0013-7227/97/$03.00/0
Endocrinology
Copyright © 1997 by The Endocrine Society
Vol. 138, No. 3
Printed in U.S.A.
Prenatal Androgens Time Neuroendocrine Puberty in
the Sheep: Effect of Testosterone Dose*
SHEPHARD S. KOSUT, RUTH I. WOOD, CRISTINA HERBOSA-ENCARNACIÓN†,
DOUGLAS L. FOSTER
AND
Reproductive Sciences Program and Departments of Biology (S.S.K., C.H.-E., D.L.F.) and Obstetrics
and Gynecology (D.L.F.), University of Michigan, Ann Arbor, Michigan 48109-0404; and the
Department of Obstetrics and Gynecology, Yale University (R.I.W.), New Haven, Connecticut
06520-8063
ABSTRACT
In sheep, prenatal exposure to androgens during a critical period
for sexual differentiation of the brain (30 –90 days of gestation; 145
days is term) can advance the timing of puberty in females and
prevent the preovulatory LH surge. The present study tests the hypothesis that in sheep, the timing of neuroendocrine sexual maturation is related to the amount of prenatal steroid exposure. In addition,
we determined if different steroid requirements exist for sexual differentiation of the tonic and surge modes of gonadotropin secretion.
Testosterone was administered weekly to three groups of pregnant
ewes from days 30 –90 of gestation at doses of 200, 80, or 32 mg/week.
The resulting androgenized female lambs together with control males
and females (n 5 5–7/group) were gonadectomized at 3 weeks of age,
and gonadal steroids were replaced with a SILASTIC brand estradiolfilled capsule. LH concentrations were measured from biweekly blood
samples. Sustained increases in circulating LH were considered to
reflect the initiation of neuroendocrine puberty. In male lambs, LH
secretion started to increase at 8.3 6 0.9 weeks of age (mean 6 SEM).
The two highest doses of prenatal androgen advanced the onset of
neuroendocrine sexual maturation in females. In the 200 mg androgenized females, the pubertal LH rise (10.2 6 2.0 weeks) began about
the same time as in males. In the 80 mg treatment group, LH concentrations increased at 16.2 6 1.5 weeks, which was later than in
males, but well before that in normal females (27.1 6 0.7 weeks). For
females treated with the lowest dose of androgen (32 mg), the pubertal
LH increase (24.6 6 1.9 weeks) began about the same time as in
normal females. To test the function of the LH surge system, LH was
measured every 2 h for 60 h after an acute increase in circulating
estradiol was produced by implanting additional estrogen capsules.
All control females produced a surge in response to acute estradiol
stimulation. LH surges did not occur in males, 200 mg androgenized
females, or 80 mg androgenized females. Of six females from the 32
mg treatment group, two produced LH surges in response to the
stimulatory feedback action of estradiol. We conclude that the greater
the amount of prenatal testosterone, the earlier the initiation of the
pubertal LH rise. Moreover, the finding that low doses of testosterone
(32 mg/week) are capable of abolishing the LH surge without significantly advancing the timing of puberty supports our hypothesis that
different steroid requirements exist for sexual differentiation of tonic
and surge modes of LH secretion. (Endocrinology 138: 1072–1077,
1997)
I
their sustained increase in tonic LH secretion at approximately 10 weeks of age, females remain hypogonadotropic
until approximately 30 weeks. This pubertal rise in LH is a
result of a decrease in sensitivity to estradiol negative feedback (reviewed in Ref. 4).
Sex differences in tonic and surge modes of gonadotropin
secretion arise from androgens acting during a prenatal critical period of development, when the brain is susceptible to
the organizing actions of gonadal steroids. The early studies
of Short (5) and Clarke et al. (6) on androgenized female sheep
determined that the critical period for sexual differentiation
of the LH surge system is 30 –90 days gestation (145 days is
term). More recent studies from our laboratory (7, 8) have
confirmed these findings (5, 6, 9). Of equal interest, we have
determined that exposure to androgens during this same
period masculinizes the control of tonic LH secretion to advance the timing of puberty.
An emerging concept of prenatal steroid action is that
separate steroid requirements exist for the differentiation of
tonic and surge modes of LH secretion in the sheep. The
developing neuroendocrine system may be sensitive to the
timing, dose, and identity (androgen vs. estrogen) of steroid
in utero. A previous study from our laboratory (8) determined
that a longer duration of androgen exposure is required for
N MAMMALIAN and avian species, sexual differentiation of the neuroendocrine system plays a critical role in
the behavioral and hormonal differences between the sexes
(reviewed in Ref. 1). In sheep (2) as well as many other species
(reviewed in Ref. 1), the preovulatory LH surge resulting
from the stimulatory feedback of estradiol is sexually differentiated. Females respond to increasing circulating estradiol with a massive discharge of LH, but this same response
is normally neither present nor inducible in males (2). We
have recently determined that tonic LH secretion is also
sexually differentiated (3). Unlike the surge system, however, tonic LH secretion is common to both sexes. Sex differences arise in the timing of increased tonic LH secretion
that initiates the onset of sexual maturity. While males begin
Received August 5, 1996.
Address all correspondence and requests for reprints to: Dr. Douglas
L. Foster, Room 1101, 300 North Ingalls Building, Ann Arbor, Michigan
48109-0404. E-mail: [email protected].
* Preliminary reports of this work were presented at the 10th International Congress of Endocrinology, San Francisco, CA, 1996. This work
was supported by USDA Grants 92– 0269 and 93– 0184, and the Office
of the Vice President for Research at the University of Michigan.
† Present address: College of Medicine, Department of Obstetrics and
Gynecology, Fifth Floor Means Hall, 1654 Upham Drive, Ohio State
University, Columbus, Ohio 43210-1228.
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BRAIN DOSE-RESPONSE TO PRENATAL ANDROGENS
masculinization of the LH surge compared to that for masculinization of tonic LH. The present study investigated the
effects of androgen dose. Female lambs were androgenized
from days 30 –90 of gestation with three doses of testosterone.
After birth, the timing of onset of neuroendocrine sexual
maturation and the responsiveness to the stimulatory feedback of estradiol were characterized in these androgenized
females and compared to those in normal males and females.
Materials and Methods
Animals
The method used to obtain masculinized female lambs was identical
to that used in our earlier study (7), except that the dose of testosterone
was varied to delineate the influence of amount of steroid on the developing brain. Three groups of pregnant Suffolk ewes with known
conception dates received nine weekly injections of testosterone cypionate at doses of 200, 80, and 32 mg/week (in 1.0 ml cottonseed oil, im;
Sigma Chemical Co., St. Louis, MO) between approximately 30 and 90
days gestation (term is 145 days). Whereas testosterone treatment at a
dose of 100 mg/week induces variable masculinization of the genitalia
and tonic LH (7), weekly injections of 200 mg testosterone are sufficient
for complete masculinization of neuroendocrine function (8). The 80and 32-mg doses of testosterone used in the present study each represent
a 2.5-fold reduction from the next highest dose. The time of testosterone
treatment in utero spanned the critical period for sexual differentiation
of the LH surge mechanism established by Short (5) and Clarke et al. (6)
and our laboratory (7, 8).
Males (n 5 7), females (n 5 5), and three groups of androgenized
females (200 mg group, n 5 5; 80 mg group, n 5 6; 32 mg group, n 5
6) with a mean (6 sem) birth date of April 5 6 1.2 days were used. At
1 week of age, these lambs along with their mothers were transported
to the Reproductive Sciences Program Sheep Research Facility in Ann
Arbor from the breeder (Wallen Farm, Hubbard Lake, MI). All lambs
were housed outdoors with their mothers until weaning. At 8 weeks of
age, the lambs were weaned and raised on a commercial pelleted diet
containing 20% protein, supplemented with vitamins, minerals, and
alfalfa hay to maintain a rapid growth rate. Water was available at all
times. Body weights were determined weekly and are plotted in Fig. 1.
Surgery and steroid replacement
To assess neuroendocrine sexual maturation, the timing of the pubertal rise in LH was determined in a model used routinely in our
1073
laboratory (gonadectomized lamb chronically treated with estradiol).
Estradiol is an important feedback hormone regulating LH secretion
before puberty in the female lamb (reviewed in Ref. 4) as well as in the
male (7, 10, 11). With the initiation of sexual maturity, a marked decrease
in sensitivity to the inhibitory feedback action of estradiol contributes to
a sustained rise in circulating gonadotropins. The pronounced increase
in LH in ovariectomized estradiol-treated females coincides with the
initiation of ovulations and estrous cycles in ovary intact females (12).
Likewise, the tonic LH secretion increases in orchidectomized estradioltreated male lambs coincident with the onset of testicular growth and
spermatogenic cycles in gonad-intact males (13). To standardize the sex
steroid environment in both males and females during development,
endogenous steroids were removed via gonadectomy, and animals were
then replaced with an estradiol-containing capsule implanted sc to provide constant steroid feedback. The capsule consisted of SILASTIC tubing (od, 0.46 cm; id, 0.34 cm; Dow Corning, Midland, MI) with a 30-mm
packed column of crystalline 17b-estradiol (Sigma Chemical Co.), which
was sealed with SILASTIC adhesive type A (Dow Corning). Estradiol
implants were inserted at 2 weeks of age; they were preincubated in
water overnight before insertion to prevent a postimplantation peak in
steroid release (14). There are no sex differences in serum concentrations
of estradiol resulting from these implants (3), and by 20 weeks of age,
each implant maintains estradiol levels in lambs at approximately 3–5
pg/ml (3, 12). At 3 weeks of age, untreated and androgenized females
were ovariectomized under anesthesia [atropine (0.2 mg/kg), xylazine
(0.1– 0.2 mg/kg), and ketamine (10 –20 mg/kg)] via a midline abdominal
incision. Testes were removed at 1 week of age under local lidocaine
anesthesia. Description and measurement of the external genitalia from
each lamb were performed at the time of gonadectomy.
Tonic LH
Beginning at 2 weeks of age, 5-ml blood samples were collected by
jugular venipuncture twice weekly throughout the 40-week experimental period to monitor changes in LH secretion. Samples were allowed to
coagulate overnight, serum was decanted and then frozen until assayed
for LH by RIA. The timing of neuroendocrine puberty in gonadectomized, estradiol-treated lambs was determined from the pattern of circulating LH concentrations according to a criterion established previously in our laboratory (15). The onset of the pubertal rise was defined
as the age when the first of six consecutive LH samples (3 weeks)
exceeded 1 ng/ml. This age was compared among the five groups using
ANOVA, and post-hoc comparisons were made using Scheffe’s F test
(Statview SE1 Graphics, Brainpower, Calabasas, CA).
LH surge
The influence of prenatal androgen dose on the positive feedback
system postpubertally was studied in androgenized females along with
control males and females using an identical paradigm as that in our
previous studies (16, 17). To maximize the amplitude of LH release, the
single estradiol capsule was removed when the lambs reached about 40
weeks of age. Three weeks later, the surge induction protocol was begun.
Blood samples (5 ml) were collected every 2 h for 12 h before and 60 h
after implantation of four 30-mm SILASTIC estradiol capsules (described above). This estrogen treatment produces high physiological
levels of the steroid (12 pg/ml) and is sufficient to induce a LH surge
in normal female lambs (18). Two criteria established previously by our
laboratory (19) were used to define a LH surge. First, circulating LH
concentrations must be sustained above the pretreatment baseline for at
least 8 h (four samples). Second, the peak concentration of LH must
exceed at least twice the average of the preestradiol treatment concentration. The incidence of surges was compared between the groups using
Scheffe’s F test (Statview SE1 Graphics, Brainpower). x2 analysis was
used to compare the proportion of animals in the various prenatal
treatment groups responding to the stimulatory feedback action of estradiol with a LH surge.
LH assay
FIG. 1. Growth of males (filled squares), females (filled circles), and
androgenized females (open symbols) during the experimental time
period.
LH was measured in duplicate 25- to 200-ml aliquots using modifications (20, 21) of a RIA developed by Niswender et al. (22, 23). The assay
sensitivity, as defined by 2 standard deviations from maximum binding,
1074
BRAIN DOSE-RESPONSE TO PRENATAL ANDROGENS
was 0.87 ng/ml for 200 ml serum (16 assays) expressed relative to NIH
LH-S12. Intraassay coefficients of variation, determined from two quality control pools of 20% and 80% on the standard curve, averaged 7.5%
and 16.7%, respectively; the interassay coefficient of variation averaged
17.5%.
Results
External genitalia
Schematic views of the external genitalia of males, females,
and representative androgenized females are presented in
Fig. 2. The 200-mg dose of testosterone masculinized the
external genitalia of females producing a penis and an empty
scrotum as in our previous study (8). However, these females
had a significant (P , 0.05) posterior displacement of the
penis compared to normal males. Females receiving the intermediate testosterone dose of 80 mg in utero were partially
masculinized. All possessed a split scrotum, with the urethral opening lying between the two empty scrotal folds. The
vulvae were considerably turgid in these females. The position of the urethral opening lay between that for normal
males and females, but significantly more posterior than that
in the 200 mg treatment group. The 32-mg dose of testos-
Endo • 1997
Vol 138 • No 3
terone produced minimal masculinization of the external
genitalia. Although the vulvae were slightly turgid, these
females were otherwise indistinguishable from untreated
females. The position of the urethral opening was similar to
that for normal females. In none of the androgenized females
were any obvious abnormalities found in the uterus or ovaries upon gross visual inspection at the time of ovariectomy.
There were no significant differences in body weight between male and female lambs during the course of this study
(Fig. 1). Moreover, as reported previously (7), prenatal exposure to androgens had no effect on body weight during the
first 30 weeks of life.
Tonic LH
Figure 3 presents the mean (6sem) circulating LH concentrations in biweekly blood samples collected from 2– 40
weeks in males, females, and androgenized females. For
males, a sustained increase in circulating LH, reflecting a
reduction in the response to estradiol negative feedback,
began at 8.3 6 0.9 weeks. This sustained increase in serum
LH concentrations above 1 ng/ml did not occur in normal
females until 27.1 6 0.7 weeks.
The two highest androgen doses advanced the initiation of
the pubertal LH rise in females. In the 200 mg androgenized
group, LH concentrations began to increase at 10.2 6 2.0
weeks, an age comparable to that for normal males (P . 0.05).
In 80 mg androgenized females, the pubertal rise in LH
occurred at 16.2 6 1.5 weeks, which was significantly earlier
than that in normal females (P , 0.05), but later than that in
normal males (P , 0.05). The 32 mg females exhibited the
beginning of the sustained LH rise at 24.6 6 1.9 weeks of age,
which was comparable to that in normal females (P . 0.05).
LH surge
FIG. 2. Effects of prenatal testosterone on the external genitalia of
postnatal female lambs viewed from the left as sagittal hind quarter
diagrams of a normal male (top), female (bottom), and representative
androgenized females (middle); the penis and scrotum are shaded.
Indicated is the mean (6SEM) position of the urethral opening (penis
or vulva) relative to the distance between the anus (0) and the navel
(1).
Figure 4 depicts the pattern of LH secretion in response to
the stimulatory feedback effects of estradiol in representative
lambs from each group. Exogenous estradiol produced LH
surges in all five normal females, and LH concentrations
increased to a peak of 131.4 6 20.2 ng/ml. In four of five
females, the peak of the LH surge occurred synchronously at
18.5 6 1.0 h, a time comparable to that in our previous studies
using the identical surge-inducing paradigm (19). However,
one female (no. 551) did not increase LH secretion until 50 h
after the beginning of estradiol treatment. She was considered to be a physiological outlier because of this long latency
period; when her data were included, the overall mean time
was 24.8 6 6.3 h. Surges were absent in all normal males (n 5
7). Like males, none of the 200 mg (n 5 5) and 80 mg (n 5
5) androgenized females produced a LH surge during the
72-h sampling period. However, the 32 mg androgenized
females showed variability in their responses to estradiol,
with most being unable to produce a LH surge. Of the six
females in this group, LH concentrations were never sustained above pretreatment levels in four animals. The remaining two females produced LH surges at 26 (92 ng/ml)
and 40 (51 ng/ml) h after estradiol treatment. As a group, the
proportion of 32 mg females responding (two of six) was not
different from that of the 200 mg androgenized females or
males (no response, P . 0.05).
BRAIN DOSE-RESPONSE TO PRENATAL ANDROGENS
FIG. 3. Timing of neuroendocrine sexual maturity in normal males
(top), females (bottom), and androgenized females (middle). LH concentrations are represented as the mean 6 SEM per group. Closed
arrowheads indicate the average age at initiation of sexual maturity
for the group. Open arrowheads indicate the timing of a sustained
increase in LH above 1 ng/ml for each individual.
Discussion
The results of the present study extend our previous findings (7, 8) that exposure to prenatal testosterone during a
critical period of brain development can advance the timing
1075
of neuroendocrine sexual maturity as well as abolish the LH
surge in females. By altering the amount of testosterone in
utero, we found that a dose-dependent relationship exists
between the amount of prenatal steroid exposure and the
timing of neuroendocrine sexual maturation. Of significant
interest is the finding that exposure to low doses of testosterone abolishes the LH surge system without advancing the
time of the pubertal rise in tonic LH secretion. These results
provide further evidence for our hypothesis that different
steroid requirements exist for sexual differentiation of tonic
and surge modes of LH secretion (8).
The developing neuroendocrine system may be sensitive
to the timing, dose, or type of steroid (androgen vs. estrogen)
in utero. In addition, tonic and surge modes of gonadotropin
secretion may be differentially responsive to each of these
factors. Our previous study of androgenized lambs investigated the importance of timing. By subdividing the putative
60-day critical period for sexual differentiation of gonadotropin secretion (30 –90 days gestation) into smaller segments, we determined that tonic and surge modes of LH
secretion can be differentially influenced by the timing of
prenatal steroid exposure (8). For lambs exposed to androgens early (30 –51 days) or late (65– 86 days) in gestation, it
was possible to advance the timing of neuroendocrine sexual
maturation without preventing the LH surge. Females exposed to androgens throughout the full 60-day period, however, had a defeminized surge system as well as an advanced
neuroendocrine puberty. However, in that study of the timing of androgen exposure, the amount of steroid was confounded with the duration of steroid treatment. That is, a
longer duration of steroid treatment resulted in a greater
total steroid exposure. Because it is reasonable to expect that
the developing neuroendocrine system is also sensitive to the
amount of steroid exposure, our current experiment focused
solely on dose.
The combined findings from our current study of dose and
our previous study of timing (8) begin to provide some
understanding about the organization of the LH surge system by prenatal steroids. From our timing study, females
treated with testosterone early (30 –51 days) or late (65– 86
days) in the critical period could produce LH surges in response to exogenous estradiol. The total amount of testosterone (800 mg) provided to those females in utero was
roughly equivalent to the total amount administered to the
current 80 mg treatment group (720 mg from days 30 –90).
However, the LH surge was abolished in all of the current 80
mg treated females. These considerations suggest that defeminization of the LH surge is highly dependent on the
duration of testosterone exposure. It seems that exposure
during the entire critical period (30 –90 days gestation) is
necessary to organize central mechanisms to render the LH
surge system inoperable after birth. The early and late treatment groups used previously were each only exposed to
testosterone during a portion of this developmental window,
and consequently, the LH surge remained functional. However, the current 80-mg treatment covered a much broader
time interval, the entire critical period, and abolished the
function of the LH surge mechanism postnatally.
On the other hand, the amount of steroid, within the range
of doses tested, is much less important for the defeminization
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BRAIN DOSE-RESPONSE TO PRENATAL ANDROGENS
Endo • 1997
Vol 138 • No 3
FIG. 4. Representative patterns of LH secretion in response to acute estradiol stimulation in gonadectomized males, females, and androgenized
females. At 0 h, four estradiol implants were inserted to raise circulating levels to approximately 12 pg/ml. Blood samples were collected for
12 h before and 60 h after steroid administration. Shading indicates the average LH concentration before estradiol treatment.
of the surge system. Both the current 200- and 80-mg testosterone treatments defeminized the surge mechanism. The
finding that the LH surge was not functional in most (four
of six) animals in the 32-mg treatment group suggests that the
steroid threshold required for defeminization is relatively
low. Small amounts of steroid are capable of abolishing the
surge as long as they are present from 30 –90 days gestation.
Therefore, it seems that the duration of exposure, and not the
dose of steroid, is critical for defeminizing the LH surge.
The prenatal organizational action of steroids on the postnatal control of tonic LH secretion seems more complex.
From our previous study (8), females treated with testosterone in utero early (30 –51 days) or late (65– 86 days) in the
critical period had an intermediate advancement in the onset
of neuroendocrine sexual maturity (19 –20 weeks of age). The
current 80 mg treatment group, with a similar steroid dose
but for the full duration of the critical period (prenatal days
30 –90), also produced an intermediate advancement in neuroendocrine puberty at approximately 16 weeks of age. Such
results suggest that neither timing nor dose plays a commanding role in the sexual differentiation of tonic LH secretion. Rather, it seems that masculinization of tonic LH is
an integration of both timing and amount. Testosterone exposure for a short duration at high doses is not sufficient for
complete masculinization of tonic LH, as shown by the early
and late treatment groups (8). Moreover, exposure to a low
dose of testosterone for an extended period is not sufficient
for masculinization, as evidenced by the failure to advance
the timing of the pubertal LH rise in the current 32 mg
treatment group. Similarly, the intermediate 80-mg treatment, although sufficiently long in duration, was also too
small an amount of steroid to completely masculinize tonic
LH mechanisms. For complete masculinization, a high
amount of testosterone over an extended time is necessary.
This is exemplified by the pubertal rise in LH secretion occurring as in males at about 10 weeks of age both in the long
term treated females from our previous study (200 mg, days
30 –90) and in the identical treatment group from the current
study. Therefore, both duration and amount of testosterone
contribute to the masculinization of the control of tonic LH
secretion.
Little is known about the identity of steroid responsible for
sexual differentiation of the surge system. The actions of fetal
testosterone can be androgenic through direct action or reduction to dihydrotestosterone or estrogenic through aromatization to estrogen (reviewed in Ref. 1). Although in the
rodent, estrogen is responsible for many aspects of brain
differentiation (reviewed in Ref. 1), some elements of masculinization in the guinea pig are more dependent on androgens (24). In sheep, the specific steroid action is not
known, although data from the rat (25) suggest that estrogen
is the key steroid in defeminizing the LH surge. Nothing is
yet known about the specific steroids responsible for masculinization of tonic LH secretion in sheep. It is tempting to
propose that estrogen defeminizes the LH surge while an-
BRAIN DOSE-RESPONSE TO PRENATAL ANDROGENS
drogens masculinize tonic LH, clearly a hypothesis worthy
of testing.
We are currently unsure where gonadal steroids are acting
to masculinize gonadotropin secretion. A key determinant of
the sex difference in the timing of puberty in sheep is the
sexual differentiation of photoperiod responsiveness (4).
Current work from our laboratory (10) and others (26, 27)
suggests that male and female lambs have different photoperiod requirements for timing puberty. To exhibit a sustained increase in tonic LH secretion, females require the
shortening days of late summer and autumn (4); males have
no such light requirements and can initiate reproductive
activity under a variety of photoperiods, including the increasing day lengths of spring (11, 26). Furthermore, androgenized female lambs treated with testosterone from days
30 – 86 of gestation have a reduced responsiveness to photoperiod cues and an advanced timing of neuroendocrine
sexual maturity (10), much like males. These and other findings have led to the hypothesis that one important organizing
action of prenatal androgens in sheep to modify the timing
of puberty is through altering the characteristics of the photoperiod response system.
From an integration of our current and previous studies,
some understanding of the sexual differentiation of gonadotropin secretion in sheep is beginning to develop. Defeminization of the LH surge mechanism is highly dependent on
the duration of prenatal steroid exposure and much less so
on the amount. Differentiation of the control of tonic LH
secretion and the timing of its increase at puberty, however,
rely on an integration of both duration and amount of prenatal steroid for masculinization. Although it is completely
uncertain how and where testosterone is acting during development, the findings that different steroid requirements
exist for tonic and surge modes of gonadotropin secretion
suggest that the underlying neural elements may arise independently within the brain.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Acknowledgments
We are grateful to Mr. Richard and Mrs. Marilyn Wallen (Hubbard
Lake, MI) for providing high quality lambs for this study; Mr. Douglas
D. Doop and Ms. Juanita Pelt for expert technical advice and assistance;
Dr. David C. Bucholtz, Dr. Christopher L. Medina, Mr. John M. Ling, and
Ms. Heather M. Patton for their help in conducting the experiment; Gary
R. McCalla of the Sheep Research Core Facility for conscientious animal
care; the Reproductive Sciences Program Assays and Reagents Core
Facility for standardization of hormone RIA reagents; Dr. Gordon D.
Niswender, Colorado State University, and Dr. Leo E. Reichert, Jr.,
Albany Medical College, for providing reagents used in the LH assay.
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