The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals

Abstract

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

Embryo cell allocation patterns are not altered by biopsy but can be linked with further development

“…In the present study, we confirm the presence and a comparable distribution of the three different cell allocation patterns in mouse blastocysts irrespective of mouse strain or embryo cryopreservation at 1-cell stage. These resuls are also in agreement with those previously reported in mouse, bovine and ovine embryos ( Hosseini et al 2016 , Sepulveda-Rincon et al 2016 ). Embryo biopsy at the 8-cell stage, whether the biopsied cell is stained or not stained (data not shown), does not seem to alter the cell allocation patterns in mouse embryos similar to what we have previously reported in bovine embryos ( Sepulveda-Rincon et al 2016 ).…”

“…It is still unclear if mammalian embryos are pre-patterned or the presence of stochastic development is just a reflex of the great plasticity of mammalian embryos. Research performed on cell allocation patterns suggest that it might be a common characteristic during pre-implantation embryo development of different mammalian species ( Park et al 2009 , Hosseini et al 2016 , Sepulveda-Rincon et al 2016 ). However, there is a lack of evidence on the mechanism(s) leading to these cell allocation patterns.…”

Totipotency segregates between the sister blastomeres of two-cell stage mouse embryos

“…Such a gradient has been described for LEPTIN and STAT3 in mouse and human oocytes 50 . Western blot analysis of bisected sheep oocytes revealed that specific maternal proteins, including NANOG, are distributed asymmetrically 51 . In mice, however, Nanog is not transcribed in early embryos until the 8-cell stage 52 , thereby excluding the possibility that a different maternal legacy of Nanog mRNA in the sister two-cell blastomeres would account for the discordant sizes of the EPI compartments of the MZ blastocysts.…”

Perfect date—the review of current research into molecular bases of mammalian fertilization

“…These actin-filled hairlike projections greatly enlarge the contact area and decrease the repulsion between the two juxtapositioned membranes [49]. In many animal species, sperms have a preferential entry point [50][51][52]. In mouse, rat, and hamster eggs, sperm do not fuse with the microvilli-free Fig.…”