Endocrine Abstracts

In rodents and other mammals, it is well established that the production of certain TGF-β members including Gdf9 and Bmp15 by oocytes is important for follicle development to proceed. It is believed that these growth factors form part of a short loop paracrine system to regulate gene expression in neighbouring follicle cells. We propose that one aspect of this feedback loop may involve the genomic modulation of TGF-β antagonists in granulosa cells by oocyte-secreted factors, yet simple models to directly test this concept in preantral follicles are currently limited. Preantral follicles with <3 complete layers of granulosa cells were freshly isolated from the ovaries of 12-day-old mice and imaged. Follicle size was related to the number of layers of granulosa cells as assessed by 3D confocal imaging. Each follicle was either mechanically oocytectomised (OOX), or manipulated under a microscope using glass micropipettes (control) and maintained in vitro. After 24 h, individual follicles were snap frozen for RNA isolation and RT-PCR analysis. Expression of Gdf9 was detectable in control follicles but was completely absent in all OOX samples. Transcripts for factors normally expressed by granulosa cells such as AMH receptor 2, inhibin-α and Kit ligand were detectable in all samples. The TGF-β antagonists Twsg1, Prdc and Htra1, which are normally highly expressed in preantral follicles, were still readily detectable in granulosa cells after oocytectomy, indicating that the oocyte does not play a major role regulating their expression. These results demonstrate that the microsurgical removal of preantral oocytes still enables the integrity and phenotype of the remaining granulosa cells to be preserved, thus providing a useful model for dissecting the effects of oocyte secreted factors on the granulosa cell transcriptome during early stages of follicle development.