Molecular studies on the neuroendocrine regulation of pubertal development in grey mullet, Mugil Cephalus

Nocillado, Josephine N. (2007). Molecular studies on the neuroendocrine regulation of pubertal development in grey mullet, Mugil Cephalus PhD Thesis, School of Integrative Biology, The University of Queensland.

       
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Author Nocillado, Josephine N.
Thesis Title Molecular studies on the neuroendocrine regulation of pubertal development in grey mullet, Mugil Cephalus
School, Centre or Institute School of Integrative Biology
Institution The University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Francis Carrick
Collection year 2008
Language eng
Subjects L
Abstract/Summary This research project was designed to advance our understanding of the molecular regulation of pubertal development in a late maturing fish, using the grey mullet as model species. The objective was addressed by the molecular cloning of key genes involved in reproductive function followed by the analysis of the regulation of selected genes at the promoter and gene expression level. The cDNAs of the genes encoding for muGPR54, muGnRH1, muGnRH2, muGnRH3, muCyp19a, muCyp19b, muIGF-I, muIGF-II, muERH, and muERI were isolated, and their expression along the BPG axis determined, using RTPCR. This was followed by the isolation of the promoter regions of mudrd2, muCyp19a and muCyp19b genes by genome walking PCR and determination of promoter functionality in vitro by reporter gene assay, using luciferase as reporter gene. The brain, pituitary and ovarian expression profile of muGPR54, muGnRH1, muGnRH2, muGnRH3, mudrd2, muCyp19a and muCyp19b genes was characterised by QPCR in female fish undergoing puberty. The pubertal stages of the experimental fish were defined according to the oocyte developmental stage and the levels of plasma vitellogenin, which was determined by ELISA for mullet vitellogenin developed in the course of the present study. The studies concerning the promoters revealed two putative promoters of the mudrd2 gene. The first putative promoter, located in the region flanking the 5’UTR, contained Sp1, progesterone receptor, CREB, GATA and STAT binding sites, GC boxes and CAAT- and TATA- like sequences. The second putative promoter, situated in the region flanking the first coding exon and is therefore an intronic promoter, contained consensus Inr and DPE elements as well as putative AP4, GR, NFkappaB, CREB, AP1, TTF-1, C/EBP, Pit1,GHF-1, Ahr/Arnt, Ptx1, RARH, GATA binding sites, GC boxes and CAAT- and TATA-like sequences. Functionality of the intronic promoter, and the serially deleted constructs derived from it, were demonstrated in COS-7, HT3 and TE671 cell lines. The distinct basal promoter activity of the different constructs indicated the functional relevance of the putative regulatory elements and the tissue-specific regulation of mudrd2 gene expression. The muCyp19a promoter sequence contained consensus TATA box and putative ½ERE, SF-1, AhR/Arnt, PR and GATA sites. The muCyp19b promoter sequence revealed consensus TATA and CCAAT boxes and putative PR, ERE and ½ERE, SP-1, GATA C/EBP, GRE, NFkappaB, STAT, PPAR/RXR, Ahr/Arnt and CRE sites. As in the mudrd2 gene promoter, the functionality of muCyp19a and muCyp19b gene promoters, observed in COS-7, HT3 and TE671 cell lines, pointed to the transcriptional roles of the putative regulatory elements identified on the promoter sequences. Additionally, for the muCyp19b gene, negative This research project was designed to advance our understanding of the molecular regulation of pubertal development in a late maturing fish, using the grey mullet as model species. The objective was addressed by the molecular cloning of key genes involved in reproductive function followed by the analysis of the regulation of selected genes at the promoter and gene expression level. The cDNAs of the genes encoding for muGPR54, muGnRH1, muGnRH2, muGnRH3, muCyp19a, muCyp19b, muIGF-I, muIGF-II, muERH, and muERI were isolated, and their expression along the BPG axis determined, using RTPCR. This was followed by the isolation of the promoter regions of mudrd2, muCyp19a and muCyp19b genes by genome walking PCR and determination of promoter functionality in vitro by reporter gene assay, using luciferase as reporter gene. The brain, pituitary and ovarian expression profile of muGPR54, muGnRH1, muGnRH2, muGnRH3, mudrd2, muCyp19a and muCyp19b genes was characterised by QPCR in female fish undergoing puberty. The pubertal stages of the experimental fish were defined according to the oocyte developmental stage and the levels of plasma vitellogenin, which was determined by ELISA for mullet vitellogenin developed in the course of the present study. The studies concerning the promoters revealed two putative promoters of the mudrd2 gene. The first putative promoter, located in the region flanking the 5’UTR, contained Sp1, progesterone receptor, CREB, GATA and STAT binding sites, GC boxes and CAAT- and TATA- like sequences. The second putative promoter, situated in the region flanking the first coding exon and is therefore an intronic promoter, contained consensus Inr and DPE elements as well as putative AP4, GR, NFkappaB, CREB, AP1, TTF-1, C/EBP, Pit1,GHF-1, Ahr/Arnt, Ptx1, RARH, GATA binding sites, GC boxes and CAAT- and TATA-like sequences. Functionality of the intronic promoter, and the serially deleted constructs derived from it, were demonstrated in COS-7, HT3 and TE671 cell lines. The distinct basal promoter activity of the different constructs indicated the functional relevance of the putative regulatory elements and the tissue-specific regulation of mudrd2 gene expression. The muCyp19a promoter sequence contained consensus TATA box and putative ½ERE, SF-1, AhR/Arnt, PR and GATA sites. The muCyp19b promoter sequence revealed consensus TATA and CCAAT boxes and putative PR, ERE and ½ERE, SP-1, GATA C/EBP, GRE, NFkappaB, STAT, PPAR/RXR, Ahr/Arnt and CRE sites. As in the mudrd2 gene promoter, the functionality of muCyp19a and muCyp19b gene promoters, observed in COS-7, HT3 and TE671 cell lines, pointed to the transcriptional roles of the putative regulatory elements identified on the promoter sequences. Additionally, for the muCyp19b gene, negative vi modulation of promoter activity by quinpirole, a DA D2-receptor type agonist, was observed. The gene expression profile of muGPR54, muGnRH1, muGnRH2 and muGnRH3, mudrd2, muCyp19a and muCyp19b genes in pubertal fish revealed their functional relevance during sexual maturation. The significantly high relative expression levels of muGPR54, muGnRH1, muGnRH2 and muGnRH3 genes in the brain at the early stage of puberty (defined by low GSI, no apparent oocyte development and low, but detectable, plasma vitellogenin) pointed to a coordinated stimulatory role of these genes at the beginning of puberty. Data also suggested that muGnRH1 has the gonadotrophic role. In contrast to the expression profiles observed in the brain, expression levels of muGPR54 and muGnRH1 in the ovary were high at the advanced stage of puberty (characterised by high GSI, yolky oocytes and high plasma vitellogenin), suggesting a role of these genes towards sustaining gonadal development. The high expression levels of mudrd2 in the brain at the early stage of puberty and in the pituitary at both the early and intermediate stages, which correspond to a period of pronounced DA inhibition of reproductive function in grey mullet, indicated the role of mudrd2 in mediating the inhibitory effect of DA. Ovarian expression of muCyp19a and brain expression of muCyp19b increased during pubertal development, which implied the functional relevance of E2 biosynthesis to the pubertal process in those tissues. In conclusion, the results provide evidence that the molecular regulation of pubertal development in female grey mullet is contingent upon the functional integration of the regulatory signals coming from a suite of genes, including, at least, the genes characterised in the present study. The molecular tools obtained and the understanding achieved from the present research work provide basis for further investigations dealing with the manipulation of puberty in late maturing species of fish.

 
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Created: Fri, 21 Nov 2008, 15:48:50 EST