OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos

Abstract

Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts. Therefore, in this study, we constructed chimeric embryos comprising blastomeres that either expressed OCT-4 normally or showed downregulated OCT-4 expression by co-injection of OCT-4-siRNA and Dx into one blastomere in 2- to 4-cell stage embryos. In control embryos, which were co-injected with control siRNA and Dx, Dx-positive cells contributed to the TE lineage in almost all the blastocysts examined. In contrast, Dx-positive cells derived from a blastomere co-injected with OCT-4-siRNA and Dx were degenerated in almost half the blastocysts. This was probably due to the inability of these cells to differentiate into the TE lineage. Real-time RT-PCR analysis revealed no difference in the levels of SOX2, TEAD4, FGF4 and FGFR1-IIIc, all of which are known to be regulated by OCT-4, between the OCT-4-siRNA-injected morulae and the control ones. However, the level of CDX2, a molecule specifically expressed in the TE lineage, was significantly higher in the former than in the latter. Our results indicate that continuous expression of OCT-4 in blastomeres is essential for TE formation of porcine embryos.

Fig. 1.

OCT-4 mRNA and protein expression in porcine embryos. (A) Relative abundance (mean ± SEM) of OCT-4 transcripts in uninjected porcine morula stage embryos (n = 5) or porcine morula stage embryos treated with control siRNA + Dx (n = 5), or with OCT-4-siRNA + Dx (n = 5). ^{a, b} Different superscripts indicate a significant difference (P < 0.001). (B) OCT-4 signals (indicated by arrows) were visible in the uninjected morula as well as the morula derived from the control siRNA-injected 1-cell stage embryo. However, such signals were absent in the morula derived from the OCT-4-siRNA-injected 1-cell stage embryo (shown by arrowheads).

Fig. 2.

Fate of OCT-4-downregulated blastomeres from embryos injected with siRNA and Dx at the 2- to 4-cell stage. (A) The percentage of TE formation in porcine blastocyst stage embryos following injection of one blastomere with control siRNA + Dx (n = 38) or with OCT-4-siRNA + Dx (n = 30) at the 2- to 4-cell stage. ^{a, b} Different superscripts indicate a significant difference. (B) Representative photographs of calcein-AM and Dx signals in porcine embryos. siRNA-injected blastomeres fluoresced red and live cells fluoresced green. All Dx-positive cells were stained by calcein-AM (TE), some Dx-positive cells were not stained by calcein-AM (TE+Deg.), and all Dx-positive cells were not stained by calcein-AM (Deg.).

Fig. 3.

Relative abundance (mean ± SEM) of (A) SOX2, (B) CDX2 and (C) TEAD4 transcripts in uninjected porcine morula (n = 5) or porcine morula treated with control siRNA (n = 5) or with OCT-4-siRNA (n = 5). ^{a, b} Different superscripts indicate a significant difference (P < 0.05).

Fig. 4.

Relative abundance (mean ± SEM) of (A) FGF4 and (B) FGFR1-IIIc transcripts in uninjected porcine morula (n = 5) or porcine morula treated with control siRNA + Dx (n = 5) or with OCT-4-siRNA + Dx (n = 5).