Coculturing Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation Affects Oocyte Maturation, Zona Hardening, Polyspermy and Oocyte GPX 1 Gene Expression.

The growth and development of mammalian oocytes is tightly regulated by a two-way functional communication axis between the oocyte and the surrounding somatic cells. The oocyte gradually and sequentially acquires meiotic and developmental competence during the antral follicular development period. The molecular and cytoplasmic machinery required for normal development are also acquired during this phase. Therefore, a close association between the oocyte and the surrounding cumulus cells (CCs) is necessary for maintaining oocyte health and development. The CCs encourage oocyte developmental competence by promoting cytoplasmic and nuclear maturation via paracrine factors and through gap junctions. Consequently, CC health is important for appropriate in vitro embryonic development and is maintained by oocyte-secreted factors (OSFs) that regulate a broad range of CC functions. The developmental competence of bovine oocytes is increased when denuded oocytes (DOs) are cocultured with cumulus oocyte complexes (COCs) during in vitro maturation (IVM). The present study examined the effect of coculturing COCs and DOs on nuclear maturation, zona hardening (ZH), in vitro fertilization (IVF) and oocyte glutathione peroxidase (Gpx1) gene expression. Twelve immature COCs derived from abattoir ovaries were matured in vitro with sixty DOs (coculture) or without DOs (control) in a 120 µL droplet of IVM medium for 22-24 h. Following IVM, COCs from cocultures and control groups were subjected to IVF and in vitro culture in a 60 µL droplet of medium at 38.5°C and 5% CO2 in a humidified atmosphere. TUNEL staining was conducted on day 8-blastocysts. Nuclear maturation was confirmed by the presence of polar bodies. The time required for complete dissolution of the zona in 0.5% pronase was used as a measurement of ZH. Oocyte mRNA (a pool of 125 zona-free) was extracted and subjected to quantitative RT-PCR. Gene expression was normalized to Actb, 18s rRna and Ywhaz. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage (39.9%), the total number of cells per blastocyst (11.7%), the percentage of oocytes reaching metaphase II (18.8%), the monospermic fertilization pattern (22.0%) and oocyte Gpx1 (3.4-folds) levels compared to controls. Moreover, coculturing significantly reduced the zona hardening of oocytes as well as polyspermy compared to controls. In conclusion, the present study shows that the synergestic effects achieved by coculturing COCs and DOs are the results of increased nuclear maturation, prevention of ZP hardening, increased monospermic fertilization and increased expression of Gpx1 in oocytes in response to endogenous oocyte secretion factors. This work was partly supported by the BK21 program, the KRF (KRF-2008-211-F00011), the IPET (108068-03-1-SB010) and the KOSEF (10525010001-05N2501-00110). (poster)