During the years of activity as a PhD student in the group of Prof. Sergio Minucci (Dept. of Experimental Medicine, Second University of Naples) I studied the expression and localization of two proteins, prothymosin alpha (Part I) and discevelled-associated activator of morphogenesis (Part III) in male gametogenesis. Moreover, I spent six months in US at the School of Medicine of Yale University (NH, Connecticut). There I focused on the possible role for Prolyl endopeptidase on spermatogenesis in mouse (Part II). Part I: PTMA expression and localization during the spermatogenesis of Danio rerio Prothymosin alpha (PTMA) is a highly acidic (Frangou-Lazaridis et al., 1988), intrinsically disordered protein (Gast et al., 1995), which was first extracted from rat thymus and characterized as an immunogenic factor (Haritos et al., 1984a) but soon detected in a variety of mammalian tissues (Haritos et al., 1984b; Clinton et al., 1989). The presence of a nuclear localization signal and the adoption of a peculiar random coil conformation are amongst the reasons behind its interaction with a number of molecular partners; hence, today PTMA is known to be a very conserved and widely-expressed molecule, involved in several and diverse biological processes, like H1 histone interaction and chromatin remodeling (Karetsou et al., 1998; Ueda et al., 2012), cell death (Enkermann et al., 2000a; Jiang et al., 2003; Malicet et al., 2006; Ueda, 2009), transcriptional regulation (Karetsou et al., 2002; Martini et al., 2000; Martini and Katzenellenbogen, 2003) cancer development (Dominguez et al., 1993; Skopeliti et al., 2006; Tsitsiloni et al., 1993; Wu et al., 1997; Zhang et al., 2014) and, as already alluded to, immunity (Baxevanis et al., 1992; Pan et al., 1986; Voutsas et al., 2000). Since 2002 our group has studied PTMA during the spermatogenesis of several vertebrate species (Aniello et al., 2002; Ferrara et al., 2009; Prisco et al., 2009; Ferrara et al., 2010; Ferrara et al., 2013): the expression of PTMA in meiotic and post-meiotic germ cells inside testicular tubules and its presence in spermatozoa (associated with the acrosome, where present) reveal a striking conservation of the pattern during phylogenesis and suggest a possible role for the protein in gametogenesis and in fertilization. Since many vertebrate models show an established association between PTMA and the acrosome (Ferrara et al., 2013), the study of the possible differences in an acrosome-less sperm model may prove very compelling. In the light of the above Danio rerio was chosen as a model for the continuation of the project (first part of thesis work), given its peculiar anastomosing tubular organization of the testis and, above all, the fact that the spermatozoa (SPZ) do not develop an acrosome system at all, being its fertilization a mechanical process (Hirai, 1988). Ptma transcript localization in the testis of Danio rerio approximately matches the pattern that we and others had previously highlighted in other species (Aniello et al., 2002, Ferrara et al., 2009; Prisco et al., 2009; Ferrara et al., 2010; Ferrara et al., 2013), with a clear predilection for meiotic and post-meiotic germ cells. The immunohistochemical data clearly show that the protein shares the same localization. Indeed, it is absent in spermatogonia, which suggests that it does not participate in the proliferation of staminal/mitotic phases, while its presence in primary spermatocytes (SPC) and in spermatids (SPT) supports its possible role during meiosis and/or during the subsequent stages of SPT differentiation into mature SPZ. Specifically, soon after its first appearance in the cytoplasm of leptotene/zygotene primary SPC, PTMA extends its localization to the membrane-free chromatin region of dividing cells and, then, it retains its nuclear distribution during SPT differentiation. The nuclear localization in the acrosome-lacking spermatozoa suggests a role for PTMA in chromatin remodeling during gamete differentiation. These data further provide a compelling starting point for the study of PTMA functions during vertebrate fertilization. Part II: PREP is associated with male reproductive function and male gametes in mice Prolyl endopeptidase (PREP) is a member of the serine peptidase group, which is widely conserved through evolution (Venäläinen et al., 2004). PREP is involved in the maturation and degradation of peptide hormones and neuropeptides (Mentlein, 1988; Wilk, 1983), but its activity is restricted to oligopeptides comprising no more than about 30 amino acid residues. Despite its common cytosolic localization, it is believed that PREP may act outside the cells by inactivating extracellular neuropeptides. In fact, it was suggested that PREP may be released from the cells (Ahmed et al., 2005), even though it lacks a secretion signal and it does not contain a transmembrane region, or a lipid anchor sequence (Venäläinen et al, 2004). PREP has been implicated in many biological processes, such as the maturation and degradation of peptide hormones and neuropeptides (Mentlein, 1988), learning and memory (Cunningham and O’Connor, 1997; D’Agostino et al., 2013), cell proliferation and differentiation (Matsubara et al., 1998; Suzuki et al., 2014) and glucose metabolism (Kim et al., 2014). A small number of reports have also suggested PREP participation in both male and female reproduction-associated processes (Kimura et al., 1998; Kimura et al., 2002). In order to improve the understanding of the possible role of PREP in male reproduction, in this second part of thesis work the effect of PREP knockdown (Prepgt/gt) on testis and sperm in adult mice was examined. After confirming PREP expression in wild type (wt) testis and spermatozoa (SPZ), as opposed to Prepgt/g samples, we performed a comparison of macroscopic parameters on wt and knockdown testes: the data show that Prepgt/gt gonads are smaller and weigh less than the wild type. Likewise, histological analysis shows that, while the general morphology appears to be preserved, tubule and lumen diameters are reduced in Prepgt/gt mice, and that the percentage of spermiated tubules is also lower than wt. Inside the gonad, the protein is localized in elongating spermatids and luminal SPZ of wt mice, as well as Sertoli cells, Leydig cells and peritubular cells. PREP is also expressed in epididymal SPZ, where it is detected in the head, as well as in the midpiece of the sperm flagellum, while the remaining tail region shows a weaker signal. Conversely, in knockdown testis and SPZ the signal is almost undetectable. These data suggest that the endopeptidase may be involved in mature sperm function; indeed, this is supported and enriched by final data on sperm parameters, which show that total count, normal morphology, and motility are altered and reduced in Prepgt/gt mice, compared to the wild type. These results suggest that PREP may play a fundamental role in mouse spermatogenesis, and sperm motility. Further experiments are required to understand whether the role played by this protein in spermatogenesis affects gonadal tissue development and gamete function directly. Part III: DAAM1 expression and localization during the spermatogenesis of rat and in human spermatozoa DAAM1 is a protein belonging to the formins, a large group of molecules which control the nucleation and assembly of actin fibers in response to several signals (Kovar, 2006; Goode and Eck, 2007). Several studies have shown that DAAM1 participates in essential biological processes, such as cytoskeletal organization, movement and adhesion during morphogenesis and organogenesis, as well as cell polarity (Zallen, 2007; Sato et al., 2006; Matusek et al., 2006; Aspenstrom et al., 2006; Lu et al., 2007; Yamashita et al., 2007). Cytoskeletal remodelling is a process that allows the cells to modulate their architecture and shape following intracellular and/or extracellular stimuli. As a formin, DAAM 1 has the ability to nucleate unbranched actin filaments and, thus, it acts as a regulator of the cytoskeleton in cell polarity and movement. Although its role during the development has been well studied, its potential activity in adult tissues is still unexplored. As of now, no reports have directly associated DAAM1 with reproductive processes, but it is known that factors which participate in the same molecular pathways are expressed during spermatogenesis: these include Dvl1, Dvl2 and 3 (Ma et al., 2006), as well as several GTPases and Rho-related proteins (Naud et al., 2003). In 2011, our group isolated a cDNA coding for DAAM1 from the testis of Pelophylax esculentus. The first studies, carried out on this model, showed that the protein localizes inside the spermatocysts. In this third part of thesis work, the study was focused on another model, Rattus norvegicus, since its testicular anatomy and the events which occur during spermatogenesis are emblematic of those of Mammals (Russell et al., 1989). First, the presence of DAAM1 was confirmed in rat testis at several time points during post-natal development (7-14-21-28-35-42-60 days post-partum, dpp), which suitably recapitulate the key events of the first spermatogenetic wave. In order to better characterize DAAM1 profile and to confirm its possible involvement during spermatogenesis, the localization of the protein during the post-natal development of the male gonad was studied by immunofluorescence analysis. A remarkable variation in DAAM1 distribution was detected through the times point studied: at 7 dpp, the protein localizes in the central region of the tubules; at 14 dpp, the signal is evident in A and B spermatogonia in the same central region, up until 21-28 dpp, when the signal is detectable inside the spermatocytes. During spermiohistogenesis (35 dpp and 42 dpp), DAAM1 signal is detectable in spermatids, with a peculiar localization in the forming acrosomal region. Finally, at 60 dpp, the signal is also present in mature spermatozoa (SPZ), in the cytoplasmic droplet. Given DAAM1 involvement in cytoskeletal remodeling, the localization profile of the formin was compared with the distribution of other cytoskeletal proteins: actin and tubulin. Actin signal is consistent with DAAM1 during the first phases of testis development, but it is also present in Sertoli cells which form the blood-testis barrier; later, at 35 dpp, the signal is evident in the now-complete barrier, as well as, during spermiohistogenesis, in the epithelial cells which rearrange their architecture to support the path of the evolving germ cells toward the lumen (42 dpp). Inside the adult testis, actin is expressed by all the cell types, including the SPZ. As for tubulin, it is also expressed in all stages, located inside the Sertoli cells which nurse the germ cells during their differentiation into SPZ. In order to deepen the knowledge on DAAM1 localization in male gametes, an immunofluorescence analysis was carried out on rat epididymal SPZ: there, the protein is mainly detectable inside the flagellum. Then, the analysis was extended to DAAM1 localization in human ejaculated SPZ, where the droplet is often physiologically retained (WHO 2010; Mortimer & Menkveld, 2001). Indeed, the signal in human gametes confirmed DAAM1 retention inside this cytoplasmic structure. Finally, a comparative analysis of the localization of cytoskeletal proteins was also performed on rat epididymal SPZ and in human ejaculated sperm: in both species actin is located in the head, in the acrosomal region, as well as in the flagellum, while tubulin is mainly distributed in the latter region. These results show, for the first time, the expression and the localization of DAAM1 during rat spermatogenesis and in rat and human SPZ, and they provide a comparative profile of its distribution versus the main germinal-compartment architectural factors, suggesting its possible involvement as an actor in morpho-functional remodeling and organization of the gonad and of male gametes