Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro

Journal of Reproductive Immunology 115 (2016) 6–13

Contents lists available at ScienceDirect

Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro Hiroshi Yoshitake a , Risako Oda a,b , Mitsuaki Yanagida a , Yu Kawasaki a,b , Mayumi Sakuraba a , Kenji Takamori a , Akiko Hasegawa c , Hiroshi Fujiwara d , Yoshihiko Araki a,b,∗ a Institute for Environmental & Gender-Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan b Department of Obstetrics and Gynecology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan c Department of Obstetrics and Gynecology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan d Department of Obstetrics and Gynecology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8640, Japan

a r t i c l e

i n f o

Article history: Received 26 December 2015 Received in revised form 26 March 2016 Accepted 1 April 2016 Keywords: Anti-sperm auto-antibody Acrosome reaction Fertilization Heparan sulfate Alpha-N-acetylglucosaminidase Oligosaccharide chain

a b s t r a c t We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting Nacetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77 kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Nagluforms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Anti-sperm antibodies (Abs) are occasionally identified in the sera of infertile patients (Isojima 1989). Some anti-sperm Abs are believed to inhibit fertility via immunological effects, such as sperm agglutination (Koide et al., 2000), reduction of sperm

∗ Corresponding author at: Institute for Environmental & Gender—Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan. E-mail address: [email protected] (Y. Araki). http://dx.doi.org/10.1016/j.jri.2016.04.001 0165-0378/© 2016 Elsevier Ireland Ltd. All rights reserved.

motility (Barratt et al., 1989), impaired cervical mucus penetration (Eggert-Kruse et al., 1993), or interference with gamete interaction (Bronson et al., 1989). Anti-sperm Abs are produced as anti-sperm allo-Abs in females and as auto-Abs in males, respectively. Although the precise mechanism underlying the induction of anti-sperm auto-Abs (ASAAbs) remains unclear, one hypothesis proposes that cross-reactive immune responses against external antigens (e.g., bacteria or viruses) induce an immune response against sperm antigens (Fijak and Meinhardt 2006). Based on this hypothesis, we previously generated a monoclonal Ab (mAb) against mouse epididymal spermatozoa, designated Ts4, using spleen cells of aged mice (>1 year

H. Yoshitake et al. / Journal of Reproductive Immunology 115 (2016) 6–13

old) maintained under conventional conditions (Yoshitake et al., 2008a). We reported that this mAb immunoreacts with several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa and the germ cells within the seminiferous tubules via binding to a common oligosaccharide (OS) chain on the molecules (Yoshitake et al., 2008a; Shirai et al., 2009). The immunoreactivity of Ts4 was not observed in the ovary including eggs nor other somatic cells of major organs (Shirai et al., 2009). We further showed that Ts4 immunoreacted to TEX101 (a germ cell-marker glycoprotein (Kurita et al., 2001; Takayama et al., 2005b)) within the mouse testis and that the antigenic determinant for Ts4 was located on the fucosylated agalacto-biantennary complex-type N-glycan with bisecting N-acetylglucosamine (GlcNAc) of TEX101 (Yoshitake et al., 2015). However, the polypeptides associated by the OS chain in the epididymal spermatozoa have not yet been identified. The acrosomal region of sperm contains a variety of glycoproteins, including acrosin, sp56, SP-10, DE, beta-1,4galactosyltransferase, fertilin, PH-20, TESP1, TESP2, equatorin, SAMP32, and IZUMO, which are thought to be involved in the interaction between sperm and the zona pellucida (Bleil and Wassarman 1990; Baba et al., 1994; Coonrod et al., 1996; Hunnicutt et al., 1996; Lu and Shur 1997; Kohno et al., 1998) or in the spermegg plasma membrane binding and fusion (Rochwerger et al., 1992; Cho et al., 1998; Yoshinaga et al., 2001; Hao et al., 2002; Nishimura et al., 2004; Inoue et al., 2005). Based on these findings, we speculated that Ts4-reactive glycoproteins in the acrosomal region may also play a role in fertilization. In this study, we identified alpha-N-acetylglucosaminidase (Naglu), as one of Ts4-reactive proteins in spermatozoa using an immunoprecipitation method combined with liquid chromatography-tandem mass spectrometry (LC–MS/MS). Furthermore, we investigated potential functions of the identified molecule on the process of fertilization and the effect of Ts4 mAb on mouse fertility in vitro. 2. Materials and methods 2.1. Animals Male ICR mice (6–12-week-old), male and female BDF1 mice (8–10- and 3–4-week-old, respectively), and female NZW rabbits (9-week-old) were purchased from Sankyo Lab Service (Tokyo, Japan) or Japan SLC (Shizuoka, Japan). They were maintained at the Animal facilities of Juntendo University or Hyogo College of Medicine under 12L:12D conditions, and given free access to food and water. All animal experiments were conducted according to the guide for care and use of laboratory animals, Juntendo University or Hyogo College of Medicine. 2.2. Antibodies and reagents The Ts4 mAb (mouse IgM) was established as previously described (Yoshitake et al., 2008a). RP-3 (an anti-rat neutrophil mAb: mouse IgM) used as normal control was generated as previously reported (Sekiya et al., 1989). An anti-Naglu polyclonal Ab (pAb) was produced by immunizing rabbits with recombinant mouse Naglu582-739 as described before (Yoshitake et al., 2008b) with a slight modification. The anti-Naglu pAb labeled with biotin was prepared as described elsewhere (Guesdon et al., 1979). The mAbs against IZUMO1 (Inoue et al., 2005) and equatorin (Toshimori et al., 1992) were generous gifts from Dr. Masaru Okabe (Osaka University, Osaka, Japan) and Dr. Kiyotaka Toshimori (Chiba University, Chiba, Japan), respectively. Other Abs and reagents were purchased from the following companies: horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rat immunoglobulin (Ig) pAb, normal control rabbit Ig, and normal control mouse IgM (DAKO,

7

Glostrup, Denmark), HRP-conjugated goat anti-mouse IgM pAb (Chemicon, Temecula, CA, USA), Alexa Fluor 488-conjugated goat anti-mouse IgM pAb and Alexa Fluor 594-conjugated goat antirabbit IgG pAb, HRP-conjugated avidin (Invitrogen, Carlsbad, CA, USA). 2.3. Preparation of mouse epididymal extracts, silver staining, and western blot analysis Mouse epididymal Triton X-100-soluble extracts were prepared as previously described (Tsukamoto et al., 2006) with a slight modification. The epididymal extract was separated by SDS-PAGE system (Laemmli 1970). Following silver staining was performed as described previously (Tsukamoto et al., 2006). The protein components resolved by SDS-PAGE were electrophoretically blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA), and then the reactivity of the transferred proteins with primary Abs (0.5–1 ␮g/ml) was assayed using HRP-conjugated secondary Abs or avidin and an ECL detection system (GE Healthcare, Buckinghamshire, UK) (Tsukamoto et al., 2006). 2.4. Identification of Ts4-reactive proteins in mouse epididymis Immunoprecipitation using Ts4 was performed essentially according to the method as described previously (Yoshitake et al., 2015). To identify mouse epididymal proteins recognized by Ts4, in-gel trypsin digestion and LC–MS/MS analysis were performed as previously described (Tsukamoto et al., 2006). The identified proteins were confirmed by Western blot analysis using the specific Abs against the molecules. 2.5. Immunofluorescent studies Cauda epididymal spermatozoa were collected as described previously (Takayama et al., 2005a), smeared onto 3aminopropyltriethoxysilane (APS)-coated slide glass (Matsunami, Osaka, Japan), and then fixed with 1% paraformaldehyde. The spermatozoa were incubated with the primary Abs at 4 ◦ C overnight and then treated with Alexa Fluor 488 or 594-conjugated secondary Ab for 1 h at room temperature. The specimens were sequentially counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen) and mounted in the ProLong Gold antifade reagent (Invitrogen). The immunostained samples were observed under a BIOREVO BZ-9000 microscope system (KEYENCE, Osaka, Japan). 2.6. In vitro fertilization (IVF) Superovulation of female BDF1 mice was induced by intraperitoneal (IP) injection of 7.5 IU gonadotropin from pregnant mares serum (PMSG) (Sigma-Aldrich, St. Louis, MO, USA) followed 48 h later by IP injection of 7.5 IU chorionic gonadotropin human (hCG) (Sigma-Aldrich). At 17 h after the hCG injection, the oviducts were isolated. The cumulus masses containing superovulated eggs were harvested from the ampullae, resuspended in 200 ␮l of CARD MEDIUM (Kyudo, Saga, Japan), and then incubated at 37 ◦ C in 5% CO2 in air for 30 min. Cauda epididymal spermatozoa were isolated from male BDF1 mice and then resuspended into 100 ␮l of FERTIUP medium (Kyudo). After preincubated with Ts4, PR-3, or anti-Naglu pAb for 60 min at 37 ◦ C in 5% CO2 in air, the spermatozoa were applied to the egg medium resulting in a target final sperm concentration of 1.0–3.0 × 105 /ml, and then incubated for 3 h at 37 ◦ C in 5% CO2 in air. After incubation, the eggs were transferred into 80 ␮l HTF medium (Kyudo) and incubated at 37 ◦ C in 5% CO2 in air. After 6 h of insemination, the eggs were mounted on slides and then fixed with 2.5% glutaraldehyde in 0.1 M phosphoric acid buffer for

8

H. Yoshitake et al. / Journal of Reproductive Immunology 115 (2016) 6–13

20 min at room temperature. The cells were stained with 1% orcein acetate (Muto Pure Chemicals, Tokyo, Japan), and then examined under a Leica DM IL microscope (Leica, Wetzlar, Germany). Fertilization were assessed according to the formation of female and male pronuclei, a fertilizing sperm tail, and a second polar body (Nohara et al., 1998).

2.7. Assessment of acrosome reaction (AR) AR was assessed by a Pisum sativum agglutinin (PSA) staining method (Choi et al., 2001). After incubated in the TYH medium (Mitsubishi Kagaku, Tokyo, Japan) for 2 h at 37 ◦ C, to induce AR, the cauda epididymal spermatozoa were treated with 1 mM of ionophore A23187 (Sigma-Aldrich) for 10 min at 37 ◦ C, or various concentrations of heparin, heparan sulfate, dermatan sulfate (Iduron, Manchester, UK), chondroitin sulfate, or hyaluronan (Sigma-Aldrich) for 4 h at 37 ◦ C. After centrifuged at 1000g for 5 min, the sediment spermatozoa were treated with fluorescein isothiocyanate (FITC)-conjugated PSA (EY Laboratories Inc., San Mateo, CA, USA) for 1 h at room temperature. The fluorescent intensity of the treated spermatozoa was measured with a FACSCalibur (Becton Dickinson, San Jose, CA, USA).

2.8. Statistical analysis Significant differences (p < 0.05) were evaluated using the Student’s t or ␹2 test. For graphical representation of data, y-axis error bars indicate the standard deviation (SD) of the data for each point on the graph.

3. Results 3.1. Identification of Ts4-reactive cauda epididymal proteins We previously reported that Ts4 mainly detected two bands between 100 and 150 kDa, and faintly reacted against additional several bands in the epididymal sperm extract (Shirai et al., 2009). To better understand Ts4 reactivity, we performed a Western blot analysis of the cauda epididymal proteins separated out via SDSPAGE on a 7.5% gel, focusing proteins with relatively high molecular mass (>50 kDa). The Ts4 mAb detected six bands in the whole epididymal extract (Fig. 1A, lane “Cont”), including the two bands between 100 and 150 kDa, consistent with our previous results (Shirai et al., 2009). Attempts were then made to identify the molecules recognized with Ts4 using an immunoprecipitation method combined with LC–MS/MS. As an initial trial, we investigated whether the six bands (shown in Fig. 1A, lane “Cont”) were detected in the Ts4 immunoprecipitant. After immunoprecipitation with Ts4, mainly four bands at approximately 170, 140, 110, and 80 kDa were detected with the same mAb (Fig. 1A, lane 1). The same immunoprecipitated proteins were separated by SDS-PAGE, and sequentially visualized by silver staining. Two specific bands at apparent molecular masses of 155 and 82 kDa were clearly observed among Ts4-precipitated proteins (Fig. 1B, lane 1), but not in the normal control IgM-precipitated proteins (Fig. 1B, lane 2), nor in the other controls (Fig. 1B, lanes 3 and 4). Subsequently, we analyzed the 155- and 82-kDa precipitated proteins using LC–MS/MS. The 82-kDa band contained Naglu (NCBI RefSeq Accession No. NP 038820), with a four-peptide match (Table 1). The 155-kDa band did not contain meaningful peptides.

3.2. Confirmation that Naglu is one of Ts4-immunoreactive proteins We next confirmed the results of the proteomic analysis. We initially established a pAb against mouse Naglu by immunizing rabbits with recombinant Naglu. Western blot analysis of epididymal extracts showed that the generated anti-Naglu pAb clearly detected two bands at approximately 82 and 77 kDa (Supplementary Fig. S1A in the online version at DOI: 10.1016/j.jri.2016.04.001). Furthermore, an immunofluorescent test revealed that immunopositive staining of this pAb was observed on the acrosomal region and along the entire flagellum of the epididymal sperm (Supplementary Fig. S1B in the online version at DOI: 10.1016/j.jri.2016.04.001). When pre-immune serum derived from the same rabbit was used in place of the pAb, the immunoreactivity was not entirely observed (data not shown). To confirm that the Ts4-recognized molecule corresponds to Naglu, we conducted a Western blot analysis of the epididymal proteins immunoprecipitated with Ts4 using the anti-Naglu pAb. As expected, a Ts4-immunoreactive band was observed in Ts4 immunoprecipitant at an apparent molecular mass of 82 kDa (Fig. 2A, lane 1), similar to the result shown in Fig. 1A, lane 2. An 82-kDa band was also detected in the same immunoprecipitant when the anti-Naglu pAb was used as a probe (Fig. 2B, lane 1). However, unlike the 82-kDa band, a 77-kDa band was not observed in the immunoprecipitant with Ts4 (Fig. 2B, lane 1). In a bidirectional approach, we examined whether the immunoprecipitant obtained with the anti-Naglu pAb contained both Naglu and the molecules reactive with Ts4. Western blot analysis revealed that a specific 82-kDa band was detected with both the anti-Naglu pAb and Ts4 (Fig. 2C and D), although the pAb seems to be not compatible as an immunoprecipitating carrier for the 77-kDa protein in the epididymal proteins because the 77-kDa band was not observed (Fig. 2C, lane 1). Mouse acrosomal glycoproteins, IZUMO1 (Inoue et al., 2005) and equatorin (Toshimori et al., 1992) were not detected in the immunoprecipitant with either Ts4 or the anti-Naglu pAb (data not shown). We performed an immunofluorescent test of cauda epididymal sperm using Ts4 and the anti-Naglu pAb. As demonstrated in our previous report (Shirai et al., 2009), only the acrosomal region of cauda epididymal spermatozoa showed immunoreactivity for Ts4 (Fig. 2E). As shown in Supplementary Fig. S1B in the online version at DOI: 10.1016/j.jri.2016.04.001, the anti-Naglu pAb showed immunoreactivity against both the acrosomal region and the flagellum (Fig. 2G). Consequently, double positive staining with these Abs was observed only in the acrosomal region of spermatozoa (Fig. 2F), indicating that the 82-kDa form of Naglu is one of the several Ts4-recognized molecules, located in the acrosome.

3.3. Effect of Ts4 and the anti-Naglu pAb on IVF To investigate whether Ts4 affected fertilization, like some ASA-Abs, we examined the fertilizing ability of spermatozoa after treatment with Ts4. When spermatozoa were pretreated with RP-3 (control mouse IgM) or Ts4 at the concentration of 70 ␮g/ml, control spermatozoa achieved a fertilization rate of 76.8%. However, spermatozoa treated with the Ts4 mAb before insemination showed a significantly decreased fertilization rate of 32.4% (Fig. 3C). The inhibitory effect of Ts4 on fertilization was not observed when spermatozoa were pretreated with lower concentrations of the mAb (10 or 50 ␮g/ml) (Fig. 3A,B). We next investigated whether the anti-Naglu pAb inhibited IVF, like Ts4. When spermatozoa were preincubated with the anti-Naglu pAb (0, 10, 50, or 75 ␮g/ml), successful fertilization rates reduced in a concentration-dependent manner of the pAb (Table 2).

H. Yoshitake et al. / Journal of Reproductive Immunology 115 (2016) 6–13

9

Fig. 1. S SDS-PAGE analysis of mouse epididymal proteins immunoprecipitated with Ts4. Western blot analysis using Ts4 (A). Epididymal proteins immunoprecipitated with either Ts4 (lane 1) or normal control mouse IgM (n.c.) (lane 2) were separated via SDS-PAGE on a 7.5% gel under reducing conditions. Control experiments were conducted under the same conditions, but in the absence of the epididymal extract (lanes 3 and 4). *The loading sample as a positive control (lane “Cont”) was a whole epididymal extract before an immunoprecipitation (IP) treatment. The separated proteins were electroblotted to a PVDF membrane and then probed with Ts4. Arrows and arrowheads indicate the specific immunoreactive bands observed in lanes 1 and 2, respectively. Visualized by silver staining (B). The same samples in (A) were separated via SDS-PAGE on a 7.5% gel under reducing conditions and the gel was silver-stained. Specific bands obtained via immunoprecipitation with Ts4 are indicated with arrowheads (lane 1). Mr, molecular mass. Table 1 Summary of LC–MS/MS proteomic analysis for the epididymal mature sperm-proteins immunoprecipitated with the Ts4 mAb. a

Exp. Mr

155 kDa 82 kDa

a b c

Protein

NCBI RefSeq Accession Number

b

Not detected Naglu

NP 038820

82,558

Cal. Mw

Coverage (%)

c

5%

78

Protein Score

Peptide identified AVLEAVPR GDTVDLSKK YDLLDVTR GSTGVAAAAGLHR

Exp. Mr, experimental molecular mass on SDS-PAGE under reducing conditions. Cal. Mw, calculated molecular weight from primary protein sequence without posttranslational modifications. Protein Score, the value was calculated by Mascot search.

Table 2 Effect of anti-Naglu pAb on IVF. Concentration (␮g/ml)

Number of used oocytes

Number of fertilized oocytes

Rate (%)

0 10 50 75

63 120 73 43

60 117 16 7

95.2 97.5 21.9* 16.3*

A successful fertilization rate of spermatozoa pretreated with various concentrations of the anti-Naglu pAb. * Significant differences (p < 0.05) compared with no pAb based on the ␹2 test.

3.4. Inhibitory effect of Ts4 on the glycosaminoglycan-induced AR To clarify the mechanism of the inhibitory fertilization effect with Ts4, we evaluated whether Ts4 treatment of spermatozoa impaired the AR using flow cytometry with PSA staining (Choi et al., 2001). After treatment with ionophore A23187, the peak of PSA-positive sperm numbers shifted to a lower-intensity region compared to non-treated sperm (Fig. 4 A), implying that the number of spermatozoa completing AR increased. When the spermatozoa were incubated with Ts4 before treatment with A23187, the peak of fluorescence partially returned to the ionophore-non-treated position (Fig. 4A; blue line). These results show that Ts4 possesses an inhibitory activity for sperm AR induced by the ionophore A23187. Naglu is known to be a critical enzyme required for the degradation of heparan sulfate, which can be categorized as

a glycosaminoglycan (GAG) (Zhao et al., 1996) along with heparin, chondroitin sulfate, hyaluronic acid, and dermatan sulfate. In bovines, some GAGs were reported to enhance the incidence of the AR in vitro (Handrow et al., 1982). We hypothesized that heparan sulfate may induce the mouse AR in vitro and that the Ts4 mAb may also inhibit the AR induced by the GAGs, like in the case of AR induction by the ionophore A23187. To elucidate these possibilities, we initially investigated the ability of heparan sulfate to induce the AR using the PSA staining methods. Heparan sulfate decreased the fluorescent intensity of spermatozoa labeled with FITC-conjugated PSA in a concentration-dependent manner (Fig. 4B), indicating that heparan sulfate induces the AR on mouse spermatozoa. The induction of AR was also observed in the presence of other GAGs, including heparin, chondroitin sulfate, and dermatan sulfate (Fig. 4B). When spermatozoa were preincubated with the Ts4 mAb before treatment with heparan sulfate, the decrease of fluorescent intensity by the GAG was suppressed (Fig. 4C). However, this change in Ts4-induced fluorescent intensity was not observed when the spermatozoa were stimulated with heparin (Fig. 4D). 4. Discussion In this study, we showed that Naglu was identified as one of the Ts4-reactive molecules in the epididymal spermatozoa (Figs. 1 and 2, and Table 1). Mouse Naglu contains 739 amino acids, with a calculated molecular weight of 82,558 and six potential N-glycosylation sites in the amino acid sequence. Although the precise molecular

10

H. Yoshitake et al. / Journal of Reproductive Immunology 115 (2016) 6–13

Fig. 2. Relation between Ts4-reactive molecules and Naglu in mouse spermatozoa. Western blot analyses of epididymal proteins immunoprecipitated with Ts4 or the antiNaglu pAb (A–D). The proteins immunoprecipitated from the epididymal extract with either Ts4 (A and B, lane 1) or negative control mouse IgM (A and B, lane 2), or either the anti-Naglu pAb (C and D, lane 1) or normal rabbit Ig (C and D, lane 2) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the extract (A–D, lanes 3 and 4). The epididymal extract was used as a positive control (A–D, lane 5). Specific bands (indicated with arrowheads) in the immunoprecipitated proteins were detected with Ts4 (A and D) or the anti-Naglu pAb (B and C). Mr, molecular mass. Immunolocalization of Ts4 and Naglu within cauda epididymal sperm (E–G). After being smeared and dried on an APS-coated slide glass, cauda epididymal spermatozoa were immunostained with Ts4 and the anti-Naglu pAb. An Alexa Fluor 488 or 594-conjugated secondary antibody was used for detection of the primary antibodies. Green: Ts4 (E), red: Naglu (G). Overlay image of E and G (F). The sample was counterstained with DAPI (blue). Arrowheads: acrosomal region. Arrows: flagellum. Bar: 20 ␮m.

characteristics of mouse Naglu have not been yet clarified, the human homologue NAGLU is produced by post-translational cleavage into two products: the 82-kDa form and the 77-kDa form (Weber et al., 1996). In this study, we showed that mouse Naglu also consists of the two forms of 82 and 77 kDa (Supplementary Fig. S1A in the online version at DOI: 10.1016/j.jri.2016.04.001). Of the two forms identified with the pAb, the Ts4 mAb detected the 82-kDa band, but not the 77-kDa band (Fig. 2). We recently found that this mAb reacted against an epitope containing a fucosylated agalacto-complex-type biantennary glycan carrying bisecting Glc-

NAc (Yoshitake et al., 2015). These findings suggest that the 82and 77-kDa forms of Naglu have each kind of N-glycosylation pattern, but that only the 82-kDa form has the N-linked OS chain reactive with Ts4. Distinct immunoreactivity between Ts4 and the anti-Naglu pAb was also observed via morphological studies. In cauda epididymal spermatozoa, Ts4 was only immunoreactive against proteins located in the acrosomal region. However, positive staining of the anti-Naglu pAb was observed in the flagellum, as well as in the acrosomal region (Fig. 2E–G). These results suggest that the structure of the N-linked OS chains on Naglu may

Fig. 3. Inhibitory effect of Ts4 concentration on IVF. Cauda epididymal spermatozoa pretreated with 10 (A), 50 (B), or 70 ␮g/ml (C) of RP-3 (an isotype matched control mAb) or Ts4 were used in the IVF examination. The numbers of eggs provided in this study: A; 115 (RP-3) and 110 (Ts4), n = 3, B; 321 (RP-3) and 308 (Ts4), n = 6, C; 186 (RP-3) and 188 (Ts4), n = 3. *, p < 0.05 based on the Student’s t test.

H. Yoshitake et al. / Journal of Reproductive Immunology 115 (2016) 6–13

11

Fig. 4. Effects of Ts4 on AR in vitro. Inhibitory effect of Ts4 on A23187-induced AR (A). After pretreated with Ts4 or normal control mouse IgM, spermatozoa were incubated with the ionophore for 10 min at 37◦ C, and then stained with FITC-conjugated PSA. The fluorescent intensity of PSA was measured using flow cytometry. The histograms show representative results of changes in fluorescence intensity of the spermatozoa incubated with the ionophore after treatment with Ts4 (blue line) or normal control mouse IgM (n.c.; red line). Black lines indicate the fluorescence intensity of the spermatozoa incubated in the absence of the ionophore. Enhancement of AR treated by GAGs (B). The cauda epididymal spermatozoa were incubated with heparan sulfate, heparin, chondroitin sulfate, dermatan sulfate, or hyaluronan (each 1, 10, or 100 ␮g/ml) for 4 h at 37 ◦ C. Spermatozoa were then treated with FITC-conjugated PSA and examined using the FACSCalibur flow cytometry system. The data are expressed as the relative intensity of mean fluorescence intensity (MFI); i.e., (MFI after incubation in the presence of each GAGs)/(MFI in the absence of the GAG) with SD (n = 3). **, p < 0.01; *, p < 0.05 compared to the control (no GAG treatment) based on the Student’s t test. Inhibitory effect of Ts4 on AR induced by GAGs (C, D). After treatment with 10 ␮g/ml of Ts4 or normal control

12

H. Yoshitake et al. / Journal of Reproductive Immunology 115 (2016) 6–13

differ between the epididymal sperm head and the flagellum of the spermatozoa. In the present study, we showed that Ts4 and the anti-Naglu pAb inhibited fertilization in vitro (Fig. 3 and Table 2), suggesting that these Abs-reactive molecules (the fucosylated agalacto-biantennary complex-type N-glycan with bisecting GlcNAc and Naglu, respectively) may be involved in the binding and fusion of spermatozoa to eggs. In cancer cells, the bisecting GlcNAc structures on cadherins or integrins were reported to play roles in cell–cell and cell-extracellular matrix interactions (Zhao et al., 2008). It showed that one of sperm glycoproteins possessing the Ts4-reactive bisected N-glycan is Naglu, but others are not yet identified. However, our present experimental data suggest the involvement of this OS chain on the sperm head glycoproteins (including Naglu) with the sperm-egg interaction. On the other hand, Naglu is an enzyme required for the degradation of heparan sulfate, which can be categorized as a GAG (Zhao et al., 1996). GAGs play pivotal roles in biological processes (e.g., cell–cell and cell-matrix interactions, regulation of growth factors, and the activation of chemokines and cytokines) (Yung and Chan 2007; Dreyfuss et al., 2009). Although the biological function of Naglu on sperm head has not been yet clarified, this molecule may influence successful IVF via the degradation of heparan sulfate. However, fertile ability of Naglu-deficient mice is within normal range (Li et al., 1999), suggesting that this molecule is not always essential for mice fertilization process in vivo. Although the precise reason(s) of the discrepancy between in vitro and in vivo experimental results is unknown, similar phenomena were also observed in other several molecules. For instance, an anti-CD9 mAb inhibited sperm-egg binding and fusion in vitro (Chen et al., 1999), whereas CD9-null male mice showed normal fertility in vivo (Miyado et al., 2000). As a possible explanation in the case of Naglu-deficient mice, other molecule(s) on the sperm head may compensate the function of Naglu in the process of fertilization in vivo. In bovine spermatozoa, heparan sulfate, heparin, and chondroitin sulfate are well known to promote capacitation and the AR in vitro (Handrow et al., 1982; Parrish et al., 1985; Therien et al., 2005; Bergqvist et al., 2007). We showed that heparan sulfate, heparin, chondroitin sulfate, and dermatan sulfate also induce the AR in mouse spermatozoa in vitro (Fig. 4B). Moreover, the heparan sulfate-induced AR was inhibited by pretreatment with Ts4 (Fig. 4C), suggesting that the binding of heparan sulfate to Ts4-recognized molecules such as Naglu may promote the AR. However, because the Ts4-reactive N-glycan binds to multiple proteins (at least six kinds of glycoproteins including Naglu) (Fig. 1A), the effects of Ts4 may cause via binding to other molecules possessing the epitope for Ts4 than Naglu. In summary, we identified Naglu as one of the Ts4-reactive molecules. Although NAGLU deficiency is known to cause Sanfilippo B syndrome (mucopolysaccharidosis III B) (Neufeld and Muenzer 2001), the precise biological function of this molecule is not fully understood. We showed that Naglu is expressed in the acrosomal region of mouse spermatozoa and the OS chains on this molecule may play a role in the process of fertilization. The next step in this line of study will be identification of the Ts4-recognized molecules in the mouse spermatozoa (other than Naglu). The production and biological effect of the Ts4 mAb clearly demonstrate that acrosomal region-specific OS chains are amongst the target antigens recognized by ASA-Abs and play an important role in fertilization. Structure and function of the various OS chains

involved (as target antigens for ASA-Abs) must be explored to gain a deep understanding of fertilization. Acknowledgements We are indebted to Dr. Masaru Okabe (Osaka University) and Dr. Kiyotaka Toshimori (Chiba University) for the gifts of the anti-IZUMO1 mAb and MN9. We deeply express our thanks to Mr. Shozo Ichinose, Mr. Daisuke Arii, and Dr. Kensuke Hamamura (Juntendo University) for their technical assistance with producing the Abs. This work was supported in part by Grants-in Aid for General Scientific Research, Nos. 21592111, 23390389, 24592609 & 25462575, and “High-Tech Research Center” Project for Private Universities: matching fund subsidy from the Minister of Education, Culture, Sports, Science, and Technology, Japan. References Baba, T., et al., 1994. Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization. J. Biol. Chem. 269, 31845–31849. Barratt, C.L., et al., 1989. Antisperm antibodies are more prevalent in men with low sperm motility. Int. J. Androl. 12, 110–116. Bergqvist, A.S., et al., 2007. Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-540. Zygote 15, 225–232. Bleil, J.D., Wassarman, P.M., 1990. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking. Proc. Natl. Acad. Sci. U. S. A. 87, 5563–5567. Bronson, R.A., et al., 1989. Effects of anti-sperm antibodies on human sperm ultrastructure and function. Hum. Reprod. 4, 653–657. Chen, M.S., et al., 1999. Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin alpha6beta1: implications for murine fertilization. Proc. Natl. Acad. Sci. U. S. A. 96, 11830–11835. Cho, C., et al., 1998. Fertilization defects in sperm from mice lacking fertilin beta. Science 281, 1857–1859. Choi, D., et al., 2001. The biological significance of phospholipase C beta 1 gene mutation in mouse sperm in the acrosome reaction, fertilization, and embryo development. J. Assist. Reprod. Genet. 18, 305–310. Coonrod, S.A., et al., 1996. Inhibition of bovine fertilization in vitro by antibodies to SP-10. J. Reprod. Fertil. 107, 287–297. Dreyfuss, J.L., et al., 2009. Heparan sulfate proteoglycans Structure, protein interactions and cell signaling. An. Acad. Bras. Cienc. 81, 409–429. Eggert-Kruse, W., et al., 1993. Antisperm antibodies in cervical mucus in an unselected subfertile population. Hum. Reprod. 8, 1025–1031. Fijak, M., Meinhardt, A., 2006. The testis in immune privilege. Immunol. Rev. 213, 66–81. Guesdon, J.L., et al., 1979. The use of avidin-biotin interaction in immunoenzymatic techniques. J. Histochem. Cytochem. 27, 1131–1139. Handrow, R.R., et al., 1982. Structural comparisons among glycosaminoglycans to promote an acrosome reaction in bovine spermatozoa. Biochem. Biophys. Res. Commun. 107, 1326–1332. Hao, Z., et al., 2002. SAMP32 a testis-specific, isoantigenic sperm acrosomal membrane-associated protein. Biol. Reprod. 66, 735–744. Hunnicutt, G.R., et al., 1996. Sperm surface protein PH-20 is bifunctional one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding. Biol. Reprod. 55, 80–86. Inoue, N., et al., 2005. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434, 234–238. Isojima, S., 1989. Human sperm antigens corresponding to sperm-immobilizing antibodies in the sera of women with infertility of unknown cause: personal review of our recent studies. Hum. Reprod. 4, 605–612. Kohno, N., et al., 1998. Two novel testicular serine proteases, TESP1 and TESP2: are present in the mouse sperm acrosome. Biochem. Biophys. Res. Commun. 245, 658–665. Koide, S.S., et al., 2000. Antisperm antibodies associated with infertility: properties and encoding genes of target antigens. Proc. Soc. Exp. Biol. Med. 224, 123–132. Kurita, A., et al., 2001. Identification cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen. Biol. Reprod. 64, 935–945. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Li, H.H., et al., 1999. Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding alpha-N-acetylglucosaminidase. Proc. Natl. Acad. Sci. U. S. A. 96, 14505–14510.

mouse IgM (n.c.), the spermatozoa were incubated with 100 ␮g/ml of heparan sulfate (C) or heparin (D) for 4 h at 37 ◦ C, and then were labeled with FITC-conjugated PSA. The data shown in panels C and D are the relative intensity the MFI: (MFI after treatment with Ts4 or normal control IgM (n.c.) following incubation in the presence of the each GAGs)/(MFI after incubation in the absence of the GAG) with SD (n = 3). *, p < 0.05; ND, not difference based on the Student’s t test.

H. Yoshitake et al. / Journal of Reproductive Immunology 115 (2016) 6–13 Lu, Q., Shur, B.D., 1997. Sperm from beta 1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly. Development 124, 4121–4131. Miyado, K., et al., 2000. Requirement of CD9 on the egg plasma membrane for fertilization. Science 287, 321–324. Neufeld, E.F., Muenzer, J., 2001. The mucopolysaccharidoses. In: Scriver, C.R., et al. (Eds.), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York, pp. 3421–3452. Nishimura, H., et al., 2004. Possible function of the ADAM1a/ADAM2 fertilin complex in the appearance of ADAM3 on the sperm surface. J. Biol. Chem. 279, 34957–34962. Nohara, M., et al., 1998. Partial characterization of the gametes and development of a successful in vitro fertilization procedure in the mastomys (Praomys coucha): a new species for reproductive biology research. Biol. Reprod. 58, 226–233. Parrish, J.J., et al., 1985. Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro. Theriogenology 24, 537–549. Rochwerger, L., et al., 1992. Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion. Dev. Biol. 153, 83–90. Sekiya, S., et al., 1989. Selective depletion of rat neutrophils by in vivo administration of a monoclonal antibody. J. Leukoc. Biol. 46, 96–102. Shirai, Y., et al., 2009. Distribution of molecular epitope for Ts4, an anti-sperm auto-monoclonal antibody in the fertilization process. J. Reprod. Dev. 55, 240–246. Takayama, T., et al., 2005a. TEX101 is shed from the surface of sperm located in the caput epididymidis of the mouse. Zygote 13, 325–333. Takayama, T., et al., 2005b. Sexually dimorphic expression of the novel germ cell antigen TEX101 during mouse gonad development. Biol. Reprod. 72, 1315–1323.

13

Therien, I., et al., 2005. Isolation and characterization of glycosaminoglycans from bovine follicular fluid and their effect on sperm capacitation. Mol. Reprod. Dev. 71, 97–106. Toshimori, K., et al., 1992. Characterization of the antigen recognized by a monoclonal antibody MN9: unique transport pathway to the equatorial segment of sperm head during spermiogenesis. Cell Tissue Res. 270, 459–468. Tsukamoto, H., et al., 2006. Testicular proteins associated with the germ cell-marker, TEX101: Involvement of cellubrevin in TEX101-trafficking to the cell surface during spermatogenesis. Biochem. Biophys. Res. Commun. 345, 229–238. Weber, B., et al., 1996. Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis IIIB). Hum. Mol. Genet. 5, 771–777. Yoshinaga, K., et al., 2001. Inhibition of mouse fertilization in vivo by intra-oviductal injection of an anti-equatorin monoclonal antibody. Reproduction 122, 649–655. Yoshitake, H., et al., 2008a. Molecular diversity of TEX101, a marker glycoprotein for germ cells monitored with monoclonal antibodies: variety of the molecular characteristics according to subcellular localization within the mouse testis. J. Reprod. Immunol. 79, 1–11. Yoshitake, H., et al., 2008b. TEX101 a germ cell-marker glycoprotein, is associated with lymphocyte antigen 6 complex locus k within the mouse testis. Biochem. Biophys. Res. Commun. 372, 277–282. Yoshitake, H., et al., 2015. Chemical characterization of N-linked oligosaccharide as the antigen epitope recognized by an anti-sperm auto-monoclonal antibody, Ts4. PLoS One 10, e0133784. Yung, S., Chan, T.M., 2007. Glycosaminoglycans and proteoglycans: overlooked entities? Perit. Dial. Int. 27 (Suppl. 2), S104–9. Zhao, H.G., et al., 1996. The molecular basis of Sanfilippo syndrome type B. Proc. Natl. Acad. Sci. U. S. A. 93, 6101–6105. Zhao, Y., et al., 2008. Branched N-glycans regulate the biological functions of integrins and cadherins. FEBS J. 275, 1939–1948.