Two mutations in TUBB8 cause developmental arrest in human oocytes and early embryos

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.

To identify and characterize amyloid-like substance (ALS) in human and mouse oocytes and preimplantation embryos. An experimental prospective pilot study. A total of 252 mouse oocytes and preimplantation embryos and 50 immature and in vitro matured human oocytes and parthenogenetic human embryos, from 11 consenting fertility patients, ages 18-45. Fluorescence intensity from immunofluorescent staining and data from confocal microscopy were quantified. Data were compared by one-way analysis of variance, with the least square-MEANS post-test, Pearson correlation coefficients (r), and bivariate analyses (t tests). ALS morphology was verified using transmission electron microscopy. Immunostaining for ALS appears throughout the zona pellucida, as well as in the cytoplasm and nucleus of mouse and human oocytes, polar bodies, and parthenogenetic embryos, and mouse preimplantation embryos. In mouse, 2-cell embryos exhibited the highest level of ALS (69000187.4 ± 6733098.07). Electron microscopy confirmed the presence of ALS. In humans, fresh germinal vesicle stage oocytes exhibited the highest level of ALS (4164.74088 ± 1573.46) followed by metaphase I and II stages (p = 0.008). There was a significant negative association between levels of ALS and patient body mass index, number of days of ovarian stimulation, dose of gonadotropin used, time between retrieval and fixation, and time after the hCG trigger. Significantly higher levels of ALS were found in patients with AMH between 1 and 3ng/ml compared to < 1ng/ml. We demonstrate for the first time the presence, distribution, and change in ALS throughout some stages of mouse and human oocyte maturation and embryonic development. We also determine associations between ALS in human oocytes with clinical characteristics.

The nuclear DNA content of intact, live or fixed, human and mouse oocytes and blastomeres has been measured rapidly and reliably. Chromosomal DNA has been stained with DAPI, the fluorescent emission from which has been measured photocytometrically. In vitro fertilised mouse oocytes and embryos at various stages of development were assessed for their DNA content. The mean values of 1C, 2C and 4C DNA content were clearly different, and it was possible to assign correctly individual values for DNA content to each class with 92%, 61% and 81% confidence respectively. Maintaining the cells as whole mounts allowed other morphological and structural features to be examined. When formation of multiple micronuclei was induced in mouse oocytes by their insemination in the presence of nocodazole, the additive signal from all the micronuclei in one zygote was equivalent to the expected DNA content. Application to early human blastomeres of this photocytometric technique for measurement of the total cellular DNA content revealed that multinucleated blastomeres contained 2C to 4C DNA levels, consistent with a diploid DNA content.

The Distribution of Mitochondria in Human Oocytes at Different Stages of Their Maturation

A sperm-specific phospholipase C zeta (PLCzeta) has been shown to trigger Ca(2+) oscillations in mouse and human oocytes and appears to be the sperm factor responsible for activation at fertilization. Previously, complementary RNA (cRNA) injection was used to introduce PLCzeta into oocytes, but it was unclear how much PLCzeta protein is required for development. Here we have injected cRNA encoding luciferase-tagged human PLCzeta (hPLCzeta-luc) into mouse oocytes and established the relationship between hPLCzeta-luc expression, Ca(2+) oscillations and development. Mouse oocytes were injected with hPLCzeta-luc cRNA and a fluorescent Ca(2+)dye to monitor hPLCzeta-luc expression and Ca(2+) oscillations, respectively. After inducing diploidy, development in vitro was monitored in hPLCzeta-luc cRNA microinjected oocytes and compared with parallel oocytes activated by incubation in Sr(2+). Repetitive Ca(2+) oscillations and oocyte activation were triggered by hPLCzeta over a wide range of luciferase expression levels. However, subsequent development of embryos to the blastocyst stage was observed only when expression of hPLCzeta-luc was optimized within a specific range. The blastocyst cell number was also affected by the level of hPLCzeta expression. Human PLCzeta can readily activate mouse oocytes, however, effective development to blastocyst stages is only achieved within a specific window of hPLCzeta-luc protein expression levels.

Tanshinone IIA, an isolated compound from Salvia miltiorrhiza Bunge, induces apoptosis in HeLa cells through mitotic arrest

Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMPs) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface.

Chemically assisted enucleation has been successfully applied to porcine and bovine oocytes to prepare recipient cytoplasts for nuclear transfer procedures. In this study, the antimitotic drugs demecolcine, nocodazole, and vinblastine were first assessed for their ability to induce the formation of cortical membrane protrusions in mouse, goat, and human oocytes. While only 2% of the treated human oocytes were able to form a protrusion, high rates of protrusion formation were obtained both in mouse (84%) and goat oocytes (92%), once the treatment was optimized for each species. None of the antimitotics applied was superior to the others in terms of protrusion formation, but mouse oocytes treated with vinblastine were unable to restore normal spindle morphology after drug removal and their in vitro development after parthenogenetic activation was severely compromised, rendering this antimitotic useless for chemically assisted enucleation approaches. Aspiration of the protrusions in mouse oocytes treated with demecolcine or nocodazole yielded 90% of successfully enucleated oocytes and allowed the extraction of a smaller amount of cytoplasm than with mechanical enucleation, but both enucleation methods resulted in the depletion of spindle-associated gamma-tubulin from the prepared cytoplasts. Treatment of mouse oocytes with demecolcine or nocodazole had no effect on their in vitro development after parthenogenetic activation, or on their ability to repolymerize a new spindle after the removal of the drug or the reconstruction of the treated cytoplasts with a somatic nucleus. Therefore, demecolcine- and nocodazole-assisted enucleation appears as an efficient alternative to mechanical enucleation, which can simplify nuclear transfer procedures.

Study question How does the Fibrous Corona (FC) behave in human and bovine oocyte meiosis and what is its function in chromosome segregation during meiosis I? Summary answer In human and bovine oocytes, FC shows extreme plasticity expanding beyond the centromeric region of the chromosomes. Pharmacological inhibition of FC’s expansion induces chromosome mis-segregation. What is known already Embryonic aneuploidy of meiotic origin is one of the major causes of implantation failure and early pregnancy loss. Erroneous microtubule (MT)-kinetochore attachments in human meiosis are the main cause of meiotic chromosome segregation errors. In somatic cells, the FC, a multiprotein complex, assembles on kinetochores and expands around the centromere during prometaphase to facilitate MT interactions and stimulate correct MT-kinetochore attachment. We have shown that in bovine oocytes FC expand beyond the centrosomes and is a potent MTs nucleator (10.1016/j.cub.2023.01.010). However, it is still unknown how FC behaves and what are its functions in MT-kinetochore interactions in human oocytes. Study design, size, duration FC morphology was studied in immature human oocytes discarded during ICSI procedures and donated (informed consent) by the patients for research (Ethical permit TCBio.21-754) during the years 2021-2024. Because of ethical concerns and reduced availability of human oocytes, functional studies on FC were performed on bovine oocytes collected from slaughterhouse ovaries. Participants/materials, setting, methods 180 immature human oocytes were matured in vitro and either fixed at different meiosis I stages or treated 2h with colcemid, a MT depolymerizing drug, to induce maximum FC expansion and fixed. The oocytes were then immuno-stained for chromatin(DAPI), FC(ZW10), MT(alpha tubulin) and kinetochores(CREST) and imaged with super-resolution confocal microscopy. Bovine immature oocytes were matured in vitro with or without lonafarnib (inhibitor of FC expansion). After maturation chromosome spreads were performed to evaluate oocyte ploidy. Main results and the role of chance In human oocytes, after nuclear envelope breakdown, before microtubule appearance when the chromosomes appear compacted, FCs are visible on the top of the kinetochores. During MT nucleation, FCs form thick mesh in the center of the compacted chromosomes. During prometaphase I, FCs expand beyond the centromeric region of the chromosomes forming a cup around the sister kinetochores of the paired homologous chromosomes. When the chromosomes starts to align at the metaphase plate, FCs start to be stripped from the kinetochores and colocalize to the MTs. At metaphase I and II, only kinetochore localization is visible. When oocytes are treated with colcemid, FCs extend to partially cover chromosome arms. We observed a wide variability in FCs expansion between and within patients. Inhibition of FC expansion in bovine oocytes increase the incidence of oocyte aneuploidy (90% in lonafarnib treated vs 37% in control oocytes). Altogether, our data reveal that in human oocytes FCs expand more compared to human somatic cells. Considering that inhibition of FC expansion induces chromosome segregation errors, we propose that the large FC size in human oocytes may play a role in achieving correct chromosome-MT attachments within their extremely large cytoplasm thus ensuring proper chromosome segregation. Limitations, reasons for caution The limited availability of human oocytes requires the use of an animal model for in depth mechanistic studies. Although bovine oocytes have similar aneuploidy rate and spindle assembly mechanisms as human oocytes, they show morphological differences in FC expansion which could underlie intrinsic functional discrepancies between the two organisms. Wider implications of the findings A better understanding of the chromosome segregation mechanisms in human meiosis will help understanding what causes the high incidence of aneuploidies in human oocytes and possibly contribute to the development of interventions or of oocyte selection procedures to reduce the incidence of oocyte aneuploidy during in vitro maturation. Trial registration number Not applicable

Heterozygous loss-of-function variants in LHX8 cause female infertility characterized by oocyte maturation arrest

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed.

It has been claimed repeatedly that gamma-tubulin is exclusively localized at the spindle poles in mitotic animal cells, where it plays a role in microtubule nucleation. In addition to this localization, we have observed a gamma-tubulin-specific staining of the mitotic spindle in several animal cells (human, kangaroo rat, mouse, Chinese hamster, Xenopus and Drosophila) using five polyclonal antibodies raised against unique gamma-tubulin sequences and four different fixation protocols. In HeLa and PtK2 cells, gamma-tubulin was detected in the mitotic spindle from late prometaphase to telophase. In contrast, in other cell types, it was detected in metaphase only. In all cases we failed to detect gamma-tubulin in the short aster microtubules at the spindle poles. Electron microscopic observation revealed that at least part of the gamma-tubulin localized on the surface of spindle microtubules with a preferential distribution along kinetochore microtubules. In HeLa cells, the amount of antigenic gamma-tubulin was fairly constant in the spindle poles during mitosis from prometaphase to telophase. In contrast, gamma-tubulin appeared in the mitotic spindles in prometaphase. The amount of gamma-tubulin decreased in telophase, where it relocalized in the interzone. In metaphase cells about 15-25% of the total fluorescence was localized at the spindle poles, while 75-85% of the fluorescence was distributed over the rest of the spindle. These results suggest that the localization and timing of gamma-tubulin during the cell cycle is highly regulated and that is physiological role could be more complex and diverse than initially assumed.

Expression of the embryonic poly(A)-binding protein (EPAB) in frog, mouse, and human oocytes and early-stage embryos is maintained at high levels until embryonic genome activation (EGA) after which a significant decrease occurs in EPAB levels. Studies on the vertebrate oocytes and early embryos revealed that EPAB plays key roles in the translational regulation, stabilization, and protection of maternal mRNAs during oocyte maturation and early embryogenesis. However, it remains elusive whether EPAB interacts with other cellular proteins and undergoes phosphorylation to perform these roles. For this purpose, we identified a group of Epab-interacting proteins and its phosphorylation status in mouse germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, and in 1-cell, 2-cell, and 4-cell preimplantation embryos. In the oocytes and early preimplantation embryos, Epab-interacting proteins were found to play roles in the translation and transcription processes, intracellular signaling and transport, maintenance of structural integrity, metabolism, posttranslational modifications, and chromatin remodeling. Moreover, we discovered that Epab undergoes phosphorylation on the serine, threonine, and tyrosine residues, which are localized in the RNA recognition motifs 2, 3, and 4 or C-terminal. Conclusively, these findings suggest that Epab not only functions in the translational control of maternal mRNAs through binding to their poly(A) tails but also participates in various cellular events through interacting with certain group proteins. Most likely, Epab undergoes a dynamic phosphorylation during the oocyte maturation and the early embryo development to carry out these functions.

Small RNAs have important functions. However, small RNAs in primate oocytes remain unexplored. Herein, we develop CAS-seq, a single-cell small RNA sequencing method, and profile the small RNAs in human oocytes and embryos. We discover a class of ~20-nt small RNAs that are predominantly expressed in human and monkey oocytes, but not in mouse oocytes. They are specifically associated with HIWI3 (PIWIL3), whereas significantly shorter than the commonly known PIWI-interacting RNAs (piRNAs), designated as oocyte short piRNAs (os-piRNAs). Notably, the os-piRNAs in human oocytes lack 2’-O-methylation at the 3’ end, a hallmark of the classic piRNAs. In addition, the os-piRNAs have a strong 1U/10 A bias and are enriched on the antisense strands of recently evolved transposable elements (TEs), indicating the potential function of silencing TEs by cleavage. Therefore, our study has identified an oocyte-specific piRNA family with distinct features and provides valuable resources for studying small RNAs in primate oocytes.

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.