Frédéric Leduc

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.

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.

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.

Genome-wide mapping of DNA strand breaks.

Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed “damaged DNA immunoprecipitation” (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

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.

Post-Meiotic DNA Damage and Response in Male Germ Cells

Spermatids are haploid cells that differentiate into spermatozoa and may be considered as an interesting model of DNA damage response and repair. Key features, such a unique set of chromosomes, radioresistance to apoptosis, the presence of known end-joining DNA repair pathways and an underlying prerogative to limit the transmission of any mutation to the next generation, make them a unique cell type to provide new insights on similar pathways in somatic cells. Although DNA damage signaling and repair mechanisms have been extensively studied during meiosis, the contribution of post-meiotic germ cells to the genetic integrity of the male gamete have been overlooked. In this chapter we present clear evidences that the haploid phase of spermatogenesis, termed spermiogenesis, may represent an even greater challenge for the maintenance of the genetic integrity of the male gamete. Since transient DNA strand breaks are intrinsic to the differentiation program of spermatids (Leduc et al., 2008a; Marcon and Boissonneault, 2004), a better understanding of DNA repair pathways involved may shed some light on their potential contribution to male-driven de novo mutations and eventually to some unresolved cases of male infertility. This chapter will mainly focus on DNA breaks occurring in the post-meiotic phase of the spermatogenesis and how germ cells deal with it.

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 post‐translational 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.

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.

Spermiogenesis in Sperm Genetic Integrity

This review suggests that spermiogenesis has probably been overlooked as an important source of genetic instability that can provide a repertoire of mutations distributed through millions of spermatozoa, each having the potential to transfer genetic alterations to the next generation. Further investigation will be needed to establish whether this could be considered as a new component of evolution.