Establishment Of A Mouse Model Of Male Infertility By Radiation&Impairment Research On Leydig Cells

Introduction The technology of nuclear power and radiation has been more and more widely used in military and in the production of all aspects of life.Under current condition,the soldiers and working staff exposed to radiation are facing inevitable damage.Genital gland is sensitive to radiatoin.Male infertility caused by radiation has been a considerably tough social problem.The study of the mechanism and the treatment of the male infertility requires economic anmimal model that has abudant genetic imformation.Rodent model is more economic and it has many inbred strains and rich imformation in genetics.It is more suitable for lab.However,there is no ideal mouse model for male infertility induced by radiation.The mechanism underlying consistant or permanent azoospermia casued by radiation is not clear.Through the establishment of the radiation-induced male infertility in mouse model and the evaluation of the model,we promote the hypothesis: while the radiation impair the germ cells,it impairs the Leydig cells which leads to decline of the serum testosterone level.The change of factors in spermatogenic microvironment causes the block in spermatogesis,which leads to the consistant azoospermia and infertility.At last,we analyzed the exposed Leydig cell strain(TM3 cell)at the level of transcription,protein and cytobiological behaviors.Section one: Establishment of a mouse model of male fertility by 60Co-γ radiationMethods:65 C57BL/6 male mice were randomly divided into seven groups: Group A(control,n=5),Group B(n=10),Group C(n=10),Group D(n=10),Group E(n=10),Group F(n=10)and Group G(n=10).Different doses of irradiation(12 Gy,6+6 Gy,10 Gy,6+4 Gy,8 Gy,4+4 Gy)were given to Group B to Group G respectively.According to the general condition and death of the mice from all groups,the survival curves were drawn,for which the longest oberservation time was 24 weeks after the treatment.The body weight and fertility rate of the mice at day 30,day 50,day 70 after the trial were recorded and analyzed.Results: The survival time of Group A,E,F,G was longer,followed by that of Group D.The survival time of Group B and C was the shortest.At day 30,day 50 and day 70 after the exposure,the fertility rate of Group D and Group E was 0%.The feritility test showed positive of Group F and G.At day 30,day 50 and day 70 after the exposure,the body weight of all the exposed groups decreased significantly.Conclusion: The double exposure of 6+4 Gy with 60Co-γ irradiation,the mouse model of male infertility was successfully established.The survival time of Goup 6+4 Gy was no difference from that of the control.Between the groups exposed to a total dose of 10 Gy,the mortality of double-irradiation group was lower than single-irradiation group.12 Gy of irradiation could cause death within 5 weeks after exposure.The exposure of a dose no more than 8 Gy was not sufficient to establish a mouse model of male infertility.The radiation afftected the body weight of the mice.Section two: Study on the impairment of the infertile mouse model established with 60Co-γ radiationMethods: 216 C57BL/6 male mice were randomly divided into four groups,Group A to Group D,54 mice for each group.Different irradiation(6+4 Gy,4+4 Gy,6 Gy,)were given to Group B to Group D respectively.At day 30,day 50,day 70 of the trial,the testis index was calculated;the testis paraffin sections stained with HE and the Johnsen scores for testes were analyzed to evaluate the effect of irradiation on spermatogenesis;the PLZF positive spermatogonial stem cells(by immunohistochemistry)were counted;the serum inhibin B,testosterone,FSH was tested(by ELISA);the expression level of 3β-HSD,the marker of Leydig cells were tested(by Western Blot).Results: The testis index of mice after exposure was all lower than that of the control.The Johnsen scores of the exposed groups were lower than that of the control.At day 30,50 and 70 after exposure,the PLZF positive cell counts of the Group B was obviously decreased than that that of the control,while other groups were not in the same manner.At day 30,50 and 70 after the exposure,the serum inhibin B level of Group B was significantly decreased compared to that of the control;the level of the other exposed groups(Group C and Group D)was of no difference compared to the control.At day 30 and day 50,the serum FSH level of Group C and Group D was higher than that of the control.At day 70 after the exposure,the serum FSH level of Group C was increased compared to that of the control.At day 30,day 50 and day 70 after the exposure,the serum testosterone level of the the exposed groups was lower than that of the control,in a dose-response manner.At day 30 after the exposure,the expression of 3β-HSD in exposed groups were all decreased in a dose-response manner.At day 50 and day 70 after the exposure,no difference was shown.Conclusion:The exposure caused the deline of the testis index and impairment of spermatogenesis in a dose-response manner in mouse.The spermatogonial stem cells still existed and continued to proliferate after the exposure of 6+4 Gy.However,there was block in spermatogenesis which caused azoospermia and male infertility.Therefore,the mouse model of male infertility could be successfully established with this does of γ-irradiation.The testosterone level of the exposed groups was all decreased in a dose-response manner,which implied that the testosterone level might be one of the microenvironmental factors which caused spermatogenic block.The serum inhibin B level could be the marker of the reproductive impairment in the model group.The protein level of 3β-HSD in the testis tissue could be the marker of the early(day 30 after the exposure)damage of the exposure.Section Three: Study on the impairment of the Effect of radiation on TM3 cellsMethods:hCG stimulation test: TM3 cells were plated in 12-well plates.Different concentrations of hCG were added to the cells,while no hCG was added to the control group.The concentration of the testosterone in the culture supermate were tested after the cells were incubated for 24 hours,48 hours and 72 hours.Irradiation test:TM3 cells were divided into four groups: a control group and three groups exposed to 3 Gy,6 Gy and 9 Gy of 60 Co γ radiation,respectively.At 24 h,48h,72 h,the percentage of apoptotic cells in all groups was measured by flowcytometry;the oxidative damage was observed by ELISA;the secretion of the testosterone by ELISA;m RNA expression of four key factors in the synthesis of testosterone,including St AR,P450 Scc,P450c17 and 3β-HSD,was measured by q PCR.At 24,48,72,96,120,144 h,cell proliferation was measured by MTS.Results: In hCG stimulation test,at 48 h after the addition of hCG,the testosterone level in the culture medium increased significantly.At 72 h,the testosterone concentration of the group with 1 IU/ml hCG increased significantly compared to that of the control.Cell apoptosis test showed that the percentage of the early apoptotic cells increased in the 3 Gy Group and the 6 Gy Group at 24 h after exposure;it also increased in the 6 Gy Group and the 9 Gy Group at 48;it increased in all exposed groups at 72 h after xposure.The percentage of the late apoptotic cells in the 9 Gy Group increased at 24 h after exposure.It increased in the 6 Gy Group increased at 48 h after exposure.It increased in the 9 Gy Group at 24 h,48h,72 h after exposure.In the cell proliferation test,the OD490 value of Goup 6 Gy and Group 9 Gy decreased after 72h;OD490 value of all exposed groups decreased after 144 h.In oxidative damage test,the 3 Gy Group had no difference from the control.At 24,48,72 h,no significant difference of 8-OHd G level was observed between 3 Gy group and the control group(P>0.008);8-OHd G level of 9 Gy group was higher than that of the control group(P<0.008).At 72 h,m RNA expression of St AR,P450 scc and P450 scc in all exposed groups was down-regulated,as compared to the control(P<0.008).However,m RNA expression of P450c17 in 3 Gy group and in control group had no difference.P450c17 m RNA level in 6 Gy group was higher than the control.It was higher than the control in 6 Gy group and was lower than the control in 9 Gy group.Conclusion: 3 Gy of 60 Co γ radiation induces cell apoptosis and decrease cell proliferation in TM3 cells.≥6 Gy of radiation causes oxidative damage.9 Gy of radiation suppress the m RNA expression of four key factors in the testosterone synthesis.These four factors are St AR,P450 scc,P450c17 and 3β-HSD.Summary and innovation: First of all,we establised a stable mouse model of male infertility by double irradiation to provide a tool for the study of radiation induced male infertility in human.Secondly,throught the analysis of the Johnsen scores and PLZF positive cell counts,we found the reason why the mice remained infertility for a long period of time after irradiation.It was because of the azoospermia due to the block in spermatogensis,not the lack of the PLZF positive spermatogonial stem cells.Thirdly,we found the cause of the block in spermatogensis was not the dysfunction of the spermatogonial stem cells,but the impairment of the spermatogenic microenvironment.At last,we found even exposed to the dose as low as 3 Gy,the proliferation and the biological functione of the Leydig cells,which suggested that one of the reasons of the block in spermatogenesis was the decreased of the synthesis and the secretion of the testosterone due to the impairment of the Leydig cells.There were controversies whether the irradiation could induce block in spermatogenesis in mice;whether the key factor was the spermatogonial stem cells or the spermatogenic microenvironment;whether the Leydig cells could tolerate low to mediate dose of irradiation or not.Our study observed and disscussed these issues from a new point of view,which laid a foundation for the study of the mechanism of the male infertility induced by irradaiton and provided some experimental data.