Mouse oocytes sequester aggregated proteins in degradative super-organelles.

Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.

Beyond simple tails: poly(A) tail-mediated RNA epigenetic regulation.

The poly(A) tail is an essential structural component of mRNA required for the latter's stability and translation. Recent technologies have enabled transcriptome-wide profiling of the length and composition of poly(A) tails, shedding light on their overlooked regulatory capacities. Notably, poly(A) tails contain not only adenine but also uracil, cytosine, and guanine residues. These findings strongly suggest that poly(A) tails could encode a wealth of regulatory information, similar to known reversible RNA chemical modifications. This review aims to succinctly summarize our current knowledge on the composition, dynamics, and regulatory functions of RNA poly(A) tails. Given their capacity to carry rich regulatory information beyond the genetic code, we propose the concept of 'poly(A) tail epigenetic information' as a new layer of RNA epigenetic regulation.

Genetic adaptations for the oceanic success of fish eggs.

Genetic adaptations of organisms living in extreme environments are fundamental to our understanding of where life can evolve. Water is the single limiting parameter in this regard, yet when released in the oceans, the single-celled eggs of marine bony fishes (teleosts) have no means of acquiring it. They are strongly hyposmotic to seawater and lack osmoregulatory systems. Paradoxically, modern teleosts successfully release vast quantities of eggs in the extreme saline environment and recorded the most explosive radiation in vertebrate history. Here, we highlight key genetic adaptations that evolved to solve this paradox by filling the pre-ovulated eggs with water. The degree of water acquisition is uniquely prevalent to marine teleosts, permitting the survival and oceanic dispersal of their eggs.

New insights into oocyte cytoplasmic lattice-associated proteins.

Oocyte maturation and preimplantation embryo development are critical to successful pregnancy outcomes and the correct establishment and maintenance of genomic imprinting. Thanks to novel technologies and omics studies in human patients and mouse models, the importance of the proteins associated with the cytoplasmic lattices (CPLs), highly abundant structures found in the cytoplasm of mammalian oocytes and preimplantation embryos, in the maternal to zygotic transition is becoming increasingly evident. This review highlights the recent discoveries on the role of these proteins in protein storage and other oocyte cytoplasmic processes, epigenetic reprogramming, and zygotic genome activation (ZGA). A better comprehension of these events may significantly improve clinical diagnosis and pave the way for targeted interventions aiming to correct or mitigate female fertility issues and genomic imprinting disorders.

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.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

A genome-wide perspective of the maternal mRNA translation program during oocyte development.

Transcriptional and post-transcriptional regulations control gene expression in most cells. However, critical transitions during the development of the female gamete relies exclusively on regulation of mRNA translation in the absence of de novo mRNA synthesis. Specific temporal patterns of maternal mRNA translation are essential for the oocyte progression through meiosis, for generation of a haploid gamete ready for fertilization and for embryo development. In this review, we will discuss how mRNAs are translated during oocyte growth and maturation using mostly a genome-wide perspective. This broad view on how translation is regulated reveals multiple divergent translational control mechanisms required to coordinate protein synthesis with progression through the meiotic cell cycle and with development of a totipotent zygote.

Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

X centromeric drive may explain the prevalence of polycystic ovary syndrome and other conditions: Genomic structure of the human X chromosome pericentromeric region is consistent with meiotic drive associated with PCOS and other conditions.

X chromosome centromeric drive may explain the prevalence of polycystic ovary syndrome and contribute to oocyte aneuploidy, menopause, and other conditions. The mammalian X chromosome may be vulnerable to meiotic drive because of X inactivation in the female germline. The human X pericentromeric region contains genes potentially involved in meiotic mechanisms, including multiple SPIN1 and ZXDC paralogs. This is consistent with a multigenic drive system comprising differential modification of the active and inactive X chromosome centromeres in female primordial germ cells and preferential segregation of the previously inactivated X chromosome centromere to the polar body at meiosis I. The drive mechanism may explain differences in X chromosome regulation in the female germlines of the human and mouse and, based on the functions encoded by the genes in the region, the transmission of X pericentromeric genetic or epigenetic variants to progeny could contribute to preeclampsia, autism, and differences in sexual differentiation.

MIB2 Functions in Oocyte Meiosis by Modulating Chromatin Configuration.

Chromatin configuration serves as a principal indicator of GV (germinal vesicle)-stage oocyte quality. However, the underlying mechanisms governing the chromatin configuration transition from NSN (non-surrounded nucleolus) to SN (surrounded nucleolus) remain unclear. In this study, by conducting a quantitative proteomic analysis, we identified an increased expression of the MIB2 (MIB E3 ubiquitin protein ligase 2) protein in SN oocytes. Specific depletion of MIB2 in SN oocytes not only leads to severe disruption of the meiotic apparatus and a higher incidence of aneuploidy but also adversely affects meiotic maturation and early embryo development. Notably, overexpression of MIB2 in NSN oocytes facilitates the chromatin configuration transition. Meantime, we observed that forced expression of MIB2 in NSN oocytes significantly mitigates spindle/chromosome disorganization and aneuploidy. In summary, our results suggest that chromatin configuration transition regulated by MIB2 is crucial for oocytes to acquire developmental competence.

The small molecule activator S3969 stimulates the epithelial sodium channel by interacting with a specific binding pocket in the channel's ß-subunit.

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969-binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of ß-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics simulations predicted critical residues in the thumb domain of ß-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the ß-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. Molecular dynamics simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of ß-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (ßR437C - γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

Disease-associated missense mutations in the pore loop of polycystin-2 alter its ion channel function in a heterologous expression system.

Polycystin-2 (PC2) is mutated in ∼15% of patients with autosomal dominant polycystic kidney disease (ADPKD). PC2 belongs to the family of transient receptor potential (TRP) channels and can function as a homotetramer. We investigated whether three disease-associated mutations (F629S, C632R, or R638C) localized in the channel's pore loop alter ion channel properties of human PC2 expressed in Xenopus laevis oocytes. Expression of wild-type (WT) PC2 typically resulted in small but measurable Na+ inward currents in the absence of extracellular divalent cations. These currents were no longer observed when individual pore mutations were introduced in WT PC2. Similarly, Na+ inward currents mediated by the F604P gain-of-function (GOF) PC2 construct (PC2 F604P) were abolished by each of the three pore mutations. In contrast, when the mutations were introduced in another GOF construct, PC2 L677A N681A, only C632R had a complete loss-of-function effect, whereas significant residual Na+ inward currents were observed with F629S (∼15%) and R638C (∼30%). Importantly, the R638C mutation also abolished the Ca2+ permeability of PC2 L677A N681A and altered its monovalent cation selectivity. To elucidate the molecular mechanisms by which the R638C mutation affects channel function, molecular dynamics (MD) simulations were used in combination with functional experiments and site-directed mutagenesis. Our findings suggest that R638C stabilizes ionic interactions between Na+ ions and the selectivity filter residue D643. This probably explains the reduced monovalent cation conductance of the mutant channel. In summary, our data support the concept that altered ion channel properties of PC2 contribute to the pathogenesis of ADPKD.

Aging-associated reduction of chromosomal histones in mammalian oocytes.

Mammalian oocytes undergo a long-term meiotic arrest that can last for almost the entire reproductive lifespan. This arrest occurs after DNA replication and is prolonged with age, which poses a challenge to oocytes in maintaining replication-dependent chromosomal proteins required for the completion of meiosis. In this study, we show that chromosomal histones are reduced with age in mouse oocytes. Both types of histone H3 variants, replication-dependent H3.1/H3.2 and replication-independent H3.3, decrease with age. Aging-associated histone reduction is associated with transcriptomic features that are caused by genetic depletion of histone H3.3. Neither the genetic reduction of chromosomal H3.1/H3.2 nor H3.3 accelerates the aging-associated increase in premature chromosome separation that causes meiotic segregation errors. We suggest that aging-associated reduction of chromosomal histones is linked to several transcriptomic abnormalities but does not significantly contribute to errors in meiotic chromosome segregation during the reproductive lifespan of mice.

Female-specific mechanisms of meiotic initiation and progression in mammalian oocyte development.

Meiosis is regulated in sexually dimorphic manners in mammals. In females, the commitment to and entry into meiosis are coordinated with the developmental program of oocytes. Female germ cells initiate meiosis within a short time window during the fetal period and then undergo meiotic arrest until puberty. However, the genetic mechanisms underlying the orchestration of oocyte development and meiosis to maximize the reproductive lifespan of mammalian females remain largely elusive. While meiotic initiation is regulated by a sexually common mechanism, where meiosis initiator and Stimulated by Retinoic Acid Gene 8 (STRA8) activate the meiotic genes, the female-specific mode of meiotic initiation is mediated by the interaction between retinoblastoma (RB) and STRA8. This review highlights the female-specific mechanisms of meiotic initiation and meiotic prophase progression in the context of oocyte development. Furthermore, the downstream pathway of the RB-STRA8 interaction that may regulate meiotic arrest will be discussed in the context of oocyte development, highlighting a potential genetic link between the female-specific mode of meiotic entry and meiotic arrest.

Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes.

In eukaryotes, DNA is housed within the cell nucleus. Molecules required for the formation of a nucleus have been identified using in vitro systems with frog egg extracts and in vivo imaging of somatic cells. However, little is known about the physicochemical factors and conditions required for nuclear formation in mouse oocytes. In this study, using a reconstitution approach with purified DNA, we aimed to determine factors, such as the amount and timing of DNA introduction, required for the formation of nuclei with nuclear transport activity in mouse oocytes. T4 phage DNA (~166 kbp) was microinjected into strontium-activated oocytes to evaluate the conditions appropriate for nuclear formation. Microinjection of 100-500 ng/µL of T4 DNA, but not 20 ng/µL, was sufficient for the formation of nucleus-like structures. Furthermore, microinjection of DNA during metaphase II to telophase II, but not during interphase, was sufficient. Electron and fluorescence microscopy showed that T4 DNA-induced nucleus-like structures had nuclear lamina and nuclear pore complex structures similar to those of natural nuclei, as well as nuclear import activity. These results suggest that exogenous DNA can form artificial nuclei with nuclear transport functions in mouse oocytes, regardless of the sequence or source of the DNA.

Peptide derived from RAGE efficiently improves oocyte development through attenuating oxidative stress in oocytes of mice with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder in reproductive-aged women that frequently leads to infertility due to poor oocyte quality. In this study, we identified a new active peptide (advanced glycation end products receptors RAGE344-355 ) from PCOS follicular fluid using mass spectrometry. We found that supplementing PCOS-like mouse oocytes with RAGE344-355 attenuated both meiotic defects and oxidative stress levels, ultimately preventing developmental defects. Additionally, our results suggest that RAGE344-355 may interact with eEF1a1 to mitigate oxidative meiotic defects in PCOS-like mouse oocytes. These findings highlight the potential for further clinical development of RAGE344-355 as a potent supplement and therapeutic option for women with PCOS. This research addresses an important clinical problem and offers promising opportunities for improving oocyte quality in PCOS patients.

Arf6 GTPase deficiency leads to porcine oocyte quality decline during aging.

Arf6 is a member of ADP-ribosylation factor (Arf) family, which is widely implicated in the regulation of multiple physiological processes including endocytic recycling, cytoskeletal organization, and membrane trafficking during mitosis. In this study, we investigated the potential relationship between Arf6 and aging-related oocyte quality, and its roles on organelle rearrangement and cytoskeleton dynamics in porcine oocytes. Arf6 expressed in porcine oocytes throughout meiotic maturation, and it decreased in aged oocytes. Disruption of Arf6 led to the failure of cumulus expansion and polar body extrusion. Further analysis indicated that Arf6 modulated ac-tubulin for meiotic spindle organization and microtubule stability. Besides, Arf6 regulated cofilin phosphorylation and fascin for actin assembly, which further affected spindle migration, indicating the roles of Arf6 on cytoskeleton dynamics. Moreover, the lack of Arf6 activity caused the dysfunction of Golgi and ER for protein synthesis and signal transduction. Mitochondrial dysfunction was also observed in Arf6-deficient porcine oocytes, which was supported by the increased ROS level and abnormal membrane potential. In conclusion, our results reported that insufficient Arf6 was related to aging-induced oocyte quality decline through spindle organization, actin assembly, and organelle rearrangement in porcine oocytes.

KAT8 functions in redox homeostasis and mitochondrial dynamics during mouse oocyte meiosis progression.

As a histone acetyltransferase, lysine acetyltransferase 8 (KAT8) participates in diverse biological processes. However, the effect of KAT8 on oocyte maturation in mice remains unclear. In this study, we found that mouse oocytes overexpressing Kat8-OE induced maturation failure manifested reduced rates of GVBD and first polar body emission. In addition, immunostaining results revealed that Kat8 overexpressing oocytes showed inappropriate mitochondrial distribution patterns, overproduction of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX, hyperacetylation of α-tubulin, and severely disrupted spindle/chromosome organization. Moreover, we revealed that Kat8 overexpression induced a decline in SOD1 proteins and KAT8's interaction with SOD1 in mouse ovaries via immunoprecipitation. Western blotting data confirmed that Kat8-OE induced downregulation of SOD1 expression, which is a key factor for the decline of oocyte quality in advanced maternal age. Also, the injection of Myc-Sod1 cRNA could partially rescue maternal age-induced meiotic defects in oocytes. In conclusion, our data demonstrated that high level of KAT8 inhibited SOD1 activity, which in turn induced defects of mitochondrial dynamics, imbalance of redox homeostasis, and spindle/chromosome disorganization during mouse oocyte maturation.

Kinesin motor KIF16A regulates microtubule stability and actin-dependent spindle migration in mouse oocyte meiosis.

Kif16A, a member of the kinesin-3 family of motor proteins, has been shown to play crucial roles in inducing mitotic arrest, apoptosis, and mitotic cell death. However, its roles during oocyte meiotic maturation have not been fully defined. In this study, we report that Kif16A exhibits unique accumulation on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Targeted depletion of Kif16A using gene-targeting siRNA disrupts the progression of the meiotic cell cycle. Furthermore, Kif16A depletion leads to aberrant spindle assembly and chromosome misalignment in oocytes. Our findings also indicate that Kif16A depletion reduces tubulin acetylation levels and compromises microtubule resistance to depolymerizing drugs, suggesting its crucial role in microtubule stability maintenance. Notably, we find that the depletion of Kif16A results in a notably elevated incidence of defective kinetochore-microtubule attachments and the absence of BubR1 localization at kinetochores, suggesting a critical role for Kif16A in the activation of the spindle assembly checkpoint (SAC) activity. Additionally, we observe that Kif16A is indispensable for proper actin filament distribution, thereby impacting spindle migration. In summary, our findings demonstrate that Kif16A plays a pivotal role in regulating microtubule and actin dynamics crucial for ensuring both spindle assembly and migration during mouse oocyte meiotic maturation.

Follicular oocyte as a potential target for severe acute respiratory syndrome coronavirus 2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in December 2019 and rapidly became a pandemic as coronavirus disease 2019 (COVID-19). Apart from other organs, presence of specific receptor angiotensin-converting enzyme (ACE2) and corresponding proteases such as transmembrane serine protease 2, basigin and cysteine protease cathepsin L make follicular somatic cells as well as oocyte as potential targets for SARS-CoV-2 infection. The SARS-CoV-2 causes inflammation and hypoxia that generate reactive oxygen species (ROS) in critically ill patients. In addition, a large number of casualties and insecurity of life due to repeated waves of SARS-CoV-2 infection generate psychological stress and cortisol resulting in the further generation of ROS. The excess levels of ROS under physiological range cause meiotic instability, while high levels result in oxidative stress that trigger various death pathways and affect number as well as quality of follicular oocytes. Although, emerging evidence suggests that the SARS-CoV-2 utilises cellular machinery of ovarian follicular cells, generates ROS and impairs quality of follicular oocytes, the underlying mechanism of viral entry into host cell and its negative impact on the follicular oocyte remains poorly understood. Therefore, this review summarises emerging evidence on the presence of cellular machinery for SARS-CoV-2 in ovarian follicles and the potential negative impact of viral infection on the follicular oocytes that affect ovarian functions in critically ill and stressed women.