日本鰻生長分化因子-9 (GDF-9) 基因選殖與其在人工誘導性成熟時卵巢發育的表現

Abstract

In captivity, the Japanese eels can not undergo further gonadal development, and the population size of wild elvers had gradually decreased year by year. In the silver Japanese eel, artificial induction of gonadal development and maturation by injection of salmon pituitary homogenate (SPH) has been applied for 40 years. One of the major problems on the eel artificial maturation is that, the development of ovarian follicles are rather asynchronous during artificial maturation by SPH injection. But according to the previous in vivo induction data in our laboratory, although both of 17α-methyltestosterone (MT) combined with SPH (SPH+MT treatment) and SPH alone (SPH treatment) induction could promote ovarian development, SPH+MT enhanced the survival and synchronized ovarian follicles development. Previous results reveal that SPH is essential, and MT may directly regulate ovarian development during induction of sexual maturation. Current research focus on growth differentiation factor 9 (GDF-9) which is one of the oocyte-secreted factors, and it is essential for early follicle development. We have already obtained the cDNA encoding for GDF-9 from female Japanese eel. The overall sequence of GDF-9 cDNA isolated from Japanese eel contained 2135 bp, and this GDF-9 cDNA encodes 430 amino acids of peptide sequence. The deduced amino acid sequence shares 38 %-62 % identity with other teleost GDF-9. We propose that MT would interact with GDF-9 to regulate the SPH action in ovarian development during SPH and MT induction in Japanese eel, and GDF-9 might be regulated by androgen receptor. Cultivated female Japanese eels obtained from local supplier were acclimated to seawater. After acclimation, eels were intraperitoneal injected respectively with different exogenous hormone for 2 times and 6 times at 1-week interval. Another treatment, eels were intraperitoneal injected with exogenous hormone above combined with anti-androgen flutamide. Real-time RT-PCR (RT-qPCR) was employed to investigate the ovarian gene expression of GDF-9 and follicle stimulating hormone receptor (FSHR). Our data showed that the ovarian GDF-9 expression in SPH alone treatment declined significantly after two injections. By contrast, the supplement of MT (SPH+MT) could maintain the expression level after two injections. MT alone treatment could significantly increase the expression level compared with control group (SW control). The gene expression of GDF-9 and FSHR in hormone induction showed the significantly positive correlation, and it implied the interaction between GDF-9 and FSHR. Flutamide could significantly inhibit the SPH-induced GDF-9 expression. In SPH+MT group after six injections, most of larger follicles with larger diameter (280~300μm) were still at the oil droplet stage, on the other hand, those of follicles by SPH tretment with similar oocyte diameter (280μm) had entered the period of yolk accumulation. In conclusion, MT might induce GDF-9 expression and then increase FSHR expression level, and thus promoted folliculogenesis in smaller follicles within the ovary, and the steroidogenesis in larger follicles would be inhibited by the amount of GDF-9 expression. As result, pre-vitellogenic stage could be prolonged, so that oil droplets could be fully cumulated, egg quality could also be improved, and the development of ovarian follicles could be more synchronized. These results imply that the interaction of SPH and MT might be mediated through GDF-9 action to affect ovarian development in Japanese eel. However, when is the initiation of GDF-9 expression in the ovary of Japanese eel, and how does GDF-9 regulate the development of ovarain follicles in Japanese eel needs further elucidation.