ANDROLOGY
ISSN: 2047-2919
ORIGINAL ARTICLE
Correspondence: Vrinda V. Khole, Department of Gamete Immunobiology, National Institute for Research in Reproductive Health, ICMR, J. M. Street, Parel, Mumbai 400012, India. E-mail: [email protected]
Keywords: acrosome reaction, leucocyte antigen-related protein, liprin a3, nidogen, rab-interacting molecule, spermatozoa, syndecan, wedge peptide
Regulation of acrosome reaction by Liprin a3, LAR and its ligands in mouse spermatozoa C. S. Joshi, S. A. Khan and V. V. Khole Department of Gamete Immunobiology, National Institute for Research in Reproductive Health, Mumbai, India
Received: 28-Sep-2013 Revised: 1-Nov-2013 Accepted: 4-Nov-2013 doi: 10.1111/j.2047-2927.2013.00167
SUMMARY Zona pellucida-based induction of acrosome reaction (AR) is a popular and well-accepted hypothesis. However, this hypothesis is being challenged in recent years and it has been proposed that the cumulus cells might be the site of AR. In our previous study, we reported the presence of a synaptic protein Liprin a3 on sperm acrosome, and proposed its role in AR. This study was designed to understand the role of Liprin a3 and its interacting proteins in regulation of AR. It is observed that the presence of anti-Liprin a3 antibody inhibits the process of AR. Colocalization experiments demonstrate the coexistence of leucocyte antigen related (LAR) protein, Rab-interacting molecule (RIM) and Liprin a3 on sperm acrosome thereby completing the identification of all the members of RIM/ MUNC/Rab3A/liprina complex required for membrane fusion. This study demonstrates the effect of LAR ligands such as Syndecans, Nidogens and LAR wedge domain peptide on AR. We could see an increase in AR in presence of these ligands. On the basis of these data, we speculate that in presence of ligands or wedge peptide, LAR undergoes dimerization leading to inhibition of phosphatase activity and increase in AR. The presence of one of the ligands Syndecan-1 on cumulus cells led us to hypothesize that it is Syndecan which induces AR in vivo and thus another site of AR could lie in cumulus.
INTRODUCTION Acrosome is a unique and special type of vesicle, designed to play a crucial role in controlling of the fertilizing ability of spermatozoa. The release of acrosomal content is a regulated exocytosis process that involves calcium and several other players. Several studies suggest that synaptic vesicle exocytosis and acrosomal exocytosis are mediated by a common set of proteins such as N-Ethylamide sensitive factor (NSF) (Zarelli et al., 2009), soluble NSF attachment protein receptor (SNARE) (Ramalho-Santos et al., 2000), soluble NSF attachment protein (Wadia & Dowdy, 2002), Synaptotagmin (Michaut et al., 2001), Rab-Interacting Molecule (RIM) (Bello et al., 2012) etc. It was proposed and also proven that RIM serves as a Rab3-dependent regulator of synapse fusion (Wang et al., 1997; Schoch et al., 2002). However, interaction between RIM and Liprin in acrosome has not been demonstrated. We recently reported the presence of synaptic protein Liprin a3 on spermatozoa and its involvement in acrosome biogenesis s (Joshi et al., 2013). Liprins were first discovered by Serra-Page et al. (1995) at focal adhesion by trapping proteins interacting © 2013 American Society of Andrology and European Academy of Andrology
with cytoplasmic region of leucocyte antigen-related (LAR) protein. C-terminus of liprins show Liprin homology (LH) domain, composed of three sterile alpha motifs (SAMs) that interacts with LAR. It has been described that Liprin a3 and LAR protein are present in the brain and neuronal tissues and function as scaffolding molecules and are involved in vesicle transport across the pre-synaptic junctions (Spangler & Hoogenraad, 2007). The function of LAR was studied extensively in PC12 cells using wedge peptides by Yang et al. (2003, 2005, 2006) and Xie et al. (2006). The wedge peptide showed inhibitory effect on LAR phosphatase activity thereby affecting its downstream potential targets like beta catenin and TrkA (Xie et al., 2006; Yang et al., 2006). The receptor–ligand-based acrosome reaction (AR) has been proposed by Benoff (1998). He proposed that the binding of glycosylated residues present on egg interact with their respective receptor present on spermatozoa and activate AR. It was found that the ectodomain of LAR is involved in ligand–receptor interactions. Sulphated proteoglycan like Syndecans and Nidogens are also known to interact with LAR (Johnson et al., 2006). These Andrology, 2014, 2, 165–174
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ligands are reported to be present on cumulus cells (Watson et al., 2012); however, their role has never been studied with reference to their counterpart present on the spermatozoa. To date, there are no reports of presence of LAR and its involvement in sperm function. In this study, we demonstrate the interaction of Liprin a3 with RIM and LAR and show the importance of interaction of Liprin a3 and LAR in AR. Our data also support the hypothesis that another site of AR could be cumulus where LAR ligands are present.
MATERIALS AND METHODS Materials Phosphate-buffered saline (PBS), Western blot and sodium dodecyl sulphate-polyacryl amide gel electrophoresis chemicals were procured from SRL India (Mumbai, India). Nitrocellulose membranes (Hybond-C Extra) and ECL Plus Western blotting detection reagent were obtained from GE Health Care (Buckinghamshire, UK). Protein molecular weight markers were procured from Fermentas (MD, USA). Non-fat dry milk (NFDM) powder was procured from Anikspray (Mumbai, India). Bovine serum albumin (BSA) fraction V was obtained from USB (Cleveland, OH, USA). Human tubal fluid (HTF) was obtained from Irvin Scientific (Santa Ana, CA, USA). ProLong Gold anti-fade agent and all Alexa fluor antibodies were purchased from Invitrogen (Eugene, OR, USA). Pisum sativum-FITC (FITC-PSA), calcium ionophore A23187 and paraformaldehyde (PFA) were obtained from Sigma Aldrich (Steinheim, Germany). Complete protease inhibitor cocktail and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Roche (Mannheim, Germany), Kodak X-ray films were purchased from Asset healthcare (Silvassa, India). Antibodies and recombinant proteins Rabbit polyclonal anti-Liprin a3, chicken polyclonal antiPTPRF (LAR) and mouse monoclonal anti-b actin were procured from Sigma Aldrich (St. Louis, MO, USA); Anti RIM antibody was purchased from Synaptic System. Mouse polyclonal anti-Liprin a3 was procured from Novus Biologicals. Recombinant Syndecan-1, 2, 3, Nidogen-1, 2 were procured from R&D systems (Minneapolis, MN, USA). Peptides Wedge peptides as described by Xie et al. (2006) were synthesized with an amide substitute at C-terminus. WLAR wedge sequence without Tat, TAT-WLAR wedge sequence with Tat peptide sequence (underlined) linked to C-terminus and SLAR-TAT scrambled wedge sequence with Tat peptide sequence (underlined) were synthesized at by USV ltd (Mumbai, India). WLAR: NH2- DLADNIERLKANDGLKFSQEYESI-NH2 TAT-WLAR: NH2- GRKKRRQRRRCDLADNIERLKANDGLKFS QEYESI-NH2 SLAR-TAT: NH2- NADKGLSLRKEFSIQNEDAYLEDIGRKKRRQ RRRC-NH2 Animals Mature Swiss male and female mice and Holtzman male rat were used for the study. Animals were maintained at a temperature of 22–23 °C, humidity of 50–55% and a cycle of 14 h light 10 h dark with food and water available ad libitum. All animal 166
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care practices and experimental procedures complied with the guidelines of the Care and Prevention Society against Cruelty of Experimental Animals (CPCSEA) and were approved by the Institutional Animal Ethics Committee (IAEC). Human spermatozoa sample We collected human semen sample from participants attending the infertility clinic at the institute. Ethical approval was obtained from the Institutional Human Ethics Committee for clinical studies. Samples were analysed as per the guideline set by WHO, 2010. Samples with normal parameters were included in this study. Methods Spermatozoa preparation We sacrificed four mature male Holtzman rat or Swiss mice by CO2 asphyxiation. Cauda epididymides were removed and placed in pre-warmed HTF. Three to four radial cuts were made to release the spermatozoa. Swim up spermatozoa population was taken and washed with 0.1 M PBS, and suspended either in lysis buffer (1% SDS and protease inhibitor tablet) for protein extraction or in 4% PFA-PBS for immunofluorescence. Tissue preparation Rats were sacrificed; testis and epididymides were dissected and subjected for protein extraction as described (Joshi et al., 2013) and protein was quantified by Bradford’s method. Human spermatozoa sample preparation Human spermaozoa were processed as described earlier (Joshi et al., 2013). Semen sample of 150 lL was layered on the bottom of a Falcon tube containing 1.5 mL washing medium (HTF). After 30 min, the upper part (1 mL) of the supernatant was aspirated. Supernatant was spun at 500 g for 10 min, and pellet was either reconstituted in medium and used either for protein extraction or fixed in PFA as mentioned above and used for IIF. Western blot analysis We used protein from rat testis and epididymis, mouse and human spermatozoa for Western blot analysis as described earlier (Joshi et al., 2013). A quantity of 40 lg of protein was loaded on 10% polyacrylamide gel and SDS PAGE was carried out. Protein was electro trans-blotted to nitrocellulose membrane. Nonspecific sites on the membrane were blocked by incubating blots with 5% NFDM-PBS for 1 h at RT. Blots were incubated with anti-PTPRF antibody diluted 1 : 2000 in 0.1 M PBS at 4 °C overnight. They were then washed thrice with 0.1% Tween 20-PBS for 10 min each. Blots were incubated with Horse Raddish Peroxidase conjugated to rabbit anti-chicken (1 in 3000 dilution in 1% NFDM-PBS) for 1 h at RT and then washed as mentioned above. Detection was carried out by using enhanced chemiluminescence kit (ECL Plus) and recorded on Kodak blue base X-ray sheets. A blot incubated with no primary antibody was treated as a ‘Negative control’. Blots were stripped and probed with beta actin antibody which served as loading control. Indirect immunofluorescence We used 4% PFA-PBS fixed spermatozoa for all immunofluorescence as described earlier (Joshi et al., 2013). Cells were © 2013 American Society of Andrology and European Academy of Andrology
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ROLE OF LIPRIN a3 AND LAR IN ACROSOME REACTION
incubated with blocking solution containing 3% BSA-PBS for 1 h at RT. Spermatozoa cells were incubated with polyclonal antiPTPRF diluted to 1 in 100 in 0.1 M PBS overnight at 4 °C. Excess of primary antibody was removed by washing the slides thrice with 0.1 M PBS. Alexa fluor 488 conjugated goat anti-chicken was used at 1 : 200 dilution and incubated for 1 h at RT along with counterstain DAPI (300 nM). Slides were washed as mentioned above to remove the excess of secondary antibodies. Slides were mounted in ProLong Gold antifade agent and examined under confocal fluorescent microscope (LSM 510; META Zeiss, Jena, Germany). A slide incubated with no primary antibody served as a ‘Negative control’. Colocalization We used PFA fixed sperm smears for colocalization of Liprin a3, PTPRF (LAR) and RIM1/2. We followed protocol for indirect immunofluorescence (IIF) of Liprin a3 as described previously by Joshi et al. (2013) with some modification. Briefly, fixed spermatozoa were permeabilized with 0.1% Triton X-100 for 15 min at RT. Non-specific sites were blocked by incubating the slides with 2% BSA-PBS for 1 h at RT. A cocktail of rabbit polyclonal anti-Liprin a3 and polyclonal chicken anti-PTPRF (both antibodies diluted to 1 in 50 in 0.1 M PBS) or mouse polyclonal anti-Liprin a3 and rabbit polyclonal anti-RIM1/2 (both antibodies diluted to 1 in 50 in 0.1 M PBS) was incubated with sperm smear overnight at 4 °C. Excess of primary antibody was removed by washing the slides thrice with 0.1 M PBS. A cocktail of Alexa fluor 488 goat anti-chicken and Alexa fluor 594 goat anti-rabbit diluted at 1 in 100 or a cocktail of Alexa fluor 488 goat anti-rabbit and Alexa fluor 594 goat anti-mouse diluted at 1 in 100 was incubated at RT for 1 h along with nuclear stain DAPI. Slides were washed as mentioned above to remove the excess of secondary antibodies. A slide incubated with no primary antibody served as a ‘Negative control’. Slides were mounted with ProLong Gold antifade and were examined under confocal microscope (LSM 510 META Zeiss). Areas showing overlap of green and red were subjected for calculation of ‘Overlap coefficient’. Cut mask images were generated. Ten random spermatozoa were selected from each set of experiments and overlap coefficient was calculated using in built software of LSM 510 META microscope. Mean and SEM was calculated. Cumulus oophorus preparation and localization of Syndecan-1 Superovulation was induced in 21-day-old female Swiss mice to obtain cumulus–oophorus complex (COCs) as per the protocol described previously by Pires et al. (2011). Briefly, 20 IU of pregnant mare serum gonadotropin (PMSG) was administered intraperitoneally. After 48 h, 20 IU of human chorionic gonadotropin was injected and after 16–18 h animals were sacrificed. Oviduct was dissected under stereo microscope (Discovery V8 SterREO; Carl Zeiss, Gottingen, Germany) and COCs were collected in small Petri dish and were fixed with 4% PFA. Blocking was carried out by incubating the COCs with 1% BSA for 1 h at RT. Anti Syndecan-1 was used at a dilution of 1 : 100 for 1 h at RT. Excess of primary antibody was washed with 0.01 M PBS. Alexa Fluor 488 goat anti-rabbit was used along with counterstain DAPI for 1 h at RT. Excess of secondary antibody was washed as described previously and mounted with ProLong Gold antifade. Slides were observed under confocal microscope (LSM 510 META Zeiss). © 2013 American Society of Andrology and European Academy of Andrology
Capacitation and acrosome reaction Three mature Swiss male mice were sacrificed and cauda epididymides were dissected out. Three to four radial cuts were made to release the spermatozoa into the medium. Spermatozoa were then suspended in the HTF medium supplemented with 0.4% BSA and capacitation was carried out for 1 h at 37 °C in an atmosphere of 5% CO2. We used capacitated spermatozoa to carry out AR. We carried out assay in presence of calcium ionophore A23187 (10 lM), wedge peptide (25, 50 and 100 lM), ligands like Syndecan 1,2,3, Nidogen 1 and 2 (10 and 50 ng of each) and Anti Liprin a3 antibody by incubating the spermatozoa with each of these individually and separately for minimum 30 min at 37 °C for each condition. At the end of an assay, spermatozoa were smeared on the glass slides, air dried and fixed with 4% PFA at RT for 5 min. Acrosomal status was assessed by staining with FITC-PSA (1 : 300) as described by Mendoza et al. (1992). At least 200 cells were counted using fluorescent microscope (Zeiss Axioskop 2 Plus). Spermatozoa with positive green signal on acrosome were considered as acrosome-intact spermatozoa whereas those without green signal were considered as acrosome-reacted spermatozoa. We included ‘stimulation by calcium ionophore’ as a positive control and ‘no calcium ionophore’ as a negative control in each experiment. For negative control of ligands based assay, ligands pre-incubated with its respective antibodies were used. Experiment was performed three times and statistical analysis was performed. Statistical analysis Acrosome exocytosis index (AE index) values were calculated by subtracting number of spontaneously reacted spermatozoa in the negative control from all test samples. Resulting values were then expressed as a percentage with respect to positive control. Student’s t-test was used for statistical analysis. Differences were considered significant at the p < 0.05 level.
RESULTS PTPRF (LAR) is present in the spermatozoa and is conserved We used protein extract of rat testis, epididymis, mouse and human spermatozoa for Western blot analysis of LAR. LAR was detected as 130, 100, 75, 50 and 30 kDa bands in testicular extract, whereas in epididymal extract we could see 100, 50 and 30 kDa bands as shown in Fig. 1A. The protein was also detected in human and mouse spermatozoa extracts and is represented in Fig. 1B. Respective negative control showed no specific bands (NC). IIF studies as shown in Fig. 1C,D depict positive reactivity (green fluorescence) with anti-LAR antibody at the anterior region of mouse (Fig. 1C) and human sperm acrosome (Fig. 1D). Insets represent respective DIC images. Negative controls did not show any reactivity (Fig. 1E,F). LAR and RIM colocalize with Liprin a3 Colocalization of RIM and LAR with Liprin a3 on anterior acrosome is represented in Fig. 2. Green fluorescence indicates positive staining of LAR, red fluorescence shows staining of Liprin a3 and yellow staining shows colocalization of LAR and Liprin a3 protein as shown in Fig. 2A. Figure 2B depicts overlap graph in which third quadrant shows overlapping pixels only, indicating Andrology, 2014, 2, 165–174
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that two proteins coexist with an overlapping coefficient of 0.88 with SEM of 0.02 as shown in Fig. 2E. Figure 2C depicts the colocalization of RIM-Liprin a3. Green fluorescence indicates positive staining of RIM, red fluorescence shows staining of Liprin a3 and yellow staining shows colocalization of RIM and Liprin a3 protein as shown in Fig. 2C. Figure 2D depicts the overlapping pixels in the third quadrant with an overlapping coefficient of 0.922 with SEM of 0.026 as shown in Fig. 2E. ‘Secondary antibody only’ served as a negative control, and did not show any signal (data not shown). Surface localization of Syndecan-1 in cumulus cells We localized Syndecan-1 in mouse cumulus cells by IIF as illustrated in Fig. 3. The optical Z stack of cells was taken at an interval 168
Figure 1 Western blot and IIF analysis of LAR. (A) Western blot analysis of LAR on rat testis epididymal protein. Rat testicular extract (T), epididymal extract (E), Arrows indicate specific bands. Lower panel represents beta actin, indicating equal load. (B) Western analysis of LAR in human spermatozoa (HSP), and Mouse caudal epididymal spermatozoa (MSP). Arrows indicate the bands of interest. Lower panel represents beta actin, indicating equal load. Negative control (NC) did not show any reactivity. Indirect immunofluorescence analysis of LAR on Mouse spermatozoa. Arrows indicate localization of LAR in the anterior region of acrosome of mouse (C) and Human spermatozoa (D). Insets represent respective DIC images. Magnification 639 with 4 9 optical zoom. Insets represent respective DIC images. Negative controls (E, F) did not show any reactivity (Scale bar represents 5 lM).
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of 0.5 lM. The surface localization of Syndecan-1 can be seen as indicated by arrows. No staining was seen in the cytoplasm. Negative control did not show any reactivity (data not shown). Anti-Liprin a3 antibody inhibits acrosome reaction Liprin proteins are known to interact with vesicle associated protein to bring about the membrane fusion reaction in the synapse. We hypothesized that Liprin alpha proteins are also involved in AR. To test this hypothesis, we incubated capacitated mouse spermatozoa in presence of Liprin a3 antibody and normal rabbit IgG (Isotype control) and checked for AR using FITC-PSA. The positive staining of FITC-PSA is an indication of intact acrosome while no FITC-PSA staining on sperm head is a sign of loss of acrosome or acrosome-reacted spermatozoa. Capacitated © 2013 American Society of Andrology and European Academy of Andrology
ROLE OF LIPRIN a3 AND LAR IN ACROSOME REACTION Figure 2 Colocalization of RIM, LAR and Liprin a3 in mouse spermatozoa. (A) Colocalization of LAR and Liprin a3 on anterior acrosome, LAR (green), Liprin a3 (Red) and colocalization (Yellow), (B) depicts an extent of overlap, pixels in the third quadrant represent overlapping pixels indicating colocalization of two signals. (C) Colocalization of RIM and Liprin a3 on anterior acrosome, RIM (green), Liprin a3 (Red) and colocalization (Yellow), (D) depicts overlapping pixels in quadrant 3, indicating colocalization of RIM and Liprin a3. (Scale bar represents 5 lM). (E) represents Overlap coefficient graph for respective colocalizations.
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spermatozoa challenged with calcium ionophore showed 90% acrosome-reacted spermatozoa as represented in Fig. 4A as AR. However, when capacitated spermatozoa were incubated with Liprin a3 antibody (1 : 10 or 1 : 20) and later challenged with calcium ionophore the number of reacted spermatozoa reduced significantly in concentration dependent manner. One in ten dilution antibody showed 30% spermatozoa undergoing AR whereas 1 in 20 diluted showed 40% spermatozoa undergoing AR, indicating a dose-dependent inhibitory effect of the antibody on AR. Normal IgG showed 60% acrosome-reacted spermatozoa, which was equivalent to number of spontaneously reacted spermatozoa in capacitated population (cap), indicating no effect of IgG on AR (p > 0.05 indicating non-significant change). © 2013 American Society of Andrology and European Academy of Andrology
Effect of LAR ligands on acrosome status In synapse function and development, LAR ligands such as Nidogens and Syndecans play a key role. Activity of LAR is largely affected and regulated by these ligands. To check its effect on AR we incubated capacitated mouse spermatozoa with these ligands at different concentrations. The data obtained show a dose-dependent effect on activation of AR as summarized in Fig. 4B. A quantity of 50 ng of ligands showed a significant increase in AE index as compared to 10 ng. Nidogen 1 and 2 (at a concentration of 50 ng) showed an increase in AE index by around 30%, whereas Syndecan 1, 2, 3 (50 ng) showed around 30–45% increase in AE index. Andrology, 2014, 2, 165–174
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ANDROLOGY Figure 3 Localization of Syndecan-1 on cumulus cells. Arrows show surface localization of Syndecan-1. (Scale bar represents 2 lM). The optical Z stack was taken at 0.5 lM interval.
Negative control did not show any effect on AR (data not shown). Effect of LAR wedge peptide on acrosome reaction To assess the effect of LAR wedge peptide on AR we used different concentrations of peptides such as 25, 50 and 100 lM. Scrambled peptide (SLAR-TAT) served as a negative control. The results obtained are summarized in Fig. 4C. Scrambled peptide did not show effect on AE index. Both WLAR and TAT-WLAR showed the effect on acrosome exocytosis index. WLAR peptide without tat sequence showed only 10% increase in AE index as compared to Tat-linked peptide, highlighting the importance of membrane-permeant Tat sequence. TAT-WLAR showed a 30% increase in AE index at 50 lM, indicating that higher number of spermatozoa undergo AR and underlies that Tat sequence facilitates the entry of peptide into the cell. Asterisk represents significant increase in AR (p < 0.05) in case of 50 lM concentration of Tat-WLAR.
DISCUSSION In our previous report, we identified Liprin a3 on sperm acrosome (Joshi et al., 2013). It has been shown that formation of RIM/MUNC/Rab3A/liprina complex is a critical component in the process of membrane fusion at synapse (Andrews-Zwilling et al., 2006). Of the molecules described in synaptic vesicle docking, the implication of RIM in sperm AR has already been reported (Bello et al., 2012). The anti- Munc antibody showed AR inhibition indicating involvement of Rab3/RIM/Munc13 complex in an early stage of membrane fusion (Bello et al., 2012). It has been shown that RIM protein activates vesicle 170
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priming by interacting with Munc (Deng et al., 2011) whereas another study carried out in brain demonstrated the association of RIM and Liprin a. It was observed that Liprin a proteins interact with RIM through its N-terminal coiled coil domain in a calcium-dependent manner (Ko et al., 2003). This study demonstrates that RIM and Liprin a3 coexist on mouse acrosome, thus providing evidence for the presence of RIM/MUNC/Rab3A/liprin a complex in acrosome as proposed earlier by us (Joshi et al., 2013). The process of AR requires a complex equilibrium between kinases and phosphatases. Studies conducted on epididymal spermatozoa demonstrated that the level of phosphorylation can be inversely correlated with spontaneous or calcium induced AR (Aitken et al., 2007). Liprins interact with LAR that belongs to phosphotyrosine phosphatase (PTPs) family. PTPs can be either cytosolic or receptor type (Pulido et al., 1995). LAR belongs to the receptor family of PTPs, which also includes PTPd, and PTPr. It was found that cytosolic tyrosine phosphatase PTP1B, is required for dephosphorylation of NSF (Zarelli et al., 2009; Aoki & Matsuda, 2000). It was found that Phospho-NSF is a substrate for PTP1B. The findings also indicated that dephosphorylation of NSF facilitates SNARE complex formation and thus increase membrane fusion activity. All these results highlight the importance of protein tyrosine phosphatase (PTPs) in AR. The presence of a partner of one such receptor PTPs (Liprin a3) on spermatozoa (Joshi et al., 2013) led us to speculate the presence of LAR on spermatozoa as well. To check the same, we performed Western blot and IIF analysis on spermatozoa. Our Western blot analysis on testicular and epididymal tissue showed varied sizes of LAR that are in agreement with data © 2013 American Society of Andrology and European Academy of Andrology
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Figure 4 Effect of various Liprin a3 antibody, LAR ligands, LAR wedge peptides on acrosome exocytosis. (A) Asterisk (*) indicates statistically significant decrease in acrosome reaction (AR) in presence of 1 in 10 diluted Anti Liprin a3 antibody (Anti Liprin 1 : 10) as compared with IgG control (*p = 0.006), **indicate significant decrease in AR in presence of 1 in 20 diluted anti-Liprin a3 antibody (Anti Liprin 1 : 20) (**p = 0.027), ***indicate significant decrease in AR (***p = 0.037). However, no significant change in AR can be seen in presence of IgG as compared with capacitated spermatozoa (cap) (+p = 0.565). (B) Effect of LAR ligands on acrosome exocytosis index. Nidogen-1 and 2, Syndecan-1, 2, 3 were used for experiment at 10 and 50 ng. Asterisk indicates statistically significant increase in AE index as compared with 10 ng concentration of same protein (p < 0.05). (C) Effect of LAR wedge peptide on acrosome exocytosis index. WLAR, TAT-WLAR and SLAR-TAT were used at 10, 50 and 100 µM concentration. Capacitated spermatozoa were incubated with these peptides and incubated for 30 min at 37 °C. Tat-WLAR at 50 lM shows statistically significant increase in AE index in comparison with other peptides (p < 0.05).
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demonstrated in liver and testis by Welham et al. (2009). They detected four bands 100, 75, 50, 30 kDa in testis using an antibody specific to trans-membrane region of LAR and attributed these bands to be processed LAR C-terminal Fragments (LARCTF). The anti-LAR antibody used by us was raised against a peptide of trans-membrane region as well, thereby suggesting the presence of LAR–CTF in testis and epididymis. It is observed that functionally relevant proteins are usually conserved across species. The presence LAR in rat, mouse and human spermatozoa as seen by us indicates its conserved nature and highlights its functional relevance in spermatozoa. The liprins were discovered in a pull down assay with LAR and therefore named as LAR-Interacting Protein 1 (LIP.1) (Serras et al., 1995). It was discovered that Liprin a interacts with Page s et al., LAR through its C-terminal LH domain (Serra-Page 1998). Both Liprins and LAR are known to be required for cargo transport and membrane fusion events at synapse (Zhen & Jin, © 2013 American Society of Andrology and European Academy of Andrology
1999; Miller et al., 2005). This study shows that LAR and Liprin a3 coexist on mouse acrosome (Fig. 5A) indicating probable interaction of these two proteins. Thus, we speculate that LAR-Liprin a3 on spermatozoa brings about a similar function as in synapse i.e. membrane fusion. On the basis of our colocalization results, we propose the interaction between LAR–Liprin a3RIM in mouse acrosome (Fig 5A). After having demonstrated the presence of LAR, Liprin a3 and RIM we decided to check the contribution of Liprin a3 to the AR. In presence of anti-Liprin a3 antibody we saw a decrease in the number of acrosome-reacted spermatozoa, indicating inhibition of AR. We speculate that the proposed interactome (Fig. 5A) for AR may be abrogated due to the anti-Liprin a3 antibody. This may lead to an incompetent configuration of molecules at acrosome leading to decreased acrosomal fusion. Leucocyte antigen related (LAR) is characterized by an extracellular domain composed of multiple immunoglobulin (Ig)-like Andrology, 2014, 2, 165–174
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Figure 5 Sperm plasma membrane: PM and acrosomal membrane: AM (A) The proposed Liprin a3 interactome involved in acrosome reaction (AR). This study shows the coexistence of Liprin a3, RIM and LAR in acrosome therefore, representing the presence of protein assembly required for AR. The colocalization of LAR and Liprin a3 indicates the importance of LAR. (B) The proposed pathway of LAR activity (a) The monomer of LAR has an accessible D1 phosphatase domain to its substrate like Akt and b-catenin. The dephosphorylation of Akt and b-catenin renders them inactive. Thus, resulting in unchanged actin cyto-architecture and no AR. (b) In presence of ligands like Syndcans and Nidogens, LAR undergoes dimerization. This makes D1 domain inaccessible to Akt and b-catenin. The active phosphorylated Akt and b-catenin can now activate AR (AR) (c) The wedge domain peptide mimics the dimerization and makes D1 catalytic domain inaccessible to its substrate leading to AR. (C) Cumulus is a site of AR (a) Spermatozoa are travelling across cumulus cells in their journey to reach an oocyte. (b) Cumulus exhibits surface Syndecans (c) Passing spermatozoa interact with Syndecans and undergo dimerization. (d) We proposed that this dimerization activates LAR downstream pathway as shown in (B) and AR is triggered and acrosomal exocytosis is brought about.
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domain and fibronectin type III repeats and two cytoplasmic catalytic domains D1 and D2. These domains have phosphatase activity. Various ligands interacting with LAR were discovered (O’Grady et al., 1998). The studies conducted in Drosophila and C. Elegans suggest that Ig domain has an important role at synapse and it interacts with extracellular ligands such as Syndecan (Sdc) and dally-like protein (Dlp) (Johnson et al., 2006). It was correlated that LAR Laminin–Nidogen interaction changes the actin cytoskeleton structure (O’Grady et al., 1998). These data suggest that the extracellular ligands interact with LAR through Ig domain, thereby regulating active zones at synapse. To unravel the mechanism, studies were conducted. It was found that upon binding with ligands, LAR demonstrates dimerization and inhibits function of catalytic domain. During dimerization, the wedge structure from D1 domain of one LAR interacts with D1 of other. This configuration makes a D1 catalytic site inaccessible to substrates thereby inhibiting LAR phosphatase activity (Bixby, 2001). This triggers various intracellular events such as TrkA and b- catenin phosphorylation (Xie et al., 2006). To study intracellular dimerization of wedge domain a peptide responsible for same needs to get incorporated into a live cell to mimic natural process. Attaching a Tat sequence to bait is the popular approach (Wadia & Dowdy, 2002). Xie et al. (2006) designed wedge peptide for LAR with a Tat peptide that would mimic the event of dimerization. It was found that in presence of wedge peptide, phosphorylation of TrkA is upregulated thus elevating the levels of phospho-Akt pathway (Xie et al., 2006). Similar effect was seen with beta catenin wherein LAR regulates the actin polymerization and actin-based cell motility (Kypta et al., 1996) (Fig. 5B a). All these reports point towards the possibility that, if LAR is present on spermatozoa then it may follow a similar pattern and activate cascade required for membrane fusion in spermatozoa. To test the same, we devised two different experiments. In the first set of experiments, spermatozoa were challenged with different concentrations of LAR ligands like Syndecans and Nidogens. We observed a dose-dependent increase in the acrosome exocytosis index. In another set of experiments, we spiked spermatozoa with membrane-permeant Tat-LAR wedge peptide and the effect was similar to that seen in the presence of ligands. We speculate that in presence of LAR ligands or wedge peptide LAR undergoes or mimics dimerization leading to inhibition of phosphatase activity (Fig 5B b and c). This could promote Akt phosphorylation and activation of the downstream signalling pathway. The elevated level of Phospho-Akt then upregulates hypermotility and the AR (Sagare-Patil et al., 2013). The LAR activity may also change actin cyto-architecture (Kypta et al., 1996) and such changes are required to promote AR (Brener et al., 2003). Hence, we could see an increase in acrosome exocytosis index. There are two different schools of thought that propose the triggering pathway of AR. According to one school, Zona pellucida (ZP) proteins mediate AR (Gupta and Bhandari, 2011), and is the most accepted site of AR. However, the other school considers that the AR probably happens before interaction of spermatozoa with ZP, perhaps in the cumulus (Cummins & Yanagimachi, 1986). The studies conducted by Inoue et al. (2011) and Jin et al. (2011) demonstrate clearly that cumulus but not ZP is essential for AR. However, these reports did not look for the cumulus proteins that are involved in AR. Our IIF © 2013 American Society of Andrology and European Academy of Andrology
data clearly show the presence of one such LAR ligand Syndecan-1 on cumulus cell surface (Fig. 5C b). This was followed by experiments, which indicate that cumulus proteins like Syndecan -1, 2, 3 may activate AR. This gives us a preliminary evidence to postulate that in in vivo situations these cumulus Syndecans may induce dimerization of LAR (Fig. 5C c) and trigger downstream pathway that activate AR in spermatozoa (Fig. 5C d). However, extensive experiments need to be carried out to prove this hypothesis.
CONCLUSION Overall, this study demonstrates that sperm AR is driven by common set of proteins like Liprin a3, LAR, RIM shown to be responsible for membrane fusion at synapse. We could also demonstrate that the ligands and wedge peptide can induce LAR dimerization and could be one of the mechanisms of stimulating acrosomal exocytosis. The observations support the hypothesis that cumulus could be another site of AR.
ACKNOWLEDGEMENTS The authors thank Ms. Shobha Sonawane and Ms. Reshma Gaonkar for their assistance at the confocal microscopy department of NIRRH. The authors also thank Dr. Rahul Gajabhiye, clinician, for providing normal semen sample from institutional clinic.
DISCLOSURES Financial assistance provided to CJ by Department of Science and Technology, New Delhi as DST-JRF, and Indian Council of Medical Research (ICMR), New Delhi for ICMR-SRF and by NIRRH, Mumbai is gratefully acknowledged.
AUTHOR CONTRIBUTIONS CJ and SK performed experiments, analysed the data and wrote the manuscript, VK designed, supervised the research study, wrote and corrected the manuscript.
REFERENCES Aitken RJ, Nixon B, Lin M, Koppers AJ, Lee YH & Baker MA. (2007) Proteomic changes in mammalian spermatozoa during epididymal maturation. Asian J Androl 9, 554–564. Andrews-Zwilling YS, Kawabe H, Reim K, Varoqueaux F & Brose N. (2006) Binding to Rab3A-interacting molecule RIM regulates the presynaptic recruitment of Munc13-1 and ubMunc13-2. J Biol Chem 281, 19720–19731. Aoki N & Matsuda TA. (2000) Cytosolic protein-tyrosine phosphatase PTP1B specifically dephosphorylates and deactivates prolactin-activated STAT5a and STAT5b. J Biol Chem 275, 39718–39726. Bello OD, Zanetti MN, Mayorga LS & Michaut MA. (2012) RIM, Munc13, and Rab3A interplay in acrosomal exocytosis. Exp Cell Res 318, 478–488. Benoff S. (1998) Modelling human sperm-egg interactions in vitro: signal transduction pathways regulating the acrosome reaction. Mol Hum Reprod 4, 453–471. Bixby JL. (2001) Ligands and signaling through receptor-type tyrosine phosphatases. IUBMB Life 51, 157–163. Brener E, Rubinstein S, Cohen G, Shternall K, Rivlin J & Breitbart H. (2003) Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction. Biol Reprod 68, 837–845. Cummins JM & Yanagimachi R. (1986) Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated Andrology, 2014, 2, 165–174
173
C. S. Joshi, S. A. Khan and V. V. Khole in vitro, in relation to the timing of the acrosome reaction. Gamete Res 15, 187–212. € dhof TC. (2011) RIM proteins activate Deng L, Kaeser PS, Xu W & Su vesicle priming by reversing auto inhibitory homo dimerization of Munc13. Neuron 69, 317–331. Gupta SK & Bhandari B. (2011) Acrosome reaction: relevance of zona pellucida glycoproteins. Asian J Androl 13, 97–105. Inoue N, Satouh Y, Ikawa M, Okabe M & Yanagimachi R. (2011) Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs. Proc Natl Acad Sci U S A 108, 20008–20011. Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA et al. (2011) Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proc Natl Acad Sci U S A 108, 4892–4896. Johnson KG, Tenney AP, Ghose A, Duckworth AM, Higashi ME, Parfitt K et al. (2006) The HSPGs Syndecan and Dally like bind the receptor phosphatase LAR and exert distinct effects on synaptic development. Neuron 49, 517–531. Joshi CS, Suryawanshi AR, Khan SA, Balasinor NH & Khole VV. (2013) Liprin a3: a putative estrogen regulated acrosomal protein. Histochem Cell Biol 139, 535–548. Ko J, Na M, Kim S, Lee J-R & Kim E. (2003) Interaction of the ERC family of RIM-binding proteins with the liprin-alpha family of multidomain proteins. J Biol Chem 278, 42377–42385. Kypta RM, Su H & Reichardt LF. (1996) Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex. J Biol Chem 134, 1519–1529. Mendoza C, Carreras A, Moos J & Tesarik J. (1992) Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 5, 755–763. Michaut M, De Blas G, Tomes CN, Yunes R, Fukuda M & Mayorga LS. (2001) Synaptotagmin VI participates in the acrosome reaction of human spermatozoa. Dev Biol 235, 521–529. Miller KE, De Proto J, Kaufmann N, Patel BN, Duckworth A & Van Vactor D. (2005) Direct observation demonstrates that Liprin-alpha is required for trafficking of synaptic vesicles. Curr Biol 15, 684–689. O’Grady P, Thai TC & Saito H. (1998) The laminin-nidogen complex is a ligand for a specific splice isoform of the transmembrane protein tyrosine phosphatase LAR. J Cell Biol 141, 1675–1684. Pires ES, Choudhury AK, Idicula-Thomas S & Khole VV. (2011) Anti-HSP90 autoantibodies in sera of infertile women identify a dominant, conserved epitope EP6 (380-389) of HSP90 beta protein. Reprod Biol Endocrinol 9, 16. s C, Tang M & Streuli M. (1995) The LAR/PTP delta/ Pulido R, Serra-Page PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1. Proc Natl Acad Sci U S A 92, 11686–11690. Ramalho-Santos J, Moreno RD, Sutovsky P, Chan AW, Hewitson L, Wessel GM et al. (2000) SNAREs in mammalian sperm: possible implications for fertilization. Dev biol 223, 54–69. Sagare-Patil V, Vernekar M, Galvankar M & Modi D. (2013) Progesterone utilizes the PI3K-AKT pathway in human spermatozoa to regulate
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ANDROLOGY motility and hyperactivation but not acrosome reaction. Mol Cell Endocrinol 374, 82–91. Schoch S, Castillo PE, Jo T, Mukherjee K, Geppert M, Wang Y et al. (2002) RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature 415, 321–326. Serra-Pag es C, Kedersha NL, Fazikas L, Medley Q, Debant A & Streuli M. (1995) The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions. EMBO J 14, 2827–2838. Serra-Pag es C, Medley QG, Tang M, Hart A & Streuli M. (1998) Liprins, a family of LAR transmembrane protein-tyrosine phosphatase-interacting proteins. J Biol Chem 273, 15611–15620. Spangler SA & Hoogenraad CC. (2007) Liprin-alpha proteins: scaffold molecules for synapse maturation. Biochem Soc Trans 35, 1278–1282. Tomes CN, Michaut M, De Blas G, Visconti P, Matti U & Mayorga LS. (2002) SNARE complex assembly is required for human sperm acrosome reaction. Dev biol 243, 326–338. Wadia JS & Dowdy SF. (2002) Protein transduction technology. Curr Opin Biotechnol 13, 52–56. € dhof TC. (1997) Rim is Wang Y, Okamoto M, Schmitz F, Hofmann K & Su a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature 388, 593–598. Watson LN, Mottershead DG, Dunning KR, Robker RL, Gilchrist RB & Russell DL. (2012) Heparan sulfate proteoglycans regulate responses to oocyte paracrine signals in ovarian follicle morphogenesis. Endocrinology 153, 4544–4555. Welham SJM, Clark AJL & Salter AM. (2009) A novel liver specific isoform of the rat LAR transcript is expressed as a truncated isoform encoded from a 5’UTR located within intron 11. BMC Mol Biol 10, 30. WHO. (2010) WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. Geneva, Switzerland. ISBN 978 92 4 154778 9. Xie Y, Massa SM, Ensslen-Craig SE, Major DL, Yang T, Tisi MA et al. (2006) Protein-tyrosine phosphatase (PTP) wedge domain peptides: a novel approach for inhibition of PTP function and augmentation of protein-tyrosine kinase function. J Biol Chem 281, 16482–16492. Yanagimachi R. (2011) Mammalian sperm acrosome reaction: where does it begin before fertilization? Biol Reprod 85, 4–5. Yang T, Bernabeu R, Xie Y, Zhang JS, Massa SM, Rempel HC et al. (2003) Leukocyte antigen-related protein tyrosine phosphatase receptor: a small ectodomain isoform functions as a homophilic ligand and promotes neurite outgrowth. J Neurosci 23, 3353–3363. Yang T, Yin W, Derevyanny VD, Moore LA & Longo FM. (2005) Identification of an ectodomain within the LAR protein tyrosine phosphatase receptor that binds homophilically and activates signalling pathways promoting neurite outgrowth. Eur J Neurosci 22, 2159–2170. Yang T, Massa SM & Longo FM. (2006) LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathways. J Neurobiol 66, 1420–1436. Zarelli VEP, Ruete MC, Roggero CM, Mayorga LS & Tomes CN. (2009) PTP1B dephosphorylates N-ethylmaleimide-sensitive factor and elicits SNARE complex disassembly during human sperm exocytosis. J Biol Chem 284, 10491–10503. Zhen M & Jin Y. (1999) The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature 401, 371–375.
© 2013 American Society of Andrology and European Academy of Andrology
ISSN: 2047-2919
ORIGINAL ARTICLE
Correspondence: Vrinda V. Khole, Department of Gamete Immunobiology, National Institute for Research in Reproductive Health, ICMR, J. M. Street, Parel, Mumbai 400012, India. E-mail: [email protected]
Keywords: acrosome reaction, leucocyte antigen-related protein, liprin a3, nidogen, rab-interacting molecule, spermatozoa, syndecan, wedge peptide
Regulation of acrosome reaction by Liprin a3, LAR and its ligands in mouse spermatozoa C. S. Joshi, S. A. Khan and V. V. Khole Department of Gamete Immunobiology, National Institute for Research in Reproductive Health, Mumbai, India
Received: 28-Sep-2013 Revised: 1-Nov-2013 Accepted: 4-Nov-2013 doi: 10.1111/j.2047-2927.2013.00167
SUMMARY Zona pellucida-based induction of acrosome reaction (AR) is a popular and well-accepted hypothesis. However, this hypothesis is being challenged in recent years and it has been proposed that the cumulus cells might be the site of AR. In our previous study, we reported the presence of a synaptic protein Liprin a3 on sperm acrosome, and proposed its role in AR. This study was designed to understand the role of Liprin a3 and its interacting proteins in regulation of AR. It is observed that the presence of anti-Liprin a3 antibody inhibits the process of AR. Colocalization experiments demonstrate the coexistence of leucocyte antigen related (LAR) protein, Rab-interacting molecule (RIM) and Liprin a3 on sperm acrosome thereby completing the identification of all the members of RIM/ MUNC/Rab3A/liprina complex required for membrane fusion. This study demonstrates the effect of LAR ligands such as Syndecans, Nidogens and LAR wedge domain peptide on AR. We could see an increase in AR in presence of these ligands. On the basis of these data, we speculate that in presence of ligands or wedge peptide, LAR undergoes dimerization leading to inhibition of phosphatase activity and increase in AR. The presence of one of the ligands Syndecan-1 on cumulus cells led us to hypothesize that it is Syndecan which induces AR in vivo and thus another site of AR could lie in cumulus.
INTRODUCTION Acrosome is a unique and special type of vesicle, designed to play a crucial role in controlling of the fertilizing ability of spermatozoa. The release of acrosomal content is a regulated exocytosis process that involves calcium and several other players. Several studies suggest that synaptic vesicle exocytosis and acrosomal exocytosis are mediated by a common set of proteins such as N-Ethylamide sensitive factor (NSF) (Zarelli et al., 2009), soluble NSF attachment protein receptor (SNARE) (Ramalho-Santos et al., 2000), soluble NSF attachment protein (Wadia & Dowdy, 2002), Synaptotagmin (Michaut et al., 2001), Rab-Interacting Molecule (RIM) (Bello et al., 2012) etc. It was proposed and also proven that RIM serves as a Rab3-dependent regulator of synapse fusion (Wang et al., 1997; Schoch et al., 2002). However, interaction between RIM and Liprin in acrosome has not been demonstrated. We recently reported the presence of synaptic protein Liprin a3 on spermatozoa and its involvement in acrosome biogenesis s (Joshi et al., 2013). Liprins were first discovered by Serra-Page et al. (1995) at focal adhesion by trapping proteins interacting © 2013 American Society of Andrology and European Academy of Andrology
with cytoplasmic region of leucocyte antigen-related (LAR) protein. C-terminus of liprins show Liprin homology (LH) domain, composed of three sterile alpha motifs (SAMs) that interacts with LAR. It has been described that Liprin a3 and LAR protein are present in the brain and neuronal tissues and function as scaffolding molecules and are involved in vesicle transport across the pre-synaptic junctions (Spangler & Hoogenraad, 2007). The function of LAR was studied extensively in PC12 cells using wedge peptides by Yang et al. (2003, 2005, 2006) and Xie et al. (2006). The wedge peptide showed inhibitory effect on LAR phosphatase activity thereby affecting its downstream potential targets like beta catenin and TrkA (Xie et al., 2006; Yang et al., 2006). The receptor–ligand-based acrosome reaction (AR) has been proposed by Benoff (1998). He proposed that the binding of glycosylated residues present on egg interact with their respective receptor present on spermatozoa and activate AR. It was found that the ectodomain of LAR is involved in ligand–receptor interactions. Sulphated proteoglycan like Syndecans and Nidogens are also known to interact with LAR (Johnson et al., 2006). These Andrology, 2014, 2, 165–174
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ligands are reported to be present on cumulus cells (Watson et al., 2012); however, their role has never been studied with reference to their counterpart present on the spermatozoa. To date, there are no reports of presence of LAR and its involvement in sperm function. In this study, we demonstrate the interaction of Liprin a3 with RIM and LAR and show the importance of interaction of Liprin a3 and LAR in AR. Our data also support the hypothesis that another site of AR could be cumulus where LAR ligands are present.
MATERIALS AND METHODS Materials Phosphate-buffered saline (PBS), Western blot and sodium dodecyl sulphate-polyacryl amide gel electrophoresis chemicals were procured from SRL India (Mumbai, India). Nitrocellulose membranes (Hybond-C Extra) and ECL Plus Western blotting detection reagent were obtained from GE Health Care (Buckinghamshire, UK). Protein molecular weight markers were procured from Fermentas (MD, USA). Non-fat dry milk (NFDM) powder was procured from Anikspray (Mumbai, India). Bovine serum albumin (BSA) fraction V was obtained from USB (Cleveland, OH, USA). Human tubal fluid (HTF) was obtained from Irvin Scientific (Santa Ana, CA, USA). ProLong Gold anti-fade agent and all Alexa fluor antibodies were purchased from Invitrogen (Eugene, OR, USA). Pisum sativum-FITC (FITC-PSA), calcium ionophore A23187 and paraformaldehyde (PFA) were obtained from Sigma Aldrich (Steinheim, Germany). Complete protease inhibitor cocktail and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Roche (Mannheim, Germany), Kodak X-ray films were purchased from Asset healthcare (Silvassa, India). Antibodies and recombinant proteins Rabbit polyclonal anti-Liprin a3, chicken polyclonal antiPTPRF (LAR) and mouse monoclonal anti-b actin were procured from Sigma Aldrich (St. Louis, MO, USA); Anti RIM antibody was purchased from Synaptic System. Mouse polyclonal anti-Liprin a3 was procured from Novus Biologicals. Recombinant Syndecan-1, 2, 3, Nidogen-1, 2 were procured from R&D systems (Minneapolis, MN, USA). Peptides Wedge peptides as described by Xie et al. (2006) were synthesized with an amide substitute at C-terminus. WLAR wedge sequence without Tat, TAT-WLAR wedge sequence with Tat peptide sequence (underlined) linked to C-terminus and SLAR-TAT scrambled wedge sequence with Tat peptide sequence (underlined) were synthesized at by USV ltd (Mumbai, India). WLAR: NH2- DLADNIERLKANDGLKFSQEYESI-NH2 TAT-WLAR: NH2- GRKKRRQRRRCDLADNIERLKANDGLKFS QEYESI-NH2 SLAR-TAT: NH2- NADKGLSLRKEFSIQNEDAYLEDIGRKKRRQ RRRC-NH2 Animals Mature Swiss male and female mice and Holtzman male rat were used for the study. Animals were maintained at a temperature of 22–23 °C, humidity of 50–55% and a cycle of 14 h light 10 h dark with food and water available ad libitum. All animal 166
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care practices and experimental procedures complied with the guidelines of the Care and Prevention Society against Cruelty of Experimental Animals (CPCSEA) and were approved by the Institutional Animal Ethics Committee (IAEC). Human spermatozoa sample We collected human semen sample from participants attending the infertility clinic at the institute. Ethical approval was obtained from the Institutional Human Ethics Committee for clinical studies. Samples were analysed as per the guideline set by WHO, 2010. Samples with normal parameters were included in this study. Methods Spermatozoa preparation We sacrificed four mature male Holtzman rat or Swiss mice by CO2 asphyxiation. Cauda epididymides were removed and placed in pre-warmed HTF. Three to four radial cuts were made to release the spermatozoa. Swim up spermatozoa population was taken and washed with 0.1 M PBS, and suspended either in lysis buffer (1% SDS and protease inhibitor tablet) for protein extraction or in 4% PFA-PBS for immunofluorescence. Tissue preparation Rats were sacrificed; testis and epididymides were dissected and subjected for protein extraction as described (Joshi et al., 2013) and protein was quantified by Bradford’s method. Human spermatozoa sample preparation Human spermaozoa were processed as described earlier (Joshi et al., 2013). Semen sample of 150 lL was layered on the bottom of a Falcon tube containing 1.5 mL washing medium (HTF). After 30 min, the upper part (1 mL) of the supernatant was aspirated. Supernatant was spun at 500 g for 10 min, and pellet was either reconstituted in medium and used either for protein extraction or fixed in PFA as mentioned above and used for IIF. Western blot analysis We used protein from rat testis and epididymis, mouse and human spermatozoa for Western blot analysis as described earlier (Joshi et al., 2013). A quantity of 40 lg of protein was loaded on 10% polyacrylamide gel and SDS PAGE was carried out. Protein was electro trans-blotted to nitrocellulose membrane. Nonspecific sites on the membrane were blocked by incubating blots with 5% NFDM-PBS for 1 h at RT. Blots were incubated with anti-PTPRF antibody diluted 1 : 2000 in 0.1 M PBS at 4 °C overnight. They were then washed thrice with 0.1% Tween 20-PBS for 10 min each. Blots were incubated with Horse Raddish Peroxidase conjugated to rabbit anti-chicken (1 in 3000 dilution in 1% NFDM-PBS) for 1 h at RT and then washed as mentioned above. Detection was carried out by using enhanced chemiluminescence kit (ECL Plus) and recorded on Kodak blue base X-ray sheets. A blot incubated with no primary antibody was treated as a ‘Negative control’. Blots were stripped and probed with beta actin antibody which served as loading control. Indirect immunofluorescence We used 4% PFA-PBS fixed spermatozoa for all immunofluorescence as described earlier (Joshi et al., 2013). Cells were © 2013 American Society of Andrology and European Academy of Andrology
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incubated with blocking solution containing 3% BSA-PBS for 1 h at RT. Spermatozoa cells were incubated with polyclonal antiPTPRF diluted to 1 in 100 in 0.1 M PBS overnight at 4 °C. Excess of primary antibody was removed by washing the slides thrice with 0.1 M PBS. Alexa fluor 488 conjugated goat anti-chicken was used at 1 : 200 dilution and incubated for 1 h at RT along with counterstain DAPI (300 nM). Slides were washed as mentioned above to remove the excess of secondary antibodies. Slides were mounted in ProLong Gold antifade agent and examined under confocal fluorescent microscope (LSM 510; META Zeiss, Jena, Germany). A slide incubated with no primary antibody served as a ‘Negative control’. Colocalization We used PFA fixed sperm smears for colocalization of Liprin a3, PTPRF (LAR) and RIM1/2. We followed protocol for indirect immunofluorescence (IIF) of Liprin a3 as described previously by Joshi et al. (2013) with some modification. Briefly, fixed spermatozoa were permeabilized with 0.1% Triton X-100 for 15 min at RT. Non-specific sites were blocked by incubating the slides with 2% BSA-PBS for 1 h at RT. A cocktail of rabbit polyclonal anti-Liprin a3 and polyclonal chicken anti-PTPRF (both antibodies diluted to 1 in 50 in 0.1 M PBS) or mouse polyclonal anti-Liprin a3 and rabbit polyclonal anti-RIM1/2 (both antibodies diluted to 1 in 50 in 0.1 M PBS) was incubated with sperm smear overnight at 4 °C. Excess of primary antibody was removed by washing the slides thrice with 0.1 M PBS. A cocktail of Alexa fluor 488 goat anti-chicken and Alexa fluor 594 goat anti-rabbit diluted at 1 in 100 or a cocktail of Alexa fluor 488 goat anti-rabbit and Alexa fluor 594 goat anti-mouse diluted at 1 in 100 was incubated at RT for 1 h along with nuclear stain DAPI. Slides were washed as mentioned above to remove the excess of secondary antibodies. A slide incubated with no primary antibody served as a ‘Negative control’. Slides were mounted with ProLong Gold antifade and were examined under confocal microscope (LSM 510 META Zeiss). Areas showing overlap of green and red were subjected for calculation of ‘Overlap coefficient’. Cut mask images were generated. Ten random spermatozoa were selected from each set of experiments and overlap coefficient was calculated using in built software of LSM 510 META microscope. Mean and SEM was calculated. Cumulus oophorus preparation and localization of Syndecan-1 Superovulation was induced in 21-day-old female Swiss mice to obtain cumulus–oophorus complex (COCs) as per the protocol described previously by Pires et al. (2011). Briefly, 20 IU of pregnant mare serum gonadotropin (PMSG) was administered intraperitoneally. After 48 h, 20 IU of human chorionic gonadotropin was injected and after 16–18 h animals were sacrificed. Oviduct was dissected under stereo microscope (Discovery V8 SterREO; Carl Zeiss, Gottingen, Germany) and COCs were collected in small Petri dish and were fixed with 4% PFA. Blocking was carried out by incubating the COCs with 1% BSA for 1 h at RT. Anti Syndecan-1 was used at a dilution of 1 : 100 for 1 h at RT. Excess of primary antibody was washed with 0.01 M PBS. Alexa Fluor 488 goat anti-rabbit was used along with counterstain DAPI for 1 h at RT. Excess of secondary antibody was washed as described previously and mounted with ProLong Gold antifade. Slides were observed under confocal microscope (LSM 510 META Zeiss). © 2013 American Society of Andrology and European Academy of Andrology
Capacitation and acrosome reaction Three mature Swiss male mice were sacrificed and cauda epididymides were dissected out. Three to four radial cuts were made to release the spermatozoa into the medium. Spermatozoa were then suspended in the HTF medium supplemented with 0.4% BSA and capacitation was carried out for 1 h at 37 °C in an atmosphere of 5% CO2. We used capacitated spermatozoa to carry out AR. We carried out assay in presence of calcium ionophore A23187 (10 lM), wedge peptide (25, 50 and 100 lM), ligands like Syndecan 1,2,3, Nidogen 1 and 2 (10 and 50 ng of each) and Anti Liprin a3 antibody by incubating the spermatozoa with each of these individually and separately for minimum 30 min at 37 °C for each condition. At the end of an assay, spermatozoa were smeared on the glass slides, air dried and fixed with 4% PFA at RT for 5 min. Acrosomal status was assessed by staining with FITC-PSA (1 : 300) as described by Mendoza et al. (1992). At least 200 cells were counted using fluorescent microscope (Zeiss Axioskop 2 Plus). Spermatozoa with positive green signal on acrosome were considered as acrosome-intact spermatozoa whereas those without green signal were considered as acrosome-reacted spermatozoa. We included ‘stimulation by calcium ionophore’ as a positive control and ‘no calcium ionophore’ as a negative control in each experiment. For negative control of ligands based assay, ligands pre-incubated with its respective antibodies were used. Experiment was performed three times and statistical analysis was performed. Statistical analysis Acrosome exocytosis index (AE index) values were calculated by subtracting number of spontaneously reacted spermatozoa in the negative control from all test samples. Resulting values were then expressed as a percentage with respect to positive control. Student’s t-test was used for statistical analysis. Differences were considered significant at the p < 0.05 level.
RESULTS PTPRF (LAR) is present in the spermatozoa and is conserved We used protein extract of rat testis, epididymis, mouse and human spermatozoa for Western blot analysis of LAR. LAR was detected as 130, 100, 75, 50 and 30 kDa bands in testicular extract, whereas in epididymal extract we could see 100, 50 and 30 kDa bands as shown in Fig. 1A. The protein was also detected in human and mouse spermatozoa extracts and is represented in Fig. 1B. Respective negative control showed no specific bands (NC). IIF studies as shown in Fig. 1C,D depict positive reactivity (green fluorescence) with anti-LAR antibody at the anterior region of mouse (Fig. 1C) and human sperm acrosome (Fig. 1D). Insets represent respective DIC images. Negative controls did not show any reactivity (Fig. 1E,F). LAR and RIM colocalize with Liprin a3 Colocalization of RIM and LAR with Liprin a3 on anterior acrosome is represented in Fig. 2. Green fluorescence indicates positive staining of LAR, red fluorescence shows staining of Liprin a3 and yellow staining shows colocalization of LAR and Liprin a3 protein as shown in Fig. 2A. Figure 2B depicts overlap graph in which third quadrant shows overlapping pixels only, indicating Andrology, 2014, 2, 165–174
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(C)
(D)
(E)
that two proteins coexist with an overlapping coefficient of 0.88 with SEM of 0.02 as shown in Fig. 2E. Figure 2C depicts the colocalization of RIM-Liprin a3. Green fluorescence indicates positive staining of RIM, red fluorescence shows staining of Liprin a3 and yellow staining shows colocalization of RIM and Liprin a3 protein as shown in Fig. 2C. Figure 2D depicts the overlapping pixels in the third quadrant with an overlapping coefficient of 0.922 with SEM of 0.026 as shown in Fig. 2E. ‘Secondary antibody only’ served as a negative control, and did not show any signal (data not shown). Surface localization of Syndecan-1 in cumulus cells We localized Syndecan-1 in mouse cumulus cells by IIF as illustrated in Fig. 3. The optical Z stack of cells was taken at an interval 168
Figure 1 Western blot and IIF analysis of LAR. (A) Western blot analysis of LAR on rat testis epididymal protein. Rat testicular extract (T), epididymal extract (E), Arrows indicate specific bands. Lower panel represents beta actin, indicating equal load. (B) Western analysis of LAR in human spermatozoa (HSP), and Mouse caudal epididymal spermatozoa (MSP). Arrows indicate the bands of interest. Lower panel represents beta actin, indicating equal load. Negative control (NC) did not show any reactivity. Indirect immunofluorescence analysis of LAR on Mouse spermatozoa. Arrows indicate localization of LAR in the anterior region of acrosome of mouse (C) and Human spermatozoa (D). Insets represent respective DIC images. Magnification 639 with 4 9 optical zoom. Insets represent respective DIC images. Negative controls (E, F) did not show any reactivity (Scale bar represents 5 lM).
(B)
(A)
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of 0.5 lM. The surface localization of Syndecan-1 can be seen as indicated by arrows. No staining was seen in the cytoplasm. Negative control did not show any reactivity (data not shown). Anti-Liprin a3 antibody inhibits acrosome reaction Liprin proteins are known to interact with vesicle associated protein to bring about the membrane fusion reaction in the synapse. We hypothesized that Liprin alpha proteins are also involved in AR. To test this hypothesis, we incubated capacitated mouse spermatozoa in presence of Liprin a3 antibody and normal rabbit IgG (Isotype control) and checked for AR using FITC-PSA. The positive staining of FITC-PSA is an indication of intact acrosome while no FITC-PSA staining on sperm head is a sign of loss of acrosome or acrosome-reacted spermatozoa. Capacitated © 2013 American Society of Andrology and European Academy of Andrology
ROLE OF LIPRIN a3 AND LAR IN ACROSOME REACTION Figure 2 Colocalization of RIM, LAR and Liprin a3 in mouse spermatozoa. (A) Colocalization of LAR and Liprin a3 on anterior acrosome, LAR (green), Liprin a3 (Red) and colocalization (Yellow), (B) depicts an extent of overlap, pixels in the third quadrant represent overlapping pixels indicating colocalization of two signals. (C) Colocalization of RIM and Liprin a3 on anterior acrosome, RIM (green), Liprin a3 (Red) and colocalization (Yellow), (D) depicts overlapping pixels in quadrant 3, indicating colocalization of RIM and Liprin a3. (Scale bar represents 5 lM). (E) represents Overlap coefficient graph for respective colocalizations.
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spermatozoa challenged with calcium ionophore showed 90% acrosome-reacted spermatozoa as represented in Fig. 4A as AR. However, when capacitated spermatozoa were incubated with Liprin a3 antibody (1 : 10 or 1 : 20) and later challenged with calcium ionophore the number of reacted spermatozoa reduced significantly in concentration dependent manner. One in ten dilution antibody showed 30% spermatozoa undergoing AR whereas 1 in 20 diluted showed 40% spermatozoa undergoing AR, indicating a dose-dependent inhibitory effect of the antibody on AR. Normal IgG showed 60% acrosome-reacted spermatozoa, which was equivalent to number of spontaneously reacted spermatozoa in capacitated population (cap), indicating no effect of IgG on AR (p > 0.05 indicating non-significant change). © 2013 American Society of Andrology and European Academy of Andrology
Effect of LAR ligands on acrosome status In synapse function and development, LAR ligands such as Nidogens and Syndecans play a key role. Activity of LAR is largely affected and regulated by these ligands. To check its effect on AR we incubated capacitated mouse spermatozoa with these ligands at different concentrations. The data obtained show a dose-dependent effect on activation of AR as summarized in Fig. 4B. A quantity of 50 ng of ligands showed a significant increase in AE index as compared to 10 ng. Nidogen 1 and 2 (at a concentration of 50 ng) showed an increase in AE index by around 30%, whereas Syndecan 1, 2, 3 (50 ng) showed around 30–45% increase in AE index. Andrology, 2014, 2, 165–174
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ANDROLOGY Figure 3 Localization of Syndecan-1 on cumulus cells. Arrows show surface localization of Syndecan-1. (Scale bar represents 2 lM). The optical Z stack was taken at 0.5 lM interval.
Negative control did not show any effect on AR (data not shown). Effect of LAR wedge peptide on acrosome reaction To assess the effect of LAR wedge peptide on AR we used different concentrations of peptides such as 25, 50 and 100 lM. Scrambled peptide (SLAR-TAT) served as a negative control. The results obtained are summarized in Fig. 4C. Scrambled peptide did not show effect on AE index. Both WLAR and TAT-WLAR showed the effect on acrosome exocytosis index. WLAR peptide without tat sequence showed only 10% increase in AE index as compared to Tat-linked peptide, highlighting the importance of membrane-permeant Tat sequence. TAT-WLAR showed a 30% increase in AE index at 50 lM, indicating that higher number of spermatozoa undergo AR and underlies that Tat sequence facilitates the entry of peptide into the cell. Asterisk represents significant increase in AR (p < 0.05) in case of 50 lM concentration of Tat-WLAR.
DISCUSSION In our previous report, we identified Liprin a3 on sperm acrosome (Joshi et al., 2013). It has been shown that formation of RIM/MUNC/Rab3A/liprina complex is a critical component in the process of membrane fusion at synapse (Andrews-Zwilling et al., 2006). Of the molecules described in synaptic vesicle docking, the implication of RIM in sperm AR has already been reported (Bello et al., 2012). The anti- Munc antibody showed AR inhibition indicating involvement of Rab3/RIM/Munc13 complex in an early stage of membrane fusion (Bello et al., 2012). It has been shown that RIM protein activates vesicle 170
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priming by interacting with Munc (Deng et al., 2011) whereas another study carried out in brain demonstrated the association of RIM and Liprin a. It was observed that Liprin a proteins interact with RIM through its N-terminal coiled coil domain in a calcium-dependent manner (Ko et al., 2003). This study demonstrates that RIM and Liprin a3 coexist on mouse acrosome, thus providing evidence for the presence of RIM/MUNC/Rab3A/liprin a complex in acrosome as proposed earlier by us (Joshi et al., 2013). The process of AR requires a complex equilibrium between kinases and phosphatases. Studies conducted on epididymal spermatozoa demonstrated that the level of phosphorylation can be inversely correlated with spontaneous or calcium induced AR (Aitken et al., 2007). Liprins interact with LAR that belongs to phosphotyrosine phosphatase (PTPs) family. PTPs can be either cytosolic or receptor type (Pulido et al., 1995). LAR belongs to the receptor family of PTPs, which also includes PTPd, and PTPr. It was found that cytosolic tyrosine phosphatase PTP1B, is required for dephosphorylation of NSF (Zarelli et al., 2009; Aoki & Matsuda, 2000). It was found that Phospho-NSF is a substrate for PTP1B. The findings also indicated that dephosphorylation of NSF facilitates SNARE complex formation and thus increase membrane fusion activity. All these results highlight the importance of protein tyrosine phosphatase (PTPs) in AR. The presence of a partner of one such receptor PTPs (Liprin a3) on spermatozoa (Joshi et al., 2013) led us to speculate the presence of LAR on spermatozoa as well. To check the same, we performed Western blot and IIF analysis on spermatozoa. Our Western blot analysis on testicular and epididymal tissue showed varied sizes of LAR that are in agreement with data © 2013 American Society of Andrology and European Academy of Andrology
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Figure 4 Effect of various Liprin a3 antibody, LAR ligands, LAR wedge peptides on acrosome exocytosis. (A) Asterisk (*) indicates statistically significant decrease in acrosome reaction (AR) in presence of 1 in 10 diluted Anti Liprin a3 antibody (Anti Liprin 1 : 10) as compared with IgG control (*p = 0.006), **indicate significant decrease in AR in presence of 1 in 20 diluted anti-Liprin a3 antibody (Anti Liprin 1 : 20) (**p = 0.027), ***indicate significant decrease in AR (***p = 0.037). However, no significant change in AR can be seen in presence of IgG as compared with capacitated spermatozoa (cap) (+p = 0.565). (B) Effect of LAR ligands on acrosome exocytosis index. Nidogen-1 and 2, Syndecan-1, 2, 3 were used for experiment at 10 and 50 ng. Asterisk indicates statistically significant increase in AE index as compared with 10 ng concentration of same protein (p < 0.05). (C) Effect of LAR wedge peptide on acrosome exocytosis index. WLAR, TAT-WLAR and SLAR-TAT were used at 10, 50 and 100 µM concentration. Capacitated spermatozoa were incubated with these peptides and incubated for 30 min at 37 °C. Tat-WLAR at 50 lM shows statistically significant increase in AE index in comparison with other peptides (p < 0.05).
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demonstrated in liver and testis by Welham et al. (2009). They detected four bands 100, 75, 50, 30 kDa in testis using an antibody specific to trans-membrane region of LAR and attributed these bands to be processed LAR C-terminal Fragments (LARCTF). The anti-LAR antibody used by us was raised against a peptide of trans-membrane region as well, thereby suggesting the presence of LAR–CTF in testis and epididymis. It is observed that functionally relevant proteins are usually conserved across species. The presence LAR in rat, mouse and human spermatozoa as seen by us indicates its conserved nature and highlights its functional relevance in spermatozoa. The liprins were discovered in a pull down assay with LAR and therefore named as LAR-Interacting Protein 1 (LIP.1) (Serras et al., 1995). It was discovered that Liprin a interacts with Page s et al., LAR through its C-terminal LH domain (Serra-Page 1998). Both Liprins and LAR are known to be required for cargo transport and membrane fusion events at synapse (Zhen & Jin, © 2013 American Society of Andrology and European Academy of Andrology
1999; Miller et al., 2005). This study shows that LAR and Liprin a3 coexist on mouse acrosome (Fig. 5A) indicating probable interaction of these two proteins. Thus, we speculate that LAR-Liprin a3 on spermatozoa brings about a similar function as in synapse i.e. membrane fusion. On the basis of our colocalization results, we propose the interaction between LAR–Liprin a3RIM in mouse acrosome (Fig 5A). After having demonstrated the presence of LAR, Liprin a3 and RIM we decided to check the contribution of Liprin a3 to the AR. In presence of anti-Liprin a3 antibody we saw a decrease in the number of acrosome-reacted spermatozoa, indicating inhibition of AR. We speculate that the proposed interactome (Fig. 5A) for AR may be abrogated due to the anti-Liprin a3 antibody. This may lead to an incompetent configuration of molecules at acrosome leading to decreased acrosomal fusion. Leucocyte antigen related (LAR) is characterized by an extracellular domain composed of multiple immunoglobulin (Ig)-like Andrology, 2014, 2, 165–174
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Figure 5 Sperm plasma membrane: PM and acrosomal membrane: AM (A) The proposed Liprin a3 interactome involved in acrosome reaction (AR). This study shows the coexistence of Liprin a3, RIM and LAR in acrosome therefore, representing the presence of protein assembly required for AR. The colocalization of LAR and Liprin a3 indicates the importance of LAR. (B) The proposed pathway of LAR activity (a) The monomer of LAR has an accessible D1 phosphatase domain to its substrate like Akt and b-catenin. The dephosphorylation of Akt and b-catenin renders them inactive. Thus, resulting in unchanged actin cyto-architecture and no AR. (b) In presence of ligands like Syndcans and Nidogens, LAR undergoes dimerization. This makes D1 domain inaccessible to Akt and b-catenin. The active phosphorylated Akt and b-catenin can now activate AR (AR) (c) The wedge domain peptide mimics the dimerization and makes D1 catalytic domain inaccessible to its substrate leading to AR. (C) Cumulus is a site of AR (a) Spermatozoa are travelling across cumulus cells in their journey to reach an oocyte. (b) Cumulus exhibits surface Syndecans (c) Passing spermatozoa interact with Syndecans and undergo dimerization. (d) We proposed that this dimerization activates LAR downstream pathway as shown in (B) and AR is triggered and acrosomal exocytosis is brought about.
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domain and fibronectin type III repeats and two cytoplasmic catalytic domains D1 and D2. These domains have phosphatase activity. Various ligands interacting with LAR were discovered (O’Grady et al., 1998). The studies conducted in Drosophila and C. Elegans suggest that Ig domain has an important role at synapse and it interacts with extracellular ligands such as Syndecan (Sdc) and dally-like protein (Dlp) (Johnson et al., 2006). It was correlated that LAR Laminin–Nidogen interaction changes the actin cytoskeleton structure (O’Grady et al., 1998). These data suggest that the extracellular ligands interact with LAR through Ig domain, thereby regulating active zones at synapse. To unravel the mechanism, studies were conducted. It was found that upon binding with ligands, LAR demonstrates dimerization and inhibits function of catalytic domain. During dimerization, the wedge structure from D1 domain of one LAR interacts with D1 of other. This configuration makes a D1 catalytic site inaccessible to substrates thereby inhibiting LAR phosphatase activity (Bixby, 2001). This triggers various intracellular events such as TrkA and b- catenin phosphorylation (Xie et al., 2006). To study intracellular dimerization of wedge domain a peptide responsible for same needs to get incorporated into a live cell to mimic natural process. Attaching a Tat sequence to bait is the popular approach (Wadia & Dowdy, 2002). Xie et al. (2006) designed wedge peptide for LAR with a Tat peptide that would mimic the event of dimerization. It was found that in presence of wedge peptide, phosphorylation of TrkA is upregulated thus elevating the levels of phospho-Akt pathway (Xie et al., 2006). Similar effect was seen with beta catenin wherein LAR regulates the actin polymerization and actin-based cell motility (Kypta et al., 1996) (Fig. 5B a). All these reports point towards the possibility that, if LAR is present on spermatozoa then it may follow a similar pattern and activate cascade required for membrane fusion in spermatozoa. To test the same, we devised two different experiments. In the first set of experiments, spermatozoa were challenged with different concentrations of LAR ligands like Syndecans and Nidogens. We observed a dose-dependent increase in the acrosome exocytosis index. In another set of experiments, we spiked spermatozoa with membrane-permeant Tat-LAR wedge peptide and the effect was similar to that seen in the presence of ligands. We speculate that in presence of LAR ligands or wedge peptide LAR undergoes or mimics dimerization leading to inhibition of phosphatase activity (Fig 5B b and c). This could promote Akt phosphorylation and activation of the downstream signalling pathway. The elevated level of Phospho-Akt then upregulates hypermotility and the AR (Sagare-Patil et al., 2013). The LAR activity may also change actin cyto-architecture (Kypta et al., 1996) and such changes are required to promote AR (Brener et al., 2003). Hence, we could see an increase in acrosome exocytosis index. There are two different schools of thought that propose the triggering pathway of AR. According to one school, Zona pellucida (ZP) proteins mediate AR (Gupta and Bhandari, 2011), and is the most accepted site of AR. However, the other school considers that the AR probably happens before interaction of spermatozoa with ZP, perhaps in the cumulus (Cummins & Yanagimachi, 1986). The studies conducted by Inoue et al. (2011) and Jin et al. (2011) demonstrate clearly that cumulus but not ZP is essential for AR. However, these reports did not look for the cumulus proteins that are involved in AR. Our IIF © 2013 American Society of Andrology and European Academy of Andrology
data clearly show the presence of one such LAR ligand Syndecan-1 on cumulus cell surface (Fig. 5C b). This was followed by experiments, which indicate that cumulus proteins like Syndecan -1, 2, 3 may activate AR. This gives us a preliminary evidence to postulate that in in vivo situations these cumulus Syndecans may induce dimerization of LAR (Fig. 5C c) and trigger downstream pathway that activate AR in spermatozoa (Fig. 5C d). However, extensive experiments need to be carried out to prove this hypothesis.
CONCLUSION Overall, this study demonstrates that sperm AR is driven by common set of proteins like Liprin a3, LAR, RIM shown to be responsible for membrane fusion at synapse. We could also demonstrate that the ligands and wedge peptide can induce LAR dimerization and could be one of the mechanisms of stimulating acrosomal exocytosis. The observations support the hypothesis that cumulus could be another site of AR.
ACKNOWLEDGEMENTS The authors thank Ms. Shobha Sonawane and Ms. Reshma Gaonkar for their assistance at the confocal microscopy department of NIRRH. The authors also thank Dr. Rahul Gajabhiye, clinician, for providing normal semen sample from institutional clinic.
DISCLOSURES Financial assistance provided to CJ by Department of Science and Technology, New Delhi as DST-JRF, and Indian Council of Medical Research (ICMR), New Delhi for ICMR-SRF and by NIRRH, Mumbai is gratefully acknowledged.
AUTHOR CONTRIBUTIONS CJ and SK performed experiments, analysed the data and wrote the manuscript, VK designed, supervised the research study, wrote and corrected the manuscript.
REFERENCES Aitken RJ, Nixon B, Lin M, Koppers AJ, Lee YH & Baker MA. (2007) Proteomic changes in mammalian spermatozoa during epididymal maturation. Asian J Androl 9, 554–564. Andrews-Zwilling YS, Kawabe H, Reim K, Varoqueaux F & Brose N. (2006) Binding to Rab3A-interacting molecule RIM regulates the presynaptic recruitment of Munc13-1 and ubMunc13-2. J Biol Chem 281, 19720–19731. Aoki N & Matsuda TA. (2000) Cytosolic protein-tyrosine phosphatase PTP1B specifically dephosphorylates and deactivates prolactin-activated STAT5a and STAT5b. J Biol Chem 275, 39718–39726. Bello OD, Zanetti MN, Mayorga LS & Michaut MA. (2012) RIM, Munc13, and Rab3A interplay in acrosomal exocytosis. Exp Cell Res 318, 478–488. Benoff S. (1998) Modelling human sperm-egg interactions in vitro: signal transduction pathways regulating the acrosome reaction. Mol Hum Reprod 4, 453–471. Bixby JL. (2001) Ligands and signaling through receptor-type tyrosine phosphatases. IUBMB Life 51, 157–163. Brener E, Rubinstein S, Cohen G, Shternall K, Rivlin J & Breitbart H. (2003) Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction. Biol Reprod 68, 837–845. Cummins JM & Yanagimachi R. (1986) Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated Andrology, 2014, 2, 165–174
173
C. S. Joshi, S. A. Khan and V. V. Khole in vitro, in relation to the timing of the acrosome reaction. Gamete Res 15, 187–212. € dhof TC. (2011) RIM proteins activate Deng L, Kaeser PS, Xu W & Su vesicle priming by reversing auto inhibitory homo dimerization of Munc13. Neuron 69, 317–331. Gupta SK & Bhandari B. (2011) Acrosome reaction: relevance of zona pellucida glycoproteins. Asian J Androl 13, 97–105. Inoue N, Satouh Y, Ikawa M, Okabe M & Yanagimachi R. (2011) Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs. Proc Natl Acad Sci U S A 108, 20008–20011. Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA et al. (2011) Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proc Natl Acad Sci U S A 108, 4892–4896. Johnson KG, Tenney AP, Ghose A, Duckworth AM, Higashi ME, Parfitt K et al. (2006) The HSPGs Syndecan and Dally like bind the receptor phosphatase LAR and exert distinct effects on synaptic development. Neuron 49, 517–531. Joshi CS, Suryawanshi AR, Khan SA, Balasinor NH & Khole VV. (2013) Liprin a3: a putative estrogen regulated acrosomal protein. Histochem Cell Biol 139, 535–548. Ko J, Na M, Kim S, Lee J-R & Kim E. (2003) Interaction of the ERC family of RIM-binding proteins with the liprin-alpha family of multidomain proteins. J Biol Chem 278, 42377–42385. Kypta RM, Su H & Reichardt LF. (1996) Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex. J Biol Chem 134, 1519–1529. Mendoza C, Carreras A, Moos J & Tesarik J. (1992) Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 5, 755–763. Michaut M, De Blas G, Tomes CN, Yunes R, Fukuda M & Mayorga LS. (2001) Synaptotagmin VI participates in the acrosome reaction of human spermatozoa. Dev Biol 235, 521–529. Miller KE, De Proto J, Kaufmann N, Patel BN, Duckworth A & Van Vactor D. (2005) Direct observation demonstrates that Liprin-alpha is required for trafficking of synaptic vesicles. Curr Biol 15, 684–689. O’Grady P, Thai TC & Saito H. (1998) The laminin-nidogen complex is a ligand for a specific splice isoform of the transmembrane protein tyrosine phosphatase LAR. J Cell Biol 141, 1675–1684. Pires ES, Choudhury AK, Idicula-Thomas S & Khole VV. (2011) Anti-HSP90 autoantibodies in sera of infertile women identify a dominant, conserved epitope EP6 (380-389) of HSP90 beta protein. Reprod Biol Endocrinol 9, 16. s C, Tang M & Streuli M. (1995) The LAR/PTP delta/ Pulido R, Serra-Page PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1. Proc Natl Acad Sci U S A 92, 11686–11690. Ramalho-Santos J, Moreno RD, Sutovsky P, Chan AW, Hewitson L, Wessel GM et al. (2000) SNAREs in mammalian sperm: possible implications for fertilization. Dev biol 223, 54–69. Sagare-Patil V, Vernekar M, Galvankar M & Modi D. (2013) Progesterone utilizes the PI3K-AKT pathway in human spermatozoa to regulate
174
Andrology, 2014, 2, 165–174
ANDROLOGY motility and hyperactivation but not acrosome reaction. Mol Cell Endocrinol 374, 82–91. Schoch S, Castillo PE, Jo T, Mukherjee K, Geppert M, Wang Y et al. (2002) RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature 415, 321–326. Serra-Pag es C, Kedersha NL, Fazikas L, Medley Q, Debant A & Streuli M. (1995) The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions. EMBO J 14, 2827–2838. Serra-Pag es C, Medley QG, Tang M, Hart A & Streuli M. (1998) Liprins, a family of LAR transmembrane protein-tyrosine phosphatase-interacting proteins. J Biol Chem 273, 15611–15620. Spangler SA & Hoogenraad CC. (2007) Liprin-alpha proteins: scaffold molecules for synapse maturation. Biochem Soc Trans 35, 1278–1282. Tomes CN, Michaut M, De Blas G, Visconti P, Matti U & Mayorga LS. (2002) SNARE complex assembly is required for human sperm acrosome reaction. Dev biol 243, 326–338. Wadia JS & Dowdy SF. (2002) Protein transduction technology. Curr Opin Biotechnol 13, 52–56. € dhof TC. (1997) Rim is Wang Y, Okamoto M, Schmitz F, Hofmann K & Su a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature 388, 593–598. Watson LN, Mottershead DG, Dunning KR, Robker RL, Gilchrist RB & Russell DL. (2012) Heparan sulfate proteoglycans regulate responses to oocyte paracrine signals in ovarian follicle morphogenesis. Endocrinology 153, 4544–4555. Welham SJM, Clark AJL & Salter AM. (2009) A novel liver specific isoform of the rat LAR transcript is expressed as a truncated isoform encoded from a 5’UTR located within intron 11. BMC Mol Biol 10, 30. WHO. (2010) WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. Geneva, Switzerland. ISBN 978 92 4 154778 9. Xie Y, Massa SM, Ensslen-Craig SE, Major DL, Yang T, Tisi MA et al. (2006) Protein-tyrosine phosphatase (PTP) wedge domain peptides: a novel approach for inhibition of PTP function and augmentation of protein-tyrosine kinase function. J Biol Chem 281, 16482–16492. Yanagimachi R. (2011) Mammalian sperm acrosome reaction: where does it begin before fertilization? Biol Reprod 85, 4–5. Yang T, Bernabeu R, Xie Y, Zhang JS, Massa SM, Rempel HC et al. (2003) Leukocyte antigen-related protein tyrosine phosphatase receptor: a small ectodomain isoform functions as a homophilic ligand and promotes neurite outgrowth. J Neurosci 23, 3353–3363. Yang T, Yin W, Derevyanny VD, Moore LA & Longo FM. (2005) Identification of an ectodomain within the LAR protein tyrosine phosphatase receptor that binds homophilically and activates signalling pathways promoting neurite outgrowth. Eur J Neurosci 22, 2159–2170. Yang T, Massa SM & Longo FM. (2006) LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathways. J Neurobiol 66, 1420–1436. Zarelli VEP, Ruete MC, Roggero CM, Mayorga LS & Tomes CN. (2009) PTP1B dephosphorylates N-ethylmaleimide-sensitive factor and elicits SNARE complex disassembly during human sperm exocytosis. J Biol Chem 284, 10491–10503. Zhen M & Jin Y. (1999) The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature 401, 371–375.
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