Mouse Germ Cells Research Articles

Germ cell responses to doxorubicin exposure in vitro

Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1mM, respectively) of Dox, and fixed 1h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells.

Genome-Wide Deletion Screening with the Array CGH Method in Mouse Offspring Derived from Irradiated Spermatogonia Indicates that Mutagenic Responses are Highly Variable among Genes.

Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10-2/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10-5/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current method is substandard for detecting small deletions.

Transcriptomic Variation during Spermiogenesis in Mouse Germ Cells.

To explore variations in the transcription activity during spermiogenesis, round and elongated spermatids were collected from ICR/CD1 model mice using laser capture microdissection (LCM) and cauda epididymal sperm samples. The transcripts were sequenced using RNA-seq, and the reads were mapped to mm9. The majority of the reads (70%) in the round and elongated spermatids were mappable to known and predicted exons, but that in sperm was only 9%. The results of the distribution of reads suggested that alternative splicing was more complicated in sperm than in round and elongated spermatids. In the 19,127 genes, we detected the expression of 5,104 de novo genes and 91,112 alternative splicing events, and 12,105 were differentially expressed. Gene ontology (GO), InterPro domains, and KEGG revealed changes in gene transcription, mitochondrial protein translation, cellular components, and energy metabolism during spermiogenesis. The results provided considerable information about alternative splicing events, differentiallly expressed genes (DEGs), and novel transcriptions during spermiogenesis in mice.

DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells

Chromatin remodeling is important for the epigenetic reprogramming of human primordial germ cells. However, the comprehensive chromatin state has not yet been analyzed for human fetal germ cells (FGCs). Here we use nucleosome occupancy and methylation sequencing method to analyze both the genome-wide chromatin accessibility and DNA methylome at a series of crucial time points during fetal germ cell development in both human and mouse. We find 116 887 and 137 557 nucleosome-depleted regions (NDRs) in human and mouse FGCs, covering a large set of germline-specific and highly dynamic regulatory genomic elements, such as enhancers. Moreover, we find that the distal NDRs are enriched specifically for binding motifs of the pluripotency and germ cell master regulators such as NANOG, SOX17, AP2γ and OCT4 in human FGCs, indicating the existence of a delicate regulatory balance between pluripotency-related genes and germ cell-specific genes in human FGCs, and the functional significance of these genes for germ cell development in vivo. Our work offers a comprehensive and high-resolution roadmap for dissecting chromatin state transition dynamics during the epigenomic reprogramming of human and mouse FGCs.

The morphological expression of an oligosaccharide chain with bisecting N-acetylglucosamine recognized by anti-sperm auto-monoclonal antibody, Ts4, in mouse germ and somatic cells

The morphological expression of an oligosaccharide chain with bisecting N-acetylglucosamine recognized by anti-sperm auto-monoclonal antibody, Ts4, in mouse germ and somatic cells

Effective production of recipient male pigs for spermatogonial stem cell transplantation by intratesticular injection with busulfan

Germ cell transplantation has facilitated spermatogonial stem cell (SSC) and spermatogenesis research and shown great potential in the seed-breeding of domestic livestock. However, little progress has been made in large animals, primarily reflecting the difficulties in preparing sterile recipients. Here, we developed a novel protocol to prepare recipient pigs through the direct injection of busulfan into the cavum vaginale of the scrotums of Landrace-Large bi-crossbreeding male pigs and Seghers male pigs, two economically-important types of pigs, to eliminate endogenous spermatogonia. No severe diseases or weight loss was observed in either pig type after the injection with busulfan. Histologic analysis showed an advanced and dose-dependent germ cell loss, with complete germ cell loss observed in the highest dose group, 3.0 mg/kg in the Landrace-Large bi-crossbreeding pigs and 2.0 mg/kg in the Seghers pigs. A smaller seminiferous tubule diameter, a vacuolized seminiferous epithelium and the overproliferation interstitial cells, frequently observed in mouse germ cell deficiency models, were present in the most of the high-dose busulfan-treated groups. Molecular markers detected in Seghers pigs further confirmed the depletion of endogenous germ cells, providing an accessible niche for exogenous SSCs. This study provides a basis to prepare the transplantation recipients of SSCs in pigs.

Human umbilical perivascular cells: a novel source of MSCs to support testicular niche regeneration.

The expansion of functional testicular biopsy-derived human spermatogonial stem cells (hSSC) ex-vivo may enable the restoration of fertility in pre-pubertal males having undergone gonadotoxic therapies or men with severe male factor infertility. Various somatic cells are known to regulate SSC homeostasis and spermatogenesis in the developing and adult testis. Prior attempts to recapitulate this niche demonstrated the requirement of feeder cells, such as endogenous testicular somatic cells, for germ cell expansion ex-vivo. However, this strategy has limitations for the expansion of hSSCs from tissue biopsies where spermatogenesis is absent or defective. Our aim was to evaluate first trimester human umbilical cord perivascular cells (FTM HUCPVCs), a novel source of mesenchymal stromal-like cells (MSCs), as potential human feeder cells for standardized hSSC expansion ex-vivo. Targeted RNA sequencing analysis demonstrated that CD90+ve FTM HUCPVCs expanded in vitro under germ cell culture conditions express a profile of targeted testicular-associated transcripts that is similar to cultured human CD90+ve testicular adherent cells (hTACs) and secrete LIF, FGF2 and BMP4, key growth factors known to regulate spermatogenesis. We also demonstrated that mitotically-inactivated FTM HUCPVCs support the expansion of mouse germ cells and putative SSCs ex-vivo, and that FTM HUCPVC transplantation promotes in vivo germ cell regeneration following mono-2- ethylhexyl phthalate (MEHP)-induced seminiferous tubule damage in a murine model, including a partial reconstitution of tubular cellular architecture and reestablishment of DAZL and acrosin+ve germ cell layers. Together, these data suggest that FTM HUCPVCs have phenotypical and functional properties that may support repair of the human testicular niche.

Radiofrequency radiation (900 MHz)-induced DNA damage and cell cycle arrest in testicular germ cells in swiss albino mice.

Even though there are contradictory reports regarding the cellular and molecular changes induced by mobile phone emitted radiofrequency radiation (RFR), the possibility of any biological effect cannot be ruled out. In view of a widespread and extensive use of mobile phones, this study evaluates alterations in male germ cell transformation kinetics following RFR exposure and after recovery. Swiss albino mice were exposed to RFR (900 MHz) for 4 h and 8 h duration per day for 35 days. One group of animals was terminated after the exposure period, while others were kept for an additional 35 days post-exposure. RFR exposure caused depolarization of mitochondrial membranes resulting in destabilized cellular redox homeostasis. Statistically significant increases in the damage index in germ cells and sperm head defects were noted in RFR-exposed animals. Flow cytometric estimation of germ cell subtypes in mice testis revealed 2.5-fold increases in spermatogonial populations with significant decreases in spermatids. Almost fourfold reduction in spermatogonia to spermatid turnover (1C:2C) and three times reduction in primary spermatocyte to spermatid turnover (1C:4C) was found indicating arrest in the premeiotic stage of spermatogenesis, which resulted in loss of post-meiotic germ cells apparent from testis histology and low sperm count in RFR-exposed animals. Histological alterations such as sloughing of immature germ cells into the seminiferous tubule lumen, epithelium depletion and maturation arrest were also observed. However, all these changes showed recovery to varied degrees following the post-exposure period indicating that the adverse effects of RFR on mice germ cells are detrimental but reversible. To conclude, RFR exposure-induced oxidative stress causes DNA damage in germ cells, which alters cell cycle progression leading to low sperm count in mice.

Cohesin Removal along the Chromosome Arms during the First Meiotic Division Depends on a NEK1-PP1γ-WAPL Axis in the Mouse

Mammalian NIMA-like kinase-1 (NEK1) is a dual-specificity kinase highly expressed in mouse germ cells during prophase I of meiosis. Loss of NEK1 induces retention of cohesin on chromosomes at meiotic prophase I. Timely deposition and removal of cohesin is essential for accurate chromosome segregation. Two processes regulate cohesin removal: a non-proteolytic mechanism involving WAPL, sororin, and PDS5B and direct cleavage by separase. Here, we demonstrate a role for NEK1 in the regulation of WAPL loading during meiotic prophase I, via an interaction between NEK1 and PDS5B. This regulation of WAPL by NEK1-PDS5B is mediated by protein phosphatase 1 gamma (PP1γ), which both interacts with and is a phosphotarget of NEK1. Taken together, our results reveal that NEK1 phosphorylates PP1γ, leading to the dephosphorylation of WAPL, which, in turn, resultsinits retention on chromosome cores to promote loss of cohesion at the end of prophase I in mammals.

Utility of Dexrazoxane for the Attenuation of Epirubicin-Induced Genetic Alterations in Mouse Germ Cells

Dexrazoxane has been approved to treat anthracycline-induced cardiomyopathy and extravasation. However, the effect of dexrazoxane on epirubicin-induced genetic alterations in germ cells has not yet been reported. Thus, the aim of this study was to determine whether dexrazoxane modulates epirubicin-induced genetic damage in the germ cells of male mice. Our results show that dexrazoxane was not genotoxic at the tested doses. Furthermore, it protected mouse germ cells against epirubicin-induced genetic alterations as detected by the reduction in disomic and diploid sperm, spermatogonial chromosomal aberrations, and abnormal sperm heads. The attenuating effect of dexrazoxane was greater at higher dose, indicating a dose-dependent effect. Moreover, sperm motility and count were ameliorated by dexrazoxane pretreatment. Epirubicin induced marked biochemical changes characteristic of oxidative DNA damage including elevated 8-hydroxy-2ʹ-deoxyguanosine levels and reduction in reduced glutathione. Pretreatment of mice with dexrazoxane before epirubicin challenge restored these altered endpoints. We conclude that dexrazoxane may efficiently mitigate the epirubicin insult in male germ cells, and prevent the enhanced risk of abnormal reproductive outcomes and associated health risks. Thus, pretreating patients with dexrazoxane prior to epirubicin may efficiently preserve not only sperm quality but also prevent the transmission of genetic damage to future generations.

α-Lipoic acid attenuates transplacental nicotine-induced germ cell and oxidative DNA damage in adult mice.

Smoking during pregnancy is associated with numerous fetal and developmental complications and reproductive dysfunctions in the offspring. Nicotine is one of the key chemicals of tobacco responsible for addiction. The present study was aimed to investigate the protective role of α-lipoic acid (ALA) during the transplacental nicotine-induced germ cell and DNA damage in the offspring of Swiss mice. Pregnant mice were treated with nicotine (20 mg/kg/day) in drinking water from 10 to 20 days of gestation period, and ALA (120 mg/kg/day) was administered orally for the same period. Endpoint of evaluation includes general observations at delivery and throughout the study, litter weight and size, sperm count and sperm head morphology, while structural damages and protein expression were assessed by histology and immunohistochemistry, respectively. Maternal nicotine exposure led to decreased growth rate, litter and testicular weight, testosterone level, 3β-HSD expression and sperm count as well as increased sperm head abnormalities, micronucleus frequency and 8-oxo-dG positive cells, and the effects have been restored by ALA supplementation. The present study clearly demonstrated that ALA ameliorates nicotine-associated oxidative stress, DNA damage and testicular toxicity in the offspring by improving steroidogenesis, spermatogenesis and sperm count.

Reciprocal localization of transcription factors YY1 and CP2c in spermatogonial stem cells and their putative roles during spermatogenesis

Maintaining stemness and permitting differentiation mediated by combinations of transcription factors (TFs) are key aspects of mammalian spermatogenesis. It has been established that yin yang 1 (YY1), a target factor of mammalian polycomb repressive complex 2 (PRC2) and a regulator of stemness, is involved in the stable maintenance of prophase stage spermatocytes. Recently, we have demonstrated that the TF CP2c partners with YY1 in some cells to antagonistically regulate the other protein’s function. To date, the functional roles of YY1 and CP2c in spermatogonial stem cells and their derived germ cells remain unclear. Here, we investigated the expression of YY1 and CP2c in mouse gonocytes and germ cells using tissue immunohistochemical and immunofluorescence analyses. At E14.5, both YY1 and CP2c were stained in gonocytes and Sertoli cells in testicular cords, showing different proportion and density of immunoreactivity. However, in adult testes, YY1 was localized in the nuclei of spermatogonial stem cells and spermatocytes, but not in spermatozoa. It was also detected in spermatogonia and spermatids in a stage–specific manner during spermatogenic cycle. CP2c could be detected mostly in the cytoplasm of spermatocytes but not at all in spermatogonial stem cells, indicating mutually exclusive expression of CP2c and YY1. Interestingly, however, CP2c was stained in the cytoplasm and nucleus of spermatogonia at elongation and release stages, and co-localized with YY1 in the nucleus at grouping, maturation, and releasing stages. Neither YY1 nor CP2c was expressed in spermatozoa. Our data indicate that YY1 strongly localizes in the spermatogonial stem cells and co-localizes heterogeneously with CP2c to permit spermatogenesis, and also suggest that YY1 is essential for stemness of spermatogonial stem cells (SCs) whereas CP2c is critical for the commitment of spermatogonia and during the progression of spermatogonia to spermatids. This evaluation expands our understanding of the molecular mechanism of spermatogonia formation as well as spermatogenesis in general.

Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male’s lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.

Potential toxicity of engineered nanoparticles in mammalian germ cells and developing embryos: treatment strategies and anticipated applications of nanoparticles in gene delivery.

Engineered nanoparticles (ENPs) offer technological advantages for a variety of industrial and consumer products as well as show promise for biomedical applications. Recent progress in the field of nanotechnology has led to increased exposure to nanoparticles by humans. To date, little is known about the adverse effects of these ENPs on reproductive health, although interest in nanotechnology area is growing. A few biocompatible ENPs have a high loading capacity for exogenous substances, including drugs, DNA or proteins, and can selectively deliver molecular cargo into cells; however, they represent a potential tool for gene delivery into gametes and embryos. Understanding the reprotoxicological aspects of these ENPs is of the utmost importance to reliably estimate its potential impact on human health. In addition, a search for protective agents to combat ENP-mediated reproductive toxicity is warranted. Therefore, in this review we summarize the toxic effects of a few ENPs (metal and metal oxides, carbon-based nanoparticles, quantum dots and chitosan) in mammalian germ cells and developing embryos, and propose some treatment strategies that could mitigate nanoparticle-mediated toxicity. In addition, we outline the anticipated applications of ENPs in transgenic animal production in order to generate models for investigations into the mechanisms for human disease. A literature search was performed using the National Center for Biotechnology Information PubMed database up until March 2016 and relevant keywords were used to obtain information regarding mammalian germ cell-specific toxicity and embryotoxicity of ENPs, possible treatment strategies, as well as the anticipated applications of nanoparticles in gene delivery in germ cells and embryos. Only English language publications were included. Here, we demonstrate the toxicological effects of ENPs in mammalian germ cells and developing embryos by considering both in vitro and in vivo experimental models based on the existing literature. The biodistribution and cellular uptake of ENPs and the observed toxicities are mostly dependent on ENP size and surface-coating agents (surface functional groups/surface charge). ENPs have been shown to induce toxicity via oxidative stress, inflammation and DNA damage in both human and mouse germ cells. Use of antioxidant, anti-inflammatory drugs and selective metal chelators would be beneficial against nanoparticle-induced toxicity. Our review provides the reproductive scientists a mechanistic insight into the reprotoxicological aspects of ENPs to reliably estimate its potential impact on human health and help to select/design protective agents to combat ENP-mediated toxicity. Furthermore, research regarding the detailed mechanism(s) of ENP toxicity in mammalian germ cells and developing embryos as well as the search for protective agents to combat ENP-mediated reproductive toxicity is warranted. Furthermore, we anticipate that investigations into the possibility of applying nanovectors to gene delivery in germ cells and early embryos will open new horizons in reproductive biology.

Characterization of Mammalian ADAM2 and Its Absence from Human Sperm.

The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans.

Comprehensive analysis of histone post-translational modifications in mouse and human male germ cells.

BackgroundDuring the process of spermatogenesis, male germ cells undergo dramatic chromatin reorganization, whereby most histones are replaced by protamines, as part of the pathway to compact the genome into the small nuclear volume of the sperm head. Remarkably, approximately 90 % (human) to 95 % (mouse) of histones are evicted during the process. An intriguing hypothesis is that post-translational modifications (PTMs) decorating histones play a critical role in epigenetic regulation of spermatogenesis and embryonic development following fertilization. Although a number of specific histone PTMs have been individually studied during spermatogenesis and in mature mouse and human sperm, to date, there is a paucity of comprehensive identification of histone PTMs and their dynamics during this process.ResultsHere we report systematic investigation of sperm histone PTMs and their dynamics during spermatogenesis. We utilized “bottom-up” nanoliquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) to identify histone PTMs and to determine their relative abundance in distinct stages of mouse spermatogenesis (meiotic, round spermatids, elongating/condensing spermatids, and mature sperm) and in human sperm. We detected peptides and histone PTMs from all four canonical histones (H2A, H2B, H3, and H4), the linker histone H1, and multiple histone isoforms of H1, H2A, H2B, and H3 in cells from all stages of mouse spermatogenesis and in mouse sperm. We found strong conservation of histone PTMs for histone H3 and H4 between mouse and human sperm; however, little conservation was observed between H1, H2A, and H2B. Importantly, across eight individual normozoospermic human semen samples, little variation was observed in the relative abundance of nearly all histone PTMs.ConclusionIn summary, we report the first comprehensive and unbiased analysis of histone PTMs at multiple time points during mouse spermatogenesis and in mature mouse and human sperm. Furthermore, our results suggest a largely uniform histone PTM signature in sperm from individual humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0072-6) contains supplementary material, which is available to authorized users.

Occlusion intestinale aiguë

Maintaining stemness and permitting differentiation mediated by combinations of transcription factors (TFs) are key aspects of mammalian spermatogenesis. It has been established that yin yang 1 (YY1), a target factor of mammalian polycomb repressive complex 2 (PRC2) and a regulator of stemness, is involved in the stable maintenance of prophase stage spermatocytes. Recently, we have demonstrated that the TF CP2c partners with YY1 in some cells to antagonistically regulate the other protein’s function. To date, the functional roles of YY1 and CP2c in spermatogonial stem cells and their derived germ cells remain unclear. Here, we investigated the expression of YY1 and CP2c in mouse gonocytes and germ cells using tissue immunohistochemical and immunofluorescence analyses. At E14.5, both YY1 and CP2c were stained in gonocytes and Sertoli cells in testicular cords, showing different proportion and density of immunoreactivity. However, in adult testes, YY1 was localized in the nuclei of spermatogonial stem cells and spermatocytes, but not in spermatozoa. It was also detected in spermatogonia and spermatids in a stage–specific manner during spermatogenic cycle. CP2c could be detected mostly in the cytoplasm of spermatocytes but not at all in spermatogonial stem cells, indicating mutually exclusive expression of CP2c and YY1. Interestingly, however, CP2c was stained in the cytoplasm and nucleus of spermatogonia at elongation and release stages, and co-localized with YY1 in the nucleus at grouping, maturation, and releasing stages. Neither YY1 nor CP2c was expressed in spermatozoa. Our data indicate that YY1 strongly localizes in the spermatogonial stem cells and co-localizes heterogeneously with CP2c to permit spermatogenesis, and also suggest that YY1 is essential for stemness of spermatogonial stem cells (SCs) whereas CP2c is critical for the commitment of spermatogonia and during the progression of spermatogonia to spermatids. This evaluation expands our understanding of the molecular mechanism of spermatogonia formation as well as spermatogenesis in general.

CTCF contributes in a critical way to spermatogenesis and male fertility.

The CCCTC-binding factor (CTCF) is an architectural protein that governs chromatin organization and gene expression in somatic cells. Here, we show that CTCF regulates chromatin compaction necessary for packaging of the paternal genome into mature sperm. Inactivation of Ctcf in male germ cells in mice (Ctcf-cKO mice) resulted in impaired spermiogenesis and infertility. Residual spermatozoa in Ctcf-cKO mice displayed abnormal head morphology, aberrant chromatin compaction, impaired protamine 1 incorporation into chromatin and accelerated histone depletion. Thus, CTCF regulates chromatin organization during spermiogenesis, contributing to the functional organization of mature sperm.

Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells.

Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3′-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells.

Genotoxic effect of the tricyclic antidepressant drug clomipramine hydrochloride in somatic and germ cells of male mice

ObjectiveTo assess the genotoxic potential of the antidepressant drug clomipramine hydrochloride (CH) through different mutagenic end points. MethodsThe study included chromosomal aberration analysis of bone marrow cells, primary spermatocytes, morphological sperm abnormalities and histopathological changes of liver cells using both light and electron microscopy. Three doses (0.195, 0.26 and 0.65 mg/20 g body weight) were tested. Each dose was given orally to mice for different periods of time (the doses are equivalent to the recommended daily intake doses in man). ResultsThe tested doses of CH applied for 5 and 30 days increased the frequencies of chromosomal aberrations with dose and time dependant manner. The two high doses, 0.26 and 0.65 mg/20 g body weight revealed significant effect in comparison to the control. Dose-dependent increase in morphological sperm abnormalities and decrease in sperm count were recorded after CH treatment for 5 consecutive days. Pathological changes in liver tissue reached to sever damage were recorded after treatment with the medium and the high doses for 30 days. Ultrastructural examination showed that the low dose had little differences in liver histological architecture as compared to the control group, while prominent pathological changes in nuclei as well as dilated rough endoplasmic reticulum were observed in mice treated with the medium or the high dose of the drug. ConclusionsIt is concluded that CH has genotoxic effect in somatic and germ cells of mice as well as damaging effect on liver tissue after treatment with the medium and the high doses. However, the usage of low dose (especially for short time, 5 days) can be utilized as a safe therapeutic dose.