The Mechanism Of Spermatogenesis Reduction Caused By Hypoxia-induced Apoptosis Of Spermatocytes

Background:Hypobaric hypoxia?HH?has a significant negative impact on male reproductive function.Spermatogenesis reduction is of note the key link of male reproduction dysfunction at high altitude.The formation of spermatogenesis reduction is a complex pathophysiological process.In vivo studies have shown that spermatocyte is the most sensitive spermatogenic cell to HH.But the mechanism of hypoxia-induced spermatocyte apoptosis in vivo is not clear.There are two important apoptotic pathways:death receptor apoptotic pathway and mitochondrial apoptotic pathway.These two apoptotic pathways have been extensively studied in tumors.However,whether these two apoptotic pathways participate in spermatocyte apoptosis induced by hypoxia is unclear.HIF-1a is closely related to varicocele,testicular torsion and oligozoospermia in the disease model of spermatogenesis reduction caused by ischemic hypoxia in vivo,but whether HIF-1a is involved in spermatocyte apoptosis induced by high altitude hypoxia and the regulation mechanism between HIF-1a and the two apoptotic pathways is still unknown.Autophagy begins with the formation of Beclin-1-linked autophagy initiation complex,which connects with each other to form a bilayer membrane structure.The bilayer membrane further lengthens to form closed-loop autophagosomes,which encapsulate damaged organelles and damaged proteins in cells and fuse with lysosomes,ultimately allowing cells to survive.Autophagy and apoptosis are two closely related pathophysiological processes.Autophagy precedes apoptosis.When external stimuli appear,autophagy appears and inhibits apoptosis.When external stimuli exceed a certain limit,apoptosis in turn inhibits autophagy and reinforces apoptosis by positive feedback.To elucidate the molecular mechanism of the interaction between autophagy and apoptosis of spermatocyte under hypoxia is of great practical significance for studying the reduction of spermatogenesis induced by hypoxia.The purpose of this study was to investigate the internal mechanism of spermatocyte apoptosis induced by hypoxia,to clarify the effect of hypoxia on spermatocyte autophagy,and to explore the molecular mechanism of interaction between autophagy and apoptosis in spermatocyte.To provide new strategies and targets for the prevention and treatment of spermatogenesis reduction caused by hypoxia.Material and Methods:1.Establish the apoptotic model of mouse pachytene-derived spermatocyte line GC-2induced by HH?1%oxygen concentration content?and make it possible apoptosis was detected by TUNEL and flow cytometry.2.To study the role of Death receptor?DR?apoptotic pathway in hypoxia-induced mouse spermatocytes apoptosis,GC-2 were exposed to 1%O2,Lactic dehydrogenase?LDH?and caspase-8 activity were detected by enzyme labeling method.DR apoptotic pathway candidates induced by hypoxia were detected by RT-qPCR and Western blot.GC-2 were exposed to 1%O2,Cell proliferation activitytreated by Z-IETD-FMK?Caspase-8 specific inhibitor?was detected by enzyme labeling method.3.To study the role of mitochondrial apoptotic pathway in hypoxia-induced mouse spermatocytes apoptosis,Caspase-3/9 activity induced by hypoxia was detected by enzyme labeling method;mitochondrial transmembrane potential induced by hypoxia was detected by JC-1 probe method;Mitochondrial apoptotic pathway candidates induced by hypoxia were detected by RT-qPCR and Western blot.4.To further explore the role of HIF-1?in DR and mitochondrial apoptosis pathway,We knocked down the expression of HIF-1?with siRNA in GC-2 cell.Apoptosis induced by hypoxia was detected by flow cytometry,caspase-3/8/9 activity induced by hypoxia was detected by enzyme labeling,Western blot was used to detect DR and mitochondrial apoptotic pathway-related proteins.5.Establish anhypoxia-inhibited autophagy model of GC-2 cell.Autophagosome and autolysosome labeled by mCherry-GFP-LC3B adenovirus were used to evaluate autophagy flow.And autophagy flow was detected by laser confocal microscopy and flow cytometry.Autophagy related proteins were detected by Western blot.6.We knocked Caspase-8 with siRNA in GC-2 cell,caspase-3/8 activity induced by hypoxiawas detected by enzyme labeling method.Autophagy flow labelled by mCherry-GFP-LC3B adenovirus was observed by laser confocal microscope.Cyto-ID and acridine orange characterized by living fluorescence dyes were used to quantitative analysis autolysosome.Autophagy related proteins induced by hypoxia were detected by Western blot.7.Over-expression of exogenous Beclin-1 transfected by lentivirus in GC-2cells.Autolysosome induced by hypoxia was detected by Cyto-ID fluorescence dye.Apoptosis induced by hypoxia was detected by flow cytometry.And TUNEL staining was used to observe apoptosis induced by hypoxia.Mitochondrial transmembrane potential induced by hypoxia was detected by JC-1 method.Western blot was used to detect DR and mitochondrial apoptotic pathway-related proteins.8.Adult male BALB/c mice were randomly divided into four groups:control,30-day?H30d?hypoxic,lentivirus?LV?control hypoxic,and LV-Beclin1 hypoxic.Hypoxic mice were raised in a hypobaric chamber,where the atmospheric pressure was reduced to simulate an altitude of 5000 m.At day 1 and day 15,LV-Beclin-1 mice were injected with 3?l of the lentiviral preparations?2.5�10⁷ TU?into the testicular tissue.HE staining and transmission electron microscope were used to observe pathology structure of seminiferous tubules.TUNEL staining was used to detect apoptosis of spermatogenic cell.Results:1.Compared with normoxia group,Exposure to 1%O₂caused an increase inchromatin condensation and nuclear fragmentationoccurred in the mouse GC-2 cells after 24hof hypoxia treatment.Hypoxia induced apoptosis of mouse GC-2 cells in a time-dependent manner.2.Compared with normoxia group,hypoxia induced an increase in the mRNA and the protein expression of DR₅,TRAIL,and Fas,and a decrease in the mRNA and the protein expression of c-FLIP and D_c R₂,which were determined by RT-qPCR and Western blot analysis.Hypoxia also decreased the mRNA expression of D_c R_1.Moreover,compared with the normoxia group,the hypoxia group had activities of caspase-8 and LDH?Lactic dehydrogenase?that significantly increased after 48 h of 1%oxygen treatment.Z-IETD-FMK,a specific caspase-8 inhibitor,significantly increased thecell proliferation rate in mouse GC-2 cells compared with thevehicle-treated cells that were exposed to the same hypoxic conditions.3.Compared with normoxia group,The results of RT-qPCR and Western blot analysis showed that hypoxia induced an increase in the mRNA and the protein expression of p53and Bax but a decrease in the mRNA and the protein expression of Bcl-₂.And the BNIP-₃protein level was significantly inhibited by hypoxia.In addition,the activities of caspase-9and caspase-3 significantly increased in GC-2 cellsthat were treated with hypoxia.Z-LEHD-FMK alone significantly increasedthecell proliferation rate in mouse GC-2 cells relative to thevehicle treated cells that were exposed to 1%O₂ concentration for48 h.The cell proliferation rate was further increased in the cells that were co-incubated with Z-IETD-FMK and Z-LEHD-FMKwith hypoxia treatment.Hypoxia treatment also caused the dissipation of the mitochondrial membrane potential.4.The apoptosisof GC-2 cells treated by HIF-1?silencing was alleviated under hypoxia culture conditions by staining with annexin V-FITC and PI.Meanwhile,depletion of HIF-1?by siRNA resulted in an evident increase in the expressions of c-FLIP,D_cR₂,Bcl-₂ andasignificant reduction inthe expressions of DR₅,TRAIL,Bax,and p53 and activitiesof caspase-3,caspase-8,and caspase-9.5.Compared with the normoxic control group,Exposure to 1%O₂ caused an increase in the number of immature autophagosomes and decrease in the number of autolysosome occurred in the mouse GC-2 cells after 24h of hypoxia treatment.hypoxia inhibited autophagy in GC-2 cells in a time-dependent manner.When GC-2 cells were exposed to1%O2 for 48 hor60 h,the autophagic flux markers Beclin-1,LC3-II,VPS₃₄,ATG₅,BNIP-₃,LAMP₂ and VMA were downregulated.However,p62?a substrate for autophagic degradation?and autophagic flux inhibitors such as mTOR were increased.6.Autophagic flux in GC-2 cells treated by caspase-8 silencingwas improvedunder hypoxia treatment culture conditions by staining with mCherry-GFP-LC3B.Flow cytometryfurther indicated that though caspase-8 siRNA�negative cellsdid not have further decreased autolysosome levels in GC-2 cells under 1%O₂,depletion of caspase-8markedly increased the autolysosome level under 1%O₂ for 48 hor60 h.In addition,depletion ofcaspase-8 by siRNA resulted in marked reduction of the levels of caspase-8,cleaved caspase-8 and caspase-3,and the activity of caspase-3/8 in GC-2 cells under 1%O₂ for 48 h or 60 h.Meanwhile,depletion of caspase-8 by siRNA resulted in an evident increase in the expression of Beclin-1,LC3-II,VPS₃₄,ATG₅,BNIP-₃,LAMP₂,and VMA and a significant reduction in the expression of mTOR and p62 as determined by Western blot.7.Activation of autophagy by Beclin-1 overexpression significantly decreased cell death at 48 h and 60 h asdetected by TUNEL staining and annexin V-FITC in GC-2 cells under 1%O₂.The JC-1monomers were assembled into aggregates?red fluorescence?in the cells that were subjected to hypoxia treatment for 48 h or 60 h when autophagy was activated by Beclin-1 overexpression.Meanwhile,activation of autophagy by Beclin-1overexpression resulted in an evident increase in the expression of c-FLIP,D_cR₂,and Bcl-₂and a significant reduction in the expression of DR₅,TRAIL,Bax,and p53.8.Mice were exposed to 5000m simulated high altitude in hypobaric chamber to establish spermatogenesis reduction model.Compared with the Lentivirus?LV?-control,delivery of LV-Beclin 1 resulted in increased expression of Beclin-1 in testis and an elevated level of autophagy.Cyto-ID analysis with testis tissues also confirmed that the autophagy percentage was increased in mice that received the LV-Beclin 1 injections compared with LV-control.Compared with the Lentivirus?LV?-control,LV-Beclin1�injected mice showed a considerable reduction in the pathological damage degree of seminiferous tubule and apoptosis of spermatogenic cells.Conclusion:1.Hypobaric hypoxia induces GC-2 cell apoptosis through DR and mitochondrial apoptosis pathway.2.The death receptor pathway and mitochondrial pathway,which arelikely mediated by HIF-1?,contribute tohypoxia-inducedGC-2 cell apoptosis.3.Hypoxia inhibits autophagy,which further enhances hypoxia-induced apoptosis of mouse spermatocytes by promoting caspase-8 activation in a time-dependent manner4.Overexpression of Beclin-1 can improve the apoptosis of GC-2 cells under hypoxia and reduce the degree of testicular seminiferous tubule lesion and spermatocyte apoptosis.5.As such,targeting Beclin-1 and caspase-8 in spermatocytes may yield therapeutic strategy for treating spermatogenesis reduction.