Effect of the coculture of porcine luteal cells and cumulus-oocyte complexes on the blastocyst development and gene expression

TEPLITZ, G.; SIRARD, MA.; LOMBARDO, DM.

Ottawa

Encuentro; 53rd SSR Annual Meeting of Society for the Study of Reproduction; 2020

Society for the Study of Reproduction

The coculture with somatic cells is an alternative to improve suboptimal in vitro culture conditions. In the pig, the in vitro fertilization (IVF) is related to poor male pronuclear formation and high rates of polyspermy, and the in vitro embryo development is still an inefficient biotechnology. In the present study, we investigated the effects of a coculture system of porcine luteal cells during in vitro maturation (IVM) on blastocyst development and gene expression. The cumulus-oocyte complexes were matured in vitro in supplemented TCM199 with human menopausal gonadotrophin (control) and in coculture with porcine luteal cells from passage 1 (PLC-1). IVF was performed for 4 h with frozen-thawed boar semen in 100 µL-drop of modified Tris-buffered medium (20 denuded oocytes per drop, 1.5x106 spermatozoa/mL). Presumptive zygotes were washed and cultured in porcine zygote medium at 39°C, 7 % O2, 5 % CO2, and humidity for 7 days. The coculture with PLC-1 significantly increased the blastocyst rate (blastocyst/cleavage) (control: 14.4%; PLC-1 21.2%; Fisher´s test). Gene expression changes were measured with a porcine embryo-specific microarray in four pools of ten blastocysts of each treatment and confirmed by RT-qPCR on six candidate genes. Transcriptome analysis in the PLC-1 versus control groups revealed differences for 530 transcripts in the PLC-1 group (333 downregulated, 197 upregulated); (symmetrical fold-change > 1.2, p< 0.05) The global transcription pattern of embryos developing after coculture treatment during oocyte maturation exhibited overall downregulation of gene expression. Following global gene expression pattern analysis, genes associated with lipid metabolism, mitochondrial function, endoplasmic reticulum stress and cell cycle and apoptosis were found downregulated, and genes associated with cell cycle and apoptosis were found upregulated. Canonical pathway and cellular function analysis by Ingenuity Pathway (IPA) revealed that differential expression transcripts were associated with the sirtuin signaling pathway ( p= 6,23E-06 and z-score= -0,72); oxidative phosphorylation pathway ( p= 2,48E-04 and z-score= 0.63); Chemoquine signaling ( p= 4,44E-04 and z-score= 0,707) and Ephrin receptor signaling (p= 1E-03 and z-score= 0,707). To conclude, the results of the present study demonstrate that a coculture system with PLC-1 during IVM has a lasting effect on the embryo until the blastocyst stage modifying the gene expression, with a positive effect on the embryo development rates. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in the pig.