CHK1-CDC25A-CDK1 regulate cell cycle progression and protect genome integrity in early mouse embryos.

After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated and transcriptionally quiescent cells into early cleavage-stage blastomeres that are transcriptionally active and totipotent. This developmental transition is accompanied by cell cycle adaptation, such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in two-cell stage mouse embryos. Finally, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.

Using ZINC08918027 inhibitor to determine Aurora kinase-chromosomal passenger complex isoforms in mouse oocytes.

OBJECTIVE: Miscarriages affect 10% of women aged 25-29, and 53% of women over 45. The primary cause of miscarriage is aneuploidy that originated in eggs. The Aurora kinase family has three members that regulate chromosome segregation. Therefore, distinguishing the roles of these isoforms is important to understand aneuploidy etiology. In meiosis, Aurora kinase A (AURKA) localizes to spindle poles, where it binds TPX2. Aurora kinase C (AURKC) localizes on chromosomes, where it replaces AURKB as the primary AURK in the chromosomal passenger complex (CPC) via INCENP binding. Although AURKA compensates for CPC function in oocytes lacking AURKB/C, it is unknown whether AURKA binds INCENP in wild type mouse oocytes. ZINC08918027 (ZC) is an inhibitor that prevents the interaction between AURKB and INCENP in mitotic cells. We hypothesized that ZC would block CPC function of any AURK isoform. RESULTS: ZC treatment caused defects in meiotic progression and spindle building. By Western blotting and immunofluorescence, we observed that activated AURKA and AURKC levels in ZC-treated oocytes decreased compared to controls. These results suggest there is a population of AURKA-CPC in mouse oocytes. These data together suggest that INCENP-dependent AURKA and AURKC activities are needed for spindle bipolarity and meiotic progression.

CDC25B is required for the metaphase I-metaphase II transition in mouse oocytes.

Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

Aurora kinase A is essential for meiosis in mouse oocytes.

The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile, and their oocytes fail to complete meiosis I. In determining AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at acentriolar microtubule organizing centers (aMTOCs; meiotic spindle poles). This activation induces fragmentation of the aMTOCs, a step essential for building a bipolar spindle. We also show that AURKA is required for regulating localization of TACC3, another protein required for spindle building. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.

The most abundant maternal lncRNA Sirena1 acts post-transcriptionally and impacts mitochondrial distribution.

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1-/- oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.

RanGTP and importin ß regulate meiosis I spindle assembly and function in mouse oocytes.

Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin ß signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin ß and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin ß in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

Genetic Interactions between the Aurora Kinases Reveal New Requirements for AURKB and AURKC during Oocyte Meiosis.

Errors in chromosome segregation during female meiosis I occur frequently, and aneuploid embryos account for 1/3 of all miscarriages in humans [1]. Unlike mitotic cells that require two Aurora kinase (AURK) homologs to help prevent aneuploidy (AURKA and AURKB), mammalian germ cells also require a third (AURKC) [2, 3]. AURKA is the spindle-pole-associated homolog, and AURKB/C are the chromosome-localized homologs. In mitosis, AURKB has essential roles as the catalytic subunit of the chromosomal passenger complex (CPC), regulating chromosome alignment, kinetochore-microtubule attachments, cohesion, the spindle assembly checkpoint, and cytokinesis [4, 5]. In mouse oocyte meiosis, AURKC takes over as the predominant CPC kinase [6], although the requirement for AURKB remains elusive [7]. In the absence of AURKC, AURKB compensates, making defining potential non-overlapping functions difficult [6, 8]. To investigate the role(s) of AURKB and AURKC in oocytes, we analyzed oocyte-specific Aurkb and Aurkc single- and double-knockout (KO) mice. Surprisingly, we find that double KO female mice are fertile. We demonstrate that, in the absence of AURKC, AURKA localizes to chromosomes in a CPC-dependent manner. These data suggest that AURKC prevents AURKA from localizing to chromosomes by competing for CPC binding. This competition is important for adequate spindle length to support meiosis I. We also describe a unique requirement for AURKB to negatively regulate AURKC to prevent aneuploidy. Together, our work reveals oocyte-specific roles for the AURKs in regulating each other's localization and activity. This inter-kinase regulation is critical to support wild-type levels of fecundity in female mice.

Molecular evidence of infection with air sac nematodes in the great tit (Parus major) and the captive-bred gyrfalcon (Falco rusticolus).

Serratospiculiasis is a parasitic disease caused by filariid nematodes of the genus Serratospiculum that parasitise the air sacs of various species of falcons, bald eagles and Cooper's hawks around the world. An infection with Serratospiculum was recently confirmed in a nonspecific host, the great tit, in Slovakia. Parasitic material from this host was fixed for molecular analysis. Nematode found in the air sacs from a captive-bred gyrfalcon was also stored. Analysis of small subunit (18S) ribosomal DNA (18S rDNA) gene indicated that sequences from Serratospiculum sp. and Serratospiculoides amaculata were closely related to a reference sequence from Serratospiculum tendo, in agreement with morphology. This study is the first to generate molecular data and infer the phylogenetic position of S. amaculata as the first representative of the genus Serratospiculoides.

Haspin kinase regulates microtubule-organizing center clustering and stability through Aurora kinase C in mouse oocytes.

Meiotic oocytes lack classic centrosomes and, therefore, bipolar spindle assembly depends on clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. However, the molecular mechanism regulating MTOC assembly into two poles is not fully understood. The kinase haspin (also known as GSG2) is required to regulate Aurora kinase C (AURKC) localization at chromosomes during meiosis I. Here, we show that inhibition of haspin perturbed MTOC clustering into two poles and the stability of the clustered MTOCs. Furthermore, we show that AURKC localizes to MTOCs in mouse oocytes. Inhibition of haspin perturbed the localization of AURKC at MTOCs, and overexpression of AURKC rescued the MTOC-clustering defects in haspin-inhibited oocytes. Taken together, our data uncover a role for haspin as a regulator of bipolar spindle assembly by regulating AURKC function at acentriolar MTOCs in oocytes.

Chalcone derivatives cause accumulation of colon cancer cells in the G2/M phase and induce apoptosis.

AIMS: Chalcones, naturally occurring open-chain polyphenols abundant in plants, have demonstrated antiproliferative activity in several cancer cell lines. In the present study, the potential anticancer activity of two synthetic analogues named Ch1 and Ch2 in colon cancer cell line was investigated. MAIN METHODS: Antiproliferative activities of both synthetic analogues were assessed by Growth Inhibition Assay (MTT) and xCELLigence cell analysis. Apoptosis was assessed by annexin V/PI staining (early stage) or by DNA fragmentation (final stage). To study the cell death mechanism induced by tested substances, we assessed a series of assays including measurements of the caspase 3 activity, membrane mitochondrial potential (MMP) changes, reactive oxygen species (ROS) production by flow cytometry and expression of important apoptosis-related genes by realtime PCR. KEY FINDINGS: We found concentration and time-dependent cytotoxicity, inhibition of proliferation of Caco-2 cells after Ch1 and Ch2 treatment in parallel with G2/M phase cell cycle arrest and increased cell proportion in subG0/G1 population with annexin V positivity. We demonstrated that both Ch1 and Ch2 induced caspase-dependent cell death associated with increased ROS production, suppressed Bcl-2 and Bcl-xL and enhanced Bax expression. Treatment of Ch1 also suppressed α-, α1- and ß5-tubulins, on the other hand Ch2 only suppressed α-tubulin expression. SIGNIFICANCE: Presented chalcones induce apoptosis by intrinsic pathways, and therefore may be an interesting strategy for cancer therapy.

ROS-dependent antiproliferative effect of brassinin derivative homobrassinin in human colorectal cancer Caco2 cells.

This study was designed to examine the in vitro antiproliferative effect of brassinin and its derivatives on human cancer cell lines. Among seven tested compounds, homobrassinin (K1; N-[2-(indol-3-yl)ethyl]-S-methyldithiocarbamate) exhibited the most potent activity with IC50 = 8.0 µM in human colorectal Caco2 cells and was selected for further studies. The flow cytometric analysis revealed a K1-induced increase in the G2/M phase associated with dysregulation of α-tubulin, α1-tubulin and ß5-tubulin expression. These findings suggest that the inhibitory effect of K1 can be mediated via inhibition of microtubule formation. Furthermore, simultaneously with G2/M arrest, K1 also increased population of cells with sub-G1 DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V/PI double staining, DNA fragmentation assay and chromatin condensation assay. The apoptosis was associated with the loss of mitochondrial membrane potential (MMP), caspase-3 activation as well as intracellular reactive oxygen species (ROS) production. Moreover, the antioxidant Trolox blocked ROS production, changes in MMP and decreased K1 cytotoxicity, which confirmed the important role of ROS in cell apoptosis. Taken together, our data demonstrate that K1 induces ROS-dependent apoptosis in Caco2 cells and provide the rationale for further in vivo anticancer investigation.

Benzylidenetetralones, cyclic chalcone analogues, induce cell cycle arrest and apoptosis in HCT116 colorectal cancer cells.

Colorectal cancer is the third most common cancer in the world, with 1.2 million new cancer cases annually. Chalcones are secondary metabolite precursors of flavonoids that exhibit diverse biological activities, including antioxidant and antitumor activities. The aim of this study was to investigate the antiproliferative effect of new synthetic chalcone derivatives on HCT116 cells. (E)-2-(2',4'-dimethoxybenzylidene)-1-tetralone (Q705) was found to be the most active (IC50 = 3.44 ± 0.25 µM). Based on these results, this compound was chosen for further analysis of its biochemical and molecular mechanisms. Our results showed that Q705 inhibited the growth and clonogenicity of HCT116 cells. The results of a flow cytometric analyses suggested that this compound caused a significant cell cycle arrest in G2/M phase and increased the proportion of cells in the subG0/G1 phase, marker of apoptosis. Q705-induced apoptosis was confirmed by TdT-mediated dUTP nick end labelling (TUNEL) assay. Treatment of HCT116 cells with this chalcone significantly increased the caspase-3,-7 activity and resulted in cleavage of poly-ADP-ribose polymerase (PARP). Changes in the nuclear morphology such as chromatin condensation were also observed. These effects were associated with a decreased expression of bcl-xL and increased overall ratio of bax/bcl-xL mRNA levels. Immunofluorescence and qRT-PCR analysis revealed that Q705 induced H2AX histone modifications characteristic of DNA damage, disruption of microtubule organization and downregulation of tubulins. In summary, these results suggest that the cyclic chalcone analogue Q705 has potential as a new compound for colorectal cancer therapy.

Brassinin and its derivatives as potential anticancer agents.

The aim of the study was to investigate the anti-proliferative activity of brassinin and its derivatives on human cancer cell lines. We found that among twenty-one tested compounds, 1- methoxybrassinin exerted the most potent anti-proliferative activity in Caco-2 cells with IC50 8.2 (±1.2)µmoll(-1). The flow cytometric analysis revealed a 1-methoxybrassinin-induced increase in the sub-G1 DNA content fraction which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by DNA fragmentation assay. Moreover, quantitative real-time PCR showed that 1-methoxybrassinin upregulated the expression of pro-apoptotic Bax and downregulated the expression of anti-apoptotic genes Bcl-2 and Bcl-xL. The compound also increased activity of caspase-3, -7, cleaved PARP and decreased intracellular GSH content. The present study has assessed the in vitro anti-proliferative potential of 1-methoxybrassinin. The results generate a rationale for in vivo efficacy studies with this compound in preclinical cancer models.