Titre de la dissertation: « Translation in mature human spermatozoa: identification of the type of ribosomes involved and of produced proteins ».

Promoteur de thèse: Madame Elise Hennebert

Co-promoteur de thèse: Monsieur Jean-François Simon (Hôpital Ambroise Paré Mons)

Résumé de la dissertation: During spermiogenesis, the final stage of spermatogenesis, the spermatid undergoes an important compaction of its DNA inside the nucleus and the release of the major part of its cytoplasm. These events have for a long time lead the scientists to think that transcription and translation are absent in mature spermatozoa. Since the 1950s, some published studies tend to suggest that spermatozoa can produce proteins during in vitro capacitation. However, the type of ribosomes involved (cytoplasmic or mitochondrial) is still debated. The aim of the present thesis was to identify potential translated proteins and the type of ribosomes involved in their synthesis.The first part of this thesis focused on solvent concentrations to use in the next experiments to avoid side effects. Indeed, ribosome inhibitors such as cycloheximide (CHX) and chloramphenicol (CP) are usually dissolved in solvents, e.g., ethanol (EtOH) or dimethyl sulfoxide (DMSO), which could influence the outcome of the experiment. Human spermatozoa were incubated for 4 h in a capacitation medium in the absence or the presence of different concentrations of EtOH and DMSO (0.1, 0.5, 1.0, and 2.0%) to assess the impact of these solvents on sperm motility, vitality, capacitation, and acrosome integrity. The presence of statistically significant relationships between increasing solvent concentrations and the investigated parameters was assessed using linear mixed models. A significant effect was observed with both solvents for total and progressive sperm motilities. The effect of time was also evaluated for these parameters and showed that the influence of the solvents was stable between 0 and 4 h, indicating an almost direct impact of the solvents. While EtOH did not influence sperm vitality and acrosome integrity, a significant effect of increasing DMSO concentrations was observed for these parameters. Finally, regarding capacitation, measured via phosphotyrosine content, although a dose-dependent effect was observed with both solvents, the statistical analysis did not allow to precisely evaluate the intensity of the effect. Based on the results obtained in the present study, and the corresponding linear mixed models, the concentration of both solvents which would result in a 5% decline in sperm parameters was caldulated. For EtOH, these concentrations are 0.9, 0.7, and 0.3% for total motility, progressive motility, and capacitation, respectively, while for DMSO they are 1.5, 1.1, >2, 0.3 and >2% for total motility, progressive motility, vitality, capacitation, and acrosome integrity, respectively. Based on these results, it can be concluded that solvent concentrations below these values should be used to dissolve the molecules to be tested on spermatozoa, in order to limit side effects.In the second part, the presence and activity of the two types of ribosomes was investigated in mature human spermatozoa. By targeting ribosomal RNAs and proteins, it was showed that both types of ribosomes are localized in the midpiece as well as in the neck and the base of the head of the spermatozoa. The impact of cycloheximide (CHX) and chloramphenicol (CP), inhibitors of cytoplasmic and mitochondrial ribosomes, respectively, was assessed on different sperm parameters. Neither CHX, nor CP impacted sperm vitality, mitochondrial activity (measured through the ATP content), or capacitation (measured through the content in phosphotyrosines). However, increasing CP concentrations induced a decrease in total and progressive motilities as well as on some kinematic parameters while no effect was observed with CHX. A quantitative proteomic analysis was performed by mass spectrometry in SWATH mode to compare the proteomes of spermatozoa capacitated in the absence or presence of the two ribosome inhibitors. Among the ~700 proteins identified in the different tested conditions, 3, 3 and 25 proteins presented a modified abundance in the presence of 1 and 2 mg/ml of CHX, and 1 mg/ml of CP, respectively. The observed abundance variations of some CP-down regulated proteins were validated using Multiple-Reaction Monitoring (MRM). Overall, the results show that the sperm motility deficits observed in the presence of CP could be linked to the observed decreased abundance of several proteins, at least FUNDC2 and QRICH2. Although other off-target effects of CP on protein abundance cannot be ruled out, a plausible assumption stands in the involvement of mitochondrial ribosomes, which are well documented targets of CP.Finally, in complement to previous experiments, two strategies were used to identify, via specific enrichment, the newly synthesized proteins. First, a ribosome profiling analysis was performed to identify the messenger RNAs (mRNAs) actively translated in the spermatozoa during their capacitation. To do so, RNAs from spermatozoa were extracted and digested with an RNase in order to degrade the free mRNA sequences (i.e. not protected by ribosomes). The resulting monosomes (each containing approximately 30 nucleotides of protected mRNA) were purified and separated on gel to isolate fragments of ~30 nucleotides. After extraction of the RNAs from the gel and depletion of contaminating rRNAs, libraries were prepared and sequencing on a high-throughput sequencing platform was performed. Bioinformatics analysis of the sequences was carried out to highlight the mRNAs that were being translated and, consequently, the proteins that were being synthesized. Secondly, protein synthesis was investigated by incubating spermatozoa with homopropargylglycine (HPG), a methionine analogue with an alkyne group, in the absence or presence of various inhibitors of ribosome activity. After extraction of the proteins, those having incorporated HPG were labelled with TAMRA-azide via “click chemistry”. The proteins were then analyzed by electrophoresis using a confocal scanner (Typhoon) for the detection of fluorescent signals. In addition, the identification of newly synthesized proteins was carried out by mass spectrometry after the enrichment of HPG-labelled proteins.