Expression Of Cx37 And Cx43 In Mouse Ovarian Follicles

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。...