Agnieszka Paradowska

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences.

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

Analysing the sperm epigenome: roles in early embryogenesis and assisted reproduction.

An understanding of the epigenetic mechanisms involved in sperm production and their impact on the differentiating embryo is essential if we are to optimize fertilization and assisted reproduction techniques in the future. Male germ cells are unique in terms of size, robustness, and chromatin structure, which is highly condensed owing to the replacement of most histones by protamines. Analysis of sperm epigenetics requires specific techniques that enable the isolation of high quality chromatin and associated nucleic acids. Histone modification, DNA methylation and noncoding RNAs have important, but so far underestimated, roles in the production of fertile sperm. Aberrations in these epigenetic processes have detrimental consequences for both early embryo development and assisted reproductive technology. Emerging computational techniques are likely to improve our understanding of chromatin dynamics in the future.

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.

The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART programme

STUDY QUESTION Could the protamine-1 to protamine-2 mRNA ratio serve as a biomarker to estimate the fertilizing capacity of sperm from men taking part in an IVF/ICSI programme? SUMMARY ANSWER The protamine mRNA ratio clearly discriminates between fertile and subfertile men and sperm with a normal protamine mRNA ratio exhibit a higher fertilizing capacity in IVF/ICSI. WHAT IS KNOWN ALREADY Aberrant sperm protamine ratios are associated with male factor infertility and mRNA ratio is comparable with protein ratio (due to transcriptional stop in elongating spermatids). STUDY DESIGN, SIZE, DURATION The study population was drawn from subfertile men, whose female partners participated in IVF or ICSI programmes between September 2010 and February 2012. Normozoospermic healthy volunteers served as controls. Sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time quantitative PCR using specific primer pairs for protamine-1 and protamine-2. Relative protamine-1 and protamine-2 mRNA levels were analysed with the Mann-Whitney U-test (two-tailed). PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative RT-PCR for protamines 1 and 2 has been performed in ejaculates from 32 normozoospermic volunteers (control, University Clinic Giessen, Germany) and 306 patients, whose female partners took part in an IVF (n = 76; University Clinic Hamburg, Germany and Shanghai Jiaotong University, China) or an ICSI (n = 230; University Clinic Munich, Germany and Kinderwunschzentrum Wiesbaden, Germany) programme. MAIN RESULTS AND THE ROLE OF CHANCE The sperm protamine mRNA ratio in normozoospermic men (0.98 ± 0.3) differed significantly from that of ICSI patients (Munich 0.81 ± 0.1; Wiesbaden 0.78 ± 0.2; P < 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0 ± 0.07; Shanghai 1.0 ± 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc > 80%; P = 0.017) and ICSI (65.1% of patients with fc > 70%; P = 0.028). LIMITATIONS, REASONS FOR CAUTION The protamine mRNA ratio in an individual sperm cell used for ICSI may be different from the overall value obtained from a semen aliquot. WIDER IMPLICATIONS OF THE FINDINGS Data are in line with current literature and suggest the protamine mRNA ratio as a diagnostic marker to estimate the fertilizing capacity of sperm. STUDY FUNDING The German Research Foundation (DFG) to K.S., W.W. and A.P. (STE 892/9-2), as well as to A.S. and H.C.O. (SP721/1-3). COMPETING INTEREST(S) None.

The interaction of modified histones with the bromodomain testis-specific (BRDT) gene and its mRNA level in sperm of fertile donors and subfertile men

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.

Presence of histone H3 acetylated at lysine 9 in male germ cells and its distribution pattern in the genome of human spermatozoa

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.

PTPIP51 mRNA and protein expression in tissue microarrays and promoter methylation of benign prostate hyperplasia and prostate carcinoma

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) shows a tissue‐specific expression pattern and is associated with cellular differentiation and apoptosis in several mammalian tissues. Overexpression of the full‐length protein enhances apoptosis. It is also expressed in various carcinomas. In this study the expression of PTPIP51 and its in vitro interaction partners was investigated in human benign prostate hyperplasia (BPH) and in prostate carcinoma (PCa).

Evaluating the localization and DNA binding complexity of histones in mature sperm.

The paternal genome in many animal taxa is efficiently packaged into the sperm nucleus by switching from a histone (nucleosome)-based chromatin configuration to one using predominantly protamines. Nonetheless, various studies have shown that some nucleosomes, often containing modified histones are retained in mature sperm and bind DNA with distinct sequence compositions. Considering the significance of histone modifications in epigenetic phenomena and the fact that sperm histones and their bound DNA must be carried into the oocyte, this chapter describes methods aimed at examining and analysing the histone composition of sperm chromatin. The focus is on both microscopic visualisation and evaluation of sequence composition of histones and histone-bound DNA in human and mouse spermatozoa. However, similar methods may be applicable to the sperm of other mammalian and even non-mammalian classes.

27 The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART program

main results and the role of chance: The sperm protamine mRNA ratio in normozoospermic men (0.98+ 0.3) differed significantly from that of ICSI patients (Munich 0.81+ 0.1; Wiesbaden 0.78+ 0.2; P , 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0+ 0.07; Shanghai 1.0+ 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc . 80%; P ¼ 0.017) and ICSI (65.1% of patients with fc . 70%; P ¼ 0.028).

50 The relevance of acetylated histone H4 at lysine 12 (H4K12ac) for RNA transcripts in human spermatozoa, mouse pronucleus formation and parthenogenetic activation of murine oocytes

Comparision of the data set of genes interacting H4K12ac in sperm of fertile and subfertile men (Chromatin Immunoprecipitation with H4K12ac in combination with promoter microarray HG18 NimbleGen) with genome wide expression profiling of mRNAs from human sperm of fertile and subfertle patients (CodeLink Human Whole Genome Bioarray, Applied Microarrays). The immunofluorscence with H4K12ac antibodies was carried out after in vitro fertilisation or parthenogenetic activation of oocytes. 140 differentially expressed genes which promoters interacting with H4K12ac in sperm chromatin were subjected to gene ontology classification and clustering with the DAVID2010 database. Functional terms such as “cellular protein molecular process” (35 out of 140 P value=3.1e-4), “positive regulation of transcription, DNA-dependent” (11 out of 140; p value=5.4e-3), “embryonic development” (11 out of 140; p value 6.4e-2) are overrepresented in the group of genes expressed possibly through acetylation of H4K12 during spermatogenesis. Immunofluorescence with H4K12ac antibodies showed a strong compact signal over the male pronuclus compared to a weaker one in female pronucleus. During pronuclei migration and at the point of pronuclei fusion, there was observed an increasing H4K12ac signal in female pronucleus as well, however, the male pronucleus remained strongly labelled After parthenogenetic oocyte stimulation, both established female pronuclei were positively labelled for H4K12ac. The relevance of acetylated histone H4 at lysine 12 (H4K12ac) for RNA transcripts in human spermatozoa, mouse pronucleus formation and parthenogenetic activation of murine oocytes