Effects of hormone-primed oviduct epithelial cell co-culture system on swine SCNT embryo development

The developmental efficiency of swine somatic cell nuclear transfer (SCNT) embryos remains limited, primarily due to the lack of physiologically relevant <i>in vitro</i> culture conditions that can fully support reprogramming and early embryogenesis. In this study, we established a co-culture system using swine oviduct epithelial cells (OECs), including untreated and hormone-pretreated OECs with estradiol and progesterone (EP-OECs), to better mimic the oviductal environment. Compared with the control group, EP-OECs exhibited elevated expression of the oviduct-specific marker OVGP1. Moreover, SCNT embryos co-cultured with EP-OECs displayed a significantly higher blastocyst formation rate (control: 18.6% ± 0.01; OEC: 24.5% ± 0.01; EP-OEC: 30.5% ± 0.03). Although the total blastocyst cell number did not increase, co-culture significantly elevated intracellular glutathione (GSH) levels and reduced oxidative stress at key developmental stages. Single-cell transcriptomics (SMART-seq2) analysis revealed that the co-culture activated multiple metabolic pathways, including the pentose phosphate pathway and lipid metabolism, thereby improving redox regulation and energy utilization. Moreover, embryonic development was enhanced through the modulation pluripotency-associated factors, including <i>SOX2</i>, and activation of the PI3K-AKT signaling cascade. Notably, OEC co-culture induced <i>PI3K</i> upregulation at the 8-cell stage and further affected <i>PDK</i> expression. OEC/EP-OEC co-culture treatment suggests PI3K-AKT signaling pathway activation in embryos, which may be a key mechanism promoting embryonic development. These findings indicate that hormone-primed OEC co-culture provides a microenvironment that closely resembles <i>in vivo</i> conditions, offering an effective strategy for improving the efficiency of swine cloning and a new insight into the molecular mechanisms underlying embryonic development <i>in vitro</i>.