Katarzyna Szczepanska

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.

Allocation of inner cells to epiblast vs primitive endoderm in the mouse embryo is biased but not determined by the round of asymmetric divisions (8→16- and 16→32-cells)

The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8→16 (primary inner cells) and 16→32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.

Isolated mouse inner cell mass is unable to reconstruct trophectoderm.

The ability of ICM to differentiate into TE is still a controversial issue. Many of authors have showed the reconstruction of TE from isolated ICMs. We showed that immunosurgical method is not 100% efficient and that the original TE cells very often remain on the surface of isolated ICMs. We also found that isolated ICM cells cultured in vitro do not express Cdx2, and that the TE is reconstituted from TE cells which have survived immunosurgery. This indicates that very soon after the formation of TE in the blastocyst, the cells of ICM lose the potency to differentiate into trophectoderm.

Oct4 protein remains in trophectoderm until late stages of mouse blastocyst development

Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During the formation of the blastocyst Oct-4 is downregulated in the trophectoderm (TE) and its expression becomes restricted to the inner cell mass (ICM). In order to determine the exact timing of the disappearance of Oct-4 protein from TE we analyzed the localization and level of Oct-4 at different stages of blastocyst development. The presence of Oct-4 protein was determined by immunohistochemistry using confocal microscopy. We found that the downregulation of Oct-4 protein in TE of mouse blastocysts progresses gradually during development, and Oct-4 protein persists in some of the TE cells at least until the expanded blastocyst (120-140 cells) stage. Our findings indicate that the switching-off of the Oct-4 expression is not necessary for the trophectoderm formation. The complete elimination of Oct-4 protein from TE occurs at the period of blastocyst implantation, when lack of Oct-4 is required for the proper functioning of the trophectoderm.

Expression of survivin in human oocytes and preimplantation embryos

OBJECTIVE To determine whether [1] survivin is expressed in human oocytes and embryos; [2] embryos grown in vitro secrete survivin protein; and [3] survivin levels are correlated with embryo cleavage rates. DESIGN Experimental. SETTING University-affiliated IVF clinic. PATIENT(S) Couples undergoing IVF-ET cycles. INTERVENTION(S) Conventional reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, immunohistochemistry, Western blot on oocytes, embryos and control choriocarcinoma JEG-3 cells, and ELISA analysis of conditioned culture media. MAIN OUTCOME MEASURE(S) Detection of survivin mRNA and protein in oocytes and preimplantation embryos and in JEG-3 cancer cells. Detection of survivin concentrations in embryo culture media. RESULT(S) Survivin mRNA and protein were expressed during human oocyte maturation, from germinal vesicle to metaphase II stage, and throughout embryo development, from pronuclear stage to blastocyst stage. Survivin was localized predominantly in the cytoplasm of all cells examined and in the oocytes on the chromatin of metaphase chromosomes and midbodies. Western blot analysis of human oocyte and cancer cell extracts detected a full-length (primary) survivin band of 16.5 kDa. Survivin was also detected in conditioned media samples from embryo cultures and showed a positive correlation with embryo cleavage rates. CONCLUSION(S) Our data have demonstrated for the first time that human oocytes/embryos not only express but also secret survivin, suggesting that survivin may play an important role in human oogenesis and embryogenesis.

Delay of polarization event increases the number of Cdx2-positive blastomeres in mouse embryo.

During preimplantation mouse embryo development expression of Cdx2 is induced in outer cells, which are the trophectoderm (TE) precursors. The mechanism of Cdx2 upregulation in these cells remains unclear. However, it has been suggested that the cell position and polarization may play a crucial role in this process. In order to elucidate the role of these two parameters in the formation of TE we analyzed the expression pattern of Cdx2 in the embryos in which either the position of cells and the time of polarization or only the position of cells was experimentally disrupted. Such embryos developed from the blastomeres that were isolated from 8-cell embryos either before or after the compaction, i.e. before or after the cell polarization took place. We found that in the embryos developed from polar blastomeres originated from the 8-cell compacted embryo, the experimentally imposed outer position was not sufficient to induce the Cdx2 in these blastomeres which in the intact embryo would form the inner cells. However, when the polarization at the 8-cell stage was disrupted, the embryos developed from such an unpolarized blastomeres showed the increased number of cells expressing Cdx2. We found that in such experimentally obtained embryos the polarization was delayed until the 16-cell stage. These results suggest that the main factor responsible for upregulation of Cdx2 expression in outer blastomeres, i.e. TE precursors, is their polarity.

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.

Mouse blastomeres acquire ability to divide asymmetrically before compaction

The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell mass (ICM) during the 8- to 16-cell stage transition, when the apico-basal polarized blastomeres undergo divisions that give rise to cells with different fate. Asymmetric segregation of polar domain at 8–16 cell division generate two cell types, polar cells which adopt an outer position and develop in TE and apolar cells which are allocated to inner position as the precursors of ICM. It is still not know when the blastomeres of 8-cell stage start to be determined to undergo asymmetric division. Here, we analyze the frequency of symmetric and asymmetric divisions of blastomeres isolated from 8-cell stage embryo before and after compaction. Using p-Ezrin as the polarity marker we found that size of blastomeres in 2/16 pairs cannot be used as a criterion for distinguishing symmetric and asymmetric divisions. Our results showed that at early 8-cell stage, before any visible signs of cortical polarity, a subset of blastomeres had been already predestined to divide asymmetrically. We also showed that almost all of 8-cell stage blastomeres isolated from compacted embryo divide asymmetrically, whereas in intact embryos, the frequency of asymmetric divisions is significantly lower. Therefore we conclude that in intact embryo the frequency of symmetric and asymmetric division is regulated by cell-cell interactions.

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.