Zhihai Lei

Expression of ghrelin in the porcine hypothalamo-pituitary-ovary axis during the estrous cycle.

Ghrelin, a 28-amino acid acylated peptide produced mainly by the stomach, has various functions. Recent studies focus on its endocrine and/or paracrine effects in the regulation of the hypothalamo-pituitary-gonadal axis, that is, the role in reproduction. Previous data have shown that variation of ghrelin depended on the phases of estrous cycle in adult rat ovary. This study was to investigate the expression of ghrelin in the cyclic porcine hypothalamo-pituitary-ovary axis and stomach by semiquantitative RT-PCR and immunohistochemical method. Twenty virginal gilts were classified into four groups as the proestrus, estrus, diestrus1 and diestrus2. Results showed that expression of ghrelin mRNA in the hypothalamus changed with the estrous cycle, i.e., with the highest level in the proestrus and the lowest in the estrus. In the pituitary, the pattern of ghrelin mRNA expression during estrous cycle markedly decreased in the estrus and diestrus1. In the ovary, ghrelin mRNA exhibited with the highest level in the diestrus2 and the lowest in the proestrus, which was different from those in the hypothalamus and pituitary. In the stomach, the expression of ghrelin mRNA had the same tendency as that of the porcine ovary. In immunohistochemical experiment, ghrelin immunoreactive cells were predominantly located in the luteal compartment and growing follicles in the luteal phase of ovary. However, only few ghrelin immunoreactive cells were found in the proestrus ovary. In gastric mucosa, ghrelin immunoreactive cells were detected in the estrus, diestrus1 and diestrus2, but few ghrelin positive cells were seen in the proestrus. Results suggest that ghrelin may play a major role in the endocrine network that integrates energy balance and reproduction.

Gonadotropin-inhibitory hormone (GnIH) and its receptor in the female pig: cDNA cloning, expression in tissues and expression pattern in the reproductive axis during the estrous cycle

Since its discovery, gonadotropin-inhibitory hormone (GnIH) has appeared to act as a key neuropeptide in the control of vertebrate reproduction. GnIH acts via the novel G protein-coupled receptor 147 (GPR147) to inhibit gonadotropin release and synthesis. To determine the physiological functions of GnIH in the pig, a study was conducted to clone and sequence the cDNA of the GnIH precursor and GPR147. Our results demonstrated that the cloned pig GnIH precursor cDNA encoded three LPXRF and that its receptor possessed typical transmembrane features. Subsequently, tissue expression studies revealed that GnIH was mainly expressed in the brain, corresponding largely with the tissue expression patterns of GPR147 in the pig. The expression patterns in the reproductive axis of the female pig across the estrous cycle were also systemically investigated. The hypothalamic levels of both GnIH and its receptor mRNA were lowest in estrus and peaked in the proestrus and diestrus phases. The highest pituitary GnIH mRNA level was detected in the metestrus, and its receptor displayed a somewhat similar pattern of expression to that of the ligand. However, the expression patterns of GnIH and GPR147 were negatively correlated in the ovary. Immunolocalization in the ovary during the estrous cycle revealed that the immunoreactivities of GnIH and GPR147 were mainly localized in the granulosa and theca cells of the antral follicles during proestrus and estrus and in the luteal cells during metestrus and diestrus. Taken together, this research provided molecular and morphological data for further study of GnIH in the pig.

Distribution of orexin B and its relationship with GnRH in the pig hypothalamus

Increasing evidence suggests that orexins--hypothalamic neuropeptides--act as neurotransmitters or neuromediators in the brain, regulating autonomic and neuroendocrine functions. Orexins are closely associated with gonadotropin-releasing hormone (GnRH) neurons in the preoptic area and alter luteinizing hormone (LH) release, suggesting that they regulate reproduction. Here, we investigated the distribution of orexin B (immunohistochemical technique) and the relationship between orexin B and GnRH containing fibres and neurons in the pig hypothalamus using double immunofluorescence and laser-scanning confocal microscopy. Orexin B immunoreactive neurons were mainly localized in the perifornical area (PeF), dorsomedial hypothalamic nucleus (DMH), zona incerta (ZI) and the posterior hypothalamic area (PH), with a sparser distribution in the preoptic and anterior hypothalamic area. Immunoreactive fibres were distributed throughout the central nervous system. Approximately 30% GnRH neurons were in close contact with orexin B immunoreactive fibres, among these approximately 6% of GnRH neurons co-localized with orexin B perikarya in the region between the caudal preoptic area and the anterior hypothalamic area. Orexin B may regulate reproduction by altering LH release in the hypothalamus.

The effects of RFRP-3, the mammalian ortholog of GnIH, on the female pig reproductive axis in vitro.

RFamide-related peptide-3 (RFRP-3) has been proposed as a key inhibitory regulator of mammalian reproduction. To further determine the potential mechanisms and sites of action of RFRP-3, we systematically investigated the direct effect of RFRP-3 on the female pig reproductive axis in vitro. Initially, we confirmed that G protein-coupled receptor 147 (GPR147) was distributed in isolated hypothalamic, anterior pituitary and ovarian granulosa cells, suggesting that RFRP-3 could act on these cells in vitro. Subsequently, the direct effects of RFRP-3 on hormone and steroid secretion, the synthesis of subunit genes and the expression of proteins related to proliferation in the hypothalamus, pituitary and ovary were evaluated. Our results demonstrate that different doses of RFRP-3 inhibited the release and synthesis of gonadotrophin releasing hormone, gonadotrophin and steroid hormones and impacted the relative gene expression of KISS1 and GnRHR and the protein expression of cyclin B1, PCNA and ERK 1/2.

Developmental changes in the role of gonadotropin-inhibitory hormone (GnIH) and its receptors in the reproductive axis of male Xiaomeishan pigs.

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.

Effects of neuropeptide S on the proliferation of splenic lymphocytes, phagocytosis, and proinflammatory cytokine production of pulmonary alveolar macrophages in the pig

Neuropeptide S (NPS), a newly identified neuropeptide, is involved in many physiological and pathological activities through the NPS receptor (NPSR). Recently, the NPS and NPSR have been detected in peripheral systems of pigs including immune tissues, suggesting that NPS may play an important role in the regulation of immune function. The aim of this study was to demonstrate the presence and function of NPS and NPSR in splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs) of pigs. By RT-PCR, the expression of NPS and NPSR mRNA was detected in the SPLs and PAMs. NPS immunoreactivity was observed in the membrane and cytoplasm of both SPLs and PAMs. We found that NPS could stimulate the proliferation of SPLs, when NPS was added at concentrations of 0.01, 0.1, 1, 10, 100 and 1000 nM alone or in combination with PHA/LPS in vitro. In macrophages from bronchoalveolar lavage (BAL) fluid of pigs, various doses of NPS (0.01, 0.1, 1, 10, 100 and 1000 nM) up-regulated the phagocytosis of PAMs in comparison to controls. In PAMs, NPS could induce the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Taken together, all data suggest that NPS is capable of inducing phagocytosis of non-opsonized E. coli. NPS might act as potent neuroimmunomodulatory factors and affects the maintenance of immune homeostasis.

The regulatory mechanism of neuromedin S on luteinizing hormone in pigs.

Neuromedin S (NMS) has been implicated in the regulation of luteinizing hormone (LH) secretion. However, the regulatory mechanism of NMS on LH in pigs remains unexplored. In the present study, we confirmed the hypothesis that the effect of NMS on LH could be mediated via hypothalamic melanocyte-stimulating hormones (MSH) neurons of ovariectomized pigs. In an immunohistological experiment, NMS receptor NMU2R-positive neurons were found in the paraventricular nucleus of hypothalamus, widely distributed in the anterior pituitary, and sparsely observed in the posterior pituitary. We also found that serum LH level was declined at between 12 and 60 min with the lowest level at 24 min after NMS injection. The decreased LH secretion induced by NMS could be completely abolished by pretreatment with melanocortin receptor-4 antagonist SHU9119, while a signal injection of 1.0 nM SHU9119 per se did not affect the serum LH level. Real time quantitative RT-PCR results showed that the expression of GnRH and LH mRNAs were down-regulated by NMS treatment, but their reduction was restored to normal level by SHU9119 treatments. The expression of NMU2R and PR mRNAs were up-regulated by NMS treatment, but their effects were blocked by SHU9119 treatments. The expression of the estrogen receptor mRNA in the pig hypothalamus and pituitary was unchanged under the NMS and SHU9119+NMS treatments. In summary, all results suggest that the inhibitory effect of NMS on LH is at least in part through its receptor NMU2R and mediated via MSH neurons in hypothalamus-pituitary axis of ovariectomized pigs.

Distribution of neuromedin S and its receptor NMU2R in pigs

Neuromedin S (NMS) has been found to be involved in the regulation of the reproductive, endocrine, and immune systems in mammals. However, its function in pigs is currently not well understood. Thus, it is essential and important to characterize the central distribution of NMS mRNA and its receptor, neuromedin U receptor-2 (NMU2R), in pigs for clarifying its physiological functions. In this study, we found that NMS mRNA were densely distributed in the hypothalamus, hypophysis, hippocampus, and brain stem of pigs by in situ hybridization. Moreover, NMS and NMU2R mRNAs was also expressed in the alimentary organs, endocrine and lymphatic organs, and ovaries by semi-Q RT-PCR. All these results suggest that the NMS/NMU2R system plays an important role in modulating various physiological functions in pigs. This research provides useful information for predicting the physiological functions of the NMS/NMU2R system in pigs.

Cloning and distribution of neuropeptide W and its receptors in pigs

Neuropeptide W (NPW), a novel hypothalamic peptide, is an endogenous ligand for the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Although several studies have implicated NPW in the regulation of feeding and energy metabolism in many species, the precise physiological function of NPW in pigs remains unclear. In this study, we cloned and sequenced NPW, GPR7, and GPR8 cDNA from pigs. NPW, GPR7, and GPR8 mRNA expression was quantified in the pig brain and peripheral tissues by semiquantitative reverse transcriptase polymerase chain reaction. Immunohistochemistry showed that NPW protein expression was limited in the brain and abundant in peripheral tissues. These results suggest that NPW is involved in the regulation of various physiological functions in pigs. The molecular and morphological data from this study provide a basis for further research on the functions of NPW in pigs.

Heat-Stress-Induced Metabolic Changes and Altered Male Reproductive Function

Heat stress can cause systemic physiological and biochemical alterations in living organisms. In reproductive systems, heat stress induces germ cell loss and poor quality semen. However, until now, little has been known about such a complex regulation process, particularly in the perspective of metabolism. In this study, serum, hypothalamus, and epididymis samples derived from male SD (Sprague-Dawley) rats being exposed to high environmental temperature (40 °C) 2 h per day for 7 consecutive days were analyzed using metabonomics strategies based on GC/TOFMS. Differentially expressed metabolites reveal that the energy metabolism, amino acid neurotransmitters, and monoamine neurotransmitters pathways are associated with heat stress, in accordance with changes of the three upstream neuroendocrine system pathways in the SNS (sympathetic adrenergic system), hypothalamic pituitary adrenal axis (HPA), and hypothalamic pituitary testis axis (HPT) axis. Many of these metabolites, especially in the epididymis, were found to be up-regulated, presumably due to a self-preserving action to resist the environmental hot irritation to maintain normal functioning of the male reproductive system.