During spermatogenesis, approximately 85% of histones are replaced by protamines. The remaining histones have been proposed to carry essential marks for the establishment of epigenetic information in the offspring. The aim of the present study was to analyse the expression pattern of histone H3 acetylated at lysine 9 (H3K9ac) during normal and impaired spermatogenesis and the binding pattern of H3K9ac to selected genes within ejaculates. Testicular biopsies, as well as semen samples, were used for immunohistochemistry. Chromatin immunoprecipitation was performed with ejaculated sperm chromatin. HeLa cells and prostate tissue served as controls. Binding of selected genes was evaluated by semiquantitative and real-time polymerase chain reaction. Immunohistochemistry of H3K9ac demonstrated positive signals in spermatogonia, spermatocytes, elongating spermatids and ejaculated spermatozoa of fertile and infertile men. H3K9ac was associated with gene promoters (CRAT, G6PD, MCF2L), exons (SOX2, GAPDH, STK11IP, FLNA, PLXNA3, SH3GLB2, CTSD) and intergenic regions (TH) in fertile men and revealed shifts of the distribution pattern in ejaculated spermatozoa of infertile men. In conclusion, H3K9ac is present in male germ cells and may play a role during the development of human spermatozoa. In addition, H3K9ac is associated with specific regions of the sperm genome defining an epigenetic code that may influence gene expression directly after fertilisation.

As histone modifications have been suggested to be involved in the regulation of gene expression after fertilisation, the present study aimed to analyze the interaction between the bromodomain testis-specific (BRDT) gene and differentially modified histones in human spermatozoa. The BRDT transcript level was studied to identify possible correlations between epigenetic changes, mRNA level and subfertility associated with impaired sperm chromatin condensation. Chromatin immunoprecipitation (ChIP) was performed with ejaculates from fertile and subfertile men using antibodies against specifically acetylated and methylated histone H3. Immunoprecipitated DNA was analysed by real-time quantitative PCR with primer pairs for BRDT. The BRDT mRNA level was screened by real-time RT-PCR. ChIP assay revealed co-localisation of acetylated and methylated histones within promoter and exon regions of the BRDT gene in fertile men. Interestingly, reduced binding of investigated modified histone modifications was observed in the BRDT promoter of subfertile patients. Different mRNA levels of BRDT have been detected in a group of infertile patients, as well as in fertile men. Enrichment of methylated histones within the BRDT promoter of fertile sperm suggests that this epigenetic mark may cause repression of BRDT after fertilisation, and may be changed in infertile patients. Our data suggest that reduced histone methylation in the promoter of BRDT may be associated with increased transcript levels in subfertile patients.

Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although an aberrant protamine ratio have been observed in subfertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the DNA fragmentation factor 40 (DFF40) ratio or caspase (Casp4, Casp6) and tumor necrosis factor superfamily member 10 (TNFSF10) ratio together with the P1/P2 ratio represents a reliable biomarker to discriminate between fertile and subfertile men. Real-time quantitative RT-PCR was used for amplification of P1, P2 and DFF40 in 49 testicular biopsies. Casp4, Casp6 and TNFSF10 have been selected from a PCR apoptosis array and were further investigated in another group of testicular biopsies (22 subfertile men versus 11 potentially fertile men). Using Spearman's rank correlation coefficient analysis, we did not find a correlation between DFF40 and P1, P2, P1/P2, score, fertilization rate and age. In addition, logistic regression analysis demonstrated no statistically significant effect of the analyzed variables on pregnancy. A two-way analysis of variance with repeated measures of relative expression of Casp4, Casp6 and TNFSF10 versus P1 or P2 in potentially fertile men and subfertile patients demonstrated statistically significant differences between both groups, all tested gene combinations and the interaction between two genes and both groups in all cases analyzed. Furthermore, significant differences in the expression of Casp4 and TNFSF10 between the groups of potentially fertile and subfertile men could be demonstrated. In addition, the means of differences of selected gene combinations revealed that the protamine to apoptotic gene ratio is statistically different between both groups. Our data suggest that Casp4, Casp 6 and TNFSF10 are differentially expressed in potentially fertile and subfertile men and represent useful biomarkers for predicting male fertility in combination with P1 and P2.

During human spermiogenesis, chromatin condensation is associated with replacement of histones by protamines. This exchange is supported by acetylated histones and chromatin remodelling factors. Ten chromatin remodelling factor protein families are known. This study aims to analyse whether a different chromatin remodelling factor expression pattern exists between normal spermatogenesis and round spermatid maturation arrest as potential reason for impaired spermatogenesis and idiopathic male infertility. Laser capture microdissection was used to excise seminiferous tubules from testicular biopsies with normal spermatogenesis and round spermatid maturation arrest. RNA was isolated, first strand cDNA synthesis and pre-amplification were performed using Epigenetic Chromatin Remodelling Factors PCR arrays with 84 genes. Applying hierarchical cluster analysis, three gene expression clusters with six subgroups were identified. The expression pattern ranges from a few high expressed genes in round spermatid maturation arrest to a multitude of genes (74) which are more highly expressed in normal spermatogenesis than in maturation arrest. A total of 22 genes showed a significant difference between normal spermatogenesis and round spermatid maturation arrest (1 gene was up-regulated and 21 genes were down-regulated in the developmental arrest). The significantly different expression of chromatin remodelling factors between normal spermatogenesis and round spermatid maturation arrest may lead to impaired epigenetic information and aberrant transcription during sperm development representing one possible reason for developmental arrest of round spermatids.

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