| Neuropeptide W (NPW), which was first isolated from the porcine hypothalamus in 2002 by Shimomura et al, exists in two forms, consisting of 23 (NPW23) or 30 (NPW30) amino acids. Because both the first and last amino acids of the 30-residue peptide are Trp, they designated the peptide as neuropeptide W. These neuropeptides bind to one of two NPW receptors, either GPR7 (otherwise known as NPBWR1) or GPR8 (NPBWR2), which belong to the G protein-coupled receptor family. GPR7 and GPR8 share 64% amino acid identity and 70% nucleotide identity with each other. GPR7 has been isolated from many species, while GPR8 has not been detected in rodents. Several studies revealed that NPW is implicated in the regulation of feeding, energy metabolism, neuroendocrine function, stress responses, and modulating inflammatory pain. Although the genetic structure, distribution and physiological function of NPW have been studied in many species, the distribution and the precise physiological function of NPW in the pig have never been reported. Thus, we used gene homology cloning, RT-PCR, immunohistochemistry (IHC) to systematically investigate the distribution of pig neuropeptide W and its receptors, and expression of their mRNAs in pig hypothalamus-pituitary-ovary axis during the estrous cycle in this study. The main contents of our study included the gene cloning and expression of pig NPW and its receptors, the distribution of NPW and GPR7 in pigs, expression pattern of NPW and its receptors mRNAs in pig reproductive axis during the estrus cycle (proestrus, estrus, metestrus and diestrus). The purpose of all the studies were to explore the distribution of pig neuropeptide W and its receptors and expression of neuropeptide W and its receptors mRNAs in pig hypothalamus-pituitary-ovary axis during the estrous cycle.1 Cloning and expression of NPW and its receptors in pigsIn this experiment, we cloned pig NPW, GPR7, and GPR8 genes by using gene homology cloning technique and analyzed sequences. Then we researched the expression of NPW, GPR7, and GPR8 mRNAs in pig tissues by RT-PCR method. The results were as follows:(1) In this study, we cloned pig NPW, GPR7, and GPR8 genes fragments using PCR-based approach, and the PCR products were 312,542, and 420 bp, respectively. These PCR products were confirmed by sequencing, and the GPR7 and GPR8 sequences were submitted to GenBank (GenBank ID:KF361314, KC441488). The putative pig GPR7 and GPR8 amino acid sequence was found to have typical transmembrane domain features. The identity at the protein and nucleotide levels of NPW, GPR7, and GPR8 were rather high with other species. NPW was located in chromosome 3, GPR7 and GPR8 were located in chromosome 4.(2) The results of RT-PCR analysis showed that NPW, GPR7, and GPR8 mRNAs were expressed in the central nervous system (CNS) and peripheral tissues, and displayed a restricted distribution in brain, but was abundant in peripheral tissues.2 The distribution of NPW and GPR7 in pigsIn this experiment, we researched the distribution of NPW and GPR7 by immunohistochemistry. The results were as follows:(1) NPW-immunoreactive (-ir) cells and fibers were exclusively found in the pig hypothalamus. A few NPW-immunoreactive cells were detected only in the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus (SON), while dense NPW-ir fibers were observed in the PVN, arcuate nucleus (ARC), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH), SON, and lateral hypothalamic area (LHA). In the peripheral tissues, NPW-ir was diffusely observed in the respiratory system, digestive system, urogenital system, endocrine system, lymphatic organs, and circulatory system.(2) Immunohistochemistry analysis indicated that although GPR7-ir was limited in the PVN in the center nervous system of pigs, it was abundant in peripheral tissues, including the respiratory system, digestive system, urogenital system, endocrine system, lymphatic organs, and circulatory system.(3) There existed both conservatism and partial interspecific difference in the distribution of NPW and GPR7.3 Expression of NPW, GPR7, and GPR8 mRNAs in pig hypothalamus-pituitary-ovary axis during the estrous cycleThe aim of this experiment was to explore the expression patterns of NPW, GPR7, and GPR8 mRNAs at each levels of pig hypothalamus-pituitary-ovary axis during the estrus cycle (proestrus, estrus, metestrus, and diestrus) by RT-PCR. The results were as follows:(1) In the hypothalamus, the lowest level of NPW expression was found during estrus, followed by a significant increase during metestrus and a peak in diestrus. The lowest GPR7 expression levels were detected in metestrus, whereas the highest expression level was found in diestrus. The lowest hypothalamic GPR8 mRNA level was detected in proestrus. Subsequently, GPR8 expression gradually increased from proestrus s to diestrus.(2) In the pituitary gland, the lowest expression level of NPW, GPR7, and GPR8 mRNAs were detected in proestrus, and then a gradual increased from the proestrus to diestrus, although the changes in GPR8 expression from estrus to metestrus were not significant. The highest NPW, GPR7, and GPR8 expression levels occurred in diestrus.(3) In the ovary, the lowest expression level of NPW mRNA was observed at proestrus, whereas its mRNA levels significantly increased at estrus. GPR8 mRNA has a similar expression profile in the ovary with predominant expression at the estrus, a low value at proestrus, and a medium level from metestrus to diestrus. The changes in GPR7 expression during the estrus cycle were not significant.These molecular and morphological data will provide basis for the further study of NPW. |
PDF Full Text Request