Ewa Borsuk

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.

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Phosphorylation of histone H3 serine 10 in early mouse embryos: Active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses

Cell division in mammalian cells is regulated by Aurora kinases. The activity of Aurora A is indispensable for correct function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation. Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase. Data concerning the Aurora B activity and H3S10Ph in embryonic cells are limited to primordial and maturing oocytes and advanced pronuclei in zygotes. In the present study we have analyzed H3S10Ph in 1- and 2-cell mouse embryos. We show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and subsequently phosphorylated de novo at late S phase of the first and second cell cycle. These results show that the H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. The behaviour of thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible for de novo H3S10 phosphorylation, most probably Aurora B, are active until G1 of the first cell cycle of mouse embryo. The inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle checkpoint remained active.

Delayed sperm incorporation into parthenogenetic mouse eggs : Sperm nucleus transformation and development of resulting embryos

In this study we examined the effect of experimentally induced asynchrony between male and female pronuclei on male pronucleus formation and developmental potential of the resulting mouse embryos. We demonstrate that when the interval between oocyte activation and sperm incorporation is up to 1.5–2 hr, the spermatozoa transform into normal pronuclei. These male pronuclei can replicate their chromosomes during the first embryonic cell cycle and are transcriptionally competent. During the first cleavage these “delayed” male pronuclei condense into discrete mitotic chromosomes and when resulting embryos are transplanted into oviducts of pregnant females at least some of them can develop to term. In contrast, when sperm nuclei are introduced into parthenogenetic eggs 3 hr or more after activation, their transformation into pronuclei is significantly impaired, and they neither replicate nor transcribe. During the first mitosis they form a group of condensed chromatin, which is displaced into one of the resulting blastomeres leading to formation of haploid/diploid mosaic embryos. These mosaic embryos have poor developmental potential: only a few can reach blastocyst stage in vitro and no full‐term development of such embryos was observed after transfer into pregnant females. We conclude that the cytoplasmic factors that make possible the transformation of a sperm nucleus into a functional male pronucleus exhaust within 1.5–2 hr after fertilization and that the male genome which had skipped the first cell cycle cannot become a functional partner in the embryonic genome. Mol. Reprod. Dev. 54:303–310, 1999.

Association of TCTP with Centrosome and Microtubules

Translationally Controlled Tumour Protein (TCTP) associates with microtubules (MT), however, the details of this association are unknown. Here we analyze the relationship of TCTP with MTs and centrosomes in Xenopus laevis and mammalian cells using immunofluorescence, tagged TCTP expression and immunoelectron microscopy. We show that TCTP associates both with MTs and centrosomes at spindle poles when detected by species-specific antibodies and by Myc-XlTCTP expression in Xenopus and mammalian cells. However, when the antibodies against XlTCTP were used in mammalian cells, TCTP was detected exclusively in the centrosomes. These results suggest that a distinct pool of TCTP may be specific for, and associate with, the centrosomes. Double labelling for TCTP and γ-tubulin with immuno-gold electron microscopy in Xenopus laevis oogonia shows localization of TCTP at the periphery of the γ-tubulin-containing pericentriolar material (PCM) enveloping the centriole. TCTP localizes in the close vicinity of, but not directly on the MTs in Xenopus ovary suggesting that this association requires unidentified linker proteins. Thus, we show for the first time: (1) the association of TCTP with centrosomes, (2) peripheral localization of TCTP in relation to the centriole and the γ-tubulin-containing PCM within the centrosome, and (3) the indirect association of TCTP with MTs.

Polarity and Asymmetry During Mouse Oogenesis and Oocyte Maturation

Cell polarity and asymmetry play a fundamental role in embryo development. The unequal segregation of determinants, cues, and activities is the major event in the differentiation of cell fate and function in all multicellular organisms. In oocytes, polarity and asymmetry in the distribution of different molecules are prerequisites for the progression and proper outcome of embryonic development. The mouse oocyte, like the oocytes of other mammals, seems to apply a less stringent strategy of polarization than other vertebrates. The mouse embryo undergoes a regulative type of development, which permits the full rectification of development even if the embryo loses up to half of its cells or its size is experimentally doubled during the early stages of embryogenesis. Such pliability is strongly related to the proper oocyte polarization before fertilization. Thus, the molecular mechanisms leading to the development and maintenance of oocyte polarity must be included in any fundamental understanding of the principles of embryo development. In this chapter, we provide an overview of current knowledge regarding the development and maintenance of polarity and asymmetry in the distribution of organelles and molecules in the mouse oocyte. Curiously, the mouse oocyte becomes polarized at least twice during ontogenesis; the question of how this phenomenon is achieved and what role it might play is addressed in this chapter.

Appearance and heterochromatin localization of HP1α in early mouse embryos depends on cytoplasmic clock and H3S10 phosphorylation.

Pericentric constitutive heterochromatin surrounds centromeric regions and is important for centromere function and chromatid cohesion. HP1 (heterochromatin protein 1), a homolog of yeast Swi6, has been shown to be indispensible for proper heterochromatin structure and function. In mammalian somatic cells, two HP1 isoforms, HP1α and HP1β, are constitutively present in pericentric heterochromatin until late G2, when they dissociate from heterochromatin. Subsequently, they re-associate with heterochromatin at late anaphase. In one-cell mouse embryos, pericentric heterochromatin has a unique configuration and features. It does not form heterochromatin clusters observed in somatic cells and known as chromocenters. Instead, in both pronuclei, it surrounds nucleolar precursor bodies (NBPs), forming ring-like structures. These regions contain HP1β but lack HP1α in both pronuclei. In subsequent interphases, HP1β is constitutively found in heterochromatin until the blastocyst stage. It is not known when HP1α appears and what is its function in early mouse embryos. Here, we show that HP1α appears for the first time at late S phase of two-cell stage, at the time when pericentric heterochromatin is replicated. Its appearance is regulated at the level of translation. In two-cell embryos, the amount of HP1α that can bind to these regions is regulated by phosphorylation of serine 10 of histone H3 (H3S10Ph). Elimination of HP1α by siRNA interfered with centromere relocation from heterochromatin surrounding NPBs to pro-chromocenters at the two-cell stage but did not affect preimplantation develoment to the blastocyst stage.

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition.

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition

Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic change in the composition of the protein complex upon M-phase exit and the oocyte to embryo transition.

DNA replication and RNA synthesis in thymocyte nuclei microinjected into the cytoplasm of artificially activated mouse eggs

Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thymocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus-like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II-dependent.