Abstract

Spermatogenesis is the biological process culminating in the production of spermatozoa. These latter undergo maturation steps that allow them to fertilize oocytes during gamete interaction. An anomaly happening during spermatogenesis, sperm maturation or gamete interaction can lead to fertility defects. Today, 15% of infertility cases remain unexplained. It is therefore essential to understand in detail the different mechanisms underlying spermatogenesis and gamete interaction.

The goal of my thesis was to identify new genes playing a role in the different stages of spermatogenesis and gamete interaction. I worked on the identification of genes responsible for the setup of the acrosome, an organelle essential for fertilization. In addition, I have studied gamete interaction by the analysis of a gene that could play a role in the fusion of gametes.

In the first part of my work, I analyzed mouse hypofertile IRCS (Interspecific Recombinant Congenic Strain) lines presenting a phenotype of partial globozoospermia characterized by an acrosome detachment. We could define a locus called Mafq1 for Male fertility Quantitative Trait Locus on chromosome 1, in which the expression of some genes in a Mus spretus version within a global Mus musculus domesticus genomic background was responsible for the observed phenotype. Within Mafq1, we first studied the Spermatogenesis Associated 3 (Spata3) gene by producing a mouse Knock-Out (KO) line. KO males have a normal fertility in vivo but are hypofertile in vitro. Their sperm have morphologic (a partial acrosome detachment) as well as metabolic (remaining cytoplasmic residues including lipid droplets) defects. The Spata3 KO line only partially reproduces the phenotype of the original IRCS. We then studied the Tex44 gene located in the Mafq1 region too. Tex44 KO males have a reduced fertility both in vivo and in vitro. Acrosomes are normal but flagellar defects have been observed including a disjunction between the midpiece and the principal piece and the absence of some microtubule doublets in the principal piece. These flagellar anomalies can be responsible for the abnormal sperm motility and hypofertility. Therefore, the analysis of an IRCS line showing partial globozoospermia allowed us to identify two new genes involved in spermatogenesis, one participating to the acrosome setup, the other to flagellar structure.

In parallel, I conducted a project dedicated to the characterization of ADAM20’s function, a protein suspected to play a role in adhesion and/or fusion of gametes. In in vitro fertilization assays in mice and humans, in the presence of antibodies directed against ADAM20, we observed a significant reduction of the fertilization index, suggesting a role of ADAM20 in gamete interaction. We generated an Adam20 KO line in which KO males have a normal in vivo fertility but a reduced in vitro fertility. We then generated a mouse line deleted for a genomic fragment encompassing the genes Adam25, 20 and 39 because these 3 neighbor genes are very homologous. The triple KO males have the same phenotype as Adam20 KO males. ADAM20 in humans and ADAM25, 20 and 39 in mice seem to play a role in gamete interaction but are not essential for this step.

Globally, my thesis work concerned anomalies of fertility whose origins could be searched during spermatogenesis and gamete interaction stages. Though the knowledge on the fertilization process is regularly improving, some uncertainties still remain and it is crucial to pursue researches to completely decipher the mechanisms involved in these fundamental processes.

Keywords: Fertility, Gamete Interaction, Knock-Out (KO), Globozoospermia, Acrosome, Flagellum, Mafq1, Spata3, Tex44, Adam20, Adam25, Adam39