Characterization, Expression Of Porcine GPR3Gene And Its Effects On Porcine Follicular Granulosa Cells

Mammalian follicular development is a very complex physiological and biochemical cascade process, including recruitment of the primitive follicle, the development of preantral follicle and antral follicle selection, growth and mature or follicle atresia. This procedure can be regulated by a lot of factors, such as hormones and growth factors. It is well known that only a few follicles can reach dominant follicles and ovulation, and more than99%follicles go atresia in mammals. Granulosa cells are key somatic cells in follicles, and play very important roles in oocyte maturation and follicular development. Follicular development is accompanied by growth, proliferation, differentiation, and maturation of granulosa cells. G protein-coupled receptor3(Gpr3) belongs to the G protein-coupled receptor superfamily. Recent study showed that Gpr3regulated follicular development and oocyte maturation through Gs-linked signal pathways in ovarian cells of mice and human. However, the expression pattern in ovary and the roles in follicular granulosa cells of Gpr3are have not yet been well documented in pigs. Therefore, it is immediately required to carry out the further study on the expression pattern of Gpr3and regulatory mechanisms of the Gpr3signaling pathway in the follicular granulosa cells of pigs, which provides possible rationale to selection of mammalian reproductive trait and has 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) and rapid amplification of cDNA ends (RACE), we cloned the full-length of porcine Gpr3gene and the complete coding sequences of porcine Gpr6and Gpr12genes. Bioinformatics methods were adopted to predict the structure and function of Gpr3, Gpr6and Gpr12protein. Real-time RT-PCR and Western blotting were performed to investigate Gpr3expression pattern in tissues and the whole cumulus-oocyte complexes (COCs) of in vitro maturation (IVM). Cellular localization of Gpr3in the ovaries of50,70and90post coitum (dpc), days1,25,35,70,140and180postpartum (dpp) of commercial pigs were examined by immunohistochemistry (IHC). Time-spatial expression of Gpr3in multi-age, multi-breed ovaries and multi-diameter follicles were detected by Real-time RT-PCR and Western blotting. Subcellular localization, constitutive activity of Gpr3and the effect of sphingolipids on its constitutive activity and ligand-induced receptor internalization were tested using the vectors of pcDNA-Gpr3and pEGFP-Gpr3in HEK293cells. In order to repress Gpr3signaling, the gene of Gpr3was silenced by RNAi, the effects of repressed Gpr3signaling on porcine follicular granulosa cells and underlying molecular mechanism were detected by MTT, FCM, RIA and real-time RT-PCR. The effects of Gpr3overexpression on porcine follicular granulosa cells and underlying molecular mechanism were also detected by MTT, FCM, RIA and real-time RT-PCR.The main results achieved were as follows:1The full-length of porcine Gpr3gene and the complete coding sequences of porcine Gpr6and Gprl2genes were cloned by RT-PCR and RACE, and were submitted to GenBank (Accession No. HQ606483, HM777010and HM77711, respectively). Using bioinformatics network resources and relevant software, we predicted that the full sequence of Gpr3gene with its length being2101bp nucleotides, including an open reading frame of993bp, encoded a330amino acid polypeptide with the molecular weight of35.2kDa, localizeg at chromosome6. Comparison of the putative amino acid of porcine orthologs with that of other species showed that porcine Gpr3shared96.4%,95.1%,94.9%,94.8%,90.0%and85.4%homology with Bos taurus (XP612644.2), Canis lupus familiaris (XP544470.1), Homo sapiens (NP005272.1), Pan troglodytes (XP001148989.1), Mus musculus (NP032180.1) and Rattus norvegicus (NP714949.1) respectively, which proved that Gpr3gene was well conserved in the process of evolution. The Gpr3protein 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. Further, on the basis of their high levels of sequence similarity and similar potential roles, Gpr3, Gpr6and Gpr12genes were identified as a subfamily of GPCRs, called Gpr3subfamily of GPCRs.2Expression patterns of Gpr3in tissues and the whole COCs during IVM were investigated by real-time RT-PCR and Western blotting. The results indicated that Gpr3gene was expressed in tissues of cerebrum, cerebellum, hypothalamus, pituitary, heart, liver, spleen, lung, kidney, muscle, fat, thymus, ovary, oviduct, uterus, testis, oocyte, and granulosa cell at different expression levels, the expression levels of this gene in brain, pituitary, liver, fat, uterus and oocyte being higher than that in other tissues. Since Gpr3mRNA and protein were detected in porcine hypothalamus-pituitary-ovary and testis, it can be speculated that this receptor may play an important role in the neural regulation of sexual and/or reproductive functions and fat metabolism in pigs. Interestingly, the mRNA and protein levels of Gpr3in the whole COCs were down-regulated, and its mRNA expression levels were significantly and negatively correlated with the degrees of cumulus expansion (r=-0.937, P<0.01) during IVM, suggesting its important roles in cumulus expansion and oocyte maturation. Maturation/M-phase promoting factor (MPF) is a complex composed of a regulatory subunit cyclin B1and a catalytic subunit CDK1. Cyclin B1transcripts was up-regulated after24h of maturation, while CDK1mRNA levels were unchanged during IVM, suggesting that MPF might play roles in the later stage of oocyte maturation.3Cellular localization and expression pattern of Gpr3were investigated by IHC, real-time PCR and Western blotting in the ovaries of pigs. The results showed that Gpr3protein was primarily expressed in the oogonia in egg nest and the oocyte in primodial and primary follicles of the fetal and neonatal pigs. In postnatal pigs, Gpr3protein was expressed mainly in the granulosa cell layer, cumulus cell and cytoplasm of oocytes. Furthermore, the immunostaining for Gpr3in cytoplasm of oocytes was the most intensive. In additional, the staining intensity was faintly presented in the corpora lutea of the ovary in adult pigs. The results of real-time PCR and Western blotting indicated that Gpr3mRNA and protein were both presented in the different ages of ovaries, and the expression levels were wave-changed from postpartum1to180days. The expression levels of Gpr3in the1-dpp-old and140-dpp-old ovaries were higher than that of other age ovaries. There was no significant difference among three tested breeds at the expression level of Gpr3in the ovary. Moreover, both mRNA and protein levels of Gpr3were up-regulated significantly during follicle growth. The stage-and cell-specific expression pattern of Gpr3in the porcine ovary suggested that Gpr3might play an important role in the process of entire follicular development and luteinization.4In order to understand the properties and functions of Gpr3, by which to screen its potential ligands, the vectors of pcDNA-Gpr3and pEGFP-Gpr3were constructed for checking the subcellular localization, constitutive activity of Gpr3and the effect of sphingolipids on its constitutive activity and ligand-induced receptor internalization in HEK293cells. The results showed that expression of porcine Gpr3in HEK293cells resulted in constitutive activation of adenylate cyclase (AC) similar in amplitude to that produced by fully stimulated Gs-coupled receptors. Moreover, sphingosine1-phosphate (SIP) could increase AC activation via the constitutively active Gpr3receptor. When expressed in HEK293cells, GFP-labeled Gpr3protein was shown to be localized in the plasmalemma and subcellular membranes. After SIP treatment, agonist-mediated intemalization could be visualized by confocal microscopy. However, sphingosine (Sph) and sphingomyelin (Sphi) had no ability like that of S1P. In short, our findings suggested that the porcine Gpr3is a constitutively active G protein-coupled receptor. Constitutive activation of AC and agonist-mediated intemalization of Gpr3receptor could be modulated by the SIP, suggesting that SIP might act as an activator for porcine Gpr3receptor.5To demonstrate the effects of Gpr3signaling pathways on growth and differentiation of porcine granulosa cells (pGCs), pGCs were isolated from3-5mm antral follicles in diameter and cultured in vitro. A strategy of RNAi-mediated gene silencing of Gpr3was used to repress endogenous Gpr3signaling in cells. The results indicated that GprJ-siRNA causes specific inhibition of Gpr3mRNA and protein expression after transfection, which successfully repressed endogenous Gpr3signaling in pGCs. Repressed endogenous Gpr3signaling significantly promoted growth and inhibited apoptosis of pGCs, but no effect was shown on steroidogenesis in pGCs. In addition, repressed Gpr3signaling significantly changed the mRNA expression of Cyclin B1, Cyclin D2, Bcl-2, Bax and Gpr12(P<0.05), but there is no change in the expression of CDK1, CDK4, P450arom, P450scc and Gpr6(P>0.05).6To further confirm the roles of Gpr3in pGCs, Gpr3overexpression was performed in pGCs. The results showed that Gpr3overexpression significantly inhibited porcine GCs proliferation, increased the proportion of G0/G1-phase cells, and decreased the cells of S-phase and the mRNA expression of Cyclin B1and CDK1; Gpr3overexpression significantly induced apoptosis of porcine GCs, decreased the expression of Bcl-2, and increased the expression of Bax; Gpr3overexpression significantly increased the expression of Gpr6. However, there was no effect of Gpr3overexpression on steroidogenesis and related genes in pGCs. These results implied that Gpr3signaling play an important role in pGCs, and the change of genes related to cell growth and apoptosis were provided a basis for further research on the regulatory mechanisms of porcine Gpr3gene in pGCs.