-
Methods in Molecular Biology (Clifton,... 2024Immunocytochemical analysis of meiotic proteins on mouse chromosome spreads is one method of choice to study prophase I chromosome organization and homologous...
Immunocytochemical analysis of meiotic proteins on mouse chromosome spreads is one method of choice to study prophase I chromosome organization and homologous recombination. In recent decades, the development of microscopic approaches led to the production of a large number of images that monitor fluorescent proteins, defined as fluorescent objects, and a major challenge facing the community is the deep analysis of these fluorescent objects (measurement of object length, intensity, distance between objects, as well as foci identification, counting, and colocalization). We propose a set of tools designed from the macro language of the widely used image analysis software ImageJ (Schindelin et al., Nat Methods 9: 676-682, 2012), embedded in the "MeiQuant" macro, which are specifically designed for analyzing objects in the field of meiosis. Our aim is to propose a unified evolutive common tool for image analysis, with a specific focus on mouse prophase I meiotic events.
Topics: Animals; Mice; Meiosis; Meiotic Prophase I; Prophase; Chromosomes
PubMed: 38351458
DOI: 10.1007/978-1-0716-3698-5_17 -
Methods in Molecular Biology (Clifton,... 2024Spermatogonial stem cells (SSCs) produce haploid sperm via mitosis and meiosis in vivo. Although the technique to culture mouse SSCs has been well established, induction...
Spermatogonial stem cells (SSCs) produce haploid sperm via mitosis and meiosis in vivo. Although the technique to culture mouse SSCs has been well established, induction of meiosis in vitro has remained a challenge. Retinoic acid (RA) is required for meiosis in vivo; however, RA alone is not sufficient to induce meiosis in vitro. Here, we describe a method in which nutrient restriction and RA synergistically induce meiotic initiation into meiotic prophase I in cultured mouse SSCs.
Topics: Male; Mice; Animals; Meiosis; Retinoids; Semen; Tretinoin; Stem Cells; Nutrients; Spermatogonia; Spermatogenesis; Cell Differentiation
PubMed: 38351450
DOI: 10.1007/978-1-0716-3698-5_9 -
MicroPublication Biology 2024COSA-1 is essential for accurate meiosis in . Two null mutants ( and ) have been notably studied. These null mutants exhibit severe meiotic defects, hindering the...
COSA-1 is essential for accurate meiosis in . Two null mutants ( and ) have been notably studied. These null mutants exhibit severe meiotic defects, hindering the observation of the subtle or dynamic nature of COSA-1 function. To overcome these limitations, we developed a strain with inducible COSA-1 degradation using the Auxin-Inducible Degron (AID) system. This strain exhibits normal fertility and COSA-1::GFP foci. Auxin treatment successfully depletes COSA-1, resulting in a 96% decrease in progeny viability and 12 univalent chromosomes in diakinesis oocytes. This strain serves as a valuable tool for studying the dynamics of COSA-1.
PubMed: 38304162
DOI: 10.17912/micropub.biology.001087 -
Journal of Cellular Physiology Apr 2024Dynamic nuclear architecture and chromatin organizations are the key features of the mid-prophase I in mammalian meiosis. The chromatin undergoes major changes,...
Dynamic nuclear architecture and chromatin organizations are the key features of the mid-prophase I in mammalian meiosis. The chromatin undergoes major changes, including meiosis-specific spatiotemporal arrangements and remodeling, the establishment of chromatin loop-axis structure, pairing, and crossing over between homologous chromosomes, any deficiencies in these events may induce genome instability, subsequently leading to failure to produce gametes and infertility. Despite the significance of chromatin structure, little is known about the location of chromatin marks and the necessity of their balance during meiosis prophase I. Here, we show a thorough cytological study of the surface-spread meiotic chromosomes of mouse spermatocytes for H3K9,14,18,23,27,36, H4K12,16 acetylation, and H3K4,9,27,36 methylation. Active acetylation and methylation marks on H3 and H4, such as H3K9ac, H3K14ac, H3K18ac, H3K36ac, H3K56ac, H4K12ac, H4K16ac, and H3K36me3 exhibited pan-nuclear localization away from heterochromatin. In comparison, repressive marks like H3K9me3 and H3K27me3 are localized to heterochromatin. Further, taking advantage of the delivery of small-molecule chemical inhibitors methotrexate (heterochromatin enhancer), heterochromatin inhibitor, anacardic acid (histone acetyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor), IOX1 (JmjC demethylases inhibitor), and AZ505 (methyltransferase inhibitor) in seminiferous tubules through the rete testis route, revealed that alteration in histone modifications enhanced the centromere mislocalization, chromosome breakage, altered meiotic recombination and reduced sperm count. Specifically, IOX1 and AZ505 treatment shows severe meiotic phenotypes, including altering chromosome axis length and chromatin loop size via transcriptional regulation of meiosis-specific genes. Our findings highlight the importance of balanced chromatin modifications in meiotic prophase I chromosome organization and instability.
Topics: Animals; Male; Mice; Chromatin; Heterochromatin; Histones; Meiosis; Meiotic Prophase I; Protein Processing, Post-Translational; Spermatocytes
PubMed: 38284481
DOI: 10.1002/jcp.31201 -
Journal of Assisted Reproduction and... Mar 2024To investigate the genetic etiology of patients with female infertility.
PURPOSE
To investigate the genetic etiology of patients with female infertility.
METHODS
Whole Exome Sequencing was performed on genomic DNA extracted from the patient's blood. Exome data were filtered for damaging rare biallelic variants in genes with possible roles in reproduction. Sanger sequencing was used to validate the selected variants and segregate them in family members.
RESULTS
A novel homozygous likely pathogenic variant, c.626G>A, p.Trp209*, was identified in the TERB1 gene of the patient. Additionally, we report a second homozygous pathogenic TERB1 variant, c.1703C>G, p.Ser568*, in an infertile woman whose azoospermic brother was previously described to be homozygous for her variant.
CONCLUSIONS
Here, we report for the first time two homozygous likely pathogenic and pathogenic TERB1 variants, c.626G>A, p.Trp209* and c.1703C>G, p.Ser568*, respectively, in two unrelated women with primary infertility. TERB1 is known to play an essential role in homologous chromosome movement, synapsis, and recombination during the meiotic prophase I and has an established role in male infertility in humans. Our data add TERB1 to the shortlist of Meiosis I genes associated with human infertility in both sexes.
Topics: Female; Humans; Azoospermia; Cell Cycle Proteins; Homozygote; Infertility, Male; Meiosis; DNA-Binding Proteins
PubMed: 38277113
DOI: 10.1007/s10815-024-03031-x -
BioRxiv : the Preprint Server For... Jan 2024During meiosis, pairing of homologous chromosomes (homologs) ensures the formation of haploid gametes from diploid precursor cells, a prerequisite for sexual...
During meiosis, pairing of homologous chromosomes (homologs) ensures the formation of haploid gametes from diploid precursor cells, a prerequisite for sexual reproduction. Pairing during meiotic prophase I facilitates crossover recombination and homolog segregation during the ensuing reductional cell division. Mechanisms that ensure stable homolog alignment in the presence of an excess of non-homologous chromosomes have remained elusive, but rapid chromosome movements during prophase I appear to play a role in the process. Apart from homolog attraction, provided by early intermediates of homologous recombination, dissociation of non-homologous associations also appears to contribute to homolog pairing, as suggested by the detection of stable non-homologous chromosome associations in pairing-defective mutants. Here, we have developed an agent-based model for homolog pairing derived from the dynamics of a naturally occurring chromosome ensemble. The model simulates unidirectional chromosome movements, as well as collision dynamics determined by attractive and repulsive forces arising from close-range physical interactions. In addition to homolog attraction, chromosome number and size as well as movement velocity and repulsive forces are identified as key factors in the kinetics and efficiency of homologous pairing. Dissociation of interactions between non-homologous chromosomes may contribute to pairing by crowding homologs into a limited nuclear area thus creating preconditions for close-range homolog attraction. Predictions from the model are readily compared to experimental data from budding yeast, parameters can be adjusted to other cellular systems and predictions from the model can be tested via experimental manipulation of the relevant chromosomal features.
PubMed: 38260664
DOI: 10.1101/2023.08.09.552574 -
Chromatin remodeler CHD8 is required for spermatogonial proliferation and early meiotic progression.Nucleic Acids Research Apr 2024Meiosis is a key step during germ cell differentiation, accompanied by the activation of thousands of genes through germline-specific chromatin reorganization. The...
Meiosis is a key step during germ cell differentiation, accompanied by the activation of thousands of genes through germline-specific chromatin reorganization. The chromatin remodeling mechanisms underpinning early meiotic stages remain poorly understood. Here we focus on the function of one of the major autism genes, CHD8, in spermatogenesis, based on the epidemiological association between autism and low fertility rates. Specific ablation of Chd8 in germ cells results in gradual depletion of undifferentiated spermatogonia and the failure of meiotic double-strand break (DSB) formation, leading to meiotic prophase I arrest and cell death. Transcriptional analyses demonstrate that CHD8 is required for extensive activation of spermatogenic genes in spermatogonia, necessary for spermatogonial proliferation and meiosis. CHD8 directly binds and regulates genes crucial for meiosis, including H3K4me3 histone methyltransferase genes, meiotic cohesin genes, HORMA domain-containing genes, synaptonemal complex genes, and DNA damage response genes. We infer that CHD8 contributes to meiotic DSB formation and subsequent meiotic progression through combined regulation of these meiosis-related genes. Our study uncovers an essential role of CHD8 in the proliferation of undifferentiated spermatogonia and the successful progression of meiotic prophase I.
Topics: Male; Cell Proliferation; Chromatin; Meiosis; Spermatogenesis; Spermatogonia; Animals; Mice
PubMed: 38224953
DOI: 10.1093/nar/gkad1256 -
Cell Reports Jan 2024Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male...
Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. Maps male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.
Topics: Humans; Male; Mice; Animals; Chromatin; Meiosis; Pachytene Stage; Phase Separation; Meiotic Prophase I; Spermatocytes; Mammals
PubMed: 38175751
DOI: 10.1016/j.celrep.2023.113651 -
Annals of Botany Apr 2024Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2n = 5x = 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis...
BACKGROUND AND AIMS
Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2n = 5x = 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis characterized by two types of chromosomes: (1) chromosomes forming bivalents and distributed in the normal sexual way; and (2) chromosomes occurring as univalents and transferred by a female gamete only. In the mature pollen of pentaploid species, seven bivalent-derived chromosomes are transmitted to offspring, and 21 unpaired univalent chromosomes are eliminated during microsporogenesis. To discriminate between bivalent- and univalent-forming chromosomes, we studied histone H3 phosphorylation patterns regulating meiotic chromosome condensation and segregation.
METHODS
We analysed histone modification patterns during male canina meiosis in two representative dogrose species, 5x Rosa canina and 5x Rosa rubiginosa, by immunohistochemical and molecular cytogenetics approaches. Immunostaining of meiotic cells included α-tubulin, histone H3 phosphorylation (H3S10p, H3S28p and H3T3p) and methylation (H3K4me3 and H3K27me3) marks. In addition, fluorescent in situ hybridization was carried out with an 18S rDNA probe.
KEY RESULTS
In the first meiotic division, univalent chromosomes underwent equational division into chromatids, while homologues in bivalents were segregated as regular dyads. In diakinesis, bivalent chromosomes displayed strong H3 phosphorylation signals in proximal regions, spreading to the rest of the chromosome. In contrast, in univalents, the H3 phosphorylation signals were weaker, occurring mostly outside proximal regions largely overlapping with the H3K4me3 signals. Reduced phosphorylation was associated with relative under-condensation of the univalent chromosomes, particularly at early diakinesis.
CONCLUSIONS
We hypothesize that the absence of pairing and/or recombination in univalent chromosomes negatively affects the histone H3 phosphorylation of their chromatin and perhaps the loading of meiotic-specific cohesins. This apparently destabilizes cohesion of sister chromatids, leading to their premature split in the first meiotic division.
Topics: Histones; Phosphorylation; In Situ Hybridization, Fluorescence; Meiosis; Chromosomes; Epigenesis, Genetic
PubMed: 38127060
DOI: 10.1093/aob/mcad198 -
FASEB Journal : Official Publication of... Jan 2024Oocyte meiotic prophase I (MI) is an important event in female reproduction. Breast cancer amplified sequence 2 (BCAS2) is a component of the spliceosome. Previous...
Oocyte meiotic prophase I (MI) is an important event in female reproduction. Breast cancer amplified sequence 2 (BCAS2) is a component of the spliceosome. Previous reports have shown that BCAS2 is critical in male germ cell meiosis, oocyte development, and early embryo genome integrity. However, the role of BCAS2 in oocyte meiosis has not been reported. We used Stra8-GFPCre mice to knock out Bcas2 in oocytes during the pachytene phase. The results of fertility tests showed that Bcas2 conditional knockout (cKO) in oocytes results in infertility in female mice. Morphological analysis showed that the number of primordial follicles in the ovaries of 2-month-old (M) mice was significantly reduced and that follicle development was blocked. Further analysis showed that the number of primordial follicles decreased and that follicle development was slowed in 7-day postpartum (dpp) ovaries. Moreover, primordial follicles undergo apoptosis, and DNA damage cannot be repaired in primary follicle oocytes. Meiosis was abnormal; some oocytes could not reach the diplotene stage, and more oocytes could not develop to the dictyotene stage. Alternative splicing (AS) analysis revealed abnormal AS of deleted in azoospermia like (Dazl) and diaphanous related formin 2 (Diaph2) oogenesis-related genes in cKO mouse ovaries, and the process of AS was involved by CDC5L and PRP19.
Topics: Male; Female; Mice; Animals; Meiotic Prophase I; Meiosis; Alternative Splicing; RNA, Messenger; Oocytes; Neoplasm Proteins
PubMed: 38085152
DOI: 10.1096/fj.202301234RR