Electrotransfer of single-chain LH gene into skeletal muscle induces early ovarian development of orange-spotted grouper (Epinephelus coioides)

Abstract

Luteinizing hormone (LH) plays important roles in regulating steroidogenesis and reproductive development of vertebrates. In the present paper, we study function of LH on early ovarian development of orange-spotted grouper by electrotransfer of single-chain LH gene into skeletal muscle for the first time. Short-term and long-term injection experiments were performed in this work, respectively. For short-term injection experiments, fish received one electrotransfer with the plasmid in skeletal muscle, then blood and muscle around the injected area were sampled 1, 3, 5 and 7 days after the injection, mRNA expression levels of LH gene relative to 18S were determined by quantitative real-time PCR (RT-PCR) assays and serum 17β–estradiol (E2) levels were quantified by ELISA method. The results showed that levels of mRNA of LH gene in muscle and serum E2 level increased from 1 day to 7 days after the injection. For long-term injection experiments, fish received electrotransfer with the plasmid 4 times at weekly intervals in skeletal muscle. 48 h after the last injection, blood, gonad and hypothalamus samples were collected. Transcripts of cyp19a1a, cyp19a1b and gnrh1 genes and levels of serum E2 were separately analyzed by RT-PCR assays and ELISA method, and ovarian tissues were made of paraffin sections and stained by hematoxylin-eosin by method and observed by optical microscopy. The results suggested that long-term injection of LH gene into muscle upregulated transcripts of cyp19a1a and cyp19a1b and downregulated that of gnrh1, and stimulated E2 production and early-stage oogenesis. Moreover, statistical data showed that 9 of 10 ovaries of injected fish with LH gene began to develop after the long-term experiments. These data suggest that single-chain LH gene introduced into skeletal muscle via electrotransfer can be expressed and induce the early ovarian development of juvenile orange-spotted grouper. This work contributes to solve reproductive dysfunctions associated with low hormone levels of teleosts, further it may represent the demonstration at regulation of LH on early ovarian development of orange-spotted grouper to a certain extent.