Studies On Function Of Gonadotropin Receptors In Reproductive Cycle Of Cynoglossus Semilaevis Günther

Half-smooth tongue sole(Cynoglossus semilaevis Gunther) is a Chinese inshore species with great potential for aquaculture. This paper mainly study on the function of gonadotropin receptors in reproductive cycle of Half-smooth tongue sole by the methods of molecular biology methods, histological staining, immunohistochemical techniques, hormone radioimmunoassay in details. The main results and conclusions are as follows:1. Cloning and expression analysis of Follicle-stimulating hormone receptor (FSHR) in Cynoglossus semilaevis GuntherFull sequnce of Follicle-stimulating hormone receptor (FSHR) gene was cloned from Cynoglossus semilaevis.The length of the cDNA is 3105 bp, and encodes a protein of about 704 amino acids, which contains the conserved seven transmembrane helix domains (TM helix), and it belongs to glycoprotein hormone receptor (GHR) family. Tissue expression analysis showed that FSHR mRNA expressed widely in Cynoglossus semilaevis. Except the high levels in gonads, strong amplification signals were also detected in extragonadal tissues including the spleen, kidney and head kidney.It is the fist study to analyze the seasonal expression changes of FSHR mRNA in ovary and brain of female tongue sole and male tongue sole's testis, brain and spleen. The results show that FSHR gene expression change seasonally in the five tissues. Combination of plasma E2 levels, T levels and GSI gene, functions of FSHR in reproductive cycle of tongue sole were discussed.2. Expression analysis of luteinizing hormonene receptor (LHR) in Cynoglossus semilaevis GuntherPartial cDNAs encoding LHR (luteinizing hormonene receptor) gene were cloned from Cynoglossus semilaevis by nested reverse transcrip-polymerase chain reaction (nested RT-PCR). The length of the fragment is 614bp, and encodes a protein of about 204 amino acids, which contains the conserved seven transmembrane helix domains (TM helix), and it belongs to glycoprotein hormone receptor (GHR) family. Tissue expression analysis showed that LHR mRNA expressed in all eleven tissues including ovary, spermary, liver, stomach, intestines, gill, heart, spleen, kidney, head kidney, and brain, from which the spleen and kidney presented the strongest amplification signal.It is the fist time to analyze the seasonal expression changes ofLHR mRNA in ovary,brain,spleen and kidney of female tongue sole and male tongue sole's testis spleen. The results show that FSHR gene expression change seasonally in the six tissues. Combination of GSI and FSHR gene expressions, functions of LHR in reproductive cycle of tongue sole were discussed.3. Immunohistochemical studies on gonadotropin receptors in gonads of Cynoglossus semilaevis Gunther3.1 Immunohistochemical study on FSHR in gonads of Cynoglossus semilaevis GuntherA weak immunopositive reaction to FSHR antibodies was detected in II phase of follicular oocytes. With the development of oocytes, immunopositive reactions express more and more intensely. Cytoplasm and follicular layer have a positive reaction to FSHR antibodies in the phaseâ…£andâ…¤phase oocyte. At the V phase oocytes, positive reaction in granulosa cells is stronger than theca cells to FSHR antibodies. By VI phase, the follicular cells response weakly to FSHR, however, strong positive reaction appears in Cytoplasm of oocytes.The FSHR expresses at spermatogonium, spermatocyte, spermid and space between folioles in each period of testis development, especially high levels in inferstitial cells of stage V of testis; but the nucleolus of spermatogonium, karyon of spermatocyte and sperm assume immunonegative reaction toFSHR antibodies.The immunopositive reaction changes to FSHR are similar to FSHR gene expression profiles in ovary and testis of half-smooth tongue sole.Taken together, these observations supports the involvement of the FSHR in the final oocyte maturation and spermiation processes.3.2 Immunohistochemical study on LHR in gonads of Cynoglossus semilaevis GuntherThere is a weak immunopositive reaction to FSHR antibodies in follicular layer ofâ…¡phase oocyte, and then, immunopositive reactions to FSHR antibodies were detected on follicular cells of oocytes inâ…¢phase. With the development of oocytes, immunopositive reactions to LHR antibodies become more and more faint, until to oocyte in V phase, immunopositive reactions to FSHR antibodies were just found on theca cells. And at the VI phase oocytes, there were weak positive reactions in follicular cells to FSHR antibodies.There are LHR immunopositive reactions at spermatogonium, spermatogonium, spermid and space between folioles in each period of development, especially high levels in inferstitial cells of stageâ…¤of testis; however, the nucleolus of spermatogonium and sperm are immunonegative to LHR.The immunopositive reaction changes to LHR are consist with LHR gene expression profiles in ovary and testis of half-smooth tongue sole. And so, the above results indicate that the half-smooth tongue sole LHR mainly plays a major role at beginning of the vitellogenesis and spermiation processes.