Molecular Cloning And Expression Analysis Of Gonadotropic Hormone Receptors And Their Responses To Exogenous Hormones And Temperature In Oreochromis Niloticus

The reproductive activity of fish is affected by environmental factors and its own brain pituitary gonad（BPG）axis.In the BPG axis of fish,gonadotropin releasing hormone（Gn RH）is produced and excreted in the brain and acts on the pituitary gland.The pituitary gland is stimulated to excrete gonadotropic hormone（GTH）.The combination of GTH and gonadal receptor（GtHR）will open the downstream pathway to participate in the reproductive activities of fish.The research object of this article is Nile tilapia（Oreochromis niloticus）.Using RACE（rapid amplification of c DNA ends）technology,the Nile tilapia luteinizing hormone receptor（LHR）and follicle stimulating hormone were obtained.Real time quantitative PCR and enzyme-linked immunosorbent assay（ELISA）were used to analyze the tissue expression,the expression of related genes after exogenous hormone injection and temperature treatment,and the changes of serum neutral steroid hormone concentration.1.Cloning and sequence analysis of FSHR and LHR of Nile tilapiaNile tilapia FSHR gene c DNA has a full length of 2577 bp（Gene Bank:MW353713）,of which the open reading frame is 2106 bp,and a total of 701 amino acids are encoded.The5’UTR is 174 bp and 3’UTR is 297bp.The full-length c DNA of Nile tilapia LHR is 3177 bp（Gene Bank:MW353714）,the open reading frame is 2082 bp,and a total of 693 amino acids are encoded.The 5’UTR is 221 bp,the 3’UTR is 874 bp.Bioinformatics analysis showed that FSHR and LHR of Nile tilapia were relatively conservative in the evolutionary stage.2.The tissues expression of FSHR and LHR in Nile tilapiaThe expression of FSHR and LHR in 13 tissues of Nile tilapia was detected by quantitative real-time PCR.The results showed that FSHR was mainly expressed in the testis and ovaries.2.Effects of exogenous hormone injection on the expression of FSHR,LHR and related factors in Nile tilapia and the changes of serum sex steroid hormone content.Within 72 hours after the injection of different exogenous hormones（DOM,LRH-A₂,DOM+LRH-A₂）,the expression of FSHR in ovaries of the injection group first increased and then decreased,reaching the maximum at 12 hours（P<0.05）;LHR reached the maximum at 72 h after injection（P<0.05）.In the brain of female fish,the expression of FSHR in the injection group reached the maximum at 12 h（P<0.05）,and decreased to the minimum at 72 h（P<0.05）;The expression of LHR was the lowest 72 h after injection（P<0.05）.In the injection group,E₂and P showed a trend of increasing first and then decreasing,and reached the maximum at 24 h and 36 h,respectively（P<0.05）.In the ovary,the expression of VTG in the injection group increased first and then decreased,and reached the maximum at 48 h（P<0.05）,In the liver of female fish,the expression of VTG in the injection group increased first and then decreased,and reached the maximum at 36 h（P<0.05）.The expression of AMH in ovaries decreased at first and then increased.The expression of AMH at 24 h and 72 h after injection was significantly lower than that in the control group（P<0.05）.In testis,FSHR in injection group increased first and then decreased,and reached the maximum at 24 h（P<0.05）;The expression of LHR reached the maximum at 72 h after injection（P<0.05）.T in the serum of male fish in the injection group showed a trend of increasing at first and then decreasing,and reached the maximum at 24 h after injection（P<0.05）.In the brain of male fish,FSHR in the injection group increased first and then decreased,and reached the maximum at 24 h（P<0.05）;The expression of LHR increased first and then decreased,and reached the maximum at 36 h（P<0.05）.The expression of AMH in testis showed a trend of first decreasing,then increasing,and then decreasing.The expression of AMH in testis was significantly lower than that in control group at 36 h and72 h after injection（P<0.05）.4.The effect of temperature on the expression of FSHR,LHR and related factors in Nile tilapia and the changes of serum sex steroid hormone content.In different temperatures（24℃,28℃,32℃）treatment for 28 days,the results showed that the E₂Content in serum of female fish showed a rising trend.On the 28th day,the E₂Content of each temperature treatment group was significantly different from that of each temperature treatment group on the 0 day（P<0.05）.On the 28th day,the P content of28℃treatment group was significantly different from that of 24℃treatment group（P<0.05）.On the 28th day,the expression of FSHR in the brain of different temperature treatment groups was significantly different from that in the brain of different temperature treatment groups on the 0th day（P<0.05）;LHR showed a downward trend and then an upward trend,On the 28th day,the expression of LHR in 28℃treatment group was significantly different from that in 24℃treatment group（P<0.05）.FSHR in ovaries showed a trend of first increasing,then decreasing and then increasing,On day 7 and 21,the expression of FSHR in ovaries of different temperature treatment groups was significantly different from that on day 0（P<0.05）,LHR showed a trend of first decreasing,then increasing and then decreasing.In ovaries,the expression of _VTGin each temperature treatment group was increased first,and the expression of VTG in the 14 day,28℃and32℃group was significantly different from that in 24℃group（P<0.05）.In the liver of female fish,the expression of VTG in different temperature groups showed a downward trend.On the 28th day,the expression of VTG in different temperature groups was significantly lower than that on the 0th day（P<0.05）.The expression of AMH in the ovary of female fish showed a downward trend,On day 14,the expression of AMH in different temperature treatment groups was significantly lower than that on day 0（P<0.05）.FSHR in the brain of male fish showed a trend of first decreasing,then increasing,and then decreasing,On the 28th day,the expression of FSHR in the brain of different temperature treatment groups was significantly different from that in the brain of different temperature treatment groups on the 0th day（P<0.05）,LHR showed a trend of first decreasing,then increasing and then decreasing.FSHR in testis showed a downward trend,after 7 days,the expression of FSHR decreased significantly（P<0.05）,LHR showed a downward trend and then an upward trend,after 14 days,the expression of LHR in testis of each temperature treatment group was significantly lower than that of 0 day（P<0.05）.The expression of AMH in testis showed a downward trend.On the 21st day,the expression of AMH in different temperature treatment groups was significantly lower than that on the 0 day（P<0.05）.