Molecular Human Reproduction, Vol.19, No.5 pp. 327–335, 2013 Advanced Access publication on January 12, 2013 doi:10.1093/molehr/gat001

ORIGINAL RESEARCH ADP-ribosylation factor-like 3, a manchette-associated protein, is essential for mouse spermiogenesis

Yujuan Qi†, Min Jiang†, Yan Yuan, Ye Bi, Bo Zheng, Xuejiang Guo, Xiaoyan Huang*, Zuomin Zhou*, and Jiahao Sha

State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, 140 Hanzhong Road, Downloaded from Nanjing, Jiangsu Province 210029, China

*Correspondence address. Tel: +86-025-86862908; Fax: +86-025-86862908; E-mail: [email protected] (X.H.); [email protected] (Z.Z.) Submitted on October 29, 2012; resubmitted on December 24, 2012; accepted on January 4, 2013 http://molehr.oxfordjournals.org/ abstract: During spermiogenesis, the manchette is an important structure for sperm head and tail formation. However, the mechan- isms responsible for this process are poorly understood. In a previous study, we established a comparative proteome profile for mouse testis during the first wave of spermatogenesis, and provided lists of proteins of potential importance in the regulation of male fertility and infertility. Here we have selected Arl3, one of these interesting proteins, and investigated its expression and function during spermiogenesis. Western blotting was used to determine Arl3 expression levels in mice at different time points from birth to adulthood. The results show Arl3 was expressed from birth, and the expression level increased significantly from Week 4, when mouse spermiogenesis begins. Immunohistochem- istry and indirect immunofluorescence were used to investigate the Arl3 expression during sperm development, and the intracellular local- ization of Arl3 in more detail. In elongating spermatids from steps 8 to 15, Arl3 was localized to the posterior section of the head, in a similar pattern to the manchette. The Arl3 signal was colocalized during spermiogenesis with a-tubulin, a marker for the manchette. To investigate at University of Queensland on June 21, 2015 the possible functional role of Arl3, mouse testes were injected with small interfering RNA (siRNA) against Arl3, or control siRNA. Western blotting showed a 60% reduction in the Arl3 expression after 72 h, and a significant increase in sperm abnormalities after 3 weeks compared with the negative control. In conclusion, we propose that Arl3 is a novel manchette-related protein with an important role in spermiogenesis.

Key words: Arl3 / manchette / RNAi / spermiogenesis

Introduction timing of manchette development is very precise: it appears during early elongation and disappears as the processes of elongation and Spermatogenesis is a biological process that involves successive condensation of the spermatid nucleus approach completion (Kiers- mitotic, meiotic and post-meiotic phases. Meiosis is unique to zenbaum, 2002; Toshimori and Ito, 2003; Matsuoka et al., 2008). gamete formation, whereas the post-meiotic phase, known as sper- Components of the manchette have been identified, including many miogenesis, only occurs during male germ cell development (Wolge- microtubular proteins, such as microtubule-dependent motor proteins muth et al., 1995; Eddy, 2002). During spermiogenesis, the and microtubule-associated proteins (Kierszenbaum and Tres, 2002; morphogenetic process, shaping and condensation of the nucleus as Yamaguchi et al., 2004; Akhmanova et al., 2007). Several findings well as the formation of the acrosome and the tail take place. suggest that the manchette may serve to convey molecules necessary These dramatic changes require a stringent, well-coordinated and for spermatid nuclear condensation and tail formation (Kierszenbaum unique system that regulates specific patterns of gene and protein ex- et al., 2002; Tovich et al., 2004). However, the detailed mechanism pression (Krausz and Sassone-Corsi, 2005; Tanaka and Baba, 2005). and the important molecules involved in this process remain to be The manchette, one of the important structures in spermiogenesis, elucidated. is a transient construct that emerges between steps 8 and 16 and In a previous study, we established a comparative proteome profile encircles part of the nuclei of elongating spermatids in mice. The for mouse testis during the first wave of spermatogenesis and

† These authors contributed equally to the work.

& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] 328 Qi et al. identified 362 differential protein spots corresponding to 257 different Materials and Methods proteins involved in the initiation of mouse spermatogenesis (Huang et al., 2008; Huang and Sha, 2011). Several proteins from this list Animals were chosen for further investigation and ADP-ribosylation factor-like Male CD-1 mice (aged 0 weeks, 1 week, 10 days, 2, 3, 4 weeks and adult) 3 (Arl3) was one of these interesting proteins. Arl3 is a Ras-related were obtained from the animal center of Nanjing Medical University small GTP-binding protein. Like other ARF family members, Arl3 (Nanjing, Jiangsu, China). All experiments requiring the use of animals binds guanine nucleotides, but it lacks ARF activity (Memon, 2004; were approved by the ethical board of Nanjing Medical University. Cavenagh et al., 1994). The aim of this study was to investigate the levels of expression and Sample preparation and protein extraction the localization of Arl3 at different time points during spermiogenesis Testes obtained from male CD-1 mice were solubilized in lysis buffer (7 M and to determine whether this protein plays a functional role in sperm urea, 2 M thiourea, 4% [w/v] CHAPS, 2% [w/v] DTT) in the presence of development. 1% (w/v) protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL, Downloaded from http://molehr.oxfordjournals.org/ at University of Queensland on June 21, 2015

Figure 1 Western blotting of Arl3 in mouse testis at different developmental stages. The upper panel indicates Arl3 as a dot in the 2DE gel. The middle panel shows western blotting of mouse testis with the anti-Arl3 antibody at the same time points. The lower panel shows the control blots using antibody to b-tubulin.

Figure 2 Localization of Arl3 in adult mouse testis by immunohistochemical analysis. Magnification is ×200 in (A), ×400 in (B) and (C). (A) Arl3 was highly expressed in the seminiferous tubules. (B) Arl3 was strongly expressed from late stage of pachytene spermatocytes to elongating spermatids in adult mouse testis. At Stage I of spermatogenic cycle, Arl3 was expressed in round spermatids (arrows) and in elongating spermatids (asterisks), but not expressed in early stage of panchytene spermatocyte. At Stage ?, Arl3 was expressed in the late stage of pachytene seprmatocyte (arrowheads). (C) Specific staining was not present in tissue incubated with normal rabbit IgG. Scale bars are all 10 mm. Arl3 location and function in mouse spermiogenesis 329

USA), then homogenized and sonicated. The mixture was placed on a blocked using blocking serum and then incubated overnight at 48C with shaker at 48C for 1 h, and the insoluble matter was removed by centrifu- anti-Arl3 antibody (1:100). Sections were then incubated with gation at 40 000 g and 48C for 1 h. The protein concentration in each HRP-conjugated secondary antibody (Beijing ZhongShan Biotechnology, sample was determined by the Bradford method, using bovine serum Beijing, China). Immunoreactive sites were visualized in brown using diami- albumin as the standard. nobenzidine and mounted for bright field microscopy (Axioskop 2 plus; Zeiss, Oberkochen, Germany). To confirm the specificity of the Western blotting anti-Arl3 antibody, negative controls were processed in an identical manner, except that the primary antibody was replaced by normal IgG Samples containing 100 mg of protein were electrophoresed on a 12% SDS (ab27478; Abcam, Cambridge, MA, USA). polyacrylamide gel and transferred onto a PVDF membrane (GE Health- care, Pittsburgh, PA, USA). Membranes were blocked in TBS containing 5% non-fat milk powder for 1 h and then incubated overnight with Indirect immunofluorescence anti-Arl3 (1:1000; No: 10961-1-AP; Proteintech, Chicago, IL, USA) or Testis tissue was embedded in optimal cutting temperature (OCT) com- anti-b-tubulin antibody (1:2000; No: ab6046; Abcam, Cambridge, MA, pound, cryosectioned into 5-mm-thick sections using a Leica Model USA) diluted in TBS containing 5% non-fat milk powder. Anti-b-tubulin CM1900 cryostat (Leica Microsystems, Wetzlar, Germany), mounted on antibody was used as a loading control. Membranes were washed and slides and left to dry before fixation. Single cell suspensions prepared then incubated for 1 h with horseradish peroxidase (HRP)-conjugated from testes using a Medimachine (Becton Dickinson, NJ, USA) were air Downloaded from goat anti-rabbit IgG (1:1000; Beijing ZhongShan Biotechnology Co., dried onto slides. The prepared slides were fixed with 4% formaldehyde Beijing, China). Specific proteins were detected using an ECL kit and in PBS for 30 min, washed three times with PBS for 5 min each time AlphaImagerTM (GE Healthcare). and then blocked with goat serum (Beijing ZhongShan Biotechnology) for 2 h at room temperature. Following incubation with primary antibodies Immunohistochemistry (Arl3, 1:100) overnight at 48C, cells were incubated with FITC-labeled sec- http://molehr.oxfordjournals.org/ Bouin’s solution-fixed, paraffin-embedded sections from mouse testis ondary antibody (Beijing ZhongShan Biotechnology) at a 1:1000 dilution were immunostained as described previously (Zhang et al., 2007). In for 1 h at room temperature. For negative controls, the primary antibody brief, after quenching the endogenous peroxidase activity, sections were was replaced with normal rabbit IgG. The nucleus was stained with at University of Queensland on June 21, 2015

Figure 3 Localization of Arl3 in adult mouse testis by indirect immunofluorescence. Magnification is ×400 in (A) and (C), ×1000 in (B). (A) Arl3 was expressed from primary spermatocyte to elongating spermatid. Arrows (in B Merge) indicate Arl3 was distributed as irregular punctate signal in the post-nucleus area in elongating spermatids. White boxed area was highlighted to show the localization. (C) Specific staining was not present in tissue incubated with normal rabbit IgG. Scale bars are all 10 mm. 330 Qi et al. Downloaded from http://molehr.oxfordjournals.org/ at University of Queensland on June 21, 2015

Figure 4 Distribution of Arl3 at each stage of germ cell development, from spermatocyte to sperm. Magnification is ×1000. Indirect immunofluor- escence shows Arl3 was expressed as punctate signal in pachytene spermatocytes. In round spermatids, the signal was still in the cytoplasm, with a similar distribution to the centrosomes. In elongating spermatids at Steps 8–15, Arl3 was located in the posterior head of spermatids, consistent with the involvement in the development of the spermatid tail. In the sperm from the caudal epididymis, Arl3 was located in the axon of the sperm tail. Arl3 location and function in mouse spermiogenesis 331 Downloaded from http://molehr.oxfordjournals.org/

Figure 5 Distribution of Arl3 in step 13 spermatids under CarlZeiss LSM710. Magnification is ×1000. Indirect immunofluorescence with anti-Arl3 antibody shows the signal was located in the post-nucleus area, consistent with association of Arl3 with the manchette. Green signals show staining with ant-Arl3 antibody, and red signals show PI staining. A–F represent six panels under microscopy scanning. at University of Queensland on June 21, 2015

5 mg/ml Hoechst H33342 (Sigma, St. Louis, MO, USA) for 30 s or 5 mg/ observation. Deformities were classified according to the existing literature ml propidium iodide (PI; Sigma, St. Louis, MO, USA) for 5 min. For step 13 (Wyrobek and Bruce, 1975) and classified by two types: sperm head ab- spermatids, six different planes were scanned using a CarlZeiss LSM710. normality and sperm tail abnormality. Three hundred sperm were counted in each slide with a double-blind method. Statistical differences between Intratesticular injection of siRNA the two groups were measured by Student’s t-test. P values of , 0.05 were considered significant. against Arl3 Double-stranded Arl3 StealthTM siRNAs against Arl3 mRNA (Invitrogen, Grand Island, NY, USA; Catalog Nos MSS226063, MSS226065 and Results MSS226067) were diluted to a final concentration of 20 mM and stored at 2208C. The intratesticular injection was similar to that previously Expression of Arl3 during the first wave described (Lu et al., 2006; Guo et al., 2010). In brief, 3-week-old male mice were anesthetized with sodium pentobarbital and testes were exter- of mouse spermatogenesis iorized through a midline abdominal incision 5-mm long. Approximately In a previous study, we constructed the proteome profile that is func- 3–5 ml of individual siRNA mixed with indicator (0.4% trypan blue) was tional during the first wave of mouse spermatogenesis (Huang et al., injected into the seminiferous tubules via the rete testis. Control testes 2008). Arl3 appeared at birth as a spot in the 2DE gels and its expres- were injected with negative control siRNA (Invitrogen; No. 12935-400). sion level increased gradually with the development of the testis Following injection, the testes were replaced into the abdomen and the (Fig. 1). To verify these results, we used mouse testis protein from incisions were sutured. The mice recovered and were raised for another the same time points as in the proteome profile (0, 1, 2, 3, 4 3 weeks. Western blotting was used to assay the relative changes in weeks and adulthood) to perform western blotting. The results protein expression between groups 72 h after injection and verify the efficiency of the three siRNAs against Arl3. Subsequent in vivo RNAi showed that the 20 kDa Arl3 protein was weakly expressed from experiments were performed under the same conditions. birth to Day 21, with a significant increase in the expression on Day 28 which was maintained until adulthood (Fig. 1). Classification of epididymal sperm morphology Location of Arl3 in adult mouse testis Spermatozoa were collected from the caudal epididymis 3 weeks after The results of immunohistochemistry showed that Arl3 was strongly siRNA injection. Spermatozoa were spread on slides, fixed with 4% paraf- expressed at all germ cell types from the pachytene spermatocyte ormaldehyde and stained with hematoxylin and eosin for morphological to the elongating spermatids in adult mouse testis (Fig. 2A and B). 332 Qi et al. Downloaded from http://molehr.oxfordjournals.org/ at University of Queensland on June 21, 2015 Figure 6 Localization of Arl3 and a-tubulin during spermiogenesis. Magnification is ×1000. Indirect immunofluorescence using anti-Arl3 and anti-a-tubulin antibodies demonstrates their signals were overlapping in step 10–15 elongating spermatids. Green signals indicate Arl3 localization. Red signals indicated a-tubulin localization and nuclear signal was blue.

In order to clearly demonstrate the location of Arl3, indirect immuno- two experiments. First, we captured spermatids at step 13 to scan fluorescence was performed in adult mouse testis, and it gave similar the positive signal under a CarlZeiss LSM710 and analyzed the results to the immunohistochemistry. In particular in elongating sper- results in six different planes. The results showed the Arl3 signal matids, Arl3 was distributed in the post-nuclear region and in these was located in the distal region of the nucleus (Fig. 5), consistent spermatids close to the lumen, the strong signal appeared as linear with the location of the manchette. We then used a-tubulin as a streaks consistent with localization to the spermatid tails (Fig. 3). marker for the manchette to compare its location with that of Arl3. The results showed that in spermatids from step 10 to 15, the Distribution of Arl3 during spermatogenesis signals of Arl3 and a-tubulin were overlapping, suggesting that Arl3 Further analysis to assess the localization of Arl3 by light microscope, was located in the manchette (Fig. 6). we prepared preparations of the major germ cell types. The results showed that Arl3 expression was detected as a punctate signal in pachytene spermatocytes, and in round spermatids the signal was Deformity of sperm after interference with still distributed in the cytoplasm, in a similar pattern to the centro- Arl3 during spermiogenesis somes. However, during spermiogenesis, we found the Arl3 signal The manchette is an essential structure during spermiogenesis and so was located in the posterior head of the elongating spermatid and we predict that Arl3 must play an important role during this process. was consistent with the development of the spermatid tail (Fig. 4). To confirm this, we carried out in vivo RNAi interference, using The distribution was similar to the manchette structure. In the double-stranded Arl3 StealthTM siRNAs. As described in our previous sperm from the caudal epididymis, Arl3 was located in the axoneme study, mouse spermiogenesis begins 3 weeks after birth (Huang et al., of the sperm tail (Fig. 4). 2008). So we used 3-week-old mice for injection. Injected siRNA materials reached a40% of the seminiferous tubules. Western blot- Arl3 is a manchette-associated protein ting demonstrated that Arl3 proteins were significantly suppressed in From the above results, we predicted that Arl3 was located in the the testis 72 h after injection with the siRNA MSS226067 (designated manchette. To verify this hypothesis, we conducted the following as R7; Fig. 7). Arl3 location and function in mouse spermiogenesis 333

Three weeks after intratesticular injection of siRNA against Arl3, the caudal epididymis in mice treated with R7 SiRNA against Arl3 had sperm from the caudal epididymis were collected for morphological an abnormal head shape, a lasso-like coiled tail or were decapitated analysis. Two students counted the rate of sperm abnormality with (Fig. 8B). In sperm from the negative control group, these abnormal- double blind and the variability and the details of the parameters mea- ities were barely detectable. sured were shown in Table I. The results showed that nearly 27% of sperm from testes treated with R7 siRNA against Arl3 were abnormal, compared with 15% in the negative control group (P , 0.05). Sperm Discussion with abnormal tail was also significantly increased in the experimental Arl3 was first cloned from human and rat tissues (Cavenagh et al., group. Although the rate of sperm with abnormal head had no statis- 1994). Eight years later, the function of the small G protein-encoding tical significance compared with the control group, the tendency was LdARL-3A gene isolated from Leishmania donovani, a homolog of the increasing (Fig. 8A). We observed that a large number of sperm from human ARL-3 gene, was reported (Cuvillier et al., 2000). When a con- stitutively ‘active’ form of LdARL-3A was overexpressed, the promasti- gotes were immobile with a very short flagellum, and the length of the flagellum was inversely proportional to mutant protein expression (Cuvillier et al., 2000). Further studies reported that Arl3 (2/2) Downloaded from mice were born at a sub-Mendelian ratio and failed to thrive (Schrick et al., 2006). These mice exhibited abnormal development of renal, hepatic and pancreatic epithelial tubule structures. Moreover, mice lacking Arl3 exhibited photoreceptor degeneration. These results were the first to show Arl3 in a ciliary disease affecting the kidney, http://molehr.oxfordjournals.org/ biliary tract, pancreas and retina. Although they did not observe the sperm of Arl3 (2/2) mice, we predict that these mice might exhibit abnormal sperm morphology because the sperm tail is a ciliary structure. In our study, Arl3 was expressed weakly during the first 3 weeks after birth, and highly expressed from 4 weeks until adulthood. It is known that at 4 weeks, during the first wave of spermatogenesis, spermiogen- esis begins with the sperm tail or flagellum and head develops. We

therefore initially predicted that Arl3 was involved in mouse spermio- at University of Queensland on June 21, 2015 genesis. Further investigation of the detailed distribution patterns of Arl3 expression at different stages during spermatocyte development and spermiogenesis, as described in the results section, were consistent with its involvement in spermiogenesis, and in particular with its location in the manchette at the posterior region of the spermatid nucleus. This was confirmed by overlapping signals obtained in indirect immunofluor- escense analysis using anti-Arl3 and anti-a-tubulin antibodies, which is the marker of manchette. Figure 7 Efficiency of one type of Arl3 siRNA (R7) in vivo 72 h after To further explore the function of Arl3, we used an in vivo RNAi- injection. Western blotting (A) and statistical analysis (B) show R7 mediated knockdown strategy. To our knowledge, transcription and reduced protein levels by 60%. Data represent a mean of triplicate translation almost cease during spermiogenesis (Steger, 1999, 2001). experiments, and the values are expressed as a percentage normal- The introduction of siRNA into germ cells degrades the target mRNA ized to the negative control (100%). and stops the cell transcribing new mRNA. The levels of protein expressed by the target gene can therefore be suppressed for a relatively

Table I The variability and the details of the parameters measured with double-blind method during sperm abnormality rate analysis.

Group Head abnormality (mean +++++ SD) Tail abnormality (mean +++++ SD) Total abnormality (mean +++++ SD) Data sources ...... Neg (%) 9.04 + 2.3 6.5 + 0.99 15.54 + 1.34 From Min 9.95 + 2.61 3.72 + 1.23 13.68 + 1.43 From Bo 9.50 + 2.45 5.11 + 1.11 14.61 + 1.39 Combined R7 (%) 14.13 + 4.24 12.06 + 0.52* 26.19 + 4.74* From Min 14.25 + 6.53 14.15 + 1.33* 28.40 + 5.19* From Bo 14.19 + 5.33 13.10 + 0.41* 27.30 + 4.92* Combined

*P , 0.05. 334 Qi et al. Downloaded from http://molehr.oxfordjournals.org/

Figure 8 Effect of in vivo siRNA against Arl3 on sperm morphology in the caudal epididymis. Abnormalities in the sperm from the caudal epididymis were counted under the microscope 3 weeks after siRNA injection and compared. (A) Rate of total sperm abnormality and tail abnormality were significantly increased in the experimental group. Significant differences are indicated by *P , 0.05. (B) The shapes of the sperm from the caudal epi- didymis of mouse testes treated with the siRNA R7 are shown. White arrows indicate an abnormal head shape. Black arrows indicate an abnormal sperm tail with a lasso-like coiled tail or decapitation. Scale bars are all 10 mm. at University of Queensland on June 21, 2015 long period of time (Aigner, 2006). Phenotypic analysis of sperm from Tres and Kierszenbaum, 1996; Brohmann et al., 1997; Kierszenbaum the first wave of spermatogenesis showed that the rate of sperm abnor- et al., 2002). The manchette is therefore a very important structure mality, particularly an abnormal head shape, a lasso-like coiled tail or de- for sperm head and tail formation. capitation, was significantly different between the Arl3 siRNA injection In this study, we present preliminary results indicating that mouse group and the control group. This phenotype was very similar to that Arl3 is a novel manchette-associated protein. However, the seen after loss or deletion of many manchette-associated proteins. in-depth function of Arl3 needs further exploration. In future studies The Hook1 gene is predominantly expressed in the manchette, and we will explore the protein interactions of Arl3 during manchette de- the loss of Hook1 function causes the azh phenotype: abnormal head velopment, and further germ cell-specific knockout of Arl3 will be con- shape and abnormal bending and looping of the sperm tail (Cole ducted to clarify the molecular mechanism of Arl3 function during et al., 1988; Mendoza-Lujambio et al., 2002). Katanin p80 is also a mouse spermiogenesis. manchette-associated protein. Perturbed katanin p80 function results in male sterility characterized by sperm with an abnormal head shape (O’Donnell et al., 2012). RIM-BP3 protein is associated with the man- Authors’ roles chette, and targeted deletion of the RIM-BP3 gene also resulted in Y.Q. and M.J. are the co-authors. They contributed to acquisition of male infertility owing to abnormal sperm heads, characterized by a data, initial analysis of data. Y.Y. and Y.B. helped Qi and Jiang to do deformed nucleus and a detached acrosome (Zhou et al., 2009). experiments. B.Z. helped count rate of abnormal sperm in the revi- During spermiogenesis, the manchette appears at the time when sion. X.G. helped in analysis of data. X.H. contributed to interpret- the spermatid nucleus begins to elongate, and disappears as elongation ation of data, draft the article and revise it critically for important and condensation approach completion. The elongated manchette is intellectual content. Z.Z. and J.S. contributed to conception and thought to be used for temporary storage and transport of structural design of the whole experiment. and signaling proteins (Rivkin et al., 1997; Kierszenbaum, 2001). This sorting mechanism is described as intramanchette transport (IMT) (Kierszenbaum, 2002). Research to date indicates that IMT cannot only transport cargo proteins to the developing sperm tail for use in Funding tail formation, but also transports cargo proteins that are involved in This work was supported by grants from the National Basic Research spermatid nuclear condensation (Burfeind and Hoyer-Fender, 1991; Program of China (2011CB944301). Arl3 location and function in mouse spermiogenesis 335

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