Effects And Mechanisms Of Action Of Cumulus Cells On Cytoplasmic Maturation Of Mouse Oocytes

In vitro matured (IVM) oocytes of high quality are crucial for successful human in vitro fertilization (IVF) and animal in vitro embryo production and cloning. Although spontaneous nuclear maturation of oocytes appears to occur normally in vitro, the quality of the IVM oocytes is generally lower than that of oocytes matured in vivo (IVO), the embryo developmental ability of oocytes matured and fertilized in vitro also is very low. This is believed to be due mainly to incomplete cytoplasmic rather than nuclear maturation in vitro. Several studies have indicated that IVM system for oocytes is contacted closely with the success of in vitro fertilization and embryo development. Therefore, it is very important for us to establish a perfect culture system of IVM oocytes .Although studies indicated that follicular cells were considered to play an important role in oocyte cytoplasmic maturation, the specific role and mechanisms by which different cell types support oocyte cytoplasmic maturation are poorly understood. In this study, we established an experimental model of mouse cumulus-denuded oocytes (DOs) co-cultured with cumulus cells for two purpose: firstly, we investigate the mechanisms of cumulus cells action on oocyte cytoplasmic maturation; secondly, we increase the abilities of DOs maturity and development as much as possible by improveing IVM system of DOs which could provide a technical approach to such procedures as germinal vesicle transfer and oocyte cryopreservation at the germinal vesicle stage. In this study, we investigated the effects of somatic cell types and junctional association of cumulus cells (CCs) with oocytes on cytoplasmic maturity of the co-cultured mouse oocytes. Effect of gonadotropin on co-culture was also observed. In addition, we observed some other parameters of oocyte cytoplasmic maturity, including oocyte meiotic nuclear progression, zona pellucida hardening, cortical granule redistribution and exocytosis, MPF activity, intracellular glutathione level, spindle assembly and mitochondrial distribution. The results were summarized as follows:1. The optimum activation age of IVM oocytes is twenty-six hours oocytes cultured. The activation protocol we optimized was exposing to 10mM SrCl2 and 5μg/mlCB for 2.5h followed by cultured in 5μg/mlCB for for 3.5h.2. Co-culture on the CC monolayer significantly increased rates of maturation and blastocysts of DOs. Co-culture on monolayers of oviduct epithelial or fetal fibroblast cells improved only nuclear maturation of cumulus-denuded oocytes (DOs). Mural granulosa cells had no effect. When co-cultured on monolayer of CCs collected from the GV stage oocytes 46 h (GVCC 46 h) or 24 h (GVCC 24 h) post PMSG injection, rates of maturation and blastocysts of DOs did not differ, but were higher (P<0.05) than those of DOs cultured on monolayer of CCs recovered from IVO oocytes (MIICC).3. The junctional association or close contact between CCs with oocytes is not necessary. Culture in medium conditioned with CC monolayers also enhanced maturation and blastulation of DOs.4. Effects of co-culture with either cumulus-oocyte complexes (COCs) or dispersed CCs increased as the density of CCs increased.5. Oocyte competence for cytoplasmic maturity reduced drastically upon shortening the duration of in vivo gonodotropin (PMSG) priming from 46 to 24 h, and CC co-culture promoted cytoplasmic maturity of both COCs and DOs recovered following 24-h priming; Omission of PMSG from maturation medium decreased the effect of CC co-culture.6. The CC co-culture also improved fertilizability (fertilization rates, cortical granule distribution and zona penetrability) and other parameters of cytoplasmic maturity (MPF activity and spindle assembly) of DOs, though the level of intracellular glutathione did not differ between COCs and DOs matured alone. The co-cultured DOs showed a timing of nuclear progression closer to that of in vivo matured oocytes than DOs cultured alone. The percentage of oocytes which occurred premature exocytosis of DOs was reduced drastically and the degree of zona pellucida hardening was alleviated by co-cultured with CCs. The percentages of oocytes which have normal spindle shapes and complete polarized mitochondrial distribution of coDOs were more than that of DOs.It is concluded that CCs produced a soluble factor(s) in a tissue-specific and hormone-dependent manner that promoted oocyte cytoplasmic maturation possibly through regulation of MPF activity and meiotic progression.