DOT1L regulates chromatin reorganization and gene expression during sperm differentiation.

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

Proteomics of human spermatozoa.

Proteomic methodologies offer a robust approach to identify and quantify thousands of proteins from semen components in both fertile donors and infertile patients. These strategies provide an unprecedented discovery potential, which many research teams are currently exploiting. However, it is essential to follow a suitable experimental design to generate robust data, including proper purification of samples, appropriate technical procedures to increase identification throughput, and data analysis following quality criteria. More than 6000 proteins have been described so far through proteomic analyses in the mature sperm cell, increasing our knowledge on processes involved in sperm function, intercommunication between spermatozoa and seminal fluid, and the transcriptional origin of the proteins. These data have been complemented with comparative studies to ascertain the potential role of the identified proteins on sperm maturation and functionality, and its impact on infertility. By comparing sperm protein profiles, many proteins involved in the acquisition of fertilizing ability have been identified. Furthermore, altered abundance of specific protein groups has been observed in a wide range of infertile phenotypes, including asthenozoospermia, oligozoospermia, and normozoospermia with unsuccessful assisted reproductive techniques outcomes, leading to the identification of potential clinically useful protein biomarkers. Finally, proteomics has been used to evaluate alterations derived from semen sample processing, which might have an impact on fertility treatments. However, the intrinsic heterogeneity and inter-individual variability of the semen samples have resulted in a relatively low overlap among proteomic reports, highlighting the relevance of combining strategies for data validation and applying strict criteria for proteomic data analysis to obtain reliable results. This mini-review provides an overview of the most critical steps to conduct robust sperm proteomic studies, the most relevant results obtained so far, and potential next steps to increase the impact of sperm proteomic data.

Histone H4 acetylation is dysregulated in active seminiferous tubules adjacent to testicular tumours.

STUDY QUESTION: Is histone H4 acetylation (H4ac) altered in the seminiferous tubules of patients affected by testicular tumours? SUMMARY ANSWER: A considerable dysregulation of H4ac was detected in the cells of the seminiferous tubules adjacent to testicular tumours of different aetiology and prior to any treatment, while no comparable alterations were observed in patients with disrupted spermatogenesis. WHAT IS KNOWN ALREADY: Altered H4ac levels have been associated with a variety of testicular pathological conditions. However, no information has been available regarding potential alterations in the spermatogenic cells adjacent to the neoplasia in testicular tumour patients. STUDY DESIGN, SIZE, DURATION: A retrospective analysis using testicular sections from 33 men aged between 21 and 74 years old was performed. Three study groups were defined and subjected to double-blind evaluation: a control group with normal spermatogenesis (n = 6), patients with testicular tumours (n = 18) and patients with spermatogenic impairments (n = 8). One additional sample with normal spermatogenesis was used as a technical internal control in all evaluations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry against H4ac and, when needed, Placental-like alkaline phosphatase and CD117, was performed on testicular sections. The H4ac H-score, based on the percentage of detection and signal intensity, was used as the scoring method for statistical analyses. Protein expression data from the Human Protein Atlas were used to compare the expression levels of predicted secreted proteins from testicular tumours with those present in the normal tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We revealed, for the first time, a dramatic disruption of the spermatogenic H4ac pattern in unaffected seminiferous tubule cells from different testicular tumour patients prior to any antineoplastic treatment, as compared to controls (P < 0.05). Since no similar alterations were associated with spermatogenic impairments and the in silico analysis revealed proteins potentially secreted by the tumour to the testicular stroma, we propose a potential paracrine effect of the neoplasia as a mechanistic hypothesis for this dysregulation. LIMITATIONS, REASONS FOR CAUTION: Statistical analyses were not performed on the hypospermatogenesis and Leydig cell tumour groups due to limited availability of samples. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first report showing an epigenetic alteration in cells from active seminiferous tubules adjacent to tumour cells in testicular tumour patients. Our results suggest that, despite presenting spermatogenic activity, the global epigenetic dysregulation found in the testicular tumour patients could lead to molecular alterations of the male germ cells. Since testicular tumours are normally diagnosed in men at reproductive age, H4ac alterations might have an impact when these testicular tumour patients express a desire for fatherhood. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the European Union Marie Curie European Training Network actions and by grants to R.O. from the 'Ministerio de Economía y Competividad (Spain)' (fondos FEDER 'una manera de hacer Europa', PI13/00699, PI16/00346 and PI20/00936) and from EU-FP7-PEOPLE-2011-ITN289880. J.C. was supported by the Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109. J.C. is a Serra Húnter fellow (Universitat de Barcelona, Generalitat de Catalunya). F.B. has received grants from the Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario (Spain) (FPU15/02306). A.d.l.I. is supported by a fellowship of the Ministerio de Economía, Industria y Competitividad (Spain) (PFIS, FI17/00224). M.J. is supported by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

H4K5 Butyrylation Coexist with Acetylation during Human Spermiogenesis and Are Retained in the Mature Sperm Chromatin.

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.

Stable-protein Pair Analysis as A Novel Strategy to Identify Proteomic Signatures: Application To Seminal Plasma From Infertile Patients.

Our aim was to define seminal plasma proteome signatures of infertile patients categorized according to their seminal parameters using TMT-LC-MS/MS. To that extent, quantitative proteomic data was analyzed following two complementary strategies: (1) the conventional approach based on standard statistical analyses of relative protein quantification values; and (2) a novel strategy focused on establishing stable-protein pairs. By conventional analyses, the abundance of some seminal plasma proteins was found to be positively correlated with sperm concentration. However, this correlation was not found for all the peptides within a specific protein, bringing to light the high heterogeneity existing in the seminal plasma proteome because of both the proteolytic fragments and/or the post-translational modifications. This issue was overcome by conducting the novel stable-protein pairs analysis proposed herein. A total of 182 correlations comprising 24 different proteins were identified in the normozoospermic-control population, whereas this proportion was drastically reduced in infertile patients with altered seminal parameters (18 in patients with reduced sperm motility, 0 in patients with low sperm concentration and 3 in patients with no sperm in the ejaculate). These results suggest the existence of multiple etiologies causing the same alteration in seminal parameters. Additionally, the repetition of the stable-protein pair analysis in the control group by adding the data from a single patient at a time enabled to identify alterations in the stable-protein pairs profile of individual patients with altered seminal parameters. These results suggest potential underlying pathogenic mechanisms in individual infertile patients, and might open up a window to its application in the personalized diagnostic of male infertility.

Characterization of Human Sperm Protamine Proteoforms through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches.

Protamine 1 (P1) and protamine 2 (P2) family are extremely basic, sperm-specific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies is not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach. By top-down MS, described and novel truncated P1 and pre-P2 proteoforms were identified. Intact P1, pre-P2, and P2 mature proteoforms and their phosphorylation pattern were also detected. Additionally, a +61 Da modification in different proteoforms was observed. By the bottom-up MS approach, phosphorylated residues for pre-P2, as well as the new P2 isoform 2, which is not annotated in the UniProtKB database, were revealed. Implementing these strategies in comparative studies of different infertile phenotypes, together with the evaluation of P1/P2 and pre-P2/P2 MS-derived ratios, would permit determining specific alterations in the protamine proteoforms and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.

Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling.

When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

Sperm proteomic changes associated with early embryo quality after ICSI.

RESEARCH QUESTION: Do alterations of human sperm protein profile affect embryo quality? DESIGN: Sperm proteins from 27 infertile couples undergoing intracytoplasmic sperm injection (ICSI) were extracted and digested. The resulting peptides were labelled using tandem mass tags, separated by two-dimensional liquid chromatography, and identified and quantified using tandem mass spectrometry. Subsequently, sperm protein and peptide abundance were statistically analysed for correlation with ICSI-derived embryo quality in the subset of idiopathic infertile couples. Detected correlations were further assessed in the subset of infertile patients with a known factor. RESULTS: The abundance of 18 individual sperm proteins was found to correlate with embryo quality after ICSI. Of note, a high percentage of poor-quality ICSI-derived embryos was associated with alterations in several components of the eight-membered chaperonin-containing T-complex, which plays an important role in the folding of many essential proteins. Additionally, the abundance of sperm proteins with known functions in embryogenesis, such as RUBVL1, also correlated with early embryo quality (r = -0.547; P = 0.028). Some of the correlations found in this study were validated using either proteomic data from infertile patients with a known factor or data from similar published studies. Analysis at the peptide level revealed the association of some correlations with specific post-translational modifications or isoforms. CONCLUSIONS: Our results support the hypothesis that the sperm proteome plays a role in early embryogenesis. Moreover, several sperm proteins have emerged as potential biomarkers that could predict the outcome of in-vitro assisted reproductive technologies, leading to the possibility of improved diagnosis of couples with idiopathic infertility.

Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI.

STUDY QUESTION: Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? SUMMARY ANSWER: New mutations in the PLCZ1 sequence are associated with FFs after ICSI. WHAT IS KNOWN ALREADY: FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. STUDY DESIGN, SIZE, DURATION: We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection. MAIN RESULTS AND THE ROLE OF CHANCE: In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. LIMITATIONS, REASONS FOR CAUTION: Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. WIDER IMPLICATIONS OF THE FINDINGS: PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

Sperm telomere length in donor samples is not related to ICSI outcome.

PURPOSE: Variations in sperm telomere length (STL) have been associated with altered sperm parameters, poor embryo quality, and lower pregnancy rates, but for normozoospermic men, STL relevance in IVF/ICSI is still uncertain. Moreover, in all studies reported so far, each man's STL was linked to the corresponding female partner characteristics. Here, we study STL in sperm donor samples, each used for up to 12 women, in order to isolate and determine the relationship between STL and reproductive outcomes. METHODS: Relative STL was determined by qPCR in 60 samples used in a total of 676 ICSI cycles. Univariable and multivariable statistical analyses were used to study the STL effect on fertilization rate; embryo morphology; biochemical, clinical, and ongoing pregnancy rates; and live birth (LB) rates. RESULTS: The average STL value was 4.5 (relative units; SD 1.9; range 2.4-14.2). Locally weighted scatterplot smoothing regression and the rho-Spearman test did not reveal significant correlations between STL and the outcomes analyzed. STL was not different between cycles resulting or not in pregnancy and LB (Mann-Whitney U test, p > 0.05). No significant effect of STL on reproductive outcomes was found, with the OR for each unit increase in STL (95% CI) of 0.94 (0.86-1-04), 0.99 (0.9-1.09), 0.98 (0.89-1.09), and 0.93 (0.8-1.06) for biochemical, clinical, and ongoing pregnancy and LB, respectively. The multilevel analysis confirmed that the effect of STL on fertilization; biochemical, clinical, and ongoing pregnancy; and LB was not significant (p > 0.05). CONCLUSION: After addressing STL independently from female variables, results show that STL measurement is not useful to predict reproductive outcomes in ICSI cycles using donor semen.

The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes.

At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline.

Advances in sperm proteomics: best-practise methodology and clinical potential.

The recent application of mass spectrometry to the study of the sperm cell has led to an unprecedented capacity for identification of sperm proteins in a variety of species. Knowledge of the proteins that make up the sperm cell represents the first step towards understanding its normal function and the molecular anomalies associated with male infertility. The present review starts with an introduction of the sperm cell biology and is followed by the consideration of the methodological key aspects to be aware of during sample sourcing and preparation, including data interpretation. It then overviews the initiatives developed so far towards the completion of the sperm proteome, with a particular focus in human but with the inclusion of some comments on different model species. Finally, all studies performing differential proteomics in infertile patients are reviewed, pointing to future potential applications.

Human sperm tail proteome suggests new endogenous metabolic pathways.

Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous pools, and thus to adapt to putative exogenous fluctuations.

Identification of proteins involved in human sperm motility using high-throughput differential proteomics.

Mammalian sperm motility is a prerequisite for in vivo fertilization, and alterations in this parameter are commonly observed in infertile males. However, we still do not have a complete understanding of the molecular mechanisms controlling it. The aim of this study was to identify proteins involved in human sperm motility deficiency by using TMT protein labeling and LC-MS/MS. Two complementary approaches were used: comparison between sperm samples differing in motility (asthenozoospermic versus normozoospermic) and comparison between sperm subpopulations of fractionated normozoospermic samples differing in motility (non-migrated versus migrated). LC-MS/MS resulted in the identification of 1157 and 887 proteins in the first and second approaches, respectively. Remarkably, similar proteomic alterations were detected in the two experiments, with 80 proteins differentially expressed in the two groups of samples and 93 differentially expressed in the two groups of subpopulations. The differential proteins were analyzed by GO, cellular pathways, and clustering analyses and resulted in the identification of core deregulated proteins and pathways associated with sperm motility dysfunction. These included proteins associated with energetic metabolism, protein folding/degradation, vesicle trafficking, and the cytoskeleton. Contrary to what is usually accepted, the outcomes support the hypothesis that several metabolic pathways (notably, mitochondrial-related ones) contribute toward regulating sperm motility.

Genomic and proteomic dissection and characterization of the human sperm chromatin.

The mammalian spermatozoon has a unique chromatin structure where the majority of DNA is packaged by protamines, while a small fraction (∼8%) remains associated with nucleosomes. However, the chromatin affinity and repertoire of the additional proteins constituting the different sperm chromatin fractions have not yet been explored. To address this we have carried out a genomic and proteomic characterization of human sperm samples subjected to chromatin fractionation using either 0.65 M NaCl extraction followed by EcoRI/BamHI DNA restriction enzyme digestion, or micrococcal nuclease digestion. DNA fractions corresponding to the nucleosome-packaged DNA were sequenced, confirming an appropriate dissection of the sperm chromatin. In addition we detected and sequenced a subnucleosomal particle. Although both fractions were highly enriched at gene promoters, some sequences were found to be exclusively associated with one of those. The results of the proteomic analyses demonstrate that there are two distinct sets of sperm proteins which differ in chromatin affinity. Histone variants, transcription factors, chromatin-associated and modifying proteins involved in regulatory roles were identified as weakly attached to the sperm DNA, whereas proteins with structural roles were identified in the condensed fraction. Many factors, such as the histone lysine demethylase PHF8 identified for the first time in the human sperm cell in this study, were identified exclusively in soluble fraction. Our results provide additional support to the possibility that all of these factors may constitute additional layers of sperm epigenetic information or have structural or regulatory roles transmitted by the sperm cell to the oocyte at fertilization.

High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction.

STUDY QUESTION: Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy? SUMMARY ANSWER: Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins. WHAT IS KNOWN ALREADY: The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins. STUDY DESIGN, SIZE, DURATION: This was a case-control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008. PARTICIPANTS/MATERIALS, SETTING, METHODS: Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy < 0.67) and 35 at higher abundance (ratio no pregnancy/pregnancy > 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values < 0.05). In addition, the differential abundance of one of the proteins (SRSF protein kinase 1) was further validated by western blotting using independent samples (P value < 0.01). LIMITATIONS, REASONS FOR CAUTION: For individual samples the amount of recovered sperm not used for IVF was low and in most of the cases insufficient for MS analysis, therefore pools of samples had to be used to this end. WIDER IMPLICATIONS OF THE FINDINGS: Alterations in the proteins involved in chromatin assembly and metabolism may result in epigenetic errors during spermatogenesis, leading to inaccurate sperm epigenetic signatures, which could ultimately prevent embryonic development. These sperm proteins may thus possibly have clinical relevance. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economia y Competividad; FEDER BFU 2009-07118 and PI13/00699) and Fundación Salud 2000 SERONO13-015. There are no competing interests to declare.

Protamine alterations in human spermatozoa.

Protamines are the major nuclear proteins in sperm cells, having a crucial role in the correct packaging of the paternal DNA. The fact that protamine haploinsufficiency in mice resulted in abnormal chromatin packaging and male infertility suggested that the protamines could also be important candidates in explaining some of the idiopathic male infertility cases in humans. The first clinical studies focused on analyzing protamines at the protein level. Various studies have found the presence of an altered amount of protamines in some infertile patients, in contrast to the normal situation in fertile individuals where the two protamines, protamine 1 and protamine 2, are both present in approximately equal quantities. Subsequently, the protamine genes were the subject of various mutational genetic screening studies in search of variants that could be associated with deregulation in the protamine expression observed. The results of these protamine mutational studies showed that the presence of high penetrant mutations is a very rare cause of male infertility. However, some variants and some haplotypes described may behave as risk factors for male infertility. More recently, the presence of RNA in the mature sperm cell has also been investigated. The present chapter will introduce the basic aspects of protamine evolution and function and review the various articles published to date on the relationship between the protamines studied at the DNA, RNA, and protein levels and male infertility.

High apolipoprotein E4 allele frequency in FXTAS patients.

PURPOSE: Fragile X-associated tremor/ataxia syndrome is a late-onset neurodegenerative disorder that occurs in FMR1 premutation carriers. It is well known that the apolipoprotein E ε4 allele is a risk factor for neurodegenerative disease. The main goal of this work was to evaluate the apolipoprotein E genotypes and allelic distribution among patients with fragile X-associated tremor/ataxia syndrome. METHODS: A total of 44 unrelated FMR1 premutation carriers (22 presenting with fragile X-associated tremor/ataxia syndrome and 22 without fragile X-associated tremor/ataxia syndrome) were genotyped. RESULTS: All the apolipoprotein E ε4/4 genotype carriers detected (100%), and six of the seven apolipoprotein E ε4/3 genotype carriers (85.7%) are patients presenting with fragile X-associated tremor/ataxia syndrome symptoms, whereas only 40% of the apolipoprotein E ε3/3 genotype carriers belong to the fragile X-associated tremor/ataxia syndrome group. The results showed that the presence of the apolipoprotein E ε4 allele increases the risk of developing fragile X-associated tremor/ataxia syndrome (odds ratio = 12.041; P = 0.034). CONCLUSION: On the basis of these results, we conclude that the presence of at least one apolipoprotein E ε4 allele might act as a genetic factor predisposing individuals to develop fragile X-associated tremor/ataxia syndrome.

Insights into the sperm chromatin and implications for male infertility from a protein perspective.

Male germ cells undergo an extreme but fascinating process of chromatin remodeling that begins in the testis during the last phase of spermatogenesis and continues through epididymal sperm maturation. Most of the histones are replaced by small proteins named protamines, whose high basicity leads to a tight genomic compaction. This process is epigenetically regulated at many levels, not only by posttranslational modifications, but also by readers, writers, and erasers, in a context of a highly coordinated postmeiotic gene expression program. Protamines are key proteins for acquiring this highly specialized chromatin conformation, needed for sperm functionality. Interestingly, and contrary to what could be inferred from its very specific DNA-packaging function across protamine-containing species, human sperm chromatin contains a wide spectrum of protamine proteoforms, including truncated and posttranslationally modified proteoforms. The generation of protamine knock-out models revealed not only chromatin compaction defects, but also collateral sperm alterations contributing to infertile phenotypes, evidencing the importance of sperm chromatin protamination toward the generation of a new individual. The unique features of sperm chromatin have motivated its study, applying from conventional to the most ground-breaking techniques to disentangle its peculiarities and the cellular mechanisms governing its successful conferment, especially relevant from the protein point of view due to the important epigenetic role of sperm nuclear proteins. Gathering and contextualizing the most striking discoveries will provide a global understanding of the importance and complexity of achieving a proper chromatin compaction and exploring its implications on postfertilization events and beyond. This article is categorized under: Reproductive System Diseases > Genetics/Genomics/Epigenetics Reproductive System Diseases > Molecular and Cellular Physiology.

Differential RNAs in the sperm cells of asthenozoospermic patients.

BACKGROUND: Alterations in RNAs present in sperm have been identified using microarrays in teratozoospermic patients and in other types of infertile patients. However, so far, there have been no reports on using microarrays to determine the RNA content of sperm from asthenozoospermic patients. METHODS: We started the present project with the goal of characterizing the RNA abundance in the sperm cells of asthenozoospermic patients when compared with controls. To reach this objective, we initially selected four normal fertile donors and four asthenozoospermic infertile patients. Equal amounts of RNA were extracted from the sperm samples, subjected to different quality controls and hybridized to the Affymetrix U133 Plus version 2 arrays. RESULTS: Several transcripts were identified that were present in different abundance in patients compared with controls. Subsequently, we validated the differential expression of three of the detected transcripts (ANXA2, BRD2 and OAZ3), using real-time PCR in a larger set of samples. A positive correlation between the expression of these transcripts and progressive motility was observed. CONCLUSIONS: The sperm cells of asthenozoospermic patients contain an altered amount of some RNAs as detected using microarray analysis and subsequently validated using real-time PCR. These results open up the possibility to investigate the implication of these genes in the pathogenic mechanisms in asthenozoospermia and to consider their potential utility as infertility biomarkers.