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BOR - Papers in Press, published online ahead of print December 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.065904
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BIOLOGY OF REPRODUCTION 78, 636–647 (2008)
DOI: 10.1095/biolreprod.107.065904
© 2008 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology; 

Developmental Programming: Deficits in Reproductive Hormone Dynamics and Ovulatory Outcomes in Prenatal, Testosterone-Treated Sheep1

A. Veiga-Lopez 3, W. Ye 4, D.J. Phillips 6, C. Herkimer 3, P.G. Knight 7, and V. Padmanabhan 2 3 5

Departments of Pediatrics3 and Biostatistics,4 and the Reproductive Sciences Program,5 University of Michigan, Ann Arbor, Michigan 48109 Monash Institute of Medical Research,6 Monash University, Clayton, Victoria 3168, Australia School of Biological Sciences,7 The University of Reading, Reading RG6 6AJ, United Kingdom

ABSTRACT

Prenatal testosterone excess leads to neuroendocrine, ovarian, and metabolic disruptions, culminating in reproductive phenotypes mimicking that of women with polycystic ovary syndrome (PCOS). The objective of this study was to determine the consequences of prenatal testosterone treatment on periovulatory hormonal dynamics and ovulatory outcomes. To generate prenatal testosterone-treated females, pregnant sheep were injected intramuscularly (days 30–90 of gestation, term = 147 days) with 100 mg of testosterone-propionate in cottonseed oil semi-weekly. Female offspring born to untreated control females and prenatal testosterone-treated females were then studied during their first two breeding seasons. Sheep were given two injections of prostaglandin F2alpha 11 days apart, and blood samples were collected at 2-h intervals for 120 h, 10-min intervals for 8 h during the luteal phase (first breeding season only), and daily for an additional 15 days to characterize changes in reproductive hormonal dynamics. During the first breeding season, prenatal testosterone-treated females manifested disruptions in the timing and magnitude of primary gonadotropin surges, luteal defects, and reduced responsiveness to progesterone negative feedback. Disruptions in the periovulatory sequence of events during the second breeding season included: 1) delayed but increased preovulatory estradiol rise, 2) delayed and severely reduced primary gonadotropin surge in prenatal testosterone-treated females having an LH surge, 3) tendency for an amplified secondary FSH surge and a shift in the relative balance of FSH regulatory proteins, and 4) luteal responses that ranged from normal to anovulatory. These outcomes are likely to be of relevance to developmental origin of infertility disorders and suggest that differences in fetal exposure or fetal susceptibility to testosterone may account for the variability in reproductive phenotypes.

fetal programming, infertility, ovulation

INTRODUCTION

Abnormal hormonal, nutritional, metabolic, or environmental conditions during pregnancy alter the developmental trajectory of the fetus, leading to morphologic and physiologic changes in many organ systems, culminating in adult diseases [1–5]. Exposure to excess testosterone during fetal life in sheep from Days 30 to 90 of gestation, as in primates [5, 6], leads to adult reproductive disruptions at neuroendocrine, ovarian, and metabolic levels [7–13] that parallel the phenotype of women with polycystic ovary syndrome (PCOS) [14–16]. At the neuroendocrine level, prenatal testosterone treatment disrupts estradiol (E2) negative feedback [10–12, 17], E2 positive feedback [10–12, 18, 19], and progesterone (P4) negative feedback [20]. At the ovarian level, prenatal testosterone excess leads to polycystic ovary morphology [7], the result of increased ovarian follicular recruitment [21] and follicular persistence [9, 22]. Reproductive defects of prenatal testosterone-treated females worsen with age [9, 23, 24].

An understanding of the nature of hormonal disruptions that occur during the reproductive cycle will help in dissecting out the relative neuroendocrine and ovarian contributions in mediating reproductive failure and aid in developing interventions to overcome reproductive dysfunction. Any disruption in the periovulatory sequence of hormonal changes leading up to ovulation would likely compromise cyclicity and ovulation. Several possible disruptions may precipitate reproductive failure in prenatal testosterone-treated females. First, reduced sensitivity to E2 negative feedback may lead to hypersecretion of LH and provide inappropriate gonadotropic stimulus during the follicular phase; namely, a disproportionate increase in LH/FSH secretion. Second, an inadequate or delayed preovulatory E2 rise stemming from ovarian follicular defects may provoke a mistimed LH surge, leading to delayed or lack of ovulation. Third, the LH surge mechanism may be compromised, leading to an inadequate ovulatory signal and short or absent luteal phases. Fourth, a compromised sensitivity to P4 negative feedback during the luteal phase may lead to an increase in LH pulse frequency, which can cause persistence of follicles. Fifth, disruption in temporal changes in regulators of FSH, such as activin, inhibin, and follistatin, may lead to suboptimal FSH drive, culminating in follicular disruptions. Finally, a compromised follicular milieu may perturb ovulation or development of a healthy corpus luteum. The objective of this study was to determine the disruptions in periovulatory hormonal dynamics and ovulatory outcomes in prenatal testosterone-treated sheep, the adult phenotype of which mimics characteristics of women with PCOS.

MATERIALS AND METHODS

Animals and Prenatal Treatment

Details of prenatal testosterone treatment, husbandry, and nutrition have been described previously [25]. In brief, pregnant Suffolk sheep were treated twice weekly with 100 mg testosterone propionate intramuscularly (Sigma-Aldrich Corp., St. Louis, MO) in cottonseed oil (2.4 ml) from 30–90 days of gestation (term = 147 days). Control females were treated intramuscularly with the same volume of cottonseed oil. All lambs were fed a pelleted diet (Shur-Gain, Elma, NY) composed of 3.6 MCal/kg digestible energy and 18% crude protein. After weaning at 8 wk of age, the lambs were maintained outdoors at the Research Facility (Ann Arbor, MI). They were fed ad libitum until they attained 40 kg body weight, when they were switched to a diet with 15% crude protein until 6 mo of age (Shur-Gain). Adult sheep were fed a ration consisting of 2.3 MCal/kg digestible energy and 11.3% crude protein.

Experimental Design

Cycle characterizations were undertaken during the first and second breeding seasons of the offspring. Five control females intramuscularly injected with vehicle (referred to as IM) and nine prenatal testosterone-treated females were studied during the first breeding season. In addition, to test for vehicle effect, additional age-matched females (n = 4) whose mothers were treated subcutaneously with the same volume of vehicle (referred to as SC), and untreated control females (n = 9) of similar age purchased from the source that provided the breeder ewes and brought to the Sheep Research Facility at ~8 wk of age (referred to as purchased), were evaluated in parallel during the first breeding season. A more detailed assessment of hormonal dynamics was undertaken during the second breeding season with 5 control (vehicle-treated) and 12 prenatal testosterone-treated females.

First Breeding Season Studies

All 20 control females (IM: n = 7; SC: n = 4; purchased: n = 9) and 9 prenatal testosterone-treated sheep were treated to synchronize estrus during the first breeding season (December 2002) with two intramuscular injections of 20 mg prostaglandin F2 (PGF2{alpha}; 5 mg/ml; Lutalyse; Pfizer Animal Health) 11 days apart. Beginning with the second PGF2{alpha} injection, blood samples were obtained every 2 h for 120 h (5 days) for determination of changes in circulating gonadotropins. Thereafter, daily blood samples were taken until Day 20. Plasma samples were stored at –20°C until assayed for LH and FSH in two hourly samples and P4 in daily samples. In addition, to determine whether feedback sensitivity to P4 was reduced in prenatal testosterone-treated females, frequent blood samples were taken every 10 min for 8 h during the presumptive midluteal phase of the synchronized cycle from five control and nine prenatal testosterone-treated females.

Second Breeding Season Studies

During the second breeding season (December 2003–January 2004), 5 control and 12 prenatal testosterone-treated females (only a subset of animals used during the first year were available for inclusion in the second year cycle characterization studies) were studied. Synchronization protocols and sample collection details were the same as those in the first breeding season (barring exclusion of 8-h frequent blood samples during the luteal phase), but a more detailed characterization of hormonal dynamics was undertaken. This included measurement of not only LH and FSH, but also E2, immunoreactive inhibin, inhibin A, activin A, and total follistatin (FST). All procedures were approved by the University of Michigan Institutional Animal Care and Use Committee and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Hormone Assays

Plasma LH and FSH concentrations were measured in duplicate in all samples using validated assays [26, 27]. The results are expressed in terms of NIH-LH-S12 and NIDDK-ovine FSH-1. The sensitivity of the LH assay was 0.45 ± 0.1 ng/ml (n = 10 assays; mean ± SEM). Mean intraassay coefficients of variation (CVs) were 4.9%, 4.9%, 5.4%, and 14.1%. The interassay CVs, based on four quality control pools measuring 1.4, 12.3, 14.1, and 21.9 ng/ml, averaged 15.8%, 8.6%, 6.4%, and 7.3%, respectively. The sensitivity of the FSH assay was 0.6 ± 0.1 ng/ml (n = 6 assays; mean ± SEM). Mean intraassay CVs based on two quality control pools measuring 4.8 and 20.2 ng/ml were 17.0% and 13.9%, respectively. The interassay CVs for the same quality control pools were 5.6% and 6.7%, respectively. Plasma E2 concentrations were measured in alternate samples using a validated radioimmunoassay (RIA) first developed by Butcher et al. [28] and modified by Tortonese et al. [29], following extraction of plasma samples with ether. The sensitivity of the E2 assay was 0.06 ± 0.01 pg/ml (n = 9 assays; mean ± SEM).

Plasma P4 concentrations were measured in daily samples using a solid-phase RIA kit (Coat-A-Count P Diagnostic Products Corp., Los Angeles, CA) as previously described [30]. All samples were assayed in duplicate 100-µl aliquots. The assay sensitivity was 0.13 ± 0.05 ng/ml (n = 8 assays; mean ± SEM), and intraassay CVs, based on the three quality control pools measuring 0.16 ± 0.01, 1.74 ± 0.14, and 13.1 ± 0.4 ng/ml, were 29.4%, 23.9%, and 9.3%, respectively. The interassay CVs for the same quality control pools were 18.8%, 8.3%, and 6.8%, respectively.

Immunoreactive inhibin was measured in all twice-hourly samples by heterologous RIA as described previously [31] using an in-house human recombinant (hr)-inhibin A standard and iodinated hr-inhibin A as tracer. The assay cross-reacts 288% with pro-alpha C, the pro-sequence of the INHA subunit [32]. The limit of detection was 0.21 ng/ml, and intraassay and interassay CVs were 5.7% and 5.4%, respectively (n = 7 assays). Dimeric inhibin A concentrations were measured in alternate samples using an ELISA specific for ovine inhibin A [33] using anti-INHA subunit monoclonal (E4)-coated plates (Oxford Bio-Innovations) and modified using a biotinylated monoclonal antibody (PPG-1) specific for ovine INHBA subunit [34]. The standard used was a pool of bovine follicular fluid calibrated in this assay against WHO 91/684 inhibin A standard. Castrate ram serum, which contained no detectable inhibin A, was used as diluent to eliminate any matrix effect of the plasma. The limit of detection for inhibin A assay was 0.062 ng/ml. The average intraplate and interplate CVs were 5.8% and 9.8%, respectively (n = 14 plates).

Dimeric activin A was measured in alternate samples using a specific ELISA [35] according to the manufacturer's instructions (Oxford Bio-Innovations) with some modifications, as described previously [36]. The limit of detection for the activin A assay was 0.008 ng/ml. The average intraplate and interplate CVs were 9.8% and 6.3%, respectively (n = 11 plates). Plasma FST concentrations were measured in all twice-hourly samples using an RIA described previously [37] that detects "total" FST (activin A-bound and free). The assay uses a dissociating reagent, which dissociates the activin A-FST complex. The samples were measured in seven assays, the limit of detection was 2.5 ng/ml, and the average intraassay and interassay CVs were 8.3% and 5.9%, respectively.

Statistical Analysis

For all analyses, values below assay sensitivity were assigned the detection limit of the assay. Serial LH data from frequent samples collected during the luteal phase of the first breeding season were subjected to pulse analysis using the Cluster algorithm [38]. The Cluster algorithm identifies pulses using criteria that define a pulse such that the peak of the pulse differs significantly from both the preceding and following nadirs according to two-sample t-tests. For analysis with Cluster, the minimum number of data points in a peak and nadir was set at 2 and 2, respectively. The t-statistic values used to identify a significant increase from preceding nadir and a decrease to following nadir were each 2.0. All Cluster-identified peaks with LH increases greater than two times the assay sensitivity from the preceding nadir were considered as pulses for further statistical analyses. The amplitude was calculated as the difference between the pulse peak value and preceding nadir value. Mean concentration, frequency, and amplitude of LH pulses and timing and dynamics of gonadotropin surges were analyzed by ANOVA after appropriate transformations to account for heterogeneity of variance. Fisher exact test was used for comparing percentages of animals responding to PGF2{alpha} and having an LH surge.

Although all 20 control females had an LH surge and a luteal P4 increase, prenatal testosterone-treated females had variable outcomes, which ranged from normal to anovulatory. For comparison of detailed hormonal dynamics and temporal relationships during the second breeding season, prenatal testosterone-treated females that had a definable LH surge were compared to control females. For determining luteal P4 increase and duration in cycling females, onset was defined as beginning when daily concentrations of P4 reached above 0.5 ng/ml and ending when they fell below 0.5 ng/ml. The cutoff of 0.5 ng/ml was based on the following information: 1) P4 concentration measured in the circulation, in general, reflects changes in corpus luteum activity; 2) detection of corpus luteum by ultrasonography as early as Day 2 after ovulation corresponds with circulating P4 levels of ~0.5 ng/ml [39]; and 3) the first progestogenic increase that marked onset of puberty in our earlier studies averaged ~0.5 ng/ml.

Because hormone levels changes across time as a result of biological variations and measurement error, and temporal changes in hormones across estrous cycle cannot be modeled by any parametric models linear, quadratic, or cubic, we used smoothing statistics to capture these curves. A smoothing statistic allows one to capture various shapes of trajectories. It also takes into account correlation between repeated measurements within subjects. Furthermore, in situations where alternate samples were used (gonadal peptides) and data then aligned to LH peak for analyses, smoothing statistic allows comparing trajectories with such sample offsets. For control and prenatal testosterone-treated cycling groups, a cubic smoothing spline [40] (R –project/smooth spline function) was fitted to each of the individual hormone time profiles. Peak LH, E2, and activin A concentrations were defined as the time points at which the smooth curves were at maximum values. For FSH, two peaks were identified, the first FSH peak coinciding with the LH peak and the second FSH occurring after the primary gonadotropin surge. The nadirs of immunoreactive inhibin and inhibin A were defined as the time points at which the smooth curves were at minimum values. All peak and nadir values used in analyses were values predicted by the smooth curve. The population mean time profiles of each hormone were also calculated for all of the groups. For LH and FSH, this was done by calculating the sample mean at each time point for each group. For E2, activin A, immunoreactive inhibin, and inhibin A (where alternate samples were measured) and FST (where all samples were measured), the data from each group were fitted to a semiparametric mixed model with regression splines [41], and predicted mean curves were obtained. This model fits the population mean curve flexibly with B-splines, which capture various shapes of trajectories, and takes into account the correlation between repeated measurements within subjects. All values were log (e-base) transformed before analysis. For analysis of LH, FSH, E2, activin A, immunoreactive inhibin, and inhibin A in control and prenatal testosterone-treated cycling groups, data were aligned according to the subject-specific peak/nadir time of the particular hormone. The time profile for FST did not show any peak or nadir, so FST was aligned relative to LH surge time. Circulating P4 was analyzed relative to the time of PGF2{alpha} injection. To compare changes in timing (peak/nadir time) and hormone dynamics between the control and prenatal testosterone-treated cycling animals, exact Wilcoxon test was used. Pearson correlation coefficients were calculated to capture potential associations among hormonal variables.

Because an increase in activin:FST or activin:inhibin or activin:inhibin + FST ratio should result in more available free activin to stimulate FSH, to provide a theoretical index of whether activin biological activity differed between the control and prenatal testosterone-treated female groups, ratios of activin A to FST, activin A to inhibin A, and activin A to inhibin A + FST were computed. Ratios were log (e-base) transformed prior to analysis. Data then were aligned relative to the LH peak time of each animal. The mean log (ratio) within five 20-h time blocks (–50 to –30 h, –30 to –10 h, –10 to +10 h, +10 to +30 h, and +30 to +50 h) was compared across treatments using repeated-measures ANOVA. All analyses were carried out using SAS for Windows release 9.1.3 (SAS Institute, Cary, NC). Significance was defined as P < 0.05. All results are presented as the mean ± SEM.

RESULTS

First Breeding Season

Figure 1 summarizes the percentages of control (composite of IM, SC, and purchased) and prenatal testosterone-treated females that exhibited synchronized estrus in response to PGF2{alpha}, circulating twice-hourly patterns of LH/FSH from four control and four prenatal testosterone-treated females and the percentages of control and prenatal testosterone-treated females showing subsequent luteal P4 increase. The mean time of onset of the LH surge and LH surge amplitude for all the control females studied averaged 44.3 ± 2.4 h and 154.9 ± 9.9 ng/ml, respectively. There were no differences in surge characteristics among the three subsets of control females (onset, IM: 46.3 ± 3.5 h, SC: 39.0 ± 7.7 h, and purchased: 45.2 ± 3.3 h; surge amplitude, IM: 145.0 ± 16.9 ng/ml, SC: 171.9 ± 31.3 ng/ml, and purchased: 154.9 ± 12.0 ng/ml). Although 100% of control females exhibited synchronized estrus in response to PGF2{alpha} and showed a luteal P4 increase, only four of the nine prenatal testosterone-treated females showed a luteal P4 increase (control vs. prenatal testosterone-treated: P < 0.05). The primary FSH surge in control females averaged 9.1 ± 0.8 ng/ml and occurred in parallel with the LH surge. Although five of the nine prenatal testosterone-treated females had increases in LH that met the definition of a surge (control vs. prenatal testosterone-treated: P < 0.005), only one prenatal testosterone-treated female had an LH surge of the magnitude seen in the control group (Fig. 1, top right panel). The LH surge in prenatal testosterone-treated females occurred at 64.8 ± 20.0 h after the second PGF2{alpha} injection (control: 44.3 ± 2.4 h; control vs. prenatal testosterone-treated, P = 0.06), with a mean LH surge amplitude of 46.9 ± 19.2 ng/ml (control: 154.9 ± 9.9 ng/ml; control vs. prenatal testosterone-treated, P < 0.01). The primary FSH surge peak, which occurred coincidentally with the LH surge, averaged 6.6 ± 2.1 ng/ml in prenatal testosterone-treated females (control: 7.7 ± 0.6 ng/ml; control vs. prenatal testosterone-treated, non significant).


Figure 01
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  		FIG. 1. Left panels: Percentage of control (C; white bars) and prenatal testosterone-treated (T; black bars) females that exhibited estrus in response to PGF2{alpha} (top) and showed a subsequent luteal response (bottom). Middle and right panels: Circulating patterns of LH (black circles) and FSH (white circles) from four representative control (left column) and four prenatal testosterone-treated (right column) females. Blood samples were taken at 2-h intervals from PGF2{alpha} administration up to 120 h. Note differences in scale between prenatal testosterone-treated females.

Luteal P4 patterns (top panel) and LH pulse patterns (bottom panels) during the luteal phase in a subset of control (IM) and prenatal testosterone-treated (n = 9) females are shown in Figure 2. To determine whether prenatal testosterone-treated females had reduced sensitivity to P4 negative feedback, they were divided into two groups on the basis of P4 concentrations achieved: those with luteal P4 greater than 1 ng/ml (n = 4) and those with P4 concentrations less than 1 ng/ml (subluteal, n = 5). Daily patterns of P4 in prenatal testosterone-treated females with levels of P4 greater than 1 ng/ml paralleled the luteal P4 increase observed in control females. The LH pulse dynamics as well as correlation between LH frequency and P4 concentrations achieved are summarized in Figure 3. LH pulse frequency was higher (P < 0.05) in both groups of prenatal testosterone-treated females than in control females. Compared with control females, mean LH concentration tended to be greater (P = 0.09) in the prenatal testosterone-treated females with normal luteal P4 increase but significantly higher in the prenatal testosterone-treated females with subluteal P4 concentrations (P < 0.001). Mean LH pulse amplitude tended to be higher in the prenatal testosterone-treated females with subluteal P4 concentrations compared with control and prenatal testosterone-treated females with normal luteal P4 increase (P = 0.05). Correlation analyses across treatment groups revealed overall negative correlations between P4 concentrations and LH pulse frequency (r = –0.53, P < 0.05) and P4 concentrations and mean LH concentrations (r = –0.59, P < 0.05). The LH pulse frequency of prenatal testosterone-treated females with comparable P4 concentrations to control females was numerically higher. Although the regression slope for P4 vs. LH pulse frequency did not differ between control and prenatal testosterone-treated females with normal luteal P4 increases (control: –0.081, testosterone-treated: –0.98; P = 0.75), the intercept did (control: 0.70, testosterone-treated: 1.06; P < 0.005), indicating differences in the requirement of P4 to suppress LH pulse frequency (higher threshold for prenatal testosterone treated). Consistent with this, the one prenatal testosterone-treated female with reduced LH frequency had the highest P4 concentration (6.9 ng/ml). The small range in P4 concentrations achieved in the subluteal prenatal testosterone-treated group precluded similar analyses.


Figure 02
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  		FIG. 2. Top row: Mean ± SEM circulating patterns of daily P4 from the control (left), prenatal testosterone (T)-treated females with luteal P4 response of >1 ng/ml (middle), and prenatal T-treated females with luteal P4 response of <1 ng/ml (right) following synchronization with PGF2{alpha}. Arrows indicate the time of frequent sampling. Bottom panels: Circulating patterns of LH from three representative control females (left), three prenatal testosterone-treated females with luteal P4 response of >1 ng/ml (middle), and three prenatal T-treated females with luteal P4 response of <1 ng/ml (right). Blood samples were taken 8 days (early to midluteal phase) after the second PGF2{alpha} administration at 10-min intervals for 8 h. Inverted triangles, LH pulses identified by the Cluster algorithm.


Figure 03
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  		FIG. 3. Top panels: Mean LH (±SEM), LH pulse frequency (pulses/8 h), and LH pulse amplitude (ng/ml) from control (n = 5; white bars), prenatal testosterone (T)-treated females with luteal P4 response of >1 ng/ml (n = 4; gray bars), and prenatal T-treated females with luteal P4 response of <1 ng/ml (n = 5; black bars). Differing letters on the histograms denote significant differences, a vs. b, P < 0.05; a vs. a', P = 0.09. Bottom panel: The relationship between circulating P4 levels (ng/ml) and LH pulse frequency on day of LH pulse determination (Day 8 after PGF2{alpha} administration). White triangles, control; gray circles, prenatal T-treated females with luteal P4 response of <1 ng/ml; black circles, prenatal T-treated females with luteal P4 response of >1 ng/ml.

Second Breeding Season

All control females had the expected cyclic hormonal changes after the estrous synchronization protocol. Prenatal testosterone-treated females had varying degrees of cycle disruptions, with some showing luteal P4 increases and others not. To assess disruptions in periovulatory hormonal dynamics, prenatal testosterone-treated females were categorized into three groups based on previous studies that correlated luteal mass with P4 plasma concentrations [39]: 1) those that had a definable LH surge and a luteal P4 increase (cycling prenatal testosterone treated; n = 5); 2) those that lacked a definable LH surge but had a P4 rise (>0.05 ng/ml; subluteal prenatal testosterone treated; n = 3); and 3) those that had P4 concentrations below 0.05 ng/ml and were considered to be anovulatory (anovulatory prenatal testosterone treated; n = 4). Because luteal P4 patterns were used as a categorization variable, luteal P4 patterns are discussed first.

Luteal P4 Dynamics

Normal increases in luteal P4 followed estrus synchronization with PGF2{alpha} in all control females (Fig. 4, left panel [control]). The onset, duration, and magnitude of luteal P4 patterns in prenatal testosterone-treated females that had a definable LH surge were similar to those in control females (Fig. 4, second panel from left [T (cycling)]. The subluteal prenatal testosterone-treated group had a delayed but sustained P4 increase to magnitudes higher than 0.5 ng/ml but lower than 1 ng/ml (Fig. 4, third panel from left [T (subluteal)]. Anovulatory prenatal testosterone-treated females had P4 levels less than 0.5 ng/ml (Fig. 4, right panel [T (anovulatory)].


Figure 04
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  		FIG. 4. Mean (±SEM, shaded area) circulating patterns of daily P4 (ng/ml) in control, cycling prenatal testosterone-treated [T (cycling)], subluteal prenatal testosterone-treated [T (subluteal)], and anovulatory prenatal testosterone-treated [T (anovulatory)] sheep relative to PGF2{alpha} administration. The vertical dotted line represents mean time of the preovulatory LH surge for the control and cycling prenatal testosterone-treated group. Means and SEMs are based on the fit of the semiparametric mixed model with regression.

Preovulatory E2 Rise

Changes in circulating E2 in control and prenatal testosterone-treated females that had normal luteal P4 responses are shown in Figure 5 (bottom panels), and summary statistics are shown in Figure 6. The control females had preovulatory E2 increases at 46.2 ± 5.6 h after the second PGF2{alpha} (Fig. 6, bottom left panel). In cycling prenatal testosterone-treated females, the preovulatory E2 peak tended to occur later (64.2 ± 8.3 h). The peak E2 rise was higher in prenatal testosterone-treated females compared with control females (control: 4.3 ± 0.2 pg/ml, prenatal testosterone-treated: 7.6 ± 1.8 pg/ml; P < 0.05). Subluteal prenatal testosterone-treated females had a much more delayed E2 rise, which began near the end of the sampling period (~120 h after the PGF2{alpha}). Anovulatory prenatal testosterone-treated females had elevated but unchanging concentrations of E2 throughout the sampling period (Fig. 5, bottom right panel).


Figure 05
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  		FIG. 5. Mean circulating patterns of twice-hourly samples of LH (ng/ml, top row), FSH (ng/ml, middle row), and E2 (pg/ml, bottom row) in control, cycling prenatal testosterone-treated [T (cycling)], subluteal prenatal testosterone-treated [T (subluteal)], and anovulatory prenatal testosterone-treated [T (anovulatory)] sheep relative to PGF2{alpha} administration. The vertical dotted line represents mean time of the preovulatory LH surge for the control and cycling prenatal testosterone-treated group. For LH and FSH, the mean patterns and SEMs are from observations at each time point. For E2, both the mean and SEM (shaded area) are based on the fit of the semiparametric mixed model with regression splines.


Figure 06
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  		FIG. 6. Top row: Mean (±SEM) time (hours) of the preovulatory E2 rise (Estradiol), LH surge (LH), and primary and secondary FSH surges (FSH Primary and FSH Secondary, respectively) relative to second PGF2{alpha} injection. Middle row: Mean (±SEM) time (hours) of the preovulatory LH and primary and secondary FSH surges relative to the E2 rise. Bottom row: Mean (±SEM) amplitude of the preovulatory E2 rise (pg/ml), LH surge (ng/ml), and primary and secondary FSH surges (ng/ml). White bars, control females; black bars, cycling prenatal testosterone-treated females. Asterisks indicate significant differences.

Gonadotropin Surge Dynamics

Changes in circulating LH and FSH in control and prenatal testosterone-treated females with normal luteal response are shown in Figure 5, top and middle panels, respectively, and summary statistics are shown in Figure 6. All control females exhibited normal preovulatory LH and FSH surges at the expected times (left panels). Primary gonadotropin surges were delayed in cycling prenatal testosterone-treated females (control: 50.8 ± 5.7 h, testosterone-treated: 74.2 ± 8.6 h; P < 0.05). Amplitudes of the LH (control: 161.1 ± 15.9 ng/ml, testosterone-treated: 33.6 ± 6.9 ng/ml; P < 0.01) and primary FSH surges (control: 6.8 ± 0.6 ng/ml; testosterone-treated: 2.9 ± 0.2 ng/ml; P < 0.01) were severely reduced in cycling prenatal testosterone-treated females compared with control females. The secondary FSH surges in control and cycling prenatal testosterone-treated females occurred at 79.0 ± 8.1 h and 99.5 ± 2.3 h, respectively, and did not differ significantly. As opposed to dampened primary FSH surges, secondary FSH surges in cycling prenatal testosterone-treated females tended to be of greater magnitude than in control females (control: 3.8 ± 0.3 ng/ml, testosterone: 4.9 ± 0.3 ng/ml; P = 0.06). A definable LH surge was not detected in the subluteal and anovulatory prenatal testosterone-treated females, although higher basal LH often was evident. Circulating FSH concentrations declined in the subluteal group from ~4 ng/ml at the start of sampling to ~1.5 ng/ml at 120 h, coincident with the E2 increase, so an LH surge in these animals may have occurred beyond the 120-h sample collection period. Low LH and unchanging FSH concentrations (~2 ng/ml) were detected in the anovulatory prenatal testosterone-treated females.

FSH Regulatory Protein Dynamics

Changes in circulating activin A, inhibin A, immunoreactive inhibin, and FST in control and cycling prenatal testosterone-treated females are shown in Figure 7 and summary statistics in Figure 8. A rise in activin A preceded the primary gonadotropin surge in both control and prenatal testosterone-treated females with normal luteal P4 dynamics (Fig. 7). The amplitude of activin A increase did not differ between the two groups (Fig. 8). A decline in inhibin A followed the primary gonadotropin surge in both control and cycling prenatal testosterone-treated females, with the lowest values coinciding with the end of the primary gonadotropin surge (Figs. 5 and 7). The nadir in inhibin A tended to occur later in cycling prenatal testosterone-treated females compared with control females (control: 74.8 ± 5.8 h, testosterone-treated: 95.0 ± 8.0 h; P = 0.056). Immunoreactive inhibin remained unchanged during the late follicular phase (Fig. 7), declining after the preovulatory LH surge in both groups and reaching a nadir coincidentally with inhibin A (control: 78.8 ± 5.6 h and testosterone-treated: 90.0 ± 5.9 h; Table 1). Total FST concentrations remained constant (around 4 ng/ml) throughout the periovulatory period in control females but showed a nonsignificant decline in cycling prenatal testosterone-treated females until the primary gonadotropin surge, and they increased thereafter (Fig. 7). Comparison of the ratios of stimulatory (activin A) and inhibitory (inhibin A and FST) FSH regulatory proteins showed that the activin A:FST ratio and activin A:FST + inhibin A ratio were both higher during the follicular phase in the cycling prenatal testosterone-treated females (Fig. 9). Subluteal prenatal testosterone-treated females failed to show the increase in activin A or fall in inhibin A (Fig. 7). Anovulatory prenatal testosterone-treated females also had unchanging patterns of the FSH regulatory proteins (Fig. 7).


Figure 07
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  		FIG. 7. Mean (±SEM) circulating patterns (ng/ml) of activin A (Activin), dimeric inhibin A (Inhibin A), immunoreactive inhibin (Immuno. Inhibin), and FST in control, cycling prenatal testosterone-treated [T (cycling)], subluteal prenatal testosterone-treated [T (subluteal)], and anovulatory prenatal testosterone-treated [T (anovulatory)] sheep relative to PGF2{alpha} administration. The vertical dotted line represents mean time of the preovulatory LH surge for the control and cycling prenatal testosterone-treated group. Means and SEMs (shaded areas) are based on the fit of the semiparametric mixed model with regression splines.


Figure 08
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  		FIG. 8. Top row: Mean (±SEM) time (hours) of the periovulatory activin A increase (Activin Peak), dimeric inhibin A (Inhibin A), and immunoreactive inhibin (Imm. inhibin) decline relative to PGF2{alpha}. Middle row: Mean (±SEM) time (hours) of the periovulatory activin A increase and dimeric inhibin A and immunoreactive inhibin decline relative to the preovulatory E2 peak. Bottom row: Mean (±SEM) amplitude (ng/ml) of the periovulatory activin A increase and dimeric inhibin A and immunoreactive inhibin decline. White bars, control females; black bars, cycling prenatal testosterone-treated females.


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  		TABLE 1. Temporal relationships (hours) between E2, gonadotropins, and FSH regulatory proteins in control and prenatal testosterone-treated cycling females during the periovulatory sequence.


Figure 09
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  		FIG. 9. Mean (±SEM) ratio of activin A to inhibin A (top panel), activin A to FST (middle panel), and activin A to FST + inhibin A (bottom panel) in control (white bars) and cycling prenatal testosterone-treated (black bars) females during the periovulatory period. Five 20-h time blocks (–50 to –30 h, –30 to –10 h, –10 to +10 h, +10 to +30 h, and +30 to +50 h) relative to the LH surge were used for ratio calculations. Asterisks indicate significant difference between control and prenatal T-treated females (P < 0.05).

Temporal Relationships Between E2, FSH Regulatory Proteins, and Periovulatory Gonadotropin Surges

The time relationships between various hormonal variables are summarized in Table 1. Primary gonadotropin surges occurred ~5 h after the preovulatory E2 rise in controls and 9 h in cycling prenatal testosterone-treated females. The activin A peak occurred 0 and 12.2 h before the preovulatory E2 peak in control and cycling prenatal testosterone-treated females, respectively. The primary gonadotropin surge also was temporally delayed from the activin A peak in cycling prenatal testosterone-treated females compared with controls (Table 1). There were no differences in the timing of secondary FSH surges in the control and cycling prenatal testosterone-treated females relative to the E2 or activin A increase or inhibin A decrease. The secondary FSH surge began ~4 h after the decline in inhibin A in both control and cycling prenatal testosterone-treated females, and 33 and 56.5 h, respectively, after the activin A peak in control and cycling prenatal testosterone-treated females, respectively.

Estrogen was negatively correlated with the secondary FSH surge, with a time lag of 16 h in both control and cycling prenatal testosterone-treated females (control: r = –0.593, testosterone-treated: r = –0.769; P < 0.001). A positive correlation was found between E2 and activin A in both control (r = 0.521; P < 0.001) and cycling prenatal testosterone-treated (r = 0.509; P < 0.001) females. Activin A also correlated positively with FSH concentrations, with a time lag of 36 h in control (r = 0.700; P < 0.001), and 60 h in cycling prenatal testosterone-treated females (r = 0.388; P = 0.06). Estrogen was positively correlated with inhibin A in control (r = 0.526; P < 0.001) but not in the cycling prenatal testosterone-treated females. The secondary FSH surge was negatively correlated with inhibin A in both control and cycling prenatal testosterone-treated females (control: r = –0.643, testosterone-treated: r = –0.514; P < 0.001). The correlation was higher when the analysis was carried out by lagging FSH by 8 h (control: r = –0.845, testosterone-treated: r = –0.649; P < 0.0001).

DISCUSSION

Findings from this study provide evidence that prenatal testosterone excess disrupts the periovulatory hormonal dynamics, leading to a delayed but amplified preovulatory E2 rise, delayed and severely dampened primary gonadotropin surges, an increase in the ratio of circulating activin A relative to FST and inhibin A, tendency toward increase in the magnitude of the secondary FSH surge, and luteal defects. Furthermore, ovulatory outcomes varied among prenatal testosterone-treated individuals, with some showing ovulatory responses and others subluteal or anovulatory conditions. The nature of defects in periovulatory events of prenatal testosterone-treated females are consistent with contributions from these defects originating at both neuroendocrine and ovarian sites. In addition to these findings, integrated determination of the changes in stimulatory and inhibitory FSH regulatory proteins (activin, inhibin, and follistatin) suggest that in addition to a reduction in E2, an overall increase in activin availability may also play a facilitatory role in the generation of secondary FSH surge.

E2 Dynamics

Clearly, prenatal testosterone excess disrupts preovulatory E2 dynamics in the offspring. This disruption was manifested at several levels, with a delayed increase in E2 rise and an increase in peak levels of E2 achieved, the latter suggestive of hyperresponsiveness of the ovary. The multifollicular ovarian morphology [7] of the prenatal testosterone-treated females provides support for this being a likely possibility. However, the increase in E2 is not likely the result of an increased number of preovulatory follicles for the following reasons. Luteal phase P4 was similar between control and cycling prenatal testosterone-treated females. Because P4 concentrations reliably reflect corpus luteum mass in mono-ovulatory [42] and polyovulatory breeds [43], similar P4 in the cycling prenatal testosterone-treated females indicates that the number of corpus lutea formed must be similar. Moreover, our earlier ultrasonographic studies monitoring follicular and CL dynamics [9, 22] found similar numbers of ovulatory follicles in control and prenatal testosterone-treated females [22]. The increase in preovulatory E2 peak in prenatal testosterone-treated females may be the result of E2 contribution from multiple compromised antral follicles that failed to proceed to the preovulatory state. This premise is supported by our earlier finding of follicular persistence in prenatal testosterone-treated females [9, 22] and the higher E2 baseline of cycling prenatal testosterone-treated females as well as the elevated but constant E2 patterns seen in the anovulatory prenatal testosterone-treated females. High periovulatory E2 concentrations have been reported to be associated with reduced fertilization and embryo development in cattle and sheep [44–46]. High E2 levels during in vitro fertilization cycles in women also have been associated with deleterious effects on either oocyte/embryo quality or endometrial receptivity [47–49]. The lower fertility rates of the prenatal testosterone-treated sheep [50] and compromised oocyte development in prenatal testosterone-treated monkeys [51, 52] provide further evidence that follicles in prenatal testosterone-treated females are abnormal.

The delayed E2 rise reflects a delay in the development of preovulatory follicle(s). This may be the result of abnormal gonadotropic drive or a compromised intrafollicular environment. Prenatal testosterone-treated females manifest increased LH [10, 17, 18] due to compromised E2 and P4 negative feedbacks [10, 17, 20]. This may also reflect a compromised intrafollicular activin environment in the antral follicles of prenatal testosterone-treated females, due to increased FST mRNA and decreased INHBB mRNA [7].

Gonadotropin Surge Dynamics

Our finding of high circulating E2 in the anovulatory prenatal testosterone-treated females and the fact that the LH surge in cycling prenatal testosterone-treated females occurred following an increase in E2 are supportive of the contention that an increase in E2 serves as the trigger for the generation of the LH surge rather than absolute levels. The severely dampened primary gonadotropin surge observed in prenatal testosterone-treated cyclic females in the face of the higher preovulatory E2 peak suggests that compromised hypothalamic or pituitary responses to the E2 positive feedback signal are the causal mechanism rather than a diminished E2 signal. This is supported by our previous findings in which insertion of an E2 implant to achieve normal preovulatory E2 concentrations resulted in severely dampened and delayed LH surges in prenatal testosterone-treated Suffolk females [18]. Other investigators using the Dorset breed of sheep reported no definable LH surge in response to exogenous E2 [19]. The dampened LH surge may reflect reduced LH storage due to LH hypersecretion manifested in these animals [10, 17, 18, 53]. The reduced responsiveness of the hypothalamic-pituitary axis in prenatal testosterone-treated females in concert with increased E2 also may reflect desensitization at the level of E2 receptors and consequent disruption of neuroendocrine signals leading up to the generation of the LH surge. Recently, we found that prenatal testosterone treatment altered the developmental trajectory of pituitary E2 receptors [53]. Desensitization at the E2 receptor level is also consistent with the requirement of an increase in E2 for overriding this process to generate the LH surge. The absence of a surge of LH in the anovulatory prenatal testosterone-treated females that exhibit high but constant circulating E2 is also supportive of this premise.

The dampened LH surge in some of the prenatal testosterone-treated animals was sufficient to induce a normal luteal response. Whether the disrupted LH surge has deleterious effects on oocyte viability, a feature seen in prenatal testosterone-treated monkeys [51, 52], remains to be elucidated. The magnitude of the LH surge was, however, not affected in sheep treated prenatally with testosterone from Days 60–90 of gestation [54]. Differences in surge dynamics of females treated prenatally with testosterone from Days 30–90 vs. Days 60–90 of gestation may reflect critical periods of differentiation of hypothalamic or pituitary mediators that are involved in amplification of the surge. In the subluteal prenatal testosterone-treated females in this study, either a very low amplitude surge occurred after the 120-h sampling period, or subtle increases such as those seen in prenatal testosterone-treated females may have induced a subluteal P4 response, perhaps from luteinized follicles.

FSH Regulatory Protein Dynamics

Based upon comparison of circulating amounts of the gonadal/pituitary regulators of FSH (activin, inhibin, and FST) in control and prenatal testosterone-treated females, the main effect of prenatal testosterone excess on preovulatory dynamics of the regulatory proteins was subtle. The temporal relationship between E2 and activin A increase and E2 rise and inhibin decrease were similar between control and prenatal testosterone-treated females, suggestive of a close temporal relationship. The parallel increases in preovulatory E2 and activin A in both control and cycling prenatal testosterone-treated females, but not in anovulatory prenatal testosterone-treated females, suggests a contribution of activin A into the circulation from preovulatory follicles.

Although other studies have addressed hormonal relationships focusing on isolated FSH regulatory proteins [33, 55], none have addressed these three protein factors in concert. Considering that FST is a neutralizer of activin and that inhibin can antagonize the actions of activin [56–58], it is advantageous that these regulators are considered together. Similar time intervals between the preovulatory E2/activin A increase or inhibin A decrease and secondary FSH surge in the control and prenatal testosterone-treated animals suggest that the relative equilibrium of these regulators may contribute to the generation of the secondary FSH surge. It is well established that circulating inhibin is ovarian derived and plays an inhibitory role in regulating FSH [59]. Most circulating FST, believed to be pituitary derived [60], modifies the action of activin. The increase in the activin:FST + inhibin ratio observed during the presurge period in the prenatal testosterone-treated females is supportive of peripheral activin contribution to amplification of the secondary FSH surge in the prenatal testosterone-treated females. However, the majority of evidence to date point to a paracrine rather than an endocrine role for activin [61], although an endocrine role has been postulated for activin in maintaining elevated FSH in older reproductive-age women [62, 63]. To our knowledge, a validated free activin assay or bioassay with sufficient sensitivity is not available to assess net stimulatory or inhibitory activity of these regulatory proteins in circulation. As such, the effect of the changes in relative equilibrium of these factors in the circulation of prenatal testosterone-treated females on pituitary FSH release remains to be tested.

Luteal Defects

Despite the proven high effectiveness of synchronization of estrus with PGF2{alpha} in sheep [64, 65], only a subset of prenatal testosterone-treated females exhibited estrus in response to PGF2{alpha}. These results suggest the existence of a PGF2{alpha}-unresponsive corpus luteum or luteinized follicles, or else the anovulatory status of the prenatal testosterone-treated females. Clearly, a wide range of luteal defects, manifested as absent or subluteal P4 patterns, were evident in prenatal testosterone-treated females. Because severely dampened LH surges such as those seen in cycling prenatal testosterone-treated females were capable of inducing a normal-magnitude luteal P4 response, the P4 response seen in the subluteal group of prenatal testosterone-treated females, which lacked a definable surge, might represent responses to small increases in LH. Complete disruption of E2 positive feedback but the presence of cyclic progesterone was a feature also reported in prenatal testosterone-treated Dorset ewes [19]. Alternatively, the presence of a persistent follicle [9, 22] and delayed primary gonadotropin surge (this study and Sharma et al. [18]) suggest a compromised follicular environment contributing to the development of a defective CL.

Another luteal-phase defect seen in prenatal testosterone-treated females relates to the negative feedback effects of P4. The increased LH pulse frequency seen during the luteal phase of cycling prenatal testosterone-treated females in the face of P4 concentrations similar to control females is supportive of a reduced responsiveness of the hypothalamic-pituitary axis to P4 negative feedback. This is a feature also seen in women with PCOS [66] and hyperandrogenic adolescents [67]. Previous studies using ovariectomized Dorset females found that prenatal testosterone-treated females had reduced LH responsiveness to P4 negative feedback [20].

Translational Significance

Considering that prenatal testosterone-treated sheep manifest oligo/anovulation [9, 23, 24], functional hyperandrogenism, LH excess [17, 18, 53], polycystic ovarian morphology [7], and insulin resistance [8], features characteristic of women with PCOS, it would be of interest to determine whether similar variability is manifested at the level of insulin resistance and ovarian morphology and if they correlate with the ovulatory status of these animals. The broad range of ovulatory outcomes that follow similar prenatal testosterone treatment suggest that the actual concentrations of testosterone or estrogen (due to aromatization of testosterone) the fetus is exposed to may vary due to changes in maternal metabolism or placental transfer or, alternatively, there may be differences in fetal susceptibility to steroid exposure, indicative of gene-environment interactions. Interestingly, earlier cordiocentesis studies in the human found that testosterone concentrations in 4 of 10 female fetuses were in the male fetal range [68]. The constellation of reproductive disruptions that follow prenatal testosterone treatment also highlights the threats posed by exposure to excess steroids, native or environmental, on the reproductive health of offspring [7, 69, 70].

Finally, because experimentally induced fetal testosterone excess induces PCOS-like traits in adult females [4, 5], it is interesting to speculate that the varying reproductive phenotypes seen in women with PCOS may be the result of differences in amount and/or susceptibility to fetal steroid exposure, native or environmental. While this speculation is consistent with the proposal that PCOS may have its origin in fetal life, due to the genetic predisposition of the fetal ovary to hypersecrete androgens [16, 71, 72], this will remain a premise until proven.

To summarize, findings from this study document disruptions at several levels in the preovulatory hormonal dynamics, culminating in varying ovulatory outcomes. They further suggest that individual susceptibility and/or inappropriate fetal steroid milieu may provide a basis for the developmental origin of the varying reproductive phenotypes seen in this study.

ACKNOWLEDGMENTS

We are grateful to Mr. Douglas Doop for providing quality care and maintenance of animals used in this study; and Dr. Mohan Manikkam, Dr. Hiren Sarma, Dr. Teresa Steckler, Mr. James Dell'Orco, Mr. James Lee, Mr. Gary McCalla, Ms. Pamela Olton, and Ms. Danielle Djoumbi for assistance with prenatal testosterone treatment, help during animal experimentation, or performance of gonadotropin/P4 assays. We also thank Ms. Anne O'Connor and Mrs. Susan Hayward for performing the activin A, inhibin A, immunoreactive inhibin, and FST assays, and Dr. E. Keith Inskeep for his helpful edits of the manuscript.

FOOTNOTES

1Supported by US Public Health Service grant P01 HD44232 (to V.P.), R01 HD 41098 (to V.P.), and the National Health and Medical Research Council of Australia GrantNet ID 334011 (to D.J.P.). Back

Correspondence: 2Vasantha Padmanabhan, Department of Pediatrics and Reproductive Sciences Program, University of Michigan, 300 North Ingalls Bldg., Rm. 1109 SW, Ann Arbor, MI 48109-0404. FAX: 734 936 8620; e-mail: vasantha{at}umich.edu

Received: 1 October 2007.

First decision: 18 October 2007.

Accepted: 24 November 2007.

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