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 1487 bp, including one open reading frame (ORF) of 1155 bp 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.