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Study of the role of the deglutamylating enzyme CCP5 in microtubules function regulation during mouse spermatogenesis

Abstract : Spermatogenesis is the process by which germ cells are transformed into spermatozoa by three sequential phases: the mitotic- and meiotic- phase followed by spermiogenesis. To allow the final maturation of haploid germ cells into spermatozoa specific structures have to be developed during the spermiogenesis: the acrosome, the manchette and the flagellum. The manchette is a MTs-based structure, located caudally to the acrosome, organizing in a skirt-like fashion. Manchette is known to participate in the shaping of the nucleus conferring it the typical hook-like shape and several studies have underlined its importance in acrosome and flagellum formation. During spermiogenesis all molecules and organelles necessary for both acrosome and flagellum formation have to be transported to their destination sites and manchettal MTs allow the movement of organelles and other proteins between the pro-acrosome region and the spermatid tail. However this MTs-based traffic has to be regulated both in space and time as it has been shown that ectopic or mislocalization of certain proteins can lead to failures in acrosome, manchette and flagellum development. The generation of posttranslationally modified MTs might explain a possible mechanism of traffic regulation since it has been demonstrated that posttranslational modifications (PTMs) can regulate the interaction between MTs and molecular motors and microtubules binding proteins. Polyglutamylation, consist in the addition of glutamate side chains of variable length on α- and β- tubulin carboxy-terminal tails. Glutamylation levels are determined by the combined action of glutamylase (TTLLs) and deglutamylase (CCPs) enzymes. Several reports have recently highlighted the importance of some of these enzymes in flagellum assembly and/or maintenance. During my PhD I investigated about the functional role of CCP5 during mouse spermatogenesis. CCP5 is the only enzyme able to remove the glutamate branching point of the added side chain. Thus, its activity might regulate the equilibrium between presence/absence of glutamate branching points, in turn interfering with polyglutamylation levels. The study of the CCP5-KO mouse reveals that CCP5 has an essential role during mouse spermiogenesis. CCP5-KO male produces 100-fold less sperm cells than controls and released sperm cells are highly defective and immotile. Moreover, haploid immature germ cells are also found in CCP5-KO semen. A deep-analysis reveals that the reduced sperm output is due to several ultrastructural defects emerging during the spermatids differentiation process. The acrosome, although is still formed, it does not appear to develop symmetrically and appears to detach from the nucleus in condensed spermatids. Another structure that is impaired in CCP5-KO spermatids in the manchette. Manchettal MTs, are seen to emanate from ectopic regions of the germ cells without running parallel to the nucleus, and are often observed within the spermatids nuclei. Altogether these defects correlate with an aberrant-shaped spermatid nucleus not showing the typical hook-like shape. Another phenotype observed in CCP5-KO elongating spermatids is the presence of supernumerary basal bodies that correlates with the presence of singlet or doublets microtubules dispersed within the germ cell cytoplasm. Interestingly sperm accessory structures are seen to chaotically organize around the microtubules. Unstable disassembling axonemes are seen together with those MTs, suggesting that CCP5-KO spermatids develop abortive unstable flagella. Interesting all these ultrastructural defects correlate with increased level of glutamylation on round spermatids’ cortical MTs and elongating spermatids’ manchettal MTs. Taken together, this study strongly suggests that CCP5-mediated glutamylation regulation is fundamental for spermatids differentiation into healthy functional spermatozoa.
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Tiziana Giordano. Study of the role of the deglutamylating enzyme CCP5 in microtubules function regulation during mouse spermatogenesis. Cellular Biology. Université Paris Saclay (COmUE), 2016. English. ⟨NNT : 2016SACLS574⟩. ⟨tel-01796055⟩

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