Identification des tyrosine kinases de la famille *Src presentes dans les spermatozoides de taureau

In order to fertilize the mammalian oocyte, ejaculated spermatozoa must undergo a complex series of membrane and intracellular events collectively called capacitation. Tyrosine phosphorylation of specific sperm proteins is required for successful capacitation and results from a change in the balance between cellular tyrosine kinase and tyrosine phosphatase activities. In spermatozoa, the key players implicated in tyrosine phosphorylation during capacitation remain unidentified. The aim of the present project was to identify the protein tyrosine kinases present in ejaculated bovine spermatozoa that could be implicated in capacitation. To do so, three approaches were taken. The first two approaches were aimed specifically at identifying tyrosine kinases of the Src family, whereas the last approach was undertaken to identify tyrosine kinases on the basis of enzymatic activity. Using the first approach, we identified an 80 kDa protein recognized by a monoclonal antibody directed against the human Src tyrosine kinase. Partial sequencing revealed that the 80 kDa protein was not related to Src but was homologous to the mammalian sperm protein PH-20, previously implicated in sperm-egg interactions. The second approach used the techniques of molecular biology. Spermatozoa are devoid of transcriptional and translational activities. The aim was then to identify the messenger RNAs (mRNAs) encoding Src family tyrosine kinases present in transcriptionally and translationally active spermatozoa precursor cells at the haploid (spermatids) and tetraploid (pachytene spermatocytes) stages. We identified mRNAs encoding four members of the src family: Yes, Lyn, Lck and Hck. Analysis of a sequence derived from haploid spermatids suggested the presence of a messenger encoding a truncated form of the tyrosine kinase Hck. The last approach used the enzymatic activity of tyrosine kinases and a proteomic identification. We detected and enriched a tyrosine kinase activity in the cytosolic fraction of nitrogen cavitated ejaculated spermatozoa. A proteomic approach identified tyrosine kinases from three families in this tyrosine kinase enriched fraction: Src (Lyn), Csk, Tec (Bmx, Btk). We also identified many other sperm proteins that could represent potential signaling partners for these tyrosine kinases. The results presented in this thesis will help in a better understanding of the signaling pathways implicated in sperm capacitation and possibly other sperm functions.