| Neuromedin B(NMB)was isolated and purified from pig spinal cord in 1983,and gastrin-releasing peptide(GRP)was isolated from porcine stomach tissue in 1979.NMB/GRP is a mammalian bombesin(BN)-like peptide,which play physiological functions via its receptor NMBR or GRPR.NMB/GRP and its receptor NMBR/GRPR are widely distributed in the central nervous system(CNS)and peripheral tissues,and demonstrate a broad spectrum of pharmacological and biological effects.Currently,the studies of NMB/GRP and its receptor are mainly concentrated on the human,rat and mouse,and there are few reports about NMB/GRP and its receptor in pigs.Therefore,in this study,Xiaomeishan pig as the object of study:cloning of NMB/GRP and its receptor gene,expression of NMB/GRP and its receptor mRNA in pigs,distribution of NMBR/GRPR in pig peripheral tissues,as well as expression patterns of NMB and its receptor mRNA on the reproductive axis of the boar postnatal development stages.In addition,in vitro,we study the effects of NMB/GRP on testosterone secretion,testosterone secretion mechanism,and cell proliferation as well as apoptosis in the cultured pig Leydig cells.Through the above study,we aimed to find out the possible physiological role of NMB/GRP in pigs,and illuminate the mechanism of on the reproductive regulation of boars.1 Cloning of NMB and its receptor in pigsIn this study,we cloned pig NMB and NMBR genes via gene cloning technique,and the gene and amino acid sequence of NMB and its receptor were analyzed by bioinformatics technology.The results were as follows:(1)The pig NMB cloned at 567 bp,including one open reading frame(ORF)of 366 bp and encodes 121 amino acids(aa),with a signal peptide of 24 aa and a mature peptide of 32 aa,with a conserved sequence of NMB-10(GNLWATGHFM).(2)The pig NMBR cloned at 1,173 bp,including one ORF of 1,173 bp and encoding 390 aa,with seven transmembrane structure for a G-protein coupled receptor,located on the pig chromosome 1.The gene sequence has been submitted to NCBI(GenBank ID:KM058699).(3)Compared with other species,the nucleotide and amino acid sequence of NMB and its receptor are highly homologous and conservative.Genetic evolution is relatively close.2 Expression of NMB and its receptor in the pig tissue,as well as distribution of NMBR in the peripheral tissues of pigsIn this study,the expression patterns of NMB and its receptor mRNA in pigs were detected by fluorescence quantitative PCR,and the distribution of NMBR in the peripheral tissues was studied by immunohistochemistry.The results are as follows:(1)NMB mRNA was mainly expressed in the central nervous system(CNS),the highest expression level in the olfactory bulb,in other parts of the brain were also found a high level of expression;in the peripheral tissues,NMB mRNA was mainly expressed in the pancreas,adrenal gland,testis and ovary.(2)NMBR mRNA was mainly expressed in the peripheral tissues,the highest expression level in the pancreas,and also in the lymphoid tissues,glands and reproductive organs;in CNS,NMBR mRNA was mainly expressed in the cerebellum and pituitary gland.(3)NMBR-ir is widely distributed in the peripheral tissues and organs of pigs,including respiratory and circulatory system,digestive system,urogenital system,lymphoid organ and endocrine gland.These results suggest that NMB may play an important role in regulating the physiological function of pigs.3 Cloning of GRP and its receptor in pigsIn this study,we cloned pig GRP and GRPR genes via electronic cloning technology and gene cloning technique,and the gene and amino acid sequence of GRP and its receptor were analyzed by bioinformatics technology.The results were as follows:(1)The electronic cloning sequence of pig GRP gene was obtained.The pig GRP cloned at 608 bp,including one open reading frame(ORF)of 441 bp and encodes 146 amino acids(aa),with a signal peptide of 23 aa and a mature peptide of 27 aa,with a conserved sequence of GRP-10.Compared with other species,the nucleotide and amino acid sequence of pig GRP are highly homologous and conservative.Pig GRP with cattle,sheep and horses on genetic evolution is most close.(2)The pig GRPR cloned at 1,487 bp,including one ORF of 1,155 bp and encoding 384 aa,with seven transmembrane structure for a G-protein coupled receptor,located on the pig chromosome X.The gene sequence has been submitted to NCBI(GenBank ID:KM 052179.1).Compared with other species,the nucleotide and amino acid sequence of pig GRPR are highly homologous and conservative.Pig GRPR with cattle,sheep and horses on genetic evoluti on is most close.4 Expression of GRP and its receptor in the pig tissue,as well as distribution of GRPR in the peripheral tissues of pigsIn this study,the expression patterns of GRP and its receptor mRNA in pigs were detected by fluorescence quantitative PCR,and the distribution of GRPR in the peripheral tissues was studied by immunohistochemistry.The results are as follows:(1)GRP mRNA was mainly expressed in the peripheral tissues,and the expression level was highest in the esophagus.In addition,there was a high level expression in the gastrointestinal tract,digestive gland,urogenital system,lymphoid organs and lungs.(2)GRPR mRNA was widely expressed in the central nervous system and peripheral tissues,the highest expression level in the pancreas,and the high expression level in the digestive tissues,reproductive organs and lymph organs,skeletal muscle and fat.In the central nervous system(CNS),GRPR mRNA was mainly expressed in the cerebellum,pituitary,hypothalamus and spinal cord.(3)The distribution of GRPR-ir in the peripheral organs and tissues of pigs was extensive,mainly in the heart,lung,digestive system,urogenital system,endocrine tissue and lymphoid organs.These results suggest that GRP may play an important role in regulating the physiological function of pigs.5 The expression patterns of NMB and its receptor in the gonad axis of male Xiaomeishan pigsIn this study,the postnatal development of NMB and its receptor mRNA was investigated in the hypothalamus-pituitary-testicular(HPT)axis of male Xiaomeishan pigs by fluorescence quantitative PCR.The results are as follows:(1)In the hypothalamus,peak expression levels of NMB mRNA were observed on day 60.Although the lowest levels of NMB expression were detected on day 30,changes in hypothalamic NMB mRNA levels found on days 30,90 and 120 were not significant.NMBR mRNA expression reached peak levels on day 30,followed by a gradual decrease from days 30 to 120.The lowest NMBR expression levels were detected on day 120.(2)In the pituitary gland,the highest expression level of NMB occurred on day 30,whereas the lowest level was observed on 'day 120.However,the lowest expression of NMBR mRNA was measured on day 3.On day 30,the pituitary NMBR expression level was slightly higher than that for day 3.Peak NMBR expression levels were detected on day 60,changes in pituitary NMBR mRNA levels on days 90 and 120 increased significantly.(3)In the testis,peak levels of NMB mRNA were found on day 3,whereas the lowest levels were detected on day 60.On day 30,NMB expression levels were slightly lower than those detected on day 3.NMB expression levels then gradually increased from day 60 to day 120.Day 3 NMBR expression levels were higher than those of day 120.Peak NMBR expression levels occurred on day 90 and were slightly higher than day 3 levels.The lowest expression of NMBR mRNA occurred on day 120,and levels significantly decreased on days 30 and 60 relative to day 3 levels.These results suggest that NMB may play an important role in regulating the reproductive function of male pigs.6 The effect of NMB on the cultured pig Leydig cells in vitroTo study the effect of NMB on boar reproductive function,in this study,the effects of NMB on testosterone secretion and testosterone secretion mechanism in cultured Leydig cells were investigated,and the effects of NMB on proliferation and apoptosis.The results are as follows:(1)NMB could promote testosterone secretion of pig Leydig cells,and NMBR antagonist BIM23127 could block the effect of NMB on testosterone secretion.(2)NMB could increase the expression of STAR,CYP11A1,CYP17A1,3?-HSD and HSD17B1 mRNA and protein of Leydig cells;and NMBR antagonist could inhibit the expression of key enzymes of testosterone synthesis.(3)NMB could promote the proliferation of Leydig cells,and increase the expression of PCNA and Cyclin B1.(4)NMB may inhibit the apoptosis of Leydig cells by decreasing the expression of Caspase-3 and Bax.These results show that NMB/NMBR system can promote the synthesis and secretion of testosterone by regulating expression of Leydig cell testosterone synthesis key enzyme,and the regulation of proliferation and apoptosis of Leydig cells through increased the expression of proliferation associated protein and decreased the expression of apoptosis related proteins.The aim of this study was to provide the experimental basis for studying the mechanism of NMB on reproductive function in boars.7 The effect of GRP on the cultured pig Leydig cells in vitroIn this study,the mechanism of GRP on the cultured pig Leydig cells was studied in vitro.The results showed that GRP could promote the synthesis and secretion of testosterone,through GRP/GRPR system regulate the expression of STAR,CYP11A1,CYP17A1,3?-HSD and HSD17B1 of pig Leydig cell testosterone synthesis key enzyme;as well as promote proliferation of Leydig cells via increased the expression of proliferation associated protein(PCNA and Cyclin B1)and decreased the expression of apoptosis related proteins(Caspase-3 and Bax).The study laid the theoretical foundation for studying the reproductive function of GRP on boar. |
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