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The timing of the M-phase is precisely controlled by a CDC6-dependent mechanism inhibiting the mitotic histone H1 kinase. Here, we describe the differential regulation of the dynamics of this mitotic kinase activity by exogenous cyclin A or cyclin B in the Xenopus laevis cycling extracts. We show that the experimental increase in cyclin A modifies only the level of histone H1 kinase activity, while the cyclin B increase modifies two parameters: histone H1 kinase activity and the timing of its full activation, which is accelerated. On the other hand, the cyclin A depletion significantly delays full activation of histone H1 kinase. However, when CDC6 is added to such an extract, it inhibits cyclin B-associated histone H1 kinase, but does not modify the mitotic timing in the absence of cyclin A. Further, we show via p9 co-precipitation with Cyclin-Dependent Kinases (CDKs), that both CDC6 and the bona fide CDK1 inhibitor Xic1 associate with the mitotic CDKs. Finally, we show that the Xic1 temporarily separates from the mitotic CDKs complexes during the peak of histone H1 kinase activity. These data show the differential coordination of the M-phase progression by cyclin A- and cyclin B-dependent CDKs, confirm the critical role of the CDC6-dependent histone H1 kinase inhibition in this process, and show that CDC6 acts differentially through the cyclin B- and cyclin A-associated CDKs. This CDC6- and cyclins-dependent mechanism likely depends on the precisely regulated association of Xic1 with the mitotic CDKs complexes. We postulate that: i. the dissociation of Xic1 from the CDKs complexes allows the maximal activation of CDK1 during the M-phase, ii. the switch between cyclin A- and cyclin B-CDK inhibition upon M-phase initiation may be responsible for the diauxic growth of mitotic histone H1 kinase activity.

Mesenchymal stem cells (MSCs) are used as therapeutic agents for the treatment of a wide spectrum of diseases, as well as for the regeneration and healing of burns and wounds. MSCs have an immunomodulatory effect and influence the phenotype and functions of immune cells, including macrophages, which in turn prime and license the MSCs. We discuss the new findings on the feedback loop between MSCs and macrophages and its consequences on the outcome of MSC therapies.

Mechanisms governing differentiation of the bipotential gonad into the testes or ovaries are complex and still vague. The primary cilium is an organelle involved in cell signaling, which controls the development of many organs, but the role of primary cilium in the sex determination and sexual differentiation of gonads is com-pletely unknown. Here we studied the expression of genes involved in primary cilium formation and function-ing in fetal mouse gonads, before, during and after sexual differentiation. We studied the expression of 175 primary cilia-related genes using microarray technique. 144 of these genes were ubiquitously expressed in all studied cell types with no significant differences in expression level. Such a high level of expression of primary cilia-related genes in developing mouse gonads suggests that the primary cilia and/or primary cilia-related genes are important for the development of both somatic and germline component of the gonads. Only 31 genes showed a difference in expression between different cell types, which suggests that they have different functions in the somatic and germ cells. These results justify further studies on the role of primary cilia and the primary cilia-related genes in gonad development.

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.

The aim of this short review is to describe the contribution of Xenopus laevis egg extracts to the discovery and understanding of the regulation and function of the serine/threonine kinase Aurora-A. The power of these extracts to recapitulate cell cycle events makes them a precious tool to decipher complex biological processes at the molecular level, including the mechanisms that affect Aurora-A (post-translational modifications) and mechanisms in which Aurora-A plays a crucial role (bipolar spindle assembly). We focus on the results obtained in cell-free extracts, but we also give an updated overview of Aurora A functions found in other systems.

During the cell cycle, cyclin dependent kinase 1 (CDK1) and protein phosphatase 2A (PP2A) play major roles in the regulation of mitosis. CDK1 phosphorylates a series of substrates triggering M-phase entry. Most of these substrates are dephosphorylated by PP2A. To allow phosphorylation of CDK1 substrates, PP2A is progressively inactivated upon M-phase entry. We have shown previously that the interplay between these two activities determines the timing of M-phase entry. Slight diminution of CDK1 activity by the RO3306 inhibitor delays M-phase entry in a dose-dependent manner in Xenopus embryo cell-free extract, while reduction of PP2A activity by OA inhibitor accelerates this process also in a dose-dependent manner. However, when a mixture of RO3306 and OA is added to the extract, an intermediate timing of M-phase entry is observed. Here we use a mathematical model to describe and understand this interplay. Simulations showing acceleration and delay in M-phase entry match previously described experimental data. CDC25 phosphatase is a major activator of CDK1 and acts through CDK1 Tyr15 and Thr14 dephosphorylation. Addition of CDC25 activity to our mathematical model was also consistent with our experimental results. To verify whether our assumption that the dynamics of CDC25 activation used in this model are the same in all experimental variants, we analyzed the dynamics of CDC25 phosphorylation, which reflect its activation. We confirm that these dynamics are indeed very similar in control extracts and when RO3306 and OA are present separately. However, when RO3306 and OA are added simultaneously to the extract, activation of CDC25 is slightly delayed. Integration of this parameter allowed us to improve our model. Furthermore, the pattern of CDK1 dephosphorylation on Tyr15 showed that the real dynamics of CDK1 activation are very similar in all experimental variants. The model presented here accurately describes, in mathematical terms, how the interplay between CDK1, PP2A and CDC25 controls the flexible timing of M-phase entry.

Analysis of cell-free extracts has allowed us to understand many of the fundamental processes of cell physiology and pathology, including those involved in embryo development and cancer. This methodology is being continuously modified and improved. Papers selected for this Special Issue will show readers the plethora of systems and applications of this methodology.

Bidder's organ is an ovary-like structure, which develops from the anterior part of the gonadal ridge in anuran amphibians belonging to the Bufonidae family. Bidder's organs form in both males and females. Because Bidder's organ contains female germ cells (oocytes), the bufonid males are de facto hermaphrodites. Due to similarity with the undeveloped ovary, Bidder's organ was, in early literature, described, inaccurately, as a structure present only in males. Due to the fact that Bidder's organ is a unique structure present only in Bufonidae, it is not well studied and its function still remains a mystery. Here we describe the development and structure of Bidder's organs, summarize the knowledge on gene expression and steroidogenic activity in these organs, and present hypotheses regarding Bidder's organ function.

Harmonious embryo development requires precise coordination between the timing of the cell cycle and the developmental program. Cyclin accumulation determines the timing of the cell cycle M-phase entry and its degradation determines the timing of the M-phase exit. It is well known that CDK1 and PP2A also govern M-phase entry. However, it is unknown how this kinase and phosphatase regulate the precise timing of events at the beginning of the M-phase and how they cooperate with cyclin metabolism. Here we use Xenopus laevis one-cell embryo cell-free extract experiments to answer this question critical for understanding the regulation of embryo development. Using, separately, low concentrations of the chemical inhibitor of CDK1, RO3306 (RO), or the inhibitor of phosphatases, okadaic acid (OA), we show that moderately diminished CDK1 or PP2A activities results in a delay and an acceleration respectively, of M-phase entry. Simultaneous diminution of CDK1 and PP2A activities results in an intermediate timing of M-phase entry, prolongs the duration of M-phase and diminishes the dynamics of cyclin B2 degradation. We thus show, for the first time, that equilibrium between CDK1 and PP2A specifies the timing of M-phase entry and exit and regulates the dynamics of cyclin B degradation upon M-phase exit in Xenopus laevis first embryonic mitosis.

The gonads develop as the metameric mesodermal structures at the ventral surface of the mesonephroi. To study the evolutionary trends for anuran gonads, we performed comparative analysis of three species from the basal paraphyletic group - Archaeobatrachia (Bombina bombina, Xenopus laevis, Pelobates fuscus) and five species from more derived monophyletic Neobatrachia group (Hyla arborea, Bufotes viridis, Rana dalmatina, Rana arvalis, Rana temporaria). Light and scanning electron microscopy analysis showed that in Archaeobtrachia the undifferentiated gonads and ovaries, but not the testes, have pronounced external metamery. In contrast, Neobatrachia lacked external gonadal metamery and the internal metamery was well pronounced in the ovaries, but only rudimentary in the testes. There was also a difference in the site of genital ridge formation between these two groups. In Archaeobatrachia, the genital ridges developed along almost the entire length of mesonephroi, while in Neobatrachia they were limited to the anterior parts of the mesonephroi. A transient form was observed in P. fuscus, where the gonads formed along the anterior half of the mesonephroi. There was also an evolutionary trend for gradual postero-anterior shortening of the gonadal anlagen. In summary, our study indicates that among anurans there is an evolutionary trend for the gradual loss of metamery and shortening of the gonad.

In vertebrates, several genes which are differentially expressed in various species, have been implicated in sex determination and gonadal differentiation. We used immunolocalization to study the expression pattern of three proteins AMH, DMRT1, RSPO1 involved in the sexual differentiation of gonads. The pattern of AMH, DMRT1 and RSPO1 expression was analyzed in X. laevis and in five other divergent anuran species: Bombina bombina, Bufo viridis, Hyla arborea, Rana arvalis and Rana temporaria during gonadal development. The pattern of expression of AMH in the developing testes of six studied anuran species was similar to that described for other vertebrates. AMH was strongly expressed in differentiating Sertoli cells. Interestingly, in B. viridis, R. arvalis and R. temporaria, AMH was also expressed in ovaries. In all studied species, DMRT1 was highly expressed in the developing testes, in both the somatic and germ cells. It was also expressed at low level in ovaries in all studied species, with the exception of H. arborea. RSPO1 was expressed in the developing ovaries, especially in the somatic cells, and was almost undetectable in developing testes in all examined anurans. These developmental expression patterns strongly suggest an involvement of AMH and DMRT1 in the development of male gonads and of RSPO1 in the female gonads. The differences in the expression patterns of these proteins in the gonads of different species might reflect the diversity of gonadal development patterns in anurans resulting from long lasting and diverged paths of their evolution.

Here, we outline the mechanisms involved in the regulation of cell divisions during oocyte maturation and early cleavages of the mouse embryo. Our interest is focused on the regulation of meiotic M-phases and the first embryonic mitoses that are differently tuned and are characterized by specifically modified mechanisms, some of which have been recently identified. The transitions between the M-phases during this period of development, as well as associated changes in their regulation, are of key importance for both the meiotic maturation of oocytes and the further development of the mammalian embryo. The mouse is an excellent model for studies of the cell cycle during oogenesis and early development. Nevertheless, a number of molecular mechanisms described here were discovered or confirmed during the study of other species and apply also to other mammals including humans.

Cyclin-dependent kinase 1 (CDK1) is the enzymatic subunit of M-phase Promoting Factor (MPF). It is positively regulated by phosphorylation on Thr-161 and association with a cyclin B molecule. The role of Thr-161 dephosphorylation upon MPF inactivation remains unclear; nevertheless, degradation of cyclin B is thought to be a direct cause of MPF inactivation. However, MPF inactivation actually precedes cyclin B degradation in Xenopus cell-free extracts. Here we study in details the temporal relationship between histone H1 kinase (reflecting MPF activity) inactivation, Thr-161 dephosphorylation, CDK1-cyclin B2 dissociation and cyclin B2 proteolysis in such extracts. We show an asynchrony between inactivation of histone H1 kinase and degradation of cyclin B2. CDK1 dephosphorylation on Thr 161 is an even later event than cyclin B2 degradation, reinforcing the hypothesis that cyclin B dissociation from CDK1 is the key event inactivating MPF. Cyclins synthesized along with MPF inactivation could deliver shortly living active MPF molecules, potentially increasing the asynchrony between histone H1 kinase inactivation and cyclin B2 degradation. We confirm this by showing that in the absence of protein synthesis, such a tendency is lower, but nevertheless, still detectable. Finally, to characterise better CDK1/cyclin B dissociation, we show that CDK1 begins to dissociate from cyclin B2 before the very beginning of cyclin B2 degradation and that the diminution in CDK1-associated cyclin B2 is faster than the decline of its total pool. Thus, neither cyclin B2 degradation nor Thr-161 dephosphorylation participates directly in CDK1 inactivation as measured by histone H1 kinase decline upon the exit from mitotic M-phase in Xenopus embryo extract.

The behavior of microtubules was studied in hybrids formed between mouse oocytes arrested in metaphase II or activated parthenogenetically and mouse embryo interphase blastomeres. In all cases the interphase blastomere's network of microtubules disassembles rapidly after fusion with oocytes. Introduction of interphase cytoplasm and nuclei to metaphase oocytes during fusion induces the polymerization of new microtubules in the cytoplasm and in the meiotic spindle. The degree and the duration of this facilitated polymerization of microtubules was positively correlated with the volume of blastomeres used for fusion. The blastomere nuclei induce the formation of microtubular frames, which become more evident when the chromatin undergoes premature condensation. Finally, spindle-like structures are formed around the prematurely condensed chromosomes. In hybrids activated around the time of fusion, the blastomere nuclei undergo pronuclear-like transformation. These hybrids develop an interphase network of microtubules typical for activated oocytes. These results are discussed with regards to the cell cycle control of microtubule behavior.