54 research outputs found
Mouse gastrocnemius muscle regeneration after mechanical or cardiotoxin injury
The goal of our study was to compare the skeletal muscle regeneration induced by two types of injury: either crushing, that causes muscle degeneration as a result of mechanical devastation of myofibers, or the injection of a cardiotoxin that is a myotoxic agent causing myolysis of myofibers leading to muscle degeneration. Regenerating muscles were analyzed at selected intervals, until the 14th day following the injury. We analyzed their weight and morphology. We also studied the expression of different myosin heavy chain isoforms as a molecular marker of the regeneration progress. Histological analysis revealed that inflammatory response and myotube formation in crushed muscles was delayed compared to cardiotoxin-injected ones. Moreover, the expression of myosin heavy chain isoforms was observed earlier in cardiotoxin-injured versus crushed muscles. We conclude that the dynamics of skeletal muscle regeneration depends on the method of injury
Phosphorylated ERK5/BMK1 transiently accumulates within division spindles in mouse oocytes and preimplantation embryos
MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early
embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic
M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus
potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show
that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase
progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells,
and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the
anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular,
vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in
the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division.
(Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 528–534
Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs
Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained arrested in M-phase, the oocytes fused with fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte activation was still present at the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involved in calcium release triggered by a sperm component at fertilization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage
Temporal regulation of embryonic M-phases.
Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis), the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein), a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit). We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of other candidates is in progress
Early Development of Mouse Embryos Null Mutant for the Cyclin A2 Gene Occurs in the Absence of Maternally Derived Cyclin A2 Gene Products
AbstractProgression through the mammalian cell cycle is regulated by the sequential activation and inactivation of the cyclin-dependent kinases. In adult cells, cyclin A2-dependent kinases are required for entry into S and M phases, completion of S phase, and centrosome duplication. However, mouse embryos lacking the cyclin A2 gene nonetheless complete preimplantation development, but die soon after implantation. In this report, we investigated whether a contribution of maternal cyclin A2 mRNA and protein to early embryonic cell cycles might explain these conflicting observations. Our data show that a maternal stock of cyclin A2 mRNA is present in the oocyte and persists after fertilization until the second mitotic cell cycle, when it is degraded to undetectable levels coincident with transcriptional activation of the zygotic genome. A portion of maternally derived cyclin A2 protein is stable during the first mitosis and persists in the cytoplasm, but is completely degraded at the second mitosis. The ability of cyclin A2-null mutants to develop normally from the four-cell to the postimplantation stage in the absence of detectable cyclin A2 gene product indicates therefore that cyclin A2 is dispensable for cellular progression during the preimplantation nongrowth period of mouse embryo development
Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs
Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained arrested in M-phase, the oocytes fused with fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte activation was still present at the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involved in calcium release triggered by a sperm component at fertilization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage
Stem cells migration during skeletal muscle regeneration - the role of Sdf-1/Cxcr4 and Sdf-1/ Cxcr7 axis
The skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite
cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and
remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as
extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies
aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous
studies we showed that Sdf-1 (stromal derived factor ¡1) increased migration of stem cells and
their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the
expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study
we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1
receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary
myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the
cells of different migration and myogenic potential. We showed that Sdf-1 altered actin
organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-
Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the
transcription profile of genes encoding factors engaged in cells adhesion and migration. As the
result, cells such as primary myoblasts or embryonic stem cells, became characterized by more
effective migration when transplanted into regenerating muscle
Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene
The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of
satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to
promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional
Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied
mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells
under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic
factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells.
Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the
expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of
functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs
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