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ER oxidoreduclin 1α (ERO1α) is an oxidase, participating in formation of secretory and membrane proteins. However, the other physiological functions ERO1α is not well known. We found that ERO1α is high in the Leydig cells of the testis. Therefore, the purposes of the current study are to explore the role of ERO1α and the possible mechanisms in regulating cell proliferation, apoptosis, and testosterone secretion of Leydig cells. ERO1α was mainly localized in Leydig cells in the adult mice testes by immunofluorescence staining. Western blot analysis showed that ERO1α was higher in Leydig cells than that in the seminiferous tubules. The effect of ERO1α on cell proliferation, apoptosis, and testosterone secretion was detected by transducing ERO1α overexpression and knockdown lentiviruses into cultured primary Leydig cells (PLCs) together with hCG exposure. Flow cytometry analysis showed that ERO1α promoted cell proliferation by increasing cell distribution at the S phase and decreasing that at the G0/G1 phase. Western bolt analysis showed that ERO1α increased CDK2 and CDK6 expression. Cell apoptosis determination found that ERO1α inhibited PLC apoptosis. Western bolt analysis showed that ERO1α increased the ratio of BCL-2/BAX, and decreased BAD and Caspase-3 expression. Enzyme-linked immunosorbent assay analysis demonstrated that ERO1α enhanced testosterone secretion. Western bolt analysis found that ERO1α increased StAR, 3β-HSD, and CYP17A1 expression. Furthermore, ERO1α could activate the PI3K/AKT/mTOR signaling pathway. In summary, these results suggest that ERO1α might play proliferation promotion and antiapoptotic roles and enhance testosterone secretion in PLC, at least partly, via activation of the PI3K/AKT/mTOR signaling pathway., (© 2020 Wiley Periodicals, Inc.)

Mammalian bombesin-related peptide, neuromedin B (NMB) action is mediated by its receptor (NMBR), and NMB/NMBR system plays a major role in regulating hormone secretions, reproduction and cell growth. Here we report the functions of NMB in regulating steroidogenesis (testosterone synthesis), cell viability and apoptosis. The primary rabbit Leydig cells were employed as the paradigm for this research. We initially confirmed that NMBR is distributed in Leydig cells of rabbit testis, and a certain dose of NMB could increase the secretion of testosterone in primary cultured rabbit Leydig cells. Subsequently, the accumulated NMBR, StAR, CYP11A1, 3β-HSD and PKC protein could be induced by a certain dose of NMB in Leydig cells. Moreover, we found that NMB could decrease the cell viability, and decreased the expression of PCNA protein in Leydig cells; meanwhile, except for 100 nM, other doses of NMB could suppress the cell apoptosis, and regulate Caspase-3 protein expression in Leydig cells, respectively. These results identify that NMB may be a key factor in regulating testosterone synthesis through taking part in NMBR/PKC/steroidogenesis signaling pathway, as well as the cell viability and proliferation in rabbit Leydig cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© 2018 Society for Endocrinology.)

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., (Copyright © 2017. Published by Elsevier Inc.)

• NMU2R distributed in hypothalamic cells, anterior pituitary cells and Leydig cells. • The direct effect of NMS in pig hypothalamus cells, pituitary cells and leydig cells. • NMS have some potential role in hypothalamic-pituitary-testicular axis. Evidence has shown that neuromedin S (NMS) and its receptor (NMU2R) are expressed in the hypothalamus, pituitary, and testis of pigs. To determine the potential mechanisms of NMS, we systematically investigated the direct effects of NMS on the hypothalamic-pituitary-testicular (HPT) axis of male pigs in vitro. We initially confirmed that NMU2R distributed in isolated hypothalamic cells, anterior pituitary cells and Leydig cells using immunocytochemistry. Subsequently we investigated the direct effects of NMS on hormone secretion from cells (anterior pituitary cells and Leydig cells) treated with different doses of NMS. The results showed that NMS increase the release of LH and FSH from anterior pituitary cells and testosterone from Leydig cells. NMS up-regulated the expression of NMU2R and GnRH mRNAs in hypothalamic cells, NMU2R , LH and FSH mRNAs in anterior pituitary cells, and NMU2R , STAR , P450 and 3β-HSD mRNAs and the expression of PCNA and Cyclin B1 protein in Leydig cells; moreover, it down-regulated the expression of GnIH mRNA in hypothalamic cells. Using immunofluorescence staining and confocal microscopy, we also demonstrated the colocalization of NMU2R and AR or GnIH in Leydig cells. These data in vitro indicated that NMS may regulate the release and/or synthesis of LH, FSH and testosterone at different levels of the reproductive axis through NMU2R, which provided novel evidence of the potential roles of NMS in regulation of pig reproduction. [ABSTRACT FROM AUTHOR]

Neuropeptide B (NPB) is an endogenous ligand for the orphan G protein-coupled receptors NPBWR1 (GPR7) and NPBWR2 (GPR8). Some reports have investigated the role of NPB in the regulation of feeding, energy metabolism and hormone secretion in many species. However, few papers reported the physiological function of NPB in the pig. In this study, we cloned and sequenced the NPB mRNA from a pig, which was found to consist of 123 bases. NPB mRNA expression was detected in central and peripheral tissues by the quantitative fluorescence method. The results showed that NPB mRNA expression was higher in hippocampus, cerebellum, spinal cord, thymus, tonsil, duodenum, cecum, colon, ovary and testis. The distribution of NPB suggested that it may be involved in the regulation of reproductive functions in the pig. Subsequently, the expression and distribution of NPBWR1 and NPBWR2 were found in Leydig cells and ovarian granular cells. We then investigated the direct effect of NPB on pig reproductive cells in vitro. The results showed that different concentrations of NPB (10 −12 , 10 −10 , 10 −8 and 10 −6 M) promoted the secretion of testosterone in Leydig cells in concentration-dependent manner. Different doses of NPB could promote the secretion of progesterone in ovarian granulosa cells in dose-dependent manner. Low concentrations of NPB (10 −8 and 10 −10 M) promoted estradiol secretion, but high concentrations of NPB (10 −6 M) inhibited its secretion. All the results suggested that the NPB/NPBWR1 or NPBWR2 system may play a role in modulating the reproductive activity in the pig. [ABSTRACT FROM AUTHOR]

Gonadotropin-inhibitory hormone (GnIH), a key regulator of vertebrate reproduction, was identified in the Japanese quail in 2000, and RFamide-related peptide-3 (RFRP-3) was found to be a mammalian GnIH ortholog. To further determine its role in the reproductive system of male Xiaomeishan pigs, we systematically investigated changes in GnIH and its receptors (GPR147 and GPR74) during the development of the reproductive axis of male pigs. We also investigated the direct effect of RFRP-3 on the synthesis and secretion of testosterone in Leydig cells in vitro . The expression patterns of GnIH in the reproductive axis of male pigs at different stages of development (postnatal 3, 30, 60, 90, and 120D) were studied using semiquantitative RT-PCR and immunohistochemistry. Our results show that hypothalamic, pituitary and testicular levels of GnIH and its receptors mRNA significantly changed on postnatal day 30 and postnatal day 90. The immunoreactivities of the GnIH proteins were mainly localized to the spermatogenic cells, sustentacular cells and interstitial cells of the testis throughout sexual development. It was confirmed that different doses of GnIH/RFRP-3 inhibited the release and synthesis of testosterone, and impacted on the gene expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and P450, enzymes that play a key role in the synthesis of testosterone. Together, this research provides molecular and morphological data on the regulation of GnIH in the reproductive development of male pigs. [ABSTRACT FROM AUTHOR]