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Journal of Visualized Experiments : JoVE Oct 2018The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase...
The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell. The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.
Topics: Animals; Chromosomes; Insecta; Male; Micromanipulation; Spermatocytes
PubMed: 30394368
DOI: 10.3791/57359 -
Current Molecular Medicine 2019Worldwide, infertility affects 8-12% of couples of reproductive age and has become a common problem. There are many ways to treat infertility, including medication,... (Review)
Review
Worldwide, infertility affects 8-12% of couples of reproductive age and has become a common problem. There are many ways to treat infertility, including medication, intrauterine insemination, and in vitro fertilization. In recent years, stem-cell therapy has raised new hope in the field of reproductive disability management. Stem cells are self-renewing, self-replicating undifferentiated cells that are capable of producing specialized cells under appropriate conditions. They exist throughout a human's embryo, fetal, and adult stages and can proliferate into different cells. While many issues remain to be addressed concerning stem cells, stem cells have undeniably opened up new ways to treat infertility. In this review, we describe past, present, and future strategies for the use of stem cells in reproductive medicine.
Topics: Adult Germline Stem Cells; Cell Differentiation; Cellular Reprogramming Techniques; Embryonic Stem Cells; Female; Humans; Induced Pluripotent Stem Cells; Infertility, Female; Infertility, Male; Male; Mesenchymal Stem Cell Transplantation; Oocytes; Ovary; Reproductive Techniques, Assisted; Spermatocytes; Stem Cell Transplantation; Stem Cells
PubMed: 31288721
DOI: 10.2174/1566524019666190709172636 -
ELife Nov 2023Spermatogenesis in the male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific...
Spermatogenesis in the male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic chromosome, and reduced transcription from the chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.
Topics: Male; Animals; Drosophila; Epigenome; X Chromosome; Chromatin; Histones; Spermatocytes
PubMed: 38032818
DOI: 10.7554/eLife.89373 -
Cells Feb 2022dividing spermatocytes offer a highly suitable cell system in which to investigate the coordinated reorganization of microtubule and actin cytoskeleton systems during... (Review)
Review
dividing spermatocytes offer a highly suitable cell system in which to investigate the coordinated reorganization of microtubule and actin cytoskeleton systems during cell division of animal cells. Like male germ cells of mammals, spermatogonia and spermatocytes undergo cleavage furrow ingression during cytokinesis, but abscission does not take place. Thus, clusters of primary and secondary spermatocytes undergo meiotic divisions in synchrony, resulting in cysts of 32 secondary spermatocytes and then 64 spermatids connected by specialized structures called ring canals. The meiotic spindles in males are substantially larger than the spindles of mammalian somatic cells and exhibit prominent central spindles and contractile rings during cytokinesis. These characteristics make male meiotic cells particularly amenable to immunofluorescence and live imaging analysis of the spindle microtubules and the actomyosin apparatus during meiotic divisions. Moreover, because the spindle assembly checkpoint is not robust in spermatocytes, male meiosis allows investigating of whether gene products required for chromosome segregation play additional roles during cytokinesis. Here, we will review how the research studies on male meiotic cells have contributed to our knowledge of the conserved molecular pathways that regulate spindle microtubules and cytokinesis with important implications for the comprehension of cancer and other diseases.
Topics: Actins; Animals; Drosophila; Drosophila Proteins; Drosophila melanogaster; Male; Meiosis; Microtubules; Spermatocytes
PubMed: 35203341
DOI: 10.3390/cells11040695 -
Cellular & Molecular Biology Letters Sep 2023In recent years, N6-methyladenosine (mA) methylation modification of mRNA has been studied extensively. It has been reported that mA determines mRNA fate and...
BACKGROUND
In recent years, N6-methyladenosine (mA) methylation modification of mRNA has been studied extensively. It has been reported that mA determines mRNA fate and participates in many cellular functions and reactions, including oxidative stress. The PLOD2 gene encodes a protein that plays a key role in tissue remodeling and fibrotic processes.
METHODS
The mA methylation and expression levels of PLOD2 were determined by mA methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative polymerase chain reaction (qPCR) in the testes of varicocele rats compared with control. To determine whether IGF2BP2 had a targeted effect on the PLOD2 mRNA, RNA immunoprecipitation-qPCR (RIP-qPCR) and luciferase assays were performed. CRISPR/dCas13b-ALKBH5 could downregulate mA methylation level of PLOD2, which plays an important role in PLOD2-mediated cell proliferation and apoptosis in GC-2 cells.
RESULTS
PLOD2 was frequently exhibited with high mA methylation and expression level in the testes of varicocele rats compared with control. In addition, we found that IGF2BP2 binds to the mA-modified 3' untranslated region (3'-UTR) of PLOD2 mRNA, thereby positively regulating its mRNA stability. Targeted specific demethylation of PLOD2 mA by CRISPR/dCas13b-ALKBH5 system can significantly decrease the mA and expression level of PLOD2. Furthermore, demethylation of PLOD2 mRNA dramatically promote GC-2 cell proliferation and inhibit cell apoptosis under oxidative stress.
CONCLUSION
As a result, we found that varicocele-induced oxidative stress promoted PLOD2 expression level via mA methylation modification. In addition, targeting mA demethylation of PLOD2 by CRISPR/dCas13b-ALKBH5 system can regulate GC-2 cell proliferation and apoptosis under oxidative stress. Taken together, our study has acquired a better understanding of the mechanisms underlying male infertility associated with oxidative stress, as well as a novel therapeutic target for male infertility.
Topics: Male; Animals; Rats; Humans; Spermatocytes; Varicocele; 3' Untranslated Regions; Adenosine; Infertility, Male; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; RNA-Binding Proteins
PubMed: 37670228
DOI: 10.1186/s11658-023-00475-4 -
Journal of Advanced Research Sep 2023The R-loop is a naturally formed three-strand nucleic acid structure that recently has been reported to participate in multiple biological processes and helped answer...
INTRODUCTION
The R-loop is a naturally formed three-strand nucleic acid structure that recently has been reported to participate in multiple biological processes and helped answer some previously unexplained scientific questions. Meiosis process involves multiple chromatin-related events such as DNA double-stranded breaks (DSB) formation, repairing and transcriptional dynamics.
OBJECTIVES
Explore the regulatory roles and physiological functions of R-loops in the mammalian meiosis process.
METHODS
In our study, using genome-wide S9.6 CUT & Tag seq, we first mapped the genomic distribution and dynamic changes of R-loop during the meiotic process in mice, from spermatogonia to secondary spermatocytes. And we further explore the role of R-loop in physiological conditions by constructing conditional knockout mice of Rnaseh1, which deleted the R-loop endonuclease before meiosis entry.
RESULTS
R-loop predominantly distributes at promoter-related regions and varies across different meiotic stages. By joint analysis with the corresponding transcriptome, we found that the R-loop was closely related to transcription during the meiotic process. The high frequency of promoter-related R-loop in meiotic cells is usually accompanied by high transcription activity, and we further verified this in the leptotene/zygotene to the pachytene transition process. Moreover, the lack of RNase H1 caused sterility in male mice with R-loop accumulation and abnormal DSB repair in spermatocytes. Further analysis showed that abnormal R-loop accumulation in the leptotene/zygotene stages influenced transcriptional regulation in the pachytene stage.
CONCLUSION
The mutual regulation of the R-loop and transcription plays an essential role in spermatogenesis. And R-loop is also important for the normal repair process of DSB during meiosis.
Topics: Male; Mice; Animals; R-Loop Structures; DNA Breaks, Double-Stranded; Meiosis; Spermatogenesis; Spermatocytes; Mice, Knockout; Mammals
PubMed: 36396044
DOI: 10.1016/j.jare.2022.10.016 -
ELife Feb 2022The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair,...
The phosphatidylinositol 3' kinase (PI3K)-related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.
Topics: Animals; Ataxia Telangiectasia Mutated Proteins; DNA Damage; DNA Repair; Male; Meiosis; Mice, Inbred C57BL; Morpholines; Phosphorylation; Proteome; Pyrimidines; RNA, Messenger; Signal Transduction; Spermatocytes; Testis; Mice
PubMed: 35133275
DOI: 10.7554/eLife.68648 -
Cells Sep 2020Estrogen signaling plays important roles in testicular functions and tumorigenesis. Fifteen years ago, it was discovered that a member of the G protein-coupled receptor... (Review)
Review
Estrogen signaling plays important roles in testicular functions and tumorigenesis. Fifteen years ago, it was discovered that a member of the G protein-coupled receptor family, GPR30, which binds also with high affinity to estradiol and is responsible, in part, for the rapid non-genomic actions of estrogens. GPR30, renamed as GPER, was detected in several tissues including germ cells (spermatogonia, spermatocytes, spermatids) and somatic cells (Sertoli and Leydig cells). In our previous review published in 2014, we summarized studies that evidenced a role of GPER signaling in mediating estrogen action during spermatogenesis and testis development. In addition, we evidenced that GPER seems to be involved in modulating estrogen-dependent testicular cancer cell growth; however, the effects on cell survival and proliferation depend on specific cell type. In this review, we update the knowledge obtained in the last years on GPER roles in regulating physiological functions of testicular cells and its involvement in neoplastic transformation of both germ and somatic cells. In particular, we will focus our attention on crosstalk among GPER signaling, classical estrogen receptors and other nuclear receptors involved in testis physiology regulation.
Topics: Cell Transformation, Neoplastic; ErbB Receptors; Estradiol; Gene Expression Regulation, Neoplastic; Humans; Leydig Cells; Male; Receptor Cross-Talk; Receptors, Estrogen; Receptors, G-Protein-Coupled; Sertoli Cells; Signal Transduction; Spermatids; Spermatocytes; Spermatogenesis; Spermatogonia; Testicular Neoplasms; Testis
PubMed: 32957524
DOI: 10.3390/cells9092115 -
American Journal of Human Genetics Feb 2021Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still...
Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.
Topics: Adult; Azoospermia; Chromosome Pairing; Codon, Nonsense; DNA-Binding Proteins; Female; Homozygote; Humans; Male; Meiosis; Middle Aged; Mutation; Nuclear Proteins; Pedigree; Primary Ovarian Insufficiency; Spermatocytes; Synaptonemal Complex; Whole Genome Sequencing
PubMed: 33508233
DOI: 10.1016/j.ajhg.2021.01.010 -
PLoS Genetics Jun 2023Transposable elements (TE) are mobile DNA sequences whose excessive proliferation endangers the host. Although animals have evolved robust TE-targeting defenses,...
Transposable elements (TE) are mobile DNA sequences whose excessive proliferation endangers the host. Although animals have evolved robust TE-targeting defenses, including Piwi-interacting (pi)RNAs, retrotransposon LINE-1 (L1) still thrives in humans and mice. To gain insights into L1 endurance, we characterized L1 Bodies (LBs) and ORF1p complexes in germ cells of piRNA-deficient Maelstrom null mice. We report that ORF1p interacts with TE RNAs, genic mRNAs, and stress granule proteins, consistent with earlier studies. We also show that ORF1p associates with the CCR4-NOT deadenylation complex and PRKRA, a Protein Kinase R factor. Despite ORF1p interactions with these negative regulators of RNA expression, the stability and translation of LB-localized mRNAs remain unchanged. To scrutinize these findings, we studied the effects of PRKRA on L1 in cultured cells and showed that it elevates ORF1p levels and L1 retrotransposition. These results suggest that ORF1p-driven condensates promote L1 propagation, without affecting the metabolism of endogenous RNAs.
Topics: Humans; Male; Mice; Animals; Retroelements; Ribonucleoproteins; Piwi-Interacting RNA; Spermatocytes; Long Interspersed Nucleotide Elements; RNA; RNA, Messenger; Cytoplasm; RNA, Small Interfering
PubMed: 37307272
DOI: 10.1371/journal.pgen.1010797