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Biochimica Et Biophysica Acta.... Sep 2022Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome...
Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome alignment, which contribute to genome stability in mitosis. However, the functions and mechanisms of CENP-E in the meiotic division of male spermatocytes remain largely unknown. In this study, by combining the use of chemical inhibitors, siRNA-mediated gene knockdown, immunohistochemistry, and high-resolution microscopy, we have found that CENP-E inhibition results in chromosome misalignment and metaphase arrest in dividing spermatocyte during meiosis. Strikingly, we have revealed that CENP-E regulates spindle organization in metaphase I spermatocytes and cultured GC-2 spd cells. CENP-E depletion leads to spindle elongation, chromosome misalignment, and chromosome instability in spermatocytes. Together, these findings indicate that CENP-E mediates the kinetochore recruitment of BubR1, spindle assembly checkpoint and chromosome alignment in dividing spermatocytes, which finally contribute to faithful chromosome segregation and chromosome stability in the male meiotic division.
Topics: Animals; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Genomic Instability; Kinesins; Kinetochores; Male; Mice; Mitosis; Spermatocytes
PubMed: 35680098
DOI: 10.1016/j.bbamcr.2022.119306 -
Yakugaku Zasshi : Journal of the... 2021Heavy metals, both toxic and essential, have long been an important research focus in life science. To investigate the intracellular actions of heavy metals at the... (Review)
Review
Heavy metals, both toxic and essential, have long been an important research focus in life science. To investigate the intracellular actions of heavy metals at the molecular level, I have been exploring protein factors involved in induction of metallothionein (MT) genes by heavy metals that specifically bind to a metal responsive element (MRE) in the region upstream of the human MT-IIA gene. Purification of a zinc-dependent MRE-binding factor, and cloning of its cDNA identified a sequence identical to that of metal-responsive transcription factor-1 (MTF-1). MTF-1, which is characterized by six tandem repeats of the CH type zinc finger motif, is indispensable for induction of MT gene expression by multiple types of heavy metal, but zinc is the only metal that can directly activate MTF-1 binding to the MRE, indicating that other heavy metal signals act through zinc as a second messenger. Functional analysis of various MTF-1 point mutants revealed several cysteine (Cys) residues critical for DNA binding and/or transactivation activity. Interestingly, six finger motifs seem to mediate several MTF-1 functions other than DNA binding. Immunohistochemical analyses of various mouse tissues revealed selective expression of MTF-1 in spermatocytes among the testicular cells, suggesting roles relevant to spermatogenesis. The zinc regulon, under the control of MTF-1, will likely provide good clues to aid in unraveling novel functions of intracellular zinc ions.
Topics: Animals; DNA-Binding Proteins; Gene Expression; Horses; Humans; Male; Metallothionein; Mice; Point Mutation; Spermatocytes; Spermatogenesis; Tandem Repeat Sequences; Transcription Factors; Zinc; Zinc Fingers; Transcription Factor MTF-1
PubMed: 34078794
DOI: 10.1248/yakushi.20-00247 -
Biochemical and Biophysical Research... Jun 2017The meiotic G2/M1 transition is mostly regulated by posttranslational modifications, however, the cross-talk between different posttranslational modifications is not...
The meiotic G2/M1 transition is mostly regulated by posttranslational modifications, however, the cross-talk between different posttranslational modifications is not well-understood, especially in spermatocytes. Sumoylation has emerged as a critical regulatory event in several developmental processes, including reproduction. In mouse oocytes, inhibition of sumoylation caused various meiotic defects and led to aneuploidy. However, the role of sumoylation in male reproduction has only begun to be elucidated. Given the important role of several SUMO targets (including kinases) in meiosis, in this study, the role of sumoylation was addressed by monitoring the G2/M1 transition in pachytene spermatocytes in vitro upon inhibition of sumoylation. Furthermore, to better understand the cross-talk between sumoylation and phosphorylation, the activity of several kinases implicated in meiotic progression was also assessed upon down-regulation of sumoylation. The results of the analysis demonstrate that inhibition of sumoylation with ginkgolic acid (GA) arrests the G2/M1 transition in mouse spermatocytes preventing chromosome condensation and disassembling of the synaptonemal complex. Our results revealed that the activity of PLK1 and the Aurora kinases increased during the G2/M1 meiotic transition, but was negatively regulated by the inhibition of sumoylation. In the same experiment, the activity of c-Abl, the ERKs, and AKT were not affected or increased after GA treatment. Both the AURKs and PLK1 appear to be "at the right place, at the right time" to at least, in part, explain the meiotic arrest obtained in the spermatocyte culture.
Topics: Animals; Cell Cycle Checkpoints; Cells, Cultured; Male; Mice; Mice, Inbred C57BL; Phosphorylation; Phosphotransferases; Receptor Cross-Talk; Small Ubiquitin-Related Modifier Proteins; Spermatocytes; Sumoylation
PubMed: 28435066
DOI: 10.1016/j.bbrc.2017.04.107 -
PloS One 2012In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y... (Comparative Study)
Comparative Study
In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI), which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.
Topics: Case-Control Studies; Chromosomal Instability; Chromosomes, Human, X; Chromosomes, Human, Y; Histones; Humans; In Situ Hybridization, Fluorescence; Male; Meiosis; Sex Chromatin; Spermatocytes; Spermatogenesis; Testis
PubMed: 22355370
DOI: 10.1371/journal.pone.0031485 -
Pharmacological Reviews Jun 2008In multicellular organisms, cell-cell interactions are mediated in part by cell junctions, which underlie tissue architecture. Throughout spermatogenesis, for instance,... (Review)
Review
In multicellular organisms, cell-cell interactions are mediated in part by cell junctions, which underlie tissue architecture. Throughout spermatogenesis, for instance, preleptotene leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier to enter the adluminal compartment for continued development. At the same time, germ cells must also remain attached to Sertoli cells, and numerous studies have reported extensive restructuring at the Sertoli-Sertoli and Sertoli-germ cell interface during germ cell movement across the seminiferous epithelium. Furthermore, the proteins and signaling cascades that regulate adhesion between testicular cells have been largely delineated. These findings have unveiled a number of potential "druggable" targets that can be used to induce premature release of germ cells from the seminiferous epithelium, resulting in transient infertility. Herein, we discuss a novel approach with the aim of developing a nonhormonal male contraceptive for future human use, one that involves perturbing adhesion between Sertoli and germ cells in the testis.
Topics: Adherens Junctions; Animals; Cell Adhesion; Cell Communication; Contraceptive Agents, Male; Humans; Intercellular Junctions; Male; Sertoli Cells; Spermatocytes; Spermatogenesis; Testis
PubMed: 18483144
DOI: 10.1124/pr.107.07105 -
Cell Research Mar 2020The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains...
The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at mA-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with mA-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of mA in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and mA-modified start codons in junction sequences.
Topics: Adenosine; Adult; Animals; Humans; Male; Mice; Mice, Inbred C57BL; Open Reading Frames; RNA, Circular; Spermatocytes; Spermatogenesis; Young Adult
PubMed: 32047269
DOI: 10.1038/s41422-020-0279-8 -
Molecular Cell Jun 2019Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a...
Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. They also have delayed and/or fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures-stochastic on autosomes, nearly absolute on X and Y-cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines a pleckstrin homology (PH) domain in REC114 and its direct intermolecular contacts with ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model in which ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, thereby regulating DSB formation.
Topics: Animals; Cell Cycle Proteins; Chromosome Pairing; Chromosome Segregation; Chromosomes; Crystallography, X-Ray; DNA Breaks, Double-Stranded; Female; Homologous Recombination; Male; Meiosis; Mice; Protein Conformation; Recombinases; Spermatocytes
PubMed: 31003867
DOI: 10.1016/j.molcel.2019.03.023 -
International Journal of Biological... 2020The male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted the effective utilization of the heterosis from crossbreeding of cattle and...
The male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted the effective utilization of the heterosis from crossbreeding of cattle and yak. Based on our previous studies, the significant divergences of the transcriptomic and proteomic sequencing between yak and cattleyak prompt us to investigate the critical roles of microRNAs in post-transcriptional regulation of gene expression during spermatogenesis. TUNEL-POD analysis presented sharply decreased spermatogenic cell types and the increased apoptotic spermatogonia in cattleyak. The STA-PUT velocity sedimentation was employed to obtain spermatogonia and spermatocytes from cattle, yak and cattleyak and these spermatogenic cells were verified by the morphological and phenotypic identification. MicroRNA microarray showed that 27 differentially expressed miRNAs were simultaneously identified both in cattleyak vs cattle and in cattleyak vs yak comparisons. Further analysis revealed that the down-regulation of bta-let-7 families, bta-miR-125 and bta-miR-23a might impair the RA-induced differentiation of spermatogonia. Target gene analysis for differentially expressed miRNAs revealed that miRNAs targeted major players involved in vesicle-mediated transport, regulation of protein kinase activity and Pathways in cancer. In addition, spermatogonia transfection analysis revealed that the down-regulation of bta-miR-449a in the cattleyak might block the transition of male germ cells from the mitotic cycle to the meiotic program. The present study provided valuable information for future elucidating the regulatory roles of miRNAs involved in spermatogenic arrest of cattleyak.
Topics: Animals; Cattle; Cells, Cultured; Male; MicroRNAs; Spermatocytes; Spermatogenesis; Spermatogonia
PubMed: 31929752
DOI: 10.7150/ijbs.38232 -
Development (Cambridge, England) Aug 2018Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in...
Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in growing clones via short cylindrical intercellular bridges (ICBs). These ICBs, which enlarge in meiotic spermatocytes, have been demonstrated to provide a conduit for postmeiotic haploid spermatids to share sex chromosome-derived gene products. In addition to ICBs, spermatogonia exhibit multiple thin cytoplasmic projections. Here, we have explored the nature of these projections in mice and find that they are dynamic, span considerable distances from their cell body (≥25 μm), either terminate or physically connect multiple adjacent spermatogonia, and allow for sharing of macromolecules. Our results extend the current model that subsets of spermatogonia exist as isolated cells or clones, and support a model in which spermatogonia of similar developmental fates are functionally connected through a shared dynamic cytoplasm mediated by thin cytoplasmic projections.
Topics: Animals; Cell Differentiation; Cytoplasm; Diffusion; Green Fluorescent Proteins; Intracellular Space; Macromolecular Substances; Male; Mammals; Meiosis; Mice, Transgenic; Papio; Rats; Spermatocytes; Spermatogonia
PubMed: 29980567
DOI: 10.1242/dev.161323 -
PloS One 2022A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously...
A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously showed that Start regulates steroidogenesis through controlling the expression of Star and Hsd3b1 genes in Leydig cells, but its function in germ cells was not known. Here we verified that a spermatocyte-specific protease gene, Prss43/Tessp-3, was downregulated in Start-knockout testes. To investigate the transcriptional regulatory activity of Start in spermatocytes, we first performed a series of reporter gene assays using a thymidine kinase promoter in spermatocyte-derived GC-2spd(ts) cells. A 5.4-kb genome sequence encompassing Start exhibited enhancer activity for this promoter, and the activity was decreased by knockdown of Start. Deletion of the Start promoter and replacement of the Start sequence abolished the enhancer activity and, consistently, the activity was detected in further experiments only when Start was actively transcribed. We then examined whether the Prss43/Tessp-3 gene could be a target of Start. A reporter gene assay demonstrated that the 5.4-kb sequence exhibited enhancer activity for a Prss43/Tessp-3 promoter in GC-2spd(ts) cells and that the activity was significantly decreased by knockdown of Start. These results suggest that Start functions in transcriptional activation of the Prss43/Tessp-3 gene in spermatocytes. Given that Start is presumed to regulate steroidogenic genes at the posttranscriptional level in Leydig cells, the function in spermatocytes is a novel role of Start. These findings provide an insight into multifunctionality of lncRNAs in the testis.
Topics: Animals; Gene Expression Regulation; Male; Mice; Promoter Regions, Genetic; RNA, Long Noncoding; Spermatocytes; Testis
PubMed: 36006924
DOI: 10.1371/journal.pone.0273279