Optimizing extended embryo culture and trophoblast derivation in sheep

Abstract

This study explores the optimization of extended embryo culture and trophoblast derivation in sheep to enhance our understanding of the early stages of embryonic development and implantation. Using a combination of feeder-free culture conditions and culture in endometrial organoids (EOs) conditioned medium, we successfully supported prolonged embryo development in vitro till day 20 post-fertilization. Morphological changes resembling early gastrulation-like events and cotyledon-like formation were observed by advanced development. Relative quantitative PCR analysis showed orchestrated fluctuations in gene expression related to pluripotency and early lineage specifications. Trophoblast cells were passaged three times using feeder-free culture conditions and immunofluorescence analysis confirmed the expression of key trophoblast markers including CDX2, GATA3, syncytin-1, β-catenin, SOX2, Fascin, SOX9, BMP4, MYC, KRT7, and KRT18. The results demonstrated that embryos cultured in EOs conditioned medium significantly improved attachment and differentiation, highlighting the critical role of maternal tissue interactions. These findings provide valuable insights into placental development and hold potential applications for assisted reproductive technologies and livestock breeding advancements.

Keywords: Development; Embryo; Extended culture; Immunofluorescence; Sheep; Trophoblast.