Meiosis is a form of specialized cell division that generates gametes, allowing recombination of alleles and halving the chromosome number. Arabidopsis and maize are the plant models that have been most extensively studied to determine the genes involved in meiosis. Here we present an RNA-seq study in which gene expression in male meiocytes isolated during prophase I was compared to that in somatic tissues of the sunflower HA89 line. We sampled more than 490 million gene tags from these libraries, assembled them de novo into a sunflower transcriptome. We obtained expression data for 36,304 sunflower genes, of which 19,574 (54%) were differentially expressed (DE) between meiocytes and somatic tissue. We also determined the functional categories and metabolic pathways that are DE in these libraries. As expected, we found large differences between the meiotic and somatic transcriptomes, which is in accordance with previous studies in Arabidopsis and maize. Furthermore, most of the previously implicated meiotic genes were abundantly and DE in meiocytes and a large repertoire of transcription factors (TF) and genes related to silencing are expressed in the sunflower meiocytes. We detected TFs which appear to be exclusively expressed in meiocytes. Our results allow for a better understanding of the conservation and differences in the meiotic transcriptome of plants.

We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase, and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development.

The nuclear accumulation of active M-phase promoting factor (MPF) during prophase is thought to be essential for coordinating M-phase events in vertebrate cells. The protein phosphatase Cdc25C, an activator of MPF, enters the nucleus to keep MPF active in the nucleus during prophase. However, the molecular mechanisms that control nuclear translocation of Cdc25C during prophase are unknown. We show that phosphorylation of a serine residue (Ser198) in a nuclear export signal sequence of human Cdc25C occurs during prophase and promotes nuclear localization of Cdc25C. We also show that Polo-like kinase 1 (Plk1) is responsible for this phosphorylation and that constitutively active Plk1 promotes nuclear localization of Cdc25C. Remarkably, a mutant Cdc25C in which Ser198 is replaced by alanine remains in the cytoplasm when wild-type Cdc25C accumulates in the nucleus during prophase. These results suggest that Plk1 phosphorylates Cdc25C on Ser198 and regulates nuclear translocation of Cdc25C during prophase.

The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase-as early as the preleptotene stage-proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts.

In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

Formation of healthy mammalian eggs from oocytes requires specialised F-actin structures. F-actin disruption produces aneuploid eggs, which are a leading cause of human embryo deaths, genetic disorders and infertility. We found that oocytes contain prominent nuclear F-actin structures that are correlated with meiotic developmental capacity. We demonstrate that nuclear F-actin is a conserved feature of healthy mammalian oocytes and declines significantly with female reproductive ageing. Actin monomers used for nuclear F-actin assembly are sourced from an excess pool in the oocyte cytoplasm. Increasing monomeric G-actin transfer from the cytoplasm to the nucleus or directly enriching the nucleus with monomers led to assembly of stable nuclear F-actin bundles that significantly restrict chromatin mobility. By contrast, reducing G-actin monomer transfer by blocking nuclear import triggered assembly of a dense cytoplasmic F-actin network that is incompatible with healthy oocyte development. Overall, our data suggest that the large oocyte nucleus helps to maintain cytoplasmic F-actin organisation and that defects in this function are linked with reproductive age-related female infertility. This article has an associated First Person interview with Federica Giannini, joint first author of the paper.

Mammalian germ cells undergo meiosis to produce sperm or eggs, haploid cells that are primed to meet and propagate life. Meiosis is initiated by retinoic acid and meiotic prophase is the first and most complex stage of meiosis when homologous chromosomes pair to exchange genetic information. Errors in meiosis can lead to infertility and birth defects. However, despite the importance of this process, germ cell-specific gene expression patterns during meiosis remain undefined due to difficulty in obtaining pure germ cell samples, especially in females, where prophase occurs in the embryonic ovary. Indeed, mixed signals from both germ cells and somatic cells complicate gonadal transcriptome studies.

Accurate centrosome separation and positioning during early mitosis relies on force-generating mechanisms regulated by a combination of extracellular, cytoplasmic, and nuclear cues. The identity of the nuclear cues involved in this process remains largely unknown. Here, we investigate how the prophase nucleus contributes to centrosome positioning during the initial stages of mitosis, using a combination of cell micropatterning, high-resolution live-cell imaging, and quantitative 3D cellular reconstruction. We show that in untransformed RPE-1 cells, centrosome positioning is regulated by a nuclear signal, independently of external cues. This nuclear mechanism relies on the linker of nucleoskeleton and cytoskeleton complex that controls the timely loading of dynein on the nuclear envelope (NE), providing spatial cues for robust centrosome positioning on the shortest nuclear axis, before nuclear envelope permeabilization. Our results demonstrate how nuclear-cytoskeletal coupling maintains a robust centrosome positioning mechanism to ensure efficient mitotic spindle assembly.

Meiosis generates four genetically distinct haploid gametes over the course of two reductional cell divisions. Meiotic divisions are characterized by the coordinated deposition and removal of various epigenetic marks. Here we propose that nuclear respiratory factor 1 (NRF1) regulates transcription of euchromatic histone methyltransferase 1 (EHMT1) to ensure normal patterns of H3K9 methylation during meiotic prophase I. We demonstrate that cyclin-dependent kinase (CDK2) can bind to the promoters of a number of genes in male germ cells including that of Ehmt1 through interaction with the NRF1 transcription factor. Our data indicate that CDK2-mediated phosphorylation of NRF1 can occur at two distinct serine residues and negatively regulates NRF1 DNA binding activity in vitro. Furthermore, induced deletion of Cdk2 in spermatocytes results in increased expression of many NRF1 target genes including Ehmt1 We hypothesize that the regulation of NRF1 transcriptional activity by CDK2 may allow the modulation of Ehmt1 expression, therefore controlling the dynamic methylation of H3K9 during meiotic prophase.

An impeccable female meiotic prophase is critical for producing a high-quality oocyte and, ultimately, a healthy newborn. SYCP3 is a key component of the synaptonemal complex regulating meiotic homologous recombination. However, what regulates SYCP3 stability is unknown.

In flowering plants, successful germinal cell development and meiotic recombination depend upon a combination of environmental and genetic factors. To gain insights into this specialized reproductive development program we used short- and long-read RNA-sequencing (RNA-seq) to study the temporal dynamics of transcript abundance in immuno-cytologically staged barley (Hordeum vulgare) anthers and meiocytes. We show that the most significant transcriptional changes in anthers occur at the transition from pre-meiosis to leptotene-zygotene, which is followed by increasingly stable transcript abundance throughout prophase I into metaphase I-tetrad. Our analysis reveals that the pre-meiotic anthers are enriched in long non-coding RNAs (lncRNAs) and that entry to meiosis is characterized by their robust and significant down regulation. Intriguingly, only 24% of a collection of putative meiotic gene orthologs showed differential transcript abundance in at least one stage or tissue comparison. Argonautes, E3 ubiquitin ligases, and lys48 specific de-ubiquitinating enzymes were enriched in prophase I meiocyte samples. These developmental, time-resolved transcriptomes demonstrate remarkable stability in transcript abundance in meiocytes throughout prophase I after the initial and substantial reprogramming at meiosis entry and the complexity of the regulatory networks involved in early meiotic processes.

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21(Cip1/Waf1/Sdi1) (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition.

Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each containing reproductive (meiocyte) and non-reproductive tissue necessary for anther development and maturation. Meiosis, where chromosomes pair and exchange their genetic material during a process called recombination, is one of the most important and sensitive stages in breeding, ensuring genetic diversity. Most anther development studies have focused on transcript variation, but very few have been correlated with protein abundance. Taking advantage of a recently published barley anther transcriptomic (BAnTr) dataset and a newly developed sensitive mass spectrometry-based approach to analyse the barley anther proteome, we conducted high-resolution mass spectrometry analysis of barley anthers, collected at six time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology, providing a robust and accurate staging mirroring our previous BAnTr dataset. We identified >6100 non-redundant proteins including 82 known and putative meiotic proteins. Although the protein abundance was relatively stable throughout prophase I, we were able to quantify the dynamic variation of 336 proteins. We present the first quantitative comparative proteomics study of barley anther development during meiotic prophase I when the important process of homologous recombination is taking place.

Rec8 is a prominent component of the meiotic prophase chromosome axis that mediates sister chromatid cohesion, homologous recombination and chromosome synapsis. Here, we explore the prophase roles of Rec8. (i) During the meiotic divisions, Rec8 phosphorylation mediates its separase-mediated cleavage. We show here that such cleavage plays no detectable role for chromosomal events of prophase. (ii) We have analyzed in detail three rec8 phospho-mutants, with 6, 24 or 29 alanine substitutions. A distinct 'separation of function' phenotype is revealed. In the mutants, axis formation and recombination initiation are normal, as is non-crossover recombination; in contrast, crossover (CO)-related events are defective. Moreover, the severities of these defects increase coordinately with the number of substitution mutations, consistent with the possibility that global phosphorylation of Rec8 is important for these effects. (iii) We have analyzed the roles of three kinases that phosphorylate Rec8 during prophase. Timed inhibition of Dbf4-dependent Cdc7 kinase confers defects concordant with rec8 phospho-mutant phenotypes. Inhibition of Hrr25 or Cdc5/polo-like kinase does not. Our results suggest that Rec8's prophase function, independently of cohesin cleavage, contributes to CO-specific events in conjunction with the maintenance of homolog bias at the leptotene/zygotene transition of meiotic prophase.

The chromosomal program of meiotic prophase, comprising events such as laying down of meiotic cohesins, synapsis between homologs, and homologous recombination, must be preceded and enabled by the regulated induction of meiotic prophase genes. This gene regulatory program is poorly understood, particularly in organisms with a segregated germline. We characterized the gene regulatory program of meiotic prophase as it occurs in the mouse fetal ovary. By profiling gene expression in the mouse fetal ovary in mutants with whole tissue and single-cell techniques, we identified 104 genes expressed specifically in pre-meiotic to pachytene germ cells. We characterized the regulation of these genes by 1) retinoic acid (RA), which induces meiosis, 2) Dazl, which is required for germ cell competence to respond to RA, and 3) Stra8, a downstream target of RA required for the chromosomal program of meiotic prophase. Initial induction of practically all identified meiotic prophase genes requires Dazl. In the presence of Dazl, RA induces at least two pathways: one Stra8-independent, and one Stra8-dependent. Genes vary in their induction by Stra8, spanning fully Stra8-independent, partially Stra8-independent, and fully Stra8-dependent. Thus, Stra8 regulates the entirety of the chromosomal program but plays a more nuanced role in governing the gene expression program. We propose that Stra8-independent gene expression enables the stockpiling of selected meiotic structural proteins prior to the commencement of the chromosomal program. Unexpectedly, we discovered that Stra8 is required for prompt down-regulation of itself and Rec8. Germ cells that have expressed and down-regulated Stra8 are refractory to further Stra8 expression. Negative feedback of Stra8, and subsequent resistance to further Stra8 expression, may ensure a single, restricted pulse of Stra8 expression. Collectively, our findings reveal a gene regulatory logic by which germ cells prepare for the chromosomal program of meiotic prophase, and ensure that it is induced only once.

A typical nucleolus structure is shaped by three components. A meshwork of fine fibers forming the fibrillar center (FC) is surrounded by densely packed fibers forming the dense fibrillar component (DFC). Meanwhile, wrapping the FC and DFC is the granular component (GC). During the mitotic prophase, the nucleolus undergoes disassembling of its components. On the contrary, throughout the first meiotic prophase that occurs in the cells of the germ line, small nucleoli are assembled into one nucleolus by the end of the prophase. These nucleoli are transcriptionally active, suggesting that they are fully functional. Electron microscopy analysis has suggested that these nucleoli display their three main components but a typical organization has not been observed. Here, by immunolabeling and electron microscopy, we show that the nucleolus has its three main components. The GC is interlaced with the DFC and is not as well defined as previously thought during leptotene and zygotene stage.

In meiotic prophase, chromosomes are organized into compacted loop arrays to promote homolog pairing and recombination. Here, we probe the architecture of the mouse spermatocyte genome in early and late meiotic prophase using chromosome conformation capture (Hi-C). Our data support the established loop array model of meiotic chromosomes, and infer loops averaging 0.8-1.0 megabase pairs (Mb) in early prophase and extending to 1.5-2.0 Mb in late prophase as chromosomes compact and homologs undergo synapsis. Topologically associating domains (TADs) are lost in meiotic prophase, suggesting that assembly of the meiotic chromosome axis alters the activity of chromosome-associated cohesin complexes. While TADs are lost, physically separated A and B compartments are maintained in meiotic prophase. Moreover, meiotic DNA breaks and interhomolog crossovers preferentially form in the gene-dense A compartment, revealing a role for chromatin organization in meiotic recombination. Finally, direct detection of interhomolog contacts genome-wide reveals the structural basis for homolog alignment and juxtaposition by the synaptonemal complex.

The nucleus plays a central role in several key cellular processes, including chromosome organisation, DNA replication and gene transcription. Recent work suggests an association between nuclear mechanics and cell-cycle progression, but many aspects of this connection remain unexplored. Here, by monitoring nuclear shape fluctuations at different cell cycle stages, we uncover increasing inward fluctuations in late G2 and in early prophase, which are initially transient, but develop into instabilities when approaching the nuclear-envelope breakdown. We demonstrate that such deformations correlate with chromatin condensation by perturbing both the chromatin and the cytoskeletal structures. We propose that the contrasting forces between an extensile stress and centripetal pulling from chromatin condensation could mechanically link chromosome condensation with nuclear-envelope breakdown, two main nuclear processes occurring during mitosis.