Studies On Physiological And Toxicological Effects Of Sperm-specific Ion Channels

Ion channels regulate intracellular calcium concentration and membrane potential,and regulate physiological functions of various cells.Sperm is a special type of cell that is produced in the male reproductive tract.Sperm ejaculated in the female reproductive tract undergo physiological changes such as capacitation,hyperactivation and acrosome reaction to have capacity of fertilizing an egg.However,there are differences in ion concentration,osmotic pressure and p H between male reproductive tract and female reproductive tract,so it is necessary for sperm to be involved in ion channels to adapt to the above new environment.Cat Sper and KSper,as sperm-specific ion channels,play a crucial role in the successful fertilization of sperm and an egg.Activation of the calcium channel Cat Sper mediates Ca²⁺influx,thereby increasing intracellular Ca²⁺([Ca²⁺]i)concentration and inducing subsequent capacitation,hyperactivation,and acrosome reaction that facilitate sperm fertilization successfully.It has been reported that Cat Sper is easy to respond to external stimuli,and some endocrine disruptors can disrupt sperm functions by acting on Cat Sper.While perfluorooctane acid?PFOA?,as a major environmental pollutant,has a certain degree of damage effect on male reproduction.However,the effect and mechanism of PFOA on human mature sperm have not been reported.In addition,ways to prevent and alleviate PFOA-induced reproductive toxicity have also not been reported.As is known to all,quercetin?Que?is a natural substance with anti-inflammatory,immune-enhancing,antioxidant and other biological effects,which has been selected to alleviate the toxicity of PFOA.Mouse sperm potassium channel KSper is mediated by the main subunit Slo3,and Slo3 channel responds to intracellular p H alkalinization opening and K⁺outflow,thus controlling membrane potential hyperpolarization.It has been reported that the sperm plasma membrane of Slo3^-/-mice could not be hyperpolarized but depolarized.Moreover,under the necessary capacitation conditions of sperm binding an egg,sperm motility was abnormal and sperm tail was similar to"hairpin structure"without acrosome reaction,and the more serious consequence was male mouse sterility.KSper has been well studied in mature sperm,but whether it plays a role in spermatogenesis has not been reported.Spermatogenesis is divided into three stages:mitosis,meiosis and spermiogenesis.The spermatogenic cells in the whole process are mainly divided into:spermatogonia,primary spermatocyte?leptotene,zygotene,pachytene,diplotene?,and spermatid?round sperm and long sperm?.If KSper plays a role in spermatogenic cells,which period?Sperm sterility caused by abnormal KSper whether be compensated by normal spermatogenic cells cultured in vitro in mice.Objective:?1?Whether Cat Sper,an ion channel,is a target and involved mechanism of PFOA toxicity;?2?To seek for a natural substance to prevent and mitigate PFOA-induced reproductive toxicity;?3?What role is KSper in spermatogenesis.Experimental methods:?1?The penetrating artificial viscous medium and acrosome reaction detection technologies were used to evaluate functions of PFOA-induced human sperm;?2?Calcium signaling technique was used to assess whether the target of PFOA toxicity was Cat Sper;?3?The technique of reactive oxygen species production detection assesses whether PFOA is involved in disrupting human sperm function;?4?Hematoxylin-eosin staining was used to observe the changes of testicular histopathology and epididymal sperm counts in mice,and to evaluate the toxicity of PFOA and the rescue effect of Que in testis;?5?The degree of oxidative stress was evaluated by real-time fluorescence quantitative nucleic acid amplification combining oxidative stress product and antioxidant enzymes;?6?Western blotting was used to determine whether apoptosis was involved in the toxic effects of PFOA;?7?Flow cytometry:Spermatogenic cells at different stages were isolated and the results were verified by GFP fluorescence mice.The changes of Slo3 expression in spermatogenic cells of Slo3^+/+?Slo3^+/-and Slo3^-/-mice,membrane potentials of sperm and intracellular alkalinization of cells with high Slo3 expression were observed;?8?Immunofluorescence was used to observe Slo3 expression and localization in spermatogenic cells;?9?Patch clamp technique:recording the currents of Cat Sper under PFOA alone and PFOA combined with progesterone?P4?irrigations;Under p H6.0 and intracellular alkalinization conditions,the Slo3 currents of pachytenes and round cells were recorded,and the Slo3 currents of p H 8.0 cells were detected.Results:?1?High doses of PFOA?25?g/ml?alone reduced the ability of human sperm to penetrate synthetic mucus,and its continuous incubation of 4 h increased the production of reactive oxygen species?ROS?;?2?PFOA activates human sperm Cat Sper and mediates extracellular Ca²⁺influx in dose-dependent,thereby increasing[Ca²⁺]i concentration.However,human sperm preconditioning PFOA?2.5-25?g/ml?significantly inhibited the extracellular Ca²⁺influx induced by P4;?3?Pretreatment with PFOA?0.25-25?g/ml?significantly inhibited the acrosome reaction and the penetrating ability of the viscous medium of sperm by P4 inducing;Compared with PFOA exposure alone in mice,Que supplementation?4?reduced the histological changes induced by PFOA,including:irregular arrangement of spermatogenic epithelial cells,decreased sperm counts in lumen,loss of spermatogonial cells and shedding of germ cells;Absolute testicular weight and epididymal sperm counts induced by PFOA were improved;?5?Testicular transcription factor NRF2 and its downstream target antioxidant genes:the expressions of superoxide dismutase?SOD?,catalase?CAT?and heme oxygenase-1?HO-1?were up-regulated,while the activities of SOD and CAT of antioxidant enzymes were also increased,and the generation of oxidative product malondialdehyde?MDA?was decreased;?6?The expression of anti-apoptotic protein BCL-2 was enhanced,while the expression of pro-apoptotic proteins p53 and BAX were decreased;?7?In Slo3^+/+mice,Slo3 expression was almost not found in spermatogonia,while high Slo3 expression was found in primary spermatocyte,and the highest Slo3 expression was found in the second meiosis?MII?,while the Slo3 expression decreased in subsequent spermatid;Compared with Slo3expression of spermatogenic cells in Slo3^+/+mice,?8?spermatogenic cells of Slo3^-/-mouse had almost no Slo3 protein expression,while Slo3^+/-spermatogenic cells had decreased Slo3 expression in anaphase?pachytene,MII,and spermatid?.?9?The addition of NH4Cl can cause intracellular alkalinization and membrane potential hyperpolarization of cells in MII period.However,the slight alkaline and the membrane potential depolarization were found by addition of NH4Cl in pachytene;?10?Slo3 was localized to the cytoplasm and membrane of the pachytene;?11?The Slo3currents were not recorded and did not respond to NH4Cl in pachytene.While the tiny Slo3 currents could be recorded in round cells,partly in response to NH4Cl under intracellular solution p H 6.0.Meanwhile,Slo3 currents also were not recorded in pachytene,but micro Slo3 current could be recorded in round cell under intracellular solution p H 8.0.Conclusion:?1?Cat Sper is the target of PFOA,and PFOA may harm human sperm function by inducing oxidative stress and disrupting P4-induced Ca²⁺signal,while Que can improve male reproductive toxicity caused by PFOA exposure by reducing oxidative stress and inhibiting cell apoptosis;?2?KSper plays a role in the second meiosis later stage.