BMP15 Regulates Porcine Cumulus Cells Apoptosis Through FAT1 And The Analysis Of FAT1-mediated Key Molecular Pathways

Porcine cumulus cells are localized around oocytes and act as a specific type of granulosa that plays essential roles in the development and maturation of oocytes, the development and atresia of follicles and the development of embryos. FAT1 belongs to transmembrane protein family, which localized on cell membranes. In mammalian animals, FAT1 plays vital roles in various developmental processes. Recent studies showed that FAT1 is a tumor-suppressed gene, and lack of FAT1 resulted in tumor cells growth and proliferation. Similar to β-catenin, a classical cadherin, FAT1 binds to β-catenin and impede its translocation activity to nucleus. Although FAT1 has been demonstrated to be able to modulate the cell-cell contact and actin dynamics, there are currently few reports regarding whether the FAT1 gene affects porcine cumulus cell apoptosis, and the mechanism of FAT1-mediated-apoptosis is still unclear.Previous high-throughput sequencing results from our group demonstrated that BMP15 increases FAT1 m RNA. In order to explore whether BMP15 regulates the expression of FAT1 gene, we added recombinant human BMP15 to porcine cumulus cells, using Flow cytometry(FCM) to analyze the apoptotic rate and RT-q PCR to examine the expression level of FAT1 gene. The results showed that after BMP15 treatment in porcine cumulus cells, the apoptotic rate increased and the m RNA expression of FAT1 increased. Several researchers believed BMP15 is secreted by oocytes, however, others believed it is specific expressed on ovarian somatic cells. Thus, to explore how BMP15 expressed in pigs, the BMP15 expressions of porcine oocytes as well as cumulus cells were examined. The results showed that BMP15 was specific expressed in porcine oocytes.To understand whether the FAT1 gene affects the apoptosis of porcine cumulus cells and to elucidate the mechanism of this potential action, FAT1 was knocked down using RNA interference. Flow cytometry(FCM) was used to detect the apoptotic rate and RT-q PCR, Western Blot, as well as Immunofluorescence were used to exam the expression levels of several target genes. The lack of FAT1 resulted in stable expression of β-catenin, enhanced expression of cleaved caspase-3 and increased BCL2/BAX ratios at both the m RNA and protein levels. These results indicate that FAT1 inhibits porcine cumulus cell apoptosis via different pathways: FAT1-β-catenin pathway,FAT1-BCL2/BAX pathway, and FAT1-caspase-3 pathway. Taken together, these data provide new insights into the mechanisms of the association between FAT1 and porcine cumulus cell apoptosis as well as other reproduction processes.