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目的了解转化生长因子-β超家族(TGF-βs)在调节卵泡发育过程中的信号转导模式,以探讨在不同发育阶段大鼠卵巢中Smad4蛋白及mRNA的表达。方法选择不同发育时期大鼠卵巢,运用免疫组化方法检测卵巢中Smad4蛋白表达,并进行图像分析;采用半定量逆转录聚合酶链反应(RT-PCR)方法检测Smad4mRNA在卵巢中的表达,以GAPDH作为内对照与特异性Smad4同时进行扩增,并计算Smad4和GAPDH的RT-PCR产物积分吸光度比值来表示Smad4mRNA的含量。结果Smad4广泛表达于卵巢组织,但主要表达在卵泡中,其表达部位和强度随卵巢的成熟度不同而变化;在卵巢发育早期,Smad4主要在原始卵泡和窦前卵泡中表达,而在间质中的表达较弱;随着卵巢的发育成熟,Smad4在间质中的表达逐渐增强,性成熟后,在窦状及成熟卵泡颗粒细胞和卵泡膜的表达与间质细胞比较无显著差异(P>0.05)。Smad4在卵泡中的表达强度也发生了变化,即随着卵泡的发育,Smad4在窦状及成熟卵泡卵母细胞的表达明显减弱,与窦前卵泡卵母细胞比较有显著差异(P<0.05,P<0.01);与窦前卵泡比较,Smad4在卵泡膜细胞的表达逐渐增强(P<0.01),而在各级卵泡颗粒细胞中的表达无显著差异(P>0.05)。RT-PCR结果显示各阶段卵巢均有mRNA的表达,从第3周起Smad4mRNA的表达明显增强,Smad4和GAPDH积分吸光度比值?
Objective To investigate the signal transduction mode of transforming growth factor-β superfamily (TGF-βs) in the regulation of follicular development in order to explore the expression of Smad4 protein and mRNA in rat ovaries at different developmental stages. Methods The ovaries of rats at different developmental stages were selected. The expression of Smad4 protein in ovary was detected by immunohistochemistry and image analysis was performed. The expression of Smad4 mRNA in ovary was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) GAPDH as an internal control and specific Smad4 simultaneous amplification, and calculated Smad4 and GAPDH RT-PCR product integral absorbance ratio to express the content of Smad4 mRNA. Results Smad4 was widely expressed in ovarian tissue but mainly expressed in follicles. The expression level and intensity of Smad4 varied with the maturation of ovary. Smad4 was mainly expressed in primordial follicles and preantral follicles in the early stage of ovarian development, The expression of Smad4 in the stroma was gradually increased with the development of the ovary. After sexual maturation, the expression of Smad4 in the granulosa cells and the follicle of the sinusoid and mature follicles was not significantly different from that of the stromal cells (P > 0.05). The expression intensity of Smad4 also changed in follicles, that is, with the development of follicles, the expression of Smad4 in the oocyte of sinusoid and mature follicles was significantly decreased compared with that of the preantral follicular oocytes (P <0.05, (P <0.01). Compared with preantral follicles, the expression of Smad4 in the follicular cells gradually increased (P <0.01), while there was no significant difference in the granulosa cells (P> 0.05). The result of RT-PCR showed that the mRNA expression of ovary in each stage was increased, the expression of Smad4 mRNA increased obviously from the third week, and the ratio of the absorbance of Smad4 and GAPDH integral?