자바리 Epinephelus bruneus 뇌에서의 KiSS1/GPR54 mRNA 발현 기작에 관한 연구

Abstract

Kisspeptin is product of the KiSS1 gene and the endogenous agonist for the GPR54 receptor. Kisspeptin has been reported to facilitate sexual maturation and ovulation by directly stimulating GnRH neurons via the GPR54 in several mammalian species. The report suggests that KiSS1/GPR54 system is play a important role of the reproductive system. Longtooth grouper, Epinephelus bruneus, a marine protogynous hemaphrodite, which is sex change from female to male begins around the age of 5 years, and it takes too much time until sexual maturation. Therefore, it makes difficult to obtain stable fertilized eggs and to understand the mature characteristics in the female longtooth grouper. The purpose of this study is understand to primary expression mechanism of KiSS1/GPR54 system, for research that KiSS1, KiSS2 and GPR54 mRNA cloning, KiSS1/GPR54 mRNA distribution in several tissues and regulation of KiSS1 mRNA expression by photoperiod, melatonin and HCG injection in the longtooth grouper.

Ⅰ. Molecular cloning of KiSS1, KiSS2, GPR54 cDNAs and tissue specific expression in the longtooth grouper, E. bruneus

The full length sequence and open reading frame of KiSS1, KiSS2 and GPR54 were 516 bp (312 amino acids), 557 bp (125 amino aicds) and 1230 bp (378 amino acids) long, respectively. Each gene showed about 70% (KiSS1), 90% (KiSS2) and 95% (GPR54) homology in amino acid sequences comparing to each other among other teleosts. Phylogenetic analysis revealed that the longtooth grouper KiSS1 and KiSS2, GPR54 was grouped with european seabass and orange-spotted grouper. KiSS1 and GPR54 mRNAs are common expression in the brain and particulary both gene expression in the hypothalamus. These results suggest of the co-existence of two kisspeptin coding genes, KiSS1 and KiSS2. Also KiSS1/GPR54 system is suggestive of control of reproduction in the longtooth grouper.

Ⅱ. Regulation of KiSS1/GPR54 mRNA expression by photoperiod and melatonin in Longtooth grouper, E. bruneus.

1. KiSS1/GPR54 mRNA expression by photoperiod and HCG injection.

Experimental group were classified into two groups, long day length (LD 16:8, light on at 04:30) and short day length (LD 8:16, lights on at 08:30), respectively. Each experimental group was injected HCG The KiSS1/GPR54 mRNA expression was high in long day length group and HCG injection group. These results suggest that KiSS1/GPR54 mRNA is upregulated by long photoperiod and positive feedback control of GTH.

2. Sensitive expression of KiSS1 by light

Longtooth groper were acclimated in LD 12:12 condition for one week. After one week, the fish were sampled at am 01:00 (dark condition) and am 03:00 (photo exposure for 2 hrs). The KiSS1 mRNA level of photo exposure group siginificantly higher than dark condition group. This result suggest that KiSS1 have photo sensitive expression.

3. KiSS1/GPR54 mRNA expression by melatonin injection

Melatonin and vehicle daily injection for 2 weeks. The KiSS1 and GPR54 mRNA expression level of melatonin injection group was lower than vehicle injection group. This result indicates that KiSS1/GPR54 mRNA expression is inhibited by melatonin in the longtooth grouper.