Postnatal growth differentiation factor 9 (GDF-9) expression in the hamster oocytes precedes the formation of primordial follicles. We examined the functional significance of GDF-9 in primordial folliculogenesis in the hamster ovary using RNA interference knockdown of GDF-9 mRNA and protein expression. Fifteen-day-old fetal ovaries were cultured for 9 d with or without 1 ng FSH, 1 μl Metafectane, 100 nM control nontargeting small interfering RNA (siRNA), GDF-9 siRNA, or GDF-9 siRNA + FSH, and the development of primordial follicles examined. The efficiency of siRNA transfecting ovarian cells in the organ culture was tested by culturing ovaries with siGlo, a nontargeting control siRNA labeled with Cy3. More than 90% of cells in the ovary were siGlo positive, and neither the Metafectane nor the siRNA-induced cellular apoptosis. Control siRNA did not affect the basal levels of GDF-9 mRNA, but GDF-9 siRNA slightly but significantly reduced the level. FSH markedly up-regulated the levels of GDF-9 mRNA and protein, and the effect was completely suppressed by GDF-9 siRNA. However, GDF-9 siRNA did not affect the levels of bone morphogenetic protein receptor IA or β-actin mRNA. GDF-9 siRNA alone also reduced GDF-9 protein expression. Concurrent with GDF-9 expression, FSH significantly augmented primordial follicle formation, but the effect was abolished by GDF-9 siRNA. These results suggest that endogenous GDF-9 plays an important role in somatic cell differentiation and the formation of primordial follicles. Furthermore, FSH, by virtue of regulating GDF-9 expression, modulates oocyte regulation of primordial follicles formation.
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