Effects of neuromedin B on steroidogenesis, cell proliferation and apoptosis in porcine Leydig cells.

Neuromedin B (NMB), a mammalian bombesin-related peptide, has numerous physiological functions, including regulating hormone secretions, cell growth, and reproduction, by binding to its receptor (NMBR). In this study, we investigated the effects of NMB on testosterone secretion, steroidogenesis, cell proliferation, and apoptosis in cultured primary porcine Leydig cells. NMBR was mainly expressed in the Leydig cells of porcine testes, and a specific dose of NMB significantly promoted the secretion of testosterone in the primary Leydig cells; moreover, NMB increased the expression of mRNA and/or proteins of NMBR and steroidogenic mediators (steroidogenic acute regulatory (STAR), CYP11A1, and HSD3B1) in the Leydig cells. In addition, specific doses of NMB promoted the proliferation of Leydig cells and increased the expression of proliferating cell nuclear antigen and Cyclin B1 proteins, while suppressing Leydig cell apoptosis and decreasing BAX and Caspase-3 protein expression. These results suggest that the NMB/NMBR system might play an important role in regulating boar reproductive function by modulating steroidogenesis and/or cell growth in porcine Leydig cells.

Distribution of the pig gastrin-releasing peptide receptor and the effect of GRP on porcine Leydig cells.

Gastrin-releasing peptide (GRP) is a mammalian bombesin (BN)-like peptide which plays a role in a number of important physiological functions via its receptor (gastrin-releasing peptide receptor, GRPR) in most animals. However, little is known about the gene encoding GRPR and its functions (especially reproduction) in pigs. In this study, we first cloned and analyzed the pig GRPR cDNA. Then we systematically investigated the expression levels of GRPR mRNA by relative real-time PCR (RT-PCR), and analyzed the distribution of the GRPR protein in pig tissues via immunohistochemistry (IHC). Finally, we studied the effect of GRP on testosterone secretion and GRPR (mRNA and protein) expression in Leydig cells. Results showed that the pig GRPR cDNA cloned at 1487bp, including one open reading frame (ORF) of 1155bp and encodes 384 amino acids. Significantly, compared with other species, the cDNA sequence and amino acid sequence of the pig GRPR were highly homologous and conservative. The RT-PCR results showed that: in the central nervous system (CNS) and the pituitary, GRPR mRNA was found in the cerebellum, hypophysis, spinal cord and hypothalamus; in the peripheral tissues, GRPR mRNA was mainly expressed in the pancreas, esophagus, ovary, testis, spleen, thymus, jejunum lymph node, muscle and fat. Moreover, the IHC results showed that GRPR immunoreactivity was widely distributed in the pig tissues and organs, such as the pancreas, esophagus, testis, ovary, spleen, pituitary gland and adrenal gland. In addition, we found that GRP promotes testosterone secretion, and increases GRPR mRNA and protein expression in cultured Leydig cells in vitro. These molecular and morphological data not only describe the anatomical locations of GRPR in pigs, but also provide the theoretical foundation for further research into its possible physiological functions in pigs. These results suggest that the GRP/GRPR system may play an important role in regulating the reproductive system of the boar.

Cloning and expression patterns of neuromedin U and its receptors in pigs.

Neuromedin U (NMU) is a highly conserved neuropeptide that performs a variety of physiological functions in animals via neuromedin U receptor-1 (NMUR1) and neuromedin U receptor-2 (NMUR2). In this study, we cloned the pig NMU, NMUR1 and NMUR2 genes. Bioinformatics analysis demonstrated that the pig NMU cDNA encoded the amino acids Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2 at the C-terminus and that the NMU receptors, which are G-protein-coupled receptors (GPCRs), contained the seven transmembrane domains typical of GPCRs. Systemic NMU, NMUR1 and NMUR2 mRNA expression was investigated in various pig tissues using real-time RT-PCR. NMU mRNA was expressed both in the central nervous system (CNS) and in peripheral tissues. NMUR1 mRNA was widely expressed in peripheral tissues, whereas NMUR2 mRNA was mainly expressed in the CNS. Immunohistochemistry (IHC) was used to determine the expression patterns of NMU and NMUR1, which were predominantly located in the gastrointestinal tract, genitourinary organs, and immune organs. This study presents molecular and morphological data to aid in additional NMU research in pigs.