| In mammals, the primordial follicle pool formed perinatally in ovary is the only source of developing follicles and oocytes throughout the entire reproductive life span and determines the reproductive potential of females. During primordial follicle formation, dysfunctional development will retard the breakdown of germline cell cysts and lead to multi-oocyte follicles (MOFs) to shorten the reproductive life span and significantlly decrease oocyte maturation rate in vitro. During primordial follicle formation, many genes are spatiotemporally expressed in germ cells and pregranulosa cells. However, the molecular mechanisms by which the essential genes are regulated coordinately to ensure primordial follicle assembly remain a mystery. Racl belongs to the small (21 kDa) RhoGTPase family that is a ubiquitous protein family that acts as molecular switches and plays many roles in cellular development. Recent studies have implicated Rac1 in the regulation of many reproductive events, However, whether Racl has a role in early folliculogenesis has not yet been reported. Racl affects transcription of many genes via STAT3, a member of the signal transducers and activators of transcription (STAT) family. Therefore, we postulated that Racl plays a physiological role in primordial follicle formation and modulates transcriptional activation of genes necessary for follicular assembly.In our study, we demonstrated that Racl was expressed in germ cells and progressively up-regulated in follicular assembly. To investigate the physiological role of Rac1 in mouse follicular assembly, we performed an in vitro ovary organ culture expriments. Results suggested that the disruption of Racl by NSC23776 or Racl siRNA resulted in a significant reduction in primordial follicles formation and a corresponding increase in germ cells that remained within nests in cortext, while the overexpression of Rac1 accelerated the formation of primordial follicle. In addition, in vivo inhibitor treatment resulted in the presence of multi-oocyte follicles in ovaries of pubertal mouse. To explore how Racl regulates primordial follicle formation, we investigated the relationship between Rac1 and the essential genes for primordial follicle formation, results imply that Rac1 regulated oocyte-enriched essential genes at the mRNA level including Jaggedl, GDF9, BMP 15 and Nobox, while Notch2 was regulated at the protein level in pregranulosa cells.Subsequent studies implied that Racl induced STAT3 nuclear trafficking through direct binding. In turn, nuclear STAT3 directly activated the transcpription of Nobox, Jaggedl, GDF9 and BMP 15 in germ cells. Further, GDF9 and BMP 15 modulated Notch2 protein translation by activating mTORC1 in pregranulosa cells. Overexression or addition of Jaggedl, GDF9 and BMP 15 not only reversed the effects of NSC23766 on primordial follicle formation, but also accelerated primordial follicle formation via Notch2 signaling pathway activation. Collectively, these results indicate that Rac1 plays important roles as a key regulator in follicular assembly. |
