| G protein-coupled receptor12(Gprl2) belongs to the G protein-coupled receptor superfamily. Recent studies showed that Gprl2is involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes. However, the expression pattern in various tissues and the roles in follicular granulosa cells of Gprl2have not yet been well documented in pigs. Therefore, further studies are immediately required on the expression pattern of Gprl2and regulatory mechanisms of the Gprl2signaling pathway in the follicular granulosa cells of pigs, which provides possible rationale to selection of mammalian reproductive trait and is of great importance to the improvement of reproductive capacity of domestic animals.In this study, using in silico approach combined with reverse transcription polymerase chain reaction (RT-PCR), we cloned the complete coding sequences of porcine Gprl2gene. Bioinformatics methods were adopted to predict the structure and function of Gpr12protein. Real-time RT-PCR was performed to investigate Gprl2expression pattern in the large, medium and small follicles. The vectors of pcDNA-Gprl2and Pegfp-Gprl2were successfully constructed, and the effects of Gprl2overexpression on porcine follicular granulosa cells were also detected by real-time RT-PCR. The main results achieved were as follows:1The complete coding sequence of porcine Gpr12gene was cloned by RT-PCR, and was submitted to GenBank (Accession No. HM77711). Using bioinformatics network resources and relevant software, we predicted that the ORF sequence of Gpr12gene with its length being1005bp nucleotides, encoded a334amino acid polypeptide with the molecular weight of36.7kDa, localized at chromosome11. Comparison of the putative amino acid of porcine orthologs with that of other species showed that porcine Gpr12shared96.41%,95.81%,95.81%,93.71%,92.81%and92.22%homology with Canis lupus familiaris (XP544470.1), Homo sapiens (NP005272.1), Pan troglodytes (XP001148989.1), Mus musculus (NP032180.1), Bos taurus (XP612644.2), and Rattus norvegicus (NP714949.1) respectively, which proved that Gprl2gene was well conserved in the process of evolution. The Gprl2protein contained the typical seven-transmembrane structure of G protein-coupled receptors (GPCRs), including several potential phosphorylation and N-glycosylation sites, suggesting that it might be involved in signal transduction and regulation of cell growth process.2Expression patterns of Gprl2in various tissues and the whole COCs were investigated by real-time RT-PCR. The results indicated that Gprl2gene was expressed in cerebrum, cerebellum, hypothalamus, pituitary, heart, liver, spleen, lung, kidney, muscle, fat, thymus, ovary, oviduct, uterus, testis, oocyte, and granulosa cell at different expression levels. However, the expression levels of this gene in brain, pituitary, liver and oocyte were higher than that in other tissues. It can be speculated that this receptor may play an important role in the neural regulation of reproductive functions and fat metabolism in pigs.3To demonstrate the effects of Gprl2signaling pathways on growth and differentiation of porcine granulosa cells (pGCs), pGCs were isolated from3-5mm antral follicles in diameter and cultured in vitro. Gprl2overexpression was performed in pGCs. The results showed that Gprl2overexpression significantly inhibited porcine GCs proliferation, and decreased the mRNA expression of Cyclin B1and CDK1; overexpression of Gprl2significantly induced apoptosis of porcine GCs, decreased the expression of Bel-2, and increased the expression of Bax. These results suggested that Gprl2signaling plays an important role in pGCs, and provided a basis for further research on the regulatory mechanisms of porcine Gprl2gene in pGCs. |
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