46 Gene expression changes in trophoblast cells after the blastocyst stage in cattle

Early embryonic deaths in cattle causing great financial loss worldwide frequently occur around day 10 to 14 after fertilisation. Exploration of gene expression changes in trophoblast cells that give rise to the embryonic part of the placenta may be useful to understand why early embryonic deaths occur at these time points. However, the absence of a reliable in vitro culture method beyond the blastocyst stage has prevented the time-dependent gene expression analyses. We previously developed an “on-gel culture” method in which bovine blastocysts are transferred onto the surface of an agarose gel filled with an optimised medium. On-gel-cultured embryos are preferred models to study the trophoblast development of bovine blastocysts as they exhibit distinct trophoblast features including the gap junction formation and the emergence of binucleate cells during culture. The present study aimed to describe the chronological changes of trophoblast marker expressions after the blastocyst stage using the on-gel-culture method. Blastocysts were produced by IVF using IVM Holstein oocytes and cryopreserved/thawed sperm, followed by the conventional microdroplet culture in vitro. At 8 days after fertilisation (Day 8), the blastocysts were transferred onto a rectangular agarose gel filled with culture medium and were incubated at 38.5°C, 5% CO₂, and 5% O₂. Mechanically dissected trophectoderm (TE) portions from 20 Day 8 embryos, five Day 10 on-gel-cultured embryos, and two Day 14 on-gel-cultured embryos per biological replicate were pooled and served for RNA extraction. The extracted RNAs were used for quantitative PCR (qPCR). Three housekeeping genes, YWHAZ, GAPDH, and ACTB, were used as the reference genes. Moreover, Day 9, Day 10, Day 11, and Day 12 on-gel-cultured embryos were fixed and used for immunostaining. qPCR analyses revealed that the expression levels of trophoblast markers were significantly down-regulated in the trophoblasts of Day 14 embryos compared with Day 8 embryos (CDX2: 4.2-, GATA2: 1.9-, GATA3: 3.0-, TEAD4: 3.1-fold down-regulation, respectively, P < 0.05). Similarly, immunostaining showed that the proportions of TEAD4- and GATA2-positive cells in the trophoblasts were decreased in Day 12 embryos compared with those in Day 8 embryos, suggesting that different subpopulations of trophoblast cells in terms of gene expression had emerged during culture. Additionally, SOX17 protein was counterstained to identify the hypoblast layer. After SOX17-positive cells were spatially separated from the inner cell mass, this subpopulation of SOX17-positive cells started to spread towards the entire embryo, indicating the formation of a distinct hypoblast layer lining the trophoblast layer in the on-gel-cultured embryos. Taken together, the present study demonstrated that expression of key trophoblast-predominant genes was down-regulated after the blastocyst stage in cattle. These results suggested that abundant expression levels of TEAD4 and GATA2 are no longer needed in functionally matured trophoblast cells although these factors are essential for trophoblast formation until the blastocyst stage. Future studies will be required for understanding different cell types within the trophoblast during placentation in single-cell resolution.