Deciphering two rounds of cell lineage segregations during bovine preimplantation development

Abstract

Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "ongel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic

Pluripotent Core in Bovine Embryos: A Review

“…Furthermore, CHIR99021 depressed expression of both NANOG and SOX2 in bovine ESCs and decreased number and percent of blastomeres positives for NANOG and SOX2 in the embryo [39]. In this line, other studies indicate that TE cells highly express transcripts related to WNT signaling [21,32], as observed that activation of WNT signaling enable the derivation of trophoblast stem cell by regulating CDX2 expression through the WNT-YAP/TAZ signaling pathway [60].…”

“…At 8-9 dpf OCT4 positive blastomeres are predominantly located in the ICM, however it is still detected within the TE cell population [7,19], where expression of OCT4 and CDX2 did not differ between ICM and TE [4]. After blastocyst hatching or 9 dpf, OCT4 is located exclusively in ICM cells [17,19,21].…”

Generation of viable calves derived from developmentally mature blastocysts produced by on-gel culture

“…From each of the two pregnant recipients into which one D10 on-gel-cultured embryo had been transferred [6], a calf was vaginally born without assistance on September 21 and November 29, 2021, respectively (Fig. 1A, C), both of which showed spontaneous respiration and were of normal appearance.…”

“…It is not possible to generate mature bovine blastocysts by day (D) 8 using a conventional culture system [ 1 ], indicating that the IVC system currently in use for bovine embryos is, in certain respects, insufficient. Given that bovine embryos undergo a second cell segregation at approximately D9.5 [ 6 ], it is necessary to develop a stable IVC system that ensures the development and differentiation of bovine embryos after D8.…”

“…In this latter respect, we recently developed a novel bovine IVC system, in which, premature D8 blastocysts were cultured on agarose gel (on-gel culture) and were thereby exposed to both gaseous and liquid phases [ 6 ]. Accordingly, it was established that on-gel-cultured embryos survived in vitro to beyond D10, during which, the second cell segregation yielding Epi and PrE was completed [ 6 ].…”