Cloning, Tissue Distribution And Feeding Regulation Of MCH,CRH And GnRH2 In Schizothorax Prenanti

To study melanin-concentrating hormone (MCH), corticotropin-releasing hormone (CRH), gonadotropin releasing hormone (GnRH) on feeding regulation in Schizothorax prenanti (S. prenanti), in this study the RT-PCR and RACE PCR were used to obtained the Ml length cDNA of MCH, CRH and GnRH2. Subsequently, real-time quantitative PCR (qPCR) was used to detect the distribution of MCH, CRH and GnRH2 in S, prenanti. Finally, the changes in expressions of MCH, CRH and GnRH2 during different feeding status were examined by qPCR.In this study, the obtained full-length MCH cDNA is 589 base pairs (bp) consist of a 66 bp 5’untranslated region (UTR), a 148 bp 3’ UTR, and a 375 bp open reading frames (ORF). The deduced amino acid sequence encoding a putative 124 amino acid (aa) protein consists of a putative 24 aa signal peptide at the N-terminus, a 17 aa mature peptide at the C-terminus and a 13 aa neuropeptide EV (NEV). The amino acid sequence highly similarity was observed with goldfish (Carassius auratus) MCH (90.3%). The full-length CRH cDNA fragment consisted of 1046 bp, including a 127 bp 5’UTR, a 430 bp 3’UTR, which contains two polyadenylation signals (AATAAA) upstream of the poly (A) tail and a 489 bp ORF. The ORF encodes the 162 aa prepro CRH, which consists of a 24 aa putative signal peptide, a cryptic region and 41 aa sequence of mature CRH. S. prenanti CRH amino acid sequence presented a high similarity with the family Cyprinidae, particularly, common carp(Cyprinus carpio) (96.3%). The full-length cGnRH Ⅱ cDNA consisted of 693 bp in length, including a 160 bp 5’UTR, a 272 bp 3’ UTR, and a 261 bp ORF. The deduced amino acid sequence encoding a putative 86 aa protein consist of a putative 24 aa signal peptide at the N-terminus, a 10 aa mature decapeptide and a 49 aa GnRH-associated peptide at the C-terminus. S. prenanti amino acid sequence identities highly similar to other Cyprinid, including the Cyprinus carpio (98.6%), Gobiocypris rarus (95.9%) and Danio rerio (90.5%).Differential expressions of mRNA for MCH, CRH and GnRH2 in various tissues of S. prenanti were detected by qPCR. The results showed MCH, CRH and GnRH2 were mainly expressed in the brain. MCH mRNA was highly expressed in the brain, moderately expressed in pituitary and very lowly expression in the other peripheral tissues. To further explore the MCH mRNA in different regions of brain, the MCH mRNA was highly expressed in hypothalamus-and very weak expressed in other regions throughout the brain. CRH mRNA was widely expressed in peripheral tissues. Furthermore, except the parencephalon, CRH mRNA was expressed in different regions of the brain, especially highest in hypothalamus. GnRH2 mRNAc was detected in all tissues tested. Moreover, the GnRH2 mRNA was expressed in different regions of the brain, particularly, highest in hypothalamus.Periprandial (pre-and post-feeding) changes of MCH, CRH and GnRH2 mRNA expressions in the hypothalamus were examined. Seven groups of weight-matched S. prenanti (average body weight 39.42±3.61 g, n=15/group) were acclimated for 2 weeks to tank conditions and fed daily at a scheduled time (11:00,0 h) for 2 weeks. Hypothalami were collected at:3 h prior to feeding (8:00 h),1 h prior to feeding (10:00 h), upon commencement of feeding (0 h),1 h after feeding (1 h) and 3 h after feeding (3 h). Two unfed groups were sampled at 1 h and 3 h and served as the fasted group. MCH mRNA was significantly decreased in the hypothalamus of fed groups compared to fasted groups after the scheduled feeding time at 1h and 3h, respectively (P< 0.05). There was no significant difference in CRH mRNA expression among the different seven groups (P> 0.05). In post-feeding, the GnRH2 mRNA expression level presented significant decrease in fasted group compared to the fed group at 1h and 3h, respectively (P<0.05).In long-term fasting experiment, six groups of fish (average body weight 36.75± 4.12g, n=20/group) were acclimated for 2 weeks in the conditions described above. Upon commencement of the study, three groups of fish continued to be fed and the other groups were fasted. Six fish of fed and fasted groups were sampled on day 1,3,5, and 7, respectively. The fasted groups were re-fed regularly on day 9 and six fish were sampled after 2 h of the re-feeding on day 9,11 and 14. MCH mRNA in fasted group showed a significant increase compared to fed group On day 1,3,5,7 (P<0.05). After re-feeding, the expression of MCH mRNA was no difference between re-fed group and fed group on day 9 and 14 (P>0.05). CRH mRNA expression level in fasted fish showed a significant decrease in the hypothalamus compared to fed fish at day 7 (P<0.05). After re-feeding, the expression of CRH mRNA showed a significant increase in fasted compared to fed fish on day 9 (P< 0.05). Subsequently, the expression of CRH mRNA was no difference in fasted and fed group on day 14. GnRH2 mRNA expression level fasting presented significant decreased in S. prenanti hypothalamus during fasting for 13,5 and 7 day (P< 0.05). After re-feeding, there was no significant change in the expression of cGnRH II mRNA between re-fed and fed group on day 9 and 14 (P>0.05).In conclusion, the present study first cloned the full-length MCH, CRH and GnRH2 cDNA from the brain of S. prenanti. In addtion, MCH, CRH and GnRH2 mRNA all high expressed in hypothalamus. Furthermore, the results suggest that MCH, CRH and GnRH2 involved in feeding regulation in S. prenanti. While, MCH might be play a orexigenic in S. prenanti, CRH and GnRH2 might be play anorexic roles.