Chromosomal and cytoplasmic context determines predisposition to maternal age-related aneuploidy: brief overview and update on MCAK in mammalian oocytes

It has been known for more than half a century that the risk of conceiving a child with trisomy increases with advanced maternal age. However, the origin of the high susceptibility to nondisjunction of whole chromosomes and precocious separation of sister chromatids, leading to aneuploidy in aged oocytes and embryos derived from them, cannot be traced back to a single disturbance and mechanism. Instead, analysis of recombination patterns of meiotic chromosomes of spread oocytes from embryonal ovary, and of origins and exchange patterns of extra chromosomes in trisomies, as well as morphological and molecular studies of oocytes and somatic cells from young and aged females, show chromosome-specific risk patterns and cellular aberrations related to the chronological age of the female. In addition, analysis of the function of meiotic- and cell-cycle-regulating genes in oogenesis, and the study of the spindle and chromosomal status of maturing oocytes, suggest that several events contribute synergistically to errors in chromosome segregation in aged oocytes in a chromosome-specific fashion. For instance, loss of cohesion may differentially predispose chromosomes with distal or pericentromeric chiasmata to nondisjunction. Studies on expression in young and aged oocytes from human or model organisms, like the mouse, indicate that the presence and functionality/activity of gene products involved in cell-cycle regulation, spindle formation and organelle integrity may be altered in aged oocytes, thus contributing to a high risk of error in chromosome segregation in meiosis I and II. Genes that are often altered in aged mouse oocytes include MCAK (mitotic-centromere-associated protein), a microtubule depolymerase, and AURKB (Aurora kinase B), a protein of the chromosomal passenger complex that has many targets and can also phosphorylate and regulate MCAK localization and activity. Therefore we explored the role of MCAK in maturing mouse oocytes by immunofluorescence, overexpression of a MCAK-EGFP (enhanced green fluorescent protein) fusion protein, knockdown of MCAK by RNAi (RNA interference) and inhibition of AURKB. The observations suggest that MCAK is involved in spindle regulation, chromosome congression and cell-cycle control, and that reductions in mRNA and protein in a context of permissive SAC (spindle assembly checkpoint) predispose to aneuploidy. Failure to recruit MCAK to centromeres and low expression patterns, as well as disturbances in regulation of enzyme localization and activity, e.g. due to alterations in activity of AURKB, may therefore contribute to maternal age-related rises in aneuploidy in mammalian oocytes.     

A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I

The Spindle Assembly Checkpoint (SAC) inhibits anaphase until microtubule-to-kinetochore attachments are formed, thus securing correct chromosome separation and preventing aneuploidy. Whereas in mitosis even a single unattached chromosome keeps the SAC active, the high incidence of aneuploidy related to maternal meiotic errors raises a concern about the lower efficiency of SAC in oocytes. Recently it was suggested that in mouse oocytes, contrary to somatic cells, not a single chromosome but a critical mass of chromosomes triggers efficient SAC pointing to the necessity of evaluating the robustness of SAC in oocytes. Two types of errors in chromosome segregation upon meiosis I related to SAC were envisaged: (1) SAC escape, when kinetochores emit SAC-activating signal unable to stop anaphase I; and (2) SAC deceive, when kinetochores do not emit the signal. Using micromanipulations and live imaging of the first polar body extrusion, as well as the dynamics of cyclin B1 degradation, here we show that in mouse oocytes a single bivalent keeps the SAC active. This is the first direct evaluation of SAC efficiency in mouse oocytes, which provides strong evidence that the robustness of SAC in mammalian oocytes is comparable to other cell types. Our data do not contradict the hypothesis of the critical mass of chromosomes necessary for SAC activation, but suggest that the same rule may govern SAC activity also in other cell types. We postulate that the innate susceptibility of oocytes to errors in chromosome segregation during the first meiotic division may not be caused by lower efficiency of SAC itself, but could be linked to high critical chromosome mass necessary to keep SAC active in oocyte of large size.     

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2 ⁺ and mad3 ⁺, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1 ⁺ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.     

Age‐dependent aneuploidy in mammalian oocytes instigated at the second meiotic division

Ageing severely affects the chromosome segregation process in human oocytes resulting in aneuploidy, infertility and developmental disorders. A considerable amount of segregation errors in humans are introduced at the second meiotic division. We have here compared the chromosome segregation process in young adult and aged female mice during the second meiotic division. More than half of the oocytes in aged mice displayed chromosome segregation irregularities at anaphase II, resulting in dramatically increased level of aneuploidy in haploid gametes, from 4% in young adult mice to 30% in aged mice. We find that the post‐metaphase II process that efficiently corrects aberrant kinetochore‐microtubule attachments in oocytes in young adult mice is approximately 10‐fold less efficient in aged mice, in particular affecting chromosomes that show small inter‐centromere distances at the metaphase II stage in aged mice. Our results reveal that post‐metaphase II processes have critical impact on age‐dependent aneuploidy in mammalian eggs.     

Lack of response to unaligned chromosomes in mammalian female gametes

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.     

Lack of response to unaligned chromosomes in mammalian female gametes

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.     

Mad2 and spindle assembly checkpoint function during meiosis I in mammalian oocytes

During mammalian mitosis, a proofreading network called the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation. An inhibitory SAC signal is deputed to inhibits mitotic cell-cycle progression in response to misaligned chromosomes until such imperfections are rectified thereby ensuring equitable chromosome partitioning to daughter cells. Amongst the cast of SAC proteins, mitotic arrest deficient 2 (Mad2) plays a leading role in transducing the SAC signal. The aneuploidy and cancer predispositions of individuals who harbour genetic mutations in SAC genes emphasise the in vivo significance of this surveillance mechanism. In humans, congenital aneuploidies such as Down's syndrome demonstrate an exponential increase with advancing female age. Although largely the result of female meiosis I errors, the molecular entities that succumb with age in oocytes remain elusive. Declining oocyte SAC function could plausibly contribute to such errors. Until recently however, convincing evidence for a functional SAC in mammalian oocytes during meiosis I was unforthcoming. Here I review the evidence regarding the SAC in female mammalian meiosis I and how our understanding of this system has evolved in recent years. This review will focus on Mad2 as this is the SAC protein that has been most comprehensively investigated.     

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.     

Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe

Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature. In a mutant dis1 background (dis1-288), loss of function of Msc1, a fission yeast homolog of the KDM5 family of proteins, suppresses the growth defect and promotes normal mitosis. Genetic analysis implicates a histone deacetylase (HDAC)–linked pathway in suppression because HDAC mutants clr6-1, clr3∆, and sir2∆, though not hos2∆, also promote normal mitosis in the dis1-288 mutant. Suppression of the dis phenotype through loss of msc1 function requires the spindle checkpoint protein Mad2 and is limited by the presence of the heterochromatin-associated HP1 protein homolog Swi6. We speculate that alterations in histone acetylation promote a centromeric chromatin environment that compensates for compromised dis1 function by allowing for successful kinetochore-microtubule interactions that can satisfy the SAC. In cells arrested in mitosis by mutation of dis1, loss of function of epigenetic determinants such as Msc1 or specific HDACs can promote cell survival. Because the KDM5 family of proteins has been implicated in human cancers, an appreciation of the potential role of this family of proteins in chromosome segregation is warranted.     

Aurora kinase B modulates chromosome alignment in mouse oocytes

The elevated incidence of aneuploidy in human oocytes warrants study of the molecular mechanisms regulating proper chromosome segregation. The Aurora kinases are a well‐conserved family of serine/threonine kinases that are involved in proper chromosome segregation during mitosis and meiosis. Here we report the expression and localization of all three Aurora kinase homologs, AURKA, AURKB, and AURKC, during meiotic maturation of mouse oocytes. AURKA, the most abundantly expressed homolog, localizes to the spindle poles during meiosis I (MI) and meiosis II (MII), whereas AURKB is concentrated at kinetochores, specifically at metaphase of MI (Met I). The germ cell‐specific homolog, AURKC, is found along the entire length of chromosomes during both meiotic divisions. Maturing oocytes in the presence of the small molecule pan‐Aurora kinase inhibitor, ZM447439 results in defects in meiotic progression and chromosome alignment at both Met I and Met II. Over‐expression of AURKB, but not AURKA or AURKC, rescues the chromosome alignment defect suggesting that AURKB is the primary Aurora kinase responsible for regulating chromosome dynamics during meiosis in mouse oocytes. Mol. Reprod. Dev. 76: 1094–1105, 2009. © 2009 Wiley‐Liss, Inc.     

Kinetochore microtubule establishment is defective in oocytes from aged mice

Errors in chromosome segregation in mammalian oocytes increase in number with advancing maternal age, and are a major cause of pregnancy loss. Why chromosome segregation errors are more common in oocytes from older females remains poorly understood. In mitosis, accurate chromosome segregation is enabled by attachment of kinetochores to microtubules from appropriate spindle poles, and erroneous attachments increase the likelihood of mis-segregation. Whether attachment errors are responsible for age-related oocyte aneuploidy is unknown. Here we report that oocytes from naturally aged mice exhibit substantially increased chromosome misalignment, and fewer kinetochore pairs that make stable end-on attachments to the appropriate spindle poles compared with younger oocytes. The profile of mis-attachments exhibited is consistent with the types of chromosome segregation error observed in aged oocytes. Loss of chromosome cohesion, which is a feature of oocytes from older females, causes altered kinetochore geometry in meiosis-I. However, this has only a minor impact upon MT attachment, indicating that cohesion loss is not the primary cause of aneuploidy in meiosis-I. In meiosis-II, on the other hand, age-related cohesion loss plays a direct role in errors, since prematurely individualized sister chromatids misalign and misattach to spindle MTs. Thus, whereas cohesion loss leading to precocious sister chromatid separation is a direct cause of errors in meiosis-II, cohesion loss plays a more minor role in the etiology of aneuploidy in meiosis-I. Our data introduce altered MT-kinetochore interactions as a lesion that explains aneuploidy in meiosis-I in older females.     

Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error

The spindle assembly checkpoint (SAC) monitors attachment to microtubules and tension on chromosomes in mitosis and meiosis. It represents a surveillance mechanism that halts cells in M-phase in the presence of unattached chromosomes, associated with accumulation of checkpoint components, in particular, Mad2, at the kinetochores. A complex between the anaphase promoting factor/cylosome (APC/C), its accessory protein Cdc20 and proteins of the SAC renders APC/C inactive, usually until all chromosomes are properly assembled at the spindle equator (chromosome congression) and under tension from spindle fibres. Upon release from the SAC the APC/C can target proteins like cyclin B and securin for degradation by the proteasome. Securin degradation causes activation of separase proteolytic enzyme, and in mitosis cleavage of cohesin proteins at the centromeres and arms of sister chromatids. In meiosis I only the cohesin proteins at the sister chromatid arms are cleaved. This requires meiosis specific components and tight regulation by kinase and phosphatase activities. There is no S-phase between meiotic divisions. Second meiosis resembles mitosis. Mammalian oocytes arrest constitutively at metaphase II in presence of aligned chromosomes, which is due to the activity of the cytostatic factor (CSF). The SAC has been identified in spermatogenesis and oogenesis, but gender-differences may contribute to sex-specific differential responses to aneugens. The age-related reduction in expression of components of the SAC in mammalian oocytes may act synergistically with spindle and other cell organelles' dysfunction, and a partial loss of cohesion between sister chromatids to predispose oocytes to errors in chromosome segregation. This might affect dose-response to aneugens. In view of the tendency to have children at advanced maternal ages it appears relevant to pursue studies on consequences of ageing on the susceptibility of human oocytes to the induction of meiotic error by aneugens and establish models to assess risks to human health by environmental exposures.     

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal‐to‐zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here, we report that porcine ssc‐miR‐205 and bovine bta‐miR‐10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc‐miR‐205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to the porcine oocyte‐to‐embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.     

New insights into the genetic regulation of homologue disjunction in mammalian oocytes

Mammalian oocytes execute a unique meiotic programme involving 2 arrest stages and an unusually protracted preamble to chromosome segregation during the first meiotic division (meiosis I). How mammalian oocytes successfully navigate their exceptional meiotic journey has long been a question of immense interest. Understanding the minutiae of female mammalian meiosis I is not merely of academic interest as 80-90% of human aneuploidy is the consequence of errors arising at this particular stage of oocyte maturation, a stage with a peculiar vulnerability to aging. Recent evidence indicates that oocytes employ many of the same cast of proteins during meiosis I as somatic cells do during mitosis, often to execute similar tasks, but intriguingly, occasionally delegate them to unexpected and unprecedented roles. This is epitomised by the master cell-cycle regulon, the anaphase-promoting complex or cyclosome (APC/C), acting in concert with a critical APC/C-targeted surveillance mechanism, the spindle assembly checkpoint (SAC). Together, the APC/C and the SAC are among the most influential entities overseeing the fidelity of cell-cycle progression and the precision of chromosome segregation. Here I review the current status of pivotal elements underpinning homologue disjunction in mammalian oocytes including spindle assembly, critical biochemical anaphase-initiating events, APC/C activity and SAC signalling along with contemporary findings relevant to progressive oocyte SAC dysfunction as a model for age-related human aneuploidy.     

Selective Disruption of Aurora C Kinase Reveals Distinct Functions from Aurora B Kinase during Meiosis in Mouse Oocytes

Aurora B kinase (AURKB) is the catalytic subunit of the chromosomal passenger complex (CPC), an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT) attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I). We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction.     

A model system for increased meiotic nondisjunction in older oocytes

For at least 5% of all clinically recognized human pregnancies, meiotic segregation errors give rise to zygotes with the wrong number of chromosomes. Although most aneuploid fetuses perish in utero, trisomy in liveborns is the leading cause of mental retardation. A large percentage of human trisomies originate from segregation errors during female meiosis I; such errors increase in frequency with maternal age. Despite the clinical importance of age-dependent nondisjunction in humans, the underlying mechanisms remain largely unexplained. Efforts to recapitulate age-dependent nondisjunction in a mammalian experimental system have so far been unsuccessful. Here we provide evidence that Drosophila is an excellent model organism for investigating how oocyte aging contributes to meiotic nondisjunction. As in human oocytes, nonexchange homologs and bivalents with a single distal crossover in Drosophila oocytes are most susceptible to spontaneous nondisjunction during meiosis I. We show that in a sensitized genetic background in which sister chromatid cohesion is compromised, nonrecombinant X chromosomes become vulnerable to meiotic nondisjunction as Drosophila oocytes age. Our data indicate that the backup pathway that normally ensures proper segregation of achiasmate chromosomes deteriorates as Drosophila oocytes age and provide an intriguing paradigm for certain classes of age-dependent meiotic nondisjunction in humans.     

miR‐125a‐5p increases cellular DNA damage of aging males and perturbs stage‐specific embryo development via Rbm38‐p53 signaling

An increasing number of men are fathering children at an older age than in the past. While advanced maternal age has long been recognized as a risk factor for adverse reproductive outcomes, the influence of paternal age on reproduction is incompletely comprehended. Herein, we found that miR‐125a‐5p was upregulated in the sperm of aging males and was related to inferior sperm DNA integrity as an adverse predictor. Moreover, we demonstrated that miR‐125a‐5p suppressed mitochondrial function and increased cellular DNA damage in GC2 cells. We also found that miR‐125a‐5p perturbed embryo development at specific morula/blastocyst stages. Mechanistically, we confirmed that miR‐125a‐5p disturbed the mitochondrial function by targeting Rbm38 and activating the p53 damage response pathway, and induced a developmental delay in a p21‐dependent manner. Our study revealed an important role of miR‐125a‐5p in sperm function and early embryo development of aging males, and provided a fresh view to comprehend the aging process in sperm.