HaiYang Wang

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos.

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.

In silico identification of conserved microRNAs and their targets in bovine fat tissue

MicroRNAs (miRNAs) represent a newly identified class of non-protein-coding ~22 nt small RNA which plays important roles in multiple biological processes by degrading targeted mRNA or repressing mRNA translation. Here we present EST (expressed sequence tags)-based and GSS (Genomic Survey Sequences)-based combined approach for the detection of conserved miRNAs of cattle. A total of 20 conserved miRNAs that belong to 18 miRNA families were detected following a range of filtering criteria; their functions were further predicted and analyzed. To confirm our prediction, a miRNA-detecting microarray was designed with probes complementary to previously known mature miRNA sequences from 131 organisms. After hybridizing with small RNAs extracted from beef cattle subcutaneous fat tissue, 219 (32.30%) miRNAs were detected in the 679 known Bos taurus miRNAs and all the miRNAs predicted above were also detected. Conformation of 22 most abundant miRNA expression by qRT-PCR indicated that they were highly accumulated not only in subcutaneous fat tissue but also in intramuscular fat tissue. Bioinformatics of KEGG pathway analysis suggested that 4 differential expression miRNAs (miR-143, miR-145, miR-2325c and miR-2361) involved in different pathways and target genes may regulate the fat deposition differently. Taken together, our results expand the number of known bovine miRNAs and provide a thorough account of the miRNA transcriptome in bovine adipose tissue.

CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development

ABSTRACT Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

DNA double‐strand breaks disrupted the spindle assembly in porcine oocytes

We used etoposide (25–100 µg/mL) to induce DNA double‐strand breaks (DSBs) in porcine oocytes at the germinal vesicle (GV) stage to determine how such damage affects oocyte maturation. We observed that DNA damage did not delay the rate of germinal vesicle breakdown (GVBD), but did inhibit the final stages of maturation, as indicated by the failure to extrude the first polar body. Oocytes with low levels of DSBs failed to effectively activate ataxia telangiectasia‐mutated (ATM) kinase, while those with severe DNA DSBs failed to activate checkpoint kinase 1 (CHK1)—the two regulators of the DNA damage response pathway—indicating that porcine oocytes lack an efficient G2/M phase checkpoint. DSBs induced spindle defects and chromosomal misalignments, leading to the arrest of these oocytes at meiotic metaphase I. The activity of maturation‐promoting factor also did not increase appropriately in oocytes with DNA DSBs, although its abundance was sufficient to promote GVBD and chromosomal condensation. Following parthenogenetic activation, embryos from etoposide‐treated oocytes formed numerous micronuclei. Thus, our results indicate that DNA DSBs do not efficiently activate the ATM/CHK1‐dependent DNA‐damage checkpoint in porcine oocytes, allowing these DNA‐impaired oocytes to enter M phase. Oocytes with DNA damage did, however, arrest at metaphase I in response to spindle defects and chromosomal misalignments, which limited the ability of these oocytes to reach meiotic metaphase II. Mol. Reprod. Dev. 83: 132–143, 2016.

Quercetin delays postovulatory aging of mouse oocytes by regulating SIRT expression and MPF activity

If no fertilization occurs at an appropriate time after ovulation, oocyte quality deteriorates rapidly as a process called postovulatory aging. Because the postovulatory aging of oocytes has detrimental effects on embryo development and offspring, many efforts have been made to prevent oocyte aging. Here we showed that quercetin prevented the decline in oocyte quality during postovulatory aging of oocytes. Quercetin treatment reduced aging-induced morphological changes and reactive oxygen species accumulation. Moreover, quercetin attenuated the aging-associated abnormalities in spindle organization and mitochondrial distribution, preventing decrease of SIRT expression and histone methylation. Quercetin also ameliorated the decrease in maturation-promoting factor activity and the onset of apoptosis during postovulatory aging. Furthermore, quercetin treatment during postovulatory aging improves early embryo development. Our results demonstrate that quercetin relieves deterioration in oocyte quality and improves subsequent embryo development.

Protein phosphatase 2A regulatory subunit B55α functions in mouse oocyte maturation and early embryonic development

Protein phosphatase 2A regulatory subunit B55α (PP2A-B55α) has been studied in mitosis. However, its functions in mammalian meiosis and early embryonic development remain unknown. Here, we report that PP2A-B55α is critical for mouse oocyte meiosis and preimplantation embryo development. Knockdown of PP2A-B55α in oocytes led to abnormal asymmetric division, disordered spindle dynamics, defects in chromosome congression, an increase in aneuploidy, and induction of the DNA damage response. Moreover, knockdown of PP2A-B55α in fertilized mouse zygotes impaired development to the blastocyst stage. The impairment of embryonic development might have been due to induction of sustained DNA damage in embryos, which caused apoptosis and inhibited cell proliferation and outgrowth potential at the blastocyst stage. Overall, these results provide a novel insight into the role of PP2A-B55α as a novel meiotic and embryonic competence factor at the onset of life.

CIP2A acts as a scaffold for CEP192-mediated microtubule organizing center assembly by recruiting Plk1 and aurora A during meiotic maturation

In somatic cells spindle microtubules are nucleated from centrosomes that act as major microtubule organizing centers (MTOCs), whereas oocytes form meiotic spindles by assembling multiple acentriolar MTOCs without canonical centrosomes. Aurora A and Plk1 are required for these events, but the underlying mechanisms remain largely unknown. Here we show that CIP2A regulates MTOC organization by recruiting aurora A and Plk1 at spindle poles during meiotic maturation. CIP2A colocalized with pericentrin at spindle poles with a few distinct cytoplasmic foci. Although CIP2A has been identified as an endogenous inhibitor of protein phosphatase 2A (PP2A), overexpression of CIP2A had no effect on meiotic maturation. Depletion of CIP2A perturbed normal spindle organization and chromosome alignment by impairing MTOC organization. Importantly, CIP2A was reciprocally associated with CEP192, promoting recruitment of aurora A and Plk1 at MTOCs. CIP2A was phosphorylated by Plk1 at S904, which targets CIP2A to MTOCs and facilitates MTOC organization with CEP192. Our results suggest that CIP2A acts as a scaffold for CEP192-mediated MTOC assembly by recruiting Plk1 and aurora A during meiotic maturation in mouse oocytes. Summary: Plk1-mediated phosphorylation at the coiled-coil domain targets CIP2A to the microtubule organizing center, where it acts independently of PP2A in spindle assembly during meiotic maturation of mouse oocytes.

The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms

In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity.

Inhibition of CDK7 bypasses spindle assembly checkpoint via premature cyclin B degradation during oocyte meiosis.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.

Correction: CIP2A acts as a scaffold for CEP192-mediated microtubule organizing center assembly by recruiting Plk1 and aurora A during meiotic maturation (doi:10.1242/dev.158584)

There was an error published in Development (2017) 144 , [3829-3839][1] (doi:[10.1242/dev.158584)][2]. In [Fig. 4][3]E, the incorrect Plk1 image was displayed for dsCIP2A+hCIP2A (it was a duplicate of the dsEGFP image). The corrected [Fig. 4][3]E appears below. ![Figure][4] This error does