Animals
Specific pathogen-free outbred ICR mice were purchased from BioLASCO Taiwan (Taipei, Taiwan). The animals were bred based on the technology derived from Charles River Laboratories (Wilmington, MA) and were maintained in the Animal Center at the Department of Medical Research, Mackay Memorial Hospital, and were treated according to institutional guidelines for the care and use of experimental animals.
They were housed under controlled lighting (12L:12D) at 21–22ºC and provided with water and NIH-31 laboratory chow ad libitum.
Protein Purification and Analysis
Adult male mice (10–12 weeks old) were killed by cervical dislocation. The SVS was successively collected, centrifuged, and fractionated by ion-exchange chromatography using a diethylaminoethyl Sephacel (GE Healthcare Life Sciences, Piscataway, NJ) column and gel filtration with a Sephadex G-75 (GE Healthcare Life Sciences) column as previously described [36]. The potential SERPINE2-containing peak II eluted from the Sephadex G-75 column was further subjected to a heparin Sepharose 6 Fast Flow (2.6- X 10-cm) column (GE Healthcare Life Sciences) preequilibrated with 0.1 M Tris-HCl, 0.01 M sodium citrate, and 0.225 M NaCl (pH 7.4). After the nonretarded fractions were washed out, the column was eluted with 0.5 M NaCl in 0.1 M Tris-HCl and 0.01 M sodium citrate at pH 7.4 at a flow rate of 0.5 ml/min; fractions (2 ml) were collected, and absorbance records are shown in Figure 1A.
The protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The N-glycoconjugate was removed from a glycoprotein using a PNGase F kit (New England Biolabs, Beverly, MA) following the manufacturer’s instructions.
Protein Identification by Mass Spectrometry
Purified protein was resolved by SDS-PAGE on a 10% slab gel. Protein bands on the SDS gel were excised and subjected to in-gel digestion with trypsin. In brief, the gel was washed in a solution of 50% (v/v) acetonitrile and 100 mM NH4HCO3 and digested by trypsin overnight at 37 ºC. The tryptic peptides were then extracted with a solution of 60% (v/v) acetonitrile and 1% (v/v) trifluoroacetic acid (TFA), lyophilized, resuspended in 0.1% (v/v) TFA, and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) equipped with an 1100 series HPLC unit (Agilent Technologies, Palo Alto, CA) and an LTQ FT hybrid mass spectrometer (Thermo Electron, San Jose, CA). MS/MS data were used for protein identification, using MASCOT search engine software (http://www.matrixscience.com), based on the International Protein Index databases (http://www.ebi.ac.uk/IPI).
Activity Assay
The inhibitory activity of SERPINE2 toward PLAU (also named uPA) was assayed using a uPA colorimetric assay kit (Millipore, Billerica, MA) according to the manufacturer’s protocol. In brief, 5 µg of purified SERPINE2 protein was incubated with 5 units of PLAU for 1 h at 37ºC. Subsequently, assay buffer and chromogenic substrate were added and incubated for 30 min at 37°C. The absorbance was read at 405 nm.
Antibody Production and Usage
Antisera against SERPINE2 were produced using New Zealand white rabbits.
Purified SERPINE2 protein in normal saline (0.4 mg/ml) was emulsified with Freund’s complete adjuvant (1:1, v/v). In total, 2 ml of the mixture was subcutaneously injected in multiple sites in individual rabbits. Two rabbits were boosted twice every 3 weeks with the mixture of the same amount of purified protein and Freund’s incomplete adjuvant (1:1, v/v). Antisera were collected 10 days after the last injection. Purified SERPINE2 protein (200 µg) was conjugated to AminoLink beads (Pierce) according to the manufacturer’s instructions. Antisera against SERPINE2 were adsorbed by the conjugated beads to remove the specific antibody against SERPINE2. The treated antiserum was used as the control antiserum. Antisera were also used to develop a custom-made sandwich-style ELISA kit by Taiwan Advanced Bio-Pharm (Taipei, Taiwan). The kit was used to estimate the SERPINE2 protein concentration in SVS, which was collected separately from 6 male mice at the age of 12 weeks.
Western Blotting
Proteins were resolved using SDS-PAGE on a 10% slab gel (8.2 × 7.3 × 0.075 cm) and stained with Coomassie Brilliant blue or transferred to a nitrocellulose
membrane for immunostaining. Membranes were blocked with 10% (w/v) skim milk in PBS (blocking solution) for 2 h and then incubated with anti-SERPINE2 antiserum or control antiserum (1:8000 dilution) in blocking solution for 1 h at room temperature.
After gentle agitation over four changes of PBS for 10 min each, membranes were immunoreacted with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (GE Healthcare Life Sciences) diluted to 1:10 000 in blocking solution for 1 h. Immunoreactive bands were revealed using an enhanced chemiluminescence substrate according to the manufacturer’s instructions (Pierce).
Immunohistochemical Staining of the Accessory Gland of Male Mice
Murine (~12 weeks old) reproductive tissues were collected, fixed in formalin, embedded in paraffin, and cut into 5-lm sections. After the slides were deparaffinized and hydrated, they were placed in a plastic slide holder filled with antigen retrieval AR-10 solution (BioGenex, San Ramon, CA), soaked in a 70°C water bath, rapidly boiled to 95°C, and maintained for 15 min. While cooling to room temperature for 30 min, the slides were treated with 3% (v/v) H2O2 in PBS for 15 min, blocked with 10%
(v/v) normal goat serum in PBS (blocking solution) for 1 h at room temperature, and then incubated with anti-SERPINE2 antiserum or the control antiserum diluted 1:1000 in the blocking solution at 4 ºC for 16 h. After slides were washed, they were treated
with biotin-conjugated goat anti-rabbit IgG (~3 µg/ml) (Zymed Laboratories, South San Francisco, CA) in blocking solution for 1 h at room temperature. Slides were washed again and then incubated with HRP-conjugated streptavidin (~1 µg/ml) (Zymed Laboratories) in blocking solution for 40 min at room temperature. Protein signals were detected by 3-amino-9-ethylcarbazole staining (Zymed Laboratories). Slides were then counterstained with hematoxylin (Vector Laboratories, Burlingame, CA) and photographed using an Olympus BX 40 microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP-70 digital camera.
Preparation of Spermatozoa
Epididymides and testes were immediately removed after male mice (;12 weeks old) were killed. Caudal epididymides were slit in prewarmed Biggers, Whitten, and Whittingham (BWW) medium and incubated at 37 ºC in 5% CO 2 for 15 min to allow motile sperm to swim upward. Motile sperm in the upper layer were collected. The caput and corpus regions of epididymides were treated in the same method as described above but were gently filtered through a 70 µm nylon cell strainer (BD Falcon, Franklin Lakes, NJ) to remove debris. Testes were decapsulated by cutting the tunica albuginea to expose seminiferous tubules. An 18-gauge needle was used to aspirate the seminiferous tubules and push them through. The dispersed seminiferous tubules were
cut into pieces and filtered through a 70-lm nylon cell strainer (BD Falcon) to collect the free seminiferous cells. To isolate ejaculated uterine and oviductal sperm, female mice (6 weeks old) were induced to superovulate by an intraperitoneal injection of 10 IU of equine chronic gonadotropin (China Chemical and Pharmaceutical, Hsinchu, Taiwan), followed by an intraperitoneal injection of 10 IU of human chorionic gonadotropin (China Chemical and Pharmaceutical) 48 h later, and were subsequently mated with male mice (~16 weeks old). Female mice with plugged vaginas were killed, and the ejaculated sperm in the uterine cavity were collected within 1 h. In brief, the semen filtered through a 70 µm nylon cell strainer (BD Falcon) was repeatedly agitated by pipetting with PBS. The sperm solution was washed by centrifuging it three times at 100 X g for 10 min. Sperm were then fixed using 4% (w/v) paraformaldehyde in an Eppendorf tube for 20 min at room temperature, transferred onto slides, and allowed to dry. Oviductal sperm were collected the next day after mating by flushing the oviduct with PBS. Sperm were transferred using a mouth pipette onto slides and fixed in 4%
(w/v) paraformaldehyde for subsequent immunostaining analysis.
Immunolocalization of SERPINE2 on Spermatozoa
To determine whether SERPINE2 protein is originally a sperm-binding protein, freshly prepared epididymal and testicular spermatozoa were fixed using 4% (w/v)
paraformaldehyde and allowed to dry on a glass slide and washed twice with PBS. After slides were incubated in blocking solution of PBS containing 10% (v/v) normal goat serum for 1 h at room temperature, they were incubated with anti-SERPINE2 antiserum or control antiserum at a dilution of 1:100 in blocking solution for 1 h. The slides were washed three times with PBS to remove excess antibodies before they were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Vector Laboratories) diluted 1:500 in blocking solution for 40 min. All slides were then washed with PBS and counterstained with 5μg/ml Hoechst 33258 stain. After three brief rinses with PBS, the slides were mounted in 100μl of ProLong Gold antifade medium (Invitrogen Molecular Probes, Eugene, OR) and photographed using an epifluorescence microscope (Olympus BX 40) equipped with an Olympus DP-70 digital camera. To determine whether exogenous SERPINE2 protein can bind to epididymal sperm, 0.5 μM SERPINE2 was incubated with living sperm in Eppendorf tubes for 20 min at 37℃.
Unbound SERPINE2 protein was washed away by centrifuging the sperm in the PBS solution at 100 x g for 5 min at room temperature. Sperm were then fixed using 4%
(w/v) paraformaldehyde in an Eppendorf tube for 20 min at room temperature, transferred onto slides, and allowed to dry. Immunostaining was done as described above, except a dilution of 1:1000 was used for anti-SERPINE2 antiserum and control antiserum. The same dilution was also used to examine SERPINE2 on ejaculated and
oviductal sperm, without incubation with the exogenous SERPINE2 protein. To examine the correlation between SERPINE2-bound sperm and sperm capacitation, oviductal sperm were double fluorescence labeled by using indirect immunofluorescence and chlortetracycline (CTC) fluorescence staining, an empirical method used to morphologically assess sperm capacitation [72,73]. In brief, sperm slides were washed twice with PBS and incubated in blocking solution, as mentioned above, for 1 h at room temperature. Then the slides were incubated with anti-SERPINE2 antiserum or control antiserum at a dilution of 1:1000 in blocking solution for 1 h. After slides were washed three times with PBS, they were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:200 in blocking solution for 40 min. All slides were then washed with PBS and counterstained with 5 µg/ml Hoechst 33258. After three brief rinses with PBS, the sperm on the slides were stained with a CTC solution prepared as previously described [72,73] by incubation at 4℃ overnight and photographed using an epifluorescence microscope (Olympus BX 40) equipped with an Olympus DP-70 digital camera.
Evaluation of Sperm Capacitation and the Acrosome Reaction
The molecular basis of sperm capacitation was examined by detecting any
capacitation-accompanied increase in protein tyrosine phosphorylation of a subset of proteins with molecular weights of 40,000–120,000, according to a previously described method [7]. In brief, about 5 x 106 spermatozoa/ml was incubated in modified Krebs-Ringer bicarbonate medium [74] with or without bovine serum albumin (BSA) (3 mg/ml), as the positive or negative control, respectively, or BSA replaced with SERPINE2 at 37℃ in an atmosphere of 5% (v/v) CO2 in humidified air for 90 min. To assess the effect of SERPINE2 on BSA-induced capacitation, SERPINE2 was preincubated with sperm under the above-described conditions for 20 min, and BSA was added thereafter. Then, the soluble fraction of sperm protein extracts was subjected to SDS-PAGE on an 8% slab gel. Proteins on the gel were electrotransferred onto nitrocellulose paper. Western blot analyses were performed using an anti-phosphotyrosine antibody according to a method described previously [75]. To evaluate sperm capacitation by the CTC fluorescence-staining method, freshly prepared epididymal spermatozoa (106 cells/ml) were capacitated in 50 µl of BWW medium with or without BSA (3 mg/ml) as the positive or negative control, respectively, at 37℃ in an atmosphere of 5% (v/v) CO2 in humidified air for 90 min. Medium was supplemented with SERPINE2 as described above, or the BSA was replaced with SERPINE2 to analyze the effects of SERPINE2 on sperm capacitation in vitro. CTC staining of sperm was carried out following the original method and examined using a
fluorescence microscope (BX 40 model; Olympus). To analyze the sperm acrosome reaction, the capacitated sperm, prepared as described above, were treated with 5 μM A23187 in dimethyl sulfoxide (DMSO) (0.2%) at 37℃ for 30 min. Sperm were smeared on the slide and fixed with methanol for 30 sec. The sperm acrosomal status was assessed by staining samples with 5 μg/ml TRITC-conjugated peanut agglutinin lectin (PNA; Sigma-Aldrich, St. Louis, MO) in the dark for 5 min and by counterstaining with 5 μg/ml Hoechst 33258. After three brief rinses in PBS, slides were mounted in 50 μl of ProLong Gold antifade medium (Invitrogen Molecular Probes) and immediately examined with a fluorescence microscope (BX 40; Olympus).
Sperm–Egg Binding and In Vitro Fertilization
Epididymal sperm (2 x 105 cells/ml) in 150μl of BWW medium under mineral oil with or without BSA and/or SERPINE2 were capacitated for 90 min at 37℃ in an atmosphere of 5% (v/v) C O 2 in humidified air. Oocyte-cumulus complexes collected by superovulation treatment, as described above, were added to the same medium. For the sperm–egg binding assay, treated sperm were inseminated with oocyte-cumulus complexes for 30 min and then gently transferred using a mouth pipette with a bore approximately 1.53 the diameter of the oocyte to 70μl of PBS under mineral oil. After allowing specimens to sit at room temperature for 5 min, the loosely bound sperm were
detached, and the tightly bound sperm on the oocyte were counted using a Zeiss Axiovert 100 microscope (Zeiss, Oberkochen, Germany). For in vitro fertilization, after 6 h of insemination, oocytes were washed with BWW medium, using a mouth pipette, fixed onto a slide with 4% (w/v) paraformaldehyde, and stained in 5μg/ml Hoechst 33258 for 3 min. Slides were observed with a fluorescence microscope (BX40;
Olympus). Two pronuclei embryos were scored as fertilized.
Cholesterol Efflux Assay
Cholesterol content in the BWW medium with or without BSA (3 mg/ ml) and/or SERPINE2 (0.2 mg/ml) was assayed following the protocol described by Roberts et al. [26]. Freshly prepared epididymal spermatozoa (2 x 10 5 cells/ml) was capacitated in 150 µl of BWW medium as described above. After incubation, sperm were centrifuged at 10,000 x g to separately collect the supernatant and sperm pellets.
Samples were mixed with chloroform and methanol in a chloroform-methanol-supernatant (or sperm) final ratio of 2:2:1.8. After vigorous vortexing, the mixture was centrifuged at 600 x g for 5 min, and the organic phase was transferred to a new Eppendorf tube and dried by speed vacuum. The cholesterol content was measured using an Amplex Red cholesterol assay kit (Invitrogen Molecular Probes) according the manufacturer’s instructions. To calculate the cholesterol content
of the samples, a cholesterol standard curve was prepared using the cholesterol reference standard provided with the kit.