occurs locally as
described by McCracken and coworkers (1972), whereas in horses the effect is via
systemic circulation.
Prostaglandin Fla has also been shown to stimulate secretion of oxytocin from the
CL in ruminants (Flint and Sheldrick, 1982; Lamsa et al., 1989). Conversely, oxytocin has
been shown to cause premature regression of the CL by stimulating the release of uterine
PGF2a (Flint and Sheldrick, 1983). Therefore, it has been hypothesized that in these
species uterine PGF2a and luteal oxytocin comprise a positive feedback loop, where
PGF2a is released from the uterine horn and acting through a counter-current transfer
mechanism, acts upon the adjacent ovary to stimulate oxytocin secretion from the CL.
This luteal oxytocin then acts back upon the uterine horn to further increase the secretion
of PGF2a, which causes the eventual regression of the CL (Flint and Sheldrick, 1983;
McCracken et al., 1984; Sheldrick and Flint, 1984). The ovarian steroids progesterone
and estradiol -1713 are also believed to be involved in regulating the secretion of PGF2a,
possibly by regulating the concentration of oxytocin receptors in the endometrium of the
uterus (Roberts et al., 1976; Robinson et al., 1976; McCracken et al., 1984).
5
The concentration of estrogen secreted from surrounding follicles begins to rise
coincidental with the decline in progesterone levels around day 13 of the ovine estrous
cycle, and attains maximal levels
24-48
hours later (Bjersing et al.,
1972).
Reduction in
progesterone and the increased production of estradiol results in increased pulsatile
secretion of LH and FSH (Goding et al.,
1970;
Scarmauzzi et al.,
1971).
It is this rise in
estradiol that causes expression of behavioral estrus and stimulates the pulsatile secretion
of LH and therefore ovulation.
SEASONAL ANESTRUS
During the time of seasonal anestrus the normal cyclicity of the female
reproductive system ceases. Ovarian and follicular activity is minimal thereby preventing
ovulation as well as behavioral estrus. The extent of seasonal anestrus varies with the
species, breed and physical environment. Anestrus occurs in such domestic species as
sheep, goats and horses as well as wild species such as deer, elk and several other
members of the cervidae family. In most of these animals, anestrus occurs during the
spring, summer and in some instances the fall, such that parturition occurs at an
environmentally desirable time. In temperate zones, spring is usually the time for delivery
of the young because food is more abundant and there is less environmental stress.
The Gonadotropin-Releasing Hormone Pulse Generator
The central mechanism controlling both external and internal cues for reproductive
activity is the GnRH pulse generator (also referred to as LH pulse generator). The GnRH
pulse generator produces an episodic pattern of GnRH release from hypothalamic nerve
terminals. These pulses in turn stimulate pulses of LH release from the anterior lobe of the
pituitary. Normally, the pulse of LH has a high amplitude but a low frequency of release.
However, near the time of ovulation, LH pulse frequency increases from one pulse every 3
6
to 4 hours to one pulse every 30 minutes (Goodman and Karsch, 1981). The overall
concentration of LH, as well as pulse frequency, increases as the time of ovulation
approaches while the amplitude of the individual LH pulse actually gets smaller.
However, if this sustained increase in pulsatile LH secretion is blocked, ovulation will be
prevented (Goodman and Karsch, 1980; Legan and Karsch, 1979). During seasonal
anestrus the pattern of LH release stays relatively constant with LH secretion having a low
frequency of release and a relatively high amplitude.
Ovarian steroids also seem to have an effect on the ability of the GnRH pulse
generator to modulate LH secretion. It has been well established that removal of
circulating gonadal steroids by ovariectomy results in a major increase of both the
frequency and the amplitude of LH pulses and that addition of exogenous estradiol and/or
progesterone can lower LH to normal levels (Foster and Ryan, 1979; Foster et al., 1986;
Goodman and Karsch, 1980). Goodman and coworkers (1982) studied both
ovariectomized (OVX) anestrous and ewes supplemented with an estradiol implant (2.7
pg/ml) 16 hours after ovariectomy. These authors found that in OVX cycling ewes, the
administration of estradiol had no effect on LH pulse frequency but did decrease pulse
amplitude and mean serum LH concentrations. In OVX anestrous ewes, the presence of
exogenous estradiol decreased mean serum LH concentrations and obliterated both LH
pulse frequency and amplitude over an 8 hour sampling period, suggesting that estradiol is
an extremely powerful inhibitor of LH secretion. This seasonal response of ewes to the
negative feedback action of estradiol was also observed by Karsch et al. (1993) who found
that positioning an implant containing a low dose of estradiol (0.3 pg/ml) into
ovariectomized anestrous or cycling ewes produced a differential response. Anestrous
ewes responded with an increase in GnRH pulse size but a decrease in both GnRH and LH
pulse frequency due to estradiol implants, while cycling ewes exhibited no effect of low
exogenous estradiol on either frequency or magnitude of GnRH or LH pulses. Taken
7
together, these results indicate that there is an increased sensitivity of the GnRH
neurosecretory system to the negative feedback actions of estradiol during anestrus.
The way in which estradiol elicits its effect on gonadotropin secretion has been
studied so extensively that its proposed mechanism of action is referred to as the
"gonadostat hypothesis". This hypothesis states that concentrations of circulating
gonadotropins are low in the immature or anestrous animal because the hypothalamus
and(or) pituitary are very sensitive to the inhibitory feedback action of ovarian estradiol.
However, as breeding season approaches, there is a decrease in sensitivity to estradiol,
allowing gonadotropin secretion to increase and reproductive activity to commence
(Foster, 1988). However, the exact mechanism by which estradiol exerts its control is still
an enigma.
Some researchers have postulated that estradiol may act directly on GnRH­
secreting neurons to depress secretion (Dyer et al., 1980) and(or) pulse amplitude of
GnRH (Dreifuss et al., 1981; Lincoln et al., 1985). Immunocytochemical techniques,
however, have shown that the target cells for estradiol in the hypothalamus are different
than the GnRH-producing neurons, although the distribution of these neurons do overlap
(Shivers et al., 1983). Despite this, McEwen et al. (1982) suggested that the absence of
identified binding sites for estradiol in GnRH neurons does not exclude these cells as
targets because steroid hormones may elicit certain neural effects through pathways other
than classical receptor-mediated genomic mechanisms.
The other possible way in which estradiol may modulate GnRH and its control
over LH and FSH secretion is by its action on the pituitary. Some researchers postulated
that the negative feedback effect of estradiol in the ewe is exerted directly at the level of
the anterior pituitary gland, and that the pituitary is more sensitive to the negative effects
of estradiol during the anestrous season (Clarke and Cummins, 1984; Goodman and
Karsch, 1980; Nett et al., 1984). The negative feedback of gonadal steroids on the
anterior pituitary, during the anestrous season, may occur by modulating GnRH membrane
8
receptor availability and thereby controlling the amount of gonadotropin release (Hauger
et al., 1977).
Gregg and Nett (1989) used ovariectomized ewes to investigate the possibility that
estradiol may be controlling pituitary GnRH receptors. Ewes were either subjected to a
pituitary stalk-section or left intact, and all ewes were treated with GnRH in the presence
or absence of estradiol. These researchers found that the presence of estradiol caused an
initial up-regulation of pituitary GnRH receptors, but only intact ewes responded with an
increase in LH secretion. This suggested that while GnRH is necessary for the secretion
of the gonadotropins, its action may be contingent upon estradiol-induced regulation of
GnRH receptors in the pituitary. Similar research was performed by Girmus and Wise
(1992) who treated ovariectomized and pituitary stalk-sectioned ewes with GnRH in the
presence or absence of estradiol, progesterone or both steroids. These researchers found
that only a combination of progesterone and estradiol lowered GnRH-induced LH surges
most efficiently. It is through this complex interaction of positive and negative control by
ovarian steroids that the GnRH pulse generator and thus ovulation is regulated.
The GnRH pulse generator in the ewe is highly susceptible to variation in the
external environment; particularly olfactory cues and changes in light patterns. For
example, introduction of pheromones from an unfamiliar ram to a group of anestrous ewes
results in an immediate induction of hourly LH pulses (Martin et al., 1983; Poindron et al.,
1980). However, this response may vary according to breed of sheep. Minton et al.
(1991) exposed both Polypay and Suffolk anestrous ewes to an unfamiliar ram, and while
both groups of ewes responded with an increase in serum LH, only Polypay ewes
exhibited serum progesterone profiles indicative of consecutive estrous cycles, while the
Suffolk ewes ovulated but had only one full length luteal phase or exhibited a short luteal
phase. Conversely, constant exposure to a familiar ram throughout the breeding season
and anestrus does not seem to have a significant effect on the timing of anestrus or
cyclicity in mature ewes. Ewes that have either constant exposure or no exposure to
9
males, have a specific timing of anestrus and reproductive seasons, which appears to be
regulated by photoperiod (Karsch et al., 1984). This suggests that the changes in
photoperiod associated with seasonality are probably the major contributor in controlling
seasonal breeding.
Effect of Photoperiod on Anestrus
The way in which photoperiod affects ovarian cyclicity has been studied by
numerous scientists. In an experiment conducted by Goodman et al. (1982), serum LH
levels from ovariectomized ewes were analyzed over the course of a year under natural
photoperiod. These authors found that during the long daylight hours of summer, LH
pulses were of a high amplitude but of low frequency. As hours of daylight decreased,
however, the secretory pattern of LH switched such that there was an increase in LH pulse
frequency but a decrease in pulse amplitude. Similar results were achieved in
ovariectomized ewes that were kept under artificial light conditions (Bittman et al., 1982;
Robinson, 1989).
As previously mentioned, however, the negative effects of ovarian hormones also
have a differential effect depending on season. This is demonstrated best by a series of
experiments by Goodman et al. (1982) who fitted ovariectomized anestrous and cycling
ewes with an estradiol implant. These authors found that while estradiol was capable of
reducing LH pulse frequency and amplitude in anestrous animals, the same dose was
ineffective in cycling ewes. A series of experiments by Legan and Karsch (1980)
demonstrated that the timing of the shift to increased sensitivity to estradiol negative
feedback during anestrus was driven by photoperiod rather than temperature. These
researchers kept intact and ovariectomized ewes (with estradiol implants) indoors and
exposed them to an artificial photoperiod, which alternated between "long" and "short"
days every 90 days. In both groups, ewes responded with increased overall concentrations
of LH normally associated with natural breeding season during the artificial "short" days.
10
This particular response was totally out of phase with the natural seasonal reproduction.
Therefore these researchers postulated that reproductive seasons are driven by changes in
photoperiod (Goodman et al., 1982). Effects of photoperiod on seasonal reproduction
appear to be mediated through the pineal gland rather than exerted directly on the GnRH
pulse generator.
The Pineal Gland and Melatonin
In both "short" and "long" day breeders, researchers have proposed that the pineal
gland mediates reproductive response to photoperiod cues through its pattern of melatonin
secretion (Bittman et al., 1983; Glass and Lynch, 1981). Melatonin is an indoleamine that
is secreted only at night such that the duration of melatonin release is roughly proportional
to the hours of darkness. The pathway for transmission of photoperiod cues, which
regulates or affects the annual reproduction cycle in ewes, includes the retinal
photoreceptors, a monosynaptic retino-hypothalamic tract, the superchiasmatic nuclei, the
superior cervical ganglia, the pineal gland and finally the secretion of melatonin (Foster,
1988).
It has been hypothesized that it is the seasonal change in duration of melatonin
secretion that coordinates reproductive activity (Wayne et al., 1988). In the ewe, as hours
of daylight decrease, the duration of melatonin secretion at night increases. This increased
duration in melatonin secretion causes a change in the sensitivity of the GnRH pulse
generator to the negative feedback of estradiol, such that the pulse generator becomes
more refractory to the inhibitory effects of ovarian steroids (Foster, 1988), thus causing a
resumption in reproductive activity. It is important to note that the patterns of melatonin
production can be similar in both "short" and "long" day breeders but the response can be
markedly different. For example, just as an increase in melatonin secretion can trigger the
onset of the breeding season in the ewe, it can also cause a cessation of reproductive
activity in the mare.
11
The influence of the pineal gland and melatonin secretion has been extensively
researched in the ewe. In an experiment conducted by Bittman et al. (1983), ewes with
intact ovaries were pinealectomized or left intact in early spring and subjected to artificial
photoperiods with 90 day shifts between "long" and "short" days for a 2 year period.
These researchers found that intact ewes responded to the artificial photoperiod
consistently such that reproductive activity commenced upon exposure to decreasing light.
In contrast, the pinealectomized ewes exhibited a reproductive pattern out of synchrony
with the imposed photoperiod while retaining some natural seasonality; however, this
endogenous seasonality became asynchronous with time. Bittman et al. (1983) also
performed a similar experiment on estradiol-treated ovariectomized and pinealectomized
ewes. Pinealectomized ewes retained an LH profile that was similar to the normal
seasonal reproductive cycle, however, this endogenous timing of seasonality eventually
became incongruent as well (Bittman et al., 1983). Based upon the results of these
experiments, it seems that the pineal gland must be present to mediate the effect of
photoperiod on ovarian cyclicity in the ewe. This function is likely to be affected through
changes in potency of estradiol in the negative feedback inhibition of tonic LH secretion.
Probably the most compelling evidence for the role of melatonin in seasonal
reproduction was demonstrated in an experiment conducted by Bittman and Karsch
(1984) in which pinealectomized ewes were kept reproductively suppressed by exposure
to "long" day melatonin infusion along with an artificial "long" day photoperiod. Ewes
were subsequently transferred to the inductive "short" day photoperiod, where control
ewes received concurrent "short" day melatonin infusion and the treated ewes received a
"long" day melatonin infusion (out of synchrony). As expected, all control ewes had
elevated serum LH concentrations typical of the secretory profile of this hormone during
normal "short" days. However, the group receiving the "long" day melatonin treatment
continued to have an attenuated LH secretory pattern even after being transferred to short
day photoperiod thus resulting in a LH secretory profile typical of long days. This
12
research provides compelling evidence that the circadian rhythm of melatonin secretion
has not merely a permissive role on the reproductive response to photoperiod, but rather
determines it.
FOLLICULOGENESIS
Ovaries of the mammalian female at birth, contain all the oogonia that the animal
will be allotted for its entire life. The ovaries contain a pool of primordial follicles, each
consisting of an oocyte arrested in Prophase I of meiosis, and surrounded by a single layer
of flattened granulosa cells (Hadley, 1992a; Fortune, 1994). In the case of most mammals
this pool develops during the fetal life, while in others such as rodents, mink, and rabbits,
the pool develops in the early neonatal period (Hirshfield, 1991; Marion and Gier, 1971).
Primordial follicles remain in this arrested state of development until puberty, when there
is an associated increase in LH production from the anterior pituitary (Hadley, 1992a).
Beginning at puberty, follicles gradually but continually leave the resting pool to
begin growth that eventually will result in either ovulation or atresia of the follicle. There
are a series of developmental stages through which follicles pass to attain an ovulatory
size; the first is the primordial follicle, followed by primary follicle, secondary follicle,
early tertiary follicle, and finally the Graafian follicle (Erickson et al., 1985; Hafez, 1987).
Granulosa cells of the primary follicle become more cubiodal in shape and surround the
mature oocyte in a single layer. These granulosa cells secrete glycoproteins which will
eventually form the zona pellucida (Erickson et al., 1985; Hadley, 1992a; Hafez, 1987).
Secondary follicles are characterized by further proliferation of the granulosa cells,
completion of the zona pellucida, an increase in blood supply, and the early development
of theca cells that are delineated from the granulosa cells by a basement membrane
(Erickson et al., 1985; Hadley, 1992a; Hafez, 1987). Follicular development, up to the
secondary follicle stage, can occur independent of gonadotropins, and most likely is
13
stimulated by a signal generated by the follicle itself (Erickson et al., 1985). However,
further development and antrum formation is dependent on LH and FSH (Hafez, 1987).
The early tertiary follicle is characterized by formation of a fluid-filled antrum, completion
of the theca interna, and beginning development of the theca externa. The final stage of
follicular development is the Graafian follicle, which contains a large antrum filled with
follicular fluid, and completed development of the theca externa (Erickson et al., 1985;
Hadley, 1992a; Hafez, 1987).
Through careful examination of blood hormone profiles, ultrasonographic
techniques, surgeries and laparoscopy, characterization of follicular development
(folliculogenesis) has been attempted in many domesticated animals. Sirois and Fortune
(1988) conducted an extensive investigation of folliculogenesis in heifers, generally
monovular animals, using real-time ultrasonography of ovaries daily throughout the
estrous cycle. Their findings confirmed some earlier reports that follicular growth occurs
in three distinct waves (Fogwell et al., 1985; Ireland and Roche, 1987). These researchers
found that during each wave one follicle is selected to become dominant, while the
remaining follicles undergo atresia. This dominant follicle will either ovulate or undergo
atresia, depending on the stage of the estrous cycle during which it is dominant (Sirois and
Fortune, 1988).
While the theory of follicular development occurring in waves in cattle has now
become central dogma, information on folliculogenesis in the ewe (a polyovular species)
remains controversial. In fact, two recent studies have been conducted, one following
follicular development by daily transrectal ultrasonography (Schrick et al., 1993), the
other by performing daily laparoscopy (Noel et al., 1993). The results of these studies are
conflicting.
Schrick et al. (1993) found that while there were two periods during the ovine
estrous cycle characterized by increased follicular activity, there was not an emergence of
a dominant follicle during these times. In fact, these researchers support the theory that
14
follicular activity is characterized by a continuous entry, growth and atresia of follicles,
which they believe fits with a lack of dominance. In essence, this allows for the presence
of several ovulatory-sized follicles necessary for multiple ovulations.
In contrast, Noel et al. (1993) examined ewes daily using non-traumatic
laparoscopy (5 mm optic fibre and manipulation probe under local anesthetic), and
concluded that follicular development occurs in three distinct waves, each lasting
approximately 6 days. They reported that during each wave, follicles ?_ 2 mm develop
from a pool, and that a few of these follicles will continue to undergo maturation and
growth to eventually become dominant follicles. These follicles either ovulate (dominant
ovulatory follicle) or become atretic (dominant non-ovulatory follicle). Therefore the
results of this study do not support the concept of continuous turnover of ovarian follicles,
but rather a pattern of follicular growth and turnover similar to that observed in the
bovine.
OVULATION OF THE DOMINANT FOLLICLE
The preovulatory surge of LH sets in motion a series of morphological and
biochemical changes resulting in the differentiation of the mature follicle into the CL
(Niswender and Nett, 1988; Niswender et al., 1994). Under stimulation of LH, the
preovulatory follicles of the ewe are capable of synthesizing progesterone (Murdoch and
Dunn, 1982) and prostaglandins (Murdoch et al., 1981). It has been suggested in other
species that progesterone and(or) prostaglandins may be mediators for follicular
collagenase activity responsible for the degradation of the basement membrane between
the theca interna and the membrana granulosa, which leads to eventual ovulation (Espey,
1974, 1980; Rondell, 1974).
It was not until an elaborate study performed by Murdoch and coworkers (1986),
that the role of these hormones in ovine ovulation was elucidated. Ovine follicles were
15
isolated from ovaries at different intervals after the initial surge of LH and analyzed for
concentrations of progesterone, prostaglandins and collagenase activity. They then
performed the same measurements in the presence a progesterone antagonist or
indomethacin (a prostaglandin biosynthesis inhibitor). These researchers found that in the
presence of either or both of these substances ovulation would not occur. Their findings
suggested that progesterone acts as an intermediate by influencing prostaglandin synthesis,
primarily PGF2, from the preovulatory follicle. They also found that PGF2c, is essential
for increased collagenase activity and ovulation.
During luteinization, the granulosa and theca cells rapidly increase in number and
any clotted blood in the follicular cavity is reabsorbed. Theca cells migrate into the
follicular cavity and become dispersed among granulosa cells (O'Shea et al., 1980; Hadley,
1992a). Granulosa cells exhibit a smooth endoplasmic reticulum, their mitochondria
become rounded with tubular cristae, and granules form within the cytoplasm (Niswender
and Nett, 1988; Niswender et al., 1994). Mitotic activity will continue to occur in theca
cells, but as the granulosa cells accumulate large amounts of cholesterol after ovulation,
cell division virtually ceases (McClellan et al., 1975; Hadley, 1992a). It is this
luteinization process that leads to the formation of the functional CL.
CHARACTERISTICS OF THE CORPUS LUTEUM IN THE NONPREGNANT
ANIMAL
The corpus luteum, or "yellow body", is a transient endocrine gland that is
required for the maintenance of pregnancy in mammals (Niswender and Nett, 1988;
Niswender et al., 1994). After the ovum is ovulated, the cells of the recently ovulated
follicle luteinize to form the CL and if the ovum is fertilized, the presence of an embryo
within the uterus results in the maintenance of the CL (Niswender et al., 1985). The CL
produces progesterone, which prepares the uterine endometrium for the implantation of
16
the fertilized ovum and maintains early pregnancy. The CL must remain functional for a
certain period of gestation in order for a successful pregnancy, however, this duration of
time varies with species. In rabbits (Browning et al., 1980), sows (Ellicott and Dzuik,
1973) and goats (Hafez, 1987), the CL are required for maintenance of pregnancy
throughout gestation. The CL in cows must be present for the first 200 days of gestation
and thereafter the adrenal gland is able to secrete enough progesterone to maintain
pregnancy (Wendorf et al., 1983). In the ewe, the CL must remain functional for only 55
days of gestation at which time the placenta assumes the role as the primary source of
progesterone and is responsible for the maintenance of pregnancy (Casida and Warwick,
1945; Cowie et al., 1963). In the mare, Holtan et al. (1979) reported that ovariectomy
after days 50-70 of gestation did not terminate pregnancy, nor did it affect gestation
length, incidence of placental retention, mammary development or milk production.
Evidently the placenta assumes the role of the CL to maintain pregnancy after this time.
These authors later discovered that the progesterone produced by the placenta is rapidly
metabolized to other progestins (Holtan et al., 1991). If fertilization or implantation does
not occur, ovulation will occur at the subsequent estrus after regression of the CL (Auletta
and Flint, 1988).
Morphology and Biochemistry of the Corpus Luteum
The corpus luteum is composed of at least two morphologically and biochemically
distinct steroidogenic cell types (Corner, 1919; 1945; Warbritton, 1934; Mossman and
Duke, 1973; Niswender et al., 1985, Niswender and Nett, 1988). The most obvious
difference between these two cell types is their size, leading to their designation as small
and large luteal cells. The precise size range used for each type of cell varies among
species and researchers. In general, ovine small luteal cells are 10-20 pin in diameter and
account for 12-18% of total volume of the CL whereas large luteal cells range from 20-35
p.m and account for 25-35% of the total luteal volume (Nett et al., 1976; Rodgers et al.,
17
1984; Stormshak et al., 1987). At the time of maximum progesterone secretion, during
the luteal phase, vascular elements account for more than 11% of luteal volume,
connective tissue 22-29%, and fibroblasts 7-11% of total volume (Nett et al., 1976;
Rodgers et al., 1984).
The structural appearance of the two cell types is also distinct and has been
reviewed at length by numerous researchers (Hansel et al, 1987; Niswender and Nett,
1988; Niswender et al., 1994; O'Shea et al., 1979; Rothchild, 1981; Stormshak et al.,
1987). Small cells appear spindle-shaped with darkly stained cytoplasm and large lipid
droplets. The nuclei are irregular in shape and convoluted, with about 10% of the small
cell nuclei appearing to contain inclusions of cytoplasm (Rodgers and O'Shea, 1982). The
Golgi complex is located along the periphery of the cell and is associated with the rough
endoplasmic reticulum. Small cells contain numerous amounts of smooth endoplasmic
reticulum and mitochondria with tubular and lamelliform cristae, both of which are
characteristic of steroid secreting cells (Christensen and Gillim, 1969; Niswender et al.,
1994).
Large cells are probably the most readily distinguishable luteal cells. They possess
a spherical or polyhedral shape and contain a distinct nucleolus. These cells also contain
elements of steroid secreting cells, abundant mitochondria and smooth endoplasmic
reticulum, as well as stacks of rough endoplasmic reticulum (Christensen and Gillim, 1969;
Niswender et al., 1994). In addition, large luteal cells contain electron dense secretory
granules that are said to contain oxytocin and relaxin in some species (Niswender et al.,
1994). Within the cytoplasm, there are some areas of clustering and exclusion of
mitochondria (Enders et al., 1973). There is also an extensive Golgi complex located at
one side of the nucleus and some indication that the smooth endoplasmic reticulum may be
in direct communication with the Golgi cisternae (Fawcett et al., 1969).
Large and small cells have been hypothesized to have differentiated from granulosa
membrana and theca interna cells, respectively. Donaldson and Hansel (1965) found that
18
while large luteal cells have a limited ability to divide, small luteal cells retained the
capacity to multiply and therefore these researchers believed it to be conceivable that small
cells are capable of growing into large luteal cells under the influence of gonadotropins.
This hypothesis was supported by Niswender et al. (1985) who found that by treating
ewes with human chorionic gonadotropin (hCG) that there was an increased number of
large luteal cells by midcycle.
By using monoclonal antibodies against theca specific and granulosa cell specific
antigens, Alila and Hansel (1984) set out to determine the origin of large and small cells of
the bovine CL during the estrous cycle and early pregnancy. They found that during the
early stages of the estrous cycle, days 4-6, the majority of large luteal cells bound
granulosa antibody while small cells bound theca antibody. However, as the age of the CL
increased, the number of large cells binding granulosa antibody decreased and more large
cells were observed binding theca antibody. In fact, by day 100 of gestation luteal cells
only bound theca antibody, suggesting that small luteal cells differentiate into large luteal
cells as the CL ages (Alila and Hansel, 1984).
Although the research by Alila and Hansel (1984) is compelling evidence that small
luteal cells may differentiate into large luteal cells, some controversy remains. For
example, research conducted by O'Shea and coworkers (1986) compared cellular
morphology of the CL by ultrastructural morphometry on days 9 and 13 of the cycle.
These researchers found no difference in luteal volume, plasma progesterone
concentration, or volume density of any component of the luteal tissue between days 9 and
13, suggesting that there is no transformation of small to large luteal cells. This supported
their earlier findings in which they compared CL from day 10 of the estrous cycle and days
15, 25, 50, 100, 125 and 140 of gestation. By using light and electron microscopy the
ultrastructure of the various CL were analyzed but no evidence was found that would
suggest transformation of small to large luteal cells (O'Shea et al., 1979).
19
Hormones Synthesized by the Corpus Luteum
Small and large luteal cells are capable of synthesizing and secreting both steroid
and protein hormones. In fact, the primary function of the CL is considered to be the
production of the steroid hormone progesterone, which prepares the uterine endometrium
for implantation and is responsible for the maintenance of early pregnancy (Auletta and
Flint, 1988). In some species estrogen, another steroid hormone, is also manufactured by
the CL and is important for up-regulating progesterone receptors in target tissues. Protein
hormones, such as oxytocin, are produced by the large luteal cells in most species, and are
essential for interacting with uterine hormones that regulate the life span of the CL.
Steroid Hormones
Steroid hormones are synthesized by complex multiple-enzyme systems located in
the mitochondria, smooth endoplasmic reticulum and cytoplasm (Hadley, 1992a; Miller,
1988). The initial substrate for luteal steroidogenesis is cholesterol, which may be
extracted from low or high density lipoproteins (LDL and HDL) in the circulation,
released from intracellular stores of cholesterol esters or synthesized de novo from
acetate. Transfer of cholesterol to the mitochondria appears to involve the cytoskeleton
and may be stimulated by cAMP and activation of protein kinase A (Wiltbank et al.,
1993). It is within the inner mitochondrial membrane where the rate-limiting step in
progesterone biosynthesis occurs: the cleavage of the cholesterol side chain by the
cytochrome P450 side-chain cleavage enzyme (Niswender et al., 1994). Further
conversion of cholesterol occurs within this organelle with the aid of various enzymes for
the production of pregnenolone. Pregnenolone is then transported to the endoplasmic
reticulum, where it is converted to the progesterone. Depending upon the stimulation and
the stage of the estrous cycle, progesterone may be the end product or further converted
by lyase to testosterone and then aromatized to estradiol -1713 (Savard, 1973). These
steroid hormones are then passively transported out of their cell of origin and are bound to
20
specific plasma proteins to protect the steroid hormones from premature degradation.
Upon reaching the target tissue, steroid hormones diffuse through the membrane and
cytosoplasm of the cell to bind to their receptors located within the nucleus. These
steroid-receptor complexes dimerize, bind to steriod hormone response elements (HRE)
on DNA and influence gene transcription (Hadley, 1992a).
There are species differences in the biosynthetic potential of the CL. For example,
whereas the human CL will secrete progesterone, androgens and estrogens, the bovine CL
appears to lack both lyase and the aromatizing enzyme system so that no significant
amounts of either androgens or estrogens are produced (Miller, 1988; Savard, 1973). The
CL of the sow (Cook et al., 1967) and ewe (Kaltenbach et al., 1967) appear to produce
17a-hydroxyprogesterone and 4-androstenedione in addition to progesterone, but not
estrogens. The two steroid hormones that will be discussed in detail in this review are
progesterone and estrogen.
Progesterone
It was once believed that secretion of progesterone from the CL was an active
process rather than a passive one. Gemmell and Stacy (1979) found that upon treatment
of the ovine CL with cyclohexamide, an inhibitor of protein synthesis, there was an
inhibition of the formation and secretion of densely stained granules of the luteal cells with
an associated decrease in progesterone secretion. Because of these findings these
researchers hypothesized that progesterone is stored in secretory granules from the Golgi
bound with a high affinity to a protein and released from the cells by exocytosis.
However, this hypothesis was challenged by Rodgers and coworkers (1983) when
they separated small and large luteal cells and measured the concentration of progesterone
produced by each cell type under the stimulation of LH. These scientists found that under
the influence of LH, small luteal cells appear to be the major source of progesterone,
secreting about three times more than large luteal cells. They concluded that because
21
small luteal cells are virtually devoid of any secretory granules, progesterone must be
stored and secreted in some other manner.
The work of Hirst et al. (1986) further supported the hypothesis that progesterone
was not stored in granules when they found that oxytocin, not progesterone, was
associated with and actively released by luteal secretory granules. This was demonstrated
by an in vitro incubation of luteal cells with or without calcium and by measuring changes
in progesterone or oxytocin secretion. Their results indicated that oxytocin secretion is
calcium dependent, which is indicative of exocytotic events, while progesterone is not.
Similar results were found by Rice et al. (1986) when they separated particle-associated
progesterone and oxytocin and subjected these hormones to treatments that stimulate
release from granules. Their results indicated that progesterone is not stored in a proteinbound form in luteal secretory granules, but rather particle-associated progesterone may
intercalate into cell membranes for ultimate release.
Secretion of progesterone is required for the growth and development of the
uterus in preparation for the fertilization of the ovum as well as to suppress further
ovulation (Hadley, 1992a). In order to fulfill this role, the CL must remain fully functional
for a specific period of time. In some species such as sheep and primates, the placenta will
take over the role as the major source of progesterone for the remainder of the pregnancy
(Auletta and Flint, 1988); in cattle the adrenal gland assumes this role (Wendorf et al.,
1983), whereas in others such as the pig, goat and rabbit, the CL remains the sole source
of progesterone throughout gestation (McCracken, 1984). To suppress ovulation,
progesterone is capable of inhibiting pituitary LH secretion either directly or through the
hypothalamus by reducing GnRH release (Girmus and Wise, 1992, Hadley, 1992b; Karsch
et al., 1987).
Progesterone administered early in the estrous cycle, prior to when it normally
reaches biologically active levels, has been shown to induce premature secretion of PGF2ct
and luteolysis in both cows and sheep (Garrett et al., 1988; Ginther, 1970; Ottobre et al.,
22
1980; Thwaites, 1971). Ginther and Woody (1970) demonstrated that the detrimental
effect of progesterone administration early in the estrous cycle was mediated by the uterus
when they treated unilaterally hysterectomized ewes and cows with progesterone. Upon
morphological analysis of the ovaries on day 14 of the cycle they found that only the ovary
ipsilateral to the remaining uterine horn responded with a decrease in luteal weight,
whereas the CL in the ovary opposite the remaining horn remained unchanged. Oxytocin
has been shown to stimulate uterine secretion of PGF2a in ovariectomized ewes and cows
only after the animal has been exposed to progesterone for 7-10 days (Homanics and
Silvia, 1988; LaFrance and Goff, 1988). Therefore, the action of progesterone on PGF2a
secretion may be indirect by affecting uterine secretory responsiveness to oxytocin during
the estrous cycle. Vallet et al. (1990) reinforced the indirect action of progesterone for
normal luteolysis when they reported that oxytocin receptor concentrations increased after
treatment of ovariectomized ewes with exogenous progesterone followed by estradiol.
Zhang et al. (1992) suggested that while progesterone priming is important for luteolysis
in the ewe, the increase in estradiol concentration after the withdrawal of progesterone
actually causes the increase in oxytocin receptor numbers. Further, active immunization of
nonpregnant ewes against progesterone has been shown to result in an erratic cycle and is
frequently associated with prolonged maintenance of the CL (French and Spennetta, 1981;
Thomas et al., 1985). In addition to the effects of progesterone on oxytocin receptor
development, this steroid also appears to affect uterine tissue more directly by changing
the uterine secretory responsiveness to oxytocin. The accumulation of lipid droplets in
uterine epithelial cells of the ewe has been demonstrated to be a progesterone-dependent
process (Brinsfield and Hawk, 1973). Further, the amount of immunoreactive
prostaglandin H endoperoxide synthase (PGH synthase) activity, the primary enzyme
required for PGF2a production in uterine epithelial cells, is increased when ovariectomized
ewes are treated with progesterone (Raw et al., 1988). Further, treatment of intact ewes
with progesterone early in the estrous cycle induces a premature increase in the
23
concentrations of mRNA coding for PGH synthase in uterine tissue as well as premature
luteolysis (Eggleston et al., 1990). These data imply that progesterone may act both
directly by stimulating synthesis of PGH synthase and indirectly by upregulating OT
receptors on the uterine endometrium to increase PGF2u production.
In contrast to the luteolytic effects of progesterone, late administration of this
steroid can actually prolong the duration of the estrous cycle (Sirois and Fortune, 1990).
Ginther (1970) demonstrated that exogenous progesterone administered on days 8 to 11,
12 to 15 or 16 to 19 of the bovine estrous cycle significantly prolonged the cycle when
compared to controls. Further, progesterone supplementation has been used by sheep and
cattle producers to compensate for inadequate luteal function in young animals or "repeat­
breeder" animals (animals that must be bred several times to achieve pregnancy), and has
been shown to increase fertility in some cases (McMillan, 1987; Robinson et al., 1989).
However, it is important to note that progesterone supplementation does not increase the
life span of the CL. Ginther (1971) found that progesterone supplementation over a 4 day
period either early or late in the ovine estrous cycle caused a decrease in number of large
follicles at necropsy, regardless of the time during the cycle supplementation occurred.
Therefore, administration of progesterone late in the estrous cycle does not appear to
prolong the life span of the CL, but rather acts at the level of the hypothalamus and(or) the
pituitary to suppress release of the gonadotropins necessary for ovulation (Karsch et al.,
1987; Girmus and Wise, 1992).
Estrogen
Just as progesterone is the major steroid hormone secreted by the ovary during the
luteal phase, estrogen is the primary steroid produced during the follicular phase. In most
species estrogens are produced primarily by ovarian follicles and high estrogen levels
reflect periods of increased follicular growth. However, in the human and non-human
24
primate, the corpus luteum contains all the enzymes needed for the both progesterone and
estrogen synthesis (Niswender and Nett, 1988).
During follicular growth, estradiol production results from the coordinated
steroidogenic activity of the theca interna and the granulosa cells. This was first
demonstrated by Falck (1959) in rat ovarian follicles and was later shown to be the case in
the follicle of pigs (Evans et al., 1981), cows (Fortune, 1986; Fortune, 1994), ewes
(Armstrong et al., 1981) and primates (McNatty et al., 1979). Various in vitro research
led to the formation of the "two-cell theory" of follicular estrogen synthesis, which states
that LH stimulates the production of androgens from the theca interna, which are then
transported to the granulosa cells where they are converted to estrogens under the
influence of FSH. According to this hypothesis, theca cells lack aromatase activity and
are unable to convert androgens to estrogens, while the granulosa cells are incapable of
producing androgens. Therefore, these cells must work together in order to produce
estrogens. In vitro research with bovine follicles further suggests that progestins produced
by the granulosa cells, primarily pregnenolone, may act back upon the theca cells and act
as a precursor for androgen production, thereby regulating estrogen synthesis (Fortune,
1986).
While there seems to be no doubt that these two follicular cell types interact
extensively for the production of estrogen, this interaction seems to vary somewhat among
different species. Evans and coworkers (1981) demonstrated that porcine theca cells, in
addition to granulosa cells, are capable of producing some estrogens in vitro. Armstrong
et al. (1981) reported similar findings in ovine medium or large preovulatory follicles.
However, regulation of granulosa estrogen production may still be regulated by theca
cells. Furthermore, human granulosa cells in culture have been shown to be capable of de
novo androgen synthesis although interaction between the two follicular cell types still
exists (McNatty et al., 1979).
25
An appropriate endocrine balance is essential for ovarian follicular development as
well as female sexual behavior. Behavioral estrus begins around the time of maximal
estradiol -17(3 production and the coincident preovulatory LH surge (Thatcher and
Chenault, 1976), and estrogens have been implicated as having an essential role in sexual
behavior. In fact, sexual behavior is inhibited by ovariectomy in domestic animals, but can
be restored in the cow and sow by progesterone pretreatment followed by estrogen
administration (Hafez, 1987). Further, administration of estradiol benzoate alone has been
shown to induce sexual receptivity in ovariectomized heifers and cows (Cook et al.,
1986). In the ewe and the sow there appears to be a linear dose-response relationship
between the duration of estrus and the logarithm of the dose of estrogen (Hafez, 1987).
In cattle, however, this does not seem to be the case, although estrogens still appear to be
responsible for estrous behavior (Glencross et al., 1981).
Saumande and Lopez-Sebastian (1982) reported that serum concentrations of
estradiol -17(3, along with other estrogens, were elevated in association with increased
ovulation rate. This increase in estrogen production may be associated with the essential
role of estradiol in triggering the preovulatory gonadotropin surge (Legan and Karsch,
1979). In fact, active immunization of ewes against estradiol blocked the preovulatory LH
surge in addition to inhibiting behavioral estrus (Rawlings et al., 1978).
The effects of estradiol on ovulatory events occur at both the level of the
hypothalamic-hypophyseal axis, as mentioned previously, and at the level of the ovary
itself. At the level of the hypothalamic-hypophyseal axis, estradiol exerts a biphasic effect
on the secretion of LH (Clarke and Cummins, 1985; Herman and Adams, 1990). Initially,
administration of estradiol reduces serum concentrations of LH and pituitary
responsiveness to GnRH (Herman and Adams, 1990; Nett et al., 1990). After the initial
inhibitory period, estradiol stimulates pituitary GnRH receptor formation (Clarke et al.,
1988; Gregg and Nett, 1989; Gregg et al., 1990) and frequency of GnRH pulses (Moenter
et al., 1990), thereby inducing a preovulatory-like surge of LH (Nett et al., 1984). At the
26
level of the ovary, estradiol has been shown to enhance the FSH stimulated LH-receptor
formation on rat granulosa cells in culture (Rani et al., 1981). Goldenberg et al. (1972)
examined the effects of diethylstilbestrol (DES) in hypophysectomized rats and found that
administration of DES alone significantly increased ovarian weights and follicular antrum
formation and that concomitant injection of FSH further enhanced ovarian development.
Further, Welsh and coworkers (1983) found that while LH or FSH increased production
of progesterone in the rat granulosa cell in vitro, addition of either DES or estradiol also
enhanced the ability of the granulosa cells to produce progesterone.
The effects of estradiol are not restricted to follicular and ovulatory events, but can
also influence the life span of the corpus luteum. Researchers determined that injection of
estradiol -1713 during the ovine luteal phase resulted in a significant decrease in luteal
weight indicative of premature luteal regression (Bolt and Hawk, 1975; Stormshak et al.,
1969). However, ewes that were totally hysterectomized did not respond to the luteolytic
effect of injected estradiol (Stormshak et al., 1969), demonstrating that estradiol may act
through the uterus. This theory was further supported by the research of Akbar et al.
(1971) who determined that in estradiol-treated unilaterally hysterectomized ewes, CL in
the ovary adjacent to the intact uterine horn were significantly smaller in size than the CL
of the other ovary. Destruction of the ovarian follicles, the source of estrogen synthesis,
has been shown to delay luteal regression in sheep (Karsch et al., 1970). Howland et al.
(1971) treated ewes with a single injection of estradiol on either day 4 or 11 of the estrous
cycle. They found that while injection of estradiol on day 4 increased CL weight,
treatment on day 11 decreased the weight of the CL significantly over controls. Caldwell
et al. (1972) found that injection of exogenous estradiol to progesterone-primed
ovariectomized ewes caused increased plasma PGF2a concentrations. Similarly, infusion
of physiological levels of estradiol into the arterial supply of an autotransplanted uterus
during the late luteal phase of the cycle resulted in elevated PGFR, release within 60-90
min of administration. Further, increased synthesis of PGF2c, from uterine tissue in vitro
27
has been found after estradiol treatment of progesterone-primed ewes. Similarly,
treatment of ovariectomized ewes with estradiol has been shown to increase uterine
synthesis of PGF2,, ( Homanics and Silvia, 1988; Ottobre et al., 1984; Sharma and
Fitzpatrick, 1974; Vincent and Inskeep, 1986).
The action of estradiol on luteolysis is most likely by facilitating the action of
oxytocin. This is supported by Holtorf et al. (1989) who found that there was a high
correlation between the amount of oxytocin secreted by bovine granulosa cells and the
estrogen concentration in the corresponding follicular fluid. In fact, both estradiol and
progesterone appear to stimulate uterine prostaglandin production indirectly by controlling
the availability of endometrial oxytocin receptors, as demonstrated by Zhang et al. (1992),
in progesterone-primed ovariectomized ewes treated with estradiol. Estradiol alone also
has been implicated to increase oxytocin receptor formation (McCracken et al., 1978).
These investigators demonstrated that when estradiol was infused into the systemic
circulation for 12 hours, formation of oxytocin receptors occurred within 6 hours, as
evidenced by a significant increase in oxytocin-induced PGF2c, secretion. This was
confirmed by Hixon and Flint (1987) who found that induction of oxytocin receptors
occurs within 12 hours of estradiol administration in nonpregnant ewes. Burgess et al.
(1990) also found that long-term infusion of estradiol into ewes enhanced uterine
responsiveness to oxytocin, possibly by increasing uterine oxytocin receptor
concentrations.
Peptide Hormones: A Focus on Oxytocin
The majority of vertebrate hormones are peptide (protein) in nature, meaning they
are made up of specific amino acid sequences. In peptide secreting tissues, hormone
biosynthesis occurs in the rough endoplasmic reticulum, specifically by membrane attached
ribosomes. Ribosomes attach to the mRNA of the protein hormone and translates the
nucleotide sequence into amino acids until the precursor protein hormone is complete
28
(Alberts et al., 1989). Most polypeptide hormones and neuropeptides are synthesized as
inactive protein precursors, called pre-pro-hormones, from which the active hormone is
derived.
The nascent pre-pro-hormone is released from the ribosome and transported into
the cisternae of the rough endoplasmic reticulum where the pre-piece, consisting of the
signal peptide, is cleaved off. The pro-hormone is then transported to the Golgi where it
is packaged with proteolytic enzymes and pinched off the terminal cisternae into secretory
vesicles which are then distributed within the cytoplasm. The conversion of the prohormone to the active peptide hormone begins within the Golgi complex and continues in
the secretory vesicles (Sheldrick and Flint, 1989; Wallis et al., 1985).
Secretion of protein hormones occurs by calcium-dependent exocytosis. With an
influx of calcium, secretory granules are transferred from the cytoplasmic vesicle pool to
the plasma membrane, where the vesicle fuses with the plasma membrane and releases its
contents. The vesicle can then be recycled back into the Golgi where it may be utilized
again (Wallis et al., 1985). The mammalian corpus luteum is the source of several peptide
hormones; the most significant is the peptide hormone oxytocin.
Oxytocin originates from a larger precursor which contains a signal sequence, the
nonapeptide oxytocin, the protein neurophysin I, and another peptide of unknown
function. Post-translational processing leads to production of the mature peptide and its
associated neurophysin I, which is thought to act as a carrier for oxytocin (Ivell and
Richter, 1984). This complex is packaged into membrane bound granules, which contain
proteolytic enzymes that will cleave oxytocin from neurophysin I (Ivell et al., 1985).
Oxytocin synthesis was originally believed to take place only in the hypothalamus
and transferred down axons to the posterior pituitary where it was subsequently stored
and released into the systemic circulation. Functions usually associated with oxytocin
include the stimulation of uterine contractions during parturition and the milk ejection
reflex (Nell et al., 1985). However, with research indicating that oxytocin may cause
29
luteolysis in cattle if administered early in the estrous cycle (Armstrong and Hansel, 1959),
along with research demonstrating that immunization against oxytocin prolongs the
duration of the ovine estrous cycle (Sheldrick et al., 1980), the importance of oxytocin in
luteal function was realized. Further evidence that oxytocin plays a role in luteal function
was provided by McCracken (1980). He proposed that oxytocin acts by stimulating
uterine PGF2c, production and that steroid hormones, such as progesterone and estradiol,
may regulate PGF2c,, secretion by controlling the number of oxytocin receptors in uterine
endometrial tissue (McCracken, 1980; Roberts et al., 1976).
It was not until an observation made by Wathes and Swann (1982), when testing
peptide extracts of the ovine CL, that a second storage site for oxytocin was revealed.
Testing ovine luteal peptide extracts for relaxin-like activity on rat uterine strips, they
observed that some fractions were stimulatory rather than inhibitory. Upon further
investigation these researchers found that a similar observation was reported for cows
(Fields et al., 1980). Wathes and Swann (1982) tested the ovine luteal extract for crossreactivity with oxytocin and found that the luteal fractions did indeed contain oxytocin.
However, it was still not known at this time whether oxytocin was produced or simply
sequestered and stored in the CL. By examining luteal cells via immunocytochemical
techniques, it was determined that neurophysin I and oxytocin are found together in
granules within the large luteal cells of the ovine (Rodgers et al., 1983) and bovine corpus
luteum (Guldenaar et al., 1984; Schams et al., 1985b). This colocalization reflects de
novo synthesis of oxytocin and neurophysin I.
Since the initial observation that the ovine CL contained oxytocin, this peptide has
been shown to be produced in the CL of many other species such as humans (Fields et al.,
1983), non-human primates (Khan-Dawood et al., 1984; Khan-Dawood, 1986), pigs
(Jarry et al., 1990) and goats (Kiehm et al., 1989). Ovarian oxytocin is synthesized and
secreted primarily by the large luteal cells and in smaller quantities by the granulosa cells
of preovulatory follicles (Guldenaar et al., 1984; Schams et al., 1985a). Oxytocin is
30
secreted into the ovarian vein (Flint and Sheldrick, 1982) where it may act via systemic
route on the adjacent uterine horn to regulate luteal life span (McCracken et al., 1972;
Sheldrick and Flint, 1983).
The capacity of these ovarian cells to synthesize oxytocin changes dramatically at
different stages of the estrous cycle (Wathes et al., 1992). In both bovine and caprine
preovulatory follicles, oxytocin is detectable in significant amounts only when sampled
after the gonadotropin surge (Kiehm et al., 1989; Voss and Fortune, 1991). Furthermore,
Voss and Fortune (1991) found that bovine granulosa cells isolated before the LH/FSH
surge responded to gonadotropins added to culture with increased oxytocin secretion,
suggesting a role for oxytocin in the regulation of luteinization and ovulatory events.
Luteal concentrations of oxytocin in cattle and sheep increase during the early part
of the luteal phase and decline thereafter (Ivell et al., 1985; Sheldrick and Flint, 1981;
Wathes et al., 1984). In the ovine CL, mRNA concentrations of oxytocin and its
prohormone peak around day 3 (Ivell et al., 1990; Jones and Flint, 1988); however, peak
luteal concentrations of oxytocin do not occur until day 6 of the cycle (Sheldrick and Flint,
1989). Similarly, in the bovine CL, the maximum amount of mRNA is attained around
day 3 of the cycle and the oxytocin content peaks around day 8 (Abdelgadir, 1987; Ivell et
al., 1985). After this time the concentrations of oxytocin and its mRNA decline, such that
low levels are present just prior to luteolysis. Flint and Sheldrick (1983) found when
taking frequent blood samples from ewes around the time of luteolysis that there are small
episodic releases of luteal oxytocin. They surmised this pulsatile release of oxytocin may
be in response to uterine release of PGF20, (Flint and Sheldrick, 1983).
Luteal oxytocin has been postulated to have two major actions: a paracrine action
on luteal tissue to regulate steroid secretion and an endocrine action on the uterus to
increase secretion of PGF2c, and hence hasten luteolysis. In porcine luteal cell cultures,
there is a dose-dependent inhibition of progesterone secretion but a stimulation of
estradiol release in response to oxytocin. The inhibitory effect of oxytocin on
31
progesterone secretion was abolished in the presence of an oxytocin antagonist, which
suggests a receptor-mediated effect (Pitzel, 1988). However, in other species the
evidence of a direct effect of oxytocin on luteal steroid secretion has been conflicting.
Pretreatment of caprine luteal tissue treated in vitro with oxytocin significantly
inhibited hCG-enhanced secretion of progesterone production. However, treatment with
an oxytocin antagonist early in the estrous cycle also inhibited progesterone production in
this species (Homeida and Al-Eknah, 1992). Studies of progesterone production by
dispersed bovine and ovine luteal cells have failed to demonstrate any inhibitory action of
oxytocin alone or with LH and hCG (Rodgers et al., 1985). Further, oxytocin receptors
have been reported in porcine, bovine and ovine luteal tissue, but research has been
equivocal as to time of development, and control or action of these receptors. Luteal
oxytocin receptors have been shown to be present in the porcine and bovine CL during the
early luteal phase and decline thereafter (Jarry et al., 1990; Okuda et al., 1992). However,
this was contradicted earlier by reports of Fuchs et al. (1990b) who found that in bovine
luteal tissue the oxytocin receptor concentration was highest late in the estrous cycle. In
addition, luteal oxytocin receptors have been found in pregnant but not cycling ewes
(Sernia et al., 1989). While evidence for the paracrine action of oxytocin in regulating
steroidogenesis remains unclear, its endocrine action and involvement in luteolysis have
been well characterized, especially in ewes and cows.
As mentioned previously, exogenous oxytocin has been shown to shorten the
estrous cycle in cows if administered between days 3-6 of the cycle (Armstrong and
Hansel, 1959; Hansel and Wagner, 1960). However, if oxytocin is given either earlier or
later it appears to have no effect on development of the CL or cycle length (Donaldson et
al., 1965; Hansel and Wagner, 1960; Harms et al., 1969). Similar findings were
demonstrated in the goat, with the daily administration of oxytocin early in the estrous
cycle resulting in the premature demise of the CL, whereas active immunization against
oxytocin prolonged the luteal phase (Cooke and Homeida, 1985). Active immunization
32
against oxytocin also increased the length of the estrous cycle in ewes (Sheldrick et al.,
1980; Wathes et al., 1989). These observations provide strong evidence that oxytocin
may act as a luteolytic factor late in the estrous cycle. Furthermore, oxytocin
administration late in the estrous cycle of ewes has been shown to increase uterine
secretion of PGF2,1 (Fairclough et al., 1984), a substance which has been identified as the
primary luteolysin (McCracken et al., 1972). Similarly, exogenous PGF2o, has been shown
to stimulate luteal oxytocin and its associated neurophysin release from the ovine CL
(Flint and Sheldrick, 1983; Watkins and Moore, 1987).
Oxytocin receptors have been reported to exist in the uterine endometrium and
myometrium of many mammals. Oxytocin has been reported to enhance myometrial
activity in the form of uterine contractions (Fitzpatrick, 1960; Roberts and McCracken,
1976). The overall concentration of bovine and ovine uterine oxytocin receptors increase
over the late luteal phase and peak on the day of behavioral estrus (Fuchs et al., 1990a;
Roberts et al., 1976; Sheldrick and Flint, 1985). In sheep, the increase in oxytocin
receptor concentrations occurs in the endometrium, caruncular stroma and myometrium,
and appears to be under the influence of estradiol because there is a strong positive
correlation between oxytocin and estradiol receptor development (Ayad et al., 1991;
Wathes and Hamon, 1993). However, in cows the concentrations of uterine oxytocin
receptors in myometrium show little variation over the estrous cycle while endometrium
concentrations do increase in association with estradiol (Fuchs et al., 1990a). The
development of oxytocin receptors in the endometrium during the time of luteolysis is of
particular interest because the endometrium has been shown to be the site of prostaglandin
synthesis, further substantiating the role of oxytocin in luteal regression (Cerini et al.,
1979; Salamonsen and Findlay, 1990).
Development and concentration of uterine oxytocin receptors seems to be
regulated primarily by the steroid hormones estrogen and progesterone. Treatment with
estradiol -1713 has been shown to promote uterine secretion of PGF2a in response to
33
oxytocin stimulus in ewes (McCracken et al., 1984; Sharma and Fitzpatrick, 1974) and to
increase the number of specific binding sites for oxytocin in the uterus of ewes (Hixon and
Flint, 1987), rabbits (Nissenson et al., 1978) and rats (Soloff, 1975). Presence of
progesterone has been demonstrated to be initially inhibitory to ovine oxytocin receptor
formation, but near the end of the luteal phase it becomes facilitory (Levitt et al., 1983;
Vallet et al., 1990). In fact, progesterone pretreatment is necessary before estrogen can
maximally stimulate oxytocin receptor production in the ewe (Homanic and Silvia, 1988;
McCracken et al., 1984; Vallet et al., 1990; Zhang et al., 1992). Additionally, maximal
responses to oxytocin in terms of PGF2a secretion in vivo are elicited by administration of
estrogen after prolonged treatment of progesterone (McCracken et al., 1981). Therefore,
progesterone alone or in the presence of estradiol causes a decrease in uterine oxytocin
receptors, while estradiol given after progesterone withdrawal increases the number of
oxytocin receptors (Zhang et al., 1992). This increase in oxytocin receptors also coincides
with an increase in the pulsatile release of PGF2a from the uterus (Sheldrick and Flint,
1988) and is similar to the hormone profile observed around the time of luteolysis.
Beard and Hunter (1994) injected estradiol into ovariectomized ewes for 2 days,
one-half of which had undergone progesterone pretreatment for 10 days, then
subsequently injected these animals with both estradiol and progesterone for either 0, 2, 3,
or 4 days. Following injections with estradiol and progesterone ewes were treated with an
oxytocin challenge and endometrial tissue oxytocin receptor numbers were analyzed.
These researchers found that both groups of ewes were able to form endometrial oxytocin
receptors, regardless of progesterone pretreatment, but that the number of endometrial
oxytocin receptors in the progesterone pretreated ewes declined rapidly after day 0 while
those non-pretreated ewes had a slower decline in oxytocin receptor concentration. These
authors hypothesized that the slow decline of endometrial oxytocin receptors in ewes, not
pretreated with progesterone, may induce premature luteolysis. This is because the
oxytocin-PGF2a positive feedback mechanism that induces luteolysis may be able to act
34
earlier in these animals. Further, they postulated it is because of this mechanism that the
first estrous cycle during seasonal anestrous or the post-partum period, when
progesterone concentrations are low, often result in premature luteolysis.
HYPOTHALAMIC -HYPOPHYSEAL HORMONES INFLUENCING THE
CORPUS LUTEUM DURING THE ESTROUS CYCLE
The life span and function of the mammalian corpus luteum are regulated by
complex interactions between the hormones secreted from the hypothalamus, pituitary,
uterus, as well as the ovary and CL themself. The hypothalamus secretes gonadotropinreleasing hormone (GnRH) which indirectly influences the life span of the CL in
domesticated animals by regulating the secretion of the pituitary gonadotropins. These
gonadotropins then act directly on the ovary and participate in regulating the development
of the CL. Additionally, ovarian hormones such as oxytocin may act to stimulate the
release of uterine hormones, which may bring about the ultimate demise of this transient
endocrine organ.
Hypothalamic Hormones: An Overview of Gonadotropin-Releasing Hormone
Of all the hypothalamic hormones known to exist, only GnRH has been implicated
in affecting ovarian function. This well conserved mammalian decapeptide hormone is
synthesized and released from the hypothalamus (Hadley, 1992b). The actions of GnRH
are essential for normal follicular growth, ovulation and the maintenance of luteal
functions via the control of progesterone synthesis by luteinizing hormone.
Many questions surround the mechanism by which GnRH regulates the secretion
of LH and FSH from gonadotropes at different times during the estrous cycle. At the time
of ovulation, both LH and FSH are secreted in response to GnRH stimulation. Shortly
after the initial surge of both gonadotropins, FSH has a secondary release that seems to be
important to increase follicle growth and possibly ensure ovulation in cyclic ewes (Wallace
35
and McNeil ly, 1985). This rebound release of FSH appears to be independent of GnRH
secretion because treatment with a GnRH antibody failed to inhibit its release (Narayana
and Dobson, 1979). As the estrous cycle continues to progress, FSH secretion controls
follicular development and recruitment without the concomitant release of LH.
Because of the differential control over the pituitary gonadotropes by GnRH, it
was initially hypothesized that the release of LH and FSH were controlled by two different
substances. These substances were termed luteinizing hormone-releasing hormone (LH­
RH) and follicle stimulating hormone-releasing hormone (FSH-RH), respectively (Schally
and Kastin, 1970). However, with improved protein purification technology, porcine LH­
RH was isolated and found to be capable of stimulating the release of both LH and FSH in
vivo or in vitro (Schally and Kastin, 1970). This purified hypothalamic extract was also
shown to stimulate the release of both LH and FSH in rats, chimpanzees and humans
(Schally and Kastin, 1970).
It is now fairly well accepted that GnRH is the sole hypothalamic peptide hormone
that induces the secretion of both LH and FSH. Nonetheless, the differential release of
these hormones suggests that other modulators, such as steroid hormones or hypothalamic
neurotransmitters may assist in controlling the release of LH and FSH (Hadley, 1992b).
The hormones that have been implicated in controlling the action of GnRH on
gonadotropin secretion are the ovarian steroid hormones progesterone and particularly
estradiol.
The mechanism of action by which GnRH affects ovarian function and luteal life
span may be either indirectly, through the regulation of the pituitary gonadotropes, or may
be a direct effect on the ovary. Rippel and Johnson (1976) were among the first
researchers to provide evidence that GnRH injection may act directly to alter luteal
function in rats. These researchers found that treatment of immature hypophysectomized
rats with GnRH inhibits hCG-induced uterine and ovarian weight gain. Additional
research demonstrating the direct effects of GnRH was conducted on adult
36
hypophysectomized diethylstilbestrol-treated female rats that were induced to ovulate with
injection of LH after FSH priming. Luteal function in these animals was subsequently
maintained by daily injections of prolactin, which increased serum progesterone level;
however, if GnRH was injected in addition to prolactin, there was a dose-dependent
decrease in progesterone concentration as well as ovarian LH receptor content (Jones and
Hsueh, 1980). These authors also conducted experiments on rat luteal cells in vitro,
incubating luteal cells with FSH, LH and prolactin in the presence or absence of a GnRH­
agonist. The control cells, incubated without GnRH-agonist, responded with a threefold
increase in progesterone secretion (in the presence of the pituitary hormones) while those
incubated in the presence of GnRH-agonist responded with a dose-dependent decrease in
progesterone secretion (Jones and Hseuh, 1980). Furthermore, receptors for GnRH have
been detected in rat luteal tissue, thus supporting the hypothesis that this peptide acts
directly at the level of the ovary to affect luteal function in this species (Clayton et al.,
1979; Hseuh and Jones, 1983).
In domesticated animals, however, the mechanism of action of GnRH in altering
luteal function appears to be indirect via the pituitary gonadotropes. The most compelling
evidence for such action was provided by Brown and Reeves (1983) who found that luteal
tissue from ewes, cows and sows were devoid of GnRH receptors. Injection of GnRH has
been demonstrated to induce ovulation in seasonal anestrous ewes by inducing a
preovulatory increase of LH (McLeod et al., 1982). Similarly, an injection of estradiol
benzoate can also cause a GnRH-induced surge of LH in anestrous animals (Clarke,
1988). In addition, Rodger and Stormshak (1986) found that injection of GnRH into beef
cows on either day 2 or 10 of the estrous cycle caused an immediate increase in LH
secretion and a subsequent decrease in serum progesterone without affecting estrous cycle
length. These authors also discovered that the administration GnRH not only caused an
increase in serum LH, but also caused a down-regulation of luteal LH receptors. Slayden
and Stormshak (1990) found that administration of GnRH to ewes on day 2 of the estrous
37
cycle resulted an immediate increase in serum LH and a decrease in serum progesterone by
day 6 of the estrous cycle. This effect could be mimicked by injecting ovine LH alone on
day 2 of the estrous cycle. Therefore, the effect of GnRH on luteal function in
domesticated animals appears to be indirect via the pituitary hormone LH.
To determine how GnRH may act through LH to compromise luteal function
Martin et al. (1990) administered GnRH to beef cows on days 2 and 8 of the estrous cycle
and similarly found that its administration increased LH while attenuating progesterone.
However, when the CL were removed and incubated on day 10 of the estrous cycle, these
authors found that luteal tissue from cows treated with GnRH had higher basal secretion
of progesterone but had a reduced response to LH when the gonadotropin was added to
the culture. A similar study in which GnRH was injected into dairy cows 12 hours after
the onset of behavioral estrus resulted in an increase rather than a decrease in serum
progesterone levels (Mee et al., 1993). Corpora lutea collected on day 10 from treated
and control cows had similar basal secretion of progesterone; however, when tissue was
incubated in the presence of LH luteal tissue from GnRH-treated cows responded with
lower production of progesterone compared to tissue from control cows. Upon
morphological examination, the CL from those cows treated with GnRH had a higher
proportion of large luteal cells than those of untreated cows, and a lower number of small
luteal cells, which contain receptors for LH (Mee et al., 1993). Therefore, it has been
postulated that LH secretion, stimulated by exogenous GnRH, may act on luteal tissue to
cause the transition of small to large luteal cells.
The quantity of LH release from the pituitary is presumably controlled, not only by
the amount of GnRH released from the hypothalamus, but also by the number of GnRH
receptors on the pituitary gonadotropes themselves. Nett et al. (1981) demonstrated the
ability of GnRH to regulate its own pituitary receptor by infusing ovariectomized ewes
with low levels of GnRH for either 12 or 24 hours. These authors found that ewes that
were exposed to a constant low level of GnRH experienced a down-regulation of pituitary
38
receptors. Further, if these ewes were challenged with a high amount of GnRH after
infusion, release of LH would occur but at attenuated levels. Gonadotropin-releasing
hormone receptors may also be down-regulated when exposed to a GnRH antagonist.
Treatment with an antagonist results in a reduction in GnRH receptor availability,
however, this reduction does not affect overall LH secretion rates unless the number of
pituitary GnRH receptors are reduced by more than 50% (Wise et al., 1984). This finding
comes as no surprise because in many target tissues only a small percentage of receptors
need to be occupied in order to achieve maximal stimulation. The relationship between a
high number of receptors available and a maximal response achieved by binding only a
fraction of the total receptors, is termed the spare receptor hypothesis. This allows
sensitivity of the cell to low amounts of circulating hormone such that even a small
quantity of hormone can provoke a biological effect. Estradiol also seems to play a role in
GnRH receptor availability. Gregg et al. (1990) found that cultured ovine pituitary cells
treated with various concentrations of estradiol increased the number of GnRH receptors
in a dose-dependent manner. Similar findings have been found in vivo (Clarke et al.,
1988). Taken together, these results indicate that GnRH pulse frequency and steroid
concentrations are important factors in regulating GnRH receptor concentration and
regulation of gonadotropin secretion.
Pituitary Hormones
The pituitary gland has been implicated as the source of luteotropic substances that
regulate luteal development and function. The importance of the pituitary gland as a
source of luteotropins in the ewe was demonstrated by Kaltenbach et al. (1968). These
researchers found that when ewes were hypophysectomized on the day after normal
ovulation a functional CL failed to form, while hypophysectomy on day 5 of the estrous
cycle caused the regression of the partially formed corpus luteum. Additional research by
Denamur et al. (1973) demonstrated that when hysterectomized ewes having maintained
39
CL were hypophysectomized, the CL regressed. However, hypophysectomy or
hypophyseal stalk-section in some rodents shortly after ovulation does not alter the normal
life span of the corpus luteum (Greenwald and Rothchild, 1968; Illingsworth and Perry,
1971), while hypophysectomy during early pregnancy, before day 10 in the mouse, results
in the termination of luteal function and pregnancy (Choudary and Greenwald, 1969).
These studies clearly indicate the pituitary gland as the source of luteotropic hormones.
Although there are numerous hormones produced by the pituitary, there are three
in particular that have been implicated in affecting luteal function, these include prolactin,
LH and FSH. Prolactin, while being the primary hormone regulating luteal function up to
implantation in many rodents (Greenwald, 1973; Smith and Neill, 1976), appears to have
no significant effect after implantation in rodents or in regulating luteal function during the
estrous or menstrual cycles of domestic animals and primates (Stormshak et al., 1987).
Oxytocin secreted from the posterior pituitary has been implicated in influencing
reproduction, however, the extent of this influence on luteal function is unclear. In the
following section, the discussion will primarily focus on the gonadotropins, LH and FSH,
and their role in regulating luteal life span. However, a brief discussion on the influences
of oxytocin and prolactin will also be presented.
Luteinizing hormone and FSH are glycoprotein hormones produced and secreted
from the gonadotropes of the anterior pituitary under the stimulation of GnRH. Both of
these peptide hormones are composed of an a and a 13 subunit; the a subunits of LH and
FSH are virtually identical whereas the amino acid sequence of the 13 subunit is distinct and
determines the biological activity of the hormone (Hadley, 1992b). The actions of FSH
have been implicated in follicular growth and recruitment, while LH is normally associated
with ovulation of the ovum and stimulation of progesterone secretion. However, it is the
coordinated activity of both LH and FSH during the follicular phase of the estrous cycle
that regulates estrogen production, follicular growth and ovulation.
40
Luteinizing Hormone
Luteinizing hormone is essential for normal ovulation and maintenance of the
corpus luteum in many mammalian species (Leers-Sucheta and Stormshak, 1991).
Administration of LH to hypophysectomized rats has been shown to cause luteinization of
granulosa cells (Richards et al., 1976). Similarly, pulsatile injections of LH has been
shown to induce ovulation in anestrous ewes (McNeilly et al., 1982). Kaltenbach et al.
(1968) demonstrated that constant infusion of crude pituitary extracts, containing LH and
FSH, maintained luteal function in both pregnant and non-pregnant hypophysectomized
ewes. Furthermore, ewes (Karsch et al., 1971) and cows (Donaldson and Hansel, 1965)
injected with LH have exhibited prolonged estrous cycles with an extended CL life span
compared with controls.
The relationship between LH and progesterone secretion is complex. Luteinizing
hormone concentrations increase only when progesterone levels are low and estrogen
levels are rising (Karsch et al., 1983, Schallenberger et al., 1984). However, it also has
been established that LH can increase progesterone synthesis by luteal cells either in vitro
or in vivo (Niswender and Nett, 1988; Niswender et al., 1994). To further complicate this
relationship, during the luteal phase of the ovine estrous cycle when progesterone
concentrations are high, there appears to be little correlation between the episodic peaks
of serum LH and systemic progesterone concentrations (Baird et al., 1976).
This obscure relationship can be explained in part by examining the characteristics
of the CL during the estrous cycle. During the luteal phase, more than 80% of the total
progesterone secreted is from large luteal cells (Rothchild, 1981) which contain only 10%
of the total number of LH receptors (Niswender et al., 1985). However, if LH is added to
ovine luteal cells in culture, it is the small luteal cells which respond with an increase in
progesterone secretion while large luteal cells exhibit little or no response (Lemon and
Loir, 1977; Rodgers et al., 1983; Ursely and Leymarie, 1979). Similarly, when LH was
introduced into the culture medium containing the luteal cells of cows (Ursely and
41
Leymarie, 1979) and sows (Lemon and Loir, 1977), the small luteal cells had a much
greater response as measured by progesterone secretion.
One possible mechanism by which LH may affect luteal function was first
proposed by Donaldson and Hansel (1965) who stated that this gonadotropin may
regulate the differentiation of small to large luteal cells in cattle. Mee et al. (1993) found
that cows that had been treated with GnRH 72 hours after the onset of estrus had an
immediate increase in LH secretion, and when the CL was examined on day 10 of the
estrous cycle it contained a higher proportion of large luteal cells. A similar shift from
small to large luteal cells has been observed in ewes treated with either hCG (a known
LH-agonist) or LH on days 5 to 10 of the estrous cycle when compared with controls
(Farin et al., 1988; Niswender et al., 1988). Taken together, this research suggests that
while secretion of progesterone by large luteal cells is not directly regulated by LH; the
number of large luteal cells may depend, at least in part, on the actions of LH.
Another possible way in which LH may influence luteal steroidogenesis is by
regulating the availability of the luteal LH receptors. Diekman et al. (1978a) were among
the first researchers to follow the profile of LH receptor availability throughout the ovine
estrous cycle. These researchers found that the total number of LH receptors per CL
increased approximately 40-fold from day 2 to 10 of the cycle, while the number of
occupied receptors increased sixfold. The number of luteal LH receptors and secretion of
progesterone during the cycle also have been shown to be correlated in cows (Garverick
et al., 1985; Spicer et al., 1981), sows (Ziecik et al., 1980), mares (Roser and Evans,
1983) and monkeys (Cameron and Stouffer, 1982). In these species, the number of
occupied and unoccupied LH receptors are highly correlated with the weight of the corpus
luteum and progesterone secretion. Therefore, in these species, it is the LH receptor
availability that regulates the secretion of progesterone rather than the circulating levels of
this hormone.
42
Because the role of LH and LH receptors in controlling luteal cell steroidogenesis
has been fairly well characterized, researchers set out to determine whether the mechanism
of PGF2a-induced luteolysis is controlled by a reduction in the number of luteal receptors
for LH or by some other mechanism. Diekman et al. (1978b), induced luteolysis in ewes
by injecting PGF2a during the luteal phase of the estrous cycle, and found that serum
concentrations of progesterone decreased before significant changes were detected in
luteal concentrations of LH receptors. Spicer et al. (1981) similarly found that PGF2a
administered to cows during midcycle was followed by a significant reduction in luteal
progesterone secretion, which attained basal levels by 12 hours post-treatment. However,
specific binding of labeled hCG to luteal homogenates remained unchanged at 12 hours
and did not reach basal concentrations until 24 hours after initial injection. However,
when these same authors examined the correlation between LH receptors and
progesterone concentrations in cows undergoing spontaneous luteolysis, they found that
they decreased concomitantly. Research with sows also indicates that when luteolysis is
induced (Barb et al., 1984) the reduced concentrations of serum progesterone and LH
receptors are highly correlated. However, when luteolysis occurs spontaneously (Ziecik et
al., 1980) a depression in luteal LH receptors actually precedes a decrease in serum
progesterone. Thus, while it is tempting to deduce that the decrease in luteal LH
receptors is the prelude to the events leading to the ultimate destruction of the CL, clearly
more research is needed to determine the exact relationship between these factors in
different species as well as in spontaneous and induced luteolysis. However, it is feasible
that a decrease in LH receptors is not the primary determinant for the reduction in luteal
function, especially since the small luteal cells contain the majority of LH receptors, while
the large cells possess the majority of PGF2a receptors, and hence are the more likely
targets for the luteolytic action of PGF2a.
43
Follicle Stimulating Hormone
While the function of FSH in regulating the life span of the CL is not fully
understood, its role in follicular dominance, growth and ovulation that leads to the
eventual formation of the CL, has been well documented. As discussed above, it is the
intimate relationship between LH and FSH that has been primarily indicated in controlling
follicular maturation.
In ewes, there is a functional relationship between surges in circulating
concentrations of FSH and the emergence of follicular waves. Adams et al. (1992; 1993)
found that each follicular wave in the ovine estrous cycle was preceded in 1-2 days by a
significant surge in serum FSH. Numerous other species also appear to require FSH to
stimulate follicular growth and recruitment. Rats exhibit a secondary surge of FSH on the
day of estrus, just before the next cohort of follicles begin development (Smith et al.,
1975). In primates, basal FSH levels increase slightly at the beginning of the follicular
phase and the subsequent duration of the FSH rise influences the number of follicles
stimulated (Goodman et al., 1977; Zeleznik and Kubik, 1986). In cattle, a secondary
surge of FSH on the day of ovulation precedes the first follicular wave of the cycle
(Dobson, 1978; Walters and Schallenberger, 1984) but slight elevations in serum FSH
have also been shown to precede the second and third waves (Adams et al., 1992).
There seems to be a temporal correlation between higher FSH secretion and
recruitment of follicles. Abolishing the preovulatory surge of FSH in ewes and cows by
injection of bovine follicular fluid, after prostaglandin-induced luteolysis, caused a delay in
the onset of behavioral estrus. McNeilly (1984) similarly demonstrated that ewes injected
with charcoal-treated bovine follicular fluid delayed the onset of estrus and ovulation by
selectively inhibiting FSH secretion without altering LH secretion. Turzillo and Fortune
(1993) investigated the importance of FSH for the development of the dominant follicle
during the first follicular wave in cows by injecting charcoal-extracted bovine follicular
fluid on days 6 and 7 of the cycle, followed by PGF2c, to induce luteolysis. These
44
researchers found that all heifers treated with follicular fluid had arrested follicular
development, additionally one-half of those treated exhibited a depression in estradiol
production and a delay in the onset of estrus (Turzillo and Fortune, 1993). Results of
several studies suggest the suppression of FSH by follicular fluid may be independent of
short-term GnRH input, because follicular fluid seems to have no effect on LH secretion
(McNeilly, 1984; Brooks et al., 1992). Treatment with a GnRH-antagonist has no effect
on the response of FSH to follicular fluid treatment (Brooks et al., 1993).
The substance in the follicular fluid that is responsible for suppressing FSH release,
is the protein hormone inhibin. Inhibin is produced by the granulosa cells of the follicle
but little is known about its control and release during the estrous cycle (Hafez, 1987). In
an effort to increase ovulation rates in domestic animals, some researchers have attempted
the immunoneutralization of inhibin. Wheaton et al. (1992) found that either active or
passive immunoneutralization in ewes resulted in an increase in FSH secretion as well as
increasing the ovulation rate.
Follicular recruitment and development occurs in waves under the influence of
FSH, but why is it that only one follicle (in monovulatory species) or a few (in
polyovulatory species) of the follicles from the original cohort ovulate while the others
undergo atresia? There are two main hypotheses to explain the mechanism by which the
dominant follicle of the cohort suppresses the growth of the remaining follicles. The
dominant follicle may either secret a substance that directly impairs further growth and
development of the subordinate follicles or the dominant follicle could cause the atresia of
subordinates indirectly by a negative feedback mechanism (Fortune, 1994). Most
evidence seems to suggest that the dominant follicle exerts its control indirectly by
secreting estradiol and(or) inhibin which would decrease FSH to levels that are unable to
support further growth of the subordinate follicles. Zeleznik and Kubik (1986)
demonstrated that in cynomologous monkeys the plasma concentration of FSH necessary
to maintain preovulatory follicular maturation is less than that necessary to initiate
45
preovulatory growth. By this mechanism, the maturing follicle inhibits the growth of the
less mature follicles by decreasing FSH concentrations, but does not succumb to its own
inhibitory influences.
Ability to continue growth and development in the presence of lower FSH levels
may be due to the increased vascularization of the dominant follicle or because of the
increased number of LH receptors in the granulosa cells (Zeleznik, 1993). However, low
levels FSH appear to remain critical to the dominant follicle, because experimental
reduction of plasma FSH during the dominance phase in cattle is correlated with cessation
of growth and sometimes the demise of the dominant follicle (Turzillo and Fortune, 1993).
Therefore, while FSH does not seem to be critical in regulating the life span of the existing
CL, its role in follicular development leading up to the formation of the CL is essential.
Oxytocin
Oxytocin, as mentioned previously, is a peptide hormone made from a larger
precursor hormone that is synthesized by both the hypothalamus and the ovary.
Hypothalamic oxytocin is produced primarily within the cell bodies of the paraventricular
and supraoptic nuclei where it is packaged into secretory vesicles (Sawchenko and
Swanson, 1985; Zimmerman et al., 1974). Once packaged, oxytocin is transported down
the axon of these magnacellular neurons, through the median eminence, and stored in the
posterior pituitary (neurohypophysis). Under the proper stimulation, oxytocin is released
from nerve terminals in the neurohypophysis and into the blood stream by exocytosis (Ivell
and Richter, 1985).
Classically, hypothalamic oxytocin has been implicated in the milk ejection reflex,
maternal recognition of pregnancy and even parturition. At parturition, oxytocin of
maternal and fetal pituitary origin as well as oxytocin from the CL has been implicated in
the induction of uterine contractions in numerous species (for review see Fuchs, 1985).
However, the role of hypothalamic oxytocin in regulating the life span the corpus luteum
46
is uncertain. Some researchers have postulated that it is the initial stimulation of oxytocin
from the neurohypophysis that stimulates the positive feedback loop between oxytocin and
PGF2a, during the terminal stages of the estrous cycle (Silvia et al., 1991). However, this
hypothesis seems somewhat unlikely because a single CL can produce approximately 250
times more oxytocin mRNA than a single bovine hypothalamus (Ivell and Richter, 1985).
Further, the posterior pituitary seems to release only minute quantities of oxytocin after
injection with prostaglandins whereas the CL of the ovary responds precipitously to an
injection of PGF2c, (Schams et al., 1985c). These findings were confirmed by Orwig et al.
(1994) who found that while administration of cloprostenol, a PGF20, analog, caused a
significant increase in plasma oxytocin in an intact cow; those cows that received an
injection of cloprostenol at the time of CL removal, did not exhibit a release of oxytocin
that was significantly greater than baseline.
Prolactin
Prolactin is a peptide hormone that is produced and released from the anterior
pituitary (adenohypophysis). Prolactin is a species specific hormone because there is only
about 40% homology in amino acid sequence among different species as well as in its
three dimensional structure (Wallis et al., 1985b). Prolactin contains between 197 to 199
amino acids depending on the species studied (Hadley, 1992b). Prolactin, like pituitary
oxytocin, is typically associated with mammary gland growth and lactation. However, its
role as a luteotropin has been extremely well documented in rodents.
Prolactin has long been recognized as a luteotropin in the rat. Prolactin secreted
by the pituitary can, by itself, sustain basal levels of progesterone during the first week of
pregnancy in the rat (Albarracin et al., 1994). During pregnancy, prolactin is secreted
from the pituitary as two pulses per day for the first 10 days of gestation (Gunnet and
Freeman, 1983). From the day of implantation to day 10 of pregnancy, the corpus luteum
is under the combined control of pituitary prolactin and placental hormones (Kelly et al.,
47
1976; Morishige and Rothchild, 1974). Gafvels et al. (1992) found that prolactin had a
stimulatory effect on luteal LH receptor mRNA. These researchers also demonstrated that
blocking prolactin secretion by injecting bromocriptine during early pregnancy resulted in
abortion; however, if exogenous prolactin was given concomitant with bromocriptine until
the time of implantation pregnancy was carried to term (Gafvels et al., 1992). Inhibition
of prolactin secretion has been shown to induce luteolysis by decreasing LH and hCG
receptor binding capacity (Chan et al., 1980; Holt et al., 1976) and by induction of luteal
20a-hydroxysteroid dehydrogenase activity, which results in the conversion of
progesterone to 20a-hydroxyprogesterone, a reduced steroid with little progestational
activity (Albarracin et al., 1994). Administration of prolactin to rats has been shown to
decrease 20a-hydroxysteroid dehydrogenase activity (Albarracin et al., 1994). In addition
to maintaining the corpus luteum during pregnancy and stimulating luteal progesterone
production, prolactin promotes luteolysis of the CL from the previous reproductive cycle
(Malven and Sawyer, 1966). Clarke and Linzer (1993) found that the mouse ovary
contains mRNA encoding four different forms of the prolactin receptor and these authors
suggested that it is through stimulation of different prolactin receptors that this hormone
can exhibit both luteotropic and luteolytic effects.
In contrast with the luteotropic effects in the rodent, prolactin appears to play no
significant role in regulating the luteal life span in most domestic animals and primates.
Injections of the prolactin inhibitor bromocriptine into ewes reduced serum concentrations
of prolactin by more than 95% for the period of an estrous cycle without affecting either
estrous cycle length or serum progesterone concentrations (Niswender, 1974).
Additionally, when excess quantities of prolactin antiserum were administered in
conjunction with bromocriptine there was still no effect on the serum concentrations of
progesterone (Niswender et al., 1976). Further, constant infusion of prolactin into intact
ewes had no significant effect on luteal function (Karsch et al., 1971). However, it is
48
noteworthy that in the sow, prolactin may be luteotropic during the terminal stages of
gestation.
Kraeling and Davis (1974) reported that hypophysectomy during late gestation
terminates pregnancy in pigs, but when prolactin was administered immediately following
hypophysectomy the pregnancy was maintained (du Mesnil du Buisson, 1961; Denamur et
al, 1973). Yangfan et al. (1989) found that when hysterectomized pigs containing CL of
110 days are hypophysectomized, serum prolactin levels decline and the CL regress.
However, if porcine prolactin is administered from the time of hypophysectomy, these
hysterectomized and hypophysectomized gilts will maintain serum progesterone levels as
well as morphology of the aging CL during the period of prolactin administration.
Similarly, injection of prolactin between days 110-120 in hysterectomized gilts caused an
enhancement in serum progesterone concentrations when compared with saline-treated
controls (Felder et al., 1988). Hyperprolactinaemia is frequently associated with an
inhibition of luteal function, such as lactational anestrus, and is commonly associated with
infertility in humans (Ginsburg, 1992). To determine whether overproduction of prolactin
could be detrimental to pregnancy, Szafranska and Tilton (1993) induced
hyperprolactinaemia in gilts by administration of haloperidol during the second one-half of
gestation and found that although there was a drastic suppression of LH secretion,
pregnancy was maintained in these animals.
The way in which prolactin affects steroidogenesis and luteal function may be by
the active role it plays in cholesterol homeostasis within the luteal cell. One of the
functions of prolactin is to increase uptake and utilization of lipoprotein-born cholesterol,
and in the few species studied, this hormone acts by increasing cellular high density
lipoprotein (HDL) binding. Prolactin maintains or increases HDL receptor numbers in the
CL (Murphy and Silavin, 1989). Menon et al. (1985) reported that incubation of rat luteal
cells with prolactin increased progesterone synthesis in the presence of HDL. Further,
incubation of pig luteal cells (Rajkumar et al., 1985) or luteinized granulosa cells
49
(Rajkumar et al., 1988) with HDL resulted in increased progesterone accumulation in the
incubation medium. Additionally, Murphy and Rajkumar (1985) demonstrated that
dissociated luteal cells isolated from pregnant pigs demonstrated increased uptake of 1251­
LDL in response to prolactin. However, the mechanism by which prolactin increases
lipoprotein uptake is not completely understood.
ROLE OF THE UTERUS IN REGULATING LUTEAL LIFE SPAN
For a number of years, researchers have been attempting to elucidate the role the
uterus may have in regulating luteal life span and function. One method that has been
employed is the surgical removal of the uterus (hysterectomy). During a hysterectomy,
the uterine horns and the body of the uterus are removed while leaving the ovarian
vasculature intact. In this fashion, researchers are able to demonstrate if luteal and ovarian
structures can function in the absence of the uterus. Another method to examine the
function of the uterus, at least in species with bipartite or bicornuate uteri, is by the
surgical procedure known as a unilateral hysterectomy. This technique results in the
removal of one uterine horn to the level of internal bifurcation of the uterus, without
impairing ovarian blood flow or the integrity of the remaining uterine horn. The following
sections will examine the role of the uterus in regulating luteal function in various species
as well as examine the role of the uterus in luteolysis.
Effects of Hysterectomy
When experiments were first initiated in the late 1800's to elucidate the role of the
uterus in luteal function, contradictory results were obtained. It was not until the
experiments by Loeb (1923) that a clear understanding as to the purpose of this structure
began to be defined. Loeb (1923) found that removal of the uterus in the guinea pig
during mid-gestation resulted in the maintenance of the CL for more than 90 days; normal
50
gestation in this species is only about 63 days. This extension of luteal life span indicated
that luteolysis is dependent upon the uterus in the guinea pig.
Since this discovery by Loeb, researchers have been able to compile a body of
evidence that better explains the role of the uterus in a variety of species. The extent in
which the uterus may aid in luteolysis, appears to be a species specific process. The uterus
appears to be most important in regulating luteal life span in ungulates and guinea pigs and
of little importance in humans and non-human primates (Anderson, 1973). To briefly
summarize the results of these research projects, removal of the uterus in rabbits (Loeb
and Smith, 1936; Tenny et al., 1955) and rats (Anderson et al., 1967; O'Shea, 1970) does
not affect the length of the estrous cycle, but does prolong the duration of
pseudopregnancy. Uterine removal in guinea pigs (Loeb, 1923; 1927), sheep (Hu et al.,
1991; Moor and Rowson, 1966; Southee et al., 1988; Wiltbank and Casida, 1956), pigs
(Felder et al., 1988; Staigmiller et al., 1972) and cows (Anderson et al., 1962; Brunner et
al., 1969; Ginther et al., 1967; LaVoie et al., 1975) result in the prolongation of the
estrous cycle that may equal or exceed the duration of gestation for that species. In the
mare, hysterectomy does delay luteal regression, however, the secretory ability of the CL
is significantly impaired (Squires et al., 1974). Conversely, hysterectomy has no
significant effects on the duration of luteal life span in humans (Beling et al., 1970) and
non-human primates (Neill et al., 1969).
As mentioned previously, the role of the uterus in regulating luteolysis in domestic
animals is quite extensive. To further examine the utero-ovarian relationship in this group
of animals, Anderson et al. (1962) performed an elegant study examining luteal function in
groups of heifers. These researchers removed portions of uterus leaving the following
structures intact: group 1) none (horns, body and cervix removed); group 2) cervix and
body; group 3) one-quarter of the uterine horns closest to the ovaries; and group 4) onehalf of the uterine horns closest to the ovaries. Only those heifers in which the entire
uterus was removed remained anestrus for the duration of the study, approximately 270
51
days. Upon morphological analysis at the conclusion of the study, the ovaries from the
group of totally hysterectomized heifers were found to have their original CL. This
confirmed earlier work by Armstrong and Hansel (1959) who found that removal of the
uterine body and horns in heifers prevented oxytocin-induced luteolysis. Felder et al.
(1988) found by comparing serum progesterone concentrations in pregnant gilts at 100
days gestation that the integrity of the CL was not compromised in hysterectomized pigs
with aging CL. However, when these gilts were injected with porcine LH,
hysterectomized gilts responded with an increase in luteal progesterone secretion whereas
there was no significant effect on the luteal function in pregnant gilts. Upon
morphological analysis, the regression of the original CL and formation of new CL had
developed in the hysterectomized gilts. This suggests that during pregnancy the conceptus
and(or) uterine-derived luteotropins may override the effect of LH in inducing ovulations
and regression of preexisting CL (Felder et al., 1988).
The first evidence suggesting that the CL of domestic animals may be influenced
by the uterus in a local manner was obtained by du Mesnil du Buisson (1961) from
experiments with unilaterally hysterectomized pigs. Ginther et al. (1967) similarly found
that the local interaction between the uterine horn and its adjacent ovary is essential for
luteolysis in cows. In this latter work, the uterine horn adjacent (ipsilateral) to or opposite
(contralateral) to, the ovary bearing the CL was removed and animals were challenged
with an injection of oxytocin. While oxytocin was effective in inducing luteolysis in both
intact and contralateral hysterectomized heifers; oxytocin was ineffective in inducing
premature luteolysis in heifers that had the ipsilateral uterine horn removed (Ginther et al.,
1967). Similarly in sheep, unilaterally ovariectomized ewes have been demonstrated to
have a normal estrous cycle unless the uterine horn ipsilateral to the remaining ovary was
removed (Moor and Rowson, 1966) Further, in unilaterally hysterectomized ewes having
intact ovaries, with each bearing CL, treatment with a luteolytic dose of estradio1-1713
resulted in the reduction of luteal weight in the ovary ipsilateral to the remaining uterine
52
horn as compared with the weight of the CL in the contralateral ovary (Akbar et al.,
1971).
Utero-ovarian Functional Interrelationships and Luteolysis
From the above studies it seems clear that in domestic animals the uterus and the
ovary bearing the CL must be in close proximity in order for normal luteolysis to occur.
Numerous researchers set out to investigate further the functional relationship of the
uterus and the ovary in luteolysis. These projects were carried out primarily with sheep,
because the reproductive system in this species had been extremely well characterized in
comparison to other experimental models (McCracken et al., 1972). The purpose of these
studies was to determine if the relationship between the uterus and the ovary in luteal
demise was dependent upon some other local factors in the area of the reproductive tract
or solely regulated by the uterus and the ovary.
Surgical procedures in sheep where the ovary is transplanted to the neck, leaving
the uterus in its original position in the abdomen, results in the persistence of luteal
function (Baird et al., 1968; Goding et al., 1967b). Further, researchers found that
transplantation of the uterus to the neck, while leaving one ovary in the abdomen, also
results in luteal retention (Goding et al., 1967a). However, when the ovary and its
adjacent uterine horn is transplanted into the neck of the ewe as a unit, normal luteal
function and regression occurs (McCracken et al., 1970a). Thus, it was clearly established
that luteal function was controlled by the local effects of the uterine horn on the adjacent
ovary, regardless of position of this unit in the body.
To determine if the effects of the uterus on the adjacent ovary bearing the CL were
by the release of some substance into the blood system late in the estrous cycle, ligation of
various vascular connections between the reproductive structures was conducted.
Kiracofe et al. (1966) demonstrated that ligation of the uterine arteries and veins resulted
in the extension of luteal function while ligation of the uterine artery alone was without
53
effect. McCracken et al. (1972) later prepared a series of elegant cross-vascularization
experiments, to examine whether the uterine venous blood contained the luteolytic factor.
In these experiments, the donor animal had a utero-ovarian transplant in the neck and the
recipient ewes had only the ovary transplanted into the neck. Utero-ovarian venous blood
was transfused from the donor animal on day 15 of the cycle to the recipient ewe which
resulted in a decrease in progesterone secretion in the recipient ewe within 48 hours.
However, regression of the CL did not occur when recipient ewes were infused with
utero-ovarian blood from donor ewes that were at either day 2 or 10 of the estrous cycle
(McCracken et al., 1972). These results suggest during the terminal stages of the estrous
cycle that a luteolytic factor is secreted by the uterus into the uterine vein to cause the
demise of the CL.
The luteolytic factor from the uterus was suggested to be PGF2c, because this
hormone is known to be a potent vasoconstrictor (Ducharme et al., 1968) as well as being
abundant in the uterus (Pickles, 1966). Pharris and Wyngarden (1969) were able to
demonstrate that in the rat, PGF2c, administration induced a significant reduction in the
length of pseudopregnancy. However, because PGF2c, is rapidly degraded to its inactive
metabolite, PGFM, with just one pass through the lungs (Piper and Vane, 1969), the
delivery of this hormone from the uterus to the ovary would have to be local. While there
are no direct vascular connections between the uterus and the ovary, uterine vein and the
ovarian artery are closely apposed to one another. Because of the close apposition of
these vessels, Barrett et al. (1971) hypothesized that there may be some kind of countercurrent exchange mechanism by which the luteolytic substance passes from the uterine
vein to the ovarian artery. To test this hypothesis, these researchers surgically separated
the ovarian artery from the surface of the utero-ovarian vein and subsequently found that
complete separation of these two vessels and inserting a portion of broad ligament as a
barrier between them resulted in maintenance of the CL (Barrett et al., 1971). These
results confirmed the hypothesis that a counter-current exchange mechanism was
54
responsible for the luteolytic substance to travel to the ovary. However, it was still yet to
be determined whether the proposed luteolysin, PGF2, could travel via this countercurrent exchange mechanism. McCracken et al. (1972) set out to determine whether
PGF2c, could travel by this mechanism by infusing [311]-PGF2c, into the uterine vein of a
ewe on day 14 of the cycle. By comparing the radioactivity in the plasma collected from
the ovarian artery to plasma and the adjacent iliac artery, these researchers found that the
levels of [311]-PGF2c, was at least 30 times higher in the ovarian arterial plasma than the
iliac arterial plasma. Taken together, this research supports the hypothesis that the
luteolysin PGF2a, is transported from the uterus to the adjacent ovary by a counter-current
exchange mechanism which induces luteolysis.
Action of Prostaglandins
Prostaglandins belong to a family of chemically related substances known as
eicosanoids. These substances are synthesized from a common precursor, arachidonic
acid, which is liberated from the plasma membrane by phospholipase activity. Once
liberated, arachidonic acid may be further converted in the cytosol of the cell to
prostaglandins. Stimulation by hormones, such as oxytocin, or other stimuli may initiate
calcium-mediated phopholipases to liberate arachidonic acid that will eventually serve as
substrate for prostaglandin biosynthesis. The rate-limiting step in the biosynthesis of
prostaglandins appears to be the availability of this substrate, the precursor arachidonic
acid (Lapetina et al., 1978).
As mentioned in the preceding section, PGF2,,, is the known luteolysin in a number
of non-pregnant animals. Pharriss and Wyngarden (1969) were the first to demonstrate
that PGF2a could induce luteolysis by injecting this substance into pseudopregnant rats
and causing subsequent luteal regression. Since this discovery, PGF2c, has been
demonstrated to be the luteolytic factor in rabbits (Pharriss, 1970), sheep (Caldwell et al.,
55
1972; McCracken et al., 1970), cows (Hafs et al., 1974; Thatcher and Chenault, 1976)
and horses (Douglas and Ginther, 1972; Hafs et al., 1974).
During luteolysis in domestic animals, PGF2a is released from the uterus in a
pulsatile fashion with a total of approximately 5-8 discrete pulses (Fredriksson et al., 1984;
McCracken et al., 1984; Peterson et al., 1976). Variability exists among species as to the
duration and magnitude of pulses, however, they typically occur at 6-8 hour intervals
(Silvia et al., 1991). Luteal tissue appears to be particularly sensitive to the luteolytic
effect of PGF2c, when administered in a pulsatile fashion versus constant infusion
(Schramm et al., 1983). Therefore, it is likely that it is the onset of the pulsatile secretion
of PGF2a that may induce the onset of luteolysis.
Prostaglandin F2a, has also been shown to stimulate secretion of oxytocin from the
ruminant CL both in vivo (Flint and Sheldrick, 1982; Lamsa et al., 1989) and in vitro
(Abdelgadir et al., 1987; Chegini and Rao, 1987). Additionally, large luteal cells, which
have been identified to be the source of ovarian oxytocin, have been demonstrated to
possess PGF2c, receptors. During luteolysis, pulses of PGF2a, and its metabolite, are
highly correlated to levels of oxytocin when sampled in the utero-ovarian vein (Hooper et
al., 1986) as well as systemic circulation (Flint and Sheldrick, 1983). Lamsa et al. (1992)
demonstrated that the PGF2c, receptor in the ovine CL is rapidly desensitized upon
stimulation with hormone, and that a minimum rest period of 6 hours was required to
restore sensitivity. These authors hypothesized that it is the desensitization and recovery
of the PGF2c, receptor that accounts for the pulsatile nature of oxytocin during luteolysis
(Lamsa et al., 1992). Similarly, refractoriness of endometrial oxytocin receptors have also
been characterized. After acute exposure to oxytocin, refractoriness is maintained for a
period of approximately 6 hours (Sheldrick and Flint, 1986). Therefore, transient uterine
refractoriness to oxytocin as well as luteal refractoriness to PGF2c,, may account for the
pulsatile nature of PGF2a.
56
Due to the strong correlation between luteal oxytocin secretion and uterine PGF2a,
secretion during luteolysis, the secretion of PGF2a may be partially dependent upon
ovarian oxytocin. Indeed, oxytocin administration during the latter part of the estrous
cycle has been shown to cause premature regression of the CL by stimulating the release
of uterine PGF2c, (Flint and Sheldrick, 1983; Kieborz et al., 1991). Therefore, it has been
hypothesized that in domestic animals uterine PGF2a, and luteal oxytocin comprise a
positive feedback loop, where PGF2a, synthesized in the uterus is released into the uterine
vein and acting through a counter-current transfer mechanism, acts upon the adjacent
ovary to stimulate oxytocin secretion from the CL. This luteal oxytocin then acts back
upon the uterine horn to further stimulate the secretion of PGF2a, which causes the
ultimate regression of the CL (Flint and Sheldrick, 1983; McCracken et al., 1984;
Sheldrick and Flint, 1984).
Role of Oxytocin
The mechanism by which oxytocin induces PGF2e, secretion from the uterus is not
well understood. The ability of oxytocin to regulate uterine PGF2c, release from the
uterus may be dependent on several factors. Some of the factors that have been indicated
in regulating PGF20, release include: the availability of arachidonic acid as a precursor for
prostaglandin synthesis in uterine tissue (Silvia et al., 1991); the quantity of prostaglandin
H endoperoxide synthase (cyclooxygenase) enzyme, which is essential for prostaglandin
synthesis; and probably most importantly, the uterine oxytocin receptor itself.
Oxytocin receptors have been reported to exist in the uterine endometrium and
myometrium of many mammals. These two receptor locations seem to have a dual action
in the uterus: a uterotonic action on myometrial cells to stimulate contractions and a
prostaglandin-releasing action on endometrial cells (Fuchs et al., 1982). Availability and
formation of the oxytocin receptors on the uterus are regulated by the concentration of
progesterone and estradiol throughout the estrous cycle (Sheldrick and Flint, 1985; Zhang
57
et al., 1992). Number of endometrial oxytocin receptors increases with the approach of
luteolysis and becomes maximal on the day of behavioral estrus (Ayad et al., 1991;
Sheldrick and Flint, 1985). The pattern of development of oxytocin receptors in the
endometrium is of particular interest because the endometrium has been shown to be the
site of prostaglandin synthesis, further substantiating the role of oxytocin in luteal
regression (Cerini et al., 1979; Salamonsen and Findlay, 1990).
It has been hypothesized that the initiation of luteolysis may begin at the level of
the uterus, because PGF2u secretion increases before any significant rise in luteal oxytocin
occurs (Moore et al., 1986). Stimulation for the initial pulse of PGF2G, is still unknown,
but oxytocin secretion from the posterior pituitary has been implicated (Silvia et al., 1991).
Regardless, this release of PGF2c, stimulates the release of luteal oxytocin which then can
bind its receptor in the uterus to further stimulate PGF2G, secretion.
Luteotropic Mechanism
While PGF2a, has been identified as the uterine luteolytic hormone, other
prostaglandins of uterine origin have been implicated in luteotropic processes. For
example, prostacyclin (PGI2) which is a metabolite of the cyclic endoperoxide PGH2.
Milvae and Hansel (1980) found that injection of PGI2 directly into the bovine CL at
midcycle produced a striking increase in systemic plasma progesterone concentrations and
that PGI2 stimulated progesterone biosynthesis by dispersed luteal cells in vitro. Later
these researchers found that incubated luteal cells in the presence of LH and arachidonic
acid, had a higher rate of secretion of PGI2 for CL collected on day 5 as compared with
CL collected on days 10, 15 or 18 of the bovine estrous cycle (Milvae and Hansel, 1983).
Additionally, progesterone production during the 2 hour incubation period decreased as
the age of the CL advanced and as concentrations of PGI2 declined. These findings
suggest that PGI2 may play an important role particularly during the first 10 days of the
bovine estrous cycle. It is noteworthy that although PGI2 can be manufactured in the
58
uterine tissues, the bovine CL has high PGI2 synthase activity (Sun et al., 1977)
implicating an autocrine or paracrine action of this hormone to maintain luteal function.
Prostaglandin E1 (PGE1) is another prostaglandin that has been found to possess
luteotropic properties. Huie et al. (1981) found that intrauterine infusions of PGE1
resulted in the prolonged maintenance of luteal function in ewes when infused into the
uterine horn ipsilateral to the ovary bearing the CL. However, treatment of ewes with
PGE1 by infusion into the contralateral uterine horn failed to have a significant effect on
luteal maintenance or the interestrous interval as determined by estrus observations and
serum progesterone profiles. These data suggest that PGE I can act in a localized manner
to maintain luteal integrity, and thus may be an important hormone during early pregnancy
because it is capable of delaying luteal regression (Huie et al., 1981).
Weems et al. (1985) found that injection (333 lag) of either PGE1 or prostaglandin
E2 (PGE2) into the ovary of nonpregnant ewes resulted an increase in progesterone
secretion compared with that of nontreated controls at 24 hours after injection. Only
those ewes treated with PGE1, however, had elevated progesterone concentrations at 48
hours after administration, suggesting that PGE1 may be a more potent luteotropin than
PGE2.
Wilson et al. (1972) identified PGE2 in the homogenate from the endometrium of
nonpregnant ewes. In fact PGE2 infused along with PGF2a, into the artery of ovaries
transplanted into the neck in ewes, prevented the reduction of progesterone observed
when PGF2c, was infused alone (Henderson et al., 1977). Silvia et al. (1981) utilized
indwelling catheters in the utero-ovarian vein in pregnant and nonpregnant ewes. These
authors found that levels of PGE2 rose dramatically on day 13 in pregnant ewes and
remained high through day 14, whereas there was no change in secretion in nonpregnant
ewes. Conversely, Lewis et al. (1978) compared the level of this hormone in uterine
venous plasma of nonpregnant and pregnant ewes on day 14 and found that the levels of
PGE2 were not significantly different, but did tend to be lower in the nonpregnant versus
59
pregnant ewes. These observations suggest that there may be a critical period during early
pregnancy that the increase in PGE2 is necessary in order to prevent luteolysis, and after
this critical period, the levels of this luteotropic substance may return to normal levels.
However, it was impossible to determine from these experiments whether the increased
secretion of PGE2 was of uterine or conceptus origin. Blastocysts in sheep (Hyland et al.,
1982), cattle (Lewis et al., 1982), pigs (Geisert et al., 1982) and rabbits (Harper et al.,
1983) also have been shown to produce PGE2 in vitro.
Weems et al. (1993) suggested that after day 55 of gestation of the ewe, the
primary source of PGE2 becomes the placentome which continues to act to regulate
steroidogenesis of the placenta as well as maintaining luteal function. It is therefore
possible that during early pregnancy, both the uterus and the conceptus are sources of the
luteotropin PGE2, which act together to maintain pregnancy. Later on in gestation,
however, the conceptus may be the primary source of this luteotropic prostaglandin.
60
STATEMENT OF THE PROBLEM
Approximately 30% of all embryos are lost during early development (Roberts et
al., 1990) and at least some of this loss has been attributed to inadequate luteal function
(Niswender and Nett, 1988). To producers, this can be a substantial financial loss
particularly when dealing with repeat-breeders. These animals must be bred several times
before maintaining a pregnancy possibly due to delayed ovulation and(or) embryonic
death. Producers have adopted strategies to improve fertilization and early pregnancy
rates, one of the most common strategies is artificial insemination along with hormone
treatments to time luteolysis and ovulation. The administration of a GnRH analog at the
time of artificial insemination has been met with moderate success to improve conception
among repeat breeders (Hansel and Fortune, 1978; Lucy and Stevenson, 1986; Stevenson
et al., 1984). Reasons for improved fertility after GnRH treatment are not clear (Lucy and
Stevenson, 1986). However, GnRH administration after PGF2c, treatment may
synchronize the timing of ovulation by inducing LH secretion such that artificial
insemination is more successful.
Research has indicated that treatment of beef heifers with GnRH (Ford and
Stormshak, 1978; Rodger and Stormshak, 1986) as well as ewes (Slayden and Stormshak,
1990) causes an attenuation of luteal progesterone secretion. Lucy and Stevenson (1986)
hypothesized that the improved fertility may be associated with delayed or slowly rising
concentrations of luteal progesterone after ovulation. However, Mee et al. (1993)
administered GnRH at the time of artificial insemination of cows and found an increase in
serum progesterone concentrations later in the estrous cycle, along with improved fertility.
However, when CL were enucleated and incubated in vitro, CL from cows treated with
GnRH had lower progesterone production in response to LH challenge (Mee et al., 1993).
These authors hypothesized that GnRH may act on the developing CL to promote the
61
conversion of small to large luteal cells (Mee et al., 1993). These results were later
confirmed by the experiments of Bertrand and Stormshak (1994) who found that CL
incubated in vitro, after injection of GnRH on day 2, had a lower progesterone response to
LH than CL from saline-treated cows. The following experiments have been conducted to
further examine the effects that GnRH may have on luteal function as well as to elucidate
whether the actions of this decapeptide are mediated through the uterus.
62
EXPERIMENTS 1 AND 2: CORPUS LUTEUM FUNCTION IN
HYSTERECTOMIZED AND UNILATERALLY
HYSTERECTOMIZED EWES TREATED WITH GONADOTROPIN­
RELEASING HORMONE
INTRODUCTION
Treatment of ewes with GnRH during metestrus increased serum concentrations of
LH and lowered subsequent serum concentrations of progesterone (Slayden and
Stormshak, 1990). It was hypothesized that GnRH acted indirectly via LH to alter luteal
function because bovine, ovine and porcine ovaries lack GnRH receptors (Brown and
Reeves, 1983). Slayden and Stormshak (1990) confirmed the validity of this hypothesis
by demonstrating that LH injected into ewes during metestrus mimicked the GnRH­
induced attenuation of progesterone (P4) secretion. This hypothesis is also supported by
the earlier research of Rodger and Stormshak (1986) who found that treatment of beef
heifers with GnRH caused down-regulation of luteal LH receptors.
Uterine prostaglandin F20, (PGF20,) is the natural luteolysin in ewes (McCracken et
al., 1972; Goding, 1974). Luteolysis in nonpregnant ewes is promoted by secretion of
luteal oxytocin (OT) that acts upon the adjacent uterine horn to induce the release of
PGF20, (McCracken et al., 1984). Treatment of dairy heifers with GnRH 12 hours after
first detected estrus increased the number of large luteal cells, which are the source of OT
in the corpus luteum (Mee et al., 1993). Voss and Fortune (1991) reported that exposure
of cultured granulosa cells to increasing quantities of LH increased secretion of OT by
these cells. Thus, we hypothesized that GnRH-induced secretion of LH may cause the
developing corpus luteum (CL) to increase production of OT, which may in turn stimulate
uterine secretion of PGF20, in sufficient quantities to suppress luteal function.
63
The present experiments were conducted to determine whether response of the
ovine corpus luteum to exogenous GnRH differed between intact, unilaterally or
completely hysterectomized ewes. Changes in systemic serum concentrations of LH, P4,
plasma OT and utero-ovarian plasma OT and PGF2a were monitored after administration
of GnRH.
MATERIALS AND METHODS
Experiment 1
Sixteen mature crossbred ewes were checked twice daily for behavioral estrus with
vasectomized rams. Ewes exhibiting at least two consecutive estrous cycles of normal
duration were assigned randomly to four groups (n = 4) in a 22 factorial arrangement.
Treatments consisted of two levels of GnRH (0 and 100 i_tg/day) and two levels of
hysterectomy (none and UHYST).
Ewes were fasted for 48 hours prior to surgery; the last 24 hours were without
water. On day 12 of the estrous cycle (day of detected estrus = day 0) a midventral
laparotomy was performed; anesthesia for laparotomy was induced by an i.v. injection of
sodium thiamylal (Biotal 2.5%) and maintained by halothane-oxygen inhalation. Surgical
procedures were conducted under aseptic conditions and all ewes were unilaterally
ovariectomized, and when present, CL in the remaining ovary were enucleated. In eight
ewes the uterine horn, ipsilateral to the remaining ovary, and a portion of the uterine body
extending to the internal bifurcation were removed. The reproductive tract of the
remaining eight ewes was externalized and manipulated before wound closure. Ewes
exhibited behavioral estrus (x ± SE) 2.2 ± .4 d after surgery.
On each of days 2 and 3 of the subsequent cycle, one-half of the intact and onehalf of the UHYST ewes were injected i.v. with 100 lig GnRH (2 ml) while the remaining
intact and UHYST ewes were injected i.v. with saline (2 ml). Jugular blood (10 ml) was
64
collected from all ewes at 0 min (prior to injection) and 15, 30, 45 and 60 min postinjection for analysis of serum LH.
On day 4 of the cycle the caudal vena cava of each ewe was catheterized via the
lateral saphenous vein as described by Benoit and Dailey (1991). Anesthesia for the
saphenous vein catheterization consisted of an i.v. injection of sodium thiamylal to relax
the ewe, and a subcutaneous injection of 2% lidocaine administered at the point of
incision, approximately 5 cm dorsal to the hock and 3 cm lateral to the Achilles tendon.
Correct placement was determined by threading the catheter into the vein and collecting
blood samples at 2 cm intervals; serum was subsequently analyzed for serum
concentrations of P4 within 24 hours. The catheter tip location at which P4 concentrations
were at least three times that of the jugular sample was determined to be at the junction of
the utero-ovarian vein with caudal vena cava. The catheter was adjusted to the
appropriate position on day 5 after estrus. Patency of the catheter was maintained by
flushing with a sterile 3.5% sodium citrate-2% liquamycin solution after each blood
collection or once daily on non-sampling days.
Jugular and caudal vena cava blood samples were collected from all ewes on days
5, 6, 7, 8, 10, 12 and 14 after estrus for jugular serum P4 and plasma OT analysis, as well
as for vena cava plasma OT and PGF2a. Samples for analysis of serum LH and P4 (10 ml)
were collected via jugular venipuncture and stored for 24 hours at 4°C before sera were
separated by centrifugation. Jugular and vena cava blood samples (5 ml), for
determination of plasma OT were collected via heparinized vacutainer tubes, placed on ice
and treated with ethylenediaminetetraacetic acid (EDTA; .5 M, 10 pi) and 1,10­
phenanthroline (5 mg/m1 in ethanol, 5
to prevent oxytocinase activity (Kumaresan et
al., 1974). Similarly, vena cava blood samples collected for determination of plasma
PGF2a (5 ml) were placed on ice and 1,10-phenanthroline (5 mg/ml in ethanol, 5 1.4.1) was
added immediately to prevent PGF2a degradation. To obtain plasma, all blood samples
65
were processed immediately upon returning to the laboratory. All sera and plasma were
separated by centrifugation (500 x g) for 10 min at 4°C and stored at -20°C until analyzed.
Experiment 2
Twenty mature crossbred ewes were checked twice daily for behavioral estrus with
vasectomized rams. Ewes exhibiting at least two consecutive estrous cycles of normal
duration were assigned randomly to four groups (n = 5) in a 22 factorial arrangement.
Treatments consisted of two levels of GnRH (0 and 100 i..tg) and two levels of
hysterectomy (none and HYST).
On day 12 of the estrous cycle, all ewes were subjected to a midventral laparotomy
as described for Exp. 1. The reproductive tract was externalized and manipulated and
corpora lutea in both ovaries were enucleated. Ten ewes were HYST after ligating the
uterine arteries, uterine veins and placing a ligature at the utero-cervical junction. The
entire uterus (horns and body) was removed leaving the ovarian vasculature intact. Ewes
exhibited behavioral estrus (x ± SE) 3.6 ± 1.4 days after surgery.
On day 2 of the subsequent cycle one-half of the intact and one-half of the HYST
ewes were injected i.v. with 100 lig GnRH (2 ml) while the remaining intact and HYST
ewes were injected similarly with 2 ml sterile saline. Only a single injection of GnRH was
given in this experiment; a treatment regimen previously shown to be effective in altering
luteal function in cows and ewes (Rodger and Stormshak, 1986; Slayden and Stormshak,
1990).
Jugular blood samples were collected from all ewes on days 4, 6, 8, 10 and 12
after estrus to examine the effect of injected GnRH on serum concentrations of P4.
Samples for analysis of P4 (10 ml) were allowed to clot for 24 hours at 4°C. All samples
were centrifuged (500 x g) for 10 min at 4°C and the resulting sera were stored at -20°C
until assayed.
66
Radioimmunoassays
Radioimmunoassay for LH was performed according to the procedure of
McCarthy and Swanson (1976) with some modifications using ovine LH that was
iodinated by the chloramine-T method. Highly purified LH (LER-1056-C2
immunochemical grade ovine LH) was weighed and solubilized to a concentration of
10 1.1g LH/25 µl distilled water. Prior to iodination 25 Ill of .5 M sodium phosphate buffer
(PB) at pH 7.5 were added to the LH and mixed. Iodination was performed by the
addition 5 ill (.5 mCi) 1251 (IMS.30, Amersham Corporation, Arlington Heights, IL) and
5 pi, of chloramine-T (.2 mg/ml .05 M PB, pH 7.5) to the solubilized LH and the mixture
was then gently agitated for 60 seconds. The iodination reaction was terminated with the
addition of 20 1.1,1 sodium metabisulfate (.25 mg/mL .05 M PB, pH 7.5)
Separation of labeled hormone from free iodine was performed by filtration
through an anion exchange column consisting of a 3 ml plastic syringe with a small glass
wool plug, filled with Analytical Grade Anion Exchange Resin (AG 2-X8, 100-200 mesh,
chloride form, Bio-Rad Laboratories, Hercules, CA). The resin bed was equilibrated with
.5 M PB (pH 7.6) and coated with 2 ml of 5% bovine serum albumin (BSA) in .05 M PB
(pH 7.5) and collected into a 12 x 75 mm tube containing 1 ml .01 M-0.1% gelatin PB
(pH 7.5). To establish the concentration of purified labeled LH, 10 µl of the final solution
were counted. Concentrations of LH were expressed using ovine LH standards
(NIADDK-oLH-25).
All serum samples for LH were assayed in duplicate in a single assay. The
intraassay coefficient of variation was 9.8%. This was determined from aliquots of a
serum pool containing a concentration of LH that was near the midpoint of the standard
curve. Sensitivity of the assay was .5 ng/tube.
Sera were assayed for P4 in duplicate after hexane:benzene (2:1) extraction.
Progesterone RIA was performed on extracted samples following the procedure of
Koligian and Stormshak (1976). [311]-progesterone (4000 cpm, NET-381, New England
67
Nuclear, Boston, MA) was added to a third tube containing an aliquot of the sample to
determine and correct for procedural loss due to extraction. Anti-progesterone-11-BSA
antibody (provided by Dr. Gordon Niswender, Colorado State University) was utilized in
all the assays. The intra- and interassay coefficients of variation were 8.3% and 18.4%
(Exp. 1) and 7.5% and 6.5% (Exp. 2), respectively. The mean extraction efficiency for
samples of Exp. 1 and 2 was 96 + 4% and 91 ± 2%, respectively. Assay sensitivity was
10 pg/tube.
Oxytocin was extracted from plasma following the method of Schams et al. (1979)
using Waters Sep-pak C-18 cartridges (Schams, 1983). Plasma was assayed for OT by
RIA as described by Abdelgadir et al. (1987) using OT antibody generously provided by
Dr. Dieter Schams, Technical University of Munich, Freising-Weihenstephan, Germany.
Mean plasma OT extraction efficiency was 61 + 5% as determined by the addition of [3H]­
OT (4000 cpm, NEX-187, New England Nuclear). Oxytocin values determined by MA
were corrected for the added [311]-0T and losses due to extraction. Plasma sample
volume was 25 pl per tube and all samples were adjusted to a final volume of 200 1.11 with
assay buffer (.05 M sodium phosphate, 50 mM EDTA and .5 mg/ml gelatin). Intra- and
interassay coefficients of variation were 9% and 8.6%, respectively. Sensitivity of the
assay was .25 pg/tube.
Enzyme Immunoassay
Prostaglandin F20,, was analyzed using an enzyme immunoassay kit (EIA)
purchased from Cayman Chemical (Ann Arbor, MI). Plasma was purified by following the
procedure described in the Cayman Kit using Waters Sep-pak C-18 Cartridges. Mean
plasma extraction efficiency was 78 ± 2% as determined by the addition of [311]-PGF2c,
(4000 cpm, NET-433, New England Nuclear). Values for PGF2c, determined by EIA
were corrected for losses due to extraction. Intra- and interassay coefficients of variation
were 1.9% and 13.2%, respectively. Sensitivity of the assay was 1.2 pg/well.
68
Statistical Analysis
Data pertaining to the effects of treatment on serum and plasma concentrations of
LH, P4 and PGF2c, were analyzed separately using a multi-way repeated measures analysis
of variance. Because of heterogeneity of variance in the samples, data were subjected to
natural log transformations for analysis but are presented in this paper using the
nontransformed values. Plasma OT data were analyzed using the nontransformed data by
a multi-way repeated measures analysis of variance. Correlation analysis between jugular
and vena cava plasma levels of OT as well as individual day comparisons for plasma OT
were performed using Statgraphics (1991).
RESULTS
Administration of GnRH to ewes on day 2 (Fig. la) and 3 (Fig. lb) of the cycle
(Exp. 1) increased serum concentrations of LH on each day compared with those of
saline-treated ewes (P = .01). Secretion of LH in GnRH-treated animals differed between
days with the quantity of LH released on day 2 being greater than on day 3 of the cycle (P
= .05). On day 2, serum concentrations of LH were still increasing at 60 min postinjection of GnRH whereas on day 3, LH levels in GnRH-treated ewes were maximal by
15 min after injection and then plateaued.
Injection of GnRH failed to affect jugular plasma concentrations of OT in intact or
UHYST ewes on days 5 through 10 of the cycle (Fig. 2). However, (x t SE) jugular
plasma concentrations of OT (ng/ml) on days 12 and 14 were greater (P = .006) in all
intact (saline, 240.4 ± 23.2; GnRH, 195.2 ± 22) than in all UHYST animals (saline, 140.7
± 5.9; GnRH, 157.7 ± 9.8). Further, averaged over both days, jugular plasma levels of OT
in saline-treated intact ewes were significantly greater than GnRH-treated intact ewes
(P = .03).
69
Failure of the saphenous vein catheter occurred in a total of four ewes; two ewes
on day 5 (GnRH-treated intact and GnRH-treated UHYST), one ewe on day 10 (saline­
treated intact), and one ewe on day 12 (GnRH-treated UHYST). Failure was due to
either development of a blood clot or by the accidental removal of the catheter by the
animals. This resulted in a reduced number of animals used in the analysis of caudal vena
cava blood samples. Because of this, as well as the tremendous variation in vena cava
plasma OT samples, there were no significant differences detected among the treatment
groups in plasma OT concentrations (Fig. 3). However, it is noteworthy that ewes that
were UHYST and received GnRH had lower levels of vena cava plasma OT during the
terminal stages of the sampling period (days 12 to 14) than ewes in the other three
treatment groups. Over all sampling periods the concentrations of OT in vena cava
plasma were positively correlated with those in jugular plasma (r = .52, P < .0001).
Vena cava plasma PGF2a did not differ significantly among treatment groups (Fig.
4). However, vena cava plasma concentrations of PGF2a, were greater in GnRH-treated
intact ewes on days 10 through 14 compared with those of saline treated or all UHYST
ewes (P = .07).
Treatment with GnRH was without significant effect on serum concentrations of
P4 in Exp. 1. However, serum levels of this steroid were greater in UHYST ewes
compared with those in intact ewes (P = .09) regardless of whether they were treated with
GnRH (Fig. 5). In Exp. 2, ewes that were HYST and treated with GnRH produced less
P4 on days 4 through 12 of the cycle (P = .04) than HYST ewes injected with saline or
intact ewes treated with either GnRH or saline (Fig. 6). In both Exp. 1 and 2, UHYST
and HYST ewes, regardless of treatment, maintained CL beyond the expected time of
normal luteolysis.
70
DISCUSSION
Results of the present experiments failed to support the initial hypothesis that
exposure of the developing CL to increased quantities of LH would provoke increased
production of OT, which in turn would stimulate increased uterine production of PGF2a
and attenuate P4 secretion. Administration of GnRH on each of days 2 and 3 after
detected estrus did stimulate the release of LH. This response to GnRH was anticipated
because injection of the decapeptide into heifers (Lucy and Stevenson, 1986; Rodger and
Stormshak, 1986; Lamming and McLeod, 1988; Martin et al., 1990) and ewes (Nett et al.,
1981; Slayden and Stormshak, 1990) early in the estrous cycle has previously been shown
to provoke the release of LH. The quantity of LH released in response to GnRH was less
on day 3 then on day 2, which may be attributable to a down-regulation of the GnRH
receptors in the anterior pituitary or to depletion of releasable pituitary LH.
Overall, plasma OT concentrations in jugular and vena cava blood were similar.
However, in previous reports OT concentrations were found to be greater in ovarian than
systemic blood of cows (Schallenberger et al., 1984) and ewes (Sheldrick and Flint, 1983;
Hooper et al., 1986). Catheterization of the caudal vena cava was performed in this study,
and correct placement of the catheter was determined by the insertion distance at which P4
concentrations were greatest. The catheter was initially anchored with suture. However,
the position of the catheter may have changed during the 10 day sampling period due to
mobility of the ewe and(or) its interaction with other ewes in the pen. This may account
for the high variability of the vena cava plasma levels of OT among ewes. Regardless, the
highly significant positive correlation between vena cava and jugular plasma
concentrations of OT indicates that jugular sampling can be an accurate measure of
ovarian OT secretion.
Treatment with GnRH did not significantly affect secretion of jugular plasma OT
from days 5 to 10 of the cycle as determined by the measurement of plasma concentrations
71
of the neuropeptide. However, there are aspects of the secretory pattern of the jugular
plasma OT that are noteworthy. First, OT levels in intact ewes on days 12 and 14 were
significantly greater than in UHYST ewes. This may be due to lack of the local
interrelationship between the ovary bearing the CL and the adjacent uterine horn in
UHYST ewes, thus precluding PGF2,a stimulation of OT secretion that normally occurs in
intact ewes. Additionally, intact ewes treated with saline had greater plasma
concentrations of OT on days 12 and 14 than GnRH-treated intact ewes. This suggests
the possibility that release of OT occurred earlier in GnRH-treated ewes thus depleting
existing stores of luteal OT.
No significant differences in vena cava plasma PGF2c, concentrations were found
among treatment groups. However, intact ewes treated with GnRH had a higher
concentration of plasma PGF2c, than ewes of other treatment groups on days 10 to 14
after estrus. Because PGF2c, promotes the release of luteal OT these data are consistent
with the lower plasma concentrations of OT detected in these same animals during days 12
to 14 of the cycle.
It has been previously reported that injection of GnRH into beef heifers (Ford and
Stormshak, 1978; Rodger and Stormshak, 1986), as well as ewes (Slayden and
Stormshak, 1986), subsequently resulted in attenuated P4 secretion by the CL. However,
in this study injection of GnRH failed to significantly suppress luteal P4 secretion in either
intact or UHYST ewes. Nevertheless, serum concentrations of P4 in UHYST ewes were
greater (P = .09) than serum levels of P4 in the intact controls. Increased serum levels of
P4 in the UHYST ewes may be related to removal of the ipsilateral uterine horn, which is
the source of PGF2,,, that normally promotes luteolysis. This possibility is supported by
the research of Robinson et al. (1976) who reported that the major site of prostaglandin
synthesis in the ipsilateral nonpregnant uterine horn is the caruncles, removal of which
resulted in a significant increase in serum concentrations of P4 over that of intact controls.
It is also possible that the difference in P4 concentrations is the consequence of higher
72
ovulation rates due to compensatory hypertrophy of the remaining ovary, thus resulting in
more CL in the UHYST than in the intact ewes.
Experiment 2 was conducted to determine whether administration of GnRH to
HYST ewes would result in suppression of luteal function. Further, because UHYST
resulted in markedly increased serum levels of P4 it was deemed important to determine
whether total hysterectomy would have a similar effect on luteal function. As in Exp. 1,
an effect of GnRH alone on luteal function was not detected. However, in this experiment
serum levels of P4 in HYST ewes receiving GnRH were significantly less than in ewes of
the remaining three treatment groups. These data are consistent with those of Southee et
al. (1988) who hysterectomized anestrous ewes 2 weeks prior to P4-priming and then
treated ewes with low doses of GnRH. These authors found that while all ewes exhibited
behavioral estrus, serum P4 in hysterectomized animals was less than that in intact ewes,
and the CL were maintained for a prolonged period of time. This research suggests that
after hysterectomy, the CL is maintained but with a limited secretory capacity.
Data on serum concentrations of P4 in UHYST ewes in Exp. 1 and HYST ewes in
Exp. 2 cannot be directly compared. However, cursory inspection of these data suggest
that P4 levels in unilaterally ovariectomized-UHYST ewes are greater then in HYST ewes.
Whether this is due to the presence of one ovary or absence of one uterine horn is
unknown. To our knowledge no studies have been conducted in which luteal function in
UHYST and HYST ewes are compared. Such an experiment is warranted, the results of
which may shed additional light on the data of our experiments.
SUMMARY
The present research was conducted to further investigate the effects of GnRH on
luteal function in intact, partially hysterectomized and totally hysterectomized ewes.
Administration of GnRH during formation of the corpus luteum did not affect subsequent
73
luteal secretion of progesterone in control, partially hysterectomized or totally
hysterectomized ewes. However, luteal function was altered by the absence of the
adjacent uterine horn or the entire uterus.
74
35
O
g) 30
I ntact+Saline
I ntact+GnRH
±. 25
UHYST+Saline
UHYST+GnRH
2
20
2 15
cu
CV
5
cu
0
0
10 20 30 40 50 60
Time after injection (min)
35
O
cm 30
I ntact+Sal ine
I ntact+GnRH
±s 25­
UHYST+Saline
UHYST+GnRH
C
2E 20
a)
15
co"
=
10
5
0 0­
co
I­
0
10 20 30 40 50 60
Time after injection (min)
FIGURE 1. Jugular serum concentrations of LH in intact or
unilaterally hysterectomized (UHYST) ewes for 60 min after
i.v. treatment with either saline or GnRH (100 pg/d) on d 2 (A)
and 3 (B) of the cycle. Time 0 is just prior to injection.
75
O
I ntact+Saline
Intact+GnRH
UHYST+Saline
UHYST+GnRH
4
6
8
10
12
14
Day of estrous cycle
FIGURE 2. Jugular plasma concentrations of OT in intact
or unilaterally hysterectomized (UHYST) ewes treated with
either saline or GnRH (100 pg on d 2 and 3) over d 5 to 14
of the cycle.
76
300
O
280
E
Intact+Saline
Intact+GnRH
UHYST+Saline
UH ST+GnRH
260
(3)
c" 240
C­
T.')
5, 220
X
o
200
cu
6: 180
>
N
0
co
°w
>
160
140
120
100
4
i
I
I
1
6
8
10
12
i
14
Day of estrous cycle
FIGURE 3. Vena cava plasma concentrations of OT in intact
or unilaterally hysterectomized (UHYST) ewes, treated with
either saline or GnRH (100 pg on d 2 and 3) over d 5 to 14
of the cycle.
77
O
280
E
I ntact+Saline
Intact+GnRH
UHYST+Saline
UHYST+GnRH
240
er)
ci.
2 200
CD
a.
co
160
co
Q.
cu
2
co
c
a)
120
80
40
4
i
I
I
1
6
8
10
12
i
14
Day of estrous cycle
FIGURE 4. Vena cava plasma concentrations of PGF2c, in
intact or unilaterally hysterectomized (UHYST) ewes treated
with either sailine or GnRH (100 pg on d 2 and 3) over d 5 to
14 of the cycle.
78
3.5
O
o
3.0
Intact+Saline
Intact+GnRH
UHYST+Saline
UHYST+GnRH
2.5
2.0
1.5
1.0
0.5
0.0
4
6
8
10
12
14
Day of estrous cycle
FIGURE 5. Jugular serum concentrations of P4 in intact or
unilaterally hysterectomized (UHYST) ewes treated with
either saline or GnRH (100 erg on d 2 and 3) over d 5 to 14
of the cycle.
79
3.5
O
3.0
O
Intact+Saline
Intact+GnRH
HYST+Saline
HYST+GnRH
2.5
2.0
1.5
1.0
0.5
0.0
4
6
8
10
12
Day of estrous cycle
FIGURE 6. Jugular serum concentrations of P4 in intact or
hysterectomized (HYST) ewes treated with either saline
or GnRH (100 tag on d 2) over d 4 to 12 of the cycle.
80
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