Multiple intersecting pathways are involved in CPEB1 phosphorylation and regulation of translation during mouse oocyte meiosis.

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.

Maternal genetic variants in kinesin motor domains prematurely increase egg aneuploidy.

The female reproductive lifespan is highly dependent on egg quality, especially the presence of a normal number of chromosomes in an egg, known as euploidy. Mistakes in meiosis leading to egg aneuploidy are frequent in humans. Yet, knowledge of the precise genetic landscape that causes egg aneuploidy in women is limited, as phenotypic data on the frequency of human egg aneuploidy are difficult to obtain and therefore absent in public genetic datasets. Here, we identify genetic determinants of reproductive aging via egg aneuploidy in women using a biobank of individual maternal exomes linked with maternal age and embryonic aneuploidy data. Using the exome data, we identified 404 genes bearing variants enriched in individuals with pathologically elevated egg aneuploidy rates. Analysis of the gene ontology and protein-protein interaction network implicated genes encoding the kinesin protein family in egg aneuploidy. We interrogate the causal relationship of the human variants within candidate kinesin genes via experimental perturbations and demonstrate that motor domain variants increase aneuploidy in mouse oocytes. Finally, using a knock-in mouse model, we validate that a specific variant in kinesin KIF18A accelerates reproductive aging and diminishes fertility. These findings reveal additional functional mechanisms of reproductive aging and shed light on how genetic variation underlies individual heterogeneity in the female reproductive lifespan, which might be leveraged to predict reproductive longevity. Together, these results lay the groundwork for the noninvasive biomarkers for egg quality, a first step toward personalized fertility medicine.

Identifying risk variants for embryo aneuploidy using ultra-low coverage whole-genome sequencing from preimplantation genetic testing.

Aneuploidy frequently arises during human meiosis and is the primary cause of early miscarriage and in vitro fertilization (IVF) failure. Individuals undergoing IVF exhibit significant variability in aneuploidy rates, although the exact genetic causes of the variability in aneuploid egg production remain unclear. Preimplantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing is a standard test for identifying and selecting IVF-derived euploid embryos. The wealth of embryo aneuploidy data and ultra-low coverage whole-genome sequencing (ulc-WGS) data from PGT-A have the potential to discover variants in parental genomes that are associated with aneuploidy risk in their embryos. Using ulc-WGS data from ∼10,000 PGT-A biopsies, we imputed genotype likelihoods of genetic variants in embryo genomes. We then used the imputed variants and embryo aneuploidy calls to perform a genome-wide association study of aneuploidy incidence. Finally, we carried out functional evaluation of the identified candidate gene in a mouse oocyte system. We identified one locus on chromosome 3 that is significantly associated with meiotic aneuploidy risk. One candidate gene, CCDC66, encompassed by this locus, is involved in chromosome segregation during meiosis. Using mouse oocytes, we showed that CCDC66 regulates meiotic progression and chromosome segregation fidelity, especially in older mice. Our work extended the research utility of PGT-A ulc-WGS data by allowing robust association testing and improved the understanding of the genetic contribution to maternal meiotic aneuploidy risk. Importantly, we introduce a generalizable method that has potential to be leveraged for similar association studies that use ulc-WGS data.

Global SUMOylation in mouse oocytes maintains oocyte identity and regulates chromatin remodeling and transcriptional silencing at the end of folliculogenesis.

Meiotically competent oocytes in mammals undergo cyclic development during folliculogenesis. Oocytes within ovarian follicles are transcriptionally active, producing and storing transcripts required for oocyte growth, somatic cell communication and early embryogenesis. Transcription ceases as oocytes transition from growth to maturation and does not resume until zygotic genome activation. Although SUMOylation, a post-translational modification, plays multifaceted roles in transcriptional regulation, its involvement during oocyte development remains poorly understood. In this study, we generated an oocyte-specific knockout of Ube2i, encoding the SUMO E2 enzyme UBE2I, using Zp3-cre+ to determine how loss of oocyte SUMOylation during folliculogenesis affects oocyte development. Ube2i Zp3-cre+ female knockout mice were sterile, with oocyte defects in meiotic competence, spindle architecture and chromosome alignment, and a premature arrest in metaphase I. Additionally, fully grown Ube2i Zp3-cre+ oocytes exhibited sustained transcriptional activity but downregulated maternal effect genes and prematurely activated genes and retrotransposons typically associated with zygotic genome activation. These findings demonstrate that UBE2I is required for the acquisition of key hallmarks of oocyte development during folliculogenesis, and highlight UBE2I as a previously unreported orchestrator of transcriptional regulation in mouse oocytes.

Retinoic acid is dispensable for meiotic initiation but required for spermiogenesis in the mammalian testis.

Retinoic acid (RA) is the proposed mammalian 'meiosis inducing substance'. However, evidence for this role comes from studies in the fetal ovary, where germ cell differentiation and meiotic initiation are temporally inseparable. In the postnatal testis, these events are separated by more than 1 week. Exploiting this difference, we discovered that, although RA is required for spermatogonial differentiation, it is dispensable for the subsequent initiation, progression and completion of meiosis. Indeed, in the absence of RA, the meiotic transcriptome program in both differentiating spermatogonia and spermatocytes entering meiosis was largely unaffected. Instead, transcripts encoding factors required during spermiogenesis were aberrant during preleptonema, and the subsequent spermatid morphogenesis program was disrupted such that no sperm were produced. Taken together, these data reveal a RA-independent model for male meiotic initiation.

Aurora kinase B inhibits aurora kinase A to control maternal mRNA translation in mouse oocytes.

Mammalian oocytes are transcriptionally quiescent, and meiosis and early embryonic divisions rely on translation of stored maternal mRNAs. Activation of these mRNAs is mediated by polyadenylation. Cytoplasmic polyadenylation binding element 1 (CPEB1) regulates mRNA polyadenylation. One message is aurora kinase C (Aurkc), encoding a protein that regulates chromosome segregation. We previously demonstrated that AURKC levels are upregulated in oocytes lacking aurora kinase B (AURKB), and this upregulation caused increased aneuploidy rates, a role we investigate here. Using genetic and pharmacologic approaches, we found that AURKB negatively regulates CPEB1-dependent translation of many messages. To determine why translation is increased, we evaluated aurora kinase A (AURKA), a kinase that activates CPEB1 in other organisms. We find that AURKA activity is increased in Aurkb knockout mouse oocytes and demonstrate that this increase drives the excess translation. Importantly, removal of one copy of Aurka from the Aurkb knockout strain background reduces aneuploidy rates. This study demonstrates that AURKA is required for CPEB1-dependent translation, and it describes a new AURKB requirement to maintain translation levels through AURKA, a function crucial to generating euploid eggs.

Predicting Infertility: How Genetic Variants in Oocyte Spindle Genes Affect Egg Quality.

Successful reproduction relies on the union of a single chromosomally normal egg and sperm. Chromosomally normal eggs develop from precursor cells, called oocytes, that have undergone accurate chromosome segregation. The process of chromosome segregation is governed by the oocyte spindle, a unique cytoskeletal machine that splits chromatin content of the meiotically dividing oocyte. The oocyte spindle develops and functions in an idiosyncratic process, which is vulnerable to genetic variation in spindle-associated proteins. Human genetic variants in several spindle-associated proteins are associated with poor clinical fertility outcomes, suggesting that heritable etiologies for oocyte dysfunction leading to infertility exist and that the spindle is a crux for female fertility. This chapter examines the mammalian oocyte spindle through the lens of human genetic variation, covering the genes TUBB8, TACC3, CEP120, AURKA, AURKC, AURKB, BUB1B, and CDC20. Specifically, it explores how patient-identified variants perturb spindle development and function, and it links these molecular changes in the oocyte to their cognate clinical consequences, such as oocyte maturation arrest, elevated egg aneuploidy, primary ovarian insufficiency, and recurrent pregnancy loss. This discussion demonstrates that small genetic errors in oocyte meiosis can result in remarkably far-ranging embryonic consequences, and thus reveals the importance of the oocyte's fine machinery in sustaining life.

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.

Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

PURPOSE/STUDY QUESTION: Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? METHODS: Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). RESULTS: Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). CONCLUSION: The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing. Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

Mathematical modeling of human oocyte aneuploidy.

Aneuploidy is the leading contributor to pregnancy loss, congenital anomalies, and in vitro fertilization (IVF) failure in humans. Although most aneuploid conceptions are thought to originate from meiotic division errors in the female germline, quantitative studies that link the observed phenotypes to underlying error mechanisms are lacking. In this study, we developed a mathematical modeling framework to quantify the contribution of different mechanisms of erroneous chromosome segregation to the production of aneuploid eggs. Our model considers the probabilities of all possible chromosome gain/loss outcomes that arise from meiotic errors, such as nondisjunction (NDJ) in meiosis I and meiosis II, and premature separation of sister chromatids (PSSC) and reverse segregation (RS) in meiosis I. To understand the contributions of different meiotic errors, we fit our model to aneuploidy data from 11,157 blastocyst-stage embryos. Our best-fitting model captures several known features of female meiosis, for instance, the maternal age effect on PSSC. More importantly, our model reveals previously undescribed patterns, including an increased frequency of meiosis II errors among eggs affected by errors in meiosis I. This observation suggests that the occurrence of NDJ in meiosis II is associated with the ploidy status of an egg. We further demonstrate that the model can be used to identify IVF patients who produce an extreme number of aneuploid embryos. The dynamic nature of our mathematical model makes it a powerful tool both for understanding the relative contributions of mechanisms of chromosome missegregation in human female meiosis and for predicting the outcomes of assisted reproduction.

Predicting embryonic aneuploidy rate in IVF patients using whole-exome sequencing.

Infertility is a major reproductive health issue that affects about 12% of women of reproductive age in the United States. Aneuploidy in eggs accounts for a significant proportion of early miscarriage and in vitro fertilization failure. Recent studies have shown that genetic variants in several genes affect chromosome segregation fidelity and predispose women to a higher incidence of egg aneuploidy. However, the exact genetic causes of aneuploid egg production remain unclear, making it difficult to diagnose infertility based on individual genetic variants in mother's genome. In this study, we evaluated machine learning-based classifiers for predicting the embryonic aneuploidy risk in female IVF patients using whole-exome sequencing data. Using two exome datasets, we obtained an area under the receiver operating curve of 0.77 and 0.68, respectively. High precision could be traded off for high specificity in classifying patients by selecting different prediction score cutoffs. For example, a strict prediction score cutoff of 0.7 identified 29% of patients as high-risk with 94% precision. In addition, we identified MCM5, FGGY, and DDX60L as potential aneuploidy risk genes that contribute the most to the predictive power of the model. These candidate genes and their molecular interaction partners are enriched for meiotic-related gene ontology categories and pathways, such as microtubule organizing center and DNA recombination. In summary, we demonstrate that sequencing data can be mined to predict patients' aneuploidy risk thus improving clinical diagnosis. The candidate genes and pathways we identified are promising targets for future aneuploidy studies.

Aurora B and C kinases regulate chromosome desynapsis and segregation during mouse and human spermatogenesis.

Precise control of chromosome dynamics during meiosis is critical for fertility. A gametocyte undergoing meiosis coordinates formation of the synaptonemal complex (SC) to promote efficient homologous chromosome recombination. Subsequent disassembly of the SC occurs prior to segregation of homologous chromosomes during meiosis I. We examined the requirements of the mammalian Aurora kinases (AURKA, AURKB and AURKC) during SC disassembly and chromosome segregation using a combination of chemical inhibition and gene deletion approaches. We find that both mouse and human spermatocytes fail to disassemble SC lateral elements when the kinase activity of AURKB and AURKC are chemically inhibited. Interestingly, both Aurkb conditional knockout and Aurkc knockout mouse spermatocytes successfully progress through meiosis, and the mice are fertile. In contrast, Aurkb, Aurkc double knockout spermatocytes fail to coordinate disassembly of SC lateral elements with chromosome condensation and segregation, resulting in delayed meiotic progression. In addition, deletion of Aurkb and Aurkc leads to an accumulation of metaphase spermatocytes, chromosome missegregation and aberrant cytokinesis. Collectively, our data demonstrate that AURKB and AURKC functionally compensate for one another ensuring successful mammalian spermatogenesis.This article has an associated First Person interview with the first author of the paper.

Acentriolar spindle assembly in mammalian female meiosis and the consequences of its perturbations on human reproduction†.

The purpose of meiosis is to generate developmentally competent, haploid gametes with the correct number of chromosomes. For reasons not completely understood, female meiosis is more prone to chromosome segregation errors than meiosis in males, leading to an abnormal number of chromosomes, or aneuploidy, in gametes. Meiotic spindles are the cellular machinery essential for the proper segregation of chromosomes. One unique feature of spindle structures in female meiosis is spindles poles that lack centrioles. The process of building a meiotic spindle without centrioles is complex and requires precise coordination of different structural components, assembly factors, motor proteins, and signaling molecules at specific times and locations to regulate each step. In this review, we discuss the basics of spindle formation during oocyte meiotic maturation focusing on mouse and human studies. Finally, we review different factors that could alter the process of spindle formation and its stability. We conclude with a discussion of how different assisted reproductive technologies could affect spindles and the consequences these perturbations may have for subsequent embryo development.

A field guide to Aurora kinase inhibitors: an oocyte perspective.

In brief: The Aurora protein kinases have critical functions in controlling oocyte meiotic maturation. In this study, we describe an assay for examining their activation state in oocytes and establish the best working doses of three commonly used inhibitors. Abstract: Several small molecule inhibitors exist for targeting Aurora kinase proteins in somatic cells. From this point of view, we evaluate the specificity of these inhibitors in mouse oocytes, and we demonstrate that MLN 8237 and AZD 1152 are specific for Aurora kinase A and Aurora kinase C, respectively, only when used at low concentrations.

Analysis of DNA variants in miRNAs and miRNA 3'UTR binding sites in female infertility patients.

Early human embryogenesis relies on maternal gene products accumulated during oocyte growth and maturation, until around day-3 post-fertilization when human zygotic genome activation occurs. The maternal-to-zygotic transition (MZT) is a tightly coordinated process of selective maternal transcript clearance and new zygotic transcript production. If MZT is disrupted, it will lead to developmental arrest and pregnancy loss. It is well established that microRNA (miRNA) mutations disrupt regulation of their target transcripts. We hypothesize that some cases of embryonic arrest and pregnancy loss could be explained by the mutations in the maternal genome that affect miRNA-target transcript pairs. To this end, we examined mutations within miRNAs or miRNA binding sites in the 3' untranslated regions (3'UTR) of target transcripts. Using whole-exome sequencing data from 178 women undergoing in vitro fertilization (IVF) procedures, we identified 1197 variants in miRNA genes, including 93 single nucleotide variants (SNVs) and 19 small insertions/deletions (INDELs) within the seed region of 100 miRNAs. Eight miRNA seed-region variants were significantly enriched among our patients when compared to a normal population. Within predicted 3'UTR miRNA binding sites, we identified 7393 SNVs and 1488 INDELs. Between our patients and a normal population, 52 SNVs and 30 INDELs showed significant association in the single-variant testing, whereas 51 genes showed significant association in the gene-burden analysis for genes that are expressed in preimplantation embryos. Interestingly, we found that many genes with disrupted 3'UTR miRNA binding sites follow gene expression patterns resembling MZT. In addition, some of these variants showed dramatic allele frequency difference between the patient and the normal group, offering potential utility as biomarkers for screening patients prior to IVF procedures.

Meiosis interrupted: the genetics of female infertility via meiotic failure.

Idiopathic or 'unexplained' infertility represents as many as 30% of infertility cases worldwide. Conception, implantation, and term delivery of developmentally healthy infants require chromosomally normal (euploid) eggs and sperm. The crux of euploid egg production is error-free meiosis. Pathologic genetic variants dysregulate meiotic processes that occur during prophase I, meiotic resumption, chromosome segregation, and in cell cycle regulation. This dysregulation can result in chromosomally abnormal (aneuploid) eggs. In turn, egg aneuploidy leads to a broad range of clinical infertility phenotypes, including primary ovarian insufficiency and early menopause, egg fertilization failure and embryonic developmental arrest, or recurrent pregnancy loss. Therefore, maternal genetic variants are emerging as infertility biomarkers, which could allow informed reproductive decision-making. Here, we select and deeply examine human genetic variants that likely cause dysregulation of critical meiotic processes in 14 female infertility-associated genes: SYCP3, SYCE1, TRIP13, PSMC3IP, DMC1, MCM8, MCM9, STAG3, PATL2, TUBB8, CEP120, AURKB, AURKC, andWEE2. We discuss the function of each gene in meiosis, explore genotype-phenotype relationships, and delineate the frequencies of infertility-associated variants.

Origins and mechanisms leading to aneuploidy in human eggs.

The gain or loss of a chromosome-or aneuploidy-acts as one of the major triggers for infertility and pregnancy loss in humans. These chromosomal abnormalities affect more than 40% of eggs in women at both ends of the age spectrum, that is, young girls as well as women of advancing maternal age. Recent studies in human oocytes and embryos using genomics, cytogenetics, and in silico modeling all provide new insight into the rates and potential genetic and cellular factors associated with aneuploidy at varying stages of development. Here, we review recent studies that are shedding light on potential molecular mechanisms of chromosome missegregation in oocytes and embryos across the entire female reproductive life span.

SIRT7 promotes chromosome synapsis during prophase I of female meiosis.

Sirtuins are NAD+-dependent protein deacylases and ADP-ribosyltransferases that are involved in a wide range of cellular processes including genome homeostasis and metabolism. Sirtuins are expressed in human and mouse oocytes yet their role during female gamete development are not fully understood. Here, we investigated the role of a mammalian sirtuin member, SIRT7, in oocytes using a mouse knockout (KO) model. Sirt7 KO females have compromised fecundity characterized by a rapid fertility decline with age, suggesting the existence of a diminished oocyte pool. Accordingly, Sirt7 KO females produced fewer oocytes and ovulated fewer eggs. Because of the documented role of SIRT7 in DNA repair, we investigated whether SIRT7 regulates prophase I when meiotic recombination occurs. Sirt7 KO pachynema-like staged oocytes had approximately twofold increased γH2AX signals associated with regions with unsynapsed chromosomes. Consistent with the presence of asynaptic chromosome regions, Sirt7 KO oocytes had fewer MLH1 foci (~one less), a mark of crossover-mediated repair, than WT oocytes. Moreover, this reduced level of crossing over is consistent with an observed twofold increased incidence of aneuploidy in Metaphase II eggs. In addition, we found that acetylated lysine 18 of histone H3 (H3K18ac), an established SIRT7 substrate, was increased at asynaptic chromosome regions suggesting a functional relationship between this epigenetic mark and chromosome synapsis. Taken together, our findings demonstrate a pivotal role for SIRT7 in oocyte meiosis by promoting chromosome synapsis and have unveiled the importance of SIRT7 as novel regulator of the reproductive lifespan.

Specialize and Divide (Twice): Functions of Three Aurora Kinase Homologs in Mammalian Oocyte Meiotic Maturation.

The aurora kinases (AURKs) comprise an evolutionarily conserved family of serine/threonine kinases involved in mitosis and meiosis. While most mitotic cells express two AURK isoforms (AURKA and AURKB), mammalian germ cells also express a third, AURKC. Although much is known about the functions of the kinases in mitosis, less is known about how the three isoforms function to coordinate meiosis. This review is aimed at describing what is known about the three isoforms in female meiosis, the similarities and differences between kinase functions, and speculates as to why mammalian germ cells require expression of three AURKs instead of two.