BIOLOGY OF REPRODUCTION (2015) 92(1):8, 1–10 Published online before print 13 November 2014. DOI 10.1095/biolreprod.114.120642

Seminal Vesicle Secretion 2 Acts as a Protectant of Sperm Sterols and Prevents Ectopic Sperm Capacitation in Mice1 Naoya Araki,3 Gyo¨rgy Trencse´nyi,4 Zoa´rd T. Krasznai,5 Enik} o Nizsalo´czki,6 Ayako Sakamoto,3 Natsuko 7 7 8 Kawano, Kenji Miyado, Kaoru Yoshida, and Manabu Yoshida2,3,9 3

Misaki Marine Biological Station, School of Science, University of Tokyo, Miura, Japan Department of Nuclear Medicine, University of Debrecen, Debrecen, Hungary 5 Department of Obstetrics and Gynecology, University of Debrecen, Debrecen, Hungary 6 Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary 7 Department of Reproductive Biology, National Center for Child Health and Development, Tokyo, Japan 8 Biomedical Engineering Center, Toin University of Yokohama, Yokohama, Japan 9 Center for Marine Biology, University of Tokyo, Miura, Japan 4

Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methylbeta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo. cholesterol efflux, seminal plasma, sperm capacitation, SVS2

INTRODUCTION Mammalian spermatozoa are ejaculated into the vagina or the uterus and become fertile after remaining in the female reproductive tract for a period of time [1, 2]. This timedependent acquisition of fertility is known as sperm capacitation [3]; capacitated spermatozoa can undergo the acrosome 1 This work was supported in part by Grants-in-Aid for Scientific Research from MEXT and Japan Society for the Promotion of Science (JSPS; KAKENHI) to K.M. and M.Y., and by JSPS and the Hungarian Academy of Sciences under the Japan-Hungary Research Cooperative Program. 2 Correspondence: Manabu Yoshida, Misaki Marine Biological Station, Graduate School of Science, University of Tokyo, 1024 Koajiro, Misaki, Miura, Kanagawa 238-0225, Japan. E-mail: [email protected]

Received: 14 May 2014. First decision: 17 June 2014. Accepted: 3 November 2014. Ó 2015 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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reaction (AR), penetrate into the zona pellucida, and bind and fuse with oocytes. Although the molecular mechanism underlying sperm capacitation is not well understood, several in vitro studies have shown that capacitated sperm decrease sterols [4]. Although the primary sterols in sperm are cholesterol and desmosterol [5], cholesterol seems to be the main factor involved in sperm capacitation. Capacitation is associated with changes in the fluidity of the sperm plasma membrane accompanied by cholesterol efflux [6]. This yields changes in intracellular ion concentrations, metabolism, tyrosine phosphorylation of sperm proteins, and sperm motility [7, 8], all of which are steps required to achieve capacitation. In contrast, in vitro-capacitated spermatozoa reversibly lose their fertility when treated with seminal plasma [9]. This phenomenon is known as sperm decapacitation and appears to be conserved among several species [10]. Decapacitation is caused by several factors in seminal plasma, including polysaccharides [11], glycoproteins [12], and membrane vesicles [13]. Some reports have shown that proteins in the seminal vesicle inhibit capacitation [14–16]. Although decapacitation by various factors has been reported, their in vivo functions and their significance in vivo are unclear. In general, the cell plasma membrane naturally forms steroland sphingolipid-enriched stabilized microdomains, commonly known as lipid rafts, which facilitate the assembly of raftophilic molecules and are further involved in various signaling and trafficking processes. The lipid and sterol composition of the sperm plasma membrane changes drastically because of two main events. First, as spermatozoa undergo epididymal maturation, they obtain sterols from their environment [17]. Second, sperm capacitation appears to be triggered by a reduction in plasma membrane sterol levels [18]. Efflux of cholesterol from lipid rafts destabilizes spermatozoa [6], resulting in capacitation [19]. This cholesterol efflux is associated with activation of the signal transduction pathway necessary for sperm capacitation [17], which reconstitutes lipid rafts and redistributes the molecules required for fertilization [20]. Albumin and methyl-b-cyclodextrin (MbCD) can act to liberate sterols from the plasma membrane, thus promoting sperm capacitation in vitro [6, 21–23]. Concomitantly, these molecules desorb ganglioside and sialic acid from the sperm plasma membrane [24, 25]. Although the importance of sterol efflux in capacitation has been recognized, the specific mechanism underlying in vivo capacitation, including the regulation of sterols in the sperm plasma membrane, is not well understood.

ABSTRACT

ARAKI ET AL.

The seminal plasma protein seminal vesicle secretion 2 (SVS2) is the primary molecule secreted from the seminal vesicle. Seminal vesicle secretions are mixed with spermatozoa and secretions from other accessory reproductive glands before being ejaculated into the vagina. Most SVS2 proteins function in the formation of the copulatory plug in the vagina. In a previous study, however, we showed that some of the SVS2 present in seminal plasma enters into the uterus and functions as a decapacitation factor [26, 27]. We recently found that SVS2/ males showed lower fertility in vivo, whereas they showed normal sperm production and were capable of in vitro fertilization [28]. Interestingly, ejaculated spermatozoa from SVS2/ males are killed in the uterus by some uterine spermicidal attack: the number of spermatozoa capable of entering into the oviduct is significantly decreased [28]. SVS2 binds to the ganglioside GM1 on the sperm plasma membrane [27], and was found attached to the surface of spermatozoa in the uterus [26, 28]. However, after entering the oviduct, spermatozoa are attached to neither SVS2 nor GM1 [26, 27]. Because sperm capacitation appears to occur in the oviduct [29], the dynamics of SVS2 may be related to the regulation of capacitation. SVS2 may prevent spermatozoa from ectopic capacitation in the uterus, resulting in protection of spermatozoa from the spermicidal action. Furthermore, because cholesterol efflux appears to trigger sperm capacitation [19], and loss of membrane cholesterol is prevented when spermatozoa are decapacitated by seminal plasma or by a decapacitation factor [24, 30], we hypothesized that SVS2 may be directly involved in regulating sterol levels in the sperm plasma membrane. In the present study, in order to clarify its role in capacitation, we examined the effect of SVS2 on levels of cholesterol, which seems to be the primary factor in capacitation, and lipid raft dynamics in the sperm plasma membrane. We found that SVS2 acts as a protectant of sperm sterol levels and regulates sperm capacitation.

Preparation of Epididymal Spermatozoa and Incubation for Capacitation To evaluate the effect of SVS2 on sperm capacitation in vitro, spermatozoa were isolated from the cauda epididymides of sexually mature male CD-1 mice (ages 915 wk), and were suspended in HTF medium at 378C in a 5% CO2 atmosphere. We used MbCD (Sigma, St. Louis, MO) at a concentration of 500 lM and bovine serum albumin (BSA; A-4503; Sigma) at concentrations described in Figure 1. SVS2 was used at a concentration of 20 lM, which is similar to the physiological concentration in the uterus [26]. To wash the spermatozoa, HTF medium (378C) was added to spermatozoa, which were gently suspended. Incubation time of each treatment is shown in the Figures. After centrifugation at 600 3 g for 3 min, the supernatant was removed. After repeating this procedure twice, the medium was replaced. Next, SVS2, BSA, MbCD, and cholesterol were added to the replaced medium at the same concentrations as described above. Cholesterol conditions were as follows: 50 mM cholesterol (Sigma) dissolved in 100% ethanol was added to obtain a final concentration of 500 lM. We determined in advance that 1% (v/v) ethanol in the medium did not affect sperm viability (Supplemental Fig. S2) or capacitation (Supplemental Fig. S3).

Observation of Sterol by Filipin Staining

Visualization of Cholesterol Dynamics in the Sperm Plasma Membrane

All mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). Mice were housed in cages under a 12L:12D cycle. All animal experiments were carried out according to the University of Tokyo Guidelines on Animal Care.

To verify that SVS2 affects sterol dynamics, intercalation of fluorescent cholesterol into the sperm plasma membrane was studied. Spermatozoa were incubated in HTF medium containing 500 lM MbCD for 45 min. After washing as described above, the spermatozoa were incubated in replaced HTF medium containing 20 lM SVS2 and 500 lM BODIPY-cholesterol (TopFluorcholesterol; Avanti Polar Lipids, Alabaster, AL) and incubated for 45 min. Spermatozoa were then stained with Hoechst 33342 (Sigma) at a final concentration of 5 lg/ml. After incubating for 5 min, the fluorescence was visualized using a fluorescent microscope (Leica DMRBE; Solms, Germany) and images were acquired using a CCD camera (MicroPublisher 5.0; QImaging, Surrey, BC).

Preparation of Recombinant SVS2 Protein

Flow Cytometry Measurements

Because we previously showed that the GST-tagged recombinant SVS2 protein has the same effect as the native SVS2 protein [26], we used recombinant SVS2 protein in this study. To increase the solubility of recombinant SVS2, we substituted the thioredoxin (Trx) tag for a GST tag. The Trx tag itself has no effect on cholesterol level in the spermatozoon or on the induced-AR rate (Supplemental Fig. S1; Supplemental Data are available online at www.biolreprod.org). The cDNA encoding SVS2 (NCBI accession no. NM_017390) was prepared by RT-PCR using RNA isolated from the mouse seminal vesicle (C57BL/6J), and the cDNA was subcloned into the pET32 vector (Takara Bio Inc., Shiga, Japan). The Trx fusion protein was expressed in Escherichia coli BL21-pLysS cells (Novagen, Madison, WI) by induction with 1 mM isopropyl b-D-1-thiogalactopyranoside. Recombinant SVS2 protein was recovered using Ni-NTA agarose (Qiagen, Hilden, Germany) under denaturing conditions according to the manufacturer’s instructions. After removing urea using a PD-10 column (GE Healthcare, Piscataway, NJ), the protein was lyophilized and stored at 208C until use. Immediately before the experiment, recombinant SVS2 was dissolved in human tubal fluid (HTF) medium without BSA (Nippon Medical & Chemical Instruments Co., Osaka, Japan). The purity of the recombinant SVS2 protein was evaluated by Western blotting with a previously produced anti-SVS2 antibody [26] (Supplemental Fig. S1A).

Cauda epididymal mouse spermatozoa were collected from sexually mature male CD-1 mice (ages 915 wk) by placing minced cauda epididymis in 200 ll of pre-equilibrated (5% CO2, 378C for 2 h) HTF medium (without BSA). Droplets of medium were covered with mineral oil in a small culture dish. After 30-min incubation, the sperm suspension was retrieved from the dish and transferred to a new dish prepared with HTF medium without BSA, HTF medium containing MbCD (500 lM), HTF medium containing SVS2 (20 lM), and HTF medium containing both MbCD and SVS2. The incubation times were as follows: 2 h in HTF medium without BSA and in HTF medium containing SVS2; and 1 h in HTF medium containing MbCD and in HTF medium containing MbCD and SVS2, in 5% CO2 atmosphere at 378C. After washing with HEPES-buffered saline, the cells were centrifuged (600 3 g, 3 min) and fixed with 0.004% (w/v) PFA in HEPES-buffered saline for 10 min at room temperature. The sperm cells were then washed twice with HEPES-buffered saline, they were centrifuged (600 3 g, 3 min), filipin (4% [w/ v] in dimethyl sulfoxide) was added to the fixed spermatozoa at a final concentration of 0.2%, and the cells were incubated for 30 min at room temperature in the dark. After washing twice with HEPES-buffered saline by centrifugation (600 3 g, 3 min), flow cytometry data were collected. The efficiency of sterol extraction by MbCD was assessed from the decrease of fluorescence intensity of Filipin III (0.1 mg/ml; Sigma). Incubation (106 cells per milliliter) for 1 h at 378C was followed by washing twice in PBS, and cells were then analyzed on a FACSAria flow cytometer (Becton Dickinson,

MATERIALS AND METHODS Animals

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To visualize cholesterol, the filipin complex (Sigma) was used, which binds to sterols [31, 32]. Spermatozoa were fixed with 0.004% (w/v) paraformaldehyde (PFA) for 10 min and washed twice with HEPES-buffered saline (HBS; 140 mM NaCl, 5 mM KCl, 0.75 mM Na2HPO4, 6 mM glucose, and 25 mM HEPES, pH 7.1). Filipin solution (4% [w/v] in dimethyl sulfoxide) was added to the fixed spermatozoa at a final concentration of 0.2%, and the solution was incubated for 30 min. After washing with HBS, fluorescence was observed under a fluorescent microscope (IX-71; Olympus, Tokyo, Japan). The fluorescence intensity of the sperm head was quantified using ImageJ software (National Institutes of Health, Bethesda, MD), and the normalized intensity of epididymal spermatozoa was set to 100. For each batch, the fluorescence intensities of 100 spermatozoa were quantified.

ROLE OF SVS2 IN SPERM CHOLESTEROL AND CAPACITATION

Franklin Lakes, NJ). Filipin III was excited by using a 375-nm laser line and detected at 450 6 20 nm. The fluorescence histograms were prepared on the basis of 10 000 individual cells, collected in the list mode.

Localization of GM1 in the Spermatozoa GM1 (Galb1 . 3GalNAcb . 4(NeuAca2.3)Galb1 . 4Glcb1 . 1 0 Cer) was visualized using FITC-conjugated cholera toxin subunit B (FITC-CTB; Sigma) as described previously [27]. Spermatozoa were fixed with 0.004% (w/ v) PFA for 10 min and washed with HBS three times. Localization of the GM1 signal was classified into four patterns based on previously described criteria [34]; 100 signals were evaluated for each batch.

Quantification of Sperm Cholesterol Lipid extraction was performed according to a modified Bligh & Dyer protocol, as described by Jakop et al. [33]. Briefly, after washing with HBS, spermatozoa incubated in various conditions were centrifuged (1000 3 g, 5 min). Around 100 ll of sperm suspensions was mixed with 400 ll of chloroform:methanol (1:2, v/v) and shaken for 30 min. About 130 ll of chloroform was added to the sperm and shaken for 0.5 min. Then, 130 ll of 40 mM acetic acid was added and the samples were shaken. After centrifugation (1000 3 g, 20 min, 48C), samples were divided into the chloroform phase and the aqueous phase. The aqueous phase was treated with chloroform and acetic acid and centrifuged as described above. After repeating this procedure two times, the chloroform phases from each step were combined and dried under reduced pressure. Extracted lipids were suspended with 2-propanol containing 10% (v/v) Triton X-100. Enzymatic quantification of cholesterol was carried out with the Amplex red cholesterol assay kit (Molecular Probes, Eugene, OR). Fluorescence intensity was measured by a multilabel reader 2030 ARVO X4 (PerkinElmer, Waltham, MA).

Evaluation of the Sterol Level of Spermatozoa In Vivo To evaluate the effect of SVS2 on the sperm sterol content in the female reproductive tract, we measured the sterol level of the spermatozoa in the uterus. A 10- to 55-wk-old male (SVS2þ/þor SVS2/) mouse was mated with a 9- to 12-wk-old C57BL/6J female mouse. SVS2/ mice were generated as described in Kawano et al. [28]. Females were killed at 1.5 h after coitus, and spermatozoa were obtained from the uterus and oviduct by flushing with HBS. Obtained spermatozoa were fixed with 0.004% PFA for 10 min and stained with filipin, and the fluorescence intensity was quantified as described above.

Immunohistochemistry of Female Reproductive Tracts Female reproductive tracts obtained from 9- to 15-wk-old female mice (CD1) at 1.5 h after coitus were fixed with 4% (w/v) PFA for 4–6 h, embedded in paraffin, and cut into 10-lm-thick sections according to standard protocols. Immunohistochemistry was performed using the anti-SVS2 antibody [26], followed by Alexa Fluor 488-conjugated secondary antibody (goat anti-rabbit immunoglobulin G; Life Technologies, Carlsbad, CA) with 0.2 lg/ml 4 0 ,6diamidino-2-phenylindole (DAPI; Wako). Anti-SVS2 antibody that passed through a HiTrap NHS-activated HP column (GE Healthcare) conjugated with recombinant SVS2 protein was used for negative controls. Stained sections were visualized using a fluorescent microscope (Leica DMRBE) and images were acquired using a CCD camera (QImaging MicroPublisher 5.0). Exposure times were 5, 15, and 0.7 sec for staining by anti-SVS2, control antibody, and DAPI, respectively.

Sperm Capacitation Assay Sperm capacitation was evaluated based on acrosomal responsiveness to progesterone and ionomycin. Spermatozoa were treated with either 100 lM progesterone (Wako, Osaka, Japan) for 15 min at 378C or 15 lM ionomycin (Sigma) for 10 min at room temperature. Treated spermatozoa were spread onto a glass slide. Spermatozoa were permeabilized with methanol for 10 min and stained with 100 lg/ml fluorescein isothiocyanate (FITC)-conjugated peanut lectin (Sigma) for 15 min at 378C. Spermatozoa were washed with HBS, and FITC fluorescence was observed using a fluorescence microscope (IX-71; Olympus). Spermatozoa with no FITC fluorescence at the acrosome region were regarded as acrosome-reacted spermatozoa. Three fields were obtained for each batch, and 100 spermatozoa per field were evaluated.

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FIG. 1. Effect of SVS2 on sterol levels of spermatozoa. A) Sterol levels in the plasma membrane of spermatozoa of SVS2/ (white bar) and wild-type (WT; gray bar) male mice collected from the uterus and oviduct of WT female mice. The graph shows the relative filipin fluorescence intensity values compared with epididymal spermatozoa (see Materials and Methods). Data are shown as the mean 6 SD; n ¼ 8 (SVS2/ spermatozoa in the uterus); n ¼ 6 (WT spermatozoa in the uterus); and n ¼ 3 (WT spermatozoa in the oviduct). No spermatozoa were found in the oviduct of the female mated with the SVS2/ male. The fluorescence intensity of the SVS2/ spermatozoa showed a significant difference compared with that of the WT spermatozoa in the uterus (asterisk; P , 0.01, Student t-test). B) Typical filipin staining of nontreated spermatozoa with cholesterol intact (Ch intact) and capacitated spermatozoa after cholesterol efflux (after Ch efflux). Bar ¼ 5 lm. C) Effect of SVS2 on sperm capacitation induced by a high concentration of BSA. Spermatozoa were collected from the epididymis and incubated in medium containing various concentrations of BSA with 20 lM SVS2 (open circle) or without SVS2 (solid circle) for 120 min, and the rate of the AR induced by progesterone was evaluated. Asterisks indicate significant differences compared with spermatozoa treated with medium without BSA (n ¼ 3; P , 0.01, Student t-test).

ARAKI ET AL.

Quantification of Albumin in Uterine Fluid The uteri of 9- to 15-wk-old female mice (CD-1) were eviscerated and their surface fluid and blood were thoroughly removed. The uterus was hung in a tube and the uterine fluid was obtained by centrifugation at 1200 3 g for 3 min. Albumin levels in uterine fluid were quantified using an ELISA kit (Shibayagi Co., Gunma, Japan).

Statistical Analysis All experiments were repeated at least three times with different individuals. Data are expressed as the mean 6 SD. Statistical significance was calculated using the Student t-test; P , 0.01 was considered significant.

RESULTS SVS2 Prevents Cholesterol Efflux In Vivo

SVS2 Prevents Sterol Efflux and Sperm Capacitation In Vitro The cholesterol level on SVS2/ spermatozoa was significantly reduced in the uterus. Thus, SVS2 appears to inhibit sterol efflux in vivo. In fact, seven consensus regions that recognize and interact with cholesterol [36, 37] are present in the SVS2 amino acid sequence (Supplemental Fig. S4). Because SVS2 is known to inhibit sperm capacitation in vitro (decapacitation effect) [26], we next examined the effect of SVS2 on sterol levels in vitro. MbCD is a potent liberator of sterols and induces capacitation of mouse spermatozoa [21]. We confirmed the effect of MbCD on sperm capacitation by assessing acrosomal responsiveness to ionomycin or progesterone (induced-AR rate); 500 lM MbCD induced the same level of capacitation as BSA (Supplemental Fig. S5). The fluorescence intensity of filipin in sperm heads treated with 500 lM MbCD for 45 min decreased to approximately 55% of that of nontreated sperm heads (Fig. 2A). Flow cytometric analysis also showed that the fluorescence intensity of the MbCD-treated spermatozoa significantly decreased in contrast to the untreated control spermatozoa (Fig. 3A). By contrast, SVS2 inhibited the decrease in cholesterol content of the sperm plasma membrane; the fluorescence intensity on sperm heads treated with SVS2 and MbCD showed no significant difference from that on nontreated sperm heads (Figs. 2A and 3A). In this case, the rate of sperm capacitation decreased significantly. When sperma-

FIG. 2. Effect of SVS2 on MbCD-treated spermatozoa. A) Effect of SVS2 and MbCD on sterol level in the sperm plasma membrane. Data are shown as the mean 6 SD of the fluorescence intensity of spermatozoa for each treatment. B) Effect of SVS2 and MbCD on sperm capacitation induced by MbCD. The rate of the AR induced by progesterone (gray bar) or ionomycin (white bar) was evaluated. Data are shown as the mean 6 SD (n ¼ 3). C) Sperm treatment conditions for A and B. MbCD: 500 lM MbCD in HTF; SVS2: 20 lM recombinant SVS2 in HTF. Asterisks indicate significant differences compared with spermatozoa incubated in media only (n ¼ 3; P , 0.01, Student t-test).

tozoa were incubated in capacitation medium containing MbCD, they became capacitated (Fig. 2B), which agrees with previously reported results [21]. By contrast, the induced-AR rate of spermatozoa treated with both SVS2 and MbCD was suppressed and showed no significant difference from control spermatozoa (Fig. 2B). When spermatozoa were preincubated with SVS2 prior to incubation with MbCD (SVS2 . MbCD in Fig. 2), the induced-AR rate was also suppressed (Fig. 2B). SVS2 maintained the cholesterol level on the sperm head to be the same as that of spermatozoa incubated in HTF medium (Fig. 2A). Furthermore, SVS2 impeded the sperm capacitation induced by MbCD; SVS2 showed a decapacitation effect in MbCD-treated spermatozoa. When SVS2 was added to the 4

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First, using the cholesterol-staining dye filipin we investigated whether sterol levels in the mouse spermatozoa were decreased after they entered into the oviduct [31, 32]. The intensity of filipin fluorescence of wild-type male spermatozoa in the uterus after mating increased by 149% 6 40% compared with nontreated epididymal spermatozoa (n ¼ 6; Fig. 1A). On the other hand, the fluorescence intensity of the spermatozoa in the oviduct was significantly reduced (35% 6 17% of that in the uterus, n ¼ 3, P , 0.01; Fig. 1A). This indicates that cholesterol levels in mouse spermatozoa are decreased when they enter the oviduct. On the other hand, the sperm cholesterol level in the uterus of females mated with SVS2/ males was significantly reduced to the level of wild-type spermatozoa in the oviduct (34% 6 7% of that of ejaculated wild-type spermatozoa in the uterus, n ¼ 8, P , 0.01; Fig. 1A). These results indicate that SVS2 reserves sterols on the sperm membrane in the female reproductive tract until entrance into the oviduct. Serum albumin is thought to be the cholesterol acceptor in uterine fluid [18], where its contents were found to range from 6 to 108 mg/ml (Supplemental Table S1). Although albumin content in the uterus was higher than that in in vitro capacitation medium (3–5 mg/ml) [35], SVS2 inhibited sperm capacitation even in the presence of 100 mg/ml BSA (Fig. 1C).

ROLE OF SVS2 IN SPERM CHOLESTEROL AND CAPACITATION

Downloaded from www.biolreprod.org. FIG. 4. Effect of SVS2 requires cholesterol. A) The sterol level of the sperm plasma membrane evaluated based on the fluorescence intensity of filipin. B) Rate of capacitated spermatozoa evaluated by the AR induced by progesterone (gray bar) or ionomycin (white bar). C) Sperm treatment conditions of A and B. MbCD: 500 lM MbCD in HTF; SVS2: 20 lM recombinant SVS2 in HTF; Wash: washing spermatozoa with HTF twice after MbCD treatment; Ch: 500 lM cholesterol in HTF with 1% ethanol; and BSA: 5 mg/ml BSA in HTF. When MbCD-treated spermatozoa were washed, the effects of SVS2 were attenuated. However, addition of extrinsic cholesterol recovered the SVS2 effects. Data are shown as the mean 6 SD (n ¼ 36). Asterisks indicate significant differences compared with the MbCD . SVS2 treatment group (P , 0.01, Student t-test).

FIG. 3. Cholesterol content in the SVS2-treated sperm. A) Filipin fluorescence intensity distribution histograms of the sperm cells incubated for 1 h in HTF medium without BSA (control: blue), HTF medium containing 20 lM SVS2 (SVS2: black), HTF medium containing 500 lM MbCD (MbCD: green), and HTF medium containing 500 lM MbCD and 20 lM SVS2 (MbCD þ SVS2: red). Mean fluorescence intensities of control, SVS2, MbCD, and MbCD þ SVS2 were 2204 6 4389, 3067 6 5585, 1360 6 3110, and 2193 6 5108, respectively. There was no significant difference between the control sperm population and MbCD þ SVS2 sperm population, whereas the MbCD sperm population was significantly different from both the control and MbCD þ SVS2 sperm populations (P , 0.01, Student t-test). B) Cholesterol content of the total sperm extracts quantified by the cholesterol assay kit. The cholesterol content of each sperm sample is shown as a relative value: the normalized cholesterol content of the sperm treated with HTF medium only was set to 100. Absolute quantity of the cholesterol in the control sperm was 142.3 6 39.5 ng per 106 sperm cells. Data are shown as the mean 6 SD, and asterisks indicate significant differences compared with spermatozoa incubated in medium only (n ¼ 4; P , 0.01, Student t-test). C) Sperm treatment conditions of B. MbCD: 500 lM MbCD in HTF; BSA: 5 mg/ml BSA in HTF; SVS2: 20 lM recombinant SVS2 in HTF; and Wash: washing spermatozoa with HTF twice after MbCD treatment.

MbCD-treated capacitated spermatozoa, on which the membrane cholesterol level was low, cholesterol levels on the sperm heads recovered to that of an incapacitated spermatozoa (MbCD . SVS2 in Fig. 2A), and the induced-AR rate decreased significantly (MbCD . SVS2 in Fig. 2B). These changes in sperm cholesterol levels were confirmed by an enzymatic quantitative analysis. Cholesterol content in the control sperm was 142.3 6 39.5 ng per 106 sperm cells. MbCD treatment decreased the sperm cholesterol by approximately 35%, whereas SVS2 treatment maintained the sperm 5

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cholesterol at control levels (MbCD þ SVS2 and MbCD . SVS2 in Fig. 3B). Thus, SVS2 appears to mediate sperm capacitation by controlling sterol levels in the plasma membrane.

sterol is required in the medium surrounding the spermatozoa for the decapacitation effect of SVS2. Thus, SVS2 appears to prevent sterol efflux from the plasma membrane, thereby returning liberated sterol to the membrane and maintaining the spermatozoa in an incapacitated state. Our results suggest that SVS2 does not only inhibit sterol efflux, but also incorporates external sterols into the sperm plasma membrane. To verify the effect of SVS2, we observed the dynamics of extrinsic BODIPY-cholesterol. When only BODIPY-cholesterol was added to the washed MbCD-treated spermatozoa, minimal BODIPY fluorescence was observed (Fig. 5A). By contrast, when washed MbCD-treated spermatozoa were incubated with BODIPY-cholesterol and SVS2, BODIPY fluorescence was observed on many spermatozoa. BODIPY-cholesterol was incorporated into 69% 6 20% of spermatozoa (Fig. 5). Furthermore, SVS2 slightly facilitated incorporation of BODIPY-cholesterol into the spermatozoa without MbCD treatment (Fig. 5). Thus, SVS2 mediated incorporation of free cholesterol from the medium into the sperm plasma membrane.

SVS2 Retrieves Sperm Membrane Cholesterol from the Medium Interestingly, the effect of SVS2 on MbCD-treated spermatozoa was not observed when sperm incubation medium was washed out following MbCD treatment. When MbCDtreated spermatozoa were washed with new HTF medium after MbCD treatment, a recovery effect of SVS2 on the cholesterol level in the sperm was not observed by filipin staining and the enzymatic quantitation assay (MbCD . wash . SVS2 in Figs. 3B and 4A). In addition, the washed MbCD-treated spermatozoa showed no decapacitation effect by SVS2; addition of SVS2 did not decrease the induced-AR rate (MbCD . wash . SVS2 in Fig. 4B). To examine whether this observation occurred because of the lack of free sterol in the medium, we performed a rescue experiment by adding extrinsic cholesterol to the washed MbCD-treated spermatozoa. Addition of extrinsic cholesterol to the medium containing SVS2 rescued the cholesterol level in the sperm head and the decapacitation effect (MbCD . wash . SVS2 þ Ch in Fig. 4). In addition, 1% ethanol, the cholesterol vehicle, had no effect on sperm capacitation (Supplemental Fig. S3). Cholesterol itself and sterol liberators, including MbCD and BSA, had no effect on washed MbCD-treated spermatozoa (Fig. 4), indicating that

Effect of SVS2 on GM1 Distribution in Spermatozoa Sterol efflux from spermatozoa changes plasma membrane fluidity and the constitution of the lipid raft organization, resulting in sperm capacitation [6, 20]. Thus, we evaluated the effect of SVS2 on the distribution of the ganglioside GM1, which is a well-known marker of lipid rafts [20]. GM1 was visualized using FITC-CTB, and the GM1 staining pattern was 6

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FIG. 5. Visualizing cholesterol uptake into the sperm plasma membrane. A) Fluorescence images of spermatozoa with BODIPY-cholesterol (green). Sperm heads were visualized by staining with Hoechst 33342 (Hoechst; blue) in the merged images (lower panels). Bar ¼ 50 lm. B) Rate of BODIPYcholesterol (B-Ch) incorporation into spermatozoa. Data are shown as the mean 6 SD (n ¼ 3). Asterisks indicate significant differences compared with the B-Ch groups (P , 0.01, Student t-test). Extrinsic cholesterol was incorporated into the sperm plasma membrane only in the presence of SVS2. C) Sperm treatment conditions of A and B. MbCD: 500 lM MbCD in HTF; SVS2: 20 lM recombinant SVS2 in HTF; Wash: washing spermatozoa with HTF twice after MbCD treatment; and B-Ch: 500 lM BODIPY-cholesterol in HTF with 1% ethanol.

ROLE OF SVS2 IN SPERM CHOLESTEROL AND CAPACITATION

ing of the MbCD-treated spermatozoa prevented the effects of SVS2 on the GM1 staining pattern; pattern D was observed in the highest percentage of the spermatozoa, which was similar to the capacitated spermatozoa (MbCD . wash . SVS2 in Fig. 6, B and C). Addition of extrinsic cholesterol to the washed MbCD-treated spermatozoa rescued the effects of SVS2; the ratio of spermatozoa showing APM/PAPM pattern increased, whereas the ratio of spermatozoa showing pattern D decreased (MbCD . wash . SVS2 þ Ch in Fig. 6, B and C). Cholesterol itself and cholesterol with MbCD showed no effect on GM1 patterns of the washed MbCD-treated spermatozoa (Fig. 6). These results suggest that the effect of SVS2 on the reconstitution of lipid organization requires free sterol in the medium that surrounds the spermatozoa. SVS2 appears to restrict sterol dynamics by altering sperm plasma membrane fluidity. Distribution of SVS2 in the Female Reproductive Tract

DISCUSSION In this study, we demonstrated that SVS2 maintains sterols in the sperm plasma membrane to arrest spontaneous sperm capacitation in the uterus. Interestingly, SVS2 intercalated exogenous cholesterol into the spermatozoa and reset the dynamics of lipid rafts, suggesting cholesterol is essential for decapacitation. These results are consistent with the results of previous studies demonstrating that cholesterol is required to prevent sperm capacitation [38, 39]. Therefore, SVS2 regulates sperm capacitation through dynamic changes in the sterol level in the sperm plasma membrane. Serine protease inhibitors SERPINE2 [15] and SPINKL [16], which are minor components of seminal vesicle secretions, also inhibit cholesterol efflux and sperm capacitation. Intriguingly, SERPINE2/ mice showed loss of SVS2 and yielded male infertility, suggesting that SVS2 is degraded by a protease [40]. Because SVS2/ males showed the same phenotype, SVS2 must have important functions in sperm fertility. In humans, SEMG, the SVS2 homologous protein in human seminal plasma, is degraded by the protease prostatespecific antigen (PSA) [41], and the serine protease inhibitor in seminal plasma inhibits degradation of SEMG by PSA [42, 43]. Because SVS2 also has many predicted cleavage sites for PSA and is degraded in the uterus [26], SERPINE2 and SPINKL may protect SVS2 from PSA-mediated degradation in the uterus. The cholesterol concentration on the sperm plasma membrane increases during epididymal transit [44, 45]. Furthermore, seminal plasma contains a high concentration of cholesterol [29], and spermatozoa appear to obtain cholesterol even after ejaculation. Thus, spermatozoa ejaculated into the uterus contain a high concentration of sterols and lose their sterols during capacitation. The cholesterol efflux triggers Ca2þ influx and acrosome exocytosis via GM1 dynamics [46]. Accordingly, SVS2 may function to maintain the sterol level of spermatozoa and suppress the capacitation of spermatozoa in the uterus.

FIG. 6. Effect of SVS2 on distribution of lipid rafts on the sperm head. A) Fluorescence images of GM1 signals observed in spermatozoa. Spermatozoa were fixed with PFA, and GM1 was visualized using FITC-CTB. Pattern AA/PA: signal over anterior acrosome and postacrosome. Pattern PAPM: signal in postacrosome. Pattern APM/PAPM: weak signal in acrosome and remarkable signal in postacrosome. Pattern D: diffused signals through head. Bar ¼ 5 lm. B) Ratio of each GM1 localizing pattern in spermatozoa incubated under various conditions. Spermatozoa were obtained from one male mouse. C) Ratio of the pattern D in spermatozoa incubated under various conditions. Data are shown as the mean 6 SD (n ¼ 3). Asterisks indicate significant differences compared with MbCDtreated sperm groups (P , 0.01, Student t-test).

classified into one of four types based on criteria described by Selvaraj et al. [34] (Fig. 6A). When noncapacitated spermatozoa were incubated in the medium only, GM1 was primarily observed in the postacrosomal region, and occasionally on the entire sperm head (pattern PAPM or pattern APM/PAPM; Fig. 6B). When spermatozoa were capacitated using MbCD, GM1 diffused through the entire head, appearing as pattern D (Fig. 6, B and C). When spermatozoa were coincubated with SVS2 and MbCD, the pattern APM/PAPM was observed in a large proportion of spermatozoa and pattern D was observed in a low percentage of spermatozoa, which was similar to the patterns observed in noncapacitated spermatozoa (Fig. 6, B and C). Thus, SVS2 arrests the reconstitution of lipid raft organization by preventing sterol efflux. Furthermore, when MbCD-treated spermatozoa were decapacitated using SVS2, GM1 was primarily distributed in the APM/PAPM pattern, similar to the noncapacitated spermatozoa, although GM1 was dispersed over the entire sperm head (pattern D) before SVS2 addition (MbCD . SVS2 in Fig. 6, B and C). This suggests that SVS2 restores sperm plasma membrane organization by retaining membrane sterols. Wash7

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To evaluate the role of SVS2 in sperm capacitation in vivo, we examined SVS2 dynamics in the female reproductive tract after copulation. We previously showed that significantly less SVS2 was present in oviductal fluid than in uterine fluid [26]. Indeed, in the present study, in the uterine tube SVS2 was found on the surface of the uterine wall and in the uterotubal junction (UTJ), but little SVS2 was observed in the oviductal isthmus (Fig. 7), suggesting that SVS2 suppresses sperm capacitation until the spermatozoa reach the isthmus.

ARAKI ET AL.

Downloaded from www.biolreprod.org. FIG. 7. Immunolocalization of SVS2 in the female reproductive tract. SVS2 in the female reproductive tract at 1.5 h after coitus was stained with antiSVS2 antibody (green). Superimposed images of nuclei stained with DAPI (blue) on the immunostaining images are shown in the lower panel. SVS2 entered the uterus and the UTJ region, but a very low amount entered the oviductal isthmus. No obvious staining was observed in the control stainings with the antibody in which anti-SVS2 antibody was removed by the recombinant SVS2-immobilized column. Bar ¼ 50 lm.

In postcoitus female mice, SVS2-bound spermatozoa were observed in the uterus and UTJ, but SVS2 was not bound to spermatozoa in the oviductal isthmus (Fig. 7) [26]. Ejaculated spermatozoa from SVS2/ male mice were killed inside the uterus and were rarely observed in the isthmus of the oviduct, and they could not reach the ampulla [28]. Thus, SVS2 appears to maintain spermatozoa in an incapacitated state in the uterus; capacitated spermatozoa in the uterus may have been killed in order to prevent their entry into the oviduct. It is likely that liberation of SVS2 from the spermatozoa in the oviduct acts to unlock sperm capacitation. Because some protease inhibitors in

Because capacitation-inducing factor(s) appear to be present throughout the entire female reproductive tract (Supplemental Table S1) [47], in vivo capacitation seems to be regulated not by a capacitation-inducing factor but by a decapacitation factor. Therefore, SVS2 may be the main factor involved in unlocking in vivo capacitation. Furthermore, because spermatozoa recovered from the oviduct possess neither SVS2 nor its receptor, GM1 [26, 27], the final event of in vivo capacitation may be removal of GM1 from the sperm plasma membrane. Indeed, GM1 appears to be liberated from spermatozoa along with SVS2 at the UTJ. 8

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bicarbonate in the regulation of sterols in the sperm plasma membrane during sperm capacitation. Furthermore, we would like to identify the effects of SVS2 on desmosterol, which is another major sterol in the sperm membrane, in the near future. In the present study, we revealed that sperm capacitation is regulated by SVS2: SVS2 actively functions to maintain sperm sterol levels. Furthermore, SVS2 is not only a capacitation inhibitor, but also appears to play an indispensable role in switching between the capacitation states of spermatozoa. In humans, spermatozoa with abnormally high cholesterol content or abnormal changes in cholesterol levels during in vitro incubation are slower to capacitate and fail to spontaneously undergo the AR, resulting in infertility [51, 52]. Thus, controlling the sterol level in spermatozoa is important for mammalian fertilization, and further studies examining sterol level regulation mediated by SVS2 may provide new insight into male infertility.

seminal plasma show similar dynamics in the female reproductive tract [15, 16], they may act to eliminate SVS2 in the oviductal spermatozoa. We suggest the following mechanism for sperm capacitation in vivo (Fig. 8). After mating, mouse spermatozoa with high membrane sterol levels enter the uterus [48]. Ejaculated semen contains SVS2, which attaches to the sperm head via GM1 [26, 27]. The female reproductive tract contains sterol liberators, such as albumin and high-density lipoprotein, which act as capacitation-inducing factors [49]. In the uterus, however, SVS2 attaches to the sperm head to protect sterols in the sperm plasma membrane, and arrests capacitation. When spermatozoa enter the oviduct, SVS2 and GM1 detach from the spermatozoa, thereby liberating sterols from the sperm plasma membrane and resulting in capacitation. Precisely how SVS2 protects sterol in the sperm plasma membrane remains unclear. We previously showed that SVS2 directly interacts with GM1 [27]. Furthermore, seven cholesterol recognition/interaction domains are present in the SVS2 amino acid sequence (Supplemental Fig. S4). Thus, SVS2 may bind both GM1 and cholesterol. GM1 may act as a membrane scaffold for SVS2, and SVS2 could inhibit sterol efflux and then intercalate liberated sterol into the sperm plasma membrane. Sterol efflux from spermatozoa changes plasma membrane fluidity [6, 20], and SVS2 appears to restrict sterol dynamics by altering sperm plasma membrane fluidity. However, the exact localization of sterols and GM1 is still obscure, because GM1-staining patterns on the sperm were easily changed by fixatives [34]. In order to reveal the relationships between the distribution of lipid raft on the spermatozoa and sperm capacitation, we would like to identify the dynamics of SVS2, GM1, and sterols in the sperm membrane precisely. On the other hand, bicarbonate, another indispensable factor for inducing sperm capacitation in vitro, also induces cholesterol redistribution and enables sterol depletion in the sperm plasma membrane [50]. Therefore, further study is needed to determine the roles of SVS2, albumin, and

ACKNOWLEDGMENT We thank Dr. Z. Krasznai, and Dr. T Ma´ria´n (University of Debrecen) for coordinating the Japan-Hungary Research Cooperative Program and providing helpful advice.

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FIG. 8. Working hypothesis for sperm capacitation in vivo regulated by SVS2. 1) In the uterus, SVS2 attaches to a spermatozoon via GM1 and protects sterol efflux to inhibit sperm capacitation. 2) When the spermatozoon enters into the oviduct, SVS2 and GM1 are detached from spermatozoa. This induces cholesterol efflux, resulting in sperm capacitation.

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