Study Of Reconstruction Of Spermatogenic Function Based On Testicular Tissue Of BALB/c Mice In Vitro

Objective:In this study,an animal model simulating human spermatogenic damage was established by different interventions in BALB/c male mice.The testicular tissue of the model mice was cultured with agarose gel method to explore the ability of induced spermatogenesis during the culture of different pathological testis tissues in vitro,and to provide evidence for the study of human spermatogenic dysfunction in vitro.Methods:1.Using a single intraperitoneal injection of different concentrations(30 mg/kg,40 mg/kg)of busulfan,which was established a model of spermatogenic dysfunction with different degrees of injury.2.Immature testis tissue fragments were cultured by agarose gel method in vitro,which was to provided this method could induce spermatogenesis.3.Sertoli cell only syndrome(SCOS),hypo-spermatogenesis(H-S1,H-S2)were cultured in vitro by agarose gel method.4.Cryopreserved and thawed immature testis tissue was cultured to explore the ability of the testis tissue to mature in vitro after freezing.5.Immunohistochemical technique was used to detect the expression of meiotic marker protein in testis tissue before,during and after culture,including pre-meiosis(STRA8),meiosis(SCP3),and post-meiotic stages(TNP1).6.TUNEL apoptosis was used to detect the apoptosis of testicular tissue before and after culture in vitro.Results:1.Aged 8 weeks of BALB/c male mice were injected intraperitoneally with different concentrations of busulfan,the spermatogenic function of mouse testis tissue was damaged,and the seminiferous tubules of 40 mg/kg concentration group became Sertoli cell only syndrome damage after 4 weeks injection of busulfan.According to the Johnsen scoring standard,the 40 mg/kg concentration group scored 3 to 4 points 4 to 8 weeks after injection of busulfan,which was statistically significant compared with the control group(P<0.05).2.Agarose gel method to culture immature testicular tissue: Before culture,the testicular tissue of neonatal mice is loosely connected between the seminiferous tubules.The cells in the lumen are arranged regularly,but the species are single,and only spermatogonial cells can be seen on the proximal basal side.The average optical density of STRA8 positive cells was 0.4148±0.0047,and the average optical density of apoptosis was 0.0065±0.0006.During the culture,the average optical density values of STRA8,SCP3 and TNP1 were dynamically changed compared with those before culture.STRA8 and SCP3 were the smallest(P<0.05)when cultured after 4 weeks,and TNP1 expression was the lowest when cultured after 7 weeks(P<0.05).After culture,compared with before culture,the seminiferous tubules were tightly connected,the cell types in the lumen increased,TNP1 positive cells were expressed,and apoptosis increased(0.0107±0.0012).3.In vitro culture of testicular tissue with different degrees of spermatogenic disorder: Before culture,the testicular tissue of normal male mice of the same age is closely connected with the seminiferous tubules,and 7-8 layers of spermatogenic cells are regularly arranged in the lumen;the experimental group has atrophy of the tubules.The vacuolization of the lumen and the lack of spermatogenic cells were significantly lower in the meiotic marker protein than in the normal control group(P<0.05).In the culture process,the H-S2 group had the best effect in the experimental group.The average optical density values of STRA8 and SCP3 showed an increasing trend(P<0.05),and TNP1 showed a few positive expressions,while the SCOS group had the worst culture effect and without TNP1 expression.After culture,the average optical density value of TUNEL apoptosis in the SCOS group was increased(0.0015±0.0001 vs 0.0070±0.0003)compared with that before the culture.4.Thawed testicular tissue culture in vitro: Before culture,the thawed tissue was destroyed compared with fresh tissue,and the structure of the seminiferous tubules was destroyed,the cells were arranged in a disorderly manner,the positive expression of STRA8 was decreased,and the apoptosis was obvious,especially 0.1M.The apoptosis of trehalose by liquid nitrogen fumigation was the most serious(0.4689±0.0126),P<0.05.After 48 hours of culture,the meiotic marker protein of the experimental group decreased and the apoptosis increased(P<0.05).Conclusion:1.In the study,we successfully established an animal model of only the Sertoli cell and spermatogenic block.2.The agarose gel method allows the immature testicular tissue to enter meiosis during culture in vitro.3.The cells with positive TNP1 expression were obtained after culture of testicular tissue of spermatogenic disorder in vitro.It is speculated that the culture system can induce spermatogenesis in vitro of testicular tissue of mice spermatogenic disorder.4.The cryopreservation tissue is severely apoptotic during in vitro culture and the culture system still needs improvement.