Coculture with porcine luteal cells during in vitro porcine oocyte maturation affects lipid content, cortical reaction and zona pellucida ultrastructure.

CONTEXT: In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. AIM: This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. METHODS: COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. KEY RESULTS: Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. CONCLUSIONS: The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. IMPLICATIONS: The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.

Coculture of porcine luteal cells during in vitro porcine oocyte maturation affects blastocyst gene expression and developmental potential.

Oocyte maturation in culture is still the weakest part of in vitro fertilization (IVF) and coculture with somatic cells may be an alternative to improve suboptimal culture conditions, especially in the pig in which maturation takes more than 44 h. In the present study, we investigated the effect of a coculture system of porcine luteal cells (PLC) during in vitro maturation (IVM) on embryo development and gene expression. Cumulus-oocyte complexes were matured in vitro in TCM-199 with human menopausal gonadotrophin (control) and in coculture with PLC. IVF was performed with frozen-thawed boar semen in Tris-buffered medium. Presumptive zygotes were cultured in PZM for 7 days. The coculture with PLC significantly increased blastocysts rates. Gene expression changes were measured with a porcine embryo-specific microarray and confirmed by RT-qPCR. The global transcription pattern of embryos developing after PLC coculture exhibited overall downregulation of gene expression. Following global gene expression pattern analysis, genes associated with lipid metabolism, mitochondrial function, endoplasmic reticulum stress, and apoptosis were found downregulated, and genes associated with cell cycle and proliferation were found upregulated in the PLC coculture. Canonical pathway analysis by Ingenuity Pathway revealed that differential expression transcripts were associated with the sirtuin signaling pathway, oxidative phosphorylation pathway, cytokines and ephrin receptor signaling. To conclude, the coculture system of PLC during IVM has a lasting effect on the embryo until the blastocyst stage, modifying gene expression, with a positive effect on embryo development. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in porcine in vitro embryo production.

Coculture of porcine cumulus-oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development.

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus-oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

The antioxidant dimethylthiourea improves IVF efficiency and decreases cumulus cell apoptosis in pigs.

Abattoir ovaries, which are the main source of oocytes for reproductive biotechnologies, arrive at the laboratory under ischaemic conditions. Reoxygenation generates reactive oxygen species (ROS) in ischaemic tissues, which could affect oocyte quality. The aim of this study was to evaluate the effect of supplementation of media with dimethylthiourea (DMTU) during the collection and washing of cumulus-oocyte complexes (COC) on ROS levels, COC apoptosis and oocyte nuclear and cytoplasmic maturation. Thus, the collection (TCM-199) and washing (TCM-199 with 10% porcine follicular fluid, sodium pyruvate and antibiotics) media were supplemented with 1 and 10mM DMTU. In the control group, the media were not supplemented with DMTU. Intracellular ROS levels decreased significantly in the DMTU-treated groups (P<0.05). Although no effects on rate of nuclear maturation were observed, DMTU significantly increased sperm penetration rates without increasing polyspermy (P<0.05). The addition of 10mM DMTU to the collection and washing media enhanced IVF efficiency. DMTU did not modify the early or late apoptosis of oocytes. Both concentrations of DMTU significantly increased viability and decreased the apoptosis of cumulus cells (P<0.05). These results suggest that the addition of 1 or 10mM of DMTU to the media during the collection and washing of porcine COCs is useful for decreasing cumulus apoptosis mediated by ROS and for optimising the IVF of porcine oocytes.