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Cell Proliferation May 2022To evaluate the expression, potential functions and mechanisms of long noncoding RNAs (lncRNAs) in the pathogenesis of varicocele (VC)-induced spermatogenic dysfunction.
OBJECTIVES
To evaluate the expression, potential functions and mechanisms of long noncoding RNAs (lncRNAs) in the pathogenesis of varicocele (VC)-induced spermatogenic dysfunction.
MATERIALS AND METHODS
We established a rat model with left experimental VC and divided rats into the sham group, the VC group, and the surgical treatment group (each group, n = 10). Haematoxylin and eosin (HE) staining and sperm quality were analysed to evaluate spermatogenesis function. LncRNA expression profiles were analysed using lncRNA-Seq (each group n = 3) and validated using quantitative real-time polymerase chain reaction (each group n = 10). Correlation analysis and gene target miRNA prediction were used to construct competing endogenous RNA network. The regulated signalling pathway and spermatogenic dysfunction of differentially expressed lncRNAs (DE lncRNAs) were validated by Western blot.
RESULTS
HE detection and sperm quality analysis showed that VC could induce spermatogenic dysfunction. Eight lncRNAs were upregulated and three lncRNAs were downregulated in the VC group compared with the sham group and surgical treatment group. The lncRNA of NONRATG002949.2, NONRATG001060.2, NONRATG013271.2, NONRATG022879.2, NONRATG023424.2, NONRATG005667.2 and NONRATG010686.2 were significantly negatively related to sperm quality, while NONRATG027523.1, NONRATG017183.2 and NONRATG023747.2 were positively related to sperm quality. The lncRNAs promote spermatogenic cell apoptosis and inhibit spermatogonia and spermatocyte proliferation and meiotic spermatocytes by regulating the PI3K-Akt signalling pathway.
CONCLUSION
DE lncRNAs may be potential biomarkers for predicting the risk of spermatogenic dysfunction in VC and the effect of surgical repair. These DE lncRNAs promote spermatogenic dysfunction by regulating the PI3K-Akt signalling pathway.
Topics: Animals; Gene Expression Profiling; Humans; Male; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Rats; Spermatogenesis; Varicocele
PubMed: 35297519
DOI: 10.1111/cpr.13220 -
Frontiers in Cell and Developmental... 2023Actin is a multi-functional protein that is involved in numerous cellular processes including cytoskeleton regulation, cell migration, and cellular integrity. In these... (Review)
Review
Actin is a multi-functional protein that is involved in numerous cellular processes including cytoskeleton regulation, cell migration, and cellular integrity. In these processes, actin's role in respect to its structure, complex mechanical, and protein-binding properties has been studied primarily in the cytoplasmic and cellular membrane compartments. However, its role in somatic cell nuclei has recently become evident where it participates in transcription, chromatin remodeling, and DNA damage repair. What remains enigmatic is the involvement of nuclear actin in physiological processes that lead to the generation of germ cells, in general, and primary spermatocytes, in particular. Here, we will discuss the possible role and nuclear localization of actin during meiotic prophase I and its interaction with chromatin remodeling complexes, the latter being essential for the control of pairing of homologous chromosomes, cross-over formation, and recombination. It is our hope that this perspective article will extend the scope of actin's nuclear function in germ cells undergoing meiotic division.
PubMed: 38078006
DOI: 10.3389/fcell.2023.1295452 -
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 -
Reproduction (Cambridge, England) Dec 2023A new allele of the senataxin gene Setxspcar3 causes meiotic arrest of spermatocytes with aberrant DNA damage and accumulation of R-loops.
IN BRIEF
A new allele of the senataxin gene Setxspcar3 causes meiotic arrest of spermatocytes with aberrant DNA damage and accumulation of R-loops.
ABSTRACT
An unbiased screen for discovering novel mouse genes for fertility identified the spcar3, spermatocyte arrest 3, mutant phenotype. The spcar3 mutation identified a new allele of the Setx gene, encoding senataxin, a DNA/RNA helicase that regulates transcription termination by resolving DNA/RNA hybrid R-loop structures. The Setxspcar3 mutant mice exhibit male infertility and female subfertility. Histology of the Setxspcar3 mutant testes revealed the absence of spermatids and mature spermatozoa in the seminiferous tubules. Cytological analysis of chromosome preparations of the Setxspcar3 mutant spermatocytes revealed normal synapsis, but aberrant DNA damage in the autosomes, defective formation of the sex body, and arrest of meiosis in mid-prophase. Additionally, Setxspcar3 testicular cells exhibit abnormal accumulation of R-loops. Transient expression assays identified regions of the senataxin protein required for sub-nuclear localization. Together, these results not only confirm that senataxin is required for normal meiosis and spermatogenesis but also provide a new resource for the determination of its role in maintaining R-loop formation and genome integrity.
Topics: Humans; Animals; Male; Female; Mice; Alleles; RNA; Spermatogenesis; RNA Helicases; Spermatocytes; Meiosis; Infertility, Male; DNA
PubMed: 37801077
DOI: 10.1530/REP-23-0166 -
Frontiers in Cell and Developmental... 2021Meiosis is a cellular division process that produces gametes for sexual reproduction. Disruption of complex events throughout meiosis, such as synapsis and homologous... (Review)
Review
Meiosis is a cellular division process that produces gametes for sexual reproduction. Disruption of complex events throughout meiosis, such as synapsis and homologous recombination, can lead to infertility and aneuploidy. To reveal the molecular mechanisms of these events, transcriptome studies of specific substages must be conducted. However, conventional methods, such as bulk RNA-seq and RT-qPCR, are not able to detect the transcriptional variations effectively and precisely, especially for identifying cell types and stages with subtle differences. In recent years, mammalian meiotic transcriptomes have been intensively studied at the single-cell level by using single-cell RNA-seq (scRNA-seq) approaches, especially through two widely used platforms, Smart-seq2 and Drop-seq. The scRNA-seq protocols along with their downstream analysis enable researchers to accurately identify cell heterogeneities and investigate meiotic transcriptomes at a higher resolution. In this review, we compared bulk RNA-seq and scRNA-seq to show the advantages of the scRNA-seq in meiosis studies; meanwhile, we also pointed out the challenges and limitations of the scRNA-seq. We listed recent findings from mammalian meiosis (male and female) studies where scRNA-seq applied. Next, we summarized the scRNA-seq analysis methods and the meiotic marker genes from spermatocytes and oocytes. Specifically, we emphasized the different features of the two scRNA-seq protocols (Smart-seq2 and Drop-seq) in the context of meiosis studies and discussed their strengths and weaknesses in terms of different research purposes. Finally, we discussed the future applications of scRNA-seq in the meiosis field.
PubMed: 34485276
DOI: 10.3389/fcell.2021.673642 -
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 -
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