Study On The Distribution Of Neuromedin S And Its Receptor And The Regulation To Reproduction In Female Pigs

Neuromedin S was a neuropeptide initially found in rat hypothalamus and discovered as the endogenous ligand of orphans G protein coupling receptors (TGR-1 and FM-3) and named after the initials S of suprachiasmatic nucleus in 2005 by Mori et al. It was reported to play many important physiological roles mediated by its functional receptor NMU2R, such as feeding, energy metabolism, neuroendocrine and sleeping-walking rhythm etc. Previous reports have confirmed the regulation of NMS in animal reproduction, but main researches focus in rats. Here, we choose the female pigs as the main research objects and used gene homology cloning, semi-quantitative RT-PCR, immunohistochemistry (IHC), in situ hybridization (ISH), real time quantitative RT-PCR, cell culture and radioactive immunoassay analysis (RIA) for the systematic research of the regulation of NMS in the pig hypothalamus-pituitary-ovary axis. The main contents of our study included the gene cloning of pig NMS and its receptors (NMU1R and NMU2R) and their expression in pig various tissues, distribution of NMS and NMU2R in pig, expression pattern of NMS and NMU2R mRNA in pig reproductive axis during the estrus cycle (proestrus, estrus, metestrus and diestrus), effect of NMS on the reproductive axis in vitro, effects of NMS on serum LH level in ovariectomized pigs and the expression of hormone or receptor genes in the pig hypothalamus-pituitary axis after lateral ventricle injection with NMS under the pretreatment with SHU9119. The purpose of all the studies were to explore the regulatory mechanism of NMS to pig reproduction in vivo and vitro, at molecular level and cell level and by morphology and physiology etc.1 Cloning and expression of pig NMS and its receptorsIn this experiment, we cloned pig NMS and its two receptor (NMU1R and NMU2R) genes by using gene homology cloning technique, and submitted their sequences to GenBank database for obtaining accession numbers:GQ502200 (NMS), FJ949607 (NMU1R) and FJ15899 (NMU2R). We also researched the expression of NMS and NMU2R mRNA in pig various tissues by semi-quantitative RT-PCR method. The results were as following:The results of semi-quantitative RT-PCR analysis showed that NMS mRNA was expressed in 23 listed tissues of pigs with high levels in the medulla oblongata, pons, spleen, mesenteric lymph node, spinal cord, ovary, heart, pituitary, hypothalamus, kidney, colon, cecum, thymus and tongue muscles; with low levels in the lung, liver, adrenal gland, stomach, duodenum, thyroid gland, parotid gland, uterus and jejunum. NMU2R mRNA was expressed only in 19 of 23 tissues. NMU2R expression was mainly concentrated in pig hypothalamus, pituitary, spinal cord, ovary, spleen, mesenteric lymph node, thymus, parotid gland, tongue muscles, liver, duodenum, thyroid gland, lung and uterus; weakly expressed in the pons, medulla oblongata, ileum and cecum; and not expressed in pig stomach, jejunum, colon, kidney and adrenal gland.2 Distribution of NMS in pigsThe results of ISH showed that NMS-positive cells typically appeared multipolar were mainly found in the central nervous system, included the hypothalamus, pituitary, hippocampus, pons and spinal cord. The bread-like NMS-positive fibers were highly confined to pig pons and weakly to the thalamus. The labeled cells were also observed in several peripheral tissues, such as digestive organ, endocrine organ, immune organ and reproductive organ.3 Distribution of NMU2R in pigsThe results of IHC showed that NMU2R positive cells were mainly distributed in the diencephalon, pituitary, hippocampus and several peripheral tissues and organs in pig. In the diencephalon, NMU2R positive neurons were mainly distributed in the PVN, PEN and Arc of the hypothalamus, lateral hypothalamic area, ventromedial nucleus of thalamus and perifornical nucleus; NMU2R positive fibers were mainly projected to the ventromedial nucleus of the thalamus, PVN, PEN, lateral hypothalamic area, Arc of the hypothalamus, tuberomammillary nucleus, optic tract and cerebral peduncle. In pig pituitary, NMU2R positive cells were mainly localized in the anterior pituitary and posterior pituitary. In the peripheral tissues and organs, NMU2R positive cells were mainly localized in the ovary, oviduct, adrenal gland, spleen, stomach, intestine and lymph node etc.4 Expression of NMS and NMU2R mRNA in pig hypothalamus-pituitary-ovary axis during the estrus cycle The aim of this experiment was to explore the expression patterns of NMS and NMU2R mRNAs at various levels of pig hypothalamus-pituitary-ovary axis during the estrus cycle (proestrus, estrus, metestrus and diestrus, n=4) by semi-quantitative RT-PCR. The results were as following:The expression of NMS and NMU2R mRNA was all detected at the hypothalamus-pituitary-ovary axis during the estrus cycle, but their expression patterns were different. In the hypothalamus, peak expression of NMS mRNA was observed at proestrus, whereas its mRNA levels significantly decreased at estrus and gradually increased from metaestrus to diestrus. NMU2R mRNA has a similar expression profile in the hypothalamus with predominant expression at the proestrus, a low value at estrus, and a medium level at metestrus and diestrus.In the pituitary, the expression of NMS and NMU2R mRNA during the estrus cycle was similar to that in the hypothalamus with maximal level in the proestrus and a minimal level in the estrus.In the ovary, the expression of NMS and NMU2R mRNA during the estrus cycle was differed from those in the hypothalamus and pituitary. The result showed that the lowest level of NMS mRNA in the proestrus and gradually increased from the estrus to the highes level of diestrus; the highest level of NMU2R mRNA was observed in metestrus and lowest in estrus.5 Effect of NMS on female pig reproductive axis in vitroTo research the target action site of NMS on the reproductive axis in vitro, we studied the effects of 1,10,100 and 1000 nM NMS on the releases of GnRH from the cultured hypothalamic explants, of LH, FSH and PRL from the anterior pituitary cells and of E2 from the ovary cells by RIA. The results were as following:Compared with the control group, all NMS doses could stimulate the release of GnRH, 100 and 1000 nM NMS could significantly increase the release of GnRH (P< 0.01), and 1 and 10 nM NMS slightly increased the release of GnRH (P> 0.05). Potassium chloride (56 mM) could significantly stimulate the release of GnRH (P< 0.01), indicating that the activity of the hypothalamic explants in each treatment group was adequate for experiment.In the NMS-treated groups, all doses of NMS does-dependently increased LH release. 10 nM doses significantly stimulated LH release (P< 0.05), while 100 nM and 1000 nM doses drastically stimulated LH release (P< 0.01). Compared with the control group, all the treatments could slightly stimulate FSH release (P> 0.05). All NMS doses could stimulate the release of PRL from pituitary cells, the release of PRL was increased by 1 nM (P< 0.01) and 10 nM (P< 0.05) NMS, and 100 and 1000 nM NMS slightly increased the release of PRL(P> 0.05).All doses of NMS could stimulate the release of E2 from the ovary cells. Compared with the control group,1,100 and 1000 nM NMS could not significantly stimulate E2 release (P> 0.05), only 10 nM NMS significantly stimulated E2 release from pig ovary cells (P<0.05).6 Effect of NMS on serum LH level after injection of NMS into the lateral ventricle in ovariectomized pigsIn order to study the effect of NMS on LH secretion in vivo, ovariectomized pigs were injected with NMS into the lateral ventricle under pretreatment with SHU9119(NMS injection group, SHU9119+NMS injection group and control group). LH level in serum was analyzed by RIA (6 min-interval for collecting blood sample). The results were as following:In ovariectomized pigs, NMS induced a dramatic decrease in serum LH level between 12 min and 60 min after the lateral ventricle injection of NMS in NMS-injection group, and the lowest serum LH level was observed at 24 min after NMS injection (P<0.01). This inhibitory effect was gradually restored 1 h after the injection of NMS (P<0.05). Accordingly, the integrated LH secretion in NMS-injected group (over the 1 h period after NMS injection) was approximately 42.66% lower than that in controls (P< 0.01).Serum LH level was not significantly changed after NMS injection under the pretreatment with SHU9119 (P>0.05) in SHU9119+NMS injection group, but LH level was significantly higher than that of NMS-injection group during this period (over the 1 h period after NMS injection) (P<0.01). Compared with the control group, LH level and the integrated LH secretion in SHU9119+NMS injection group (over the 1 h period after NMS injection) were slightly increased and similar to that of the control group (P>0.05).7 Effect of NMS on the expression of hormone or receptor mRNA relative to reproduction on the hypothalamus-pituitary axis after injection of NMS into the lateral ventricle in ovariectomized pigsThe aim of this study was to explore the effect of NMS on the expression of hormone and receptor mRNA relative to reproduction on the hypothalamus-pituitary axis, we researched the expression pattern of GnRH, LH, NMU2R, PR and ER mRNA in pig hypothalamus-pituitary axis after injection of NMS into the lateral ventricle by real time RT-PCR method, the results were as following:Expression of GnRH (hypothalamus), LH (pituitary), NMU2R, PR, and ER (hypothalamus and pituitary) transcripts in all pigs were persistently examined, yet, their expression levels significantly changed among all the treatments (exclude ER mRNA). Expression level of hypothalamic GnRH mRNA in NMS-injection group was significant higher than that of control and SHU9119+NMS injection groups (P<0.01). Expression level of hypothalamic GnRH mRNA in SHU9119+NMS injection group was similar to that of control group (P> 0.05). Among all the experimental pigs, NMS treatment markedly increased the expression of PR and NMU2R mRNA in the hypothalamus and pituitary, but the effect of NMS on PR and NMU2R mRNA was blocked by SHU9119 pretreatment, and PR and NMU2R mRNA in SHU9119+NMS group remained at the similar level to those in controls (P> 0.05). LH mRNA level was significantly reduced in pig pituitary after NMS injection, but the reduction could be reversed by SHU9119 pretreatment (P<0.01 vs. that of vehicle-injection pigs). Under the three different treatments, the expression level of ER mRNA in the hypothalamus and pituitary of ovariectomized pig was unchanged (P 0.05).All of the above mentioned results showed that the distribution of NMS and NMU2R in pig hypothalamus, pituitary and ovary can provide morphology data for the study of the regulatory mechanism of NMS in the reproductive axis. The relationship between the expression of NMS and NMU2R mRNA in the reproductive axis during the estrus cycle and the secretion of the reproductive hormone suggests that NMS may be involved in the regulation of the secretions of these reproductive hormones. The effects of lateral ventricle injection of NMS in ovariectomized pig on serum LH level and the expression of related hormones or receptors mRNA in the hypothalamus and pituitary could be blocked by pretreatment with SHU9119, which suggest the inhibitory effect of NMS on the ovariectomized pig reproduction may be mediated by MSH combined with NMU2R centrally. Together, these studies provide morphological and physiological experimental data for the regulation of NMS in the pig reproduction.