Involvement of connexin 43 in meiotic maturation of bovine oocytes

Abstract

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

Impact of anisosmotic conditions on structural and functional integrity of cumulus–oocyte complexes at the germinal vesicle stage in the domestic cat

“…Although not always comparable to results achieved under less extreme conditions, there was rather striking success in these oocytes having the capacity to mature, fertilize and advance to blastocyst stages in vitro. Interestingly, exposure of cat oocytes to the excessive 2,000 mOsm treatment was not as detrimental to COC functionality as denuding all cumulus cells or artificially disrupting communications with the oocyte by other artificial means (Vozzi et al, 2001;Atef et al, 2005;Luciano et al, 2005). This shows that a minimum of communications (not detectable by Lucifer Yellow diffusion) were retained within the COC exposed to 2,000 mOsm which allowed producing viable appearing embryos.…”

The Nuclear Maturation and Embryo Development of Mice Germinal Vesicle Oocytes with and without Cumulus Cell after Vitrification

“…In the single-step method, as in the previous report [26] treatments of EFS30 showed that the GV-COCs after vitrification had partly dispersed cumulus cells and oocytes with shrunken cytoplasm. The connection between cumulus cells and the oocyte in the GVCOCs is shown to be important for completion of normal oocyte maturation in vitro [27]. Hochi, et al, [28] vitrified immature bovine oocytes in straws by using a mixture of 40% EG, ficoll and sucrose as a vitrification medium.…”

Studies on Gene Expression and Developmental Competence of Bovine Embryos Produced Under Different Conditions of Heat Stress

“…Therefore, the results reported indicated that increased expression of Cx43 temporally correlates with the activation of follicular development and early differentiation of granulosa cells in follicles of prepubertal pig ovaries. Vozzi et al [81] indicated that in ovarian follicles, oocyte is provided by cumulus cells with small molecules that license control maturation and growth. The germinal cell gets these nutrients through gap junction channels, present between the oocyte and cumulus cells.…”