Reproductive endocrinology
Gonadal development:
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Both the testes and the ovaries are derived from the same
gonadal primordium.
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There are two sets of ducts, the Wolfian duct and the Mullarian
duct.
Development of the primary sexual characteristics depends
directly on the endocrine environment during
development.
An individual can be forced into either a female
development or a male development by application of the
appropriate hormones, regardless of genetic makeup.
In the absence of hormonal stimulation, the gonadal
primordium will develop into ovaries and the Mullarian
ducts will develop into the uterine ducts, uterus and vagina.
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Development:

The sex organs themselves, along with all their
associated ducts and glands are referred to as the
Primary sexual characters
Secondary sexual characteristics are structures
which will enhance reproduction, but are not
necessarily required. For example, beard growth in
men.
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Without hormonal stimulation, the Wolffian
duct regresses.
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In males, the gonadal primordium begins to
secrete testosterone and Mullarian Inhibiting
Substance (MIS).
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Testosterone stimulates the development of the
Wolffian ducts, which subsequently differentiate
into the vas deferens, epididymis and seminal
vesicles.
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MIS causes the Mullarian ducts to degenerate
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Estradiol can prevent MIS from stimulating
Mullarian duct regression.
Testosterone is converted into
dihydrotestosterone (DHT) by the enzyme 5αreductase.
DHT influences the development of the external
genitalia.
 The genital tubercle becomes the penis.
 The genital folds become the shaft of the penis.
 The genital swellings become the scrotum.
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Without DHT, the external genitalia are feminized.
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The genital tubercle becomes the clitorus.
The genital folds become the labia minora.
The genital swelling becomes the labia majora.
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MIS is a 140 kDa glycoprotein in the TGF-B
superfamily.
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It activates a Serine-theonine receptor
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Circulating levels of androgens (and possibly estrogens)
also trigger differential development in the brain.
Animals exposed to androgens during a specific critical
window will develop male reproductive behavior,
regardless of the genotype or the physical phenotype.
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Development of secondary sexual characteristics:
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This usually coincides with the final maturation of the
gonads. In humans, this is referred to as puberty.
Mechanism controlling onset is unclear, but appears to
involve the loss of inhibition of gonadal development.
One potential candidate (at least in males) is melatonin.
During childhood, melatonin is produced in the pars
intermedia of the pituitary gland.
However, after childhood the pars intermedia stops
producing melatonin. Melatonin synthesis and secretion
are taken over by the pineal gland, but at a much
reduced rate.
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This drastic drop in melatonin secretion (>75%)
may trigger the secretion of sex steroids by the
adrenal glands and/or the testes.
In females, the situation may be different.
There is good evidence that the hormone leptin
is also involved.
Leptin is a hormone released by adipose tissue.
 Circulating leptin levels may reflect total body fat
storage by the body.
 In females, a certain minimum total-body fat content
is required for puberty to progress and for
maintenance of the menstrual cycle.
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Male reproductive system:
Spermatogenesis I:
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The immature germ cell in the male is referred to as the
spermatogonium.
These cells are located just under the basement membrane of
the seminiferous tubules, between adjoining sustentacular
(Sertoli) cells.
Since sperm production continues throughout adult life and at
the peak, 100-200 million sperm can be produced daily, the
spermatogonia are constantly renewed.
The first step in spermatogenesis is a mitotic division of the
spermatogonium. One of the daughter cells remains, to replace
the original spermatogonium, while the other cell (now called a
primary spermatocyte) undergoes meiosis.
Spermatid migration:
Spermatogenesis II:
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The first meiotic division yields two secondary spermatocytes. Usually, these secondary
spermatocytes do not fully separate during cell division, leaving a direct cytoplasmic
connection between the cells.
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Following the second meiotic division (again, an incomplete division), the cells are known
as spermatids. As the germ cells are undergoing meiosis, they also migrate towards the
lumen of the seminiferous tubule.
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As they approach the lumen, they shed much of their cytoplasm. They are attached to the
Sustentacular cells, via specialized junctions, which provide nutrients.
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When the spermatids reach the lumen, they remain embedded within the sustentacular cells,
where they undergo tail development, acrosome formation and nuclear condensation.
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Finally, the fully-formed spermatozoa are shed into the lumen of the seminiferous tubule,
where they are carried to the epididymus. This whole process takes between 60 and 70 days.
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The end of each ductus deferens (two) enlarges
to form ampullae, where sperm are stored until
ejaculation.
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The prostate contains the first part of the
urethra (prostatic urethra) which is where the
ejaculatory ducts merge with the urethra.
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The urethra exits the prostate, penetrated the
urogenital diaphragm and runs the length of the
penis.
Male sexual response:
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Erection
The first phase of the male
sexual response is erection of
the penis, which allows it to
penetrate the female vagina.
This occurs when the erectile
tissue of the penis becomes
engorged with blood.
When a male is not sexually
aroused, the arterioles
supplying the erectile tissues
are constricted.
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During sexual excitement, a
parasympathetic reflex is
triggered that causes these
arterioles to dilate (NO2).
As a result, the vascular spaces
of the penis fill with blood
causing the penis to become
enlarged and rigid.
Expansion of the penis also
compresses the veins retarding
the outflow of blood and
further contributing to the
swelling of the penis.
This reflex is initiated by a
variety of stimuli ranging from
thought to touch.
Ejaculation
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A spinal reflex is initiated, producing a sympathetic
discharge to the genital organs.
As a result, the reproductive ducts and accessory glands
contract peristaltically discharging their contents into
the urethra.
The muscles of the penis undergo a rapid series of
contractions propelling semen from the urethra.
This is followed by muscular and psychological
relaxation and vasoconstriction of the arterioles serving
the penis, allowing blood to drain out of the erectile
tissue, which subsequently causes the penis to become
flaccid again.
Role of the Accessory Glands:
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The seminal vesicles are paired glands that
produce about 60% of the semen.
Their secretions contain fructose sugar, ascorbic
acid and prostaglandins.
These are sac shaped glands, approximately 5
centimeters long, which lie along side the
ampullae of the ductus deferens.
They each empty into a short duct, the
ejaculatory duct, which merges with the terminal
end of the ductus deferens.
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These, in turn, fuse with the prostatic urethra which
runs from the bladder through the prostate gland.
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The alkalinity of the fluid serves to neutralize the
normally acidic environment in the distal urethra and in
the vagina.
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The fructose is supplied as an energy source for the
sperm, and the prostaglandins serve to stimulate
smooth muscle contractions in the vagina and cervix.
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This is thought to facilitate the uptake of sperm into the
uterus.
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The bulbourethral glands are paired glands that
secrete a small amount of thick clear mucus. This
secretion is released prior to ejaculation and is believed
to neutralize traces of acidic urine in the urethra.
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The prostate gland is a single gland, which secretes
about one third of the semen volume. It secretes a
milky, slightly acidic fluid containing citrate, acid
phosphatase and several proteolytic enzymes. These
enzymes are probably involved in breaking down the
mucus plug in the cervix. They also appear to
contribute to the motility and viability of the sperm
Semen Production
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Remember, Sperm + seminal fluid = semen.
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Semen provides a transport medium for the
sperm. It also provides nutrients for the sperm
and chemicals that protect them, activate them
and facilitate their movement.
The amount of semen released during
ejaculation is relatively small, about 2-6 ml but it
contains 50-100 million sperm per ml.
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The proteolytic enzymes are probably involved
in breaking down the mucus plug in the cervix.
They also appear to contribute to the motility
and viability of the sperm.
After ejaculation, SgI, SgII and fibronectin
aggregate to form a gelatinous mass, which is
believed to trap the spermatozoa within the
vagina.
Liquefaction occurs 5-20 minutes later, through
cleavage of the semenogelins by PSA prostatespecific antigen).
Brain-testicular axis:
Female reproductive system:
OOGENESIS I:
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This process is the equivalent of spermatogenesis in the
male. However, the two processes are vastly different.
In females, much of the process occurs during fetal
development.
The primitive germ cells undergo numerous rounds of
mitosis, which produces millions of oogonia (2n).
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Most of these oogonia are resorbed (through a process called
atresia).
However, a few hundred thousand begin meiosis and
enter prophase I. These are now referred to as primary
oocytes.
OOGENESIS II:
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There are no oogonia present in the adult female.
The primary oocytes are arrested in prophase I and
become quiescent until puberty.
Cyclical changes in LH and FSH will trigger three or
four primary oocytes to finish meiosis each uterine
cycle.
During the two meiotic divisions, all the cytoplasm will
stay with a single daughter cell, which is destined to
become the ovum.
The other three daughter cells simply develop as small
polar bodies that are eventually degraded and resorbed.
Fertilization and pregnancy:
Implantation:
Placental hormones:
During early
pregnancy, HCG is
secreted by the
syncitial trophoblasts.
Later, the placenta
secretes estradiol,
progesterone, relaxin
and somatomammotropin.
Function of placental hormones:
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HCG is similar to LH (and FSH and TSH)
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Maintains the corpus luteum in a functional state for 34 months.
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Shares the common alpha chain.
This keeps progesterone levels high and they maintain the
functional endometrium.
Progesterone keeps the uterine wall intact.
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Initially the progesterone comes from the corpus luteum in
the mother, but eventually the developing placenta secrets its
own progesterone.
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Estrogen initially comes from CL, but then is
secreted by placenta.
Estrogen softens pelvic ligaments to allow for
stretching of the birth canal during delivery.
Estrogen increases the sensitivity of the
myometrium to mechanical irritation, as well as
oxytocin stimulation.
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Stimulates the expression of oxytocin and
prostaglandin receptors.
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Relaxin is an insulin-like peptide.
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Relaxin increases flexibility in the pelvic joints.
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Synergistic with estrogen.
Suppressing release of oxytocin.
Relaxes smooth muscle of the uterus.
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Consists of two peptide chains joined with a
disulphide bridge.
This prevents uterine contractions.
Alters collagen in cervix to allow for the
stretching during delivery.
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Somatomammotropin is a protein hormone.
Acts like prolactin and triggers the mammary
glands to develop.
Stimulates development of mammary gland
tissue in preparation for lactation.
Also stimulates lipolysis in the mother.
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This mobilizes fatty acids for the embryo.
Also triggers elevated plasma glucose in mother,
by antagonistic action to insulin.
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Makes glucose available to embryo.
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Estrogen is stimulating expression of receptors
for oxytocin and prostaglandins.
Relaxin is inhibiting uterine contractions.
Thus, although the uterus is primed to respond
to oxytocin, early labour is inhibited.
Late in pregnancy, relaxin levels decrease and
the uterus becomes irritable.
Induction of labour is still not fully understood,
but is believed to be triggered by release of fetal
oxytocin.
Labour:
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Towards the end of
pregnancy, relaxin
secretion falls off, thus, the
uterus becomes more
sensitive to oxytocin.
Initially, the fetus secretes
oxytocin into the maternal
circulation.
The oxytocin stimulates
contractions, which push
the head down against the
cervix.
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This pressure on the
cervix stimulates the
release of oxytocin
from the maternal
pituitary gland.
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The maternal oxytocin
causes more
contractions of the
uterus, forcing the
head of the fetus
against the cervix even
harder.
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This is a positive
feedback system.
Nursing:
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Two hormones are
involved, PRL and
oxytocin.
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PRL stimulates milk
production, while
oxytocin is required for
the expression of milk
from the breast.
Comparative aspects of sex
determination:
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Mammals have genetically determined sex.
Mammals have an X and Y chromosome.
 Y chromosome confers male characteristics.
 XX – female:
Homogametic sex.
 XY – male:
Heterogametic sex.
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Homogametic sex is not always female.
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In birds and urodel amphibians: ZZ is male
ZW is female
Typically, the homogametic sex is the default
sex.
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The default sex is the one that will develop if NO
sex steroid exposure occurs.
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Chromosomally determined sexual differentiation is
referred to as genotypic sex determination (GSD).
Not all species with GSD have morphologically distinct
sex chromosomes.
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Temperature-dependent sex determination (TSD):
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Occurs in a number or reptiles, some teleosts and possibly in
some amphibians.
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Behavioral sex determination (BSD):
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Most often seen in coral reef fishes.
Usually controlled by social situations.
Usually seen is populations that have a well-defined social
hierarchy.
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Two major patterns are seen.
Protandry: individuals start as males, but change to
females.
 Protogyny: individuals start as female, but change to
male.
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Usually the change is determined by some
change is social status.
Most common pattern is protogyny.
 Dominant animal is male, most subordinate animals
and female.
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Reproduction in agnathans:
Gonad develops from embryonic cortex, regardless
of sex.
 Circulating levels of sex steroids are very low, due to
a lack of binding proteins.
 Male lampreys:
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Lobular polycystic testes.
 Only contain primary spermatocytes.
 Spermatocytes transform quickly into spawning sperm
masses.
 Interstitial cell masses (between lobules) store
cholesterol-positive lipids prior to spawning.
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Interstitial hydroxysteroid dehydrogenase (3b-HSD)
activity increases during spawning.
Female Lampreys:
Mature follicle rupture shortly before spawning and
collect in the coelom.
 Thecal cell shave elevated 3b-HSD activity and are
steroidogenic.
 Vitellogenesis is stimulated by estrogen.
 Vitellin and other vitellogenic proteins are synthesized in
the liver under the control of estrogen.
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Endocrine axis in lampreys appears to follow the
typical vertebrate plan.
Hagfish:
Very little is known about deep-sea hagfish.
 Believed to be able to spawn nor than one time,
unlike lampreys.
 Formation of “pre-ovulatory” corpora lutea and
post-ovulatory corpora lutea have been seen.
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However, the role of these structures is unknown.
 Don’t see sex change in agnathans; however, some
hermaphrodites occur. Usually due to incomplete sex
differentiation.
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Chondrichthyes:
See both oviparity and viviparity.
 All chondrichthyean fish have internal fertilization.
 Males:
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Paired testes of the cystic type.
 Sertoli cells are present but undergo cycling.
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Cells involved in steroidogenesis and sperm development are
resorbed after spawning. Next generation of Sertoli cells is
recruited from fibroblasts in the connective tissue surrounding
testes.
Sertoli cells have relatively high 3b-HSD activity.
There are nests of undifferentiated germ cells that proliferate and
give rise to spermatogonia each reproductive cycle.
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Females:
Ovary is covered with a germinal epithelium.
 May contain a large cavity.
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Cavity is derived from large lymph spaces.
As each oocyte matures, a layer of cells around it
differentiates into granulosa cells.
 The cells in the connective tissue covering the
oocyte will develop into thecal cells.
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Again, granulosa cells will develop into pre- and
post-ovulatory corpora lutea.
 Atresia of oocytes is common in selachians
 Estradiol stimulates vitellogenin synthesis and
oviduct growth (where development will occur in
viviparous species.
 In viviparous sharks, the ovaries produx a relaxinlike molecule that is very similar to mammalian
relaxin in function.
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Resembles insulin in form however.
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Teleost fishes:
In the bony fishes we see every possible
reproductive strategy.
 Teleosts arose in the devonian period, approx. 400
million ago.
 Some groups show GSD, some TSD and many
show BSD.
 Both internal and external fertilization patterns are
seen, along with oviparety and viviparety.
 Also see synchronous and asynchronous breeding
patterns.
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Testosterone
11-keto-testosterone
17,20 b-dihydroxy-4pregnen-3-one
Volume of sperm
produced
Gonadal-somatic
index
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Testosterone
Estradiol
17,20 b-dihydroxy-4pregnen-3-one
Plasma vitellin
gonadotropin
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Most teleosts have 2 forms of GnRH (1 and 2).
Most teleost fish have 2 gonadotrophs.
GTH I and GTH II, which don’t structurally
resemble LH or FSH.
 GTH I stimulates synthesis of estrogens.
 GTH II receptors are only found in the granulosa
cells of the follicles.
 GTH II stimulates production of 17,20 b-dihydroxy4-pregnen-3-one.
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This enzyme converts some steroids responsible for the
final oocyte amturatoin
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Females:
Oviparous and viviparous types.
 Ovaries are hollow – an adaptation to production of
many eggs.
 Anywhere from 10 – 10,000 at a time.
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Certainly true in oviparous fish.
 May not be quite so true for viviparous fish (for example
the ceolacanth).
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Mammalian GnRH and LH will induce ovulation.
 Mammalian FSH has no effect on ovulation.
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Males:
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Testes are of the cystic/lobular type.
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Cyclical spawners resume spermatogenesis immediately after
spawning.
Sustentacular cells have been identified in a large number of
species.
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Adapted for production of large volumes of sperm.
Have High 3 b HSD activity (produces androgens).
Major circulating androgen is 11 keto-testosterone.
In many teleosts, the testes lack interstitial cells.
There is a different cell type called the lobule boundary cells
which secrete some androgen.
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As leydig cells in mammals.
Treating each in turn: 3.) Sex ID is
permanent
Sequential Hermaphrodites:
Sex 1
Sex 2
RS
Small
Large
male
female
RS
Small
Large
Usually, this produces protogyny
(‘female-first’).
male
female
RS
Small
Large
Protogyny: Labroides dimidiata, a
cleaner fish on Australian Great
Barrier Reef
male
female
RS
Small
Large
Protogyny: Labroides dimidiata, male
displays suppress female sex change to
male. If he dies (or is removed
experimentally), largest female
changes.
male
female
RS
Small
Large
Protogyny: Thalassoma bifasciatum, blueheaded wrasse. Lek system with 2 male morphs.
Tiny ‘sneaker’ male (permanent). Others =
female till big enough to compete as dominant
lek males.
female
RS
male
Small
Large
Protandry: Coral reef fish (Amphiprion).
Monogamous pairs live under sea
anemone tentacles, joined by genderneutral immatures.
female
RS
male
Small
Large
Protandry: Coral reef fish (Amphiprion).
If resident female dies (or is experimentally
removed), partner switches to female and the
largest juvenile becomes male.
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Reproductive behaviors:
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Wide range of behaviors exhibited
Migration
 Courtship
 Nesting
 Parental care
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Both sexes can exhibit these behaviors
 Most behavioral work has been done on males
 Male behavior can be elicited by injection of
androgens.
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RE Peter, U of Alberta
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Male behaviors can be abolished by
gonadectomy.
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This also causes regression of secondary sexual
charateristics.
Courtship behavior:
Example: 3 spine stickleback.
 Normally the males are a non-descript brownishgreen colour dorsally, with a silver belly.
 During the breeding season, the male develops a red
belly.
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At the same time, the males become aggressively
territorial.
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If the males are castrated less than 1 week prior
to nest building, aggressiveness persists for 3-4
weeks.
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This is followed by nest building.
Suggests that territoriality and aggressiveness are not
directly controlled by androgens, but initiated by
them.
If castration is performed more than 1 week
before nest building, then all territoriality and
aggression is abolished.
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Diandretic species:
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As mentioned, have 2 types of males.
Primary phase males.
 Secondary phase males.
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In many species, the primary phase males are
determined genetically, while the secondary phase
are sex changed females.
 The trigger is unknown, but may be behavioral.
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Prolactin:
In fish, prolactin stimulates parental behavior.
 Usually is seen in controlling male behavior, rather
than female.
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This may be due to difficulty in assessing female behavior.
In Discus (Symphysodon nequifasciata), the males
secrete mucus which is then eaten by the fry.
 PRL appears to stimulate nest-building.
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As mentioned, circulating androgen levels
correlate to sperm production, but not
necessarily with spawning.
Some species will produce sperm in the fall, but
spawn in spring. (Several pacific salmon species do
this)
 Not yet known what triggers spawning behaviour.
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Although photoperiod may be involved, it is not the
trigger.
Parrot fish spawn every day at sunset (predator
avoidance)
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Seasonal spawners may also respond to
photoperiod.
Eg. Jenynsia lineata (a poecilid)
 Transplanted from South America to North
America.

Normal spawning in southern hemisphere: Jan.-Feb.
 Transplanted fish spawn in July-Aug.
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Reproduction in Amphibians:
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Amphibians have a more complex lifecycle than fish

Male Urodels:
Testes are of the cystic type.
 Structurally very similar to teleosts.
 Germinal epithelia is divided into lobes, then
ampullae, then germinal cysts.
 All the spermatogonia in a cyst mature
synchronously.
 Sertoli cells are recruited from fibroblasts each
breeding season.
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Urodel Testicular Structure
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As in teleosts, the cells
surrounding each lobe consists
of lobule boundary cells.

These cells undergo seasonal
lipid accumulation cycles.
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Lobule boundary cells and
Sertoli cells have 3b-HSD
activity, indicating
steroidogenesis.

Both are probably influenced by
gonadotropins.
Unlike teleosts, the principle androgen is
testosterone (not 11-keto-testosterone).
 Many are dissociated breeders.
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Androgen levels are low during breeding, but high during
testicular development. Sperm are stored until breeding.
In species where secondary sexual characteristics
develop, androgens are essential for development.
However, androgens alone are not enough.
 In the newt N. viridescens, nuptial pads develop in response
to testosterone and PRL.
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In addition, several skin glands (which secrete
pheromones) are stimulated by both hormones.

Unlike teleosts, it is the males that emit pheromones.

Complex courtship and mating displays.

In both male and female urodels, PRL stimulates
migratory behavior known as the “water drive”.
Testosterone primes the CNS for mating behavior.
 After priming, argenine vasotocin and GnRH act
synergistically to stimulate clasping behavior.
 Pheromones from hedonic and cloacal glands
stimulate courtship behavior in females.
 In some species there are “chin” glands which the
male rubs over the female. The secretions are
believed to contain pheromones which make the
female receptive.
 Abdominal glands release female attractants into the
water stream in front of the female
 In some species there may also be airborne cues
released by the females that attract the males.
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Male anurans:

Unlike urodels, the anuran testes is structurally like
the testes of the higher vertebrates.
They consist of a homogeneous mass of semeniferous
tubules.
 Contain significant numbers of interstitial cells.
 Interstitial cells have seasonal lipid cycles and have
seasonal cycles of 3b-HSD activity.
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In Bufonid toads, both sexes have ovarian tissue. In
the males, the rudimentary ovaian tissue is called the
Bidders organ

Bidder’s organ is composed of small oocytes.

However, these oocytes never reach the vitellogenic stage
and never mature.
Bidder’s organ undergoes seasonal regression and
recrudescence correlated with the testicular cycle.
 They appear to be steroidogenic as indicated by the
elevated 3b-HSD levels.
 After castration, Bidder’s organ will hypertrophy, but
never becomes a functional ovary.
 Some frog species do undergo sex reversal.
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Male Apodans:
Very little is known about apodan reproduction.
 Apodans don’t reproduce in water.

Internal fertilization and viviparity.
 Males have an intromittent organ.
 Males have retained the lower portion of the Mullerian
duct, modified into Mullerian glands, which functions
somewhat as a prostate, producing seminal fluid.


Female Anurans and Urodels:
Ovaries are hollow saclike structures.
 Layer of germinal epithelium composed of granulosa
cells with nests of oocytes.
 There is a very thin layer of thecal cells around each
follicle.
 Granulosa cells contain high levels of 3b-HSD.
 Corpora lutea form in both oviparous and
viviparous species; however, no endocrine role has
been identified in oviparous species.


Fat bodies:
Generally speaking, amphibians invest more in
vitellogenesis.
 Like the teleosts, the liver is the main site of
vitellogenic protein synthesis (stimulated by
estrogen).
 However, the fat body is involved in processing the
lips that will be incorporated in the yolks.


Most amphibians are oviparous and have
external fertilization.

Two species of European salamanders have true
viviparous breeding.
Young develop in the posterior portion if the
oviduct.
 Undergo metamorphosis in utero during a 4 year
gestation period.


Viviparity in urodels is not true viviparity.
Usually involves development of a pouch which
allows development of the eggs on the body of the
adult.
 Marsupial frogs
 Swallowing frogs
 Oviductal incubation.



Viviparity is common in Apodans
In all 3 groups there is a tendency to reduce the
dependency on water.
Midwife toad
Suriman toad
Reproduction in Reptiles:



The order is fairly diverse, so generalizations are
difficult to make.
Also, little work has been done.
Different types of sex determination.
Genetic sex determination.
 Temperature-dependent sex determination.
 Although there are some parthenogenic species,
behavioral sex determination is not seen.




For all reptiles, fertilization is internal.
Males possess an intromittent organ for
copulation.
Males:

Testes structure is similar to mammals.
This form is common to all amniotes.
 Consists of tightly coiled seminiferous tubules.
 Tubules are composed of germ cells and Sertoli cells.
 Prominent interstitial cells which are believed to secret
androgens.
 Androgens are responsible for development of secondary
sexual characteristics.


Females:
Have paired ovaries which are hollow.
 Generally speaking, reptiles lay many fewer eggs than
amphibians, but these eggs are heavily yolked.


Mainly because the young must emerge fully developed.
Follicle is differentiated into an internal thecal layer,
the cells of which are granular in appearance, and a
fibrous theca externa.
 Granulosa cells are present but do not appear to be
involved in steroid secretion.
 It is the thecal cells which appear to secrete estrogen.


Males:
Testes is the same as all other amniotes, a tightly
coiled mass of seminiferous tubules.
 The rest of the ductal system is basically the same as
seen in the mammals.
 In sexually mature squamates, a portion of the
kidney tubules undergo a hypertrophic modification.

Sperm are stored in these modified tubules.
 Epithelial cells secrete a fluid which helps maintain the
stored sperm until mating occurs.




Crocodilians and turtles produce two distinct
gonadotropins, while the squamata produce only
and FSH-like hormone.
The hypothalamus produces GnRH which
controls the release of gonadotropins by the
pituitary.
As in amphibians, there is a distinct fat boby
that appears to undergo lipid cycling in
synchrony with oogenesis and vitellogenesis.


Most reptile show very little in the way of
parental behavior, although there are some
notable exceptions.
Many crocodilians tend the nest and transport
the hatching young to the water after
emergence.

The mother stays with the young and protects them
for a significant time period.


Many secondary sexual characteristics and
reproductive behaviors are stimulated by a
combination of androgens and PRL.
In Anolis carolinensis have a moderately complex
mating pattern.
Development of a dewlap, under the chin, is
stimulated by androgens.
 Induction of the red and black colouration is
stimulated by PRL
 The characteristic black eyespots and dewlap spot is
intensified by epinephrine.


Territoriality is declared by vigorous head
bobbing, with extension of the dewlap.
In terretoriality displays against other males,
circulating EPI levels are elevated.
 This EPI causes the dewlap spot and the eyespot to
darken.


In courtship behavior, head bobbing and dewlap
displays are also involved. However, EPI is not
elevated, therefore the black spots are fainter.
Reproduction in Birds:




Avian reproduction is on of extremes.
All the different aspects of reproduction are
greatly modified in consideration of weight
reduction.
All birds are oviparous. Eggs are heavy and
cannot be carried internally for long.
Due to complex nature of the avian lifestyle,
significant parental investment is the norm.



By necessity, birds lay many fewer eggs than
reptiles (again, eggs are heavy).
Because of this, birds invest in parental care to
maximize the survival of so few eggs.
Major concessions are made in the reproductive
organs in order to conserve weight.

Most birds are migratory to some extent, so lugging
around fully developed gonads is metabolically
foolhardy.

Ovarian development:

Only one (in most species the right) develops.
During the migration of the germ cells, distribution is
asymmetric. One primordial ovary has more germ cells
than the other.
 The ovarian cells start to secrete MIF.
 However, the larger of the two ovaries (the one with the
greater number of germ cells) secretes estrogen into the
oviduct on that side.
 This estrogen desensitizes the oviduct (a Mullerian duct
derivative) and it will not degenerate.
 However, the contra lateral oviduct regresses and the
smaller ovary is resorbed.


Gonadal cycle in males:
In all birds, male or female, there is a marked
gonadal cycle.
 In males, this cycle is nothing less than dramatic.
 Between breeding seasons, the testes almost
completely break down.


In extreme cases, the regressed testes are 500 times
smaller than the fully developed testes.



Seasonal testicular development is called
recrudescence.
There are three phases to the testicular cycle.
Photoperiod strongly controls the testicular
cycle in most species.

Preparatory phase:
Begins immediately after the reproductive period.
 Marked by an almost total collapse of the testes.
 Males in this phase are insensitive to photoperiodic cues.



Referred to as photo-refractory.
Testes are little more than sacs of connective tissue with
interstitial cells.
The end if the preparatory phase is marked by the
onset of photosensitivity.
 The second phase is called the progressive phase.

Lengthening photoperiod initiates an increase in activity
of the hypothalamic-hypophyseal-testicular axis, resulting
in increased gonadotropin secretion.
 The endocrine basis for photorefractivity is not
understood, nor is the induction of photosensitivity.
 Some evidence suggests that the testosterone surge seen
during breeding feeds back on the hypothalamus, causing
prolonged suppression of LH.
 During this phase, there is marked spermatogenesis and
increased androgen secretion by interstitial cells.

Recrudescing testis
Maturing Ovary

During this phase, there is increased PRL
secretion and there is induction of secondary
sexual characteristics and reproductive behavior.




For example, territorial displays occur, singing (in
songbirds) increases, courtship behavior is induced,
etc.
There is increased conversion of steroid in the
brain (increased aromatase activity can be seen.
Brood patches begin to develop.
Long periods of cold temperature can block or
delay the progressive phase.
Aromatase Distribution in Brain
Brooding

Third phase is the culmination phase.
This coincides with ovulation in the females.
 Usually, the male is ready slightly before the female.
 Ovulation is probably induced by behavioral cues
and/or male-female interactions.
 Spermiation (release of sperm from testes) can occur
once, or several times in rapid succession.
 Upon completion of the culmination phase, the
testes undergo the collapse associated with the
preparatory phase onset.


Interstitial cells:
During the winter, the interstitial cells are small and
contain little lipid.
 Proportionally, they make up a large amount of the
testicular volume, simply because the seminiferous
tubules are so regressed.
 During the progressive stage, they become more
lipoidal and start to express aromatase activity,
reflecting the increase in androgen secretion.
 At the end of spermeation, there is a rapid depletion
in lipid stores.

Also at the end of spermeation, there is massive
autolysis of the interstitial cells, resulting in an
almost complete die off.
 New interstitial cells are recruited from fibroblasts.




Recruitment and hyperplasia are stimulated by LH.
Differentiation occurs early on and then the cells lie
dormant until the next preparatory phase.
Sertoli cells:
Do not turn over like the interstitial cells.
 Have a marked lipid cycle.


Become densely lipoidal at the end of breeding and store
the lipid until the next round of spermatogenesis.
The tremendous growth in the testes results in
damage to the tunica albuginea.
 After each reproductive season, fibroblasts are
recruited to build a new tunica albuginea.


Female reproductive cycle:
Ovarian cycle is not as marked as testicular cycle.
 There is seasonal regression of the oviducts and
assorted glands.
 Increased estrogen stimulates vitellogenesis.
 Estrogen also mobilizes calcium.



Follicular structure is similar to mammals.
However, after ovulation, the ruptured follicle
does not reorganize into a corpus luteum.
This is probably associated with the total absence of
viviparity.
 The remaining granulosa cells do secrete
progesterone, which seems to be associated with
regulating movement of the egg through the oviduct
and shell gland.
 FSH stimulates follicular development, as in
mammals, while LH stimulates ovulation.


Androgens in both sexes:
Androgens are important in females as well as males.
 Generally, if there is a change in plumage associated
with reproduction (not always restricted to the
males), that change is stimulated by androgens.
 Androgens inhibit brood patch formation (helps to
tightly regulate timing.
 Aggression and territoriality in males is usually
controlled by androgens, but can also be stimulated
by FSH (or a combination).


Thyroid function in reproduction:
Most birds migrate to reproduce.
 In preparation for migration, there is a spike in
thyroid hormone levels.



This occurs twice, there is also a spike before the
migration to the ‘wintering’ grounds.
This first spike is also required in order to support
the rapid mobilization of reserves seen in
reproduction (birds have very condensed
reproductive seasons generally).
Control of Gonadotropin Releasing Hormone
(GnRH) by the hypothalamus.

Release is pulsatile in all mammals looked at (probably
true for all vertebrates).

Disruption of pulsatile secretion is associated with
reproductive disorders in humans.

In humans there is only one form of GnRH. Often
referred to as LHRH due to sequence homology with
one of the two GnRHs found in other vertebrates.
In Rhesus monkeys



There are about 2000 neurons in hypothalamus
that contain LHRH.
Early studies have suggested that the pulse
generator is physically located in the medialbasal hypothalamus.
Critical experiment
complete deafferentation of that region does not
block pulses.

Cells taken from this region and put into cell
culture show pulsatile release of GnRH.

Pulses are also seen in cultured basal
hypothalamus both rat and guinea pig.

Electrophysiological evidence also supports the
idea that the pulse generator is from this region.

Volleys of action potentials (extracellular electrodes)
have been recorded in this region and these volleys
coincide with the pulses of GnRH.

These volleys have only been observed in the basal
hypothalamus and nowhere else, suggesting that the
pulses do not originate from outside the basal
hypothalamus.

Pulsatile LHRH release is seen in tissue fragments of
median eminence from the rat.


This region doesn’t have the cell bodies, suggesting that the
pulses are initiated in the axons of the LHRH neurons, not
up at the cell bodies.
This suggests that other signals must influence LHRH
release.

One candidate in Neuropeptide Y (NPY).

NPY has been shown to stimulate LHRH release from
median eminence fragments in culture.

NPY is also released in a pulsatile fashion in the stalk
median eminence of the rhesus monkey and these
pulses appear to be synchronous with LHRH pulses.

Note, this is not proof that the NPY is causing LHRH
release.

There are other possible candidates.

γ aminobutyric acid (GABA), dopamine and
Nitric Oxide (NO) are also found in the same
area and all seem to be released in pulses.
Focus on the endogenous pulse generator.

LHRH neurons appear to have intrinsic pacemaker
activity

Some studies have been done on the GT-1 cell line, a
mouse line that expresses the mouse GnRH transcript.

GT-1 cells release LHRH in pulses with intervals of
about 22-30 minutes.

These intervals are similar to the intervals seen in the
mouse in vivo.

This interval is not the same as seen in primates.

Primate LHRH neurons in culture also release
LHRH in pulses.

Interval of around 43-44 minutes.

LHRH neurons from 2 sources in rhesus brain
had same interval.

This suggests that there is an endogenous frequency.

Frequency in cultured cells is approximately the same as
in adult animals in vivo.

In cultured LHRH neurons, electrophysiological studies
have demonstrated that they show oscillatory bursting
activity (i.e. trains of action potential).

However, it is not know for certain if the APs are
directly associated with LHRH release.

In addition to the electrical activity, cultured
LHRH neurons show oscillatory Ca2+ activity.

When cultured individually, these neurons all
have individual frequencies.

However, when cultured together they
synchronize their Ca2+ oscillations.

The synchronous activity has a frequency similar
to the in vivo frequency.




A similar system of activity has been shown to
correlate to insulin release in β cells.
However, a link still needs to be established in
LHRH neurons.
Mechanism of communication between LHRH
neurons:
Possibilities:
Some sort of synaptic transmission?
 Electrical coupling (i.e. gap junctions)?
 Some diffusible messenger (i.e. paracrine signaling)?


GT-1 cells have been demonstrated to form
both synapses and gap junctions in culture.

However, it has been shown that LHRH
neurons grown on separate coverslips (no
physical contact) also synchronize.

Thus, we can rule out synaptic transmission and
gap junctions as potential methods of cell
communication.

This leaves paracrine signaling.

NO synthase mRNA has been found in GT-1
cells.

NO would be an ideal candidate.
Small molecule.
 Diffuses rapidly.
 Highly soluble.
 Penetrates membranes easily.

In LHRH neurons:

Show characteristics of neuroendocrine cells.

Depolarization causes release of LHRH
(induced depolarization).

Depolarization also causes Ca2+ oscillations.
In GT-1 cells:

Depolarization by high extracellular K+ causes LHRH
release.

Treatment with Veratridine (VG Na+ channel opener)
causes LHRH release.

Both these treatments also cause LHRH release from
cultured fetal rhesus monkey LHRH neurons.

Na+ channel blockers (i.e. tetrodotoxin) will block
LHRH release.

LHRH release is dependent on Ca2+ influx.

Suggests the possibility that Ca2+ influx may be
involved in the pulse generation (rather than
being a result of pulse generation).

L-type Ca2+ channels have been found in GT-1
cells as well as monkey LHRH cells.
This is a voltage-gated Ca2+ channel.
 Characterized by nifedipine blockade, but not
blockade by ώ conotoxin. Also, they are activated by
Bay-K8644.


Bay-K8644 does stimulate LHRH release in both cell systems.

L-type channels have been shown to be involved in Ca2+activated Ca2+ influx.

Ca2+-activated Ca2+ influx has been identified in GT-1 cells.

Treatment with thapsigargan or cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP; induces Ca2+ release from
mitochondria) increased [Ca2+]i and induced oscillations.

Similar treatments (i.e. thapsigargan or ryanodine) induced
calcium oscillations in fetal monkey LHRH neurons.

Other potential influences:

Neuropeptide Y (NPY)

36 aa peptide
These studies were done in vivo on monkeys, using
the push-pull cannula technique.
Perfusate
In
Under pressure
Effluent out
Skull
Push-pull cannula
Doublebarreled
cannula
Perfusate percolates
Through interstitial space

Infusion (8 hour) of antisense oligonucleotide
against NPY blocks both the NPY pulses and
LHRH pulses.

Infusion of anti-NPY antisera will also block
both LHRH and NPY pulses.

Thus, good evidence that NPY is playing a role
in at least regulating the LHRH pulses, but is it
the only factor?

Norepinephrine (NE) may also be involved.

There are NE containing neurons in the same region of
the stalk median eminence.

Adrenergic input has been shown to modulate LH and
LHRH release.

LH pulses (from pituitary) and intermittent bursts of multiple
unit activity (in ME) occur in close association with LHRH
pulses and can be suppressed with α-adrenergic blockers
phentolamine and prazosin (α1-specific).

Push-pull cannula results show that release of
NE from stalk-ME is pulsatile and the pulses are
synchronous with LHRH pulses.

Direct infusion of NE, or α-adrenergic agonists,
into stalk-ME stimulates release of LHRH.

The α1-blocker prazosin reduces pulse
amplitude, but not pulse frequency.

β- and α2- antagonists have no effect.

Infusion of NE into stalk-ME also stimulates
the release of prostaglandin E2 (PGE2).

PGE2 infusion will also stimulate LHRH
release.

Thus, NE may work directly, or through PGE2 ,
or both.

Organ culture on ME fragments has shown that
NE effects are mediated through PGE2 .

Interactions between NPY and NE/ PGE2

NPY infused into stalk-ME of prazosin-treated
monkeys stimulated LHRH release.

This suggests that NPY is working
independently of NE/ PGE2.

Also, α1- adrenergic stimulation with
methoxyamine in monkeys treated with
antisense oligonucleotide for NPY still had
LHRH release suggesting that NE effects are
not working through NPY neurons.

Thus, it appears that NPY and NE are working
independently to modulate LHRH release.
γ-aminobutyric acid (GABA)

This is the major inhibitory neurotransmitter in
the CNS and it is found in the hypothalamus.

Early studies, involving infusion of GABA into
the CSF in the third ventricle, stimulated LHRH
release

This was also observed in explants of
hypothalamic tissue kept in culture.

However, other studies were contradictory.

A more recent observation is that GABA may change
in activity with changing age or developmental status.

In rats, GABA appears to stimulate LHRH release in
juveniles, but becomes inhibitory at puberty.

More recently, studies of GT-1 cells and LHRH
neurons (from mouse olfactory placode) show that
GABA actually stimulates LHRH release, as well as
increasing intracellular Ca2+ oscillations and membrane
potentials (depolarization).

In monkeys (using the push-pull cannula)…

GABA tonically inhibits LHRH release before
puberty.

GABA levels in ME are much higher in prepubertal animals and levels drop by mid-puberty

Another neurotransmitter found in the ME region is
glutamate.

It is an agonist of the excitatory amino acid system,
working on NMDA receptors.

It has also been shown to stimulate LHRH release.

Stimulation of N-methyl-D-aspartate (NMDA)
receptors can induce precocious puberty in rats.

There are potential interactions between GABA and
glutamate.

GABA is synthesized from glutamate.

This raises the possibility that GABA and glutamate
levels may be related.

Infusion of antisense oligonucleotides for enzymes
involved in the synthesis of GABA from glutamate
cause an increase in LHRH in pre-pubertal monkeys.

i.e. when GABA synthesis was inhibited, LHRH levels
rose.

Was this due to a local increase of glutamate,
once GABA synthesis was stopped?

Or, did reduction of GABA simply unmask an
ongoing glutamate stimulation?

Several studies suggest the former.

In pre-pubertal monkeys, the glutamate levels in
the stalk ME are very low.

When GABA synthesis is blocked, these levels
rise.

However, there are studies that suggest the
former may also be occurring.

The probability is that both occur to some
extent (and there may be variability between
species).


As mentioned before, NO may be playing a role and is an ideal
candidate.

Pulsatile LHRH release occurs even when the cell bodies are not present.

This suggests some form of pre-synaptic stimulation at the
neuroterminals of the LHRH neurons.

Histological studies have failed to show the presence of pre-synaptic
synapses in the right area of the hypothalamus.

Similar results are seen in cultures cells. Cells that are physically
separated, yet cultured in the same dish, show synchrony of LHRH pulses
and Ca2+ oscillations.
This is strong evidence that there is a chemical mediator
(i.e.some kind of paracrine signaling), which coordinates the
LHRH release.

In push-pull cannula experiments, infusion of the NO
precursor L-argenine stimulated both NPY and LHRH.

Infusion of D-argenine had no effect (the dextrorotary
form cannot be converted into NO).

The enzymes involved in NO synthesis are present in
an adjacent area of the hypothalamus.

This means that NO is available in the area in question.

Finally, glial cells may also be playing a role.

In other systems, glial cells have been shown to
modify, or affect the release of neurotransmitters
and neurohormones.

In this system, circumstantial evidence suggests
this possibility.

The endogenous pacemaker seems to be located in,
or near the neuroterminals of the LHRH cells (and
not near the cells bodies).

No pre-synaptic synaptic connections have been
identified in the area.

Glial cells ARE present around the neuroterminals.

It is known that glial cells play an important role in
regulating release of the hormones of the pars nervosa, an
analogous system to that of the stalk ME.

The proposed mechanism for this interaction is that
glial cells may release the kallekrein bradykinin.

Bradykinin, in turn, would stimulate glutamate release
from astrocytes located around the neuroterminals.

This would have an effect on LHRH release from the
neuroterminals themselves.
NPY
NE
LHRH
GABA
GABA
Glu
Glu
NO
Master
Pacemaker?
GABA
Glia
Glia
Portal blood
Have a good summer!