Smads蛋白在大鼠睾丸发育不同阶段的表达和定位

EXPRESSION OF SMADS PROTEINS IN RAT TESTIS DURING POSTNATAL DEVELOPMENT

探讨转化生长因子 β超家族肽类的细胞内信号转导分子Smads蛋白在发育不同阶段大鼠睾丸的表达及在精子发生中的作用机理。分别选用出生后 3、 7、 14和 2 8d以及成年大鼠 ,应用蛋白质免疫印迹杂交技术及免疫组织化学ABC法结合葡萄糖氧化酶 DAB 硫酸镍铵增强技术 ,检测Smad 1、Smad 2、Smad 4和Smad 5蛋白在大鼠睾丸的表达、定位和发育变化 ,并通过图像分析技术对免疫组织化学结果进行统计学分析。免疫印迹杂交发现 ,Smad 1、Smad 2和Smad 4在 3、 7、 14和 2 8d以及成年大鼠睾丸均有蛋白质表达 ,Smad 5蛋白则仅表达于 2 8d和成年大鼠睾丸。免疫组织化学结果显示 ,Smad 1在 14d大鼠睾丸的生精细胞中即可见免疫阳性反应 ;Smad 2从 7d起开始表达 ,免疫阳性产物位于各级生精细胞的胞质内 ;Smad 4在发育的各个阶段的间质细胞中均有较强的表达 ;Smad 5仅在 2 8d和成年大鼠的间质细胞中有弱的表达。结果提示 :Smads蛋白在睾丸中的分布不同 ,表达量存在差异 ,为揭示TGF

Testis is a complex organ, and normal testicular development and maintenance of spermatogenesis during fetal and postnatal life is controlled by intratesticular modulators. One important local regulator of the testicular functions is the transforming growth factor β(TGF β) superfamily members. The superfamily members include TGF βs, activins/inhibins and the bone morphogenetic proteins (BMPs). Smads are recently identified proteins that mediate intracellular signaling of the TGF β superfamily from the cell surface to the nucleus. TGF β and activin typeⅠreceptors activate Smad 2 and Smad 3, whereas the BMP typeⅠreceptors target Smad 1, -5, and -8. Smad 4 is the only vertebrate common mediator Smads and can combine with the other Smads to control gene transcription. Despite the fact that members of the TGF β superfamily are expressed in the mammalian testes, their roles in spermatogenesis and testicle development are far from clear. In order to shed light on the mechanisms of TGF β action in spermatogenesis, it is crucial to determine whether and where their downstream signaling molecules are expressed in the testis. We have investigated the expression and function of Smad 1, Smad 2, Smad 4, and Smad 5 proteins in rat testes during postnatal development. In this study, whole testes were collected from male SD rats aged 3 days, 7 days, 14 days and 28 days, and adult. We examined, by means of Western Blots, the proteins expression of Smads during rat testicular development and its cellular localization by immunohistochemical ABC method with glucose oxidase DAB nickel enhancement technique. The immunostaining of Smads in testes of developing rats was performed using densitometric test by the image anslysis system. Then, the data were analyzed by one way ANOVA, using SPSS 10 0 for Windows. The Western Blots showed that Smad 1, Smad 2 and Smad 4 were present throughout testicular development, whereas Smad 5 was detected in the 28 d and adult testis. The immunostaining of Smad 1 and Smad 2 were present in spermatogenic cells, positive substance was located in cytoplasm with negative nuclei. The immunolocalization of Smad 1 was in the 14 d, 28 d and adult testis, while Smad 2 was in the 7 d, 14 d, 28 d and adult testis, and there was positive substance in Sertoli cells and Leydig cells, too. The immunolocalization of Smad 4 was exclusively localized to the cytoplasm of Leydig cells with negative nuclei throughout testicular development. Smad 5 was weakly expressed in cytoplasm of interstitial cells in the 28 d and adult rat testes. No expression was detected in germ cells. The result of image and statistical analysis showed that generally, there was a tendency that the expression of Smad 1, Smad 2, Smad 4 and Smad 5 in the testis increased gradually with the rat development. In the present study, we systemically investigated the expression and localization of the four of Smads proteins on developing and adult rat testes. The immunostaining of Smad 1 and Smad 2 were present in germ cells, which were related to the stage of the cycling of the seminiferous epithelium. So Smad 1 and Smad 2 directly influence and regulate the proliferation of spermatogonia, the meiosis of spermatocytes, and the differentiation of spermatids. Our findings of Smad 4 and Smad 5 expression in Leydig cells can speculate that both of them indirectly influence spermatogenesis and the function of the testis by regulating the function of Leydig cells. The different expression patterns of Smad 1, Smad 2, Smad 4, and Smad 5 suggest that TGF βs, activin and BMPs may affect different aspects of spermatogenesis and testis development, which provides direct evidence for the molecular mechanism of transforming growth factor β superfamily members action in rat testes during postnatal development and spermatogenesis of rats.