0013-7227/91/1285-2457$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 5 Printed in U.S.A.

Prenatal Androgens Time Neuroendocrine Sexual Maturation* RUTH I. WOOD, FRANCIS J. P. EBLINGt, HELEN FANSON, DAVID C. BUCHOLTZ, STEVEN M. YELLON, AND DOUGLAS L. FOSTER Reproductive Sciences Program (R.I.W., F.J.P.E., H.I., D.C.B., D.L.F.), and Departments of Physiology (R.I.W., D.C.B.), Obstetrics and Gynecology (H.I., D.L.F.), and Biology (D.L.F.), The University of Michigan, Ann Arbor, Michigan 48109-0404; and Division of Perinatal Biology, Departments of Physiology and Pediatrics (S.M. Y.), Loma Linda University School of Medicine, Loma Linda, California 92350

ABSTRACT. The present study determined whether exposure to gonadal steroids in utero dictates the postnatal control of gonadotropin secretion in the lamb. There is a marked sex difference in the timing of neuroendocrine sexual maturation in sheep; while male lambs undergo a reduction in sensitivity to inhibitory gonadal steroid feedback by 10 weeks of age, females remain hypersensitive until 30 weeks. The hypothesis was tested that prenatal androgens advance the time of the decrease in feedback sensitivity, and hence the pubertal increase in pulsatile gonadotropin secretion. Pregnant ewes were injected each week with 100 mg testosterone cypionate im from 30-90 days of gestation (term is ~150 days). Five female lambs were born with masculinized external genitalia (penis and scrotum). These females, together with eight androgenized males, eight control males, and eight control females, were gonadectomized at 2 weeks of age and implanted with a Silastic capsule of estradiol to produce a constant steroid feedback signal. Blood samples were collected twice weekly to monitor trends in LH secretion. For determination of LH pulse frequency, samples were collected frequently (every 12 min for 4 h) at various intervals between 5

and 32 weeks of age. In males, a sustained increase in LH from biweekly blood samples, indicative of reduced sensitivity to inhibitory steroid feedback, began at 10.1 ± 1.4 weeks (mean ± SE) of age in control males and at 5.4 ± 0.1 weeks in androgenized males. By contrast, control females remained hypersensitive much longer as evidenced by the delay in the LH rise until 27.2 ± 0.8 weeks. The response of the five androgenized females was intermediate; LH increased at 4, 7, 16, 20, and 21 weeks of age with an early increase of LH being associated with more pronounced masculinization of the genitalia. Patterns of pulsatile LH secretion reflected differences in serum LH measured from biweekly blood samples. For example, at 20 weeks of age, before the pubertal LH rise in female lambs, no pulses were evident in control females, whereas LH pulse frequency averaged 1.6 ± 0.7 pulses/4 h in androgenized females. At this age, postpubertal males had 2.8 ± 0.5 LH pulses/4 h. These results lead to the conclusion that in the sheep, prenatal androgens can masculinize patterns of gonadotropin secretion, and that the timing of reproductive neuroendocrine maturation after birth is programmed by androgens in utero. (Endocrinology 128: 2457-2468, 1991)

T

HE OBJECTIVE of this study was to determine whether the reproductive neuroendocrine system is organized prenatally by gonadal steroids to alter the physiological control of gonadotropin secretion after birth. In this regard, recent studies from our laboratory have identified a marked sex difference in the timing of neuroendocrine sexual maturity in sheep (1). In both male and female lambs, the ability to produce the high frequency pulses of LH required for puberty is attained Received November 26, 1990. Address all reprint requests and correspondence to: Douglas L. Foster, Room 1101, 300 North Ingalls Building, The University of Michigan, Ann Arbor, Michigan 48109-0404. * This work was supported by research and training grants from the NIH (Grants HD-07048, HD-18258, and HD-18394), Biomedical Support to the Vice President for Research at The University of Michigan, the United States Department of Agriculture (Grant 89-37240-4561), and a Regent's Fellowship from The University of Michigan (to R.I.W.). Preliminary reports of this work were presented at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990. t Present address: Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DT, U.K.

within a few weeks after birth (2). These are not expressed immediately, however, because the system is highly sensitive to the inhibitory feedback actions of gonadal steroids (3). The duration of the period of hypersensitivity is dependent upon the sex of the lamb. In females, it is prolonged and lasts until about 30-35 weeks of age. At this time, sensitivity to steroid feedback becomes reduced to allow expression of high frequency LH pulses that drive the follicle to the preovulatory stage, thereby initiating puberty (1, 4). In the male, the period of hypersensitivity to steroids is abbreviated; by 10-15 weeks of age feedback sensitivity becomes reduced, and more intense stimulation of the testes begins (1, 5). The factors responsible for differentiation of neuroendocrine sexual maturation have not yet been determined. The present study tested the hypothesis that exposure of the brain to testosterone during a critical period of fetal development alters the control of tonic LH secretion, leading to a younger age of puberty in the male. Our

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approach was to administer testosterone to pregnant sheep and monitor the pattern of LH secretion in their female lambs.

utero masculinization of female lambs. The paradigm of testosterone cypionate injections in the present study provided half the amount of steroid per injection, but at twice the frequency used by previous investigators (9, 10).

Materials and Methods

Measurement of testosterone in fetal and maternal circulation (Fig. 1, left). To monitor the levels of androgens in maternal circulation, blood samples were collected from each ewe by jugular venipuncture and assayed for testosterone. Daily samples were collected for 3 days before testosterone treatment, and for 3-4 days after the first (30 days of gestation), the fifth (60 days), and last testosterone injection (90 days of gestation) to determine serum concentrations of testosterone achieved by exogenous androgens. Samples were also collected biweekly throughout the entire treatment period to assess changes in circulating androgens over time. In addition, to estimate transplacental transfer of testosterone to the fetus, samples of maternal and fetal blood were collected daily from a separate group of four chronically catheterized pregnant ewes and their fetuses (one female, three males) for 2 days before, and 1 week after a single maternal testosterone injection during late gestation (130-137 days). Late gestation fetuses were used because of the technical difficulty of obtaining repeated serum samples from fetal lambs early in gestation. Venous and arterial catheters were surgically implanted in both the fetus and the mother as described previously (13).

Androgenization of female lambs in utero Synchronization of breeding activity. To provide androgenized lambs of similar birth date, breeding was synchronized in 15 Suffolk ewes by a method described elsewhere (6). Briefly, during the autumn breeding season, adult females with evidence of at least one estrus period (as determined by the use of a vasectomized ram with marking paint) received an implant impregnated with progesterone in the axillary region to synchronize estrus. Each progesterone implant (length, 5 cm; diameter, 0.75 cm), composed of crystalline progesterone (Sigma Chemical Co., St Louis, MO) and medical grade Elastomer

(Factor II, Lakeside, AZ) in a 1:9 ratio, produces about 1 ng/ ml circulating progesterone (7). After 2 weeks, the implants were removed, and the ewes were mated with a Suffolk ram at the resultant estrus. Testosterone treatment (Fig. 1, left). To induce masculinization of female lambs, pregnant ewes received nine weekly injections of testosterone cypionate (100 mg in 0.5 cc cottonseed oil im, Sigma) in the adductor magnus muscle, beginning 30 days after conception. Maternal testosterone treatment extended until 90 days of gestation (~150 days is term), encompassing the period when the developing fetus is sensitive to the masculinizing effects of gonadal steroids (8). Previous studies have used repeated im injections of testosterone cypionate, a long acting androgen (9, 10) or implants of testosterone (8, 11, 12) for in 80

PRENATAL TREATMENT

TREATMENT

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GESTATIONAL AGE (days)

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FIG. 1. Left, Testosterone treatment of pregnant ewes. Arrows represent weekly injections of testosterone cypionate. Horizontal lines indicate duration of blood sampling to determine maternal and fetal testosterone concentrations. Right, Growth of males (open circles), females (triangles), and androgenized male (squares) and female lambs (closed circles) in relation to timing of experiments.

Postnatal treatment General methods. Five androgenized females and eight androgenized male lambs were born in spring (April 12 ± 2 birthdate, mean ± SE, range April 1-21). Additional male and female Suffolk lambs (n = 8 each, April 10 ± 1 birthdate, range April 1-15) were purchased (Breasbois Farms, Freeland, MI) to serve as controls. These lambs were transported with their mothers to the Reproductive Sciences Program Sheep Research Facility in Ann Arbor at 1 week of age. Lambs were housed outdoors with their mothers until weaning at 8 weeks of age. Water was available at all times. From weaning, they were fed a commercial pelleted diet containing 20% protein supplemented with vitamins, minerals, and alfalfa hay to maintain a rapid rate of growth. Lambs were weighed weekly. Weights are plotted in Fig. 1 (right). Gonadectomy and steroid replacement. To standardize the hormonal milieu in male and female lambs, the gonads were removed, and steroids were replaced with an estradiol implant to provide a constant steroid feedback signal. At 2 weeks of age, untreated females and androgenized females were ovariectomized via midventral laparotomy under acepromazine-ketamine (0.4 mg/kg and 10 mg/kg, respectively) anesthesia to remove inhibitory gonadal steroid feedback. Untreated males and androgenized males were orchidectomized at 2 weeks of age. After gonadectomy, each lamb was implanted sc with a small Silastic capsule (od 0.46 cm, id 0.34 cm, Dow Corning, Midland MI) containing a 30-mm packed column of crystalline 17j8-estradiol (Sigma), and sealed with Silastic adhesive type A (Dow Corning). Before implantation, all estradiol capsules were preincubated in water for 16-18 h to prevent a peak in postimplantation steroid release (14). This implant maintains estradiol in lambs at a constant level of approximately 3-5 pg/

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PRENATAL ANDROGENS ADVANCE PUBERTY ml (1, 4). Estradiol was used in both male and female lambs because males have a similar degree of responsiveness to estradiol and testosterone with regard to the regulation of LH pulse frequency (5), and because estradiol is considered to serve an important role in inhibitory steroid feedback in the ram (15). Steroid-dependent LH secretion. Beginning at 4 weeks of age, 5 ml blood samples were collected from each lamb by jugular venipuncture twice each week to monitor continuous changes in LH secretion throughout the 30-week experimental period. In the estradiol-treated gonadectomized lamb, a sustained rise in serum LH concentration from samples collected infrequently reflects an increase in LH pulse frequency and results from the reduction in sensitivity to inhibitory steroid feedback at puberty (Ref. 16 for review). In addition to infrequent estimates of LH, we examined the underlying changes in LH pulse frequency at discrete times throughout the peripubertal period. Blood samples (2.5 ml) were collected frequently (every 12 min for 4 h) at 5, 8, 11, 20, 23, and 32 weeks of age. Samples were allowed to clot overnight, and the serum was decanted and frozen until assayed for LH by RIA. Evaluation of the gonadotropin surge mechanism. We previously determined that physiological doses of estradiol exert no stimulatory feedback effect on the secretion of LH in the adult ram (17). Moreover, Short (11) and Clarke and associates (18, 19) have reported that many androgenized females fail to produce an LH surge in response to exogenous estradiol. To evaluate the effectiveness of our androgenization paradigm, and to confirm previous reports on the inability of androgenized female lambs to generate an LH surge, we attempted to induce surges in males, females, and androgenized female lambs at 20 weeks of age. Although females are still prepubertal at this age, the

amplitude of an induced LH surge by 20 weeks is maximal (20). Accordingly, all lambs were treated with three additional implants of estradiol to raise serum concentrations to approximately 12 pg/ml, a treatment known to evoke an LH surge in the female lamb, based upon studies of sensitivity to estradiol before puberty (21). To monitor LH levels during surge induction, blood samples were collected every 2 h for 48 h. Steroid-independent LH secretion. We recently reported that the control of tonic LH in the absence of inhibitory steroid feedback is also sexually differentiated in sheep (1). In the absence of steroids, LH pulses from 5 weeks through at least 35 weeks of age were more rapid in males (males, 5.3 ± 0.2 pulses/4 h; females, 3.9 ± 0.2 pulses/4 h). To determine whether this higher steroid-independent LH pulse frequency in the male is due to prenatal androgens, we measured the pulse frequency of LH in agonadal androgenized females and compared it to that of untreated agonadal males and females. Because the primary aim of this study was to determine patterns of LH secretion in the presence of steroid feedback, we determined steroid-independent LH pulse frequency at the close of the study, after both males and females had reached puberty. At 33 weeks of age, estradiol implants were removed from all lambs in late autumn; 3 weeks later, when the lambs were 36 weeks of age, samples were collected every 6 min for 4 h.

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Assays LH assay. LH was measured in duplicate 25-200 ix\ aliquots of serum using modifications (22, 23) of a RIA developed by Niswender et al. (24). The mean sensitivity, defined as 2 SD above maximum binding, was 0.55 ± 0.05 ng/ml for 200 ^1 serum (14 assays), expressed relative to NIH-LH-S12. Intraassay coefficients of variation (CV), determined from 2 quality control pools of 53% and 22% on the standard curve, averaged 8.0% and 9.7%, respectively; interassay CV averaged 12.8%. Testosterone assay. Testosterone was measured in duplicate 50 fd serum aliquots using a commercial solid-phase RIA kit (CoatA-Count, Diagnostic Products Corporation, Los Angeles, CA). Assay sensitivity averaged 0.03 ± 0.01 ng/ml (three assays). To determine whether this kit was suitable for use in sheep, we compared the values of three quality control sera that had been determined previously in an assay validated for measurement of testosterone in sheep serum (5). In the Coat-A-Count assay, these pools of 1.2 ± 0.03, 6.2 ± 0.2, and 11.7 ± 0.8 ng/ml averaged 55%, 28%, and 20% on the standard curve, respectively. These values were similar to those reported previously (1.3 ± 0.07, 5.54 ± 0.3, and 9 ng/ml). Intraassay CV in the Coat-A-Count kit averaged 13%, and interassay CV averaged 7%. Data analysis Biweekly samples for LH and neuroendocrine sexual maturation. We used the pattern of LH from samples collected biweekly to specify the time of neuroendocrine sexual maturation in gonadectomized estradiol-treated lambs. According to criteria established in our laboratory (25), the first of six consecutive LH samples exceeding 1 ng/ml marks the onset of neuroendocrine sexual maturity. Using this criterion, neuroendocrine sexual maturity in gonadectomized lambs bearing an implant of estradiol coincides with the onset of ovarian cycles or spermatogenesis in gonad-intact male and female lambs, respectively (1). LH pulse detection. LH pulses in samples collected at 12-min intervals for 4 h were identified using the Pulsefit algorithm (26). Pulsefit has been used previously for detection of LH pulses in sheep serum (27). Briefly, this algorithm is based upon a mathematical model that describes the variation of LH concentration in serum over time. It assumes that the release of LH by the pituitary gland occurs at a fairly steady rate and is a relatively rapid event compared to the time between blood samples, that the decline of the LH concentration after a release event is well modeled by an exponential decay curve, that the baseline concentration of LH that would be found in the absence of pulses is constant over time but not necessarily zero, and that the error or uncertainty of determination of the actual LH concentration is proportional to the concentration. Based upon these assumptions, Pulsefit uses a combination of nonlinear parameter estimation to determine the exponential decay rate, and stepwise linear regression to determine the timing and the sizes of the release events. The algorithm is iterative in nature; it starts with a trial solution that is successively refined to provide a best fit to the observed data series. We compared the results of the Pulsefit analysis to one historically used by our laboratory, which detects pulses ac-

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Endo • 1991 Voll28«No5

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cording to criteria modified from those established by Goodman and Karsch (28). A total of 194 profiles were analyzed with both pulse detection methods. In 124 profiles (66%), the 2 techniques identified the same number of pulses. In 35 profiles (18%), Pulsefit identified fewer pulses than Goodman-Karsch. Conversely, in 31 profiles (16%), Pulsefit identified more pulses. LH surge identification. LH surges induced by increasing circulating estradiol were identified using the criteria of Legan et al. (7, 29). According to this method, LH levels must be sustained above 5 ng/ml for at least 8 h. Statistical analyses. LH pulse frequency, derived from the Pulsefit analysis, was analyzed using between subjects mixed twofactor analysis of variance (ANOVA) with repeated measures (Statview SE+ Graphics, Brainpower Inc., Calabasas, CA). To equalize variance, the values for LH pulse frequency were square-root transformed. After ANOVA, comparisons between groups at specific times were made by Scheffe F-test. Comparison of LH pulse amplitude between groups and over time was carried out using ANOVA for those profiles in which one or more pulses was detected. Changes in body weight were likewise compared using ANOVA. In all analyses, P < 0.05 was considered significant. Undetectable LH concentrations were assigned the limit of detection of the assay. Results are presented as the mean ± SE.

Results Effects of testosterone treatment upon pregnant ewes (Fig. 2) Maternal testosterone concentrations throughout midgestation are shown in Fig. 2. Before the first injection at 30 days of gestation, maternal testosterone remained at or below the limit of assay sensitivity (0.03 ± 0.01 ng/ ml). Testosterone concentrations in maternal circulation reached a peak of 3.1 ± 0.3 ng/ml on the day after the first injection of testosterone cypionate. Serum testosterone decreased slowly during the next week, reaching a nadir of 1.7 ± 0.1 ng/ml on the day of the second injection. As determined by twice weekly samples, maternal testosterone was maintained between 1.7 ± 0.1 and 4.1 ± 0.6 ng/ml throughout the treatment period. Average daily testosterone concentrations were greater after the fifth testosterone cypionate injection than after the first or last injection (P < 0.05). There was no effect of fetal sex upon maternal testosterone concentrations (P > 0.05), and the degree of androgenization of female lambs was unrelated to testosterone concentration in maternal circulation. Transplacental transfer of testosterone cypionate In chronically catheterized late gestation fetuses, testosterone in fetal circulation averaged 0.3 ± 0.05 ng/ml for females (n = 1) and males (n = 3). Maternal androgens were undetectable ( 0.05) but was greater than that of control males (1.9 ± 0.2 kg/week) and females (1.7 ± 0.1 kg/week; P < 0.05). Growth after weaning, averaging 1.8 ± 0.1 kg/week in all lambs, was not different between groups.

.9

.6

Biweekly LH measurements (Fig. 4)

.6

I

H

.5 .4

1 vulva

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FIG. 3. Effects of testosterone in utero on external genitalia of female lambs. Left, Diagrammatic sagittal section through the hind-quarters of a normal male (top), normal female (bottom), and each androgenized female lamb (middle) in descending order of masculinization; penis and scrotum are shaded. Right, Position of penis and scrotum along abdominal wall relative to the distance between the anus (0, right) and the navel (1, left).

scrotum was largest in the most masculinized females (nos. 906 and 913) and became progressively smaller in less masculinized individuals (nos. 912 and 902). The least-masculinized female (no. 907) had a small bifid scrotum, with the urethral opening directed backward between the two sacs of the scrotum. In the two most masculinized females, the penis lay immediately caudal to the navel. The penis was nearer to the scrotum in the remaining females. Although the urethral opening appeared similar to that of a normal male, the prepuce could not be drawn back from the glans penis, and the preputial cavity was shallow. In all androgenized females, the gross appearance of the ovaries was normal upon their removal at 2 weeks of age, an observation previously

LH measurements from blood samples collected twice weekly were used to estimate the time of neuroendocrine sexual maturity. Figure 4 illustrates mean serum LH from control males (mean ± SE, n = 8, top), control females (n = 6, bottom), and individual profiles from androgenized females (middle five panels) from 4-32 weeks of age. The reduction in sensitivity to inhibitory steroid feedback in males, measured as an increase in LH in the presence of a constant steroid feedback signal, averaged 10.1 ± 1.4 weeks of age. In females, LH remained below 1 ng/ml until 27.2 ± 0.2 weeks of age. Neuroendocrine sexual maturity in androgenized females was advanced by in utero masculinization. For the five androgenized females, LH increased at 4, 4, 16, 21, and 24 weeks of age (mean, 13.8 ± 4.2 weeks of age), with an early increase of LH associated with more pronounced masculinization of the genitalia. Pulsatile patterns of LH (Figs. 5 and 6) The increase in serum LH measured from biweekly blood samples (Fig. 4) reflects an increased frequency of LH pulses, rather than heightened pulse amplitude. Figure 5 illustrates patterns of pulsatile LH release in representative male (top) and female lambs (bottom) and in the five individual androgenized females (middle) at 5, 20, and 32 weeks of age. Figure 6 presents mean LH pulse frequency and amplitude in males, females, and androgenized females throughout the experimental period. At 5 weeks of age (Figs. 5 and 6, left), all lambs were prepubertal, and pulses of LH were infrequent (males,

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FIG. 5. Pulsatile LH secretion at 5, 20, and 32 weeks of age in a representative orchidectomized, estradiol-treated male lamb (top), a representative ovariectomized, estradiol-treated female (bottom), and from five ovariectomized, androgenized females chronically treated with estradiol {middle). 0

10

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AGE (weeks) FlG. 4. LH concentrations in gonadectomized, estradiol-treated lambs from samples collected twice weekly. Top, LH in control males (mean ± SE, n = 8). Dotted line indicates 1 ng/ml limit to define neuroendocrine sexual maturity (see Materials and Methods). Arrowheads indicate neuroendocrine sexual maturity in each lamb. Bottom, LH in control females (mean ± SE, n = 6). Middle, Individual LH profiles and the time of neuroendocrine sexual maturity for five androgenized females in descending order of masculinization.

0.8 ± 0.4 pulses/4 h; females, 0; androgenized females, 1.0 ± 0.5). However even at this young age, a sex difference in LH pulse frequency was apparent. LH pulse frequency was higher (P < 0.05) in males than in control females until 32 weeks of age in early autumn, when the control females began neuroendocrine sexual maturity. Likewise, pulses of LH were more frequent in androgenized females than in control females until 32 weeks of age (P < 0.05). For example, at 20 weeks of age (Fig. 5), no pulses were evident in prepubertal control females, whereas LH pulse frequency averaged 1.6 ± 0.7 pulses/4

h in androgenized females, and 2.63 ± 0.5 pulses/4 h in postpubertal control males. As with infrequent measures of LH, the frequency of LH pulses in androgenized females at 20 weeks of age reflected the degree of masculinization of the external genitalia. The most masculinized females had the highest LH pulse frequency (no. 906, two pulses, no. 913, four pulses), the moderately masculinized females each had one LH pulse (nos. 912 and 902), and the least masculinized female (no. 907) did not produce an LH pulse during the sampling period. At 32 weeks of age (Figs. 5 and 6, right), in late autumn, LH pulse frequency was high in males (3.1 ± 1.0 pulses/ 4 h), females (4.7 ± 1.3 pulses/4 h), and androgenized females (5.0 ± 0.7 pulses/4 h). In some cases, the very rapid LH pulses were not readily identifiable using a 12min sampling interval (note no. 902, Fig. 5, right). During the experimental period, LH pulse amplitude varied considerably both within and between individuals. Importantly, the pubertal increase in LH from samples collected twice per week was associated with increased

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L H Dulse 4,~J? ~~~ frequency

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control males, mean ± SE, n=8& androgenized females, n=5m control females, n=6u #

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FIG. 6. Top: Mean (±SE) LH pulse frequency at 5, 8, 11, 20, 23, and 32 weeks of age in gonadectomized, estradiol-treated males (shaded bars), females (open bars), and androgenized female lambs (solid bars). Bottom, LH pulse amplitude.

LH pulse frequency, not pulse amplitude. Between 5 and 23 weeks of age, LH pulse amplitude in males and androgenized females did not differ (Fig. 6, P > 0.05). Likewise, at 32 weeks of age, pulse amplitude of LH in sexually mature females was not different from that of males and androgenized females {P > 0.05). Evaluation of the LH surge mechanism (Figs. 7 and 8) The ability of prenatally androgenized females and untreated male and female lambs to produce a preovulatory surge of LH at 20 weeks of age was evaluated. Treatment with additional Silastic implants to elevate estradiol to high follicular phase levels [~12 pg/ml (21)] induced an LH surge in five of six control female lambs within 48 h (Fig. 7, bottom, representative). In these females, LH remained low or undetectable until 22 ± 1.7 h after insertion of estradiol implants. According to the criteria of Legan et al. (7, 29), in which LH must exceed 5 ng/ml for 8 h, the LH surge in control females lasted 14 ± 2.1 h and reached a peak amplitude of 58.6 ± 28 ng/ml. Estradiol neither induced a surge in control males, nor did it suppress tonic LH release, as evidenced by mean levels of LH before (5.3 ± 0.3 ng/ml) and after (4.9 ± 0.4 ng/ml) estradiol treatment (Fig. 8, top). In androgenized females, prenatal testosterone abolished the stimulatory feedback actions of estradiol on LH secretion. Like the control males, none of the androgenized females produced an LH surge during the 48-h sampling period. However, unlike control males, estradiol inhibited secretion of LH in the androgenized females. Mean LH, in samples collected every 2 h, decreased during

12

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36 48

TIME (h) FIG. 7. LH response to follicular phase levels of estradiol in a representative gonadectomized, estradiol-treated male (top), female (bottom), and in five androgenized female lambs (middle). To induce LH surges, lambs were given additional estradiol to raise circulating concentrations to approximately 12 pg/ml (21). Samples were collected every 2 h for 48 h.

estradiol treatment in those lambs with elevated LH before estradiol treatment (Fig. 8, middle), indicating that pulsatile secretion of LH was suppressed. Neuroendocrine sexual maturity in androgenized males (Fig. 9) Neuroendocrine sexual maturity in androgenized males, as determined in samples collected twice weekly for measurement of LH, was earlier (5.7 ± 0.5 weeks of age; Fig. 9, top) than in control males (10.1 ± 1.4 weeks of age, P < 0.05). In a similar manner, pulse frequency of LH was greater in androgenized males than in control males at 5 and 8 weeks of age (Fig. 9, bottom).

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^100

males, n=8 mean±SE

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HOURS FROM ESTRADIOL FIG. 8. Mean (±SE) LH secretion before {left), and after (right) elevating estradiol to follicular phase levels [~12 pg/ml (21)] in male (top), female (bottom), and androgenized female lambs (middle). Note, the LH surge induced in female lambs between hours 24 and 48.

Steroid-independent LH secretion (Fig. 10) At 36 weeks of age, 3 weeks after removal of estradiol implants, frequent blood samples were collected to establish the pulse frequency of LH in the absence of inhibitory steroid feedback. Steroid-independent LH pulse frequency in males and females did not differ. Pulse frequency of LH averaged 6.5 ± 0.2 pulses/4 h in control females, 6.0 ± 0.9 pulses/4 h in control males (P > 0.05; Fig. 10, middle). In androgen-treated males and females, LH pulse frequency averaged 6.67 ± 0.5 and 7.75 ± 0.5 pulses/4 h, respectively.

Discussion The timing of puberty is sexually differentiated in the sheep (1) and many other species, e.g. ferret (30), red kangaroo (31), eland, nyala (32). In sheep, the young male begins neuroendocrine sexual maturation, increas-

FIG. 9. Effects of prenatal testosterone supplementation on neuroendocrine sexual maturation in male lambs. Top, Mean (±SE) LH concentrations in androgenized, gonadectomized, estradiol-treated males (left) and in castrated males bearing an estradiol implant (right) from samples collected twice weekly. Dotted line indicates 1 ng/ml limit to define neuroendocrine puberty (see Materials and Methods). Arrowheads indicate neuroendocrine puberty in each lamb. Middle, Serum LH in a representative androgenized male (left) and untreated male (right) at 5 weeks of age. Bottom, LH pulse frequency in androgenized males (solid bars) and untreated males (open bars) at 5, 8, 11, 20, and 32 weeks of age.

ing the frequency of LH pulses, some 20 weeks before the young female lamb (1). However, the control of the sex difference in the timing of puberty is not well understood. The present study determined whether neuroendocrine sexual maturation can be advanced by testosterone in utero. We monitored the pubertal increase in LH release in the presence of a constant steroid feedback signal in prenatally androgenized female lambs to identify the timing of the reduction in sensitivity to inhibitory steroid feedback. The focus was on the increase in pulsatile LH secretion because our current working hypothesis is that an increase in the frequency of episodic LH secretion initiates puberty in male and female lambs (2, 33, 34). On the basis of both continuous (biweekly samples) and discrete (pulse pattern) estimates of LH, the control of tonic pulsatile LH secretion can be masculinized by prenatal androgens. The time of the reduction in sensitivity to inhibitory steroid feedback, resulting in an increase in tonic LH, in the androgenized female was not

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PRENATAL ANDROGENS ADVANCE PUBERTY ANDROGENIZED MALES

CONTROL MALES

ANDROGENIZED FEMALES

FIG. 10. Steroid-independent LH secretion in postpubertal, gonadectomized lambs 3 weeks after removal of estradiol implants. Top, Serum LH from three representative agonadal, androgenized males (left), control males (middle), androgenized females (middle, shaded) and female lambs (right). Middle, Mean (±SE) LH pulse frequency over the 4-h sampling period.

different from that of the normal male, and it began some 15 weeks before that in the normal female lamb. Previous studies in gonad-intact androgenized females did not identify the time of puberty. In these females, the LH surge is attenuated (8, 11, 19, 35), ovulation is sporadic (12, 36), and sexual behavior is masculinized (19, 36). An earlier study by Wilson and Tarttelin (9) concluded that LH measured infrequently (once per month) from gonad-intact androgenized female lambs was depressed during the first 6 months of life. By contrast, masculinization of tonic LH in our study led to an increase in LH pulse frequency even earlier than normal. However, the infrequent measures of LH in that earlier study (9) may have been insufficient to detect changes in LH secretion. Thus, in general, these results extend the findings of previous investigators on the effects of prenatal androgens on reproductive function by demonstrating that the pubertal LH rise is advanced by the actions of androgens before birth on mechanisms that control the reproductive neuroendocrine system. Perhaps the most unexpected aspect of this study was the varying degree to which prenatal androgens masculinized individual female lambs. One might postulate that all androgenized females would reach neuroendocrine sexual maturity at the same age, if all were heavily masculinized. Although our androgenization paradigm was designed to heavily masculinize the external genitalia, the appearance of the five androgenized females was not uniform. This effect may possibly be due to

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individual variation in the uptake or metabolism of testosterone by the fetus. However, no individual differences were observed in circulating maternal testosterone that could account for the variation in masculinization of their female lambs. All pregnant ewes received the same amount of testosterone during the same period of gestation, and all but one of the five androgenized females were singletons. (The female with two male siblings (no. 912) was only moderately masculinized.) The relationship between the effects of testosterone on the external genitalia and on the timing of neuroendocrine sexual maturation was striking; those females with highly masculinized genitalia (nos. 906 and 913) also began puberty earlier. One concept emerging from this and other studies is that the critical period for sexual differentiation is not one single entity. Instead, each sexually dimorphic trait may have a distinct period during which it is sensitive to the masculinizing effects of gonadal steroids. Unique periods of sensitivity to steroids for the control of anatomy, behavior, and neuroendocrine function in sheep have already been noted (8, 11, 37). Importantly, the results of the present study indicate that the critical period for masculinization of reproductive neuroendocrine function may be further subdivided. Although the LH surge was abolished in all five androgenized female lambs, control of tonic LH was not equally affected by prenatal androgens. This implies that defeminization of surge LH secretion may be more sensitive to testosterone than masculinization of tonic LH. It may be relatively easier to abolish a feminine trait than to establish a masculine quality. Before this study, we did not know the critical period for sexual differentiation of tonic LH secretion. On the basis of our results, it appears to be similar to the critical period for masculinization of the genitalia [30-80 days of gestation (8)]. The critical period for masculinization of the LH surge is protracted and is similar to that of behavioral sexual differentiation (30120 days), as defined by Clarke (37). In this regard, the brain of the developing fetus must be sensitive to minute changes in testosterone because androgens in fetal circulation only doubled after maternal treatment with testosterone cypionate. Whether the differences between tonic and surge LH sensitivity is related to the amount of testosterone reaching the developing fetal brain or the duration of exposure to androgens was not resolved in the present study. Prenatal androgenization prevented the positive feedback actions of estradiol to induce an LH surge in our female lambs. However, it appears that androgenized females do retain the initial negative feedback response to supplemental estradiol, characteristic of females, but not observed in our male lambs. The significance of this finding in the androgenized female is not clear, although

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PRENATAL ANDROGENS ADVANCE PUBERTY

it may be related to the relative sensitivity to androgens prenatally of mechanisms controlling the inhibitory and stimulatory feedback actions of estradiol postnatally. Treatment with additional estradiol in the present study did not affect LH secretion in males, although other studies have observed a suppression of LH secretion in adult males in response to acute increases in estradiol (11, 38), albeit with much higher doses of estradiol. Perhaps the male is simply less sensitive to estradiol, and higher levels of estradiol are required to suppress gonadotropins. We recently reported a more rapid LH pulse frequency in the absence of inhibitory steroid feedback in male lambs (6.25 ± 0.0.5 pulses/4 h) compared with females (4.62 ± 0.0.3 pulses/4 h) at 36 weeks of age (1). In male and female rhesus monkeys, the pulse frequency of LH is also sexually differentiated in the early neonatal period in the absence of inhibitory steroid feedback (39). Although in the present study, the pulse frequency of LH in agonadal males and females did not differ, it was greater in both groups than that which we previously reported (1). Both studies were conducted 3 weeks after removal of estradiol, and both groups of lambs had initially received the same amount of estradiol, although estradiol was present for a much longer duration in the more limited present study. Possibly the detection of a more rapid pulse frequency in the present study was a result of the increased frequency of sample collection in this study (every 6 min vs. every 12 min in the previous study). Effects of prenatal testosterone on male lambs Neuroendocrine puberty in androgenized males was not delayed relative to untreated males. In fact, the increase in tonic LH occurred earlier in androgenized males. This observation runs counter to previous studies of androgenized males. In sheep (9) and rats (40), testosterone in androgen-treated males was depressed relative to untreated males. Furthermore, LH in those gonadintact, androgen-treated male lambs measured from samples collected once per month also remained low throughout the first 6 months of life (9). Although gonadal activity may be affected by supplemental testosterone in utero, reproductive neuroendocrine function in our androgen-treated males was not compromised. This leads to the conclusion that exposure to supraphysiological levels of testosterone in utero does not adversely affect reproductive neuroendocrine development in males. It was not possible to determine whether prenatal androgen treatment actually advanced the timing of neuroendocrine puberty in our lambs because the androgen-treated male lambs grew faster than their untreated counterparts. This was most likely due to the effects of twinning.

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All the control lambs were twins, and all but one of the androgenized males were singletons. Physiological mechanisms of testosterone action Masculinization of reproductive function most likely occurs in the neural control of GnRH secretion. This is suggested by the effect of prenatal androgens to increase the frequency rather than the amplitude of LH pulses, i.e. an effect presumably mediated by GnRH release, rather than by altered pituitary sensitivity to GnRH stimulation (41). Furthermore, pituitary response to GnRH challenge is neither affected by androgenization, nor is it sexually differentiated in the sheep (36, 42). However, hypothalamic GnRH neurons themselves are not sexually differentiated in the developing sheep with regard to number of neurons or their location within hypothalamic nuclei (43). It is unknown at the present time where in the brain prenatal androgens act upon the developing reproductive neuroendocrine system because the neural pathways controlling the negative and positive feedback actions of estradiol have not been identified. Although we do not know the anatomical sites of action of prenatal steroids, we can postulate some of the effects of androgens upon the neuroendocrine control of reproduction by internal and external signals. In this regard, puberty in sheep is dependent upon information about both body size and time of year (for review, see Ref. 16). Compared to the developing female, the male and androgenized female begin reproductive activity at a smaller size and under different daylength. Thus, exposure to androgens in utero may alter the growth and/or photoperiodic requirements for puberty in males and androgenized females. With regard to the control of puberty by body size, gonadotropin secretion can be suppressed in both male and female lambs when growth is restricted by level of nutrition (44), indicating that the neuroendocrine reproductive system of both sexes is sensitive to growthrelated cues. However, under natural conditions, control females reach puberty many weeks after they achieve the minimum body size to begin reproductive maturation [30-35 kg (45)]. This suggests that, ultimately, metabolic signals do not time puberty in the normally growing female lamb. Thus, it appears that the sex difference in the timing of puberty is dictated principally by signals that are unrelated to growth. The composite information from studies conducted in male and female lambs leads to the hypothesis that the sex difference in the normal timing of puberty is governed by sex differences in the response to photoperiod information. Whereas the female lamb requires a decrease in daylength to time puberty to the early autumn (46), photoperiod has relatively little influence on the timing of puberty in the male lamb. Males begin sexual

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PRENATAL ANDROGENS ADVANCE PUBERTY maturation with minimal delay under diverse photoperiods (47-49). Our androgenized females raised outdoors under natural photoperiod achieved neuroendocrine sexual maturity during the long days of midsummer, a photoperiod not permissive to puberty in the normal female (25). Furthermore, as adults the next year, entry into the first breeding season after puberty, which is solely a photoperiodically modulated transition (41), was advanced in androgenized females relative to untreated females (Wood, R. I., and D. L. Foster, unpublished data). Overall, it appears that testosterone alters the photoperiod requirements for puberty in males and androgenized females to permit sexual maturation at an early age. Thus, in the control of sexual differentiation, another important element of the organizational actions of testosterone in utero on neural function is to alter the response to environmental cues timing onset of reproduction.

9.

10. 11. 12.

13. 14.

15. 16.

Acknowledgments We are grateful to Mr. Lee H. Breasbois (Freeland, MI) for providing high quality lambs for experimentation; Mr. Kirk C. Van Natter and Mr. Gary McCalla of the Reproductive Endocrinology Program Sheep Research Facility for conscientious animal care; Mr. Douglas D. Doop and Ms. Tovaghgol E. Adel for expert technical advice and assistance; Dr. Robert H. Kushler for Pulsefit pulse analysis software; Dr. Morton B. Brown for assistance with statistical analyses; Mr. Michael R. Muha for administrative support; the Reproductive Endocrinology Program Standards and Reagents Core Facility for standardization of hormone RIA reagents; Dr. Gordon D. Niswender, Colorado State University (Denver, CO) and Dr. Leo E. Reichert, Jr., Albany Medical College of Union University (Albany, NY) for providing reagents used in the LH

17. 18. 19. 20. 21.

assay. 22.

References 1. Claypool LE, Foster DL 1990 Sexual differentiation of the mechanism controlling pulsatile secretion of luteinizing hormone contributes to sexual differences in the timing of puberty in sheep. Endocrinology 126:1206-1215 2. Foster DL, Karsch FJ, Olster DH, Ryan KD, Yellon SM 1986 Determinants of puberty in a seasonal breeder. Recent Prog Horm Res 42:331-384 3. Foster DL, Ryan KD, Goodman RL, Legan SJ, Karsch FJ, Yellon SM 1986 Delayed puberty in lambs chronically treated with estradiol. J Reprod Fertil 78:111-117 4. Foster DL, Ryan KD 1979 Endocrine mechanisms governing the transition into adulthood: a marked decrease in the inhibitory feedback action of estradiol on tonic secretion of luteinizing hormone in the lamb during puberty. Endocrinology 105:896-904 5. Olster DH, Foster DL 1986 Control of gonadotropin secretion in the male during puberty: a decrease in response to steroid inhibitory feedback in the absence of an increase in steroid-independent drive in the sheep. Endocrinology 118:2225-2234 6. Dzuik PJ, Cook B, Niswender GD, Kaltenbach CC, Doane BB 1968 Inhibition and control of estrus and ovulation in ewes with a subcutaneous implant of silicone rubber impregnated with a progestagen. Am J Vet Res 29:2415-2417 7. Legan SJ, I'Anson H, Fitzgerald BP, Akaydin Jr MS 1985 Importance of short luteal phases in the endocrine mechanism controlling initiation of estrous cycles in anestrous ewes. Endocrinology 117:1530-1536 8. Clarke IJ, Scaramuzzi R, Short RV 1976 Effects of testosterone

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implants in pregnant ewes on their female offspring. J Embryol Exp Morph 36:87-99 Wilson PR, Tarttelin MF 1978 Studies of sexual differentiation in sheep. I. Foetal and maternal modifications and postnatal plasma LH and testosterone content following androgenization early in gestation. Acta Endocrinol (Copenh) 89:182-189 Klindt J, Jenkins TG, Ford JJ 1987 Prenatal androgen exposure and growth and secretion of growth hormone and prolactin in ewes postweaning. Proc Soc Exp Biol Med 185:201-205 Short RV 1974 Sexual differentiation of the brain of the sheep. INSERM 32:121-142 DeHaan KC, Berger LL, Kesler DJ, McKeith FK, Thomas DL, Nash TG 1987 Effect of prenatal androgenization on lamb performance, carcass composition and reproductive function. J Anim Sci 65:1465-1470 Yellon SM, Longo LD 1987 Melatonin rhythms in fetal and maternal circulation during pregnancy in the sheep. Am J Physiol 252:E799-E802 Karsch FJ, Dierschke DJ, Weick RF, Yamaji T, Hotchkiss J, Knobil E 1973 Positive and negative feedback control by estrogen of luteinizing hormone secretion in the rhesus monkey. Endocrinology 92:799-804 Schanbacher BD 1984 Regulation of luteinizing hormone secretion in male sheep by endogenous estrogen. Endocrinology 115:944-950 Foster DL 1988 Puberty in the female sheep. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 1739-1762 Karsch FJ, Foster DL 1975 Sexual differentiation of the mechanism controlling the preovulatory discharge of luteinizing hormone in sheep. Endocrinology 97:373-379 Clarke IJ, Scaramuzzi RJ, Short RV 1976 Sexual differentiation of the brain: endocrine and behavioural responses of androgenized ewes to estrogen. J Endocrinol 71:175-176 Clarke IJ, Scaramuzzi RJ 1978 Sexual behaviour and LH secretion in spayed androgenized ewes after a single injection of testosterone or estradiol. J Reprod Fertil 52:313-320 Foster DL, Karsch FJ 1975 Development of the mechanism regulating the preovulatory surge of luteinizing hormone in sheep. Endocrinology 97:1205-1209 Foster DL 1984 Preovulatory gonadotropin surge system of prepubertal female sheep is exquisitely sensitive to the stimulatory feedback action of estradiol. Endocrinology 115:1186-1189 Hauger RL, Karsch FJ, Foster DL 1977 A new concept for control of the estrous cycle of the ewe based upon temporal relationship between luteinizing hormone, estradiol, and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology 101:807-817 Ebling FJP, Schwartz ML, Foster DL 1989 Endogenous opioid regulation of pulsatile luteinizing hormone secretion during sexual maturation in the female sheep. Endocrinology 125:369-383 Niswender GD, Reichert Jr LE, Midgley AR, Nalbandov AV 1969 Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166-1173 Ebling FJP, Foster DL 1988 Photoperiod requirements for puberty differ from those for the onset of the adult breeding season in the female sheep. J Reprod Fertil 84:283-293 Kushler RH, Brown MB, A model for the identification of hormone pulses. Statistics Med, in press Ebling FJP, Kushler RH, Foster DL 1990 Pulsatile LH secretion during sexual maturation in the female sheep: photoperiodic regulation in the presence and absence of ovarian steroid feedback as determined in the same individual. Neuroendocrinology 52:229237 Goodman RL, Karsch FJ 1980 Pulsatile secretion of luteinizing hormone: differential suppression by ovarian steroids. Endocrinology 107:1286-1290 Legan SJ, I'Anson H, Fitzgerald BP, Fitzovich D 1985 Does the seasonal increase in estradiol negative feedback prevent luteinizing hormone surges in anestrous ewes by suppressing hypothalamic gonadotropin-releasing hormone pulse frequency? Biol Reprod 33:117-131 Sisk CL 1987 Evidence that a decrease in testosterone negative

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feedback mediates the pubertal increase in luteinizing hormone pulse frequency in male ferrets. Biol Reprod 37:73-81 Frith HJ, Sharman GB 1964 Breeding in wild populations of the red kangaroo Megaleia rufa. CSIRO Wildl Res 9:86-114 Georgiadis N 1985 Growth patterns, sexual dimorphism and reproduction in African ruminants. Afr J Ecol 23:75-87 Huffman LJ, Innskeep EK, Goodman RL 1987 Changes in episodic luteinizing hormone secretion leading to puberty in the lamb. Biol Reprod 37:754-760 Foster DL, Ryan KD, Papkoff H 1984 Hourly administration of luteinizing hormone induces ovulation in prepubertal female sheep. Endocrinology 115:1179-1185 Clarke IJ, Scaramuzzi RJ 1978 Release of luteinizing hormone in androgenized ewes after prostaglandin-induced luteolysis or luteinizing hormone releasing hormone. J Endocrinol 77:261-262 Clarke IJ, Scaramuzzi RJ, Short RV 1977 Ovulation in prenatally androgenized ewes. J Endocrinol 73:385-389 Clarke IJ 1977 The sexual behaviour of prenatally androgenized ewes observed in the field. J Reprod Fertil 49:311-315 Sakurai H, Adams TE, Gonadotropin secretion during continuous infusion of estradiol in oophorectomized sheep: effect of pulsatile delivery of GnRH. Program of the 23rd Annual Meeting of the Society for the Study of Reproduction, Knoxville, TN, 1990 (Abstract 186) Plant TM 1986 A striking sex difference in the gonadotropin response to gonadectomy during infantile development in the rhesus monkey (Macaca mulatto). Endocrinology 119:539-545 Piacsek BE, Hostetter MW 1984 Neonatal androgenization in the male rat: evidence for central and peripheral defects. Biol Reprod 30:344-351 Karsch FJ, Bittman EL, Foster DL, Goodman RL, Legan SJ,

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Robinson JE 1984 Neuroendocrine basis of seasonal reproduction. Recent Prog Horm Res 40:185-232 Wilson PR, Tarttelin MF 1978 Studies of sexual differentiation in sheep. II. Evidence of postnatal hypothalamic hypophysiotrophic depression of basal LH secretion following prenatal androgenization. Acta Endocrinol (Copenh) 89:190-195 Wood RI, Newman SW, Lehman MN, Foster DL, GnRH neurons in the fetal lamb hypothalamus are not sexually differentiated. Program of the 20th Annual Meeting of the Society for Neuroscience, St Louis, MO, 1990 (Abstract 4217) Wood RI, Ebling FJP, Foster DL 1991 Sex differences in nutritional modulation of gonadotropin secretion during development: studies in the growth-retarded lamb. Biol Reprod, in press Foster DL, Ryan KD 1990 Puberty in the lamb: sexual maturation of a seasonal breeder in a changing environment. In: Sizonenko PC, Aubert ML, Grumbach MM (eds) Control of the Onset of Puberty II. Williams & Wilkins, Baltimore, MD, pp 108-142 Foster DL, Ebling FJP, Claypool LE 1989 Photoperiodic timing of puberty in sheep. In: Reppert SM (ed) Development of Circadian Rhythmicity and Photoperiodism in Mammals: Research in Perinatal Medicine (IX). Perinatology Press, Ithaca, NY, pp 104-148 Courot M, de Reviers MM, Pelletier J 1975 Variations in pituitary and blood LH during puberty in the male lamb. Relation to the time of birth. Ann Biol Anim Biochim Biophys 15:509-516 Claypool LE 1985 Sexual maturation of sheep: photoperiod that delays puberty in the female does not retard prepubertal testicular growth. Program of the 18th Annual Meeting of the Society for the Study of Reproduction, Montreal, Quebec, Canada. Biol Reprod 32:[Suppl 1]176 (Abstract 278) Wood RI, Ebling FJP, I'Anson H, Foster DL 1991 The timing of neuroendocrine puberty in the male lamb by photoperiod. Biol Reprod, in press

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Prenatal androgens time neuroendocrine sexual maturation.

The present study determined whether exposure to gonadal steroids in utero dictates the postnatal control of gonadotropin secretion in the lamb. There...
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