[Spermiogenesis: histone acetylation triggers male genome reprogramming]. / Spermiogenèse: l'acétylation des histones déclenche la reprogrammation du génome mâle.

During their post-meiotic maturation, male germ cells undergo an extensive reorganization of their genome, during which histones become globally hyperacetylated, are then removed and progressively replaced by transition proteins and finally by protamines. The latter are known to tightly associate with DNA in the mature sperm cell. Although this is a highly conserved and fundamental biological process, which is a necessary prerequisite for the transmission of the male genome to the next generation, its molecular basis remains mostly unknown. We have identified several key factors involved in this process, and their detailed functional study has enabled us to propose the first model describing molecular mechanisms involved in post-meiotic male genome reprogramming. One of them, Bromodomain Testis Specific (BRDT), has been the focus of particular attention since it possesses the unique ability to specifically induce a dramatic compaction of acetylated chromatin. Interestingly, a mutation was found homozygous in infertile men which, according to our structural and functional studies, disrupts the function of the protein. A combination of molecular structural and genetic approaches has led to a comprehensive understanding of new major actors involved in the male genome reprogramming and transmission.

Fine mapping of re-arranged Y chromosome in three infertile patients with non-obstructive azoospermia/cryptozoospermia.

BACKGROUND: Cytogenetically detectable aberrations of the Y chromosome, such as isodicentrics, rings or translocations are sometimes associated with male non-obstructive infertility. This report presents a detailed analysis of the clinical, cytogenetic and molecular data in three patients with a re-arranged Y chromosome. METHODS: Patients A and B were azoospermic, whereas patient C was cryptozoospermic. All had a somatic mosaic karyotype including a population of 45,X cells and a cell line with a re-arranged Y chromosome. A molecular and FISH analysis of their re-arranged Y was undertaken, which specifically focussed on the presence of the AZFa, b and c regions. RESULTS: The AZFa region was present in all the three patients. The AZFb and AZFc regions were absent in patients A and B, whereas, in patient C, the distal part of AZFb and the whole AZFc region were deleted. Moreover, in this patient, the AZF FISH analysis revealed a mosaicism for the size of the AZF deletion within the re-arranged Y, suggesting a progressive enlargement of the deletion during cell mitotic divisions. CONCLUSIONS: This investigation allowed not only a more precise description of the abnormal Y, but also shed light on how this re-arrangement could be involved in the infertility phenotype.

Predictive factors for an increased risk of sperm aneuploidies in oligo-astheno-teratozoospermic males.

Patients with severe spermatogenesis impairment can now successfully father a child thanks to the use of intracytoplasmic sperm injection (ICSI). In oligozoospermic patients, many studies have reported significantly higher sperm aneuploidy rates and therefore an increased risk of transmitting a chromosomal abnormality via the injection of abnormal spermatozoa. However, the frequency of aneuploidy is highly variable between patients. The aim of the present work was to identify clinical and biological factors, which, together with non-obstructive oligozoospermia, could be predictive of elevated sperm aneuploidies. The sperm aneuploidy rates for chromosomes X, Y, 13, 18 and 21 were assessed in 31 infertile men with well-characterized spermatogenesis impairment, and in a population of control men with proven fertility. The frequency of sperm aneuploidy was compared between several patient subgroups according to their clinical and biological factors. Nearly half of the oligozoospermic males (15/31) had a significantly increased disomy rate for at least one of the five chromosomes compared with that observed in the control population (mean disomy rates + 1.96 standard deviation). Factors significantly associated with higher numbers of aneuploid sperm were cigarette smoking, an elevated follicle-stimulating hormone level, a sperm concentration less than 1 m/mL, and a severe teratozoospermia. Hence, several factors predictive of an increased risk of sperm aneuploidy rates were identified in ICSI male candidates with a non-obstructive oligozoospermia.

[Epigenetics of the sperm cell]. / Epigénétique du spermatozoïde.

In addition to genetic information, the spermatozoon carries another type of information, named epigenetic, which is not associated with variations of the DNA sequence. In somatic cells, it is now generally admitted that epigenetic information is not only regulated by DNA methylation but also involves modifications of the genome structure, or epigenome. During male germ cell maturation, the epigenome is globally re-organized, since most histones, which are associated to DNA in somatic cells, are removed and replaced by sperm specific nuclear proteins, the protamines, responsible for the tight compaction of the sperm DNA. However, a small proportion of histones, and probably other proteins, are retained within the sperm nucleus, and the structure of the sperm genome is actually heterogeneous. This heterogeneity of the sperm epigenome could support an epigenetic information, transmitted to the embryo, which could be crucial for its development. Although it is nowadays possible to appreciate the global structure of the sperm genome, the precise constitution of the sperm epigenome remains unknown. In particular, very recent data suggest that specific regions of the genome could be associated with particular proteins and define specific structures. This structural partitioning of the sperm genome could convey important epigenetic information, crucial for the embryo development.

[Organizing the sperm nucleus]. / Organisation nucléaire du spermatozoïde.

Thanks to the success of new assisted reproductive technology, including sperm microinjection (i.c.s.i.), men with severe spermatogenesis impairments can now become biological fathers. Whether the germinal cell used for i.c.s.i. is conveying appropriate genetic and epigenetic information is an important concern. However, to date, there is a huge lack of data on which information is epigenetically conveyed to the offspring and how. The basic support for epigenetic marks is the nucleus structure. During spermatogenesis, a major re-organization of the male germ cells nucleus structure occurs, which includes a global condensation associated with a removal of most core somatic histones and their replacement by sperm-specific nuclear proteins. The available data on the molecular mechanisms involved in this process and how it could relate to the setting of male-specific epigenetic information is reviewed and discussed in light of our current knowledge about nuclear structure and functions.

Misregulation of histone acetylation in Sertoli cell-only syndrome and testicular cancer.

In many species, including humans, chromatin remodelling during spermiogenesis is initiated with a marked increase in histone acetylation in elongating spermatids. We have investigated whether this process is disturbed when spermatogenesis is defective or in human testicular tumours. For this purpose, the presence of highly acetylated histone H4 was detected on testicular sections from men with a severe impairment of spermatogenesis of several origins, as well as in different types of testicular tumours. In most tubules devoid of germinal cells (including SCO, Sertoli cell only syndromes) or lacking spermatocytes and spermatids, the Sertoli cells' nuclei showed a global increase in histone H4 acetylation. A similar observation was made in the peritumoral seminiferous tubules of testicular tumour tissues, whenever they were lacking germinal cells, with carcinoma in situ (CIS) cells being hypoacetylated. The global hyperacetylation of elongating spermatids during spermatogenesis could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be disturbed in cases of severe spermatogenesis impairment, as well as in tubes surrounding germ cells in testicular tumours.

Genome organization in the human sperm nucleus studied by FISH and confocal microscopy.

The sperm nucleus has a unique chromatin structure where the DNA is highly condensed and associated with specific proteins, the protamines. It is a nondividing cell which is also transcriptionally inactive. After fusion with an oocyte, the sperm nucleus undergoes decondensation and, in the same time, starts replication and transcription. It has been suggested that somatic chromosomes during interphase are organized in territories which display a cell type and cell cycle specific distribution. The purpose of this work was to investigate whether chromosomes would also have a specific distribution in the sperm nucleus, which could be related to its inactive state, and have implications on the early stages of fertilization. In the present study, centromeric and telomeric sequences were detected by fluorescent techniques performed on human decondensed spermatozoa. Chromosome painting probes were used to detect the chromosome X and chromosome 13 on interphase sperm nuclei. The fluorescent signals were captured in 3D with a confocal microscope. For each of these chromatin structures, the volume, position, and distribution of the signals were analyzed in samples of 30 nuclei with the help of image analysis software. The centromeres appeared grouped in several foci that were randomly distributed within the sperm nucleus. The telomeres gave an approximately haploid number of small signals, evenly distributed throughout the nucleus. The chromosomes X and 13 occupied 4.7% and 3. 7% of the total nuclear volume, respectively. Interestingly, the X chromosome territory showed a preferential position in the anterior half of the volume of the nucleus, whereas chromosome 13 had a random position. This work shows a particular distribution of chromosome territories in the human sperm nucleus that could be related to mechanisms implicated in its specific functions. The analysis of more chromosomes and chromosomal structures, including the Y chromosome, would help to understand the structure of the human sperm chromatin, and its fundamental and clinical implications.

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases.

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.

Lack of value of specific IgA detection in the postnatal diagnosis of congenital toxoplasmosis.

To improve the performance of the postnatal diagnosis of congenital toxoplasmosis, we assessed the detection of IgA antibodies to Toxoplasma gondii by ELISA, compared with that of IgM by ELISA, ISAGA, and IFAT and neosynthesized antibodies using Western blot. From 1993 to 1996, IgA antibodies were detected using the Toxo IgA test (SFRI, Société Française de Recherches et d'Investissements, Bordeaux, France), in 195 serum and cord blood samples from 63 infants born to mothers who seroconverted during pregnancy. Eighteen infants had proven congenital toxoplasmosis (confirmed by the presence of IgG after 12 months of life) and 45 had no congenital toxoplasmosis (negativity of IgG after 6-12 months of life). The sensitivity of IgA detection by ELISA on serum and cord blood samples was 38.9 and 54.5% respectively, which is low when compared with the sensitivity of IgM detection by ISAGA (66.7% on serum samples, 90.9% on cord blood), ELISA (61.1% on sera, 81.8% on cord blood) and Western blot (83.3% on sera, 72.7% on cord blood). IgA antibodies were never detected by ELISA earlier than IgM or neosynthesized Ig (antibodies synthesized by infants). Thus, the detection of IgA antibodies by Toxo IgA is not useful in improving the diagnosis of congenital toxoplasmosis.