No abstract

Androgens have distinct physiological functions within the ovary. The biological action of androgens is primarily exerted through transcriptional regulation by the nuclear androgen receptor (AR), but the molecular cascades governed by AR remain largely unknown. At present, there is imminent concern that environmental man-made chemicals with antiandrogenic properties, among others, are capable of modulating hormonal responses, thereby interfering with normal physiological processes that are critical to fertility. In the present study, we aimed to further characterize a standardized and reproducible follicle culture system in terms of AR expression during in vitro folliculogenesis to be able to use it as a bioassay to study effects of antiandrogens on follicular and oocyte growth, steroid secretion profile, and oocyte meiotic maturation capacity. Immunohistochemical analysis revealed that cytoplasmic AR protein was translocated to the nucleus of granulosa and theca cells in response to endogenous androgen production in theca cells during preantral follicular development. During the antral phase in vitro, AR was differentially expressed in mural and cumulus cells, implying an oocyte-mediated regulation. Treatment of follicles with hydroxyflutamide or bicalutamide, two model antiandrogenic compounds, resulted in reduced follicular growth during the preantral phase, altered steroidogenic environment, and arrest in oocyte meiotic maturation in response to human chorionic gonadotropin. Androgen receptor expression in the culture model corresponded well to what is described in vivo, and this system revealed several ovarian functions targeted by AR antagonists that can be further investigated using more in-depth molecular techniques.

A reproducible two-step culture system for isolated mouse ovarian follicles smaller than 100 microm (type 3a follicles) was designed. First, isolated follicles were grown in single droplets of alpha-minimal essential medium (MEM) without (deoxy)ribonucleosides at a lower concentration of fetal bovine serum (FBS; 1%) for 6 days with mechanical prohibition of thecal cell attachment. Growing follicles reaching at least 100 microm were transferred to alpha-MEM medium enriched with a higher concentration (5%) of FBS to allow attachment and were cultured subsequently for an additional 12 days. Overall, more than 85% of the follicles survived the first culture step, and oocyte growth and granulosa cell proliferation had increased by 25% (P < 0.05). Follicle survival at Day 18 was related to initial follicle diameters at isolation. Average meiotic maturation rates and estrogen secretion were lower compared to those of cultures starting with early preantral follicles of 100-130 microm. Although reverse transcription-polymerase chain reaction analysis revealed the presence of LH-receptor mRNA in thecal cells, an exogenous androstenedione replacement resulted in an increase of estrogen production, suggesting substrate insufficiency. The time needed to grow from early preantral stages to in vitro ovulation is strongly dependent on the initial follicle diameter at isolation. Morphological characteristics of cultured follicles were suggestive for combined transforming growth factor beta deficiencies during in vitro culture.

No abstract

Estrogens play an important role in the growth, differentiation, and function of female reproductive tissues. Estrogen signals through estrogen receptors (ERs), members of the nuclear receptor superfamily. The two major forms, ERalpha and ERbeta, are expressed in the mouse ovary, where ERbeta is predominantly expressed in granulosa cells, and ERalpha in theca cells. In this study, we determined the expression pattern of ER subtypes within mouse follicles cultured from the early preantral stage up to the preovulatory stage and after an ovulatory stimulus in different culture conditions. Immunohistochemical studies performed at different time points of culture revealed that ERbeta was found exclusively in granulosa cell nuclei regardless of follicular growth stage or culture conditions. In contrast, ERalpha was found in oocyte, granulose, and theca cells, and its subcellular localization differed between follicular growth stages and culture conditions. A shift from a predominant cytoplasmic to a predominant nuclear immunolocalization was observed in granulosa cells as follicles reached the antral growth phase, and was postponed in culture conditions with minimal growth factor supplementation. In response to hCG, ERbeta protein levels in luteinized granulosa cells spectacularly declined to undetectable levels, while ERalpha immunostaining again shifted to cytoplasmic regions, but not in theca cells.