白消安对仔猪睾丸曲精小管内增殖分化相关蛋白表达与定位的影响

Effects of Busulfan on Expression and Localization of Proliferation and Differentiation Related Proteins in Seminiferous Tubules in Testis of Piglets

为了研究白消安对仔猪睾丸曲精小管内增殖分化相关蛋白PCNA、PLZF和GATA4表达与定位的影响,试验选取体外培养仔猪曲精小管为研究对象,分为对照组和试验组,其中试验组各添加5 mg/kg白消安,分别培养1、2、3 d,离心获取曲精小管,采用HE染色检测形态变化,采用免疫组化、qRT-PCR和Western blot法分别检测PCNA、PLZF和GATA4表达和定位的变化。HE染色结果显示,各试验组曲精小管内均含有支持细胞和生殖细胞;免疫组织化学结果显示,各试验组的PCNA蛋白定位于生殖细胞和支持细胞的细胞核,白消安作用可导致PCNA染色变深,表达量升高。各试验组GATA4蛋白定位于支持细胞的细胞核,且与对照组相比无明显差异;各试验组的PLZF蛋白定位于生殖细胞的细胞核,与对照组相比,白消安作用2 d时PLZF染色无显著差异,白消安作用1、3 d时PLZF染色变深,表达量升高。qRT-PCR和Western Blot结果显示,与对照组相比,试验组PCNA和GATA4 mRNA及其蛋白表达量均显著升高;白消安作用2 d的PLZF mRNA和蛋白表达量差异不显著,白消安作用1、3 d的PLZF mRN以及蛋白表达量均显著升高。综上所述,PCNA、PLZF和GATA4均表达于体外培养仔猪曲精小管,白消安作用可导致PCNA、PLZF和GATA4在曲精小管内表达水平改变,提示白消安通过影响增殖分化相关蛋白的表达导致猪睾丸精子发生受损。

To study the effect of busulfan on the expression and localization of the proliferation and differentiation related proteins PCNA,PLZF,and GATA4 in seminiferous tubules in testis of piglets,in this study,seminiferous tubules of piglets cultured in vitro were selected as the research subjects and divided into control group and experimental group.The experimental group was supplemented with 5 mg/kg of busulfan for 1,2 and 3 days respectively,and seminiferous tubules were obtained by centrifugation.HE staining was used to detect the morphological changes of chromosomes.Immunohistochemistry,qRT-PCR,and Western blot were used to detect the changes in the expression and localization of PCNA,PLZF,and GATA4.HE staining showed that there were both Sertoli cells and germ cells existing in the seminiferous tubules in all experimental groups.Immunohistochemical studies showed that PCNA protein was localized in the nuclei of germ cells and Sertoli cells in all experimental groups.The staining of PCNA became darker and the expression level increased due to the function of busulfan.In the experimental groups,GATA4 protein was located in the nucleus of Sertoli cell,and there was no significant difference in GATA4 protein staining between the experimental group and the control group.PLZF protein was located in nucleus of germ cells of experimental groups.Compared with the control group,PLZF staining showed no significant difference after 2 days of busulfan treatment,while PLZF staining became darker and expression increased after 1 and 3 days of busulfan treatment.The results of qRT-PCR and Western Blot showed that the mRNA and protein expressions of PCNA and GATA4 in the experimental groups were significantly increased compared with the control group.The expression of PLZF mRNA and protein had no significant differences after 2 days of busulfan treatment.The expression of PLZF mRNA and protein was significantly increased after 1 and 3 days of busulfan treatment.In conclusion,PCNA,PLZF,and GATA4 were expressed in seminiferous tubules of piglets cultured in vitro.The expression levels of PCNA,PLZF,and GATA4 in seminiferous tubules were changed due to the effect of busulfan,suggesting that busulfan damage spermatozoa of porcine testis by affecting the expressions of proteins related to proliferation and differentiation.