Effects Of BMP/Smad Signaling On Porcine Follicular Granulosa Cells

Prolificacy is one of the most important factors which influence the development of pig industry and it is also the main limiting factor to decide the efficiency of pig industry development. Fertility depends on the level of maternal growth and development of ovarian follicles as ovulation follicle development determines the number of ovum ovulated which is the key factor affecting the fecundity. To master sow follicular development and its impact factors are conducive to further improve sow reproductive capacity and enhance the economic benefits of survival pigs. Mammalian follicular development is a very complex process, including recruitment of the primitive follicle, the development of preantral follicles and antral follicle selection, growth and mature or follicle atresia. It was found that follicular growth and development is a highly coordinated and physiological and biochemical cascade processes affected and regulated by acting downstream of complex signaling pathways that integrate signals from the surrounding microenvironment but not an independent event with the development of post-genomics and bioinformatics.Granulosa cells are key somatic cells in follicules, and play very important roles in oocyte maturation and follicular development. Follicular development is accompanied by growth, proliferation, differentiation, and maturation of granulosa cells. Bone morphogenetic proteins (BMPs) belong to the transforming growth factorβ(TGFβ) superfamily and have implicated in the control and regulation of follicular development and female fertility. Recently, there are lots of research about the role of BMP on reproduction, including to follicular growth and development, granulosa cells proliferation and apotosis, steroidogenesis, oocyte maturation, ovulation and luteinization. However, the roles and the molecular mechanisms of BMP/Smad signaling alters follicular development have not been fully investigated in pigs. To further research the roles and mechanisms of BMP/Smad signaling in follicular granulosa cells provide possible rationale to select of mammalian reproductive trait and have great importance to the improvement of reproductive capacity of domestic animal. In this study, the time-spatial expression of BMP receptors and Smads in porcine follicular granulosa cells (GCs) in different period were investigated, by quantitative real-time PCR and cellular localization of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was studied in porcine ovaries by immunohistochemistry (IHC). In order to repress BMP/Smad signaling, the gene of Smad4 was silenced by RNAi, the effects of repressed BMP/Smad signaling on porcine follicular granulosa cells and underlying molecular mechanism were detected by MTT, FCM, CLIA and real time RT-PCR. Based on the repressed signaling, cells were treated with FSH, and the effects of repressed BMP/Smad signaling on the role FSH to GCs were investigated by MTT, FCM, CLIA and real time RT-PCR. Cells were treated with BMP-6, a activator of BMP/Smad signaling, as well as the Smad4-RNAi which repressed the BMP/Smad signaling, then the survival rate, proliferation and secretions of E2 and P4 of GCs were detected by MTT, FCM and CLIA; Microarray analysis was performed to capture the difference in gene expression in porcine GCs of between repressed the signaling and repressed then activated signaling.The main results achieved were as follows:1 The mRNA expression of BMP receptors and Smads in granulosa cells was detected by real time RT-PCR. The results indicated that endogenous BMP receptors and Smads expressed in porcine GCs, and ActRIA and BMPR2 mRNA levels were significantly higher in DF than that in SF(P<0.05). The mRNA levels of BMPR1A, ActR2, Smadl, Smad5 and Smad8 presented increased tendency, but there’s no significant difference in DFs and SFs. Whereas BMPR1B, Smad4 and Smad7 expression had tended to decrease from SF to DF. Together, the stage-specific expression pattern of BMP receptors and Smads suggested that the BMP/Smad signaling might play potential roles in the follicle GCs growth and differentiation.2 Cellular localization and expression of Smad4 proteins were studied by immunohistochemistry (IHC) in the ovaries of pigs. The results indicated that Smad4 localized in all kinds of cells of follicles at all stages. Smad4 localized in the cytoplasm of oocyte of primordial and primary follicles but not in granulosa cells; in pre-antral follicles, Smad4 localized in the oocytes and granulosa cells; Smad4 mainly localized in granulosa cells and follicular theca cells in antral follicles, and in oocyte, the expression of Smad4 was low. 3 To demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs, granulosa cells were isolated from 3-5mm antral follicles in diameter and cultured in vitro. A strategy of RNAi-mediated ’gene silencing’ of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to repress endogenous BMP/Smad signaling in cells. Results showed that siRNA-Smad4 causes specific inhibition of Smad4 mRNA and protein expression after transfection, with the Smad4 transcript level reduced by approximately 89% and the level of Smad4 protein also markedly decreased 56.6%. Repressed endogenous BMP/Smad signaling significantly inhibited growth and induced apoptosis of porcine GCs, and decreased E2 production. In addition, repressed BMP/Smad signaling significantly changed the mRNA expression of Cyclin D2, CDK4, Bcl-2, and P450 aromatase (P450arom). These results implied that BMP/Smad signaling have key regulation in porcine GCs.4 To demonstrate the effects of BMP/Smad signal on FSH-induced porcine GCs, we repress endogenous BMP/Smad signaling in cells while FSH was supplied in the medium of GCs. The proliferation and cycle of cells, secretions of E2 and P4, and the expression of relating genes were detected by MTT, FCM, CLIA and real time RT-PCR, respectively. The results showed that repressed endogenous BMP/Smad signaling significantly decreased the expression of FSHR, inhibited FSH-induced porcine granulosa cell proliferation, and signaificantly increased the proportion of G1-phase cells even with the existence of FSH; the repressed endogenous BMP/Smad signaling significantly decreased the expression of Cyclin D2, and inhibited FSH-induced augmentation of its expression; the repressed the signaling significantly decreased the expression of CDK4 even with the existence of FSH; besides, the repressed the signaling degraded FSH-induced E2 production, but not P4;the repressed the signaling decreaseed expression of P450 aromatase (P450arom), and inhibited FSH-induced expression of P450arom and cholesterol side-chain cleavage enzyme P450 (P450scc).5 To demonstrate the effects of BMP/Smad signal on porcine GCs, we repressed endogenous BMP/Smad signaling in cells, besides, we activated the signaling by supplyling BMP (BMP-6) in the medium, investigated the effects of repressed and activated BMP/Smad signaling on porcine GCs. The results showed that repressed endogenous BMP/Smad signaling significantly inhibited granulosa cell proliferation, increased the proportion of G1-phase cells, and decreased the cells of S-phase. Activated signaling significantly decreased the proportion of cells in G1-phase, increased the cells of S-phase, and repressed endogenous BMP/Smad signaling significantly inhibited secretion of E2, but not P4;activated signaling significantly inhibited the two steroid hormone production.37 probes showed significant differences in the GCs of between repressed the signaling and repressed then activated signaling, among which 25 were up-regulated, while 12 were down-regulated. By the GO analysis,24 genes were found in biological process, 23 genes in molecular function, and 13 genes in cellular component.