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Regular Article |
a Departments of Pediatrics
b Biostatistics and
c the Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109
d Laboratory of Neuroendocrinology, Babraham Institute, Cambridge CB2 4AT, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, follicle-stimulating hormone, luteinizing hormone, neuroendocrinology, ovulatory cycle
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Depending on the extent of androgenization, prenatal exposure to testosterone disrupts ovarian morphology [10, 11], leads to virilization of external genitalia [5, 9, 12, 13], and induces behavioral changes [14–17], hyperinsulinemia [18], reduced sensitivity to progesterone feedback [19], and reproductive failures [10, 20]. Detailed studies conducted using the gonadectomized, estradiol-treated lamb (ovx+E) model have demonstrated that prenatal androgenization advances neuroendocrine puberty in females [21] and that the degree of advancement depends upon the timing, duration and extent of androgenization. Furthermore, studies with this model have indicated that prenatally androgenized sheep have a compromised LH surge system, the extent of disruption being dependent on the timing, duration, and extent of androgenization [9, 13].
In the ovx+E model, prenatal androgen treatment from Day 30 to Day 90 produces an early pubertal LH rise (tonic) but renders the LH surge system inoperative [9]. In females treated from Day 60 to Day 90, the pubertal LH rise is also advanced, but the surge system remains functional, albeit with a long latent period. However, predictions about the advancement of ovarian cycles and the disruption of the surge system from this model must be tested in the ovary-intact animal with a complete feedback system and ovarian target tissue. The present study was conducted to test whether 1) intact prenatally androgenized female lambs show an early rise in LH secretion, 2) the onset of reproductive (progesterone) cycles parallels the increase in LH secretion, and 3) intact prenatally androgenized sheep have a compromised gonadotropin surge system, with the extent of disruption dependent on the time/duration of androgen exposure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was conducted with Suffolk sheep maintained under normal husbandry conditions at the Sheep Research Facility (Ann Arbor, MI; 42°18'N). Pregnant ewes were injected i.m. twice weekly in the hind leg with 100 mg testosterone propionate (Sigma, St. Louis, MO) suspended in cottonseed oil at a concentration of 200 mg/ml. Injections were given either between 30 and 90 days (D30–90) or between 60 and 90 days (D60–90) of gestation. The androgenization paradigm was based on an earlier study [19] that showed early onset of neuroendocrine puberty similar to that observed in previous studies [21] where testosterone was given as a single 200-mg injection. A larger number of pregnant sheep (n = 20) were administered testosterone during gestation Days 60–90 because these D60–90 androgenized animals were to be maintained for long-term assessment of fertility parameters. This protocol resulted in 14 D60–90 androgenized female lambs (one died). Another set of pregnant sheep (n = 7) was injected with testosterone propionate during gestation Days 30–90. The external genitalia of D30–90 androgenized sheep were virilized, thus preventing mating and assessment of fertility parameters. This group yielded half of the expected number of female lambs; only three female lambs were born.
Androgenized female lambs (D30–90: n = 3; D60–90: n = 13) and control lambs (n = 10) were raised in the Sheep Research Facility. Pasture was available during the spring and summer months. Lambs were also given daily rations of pellets made of alfalfa. During the winter, hay and alfalfa pellets were available in covered areas. Lambs had ad libitum access to water and minerals and were regularly treated with anthelmintics to reduce parasitic infestations.
Experiment 1: Effect of Prenatal Androgenization on the Onset of Puberty and the First Breeding Season
This study was conducted to determine 1) whether ovary-intact prenatally androgenized animals exposed to androgen either during 60–90 (D60–90) or 30–90 (D30–90) days of gestation would have early pubertal LH and FSH increases and 2) whether the onset and maintenance of reproductive (progesterone) cycles is altered differentially under these two conditions. Effect of prenatal androgenization on the onset of puberty, maintenance of ovarian cycles, and the end of the first breeding season were studied by monitoring plasma progesterone concentrations biweekly. Starting prior to the onset of the first breeding season (August) through the end of the first breeding season (April), blood samples were collected from the jugular vein by venipuncture twice each week. To determine whether the intact prenatally androgenized sheep, like the ovx+E model, would have early pubertal increases in gonadotropins, plasma LH and FSH levels were also measured in biweekly samples collected during the prepubertal period (the first animal to become pubertal did so at 25 wk of age). In addition to measuring plasma progesterone as an indicator of cyclic activity, raddled vasectomized rams were also kept with the flock throughout the breeding period to determine the start and end of the breeding season as assessed by paint marks from the rams on the rumps of the ewes. Keeping the males with the flock also provided an index of the mating preference of the males for females of the different treatment groups. Body weights were recorded every 2 wk from 10 wk through 43 wk of age.
Experiment 2: Effect of Prenatal Androgenization on the Competency of the LH Surge System
The purpose of this experiment was to determine specifically whether the functions of the preovulatory surge system are differentially organized by prenatal treatment with androgens. Competency of the surge system to respond to positive feedback effects of estradiol was tested in both the presence and absence of progesterone priming. Testing the estradiol positive feedback without progesterone priming allowed us to determine how the LH surge system responds physiologically at the onset of puberty or during the first cycle of the breeding season when animals are not primed with progesterone. Testing the system in the presence of progesterone provided a physiological measure of what happens during the breeding season when animals are cycling and exposed to progesterone during the luteal phase. In earlier studies in cystic cows [22], progesterone treatment corrected the deficits in the LH surge system. Because prenatally androgenized animals show cystic/multifollicular ovaries, we postulated that progesterone pretreatment would overcome any abnormalities in LH surge generation, as it did in the cystic cows.
This experiment was conducted in two parts using the same animals from experiment 1 during the nonbreeding season following the first breeding season when the control animals are naturally anovulatory. All animals in all three groups were sexually mature at the time of this study and had all gone through one breeding season. In part 1 of this experiment, ewes were implanted with four 3-cm silastic estradiol implants s.c. to provide follicular phase levels of estradiol. The implants were soaked in distilled water for 24 h followed by 30 min in 70% ethanol before insertion to avoid brief peaks in circulating levels of estradiol. Details of this procedure were published previously [23, 24]. Blood samples were obtained by venipuncture from the jugular vein and collected into heparinized glass tubes every 2 h starting 4 h before and ending 72 h after placement of estradiol implants. Implants were removed immediately after collection of the last sample. Plasma was harvested and stored at -20°C until analyzed.
Part 2 of the experiment was conducted 10 wk after part 1, toward the end of the nonbreeding season. All ewes were implanted s.c. in the front axillary area with two controlled internal drug-releasing (CIDR) devices containing progesterone (InterAg, Hamilton, New Zealand). A subcutaneous site was chosen because the D30–90 androgenized females do not have a vaginal opening. Two CIDR devices provided luteal phase concentrations of progesterone in previous studies [19]. CIDR devices were removed after 12 days of implantation. Five of the D60–90 androgenized and four of the control ewes had lost one or both of the implants during this 12-day treatment period. Therefore, these ewes were excluded from the study. Sixteen hours after implant removal, four 3-cm estradiol implants were inserted s.c. as in part 1 (controls: n = 6; D60–90: n = 8; D30–90: n = 3). Jugular blood was collected every 2 h starting at -4 h through 60 h relative to placement of estradiol implants (0 h is the time of estradiol implantation). The duration of blood sampling was reduced from 72 h to 60 h in part 2 based on the results of part 1, where none of the animals initiated an LH surge later than 38 h after insertion of the estradiol implants. All procedures were approved by the University of Michigan Committee for the Use and Care of Animals.
Hormone Assays
Plasma concentrations of LH were measured in 10–200 µl of plasma by a previously validated assay [25]. The sensitivity of this assay at 200 µl plasma volume was 0.73 ± 0.03 ng/ml (mean ± SEM, n = 22 assays). The intra- and interassay coefficients of variation (CVs) based on two quality control pools were 7.5% ± 0.8% and 6.1% at 13 ng/ml and 5.4% ± 0.7% and 4.5% at 23 ng/ml, respectively. Plasma concentrations of FSH were measured using a previously validated RIA [26]. The sensitivity of the assay at 100 µl plasma volume was 0.7 ± 0.1 ng/ml (n = 22 assays). The intra- and interassay CVs were 13.2% ± 1.8% and 13.6% at 4.2 ng/ml and 5.3% ± 0.7% and 7.8% at 25.5 ng/ml, respectively. Plasma concentrations of progesterone were measured using a commercial RIA kit (Coat-A-Count P4; Diagnostic Products Corporation, Los Angeles, CA). Validation of this assay for sheep plasma has been described elsewhere [27]. Sensitivity of this assay averaged 0.04 ± 0.001 ng/ml (n = 11 assays). The intra- and interassay CVs measured using two quality control pools were 9.7% ± 0.8% and 3.1% at 0.9 ng/ml and 6.1% ± 0.9% and 1.9% at 13.5 ng/ml.
Statistical Analysis
The onset of LH and FSH surges was determined based on previously established criteria [28, 29]. The onset of an LH/FSH surge was defined as 1) elevation of circulating LH/FSH above baseline by 2 SD and lasting at least 8 h and 2) peak concentration of LH/FSH exceeding at least twice the average levels of LH/FSH during pre-estradiol periods. The end of the surge was defined as the time when LH/FSH levels fell below the established criteria of surge onset. Data from biweekly measurements of LH and FSH prior to onset of puberty were divided into three 4.5-wk blocks and averaged for analysis. Data on magnitude of LH/FSH surges were log transformed before analysis. Repeated measures ANOVA was used to determine whether there were any effects of time or treatment on the rate of body weight gain, circulating concentrations of LH and FSH, and LH:FSH ratio. Statistical comparisons for all variables used to define surge characteristics were performed only between controls and the D60–90 group. Data for the D30–90 group were not included in the analysis because only two of the three animals showed definable surges. Data are presented as means and ranges to show the tightness of responses between these two animals. Changes in surge characteristics (onset, end, peak time, magnitude, and duration) within each part of the study were analyzed using a two-sample Student t-test assuming unequal variances. Changes in surge characteristics between part 1 (no progesterone priming) and part 2 (progesterone priming) of experiment 2 were analyzed using a paired t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Regression analysis revealed similar rates of body weight gain among the three groups (Fig. 1). Controls and both groups of prenatally androgenized sheep (D60–90 and D30–90) had similar body weights at the time of pubertal onset (start of first progestogenic cycle) (Fig. 1). There were no differences in the age of onset of puberty, beginning and end of the first breeding season (defined by the start and end of progesterone cycles), total number of progesterone cycles in the first breeding season, or peak progesterone concentrations achieved among treatment groups (Fig. 2 and Table 1). One of the D30–90 androgenized females stopped cycling after exhibiting three estrous cycles. Circulating LH and FSH levels measured in biweekly samples in all three groups remained constant from 8 wk of age until puberty and showed no discernable prepubertal increase (serial data not shown). Mean plasma concentrations of LH and FSH and the LH:FSH ratio divided in three 4.5-wk blocks were also not significantly different among the three groups, although values for the androgenized animals tended to be higher (Fig. 3).
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Mean estrus detection as indicated by raddled ram paint markings on the rumps of the lambs during the entire first breeding season were 3.9 ± 0.4 markings in controls and 2.4 ± 0.4 markings in the D60–90 group. The rams did not mark any of the D30–90 androgenized animals throughout the first breeding season.
Effect of Prenatal Androgenization on the Competency of the Gonadotropin Surge System
LH surge Figures 4 and 5 indicate the positive feedback responses of LH to surge-inducing levels of estradiol in the absence and presence of progesterone. In the absence of progesterone priming, both groups of prenatally androgenized females had delayed onset of LH surges as compared with controls (Figs. 4 and 5). In control females, the LH surges started at 15.4 ± 1.2 h after the insertion of the estradiol implants. Peak LH levels were achieved 3.0 ± 0.6 h after the onset of the LH surge (Fig. 4). LH surge characteristics in the D60–90 group differed from those in the control group (Fig. 5). These animals started their LH surges 6 h later (21.4 ± 1.0 h; P < 0.001) than did the control group and took longer to achieve LH surge peak from the time of onset (4.4 ± 0.4 h vs. 3.0 ± 0.6 h in D60–90 and controls, respectively; P < 0.001). In addition, the LH surge lasted longer (P < 0.001) in the D60–90 animals compared with the controls (Fig. 5). There were no differences in the magnitude of LH surges in the control and D60–90 groups (Fig. 5).
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There was a longer delay in the timing of the onset of the LH surges in the D30–90 androgenized ewes (27 ± 1.0 h) compared with the D60–90 androgenized females, although the small number of animals in the D30–90 group did not permit a valid statistical comparison. The duration of LH surges in the D30–90 animals was longer than that in the control group but similar to that in the D60–90 group. One of the D30–90 androgenized ewes did not show a definable surge. The magnitude of the LH surge in the two D30–90 ewes that produced surges was highly reduced (one third of the control and D60–90 group values) (Fig. 5).
In part 2 of the experiment (progesterone pretreatment), the beginning time, ending time, duration, and magnitude of the LH surge were not different between the control and D60–90 groups (Fig. 5). Although the LH surges started at the same time in the D60–90 and control animals, the LH surge peak was achieved later in the D60–90 group compared with the control group (P < 0.005). The interval between the onset and the peak of LH surge in the D60–90 group was approximately 2.5 h longer (P < 0.001) than that in the controls (Fig. 4). In the D30–90 androgenized group, one of the ewes (same ewe that did not show any surge in part 1 of the experiment) showed no LH surge. The other two ewes showed delay in the onset of the LH surge as compared with the controls and the D60–90 androgenized group. As in part 1 of the experiment, the magnitude of the LH surge was reduced in the D30–90 androgenized ewes, but the D60–90 androgenized ewes did not differ from the controls (Figs. 4 and 5).
Paired analysis of data from parts 1 and 2 (test for positive feedback without and with progesterone pretreatment) using data from only those animals that were studied in both parts of the experiment revealed that progesterone pretreatment delayed the onset of LH surges in both the control and the D60–90 groups (Fig. 4). The onset of LH surge was also delayed in the D30–90 animals (Fig. 4).
FSH surge In general, the FSH responses to surge-inducing levels of estradiol were qualitatively similar to those of LH, although there were some subtle differences. In part 1 of the experiment when the competency of the surge system was tested in the absence of progesterone priming, the D60–90 ewes started their FSH surges 6 h later (P < 0.001), exhibited surge peak 7 h later (P < 0.01), and ended their surge 8 h later (P < 0.001) than did the controls (Figs. 6 and 7). D30–90 animals showed a greater delay in the time of onset, peak, and end of FSH surges relative to the D60–90 animals and the controls (Figs. 6 and 7). The small number of animals in this group precluded statistical analysis. In part 2 of the experiment (with progesterone priming), the time of onset of FSH surges in the D60–90 ewes was comparable to that of controls, although there was a delay in the time to peak (P < 0.001, Fig. 6) and end (P < 0.05, Fig. 7) of the FSH surges. One of the D30–90 androgenized ewes that did not produce an LH surge also failed to produce an FSH surge in both parts of the experiment. The magnitude of the FSH surge in the two D30–90 ewes that had FSH surges was reduced by approximately one half, and onset was delayed relative to controls and D60–90 androgenized animals (Figs. 6 and 7) when the competency was tested with and without progesterone priming. As with LH, progesterone pretreatment delayed the time of onset of FSH surges in the control (P < 0.01), D60–90 (P < 0.05), and D30–90 animals (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Prenatal Androgenization on Tonic Gonadotropin Secretion
In intact sheep, during the prepubertal period GnRH/LH secretion is highly sensitive to the inhibitory effects of gonadal steroids. As such, circulating gonadotropin concentrations remain low during this period. As the animal approaches puberty, there is an escape from the inhibitory effect of steroids, and pubertal increase in gonadotropin secretion occurs around 1–2 wk before first ovulation [30]. This increase in pubertal increase in LH was evident only in studies where changes in circulating LH were monitored frequently. In the ovx+E model, the increase in circulating LH was evident at 27 wk of age in control females, at around 10 wk of age in male lambs, and at 10 wk of age in the prenatally androgenized animals, as determined by measurement of LH in biweekly samples [9, 31].
In contrast to what was predicted from the ovx+E model [21], the D60–90 and D30–90 ovary-intact animals showed no discernable pubertal increases in gonadotropin secretion. The patterns of LH and FSH in ovary-intact D60–90 and D30–90 animals in general paralleled that of the controls, although levels in the androgenized animals tended to be higher. There are three possible explanations for the dissociation in onset of neuroendocrine puberty (increase in LH secretion) between the intact androgenized and ovx+E model. First, biweekly blood sampling, although sufficient to reliably measure the pubertal rise in LH in the ovx+E model (because of the pronounced suppression in LH during the prepubertal period) may have been insufficient to do so in the intact androgenized lambs. Second, constant postnatal estradiol in the ovx+E model (1-cm constant-release estradiol implant inserted at time of ovariectomy and maintained in place throughout the study) may have exerted organizational effects at the brain level, leading to advancement of neuroendocrine puberty. Third, other ovarian factors may serve to protect the brain from postnatal organization.
Although the differences were not statistically significant, the androgenized animals tended to have higher LH and FSH levels and LH:FSH ratios than did controls. A possibility that remains to be explored is whether these animals develop hypergonadotropism as they advance reproductively. Fabre-Nys and Venier [14] determined that follicular phase levels of LH are higher in prenatally androgenized than in control animals. This observation, in conjunction with the multifollicular ovarian morphology [5, 11] and anovulatory condition [10, 20] that these sheep develop with advancing age, suggests that prenatally androgenized sheep can be used as a cost-effective model for understanding the pathophysiology of polycystic ovarian disease, the major cause of infertility in women of reproductive age. These women manifest, hypergonadotropism, polycystic ovaries, hyperandrogenism, and anovulation [32].
Effect of Prenatal Androgenization on Onset of Reproductive Cyclicity
The D60–90 and the D30–90 androgenized animals attained puberty (onset of progestogenic cycles) at the same time as did the control animals. These findings are paradoxical and clearly were not predicted from the findings in the estradiol-clamped ovariectomized lamb model. Based on the early onset of pubertal LH rise [21] and the existence of an intact surge system [33] in the D60–90 ovx+E model, we expected the D60–90 ovary-intact females to show early onset of reproductive cycles. However, none of the intact D60–90 lambs treated prenatally with androgens had an early puberty. Because prenatal androgen exposure from Day 30 to Day 90 abolishes the surge in the ovx+E model [33], we predicted that the ovary-intact lamb treated similarly before birth would not attain puberty. The onset of reproductive cyclicity in the D30–90 animals is however consistent with 1) our findings that two of the three D30–90 androgenized animals had an intact (although highly compromised) surge system and 2) early reports of the existence of reproductive cycles in more than half of the D30–80 androgenized sheep studied [10].
There are at least two possible explanations for the observed differences in the ovx+E and ovary-intact models. First, such differences may relate to the nature and time of estradiol exposure and the subsequent organizational effects of estradiol. In the ovariectomized model [21], females are constantly exposed to exogenous estradiol during the postnatal period. The ovary-intact model used in this study is arguably different because these animals are not exposed to constant estradiol input but rather to changing levels of estradiol, which wax and wane with the follicular waves. The presence of continuous circulating estradiol during postnatal growth may have contributed to the masculinization/defeminization from tonic LH secretion during the prepubertal period along with defeminization of the LH surge system in the ovx+E model [21]. Thus, the organizational effects of estradiol on the surge generating system might extend beyond the prenatal period. This hypothesis is supported by the fact that continuous exposure to estrogens in adult cows and sheep leads to disruption of the surge mechanism [22, 34], suggesting that the surge system is quite vulnerable to such postnatal disruptions, even in the adult animals.
The second possibility is that other ovarian factors may play a role in protecting the brain from the possible defeminizing effects of postnatal estradiol. Although there seems to be a role for ovarian-derived inhibin in augmenting GnRH-induced release of LH in sheep [35] and the LH surge in humans [36], no information is available in support of a role for inhibin or other ovarian factors in mediating organizational effects at the level of the brain.
Effect of Prenatal Androgenization on Estradiol Positive Feedback
Based on results of earlier studies using the ovx+E model, we predicted that the gonadotropin surge system would be intact in the D60–90 androgenized animals but absent in the D30–90 animals [9, 33]. In contrast to the findings in ovx+E model, the D30–90 ovary-intact animals had an intact surge system like that of the D60–90 group, although the magnitude of the LH and FSH surges generated was markedly reduced. Both groups of androgenized animals showed delayed responses to the positive feedback actions of estradiol (evidenced by the delayed onset of the LH and FSH surges), with the delay being more pronounced in the D30–90 than in the D60–90 females. Although the number of animals in the D30–90 group was small, the delayed induction of the surge is consistent with results from other studies of D60–90 animals where sufficiently large numbers of animals were available for testing. Together, these findings provide clear evidence in support of the disruptive effects of prenatal androgenization on the positive feedback actions of estradiol in ovary-intact animals. This impairment in estradiol positive feedback, manifested as delayed and low-magnitude LH and FSH surges, may culminate in ovulatory defects. Disruptive effects of prenatal exposure to testosterone on the reproductive capability of ewes to ovulate or to exhibit estrous cyclicity have been reported previously [10, 15].
As in sheep, prenatal androgenization in guinea pigs leads to failure of the LH surge system [37]. However, prenatal treatment of rats with testosterone had no effect on the LH surge system, although this treatment resulted in masculinization of genitalia and defeminization of behavior centers in the brain [38]. In contrast, postnatal exposure of rats to a single injection of testosterone during Days 1–5 after birth was sufficient to disrupt the LH surge system and lead to sterility [38, 39]. These findings suggest that the critical period for sexual differentiation of the LH surge system in rats lies in early postnatal life, whereas in sheep and guinea pigs such deleterious effects on the surge system follow prenatal androgen exposure. Prenatal exposure of calves to androgens during Days 80–260 of gestation also failed to disrupt estrous cyclicity [40, 41]. In contrast, the LH surge system is not sexually differentiated in nonhuman primates, and males can generate an LH surge in response to estrogen challenge [42, 43].
Contrary to our findings of the existence of a surge system in the D30–90 ovary-intact sheep (albeit with a latency), a majority of the androgenized adult females studied long term by other investigators had no estradiol positive feedback response [5, 15], although the majority of animals showed progestogenic cycles [10]. In those studies, induction of surges was monitored only for 25 or 36 h after estradiol treatment. In contrast, in our studies the positive feedback effects of estradiol were monitored for periods up to 72 h, and surge induction was clearly evident in all but one animal. The animal that did not show a definable surge had an increase in LH but did not meet the surge criteria. It is therefore possible that earlier studies may have missed the induction of surges because of shorter monitoring periods.
A second issue that needs attention relates to the criteria set forth for detection of LH and FSH surges. From a biologic standpoint, we must determine whether the small increases in LH, even though they do not meet surge criteria, are sufficient to induce ovulation. The similarities in progestogenic cycles in the control and androgenized sheep observed during the first breeding season raise the possibility that such small-magnitude surges may be sufficient to induce ovulation. The dissociation in the presence or absence of the surge system in the D30–90 androgenized intact (this study) and ovx+E model [33] possibly relate to the postnatal exposure to estradiol (from the implant) in the ovx+E model, culminating in complete defeminization of the surge system. We can also assume that the D30–90 androgenized animals showed a higher degree of defeminization because of the longer exposure to estrogen (stemming from aromatization of androgen to estrogen). Our studies with the ovx+E model have shown that when pregnant sheep are androgenized with a nonaromatizable metabolite of testosterone (i.e., dihydrotestosterone), the LH surge system remains intact [44]. Future studies using aromatase inhibitors during the period of prenatal treatment with testosterone are required to investigate this possibility.
As expected and in agreement with earlier studies [45, 46], progesterone treatment delayed the onset of LH surge in all groups when compared with the timing observed in sheep from part 1 of experiment 2, where estradiol positive feedback was tested in the absence of progesterone priming. Progesterone pretreatment protects against the detrimental effects on the LH surge system of prolonged estradiol exposure in cystic cows and sheep [22, 34]. Furthermore, progesterone increases the sensitivity of the surge system to estradiol feedback effects [45]. Thus, we expected progesterone pretreatment to normalize the positive feedback effects of estradiol in the prenatally androgenized females. To a certain extent, this prediction was true; the D60–90 androgenized animals did not manifest any delay in surge generation as compared with the controls. However, progesterone treatment failed to normalize the timing and magnitude of the gonadotropin surges in the D30–90 group. One possible explanation for this discrepancy is that the LH stores may be depleted in the D30–90 prenatally androgenized group because of the hypergonadotropic tendency of these animals. Alternatively, the magnitude of the LH and FSH surges may not be a function of estradiol sensitivity. Further studies are necessary to resolve these issues.
Effects of Prenatal Androgenization on Estrous Behavior
As expected, rams used for detection of estrus preferred normal females to the prenatally androgenized females. The differential display of sexual behavior by vasectomized males may result from failure of androgenized sheep to exhibit proper estrous behavior and receptivity. Although we did not investigate this question, other investigators have suggested that these animals lack the ability to exhibit normal female sexual behavior [5, 14–17].
Prenatal androgenization drastically affects the LH surge system and mating behavior without altering the time of onset of pubertal onset in intact female sheep. These findings in conjunction with those of earlier studies suggest that prenatally androgenized sheep could serve as model systems for understanding the fetal origin of adult reproductive diseases, the etiology of polycystic ovarian disease, and the programming of sexual orientation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH HD 41098 and an educational grant from Parke Davis (Parke Davis merged with Pfizer in June 2000). 
2 Correspondence: Vasantha Padmanabhan, Reproductive Sciences Program, 300 N. Ingalls Bldg., Rm. 1109 SW, Ann Arbor, MI 48109-0404. FAX: 734 936 8620; vasantha{at}umich.edu
3 Contributed equally to this work
4 Current address: Pfizer Global Research and Development, Ann Arbor, MI 48105
5 Current address: Department of Reproductive Science and Medicine, Institute of Reproductive and Developmental Biology, Imperial College, Hammersmith Hospital, London W12 ONN, UK
Accepted: October 31, 2001.
Received: August 14, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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K. D. Malcolm, L. M. Jackson, C. Bergeon, T. M. Lee, V. Padmanabhan, and D. L. Foster Long-Term Exposure of Female Sheep to Physiologic Concentrations of Estradiol: Effects on the Onset and Maintenance of Reproductive Function, Pregnancy, and Social Development in Female Offspring Biol Reprod, December 1, 2006; 75(6): 844 - 852. [Abstract] [Full Text] [PDF]
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J. Robinson Prenatal programming of the female reproductive neuroendocrine system by androgens. Reproduction, October 1, 2006; 132(4): 539 - 547. [Abstract] [Full Text] [PDF]
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M. Manikkam, T. L. Steckler, K. B. Welch, E. K. Inskeep, and V. Padmanabhan Fetal Programming: Prenatal Testosterone Treatment Leads to Follicular Persistence/Luteal Defects; Partial Restoration of Ovarian Function by Cyclic Progesterone Treatment Endocrinology, April 1, 2006; 147(4): 1997 - 2007. [Abstract] [Full Text] [PDF]
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 C. Guzman, R. Cabrera, M. Cardenas, F. Larrea, P. W. Nathanielsz, and E. Zambrano Protein restriction during fetal and neonatal development in the rat alters reproductive function and accelerates reproductive ageing in female progeny J. Physiol., April 1, 2006; 572(1): 97 - 108. [Abstract] [Full Text] [PDF]
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 S. E. Recabarren, V. Padmanabhan, E. Codner, A. Lobos, C. Duran, M. Vidal, D. L. Foster, and T. Sir-Petermann Postnatal developmental consequences of altered insulin sensitivity in female sheep treated prenatally with testosterone Am J Physiol Endocrinol Metab, November 1, 2005; 289(5): E801 - E806. [Abstract] [Full Text] [PDF]
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 D.H. Abbott, D.K. Barnett, C.M. Bruns, and D.A. Dumesic Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome? Hum. Reprod. Update, July 1, 2005; 11(4): 357 - 374. [Abstract] [Full Text] [PDF]
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) and D60–90 (•) and D30–90 (
) androgenized ewes from 10 to 45 wk of age. Arrow indicates the mean age of puberty for all animals (see 
), D60–90 (
), and D30–90 (
) animals. For analysis, serial values from biweekly samples were divided into three 4.5-wk blocks (8–12.5, 13–17.5, 18–22.5 wk) and averaged
