The Role Of Calcium Ion In The Apoptosis Of Male Germ Cells Induced By Microcystin

Objective Microcystins can cause damage to the reproductive system of male mammals and cause testicular cell apoptosis in mice.The aim of this study is to investigate the apoptosis and molecular mechanism of MC-LR induced male reproductive cells in mice through calcium ion,and to provide experimental basis for the prevention and treatment of MC-LR exposure in water environment.Research methods1.MC-LR exposure induces testis germ cell apoptosis in miceThirty healthy 8-week-old SPF male ICR mice were selected.After one week of adaptive feeding,the mice were randomly divided into the control group,the 10 ?g/kg MC-LR exposure group and the 25 ?g/kg MC-LR exposure group,with 10 mice in each group.Exposure to tracheal drip for acute exposure of 36 h.After the exposure,the mice were killed,the testis of mice were collected,weighed,partially fixed in 10%formaldehyde,and the rest was stored in liquid nitrogen for-80?.HE staining was used to observe the histopathological changes in the testis of mice,and the apoptosis of germ cells in the testicular tissue was detected by TUNEL,and the expression of Caspse-3,GRP78,IRE1,CHOP,PERK,Caspase-12 in the testicular tissue of mice was detected by immunohistochemistry.2.Role of calcium ion in MC-LR-induced sustentacular cell apoptosis in miceTo investigate the effect of calcium ion on the apoptosis of male reproductive cells induced by MC-LR,TM4 cells were pretreated with Bapta,an intracellular calcium chelator for 30 min,then exposed to MC-LR,and the concentration of calcium ion,endoplasmic reticulum stress response,mitochondrial membrane potential and apoptosiswere detected.Result1.MC-LR exposure induces germ cell apoptosis in mice testis(1)MC-LR exposure induced a significant increase in testicular tissue injury and apoptosis in mice: there was a slight atrophy of testicle in group 10 ?g/kg MC-LR,degeneration and vacuolation between spermatogenic cells,and the disorder and decrease of spermatogenic cells in the seminiferous tubules of 25 ?g/kg MC-LR group.Compared with the control group,the incidence of positive tubules in the testicular tissue and the average number of apoptotic cells in the MC-LR treatment group were higher(P<0.01).(2)The expression of apoptosis related protein in mice testis tissue: compared with the control group,the expression of Caspase-3 protein in the testis tissues of the MC-LR exposure group was increased(P<0.05),while the expression level of Caspase-9 protein was not significantly changed.(3)The expression of stress related protein in the endoplasmic reticulum of mice testis: compared with the control group,the Caspase-12 and CHOP protein in the testis of MC-LR mice in each group and the expression level of GRP78 protein in testis of 25?g/kg MC-LR group were all increased(P<0.05).2.Role of calcium ion in MC-LR-induced sustentacular cell apoptosis in mice(1)MC-LR exposure induced apoptosis of TM4 cells: compared with the control group,the MC-LR exposure group promoted the apoptosis of TM4 cells(P<0.01),and Bapta antagonized the apoptosis promoting effect of MC-LR(P<0.05).(2)MC-LR exposure induced the decrease of mitochondrial membrane potential in TM4 cells: compared with the control group,the membrane potential of MC-LR exposure group decreased(P<0.05).Compared with 30 ?g/mL MC-LR,Bapta inhibited the decrease of membrane potential to a certain extent(P<0.05).(3)MC-LR exposure up-regulated the expression of apoptosis related protein inTM4 cells: compared with the control group,the expression level of Caspase-3 protein in the testis tissues of mice exposed to MC-LR was increased(P<0.05).After treatment with MC-LR and Bapta,the expression of Caspase-3 protein in the MC-LR group of TM4 cells was significantly lower than that in the 30 ?g/mL MC-LR group(P <0.05).(4)MC-LR exposure up-regulated the expression of ERs-related protein in TM4cells: compared with the control group,the expression levels of Caspase12,Chop and Grp78 in the testis tissues of the mice exposed to MC-LR were all increased(P<0.05).After treatment with MC-LR and Bapta,the protein expression levels of Caspase-12,Chop and GRP78 in TM4 cells were significantly lower than those in the 30 ?g/mL MC-LR group(P <0.05).(5)MC-LR exposure up-regulated the expression of calcium ion regulation related protein in TM4 cells: compared with the control group,the expression level of p-p38,p-Erk1/2,Erk1/2 and p-CaMK II protein in the testis tissues of MC-LR exposure groups were all increased(P<0.05).After treatment with MC-LR and Bapta,the expression levels of p-p38,p-Erk1/2 and p-CaMK II in TM4 cells were significantly lower than those in the 30 ?g/mL MC-LR group(P <0.05).(6)MC-LR exposure up-regulated the expression of mitochondrial related proteins in TM4 cells: compared with the control group,the expression level of Bcl-2,Bax and Cyt C in the testis tissues of MC-LR exposed mice were all increased(P<0.05).After pretreatment with Bapta,the protein expression levels of Bcl-2,Bax and Cyt C in TM4 cells were significantly lower than those in the 30 ?g/mL MC-LR group(P<0.05).Conclusion(1)MC-LR induces apoptosis of germ cells in mice testis through endoplasmic reticulum stress.(2)Microcystin MC-LR exposure plays an important role in inducing apoptosis in mice testis sustentacular cells through endoplasmic reticulum stress and mitochondrial pathway.(3)Calcium ion plays an important role in MC-LR exposure induced apoptosis in mice testis sustentacular cells.