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

Abstract

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.

Developmental tracing of luteinizing hormone β‐subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function

“…Zebrafish pituitary gonadotropes are determined to differentiate early in larval development by 32 hpf (Pogoda and Hammerschmidt, 2009), but it is unknown at what point these primordial gonadotropes express lhb or fshb. In zebrafish transiently expressing GFP under the control of the 5 0 -flanking region of lhb, GFP signal was detected in the area of the pituitary by 48 hpf (Chen and Chiou, 2010), although this signal was not confirmed by colocalization with the endogenous lhb gene or generation of a stable transgenic line. Similarly, down-regulation of lhb in eya1 mutant zebrafish suggests that lhb is being produced in gonadotropes by 72 hpf, although cell-specific expression could not be confirmed by in situ hybridization (Nica et al, 2006).…”

Extrapituitary production of anterior pituitary hormones: an overview

“…Transcripts for LH receptors additionally occur in oocytes and pre-implantation embryos, suggesting further paracrine roles for LH in embryogenesis [133]. A role for extrapituitary LH in embryogenesis is similarly indicated in zebra fish embryos, in which antisense morpholino oligonucleotides suppress LH gene expression and embryo development [134].…”

Fishing for neuroendocrine tumors

“…This last is a member of the GH/PRL family, unique to bony fish, implicated in several physiological processes (energy homeostasis, stress response, reproduction, fat or ion metabolism, acidosis, pigmentation, etc.) (Gonzalez-Nunez et al 2003, Liu et al 2003, Zhu et al 2004, So et al 2005, Lopez et al 2006, Chen & Chiou 2010, Lohr & Hammerschmidt 2011. Classical feedback mechanisms, involving signals from peripheral organs, contribute to the regulation of hypothalamic and anterior pituitary hormone secretion.…”