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Current Protocols in Cytometry Apr 2015Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are...
Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back-gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis-defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis.
Topics: Aging; Animals; Cell Nucleus; Cell Separation; Flow Cytometry; Fluorescent Antibody Technique; Male; Meiotic Prophase I; Mice; Single-Cell Analysis; Spermatocytes; Spermatogenesis; Staining and Labeling; Testis
PubMed: 25827485
DOI: 10.1002/0471142956.cy0744s72 -
Cell Death and Differentiation Nov 2019Lethal (3) malignant brain tumor like 2 (L3MBTL2) is a member of the MBT-domain proteins, which are involved in transcriptional repression and implicated in chromatin...
Lethal (3) malignant brain tumor like 2 (L3MBTL2) is a member of the MBT-domain proteins, which are involved in transcriptional repression and implicated in chromatin compaction. Our previous study has shown that L3MBTL2 is highly expressed in the testis, but its role in spermatogenesis remains unclear. In the present study, we found that L3MBTL2 was most highly expressed in pachytene spermatocytes within the testis. Germ cell-specific ablation of L3mbtl2 in the testis led to increased abnormal spermatozoa, progressive decrease of sperm counts and premature testicular failure in mice. RNA-sequencing analysis on L3mbtl2 deficient testes confirmed that L3MBTL2 was a transcriptional repressor but failed to reveal any significant changes in spermatogenesis-associated genes. Interestingly, L3mbtl2 deficiency resulted in increased γH2AX deposition in the leptotene spermatocytes, subsequent inappropriate retention of γH2AX on autosomes, and defective crossing-over and synapsis during the pachytene stage of meiosis I, and more germ cell apoptosis and degeneration in aging mice. L3MBTL2 interacted with the histone ubiquitin ligase RNF8. Inhibition of L3MBTL2 reduced nuclear RNF8 and ubH2A levels in GC2 cells. L3mbtl2 deficiency led to decreases in the levels of the RNF8 and ubH2A pathway and in histone acetylation in elongating spermatids, and in protamine 1 deposition and chromatin condensation in sperm. These results suggest that L3MBTL2 plays important roles in chromatin remodeling during meiosis and spermiogenesis.
Topics: Acetylation; Animals; Apoptosis; Chromatin; Chromatin Assembly and Disassembly; Histones; Male; Meiotic Prophase I; Mice; Mice, Knockout; Nuclear Proteins; Pachytene Stage; Polycomb-Group Proteins; Sperm Count; Spermatocytes; Spermatogenesis; Testis; Transcription Factors; Ubiquitin-Protein Ligases
PubMed: 30760872
DOI: 10.1038/s41418-019-0283-z -
Scientific Reports May 2020Darevskia rock lizards is a unique complex taxa, including more than thirty species, seven of which are parthenogenetic. In mixed populations of Darevskia lizards, tri-...
Darevskia rock lizards is a unique complex taxa, including more than thirty species, seven of which are parthenogenetic. In mixed populations of Darevskia lizards, tri- and tetraploid forms can be found. The most important issues in the theory of reticulate evolution of Darevskia lizards are the origin of parthenogenetic species and their taxonomic position. However, there is little data on how meiosis proceeds in these species. The present work reports the complex results of cytogenetics in a diploid parthenogenetic species - D. unisexualis. Here we detail the meiotic prophase I progression and the specific features оf mitotic chromosomes organization. The stages of meiosis prophase I were investigated by immunocytochemical analysis of preparations obtained from isolated primary oocytes of D. unisexualis in comparison with maternal species D. raddei nairensis. It has been shown that in D. unisexualis at the leptotene-zygotene stages the axial elements and the synaptonemal complex (SC) form typical "bouquets". At the pachytene-diplotene stage, 18 autosomal SC-bivalents and thickened asynapted sex Z and w univalents were observed. The presence of SYCP1 protein between the lateral elements of autosomal chromosomes proved the formation of assembled SCs. Comparative genomic hybridization (CGH) on the mitotic metaphase chromosomes of D. unisexualis was carried out using the genomic DNA isolated from the parental species D. raddei nairensis and D. valentini. In the pericentromeric regions of half of the mitotic chromosomes of D. unisexualis, specific regions inherited from maternal species have been found. Following our results, we suggest a model for diploid germ cells formation from diploid oocytes without premeiotic duplication of chromosomes in the oogenesis of diploid parthenogenetic lizards D. unisexualis. Taken as a whole, our findings confirm the hybrid nature of D. unisexualis and shed light on heterozygosity and automixis in diploid parthenogenetic forms.
Topics: Animals; Chromosomes; Comparative Genomic Hybridization; In Situ Hybridization, Fluorescence; Karyotype; Lizards; Meiosis; Oocytes; Oogenesis
PubMed: 32457493
DOI: 10.1038/s41598-020-65686-7 -
Nucleus (Austin, Tex.) Nov 2017Meiosis is a specialized cellular division occurring in organisms capable of sexual reproduction that leads to the formation of gametes containing half of the original... (Review)
Review
Meiosis is a specialized cellular division occurring in organisms capable of sexual reproduction that leads to the formation of gametes containing half of the original chromosome number. During the earliest stage of meiosis, prophase I, pairing of homologous chromosomes is achieved in preparation for their proper distribution in the coming divisions. An important question is how do homologous chromosomes find each other and establish pairing interactions. Early studies demonstrated that chromosomes are dynamic in nature and move during this early stage of meiosis. More recently, there have been several studies across different models showing the conserved nature and importance of this chromosome movement, as well as the key components involved in chromosome movement. This review will cover these major findings and also introduce unexamined areas of regulation in meiotic prophase I chromosome movement.
Topics: Animals; Chromosome Pairing; Chromosomes; Cytoskeleton; Humans; Meiosis; Movement; Telomere
PubMed: 28892406
DOI: 10.1080/19491034.2017.1358329 -
Sexual Development : Genetics,... 2022In eutherian mammals, the sex chromosome complement, XX and XY, determines sexual differentiation of gonadal primordia into testes and ovaries, which in turn direct... (Review)
Review
BACKGROUND
In eutherian mammals, the sex chromosome complement, XX and XY, determines sexual differentiation of gonadal primordia into testes and ovaries, which in turn direct differentiation of germ cells into haploid sperm and oocytes, respectively. When gonadal sex is reversed, however, the germ cell sex becomes discordant with the chromosomal sex. XY females in humans are infertile, while XY females in the mouse (Mus musculus) are subfertile or infertile dependent on the cause of sex reversal and the genetic background. This article reviews publications to understand how the sex chromosome complement affects the fertility of XY oocytes by comparing with XX and monosomy X (XO) oocytes.
SUMMARY
The results highlight 2 folds disadvantage of XY oocytes over XX oocytes: (1) the X and Y chromosomes fail to pair during the meiotic prophase I, resulting in sex chromosome aneuploidy at the first meiotic division and (2) expression of the Y-linked genes during oocyte growth affects the transcriptome landscape and renders the ooplasmic component incompetent for embryonic development.
KEY MESSAGE
The XX chromosome complement gives the oocyte the highest competence for embryonic development.
PubMed: 35235936
DOI: 10.1159/000521151 -
International Journal of Molecular... Jul 2023In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is... (Review)
Review
In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is often accompanied by the cessation of its transcriptional activity. In many mammals, including mice and humans, chromatin condenses around a special nuclear organelle called the atypical nucleolus or formerly nucleolus-like body. Chromatin configuration is an important indicator of the quality of GV oocytes and largely predicts their ability to resume meiosis and successful embryonic development. In mice, GV oocytes are traditionally divided into the NSN (non-surrounded nucleolus) and SN (surrounded nucleolus) based on the specific chromatin configuration. The NSN-SN transition is a key event in mouse oogenesis and the main prerequisite for the normal development of the embryo. As for humans, there is no single nomenclature for the chromatin configuration at the GV stage. This often leads to discrepancies and misunderstandings, the overcoming of which should expand the scope of the application of mouse oocytes as a model for developing new methods for assessing and improving the quality of human oocytes. As a first approximation and with a certain proviso, the mouse NSN/SN classification can be used for the primary characterization of human GV oocytes. The task of this review is to analyze and discuss the existing classifications of chromatin configuration in mouse and human GV oocytes with an emphasis on transcriptional activity extinction at the end of oocyte growth.
Topics: Humans; Animals; Mice; Chromatin; Meiosis; Meiotic Prophase I; Oocytes; Cell Nucleus; Mammals
PubMed: 37511273
DOI: 10.3390/ijms241411517 -
Cytogenetic and Genome Research 2016The cytological analysis of meiotic chromosomes is an exceptional tool to approach complex processes such as synapsis and recombination during the division. Chromosome... (Review)
Review
The cytological analysis of meiotic chromosomes is an exceptional tool to approach complex processes such as synapsis and recombination during the division. Chromosome studies of meiosis have been especially valuable in birds, where naturally occurring mutants or experimental knock-out animals are not available to fully investigate the basic mechanisms of major meiotic events. This review highlights the main contributions of synaptonemal complex and lampbrush chromosome research to the current knowledge of avian meiosis, with special emphasis on the organization of chromosomes during prophase I, the impact of chromosome rearrangements during meiosis, and distinctive features of the ZW pair.
Topics: Animals; Birds; Crossing Over, Genetic; Female; Genetic Markers; Meiosis; Meiotic Prophase I; Sex Chromosomes; Synaptonemal Complex
PubMed: 28030854
DOI: 10.1159/000453541 -
Methods in Molecular Biology (Clifton,... 2021Crossing-over between homologous chromosomes is essential for accurate chromosome segregation at anaphase-I of meiosis. Defective crossing-over is associated with...
Crossing-over between homologous chromosomes is essential for accurate chromosome segregation at anaphase-I of meiosis. Defective crossing-over is associated with infertility, pregnancy miscarriage, and congenital disease. This chapter presents optimized protocols for the analysis of meiotic crossovers at the cytological level in spermatocytes and oocytes from mouse. The first approach employs immunocytology to detect MLH1, a DNA mismatch-repair protein that specifically marks crossover sites in the pachytene stage of meiotic prophase-I. These immunocytological methods have general utility for the analysis of other recombination steps, such as initiation and DNA strand exchange. The second approach visualizes chiasmata, the points of physical exchange between homologous chromosomes that are present during the diakinesis and metaphase-I stages. Both approaches are readily adaptable to the analysis of crossing over in other vertebrate species.
Topics: Aneuploidy; Animals; Cells, Cultured; Chromosomes, Mammalian; Crossing Over, Genetic; Female; Immunohistochemistry; Male; Mice; MutL Protein Homolog 1; Oocytes; Pachytene Stage; Spermatocytes
PubMed: 32840786
DOI: 10.1007/978-1-0716-0644-5_19 -
Journal of Experimental Botany Mar 2022Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each...
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.
Topics: Flowers; Hordeum; Meiosis; Meiotic Prophase I; Plant Proteins; Proteome
PubMed: 34758083
DOI: 10.1093/jxb/erab494 -
Scientific Reports Sep 2022Spatiotemporal regulation of proteins and RNAs is essential for the precise development of reproductive tissues in many organisms. The anther, a prominent part of the...
Spatiotemporal regulation of proteins and RNAs is essential for the precise development of reproductive tissues in many organisms. The anther, a prominent part of the male reproductive organ in plants, contains several somatic cell layers named the anther wall and, within it, the germ cells. Here, we successfully developed a simple 3D organ-immunoimaging technique for rice anthers, which distinguishes each individual cell from the four somatic cell layers and germ cells without the need for transformation, embedding, sectioning, or clearing. The 3D immunostaining method is also applicable to the intracellular localization of meiosis-specific proteins in meiocytes, as exemplified by MEL1, a germ cell-specific ARGONAUTE in the cytoplasm, and ZEP1, a pachytene marker on meiotic chromosomes. Our 3D multiple immunostaining method with single-cell and intracellular resolution will contribute to a comprehensive organ-level elucidation of molecular mechanisms and cellular connectivity.
Topics: Argonaute Proteins; Meiosis; Meiotic Prophase I; Oryza; Pollen
PubMed: 36104379
DOI: 10.1038/s41598-022-19373-4