Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer.

The research article investigates if co-culture of horse oocytes (egg cells) with theca cells improves their maturation rate and evaluates their competence by checking their fusion, activation and cleavage rates after nuclear transfer. The results indicate that the co-culture with theca cells does not enhance the maturation of these cells. Still, the use of a compound named Cytochalasin B can increase the survival rate of horse oocytes during enucleation.

Research Methodology

• The researchers obtained oocytes from ovaries derived from a slaughterhouse via a scraping method. The oocytes were split into two categories – those having expanded cumuli and those with compact cumuli.
• Expanded oocytes were matured in M199 (a specific medium used in cell cultures) supplemented with 10% FBS and 5 microU/ml FSH for 24 hours. Compact oocytes were grown in similar conditions or co-cultured in the same medium with theca interna (a layer of the ovarian follicle) for either 24 or 42 hours.
• Oocytes were fused with donor fibroblasts through electric pulsing after holding with or without 10 microg/ml cytochalasin B and subsequent washing and micromanipulation.
• The fused oocytes were then activated with Ca ionophore/cycloheximide, cultured for five days, and stained with a compound called Hoechst to determine nuclear development.

Research Findings

• The maturation rate of compact oocytes cultured with and without theca cells showed no significant difference. However, when the two groups of compact oocytes were combined, a significant increase in oocytes reaching MII (the second stage of meiosis) was observed between 24 and 42 hours.
• Expanded oocytes had a substantially higher maturation rate than the compact oocytes.
• The rate of successful enucleation (removal of the cell nucleus), fusion, activation, or cleavage (splitting of an organism into two through cell division) did not display significant differences between compact control and compact + theca oocytes, as well as between compact and expanded oocytes.
• Expanded oocytes treated with cytochalasin B showed a significantly higher surviving rate post-enucleation than untreated expanded oocytes.
• Three embryos developed to a maximum of 10 cells from recombined oocytes, demonstrating that in vitro matured equine oocytes have the potential to develop as embryos after nuclear transfer.

Conclusions

• The research concludes that the co-culture of horse oocytes with theca cells does not enhance either nuclear or cytoplasmic maturation of compact oocytes.
• The findings recommend the use of Cytochalasin B to increase the survival rate of horse oocytes during the enucleation process.
• It also affirms that in vitro matured equine oocytes have potential as embryos after nuclear transfer, making this research the first full report of cloned embryo production in horses.