Proteomics and the genetics of sperm chromatin condensation

Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies

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

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

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

Caracterització del contingut proteic del nucli de l’espermatozoïde humà i el seu potencial epigenètic

[cat] L’espermatozoide és una cèl•lula molt especialitzada amb una estructura de la cromatina única formada per dos dominis principals: el domini nucli-protamínic on la gran majoria del DNA es troba empaquetat per protamines en toroides molt compactes, i el domini nucli-histònic on una petita proporció del DNA roman empaquetat per histones, d’una manera més accessible. Cada vegada són més les evidències que suggereixen que l’espermatozoide madur presenta una funció més enllà de la simple transmissió del material genètic patern a la següent generació. La hipòtesi principal d’aqueta tesi consisteix en que aquesta contribució paterna no és solament basada en la transmissió de patrons de metilació de DNA o modificacions d’histones concretes, si no que també es troba influïda per l’estat de la cromatina espermàtica i, en especial, per la seva composició proteica. Per aquest motiu, en aquesta tesi doctoral s’ha caracteritzat detalladament el contingut proteic del nucli de l’espermatozoide humà normal mitjançant dues estratègies diferents. Per una banda, les proteïnes majoritàries del nucli espermàtic, les protamines, han sigut analitzades per extraccions i electroforesis àcides per tal d’estudiar la seva implicació en la conservació de la cromatina. S’ha observat una relació negativa significativa entre la quantitat de protamines i el dany al DNA espermàtic (mesurat per la tècnica COMET) en pacients infèrtils que cursen tècniques de reproducció assistida. Aquests resultats han suggerit una funció de les protamines referent a la protecció del material genètic patern. A més a més, mitjançant tècniques basades en espectrometria de masses (Top-down proteomics), s’ha observat com les protamines podrien contenir modificacions post-traduccionals en la seva seqüència (fosforilacions, acetilacions, metilacions), el que suggeriria també una funció reguladora per aquestes proteïnes espermàtiques. En segon lloc, mitjançant espectrometria de masses també s’han caracteritzat la resta de proteïnes presents en el nucli del gàmeta masculí madur. S’han arribat a identificar centenars de proteïnes, la meitat de les quals corresponen a histones, variants d’histones, modificadors d’histones, factors de transcripció, proteïnes addicionals associades a la cromatina i proteïnes modificadores de la cromatina espermàtica. D’aquesta manera, mitjançant anàlisis bioinformàtics es pot observar com el nucli de l’espermatozoide humà madur es trobaria enriquit en proteïnes involucrades en processos reguladors com la modificació de la cromatina, modificació d’histones, expressió gènica i transcripció. Aquestes tendències també es trobarien presents en altres espècies de mamífers, com per exemple el ratolí, la rata o el macaco. La presència d’aquests grups de proteïnes intactes en una cèl•lula considerada transcripcionalment inerta ha suggerit una funció epigenètica/reguladora durant els primers estadis del desenvolupament embrionari. Addicionalment, en aquesta tesi doctoral s’ha observat que les proteïnes espermàtiques nuclears es poden diferenciar en dos grups diferents segons la seva afinitat per la cromatina espermàtica. D’aquesta manera, les proteïnes unides més fortament al DNA es trobarien majoritàriament implicades en funcions estructurals, mentre que les proteïnes unides al DNA d’una forma més feble es trobarien relacionades amb processos reguladors o epigenètics. Aquesta observació, juntament amb el fet conegut de que la regió més accessible de la cromatina espermàtica (associada a les histones) es troba especialment enriquida en promotors i gens del desenvolupament, suggeriria que part de les proteïnes nuclears espermàtiques podrien estar relacionades amb la regulació de la transcripció d’aquests gens, a més a més d’altres processos com la remodelació de la cromatina en el pronucli masculí. Per tant, mitjançant la caracterització del contingut proteic del nucli de l’espermatozoide humà, s’ha augmentat el coneixement d’aquesta cèl•lula i de la seva funcionalitat, recolzant la hipòtesi de que a més a més del propi material genètic patern, l’espermatozoide contribueix en la fertilització amb informació epigenètica, la qual inclou una composició proteica específica de la cromatina.[eng] The mammalian spermatozoon is a very specialized cell with a unique chromatin structure: while the vast majority of the DNA is condensed by high compact toroids (avoiding any transcriptional and translational activity), a small part of the DNA remains packaged by nucleosomes. An increasing body of evidence is showing that, in addition to the transmission of the paternal genetic message to the next generation, the sperm cell is also involved in the transmission of epigenetic information. The main hypothesis of the present doctoral thesis is that, in addition to histone modifications, DNA methylation patterns and RNA content; protein composition of the sperm chromatin is also essential in epigenetic inheritance. Therefore, the protein content of the human sperm nucleus has been characterized using proteomic techniques. Firstly, by acid extraction and electrophoresis, it has been shown a negative relationship between protamine quantities and DNA damage in infertile patients who undergo assisted reproduction techniques, confirming an involvement of those proteins in the protection of the paternal DNA. In addition, by top-down proteomics, it has been detected several post-translational modifications in protamine sequences, which have suggested a potential regulatory role for those major sperm nuclear proteins. Secondly, it has been described the human sperm nuclear proteome using mass spectrometry techniques. A rich network of histones, histone variants, histone modifiers, transcription factors, chromatin-associated proteins and chromatin modifying proteins has been identified in the human sperm nucleus. In addition, based in Gene Ontology information, the human sperm nucleus seemed to be enriched in processes as chromatin remodelling, histone modification, transcription and gene expression. Furthermore, proteomic dissection of the sperm chromatin results in two groups of proteins differing in chromatin affinity. Higher-affinity sperm chromatin proteins seemed to be mainly involved in structural roles, while proteins with potential regulatory/epigenetic roles were identified with a lower sperm chromatin affinity. Altogether, the results obtained in this doctoral thesis have contributed in the knowledge of the nucleus of the male gamete and have suggested an involvement of the sperm nuclear proteins in post-fertilization events, as the chromatin remodelling in the male pronucleus or the transcription regulation of histone-bound paternal developmental genes

Proteomic changes in human sperm during sequential in vitro capacitation and acrosome reaction

The male gamete is not completely mature after ejaculation and requires further events in the female genital tract to acquire fertilizing ability, including the processes of capacitation and acrosome reaction. In order to shed light on protein changes experienced by the sperm cell in preparation for fertilization, a comprehensive quantitative proteomic profiling based on isotopic peptide labeling and liquid chromatography followed by tandem mass spectrometry was performed on spermatozoa from three donors of proven fertility under three sequential conditions: purification with density gradient centrifugation, incubation with capacitation medium, and induction of acrosome reaction by exposure to the calcium ionophore A23187. After applying strict selection criteria for peptide quantification and for statistical analyses, 36 proteins with significant changes in their relative abundance within sperm protein extracts were detected. Moreover, the presence of peptide residues potentially harboring sites for post-translational modification was revealed, suggesting that protein modification may be an important mechanism in sperm maturation. In this regard, increased levels of proteins mainly involved in motility and signaling, both regulated by protein modifiers, were detected in sperm lysates following incubation with capacitation medium. In contrast, less abundant proteins in acrosome-reacted cell lysates did not contain potentially modifiable residues, suggesting the possibility that all those proteins might be relocated or released during the process. Protein-protein interaction analysis revealed a subset of proteins potentially involved in sperm maturation, including the proteins Erlin-2 (ERLIN2), Gamma-glutamyl hydrolase (GGH) and Transmembrane emp24 domain-containing protein 10 (TMED10). These results contribute to the current knowledge of the molecular basis of human fertilization. It should now be possible to further validate the potential role of the detected altered proteins as modulators of male infertility