Developmental Programming: Prenatal Testosterone Excess Disrupts Expression of Oocyte and Granulosa Cell Growth Factors in Sheep.

Abstract

Prenatal testosterone (T) excess in sheep leads to ovarian dysfunction including increased ovarian follicular recruitment and persistence culminating in an adult Polycystic Ovary Syndrome (PCOS)-like reproductive phenotype. Growth factors expressed in granulosa cells such as antimullerian hormone (AMH) and c-kit ligand (KL) and the oocyte growth differentiation factor-9 (GDF-9) have been implicated in follicular differentiation. We hypothesized that the prenatal T excess would alter the relative expression of these growth factors thus contributing to aberrant follicular differentiation. Pregnant Suffolk sheep were given T propionate (100 mg, i.m. twice weekly) in cottonseed oil from days 30-90 of gestation (term is ~147 d). Controls (C) received vehicle. Changes in developmental expression of AMH and KL were determined in two ovarian sections (5μm thickness) <5mm apart from each of 6 animals/age/treatment group at fetal (days 90 and 140), postpubertal (10 months), and adult (21 months) ages by immunohistochemistry (IHC). Changes in oocyte GDF-9 expression were evaluated only during postpubertal and adult ages. Consecutive sections were used to relate changes in AMH and KL expression within the same follicle. Follicles were classified as primordial, transitional, primary, small preantral (SPF), large preantral (LPF), small antral (SAF) and large antral (LAF). Degree of expression was scored in a 0 to 5 scale and analyzed using T-test and linear regression. Fetal day 90 ovaries had only primordial to primary follicles while day 140 ovaries had few SPF (range/animal: 2-13), LPF (1-15), SAF (0-9), and LAF (0-1) follicles. All follicles at both ages expressed KL with AMH expressed only in the preantral and antral follicles. Number of preantral follicles studied ranged from 10-45/animal in 10 mo-old and 15-79 in 20 mo-old animals. Consistent with increased follicular recruitment, number of follicles beyond the SPF stage was higher in T females compared to C (P<0.05). Prenatal T treatment did not affect KL expression at any follicular stage. AMH expression increased with follicular stage from SPF to SAF in both groups of females. There was an age effect in AMH expression with it being higher at 20 mo compared to 10 mo in LPF and LAF in C and SPF, LPF and LAF in T females (P<0.05). AMH expression decreased between SAF and LAF (P<0.05) in C but not T females at 20 mo consistent with the altered follicular growth seen in T females. As a result, AMH expression was relatively low in LPF and high in LAF in T females compared to C (P<0.01). The percentage of LPF that had lower AMH expression (scale 0-1) was 1.8 times higher in the T group (C: 15.8 vs. T: 28.4%; P<0.05) while percentage of LAF that had lower AMH expression (0-1) was 1.4 times higher in the C group (C: 52.5 vs. 37.1%; P=0.057). Because AMH is known to reduce follicular sensitivity to FSH, the increased AMH expression in LAF in T females may contribute to the follicular persistence seen in T females. Changes in GDF-9 were evident only at 10 mo with GDF-9 expression being lower in LPF of T compared to the C group (P<0.01) suggestive of altered oocyte quality. The small sample size precluded statistical comparison of GDF-9 expression in LAF. Overall, the imbalance in paracrine environment stemming from altered AMH and GDF-9 expression in T females may underlie the follicular disruptions namely, recruitment and persistence, seen in the T females. Supported by P01 HD44232. (platform)