Guylain Boissonneault

Transient DNA Strand Breaks During Mouse and Human Spermiogenesis:New Insights in Stage Specificity and Link to Chromatin Remodeling

Abstract In the course of mammalian spermiogenesis, a unique chromatin remodeling process takes place within elongating and condensing spermatid nuclei. The histone-to-protamine exchange results in efficient packaging and increased stability of the paternal genome. Although not fully understood, this change in chromatin architecture must require a global but transient appearance of endogenous DNA strand breaks because most of the DNA supercoiling is eliminated in the mature sperm. To establish the extent of DNA strand breakage and the stage specificity at which these breaks are created and repaired, we performed a sensitive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay to detect in situ DNA strand breaks on both mice and human testis cross sections. In the mouse, we established that DNA strand breaks are indeed detected in the whole population of elongating spermatids between stages IX and XI of the seminiferous epithelium cycle perfectly coincident with the chromatin remodeling as revealed by histone H4 hyperacetylation. Similarly, TUNEL analyses performed on human testis sections revealed an elevated and global increase in the levels of DNA strand breaks present in nuclei of round-shaped spermatids also coincident with chromatin remodeling. The demonstration of the global character of the transient DNA strand breaks in mammalian spermiogenesis suggests that deleterious consequences on genetic integrity of the male gamete may arise from any disturbance in the process. In addition, this investigation may shed some light on the origin of the low success rate that has been encountered so far with intracytoplasmic injection procedures making use of round spermatids in humans.

On the Nature and Origin of DNA Strand Breaks in Elongating Spermatids

Abstract Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.

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.

DNA damage response during chromatin remodeling in elongating spermatids of mice.

Abstract A precise packaging of the paternal genome during spermiogenesis is essential for fertilization and embryogenesis. Most of the nucleosomal DNA supercoiling must be eliminated in elongating spermatids (ES), and transient DNA strand breaks are observed that facilitate the process. Topoisomerases have been considered as ideal candidates for the removal of DNA supercoiling, but their catalytic activity, in the context of such a major chromatin remodeling, entails genetic risks. Using immunofluorescence, we confirmed that topoisomerase II beta (TOP2B) is the type II topoisomerase present in ES between steps 9 and 13. Interestingly, the detection of TOP2B was found coincident with detection of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme known to resolve topoisomerase-mediated DNA damage. The presence of gamma-H2AX (also known as H2AFX) coincident with DNA strand breakage was also confirmed at these steps and indicates that a DNA damage response is triggered. Active DNA repair in ES was demonstrated using a fluorescent in situ DNA polymerase activity assay on squash preparations of staged tubules. In the context of haploid spermatids, any unresolved double-strand breaks, resulting from a failure in the rejoining process of TOP2B, must likely rely on the error-prone nonhomologous end joining, because homologous recombination cannot proceed in the absence of a sister chromatid. Because this process is part of the normal developmental program of the spermatids, dramatic consequences for the genomic integrity of the developing male gamete may arise should any alteration in the process occur.

Stimulation of DNA repair by the spermatidal TP1 protein.

The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single‐strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid‐spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV‐induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid‐spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis. Mol. Reprod. Dev. 58:437–443, 2001.

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.

Spermiogenesis and DNA Repair: A Possible Etiology of Human Infertility and Genetic Disorders

This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear events associated with spermiogenesis as a potential source of genetic instability and reduced fertilizing potential of the mature male gamete. Recent findings suggest a programmed DNA fragmentation and DNA damage response during the chromatin remodeling steps in spermatids. We also discuss the spermatid DNA repair mechanisms and the possible involvement of condensing proteins, such as transition proteins and protamines, in the process, as this DNA fragmentation is normally not found in late spermatids. We propose that alterations in the chromatin remodeling steps or DNA repair in elongating spermatids may lead to persistent DNA breaks. This vulnerable step of spermiogenesis may provide a clue to the etiology of sperm DNA fragmentation associated with infertility in humans. This vulnerability is further emphasized given the haploid character of spermatids that must resolve programmed double-stranded breaks by an error-prone DNA repair mechanism. Therefore, spermiogenesis has probably been overlooked as an important source of genetic instability.

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.

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.

The testis-specific high-mobility-group protein, a phosphorylation-dependent DNA-packaging factor of elongating and condensing spermatids.

Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.