122. REGULATION OF GDF-9 AND GDF-9B BY FSH IN PREANTRAL FOLLICLE CULTURES

Abstract

GDF-9 and -9B (BMP-15) are oocyte-derived members of the TGF-β superfamily. In the mouse ovary, the absence of GDF-9 leads to an arrest of follicle development at the primary/preantral stage. As a result GDF9 deficient mice are infertile¹ because follicle development does not reach a stage where ovulation and oocyte release can occur. In contrast, GDF-9B knockout mice are subfertile². GDF-9 was shown to act via TGFβRI and BMPRII and GDF-9B via BMPRIB and BMPRII. We have much to learn about what regulates the production of GDF-9, GDF-9B and the expression of its receptors. These studies investigated whether FSH, an important mediator of folliculogenesis, plays a role in this regulation. Preantral follicles (110–135 µm in diameter) were isolated from 18 day old C57BL/6 mice using fine needles. Follicles were cultured (30–35 per well) for 7 days with varying doses of FSH (0–100ng/ml). RNA was extracted, reverse transcribed and real time PCR was carried out with primer sets for GDF-9, GDF-9B, TGFβRI, BMPRII and GAPDH. Immunohistochemistry was conducted on sections of formalin-fixed 18 day old mouse ovary using antisera directed at TGFβRI and BMPRII. GDF-9 and GDF-9B mRNAs were downregulated by FSH treatment (compared to untreated control). There was no effect of FSH on the expression of either receptor. TGFβRI and BMPRII receptor proteins were localised to the cytoplasm of oocytes and granulosa cells in 18 day old mouse ovary. Both were mostly localised to secondary follicles, lighter TGFβRI staining was found in less mature follicles. Receptors for GDF9 signalling were both present consistent with direct effects of GDF9 on ovarian function. GDF9B might also have an effect although it remains to be seen if the type 1 receptor is localised to the mouse ovary. Further studies are required to investigate receptor regulation.