PARENT SESSION

(T440) siRNA SUPPRESSION OF GDF-9 mRNA IN VITRO BLOCKS THE FORMATION OF PRIMORDIAL FOLLICLES IN THE HAMSTER.

Wang, Cheng¹, Roy, Shyamal K¹, ¹ University of Nebraska Medical Center, Omaha, NE

ABSTRACT- Exact factors or mechanisms that signal somatic cell differentiation into granulosa cells leading to primordial follicle formation are still not fully understood. Evidence suggests that GDF-9, one of the members of transforming growth factor- superfamily of ligands, plays crucial roles in early folliculogenesis. RNAi provides a new and powerful tool for the study of the function and mechanisms of genes of interest. The objective of this study was to investigate the physiological role of GDF-9 on early folliculogenesis by silencing the expression of GDF-9 mRNA in fetal hamster ovaries in vitro. siRNA was designed from the nucleic acid sequence of the ORF of hamster GDF-9, which was cloned in our laboratory, and synthesized by Dharmacon. Fetal hamster ovaries at 15^th day of gestation were cultured in vitro in the absence or presence of 150nM GDF-9 siRNA delivered by Metafectene with or without 1 ng/ml ovine-FSH-20. Ovaries were cultured for 9 days because primordial follicles appeared by that time both in vivo and in vitro. Ovaries were processed for morphological assessment of primordial and primary follicle development. Some ovaries were cryosectioned for localization of GDF-9 protein by fluorescence immunohistochemistry and apoptotic by TUNEL protocol. Total RNA was extracted from a third set of ovaries for real-time RT-PCR quantification of the levels of GDF-9, -actin, TbRII and BMPR. The number of apoptotic cells did not vary appreciably between different treatment groups indicating that neither the transfection agent nor the dosage of siRNA had any generalized toxic effect on ovarian cells. Compared to the siRNA untreated group, GDF-9 mRNA levels increased markedly in the presence of FSH; however, siRNA treatment completely suppressed the FSH effect. In contrast, siRNA had no effect on the levels of -actin mRNA or FSH-induced increase in the levels TbRII or BMPR. Most importantly, siRNA treatment resulted in a complete block in FSH-induced primordial follicle formation and their subsequent development into the primary stage. These results indicate that GDF-9 has important roles in primordial and primary follicle formation in the hamster and GDF-9 gene expression is regulated by FSH.

KEY WORDS: GDF-9, siRNA, Primordial follicle