Study On The Mechanism Of HIF-1α In The Male Infertility

BackgroundAlong with the social development, the incidence of infertility is increasing.According to the data from the China Population Association, the number of thepeople suffering from the infertility and sterility exceeds40million. It accounts for12.5%of the population at fertile age. There is one infertile couple in eight couples inthe average. The male infertility accounts for50%of the cause of infertility and it isincreasing every year. The male infertility is complicated. Besides the productivesystem diseases, Bisphenol A (bpa), heavy metals, smoking and other environmentaltoxins and diseases such as diabetes are likely to damage testicular sperm production,reduce sperm count and quality, leading to male infertility, sexual dysfunction andabortion, fetal abnormalities. It does serious damages to the harmony of family andthe quality and quantity of the population.Leydig cells were known as testis interstitial cells. Its main function is theandrogen secretion and synthesis, including pregnenolone, testosterone, etc. and thematuration of the male genital organs. Dysfunction,of leydig cell will lead toabnormal function of the sex gland and result in male infertility. Oxidative stress (OS)is the key step of male reproductive damage by a variety of physical chemical andbiological factors. So exploring different degrees of damage of reproductive cells bycause, finding effective methods to control the reproductive cell oxidative stress is oneof the most promising strategy in the treatment of male infertility. HIF-1alpha is animportant factor adjusting adaption to the low oxygen environment. Furtherinvestigation of the regulatory mechanism of oxidative stress in leydig cells willprovide a certain theoretical basis for the treatment of male infertility.ObjectiveTo investigate the role HIF-1alpha t in male infertility. To explore the treatmentof the male infertility based on the mechanism involed in it.Methods（1） The research included five parts:Ⅰ:Mouse leydig (TM3) cells were treated with50g/ml AGES; The control cells were only with complete medium.Aftercultured for48hours.Ⅱ:TM3cells were transfected with shRNA of HIF-1αand negative shRNA for48H. Ⅲ: After treated with DMOG or0.1‰DMSO for24H,TM3cells were given50g/ml AGES for48H.Ⅳ: TM3cells wrer treated withDMOG or0.1‰DMSO for48H.Ⅴ:After transfected with shRNA of HIF-1α gene ornegative shRNA for24H, TM3cells were treated with50g/ml AGES culture for24H.The cells in five parts were all harvested for Western blotting, detection ofapoptosis and ROS.（2） The expression of HIF1α， Caspase3，HO-1，sTAR and CYP17A1ofcells in each group were detected by Western blotting.（3） The apoptosis of cells in each group were detected by Annexin V-FITCKit using flow cytometric analysis.（4） To determine the ROS of cells in each group by DCFH-DA fluorescentprobe.Results1The ROS levels:The ROS of AGES treated group(11.01±0.39) was significantly higher than the controlgroup (9.73±0.25), P=0.0189. The ROS of HIF-1αsiRNA+AGES group (9.38±1.25) wassignificantly higher than that of the control group (6.931±0.532),P=0.0354. The ROS ofHIF-1αsiRNA group (15.03±0.3861) was significantly higher than the controlgroup (9.733±0.2471),P=0.0003, showed that knockdown of HIF can increase thecontent of ROS in TM3cells.The ROS of DMOG+AGES group (5.513±0.402) was significantly lowerthan that of the control group (10.014±0.694),P=0.0006. DMOG treatment (5.445±1.993) decreased the ROS of TM3cells compared to the control group (9.733±0.2471), while the difference was not significant,P=0.0996. The rate of earlyapoptosis in AGES group (7.440±0.54)%was significantly higher than that ofcontrol group (1.370±0.0600)%, P=0.0079. The rate of late apoptosis in AGES group(18.79±1.09)%was significantly higher than that of the control group (10.42±0.48)%,P=0.0197. The total apoptosis rate of AGES group (28.68+0.82)%wassignificantly higher than that of control group (11.84±0.465)%, P=0.0031. The rateof early apoptosis in DMOG group (0.745±0.035)%was was significantly lower than that of control(1.370±0.06)%,P<0.001. There was no significant difference of thelate and total apoptosis rate between DMOG group and the control group.2Apoptosis rate of each group:The rate of early apoptosis in DMOG+AGEs group(1.547±0.029)%was significantlyincreased than in the control group (1.267±0.081)%,P=0.0314. The rate of late apoptosis(11.36±0.44)%was decreased compared to the control group (17.37±0.59)%,P=0.0012. T hetotal apoptosis rate (12.91±0.415)%was significantly decreased compared to the controlgroup (18.64+0.669)%,P=0.0019. There was no significant difference of the early apoptosisrate between HIF-1αsiRNA group (1.25±0.06)%and the control group (0.745±0.035)%,P=0.2929.While the late apoptosis rate HIF-1αsiRNA group (3.75±0.15)%wassignificantly lower than that of the control group (10.42±0.48)%,P=0.0056. The totalapoptosis rate (4.595±0.495)%was significantly lower than that of the control group (11.84±0.465)%,P=0.0087.The early apoptosis rate of HIFsiRNA+AGES group (5.84±0.5)%was wassignificantly lower than that of the control group (8.155±0.175)%,P=0.0486. Thelate apoptosis rate (11.02±0.30)%was significantly lower than that of the controlgroup (18.55±1.335)%, P=0.0316. The total apoptosis rate (16.86±0.19)%wassignificantly lower than that of thecontrol group (26.7±1.16)%, P=0.014.3The expression of cell proteinThe expression of HIF-1α protein in AGES group(0.1999±0.02627) wassignificantly lower than the control group (0.3487±0.01257),P=0.0069,which in theDMOG group (0.4367±0.01449) was significantly higher than the control group(0.3487±0.01257), P=0.0101. Expression of HIF-1α protein in HIF-1siRNA groupwas successfully45%reduced than negative control with a transfection efficiencyof70%. There was no significant difference of expression of HIF-1α proteinbetweenthe DMOG+AGES group(0.2548±0.01085) and the control group (0.1999±0.02627),P=0.1253, and between HIF-1α siRNA+AGES group (0.2011±0.01438)and the control group (0.1999±0.02627),P=0.9697.There was no significant difference of the expression of Caspase3protein between the AGESgroup (0.3410±0.0131)and the control group (0.4297±0.0293),P=0.0503, and between the theDMOG group (0.4758±0.0269) and the control group (0.4297±0.0293), P=0.3109. The relativeexpression of Caspase3in the HIF-1α siRNA group (0.3003±0.02016was significantly lowerthan the control group (0.6539±0.04475), P=0.0020. There was no significant difference of the expression of Caspase3protein between the DMOG+AGES group (0.4772±0.0492) and the controlgroup(0.5525±0.03144), P=0.2665. The relative expression of Caspase3protein in HIF-1αsiRNA+AGES group (0.2855±0.022) was lower than that of the control group (0.341±0.13), P=0.0948.The expression HO-1protein in the AGES group (0.2265±0.01211) was significantlydecreased than the control group (0.3852±0.02199),P=0.0032. There was no significantdifferencebetween the DMOG group (0.4223±0.008370) and the control group (0.3852±0.02199),P=0.1907, and between the HIF-1α siRNA group (0.3418±0.02062) and the control group (0.3852±0.02199),P=0.2231. The relative expression of HO-1gene protein in DMOG+AGES group(0.4638±0.05544) was significantly higher compared to the control group (0.2265±0.01211),P=0.0139. HO-1protein expression in HIF-1αsiRNA+AGES group (0.3086±0.0154) wassignificantly higher compared tothe control group(0.2265±0.01211), P=0.0138.The expression of CYP17A1protein in the AGES group (0.7441±0.03956) showed nosignificant difference compared to the control group (0.7462±0.02578),P=0.9664). There was alsono significant difference of CYP17A1expression between the DMOG group (0.8273±0.02107) andthe control group (0.7462±0.02578), P=0.0715. The CYP17A1expression expression in HIF-1αsiRNA group (0.5158±0.01579) was significantly lower than the control group(0.7462±0.02578), P=0.0016. The CYP17A1expression of DMOG+AGES group (0.7164±0.02530)showed no significant difference compared to the control group (0.7441±0.03956),P=0.5864. While that of HIF-1αsiRNA+AGES group (0.3547±0.02325) was significantlydecreased than the control group (0.7441±0.03956), P=0.0011.The expression of StAR protein in the AGES group (0.6734±0.04594) showed no significantdifference compared to the control group (0.7984±0.03910),P=0.1069. There was no significantdifference between the DMOG group (0.7951±0.04411) and control group (0.7984±0.03910),P=0.9578,and between the HIF-1αsiRNA group (0.6420±0.05320) and the control group(0.7984±0.03910),P=0.0769. The StAR protein expression of DMOG+AGES group (0.6291±0.05480)showed no significant difference compared to the control group (0.6734±0.04594),P=0.3437. TheStAR expression of HIFsiRNA+AGES group(0.4163±0.02377) was significantly lower than thecontrol group (0.6734±0.04594), P=0.010.Conclusion(1) HIF-1α reduces the production of ROS and plays a regulatory role in maleinfertility caused by oxidative stress.(2) Oxidative stress injury caused by AGES does not affect the CYP17A1andStAR gene expression of TM3cells, but can mediate the expression of CYP17A1and StAR by HIF-1α.(3) AGES decreased the expression of HO-1and mediated the regulation ofHO-1by HIF-1α(4) HIF-1α promoted the expression of Caspase3and TM3apoptosis.(5)The role of HIF-1α in oxidative stress injury to the leydig cells providesstrategies for the diagnosis and treatment of male infertility.