Zebrafish Gonadotropins and Their Receptors: II. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Subunits—Their Spatial-Temporal Expression Patterns and Receptor Specificity1

Abstract

Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) play critical roles in vertebrate reproduction. In the present study, we cloned and characterized zebrafish FSHbeta (fshb), LHbeta (lhb), and GTHalpha (cga) subunits. Compared with the molecules of other teleosts, the cysteine residues and potential glycosylation sites are fully conserved in zebrafish Lhb and Cga but not in Fshb, whose cysteines exhibit unique distribution. Interestingly, in addition to the pituitary, fshbeta, lhbeta, and cga were also expressed in some extrapituitary tissues, particularly the gonads and brain. In situ hybridization showed that zebrafish fshbeta and lhbeta were expressed in two distinct populations of gonadotrophs in the pituitary. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that all the three subunits increased expression before ovulation (0100-0400) when the germinal vesicles in the full-grown follicles were migrating toward the periphery, but the levels dropped at 0700, when ovulation occurred. Recombinant zebrafish FSH (zfFSH) and LH (zfLH) were produced in the Chinese hamster ovary (CHO) cells and their effects on the cognate receptors (zebrafish Fshr and Lhr) tested. Interestingly, zfFSH specifically activated zebrafish Fshr expressed together with a cAMP-responsive reporter gene in the CHO cells, whereas zfLH could stimulate both Fshr and Lhr. In conclusion, the present study systematically investigated gonadotropins in the zebrafish in terms of their structure, spatial-temporal expression patterns, and receptor specificity. These results, together with the availability of recombinant zfFSH and zfLH, provide a solid foundation for further studies on the physiological relevance of FSH and LH in the zebrafish, one of the top biological models in vertebrates.

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

“…For example, in mammals gpa expression precedes lhb and fshb expression in mouse (Japon et al, 1994) and human gonadotropes (Pope et al, 2006), which may explain the inability to detect lhb transcripts in the pituitary of zebrafish when gpa could be detected. In addition, the presence of two lhb genes in zebrafish (So et al, 2005) could lead to partially divergent function or tissue distribution, possibly explaining the proposed earlier onset of lhb gene expression relative to medaka.…”

“…Upon release, LH and FSH bind to their specific receptors, LHR and FSHR, in the gonads, stimulating steroidogenesis and gametogenesis. Gonadotropin receptors, however, are expressed in multiple tissues also outside the b-p-g axis (Kumar et al, 2001;Vischer and Bogerd, 2003;Wong and Van Eenennaam, 2004;So et al, 2005;Rocha et al, 2007;Mittelholzer et al, 2009), suggesting LH and FSH could be involved in many physiological processes, although nonreproductive functions are largely unknown.…”

Fsh and Lh have common and distinct effects on gene expression in rainbow trout testis

“…Not only does receptor localization differ, but cross-reactivity has also been described in binding assays with purified fish gonadotropins (Yan et al 1992) and in functional studies using mammalian cell lines expressing fish receptors separately. In African catfish (Bogerd et al 2001, zebrafish (So et al 2005), and Atlantic salmon (Andersson et al 2009), a promiscuous activation of Fsh receptors has been reported using high Lh concentrations. However, in two salmonid species, amago salmon and rainbow trout, only Fsh was able to activate Fshr (Oba et al 1999, Sambroni et al 2007).…”

Fishing for gene function – endocrine modelling in the zebrafish

“…Nica et al (2004) cloned a zebrafish homologue of the gsu and found two distinct populations of cells; those that coexpressed pit1 (presumptive thyrotrophs) and those that did not (presumptive gonadotrophs). Most recently, the D. rerio genes for the gonadotrophins have been cloned (So et al 2005). Two LH isoforms (lh 1 and lh 2), fsh and GTH sequences were cloned, and their distribution was restricted to the pituitary, testis and ovaries.…”

“…Two LH isoforms (lh 1 and lh 2), fsh and GTH sequences were cloned, and their distribution was restricted to the pituitary, testis and ovaries. Sequence homology of LH and GSU with other fish species is reasonably high (70%), but less conserved for FSH (37-64%) (So et al 2005). In mammals, one of the key transcription factors responsible for the differentiation of gonadotroph lineage cells is steroidogenic factor-1 (SF-1), an ftz-f1 homologue.…”