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Am J Physiol Endocrinol Metab 302: E4–E18, 2012.
First published October 25, 2011; doi:10.1152/ajpendo.00488.2011.
Perspectives
Hirsutism, virilism, polycystic ovarian disease, and the
steroid-gonadotropin-feedback system: a career retrospective
Virendra B. Mahesh
Department of Physiology and Endocrinology, Georgia Health Sciences University, Augusta Georgia
Submitted 19 September 2011; accepted in final form 20 October 2011
estrogens; androgens; progesterone
IN THIS CAREER RETROSPECTIVE ARTICLE,
the author describes
briefly how he started working in the field of steroid hormones,
which led to work in the area of hirsutism, virilism, and
polycystic ovarian disease. These initial studies led to a number of interlocking questions resulting in a detailed study of the
steroid-gonadotropin feedback system, leading into a lifelong
career of scientific investigation from 1956 to 2010. The
approach for solving the questions generated was inspired by
the literature at the time the research was done as well as the
incorporation of newer techniques as they developed. This
article covers an extensive area of reproductive biology, and it
is not possible to refer to all the literature surrounding the
Address for reprint requests and other correspondence: V. B. Mahesh, Dept.
of Physiology and Endocrinology, Georgia Health Sciences University, 1120
15th St., Augusta Georgia 30912 (E-mail: [email protected]).
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research due to the breadth of the area covered. However, it is
fully referenced in the articles cited and the periodic reviews on
the subject.
In the mid 1950s, there was no clear understanding of how
steroid hormones exerted their biological action. In the area of
glucocorticoids, based on the metabolism of cortisol (11␤,17␣,21trihydroxy-4-pregnen-3,20-dione) as well as the effectiveness of
cortisol compared with cortisone (17␣,21-dihydroxy-4-pregnen-3,11,20-trione) in tissues in which the conversion of cortisone to cortisol did not take place, it was postulated that
cortisol might be the active hormone (51, 82, 183). On the
other hand, working with placental isocitric dehydrogenase in
vitro, Talalay and Williams-Ashman in 1958 (212) explained
the action of estradiol as a coenzyme in a redox system. To
investigate whether a hormone was required to participate in an
oxidation-reduction cascade to exert biological action, Bush
and Mahesh (52) studied the factors that governed the reduction of the 11-keto group to the 11␤-hydroxyl group in corticosteroids. It was postulated that since the ␣-side of the
molecule is flat in the 3-keto-4-ene and 3␣,5␣-reduced steroids, the enzyme involved in reducing the 11-keto group could
approach the steroid from the ␣-side with ease and reduce it to
the 11␤-hydroxyl group. On the contrary, in the 3␣,5␤-steroids, Ring A of the steroid is at a right angle to the rest of the
molecule and thus would provide steric hindrance in reduction
at the 11-position. Thus, the synthesis 3␣,11␤-dihydroxy-5␣androstan-17-one; 3␣-hydroxy-5␣-androstan-11, 17-dione;
3␣,11␤-dihydroxy-5␤-androstan-17-one; 3␣-hydroxy-5␤-androstan-11,17-dione, and 4-androsten-3,11,17-trione was carried out and their metabolism studied in the human. The results
clearly showed the reduction of the 11-ketone to the 11hydroxyl compounds in 3-keto-4-ene and 3␣,5␣-steroids but
not in the 3␣,5␤-steroids. To slow down the metabolism of
cortisol in Ring A of the steroid, 2␣-methyl-cortisol and
2␣-methyl-cortisone were synthesized and tested for biological
activity (70). The 2␣-methyl-cortisol was found to be very
active biologically and the 2␣-methyl-cortisone had very little
biological activity. Based on the structural requirements for
reduction of the 11-ketone to the 11-hydroxyl group as determined by Bush and Mahesh (52), the 2␣-methyl group would
provide enough steric hindrance to prevent reduction of the
11-ketone to the 11␤-hydroxyl group. This was verified experimentally by Bush and Mahesh (53), leading to the conclusion
that the 11␤-hydroxyl group of cortisol did not have to undergo
any metabolism for the exertion of biological activity. The
higher biological activity of 9␣-fluoro steroids was also explained, in part, due to a greater persistence of the 11␤hydroxyl group during metabolism (55). Jensen also provided
proof that estradiol exerted its biological activity without being
converted to estrone (102). Further examination of the metabolism of cortisol in various rat tissues identified the kidney as
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Mahesh VB. Hirsutism, virilism, polycystic ovarian disease, and
the steroid-gonadotropin-feedback system: a career retrospective. Am
J Physiol Endocrinol Metab 302: E4 –E18, 2012. First published
October 25, 2011; doi:10.1152/ajpendo.00488.2011.—This career retrospective describes how the initial work on the mechanism of
hormone action provided the tools for the study of hirsutism, virilism,
and polycystic ovarian disease. After excessive ovarian and or adrenal
androgen secretion in polycystic ovarian disease had been established,
the question whether the disease was genetic or acquired, methods to
manage hirsutism and methods for the induction of ovulation were
addressed. Recognizing that steroid gonadotropin feedback was an
important regulatory factor, initial studies were done on the secretion
of LH and FSH in the ovulatory cycle. This was followed by the study
of basic mechanisms of steroid-gonadotropin feedback system, using
castration and steroid replacement and the events surrounding the
natural onset of puberty. Studies in ovariectomized rats showed that
progesterone was a pivotal enhancer of estrogen-induced gonadotropin release, thus accounting for the preovulatory gonadotropin surge.
The effects of progesterone were manifested by depletion of the
occupied estrogen receptors of the anterior pituitary, release of hypothalamic LHRH, and inhibition of enzymes that degrade LHRH.
Progesterone also promoted the synthesis of FSH in the pituitary. The
3␣,5␣-reduced metabolite of progesterone brought about selective LH
release and acted using the GABAA receptor system. The 5␣-reduced
metabolite of progesterone brought about selective FSH release; the
ability of progesterone to bring about FSH release was dependent on
its 5␣-reduction. The GnRH neuron does not have steroid receptors;
the steroid effect was shown to be mediated through the excitatory
amino acid glutamate, which in turn stimulated nitric oxide. These
observations led to the replacement of the long-accepted belief that
ovarian steroids acted directly on the GnRH neuron by the novel
concept that the steroid feedback effect was exerted at the glutamatergic neuron, which in turn regulated the GnRH neuron. The neuroprotective effects of estrogens on brain neurons are of considerable
interest.
Perspectives
STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
the major organ for the oxidation of cortisol to cortisone (139,
140), a finding that proved in later years to be of great
importance in the action of mineralocorticoids.
These initial studies in the mechanism of steroid hormone
action required the development of methods for the separation,
characterization, and measurement of steroid hormone metabolites in human biological fluids and made studies in hirsutism,
virilism, and polycystic ovarian disease possible.
HIRSUTISM, VIRILISM AND POLYCYSTIC OVARIAN DISEASE
Steroid Secretion Patterns
vein, and ovarian vein steroids were measured before and after
ACTH and human chorionic gonadotropin (HCG) to further
establish the adrenal or ovarian source of excessive androgens
(176).
Even before the availability of radioimmunoassay (RIA) for
the determination of serum gonadotropins, levels of luteinizing
hormone (LH) secretion in urine were measured throughout the
menstrual cycle using an anti-HCG antiserum. Levels of LH
were found to show a peak either coinciding with or preceding
a rise in basal body temperature which was then considered to
be an indication of ovulation (203). With the availability of
RIA for measuring serum LH, our group as well as others
demonstrated a high pulsatile level of LH in patients with
polycystic ovary syndrome (80).Initially, there was no explanation for this high pulsatile secretion of LH, and a hypothalamic defect was postulated. Most patients with polycystic
ovary syndrome show good estrogenic vaginal smears due to
ovarian secretion of estrogens and the peripheral conversion of
androgens to estrogens. Using pituitary stalk-sectioned rats in
which aluminum foil was placed after stalk resection to prevent
regeneration of the hypothalamic blood supply, Greeley et al.
(87) showed that the rats still responded to luteinizing hormone-releasing hormone [LHRH; also referred to as gonadotropin-releasing hormone (GnRH)] in the secretion of LH and
FSH. Furthermore, if such rats were treated with estradiol, the
pituitary showed enhanced sensitivity to the release of LH (88).
Studies of Legan and Karach (110) showed that in long-term
ovariectomized rats, a single injection of estradiol brought
about daily LH surges, whereas a single injection of progesterone initially enhanced the estrogen-triggered surge of gonadotropins and then promptly brought about the extinction of
the estrogen-induced LH surge. There were no multiple surges
of gonadotropins on subsequent days of progesterone administration after the first surge took place. Thus, it appeared that
persistent estrogen stimulation to the hypothalamic-pituitary
axis could cause persistent LH surges in the human, which
could only be dampened by luteal levels of progesterone. This
was shown to occur in one patient with polycystic ovary
syndrome who underwent wedge resection of the ovary; the
high pulsatile levels of LH persisted only until the luteal rise of
progesterone occurred after the first ovulation (138). This
change in hormonal profile tends to explain the beneficial
effects of wedge resection on the ovulatory process. The
pivotal role of progesterone in initiating the surge of LH and
FSH secretion and also the termination of the surge will be
discussed in detail later in this article.
Other studies on hirsutism, virilism, and steroid secretion
carried out were in cases with ovarian stromal hyperplasia
(131, 133), arrhenoblastoma (93, 132), adrenal rest tumor of
the ovary (181), virilizing adrenal tumors (124, 127), and
delayed onset of congenital adrenal hyperplasia (128). 11␤Hydroxyestrone was also isolated in a case of feminizing
adrenal carcinoma (130).
Three overall questions emerged from the aforementioned
studies. The first question was whether there was a treatment to
induce ovulation in patients with polycystic ovarian syndrome
without undergoing surgical procedures such as the wedge
resection of the ovary. The second question concerned the
methods that could be employed for the management of hirsutism. The third question was whether polycystic ovary syndrome was a genetic or an acquired disorder. Initial attempts to
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The earliest case studied by the author was a case of twin
sisters, one of whom had undergone severe psychological
stress and had a sudden onset of hirsutism (54). The sister with
hirsutism had a very high excretion of androgen metabolites in
her urine compared with her normal sister and was hyperresponsive to adrenocorticotropic hormone (ACTH) stimulation
in androgen production. That the excessive androgens were
coming from the adrenal gland was demonstrated by large
quantities of dehydroepiandrosterone (3␤-hydroxy-5-androsten-17-one) and androstenedione (4-androsten-3,17-dione) in
the adrenal vein blood. In the mid 1950s, wedge resection of
the polycystic ovaries was the standard way to treat polycystic
ovarian disease. The ovarian wedges obtained from such patients, after obtaining informed consent, were extracted, and
the steroids obtained were characterized. Several ovaries contained large quantities of dehydroepiandrosterone that was
characterized by chromatographic properties and infrared spectroscopy along with 17␣-hydroxy-5-pregnenolone (3␤,17␣dihydroxy-5-pregnen-20-one). Some ovaries contained large
quantities of androstenedione compared with normal ovaries
(119, 120, 125). This was the first demonstration of androgen
secretion by the polycystic ovary, and the background references are provided in two reviews (122, 125). In vitro incubation studies showed that the ovaries containing large quantities
of dehydroepiandrosterone converted less substrate to androstenedione compared with normal ovaries, suggesting diminished 3␤-hydroxysteroid dehydrogenase activity, and those
containing large quantities of androstenedione showed lower
aromatase activity. Since androstenedione and dehydroepiandrosterone are weak androgens and we were not able to
extract significant amounts of testosterone from the polycystic
ovaries studied, it was of interest to determine whether these
weak androgens could be converted peripherally into testosterone. Oral administration of dehydroepiandrosterone and
androstenedione to women resulted in elevation of plasma
testosterone levels (121).
Since in our earlier study (54) the adrenal was the source of
excessive androgens, a finding that had also been reported by
others (122, 125), it was important to determine whether, in a
particular patient, the adrenal or the ovary or both were the
source of excessive androgens. Thus, urinary androgen metabolites were measured before and after adrenal and ovarian
suppression (126). The steroids were also measured after
ovarian stimulation with human pituitary follicle-stimulating
hormone (FSH) (123). The results showed that different patients could have adrenal oversecretion of androgens, ovarian
oversecretion of excessive androgens, or both. Human FSH
enhanced ovarian hypersecretion of androgens and also caused
multiple ovulations. In a subsequent study, peripheral, adrenal
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answer the third question were to determine whether there was
a chromosomal abnormality in polycystic ovary syndrome
patients. No chromosomal abnormalities were found in patients
with polycystic ovary syndrome (56); therefore, animal models
were constructed in an attempt to answer that question.
Induction of Ovulation in Patients with Polycystic Ovary
Syndrome
Studies on the Use of Antiandrogens for the Management
of Hirsutism
Management of hirsutism in patients with polycystic ovary
syndrome has been difficult, as the suggested approaches were
ovarian suppression by use of contraceptive steroids when the
source of excessive androgens was the ovary, or adrenal
suppression when the source of androgens was the adrenal, or
a combination of both treatments when the source of excessive
androgens were the ovary and the adrenal. Establishment of an
ovarian or adrenal source required extensive diagnostic tests,
and long-term suppression of the adrenal or the ovary was not
an easily acceptable choice. Thus, another approach was the
use of an antiandrogen, 17␣-methyl-B-nor-testosterone. In
male rats, 17␣-methyl-B-nor-testosterone reduced seminal ves-
Animal Models for Polycystic Ovarian Disease in Humans
The question whether human polycystic ovary syndrome
was a genetic disorder or whether it could be caused by
excessive androgen secretion is a difficult one and it is still
being debated. The possibility of an androgen-related disorder
was indicated by our early experiments that showed that the
administration of large quantities of dehydroepiandrosterone or
androstenedione in immature rats resulted in ovulatory failure
and the presence of polycystic ovaries (202). A detailed examination of the effects of dehydroepiandrosterone on ovulatory
failure was thus carried out (106). Administration of dehydroepiandrosterone to 27-day-old female rats resulted in ovulatory
type serum FSH and LH surge on day 30 of life, and the
animals exhibited either constant estrous or constant diestrous
vaginal smears with either polycystic ovaries or ovaries containing corpus luteum-like structures. With time, all ovaries
became polycystic. Serum FSH levels were elevated compared
with control rats, serum LH levels were similar to those in
control rats, and serum prolactin was elevated. The ovary was
responsive to gonadotropin treatment, and the pituitary was
responsive to LHRH stimulation. Discontinuation of the dehydroepiandrosterone treatment resulted in a few irregular ovulatory cycles followed by normal cyclicity. Thus, this animal
model was very similar to the human one, with the exception
that prolactin rather than LH was elevated. Further studies with
precocious ovulation showed that the gonadotropin surge resulting in ovulation could be blocked with central nervous
system blocking agents such as phenobarbital and reserpine
(106). Precocious ovulation appeared to be mediated by the
conversion of dehydroepiandrosterone to estrogens, because
the nonaromatizable androgen 5␣-dihydrotestosterone did not
cause such an ovulation (107). Furthermore, cyanoketone, an
inhibitor of 3␤-hydroxysteroid dehydrogenase, which blocks
the conversion of dehydroepiandrosterone to estrogen, prevented vaginal patency, and dehydroepiandrosterone-induced
precocious ovulation (107). A study of the steroid profile after
dehydroepiandrosterone administration showed an elevation in
blood estradiol followed by depletion of the cytoplasmic estradiol receptors in the hypothalamus and the pituitary and the
gonadotropin surge leading to ovulation (180). That these
events occur in the natural process of ovulation will be discussed later in this article. The restoration of normal cyclicity
in dehydroepiandrosterone-treated rats occurred only when the
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In attempts to search for agents other than human pituitary FSH
or human postmenopausal gonadotropins that were very expensive and difficult to obtain at that time, a variety of compounds
were tested for their ability to induce ovulation. Clomiphene
{1-[p(␤-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene} was sent to us to be tested as a contraceptive agent by a
drug company. In immature female rats, this compound was
found to stimulate uterine weight, and in male rats the seminal
vesicle and ventral prostate weights, in very low doses,
whereas it inhibited them at high doses (200). In the absence of
estrogens in the ovariectomized rat, Clomiphene acted as a
weak estrogen. It acted as an antiestrogen in the presence of
estrogens. In unilateral ovariectomized rats, Clomiphene in low
doses increased ovarian weight, thus indicating antagonism of
estrogen suppression on the hypothalamic-pituitary axis and
the stimulation of gonadotropin secretion (198). The antiestrogenic activity of Clomiphene was further demonstrated by its
ability to inhibit the uptake of tritiated estradiol in the rat uterus
and pituitary gland (201). In the human, Clomiphene was able
to induce ovulation in a variety of unovulatory patients, including patients with polycystic ovary syndrome (94, 199). It
was also able to stimulate spermatogenesis in men (103).
Clomiphene was also able to induce ovulation in ChiariFrommel syndrome, in which estrogen levels are very low (92).
Thus, Clomiphene became the accepted first treatment of
choice for the induction of ovulation prior to the use of human
menopausal gonadotropins followed by HCG (95). With the
availability of RIA for gonadotropin secretion, Clomiphene
treatment was shown to result in an ovulatory type of LH and
FSH secretion in a variety of anovulatory patients (78, 79). It
was also demonstrated that, in patients that were resistant to
Clomiphene in the induction of ovulation, 11 of the 13 patients
treated ovulated, and there were five pregnancies if the Clomiphene treatment was preceded by the synthetic glucocorticoid dexamethasone (213). It was shown previously in experimental animals that dexamethasone was able to bring about
FSH release, and this perhaps aided in the ovulation (36)
icle and ventral prostate weights and counteracted the growthpromoting effects of testosterone on these organs (142). However, in castrated male rats, it stimulated the growth of seminal
vesicles and ventral prostate, indicating weak androgenic effects. 17␣-Methyl-B-nor-testosterone also enhanced the effect
of testosterone in suppressing testicular weight, indicating a
weak antigonadotropic effect. The antigonadotropic effect was
confirmed in female rats, in which it decreased ovarian weight
and the number of ovulations (214). In the human, it brought
about a significant decrease in sebum production rate in the
forehead as well as a decrease in facial hair growth, as
determined by the weight of hair shaved, starting at 30 days
after it was administered (65, 221). However, the concern that
a patient on an antiandrogen might become accidentally pregnant, causing fetal abnormalities, has prevented the use of
antiandrogens in the management of hirsutism.
Perspectives
STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
THE STEROID-GONADOTROPIN FEED BACK SYSTEM
The abovementioned experimental studies showed that, in
animals with normal ovaries and a normal hypothalamicpituitary axis, the administration of androgens resulted in the
formation of polycystic ovaries and an altered gonadotropin
secretion pattern. Therefore, a detailed examination of the
steroid-gonadotropin feedback system was undertaken. These
studies included examining the role of FSH and LH in the
ovulatory cycle, steroid-gonadotropin feedback before and after puberty, steroid and gonadotropin changes during puberty,
and the ovarian signals for the preovulatory gonadotropin
surge.
Early Work on the Role of FSH and LH in the Ovulatory
Process
Before the advent of RIA for pituitary FSH and LH in
serum, bioassays were used for their determination. In the
cycling rat there was an 50% decrease in pituitary LH and FSH
content from the morning to the late evening on the day of
proestrus (84). Such a decrease was also observed in pubertal
rats preceding their first ovulation. In immature rats in which
follicular development was initiated with a small dose of
PMSG and the endogenous gonadotropin surge blocked by
phenobarbital, ovulation could be induced with purified pituitary FSH that did not contain enough LH contaminant to cause
ovulation. In the hamster, antiserum prepared against a preparation of LH with ample FSH contaminant blocked ovulation
(85). However, when the anti-FSH component was adsorbed
out, the ovulation blocking potency of the antiserum was
greatly reduced. In addition, ovulation blocked by anti-LH
could be reinstituted by injection of an FSH preparation that
contained very little or no LH contamination. (85). These
results showed a prominent role of FSH in ovulating the mature
ovarian follicle. In view of a common ␣-subunit in FSH, LH,
and TSH, the ability to ovulate a mature ovarian follicle by
TSH, prolactin, and the two LH subunits was also tried, but
they did not induce ovulation (129). Although in the rodent the
need for the secretion of FSH as a part of the preovulatory
gonadotropin surge can be explained on the basis of the growth
of the ovarian follicles for the next cycle, the physiological role
of FSH at the time of the preovulatory gonadotropin surge, if
any, in the human is still unclear. In this regard, Strott et al.
(211) have reported several cases of short luteal phase in
women in whom the only abnormality was a dulled or misplaced FSH peak during the ovulatory surge.
The role of the ovary in regulating the preovulatory surge of
gonadotropins was shown by the fact that removing the ovary
on the morning of diestrus II blocked the preovulatory LH
surge (86). Ovariectomy at 6:00 PM on diestrus II or 9:00 AM
on proestrus resulted in a lower LH surge at proestrus.
Injection of progesterone on proestrus before the preovulatory surge of gonadotropins postponed the preovulatory
surge for one day (86).
Examination of the Steroid-Gonadotropin Feedback System
by Castration and Steroid Replacement
Immature rats castrated at day 26 of age showed a rise of LH
and FSH by 8 h in male rats and in 24 h for LH and 48 h for
FSH in female rats (72). These levels could be suppressed by
the administration of estradiol or testosterone. In the female rat,
ovariectomy on day 26 of age and treatment with increasing
doses of estradiol for 5 days showed a return to intact gonadotropin levels within the physiological dose range of estradiol
as judged by uterine weight (145). Increasing the dose of
estrogens showed an increase in gonadotropin secretion due to
the positive feedback effect, followed by suppression at higher
dose levels. This method also provided a means of evaluating
the biological activity of several synthetic estrogens. Progesterone by itself did not suppress gonadotropins in the ovariectomized rat (144). Using seminal vesicle and ventral prostate
weights, similar studies tested the steroid feedback system in
male rats (73). Ovariectomy and estrogen replacement in 26-,
45-, and 95-day-old rats showed that the dose of estradiol
required to decrease serum gonadotropins was four- to sixfold
higher in 45- and 95-day-old rats compared with 26-day-old
rats, indicating that a maturation of the feedback system after
puberty even though the increase in uterine weight caused by
estrogen administration was comparable in the three groups
(74). This change in sensitivity to the negative feedback
effect of estradiol may in part be mediated by endogenous
endorphins that are suppressive of gonadotropin secretion
(188). An endorphin antagonist, naloxone, brings about a
large increased release of LH at all times in the rat ovulatory
cycle except at the time of the gonadotropin surge. Measurement of the ␤-endorphin content in the hypothalamus of
the immature ovariectomized rat treated with estradiol and
progesterone or triamcinilone acetonide (9␣-fluoro-11␤,21dihydroxy-1,4-pregnadiene-3,20-dione-16␣,17␣-acetonide)
to induce the preovulatory type surge of gonadotropins
resulted in an estradiol-induced decrease in ␤-endorphin
preceding and during the LH surge, which was maintained
the following morning (37). However, progesterone and
triamcinilone acetonide brought back the ␤-endorphin levels
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high levels of dehydroepiandrosterone in blood were reduced
(178). Because the earlier studies were done with immature
rats, it was important to establish that the administration of
dehydroepiandrosterone caused polycystic ovaries and ovulatory failure in adult rats as well. The effect of dehydroepiandrosterone in causing polycystic ovaries in the adult rat was
shown by Ward et al. (217).
Since polycystic ovary syndrome in women is often associated with obesity and insulin resistance and insulin type growth
factors hyperstimulate theca cells to produce androgens, the
direct effect of androgens on the ovary were examined. Hypophysectomized immature rats, when ovulated with pregnant
mare’s serum gonadotropin (PMSG) and HCG, showed follicular atresia and decreased ovulation rate when treated with the
nonaromatizable androgen 5␣-dihydrotestostrone (2). That the
effect of the androgen was on the ovary and not gonadotropin
secretion was demonstrated by the fact that in the PMSGtreated rat the preovulatory gonadotropin surge was unaffected
even though the number of ovulations was reduced (62).The
effect of the androgen could be reduced by pretreatment with
estrogens. Thus, androgens could cause abnormal follicular
development, altered ovarian steroidogenesis, and ovulatory
failure. Evidence was also found for an autoregulatory effect of
androgens on ovarian theca cell androgen production (207,
208).
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STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
to control levels on the morning after the gonadotropin
surge, thus appearing to reinstate the opioid inhibition.
Changes Occurring in Steroid Levels During the
Preovulatory Gonadotropin Surge and Initiation Of Puberty
Effect of Progesterone and Corticoids in Inducing the
Preovulatory Type of Surge of Gonadotropins
Extensive work done in this area showed that progesterone
played an important part in the preovulatory gonadotropin
surge. The work consisted of defining the role of progesterone
and its mechanism of action through depletion of occupied
estrogen receptors in the anterior pituitary; hypothalamic control of the secretion of LHRH, regulation of peptidase activity
that degrades LHRH, regulation of LH-␤ and FSH-␤ mRNA
levels, and selective secretion of FSH and LH by progesterone
metabolites. The work has been the subject of periodic reviews
Fig. 1. Stimulatory effects of progesterone on the hypothalamus in the release
of LHRH and stimulation of 17␤-hydroxysteroid dehydrogenase activity in the
pituitary. It also had direct action on the pituitary in increasing the sensitivity
of the pituitary to LHRH in release of LH and synthesis of new LH and FSH.
Progesterone also inhibited peptidase activity in the hypothalamus and pituitary, slowing degradation of LHRH and increasing greater availability of
LHRH for LH secretion. Enhanced 17␤-hydroxysteroid dehydrogenase activity brings about increased conversion of estradiol to estrone in the pituitary,
resulting in decreased nuclear occupancy of the estrogen receptor. Progesterone thus decreased the inhibitory action of estrogens on the pituitary on LH
release and decreased progesterone receptor synthesis. Decreased progesterone
receptors prevent the stimulatory effect of progesterone on LH secretion.
Stimulatory pathways are shown in green, inhibitory pathways in red.
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Ovarian and uterine histology and serum gonadotropins
were measured in Holtzman rats from days 22 to 40 of age, in
which ovulation occurred most consistently on day 38 of age
(64).The percentage of large follicles showed a linear growth
from day 22 onward. The endometrial stroma, myometrium,
and luminal epithelium started growing on day 32 of age along
with an increase in uterine weight. These changes in uterine
histology were early indicators of ovarian estrogen secretion.
An early small increase in serum LH occurred on day 34, with
the preovulatory LH and FSH surge on day 38. These observations suggested that ovarian estrogens may be the trigger for
the initiation of puberty. Measurement of steroids during the
natural onset of puberty showed an increase in blood estradiol,
progesterone, and testosterone preceding vaginal opening and
an increased sensitivity of the pituitary to LHRH (179). Correlated with the gonadotropin surge was a fall in the cytoplasmic estrogen receptors of the pituitary and the hypothalamus
due to nuclear translocation by the secreted estradiol. The rise
in estradiol and the depletion of cytoplasmic estrogen receptors
in the pituitary and hypothalamus were similar to what occurred in normally ovulating rat (89) and immature rats induced to ovulate by the administration of PMSG (177). Similar
events also took place in the precocious ovulation induced by
the administration of dehydroepiandrosterone (180). In the
estrogen-primed ovariectomized rat, an injection of 2 ␮g of
estradiol brought about a decrease in the estrogen receptor
content of the pituitary without altering the estrogen receptor
mRNA levels, which only increased 12 h after the estrogen
injection in preparation for the estrogen receptor replenishment
that started at 18 h. Thus, the initial decline in estrogen receptor
content was not due to loss of estrogen receptor mRNA but to
accelerated estrogen receptor processing (61). In the uterus,
similar changes took place, and progesterone was shown to
delay estrogen receptor replenishment (223, 224). Pituitary
ultrastructure of the gonadotropes during the preovulatory
surge also showed degranulation of LH-containing gonadotropes a few hours prior to the degranulation of FSH-containing gonadotropes, which was consistent with the start of the
LH surge a little earlier than the FSH surge. (63). Extensive
work was also done on the role of androgens and LHRH in the
initiation of puberty in the male rat (135, 160, 161–164). A
detailed description, however, is outside the scope of this
article.
on the subject (14, 16, 17 113–115, 134). The results of these
studies are also summarized in Figs. 1 and 2.
Defining the role of progesterone in the preovulatory surge
of gonadotropins. Even though the administration of estrogens
to ovariectomized rats was able to induce an LH surge, the LH
surge was only of a magnitude of ⬃10% of the preovulatory
gonadotropin surge. Thus, experiments were done using doses
of estrogens that were able to reduce the postcastration rise of
gonadotropins but not prevent it, in combination with various
doses of progesterone (146). The dose of estradiol used was
enough to induce progesterone receptors but only cause a
minimal if any estrogen-induced LH surge. The doses of
progesterone used were able either to stimulate the release of
LH and FSH or to suppress them with the next higher dose.
The pattern of the lower dose being stimulatory and the next
higher dose being inhibitory was persistent in several protocols
and several experiments. Such a pattern has been shown to be
important in the human menstrual cycle where a low level of
progesterone enhanced the preovulatory LH surge while the
higher level helped terminate the surge (101). The stimulatory
dose of progesterone was able to mount a preovulatory-type
gonadotropin surge of LH and FSH similar to what is seen at
the time of proestrus. The sensitivity of the pituitary to LHRH
was enhanced by the stimulatory dose of progesterone and
suppressed by the inhibitory dose of progesterone (148). This
pattern was also exhibited by the ability of progesterone to
decrease occupied estrogen receptors of the anterior pituitary
and the effect of progesterone and 5␣-dihydroprogesterone to
attenuate estrogen-induced prolactin release, which will be
discussed later in this article.
In the immature castrated male rat, the dose of estradiol
needed to reduce the levels of gonadotropins comparable to
what was achieved with the 0.1 ␮g/kg body wt dose of
estradiol in female rats was 2.0 ␮g/kg body wt (149). This was
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STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
In the adult rat, measurement of both occupied and unoccupied estrogen receptor content was determined in the pituitary
and the uterus after 0.8, 2.0, and 4.0 mg/kg body wt progesterone. The 0.8 and 4.0 mg/kg body wt dose of progesterone
was stimulatory to gonadotropin release, whereas the 2.0
mg/kg body wt dose was inhibitory. The finding in the immature rat (146, 148) and the adult rat (57, 75) of a smaller dose
of progesterone to be stimulatory to gonadotropin release and
the next higher dose to be inhibitory was of great interest. It
was shown that in the human menstrual cycle the initial
increase in progesterone around the time of ovulation was
stimulatory to gonadotropin release, whereas a secondary rise
coincided with the termination of the LH surge (101). High
pulsatile levels of LH were also inhibited by rising levels of
progesterone after ovulation in a patient with wedge resection
of the ovary (138).
In the pituitary and the uterus, the decrease in estrogen receptors occurred only in the occupied form and not the unoccupied
estrogen receptors (75). However, whereas the pituitary showed a
dose dependency of progesterone of the effect, the 0.8 and 4.0
mg/kg body wt doses being more effective than the 2.0 mg/kg
body wt dose in reducing occupied estrogen receptors, the uterus
showed a systematic dose response. This was an interesting
tissue difference. In the estrogen-primed progesterone-treated
group, 0.8 and 4.0 mg/kg body wt progesterone increased
estrogen receptor mRNA levels in the pituitary within 1 h of
administration, whereas the 2.0 dose was ineffective. Despite
this early increase in estrogen receptor mRNA levels by the 0.8
and 4.0 mg/kg body wt progesterone, these doses still depleted
the occupied estrogen receptors of the pituitary. In the uterus,
progesterone treatment in estrogen-primed rats did not alter the
estrogen receptor mRNA levels but depleted the occupied
estrogen receptors (197). The mechanism for the decrease of
occupied estrogen receptors of the pituitary and the uterus was
by the stimulation of 17␤-hydroxysteroid dehydrogenase by
progesterone (71, 76). This converted estradiol to estrone,
which had a lower affinity for the estrogen receptor than
estradiol, thereby reducing the suppressive effect of estrogens
on gonadotropin secretion at the level of the pituitary. Accelerated estrogen receptor processing was also indicated.
The antagonism of estrogen action by progesterone and
5␣-dihydrotestosterone and their dose dependency was also
shown in the inhibition of estrogen-induced prolactin release
(30, 31, 38). Such dose dependence was also found in the
inhibition of estrogen-induced prolactin release by 5␣-dihydroprogesterone (34). Surprisingly, the antiandrogen flutamide
could not only block the action of 5␣-dihydrotestosterone but
that of progesterone as well (32). This was also true for
RU-486, which blocked progesterone as well as 5␣-dihydrotestosterone effects.
HYPOTHALAMIC REGULATION OF LHRH SECRETION REGULATED
BY PROGESTERONE. It has already been stated that progesterone
given to estrogen-primed ovariectomized rats increases the
sensitivity of the pituitary to LHRH in the release of LH (148).
Progesterone also stimulates the secretion of LHRH from the
hypothalamus in the estrogen-primed rat (182). The regulators
of LHRH secretion, catecholamine as well as neuropeptide Y,
are also released by progesterone in the medial basal hypothalamus by progesterone and the glucocorticoid triamcinilone
acetonide in the estrogen-primed ovariectomized rat (15, 26).
The release of the above are more acutely related to LH
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perhaps due to the masculinization of the hypothalamus neonatally by estrogens, resulting in the change in sensitivity to
estrogens. With this dose of estradiol, progesterone was able to
induce a preovulatory-type gonadotropin surge even in the
male rat (149).
That progesterone played a pivotal role in ovulation was further
demonstrated by the use of the progesterone and glucocorticoid
antagonist RU-486 (17␤-hydroxy-11␤-[4-dimethylaminopgenyl]17␣-[prop-1-ynyl]-estra-4,9-diene-3-one) (193). RU-486 abolished the preovulatory gonadotropin surge in PMSG-treated
immature rat and in the normally cycling adult rat. In this
regard, it is of interest to note that Goldman et al. (83) found
an elevation of progesterone in ovarian vein blood at 1400 h on
proestrus prior to the LH surge. Progesterone and LH levels
also showed an increase at 1400 h in the study of Nequin et al.
(165), although several other investigators found no rise in
preovulatory progesterone, possibly because they did not sample blood at 1400 h. A preovulatory increase in serum progesterone has been found in women 12 h before the initiation of
the rise of LH (101) and 6 h before the rise of LH in rhesus
monkeys (204).
In the estradiol-primed immature rat, deoxycorticosterone
(21-hydroxy-4-pregnene-3,20-dione) and the synthetic glucocorticoid triamcinilone acetonide were also able to induce a
preovulatory type of gonadotropin surge, whereas cortisol was
unable to do so (35, 36). The ability of deoxycorticosterone and
triamcinilone acetonide to induce the surge was considered to
be due to their interaction with the progesterone receptor.
These steroids, like progesterone, were also very effective in
stimulating fluid loss from the distended uterus caused by
estrogen treatment. Low doses of the synthetic steroid dexamethasone were able to selectively bring about FSH release in
the estrogen-primed rat (36). Deoxycorticosterone and triamcinilone acetonide were also able to induce ovulation in
PMSG-primed rats, similar to progesterone, which served as a
biological test for their effect (35). In the estrogen-primed
ovariectomized rat, acute administration of ACTH also resulted in the preovulatory type of gonadotropin release. The
surge required the presence of the adrenal glands and was
antagonized by the progesterone and glucocorticoid antagonist
RU-486. ACTH increased both progesterone and deoxycorticosterone, and the gonadotropin surge was considered to be
mediated by these steroids (190).
Mechanism of action of progesterone on the release of
gonadotropins. ANTAGONISM OF ESTROGEN ACTION ON THE
PITUITARY. An examination of the effects of progesterone on
the estrogen receptor of the anterior pituitary in ovariectomized
estrogen-primed rats showed that progesterone brought about a
decrease in the estrogen receptors of the anterior pituitary
(210). This created a controversy in the literature, as several
other investigators had failed to find an effect of progesterone
on estrogen receptors of the anterior pituitary. A detailed
examination of the progesterone effect showed that it occurred
only during the period of the nuclear occupancy of the progesterone receptor and not before or after the event (57). Furthermore, only during the period of nuclear occupancy of the
progesterone receptor in the pituitary did progesterone attenuate the effect of a second injection of estradiol on the increase
in cytosolic progesterone receptors. The effect of progesterone
on estrogen receptors was also confirmed in in vitro experiments and in the adult rats (75, 209).
E9
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STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
secretion than to FSH secretion. The direct effect of neuropeptide Y on anterior pituitary cells in culture in sensitizing the
pituitary to LHRH has also been demonstrated (168). It was
also found that progesterone modulates neuropeptide Y levels
in the anterior pituitary during the progesterone-induced surge
in the estrogen-primed rat (169). Changes in galanin mRNA
also correlate to the progesterone-induced gonadotropin surge
(8). The above-mentioned experiments clearly indicate that
progesterone in estrogen-primed rats can bring about LHRH
secretion, which is responsible for the preovulatory surge of
gonadotropins.
REGULATION OF PEPTIDASE ACTIVITY INVOLVED IN THE DEGRADATION OF LHRH IN THE HYPOTHALAMUS AND THE PITUITARY. The
REGULATION OF LH-␤ MRNA AND FSH-␤ MRNA LEVELS BY PROGESTERONE DURING THE PREOVULATORY GONADOTROPIN SURGE.
Initial experiments did not show any consistent effect of
progesterone on the pituitary LH-␤ mRNA and FSH-␤ mRNA
levels in the ovariectomized estrogen-primed rat treated with
progesterone, whereas dexamethasone increased the level of
FSH-␤ mRNA before the rise of serum FSH (27). This may be
due to the fact that the gonadotropin subunit levels in ovariectomized estrogen-primed rats were severalfold higher than
those in intact estrogen-primed rats. In the estrogen-primed
intact rat, progesterone administration brought about an elevation of LH-␤ mRNA and FSH-␤ mRNA in parallel with the
preovulatory gonadotropin surge (28). This also occurred in the
PMSG-primed immature rat. The changes in the mRNA levels
were blocked by the antiprogestin RU-486 (28). Further work
showed that a 361 base pair region of the FSH promoter gene
contained several progesterone response elements and these
mediated the progesterone effect on the FSH gene (170, 171).
SELECTIVE RELEASE OF LH AND FSH BY PROGESTERONE
METABOLITES. In the estrogen-primed immature ovariecto-
mized rat, the administration of the progesterone metabolite
5␣-dihydroprogesterone brought about the selective release of
FSH (155) and the progesterone metabolite 3␣,5␣-tetrahydroprogesterone brought about a selective release of LH (156).
The selective release of LH and FSH by the above-mentioned
progesterone metabolites also took place in the PMSG-treated
immature rats exposed to constant light (157). Initially, the
effect of 3␣,5␣-tetrahydroprogesterone was unexpected, as the
Fig. 2. Progesterone is converted into two major metabolites, 5␣-dihydroprogesterone and 3␣,5␣-tetrahydroprogesterone. The former uses the progesterone receptor and brings about selective release of FSH; the latter uses the
GABAA receptor and brings about selective release of LH. Administration of
a 5␣-reductase inhibitor in vivo or in vitro reduced FSH release without
affecting LH release. Stimulatory pathways are shown in green; inhibitory
pathways in red.
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biological activity of LHRH in the release of LH and FSH is
also dependent upon the amount of LHRH that survives degradation by the peptidase activity in the hypothalamus and the
pituitary. The peptidase activity of the hypothalamus and the
pituitary was lower in the hypothalamus and the pituitary on
proestrus and diestrus 1 compared with other parts of the
ovulatory cycle, thus leaving the maximum amount of LHRH
available during that time for biological action (166). To
examine the role of progesterone on changes in the peptidase
activity in the hypothalamus, pituitary and serum, the activity
was measured in estrogen-primed ovariectomized rats before
and after progesterone administration. Estrogen administration
increased the peptidase activity of the hypothalamus, which
was antagonized by progesterone (167). Progesterone similarly
decreased the peptidase activity of serum but had no effect on
the peptidase activity of the pituitary. These results indicate
that progesterone administration brings about a suppression of
hypothalamic peptidase activity, permitting the availability
more LHRH for mounting the preovulatory gonadotropin
surge.
compound does not interact with the progesterone receptor.
However, several steroidal anesthetics that have a 3␣,5␣reduced Ring A structure had been shown to use the ␥-amino
butyric acid A (GABAA) receptor system for their biological
action. Further work showed that the action of 3␣,5␣-tetrahydroprogesterone on LH release could be blocked by GABAA
receptor antagonists but not by RU-486 (33). Progesterone
metabolites were also able to dampen the estrogen-induced
uterine contractions. Once again, similar to the FSH and LH
release, the dampening effect of 5␣-dihydroprogesterone was
blocked by the progesterone receptor antagonist RU-486,
whereas the 3␣,5␣-tetrahydroprogesterone effect was blocked
by the GABAA antagonist (189). That the effect of progesterone in the release of FSH was mediated by its 5␣-reduction to
5␣-dihydroprogesterone was demonstrated by the use of a
5␣-reductase inhibitor N,N-diethyl-4-methyl-3oxo-4-aza-5␣androstane-17␤-carboxamide. The use of this inhibitor blocked
the reduction of progesterone in the 5␣ position and the
progesterone induced a FSH surge without affecting the progesterone-induced LH surge (Fig. 2) (191). 5␣-Dihydroprogesterone also brought about the depletion of occupied estrogen
receptors of the anterior pituitary and the uterus similar to
progesterone (77). A significant amount of the stimulatory and
inhibitory effects of LHRH on gonadotropin secretion by
progesterone occurred at the level of the pituitary, as shown in
pituitary cell cultures (112). In estrogen-primed pituitary cells,
progesterone exposure for 1– 6 h brought about the potentiation
of LHRH in the release of LH and FSH, whereas progesterone
exposure of 12 h or more was inhibitory. The 5␣-reductase
inhibitor blocked the effect of progesterone on FSH release. It
was also of interest to note that in the estrogen-primed rat the
administration of the opioid antagonist naloxone brought about
an increase in LH secretion. An inhibitor of GABA degradation aminooxyacetic acid and both GABAA and GABAB agonists blocked this action (44). In this regard, a decreased opioid
response to naloxone was observed in patients with premenstrual tension (196). 3␣,5␣-reduced steroids have an anesthet-
Perspectives
STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
ic- and anxiety-reducing effect. In women with premenstrual
tension, the metabolism of progesterone to 3␣,5␣-tetrahydroprogesterone was compromised (195).
EXCITATORY AMINO ACIDS AS MEDIATORS OF THE
STEROID-GONADOTROPIN FEED BACK SYSTEM
Estabilishing the Possible Role of Excitatory Amino Acids in
Gonadotropin Secretion
rat showed a decline in pituitary NMDA mRNA. However,
progesterone treatment to the estrogen-primed rat resulted in an
increase in pituitary NMDA mRNA, which is in keeping with
the direct effects of progesterone on the pituitary during the
gonadotropin surge mentioned earlier in this article (9). During
the rat ovulatory cycle, NMDA receptor 1 (NMDAR1) and
glutamate receptor 1 (GluR1) did not change much, but the
levels of the kainite receptor GluR6 decreased just preceding
the surge. This decrease was demonstrated to be due to progesterone action (42)
Administration of NMDA advanced puberty in the rat by 2.5
days, whereas the non-NMDA agonist kainite or the nonNMDA antagonist had no effect on puberty, indicating that
NMDA was primarily involved in the process of puberty (43).
The NMDA receptors did not change significantly in the
pituitary during puberty, whereas the AMPA receptors showed
an increase (219). This indicated selective excitatory amino
acid receptor involvement during puberty.
Determining Whether Glutamate Was Actually Secreted
During the Gonadotroipn Surge
The next important question was to determine whether the
role of glutamate, which was indicated to be important in the
gonadotropin surge by the study of various agonist and antagonist studies of various receptors of glutamate action, could be
established by the actual secretion of glutamate during the
ovulatory surge of gonadotropins. To achieve this, microdialysis was done in vivo from the preoptic area through a cannula
implanted in the estrogen-primed rat given progesterone to
induce ovulation. There was a significant increase in the
preoptic area release of glutamate and aspartate immediately
preceding the preovulatory gonadotropin surge (187).
Naloxone has been shown to bring about LH release by
inhibiting the opioid inhibitory mechanism in several studies.
In male rats, administration of naloxone brought about an
increase in serum LH and an increase in nitric oxide synthase
(NOS) in the preoptic area and medial basal hypothalamus
within 20 min (5). This increase was blocked by the NMDA
antagonist MK801. Microdialysis experiments in vivo of the
preoptic area also showed a significant increase in glutamate
secretion within 15 min of naloxone administration. Thus, the
opioid block of gonadotropin secretion appeared by their suppression of glutamate release.
Localization of NMDAR1 Receptors in the Hypothalamus
and the Role of NOSs
Physiochemical localization of NMDAR1-containing neurons showed extensive distribution in the hypothalamus, including the organum vasculosum of the lamina terminalis
(OVLT), median preoptic nucleus, and median preoptic area
(4). This NMDAR1 staining area did not colocalize with the
GnRH neuron but surrounded several of them; they colocalized
with NOS-1 activity. Central administration of a NOS inhibitor
abolished the steroid-induced preovulatory surge, indicating
that NMDA acted through NO in the release of LHRH.
NMDAR1 was also found in almost all cell types of the
anterior pituitary, suggesting some pituitary action of NMDA
(6). Further work showed that the hypothalamus contained all
three isoforms of NOS, namely brain NOS (NOS-1), microphage NOS (NOS-2), and endothelial NOS (NOS-3). Of
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Early work had suggested that glutamate may have a role in the
release of LH (175). To determine whether excitatory amino acids
mediated the steroid-induced gonadotropin surge, estrogenprimed immature rats treated with progesterone or triamcinilone
acetonide were injected with the N-methyl-D-aspartate (NMDA)
antagonist MK801 [(⫹)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)
cyclohepten-5,10-imine maleate] (18). MK801 blocked the progesterone- or triamcinilone acetonide-induced LH and FSH surge.
MK801 also reduced the number of ova ovulated in the
PMSG-treated rat along with a decrease in the LH and FSH
surge (19). MK801 also decreased serum LH levels in the adult
cycling rat. The excitatory amino acid agonist NMDA given to
estrogen-primed rats brought about prompt elevations of LH
and FSH. The effect seemed to be at the hypothalamus,
because medial basal hypothalamus/preoptic area fragments
showed an elevated release of LHRH in vitro 5 and 7 min after
the administration of NMDA (19 –21). These findings are of
considerable importance, as it was shown that the GnRH
neuron did not appear to have steroid receptors whereas there
were steroid receptors in glutamate-containing neurons (100,
111). Thus, the GnRH neuron was regulated by other neurons
in the hypothalamus that had steroid receptors. The subject has
been reviewed extensively (22–24). To determine whether
non-NMDA neurotransmission also regulated the gonadotropin surge, the non-NMDA receptor antagonist DNQX (6,7dinitroquinoxaline-2,3-dione) was administered via a third
ventricular cannula in estrogen-primed adult rats treated with
progesterone and PMSG-primed immature rats. DNQX attenuated the LH and the prolactin surges without much effect on
the FSH surge (29). Both NMDA and non-NMDA receptors
played a role in pulsatile LH release, as shown by injection of
the specific NMDA receptor antagonist AP5 (2-amino-5-phosphono-pentanoic acid) and the non-NMDA antagonist DNQX
in adult rats ovariectomized for 2 wk via a third ventricular
cannula (185). Similar results were obtained in the male rat as
well, although the NMDA receptor antagonist was more effective than the non-NMDA receptor antagonist (186). AMPA
(␣-amino-3-hydroxy-5-methylisooxazole-4-propionoc acid)
also brought about LH release in the estrogen-primed rat,
indicating a role for AMPA receptors. The steroid-induced
gonadotropin surge was blocked by the selective AMPA receptor antagonist NBQX (184).
Since NMDA was more effective in gonadotropin release in
the estrogen-primed female rat compared with the ovariectomized rat, the NMDA receptor in the hypothalamus was
characterized (141), and the NMDA receptor binding and
NMDA receptor mRNA weremeasured in the hypothalamus
upon castration and after estrogen priming (41). No change
was observed in castrated female rats after estrogen treatment
or in castrated male rats with testosterone treatment. The effect
of steroids on NMDA receptor mRNA levels in the pituitary
was also examined. Estrogen treatment of the ovariectomized
E11
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STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
these, NOS-1 was the major isoform and acted through the
activation of cGMP (3). The role of NOS-1 in the release of
steroid-induced gonadotropin surge was further confirmed by
the administration of antisense nucleotides to NOS-1, which
attenuated the steroid-induced gonadotropin surge (1). NOS
levels were also shown to increase during the proestrus gonadotropin surge in the rat (108). A regulatory role of carbon
monoxide on LH release has also been suggested (109). The
role of gaseous neurotransmitters in the release of gonadotropins has been reviewed (7).
Mechanism of Action of Progesterone in Releasing
Glutamate and Suppressing GABA
ROLE OF HYPOTHALAMIC ASTROCYTES IN
STEROID-GONADOTROPIN FEEDBACK
To determine whether astrocytes played any role in stimulating hypothalamic LHRH secretion, conditioned medium
from hypothalamic astrocytes was incubated with either hypothalamic fragments or the immortalized GnRH neuronal cells
the GT1-7 cells (49), The hypothalamic conditioned medium
brought about the release of LHRH. Ultra filtration of the
astrocytes conditioned medium to remove peptides greater than
10 kDa resulted in loss of activity. The peptide responsible for
the release of LHRH was found to be transforming growth
factor-␤ (TGF␤). Antibodies to TGF␤ abolished the ability of
OTHER RELATED STUDIES
This author has been involved in a number of related studies,
a detailed description of which is outside the scope of this
article. They are studies of steroid gonadotropin feedback in
aging (25, 42, 104, 147, 225), testicular feminization (154),
gonadal dysgenesis (90, 91, 143), metabolism of oral contraceptives (47, 48, 151, 152, 153), heterogeneity in granulosa
cells (192, 194), regulation of follicular development by diethyl stilbesterol (59, 60), estrogen and progesterone receptors
in the human endometrium (158, 159), and pregnancy in the rat
(172–174). Other studies refer to the role of kinins in the
ovulatory cycle and pregnancy and the possible role of bradykinin in the preovulatory gonadotropin surge (12, 81, 205,
206). The role of leptin in reproduction is also of considerable
interest (10, 40, 50, 58, 218, 220). Finally, computer modeling
of partially unwound DNA structures caused by receptor binding shows a stereochemical fit of estradiol in the cavity in the
center of the hormone response element. The fit in the DNA
cavity is a much more accurate prediction of biological activity
than estrogen binding to its receptor. The technique has led to
the development of search engines that can be used in drug
design (46, 96 –99, 117, 118, 136, 137)
SUMMARY AND CONCLUSIONS
Fig. 3. Progesterone inhibition of glutamic acid dehydrogenase-67 (GAD67)
permits more glutamate release and less GABA release, thus permitting an LH
surge. Stimulatory events are shown in green, inhibitory events in red.
Early work on polycystic ovary syndrome was a major
breakthrough, as it established the ovary with or without the
involvement of the adrenal as a source of androgens. The
reasons for the occurrence of hirsutism and virilism in a variety
of endocrine patients was also established. Experimental models for creating polycystic ovaries as well as other experiments
established the detrimental effects of excessive androgens on
follicular development that resulted in cystic ovaries. The
studies also resulted in establishing the use of Clomiphene for
the induction of ovulation in anovulatory women. Animal
studies showing the ability of dexamethasone in releasing FSH
resulted in the use of dexamethasone in combination with
Clomiphene to induce ovulation in women not shown to have
excessive androgen secretion but who had failed to ovulate on
treatment with Clomiphene alone. A link between premen-
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The next question was how progesterone brought about the
release of glutamate during the preovulatory gonadotropin
surge. This was shown to be due to the suppression of glutamic
acid decarboxylase-67 (GAD67) by progesterone in the estrogen-primed rat, resulting in the decrease of glutamate converted to GABA, thus permitting more glutamate and less
GABA to be secreted (Fig. 3) (215). The suppression of GAD67
during the preovulatory surge of gonadotropins was also shown to
occur in the adult rat (39). In addition, the synaptic terminals’
appositions to the GnRH neuron of the vesicular GABA transporter (VGAT) was decreased while the vesicular glutamate
transporter 2 (VGLUT2) was increased during the proestrus gonadotropin surge, thus further confirming the role of glutamate in
the surge (104).
the conditioned medium to release LHRH. Furthermore, the
hypothalamus and the GT1-7 cells possessed TGF␤ receptors.
Estrogens stimulated astrocytes to release TGF␤, and this
action was attenuated with estrogen receptor antagonists. Astrocytes also possessed estrogen receptors. Astrocytes’ conditioned medium also protected GT1-7 cell cultured in serumfree medium from death, and this was attributed to TGF␤. The
neuroprotective pathway appeared to be via the activation of
the c-Jun/AP-1 pathway (66). Similar to estradiol, tamoxifen
was also able to reduce the extent of stroke in experimental
animals in which the medial carotid artery was blocked (150).
The neuroprotective effects of estrogens were exerted in part
by the stimulation of TGF␤ secretion using a nongenomic
mechanism (68). This was another example of the nongenomic
effect of steroids (13). The role of astrocytes in reproduction
and neuroprotection has also been reviewed (116). Extensive
work on the mechanisms involved in estrogen effects on
neuroprotection has continued but is outside the scope of this
article but is described in detail elsewhere (11, 45, 67, 69, 105,
216, 222).
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STEROID-GONADOTROPIN-FEEDBACK SYSTEM: CAREER RETROSPECTIVE
ACKNOWLEDGMENTS
I wish to express my gratitude to numerous graduate students, postdoctoral
fellows, and faculty colleagues for their untiring collaboration and help in
introducing new techniques in the laboratory from time to time.
GRANTS
This research was supported by Grants AM-04429, HD-04626, HD-10795,
HD-16688, HD-14196 and HD-16396 from the National Institutes of Health.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: V.B.M. conception and design of research; V.B.M.
performed experiments; V.B.M. analyzed data; V.B.M. interpreted results of
experiments; V.B.M. drafted manuscript; V.B.M. edited and revised manuscript; V.B.M. approved final version of manuscript.
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3. Bhat G, Mahesh VB, Aguan K, Brann DW. Evidence that brain-nitric
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hypothalamic cGMP levels. Neuroendocrinology 64: 93–102, 1996.
4. Bhat GK, Mahesh VB, Lamar CA, Ping L, Aguan K, Brann DW.
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16. Brann DW, Mahesh VB. Validation of the mechanisms proposed for the
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strual tension and abnormal metabolism of progesterone was
also established. The above are excellent examples of translational applications of basic science research.
There is no clear understanding of the importance of the
FSH surge at the time of ovulation in the human. The finding
that FSH can bring about the rupture of the mature ovarian
follicle and that several women with short luteal phase have a
decreased or displaced FSH surge is intriguing, and further
work needs to be done in this area.
The studies described in this article also provide the sequence of events starting with follicular growth and an increase
in estrogens resulting in the occurrence of puberty in the
female and similar effects with androgens in the male rat.
Although the studies provide a solid foundation of the events
that take place during puberty, the mechanisms that trigger the
initial increase in follicular growth and estrogen secretion in
female rats and androgen secretion in male rats are still obscure
and require further investigation.
The role of estradiol in triggering the preovulatory surge of
gonadotropins has been well recognized in the literature. The
studies described in this article clearly show the pivotal role of
progesterone in the process of modulating the preovulatory
surge of gonadotropins. The dependence of progesterone action on estrogens is due to the synthesis of progesterone
receptors by estrogen action. Progesterone acts by stimulating
hypothalamic release of LHRH, suppressing the peptidase
activity and thus making more LHRH available, increasing the
pituitary sensitivity to LHRH along with synthesis of new LH
and FSH, and decreasing the occupied nuclear estrogen receptors of the pituitary, thus overriding the suppressive effects of
estrogens on the pituitary in gonadotropin release. The differential regulation of FSH and LH has also been a subject of
considerable interest. The demonstration of selective effects of
progesterone metabolites on FSH and LH secretion and the use
of not only the progesterone receptor but the GABAA receptor
as well in this process is therefore of considerable importance.
The work described in this article has resulted in the replacement of the long-held belief, that gonadal steroids act directly
on the GnRH neuron to regulate gonadotropin secretion, with
the novel concept that estrogens and progesterone act on the
glutamatergic neurons of the hypothalamus that contain steroid
receptors and that these neurons in turn regulate the GnRH
neuron via NOS-1. Thus, this new pathway adds to the multiple
pathways known or currently under discovery of the regulation
of reproduction, which is essential for the preservation and
continuation of the species.
Finally, the concept of the neuroprotective effect of estrogens on brain function is of considerable importance. This
neuroprotective effect is lost during years of estrogen deprivation during menopause, leading to increased incidence of
stroke and dementia. Studies on the mechanism of loss of the
neuroprotective effect of estrogens during estrogen deprivation
and methods to circumvent it would be of considerable value
for the health and well-being of our aging population.
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E17
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E18
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