PARENT SESSION

(T737) DIFFERENTIAL REGULATION OF ZEBRAFISH FSH (fshb) AND LH (lhb) PROMOTER ACTIVITIES BY ACTIVIN/SMADs AND ESTROGEN/ERs PATHWAYS.

Lin, Sze-Wah¹, Ge, Wei¹, ¹ The Chinese University of Hong Kong, Shatin, Hong Kong

ABSTRACT- Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are both important for regulating vertebrate ovarian development and function, and their expression and secretion are in turn subject to the control by multiple neuroendocrine and endocrine factors. Apart from gonadotropin-releasing hormone (GnRH), steroids such as estrogens from the gonads and local growth factors such as activin also play important roles in the differential regulation of gonadotropin subunits. Although the involvement of activin and estrogens in gonadotropin biosynthesis has been well documented, the information about their action mechanisms is rather limited, particularly in lower vertebrates. The present project therefore aims at investigating the involvement of activin transcription factors Smads and estrogen receptors (ERs) in controlling zebrafish FSH (fshb) and LH (lhb) promoter activity and localizing the potential cis-regulatory elements that respond to their signaling. The 5′ flanking promoter regions of zebrafish FSH (4500 bp) and LH (1700 bp) genes were first cloned by genome walking. The functionality of the promoters was then tested by transfecting the reporter constructs expressing SEAP (secreted human placental alkaline phosphatase) into the LT2 cells, a mouse gonadotroph cell line, together with the constructs expressing Smads (Smad2, 3, 4 and 7) and/or ERs (ER, A and B). The results demonstrated that the activin/Smads and estrogen/ERs signaling pathways differentially regulated the promoter activities of zebrafish FSH and LH gene. Activin/Smads pathway stimulated FSH promoter but slightly suppressed that of LH. In contrast, the estrogen/ERs pathway inhibited FSH but enhanced LH promoter activity. Promoter analysis and site-directed mutagenesis have localized several potential cis-regulatory Smad-response (SRE) and estrogen-response elements (ERE) in the two promoters. These results have not only confirmed our previous findings in the goldfish on activin regulation of FSH and LH expression, but also provided a platform for further analysis of the molecular mechanisms underlying the regulation of gonadotropin expression and biosynthesis in this model organism.

KEY WORDS: gonadotropin, promoter, zebrafish