高温热应激构建小鼠精原干细胞移植受体模型

Construction of a mouse model of spermatogonial stem cell transplant recipient by high-temperature heat stress

目的:探讨高温热应激方法构建小鼠精原干细胞(SSCs)移植受体的可行性。方法:将4周龄的C57BL/6雄鼠和B6(Cg)-Tyrc-2J/J毛色基因纯合突变雄鼠置于恒温箱内,43℃热应激处理1 h,通过HE染色、免疫组化染色和TUNEL凋亡检测,寻找最佳移植时间;随后将SSCs移植入小鼠生精小管,定期观察移植后受体小鼠睾丸内SSCs增殖、分化及形成精子的情况,并将受体小鼠与正常同周龄雌鼠交配,观察其后代表观遗传学特征。结果:高温热应激3~5 d后受体小鼠睾丸生精小管内生精细胞层数减少,排列紊乱、疏松且大量缺失,间质细胞数量明显减少,生精细胞凋亡明显,自噬水平升高,12 d左右基本恢复。在分离纯化后的SSCs移植8周后,受体小鼠睾丸生精小管内分化形成生精细胞及具有遗传功能的精子,经过自然交配产生正常的后代。结论:高温热应激方法可快速、高效构建小鼠精原干细胞移植受体模型。

Objective:To investigate the feasibility of constructing a mouse model of spermatogonial stem cell(SSC)transplant recipient by high-temperature heat stress.Methods:Four-week-old C57 BL/6 male mice and B6(Cg)-Tyrc-2 J/J coat color gene homozygous mutant male mice were heat-treated at 43℃for an hour in the incubator.The best transplantation time was determined by HE staining,immunohistochemistry and TUNEL and the SSCs were transplanted into the seminiferous tubules of the mice followed by regular observation of the proliferation,differentiation and spermiogenesis of the SSCs in the testis of the recipient mice.Then the recipients were mated with age-matched normal female mice and the epigenetic features of their offspring were observed.Results:After3-5 days of high-temperature heat stress,the spermatogenic cells in the testicular seminiferous tubules of the recipient mice showed obviously decreased layers,disordered and loose arrangement,massive deletion,significant apoptosis,reduced mesenchymal cells and increased autophagy,which were basically recovered in about 12 days.At 8 weeks after transplantation,the isolated and purified SSCs were differentiated into spermatogenic cells and sperm with genetic function in the testicular seminiferous tubules of the recipient mice,and normal offspring were reproduced after natural mating.Conclusion:High-temperature heat stress can be used as an efficient method for rapid construction of the mouse model of spermatogonial stem cell transplantation recipient.