Abstract
The ovarian follicle is a major site of steroidogenesis, crucially required for normal ovarian function and female reproduction. Our understanding of androgen synthesis and metabolism in the developing follicle has been limited by the sensitivity and specificity issues of previously used assays. Here we used liquid chromatography–tandem mass spectrometry to map the stage-dependent endogenous steroid metabolome in an encapsulated in vitro follicle growth system, from murine secondary through antral follicles. Furthermore, follicles were cultured in the presence of androgen precursors, nonaromatizable active androgen, and androgen receptor (AR) antagonists to assess effects on steroidogenesis and follicle development. Cultured follicles showed a stage-dependent increase in endogenous androgen, estrogen, and progesterone production, and incubations with the sex steroid precursor dehydroepiandrosterone revealed the follicle as capable of active androgen synthesis at early developmental stages. Androgen exposure and antagonism demonstrated AR–mediated effects on follicle growth and antrum formation that followed a biphasic pattern, with low levels of androgens inducing more rapid follicle maturation and high doses inhibiting oocyte maturation and follicle growth. Crucially, our study provides evidence for an intrafollicular feedback circuit regulating steroidogenesis, with decreased follicle androgen synthesis after exogenous androgen exposure and increased androgen output after additional AR antagonist treatment. We propose that this feedback circuit helps maintain an equilibrium of androgen exposure in the developing follicle. The observed biphasic response of follicle growth and function in increasing androgen supplementations has implications for our understanding of polycystic ovary syndrome pathophysiology and the dose-dependent utility of androgens in in vitro fertilization settings.
Highlights
Female reproductive health relies on the proper development of the follicle, the fundamental unit of the ovary
Administration of DHT in combination with the androgen receptor (AR) antagonist MDV resulted in a significant upregulation of follicular androgen synthesis at day 6 (490 6 256 nmol/L with DHT + MDV vs 80 6 15 nmol/L with DHT alone; P, 0.05) [Fig. 3(c)]. These differences in androgen production were not mirrored by significant changes in steroidogenic enzyme expression at the Messenger RNA (mRNA) level. These results suggest that in vitro cultured follicles are capable of autonomously adapting endogenous androgen synthesis in response to changes in AR activation status, possibly indicating an intrafollicular AR-mediated autocrine feedback circuit involved in steroidogenesis
Previous studies have proven the importance of androgen action in follicular development, our study extends this knowledge by approaching the follicle as a coordinated steroidogenic unit
Summary
Female reproductive health relies on the proper development of the follicle, the fundamental unit of the ovary. As waves of follicles grow, they produce sex steroid hormones that regulate maturation in an autocrine/paracrine manner, supply endocrine feedback. Another advantage of eIVFG is the possibility of directly studying the dose- and stage-dependent effects of exogenous factors on individual follicle development and function. Recent work has shown that nuclear and extranuclear ARmediated signaling pathways are crucially involved in promoting follicle growth and survival [27]. These fundamental studies are important because alterations in androgen homeostasis in women may result in infertility and anovulation. We have used the murine eIVFG system and steroid analysis by LC-MS/MS to comprehensively map the stagedependent endogenous steroid metabolome of the follicle during development and to directly examine the dosedependent effects of the nonaromatizable potent androgen 5a-dihydrotestosterone (DHT), the sex steroid precursor DHEA, and the selective AR antagonist enzalutamide (MDV) on follicular function and steroidogenesis
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