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.

Mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mediates the LH-induced stimulation of progesterone synthesis in the porcine corpus luteum

Progesterone (P4) synthesized by the corpus luteum (CL) plays a key role in the establishment and maintenance of pregnancy. The LH signal is important for luteinisation and P4 synthesis in pigs. In a previous study, we demonstrated that mitogen-activated protein kinase kinase kinase 8 (MAP3K8) regulates P4 synthesis in mouse CL, but whether the function and mechanism of MAP3K8 in the pig is similar to that in the mouse is not known. Thus, in the present study we investigated the effects of MAP3K8 on porcine CL. Abundant expression of MAP3K8 was detected in porcine CL, and, in pigs, MAP3K8 expression was higher in mature CLs (or those of the mid-luteal phase) than in regressing CLs (late luteal phase). Further functional studies in cultured porcine luteal cells showed that P4 synthesis and the expression of genes encoding the key enzymes in P4 synthesis are significantly reduced when MAP3K8 is inhibited with the MAP3K8 inhibitor Tpl2 kinase inhibitor (MAP3K8i, 10μM). After 12–24h treatment of luteal cells with 100ngmL−1 LH, MAP3K8 expression and P4 secretion were significantly upregulated. In addition, the 10μM MAP3K8 inhibitor blocked the stimulatory effect of LH on P4 synthesis and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in porcine luteal cells. The LH-induced increases in MAP3K8 phosphorylation and expression, ERK1/2 phosphorylation and P4 synthesis were all blocked when protein kinase A was inhibited by its inhibitor H89 (20 μM) in porcine luteal cells. In conclusion, MAP3K8 mediates the LH-induced stimulation of P4 synthesis through the PKA/mitogen-activated protein kinase signalling pathway in porcine CL.