Renata Czolowska

Blastomeres of the mouse embryo lose totipotency after the fifth cleavage division: expression of Cdx2 and Oct4 and developmental potential of inner and outer blastomeres of 16- and 32-cell embryos.

Sixteen inner or outer blastomeres from 16-cell embryos and 32 inner or outer blastomeres from 32-cell embryos (nascent blastocysts) were reaggregated and cultured in vitro. In 24 h old blastocysts developed from blastomeres derived from 16-cell embryos the expression of Cdx2 protein was upregulated in outer cells (new trophectoderm) of the inner cells-derived aggregates and downregulated in inner cells (new inner cell mass) of the external cells-derived aggregates. After transfer to pseudopregnant recipients blastocysts originating from both inner and outer blastomeres of 16-cell embryo developed into normal, fertile mice, but the implantation rate of embryos formed from inner cell aggregates was lower. The aggregates of external blastomeres derived from 32 cell embryo usually formed trophoblastic vesicles accompanied by vacuolated cells. In contrast, the aggregates of inner blastomeres quickly compacted but cavitation was delayed. Although in the latter embryos the Cdx2 protein appeared in the new trophectoderm within 24 h of in vitro culture, these embryos formed only very small outgrowths of Troma1-positive giant trophoblastic cells and none of these embryos was able to implant in recipient females. In separate experiment we have produced normal and fertile mice from 16- and 32-cell embryos that were first disaggregated, and then the sister outer and inner blastomeres were reaggregated at random. In blastocysts developed from aggregates, within 24 h of in vitro culture, the majority of inner and outer blastomeres located themselves in their original position (internally and externally), which implies that in these embryos development was regulated mainly by cell sorting.

Cytoplasmic control of nuclear maturation in mouse oocytes.

Abstract Oocytes with germinal vesicles were cut into anucleate and nucleate fragments. At the time of germinal vesicle breakdown (GVBD) in nucleate fragments (after 2–3 h of culture) sister anucleate fragments were fused with the help of inactivated Sendai virus with interphase blastomeres from 2-cell embryos. The hybrid cells were examined after 1 1 2 –3 1 2 h and 20 h. The anucleate fragments induced chromosome condensation in the nuclei of interphase blastomeres immediately after fusion. On this basis it may be concluded that GVBD and nuclear maturation in mouse oocytes is induced by a cytoplasmic factor which is produced or unmasked independently of the nucleus.

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice.

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.

The fine structure of the “germinal cytoplasm” in the egg ofXenopus laevis

Summary1.The fine structure of the „germinal cytoplasm“ of the dividing egg ofXenopus laevis has been studied. Mitochondria which occur in great numbers and electron-dense bodies associated with them are the two characteristic components of the so-called “germinal cytoplasm”.2.The electron-dense bodies are composed of granular and fibrillar elements and often contain an internal light area. Long fibrillar elements have been found within the light area. The organization of the electron-dense bodies is strongly reminiscent of that of the polar granules inDrosophila.

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study.

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certains maturation‐linked changes were noted. Sperm apposition and sperm‐oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrouded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck‐piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg‐cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients).

Factors engaged in reactivation of DNA replication in the nuclei of growing mouse oocytes introduced into the cytoplasm of parthenogenetic one-cell embryos.

Mammalian primary oocytes are arrested in the post-replicative G2 phase of the cell cycle. In contrast to other G2 nuclei, the nucleus of the growing mouse oocyte can reinitiate DNA synthesis after transfer by cell fusion under favorable cytoplasmic conditions, created by the parthenogenetic one-cell embryo. In the present study, we used the cell hybrid system to analyze the distribution of proteins involved in DNA re-replication in the oocyte nucleus. We show that this process is preceded by an extensive rearrangement of the insoluble fractions of minichromosome maintenance (MCM) proteins (Mcm2, -6 and 7). We also demonstrate that Cdc6 protein is present in primary growing mouse oocytes freshly isolated from the ovary, in a soluble and insoluble form. In contrast to MCM proteins, the insoluble fraction of Cdc6 was not rearranged in oocyte nuclei reinitiating DNA replication in hybrid cells. The rearrangement of MCM proteins and reinitiation of DNA synthesis occurred in the nuclei, in which the nuclear envelope remained intact. Reinitiation of DNA replication in the oocyte nucleus was sensitive to the inhibition of both CDK activity and polyadenylation of maternal mRNAs, indicating a role of proteins synthesized de novo by the embryo. These results allow us to understand better the mechanisms involved in the reinitiation of DNA replication in growing oocytes.

In Memoriam - Prof. Andrzej Krzysztof Tarkowski (1933-2016)

Professor Andrzej Krzysztof Tarkowski passed away last September (2016) at the age of 83. His findings, have become indispensable tools for immunological, genetic, and oncological studies, as well as for generating transgenic animals which are instrumental for studying gene function in living animals. His work and discoveries provided a tremendous input to the contemporary developmental biology of mammals.