CAP1-mediated actin cycling via ADF/cofilin proteins is essential for asymmetric division in mouse oocytes.

Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cellular events, and various actin-regulatory proteins modulate actin polymerization and depolymerization. Adenylyl cyclase-associated proteins (CAPs), highly conserved actin monomer-binding proteins, have been known to promote actin disassembly by enhancing the actin-severing activity of the ADF/cofilin protein family. In this study, we found that CAP1 regulated actin remodeling during mouse oocyte maturation. Efficient actin disassembly during oocyte maturation is essential for asymmetric division and cytokinesis. CAP1 knockdown impaired meiotic spindle migration and asymmetric division, and resulted in an accumulation of excessive actin filaments near the spindles. In contrast, CAP1 overexpression reduced actin mesh levels. CAP1 knockdown also rescued a decrease in cofilin family protein overexpression-mediated actin levels, and simultaneous expression of human CAP1 (hCAP1) and cofilin synergistically decreased cytoplasmic actin levels. Overexpression of hCAP1 decreased the amount of phosphorylated cofilin, indicating that CAP1 facilitated actin depolymerization via interaction with ADF/cofilin during mouse oocyte maturation. Taken together, our results provide evidence for the importance of dynamic actin recycling by CAP1 and cofilin in the asymmetric division of mouse female gametes.This article has an associated First Person interview with the first author of the paper.

CIP2A acts as a scaffold for CEP192-mediated microtubule organizing center assembly by recruiting Plk1 and aurora A during meiotic maturation.

In somatic cells spindle microtubules are nucleated from centrosomes that act as major microtubule organizing centers (MTOCs), whereas oocytes form meiotic spindles by assembling multiple acentriolar MTOCs without canonical centrosomes. Aurora A and Plk1 are required for these events, but the underlying mechanisms remain largely unknown. Here we show that CIP2A regulates MTOC organization by recruiting aurora A and Plk1 at spindle poles during meiotic maturation. CIP2A colocalized with pericentrin at spindle poles with a few distinct cytoplasmic foci. Although CIP2A has been identified as an endogenous inhibitor of protein phosphatase 2A (PP2A), overexpression of CIP2A had no effect on meiotic maturation. Depletion of CIP2A perturbed normal spindle organization and chromosome alignment by impairing MTOC organization. Importantly, CIP2A was reciprocally associated with CEP192, promoting recruitment of aurora A and Plk1 at MTOCs. CIP2A was phosphorylated by Plk1 at S904, which targets CIP2A to MTOCs and facilitates MTOC organization with CEP192. Our results suggest that CIP2A acts as a scaffold for CEP192-mediated MTOC assembly by recruiting Plk1 and aurora A during meiotic maturation in mouse oocytes.

Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.

Zinc plays an essential role in mammalian oocyte maturation, fertilization, and early embryogenesis, and depletion of zinc impairs cell cycle control, asymmetric division, and cytokinesis in oocyte. We report that zinc, via the actin nucleator Spire, acts as an essential regulator of the actin cytoskeleton remodeling during mouse oocyte maturation and fertilization. Depletion of zinc in the mouse oocyte impaired cortical and cytoplasmic actin formation. Spire is colocalized with zinc-containing vesicles via its zinc finger-containing Fab1, YOTB, Vac 1, EEA1 (FYVE) domain. Improper localization of Spire by zinc depletion or mutations in the FYVE domain impair cytoplasmic actin mesh formations and asymmetric division and cytokinesis of oocyte. All 3 major domains of the Spire are required for its proper localization and activity. After fertilization or parthenogenetic activation, Spire localization was dramatically altered following zinc release from the oocyte. Collectively, our data reveal novel roles for zinc in the regulation of the actin nucleator Spire by controlling its localization in mammalian oocyte.-Jo, Y.-J., Lee, I.-W., Jung, S.-M., Kwon, J., Kim, N.-H., Namgoong, S. Spire localization via zinc finger-containing domain is crucial for the asymmetric division of mouse oocyte.

Acentriolar microtubule organization centers and Ran-mediated microtubule formation pathways are both required in porcine oocytes.

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran-mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC- and Ran-mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo-like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the RanGTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC- and Ran-mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.

Distinct roles of Cep192 and Cep152 in acentriolar MTOCs and spindle formation during mouse oocyte maturation.

Mammalian oocytes lack a centriole that acts as a microtubule organization center (MTOC) in most somatic cells. During oocyte maturation, MTOCs undergo remodeling processes, including decondensation, fragmentation, and self-organization. However, the underlying mechanisms of MTOC remodeling in mouse oocytes are not well understood. We showed that two pericentriolar proteins, Cep192 and Cep152, play crucial roles during MTOC remodeling in mouse oocytes. Cep192 is present in MTOCs at all stages of oocyte maturation, and its depletion induces ablation of MTOCs, delay in spindle formation, and abnormal chromosomal alignment in spindles. In the case of Cep152, its localization on MTOCs is limited at the germinal vesicle stage and then disappears from the MTOCs after the germinal vesicle breakdown stage. Cep152 exclusion from MTOCs is involved in the fragmentation of MTOCs, and it is regulated by cyclin-dependent kinase 1 activity. Our results demonstrate the different roles of Cep192 and Cep152 in MTOC remodeling and a novel regulatory mechanism during meiotic spindle formation in mouse oocytes.-Lee, I.-W., Jo, Y.-J., Jung, S.-M., Wang, H.-Y., Kim, N.-H., Namgoong, S. Distinct roles of Cep192 and Cep152 in acentriolar MTOCs and spindle formation during mouse oocyte maturation.

Meiotic spindle formation in mammalian oocytes: implications for human infertility.

In the final stage of oogenesis, mammalian oocytes generate a meiotic spindle and undergo chromosome segregation to yield an egg that is ready for fertilization. Herein, we describe the recent advances in understanding the mechanisms controlling formation of the meiotic spindle in metaphase I (MI) and metaphase II (MII) in mammalian oocytes, and focus on the differences between mouse and human oocytes. Unlike mitotic cells, mammalian oocytes lack typical centrosomes that consist of two centrioles and the surrounding pericentriolar matrix proteins, which serve as microtubule-organizing centers (MTOCs) in most somatic cells. Instead, oocytes rely on different mechanisms for the formation of microtubules in MI spindles. Two different mechanisms have been described for MI spindle formation in mammalian oocytes. Chromosome-mediated microtubule formation, including RAN-mediated spindle formation and chromosomal passenger complex-mediated spindle elongation, controls the growth of microtubules from chromatin, while acentriolar MTOC-mediated microtubule formation contributes to spindle formation. Mouse oocytes utilize both chromatin- and MTOC-mediated pathways for microtubule formation. The existence of both pathways may provide a fail-safe mechanism to ensure high fidelity of chromosome segregation during meiosis. Unlike mouse oocytes, human oocytes considered unsuitable for clinical in vitro fertilization procedures, lack MTOCs; this may explain why meiosis in human oocytes is often error-prone. Understanding the mechanisms of MI/MII spindle formation, spindle assembly checkpoint, and chromosome segregation, in mammalian oocytes, will provide valuable insights into the molecular mechanisms of human infertility.

Filamin A is required for spindle migration and asymmetric division in mouse oocytes.

Dynamic changes in the actin network are crucial for the cortical migration of spindles and establishment of polarity, to ensure asymmetric division during meiotic maturation. In this study, filamin A (FLNA) was found to be an essential actin regulator that controlled spindle migration and asymmetric division during oocyte meiosis. FLNA was localized in the cytoplasm and enriched at the cortex and near the chromosomes. Knockdown of FLNA impaired meiotic asymmetric division and spindle migration with a decrease in the amount of cytoplasmic actin mesh and cortical actin levels. Moreover, FLNA knockdown reduced the phosphorylation of cofilin and Rho kinase (ROCK) near the spindle. Similar phenotypes, such as decreased filament actin levels, impaired spindle migration and polar body extrusion, were observed when active cofilin (S3A) was overexpressed or ROCK was inhibited. Notably, we found that FLNA and ROCK interacted directly in mouse oocytes. Taken together, our results show that FLNA plays crucial roles in asymmetric division during meiotic maturation by regulating ROCK-cofilin-mediated actin reorganization.-Wang, H., Guo J., Lin, Z., Namgoong, S., Oh, J. S., Kim, N.-H. Filamin A is required for spindle migration and asymmetric division in mouse oocytes.

Inhibition of CDK7 bypasses spindle assembly checkpoint via premature cyclin B degradation during oocyte meiosis.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.

Actin-capping proteins play essential roles in the asymmetric division of maturing mouse oocytes.

Actin polymerization is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion and cytokinesis. The heterodimeric actin-capping protein is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. However, the roles of capping protein in mammalian oocyte maturation are poorly understood. We investigated the roles of capping protein in mouse oocytes and found that it is essential for correct asymmetric spindle migration and polar body extrusion. Capping protein mainly localized in the cytoplasm during maturation. By knocking down or ectopically overexpressing this protein, we revealed that it is crucial for efficient spindle migration and maintenance of the cytoplasmic actin mesh density. Expression of the capping-protein-binding region of CARMIL (also known as LRRC16A) impaired spindle migration and polar body extrusion during oocyte maturation and decreased the density of the cytoplasmic actin mesh. Taken together, these findings show that capping protein is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation, presumably by controlling the cytoplasmic actin mesh density.

Distinct subcellular localization and potential role of LINE1-ORF1P in meiotic oocytes.

LINE-1 is an autonomous non-LTR retrotransposon in mammalian genomes and encodes ORF1P and ORF2P. ORF2P has been clearly identified as the enzyme supplier needed in LINE-1 retrotransposition. However, the role of ORF1P is not well explored. In this study, we employed loss/gain-of-function approach to investigate the role of LINE1-ORF1P in mouse oocyte meiotic maturation. During mouse oocyte development, ORF1P was observed in cytoplasm as well as in nucleus at germinal vesicle (GV) stage while was localized on the spindle after germinal vesicle breakdown (GVBD). Depletion of ORF1P caused oocyte arrest at the GV stage as well as down-regulation of CDC2 and CYCLIN B1, components of the maturation-promoting factor (MPF). Further analysis demonstrated ORF1P depletion triggered DNA damage response and most of the oocytes presented altered chromatin configuration. In addition, SMAD4 showed nuclear foci signal after Orf1p dsRNA injection. ORF1P overexpression held the oocyte development at MI stage and the chromosome alignment and spindle organization were severely affected. We also found that ORF1P could form DCP1A body-like foci structure in both cytoplasm and nucleus after heat shock. Taken together, accurate regulation of ORF1P plays an essential role in mouse oocyte meiotic maturation.

Anillin controls cleavage furrow formation in the course of asymmetric division during mouse oocyte maturation.

Anillin is a scaffold protein that recruits several proteins involved in cleavage furrow formation during cytokinesis. The role of anilllin in symmetric cell divisions in somatic cells has been intensively studied, yet its involvement in cleavage furrow formation is still elusive. In this study, we investigated the role of anillin in mammalian oocyte maturation and cytokinesis. We found that anillin is localized around the nucleus during the oocyte germinal-vesicle stage, and spreads to the cytoplasm after germinal vesicle breakdown. Thereafter, anillin concentrates at the site of the cleavage furrow from anaphase I to metaphase II. Disruption of anillin activity by microinjecting oocytes with specific siRNAs resulted in a failure of polar body extrusion and asymmetric division, and caused abnormal chromosome segregation during anaphase I. Furthermore, pharmacological inhibition of myosin light chain using Y-27632 or ML-7 resulted in decreased anillin expression. Collectively, our data suggest that anillin is an essential intracellular component that maintains the integrity of asymmetric division in mouse oocytes. Mol. Reprod. Dev. 83: 792-801, 2016 © 2016 Wiley Periodicals, Inc.

Autophagy and ubiquitin-mediated proteolysis may not be involved in the degradation of spermatozoon mitochondria in mouse and porcine early embryos.

The mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI).

The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation.

Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development.

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.

Superovulation induces defective methylation in line-1 retrotransposon elements in blastocyst.

BACKGROUND: Series of epigenetic events happen during preimplantation development. Therefore assistant reproduction techniques (ART) have the potential to disrupt epigenetic regulation during embryo development. The purpose of this study was to investigate whether defects in methylation patterns in blastocyst due to superovulation originate from abnormal expression of Dnmts. METHODS: Low- (6 IU) and high- (10 IU) dosage of PMSG was used to stimulate the female mice. The metaphase II(MII) oocytes, zygotes and blastocyst stage embryos were collected. Global methylation and methylation at H3K9 in zygote, and methylation at repeated sequence Line 1 and IAP in blastocysts were assayed. In addition, expression of Dnmts was examined in oocytes and zygotes. RESULTS: Global DNA methylation and methylation at H3K9 in zygotes derived from females after low- or high-dosage hormone treatment were unaltered compared to that in controls. Moreover, DNA methylation at IAP in blastocysts was also unaffected, regardless of hormone dosage. In contrast, methylation at Line1 decreased when high-dose hormone was administered. Unexpectedly, expression of Dnmt3a, Dnmt3b, Dnmt3L as well as maintenance Dnmt1o in oocytes and zygotes was not disrupted. CONCLUSIONS: The results suggest that defects in embryonic methylation patterns do not originate from the disruption of Dnmt expression.

WHAMM is essential for spindle formation and spindle actin polymerization in maturing mouse oocytes.

WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

Functional roles of hnRNPA2/B1 regulated by METTL3 in mammalian embryonic development.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) plays an important role in RNA processing via in m6A modification of pre-mRNA or pre-miRNA. However, the functional role of and relationship between m6A and hnRNPA2/B1 in early embryonic development are unclear. Here, we found that hnRNPA2/B1 is crucial for early embryonic development by virtue of regulating specific gene transcripts. HnRNPA2/B1 was localized to the nucleus and cytoplasm during subsequent embryonic development, starting at fertilization. Knockdown of hnRNPA2/B1 delayed embryonic development after the 4-cell stage and blocked further development. RNA-Seq analysis revealed changes in the global expression patterns of genes involved in transcription, translation, cell cycle, embryonic stem cell differentiation, and RNA methylation in hnRNPA2/B1 KD blastocysts. The levels of the inner cell mass markers OCT4 and SOX2 were decreased in hnRNPA2/B1 KD blastocysts, whereas that of the differentiation marker GATA4 was decreased. N6-Adenosine methyltransferase METTL3 knock-down caused embryonic developmental defects similar to those in hnRNPA2/B1 KD embryos. Moreover, METTL3 KD blastocysts showed increased mis-localization of hnRNPA2/B1 and decreased m6A RNA methylation. Taken together, our results suggest that hnRNPA2/B1 is essential for early embryogenesis through the regulation of transcription-related factors and determination of cell fate transition. Moreover, hnRNPA2/B1 is regulated by METTL3-dependent m6A RNA methylation.

Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes.

Inhibition of both MEK1/2 and glycogen synthase kinase-3 (GSK3; 2i system) facilitates the maintenance of naïve stemness for embryonic stem cells in various mammalian species. However, the effect of the inhibition of the 2i system on porcine early embryogenesis is unknown. We investigated the effect of the 2i system on early embryo development, expression of pluripotency-related genes, and epigenetic modifications. Inhibition of MEK1/2 (by PD0325901) and/or GSK3 (by CHIR99021) did not alter the developmental potential of porcine parthenogenetic embryos, but improved blastocyst quality, as judged by the blastocyst cell number, diameter, and reduction in the number of apoptotic cells. The expression levels of octamer-binding transcription factor 4 and SOX2, the primary transcription factors that maintain embryonic pluripotency, were significantly increased by 2i treatments. Epigenetic modification-related gene expression was altered upon 2i treatment. The collective results indicate that the 2i system in porcine embryos improved embryo developmental potential and blastocyst quality by regulating epigenetic modifications and pluripotency-related gene expression.

Reconstruction of bovine spermatozoa substances distribution and morphological differences between Holstein and Korean native cattle using three-dimensional refractive index tomography.

Measurements of the three-dimensional (3D) structure of spermatozoon are crucial for the study of developmental biology and for the evaluation of in vitro fertilization. Here, we present 3D label-free imaging of individual spermatozoon and perform quantitative analysis of bovine, porcine, and mouse spermatozoa morphologies using refractive index tomography. Various morphological and biophysical properties were determined, including the internal structure, volume, surface area, concentration, and dry matter mass of individual spermatozoon. Furthermore, Holstein cows and Korean native cattle spermatozoa were systematically analyzed and revealed significant differences in spermatozoa head length, head width, midpiece length, and tail length between the two breeds. This label-free imaging approach provides a new technique for understanding the physiology of spermatozoa.