Effects Of Leptin On HPG Axis And Reproductive Function In Male Rats Under Simulated Altitude Of 5500m Hypoxia Environment

OBJECTIVE:Studies have shown that high altitude hypoxia affects male reproductive function and body metabolism.In addition,male reproductive function is closely related to metabolism,but the relationship between hypoxia,male reproductive function and metabolism remains to be elucidated.Therefore,this study will investigate the change of leptin in male SD rats at simulated altitude of 5500 m hypoxia environment and its probable effects on hypothalamic-pituitary-gonadal axis(HPG axis).METHODS:We used hypobaric oxygen chamber to establish a hypoxic animal model.11 weeks-old male Sprague-Dawley rats were randomly divided into 1,7,14,and 28 days of hypoxic group and their corresponding normoxia control groups,14 days of hypoxia+ 14 days of normoxia recovery group.The rats' food intake and body weight changes were recorded daily.Blood was taken from the left ventricle after being narcotized.The total number of sperm,deformity rate and motility rate were counted or calculated.We calculated the testis and epididymis index right after the anesthetization.The morphology of epididymis and testis were observed by HE staining.The number of apoptotic spermatogenic cells in testis was measured by TUNEL staining.The expression of HIF-1?,GnRH and LepR in hypothalamus and the expression of HIF-l?,LepR,and Leptin in testicular were detected by immunohistochemistry.Flow cytology was performed to determine the percentage of germ cells of different ploidy.Immunofluorescence was used to detect the relative expression of sperm LepR.The concentrations of Testosterone(T),Free Testosterone(FT),Hypoxia-inducible factor(HIF-1?),Estradiol(E2),Cortisol,Gastrin,Ghrelin,Leptin,LH and FSH in serum were determined by ELISA.The expression of Leptin and LepR protein in testis tissue was measured by Western Blot.RESULTS:Compared with the corresponding control groups,the body weight and food intake of 1,7,14,and 28 days hypoxia groups decreased(P<0.01),and the body weight of the recovery hypoxia group decreased after entering the hypobaric oxygen chamber(P<0.05),but recoverd on the 12th day after the being released.Testis and epididymis HE staining showed that hypoxia resulted in a reduction of testicular sperm density and spermatogenesis,the hypothalamus HE staining showed hypoixa-induced reduction of neuroendocrine cells.Testis immunohistochemistry showed that the expression of HIF-l?,Leptin and LepR increased under hypoxic conditions,and the hypothalamus immunohistochemistry showed that the expression of HIF-1? increased while the expression of GnRH and LepR decreased upon exposing to hypoxic environment.TUNEL staining showed that the spermatogenic cell apoptosis index(AI)of hypoxia groups increased significantly(P<0.05).The results of flow cytometry showed that the percentage of haploid in 1 day,7 days and 28 day hypoxia groups increased(P<0.05),and the proportion of diploid decreased in 28 days hypoxia group(P<0.05).Immunofluorescence showed that the expression of sperm LepR increased in the 1,14,and 28 day hypoxia groups(P<0.01).The ELISA results showed that in 1 day and 28 day hypoxia groups,the T concentration decreased(P<0.05);in 1 day,14 day hypoxia groups,FT concentration decreased(P<0.05);the E2 concentration in the hypoxic group of the recovery hypoxia group decreased(P<0.01);the concentration of Leptin decreased in the 7 day hypoxic group;in 14 day hypoxia group,the concentration of acylated-ghrelin increased(P<0.05),while the acidylated-ghrelin in the recovery hypoxia group was significantly decreased(P<0.05).Western Blot showed that the expression of Leptin and LepR in testis tissue increased in 1 day,7 day and 14 days hypoxia group,and the expression of LepR increased in recovery hypoxia group.CONCLUSION:Simulated high altitude hypoxia environment decreased reproductive function,depressed HPG axis activity and altered the serum concentration of hormones related to food intake,namely,acylated-Ghrelin and Leptin,in adult male rats.Additionally,hypoxia induced the expression of Leptin and LepR in adult males testis and sperm,suggesting Leptin-LepR signaling may mediate hypoxia-induced damage in male rats' reproductive systerm.