Cloning And Localization Of Rabbit Bone Morphogenetic Protein 15 Gene

Bone morphogeneticprotein 15(BMP15) has identified as important components of oocyte-secreted factors.It was demonstrated to be important in folliculogenesis and oocyte maturation. Recently, the studies were mainly investigated in human, rat, goat, cow, procine and so on. However, up until now, no report of rabbit BMP15 can be found. Thus, in order to research the biological functions of BMP15 in rabbit reproductive system, cloning, location and its expression level during IVM of rabbit oocytes were analyzed.According to BMP15 gene sequences of many other animals in Genbank, the specific primers for PCR were designed. The BMP15 gene partial cDNA sequence was amplified by RT-PCR from rabbit oocytes, then were cloned into pMD19-T-simple vector and were sequenced. The results showed that the sequence was consisted of 919 nucleotides that crossed the exonâ… andâ…¡. The sequence shared 83%, 82%, 82%, 82%, 80% and 75% nucleotide homology with the published mRNA of pig, cow, sheep, ovine, human and mouse separately. This was the first report of rabbit BMP15 gene. It established the way for future study on biological function, activity and application of rabbit BMP15 gene.According to rabbit GAPDH cDNA sequence, we designed the primers and regard the PCR productions as the internal standard. Using semi-quantitative RT-PCR analysis, BMP15 mRNA expression could be seen in ovary and testis, and also detected in hypothalamus and pituitary. The datas suggested that the gene may play a role in non-gonad tissues.To study the distribution of BMP15 protein in rabbit ovary and testis, immunohistochemical analysis was carried out on frozen sections of tissues. The results showed that BMP15 protein were highly expressed in oocytes of follicles from the primary stage onward, but not in primordial follicles. It indicated that it play an important role in folliclar development. Meanwhile, we found BMP15 protein in folliclar antrum that may be secreted by oocytes to regulate the development of follicles. In addition, BMP15 protein was firstly observed in corpora lutea. We presumed that the biological activity of BMP15 may be blocked by follistatin expressed in corpora lutea. The findings in testis showed that BMP15 protein was expressed in spermatogonium and round spermatid. It suggested that it may play role in the testis development and spermatogenesis.