| Melanocortin 3 and 4 receptors(MC3R and MC4R)are member of MCRs and classified as G protein-coupled receptors(GPCRs)that belong to the rhodopsin-like family A,with seven transmembrane domains(TMDs).In vertebrates,five melanocortin receptors(MC1R-MC5R)have been characterized by molecular cloning.The MCRs system has been shown to be involved in the regulation of multiple physiological functions.Such as pigmentation,feed intake,stress,energy homeostasis,steroid production,lipolysis,immunomodulation,sexual function and cardiovascular regulation.Megalobrama amblycephala,commonly known as the blunt snout bream or Wuchang bream,is a commercially important freshwater fish species that is native to China.It is widely distributed in the middle and lower reaches of the Yangtze River and its affiliated rivers,where aquaculture production has greatly increased since the 1960s.It has a high potential for intensive aquaculture because it shares several desirable characteristics such as rapid growth,excellent flesh quality,and high larval survival rate.In this study,the full-length cDNA of mc3r and mc4r were obtained by transcriptome sequencing,the database of GeneBank and RACE(rapid-amplification of cDNA ends).The protein structure was predicted by several bioinformatics software.The expression patterns and expression changes under fasting of mc3r and mc4r were determined by real-time PCR.By antibody preparation and immunohistochemical analysis,the expression of MC4R in the tissues were detected.Results:From the transcriptome sequencing,RT-PCR and RACE,the obtained full-length cDNA of the bream mc3rwas 1729 bp.The length of mc3r contained a 109 bp of 5’-untranslated region(UTR),a 636 bp of 3’-UTR and a 984 bp of coding sequence encoding 327 amino acids.Sequence analysis revealed that Megalobrama amblycephala MC3R(MamMC3R)was highly homologous(>94%)at amino acid sequences to Danio rerio,Cyprinus carpio,and Carassius auratus MC3Rs.MamMC3R shared only 69%-76%identity with other mammal MC3R sequences,including Homo sapiens,Mus musculus and Bos taurus.Phylogenetic analysis showed that MamMC3R was closely related to piscine MC3Rs.Using real-time RT-PCR,mc3r was mainly expressed in hypothalamus and pituitary,followed by the ovary,testis,muscle,liver and spleen,weakly expressed in gut,eyes,skin,gills,head kidney,heart,kidney.An experiment was conducted to determine the expression profile of mc3r during fasting of the hypothalamus,pituitary,liver and ovary.The results showed that within 24 hours of fasting,the mc3r expression first increased and then decreased,showing a change of circadian rhythm.After 48 h of fasting,the expression of mc3r in pituitary and ovary significantly increased.The abundance of Mammc4r transcripts in unfed fish significantly increased at 72 h and 14 d of fasting than in fed fish.In the pituitary,mc4r expression in unfed fish was higher at 48 h and 14 d than in fed fish.In the liver,mc4r expression in unfed fish was higher at 72 h,and 14 d than in fed fish.After 72 h re-feeding,mc4r expression significantly decreased in the hypothalamus and liver.After 14 days of fasting,plasma cortisol levels significantly increased,and blood glucose levels significantly decreased.The 3’ and 5’ RACE primers were designed based on the 887 bp intermediate sequence that was downloaded from GeneBank.The obtained full-length cDNA of the bream mc4r gene was 1575 bp in size and contained a 5’-UTR of 15 bp in length,a 3’-UTR of 579 bp in length,and an ORF of 981 bp that encoded a protein of 326 amino acids.Sequence analysis revealed that MamMC4R was more closely related to the MC4R of Culter alburnus,Danio rerio,and Schizothorax prenanti,with 99%,98%,and 99%similarity.MamMC4R shared only 67%-72%identity with other mammal MC4Rs,including Homo sapiens,Mus musculus and Bos taurus.Phylogenetic tree showed that MamMC4R belonged to the clade of cyprinids.All of the analyzed tissues showed mc4r expression,with the strongest(P<0.05)signals in the hypothalamus,followed by the ovary and eyes,slightly weaker signals in the pituitary,liver,and gut,and weakest signals in the skin,gills,head kidney,heart,kidney,spleen,muscle,and testis.In the hypothalamus,mc4r expression initially decreased and subsequently increased.The abundance of Mammc4r transcripts in unfed fish significantly increased at 72 h and 14 d of fasting than in fed fish.In the pituitary,mc4r expression in unfed fish was higher at 48 h and 14 d than in fed fish.In the liver,mc4r expression in unfed fish was higher at 48 h,72 h,and 14 d than in fed fish.After 72 h re-feeding,mc4r expression significantly decreased in the hypothalamus,pituitary,and liver.No differences in mc4r expression in the ovary between fasted and fed control fish were observed.By antibody preparation and immunohistochemical analysis,we also observed the positive expression of MamMC4R in the tissue of hypothalamus,pituitary,eye,liver,gut,testis and ovary.Positive reactions were mainly observed in the submucosa and lamina propria,serous membrane,and annular myometrium,and the intestinal epithelia also depicted a relatively low level of expression.Positive staining was observed in the membranes of liver cells,the hepatic duct,the connective area connective tissue,and epithelial cells.In addition,the white blood cells near the central vein and other blood vessels showed weak staining.In the testis and ovary,positive staining was mainly observed in interstitial tissue,and no positive responses were observed between spermatocytes and sperm cells.Positive staining was observed in the nuclear membrane,follicular membrane,and around yolk granules.Except for the retina that was positive outside the nuclear layer,the choroid and scleral lateral epithelium was also positive.In the hypothalamus,positive expression was observed in the rear,ventral,and medial longitudinal bundles.The neurohypophysis and pituitary gland also showed positive staining. |
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