Genetic interaction mapping of Aurora protein kinases in mouse oocytes.

The Aurora Kinases (AURKs) are a family of serine-threonine protein kinases critical for cell division. Somatic cells express only AURKA and AURKB. However, mammalian germ cells and some cancer cells express all three isoforms. A major question in the field has been determining the molecular and cellular changes when cells express three instead of two aurora kinases. Using a systematic genetic approach involving different Aurora kinase oocyte-specific knockout combinations, we completed an oocyte-AURK genetic interaction map and show that one genomic copy of Aurka is necessary and sufficient to support female fertility and oocyte meiosis. We further confirm that AURKB and AURKC alone cannot compensate for AURKA. These results highlight the importance of AURKA in mouse oocytes, demonstrating that it is required for spindle formation and proper chromosome segregation. Surprisingly, a percentage of oocytes that lack AURKB can complete meiosis I, but the quality of those eggs is compromised, suggesting a role in AURKB to regulate spindle assembly checkpoint or control the cell cycle. Together with our previous studies, we wholly define the genetic interplay among the Aurora kinases and reinforce the importance of AURKA expression in oocyte meiosis.

A conserved germline-specific Dsn1 alternative splice isoform supports oocyte and embryo development.

The chromosome segregation and cell division programs associated with somatic mitosis and germline meiosis display dramatic differences such as kinetochore orientation, cohesin removal, or the presence of a gap phase.1,2,3,4,5,6 These changes in chromosome segregation require alterations to the established cell division machinery.5,6 It remains unclear what aspects of kinetochore function and its regulatory control differ between the mitotic and meiotic cell divisions to rewire these core processes. Alternative RNA splicing can generate distinct protein isoforms to allow for the differential control of cell processes across cell types. However, alternative splice isoforms that differentially modulate distinct cell division programs have remained elusive. Here, we demonstrate that mammalian germ cells express an alternative mRNA splice isoform for the kinetochore component, DSN1, a subunit of the MIS12 complex that links the centromeres to spindle microtubules during chromosome segregation. This germline DSN1 isoform bypasses the requirement for Aurora kinase phosphorylation for its centromere localization due to the absence of a key regulatory region allowing DSN1 to display persistent centromere localization. Expression of the germline DSN1 isoform in somatic cells results in constitutive kinetochore localization, chromosome segregation errors, and growth defects, providing an explanation for its tight cell-type-specific expression. Reciprocally, precisely eliminating expression of the germline-specific DSN1 splice isoform in mouse models disrupts oocyte maturation and early embryonic divisions coupled with a reduction in fertility. Together, this work identifies a germline-specific splice isoform for a chromosome segregation component and implicates its role in mammalian fertility.

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.

The retrotransposon-derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

The human genome contains 24 gag-like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag-like genes PNMA1 and PNMA4 support reproductive capacity during aging. Analysis of donated human ovaries shows that expression of both genes declines normally with age, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building.

Meiotic spindles are critical to ensure chromosome segregation during gamete formation. Oocytes lack centrosomes and use alternative microtubule-nucleation mechanisms for spindle building. How these mechanisms are regulated is still unknown. Aurora kinase A (AURKA) is essential for mouse oocyte meiosis because in pro-metaphase I it triggers microtubule organizing-center fragmentation and its expression compensates for the loss of the two other Aurora kinases (AURKB/AURKC). Although knockout mouse models were useful for foundational studies, AURK spatial and temporal functions are not yet resolved. We provide high-resolution analyses of AURKA/AURKC requirements during meiotic spindle-building and identify the subcellular populations that carry out these functions: 1) AURKA is required in early spindle assembly and later for spindle stability, whereas 2) AURKC is required in late pro-metaphase, and 3) Targeted AURKA constructs expressed in triple AURK knockout oocytes reveal that spindle pole-localized AURKA is the most important population controlling spindle building and stability mechanisms.

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

The female reproductive lifespan depends on egg quality, particularly euploidy. Mistakes in meiosis leading to egg aneuploidy are common, but the genetic landscape causing this is not well understood due to limited phenotypic data. We identify genetic determinants of reproductive aging via egg aneuploidy using a biobank of maternal exomes linked with maternal age and embryonic aneuploidy data. We found 404 genes with variants enriched in individuals with high egg aneuploidy rates and implicate kinesin protein family genes in aneuploidy risk. Experimental perturbations showed that motor domain variants in these genes increase aneuploidy in mouse oocytes. A knock-in mouse model validated that a specific variant in kinesin KIF18A accelerates reproductive aging and diminishes fertility. These findings suggest potential non-invasive biomarkers for egg quality, aiding personalized fertility medicine. One sentence summary: The study identifies novel genetic determinants of reproductive aging linked to egg aneuploidy by analyzing maternal exomes and demonstrates that variants in kinesin genes, specifically KIF18A , contribute to increased aneuploidy and accelerated reproductive aging, offering potential for personalized fertility medicine.

The retrotransposon - derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

The human genome contains 24 gag -like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag -like genes PNMA1 and PNMA4 support reproductive capacity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. Analysis of donated human ovaries shows that expression of both genes declines normally with aging, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

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.

A conserved germline-specific Dsn1 alternative splice isoform supports oocyte and embryo development.

Alternative mRNA splicing can generate distinct protein isoforms to allow for the differential control of cell processes across cell types. However, alternative splice isoforms that differentially modulate distinct cell division programs have remained elusive. Here, we demonstrate that mammalian germ cells express an alternate mRNA splice isoform for the kinetochore component, DSN1, a subunit of the MIS12 complex that links the centromeres to spindle microtubules during chromosome segregation. This germline DSN1 isoform bypasses the requirement for Aurora kinase phosphorylation for its centromere localization due to the absence of a key regulatory region allowing DSN1 to display persistent centromere localization. Expression of the germline DSN1 isoform in somatic cells results in constitutive kinetochore localization, chromosome segregation errors, and growth defects, providing an explanation for its tight cell type-specific expression. Reciprocally, precisely eliminating expression of the germline DSN1 splice isoform in mouse models disrupts oocyte maturation and early embryonic divisions coupled with a reduction in fertility. Together, this work identifies a germline-specific splice isoform for a chromosome segregation component and implicates its role in mammalian fertility.

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.

Whole transcriptome screening for novel genes involved in meiosis and fertility in Drosophila melanogaster.

Reproductive success requires the development of viable oocytes and the accurate segregation of chromosomes during meiosis. Failure to segregate chromosomes properly can lead to infertility, miscarriages, or developmental disorders. A variety of factors contribute to accurate chromosome segregation and oocyte development, such as spindle assembly and sister chromatid cohesion. However, many proteins required for meiosis remain unknown. In this study, we aimed to develop a screening pipeline for identifying novel meiotic and fertility genes using the genome of Drosophila melanogaster. To accomplish this goal, genes upregulated within meiotically active tissues were identified. More than 240 genes with no known function were silenced using RNA interference (RNAi) and the effects on meiosis and fertility were assessed. We identified 94 genes that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, we identified novel genes that are crucial for meiosis and oocyte development but have not been extensively studied in human or model organisms. Understanding the function of these genes will be an important step towards the understanding of their biological significance during reproduction.

Multiple intersecting pathways are involved in the phosphorylation of CPEB1 to activate translation during mouse oocyte meiosis.

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in the regulation of mRNA translation in oocytes. However, the nature of protein kinase cascades modulating the activity of CPEB1 is still a matter of controversy. Using genetic and pharmacological tools and detailed time courses, here we have reevaluated the relationship between CPEB1 phosphorylation and the activation of translation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on the phosphorylation of CPEB1 during prometaphase. Only inactivation of the CDK1/MAPK pathway disrupts translation, while inactivation of either pathway leads to CPEB1 stabilization. However, stabilization of CPEB1 induced by inactivation of the AURKA/PLK1 does not affect translation, indicating that destabilization/degradation can be dissociated from translational activation. The accumulation of the endogenous CCNB1 protein closely recapitulates the translation data. These findings support the overarching hypothesis that the activation of translation in prometaphase in mouse oocytes relies on a CDK1-dependent CPEB1 phosphorylation, and this translational activation precedes CPEB1 destabilization.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies. During cytokinesis in somatic cells, the midbody and subsequent assembly of the midbody remnant, a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the midbody and midbody remnant in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic midbodies (mMB) and mMB remnants using mouse oocytes and demonstrate that mMBs have a specialized cap structure that is orientated toward polar bodies. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

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.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies (PB). During cytokinesis in somatic cells, the midbody (MB) and subsequent assembly of the midbody remnant (MBR), a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the MB and MBR in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic MBs (mMB) and mMB remnants (mMBRs) using mouse oocytes and demonstrate that mMBs have a specialized meiotic mMB cap structure that is orientated toward PBs. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

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.

Identifying risk genes for embryo aneuploidy using ultra-low coverage whole-genome sequencing.

Background: Aneuploidy, the state of a cell containing extra or missing chromosomes, frequently arises during human meiosis and is the primary cause of early miscarriage and maternal age-related in vitro fertilization (IVF) failure. IVF patients 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 ultra-low coverage whole-genome sequencing (ulc-WGS) is a standard test for identifying and selecting IVF-derived embryos with a normal chromosome complement. The wealth of embryo aneuploidy data and ulc-WGS data from PGT-A has potential for discovering variants in paternal genomes that are associated with aneuploidy risk in their embryos. Methods: Using ulc-WGS data from ∼10,000 PGT-A biopsies, we imputed genotype likelihoods of genetic variants in parental genomes. We then used the imputed variants and aneuploidy calls from the embryos 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. Results: We identified one locus on chromosome 3 that is significantly associated with maternal 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. Conclusions: 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 can be leveraged for similar association studies using ulc-WGS data.

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.