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Cell Reports Feb 2017Meiotic homologous recombination (HR) is important for proper chromosomal segregation during gametogenesis and facilitates evolutionary adaptation via genomic...
Meiotic homologous recombination (HR) is important for proper chromosomal segregation during gametogenesis and facilitates evolutionary adaptation via genomic reshuffling. In most eukaryotes, HR is mediated by two recombinases, the ubiquitous RAD51 and the meiosis-specific DMC1. The role of RAD51 in mammalian meiosis is unclear and study of its function is limited due to embryonic lethality of RAD51 knockouts. Here, we developed an in vivo meiotic knockdown and protein complementation system to study RAD51 during mouse spermatogenesis. We show that RAD51 is crucial during meiotic prophase and its loss leads to depletion of late prophase I spermatocytes through a p53-dependent apoptotic pathway. This phenotype is distinct from that observed in the DMC1 knockdown. Our meiotic knockdown and complementation system establishes an experimental platform for mechanistic studies of meiotic proteins with unknown functions or essential genes for which a testis-specific knockout is not possible.
Topics: Animals; Apoptosis; Cell Cycle Proteins; Chromosome Segregation; Homologous Recombination; Male; Meiosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitosis; Rad51 Recombinase; Recombinases; Spermatocytes; Spermatogenesis
PubMed: 28178517
DOI: 10.1016/j.celrep.2017.01.024 -
Cell Reports Sep 2012Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into...
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.
Topics: Animals; Cell Differentiation; Cell Line; Haploidy; Humans; Male; Mice; Pluripotent Stem Cells; Spermatids; Spermatocytes; Spermatogenesis; Transcription Factors
PubMed: 22921399
DOI: 10.1016/j.celrep.2012.07.015 -
Basic & Clinical Pharmacology &... Feb 2020Methotrexate (MTX) is widely used to treat cancers and systemic autoimmune diseases. However, it is severely toxic to healthy cells, especially those of the reproductive...
Methotrexate (MTX) is widely used to treat cancers and systemic autoimmune diseases. However, it is severely toxic to healthy cells, especially those of the reproductive system, and therefore poses a great risk to patient fertility. In addition, the underlying mechanism of MTX-induced reproductive toxicity has not yet been fully elucidated. Here, a spermatocyte cell line (GC2) was used as an in vitro model system to determine whether MTX induces autophagy and apoptosis, and to elucidate the role of reactive oxygen species (ROS) and Ca in these two processes. Treatment with MTX resulted in a dramatic decrease in cell viability, inhibition of cell proliferation, collapse of the mitochondrial membrane potential and activation of caspase 3, suggesting that MTX induced apoptosis. Moreover, MTX activated autophagy, as indicated by conversion of LC3-I to LC3-II (microtubule-associated protein 1 light chain 3) and an increase in the number of LC3 puncta. Furthermore, MTX triggered ROS overproduction, rather than a Ca burst. Intriguingly, eliminating excess ROS significantly alleviated MTX-induced apoptosis and autophagy. In addition, inhibiting autophagy significantly reversed apoptosis and promoted cell survival, indicating that autophagy aggravated MTX-induced apoptosis in GC2 cells. Taken together, these results suggest that ROS signalling, not Ca , is critical in mediating MTX-induced autophagy and apoptosis and autophagy serves as a promoted partner of apoptosis to deteriorate MTX-induced cytotoxicity in GC2 cells. The findings from this study provide new perspectives for evaluating the reproductive toxicity of MTX.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Calcium; Cell Line; Cell Proliferation; Cell Survival; Male; Membrane Potential, Mitochondrial; Methotrexate; Mice; Reactive Oxygen Species; Signal Transduction; Spermatocytes
PubMed: 31420979
DOI: 10.1111/bcpt.13306 -
Cell Reports Dec 2018Heat shock factors (Hsfs) are transcription factors that regulate responses to heat shock and other environmental stimuli. Four heat shock factors (Hsf1-4) have been...
Heat shock factors (Hsfs) are transcription factors that regulate responses to heat shock and other environmental stimuli. Four heat shock factors (Hsf1-4) have been characterized from vertebrates to date. In addition to stress response, they also play important roles in development and gametogenesis. Here, we study the fifth member of heat shock factor family, Hsf5, using zebrafish as a model organism. Mutant hsf5 males, generated by CRISPR/Cas9 technique, were infertile with drastically reduced sperm count, increased sperm head size, and abnormal tail architecture, whereas females remained fertile. We show that Hsf5 is required for progression through meiotic prophase 1 during spermatogenesis as suggested by the accumulation of cells in the leptotene and zygotene-pachytene stages and increased apoptosis in post-meiotic cells. hsf5 mutants show gonadal misregulation of a substantial number of genes with roles in cell cycle, apoptosis, protein modifications, and signal transduction, indicating an important role of Hsf5 in early stages of spermatogenesis.
Topics: Animals; Apoptosis; Female; Fertility; Heat Shock Transcription Factors; Male; Meiosis; Mutation; Sex Characteristics; Spermatocytes; Spermatogenesis; Spermatozoa; Testis; Transcriptome; Zebrafish; Zebrafish Proteins
PubMed: 30566854
DOI: 10.1016/j.celrep.2018.11.090 -
Birth Defects Research. Part B,... Feb 20152-Hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV) absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing...
BACKGROUND
2-Hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV) absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing UV-induced photodecomposition. HMB has been detected in over 95% of randomly collected human urine samples from adults and from premature infants, and it may have estrogenic potential.
METHODS
To determine the effects of maternal and lactational exposure to HMB on development and reproductive organs of offspring, time-mated female Harlan Sprague-Dawley rats were dosed with 0, 1000, 3000, 10,000, 25,000, or 50,000 ppm HMB (seven to eight per group) added to chow from gestation day 6 until weaning on postnatal day (PND) 23.
RESULTS AND CONCLUSION
Exposure to HMB was associated with reduced body and organ weights in female and male offspring. No significant differences were observed in the number of implantation sites/litter, mean resorptions/litter, % litters with resorptions, number and weights of live fetuses, or sex ratios between the control and HMB dose groups. Normalized anogenital distance in male pups at PND 23 was decreased in the highest dose group. Spermatocyte development was impaired in testes of male offspring in the highest dose group. In females, follicular development was delayed in the highest dose group. However, by evaluating levels of the compound in rat serum, the doses at which adverse events occurred are much higher than usual human exposure levels. Thus, exposure to less than 10,000 ppm HMB does not appear to be associated with adverse effects on the reproductive system in rats.
Topics: Animals; Animals, Newborn; Benzophenones; Body Weight; Cell Count; Embryonic Development; Female; Lactation; Male; Organ Size; Pregnancy; Prenatal Exposure Delayed Effects; Rats, Sprague-Dawley; Reproduction; Seminiferous Tubules; Spermatocytes; Testosterone
PubMed: 25707689
DOI: 10.1002/bdrb.21137 -
STAR Protocols Sep 2022Germline development is challenging to study due to the diversity of cell types in mammalian testis. Here, we present an optimized protocol, namely centrifugal...
Germline development is challenging to study due to the diversity of cell types in mammalian testis. Here, we present an optimized protocol, namely centrifugal elutriation, that allows the simultaneous isolation of mouse germ cells at different stages with high purity within ∼2 h. This approach exploits the JE-5.0 centrifugal elutriation system that fractionates cells based on differential sedimentation gravity. We herein provide the optimized parameters and procedures for isolation of elongating spermatids, round spermatids, and pachytene spermatocytes from adult mouse testes. For complete details on the use and execution of this protocol, please refer to Bao et al. (2018).
Topics: Animals; Male; Mammals; Mice; Spermatids; Spermatocytes; Testis
PubMed: 35834386
DOI: 10.1016/j.xpro.2022.101540 -
BMC Genomics Apr 2015In mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable...
BACKGROUND
In mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable associations form through homologous chromosome synapsis. In male mouse meiosis, the largely heterologous X and Y chromosomes synapse only in their short pseudoautosomal regions (PARs), and DSBs persist along the unsynapsed non-homologous arms of these sex chromosomes. Asynapsis of these arms and the persistent DSBs then trigger transcriptional silencing through meiotic sex chromosome inactivation (MSCI), resulting in formation of the XY body. This inactive state is partially maintained in post-meiotic haploid spermatids (postmeiotic sex chromatin repression, PSCR). For the human, establishment of MSCI and PSCR have also been reported, but X-linked gene silencing appears to be more variable compared to mouse. To gain more insight into the regulation and significance of MSCI and PSCR among different eutherian species, we have performed a global analysis of XY pairing dynamics, DSB repair, MSCI and PSCR in the domestic dog (Canis lupus familiaris), for which the complete genome sequence has recently become available, allowing a thorough comparative analyses.
RESULTS
In addition to PAR synapsis between X and Y, we observed extensive self-synapsis of part of the dog X chromosome, and rapid loss of known markers of DSB repair from that part of the X. Sequencing of RNA from purified spermatocytes and spermatids revealed establishment of MSCI. However, the self-synapsing region of the X displayed higher X-linked gene expression compared to the unsynapsed area in spermatocytes, and was post-meiotically reactivated in spermatids. In contrast, genes in the PAR, which are expected to escape MSCI, were expressed at very low levels in both spermatocytes and spermatids. Our comparative analysis was then used to identify two X-linked genes that may escape MSCI in spermatocytes, and 21 that are specifically re-activated in spermatids of human, mouse and dog.
CONCLUSIONS
Our data indicate that MSCI is incomplete in the dog. This may be partially explained by extensive, but transient, self-synapsis of the X chromosome, in association with rapid completion of meiotic DSB repair. In addition, our comparative analysis identifies novel candidate male fertility genes.
Topics: Animals; Animals, Domestic; Chromosomes, Mammalian; DNA Breaks, Double-Stranded; DNA Repair; Dogs; Humans; Male; Meiosis; Mice; Sex Chromosomes; Spermatocytes; Spermatogenesis; Testis; X Chromosome Inactivation
PubMed: 25884295
DOI: 10.1186/s12864-015-1501-9 -
Journal of Molecular Biology Aug 2014Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we...
A conserved noncoding sequence can function as a spermatocyte-specific enhancer and a bidirectional promoter for a ubiquitously expressed gene and a testis-specific long noncoding RNA.
Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5'-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.
Topics: Animals; Base Sequence; Cell Adhesion Molecules; Chromosomal Proteins, Non-Histone; Conserved Sequence; Gene Expression Regulation; Histones; Liver; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Muscle Proteins; NIH 3T3 Cells; Organ Specificity; Promoter Regions, Genetic; Protein Processing, Post-Translational; RNA, Long Noncoding; Spermatocytes
PubMed: 25020229
DOI: 10.1016/j.jmb.2014.06.018 -
Methods in Molecular Biology (Clifton,... 2018A distinct form of X chromosome inactivation takes place during male meiosis, when the male sex chromosomes undergo a phenomenon known as meiotic sex chromosome...
A distinct form of X chromosome inactivation takes place during male meiosis, when the male sex chromosomes undergo a phenomenon known as meiotic sex chromosome inactivation (MSCI). MSCI is directed by DNA damage response signaling independent of Xist RNA to silence the transcriptional activity of the sex chromosomes, an essential event in male germ cell development. Here, we present protocols for the preparation and analyses of chromosome spread slides of mouse meiotic spermatocytes, thereby enabling a quick, inexpensive, and powerful cytological method to complement gene expression studies.
Topics: Animals; Epigenomics; Fluorescent Antibody Technique; Male; Meiosis; Mice; Microscopy, Fluorescence; Sex Chromosomes; Spermatocytes; X Chromosome Inactivation
PubMed: 30218364
DOI: 10.1007/978-1-4939-8766-5_10 -
ELife Mar 2020Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes...
Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes to produce sperm. Using live imaging and tomographic reconstructions of spermatocyte meiotic spindles in , we find the lagging X chromosome, a distinctive feature of anaphase I in males, is due to lack of chromosome pairing. The unpaired chromosome remains tethered to centrosomes by lengthening kinetochore microtubules, which are under tension, suggesting that a 'tug of war' reliably resolves lagging. We find spermatocytes exhibit simultaneous pole-to-chromosome shortening (anaphase A) and pole-to-pole elongation (anaphase B). Electron tomography unexpectedly revealed spermatocyte anaphase A does not stem solely from kinetochore microtubule shortening. Instead, movement of autosomes is largely driven by distance change between chromosomes, microtubules, and centrosomes upon tension release during anaphase. Overall, we define novel features that segregate both lagging and paired chromosomes for optimal sperm production.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Chromosome Pairing; Chromosome Segregation; Male; Meiosis; Spermatocytes; Spindle Apparatus; X Chromosome
PubMed: 32149606
DOI: 10.7554/eLife.50988