Sperm chromatin reveals two characteristic features in that protamines are the predominant nuclear proteins and remaining histones are highly acetylated. histone h4 acetylated at lysine 12 (h4K12ac) is localized in the post-acrosomal region, while protamine-1 is present within the whole nucleus. chromatin immunoprecipitation in combination with promoter array analysis allowed genome-wide identification of h4K12ac binding sites. previously, we reported enrichment of h4K12ac at cTcF binding sites and promoters of genes involved in developmental processes. here, we demonstrate that h4K12ac is enriched predominantly between ± 2 kb from the transcription start site. In addition, we identified developmentally relevant h4K12ac-associated promoters with high expression levels of their transcripts stored in mature sperm. The highest expressed mRNa codes for testis-specific phD finger protein-7 (phF7), suggesting an activating role of h4K12ac in the regulatory elements of this gene. h4K12ac-associated genes revealed a weak correlation with genes expressed at 4-cell stage human embryos, while 23 h4K12ac-associated genes were activated in 8-cell embryo and 39 in the blastocyst. Genes activated in 4-cell embryos are involved in gene expression, histone fold and DNadependent transcription, while genes expressed in the blastocyst were classified as involved in developmental processes. Immunofluorescence staining detected h4K12ac from the murine male pronucleus to early stages of embryogenesis. aberrant histone acetylation within developmentally important gene promoters in infertile men may reflect insufficient sperm chromatin compaction, which may result in inappropriate transfer of epigenetic information to the oocyte.

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.

Toxoplasma gondii is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various mechanisms to maximize their own reproductive success. Mice and humans are both the intermediate hosts for Toxoplasma gondii, which forms specialized vacuoles containing reproductive cysts in the formers’ tissue. As half of the human population is infected, developing a disease called toxoplasmosis, along with an ever-growing number of couples suffering with idiopathic infertility, it is therefore surprising that there is a lack of research on how Toxoplasma gondii can alter reproductive parameters. In this study, a detailed histometric screening of the testicular function along with the levels of the pituitary luteinizing hormone (LH) were analysed in infected mice. Data on relative testis and epididymis weight, and sperm count were also collected. Based on the results obtained, the level of LH in the urine of Toxoplasma gondii infected mice was lower compared to the control. In direct correlation with the hormone level, testicular function and sperm production was also significantly lower in Toxoplasma gondii positive group using sperm count and histometric analysis as a marker. Not only were the number of leptotene primary spermatocytes and spermatids lowered, but the number of Sertoli cells and the tubule diameter were elevated. In parallel, a pilot epigenetic study on global testicular methylation, and specific methylation of Crem, Creb1 and Hspa1genes essential for successfully ongoing spermatogenesis was performed. Global methylation was elevated in Toxoplasma infected mice, and differences in the DNA methylation of selected genes were detected between the Toxoplasma positive and control group. These findings demonstrate a direct relation between Toxoplasma gondii infection and the decrease of male reproductive fitness in mice, which may contribute to an increase of idiopathic infertility in humans.

Background: Histone to protamine exchange and the hyperacetylation of the remaining histones are hallmarks of spermiogenesis. Acetylation of histone H4 at lysine 12 (H4K12ac) was observed prior to full decondensation of sperm chromatin after fertilization suggesting an important role for the regulation of gene expression in early embryogenesis. Similarly, DNA methylation may contribute to gene silencing of several developmentally important genes. Following the identification of H4K12ac-binding promoters in sperm of fertile and subfertile patients, we aimed to investigate whether the depletion of histone-binding is associated with aberrant DNA methylation in sperm of subfertile men. Furthermore, we monitored the transmission of H4K12ac, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) from the paternal chromatin to the embryo applying mouse in vitro fertilization and immunofluorescence.

SUMMARYHighly compacted sperm DNA in protamine toroids and a minor fraction of nucleohistones are prerequisites for the efficient transmission of the paternal genome into the oocyte at fertilization. The objective of this study was to evaluate whether protamines might serve as a prognostic factor for stallion fertility. In situ hybridization detected specific expression of P1 mRNA in the cytoplasm of stage I to VII spermatids, whereas comparable immunohistochemical stainings showed that protein expression was delayed till elongating spermatids in differentiation stages III to VIII. No staining was detectable in cryptorchid testis because of the lack of spermatids in the seminiferous tubules. Using quantitative real-time polymerase chain reaction, we identified mRNA transcripts of P1 and 2 variants of protamine-2 (P2, P3) in ejaculated spermatozoa from 45 thoroughbred stallions. According to the mare fertility descriptor (i.e. the 'none-return-rate 28 percentage' or NRR28%), stallions were divided into three groups (i.e. high, reduced and low fertility). The P2/P1 mRNA ratio was found to be significantly reduced in the group with lower fertility (p = 0.016) and was slightly correlated with sperm concentration (correlation coefficient r = 0.263). Furthermore, morphologically abnormal sperm count negatively correlated with P2/P1 mRNA ratio, indicating that spermatozoa carrying head defects display a diminished protamine ratio (r = À0.348). Conversely, the P2/P1 ratio was positively correlated with mare fertility or NRR28% (r = 0.274). Interestingly, P3/P1 mRNA ratio remained unaltered in the investigated groups indicating that this variant plays a minor role in equine sperm chromatin compaction. Aberrant protamine transcripts content in equine spermatozoa was not associated with DNA defragmentation rate as measured by flow cytometric acridine orange test. On the basis of these results, we suggest that, similar to human, equine protamine expression constitutes a checkpoint of spermatogenesis and as a corollary the level of protamine mRNA may reflect the quality of spermatogenesis and spermatozoa's fertilizing capacity.