-
BioRxiv : the Preprint Server For... Jan 2023Post-transcriptional regulation of gene expression by RNA-binding proteins helps facilitate fast, clean transitions from one cell state to the next during germ cell...
Post-transcriptional regulation of gene expression by RNA-binding proteins helps facilitate fast, clean transitions from one cell state to the next during germ cell differentiation. Previously we showed that the RNA helicase YTHDC2 is required for germ cells to properly switch from mitosis to meiosis (Bailey et al., 2017). While YTHDC2 protein is first expressed as male germ cells enter meiosis, when it is needed to shut down the mitotic program, YTHDC2 expression continues to increase and reaches its highest levels later in meiotic prophase, in pachytene spermatocytes. Here we show that YTHDC2 has an additional essential role regulating meiotic progression in late spermatocytes during mouse germ cell differentiation. Inducing conditional knockout of during the first wave of spermatogenesis, after the germ cells have already initiated meiotic prophase, allowed -deficient germ cells to successfully reach the pachytene stage and properly express many meiotic markers. However, instead of continuing through meiotic prophase and initiating the meiotic divisions, late pachytene spermatocytes failed to transition to the diplotene stage and quickly died. Loss of function of in spermatocytes resulted in changes in transcript levels for a number of genes, some up-regulated and some down-regulated, compared to control mid-stage spermatocytes. YTHDC2 interacts with different proteins in early and late spermatocytes, with many of the interacting proteins involved in post-transcriptional RNA regulation and present in RNA granules, similar to YTHDC2. Our findings suggest that YTHDC2 facilitates proper progression of germ cells through multiple steps of meiosis, potentially via several mechanisms of post-transcriptional RNA regulation.
PubMed: 36747642
DOI: 10.1101/2023.01.23.525146 -
Animals : An Open Access Journal From... Dec 2022This brief review is focused on the viviparous lizard (Lichtenstein, 1823), of the family Lacertidae, which possesses female heterogamety and multiple sex chromosomes... (Review)
Review
A Brief Review of Meiotic Chromosomes in Early Spermatogenesis and Oogenesis and Mitotic Chromosomes in the Viviparous Lizard (Squamata: Lacertidae) with Multiple Sex Chromosomes.
This brief review is focused on the viviparous lizard (Lichtenstein, 1823), of the family Lacertidae, which possesses female heterogamety and multiple sex chromosomes (male 2 = 36, ZZZZ/ZZW, female 2 = 35, with variable W sex chromosome). Multiple sex chromosomes and their changes may influence meiosis and the female meiotic drive, and they may play a role in reproductive isolation. In two cryptic taxa of with different W sex chromosomes, meiosis during early spermatogenesis and oogenesis proceeds normally, without any disturbances, with the formation of haploid spermatocytes, and in female meiosis with the formation of synaptonemal complexes (SCs) and the lampbrush chromosomes. In females, the SC number was constantly equal to 19 (according to the SC length, 16 SC autosomal bivalents plus three presumed SC sex chromosome elements). No variability in the chromosomes at the early stages of meiotic prophase I, and no significant disturbances in the chromosome segregation at the anaphase-telophase I stage, have been discovered, and haploid oocytes ( = 17) at the metaphase II stage have been revealed. There should be a factor/factors that maintain the multiple sex chromosomes, their equal transmission, and the course of meiosis in these cryptic forms of
PubMed: 36611629
DOI: 10.3390/ani13010019 -
Journal of Visualized Experiments : JoVE Nov 2022The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the...
The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.
Topics: Mice; Animals; Microtubule-Organizing Center; Spindle Apparatus; Sexual Maturation; Oocytes; Chromosome Segregation; Laser Therapy; Mammals
PubMed: 36440837
DOI: 10.3791/64439 -
Proceedings of the National Academy of... Nov 2022In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper...
In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper segregation. These events can vary drastically between species and between males and females of the same species. In in contrast to females, males do not form synaptonemal complexes (SCs), do not recombine, and have no crossing over; yet, males are able to segregate their chromosomes properly. Here, we investigated the early steps of homolog pairing in males. We found that homolog centromeres are not paired in germline stem cells (GSCs) and become paired in the mitotic region before meiotic entry, similarly to females. Surprisingly, male germline cells express SC proteins, which localize to centromeres and promote pairing. We further found that the SUN/KASH (LINC) complex and microtubules are required for homolog pairing as in females. Chromosome movements in males, however, are much slower than in females and we demonstrate that this slow dynamic is compensated in males by having longer cell cycles. In agreement, slowing down cell cycles was sufficient to rescue pairing-defective mutants in female meiosis. Our results demonstrate that although meiosis differs significantly between males and females, sex-specific cell cycle kinetics integrate similar molecular mechanisms to achieve proper centromere pairing.
Topics: Animals; Male; Female; Chromosome Pairing; Drosophila; Synaptonemal Complex; Centromere; Meiosis; Chromosomes; Chromosome Segregation
PubMed: 36375065
DOI: 10.1073/pnas.2207660119 -
PLoS Genetics Oct 2022Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and...
Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.
Topics: Animals; Male; Drosophila melanogaster; Drosophila; Drosophila Proteins; Polytetrafluoroethylene; Chromosome Segregation; Meiosis; Sex Chromosomes; Chromosome Pairing
PubMed: 36251690
DOI: 10.1371/journal.pgen.1010469 -
Coherent anti-Stokes Raman scattering cell imaging and segmentation with unsupervised data analysis.Frontiers in Cell and Developmental... 2022Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and...
Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.
PubMed: 36051442
DOI: 10.3389/fcell.2022.933897 -
Toxicological Sciences : An Official... Oct 2022Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are products of incomplete combustion. In female mouse embryos primordial germ cells proliferate before...
Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are products of incomplete combustion. In female mouse embryos primordial germ cells proliferate before and after arriving at the gonadal ridge around embryonic (E) 10 and begin entering meiosis at E13.5. Now oocytes, they arrest in the first meiotic prophase beginning at E17.5. We previously reported dose-dependent depletion of ovarian follicles in female mice exposed to 2 or 10 mg/kg-day BaP E6.5-15.5. We hypothesized that embryonic ovaries are more sensitive to gestational BaP exposure during the mitotic developmental window, and that this exposure results in persistent oxidative stress in ovaries and oocytes of exposed F1 female offspring. We orally dosed timed-pregnant female mice with 0 or 2 mg/kg-day BaP in oil from E6.5-11.5 (mitotic window) or E12.5-17.5 (meiotic window). Cultured E13.5 ovaries were utilized to investigate the mechanism of BaP-induced germ cell death. We observed statistically significant follicle depletion and increased ovarian lipid peroxidation in F1 pubertal ovaries following BaP exposure during either prenatal window. Culture of E13.5 ovaries with BaP induced germ cell DNA damage and release of cytochrome c from the mitochondria in oocytes, confirming that BaP exposure induced apoptosis via the mitochondrial pathway. Mitochondrial membrane potential, oocyte lipid droplet (LD) volume, and mitochondrial-LD colocalization were decreased and mitochondrial superoxide levels were increased in the MII oocytes of F1 females exposed gestationally to BaP. Results demonstrate similar sensitivity to germ cell depletion and persistent oxidative stress in F1 ovaries and oocytes following gestational BaP exposure during mitotic or meiotic windows.
Topics: Pregnancy; Female; Mice; Animals; Benzo(a)pyrene; Ovary; Meiosis; Oocytes; Mitochondria; Apoptosis
PubMed: 35993611
DOI: 10.1093/toxsci/kfac086 -
Medicine Aug 2022Adenocarcinoma is the most common pathological type of lung cancer. The E2F7 transcription factor has been confirmed to be related to the occurrence and development of a...
Adenocarcinoma is the most common pathological type of lung cancer. The E2F7 transcription factor has been confirmed to be related to the occurrence and development of a variety of solid tumors, but the relationship with the prognosis of lung cancer is still unclear. Therefore, we conducted this study to explore the prognostic value of E2F7 for lung adenocarcinoma (LUAD) patients. In this study, we analyzed samples from the Cancer Genome Atlas (TCGA) to study the correlation between the expression of E2F7 and clinical features, the difference in expression between tumors and normal tissues, the prognostic and diagnostic value, and Enrichment analysis of related genes. All statistical analysis uses R statistical software (version 3.6.3). The result shows that the expression level of E2F7 in LUAD was significantly higher than that of normal lung tissue (P = 1e-34). High expression of E2F7 was significantly correlated with gender (P = .034), pathologic stage (P = .046) and M stage (P = .025). Multivariate Cox analysis confirmed that E2F7 is an independent risk factor for OS in LUAD patients (P = .027). Genes related to cell cycle checkpoints, DNA damage telomere stress-induced senescence, DNA methylation, chromosome maintenance and mitotic prophase showed differential enrichment in the E2F7 high expression group. In short, high expression of E2F7 is an independent risk factor for OS in LUAD patients and has a high diagnostic value.
Topics: Adenocarcinoma of Lung; E2F7 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Prognosis
PubMed: 35984189
DOI: 10.1097/MD.0000000000029253 -
Life Science Alliance Oct 2022Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases....
Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process-at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.
Topics: Acetylation; Histones; Mitosis; Protein Processing, Post-Translational; Sirtuin 1
PubMed: 35981887
DOI: 10.26508/lsa.202201433 -
Life (Basel, Switzerland) May 2022Variants of linker histone H1 are tissue-specific and are responsible for chromatin compaction accompanying cell differentiation, mitotic chromosome condensation, and...
The Highest Density of Phosphorylated Histone H1 Appeared in Prophase and Prometaphase in Parallel with Reduced H3K9me3, and HDAC1 Depletion Increased H1.2/H1.3 and H1.4 Serine 38 Phosphorylation.
BACKGROUND
Variants of linker histone H1 are tissue-specific and are responsible for chromatin compaction accompanying cell differentiation, mitotic chromosome condensation, and apoptosis. Heterochromatinization, as the main feature of these processes, is also associated with pronounced trimethylation of histones H3 at the lysine 9 position (H3K9me3).
METHODS
By confocal microscopy, we analyzed cell cycle-dependent levels and distribution of phosphorylated histone H1 (H1ph) and H3K9me3. By mass spectrometry, we studied post-translational modifications of linker histones.
RESULTS
Phosphorylated histone H1, similarly to H3K9me3, has a comparable level in the G1, S, and G2 phases of the cell cycle. A high density of phosphorylated H1 was inside nucleoli of mouse embryonic stem cells (ESCs). H1ph was also abundant in prophase and prometaphase, while H1ph was absent in anaphase and telophase. H3K9me3 surrounded chromosomal DNA in telophase. This histone modification was barely detectable in the early phases of mitosis. Mass spectrometry revealed several ESC-specific phosphorylation sites of H1. HDAC1 depletion did not change H1 acetylation but potentiated phosphorylation of H1.2/H1.3 and H1.4 at serine 38 positions.
CONCLUSIONS
Differences in the level and distribution of H1ph and H3K9me3 were revealed during mitotic phases. ESC-specific phosphorylation sites were identified in a linker histone.
PubMed: 35743829
DOI: 10.3390/life12060798