Expression of p97/VCP (Valosin containing protein) and Jab1/CSN5 in rat testis and epididymis during the postnatal development

Sevil Caylia, Gulsen Arslan Ataya, Fikret Erdemirb, Tamer Yenerc, Hakan Kesicia,
a

Department of Histology and Embryology, Faculty of Medicine, Gaziosmanpasa University,

10Tokat, Turkey
b

Department of Urology, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey Experimental Animal Center, Faculty of Medicine, Gaziosmanpasa University, Tokat,

Turkey

15*Corresponding author: Sevil Cayli, Assist Prof. Dr, Department of Histology and Embryology, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey Phone: +903562129500/1231 ; Fax: +903562133179 E-mail: sevilcayli@yahoo.com (S.Cayli) 20Running title: p97/VCP and Jab1/CSN5 expressions during the testicular and epididymal development

Abstract
The ubiquitin proteasome system (UPS) is a key player in regulating the many cellular 25processes via proteasomal degradation of ubiquitinlated proteins. Recently published data show that the Jab1/CSN5 interacts with p97/VCP and controls the ubiquitinlation status of proteins bound to p97/VCP in mouse and human cells. However, the expression of p97/VCP and Jab1/CSN5 in the developing rat testis and epididymis remains unclear. Here, we show the cellular localization and expressions of p97/VCP and Jab1/CSN5 in rats along postnatal 30testis and epididymis development. Testicular and epididymal tissues from 5-, 15-, 30-, and 60-day-old rats were examined by immunohistochemistry and Western blotting techniques. In 5 days old rat testis, p97/VCP and Jab1/CSN5 are specifically expressed in gonocytes. p97/VCP and Jab1/CSN5 expression significantly increased at day 15 after birth and found in spermatogonia, Sertoli cells and spermatocytes. p97/VCP expression was overlapped with 35Jab1/CSN5 expression in gonocytes, spermatogonia, spermatocytes, spermatids and Sertoli cells in 5, 15 and 30 days of rat testis. In 60 days old rat testis, p97/VCP was strongly expressed in Sertoli cells however, moderate to weak expression was observed in spermatogonia, spermatocytes, round and elongating spermatids. Jab1/CSN5 showed strong expression in spermatogonia and spermatocytes while relatively weak expression was 40observed in round and elongating spermatids in 30 and 60 days old testis. On the other hand, in epididymis, expression of both proteins was gradually increased from 5 to 60 day of age. Both the basal and the principal cells of epididymis showed p97/VCP and Jab1/CSN5 immunoreactivity. After 2 weeks of age, expression of both proteins was mostly restricted to the caput epididymis. Consequently, our study suggests that p97/VCP and Jab1/CSN5 could 45be an important part of the UPS in the developing rat testis and epididymis and both proteins may be involved in the regulation of spermatogenesis and epididiymal epithelial functions.

50Key words: p97/VCP, Jab1/CSN5, rat, testis, epididymis

1. Introduction
55 The testis has the specific function of generating spermatozoa from precursors called spermatogonia after a complex series of divisions (Clermont and Huckins, 1961; de Kretser, 1994). This process takes place within the seminiferous epithelium, which is a complex structure composed of germ cells with radially oriented supporting cells termed Sertoli cells. On the other hand, the epididymis provides an an adequate environment for the final 60maturation of sperm (Orgebin-Crist, 1969; Hinton et al., 1995; Cooper, 1998). During embryonic and postnatal development of testis and epididymis, regulated proteolysis and organelle degradation are required (Sutovsky et al., 2001; Glickman and Ciechanover, 2002; Sutovsky, 2003). The ubiquitin proteasome system (UPS) is developmentally regulated and highly substrate spesific pathway for the removal of the damaged and aberrant proteins. It is 65well known that the UPS fulfills necessary requirements for sperm cell differentiation inside the testicular seminiferous tubules and cell cycle control throughout spermatogenesis and fertilization in adult males. Moreover, ubiquitin ligases E1, E2, E3,UBC4 and some proteasomal subunits were expressed during spermatogenesis and postnatal testis development (Roest et al., 1996; Wing, 2003; Bedard et al., 2005; Liu et al., 2005). 70 In the male reproductive system, the UPS contributes to gamete quality control mechanism, providing the selective spermatogonial removal at the haploid phase of spermatogenesis (Kwon et al., 2005), protein and organelle degradation during spermiogenesis (Baarends et al., 1999; Sutovsky, 2003), and the tagging of a ubiqutin to defective spermatozoa in the epididymis (Sutovsky et al., 2001; Baska et al., 2008). For these 75reasons, exploring expression of proteins that are the components of the UPS may lead to advances in the biology of the testis and epididymis. In the ATP-dependent ubiquitin pathway, ubiquitin attached to a target protein, referred to as ubiquitination, is carried out by the ubiquitin-activating enzymes (E1), the ubiquitin-conjugating enzymes (E2) and ubiquitin ligase (E3) (Hochstrasser, 1995). The main 80purpose of ubiquitination is to deliver the ubiquitinated proteins to a cellular trash bin, a lysosome, an autophagosomal vacuole, or a 26 S proteasome. Ubiquitinated proteins can either be transferred directly to the proteasome or indirectly via p97/Valosin-containing protein (VCP), a member of the AAA-ATPase (ATPases associated with diverse cellular activities) super-family. p97/VCP has been associated with a wide variety of essential cellular 85protein pathways comprising nuclear envelope reconstruction, cell cycle regulation, Golgi

3 reassembly, suppression of apoptosis, DNA-damage response, maturation of

aoutophagosomes and sperm capacitation ( Kondo et al., 1997; Meyer et al., 2000; Dai and Li, 2001; Hetzer et al., 2001; Hirabayashi et al., 2001; Rabinovich et al., 2002; Ficarro et al., 2003; Krick et al., 2010 ; Tresse et al., 2010). In addition, during endoplasmic reticulum90associated degradation (ERAD), p97/VCP dislodges ubiquitinated proteins from the endoplasmic reticulum (ER) and chaperones them to the cytosol for proteasomal degradation (Meusser et al., 2005). For ubiquitination of misfolded proteins in the ER the interaction with p97/VCP is required (Klein et al., 2005). Moreover, we have recently shown that the COP9 signalosome interacts ATP-dependently with p97/VCP and controls the ubiquitinlation status 95of proteins bound to p97/VCP (Cayli et al., 2009). The COP9 signalosome (CSN) involved in the ubiquitin/proteasome system contains eight core subunits (CSN1-8) like the proteasome lid complex. CSN5 (also known as Jab1) facilitates the 26S proteasome-dependent degradation of several proteins, including p27Kip, luteinizing hormone receptor (LHR), p53, estrogen receptor, Smad4, Smad7, Id1, Id3, and 100IB (Li et al., 2000; Wan et al., 2002; Berse et al., 2004; Kim et al., 2004; Yun et al., 2004; Callige et al., 2005). JAMM (JAB1/MPN/Mov34 metalloenzyme) domain present within Jab1/CSN5 has deubiquitinase activity, which regulates ubiquitinlated protein sorting when associated with CSN (Cope et al., 2002). In addition to its role in UPS, Jab1/CSN5 regulates many signaling pathway such as TGF- signaling, cell proliferation, apoptosis and DNA 105repair (Shackleford and Claret, 2010). Although it seems clear that the UPS is fundamentally required for male reproductive system, to date there exists no information on the expression patterns of p97/VCP and Jab1/CSN5 and their possible roles in developing rat testis and epididymis. Therefore, the goal of the current study was to assess the developmental expression of p97/VCP and 110Jab1/CSN5 and to show a cellular localization of both complexes in rat testis and epididymis. Thus, we analyzed the expression of p97/VCP and Jab1/CSN5 in rat testis and epididymis during postnatal development using immunohistochemistry and Western blotting.

2. Materials and Methods

Animals and Tissue Preparation 115After obtaining approval from the local ethics committee (2010-HADYEK-012) twentyfour male wistar albino rats at postnatal ages of 5, 15, 30 and 60 days (six rats per group) were obtained from Gaziosmanpasa University Experimental Animal Research Laboratory. All rats were observed for several days to ascertain the health before sample collection. Pups were reared with their dams. They were kept in a temperature controlled room (2023 C), on a 12 120h light/dark cycle with food (commercial rat chow) and fresh water available adlibitum. Six rats at the same age were killed by administration of an overdose of sodium pentobarbital (150 mg/kg, i.p.) before removal of the testis and epididymis. Epididymis were dissected and subdivided into three anatomical regions as follow: caput (CT), corpus (CS) and cauda (CA) epididymis. One testis and epididymis from each 125animal were fixed in Bouins fluid for 12 hours immediately upon collection, dehydrated, and embedded in paraffin for histochemistry and immunohistochemistry (see below). The contralateral testis and epididymal regions from each animal were frozen for protein analysis. Immunohistochemical analysis 130For immunohistochemical analysis, serial sections, 5 m thick, were collected on poly-Llysine coated slides (Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at 56C. Tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Sections were then treated in a microwave oven in 10 mM citrate buffer, pH 6.0, for 5 min twice and left to cool for 20 min. After three washes in phosphate buffered saline (PBS), 135endogenous peroxidase activity was quenched by 3% hydrogen peroxide in PBS for 20 min and again washed three times in PBS. Sections were then incubated in a blocking serum (Ultra V Block, TP-060-HL; NeoMarker, Fremont, CA, USA) for 10 min in order to block non-specific binding. Subsequently, sections were incubated 1 hour at RT with mouse monoclonal p97/VCP (1: 500, Affinity BioReagent, Germany) and Jab1/CSN5 (1: 200, Santa 140Cruz Tech, USA) in a humidified chamber. Sections were washed three times in PBS and incubated with biotinylated anti-mouse (BA-9200; 1:400 Dilution; Vector Laboratories, Burlingame, CA) and biotinylated antirabbit (BA-1000; 1:400 Dilution; Vector Laboratories) secondary antibodies for 45 min. at room temperature. After three washes with PBS, the antigenantibody complexes were detected by using a streptavidinperoxidase complex (TP145060-HL; LabVision, Fremont, CA, USA), for 15 min. followed by three rinses with PBS.

5 Bound peroxidase was developed with 3-amino-9-ethylcarbazol (AEC) (ScyTek Laboratories, USA) chromogen and sections were counterstained with Mayers hematoxylin (ScyTek Laboratories, Utah, USA) and mounted with Permount (Fisher Chemicals, Springfield, NJ, USA) on glass slides. For controls, sections were treated with the appropriate isotype mouse 150IgG or normal rabbit serum depending on the primary antibody used that was diluted to the same final protein concentration as the primary antibody. Photomicrographs were taken with a Leica microscope (Leica DM2500, Nussloch, Germany). Double immunostaining 155 For double labeling, the horseradish peroxidase (HRP) and alkaline phosphatase (AP) polymerization kit (MACH 2 Double stain, Biocare, CA, USA) was used. An antibody cocktail with rabbit polyclonal Jab1/CSN5 (1: 200, Santa Cruz Biotechnology, USA) and mouse monoclonal p97/VCP (1: 500, Affinity BioReagent, Germany) were mixed and sections were incubated overnight with this antibody mixture at 40C. After washing with TBS 160at room temperature, sequential incubations were performed with mouse-AP and rabbit-HRP polymer detection kit (MRCT523, MACH 2 Double stain, Biocare, USA) for 30 min at room temperature. After rinsed in TBS, sections were subsequently incubated with diaminobenzidine (DAB) for 3 min. and Vulcan Fast Red chromogen for 5 min. Thereafter, all sections were counterstained with Mayers hematoxylin (ScyTek Laboratories, Utah, 165USA) and mounted with Permount (Fisher Chemicals, Springfield, NJ, USA) on glass slides. The evaluation of the immunohistochemistry The evaluation of the immunohistochemical labelling was performed using H-SCORE analyses (Kayisli et al., 2006). The intensities of p97/VCP and Jab1/CSN5 immunoreactivities 170were semi-quantitatively evaluated using the following intensity categories: 0 (no staining), 1+ (weak but detectable staining), 2+ (moderate or distinct staining), and 3+ (intense staining). For each tissue, an H-SCORE value was derived by calculating the sum of the percentages of cells that stained at each intensity category and multiplying that value by the weighted intensity of the staining, using the Formula H-SCORE: Pi(i+ l), where i represents 175the intensity scores and Pi is the corresponding percentage of the cells. In each slide, five randomly selected areas were evaluated under a light microscope (40x objective), and the percentage of the cells for each intensity within these areas was determined at different times by two investigators who were not informed about type and source of the tissues. The average score of both observers was used.

6 180 SDS-PAGE and Western blot analyses Total testicular and epididymal proteins were extracted modified RIPA buffer (1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 mg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM Na3VO4; and 1 mM NaF in 50 mM Tris-Cl, pH 1857.4) and quantitated using the Bradford procedure (Bio-Rad, Hercules, CA). 40 g samples were separated by 8 % SDS-PAGE and electroblotted onto nitrocellulose membrane (Bio-Rad Laboratories). The membrane was blocked with 5% non-fat dry milk in TBS containing 0.1% Tween 20 (TBS-T) for 1 h to reduce non-specific binding. Subsequently, the membrane was incubated for 1 h with primary antibody against p97/VCP (MA3-004, Affinity BioReagent, 190Germany, 1: 10,000 in 5% non-fat dry milk), Jab1/CSN5 (sc-90741, Santa Cruz Biotechnology, 1: 500 in 5% non-fat dry milk) and -actin (sc-47778, C4; Santa Cruz Biotechnology Inc., 0.05 mg/ml in 5% non-fat dry milk). The membrane was washed with TBS-T for 1 h and incubated with horseradish peroxidase conjugated anti-mouse and antirabbit secondary antibodies (Vector Laboratories) diluted in 5% non-fat dry milk in TBS-T. 195Bound secondary antibodies were visualized by enhanced chemiluminescence substrate (GE Healthcare). Immunoblot bands for p97/VCP, JAb1/CSN5 and -actin were quantified using an Alpha DigiDoc 1000 gel documentation unit (Alpha Innotech Corporation, CA, USA). The optical density (OD) values for p97/VCP and JAb1/CSN5 bands were divided by the OD values of cognate -actin bands in order to normalize OD values for loading differences. 200 Statistical analysis The Mann-Whitney U-test was employed for comparison of independent groups of samples, and the Kruskall-Wallis analysis with the Dunn posthoc test was performed for multiple 205comparison of independent groups of samples. P value of less than 0.05 was considered to indicate a statistically significant difference. Statistical calculations were performed using SigmaStat for Windows, version 3.5 (Jandel Scientific Corp., San Rafael, CA).

7 2103. Results 3.1. Cellular localization of p97/VCP in the developing rat testis and epididymis Immunohistochemical analysis of 5 day-old-rat testis revealed that p97/VCP was mainly localized to gonocytes in the lumen of the seminiferous tubules (Figure 1A-B). No immunoreaction was detected in somatic cells. In the 15-day-old rat testis, spermatogonia at 215the basal membrane, Sertoli cells and spermatocytes were strongly positive for p97/VCP (Figure 1C-D, Table 1). Additionally, peritubuler myoid cells and fetal Leydig cells showed weak to moderate immunostaining for p97/VCP at day 15 (Figure 1D). The staining intensity and the number of positively stained cells for p97/VCP significantly increased by day 15, coinciding with the apperance of leptotene spermatocytes (Malkov et al., 1998) (Figure 1C, 220D and I). The other cell types present in the 30-day-old rat testis, namely round and elongating spermatids also expressed p97/VCP (Figure 1E-F). Besides, supporting Sertoli and peritubuler myoid cells, Leydig cells showed relatively a moderate expression of p97/VCP (Figure 1F). In adult rat (60 day) testis, p97/VCP was evident in the Sertoli cells, reaching the highest expression level, however; spermatogonia, spermatocytes, round and elongating 225spermatids showed weak to moderate expression of p97/VCP (Figure 1G-H, Table 1). The staining intensity and the number of the p97/VCP posivity were found the same in all stages of seminiferous tubules in adult rat testis. There was no immunoreaction in negative control slides that were treated with normal mouse serum instead of p97/VCP primary antibody at the same final concentration (Figure 2301C, G, insets). H-SCORE analysis of p97/VCP expression revealed significant increase from day 5 to day 60 (p<0.05), while no significant difference was observed between day 30 and day 60 (Figure 1I). In the 5-day-old rat epididymis, p97/VCP immunostaining was found in corpus, cauda and caput epididymis. Epithelial cells of epididymis were positively labeled for p97/VCP 235(Figure 3A). p97/VCP immunoreactivity showed no uniformity in different region of epididymis and highly expressed in corpus at day 5. However, p97/VCP expression was higher in caput epididymis compared to corpus and caudal epididymis from day 15 to day 60 (Figure 5B). In addition to epithelial cells of epidiymis, spermatids were found to be strongly immunpositive for p97/VCP in the lumen of 30-day-old rats (Figure 3E, Table 1). p97/VCP 240immunostaining was seen both in the basal and principal cells of epididymis in adult epididymis (Figure 3G, Table 1). Moreover, in the lumen of epididymis spermatozoa were

8 weakly stained with p97/VCP. H-SCORE analysis showed that p97/VCP expression was gradually increased from 5 to 60 day of age epididymis (Figure 3I).

10 3.2. Cellular localization of Jab1/CSN5 in the developing rat testis and epididymis 245

In the 5-day-old rat testis, Jab1/CSN5 showed moderate immunolabeling in gonocytes (Figure 2A-B). The staining intensity and the number of the Jab1/CSN5 positivity were highly increased in the 15-day-old testis compared to the 5-day-old rat testis (Figure 2I). In the 15day-old testis, Jab1/CSN5 localized strongly in spermatogonia and spermatocytes but Sertoli cells indicated a moderate immunostaining (Figure 2C, D, and Table 1). In the 30-day-old rat

250testis, spermatogonia, spermatocytes, round and elongating spermatids, Sertoli and Leydig cells were positivetly labeled with Jab1/CSN5 (Figure 2 E, F). In the 60-day-old rat testis, Jab1/CSN5 showed stronger nuclear staining in spermatogonia and moderate cytoplasmic staining in spermatocytes at stages 8-12 (Figure 2G, H). However, acrosomes of round spermatids at stages 6-8 and the head of elongating 255spermatids at stages 1-5 and 9-14 displayed relatively weak immunostaining for Jab1/CSN5 (Figure 2G, H). A few interstitial cells and Sertoli cells indicated weak labeling for Jab1/CSN5. No immunoreaction was detected in control slides (Figure 2A, C, insets). According to H-SCORE analysis, Jab1/CSN5 expression significantly increased from day 5 to day 60 testis, whereas no significant differences were found between day 30 and day 60 testis 260(Figure 2I). In day 5 epididymis, Jab1/CSN5 immunopositivity was detected in the caput, corpus and caudal region of epididymis (Figure 3B). Although some epithelial cells showed no immunreactivity, Jab1/CSN5 is strongly expressed both in the principal and basal cells in day 15, 30 and 60 epididymis (Figure 3 D, F, H). In day 5 and 15, Jab1/CSN5 expression was 265relatively higher in corpus epididymis however, caput epididymis indicated increased expression in day 30 and 60 testis (Figure 5B). Additionally, spermatids in the lumen of epididiymis were strongly labelled with Jab1/CSN5 in day 30 (Figure 3F), however spermatozoa showed weak immunostaining in the lumen of day 60 epididymis (Figure 3H). Overall, the expression of Jab1/CSN5 was gradually increased from 5 to 60 days of 270epididymis (Figure 3I). 3.3. Co-localization of Jab1/CSN5 and p97/VCP in the developing rat testis and epididymis In order to determine Jab1/CSN5 and p97/VCP co-localizations, double 275immunostaining was performed in the testicular and epididymal tissues (Figure 4). In the 5day-old rat testis, Jab1/CSN5 and p97/VCP were found to be co-localized in gonocytes. In the 15-day-old rat testis, p97/VCP immunostaining was overlapped with Jab1/CSN5 in

10 spermatogonia, spermatocytes and Sertoli cells (Figure 4B and C). Although p97/VCP and Jab1/CSN5 staining intensities indicated differences in day 30, elongating and round 280spermatids, spermatocytes and Sertoli cells were found to be immunopositive for Jab1/CSN5 and p97/VCP. Additionally, spermatids in the lumen of epididymis and some epithelial cells were double labeled (Figure 4D). In the 60-day-old rat testis, spermatogonia, spermatocytes and spermatids but not Sertoli and Leydig cells were found to be double immunopositive for Jab1/CSN5 and p97/VCP (Figure 4E, F). 285 3.4. Changes in protein expression levels of p97/VCP and Jab1/CSN5 in the developing rat testis and epididymis In aggrement with immunohistochemistry, Western blot results confirmed the presence of p97/VCP and Jab1/CSN5 in the developing rat testis and epididymis. Western 290blot analyses revealed a specific band at 97 kDa for p97/VCP and at 38 kDa for Jab1/CSN5. The intensity of both bands was quantified and normalized for the intensity of -actin controls (Figure 5A, B lower panels). The expression level of p97/VCP was low on day 5 of testis and epididymis but incrased and reached the peak on day 60. However no significant changes were observed between 30 and 60 days for p97/VCP expression (Figure 5A). Jab1/CSN5 295expression was also increased gradually but reached the peak on day 30 in testis and on day 60 in the epididymis (Figure 5A, B).

11

4. Discussion
To our knowledge, this is the first study demonstrating the expression of UPS 300components, p97/VCP and Jab1/CSN5, in the postnatal development of rat testis and epididymis by using immunohistochemistry and Western blotting. Our results indicate that the expression of both proteins was gradually increased from 5 to 60 day of age in testis and epididymis. Additionally, in the 5-, 15-, 30-and 60 day-old rat testis, p97/VCP expression was overlapped with Jab1/CSN5 expression in gonocytes, spermatogonia, spermatocytes, 305spermatids, and Sertoli cells. To define specific sites where p97/VCP and Jab1/CSN5 expressions might be important, we explored cell-specific expression of p97/VCP and Jab1/CSN5 in developing rat testis and epididymis. In the 5-day-old rat testis, it was detected that p97/VCP and Jab1/CSN5 were specifically expressed in gonocytes and the expression levels of these proteins were 310lower compared to other ages. The low levels of p97/VCP and Jab1/CSN5 expressions in neonatal testis suggest that both proteins dont have a major role in the very early postnatal stages. However, from day 5 after birth to day 30, the levels of the p97/VCP and Jab1/CSN5 expression increased significantly. Previously, authors have been shown that the period of 4 days after birth to 6 weeks age corresponds to period of rapid cell proliferation and the growth 315of the rat testis and epididymis (Clermont and Perey, 1957). Therefore, high level expression of p97/VCP and Jab1/CSN5 coincides with the testicular growth, suggesting that p97/VCP and Jab1/CSN5 proteins could play important roles in cell proliferation of testis and epididymis. In fact, it is well known that Jab1/CSN5 plays an essential role in cell growth and strong expression of Jab1/CSN5 is connected with accelerated proliferation (Bounpheng et 320al., 2000). In mice, Jab1/CSN5 gene-knockout results in embryonic lethality largely due to impaired proliferation of embryonic cells (Tomoda et al., 2004). In the 5-day-old rat testis, p97/VCP was only expressed in gonocytes localized to tubule lumen. By day 15, spermatocytes within the tubule lumen was positive and spermatogonia were also immunostained for p97/VCP. In adult rat, p97/VCP 325immunoreactivity appeared strongly in Sertoli cells but very weakly in maturing spermatogonial cells. The shift in the expression of gonocytes and spermatocytes in juvenile animals to Sertoli cells in mature males suggest that the role of p97/VCP may change as the testis mature to produce supporting cells. However, cellular localization of Jab1/CSN5 in juvenile animals and mature animals were similar. For example; by day 15, spermatocytes

12 330and spermatogonia were immunopositive for Jab1/CSN5. In adult rat, Jab1/CSN5 expression was mostly observed in spermatogonia and spermatocytes. The ultrastructural study of the Sertoli cell revealed that numerous phagosomes were observed in the cytoplasm of the Sertoli cells which indicate phagocytic activity. Fawcett (1975) indicated that the Sertoli cells are greatly involved in the digestion of germ cells which 335degenerate during spermatogenesis and lobules of residual spermatid cytoplasm left during spermiation. Some researchers also reported that Sertoli cells might be involved in the exchange and elimination of excessive sperm cellular substances (Russell et al., 1983; Sakai et al., 1988; Park et al., 1993). Recently, p97/VCP was found to be essential to autophagosome maturation suggesting that p97/VCP might be selectively required for 340autophagic degradation of ubiquitinated substrates (Krick et al., 2010; Tresse et al. 2010). Based on the localizated expression of p97/VCP in the immature and mature Sertoli cells, our results support the previous observations and we suggest p97/VCP may be necessary for the processing of ubiqutinylated and misfolded proteins from larger protein complexes or membranes in Sertoli cells. Further functional studies will be required to clarify the 345physiological role of p97/VCP in Sertoli cells. In the present study, it was reported that expression of p97/VCP and Jab1/CSN5 was gradually increased during the postnatal development of epididymis. Moreover, both proteins were noticed in the epididymal epithelium and produced by all region of epididymis, but is highly expressed in the caput and corpus epididymis. These findings also supports the idea 350that p97/VCP and Jab1/CSN5 are most probably contributes to sperm maturation process in the epididymis. On the other hand, there are number of evidence that removal and degradation of defected spermatozoa occurs during epididymal passage (Sutovsky, 2003; Tengowski et al., 2007; Baska et al., 2008). However, it is not clear how the defective spermatozoa removed or 355what are the components of UPS contributed the proteasomal proteolysis in the epididymis. One possibility arised from current study is that Jab1/CSN5 together with p97/VCP may be important mediators during the proteasomal degradation of defected spermatozoa. It is well known that JAMM domain of Jab1/CSN5 has deubiquitinase activity when associated with the CSN (Cope et al., 2002). This activity of Jab1/CSN5 might be critical for protein 360degradation in the epididymis. In fact, it was previously shown when the CSN was inactivated by knockdown of Jab1/CSN5, the amount of polyubiquitinated proteins bound to p97/VCP increased indicating that the CSN is required for proper processing of substrate proteins bound to p97/VCP (Cayli et al., 2009). Obviously, p97/VCP and Jab1/CSN5 may also involve

13 in the proper processing of polyubiquitinated substrates in the epididymis. Moreover, co365localization of p97/VCP and Jab1/CSN5 in 5-, 15-, 30 day-old rat testis and epididymis also supports the previous findings that Jab1/CSN5 may regulate the ubiquitination status of proteins which are bound to p97/VCP. Collectively, p97/VCP and Jab1/CSN5 show a notable expression in the male reproductive tract and are present at virtually every phase of germ cell development and 370maturation. Therefore, it is hard to believe they dont have an important role in the developing rat testis and epididymis. For example: as shown by the staining (Figure 1, 2 and 4), p97/VCP and Jab1/CSN5 were localized in round and elongating spermatids, where the proteins included into the developing acrosome and the sperm tail. Additionally, both proteins were expressed in maturing spermatocytes and spermatogonia, suggesting that these proteins may 375also critical for normal spermatocyte development. In conclusion, we report here the presence and the developmental expression of the UPS components, p97/VCP and Jab1/CSN5 in the rat testis and epididymis. Further research might contribute to the clarification of the exact functions of these proteins in the developmental process of rat testis and epididymis. 380

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16 in mice causes male sterility associated with chromatin modification. Cell. 1996; 86:799-810. 485Russell LD, Tallon-Doran M, Weber JE, Wong V, Peterson RN. Three-dimensional reconstruction of a rat stage V Sertoli cell: III. A study of specific cellular relationships. Am J Anat. 1983; 167:181-192. Sakai Y, Nakamoto T, Yamashina S. Dynamic changes in Sertoli cell processes invading spermatid cytoplasm during mouse spermiogenesis. Anat Rec. 1988;220:51-57. 490Shackleford TJ, Claret FX. JAB1/CSN5: a new player in cell cycle control and cancer. Cell Div. 2010; 5:26-40. Sutovsky P. Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Microsc Res Tech. 2003; 61:88-102. 495Sutovsky P, Moreno R, Ramalho-Santos J, Dominko T, Thompson WE, Schatten G. A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis. J Cell Sci. 2001;114:1665-1675. Tengowski MW, Feng D, Sutovsky M, Sutovsky P. Differential expression of genes encoding constitutive and inducible 20S proteasomal core subunits in the testis and epididymis 500 of theophylline- or 1,3-dinitrobenzene-exposed rats. Biol Reprod. 2007;76:149-163. Tomoda K, Yoneda-Kato N, Fukumoto A, Yamanaka S, Kato JY. Multiple functions of Jab1 are required for early embryonic development and growth potential in mice. J Biol Chem. 2004;279:43013-43018. Tresse E, Salomons FA, Vesa J, Bott LC, Kimonis V, Yao TP, Dantuma NP, Taylor JP. 505 VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD. Autophagy. 2010;6:217-227. Wan M, Cao X, Wu Y, Bai S, Wu L, Shi X, Wang N. Jab1 antagonizes TGF-beta signaling by inducing Smad4 degradation. EMBO Rep. 2002;3:171-176. Wing SS. Deubiquitinating enzymes--the importance of driving in reverse along the ubiquitin510 proteasome pathway. Int J Biochem Cell Biol. 2003;35:590-605. Yun J, Tomida A, Andoh T, Tsuruo T. Interaction between glucose-regulated destruction domain of DNA topoisomerase IIalpha and MPN domain of Jab1/CSN5. J Biol Chem. 2004;279:31296-31303. 515

17 Figure legends: Figure 1: Localization of p97/VCP in the developing rat testis. Immunohistochemistry was used to show the cellular localization of p97/VCP in the testis at day 5 (A, B), 15 (C, D), 30 (E, F), 60 (G, H) after birth. No significant staining was observed 520in the negative controls (at the upper corner of panel C and G). A, B: Positive staining of p97/VCP expression was demonstrated at day 5 after birth and was confined to nuclear and cytoplasmic regions of gonocytes (GC) which remain in the centeral region of the tubules. Representative mesenchymal cells (M) and peritubuler myoid cells (P) are indicated no immunoreactivity (B). C, D: p97/VCP is highly expressed in spermatogonia (Sg), 525spermatocytes (Sc) and Sertoli cells (Se) at day 15. Some interstial cells (fetal Leydig cell, FLC) and peritubuler myoid cells (P) show moderate to strong immunostaining for p97/VCP. E, F: Elongating (eSt), round (rSt) spermatids, Sertoli cells (Se), spermatogonia (Sg) and Leydig cells (LC) are shown moderate to strong immunostaining for p97/VCP, while spermatocytes (Sc) are weakly labeled with p97/VCP. G, H: 530p97/VCP is weakly expressed in spermatocytes (Sc), In adult (day 60) testis, round spermatid (rSt),

spermatagonia(Sg) and Leydig cells (LC) around the vessels (V) whereas Sertoli cells (Se) are strongly immunpositive for p97/VCP. is also weakly labeled for p97/VCP. Roman numerals indicate stages of seminiferous epithelial cycle. I: The H-SCORE of p97/VCP immunostaining intensities in the developing rat testis. The data are represented as means 535SEM. a: p < 0.05, day 5 vs. day 15, 30 and 60, b: day 15 vs. day 30 and 60, c: day 30 vs. 5 and 15. Scale bars: 50 m Figure 2: Localization of Jab1/CSN5 in the developing rat testis. Immunohistochemistry was used to determine the Jab1/CSN5 immunoreactivity in the testis 540at day 5 (A, B), 15 (C, D), day 30 (E, F) day 60 (G, H) after birth. No significant staining is observed in the negative controls (at the upper right corner of panel A and C). A, B: Moderate Jab1/CSN5 expression is seen at day 5 after birth in gonocytes (GC). C, D: Jab1/CSN5 is strongly expressed in spermatogonia (Sg), spermatocytes (Sc) and Sertoli cells (Se) at day 15. E, F: Spermatogonia (Sg), spermatocytes (Sc), elongating (eSt) spermatids, 545round (rSt) spermatids, Sertoli cells (Se), Leydig cells (LC) are shown moderate to strong immunostaining for Jab/CSN5. G, H: In adult (day 60) testis, Jab1/CSN5 is strongly expressed in spermatagonia (Sg) and spermatocytes (Sc) while round (rSt) and elongating spermatids (eSt) show weak immunostaining for Jab1/CSN5. I: The H-SCORE of Jab1/CSN5 immunostaining intensities in the developing rat testis. The data are represented as means

18 550SEM. a: p < 0.05, day 5 vs. day 15, 30 and 60, b: day 15 vs. day 5 and 60, c: day 30 vs. 5 and 15. Scale bars: 50 m. Figure 3: Distribution of p97/VCP and Jab1/CSN5 in different regions of rat epididymis during the postnatal development. 555p97/VCP and Jab1/CSN5 immunopositivity are detected in the caput, corpus and caudal region of epididymis at day 5 (A, B), 15 (C, D), 30 (E, F) and 60 (G, H). p97/VCP and Jab1/CSN5 expression are demonstrated on the epithelial cells (EC) of corpus epididymis at day 5 (A, B). No significant staining is observed in the negative controls (at the upper corner of panel B, D, F and H). p97/VCP and Jab1/CSN5 is localized both in the basal (BS) and 560principal cells (PC) cells of corpus epididymis at day 15 (C, D). In addition to epithelial cells (EC), p97/VCP is highly expressed in the spermatids (St) in the caput (CT) and corpus (CS) epididymis at day 30 (E). Jab1/CSN5 is also highly expressed in the principal cells of corpus epididymis and spermatids (St) (F). Immunopositivity for p97/VCP and Jab1/CSN5 is detected both in the principal cells and basal epithelial cells while clear cells (C) showed no 565immunoreactivity at day 60 (G, H). Spermatozoa (Sp) are weakly immunopositive for p97/VCP and Jab1/CSN5. I: The H-SCORE of p97/VCP and Jab1/CSN5 immunostaining intensities in the developing rat epididymis. The data are represented as means SEM. The H-SCORE value is significantly increased from day 5 to day 60 (p < 0.05). Scale bars: 50 m 570Figure 4: Double immunohistochemistry representing the co-localizations of p97/VCP and Jab1/CSN5 in the developing rat testis and epididymis (A-H). 60 gunluk epddms eklenebilir? A: Both p97/VCP and Jab1/CSN5 positive cells (double arrow) are stained as light brown (Fastred + DAB). Gonocytes are double labeled with p97/VCP and Jab1/CSN5 in day 5. B 575and C: p97/VCP positive cells (black arrow) are stained as red (Fastred) in the interstitial region of 15-day-old testis. Spermatocytes, spermatogonia and Sertoli cells are double labeled (double arrow). D: Spermatids (double arrow) in the lumen of 30 day epididymis are shown immunopositivity for p97/VCP and Jab1/CSN5. p97/VCP positivity is seen on the epithelial cells (EC) of corpus epididymis. The upper layer of the epithelial cells (EC) is double labelled 580(double arrow) with p97/VCP and Jab1/CSN5. E, F: p97/VCP positive Sertoli cells (black arrow) and Jab1/CSN5 positive spermatocytes (red arrow) are seen in 60-day-old testis. Double labeling (double arrow) is also observed in the cytoplasm of the spermatocytes (E). Scale bars: 50 m

20

19

585Figure 5. Western blot analysis of p97/VCP and Jab1/CSN5 in the developing rat testis (A) and epididymis (B). p97/VCP (97 kDa) and Jab1/CSN5 (38 kDa) were detected by western blotting. -actin (43 kDa) was used as loading control. Immunoblot bands were quantified by an optical densitometer. The OD (optical density) values of p97/VCP and Jab1/CSN5 bands were 590normalized to the OD values of -actin bands. The data in the graphs are presented as means SEM. p97/VCP and Jab1/CSN5 expressions are significantly increased from 5 to 30 days of testis (p<0,05), however no differences are observed between 30 days and 60 days of testis (A). p97/VCP and Jab1/CSN5 expression are significantly higher in caput epididymis than corpus and cauda epididymis at day 30 and 60 (B). CT: Caput, CS: Corpus, CA: Cauda. 595Asterisks indicate p<0,05, day 5 corpus vs. day 5 cauda and caput epididymis, day 30 and 60 caput vs. day 30 and 60 cauda and corpus epididymis.

20

Table 1: Localizations of Jab1/CSN5 and p97/VCP in rat testis and epididymis Postnatal TESTIS day GC Sg Sc rSt eSt Se
Jab1 5 15 30 60 VCP Jab1
__

LC
Jab1
__

VCP
__

Jab1
__

VCP
__

Jab1
__ __

VCP
__ __

Jab1
__ __

VCP
__ __

Jab1
__

VCP
__

VCP
__

++
__ __ __

++
__ __ __

+++ ++ +++

++ ++ +

+++ ++ ++

++ + +

++ +

++ +

++ +

++ +

++ ++ +

++ ++ +++

++ ++ +

++ ++ ++

Postnatal day
5 15 30 60

PR
Jab1 VCP

EPIDIDYMIS BS St
Jab1 VCP Jab1
__ __

Sp
Jab1
__ __ __

VCP
__ __

VCP
__ __ __

+ ++ +++ +++

+ ++ +++ +++

+ ++ +++ +++

+ ++ ++ +++

+++
__

+++
__

600 Semiquantitive evaluation of Jab1/CSN5 and p97/VCP immunoreactive cells in the developing rat testis and epididymis. GC: Gonocyte, Sg: Spermatogonia, Sc: Spermatocyte, rSt: Round spermatid, eSt: Elongating spermatid Se: Sertoli cell, LC: Leydig cell, PR: 605Principal cell, BS: Basal cell, Sp: Spermatazoa. weak immunreactivity: +, moderate immunreactivity : ++, strong immunreactivity: +++

21 Figure 1:

610

22 Figure 2:

615

23

Figure 3:

620

25

24

Figure 4 625

630

635

640

645

650

25

Figure 5: 655

660

665

670