Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

Dnmt1 activity is dispensable in δ-cells but is essential for α-cell homeostasis

In addition to β-cells, pancreatic islets contain α- and δ-cells, which respectively produce glucagon and somatostatin. The reprogramming of these two endocrine cell types into insulin producers, as observed after a massive β-cell ablation in mice, may help restoring a functional β-cell mass in type 1 diabetes. Yet, the spontaneous α-to-β and δ-to-β conversion processes are relatively inefficient in adult animals and the underlying epigenetic mechanisms remain unclear. Several studies indicate that the conserved chromatin modifiers DNA methyltransferase 1 (Dnmt1) and Enhancer of zeste homolog 2 (Ezh2) are important for pancreas development and restrict islet cell plasticity. Here, to investigate the role of these two enzymes in α- and δ-cell development and fate maintenance, we genetically inactivated them in each of these two cell types. We found that loss of Dnmt1 does not enhance the conversion of α- or δ-cells toward a β-like fate. In addition, while Dnmt1 was dispensable for the development of these two cell types, we noticed a gradual loss of α-, but not δ-cells in adult mice. Finally, we found that Ezh2 inactivation does not enhance α-cell plasticity, and, contrary to what is observed in β-cells, does not impair α-cell proliferation. Our results indicate that both Dnmt1 and Ezh2 play distinct roles in the different islet cell types.     

The PKC pathway and in particular its beta1 isoform is clearly involved in meiotic arrest maintenance but poorly in FSH-induced meiosis resumption of the mouse cumulus cell enclosed oocyte

PKC modulators were used to investigate the role of the PKC pathway either on the maintenance of meiotic arrest or on FSH-induced maturation of mouse cumulus cell enclosed oocytes (CEOs). (1) Whereas PKC activation (PMA 8 microM) overcomed clearly the HX-maintained meiotic arrest (83.7 +/- 3.6% vs. 16.1 +/- 10.6% GVBD oocytes), PKC inhibition (Calphostin C 100 nM) did not. On the contrary, it better maintained the meiotic arrest than HX alone. (2) No significant effect of PKC activation or inhibition was observed. (3) HX alone maintained PKCbeta1 in the cytoplasm, whereas FSH and PKC activation induced partly its translocation into the nucleus. The results show that whereas the PKC pathway is clearly involved in maintenance of the meiotic arrest through PKCbeta1, it is not involved in FSH-induced meiosis of CEOs.     

Notch signaling in Sertoli cells regulates cyclical gene expression of Hes1 but is dispensable for mouse spermatogenesis

Mammalian spermatogenesis is a highly regulated system dedicated to the continuous production of spermatozoa from spermatogonial stem cells, and the process largely depends on microenvironments created by Sertoli cells, unique somatic cells that reside within a seminiferous tubule. Spermatogenesis progresses with a cyclical program known as the "seminiferous epithelial cycle," which is accompanied with cyclical gene expression changes in Sertoli cells. However, it is unclear how the cyclicity in Sertoli cells is regulated. Here, we report that Notch signaling, which is known to play an important role for germ cell development in Drosophila and Caenorhabditis elegans, is cyclically activated in Sertoli cells and regulates stage-dependent gene expression of Hes1. To elucidate the regulatory mechanism of stage-dependent Hes1 expression and the role of Notch signaling in mouse spermatogenesis, we inactivated Notch signaling in Sertoli cells by deleting protein O-fucosyltransferase 1 (Pofut1), using the cre-loxP system, and found that stage-dependent Hes1 expression was dependent on the activation of Notch signaling. Unexpectedly, however, spermatogenesis proceeded normally. Our results thus indicate that Notch signaling regulates cyclical gene expression in Sertoli cells but is dispensable for mouse spermatogenesis. This highlights the evolutionary divergences in regulation of germ cell development.     

The hSSB1 orthologue Obfc2b is essential for skeletogenesis but dispensable for the DNA damage response in vivo

Human single-stranded DNA-binding protein 1 (hSSB1), encoded by OBFC2B, was recently characterized as an essential factor for the initiation of DNA damage checkpoints and the maintenance of genomic stability. Here, we report that loss of Obfc2b in mice results in perinatal lethality characterized by growth delay and skeletal abnormalities. These abnormalities are associated with accumulation of γH2ax, apoptosis and defective pre-cartilage condensation, which is essential for normal bone formation. However, deficiency of Obfc2b does not affect the initiation of DNA damage checkpoints, Atm activation, or the maintenance of genomic stability in B lymphocytes and primary fibroblasts. Loss of Obfc2b results in increased expression of its homologue Obfc2a (hSSB2). In contrast to Obfc2b deficiency, depletion of Obfc2a in fibroblasts results in impaired proliferation, accumulation of γH2ax and increased genomic instability. Thus, the hSSB1 orthologue Obfc2b has a unique function during embryogenesis limited to cell types that contribute to bone formation. While being dispensable in most other cell lineages, its absence leads to a compensatory increase in Obfc2a protein, a homologue required for the maintenance of genomic integrity.     

Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis

Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2^−/− and wild-type mice. However, the testes of RA-GEF-2^−/− male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2^−/− males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2^−/− mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2^−/− testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice.     

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.     

The auxin-binding protein 1 is essential for the control of cell cycle

The phytohormone auxin has been known for >50 years to be required for entry into the cell cycle. Despite the critical effects exerted by auxin on the control of cell division, the molecular mechanism by which auxin controls this pathway is poorly understood, and how auxin is perceived upstream of any change in the cell cycle is unknown. Auxin Binding Protein 1 (ABP1) is considered to be a candidate auxin receptor, triggering early modification of ion fluxes across the plasma membrane in response to auxin. ABP1 has also been proposed to mediate auxin-dependent cell expansion, and is essential for early embryonic development. We investigated whether ABP1 has a role in the cell cycle. Functional inactivation of ABP1 in the model plant cell system BY2 was achieved through cellular immunization via the conditional expression of a single-chain fragment variable (scFv). This scFv was derived from a well characterized anti-ABP1 monoclonal antibody previously shown to block the activity of the protein. We demonstrate that functional inactivation of ABP1 results in cell-cycle arrest, and provide evidence that ABP1 plays a critical role in regulation of the cell cycle by acting at both the G1/S and G2/M checkpoints. We conclude that ABP1 is essential for the auxin control of cell division and is likely to constitute the first step of the auxin-signalling pathway mediating auxin effects on the cell cycle.     

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C‐zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ‐related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid‐binding ability, and PLCζ level along with its distribution were evaluated using computer‐assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid‐binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.     

The transmembrane protein AaSho1 is essential for appressorium formation and secondary metabolism but dispensable for vegetative growth in pear fungal Alternaria alternata

The high-osmolarity glycerol response (HOG) pathway is pivotal in environmental stress response, differentiation and virulence of Alternaria alternata. The synthetic high osmolarity sensitive sensor Sho1 has been postulated to regulate the HOG pathway. To determine the regulatory role of transmembrane protein Sho1 on vegetative growth, secondary metabolism and infection structure formation, a gene (AaSho1) encoding Sho1 was cloned and characterized from A. alternata (JT-03). Sequence analysis showed that AaSho1 has all four characteristic transmembrane domains and the SH3 domain present in another Sho1 gene from several filamentous fungal. The quantitative RT-PCR analysis showed that fruit wax extract significantly up-regulated AaSho1 gene expression in vitro. Pharmacological experiments showed that A. alternata treated with nystatin, a specific AaSho1 inhibitor, had no significant effect on the morphology of A. alternata and the invasive growth in pear fruit. However, nystatin treatment significantly reduced spore germination rates on different wax-coated hydrophobic surfaces, with 58.00, 46.70 and 83.72% reduced for fruit wax, beeswax and paraffin coated. Meanwhile, the secondary metabolism altenuene (ALT), tentoxin (TEN) toxin, and melanin content were also affected by nystatin treatment. These findings suggest that AaSho1 is required for the infection structure differentiation and secondary metabolism of A. alternata in response to physiochemical signals on the host surfaces.     

The APC regulator CDH1 is essential for the progression of embryonic cell cycles in Xenopus

The orderly progression of cell cycle depends on timely destruction of key regulators through ubiquitin-mediated proteolysis. The anaphase-promoting complex (APC) is a major component of this degradation machinery and its activation is regulated by CDC20 and CDH1. We demonstrate here that CDH1 mRNA is ubiquitously expressed in Xenopus embryos of all developmental stages. Loss of CDH1 function during early embryonic cell cycles leads to an immediate and prolonged arrest with low cyclin-dependent kinase activity. In contrast, ectopic overexpression of CDH1 induces cell cycle arrest during the first G(1) phase at the midblastula transition. CDH1-dependent degradation of cyclin A is likely involved in this G(1) arrest. Our findings establish the essential roles of CDH1 in embryonic cell cycles.     

The use of tissue microarrays for semiquantitative evaluation of ATPaseC1 expression is ineffective

Abstract

We described earlier the possible role of ATPaseC1 expression as a diagnostic and prognostic marker for oral cancer; others have reported its use for tumors of the lung and breast. We assessed ATPaseC1 expression in a sample of oral squamous cell carcinoma (OSCC) using tissue microarrays (TMAs) to analyze the relation between ATPaseC1 expression and clinical, histopathological and prognostic parameters. We performed a retrospective study of 48 cases of OSCC. We constructed TMAs using two different regions of each tumor. V-ATPaseC1 immunohistochemistry was performed and assessed semiquantitatively. ATPaseC1 staining was observed in most of the neoplastic cells in all tumors. Staining was diffusely cytoplasmic and, to a lesser extent, nuclear. The degree of concordance between the measurements performed in tissue microarray 1 (TMA1) and tissue microarray 2 (TMA2), as evaluated using the intra-class correlation coefficient (ICC), was low. We found great variability in the immunohistochemical staining of the different regions of each tumor. We found 16 cases with mild expression (33.3%), 20 with moderate expression (41.7%) and 12 with intense expression (25%). Differences in the clinical-pathological variables studied were not statistically significant. The difficulty of immunohistochemical evaluation, the heterogeneity of the carcinomas and the fact that evaluation of expression requires semiquantitative analysis render the reliability of the results obtained from TMA-based techniques questionable.     

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF 65) is essential in vivo but dispensable for activity in vitro

The general splicing factor U2AF(65) recognizes the polypyrimidine tract (Py tract) that precedes 3' splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein-protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF(65) activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF(65) in vitro. However, deletion of the RRM3 domain of S. pombe U2AF(59) abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded beta-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.     

The transcription factor C/EBPbeta is essential for inducible expression of the cox-2 gene in macrophages but not in fibroblasts

Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme for the inducible synthesis of prostaglandins, and its up-regulated activity is thought to play a pathological role in diseases such as inflammatory bowel disease, rheumatoid arthritis, and cancer. Regulation of COX-2 expression is complex and appears to involve diversified mechanisms in different cell types and conditions. Here we make use of immortalized macrophages and fibroblasts that we have generated from C/EBPbeta-deficient mice to directly test and compare the specific role played by this factor in inducible COX-2 expression in these two cell types. We could demonstrate that COX-2 mRNA induction and promoter activity were profoundly impaired in C/EBPbeta(-/-) macrophages and could be rescued by expression of C/EBPbeta. The obligatory role of C/EBPbeta in COX-2 expression appeared to be mediated exclusively by the C/EBP element located at positions -138/-130 of the murine cox-2 promoter, and did not involve altered activity at the level of the other promoter elements described previously (the -402/-392 NF-kappaB site, the -59/-48 CRE/E box element, and a potential second C/EBP site located at positions -93/-85). In contrast, COX-2 induction was completely normal in C/EBPbeta-deficient fibroblasts, thus highlighting the diversity of cell-specific molecular mechanisms in determining inducible COX-2 expression and prostaglandins production.     

The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.