Excess Testosterone Exposure Alters Hypothalamic-Pituitary-Testicular Axis Dynamics and Gene Expression in Sheep Fetuses

Abstract

Prenatal exposure to excess androgen may result in impaired adult fertility in a variety of mammalian species. However, little is known about what feedback mechanisms regulate gonadotropin secretion during early gestation and how they respond to excess T exposure. The objective of this study was to determine the effect of exogenous exposure to T on key genes that regulate gonadotropin and GnRH secretion in fetal male lambs as compared with female cohorts. We found that biweekly maternal testosterone propionate (100 mg) treatment administered from day 30 to day 58 of gestation acutely decreased (P < .05) serum LH concentrations and reduced the expression of gonadotropin subunit mRNA in both sexes and the levels of GnRH receptor mRNA in males. These results are consistent with enhanced negative feedback at the level of the pituitary and were accompanied by reduced mRNA levels for testicular steroidogenic enzymes, suggesting that Leydig cell function was also suppressed. The expression of kisspeptin 1 mRNA, a key regulator of GnRH neurons, was significantly greater (P < .01) in control females than in males and reduced (P < .001) in females by T exposure, indicating that hypothalamic regulation of gonadotropin secretion was also affected by androgen exposure. Although endocrine homeostasis was reestablished 2 weeks after maternal testosterone propionate treatment ceased, additional differences in the gene expression of GnRH, estrogen receptor-β, and kisspeptin receptor (G protein coupled receptor 54) emerged between the treatment cohorts. These changes suggest the normal trajectory of hypothalamic-pituitary axis development was disrupted, which may, in turn, contribute to negative effects on fertility later in life.

Early prenatal androgen exposure reduces testes size and sperm concentration in sheep without altering neuroendocrine differentiation and masculine sexual behavior

“…Recent studies have shown that exposing developing male lamb fetuses to excess androgen prior to midgestation inhibits pituitary gonadotropin secretion and suppresses expression of testicular enzymes catalyzing T production. Using the same treatment paradigm as the current study, we found previously that excess T suppressed LH levels and androgen synthesis enzyme expression in the testis as early as day 60 of gestation [12]. This effect was reversed two weeks after treatment stopped suggesting that the testicular disruption was shortlived.…”

“…USA) following the manufacturer's instructions. RNA sample concentration and purity were evaluated as described previously [12]. RNA was reverse transcribed to cDNA using Oligo dT primers with the First Strand Superscript III Kit (Invitrogen, Carlsbad, CA.…”

“…USA). Primer sets for the ovine genes evaluated have been validated and published previously [12]. All reactions were run on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Life Technologies).…”

“…Few studies have evaluated the effects of limiting the timing of exposure in males to the early part of the critical period when the gonads and genitalia differentiate and functional connections are established between the hypothalamus and pituitary [26, 27]. We reported that early exposure to exogenous T between days 30 and 60 of gestation (term = 147 days) acutely suppresses LH secretion and Leydig cell steroidogenesis in a manner consistent with enhanced negative feedback, but found that hypothalamic gene expression is altered even after exposure ended [12]. Early excess T also paradoxically reduced the volume of the ovine sexually dimorphic nucleus (oSDN) which is correlated with sexual partner preferences in sheep [28].…”

The influence of maternal androgen excess on the male reproductive axis

“…These findings highlight the exquisite sensitivity of the developing female brain, which ordinarily is exposed to very low androgens during development, and suggest that the male brain, which in late gestation is exposed to elevated gonadal testosterone, is able to compensate. The male reproductive axis is functional before birth in male foetuses, and maintains homeostatic levels of serum testosterone via negative feedback mechanisms 12,57,58 . Therefore, it is plausible that in males the active hypothalamic-pituitary-testicular axis is able to compensate for exogenous androgen exposure during prenatal development by adjusting LH secretion from the anterior pituitary and downstream testosterone synthesis to consequently restore homeostasis to the androgen milieu in utero .…”