Assessment of transcript and protein levels contributing to cell cycle control and gap junction connections in morphologically variable groups of porcine cumulus-oocyte complexes

摘要

Oocytes and somatic cumulus cells are connected by an extensive network of gap junctions. These connections contribute in a major way to oocyte maturation and developmental competence. Cumulus-oocyte complexes (COCs) were cultured in standard porcineIVM culture medium (TCM 199) for 44 h. The morphological classification of COCs is based on the number of cumulus cell layers and the degree of their compaction, as well as on cytoplasm composition (homogenous, heterogeneous). The obtained COCs were divided into four grades according to this classification system. By assessing the activity of glucose-6-phosphate dehydrogenase (G6PDH) using the brilliant cresyl blue (BCB) test, real-time quantitative PCR (RQ-PCR) reaction methods and confocal microscopicobservations, we determined the transcript levels of connexins 43 and 45, cyclin dependent kinases (cdk5 and cdk5r), and cdk inhibitors 1 and 3 (p27kipl and cdknS) as well as cdk4 protein in morphologically different groups of porcine oocytes isolated from puberal gilts. To assess their nuclear status the completely denuded oocytes were subjected to DA PI staining. We found statistically increased cdkn3, cdk5 and connexin 45 mRNA levels in oocytes graded as I as compared to II, III, and IV. The cdknl,cdk5r and connexin 43 transcript contents were higher only when comparing between oocytes graded as I, III and IV. The cdk4 protein in oocytes graded I and II is localized mainly in the zona pellucida, although in grade III COCs the expression of this protein is decreased and observed only in the cytoplasm. Grade IV COCs do not demonstrate a significant presence of cdk4 protein. With regards to nuclear maturation, the percentage of Mil stage oocytes was significantly (P < 0.05) higher in grade I and IIoocytes as compared to grade III and IV oocytes. Our results demonstrate for the first time that cdk4 protein localization and all of the investigated transcript levels are associated with COC morphology and may be related to further maturation ability as well as developmental competence of oocytes.