Polo-like kinase 1 promotes Cdc42-induced actin polymerization for asymmetric division in oocytes.

Polo-like kinase I (Plk1) is a highly conserved seronine/threonine kinase essential in meiosis and mitosis for spindle formation and cytokinesis. Here, through temporal application of Plk1 inhibitors, we identify a new role for Plk1 in the establishment of cortical polarity essential for highly asymmetric cell divisions of oocyte meiosis. Application of Plk1 inhibitors in late metaphase I abolishes pPlk1 from spindle poles and prevents the induction of actin polymerization at the cortex through inhibition of local recruitment of Cdc42 and Neuronal Wiskott-Aldrich Syndrome protein (N-WASP). By contrast, an already established polar actin cortex is insensitive to Plk1 inhibitors, but if the polar cortex is first depolymerized, Plk1 inhibitors completely prevent its restoration. Thus, Plk1 is essential for establishment but not maintenance of cortical actin polarity. These findings indicate that Plk1 regulates recruitment of Cdc42 and N-Wasp to coordinate cortical polarity and asymmetric cell division.

Chromosome-dependent aneuploid formation in Spo11-less meiosis.

Deficiency in meiotic recombination leads to aberrant chromosome disjunction during meiosis, often resulting in the lethality of gametes or genetic disorders due to aneuploidy formation. Budding yeasts lacking Spo11, which is essential for initiation of meiotic recombination, produce many inviable spores in meiosis, while very rarely all sets of 16 chromosomes are coincidentally assorted into gametes to form viable spores. We induced meiosis in a spo11∆ diploid, in which homolog pairs can be distinguished by single nucleotide polymorphisms and determined whole-genome sequences of their exceptionally viable spores. We detected no homologous recombination in the viable spores of spo11∆ diploid. Point mutations were fewer in spo11∆ than in wild-type. We observed spo11∆ viable spores carrying a complete diploid set of homolog pairs or haploid spores with a complete haploid set of homologs but with aneuploidy in some chromosomes. In the latter, we found the chromosome-dependence in the aneuploid incidence, which was positively and negatively influenced by the chromosome length and the impact of dosage-sensitive genes, respectively. Selection of aneuploidy during meiosis II or mitosis after spore germination was also chromosome dependent. These results suggest a pathway by which specific chromosomes are more prone to cause aneuploidy, as observed in Down syndrome.

Strategies for meiotic sex chromosome dynamics and telomeric elongation in Marsupials.

During meiotic prophase I, homologous chromosomes pair, synapse and recombine in a tightly regulated process that ensures the generation of genetically variable haploid gametes. Although the mechanisms underlying meiotic cell division have been well studied in model species, our understanding of the dynamics of meiotic prophase I in non-traditional model mammals remains in its infancy. Here, we reveal key meiotic features in previously uncharacterised marsupial species (the tammar wallaby and the fat-tailed dunnart), plus the fat-tailed mouse opossum, with a focus on sex chromosome pairing strategies, recombination and meiotic telomere homeostasis. We uncovered differences between phylogroups with important functional and evolutionary implications. First, sex chromosomes, which lack a pseudo-autosomal region in marsupials, had species specific pairing and silencing strategies, with implications for sex chromosome evolution. Second, we detected two waves of γH2AX accumulation during prophase I. The first wave was accompanied by low γH2AX levels on autosomes, which correlated with the low recombination rates that distinguish marsupials from eutherian mammals. In the second wave, γH2AX was restricted to sex chromosomes in all three species, which correlated with transcription from the X in tammar wallaby. This suggests non-canonical functions of γH2AX on meiotic sex chromosomes. Finally, we uncover evidence for telomere elongation in primary spermatocytes of the fat-tailed dunnart, a unique strategy within mammals. Our results provide new insights into meiotic progression and telomere homeostasis in marsupials, highlighting the importance of capturing the diversity of meiotic strategies within mammals.

METTL21A, a Non-Histone Methyltransferase, Is Dispensable for Spermatogenesis and Male Fertility in Mice.

Protein methyltransferases play various physiological and pathological roles through methylating histone and non-histone targets. Many histone methyltransferases have been reported to regulate the development of spermatogenic cells. However, the specific function of non-histone methyltransferases during spermatogenesis remains unclear. In this study, we found that METTL21A, a non-histone methyltransferase, is highly expressed in mouse testes. In order to elucidate the role of METTL21A in spermatogenesis, we generated a Mettl21a global knockout mouse model using CRISPR/Cas9 technology. Unexpectedly, our results showed that knockout males are fertile without apparent defects in the processes of male germ cell development, including spermatogonial differentiation, meiosis, and sperm maturation. Furthermore, the ablation of METTL21A does not affect the expression and localization of its known targeting proteins in testes. Together, our data demonstrated that METTL21A is not essential for mouse spermatogenesis and male fertility.

Signaling mechanisms and their regulation during in vivo or in vitro maturation of mammalian oocytes.

In vitro fertilization (IVF) is currently one of the most effective methods of infertility treatment. An alternative to commonly used ovarian hyperstimulation can become extracorporeal maturation of oocytes (in vitro maturation; IVM). Fertilization and normal development of the embryo depends on the cytoplasmic, nuclear and genomic maturity of the oocyte. The microenvironment of the ovarian follicle and maternal signals, which mediate bidirectional communication between granulosa, cumulus and oocyte cells, influence the growth, maturation and acquisition of oocyte development capability. During oogenesis in mammals, the meiosis is inhibited in the oocyte at the prophase I of the meiotic division due to the high cAMP level. This level is maintained by the activity of C-type natriuretic peptide (CNP, NPPC) produced by granulosa cells. The CNP binds to the NPR2 receptor in cumulus cells and is responsible for the production of cyclic guanosine monophosphate (cGMP). The cGMP penetrating into the oocyte through gap junctions inhibits phosphodiesterase 3A (PDE3A), preventing cAMP hydrolysis responsible for low MPF activity. The LH surge during the reproductive cycle reduces the activity of the CNP/NPR2 complex, which results in a decrease in cGMP levels in cumulus cells and consequently in the oocyte. Reduced cGMP concentration unblocks the hydrolytic activity of PDE3A, which decreases cAMP level inside the oocyte. This leads to the activation of MPF and resumption of meiosis. The latest IVM methods called SPOM, NFSOM or CAPA IVM consist of two steps: prematuration and maturation itself. Taking into account the role of cAMP in inhibiting and then unblocking the maturation of oocytes, they have led to a significant progress in terms of the percentage of mature oocytes in vitro and the proportion of properly developed embryos in both animals and humans.

Microtubule organizing centers regulate spindle positioning in mouse oocytes.

During female meiosis I (MI), spindle positioning must be tightly regulated to ensure the fidelity of the first asymmetric division and faithful chromosome segregation. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known about whether microtubules (MTs) participate in regulating these processes. Using mouse oocytes as a model system, we characterize a subset of MT organizing centers that do not contribute directly to spindle assembly, termed mcMTOCs. Using laser ablation, STED super-resolution microscopy, and chemical manipulation, we show that mcMTOCs are required to regulate spindle positioning and faithful chromosome segregation during MI. We discuss how forces exerted by F-actin on the spindle are balanced by mcMTOC-nucleated MTs to anchor the spindle centrally and to regulate its timely migration. Our findings provide a model for asymmetric cell division, complementing the current F-actin-based models, and implicate mcMTOCs as a major player in regulating spindle positioning.

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast.

Sexual reproduction requires genome haploidization by the two divisions of meiosis and a differentiation program to generate gametes. Here, we have investigated how sporulation, the yeast equivalent of gamete differentiation, is coordinated with progression through meiosis. Spore differentiation is initiated at metaphase II when a membrane-nucleating structure, called the meiotic plaque, is assembled at the centrosome. While all components of this structure accumulate already at entry into meiosis I, they cannot assemble because centrosomes are occupied by Spc72, the receptor of the γ-tubulin complex. Spc72 is removed from centrosomes by a pathway that depends on the polo-like kinase Cdc5 and the meiosis-specific kinase Ime2, which is unleashed by the degradation of Spo13/Meikin upon activation of the anaphase-promoting complex at anaphase I. Meiotic plaques are finally assembled upon reactivation of Cdk1 at entry into metaphase II. This unblocking-activation mechanism ensures that only single-copy genomes are packaged into spores and might serve as a paradigm for the regulation of other meiosis II-specific processes.

Interfered chromosome pairing at high temperature promotes meiotic instability in autotetraploid Arabidopsis.

Changes in environmental temperature affect multiple meiotic processes in flowering plants. Polyploid plants derived from whole-genome duplication (WGD) have enhanced genetic plasticity and tolerance to environmental stress but face challenges in organizing and segregating doubled chromosome sets. In this study, we investigated the impact of increased environmental temperature on male meiosis in autotetraploid Arabidopsis (Arabidopsis thaliana). Under low to mildly increased temperatures (5°C-28°C), irregular chromosome segregation universally occurred in synthetic autotetraploid Columbia-0 (Col-0). Similar meiotic lesions occurred in autotetraploid rice (Oryza sativa L.) and allotetraploid canola (Brassica napus cv Westar), but not in evolutionarily derived hexaploid wheat (Triticum aestivum). At extremely high temperatures, chromosome separation and tetrad formation became severely disordered due to univalent formation caused by the suppression of crossing-over. We found a strong correlation between tetravalent formation and successful chromosome pairing, both of which were negatively correlated with temperature elevation, suggesting that increased temperature interferes with crossing-over predominantly by impacting homolog pairing. We also showed that loading irregularities of axis proteins ASY1 and ASY4 co-localize on the chromosomes of the syn1 mutant and the heat-stressed diploid and autotetraploid Col-0, revealing that heat stress affects the lateral region of synaptonemal complex (SC) by impacting the stability of the chromosome axis. Moreover, we showed that chromosome axis and SC in autotetraploid Col-0 are more sensitive to increased temperature than those in diploid Arabidopsis. Taken together, our data provide evidence suggesting that WGD negatively affects the stability and thermal tolerance of meiotic recombination in newly synthetic autotetraploid Arabidopsis.

Asymmetry is defined during meiosis in the oocyte of the parthenogenetic nematode Diploscapter pachys.

Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans, and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans.

Loss of sister kinetochore co-orientation and peri-centromeric cohesin protection after meiosis I depends on cleavage of centromeric REC8.

Protection of peri-centromeric (periCEN) REC8 cohesin from Separase and sister kinetochore (KT) attachment to microtubules emanating from the same spindle pole (co-orientation) ensures that sister chromatids remain associated after meiosis I. Both features are lost during meiosis II, resulting in sister chromatid disjunction and the production of haploid gametes. By transferring spindle-chromosome complexes (SCCs) between meiosis I and II in mouse oocytes, we discovered that both sister KT co-orientation and periCEN cohesin protection depend on the SCC, and not the cytoplasm. Moreover, the catalytic activity of Separase at meiosis I is necessary not only for converting KTs from a co- to a bi-oriented state but also for deprotection of periCEN cohesion, and cleavage of REC8 may be the key event. Crucially, selective cleavage of REC8 in the vicinity of KTs is sufficient to destroy co-orientation in univalent chromosomes, albeit not in bivalents where resolution of chiasmata may also be required.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes.

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V-/- oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V-/- oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V-/- oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Meiotic Chromosome Synapsis and XY-Body Formation In Vitro.

To achieve spermatogenesis in vitro, one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, in vitro derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our in vitro culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the in vitro-generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in vivo in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional in vitro, meiotic crossover formation should be closely monitored when mimicking gametogenesis in vitro to prevent generation of aneuploid gametes.

Stage-resolved Hi-C analyses reveal meiotic chromosome organizational features influencing homolog alignment.

During meiosis, chromosomes exhibit dramatic changes in morphology and intranuclear positioning. How these changes influence homolog pairing, alignment, and recombination remain elusive. Using Hi-C, we systematically mapped 3D genome architecture throughout all meiotic prophase substages during mouse spermatogenesis. Our data uncover two major chromosome organizational features varying along the chromosome axis during early meiotic prophase, when homolog alignment occurs. First, transcriptionally active and inactive genomic regions form alternating domains consisting of shorter and longer chromatin loops, respectively. Second, the force-transmitting LINC complex promotes the alignment of ends of different chromosomes over a range of up to 20% of chromosome length. Both features correlate with the pattern of homolog interactions and the distribution of recombination events. Collectively, our data reveal the influences of transcription and force on meiotic chromosome structure and suggest chromosome organization may provide an infrastructure for the modulation of meiotic recombination in higher eukaryotes.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Direct evidence of sex and a hypothesis about meiosis in Symbiodiniaceae.

Dinoflagellates in the family Symbiodiniaceae are obligate endosymbionts of diverse marine invertebrates, including corals, and impact the capacity of their hosts to respond to climate change-driven ocean warming. Understanding the conditions under which increased genetic variation in Symbiodiniaceae arises via sexual recombination can support efforts to evolve thermal tolerance in these symbionts and ultimately mitigate coral bleaching, the breakdown of the coral-Symbiodiniaceae partnership under stress. However, direct observations of meiosis in Symbiodiniaceae have not been reported, despite various lines of indirect evidence that it occurs. We present the first cytological evidence of sex in Symbiodiniaceae based on nuclear DNA content and morphology using Image Flow Cytometry, Cell Sorting and Confocal Microscopy. We show the Symbiodiniaceae species, Cladocopium latusorum, undergoes gamete conjugation, zygote formation, and meiosis within a dominant reef-building coral in situ. On average, sex was detected in 1.5% of the cells analyzed (N = 10,000-40,000 cells observed per sample in a total of 20 samples obtained from 3 Pocillopora colonies). We hypothesize that meiosis follows a two-step process described in other dinoflagellates, in which diploid zygotes form dyads during meiosis I, and triads and tetrads as final products of meiosis II. This study sets the stage for investigating environmental triggers of Symbiodiniaceae sexuality and can accelerate the assisted evolution of a key coral symbiont in order to combat reef degradation.

The organization, regulation, and biological functions of the synaptonemal complex.

The synaptonemal complex (SC) is a meiosis-specific proteinaceous macromolecular structure that assembles between paired homologous chromosomes during meiosis in various eukaryotes. The SC has a highly conserved ultrastructure and plays critical roles in controlling multiple steps in meiotic recombination and crossover formation, ensuring accurate meiotic chromosome segregation. Recent studies in different organisms, facilitated by advances in super-resolution microscopy, have provided insights into the macromolecular structure of the SC, including the internal organization of the meiotic chromosome axis and SC central region, the regulatory pathways that control SC assembly and dynamics, and the biological functions exerted by the SC and its substructures. This review summarizes recent discoveries about how the SC is organized and regulated that help to explain the biological functions associated with this meiosis-specific structure.

FAM9B serves as a novel meiosis-related protein localized in meiotic chromosome cores and is associated with human gametogenesis.

Meiosis is a complex process involving the expression and interaction of numerous genes in a series of highly orchestrated molecular events. Fam9b localized in Xp22.3 has been found to be expressed in testes. However, FAM9B expression, localization, and its role in meiosis have not been previously reported. In this study, FAM9B expression was evaluated in the human testes and ovaries by RT-PCR, qPCR, and western blotting. FAM9B was found in the nuclei of primary spermatocytes in testes and specifically localized in the synaptonemal complex (SC) region of spermatocytes. FAM9B was also evident in the follicle cell nuclei and diffusely dispersed in the granular cell cytoplasm. FAM9B was partly co-localized with SYCP3, which is essential for both formation and maintenance of lateral SC elements. In addition, FAM9B had a similar distribution pattern and co-localization as γH2AX, which is a novel biomarker for DNA double-strand breaks during meiosis. All results indicate that FAM9B is a novel meiosis-associated protein that is co-localized with SYCP3 and γH2AX and may play an important role in SC formation and DNA recombination during meiosis. These findings offer a new perspective for understanding the molecular mechanisms involved in meiosis of human gametogenesis.

Heterologous Complementation of SPO11-1 and -2 Depends on the Splicing Pattern.

In the past, major findings in meiosis have been achieved, but questions towards the global understanding of meiosis remain concealed. In plants, one of these questions covers the need for two diverse meiotic active SPO11 proteins. In Arabidopsis and other plants, both meiotic SPO11 are indispensable in a functional form for double strand break induction during meiotic prophase I. This stands in contrast to mammals and fungi, where a single SPO11 is present and sufficient. We aimed to investigate the specific function and evolution of both meiotic SPO11 paralogs in land plants. By performing immunostaining of both SPO11-1 and -2, an investigation of the spatiotemporal localization of each SPO11 during meiosis was achieved. We further exchanged SPO11-1 and -2 in Arabidopsis and could show a species-specific function of the respective SPO11. By additional changes of regions between SPO11-1 and -2, a sequence-specific function for both the SPO11 proteins was revealed. Furthermore, the previous findings about the aberrant splicing of each SPO11 were refined by narrowing them down to a specific developmental phase. These findings let us suggest that the function of both SPO11 paralogs is highly sequence specific and that the orthologs are species specific.

Mitochondrial Calcium uniporters are essential for meiotic progression in mouse oocytes by controlling Ca2+ entry.

OBJECTIVES: The alteration of bioenergetics by oocytes in response to the demands of various biological processes plays a critical role in maintaining normal cellular physiology. However, little is known about the association between energy sensing and energy production with energy-dependent cellular processes like meiosis. MATERIALS AND METHODS: We demonstrated that cell cycle-dependent mitochondrial Ca2+ connects energy sensing to mitochondrial activity in meiosis progression within mouse oocytes. Further, we established a model in mouse oocytes using siRNA knockdowns that target mitochondrial calcium uniporters (MCUs) in order to inhibit mitochondrial Ca2+ concentrations. RESULTS: Decreased numbers of oocytes successfully progressed to the germinal vesicle stage and extruded the first polar body during in vitro culture after inhibition, while spindle checkpoint-dependent meiosis was also delayed. Mitochondrial Ca2+ levels changed, and this was followed by altered mitochondrial masses and ATP levels within oocytes during the entirety of meiosis progression. Abnormal mitochondrial Ca2+ concentrations in oocytes then hindered meiotic progress and activated AMP-activated protein kinase (AMPK) signalling that is associated with gene expression. CONCLUSIONS: These data provide new insight into the protective role that MCU-dependent mitochondrial Ca2+ signalling plays in meiotic progress, in addition to demonstrating a new mechanism of mitochondrial energy regulation by AMPK signalling that influences meiotic maturation.