OCT4/POU5F1 is indispensable for the lineage differentiation of the inner cell mass in bovine embryos

Abstract

The mammalian blastocyst undergoes two lineage segregations, that is, formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) as the remaining pluripotent lineage. To clarify the expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9, and 12 blastocysts completely produced in vivo by staining for OCT4, NANOG, SOX2 (EPI), and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4‐deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4‐deficient cells. Our data show that OCT4 is required cell autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway

“…During mammalian preimplantation development, OCT4/POU5F1 plays a critical role as a transcription factor, with diverse functions [53], encompassing the maintenance of pluripotency and the regulation of differentiation events [54]. Our results showed that the pluripotency factor OCT4/POU5F1 is consistently expressed across the entire bovine Day 7 blastocysts, similar to the observations by Simmet et al [55,56] and Kirchhof et al [57] in both in vivo and in vitro produced bovine embryos on days 7, 9, and 12. Furthermore, we observed an increased expression of POU5F1 and NANOG transcripts in blastocysts produced with miR-148b supplemented throughout the entire period of IVC or during days 1 to 4 of IVC to simulate the miRNA effect in the oviduct.…”

“…Previous studies have indicated that NANOG activation does not rely on the embryonic activation of OCT4 [53,58]. However, the absence or very low levels of the epiblast marker NANOG in OCT4 knockout (KO) blastocysts on day 7 suggests that the maintenance of epiblast cells during the early stages of the second lineage differentiation fails in the absence of OCT4, thereby confirming the requirement of OCT4/POU5F1 for NANOG expression in bovine blastocysts [55]. In another study by the same group, OCT4 KO blastocysts generated through somatic cell nuclear transfer and zygote injection demonstrated that both epiblast maintenance and hypoblast differentiation rely on OCT4.…”

“…In another study by the same group, OCT4 KO blastocysts generated through somatic cell nuclear transfer and zygote injection demonstrated that both epiblast maintenance and hypoblast differentiation rely on OCT4. Consequently, it was concluded that OCT4 is necessary for the maintenance of pluripotency in the epiblast and the differentiation of the hypoblast during the second lineage differentiation in bovine preimplantation embryos [55,56].…”

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes

“…Previous research found that knocking down POU5F1 in mouse embryos did not influence cleavage or blastocyst development rate but did reduce the expression of TE-specific genes in the ICM [ 38 , 39 , 40 ]. Simmet et al (2022) [ 41 ] showed that POU5F1 is not required for blastocyst expansion and survival. In contrast, disruption of the POU5F1 locus in bovine hampered blastocyst development and demonstrated embryonic arrest at the morula stage [ 24 ].…”

“…The OCT4 frequently interacts with the SRY-box-containing transcription factor SOX2 to control the expression of several target genes, such as important transcription factors like NANOG [ 43 ]. In a study, embryos with the POU5F1-KO mutation reached the blastocyst stage by day 8 but lost NANOG expression, while SOX2 and GATA6 were unaffected [ 41 ]. SOX2 expression was found to be downregulated when POU5F1 expression was regulated by RNA interference [ 44 ].…”