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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Testosterone is essential to maintain qualitative spermatogenesis. Nonetheless, no studies have been yet performed in humans to analyze the testosterone-mediated expression of sperm proteins and their importance in reproduction. Thus, this study aimed to identify sperm protein alterations in male hypogonadism using proteomic profiling. We have performed a comparative proteomic analysis comparing sperm from fertile controls (a pool of 5 normogonadic normozoospermic fertile men) versus sperm from patients with secondary hypogonadism (a pool of 5 oligozoospermic hypogonadic patients due to isolated LH deficiency). Sperm protein composition was analyzed, after peptide labelling with Isobaric Tags, via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) on an LTQ Velos-Orbitrap mass spectrometer. LC-MS/MS data were analyzed using Proteome Discoverer. Criteria used to accept protein identification included a false discovery rate (FDR) of 1% and at least 1 peptide match per protein. Up to 986 proteins were identified and, of those, 43 proteins were differentially expressed: 32 proteins were under-expressed and 11 were over-expressed in the pool of hypogonadic patients compared to the controls. Bioinformatic analyses were performed using UniProt Knowledgebase, and the Gene Ontology Consortium database based on PANTHER. Notably, 13 of these 43 differentially expressed proteins have been previously reported to be related to sperm function and spermatogenesis. Western blot analyses for A-Kinase Anchoring Protein 3 (AKAP3) and the Prolactin Inducible Protein (PIP) were used to confirm the proteomics data. In summary, a high-resolution mass spectrometry-based proteomic approach was used for the first time to describe alterations of the sperm proteome in secondary male hypogonadism. Some of the differential sperm proteins described in this study, which include Prosaposin, SMOC-1, SERPINA5, SPANXB1, GSG1, ELSPBP1, fibronectin, 5-oxoprolinase, AKAP3, AKAP4, HYDIN, ROPN1B, ß-Microseminoprotein and Protein S100-A8, could represent new targets for the design of infertility treatments due to androgen deficiency. [ABSTRACT FROM AUTHOR]

Background: Knowledge of the proteomic composition of the gametes is essential to understand reproductive functions. Most of the sperm proteins are related to spermatogenesis and sperm function, but less abundant protein groups with potential post-fertilization roles have also been detected. The current data are challenging our understanding of sperm biology and functionality, demanding an integrated analysis of the proteomic and RNA-seq datasets available for spermatozoa, oocytes and early embryos, in order to unravel the impact of the male gamete on the generation of the new individual.Objective and Rationale: The aim of this review is to compile human sperm proteins and to identify and infer their origin and discuss their relevance during oocyte fecundation, preimplantation embryogenesis and epigenetic inheritance.Search Methods: The scientific literature was comprehensively searched for proteomic studies on human sperm, oocytes, embryos, and additional reproductive cells and fluids. Proteins were compiled and functionally classified according to Gene Ontology annotations and the mouse phenotypes integrated into the Mouse Genome Informatics database. High-throughput RNA datasets were used to decipher the origin of embryo proteins. The tissue origin of sperm proteins was inferred on the basis of RNA-seq and protein data available in the Human Protein Atlas database.Outcomes: So far, 6871 proteins have been identified and reported in sperm, 1376 in the oocyte and 1300 in blastocyst. With a deeper analysis of the sperm proteome, 103 proteins with known roles in the processes of fertilization and 93 with roles in early embryo development have been identified. Additionally, 560 sperm proteins have been found to be involved in modulating gene expression by regulation of transcription, DNA methylation, histone post-translational modifications and non-coding RNA biogenesis. Some of these proteins may be critical for gene expression regulation after embryo genome activation, and therefore, may be potentially involved in epigenetic transmission of altered phenotypes. Furthermore, the integrative analysis of the sperm, oocyte and blastocyst proteomes and transcriptomes revealed a set of embryo proteins with an exclusive paternal origin, some of which are crucial for correct embryogenesis and, possibly, for modulation of the offspring phenotype. The analysis of the expression of sperm proteins, at both RNA and protein levels, in tissues not only from the male reproductive tract but also from peripheral organs, has suggested a putative extra-testicular origin for some sperm proteins. These proteins could be imported into sperm from the accessory sex glands and other tissues, most likely through exosomes.Wider Implications: These integrative proteome and transcriptome analyses focused on specific groups of proteins, rather than on enriched pathways, identified important sperm proteins which may be involved in early embryogenesis and provided evidence which could support the hypothesis of paternal epigenetic inheritance. The putative extra-testicular origin of some sperm proteins suggests not only the involvement of accessory sex glands in fertilization and epigenetic information transmission, but also that some proteins from additional organs could possibly contribute information to the offspring phenotype. These findings should stimulate further research in the field. [ABSTRACT FROM AUTHOR]