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Methods in Cell Biology 2021Drosophila spermatocyte centrioles are ideal for imaging studies. Their large, characteristic V conformation is both easy to identify and measure using standard imaging...
Drosophila spermatocyte centrioles are ideal for imaging studies. Their large, characteristic V conformation is both easy to identify and measure using standard imaging techniques. However, certain detailed features, such as their ninefold symmetry, are only visible below the diffraction limit of light. This is therefore a system that can benefit from the increased effective resolution potentially achievable by expansion microscopy. Here, I provide detailed protocols of two types of expansion microscopy methodologies applied to Drosophila spermatocyte centrioles, and discuss which is able to achieve the highest effective resolution in this system. I describe how to precisely measure these organelles post-expansion, and discuss how they can therefore be used as "molecular rulers" to troubleshoot and compare expansion techniques. I also provide protocols to combine expansion microscopy with super-resolution imaging in this tissue, discussing potential pitfalls. I conclude that expansion microscopy provides an effective alternative for thick tissues that are not amenable for traditional super-resolution techniques.
Topics: Animals; Centrioles; Drosophila; Male; Microscopy; Spermatocytes
PubMed: 33478691
DOI: 10.1016/bs.mcb.2020.06.008 -
Cytoskeleton (Hoboken, N.J.) 2023In the model organism insect Drosophila melanogaster short cilia assemble on spermatocytes that elaborate into 1.8 mm long flagella during spermatid differentiation. A...
In the model organism insect Drosophila melanogaster short cilia assemble on spermatocytes that elaborate into 1.8 mm long flagella during spermatid differentiation. A unique feature of these cilia/flagella is their lack of dependence on intraflagellar transport (IFT) for their assembly. Here, we show that in the common butterfly Pieris brassicae, the spermatocyte cilia are exceptionally long: about 40 μm compared to less than 1 μm in Drosophila. By transmission electron microscopy, we show that P. brassicae spermatocytes display several features not found in melanogaster, including compelling evidence of IFT structures and features of motile cilia.
Topics: Male; Animals; Cilia; Spermatocytes; Butterflies; Drosophila melanogaster; Biological Transport; Flagella; Drosophila
PubMed: 37036073
DOI: 10.1002/cm.21755 -
The Science of the Total Environment May 2023Fine particulate matter (PM) has been reported to cause various types of damage to male reproductive system, but the research on the underlying mechanisms is still...
Fine particulate matter (PM) has been reported to cause various types of damage to male reproductive system, but the research on the underlying mechanisms is still insufficient. This study attempted to explore the underlying mechanisms of this widely concerning environmental health problem through in vivo and in vitro exposure models. Significant pathological damage and abnormal mitochondria in spermatocytes were observed in the real-time PM exposure animal model. In addition, significant alterations in key biomarkers of iron metabolism and ferroptosis were found in testis tissues. Notably decreased cell viability was found in vitro. Moreover, the ferroptosis pathway was significantly enriched in the transcriptome enrichment analysis. Subsequent experiments showed that the two core events of ferroptosis, iron overload and lipid peroxidation, occurred in spermatocytes after PM treatment. Moreover, lipid metabolic genes (Acsl4 and Aloxe3) and the antioxidant gene Gpx4 were found to be key target genes of ferroptosis caused by PM in spermatocytes. Importantly, further studies showed that the damaging effect could be reversed by the iron chelator deferoxamine mesylate (DFOM) and the lipid peroxidation inhibitor ferrostatin-1 (Fer-1), which further confirmed the role of ferroptosis in PM toxicity. Our study revealed the vital role of ferroptosis in PM-induced male reproductive damage, providing novel insights into the air pollution-induced decrease in male fertility.
Topics: Animals; Male; Iron; Ferroptosis; Spermatocytes; Oxidation-Reduction; Lipid Peroxidation; Homeostasis; Particulate Matter
PubMed: 36781135
DOI: 10.1016/j.scitotenv.2023.162089 -
Nature Communications Mar 2023N6-methyladenosine (m6A) and its reader proteins YTHDC1, YTHDC2, and YTHDF2 have been shown to exert essential functions during spermatogenesis. However, much remains...
N6-methyladenosine (m6A) and its reader proteins YTHDC1, YTHDC2, and YTHDF2 have been shown to exert essential functions during spermatogenesis. However, much remains unknown about m6A regulation mechanisms and the functions of specific readers during the meiotic cell cycle. Here, we show that the m6A reader Proline rich coiled-coil 2A (PRRC2A) is essential for male fertility. Germ cell-specific knockout of Prrc2a causes XY asynapsis and impaired meiotic sex chromosome inactivation in late-prophase spermatocytes. Moreover, PRRC2A-null spermatocytes exhibit delayed metaphase entry, chromosome misalignment, and spindle disorganization at metaphase I and are finally arrested at this stage. Sequencing data reveal that PRRC2A decreases the RNA abundance or improves the translation efficiency of targeting transcripts. Specifically, PRRC2A recognizes spermatogonia-specific transcripts and downregulates their RNA abundance to maintain the spermatocyte expression pattern during the meiosis prophase. For genes involved in meiotic cell division, PRRC2A improves the translation efficiency of their transcripts. Further, co-immunoprecipitation data show that PRRC2A interacts with several proteins regulating mRNA metabolism or translation (YBX1, YBX2, PABPC1, FXR1, and EIF4G3). Our study reveals post-transcriptional functions of PRRC2A and demonstrates its critical role in the completion of meiosis I in spermatogenesis.
Topics: Male; Humans; Spermatogenesis; Meiosis; Prophase; Spermatocytes; Sex Chromosomes; RNA
PubMed: 36964127
DOI: 10.1038/s41467-023-37252-y -
Reproduction (Cambridge, England) Apr 2015Spermatogenesis is a continuous and productive process supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs), which arise from... (Review)
Review
Spermatogenesis is a continuous and productive process supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs), which arise from undifferentiated precursors known as gonocytes and are strictly controlled in a special 'niche' microenvironment in the seminiferous tubules. Sertoli cells, the only somatic cell type in the tubules, directly interact with SSCs to control their proliferation and differentiation through the secretion of specific factors. Spermatocyte meiosis is another key step of spermatogenesis, which is regulated by Sertoli cells on the luminal side of the blood-testis barrier through paracrine signaling. In this review, we mainly focus on the role of Sertoli cells in the regulation of SSC self-renewal and spermatocyte meiosis, with particular emphasis on paracrine and endocrine-mediated signaling pathways. Sertoli cell growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), as well as Sertoli cell transcription factors, such as ETS variant 5 (ERM; also known as ETV5), nociceptin, neuregulin 1 (NRG1), and androgen receptor (AR), have been identified as the most important upstream factors that regulate SSC self-renewal and spermatocyte meiosis. Other transcription factors and signaling pathways (GDNF-RET-GFRA1 signaling, FGF2-MAP2K1 signaling, CXCL12-CXCR4 signaling, CCL9-CCR1 signaling, FSH-nociceptin/OPRL1, retinoic acid/FSH-NRG/ERBB4, and AR/RB-ARID4A/ARID4B) are also addressed.
Topics: Humans; Male; Meiosis; Sertoli Cells; Signal Transduction; Spermatocytes; Spermatogenesis; Spermatogonia; Stem Cells
PubMed: 25504872
DOI: 10.1530/REP-14-0481 -
The Journal of Experimental Zoology Oct 1999The consequences of error during meiotic division in spermatogenesis can be serious: aneuploid spermatozoa, embryonic lethality, and developmental abnormalities.... (Review)
Review
The consequences of error during meiotic division in spermatogenesis can be serious: aneuploid spermatozoa, embryonic lethality, and developmental abnormalities. Recombination between homologs is essential to ensure normal segregation; thus the spermatocyte must time division precisely so that it occurs after recombination between chromosomes and accumulation of the cell-cycle machinery necessary to ensure an accurate segregation of chromosomes. We use two systems to investigate meiotic division during spermatogenesis in the mouse: pharmacological induction of meiotic metaphase in cultured spermatocytes and transillumination-mediated dissection of stage XII seminiferous tubule segments to monitor progress through the division phase. By these approaches we can assess timing of acquisition of competence for the meiotic division phase and the temporal order of events as division proceeds. Competence for the meiotic division arises in the mid-pachytene stage of meiotic prophase, after chromosomes have synapsed and coincident with the accumulation of the cell-cycle regulatory protein CDC25C. The activity of both MPF and topoisomerase II are required. The earliest hallmarks of the division phase are nuclear envelope breakdown, followed by phosphorylation of histone H3 and chromosome condensation. These events are likely to be monitored by checkpoint mechanisms since checkpoint proteins can be localized in nuclei and DNA-damaging agents delay entry into the meiotic division phase. Understanding how the spermatocyte regulates its entry into the meiotic division phase can help clarify the natural mechanisms ensuring accurate chromosome segregation and preventing aneuploidy. J. Exp. Zool. (Mol. Dev. Evol.) 285:243-250, 1999.
Topics: Animals; Cells, Cultured; Chromosome Aberrations; Chromosome Disorders; Chromosome Segregation; Humans; Male; Meiosis; Mesothelin; Mice; Seminiferous Tubules; Spermatocytes; Time Factors
PubMed: 10497323
DOI: No ID Found -
Biochemical and Biophysical Research... Jan 2021The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16...
The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16 gene as the chromatin highly marked with epigenetic modifications of both enhancers (H3K4me1 and H3K27ac) and silencers (H3K27me3) in mouse spermatocytes. In vitro reporter gene assay demonstrated that DES-K16 exhibited significant enhancer activity in spermatocyte-derived GC-2spd(ts) and hepatic tumor-derived Hepa1-6 cells, and a deletion of this sequence in GC-2spd(ts) cells resulted in a decrease and increase of Yipf5 and Kctd16 expression, respectively. This was consistent with increased and decreased expression of Yipf5 and Kctd16, respectively, in primary spermatocytes during testis development. While known dual enhancer-silencers exert each activity in different tissues, our data suggest that DES-K16 functions as both enhancer and silencer in a single cell type, GC-2spd(ts) cells. This is the first report on a dual enhancer-silencer element which activates and suppresses gene expression in a single cell type.
Topics: Animals; CRISPR-Cas Systems; Cell Line; Gene Editing; Histone Code; Male; Mice; Mice, Inbred C57BL; Silencer Elements, Transcriptional; Spermatocytes
PubMed: 33121685
DOI: 10.1016/j.bbrc.2020.10.049 -
Chemosphere Aug 2019Silica nanoparticles (SiNPs) are found in the environmental particulate matter and have been proved to pose an adverse effect on fertility. However, the relationship...
Silica nanoparticles (SiNPs) are found in the environmental particulate matter and have been proved to pose an adverse effect on fertility. However, the relationship between miRNA and apoptosis induced by SiNPs in spermatogenesis and its underlying mechanism remains confusing. Therefore, the present study was designed to investigate the toxic effects of SiNPs on spermatogenic cells mediated through miRNAs. Spermatocyte cells were divided into 0 μg/mL and 5 μg/mL SiNPs groups, and the cells were collected and analyzed after passaging for 1, 10, 20, and 30 generations. miRNA profile and mRNA profile of spermatocyte cells were measured after exposure to SiNPs for 30 generations. Further, mimics and inhibitors of miRNA were used to verify the relationship between miRNA and their predicted target genes in the 30th-generation cells. The results showed that the degree of cell apoptosis in the SiNPs group significantly increased in the 30th generation. After exposure to SiNPs for 30 generations, the expression of 15 miRNAs was altered, including 5 upregulated miRNAs and 10 downregulated miRNAs. Of the 15 miRNAs, miR-138 and miR-2861 were related to the death receptor pathway. The miR-2861 mimic could target to regulate the mRNA expression of fas/fasl/ripk1 and increase the protein expression of Fas/FasL/RIPK1/FADD/caspase-8/caspase-3 of spermatogenic cells in the 30th generation, while the miR-138 inhibitor could not. In conclusion, SiNPs could cause apoptosis of spermatocyte cells by inhibiting the expression of miRNA-2861, thereby resulting in the upregulation of mRNA expression of fas/fasl/ripk1 and activating the death receptor pathway of spermatocyte cells. miRNA-2861 could be considered a biomarker of the toxic effect of SiNPs on spermatocyte cells. The main finding: Silica nanoparticles induce apoptosis in spermatocyte cells through microRNA-2861 inhibition, thereby upregulating mRNA expression of fas/fasl/ripk1 and activating the death receptor pathway of spermatocyte cells.
Topics: Animals; Apoptosis; Cells, Cultured; Gene Expression Regulation; Male; Metabolic Networks and Pathways; Mice; MicroRNAs; Nanoparticles; Receptors, Death Domain; Silicon Dioxide; Spermatocytes; Spermatogenesis
PubMed: 31071558
DOI: 10.1016/j.chemosphere.2019.04.116 -
Theriogenology Oct 2022DNA cytosine methylation modification in the germline is of particular importance since it is a highly heritable epigenetic mark. Although cytosine methylation has been...
DNA cytosine methylation modification in the germline is of particular importance since it is a highly heritable epigenetic mark. Although cytosine methylation has been analyzed at the genome-scale for several mammalian species, our knowledge of DNA methylation patterns and the mechanisms underlying male hybrid sterility is still limited in domestic animals such as cattleyak. Here we for the first time show the genome-wide and single-base resolution landscape of methylcytosines (mC) in the primary spermatocyte (PSC) genome of yak with normal spermatogenesis and the inter-specific hybrid cattleyak with male infertility. A comparative investigation revealed that widespread differences are observed in the composition and patterning of DNA cytosine methylation between the two methylomes. Global CG or non-CG DNA methylation levels, as well as the number of mC sites, are increased in cattleyak compared to yak. Notably, the DNA methylome in cattleyak PSC exhibits promoter hypermethylation of meiosis-specific genes and piRNA pathway genes with respect to yak. Furthermore, major retrotransposonson classes are predominantly hypermethylated in cattleyak while those are fully hypomethylated in yak. KEGG pathway enrichment indicates Rap1 signaling and MAPK pathways may play potential roles in the spermatogenic arrest of cattleyak. Our present study not only provides valuable insights into distinct features of the cattleyak PSC methylome but also paves the way toward elucidating the complex, yet highly coordinated epigenetic modification during male germline development for inter-specific hybrid animals.
Topics: Animals; Cytosine; DNA; DNA Methylation; Epigenesis, Genetic; Epigenome; Infertility, Male; Male; Mammals; Spermatocytes
PubMed: 35988507
DOI: 10.1016/j.theriogenology.2022.08.016 -
Journal of Proteome Research Nov 2022Meiotic prophase I (MPI) is the most important event in mammalian meiosis. The status of the chromosome-binding proteins (CBPs) and the corresponding complexes and their...
Meiotic prophase I (MPI) is the most important event in mammalian meiosis. The status of the chromosome-binding proteins (CBPs) and the corresponding complexes and their functions in MPI have not yet been well scrutinized. Quantitative proteomics focused on MPI-related CBPs was accomplished, in which mouse primary spermatocytes in four different subphases of MPI were collected, and chromosome-enriched proteins were extracted and quantitatively identified. According to a stringent criterion, 1136 CBPs in the MPI subphases were quantified. Looking at the dynamic patterns of CBP abundance in response to MPI progression, the patterns were broadly divided into two groups: high abundance in leptotene and zygotene or that in pachytene and diplotene. Furthermore, 152 such CBPs were regarded as 26 CBP complexes with strict filtration, in which some of these complexes were perceived to be MPI-dependent for the first time. These complexes basically belonged to four functional categories, while their dynamic abundance changes following MPI appeared; the functions of DNA replication decreased; and transcription and synapsis were activated in zygotene, pachytene, and diplotene; in contrast to the traditional prediction, condensin activity weakened in pachytene and diplotene. Profiling of protein complexes thus offered convincing evidence of the importance of CBP complexes in MPI.
Topics: Male; Mice; Animals; Spermatocytes; Meiotic Prophase I; Meiosis; Carrier Proteins; Chromosomes; Mammals
PubMed: 36223561
DOI: 10.1021/acs.jproteome.2c00414