DNA methylation dynamic in male rat germ cells during gametogenesis.

In mammals, a near complete resetting of DNA methylation (DNAme) is observed during germline establishment. This wave of epigenetic reprogramming is sensitive to the environment, which could impair the establishment of an optimal state of the gamete epigenome, hence proper embryo development. Yet, we lack a comprehensive understanding of DNAme dynamics during spermatogenesis, especially in rats, the model of choice for toxicological studies. Using a combination of cell sorting and DNA methyl-seq capture, we generated a stage-specific mapping of DNAme in nine populations of differentiating germ cells from perinatal life to spermiogenesis. DNAme was found to reach its lowest level at gestational day 18, the last demethylated coding regions being associated with negative regulation of cell movement. The following de novo DNAme displayed three different kinetics with common and distinct genomic enrichments, suggesting a non-random process. DNAme variations were also detected at key steps of chromatin remodeling during spermiogenesis, revealing potential sensitivity. These methylome datasets for coding sequences during normal spermatogenesis in rat provide an essential reference for studying epigenetic-related effects of disease or environmental factors on the male germline.

DNA Repair in Haploid Context.

DNA repair is a well-covered topic as alteration of genetic integrity underlies many pathological conditions and important transgenerational consequences. Surprisingly, the ploidy status is rarely considered although the presence of homologous chromosomes dramatically impacts the repair capacities of cells. This is especially important for the haploid gametes as they must transfer genetic information to the offspring. An understanding of the different mechanisms monitoring genetic integrity in this context is, therefore, essential as differences in repair pathways exist that differentiate the gamete's role in transgenerational inheritance. Hence, the oocyte must have the most reliable repair capacity while sperm, produced in large numbers and from many differentiation steps, are expected to carry de novo variations. This review describes the main DNA repair pathways with a special emphasis on ploidy. Differences between Saccharomyces cerevisiae and Schizosaccharomyces pombe are especially useful to this aim as they can maintain a diploid and haploid life cycle respectively.

The DNA double-strand "breakome" of mouse spermatids.

De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.

Instability of trinucleotidic repeats during chromatin remodeling in spermatids.

Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.

Male-driven de novo mutations in haploid germ cells.

At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA end-joining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.

Oxidative versus reductive stress: a delicate balance for sperm integrity.

Despite the long-standing notion of "oxidative stress," as the main mediator of many diseases including male infertility induced by increased reactive oxygen species (ROS), recent evidence suggests that ROS levels are also increased by "reductive stress," due to over-accumulation of reductants. Damaging mechanisms, like guanidine oxidation followed by DNA fragmentation, could be observed following reductive stress. Excessive accumulation of the reductants may arise from excess dietary supplementation over driving the one-carbon cycle and transsulfuration pathway, overproduction of NADPH through the pentose phosphate pathway (PPP), elevated levels of GSH leading to impaired mitochondrial oxidation, or as a result NADH accumulation. In addition, lower availability of oxidized reductants like NAD+, oxidized glutathione (GSSG), and oxidized thioredoxins (Trx-S2) induce electron leakage leading to the formation of hydrogen peroxide (H2O2). In addition, a lower level of NAD+ impairs poly (ADP-ribose) polymerase (PARP)-regulated DNA repair essential for proper chromatin integrity of sperm. Because of the limited studies regarding the possible involvement of reductive stress, antioxidant therapy remains a central approach in the treatment of male infertility. This review put forward the concept of reductive stress and highlights the potential role played by reductive vs oxidative stress at pre-and post-testicular levels and considering dietary supplementation.

Functional Characterization of a Phf8 Processed Pseudogene in the Mouse Genome.

Most pseudogenes are generated when an RNA transcript is reverse-transcribed and integrated into the genome at a new location. Pseudogenes are often considered as an imperfect and silent copy of a functional gene because of the accumulation of numerous mutations in their sequence. Here we report the presence of Pfh8-ps, a Phf8 retrotransposed pseudogene in the mouse genome, which has no disruptions in its coding sequence. We show that this pseudogene is mainly transcribed in testis and can produce a PHF8-PS protein in vivo. As the PHF8-PS protein has a well-conserved JmjC domain, we characterized its enzymatic activity and show that PHF8-PS does not have the intrinsic capability to demethylate H3K9me2 in vitro compared to the parental PHF8 protein. Surprisingly, PHF8-PS does not localize in the nucleus like PHF8, but rather is mostly located at the cytoplasm. Finally, our proteomic analysis of PHF8-PS-associated proteins revealed that PHF8-PS interacts not only with mitochondrial proteins, but also with prefoldin subunits (PFDN proteins) that deliver unfolded proteins to the cytosolic chaperonin complex implicated in the folding of cytosolic proteins. Together, our findings highlighted PHF8-PS as a new pseudogene-derived protein with distinct molecular functions from PHF8.

The sperm nucleus: chromatin, RNA, and the nuclear matrix.

Within the sperm nucleus, the paternal genome remains functionally inert and protected following protamination. This is marked by a structural morphogenesis that is heralded by a striking reduction in nuclear volume. Despite these changes, both human and mouse spermatozoa maintain low levels of nucleosomes that appear non-randomly distributed throughout the genome. These regions may be necessary for organizing higher order genomic structure through interactions with the nuclear matrix. The promoters of this transcriptionally quiescent genome are differentially marked by modified histones that may poise downstream epigenetic effects. This notion is supported by increasing evidence that the embryo inherits these differing levels of chromatin organization. In concert with the suite of RNAs retained in the mature sperm, they may synergistically interact to direct early embryonic gene expression. Irrespective, these features reflect the transcriptional history of spermatogenic differentiation. As such, they may soon be utilized as clinical markers of male fertility. In this review, we explore and discuss how this may be orchestrated.

Electron microscopy analysis of histone acetylation and DNA strand breaks in mouse elongating spermatids using a dual labelling approach.

Chromatin remodelling steps in mammalian spermatids include posttranslational modifications of histones and DNA fragmentation. Histone H4 hyperacetylation (AcH4) establishes a chromatin state that facilitates DNA repair in somatic cells. So we sought to determine whether a similar link exists in spermatids by combining immunogold labelling with detection of DNA strand breaks, making use of gold particles of different sizes. DNA strand breaks were not detected in the vicinity of AcH4 chromatin, suggesting that this modified histone may not be involved in the aetiology of DNA fragmentation and repair in spermatids. The AcH4 reactivity, however, indicates that chromatin remodelling is distributed throughout the nucleus.

pH-induced variations in the TK1 gene model.

A physiological decrease in extracellular pH (pHe) alters the efficiency of DNA repair and increases formation of DNA double-strand breaks (DSBs). Whether this could translate into genetic instability and variations, was investigated using the TK6 cell model, in which positive selection of the TK1 gene loss-of-function mutations can be achieved from resistance to trifluorothymidine. Cell exposure to suboptimal pH (down to 6.9) for 3 weeks resulted in the 100 % frequency of a stronger frameshift mutation that has spread to both TK1 alleles, whereas weaker frameshift mutations within the 3'exon were eliminated during the selection. Suboptimal pHe values were also found to alter the proportion of the TK1 splicing variant expressed as percent spliced in index values and promote selection of truncated exons as well as intron retention. Although recovery at pH 7.4 did not reverse the selected frameshift mutation, reversal of splice variants and exon truncation towards control values were observed. Hence, suboptimal pHe can induce a combination of mutational events and splicing alterations within the same gene in the resistant clones. This model of positive selection for loss-of-function clearly demonstrates that suboptimal pHe may confer a similar growth advantage when such instability occurs within tumor suppressor genes.

Spermatozoal transcriptome profiling for bull sperm motility: a potential tool to evaluate semen quality.

Regarding bull fertility, establishing an association between in vitro findings and field fertility requires a multi-parametric approach that measures the integrity of various structures and dynamic functions, such as motion characteristics, among others. The heterogeneous RNA pattern of spermatozoa could be used in genomic analysis for evaluating both spermatogenesis and fertility potential of semen, mainly because of the static status of the transcriptome of this particular differentiated cell. In a previous study, we determined that some spermatozoal transcripts identified by PCR-based cDNA subtraction are associated with non-return rate, a field fertility index. In the present study, the microarray technology was used in conjunction with differential RNA transcript extraction. We have shown that among these genes, some transcripts are also associated with the motility status of a population of sperm cells fractionated from the same ejaculate. We highlighted a systematic data analysis and validation scheme important for the identification of significant transcripts in this context. With such an approach, we found that transcripts encoding a serine/threonine testis-specific protein kinase (TSSK6) and a metalloproteinase non coding RNA (ADAM5P) are associated with high-motility status (P<0.001), also confirmed by quantitative PCR (P=0.0075). This association was found only when transcripts were extracted using the hot-TRIzol protocol, whereas the cold-TRIzol RNA extract comprised mitochondrial transcripts. These results demonstrate that some transcripts previously identified in association with field fertility are also found associated with in vitro motility provided that a stringent RNA extraction protocol is used.

Genetic Instability and Chromatin Remodeling in Spermatids.

The near complete replacement of somatic chromatin in spermatids is, perhaps, the most striking nuclear event known to the eukaryotic domain. The process is far from being fully understood, but research has nevertheless unraveled its complexity as an expression of histone variants and post-translational modifications that must be finely orchestrated to promote the DNA topological change and compaction provided by the deposition of protamines. That this major transition may not be genetically inert came from early observations that transient DNA strand breaks were detected in situ at chromatin remodeling steps. The potential for genetic instability was later emphasized by our demonstration that a significant number of DNA double-strand breaks (DSBs) are formed and then repaired in the haploid context of spermatids. The detection of DNA breaks by 3'OH end labeling in the whole population of spermatids suggests that a reversible enzymatic process is involved, which differs from canonical apoptosis. We have set the stage for a better characterization of the genetic impact of this transition by showing that post-meiotic DNA fragmentation is conserved from human to yeast, and by providing tools for the initial mapping of the genome-wide DSB distribution in the mouse model. Hence, the molecular mechanism of post-meiotic DSB formation and repair in spermatids may prove to be a significant component of the well-known male mutation bias. Based on our recent observations and a survey of the literature, we propose that the chromatin remodeling in spermatids offers a proper context for the induction of de novo polymorphism and structural variations that can be transmitted to the next generation.

Post-meiotic DNA double-strand breaks are conserved in fission yeast.

In mammals, spermiogenesis is characterized by transient formation of DNA double-strand breaks (DSBs) in the whole population of haploid spermatids. DSB repair in such haploid context may represent a mutational transition. Using a combination of pulsed-field gel electrophoresis and specific labelling of DSBs at 3'OH DNA ends, we showed that post-meiotic, enzyme-induced DSBs are also observed in the synchronizable pat1-114 mutant of Shizosaccharomyces pombe as well as in a wild-type strain, while DNA repair is observed at later stages. This transient DNA fragmentation arises in the whole cell population and is seemingly independent of the caspase apoptotic pathway. Because histones are still present in spores, the transient DSBs do not require a major change in chromatin structure. These observations confirm the highly-conserved nature of the process in eukaryotes and provide a powerful model to study the underlying mechanism and its impact on the genetic landscape and adaptation.

Suboptimal extracellular pH values alter DNA damage response to induced double-strand breaks.

Conditions leading to unrepaired DNA double-stranded breaks are potent inducers of genetic instability. Systemic conditions may lead to fluctuation of hydrogen ions in the cellular microenvironment, and we show that small variations in extracellular pH, termed suboptimal pHe, can decrease the efficiency of DNA repair in the absence of intracellular pH variation. Recovery from bleomycin-induced DNA double-stranded breaks in fibroblasts proceeded less efficiently at suboptimal pHe values ranging from 7.2 to 6.9, as shown by the persistence of repair foci, reduction of H4K16 acetylation, and chromosomal instability, while senescence or apoptosis remained undetected. By allowing escape from these protective mechanisms, suboptimal pHe may therefore enhance the genotoxicity of double-stranded breaks, leading to genetic instability.

Torsional stress promotes trinucleotidic expansion in spermatids.

Trinucleotide repeats are involved in various neurodegenerative diseases and are highly unstable both in dividing or non-dividing cells. In Huntington disease (HD), the age of onset of symptoms is inversely correlated to the number of CAG repeats within exon 1 of the HTT gene. HD shows paternal anticipation as CAG repeats are increased during spermatogenesis. CAG expansion were indeed found to be generated during the chromatin remodeling in spermatids where most histones are evicted and replaced by protamines. This process involves striking change in DNA topology since free supercoils must be eliminated. Using an in vitro CAG repeat reporter assay and a highly active nuclear extracts from spermatids, we demonstrate that free negative supercoils result in CAG TNR expansion at a stabilized hairpin. We also suggest a possible role for protamines in promoting localized torsional stress and consequently TNR expansion. The transient increase in torsional stress during spermiogenesis may therefore provide an ideal context for the generation of such secondary DNA structures leading to the paternal anticipation of trinucleotidic diseases.

Quantification and genome-wide mapping of DNA double-strand breaks.

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.

Immuno-capture of UVDE generated 3'-OH ends at UV photoproducts.

A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.

Step-specific Sorting of Mouse Spermatids by Flow Cytometry.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair.

An important chromatin remodeling process is taking place during spermiogenesis in mammals and DNA strand breaks must be produced to allow the accompanying change in DNA topology. Endogenous DNA strand breaks are indeed detected at mid-spermiogenesis steps but are no longer present in mature sperm. Both in vitro and in vivo evidence suggests that the DNA-binding and condensing activities of a set of basic nuclear "transition proteins" may be crucial to the integrity of the chromatin remodeling process. We propose that these proteins are necessary for the repair of the strand breaks so that DNA fragmentation is minimized in the mature sperm.

Dynamics of reporter gene stimulation by HMG box proteins.

Overcoming local DNA rigidity is required to perform three-dimensional DNA-protein configuration at promoter regions. The abundant architectural nonhistone chromosomal HMG box proteins are nonsequence-specific; however, they have been established to specifically recognize distorted DNA. Using transient transfection to overexpress two different members of the HMGB-1/2 family of DNA architectural factors, we demonstrate that these proteins provide a general enhancement in reporter gene expression irrespective of the promoter being considered. Evidences are also provided indicating that stimulation may not be achieved by recruitment of the proteins by regulatory factors or as a consequence of major chromatin unfolding as previously suggested. Interestingly, the influence of the HMG box proteins under study was overridden when the promoters were either induced or stimulated by Trichostatin A (TSA) but recovered upon extended induction period. These results also support the concept that the architectural role of these proteins can contribute to the preinitiation complex assembly required for basal transcription, but to a much lesser extent to the poised promoter scaffolding characteristic of activated transcription.