Metaphase I Arrest Research Articles

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclinB1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosisI. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphaseI (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclinB1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphaseII (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

Single-cell RNA sequencing of human oocytes reveals a differential transcriptomic profile associated with agar-like zona pellucida

BackgroundAgar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP.MethodsHuman metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software.ResultsWe identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP.ConclusionsOur findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest.

Selective utilization of non-homologous end-joining and homologous recombination for DNA repair during meiotic maturation in mouse oocytes.

DNA double-strand breaks (DSBs) are highly toxic lesions that can cause genomic instability and can be repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. Despite extensive studies about DSB repair pathways, the roles of each pathway during meiotic maturation in oocytes are not well understood. Here we show that oocytes selectively utilize NHEJ and HR to repair DSBs during meiotic maturation. Inhibition of NHEJ impaired the meiotic maturation of oocytes with DNA damage by activating the spindle assembly checkpoint (SAC) with a concomitant increase in metaphase I (MI) arrest and DNA damage levels. In contrast, oocytes with DNA damage bypassed SAC-mediated MI arrest despite the presence of fragmented DNA when HR was inhibited. Notably, this bypass of SAC arrest by HR inhibition was associated with a loss of centromere integrity and subsequent impairment of chromosome architecture. Our results demonstrate that, while NHEJ is critical for the meiotic maturation of oocytes with DNA damage, HR is essential to maintain centromere integrity against DNA damage during meiotic maturation, revealing distinct roles of NHEJ and HR during meiotic maturation in mouse oocytes.

Topoisomerase II dysfunction causes metaphase I arrest by activating Aurora B, SAC and MPF and prevents PB1 abscission in mouse oocytes†.

Oocyte aneuploidy is caused mainly by chromosome nondisjunction and/or unbalanced sister chromatid pre-division. Although studies in somatic cells have shown that topoisomerase II (TOP2) plays important roles in chromosome condensation and timely separation of centromeres, little is known about its role during oocyte meiosis. Furthermore, because VP-16, which is a TOP2 inhibitor and induces DNA double strand breaks, is often used for ovarian cancer chemotherapy, its effects on oocytes must be studied for ovarian cancer patients to recover ovarian function following chemotherapy. This study showed that inhibiting TOP2 with either ICRF-193 or VP-16 during meiosis I impaired chromatin condensation, chromosome alignment, TOP2α localization, and caused metaphase I (MI) arrest and first polar body (PB1) abscission failure. Inhibiting or neutralizing either spindle assembly checkpoint (SAC), Aurora B or maturation-promoting factor (MPF) significantly abolished the effect of ICRF-193 or VP-16 on MI arrest. Treatment with ICRF-193 or VP-16 significantly activated MPF and SAC but the effect disappeared when Aurora B was inhibited. Most of the oocytes matured in the presence of ICRF-193 or VP-16 were arrested at MI, and only 11-27% showed PB1 protrusion. Furthermore, most of the PB1 protrusions formed in the presence of ICRF-193 or VP-16 were retracted after further culture for 7h. In conclusion, TOP2 dysfunction causes MI arrest by activating Aurora B, SAC, and MPF, and it prevents PB1 abscission by promoting chromatin bridges.

Iodoacetic acid disrupts mouse oocyte maturation by inducing oxidative stress and spindle abnormalities

Disinfection by-products (DBPs) are compounds produced during the water disinfection process. Iodoacetic acid (IAA) is one of the unregulated DBPs in drinking water, with potent cytotoxicity and genotoxicity in animals. However, whether IAA has toxic effects on oocyte maturation remains unclear. Here, we show that IAA exposure resulted in metaphase I (MI) arrest and polar-body-extrusion failure in mouse oocytes, indicating that IAA had adverse effects on mouse oocyte maturation in vitro. Particularly, IAA treatment caused abnormal spindle assembly and chromosome misalignment. Previous studies reported that IAA is a known inducer of oxidative stress in non-germline cells. Correspondingly, we found that IAA exposure increased the reactive oxygen species (ROS) levels in oocytes in a dose-dependent manner, indicating IAA exposure could induce oxidative stress in oocytes. Simultaneously, DNA damage was also elevated in the nuclei of these IAA-exposed mouse oocytes, evidenced by increased γ-H2AX focus number. In addition, the un-arrested oocytes entered metaphase II (MII) with severe defects in spindle morphologies and chromosome alignment after 14-h IAA treatment. An antioxidant, N-acetyl-L-cysteine (NAC), reduced the elevated ROS level and restored the meiotic maturation in the IAA-exposed oocytes, which indicates that IAA-induced maturation failure in oocytes was mainly mediated by oxidative stress. Collectively, our results indicate that IAA exposure interfered with mouse oocyte maturation by elevating ROS levels, disrupting spindle assembly, inducing DNA damage, and causing MI arrest.

Chk2 regulates cell cycle progression during mouse oocyte maturation and early embryo development.

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein γ-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.

Involvement of mitogen‐activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning

The metaphase I (MI) arrest of starfish oocytes is released after spawning. In this study using starfish Asterina pectinifera, the duration of MI after spawning was ~20 min and approximately 30 min in fertilized and unfertilized oocytes, respectively. This prolongation of MI in unfertilized oocytes, referred to as the MI pause, was maintained by mitogen-activating protein kinase (MAPK) as well as low intracellular pH (approximately 7.0). Contrary to previous reports, MI arrest was not maintained by MAPK, since it was inactive in the oocytes arrested at MI in the ovary and activated immediately after spawning. Also, cyclin B was not degraded at pH 6.7 in the cell-free preparation without MAPK activity, whereas it was degraded at pH 7.0, suggesting that MI arrest was solely maintained by lower pH (< 7.0). Normal development occurred when the spawned oocytes were fertilized before the first polar body formation, whereas fertilization after the first polar body formation increased the rate of abnormal development. Thus, due to MI pause and MI arrest, the probability for fertilization before the polar body formation might be increased, leading to normal development.

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH.

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.

Metaphase I Arrest and Spontaneous Parthenogenetic Activation of Strain LTXBO Oocytes: Chimeric Reaggregated Ovaries Establish Primary Lesion in Oocytes

Oocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF1) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte. Nevertheless, the oocyte's companion somatic cells play crucial roles in the expression of these lesions.

Analysis of the mechanism(s) of metaphase I-arrest in strain LT mouse oocytes: delay in the acquisition of competence to undergo the metaphase I/anaphase transition.

Fully grown oocytes of most laboratory mice progress without interruption from the germinal vesicle (GV) stage to metaphase II, where meiosis is arrested until fertilization. In contrast, many oocytes of strain LT mice arrest precociously at metaphase I and often undergo subsequent spontaneous parthenogenetic activation. Cytostatic factor (CSF), which prevents the degradation of cyclin B and maintains high maturation-promoting factor (MPF) activity, is required for maintenance of metaphase I-arrest in LT oocytes, similar to its requirement for maintaining metaphase II-arrest in normal oocytes. However, CSF does not instigate metaphase I-arrest since a temporary metaphase I-arrest occurs in MOS-null LT oocytes. This paper addresses the mechanism(s) that may instigate metaphase I-arrest and tests the hypothesis that there may be one or more defects in LT oocytes that delay their acquisition of competence to trigger the cascade of processes that normally drive entry into and progression through anaphase I. To test this hypothesis, MPF activity was artificially abrogated by treating oocytes with a general protein kinase inhibitor, 6-DMAP, at various times during the progression of meiosis I. This allowed a comparison of the time at which LT and normal oocytes become competent to undergo the metaphase I/anaphase transition even if oocytes were arrested at metaphase I when 6-DMAP-treatment was begun. There were no differences between LT and control oocytes in the kinetics of MPF suppression by 6-DMAP. However, it was found that LT oocytes do not acquire competence to undergo the metaphase I/anaphase transition in response to 6-DMAP until 50-60 min after normal oocytes. A similar delay was observed in strain CX8-4 oocytes, which also have a high incidence of metaphase I-arrest, but not in strain CX8-11 oocytes, which exhibit a low incidence of metaphase I-arrest. MOS-null LT oocytes also exhibit a delay in acquisition of competence to undergo the metaphase I/anaphase transition. Thus, a delay in competence to undergo the metaphase I/anaphase transition in response to 6-DMAP-treatment correlates with metaphase I-arrest. It is therefore hypothesized that the observed delay in acquisition of competence to enter anaphase I may instigate the sustained metaphase I-arrest in LT oocytes by allowing CSF activity to rise to a level that prevents cyclin B degradation and maintains high MPF activity before anaphase can be initiated by normal triggering mechanisms.

Essential role for gamma -tubulin in the acentriolar female meiotic spindle of Drosophila

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.

Serotonin-Induced Meiosis Reinitiation from the First Prophase and from the First Metaphase in Oocytes of the Marine Bivalve Hiatella flaccida: Respective Changes in Intracellular Ca2+ and pH

In the marine bivalve Hiatella flaccida, full-grown oocytes in ovaries are arrested at the first prophase (prophase-I) of meiosis, whereas spawned oocytes have reinitiated meiosis from prophase-I and are again arrested at the first metaphase (metaphase-I). The neurohormone serotonin (5-hydroxytryptamine, 5-HT) was able to trigger meiosis reinitiation both from prophase-I and from metaphase-I. Exposure of prophase-I oocytes to 5-HT caused an increase in intracellular Ca2+ ([Ca2+]i) composed of an initial towering transient and a following lower but sustained elevation. 5-HT-stimulated prophase-I oocytes also showed a gradual rise in intracellular pH (pHi), reaching a plateau level. None of these 5-HT-induced responses was affected by the complete absence of external Ca2+. On the other hand, these responses were suppressed by preinjection of heparin, an antagonist of inositol 1,4,5-trisphosphate-sensitive receptors. Metaphase-I oocytes also exhibited a [Ca2+]i increase in response to 5-HT; the initial [Ca2+]i transient was larger than that in prophase-I oocytes when stimulated with the same 5-HT concentration. Furthermore, after the initial transient, the elevated [Ca2+]i was not sustained but sometimes returned to the prestimulus level and then increased again. Metaphase-I oocytes had higher resting pHi levels than prophase-I oocytes and showed no significant pHi changes after addition of 5-HT. These results suggest that both a [Ca2+]i increase and a pHi rise are responsible for the release from prophase-I arrest, while a [Ca2+]i increase alone is concerned with the release from metaphase-I arrest.

Pattern and frequency of nocodazole induced meiotic nondisjunction in oocytes of mice carrying the ‘tobacco mouse’ metacentric Rb(16.17)7Bnr

Oocytes from (C3H/HeH x 101/H)F1 and Rb(16.17)7Bnr homozygous females were exposed to a range of doses of nocodazole in vitro. The spindle poison caused a dose dependent increase in metaphase I (MI) arrest and hyperploidy. A concentration of 0.03 microgram/ml was found to induce a maximum hyperploid frequency of 3.1% and 11.6% respectively without a high level of MI arrest. Between 0.03 and 0.05 microgram/ml MI arrest increased substantially and reached a frequency of approximately 90%. In a further experiment oocytes from Rb7 homozygous, heterozygous and 3H1 females were exposed to 0.03 microgram/ml nocodazole 4, 6 or 8 h after the onset of maturation. The phase at which the spindle was inhibited resulted in a specific pattern of nondisjunction which in turn was dependent on whether the female carried an Rb metacentric. 3H1 oocytes gave a normally distributed pattern of increase in aneuploid frequency (over the spontaneous value) centering around a 6 h application. This was thought to be due to the interaction of chromosomes with the microtubules of the spindle during attachment and/or alignment. In contrast both Rb homozygotes and heterozygotes gave the same biphasic response, with a high frequency of aneuploidy in the oocytes when nocodazole was applied 4 and 8 h after the onset of maturation. In Rb homozygotes we demonstrated that the Rb bivalent underwent nondisjunction more frequently than the average acrocentric, when nocodazole was administered early.(ABSTRACT TRUNCATED AT 250 WORDS)

Unusual cytoskeletal and chromatin configurations in mouse oocytes that are atypical in meiotic progression

Meiotic maturation progresses atypically in oocytes of strain LT/Sv and I/LnJ mice. LT/Sv occytes show a high frequency of metaphase I-arrest and parthenogenetic activation. I/LnJ oocytes display retarded kinetics of meiotic maturation and a high frequency of metaphase I-arrest. Some I/LnJ oocytes fail to resume meiosis. Changes in the configuration of chromatin, microtubules, and centrosomes are associated with specific stages of meiotic progression. In this study, the configuration of these subcellular components was examined in LT/Sv, I/LnJ, and C57BL/6J (control) oocytes either freshly isolated from large antral follicles or after culture for 15 hr to allow progression of spontaneous meiotic maturation. Differences were found in the organization of chromatin, microtubules, and centrosomes in LT/Sv and I/LnJ oocytes compared to control oocytes. For example, rather than exhibiting multiple cytoplasmic and nuclear centrosomes as in the normal germinal vesicle-stage oocytes, LT/Sv oocytes typically contain a single large centrosome. In contrast, I/LnJ oocytes displayed many small centrosomes. The microtubules of normal germinal vesicle-stage oocytes were organized as arrays or asters, but microtubules were shorter in LT/Sv oocytes and absent from I/LnJ oocytes. After a 15-hr culture, centrosomal material of normal metaphase II oocytes was organized at both spindle poles. In contrast, metaphase I-arrested LT/Sv oocytes exhibited an elongated spindle with centrosomal material appearing more organized at one pole of the spindle. Both control and LT/Sv oocytes displayed cytoplasmic centrosomes.(ABSTRACT TRUNCATED AT 250 WORDS)