Abstract
Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility affecting more than 100 million women worldwide, yet its etiology remains unclear. Fetal exposure to elevated androgen levels has, however, become increasingly implicated in the development of the syndrome. It is hypothesized that increased exposure to androgens during fetal development affects the steroid receptors in the ovary. The aim of this study was to use a sheep model to determine the effect testosterone treatment to pregnant ewes has on the steroid receptors, ERα, ERβ and AR in the fetal ovary.
Control and testosterone treated ovarian samples were collected at 90 days of gestation. Ovarian sections were examined histologically to determine the cell types and tissue organization at this stage of development and to observe if this was affected by testosterone treatment. Immunohistochemical staining was used for the protein localization of each steroid receptor. A radioactive in situ hybridisation (ISH) was undertaken to determine the localization of the mRNA transcripts. A quantitative analysis of the mRNA expression levels of each receptor was done using real-time quantitative PCT (qRT-PCR) (utilizing the fluorogenic dye, SYBR green).
The histological organization of ovaries in this study was similar to previous descriptions that have investigated fetal ovarian histology in sheep. The day 90 ovary contained ovigerous cords with well-pronounced cell types both within and outside the cords. No differences in histology were observed between the control and testosterone treated ovaries.
Immunohistochemistry (IHC) provided visualization of clear nuclear staining for each receptor. The staining was predominately observed in the ovarian cortex, specifically within the cells of the ovigerous cords. No observable were seen differences in the patterns of protein expression between control and testosterone treated ovaries, nor were differences in the intensities of staining apparent. This was consistent with all three steroid receptors (ERα, ERβ and AR). In situ hybridization provided evidence for the presence of mRNA transcripts of all three steroid receptors within the ovarian tissue but this method did not have the resolution to define specific cell expression. These findings were similar in both control and testosterone treated ovaries. High quality RNA was isolated from the ovaries and statistical analysis of the results obtained from the qRT-PCR provided evidence that there were no differences in mRNA expression levels of the receptors in control and testosterone treated ovaries.
The results in the current study provided evidence that testosterone treatment does not effect the distribution or expression of the steroid receptors ER𝛼, ER𝛽 and AR in fetal ovaries and thus does not support the hypothesis. This study provided further insight into the potential function of these steroid receptors in ovarian development. Specifically this highlighted their importance in the development of pre-granulosa cells from the ovarian surface epithelial cells, oogonia and their subsequent association to form primordial follicles.