REPRODUCTION

© 2016 Society for Reproduction and Fertility DOI: 10.1530/REP-15-0551ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org

ReseaRch

seminal vesicle proteins sVs3 and sVs4 facilitate sVs2 effect on sperm capacitation

Naoya Araki1, Natsuko Kawano2,3, Woojin Kang2,3, Kenji Miyado3, Kaoru Yoshida4 and Manabu Yoshida1,5

1Misaki Marine Biological Station, School of Science, The University of Tokyo, Miura, Kanagawa, Japan, 2Department of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan, 3Department of Reproductive Biology, National Center for Child Health and Development, Setagaya, Tokyo, Japan, 4Biomedical Engineering Center, Toin University of Yokohama, Yokohama, Kanagawa, Japan, and 5Center for Marine Biology, The University of Tokyo, Miura, Kanagawa, Japan

Correspondence should be addressed to M Yoshida; Email: yoshida@mmbs.s.u-tokyo.ac.jp

abstract

Mammalian spermatozoa acquire their fertilizing ability in the female reproductive tract (sperm capacitation). On the other hand, seminal vesicle secretion, which is a major component of seminal plasma, inhibits the initiation of sperm capacitation (capacitation inhibition) and reduces the fertility of the capacitated spermatozoa (decapacitation). There are seven major proteins involved in murine seminal vesicle secretion (sVs1-7), and we have previously shown that sVs2 acts as both a capacitation inhibitor and a decapacitation factor, and is indispensable for in vivo fertilization. however, the effects of sVss other than sVs2 on the sperm have not been elucidated. since mouse Svs2–Svs6 genes evolved by gene duplication belong to the same gene family, it is possible that sVss other than sVs2 also have some effects on sperm capacitation. In this study, we examined the effects of sVs3 and sVs4 on sperm capacitation. Our results showed that both sVs3 and sVs4 are able to bind to spermatozoa, but sVs3 alone showed no effects on sperm capacitation. On the other hand, sVs4 acted as a capacitation inhibitor, although it did not show decapacitation abilities. Interestingly, sVs3 showed an affinity for sVs2 and it facilitated the effects of sVs2. Interaction of sVs2 and spermatozoa is mediated by the ganglioside GM1 in the sperm membrane; however, both sVs3 and sVs4 had weaker affinities for GM1 than sVs2. Therefore, we suggest that separate processes may cause capacitation inhibition and decapacitation, and sVs3 and sVs4 act on sperm capacitation cooperatively with sVs2.Reproduction (2016) 152 313–321

10.1530/REP-15-0551

IntroductionMammalian spermatozoa need to go through a specific change called sperm capacitation in the female reproductive tract in order to acquire the fertilizing ability (Austin 1951, Chang 1951). Capacitated spermatozoa are capable of acrosome reaction, penetration into the zona pellucida and fusion with oocytes. Several factors from the male reproductive tract such as seminal vesicle autoantigen (SVA) regulate these processes (see review: Yoshida et  al. 2008). The sperm capacitation requires sterol efflux from sperm plasma membrane, which alters sperm plasma membrane physiologically and biochemically (Shadan et al. 2004, Visconti et al. 2011).

Seminal plasma has inhibitory effects on sperm capacitation, and in vitro-capacitated spermatozoa reversibly lose their fertility when treated with seminal plasma (Chang 1957, Bedford & Chang 1962). This phenomenon is known as sperm decapacitation and appears to be conserved among several species (Bedford & Chang 1962). Although decapacitation by various factors

has been reported (Dukelow et  al. 1966, Davis 1974, Reyes et al. 1975, Huang et al. 1999, Lu et al. 2011, Tseng et al. 2013), their in vivo functions remain unclear. On the other hand, the seminal vesicle plays an important role in sperm fertility, and the removal of seminal vesicles from mice results in the decline of fertility (Pang et al. 1979, Bromfield et al. 2014, Kawano et al. 2014). Previously, we reported that seminal vesicle secretory protein 2 (SVS2), which is one of the secreted proteins from the seminal vesicle (SVSs), has two effects on sperm capacitation: an inhibitory effect on initiation of capacitation (capacitation inhibitor role) and cancellation of fertility of the capacitated spermatozoa (decapacitation factor role) (Kawano & Yoshida 2007, Kawano et al. 2008). Furthermore, Svs2-/- males showed lower fertility rates due to spermicidal attack in the uterus (Kawano et al. 2014). SVS2 binds to the sperm plasma membrane in order to inhibit sterol efflux that accompanies capacitation, resulting in the suppression of ectopic capacitation (Kawano & Yoshida 2007, Araki et al. 2015). In mice, there are seven major

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SVS proteins (Fawell et al. 1987), and Svs2–Svs6 genes are paralogous genes evolved by duplication from an ancestor gene of WAP-type four-disulfide core (WFDC)-type protease inhibitors (Clauss et al. 2005, Karn et al. 2008). Svs genes have evolved rapidly under selective pressure (Ramm et  al. 2009), demonstrating their adaptability to various reproductive strategies across species. Even though the same gene family encodes SVSs, they have various functions. SVS1–SVS3 are cross-linked with each other by transglutaminase, forming the copulatory plug (Lin et al. 2002); SVS1 is a putative copper amine oxidase (Lundwall et  al. 2003), SVS4 shows immunoregulatory activity (Romano-Carratelli et al. 1995), SVS5 and SVS6 may act as serine proteinase inhibitors (Clauss et al. 2005) and SVS7 enhances sperm motility (Luo et  al. 2001). Other than SVS2, it is not known whether any of the other SVSs are involved in sperm capacitation. SVS3 and SVS4 compose the copulatory plug (Lin et al. 2002, Mangels et  al. 2015), and SVS3 show the nucleotide sequence similarity with SVS2 (Lin et  al. 2002). Thus, SVSs other than SVS2 may also have functions in sperm capacitation. Here, in order to elucidate function of SVSs other than SVS2 in the regulation of sperm capacitation, we examined the behavior of SVS3 and SVS4 after copulation, and their effect on sperm capacitation.

Materials and methods

Animals

All mice (CD-1) were purchased from Charles River Laboratories Japan (Yokohama, Japan). Mice were housed in cages under a 12 h light:12 h darkness cycle. All animal experiments were approved by the Animal Care and Use Committee of the School of Science, the University of Tokyo, and carried out according to the University of Tokyo Guidelines on Animal Care.

Preparation of recombinant SVS proteins

We used recombinant SVS proteins in our study. Recombinant SVS2 protein with a thioredoxin (Trx) tag (MW 11.8 kDa) was prepared as described previously (Araki et  al. 2015). cDNAs encoding SVS3b (NCBI accession #NM_173377; MW 27.8 kDa, pI 9.26) and SVS4 (NCBI accession #NM_009300; MW 10.3 kDa, pI 8.25) were prepared by reverse transcription-polymerase chain reaction (RT-PCR) using RNA isolated from the mouse seminal vesicles (C57BL/6J strain), and these cDNAs were subcloned into the pET-32 vector (Takara Bio). The Trx fusion proteins were expressed in Escherichia coli BL21-pLysS cells (Novagen; Madison, WI, USA) by induction with 1 mmol/L isopropyl β-D-1-thiogalactopyranoside at 20°C. Recombinant SVS3 and SVS4 proteins were recovered using Ni-NTA agarose (Qiagen) under denaturing conditions with 8 M urea, according to the manufacturer’s instructions. After removing urea using a PD-10 column (GE Healthcare), proteins were lyophilized and stored at −20°C until further use. Immediately before the experiment, recombinant SVS3

and SVS4 proteins were dissolved in HTF medium (Nippon Medical & Chemical Instruments Co; Osaka, Japan) without BSA at 30 µM and 60 µM respectively.

Purity of recombinant SVS3 and SVS4 proteins was evaluated by Western blotting, using anti-SVS3, anti-SVS4 and anti-Trx tag antibodies. Anti-SVS3 rabbit polyclonal antibody was produced by Operon Biotechnologies (Tokyo, Japan) by SVS3b peptide immunization (amino acids 75–88 of SVS3b: DADADMGGALSSQE), and purified by affinity column chromatography (HiTrap NHS-activated HP; GE Healthcare), with the recombinant SVS3 protein as a ligand. Anti-SVS4 rabbit polyclonal antibody was produced by Japan-Lamb Co. (Hiroshima, Japan) by SVS4 peptide immunization (amino acids 48–62 of SVS4: SRSSEPEVFVRPQDS), and purified by Protein A column (HiTrap Protein A; GE Healthcare). Anti-Trx tag antibody was purchased from MBL Co. (Nagoya, Japan).

Western blotting

Recombinant SVSs proteins were dissolved in Laemmli sample buffer and separated by Laemmli SDS-PAGE using a 12% (w/v) polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% (w/v) skimmed milk (Nacalai Tesque; Kyoto, Japan) in phosphate-buffered saline (PBS) with 0.1% (w/v) Tween 20 (PBS-T) (Sigma-Aldrich) for 30 min at room temperature. The membranes were treated with anti-SVS3 antibody (2 μg/mL), anti-SVS4 antibody (10 μg/mL) or anti-Trx tag antibody (1 μg/mL) in PBS-T containing 5% (w/v) skimmed milk at 4°C for 12 h. After washing the primary antibodies with PBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000) (Bethyl Laboratories; Montgomery, TX, USA) or anti-mouse IgG antibody (1:5000) (Sigma-Aldrich) in PBS-T containing 5% (w/v) skimmed milk for 1 h at room temperature, and washed with PBS-T three times. Protein detection was performed using ECL Prime Western Blotting Detection Reagent (GE Healthcare), and observed with a Chemiluminescence Imager (LuminoGraphI WSE-6100H, ATTO, Tokyo, Japan). Proteins were also visualized using Coomassie Brilliant Blue R250 staining.

Acrosomal responsiveness assay

In order to examine the effects of SVS3 and SVS4 on sperm capacitation, we used epididymal spermatozoa, since ejaculated spermatozoa are coated with seminal plasma proteins. Spermatozoa were isolated from the cauda epididymis of sexually mature male CD-1 mice (9–15 weeks old), suspended in HTF medium containing 5 mg/mL BSA (A-4503; Sigma-Aldrich), and incubated at 37°C in a 5% (v/v) CO2 atmosphere for 2 h. SVS proteins were added to the medium as indicated in the figure and/or figure legends.

Sperm capacitation was evaluated based on acrosomal responsiveness to progesterone and ionomycin as described previously (Araki et al. 2015). Briefly, spermatozoa were treated with 100 μmol/L progesterone (Wako) (and 1% ethanol as a vehicle) in the medium for 15 min at 37°C or with 15 μmol/L ionomycin (Sigma-Aldrich) (and 0.5% dimethylsulfoxide as a vehicle) in the medium for 10 min at room temperature.

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Spermatozoa were fixed with 100% methanol and stained with 100 μg/mL fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) (Sigma-Aldrich) for 15 min at 37°C. FITC fluorescence was observed using a fluorescence microscope, and spermatozoa with no fluorescence signal at the acrosome region were regarded as acrosome-reacted spermatozoa. Three fields were observed in each batch, and 100 spermatozoa per field were selected and evaluated for acrosomal reaction.

Molecular interaction assay

Interactions between GM1 and SVS proteins were investigated by a highly sensitive 30 MHz quartz crystal

microbalance (QCM; NAPiCOS; Nihon Dempa Kogyo Co, Tokyo, Japan). The first channel of a sensor chip was coated with 1000 pmol SVS3 or SVS4 dissolved in PBS (pH 7.4). The second channel of the same sensor chip was coated with 1000 pmol Trx dissolved in PBS, as a reference. The sensor chip was washed three times with PBS, placed into the chamber, perfused with PBS until the frequency was stabilized and blocked by perfusing it twice with 1 mg/mL Block Ace (Dainippon Pharmaceutical Co; Osaka, Japan) dissolved in PBS. Subsequently, the sensor chip was perfused with GM1 (2.5 nmol for SVS3; 5 nmol for SVS4) dissolved in PBS, and the change in frequency was recorded.

In order to examine SVS2 and SVS3 interactions, the first channel of a sensor chip was coated with 1000 pmol SVS3 dissolved in PBS. The second channel on the same sensor chip was coated with 1000 pmol Trx dissolved in PBS, as a reference. After washing, perfusion and blocking, the sensor chip was perfused with 100 pmol SVS2, and the change in frequency was recorded. All experiments were performed at 25°C, with a flow rate of 50 μL/min.

To evaluate the binding affinity, changes in the frequency of cumulative perfusion were analyzed. Affinities were evaluated by the Michaelis–Menten equation, and fitted curves were obtained by calculating the average of five experimental values. The dissociation constant (Kd) and maximum binding amounts (ΔMmax) were calculated with NAPiCOS Analysis software (Nihon Dempa Kogyo Co; Tokyo, Japan).

Localization of SVS3 and SVS4 in spermatozoa

Epididymal spermatozoa were isolated from sexually mature male CD-1 mice (9–15 weeks old). Spermatozoa were suspended in HTF medium (without BSA) containing 20 μmol/L recombinant Trx-SVS3, Trx-SVS4 or Trx proteins at 2.0 × 105 sperm cells/mL. After incubation at 37°C in 5% CO2 atmosphere for 2 h, spermatozoa were fixed with 2% paraformaldehyde for 10 min on ice. Spermatozoa were washed three times with PBS and blocked with PBS containing 1% (w/v) BSA for 1 h at room temperature.

Figure 1 SVS3 and SVS4 protein expressions. Mouse seminal vesicle fluid (SVF), purified recombinant Trx-SVS3 and purified recombinant Trx-SVS4 were analyzed by 12% SDS-PAGE. (A) Coomassie Brilliant Blue R250 (CBBR) staining. (B) Western blotting with anti-SVS3 antibody. (C) Western blotting with anti-SVS4 antibody. (D) Western blotting with anti-Trx tag antibody. Both anti-SVS3 and anti-Trx tag antibodies detected SVS3 in SVF (predicted MW: 28 kDa) and recombinant Trx-SVS3 protein (predicted MW: 45 kDa) at 37 and 55 kDa respectively (closed arrowheads); both anti-SVS4 and anti-Trx antibodies detected SVS4 in SVF (predicted MW: 10 kDa) and recombinant Trx-SVS4 protein (predicted MW: 28 kDa) at 13 and 31 kDa respectively (open arrowheads). The anti-Trx tag antibody detected only recombinant Trx-SVS3 and Trx-SVS4 proteins, and did not cross-react with any SVF proteins.

Figure 2 SVS3 (predicted MW: 28 kDa) and SVS4 (predicted MW: 10 kDa) in the female reproductive tract after copulation. Intratubular fluids from the vagina (V), the uterine region near the vagina (Uv), the uterine region near the oviduct (Uo) and the oviduct (O) were collected 1.5 h after copulation. Samples were also collected from the seminal vesicle fluid (SVF), the copulatory plug (CP) and the uterine fluid of virgin females in estrus (NC), to be used as controls. (A) Coomassie Brilliant Blue R250 (CBBR) staining. (B and C) Western blotting with the anti-SVS3 antibody (B) and the anti-SVS4 antibody (C). SVS3 was detected in SVF (closed arrowhead), but not in the entire female reproductive tract. SVS4 was detected in SVF, CP, Uv and Uo (open arrowhead), but not in the oviduct.

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Samples were treated with 10 μg/mL anti-Trx antibody (MBL, Nagoya, Japan) in PBS containing 1% BSA for 12 h at 4°C. After washing with PBS, spermatozoa were treated with Alexa 488-conjugated secondary antibody (anti-mouse IgG; Thermo Fisher Scientific) (1:100 in PBS containing 1% BSA) and 10 μg/mL Hoechst 33342 (Thermo Fisher) for 12 h at 4°C. Fluorescent images were acquired using a fluorescence microscope system (BIOREVO BZ-9000, KEYENCE, Tokyo, Japan). Images were taken by a monochrome camera and pseudocolored by a software of the microscope system. Exposure time was 1 s for Alexa 488 and 1/15 s for Hoechst 33342.

Detection of SVS proteins in the female reproductive tract

Female mice 9–15 weeks old (CD-1; Charles River Japan) were mated with 9–15-week-old male mice. Females were killed at 1.5 h post coitus, and intratubular fluids were collected by flushing the following parts of the female reproductive tract with PBS: the vagina, the uterine region near the vagina, the uterine region near the oviduct and the oviduct. Control samples were collected from the seminal vesicles, the copulatory plugs and estrus-stage uterine fluids. The fluids were precipitated with acetone and dissolved in Laemmli sample buffer. Western blotting with anti-SVS3 or anti-SVS4 antibodies was performed as described above.

Statistical analysis

All experiments were repeated at least three times independently. Data are expressed as mean ± s.d. Statistical significance was calculated using the Student’s t-test; P < 0.01 was considered statistically significant.

Results

Distribution of SVS3 and SVS4 in the female reproductive tract after copulation

SVS3 and SVS4 are seminal plasma proteins originating from seminal vesicle secretions (Fawell et  al. 1987). Here, we showed that SVS3 and SVS4 in the seminal vesicle fluid were existed as 37 and 13 kDa proteins respectively (Fig. 1). Recombinant Trx-tagged SVS3 and SVS4 proteins were also recognized by the antibodies and the anti-Trx tag antibody (Fig. 1). In the Trx-SVS3, there are some low (about 50 kDa) and high (110 kDa) extra bands recognized with anti-SVS3 and anti-Trx tag antibodies. Probably they are degraded and coagulated Trx-SVS3.

After copulation, SVS2 enters the uterus and attaches to the sperm head in order to protect sperm (Kawano & Yoshida 2007, Kawano et  al. 2014, Araki et  al. 2015). To examine whether this is also true for SVS3 and SVS4, we measured SVS3 and SVS4 quantities in each part of the female reproductive tract we collected,

Figure 4 Effects of SVS3 on sperm. Rates of capacitated spermatozoa were evaluated by the acrosome reaction (AR) induced by progesterone (gray bars) or by ionomycin (white bars). (A) Effects of SVS3 on the sperm. Spermatozoa were incubated in the HTF medium containing 5 mg/mL BSA and 10–30 μmol/L Trx-SVS3 for 2 h, and the induced-AR rate of the sperm was examined. (B) Effects of SVS3 on the capacitated spermatozoa. Sperm treatment conditions are shown in the right diagram. Length of bar shows incubation time. Spermatozoa were incubated with HTF medium with reagents shown in the bar. BSA: 5 mg/mL bovine serum albumin in HTF, SVS3: 20 µmol/L recombinant Trx-SVS3 in HTF. Data are shown as mean ± s.d. from three individual experiments. Asterisks indicate significant differences in both progesterone- and ionomycin-induced AR rates (P < 0.01, Student’s t-test).

Figure 3 Binding patterns of SVS3 and SVS4 to epididymal spermatozoa. Epididymal spermatozoa incubated in HTF medium with Trx-SVS3 (upper row), Trx-SVS4 (middle row) and Trx (lower row) were stained with anti-Trx antibodies (anti-Trx: green). Sperm nuclei were stained with Hoechst 33342 (Hoechst33342: blue) for showing SVSs binding region. Magnified merged images of the sperm heads were shown at the right. Images were taken by a monochrome camera and pseudocolored by a software of the microscope (see ‘Materials and methods’ section). Although faint signals on the entire sperm were detected in all samples, pronounced signals were detected in samples incubated with Trx-SVS3 or Trx-SVS4, in the postacrosomal region of the sperm head.

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1.5 h after copulation: the copulatory plug, the vagina, the uterine region near the vagina, the uterine region near the oviduct and the oviduct. Anti-SVS3 antibody showed that there were no SVS3 proteins in the entire female reproductive tract (Fig. 2), whereas the anti-SVS4 antibody showed a 13 kDa band of SVS4 accompanied with many nonspecific bands in the uterus, but not in the oviduct (Fig. 2). This suggests that SVS4 enters the uterus similarly to SVS2, while SVS3 does not enter the female reproductive tract.

Binding patterns of SVS3 and SVS4 to epididymal spermatozoa

SVS2 binds to the postacrosomal region of the sperm head (Kawano & Yoshida 2007). We examined whether SVS3 and SVS4 proteins also bind to the sperm surface. When epididymal spermatozoa were incubated with Trx-SVS3 and then immunostained with the anti-Trx tag antibody, a fluorescence signal was observed in the postacrosomal region (Fig. 3 upper row). Likewise, anti-Trx-tag antibody immunostaining of epididymal spermatozoa incubated with Trx-SVS4 showed signal in the head, probably postacrosomal region (Fig. 3 middle row). The spermatozoa incubated with Trx showed no noticeable signal in the head region (Fig.  3 lower row). Similar results were obtained when we immunostained with the anti-SVS3 and anti-SVS4 antibodies: fluorescence signals of SVS3

and SVS4 were observed in the postacrosomal region (Supplementary Fig.  1, see section on supplementary data given at the end of this article). These results indicate that both SVS3 and SVS4 can bind to the postacrosomal region of the spermatozoa, following the same interaction pattern as SVS2.

Effects of SVS3 on sperm

Our results suggest that SVS3 proteins do not enter the uterus with the semen, but they are attached to the sperm head. Thus, we examined whether SVS3 can regulate sperm capacitation. Status of sperm capacitation was evaluated by measuring the induced acrosome reaction (AR) rate. Incubation of the spermatozoa with 5 mg/mL BSA for 2 h increased the induced-AR rate significantly, which agrees with previously described results (Fig. 4A) (Kawano & Yoshida 2007, Araki et  al. 2015). The spermatozoa incubated with BSA and 10–30 μmol/L Trx-SVS3 showed no significant difference in the induced-AR rate in comparison with the spermatozoa treated with

Figure 5 Cooperative effects of SVS2 and SVS3 on sperm. Spermatozoa were incubated in the HTF medium containing 5 mg/mL BSA, 0–20 μmol/L Trx-SVS2 and 0–20 µmol/L Trx-SVS3, and the rates of acrosome reaction (AR) induced by progesterone (gray bars) or ionomycin (white bars) were evaluated. Data are shown as mean ± s.d. of values obtained in three to six individual experiments. Asterisks show significant differences in both progesterone- and ionomycin-induced AR rates (P < 0.01, Student’s t-test).

Figure 6 Effects of SVS4 on sperm. Acrosome reaction (AR) rate induced by progesterone (gray bars) or ionomycin (white bars) was evaluated. (A) Effects of SVS4 on the sperm. Spermatozoa were incubated in the HTF medium containing 5 mg/mL BSA and 10–30 μmol/L Trx-SVS4, and the induced acrosome reaction rate was examined. (B) Effects of SVS4 on capacitated spermatozoa. Sperm treatment conditions are shown in the right diagram. Length of bar shows incubation time. Spermatozoa were incubated with HTF medium with reagents shown in the bar. BSA: 5 mg/mL bovine serum albumin in HTF, SVS4: 20 µmol/L recombinant Trx-SVS4 in HTF. Data are shown as mean ± s.d. of three individual experiments. Asterisks indicate significant differences in both progesterone- and ionomycin-induced AR rates (P < 0.01, Student’s t-test).

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BSA alone (Fig. 4A). Previous studies showed that the treatment of the capacitated spermatozoa with SVS2 decreases acrosomal responsiveness of the sperm, which was designated as decapacitation effect (Kawano & Yoshida 2007, Araki et al. 2015). However, SVS3 did not show this decapacitation effect: when we incubated

spermatozoa with Trx-SVS3 following BSA treatment, the induced-AR rate did not change (Fig. 4B).

Next, we examined the influence of SVS3 on the SVS2 effect. The sperm incubated with 20 μmol/L Trx-SVS2 significantly decreased the induced-AR rate, whereas 10 μmol/L Trx-SVS2 did not show significant decrease in the induced-AR rate (Fig. 5). Interestingly, the coincubation of spermatozoa with 10 μmol/L Trx-SVS2 and 10–20 μmol/L Trx-SVS3 led to the decrease in induced-AR rate to the level of the sperm incubated with 20 μmol/L Trx-SVS2 (Fig. 5). On the other hand, the induced-AR rate of the sperm incubated with 20 μmol/L Trx-SVS2 and 20 μmol/L Trx-SVS3 was equal to that of sperm incubated with 20 μmol/L Trx-SVS2 alone (Fig. 5). These results indicate that SVS3 itself has neither capacitation-inhibiting effect nor decapacitation effect, but that SVS3 is able to potentiate the effect of SVS2 on sperm capacitation.

Effects of SVS4 on sperm

Our results show that SVS4 enters the uterus with semen and attaches to the ejaculated sperm head. Following these results, we examined the effect of SVS4 on sperm capacitation. Incubation with SVS4 inhibited the sperm capacitation: the induced-AR rate of the sperm incubated with BSA and 20–30 μmol/L Trx-SVS4 decreased significantly compared with that of the spermatozoa incubated with BSA alone (Fig.  6A). However, the induced-AR rate was not reduced when the spermatozoa were incubated with Trx-SVS4 after BSA treatment (Fig. 6). These results indicate that SVS4 can inhibit sperm capacitation but does not have decapacitation effect.

GM1 interactions with SVS3 or SVS4

SVS2 selectively binds to ganglioside GM1 (Kd = 4.8 ± 0.5 μmol/L; ΔMmax = 972.2 ± 35.1 ng) and

Figure 7 The interactions between GM1-SVS3 (A), GM1-SVS4 (B) and SVS2-SVS3 (C). (A) GM1 was continuously added to quartz-immobilized Trx-SVS3 (closed circles) or thioredoxin (Trx; open circles), and fitted curves (solid, SVS3; dashed, Trx) were obtained from five individual experiments. (B) GM1 was continuously added to the quartz-immobilized Trx-SVS4 (closed circles) or thioredoxin (Trx; open circles). Fitted curves (solid, SVS4; dashed, Trx) were obtained from five individual experiments. (C) SVS2 was continuously added to the quartz-immobilized Trx-SVS3 (closed circles) or thioredoxin (Trx; opened circles). Fitted curves (solid, SVS3; dashed, Trx) were obtained from five individual experiments. The affinities of GM1 for SVS3, GM1 for Trx-SVS4 and Trx-SVS2 for Trx-SVS3 were evaluated by the Michaelis–Menten equation, and it was demonstrated that their interactions are significantly different compared with the interactions with Trx.

Figure 8 Model of roles of each SVS on sperm. SVSs seem to have two roles on sperm. The first is interaction with the sperm plasma membrane probably via GM1, which may be indispensable for the inhibition of sperm capacitation. The second is interaction to cholesterol via cholesterol-interacting domains, which may be required for decapacitation. SVS2 has both interactions. SVS3 has stronger interaction to SVS2 than to spermatozoa and cholesterol, which yields facilitating effect on SVS2. SVS4 does not interact with cholesterol but interact with spermatozoa, which does not yield inhibition of capacitation but decapacitation.

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attaches to the sperm plasma membrane (Kawano et al. 2008). To examine whether SVS3 and SVS4 also attach to sperm via GM1, interactions between GM1 and SVS3/SVS4 were evaluated by the QCM assay. The Kd value of GM1 and Trx-SVS3 interaction was 94.1 ± 25.7 μmol/L with ΔMmax = 808.3 ± 74.9 ng, and GM1 and Trx-SVS4 interaction was 105.1 ± 23.2 μmol/L, with ΔMmax = 1599.7 ± 206.8 ng (Fig. 7). On the contrary, the Kd value of GM1 to Trx binding was 285.8 ± 55.5 μmol/L, with ΔMmax = 527.3 ± 86.5 ng. These results indicate that SVS3 and SVS4 have affinity for GM1, although these are weaker than SVS2 affinities.

Interactions between SVS2 and SVS3

As described above, SVS4 has inhibitory effect on sperm capacitation, whereas SVS3 itself has no effect on sperm capacitation. Since SVS3 facilitates the inhibitory effect of SVS2 on sperm capacitation, SVS3 may directly interact with SVS2. Thus, the interactions between SVS2 and SVS3 were analyzed by the QCM assay. The Kd value of Trx-SVS2 to Trx-SVS3 binding was 1.4 ± 0.2 μmol/L, with ΔMmax = 2395.9 ± 646.5 ng (Fig.  7C), whereas the Kd value of Trx-SVS2 and Trx interactions was 9.9 ± 3.3 μmol/L, with ΔMmax = 536.4 ± 145.2 ng. As affinity of SVS3 for SVS2 is higher than for GM1, this indicates that SVS3 interacts more tightly with SVS2 than with the sperm plasma membrane.

Discussion

Secretion of seminal vesicles is essential for fertilization in vivo (Kawano et al. 2014), and SVS2, one of the major seminal vesicle secretory proteins, was shown to play an important role in the control of sperm capacitation (Kawano & Yoshida 2007, Kawano et  al. 2008, Araki et al. 2015). Interestingly, removal of the seminal vesicles causes complete infertility, and SVS2-/- mice show a decrease in litter size (Kawano et al. 2014). Furthermore, spermatozoa treated with the seminal vesicle fluid have thicker coatings than spermatozoa treated with SVS2 only, which may contribute to protection against uterus-derived cytotoxic factors (Kawano et  al. 2014). This suggests that the main factor regulating in vivo capacitation may be SVS2, but other SVSs potentially support these effects. In this study, we showed that SVSs other than SVS2 also affect sperm capacitation. We demonstrated that SVS3 has affinity for spermatozoa and SVS2, and facilitates the effects of SVS2 on sperm capacitation. Furthermore, our results show that SVS4 binds to spermatozoa and inhibits capacitation, although the decapacitation effect was not demonstrated. It was shown previously that SVS3 has a transglutaminase cross-linking site (Lin et al. 2002) and forms oligomers with SVS1 and SVS2 (Wagner & Kistler 1987), and that SVS4 suppresses immune responses to cellular antigens in rats (Romano-Carratelli et al. 1995). We demonstrated

that SVS3 directly interacts with SVS2, enhancing the inhibitory effects of SVS2 on sperm capacitation. Our study revealed that SVS3 and SVS4 are partly involved in protecting spermatozoa from spontaneous capacitation in the uterus.

In this study, we showed that SVS4 acts as a capacitation inhibitor, but it did not show any decapacitation factor activity (Fig.  8). We suggest that separate processes may cause these two effects. The effects of SVS2 are a consequence of sterol maintenance in the sperm plasma membrane, where SVS2, which has seven cholesterol recognition/interaction domains (Epand 2006, Scolari et  al. 2010), inhibits sterol efflux from the plasma membrane and incorporates liberated sterol in the plasma membrane (Araki et  al. 2015) (Table  1). SVS4 has no cholesterol recognition/interaction domains (Table  1), suggesting that SVS4 cannot interact with sterols directly. However, since SVS4 has an ability to bind to the postacrosomal region of the spermatozoa and a weak affinity for GM1, it may inhibit cholesterol efflux via interaction with GM1 or with another unknown molecule located in this region. Lack of decapacitation effects of SVS4 might be due to its inability to retrieve sterols to the sperm plasma membrane and also to its low affinity for the sialic acid of GM1, which is the consequence of the low isoelectric point of SVS4 (pI calculated from amino acid sequence: 8.25) compared with that of SVS2 (calculated pI: 9.87). The concentrations of SVS4 and SVS2 in seminal vesicle fluid are the same, and SVS4 can be detected in the uterus after copulation. These results indicate that SVS4 is a complementary factor to SVS2, which may participate in the regulation of in vivo capacitation.

We were not able to find any effect of SVS3 on the sperm capacitation, even though SVS3 has two

Table 1 Predicted cholesterol binding domains in amino acid sequences of SVS proteins.

Name Position sequence

SVS1 124 VEQPMYR250 VWYNGK415 VPHYRLK599 LLYHSR655 LDKTQYSEWER725 VTKYQESER797 LQPFDFYNSFR

SVS2 30 VGQYGATK138 VSQIKSYGQLK196 VSQIKSYGQLK212 VKSYGQTK240 LKSYGQQK264 LKSYGQQK340 LEQYRK

SVS3a 123 VKSYAAQLKSVS3b 123 VKSYAAQJK

156 LKSYKARSVS4 NoneSVS5 NoneSVS6 49 VHEEVYEEKSVS7 59 VGTKHVYSK

79 LIYIMCCEK

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320 N Araki and others

Reproduction (2016) 152 313–321 www.reproduction-online.org

cholesterol-recognizing domains (Table  1). This suggests that SVS3 shows lower affinity for sterols than SVS2. A possible explanation for the interaction of SVS3 with SVS2 is that the effect of SVS3 on capacitation is not a consequence of direct interaction with sterols but a consequence of interaction with SVS2, which might enhance the effects of SVS2. SVS3 has a transglutaminase cross-linking site and acts as a main copulatory plug protein. Furthermore, we were not able to detect SVS3 in the female reproductive tract post coitus. This can be explained by a small amount of SVS3 that enters into the female reproductive tract bound to sperm through SVS2 interactions. Compared with this, SVS4 and SVS1–SVS3 are not cross-linked by transglutaminase, and without this covalent binding, SVS4 can easily enter into the female reproductive tract after copulation.

In humans, proteins secreted from the seminal vesicle, semenogelins (SEMG1 and SEMG2; SEMGs), suppress sperm motility and capacitation (de Lamirande et al. 2001, de Lamirande 2007). SEMG1 and SEMG2 genes are paralogous genes evolved by duplication and show no differences in their physiological roles in the control of fertility. Although mouse SVS2 shows considerable homology with SEMGs, it does not inhibit sperm motility (data not shown). Conversely, the interaction of SEMGs with GM1 has not been reported. These differences between SVS2 and SEMGs may be due to the differences in amino acid sequences, not including N-terminal regions. The chromosomal region occupied by SEMG1–SEMG2 in humans is substituted by Svs2−Svs6 in mice (Clauss et al. 2005), suggesting that SVSs also substitute the SEMG function. Therefore, the roles of other SVSs in the regulation of sperm function, including capacitation, should be further elucidated.

We demonstrated here that SVS3 and SVS4 could facilitate the inhibition of sperm capacitation by SVS2. Even though we elucidated here the roles of SVS3 and SVS4 in sperm capacitation, the male reproductive tract and the seminal plasma may contain other decapacitation factors. It is still unknown whether all of these factors also remain active in vivo, but in order to acquire fertilizing ability, spermatozoa need to inhibit their effects. The use of SVS proteins to regulate the acrosome reaction may be important for semen transport in large domesticated species (and others) where the maintenance of acrosome integrity is crucial for the success of assisted reproduction. Further studies are required in order to evaluate the function of these factors in vivo, and reveal the complete process of regulation of fertilization in mammalian spermatozoa.

supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-15-0551.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported

Funding

This work was supported by JSPS KAKENHI to M Y (grant number 15H04398) and by a grant from the National Center for Child Health and Development to K M and M Y (grant number 26-10).

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Received 19 November 2015First decision 22 December 2015Revised manuscript received 26 July 2016Accepted 1 August 2016

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