Molecular cloning and functional analysis of the zebrafish luteinizing hormone beta subunit (LH<beta>) promoter.

The luteinizing hormone (LH) plays important roles in vertebrate reproduction. In the present study, we cloned and characterized the zebrafish (Danio rerio) LH<beta> subunit gene structure and promoter region. Analysis of 3.0 kb (LH3.4K~5'UTR) of the LH<beta> subunit proximal promoter region displayed maximal promoter activity in a tilapia ovary cell line (TO2 cells) after treatment with gonadotropin-releasing hormone (GnRH). Transient expression experiments with a 5'-deletion revealed at least 10 regulatory regions in the zebrafish LH<beta> subunit gene. Compared to the molecular mechanisms of other vertebrates, GnRH treatment led to the activation of zebrafish LH<beta> subunit gene transcription in ovary cells. We demonstrated that LH<beta> subunit gene transcription increased with 6 h of treatment with GnRH but was repressed by protein kinase C, mitogen-activated protein kinase, and calcium in the TO2 cell line. To study promoter-specific expression, we constructed an LH<beta> subunit (LH3.4k~5'UTR) promoter region-driven green fluorescent protein (GFP), and the results indicated that LH<beta> promoter-driven GFP transcripts appeared in the pituitary gland. For the gene knockdown study, we targeted knockdown of the LH<beta> subunit gene by two antisense morpholino oligonucleotides that resulted in serious abnormalities and death during zebrafish embryogenesis. These results suggest that the LH plays important roles in reproduction and general embryonic development in zebrafish.