【作者】 尹宝英；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2010， 博士

【摘要】 作为细胞间直接通讯的一种重要的方式,间隙连接在卵泡形成发育过程中起着重要作用。Cx37和Cx43是哺乳动物卵巢卵泡表达的最重要的两种间隙连接蛋白。Cx37是卵母细胞分泌的与其周围颗粒细胞间形成间隙连接的主要连接蛋白,Cx43是颗粒细胞之间表达的主要间隙连接蛋白,通过基因敲除技术已确定这两种连接蛋白对维持卵母细胞的生长和卵泡发育起着重要作用。然而Cx37和Cx43的在小鼠卵泡内的表达部位仍存在争议,而且其调节机制还不是很清楚。本文对小鼠卵泡体内外发育过程中Cx37和Cx43这两种连接蛋白的表达情况进行检测,为研究间隙连接蛋白在卵泡发育过程中的作用及其调节机制打好理论基础。1.应用免疫化学、cDNA扩增和原位杂交方法确定Cx37在小鼠卵巢内发育各阶段卵泡中的定位及表达情况。结果发现在发育各阶段的卵泡内的卵母细胞和颗粒细胞内均能检测到Cx37的表达。在腔前卵泡发育阶段,卵泡内Cx37随着卵泡发育而增加,腔体形成期达到最大值,而后随着腔体的增加其表达下降。表明Cx37在卵泡发育不同阶段发挥着不同的重要作用。颗粒细胞能分泌Cx37说明卵母细胞与颗粒细胞之间和颗粒细胞间可能存在Cx37构成的间隙连接通道。2.应用免疫化学、cDNA扩增方法确定Cx43在小鼠卵巢内发育各阶段卵泡中的定位及表达情况。结果显示,在发育各阶段的卵泡内的颗粒细胞内均能检测到Cx43的表达,卵母细胞内未发现其表达。卵泡内Cx43蛋白表达总体趋势是随着卵泡发育而升高,尤其是腔体形成后Cx43上升较快。结果表明Cx43在卵泡发育不同阶段发挥着不同的重要作用。3.应用免疫组织化学方法和实时定量PCR技术检测小鼠整个动情周期和妊娠过程中卵巢内各级卵泡中Cx37和Cx43的表达,探讨其在小鼠动情周期和妊娠过程可能存在的调控。结果显示,Cx37和Cx43两种间隙连接蛋白在动情周期、妊娠期各阶段的小鼠卵巢卵泡上的表达部位没有明显改变。这两种连接蛋白表达量在次级卵泡的变化较大,且基因与蛋白水平变化一致。表现为动情前期较动情期、动情后期和动情间期表达丰富,妊娠前期较妊娠中后期表达丰富。结果表明Cx37和Cx43的表达自基因水平受动情周期及妊娠周期中各种因素的调控。4.采用real-time PCR的方法,以体内卵泡发育为对照检测小鼠腔前卵泡体外发育过程中卵泡中Cx37和Cx43 mRNA的表达情况。结果显示体外Cx37和Cx43 mRNA的表达趋势与体内一致。Cx37基因在腔前卵泡发育阶段表达随着卵母细胞发育而增加,腔体形成阶段达到最大值(P<0.01),腔体形成后Cx37表达下降。Cx43基因表达随着卵泡的发育而增加,腔体形成阶段有少许下降,腔体形成后迅速增加。体外发育的卵泡Cx37和Cx43 mRNA的表达量远远高于体内发育组,而且异常发育卵泡内Cx37和Cx43 mRNA的表达显著高于体外正常发育卵泡(P<0.01)。结果表明Cx37和Cx43基因的表达量可能依赖于卵泡发育阶段和细胞骨架介导的细胞连接调节的。另外,本试验数据提示卵泡体外培养过程中卵泡间接触连接的破坏不是低水平连接蛋白引起的细胞间连接减少所致的,相反间隙连接蛋白基因异常高表达可能暗示细胞间连接接触发生了中断。5.应用real-time PCR和westernblot技术检测冻存腔前卵泡体外短期培养过程中Cx37和Cx43基因和蛋白的表达情况。结果显示,在体外短期培养过程中冷冻卵泡与新鲜卵泡在体外生长发育形态无明显差异。只是冷冻过的卵泡凋亡数量较多。Cx43和Cx37基因和蛋白在体外短期培养的冷冻卵泡表达量明显高于新鲜卵泡体外发育组。结果表明间隙连接蛋白异常表达所指示的连接接触丧失可能是冻存卵泡体外培养死亡的重要原因。更多还原

【Abstract】 The junction between cells is the basis of follicular development. Gap junction communication is considered to play an important role during the process of oocyte growth and follicle formation and development in mamalian ovary. Cx37 and Cx43 in ovarian follicles are the two most predominant connexins, which of them play important roles in the maintenance of oocyte growth and follicular development. The role of these two gap junction proteins in the process of ovarian follicular development were identified by gene knockout technology. In mouse, the localization of Cx37 and Cx43 is still disputed, and their regulatory mechanism is not clear. For the better understanding of the mechanism of gap junction proteins in the regulation of follicular development, the present work focus on the expression of Cx37 and Cx43 in mouse ovarian follicles in vivo and in vitro1. The Cx37 was detected in mouse follicle during various development stages by methods of immunochemistry, cDNA amplification, in situ hybridization. Cx37 could be detected in the oocyte and granulosa cells from primodial follicles and a lower level was detected in mouse ovarian stroma. Cx37 in follicles increased along with follicle development, reached the highest point at the onset of antrum cavity formation and decreased after antrum formation. These data further confirmed that Cx37 played an important role during the process of follicle development. Cx37 mRNA and protein were detected in granulosa cells co-cultured with oocyte. However, specific expression of Cx37 protein was not found in granulosa cells by monolayer culture. The results indicated Cx37 expressed in granulosa cell may participate in the formation of Cx37-based homotypic gap junction couple between oocyte and granulosa cells and between granulosa cells.2. The Cx43 could be found in mouse follicle during various development stages by methods of immunochemistry and cDNA amplification. Cx43 was detected in graulosa cells and thecal cells from primordial follicles. There was no staining in oocytes. Cx43 in graulosa cells increased along with follicles growth, reached the highest point at the onset of ovulation. These data further confirmed that Cx43 played an important role during the process of follicle development.3. To explore the regulation mechanism of Cx37 and Cx43 in sencond follicles from mouse ovary during estrous cycle and pregnancy periods, the technology of immunohistochemistry and real-time quantitative PCR were used to detect the Cx37 and Cx43 mRNA and protein expression in mouse ovarian follicles during the estrous cycle and pregnancy period. The results showed the expression of Cx37 and Cx43 in second follicles varied: the expression of them was higher in mouse ovary during the proestrus period and early trimester of pregnancy, while the expression of the two connexins decreased during estrus, diestrus, mid and later trimester of pregnancy. Our results confirm that Cx37 and Cx43 expression in follicles at gene level were regulated by hormone during the estrous cycle and pregnancy period.4. To evaluate gene expression of Cx37 and Cx43 in follicles in vitro, real-time PCR method was used to detect the transient expression of Cx37 and Cx43 mRNA in developmental follicles in vitro compared with in vitro follicles. The results showed the expression of the two connexins was similar: Cx37 mRNA increased along with follicle development, reached the highest point at the onset of antrum cavity formation and decreased after antrum formation in both in vivo and in vitro mouse oocytes. Cx43 mRNA increased along with follicle growth, reached the highest point before the preovulatory, lightly decreased during antrum formation. However, Cx37 and Cx43 mRNA was significant higher (P<0.01) in in vitro cultured follicles than in vivo follicles. Moreover, significantly higher Cx37 and Cx43 mRNA were showed in in vitro abnormal developmental follicles (P<0.01). The results showed temporal gene expression of Cx37 and Cx43 in oocytes from follicules at different stages and indicated that expression of Cx37 and Cx43 gene depended on the stage of follicle development and on cell-to-cell contact depended on cytoskeleton’s situation. And, the experimental data suggest that the abnormal development of follicle that connect disrupt was not caused by low levels of connexin. Instead, our results suggested abnormal expression of connexin possibly suggests cell-to-cell connection between follicular cells was disrupted.5. Expression of Cx37 and Cx43 was assessed by real-time PCR and western blot in cultured cryopreserved mouse ovarian follicles compared with that of normal follicles. The results showed that the contaction in follicles were disrupted. The number of died was increased in granulosa cells from preantral follicles of the cryopreserved follicles. After subcutaneous culture, Cx37 and Cx43 were significantly higher in cryopreserved than in normal ovarian follicles. This results indicated that abnormal expression of gap junction proteins is an important factor which cyroperserved follicles died。更多还原