Methylation analysis of histone H4K12ac-associated promoters in sperm of healthy donors and subfertile patients.

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. RESULTS: Chromatin immunoprecipitation (ChIP) with anti-H4K12ac antibody was performed with chromatin isolated from spermatozoa of subfertile patients with impaired sperm chromatin condensation assessed by aniline blue staining. Fertile donors were used as control. DNA methylation analysis of selected H4K12ac-interacting promoters in spermatozoa was performed by pyrosequencing. Depletion of binding sites for H4K12ac was observed within the following developmentally important promoters: AFF4, EP300, LRP5, RUVBL1, USP9X, NCOA6, NSD1, and POU2F1. We found 5% to 10% hypomethylation within CpG islands of selected promoters in the sperm of fertile donors, and it was not significantly altered in the subfertile group. Our results demonstrate that the H4K12ac depletion in selected developmentally important promoters of subfertile patients was not accompanied by a change of DNA methylation. Using a murine model, immunofluorescence revealed that H4K12ac co-localize with 5mC in the sperm nucleus. During fertilization, when the pronuclei are formed, the paternal pronucleus exhibits a strong acetylation signal on H4K12, while in the maternal pronucleus, there is a permanent increase of H4K12ac until pronuclei fusion. Simultaneously, there is an increase of the 5hmC signal and a decrease of the 5mC signal. CONCLUSIONS: We suggest that aberrant histone acetylation within developmentally important gene promoters in subfertile men, but not DNA methylation, may reflect insufficient sperm chromatin compaction affecting the transfer of epigenetic marks to the oocyte.

Toxoplasma gondii decreases the reproductive fitness in mice.

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.

Genome wide identification of promoter binding sites for H4K12ac in human sperm and its relevance for early embryonic development.

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 DNA-dependent 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.