Effects of RAC1 on Proliferation of Hen Ovarian Prehierarchical Follicle Granulosa Cells

SIMPLE SUMMARY: The growth and development of ovary follicles is an intricate, highly organized process involving many local intra-ovarian factors. Ras-related C3 botulinum toxin substrate1 (RAC1) is speculated to be associated with prehierarchical follicle development of hen ovaries. The current study initially revealed RAC1 mRNA to be expressed in varied-size follicles and stroma and its expression levels in the prehierarchical follicles of 1.0–3.9 mm, 6.0–6.9 mm and 7.0–8.0 mm in diameter were remarkably higher than the other groups. Moreover, RAC1 protein was mainly expressed in the oocytes and granulosa cells (GC), as well as in stromal tissues of the follicles. To understand the exact roles of the RAC1 gene in regulation of follicular GC proliferation and differentiation, siRNA interference and overexpression of the RAC1 gene were conducted. Our experiments demonstrated that the RAC1 gene can significantly promote the expression of mRNA and proteins of FSHR, CCND2, CYP11A1, PCNA and StAR genes in GC and directly elevate the proliferation of GC in vitro. These results indicated RAC1 played a crucial role in regulation of GC proliferation and differentiation and steroidogenesis during the development of prehierarchical follicles. This study provided a base for elucidating the molecular mechanisms underlying the biological effect of RAC1 on the hen ovary follicle growth and development. ABSTRACT: RAC1 belongs to the small G protein Rho subfamily and is implicated in regulating gene expression, cell proliferation and differentiation in mammals and humans; nevertheless, the function of RAC1 in growth and development of hen ovarian follicles is still unclear. This study sought to understand the biological effects of RAC1 on granulosa cell (GC) proliferation and differentiation of hen ovarian prehierarchical follicles. Firstly, our results showed expression levels of RAC1 mRNA in the follicles with diameters of 7.0–8.0 mm, 6.0–6.9 mm and 1.0–3.9 mm were greater than other follicles (p < 0.05). The RAC1 protein was mainly expressed in oocyte and its around GCs and stromal tissues of the prehierarchical follicles by immunohistochemistry. Further investigation revealed the RAC1 gene remarkably enhanced the mRNA and protein expression levels of FSHR (a marker of follicle selection), CCND2 (a marker of cell-cycle progression and GC differentiation), PCNA (a marker of GC proliferation), StAR and CYP11A1 (markers of GC differentiation and steroidogenesis) (p < 0.05). Furthermore, our data demonstrated siRNA interference of RAC1 significantly reduced GC proliferation (p < 0.05), while RAC1 gene overexpression enhanced GC proliferation in vitro (p < 0.05). Collectively, this study provided new evidence that the biological effects of RAC1 on GC proliferation, differentiation and steroidogenesis of chicken ovary follicles.