Developmental programming: exogenous gonadotropin treatment rescues ovulatory function but does not completely normalize ovarian function in sheep treated prenatally with testosterone

Abstract

Prenatal testosterone treatment leads to LH excess as well as ovarian follicular and ovulatory defects in the adult. These disruptions may stem from LH excess, abnormal FSH input, compromised ovarian sensitivity to gonadotropins, or intrinsic ovarian defects. To determine if exogenous gonadotropins rescue ovarian and ovulatory function of testosterone-treated sheep, the release of endogenous LH and biopotent FSH in control and prenatal testosterone-treated sheep was blocked with a GnRH antagonist during the first two breeding seasons and with LH/FSH coadministered in a manner approximating natural follicular phase. An acidic mix of FSH was administered the first 36 h at 2-h intervals and a less acidic mix for the next 12 h at 1-h intervals (different FSH preparations were used each year), and ovulation was induced with hCG. Circulating FSH and estradiol responses to gonadotropins measured in 2-h samples differed between treatment groups in Year 1 but not in Year 2. Ovarian follicular distribution and number of corpora lutea (in ewes that ovulated) tracked by ultrasonography and luteal progesterone responses were similar between control and prenatal testosterone-treated females but differed between years. Furthermore, hCG administration induced large cystic and luteinized follicles in both groups of females in Year 2, although the growth rate differed between control and prenatal testosterone-treated females. Our findings provide evidence that 1) ovulatory response in prenatal testosterone-treated females can be rescued with exogenous gonadotropins, 2) resultant follicular response is dependent on the nature of gonadotropic input, and 3) an abnormal follicular milieu may underlie differences in developmental trajectory of cystic follicles in prenatal testosterone-treated females.

FIG. 1.

Top) Duration of suppression of LH in ovariectomized control females following a single administration of 5 or 25 μg/kg of the GnRH antagonist, acyline. Bottom) Efficacy of 10 μg/kg of GnRH antagonist administered every 12 h to ovariectomized control (OVX control) and ovary-intact prenatal testosterone-treated (T-treated) females in achieving prolonged suppression of LH. Arrows indicate time of administration of the GnRH antagonist.

FIG. 2.

Schematic detailing the experimental design of the two studies conducted during the first two breeding seasons. The GnRH antagonist was given every 12 h (for 5 days in study 1 and 9 days in study 2) to block endogenous LH and the GnRH-induced release of less acidic FSH. Starting 3 and 7 days after the start of GnRH antagonist treatment, LH/C-FSH was given every 2 h (denoted as C) for 36 h, followed by administration of LH/N-FSH or LH/C-FSH/N-FSH hourly for next 12 h in study 1 and study 2, respectively (denoted as N). Ovulation was induced by administering 1500 IU of hCG. Starting from 4 h before the start of LH/FSH administration, blood samples were collected at 2-h intervals until 96 h (Year 1) or 116 h (Year 2). Black boxes represent the period of frequent sampling (12-min intervals) conducted to characterize the profile of LH and FSH following an injection. Daily blood samples were taken for 15 and 19 days in study 1 and study 2, respectively, for measurement of circulating levels of progesterone to assess function of corpora lutea. Transrectal ultrasonography (US) was performed to characterize ovarian follicular response to the exogenous pulses of LH/FSH and CL development. See text for complete description of dosage and timing of treatments.

FIG. 3.

A) On the left are elution patterns of FSH isoforms from study 1 (top) and study 2 (bottom) following chromatofocusing separation of C-FSH and N-FSH preparations. Bar graph on the right shows the percentage of FSH eluting in fractions with pH greater than 5.4, pH less than 5.4, and salt peaks. Note the differences in distribution of FSH isoforms used in the two studies. B) Circulating patterns of LH and FSH during the natural follicular phase (n = 18, composite data from earlier studies). Arrows indicate timing of the LH surge. C) FSH eluting in fractions with pH greater than 5.4, pH less than 5.4, and salt peaks following chromatofocusing separation of circulating FSH from prepubertal and pubertal lambs from a previous study [40].

FIG. 4.

A) Mean circulating patterns of LH and FSH achieved in control (C) and prenatal testosterone-treated (T) females from study 1, conducted during the first breeding season. Delivery patterns of LH/FSH assessed from frequent samples are shown as insets. Period 1 represents the LH/FSH injection period at 2-h intervals and period 2 the injection period at 1-h intervals. B) Mean circulating patterns of estradiol achieved in control and prenatal testosterone-treated females. Circulating patterns of estradiol in the subset of prenatal testosterone-treated females that were anovulatory before the start of study are shown on the right. C) Estradiol summary statistics, amount of estradiol produced during the C-FSH and N-FSH administration, peak levels achieved during gonadotropin treatment, and total estradiol produced during the gonadotropin treatment. Asterisks indicate significant differences from control females (*P < 0.05).

FIG. 5.

A) Mean circulating patterns of LH and FSH in control (C) and prenatal testosterone-treated (T) females from study 2, conducted during the second breeding season. The LH/FSH delivery patterns from the frequent sampling time periods (period 1, LH/C-FSH administered every 2 h; period 2, LH and C-FSH+N-FSH administered every 1 h) are shown as insets. B) Mean circulating patterns of estradiol achieved in control and prenatal testosterone-treated females. Results for the subset of prenatal testosterone-treated females that were anovulatory before the start of study are shown on the right. C) Estradiol summary statistics (amount of estradiol produced during the C-FSH and C-FSH+N-FSH administration, peak levels achieved during gonadotropin treatment, and total estradiol produced during the gonadotropin treatment). Asterisks indicate significant differences from control females (*P < 0.05, **P < 0.01).

FIG. 6.

Left) Percentage of ewes that showed a rise in P₄ following hCG administration, number of CL per ewe, and total progesterone production from both study 1 and study 2. Right) Mean circulating patterns of progesterone in control (C) and prenatal testosterone-treated (T) females following ovulation induction with hCG at the end of LH/FSH treatment from study 1 (top) and study 2 (bottom). Inset shows circulating patterns of hCG achieved following administration of 1500 IU of hCG (not determined in study 1).

FIG. 7.

Total number of follicles in size classes of ≤3 mm, greater than 3 to 4 mm (gonadotropin-dependent recruited follicles), greater than 4 to 8 mm (follicles selected to become ovulatory sized), and greater than 8 mm as detected by ultrasonography in both ovaries of control (C) and prenatal testosterone-treated (T-treated) sheep from study 1 and study 2. Ultrasonography was performed before initiation of the study, after 2 days of GnRH antagonist treatment, after 48 h of LH/FSH injections, and 24 and 48 h after induction of ovulation with hCG in study 1 and before initiation of the study, after 6 and 7 days of GnRH antagonist treatment, after 24 and 48 h of LH/FSH injections, and 24, 48, 72, and 96 h after hCG administration in study 2. Gray-shaded areas correspond with LH/FSH injections. Double asterisks indicate significant differences (**P < 0.01).

FIG. 8.

Ultrasonographic images of ovary from one control and one prenatal testosterone-treated (Prenatal T-treated) ewe before and after GnRH antagonist (GnRH-A) administration, 24 and 48 h after start of LH/FSH treatment, and 24, 48, and 96 h after hCG administration. Scale shown at top right applies to all images.