Chemicals and animals
Unless otherwise stated, all reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Animal experiments were conducted according to the International Guiding Principles for Biomedical Research Involving Animals, as promulgated by the Society for the Study of Reproduction. White New Zealand rabbits (male: ~6 months old, body weight ~2.5 kg) and BALB/c mice (male: aged 9-11 weeks, body weight 22-24 g; female: aged 7-9 weeks, body weight 18-20 g) were purchased from Shanghai SLAC Laboratory Animal Corporation (Jiu-Ting, Shanghai, China) and accommodated in the animal facility for at least for 1 week prior to experimentation.
Preparation of epididymal spermatozoa, sperm protein and membrane protein
Isolation of mouse epididymal caput and caudal spermatozoa, and protein extraction were performed as described . Sperm membrane protein was isolated with the ReadyPrep Protein Extraction Kit (Pierce, Rockford, IL, USA). Protein concentration was determined by BCA™ Protein Assay Kit (Pierce), using bovine serum albumin (BSA) as a protein standard.
Western blot analysis
Protein samples (20 μg per well) were separated under denaturing conditions using SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Waukesha, WI, USA) using a semi-dry transfer apparatus (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% BSA. Immunoblotting was performed with rabbit polyclonal ERp29 antibody (Abcam, Cambridge, MA, USA) at a 1:1000 dilution, followed by incubation with secondary antibody conjugated to HRP (Abgent, San Diego, CA, USA) at a 1:5000 dilution. The blotting signals were detected by enhanced chemiluminescence (ECL) (ECL Plus, GE Healthcare), following the manufacturers' protocol. In the meantime, β-actin served as the internal control. Western blot analysis was repeated three times and then the results were scanned by Calibrated Densitometer (Bio-Rad GS-800). Finally, the averages were calculated with Quantity One (Version 4.6.1) software.
ERp29 immunofluorescent staining
Mouse caput and caudal spermatozoa were isolated and immediately smeared onto individual slides. ERp29 immunofluorescent staining was performed as described . Slides were blocked with 5% BSA and incubated with rabbit anti-mouse ERp29 antibodies (Abcam) or normal rabbit IgG (Upstate, Temecula, CA, USA) (1:500 dilution each). Goat anti-rabbit antibody linked with fluorescein isothiocyanate (FITC) (Rockland, Gilbertsville, PA, USA) was used as the secondary antibody at 1:300 dilution. Finally, the fluorescent-stained sperm samples were viewed under LSCM (Carl Zeiss LSM-510, Jena, Germany) and the graphs were processed with its accompanying AIM (Release Version 4.0 SP2) software. Indirect immunofluorescence analysis was repeated at least three times.
Prokaryotic expression and purification of recombinant ERp29
Total RNA was extracted from mouse liver with RNA Easy (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol. Then, RNA was transformed into cDNA with RT-PCR. A segment of ERp29 was amplified with the forward primer 5'-GCGGATCC TTCTACAAGGTCATTCCC-3'(containing BamH I site, bolded) and the reverse primer 5'-GCAAGCTT TAGCCCACTTCTTCTCTG-3'(containing Hind III site, bolded). Prokaryotic expression and purification of rERp29 were performed as previously described . The new recombinant prokaryotic expression vector was named pET-28a(+)/ERp29, and propagated in E. coli BL21 (DE3) host cells cultured with 0.8 mM isopropyl-b-Dthiogalactopyranoside (IPTG) for 12 hours at 37°C with gentle shaking. The purified protein was freeze-dried in a Heto Freeze Dryer (Thermo Electron Corporation, Waltham, MA, USA), and its identity was confirmed with 15% SDS-PAGE analysis and Western blot using anti-His monoclonal antibody (Abgent).
Production of polyclonal rabbit anti-rERp29 antibodies
Production of polyclonal rabbit anti-mouse rERp29 antibodies was performed as described . The titer of the antiserum was measured using an indirect ELISA at 450 nm in conjunction with an Anthos Zenyth 1100 multimode detector (Anthos Labtec Instruments GmbH, Wals, Austria) with a 5-s pre-read shaker. Finally, the immunized rabbit IgG (including anti-rERp29 IgG) was purified through immunoaffinity chromatography from crude rabbit sera, using the ImmunoPure (G) IgG Purification kit (Pierce). In the meantime, normal rabbit IgG was purified from pre-immunized rabbit sera.
Female BALB/c mice were superovulated by abdominal injection of 10 IU of pregnant mare's serum gonadotropin (PMSG; PROSPEC, Rehovot, Israel), followed 46-48 hours later by 10 IU of human chorionic gonadotropin (hCG; Li Zhu drug plant, Zhuhai, China). Cumulus enclosed-egg complexes were collected from the oviducts 15-16 hours after post-hCG injection, and treated with 0.1% hyaluronidase in Medium 16 (M16) to disperse the cumulus cells. The ZP was removed with 0.6% proteinase K, and then the eggs were incubated with M16 containing 3% BSA at 37°C, 5% CO2 for 30-60 minutes.
Mature spermatozoa were collected from epididymal cauda of BALB/c male mice by rough cutting in 1.5 ml of M16 containing 3% BSA; Motile spermatozoa were allowed to swim out for 10-15 minutes, and then the tissue was removed from the medium. One part of spermatozoa were cultured for 2 hours in M16 (containing 3 mg/ml BSA and 5 μmol/l ionophore A23187) at 37°C and 5% CO2, then used for localization of ERp29 in acrosome-reacted spermatozoa. The resting spermatozoa were cultured for 90 minutes in M16 containing 3 mg/ml BSA at 37°C, 5% CO2, allowing them to capacitate and undergo spontaneous AR. Following that, spermatozoa (adjusted to 107 sperm/ml) were incubated in the presence or absence of prepared and purified rabbit anti-mouse rERp29 antibodies (without sodium azide) or pre-immunized rabbit IgG (normal rabbit IgG) at different concentrations (20, 50, 100, 200 μg/ml), or rabbit anti-mouse rBPI antibodies (100 μg/ml, the antibodies were a generous gift from Dr Zhongping Zhou form at the Shanghai Key Laboratory for Reproductive Medicine, Shanghai, China), or washing buffer (control) for 40 minutes, and then diluted (1: 40) with M16 containing 3% BSA for use in the sperm-oocyte fusion assay and the final sperm concentration was 2.5 × 105 sperm/ml.
Sperm-oocyte fusion assay
For every sperm-oocyte fusion assay, all the oocytes and spermatozoa used in an experiment were combined from several female and male mice and then divided into each group randomly. ZP-free oocytes were added directly to the sperm suspensions prepared as described above, and the gametes were co-incubated for 3 hours at 37°C, 5% CO2. Then, loosely bound spermatozoa were removed from the oocytes by gentle pipetting. After that, the oocytes were fixed with 4% paraformaldehyde for 15 minutes, and then treated with Hoechst 33342 for 10 min to stain the chromatin. Finally, images were collected using the LSCM and results were evaluated as the percentage of oocytes penetrated by sperm (FR) and the average number of penetrated sperm per oocyte (FI). Each assay was repeated at least three times at each concentration of IgG used in every experiment.
Assessment of sperm motility and acrosome reaction
Spermatozoa from epididymal cauda were cultured for 90 minutes in M16 containing 3 mg/ml BSA at 37°C and 5% CO2, and then incubated either with prepared anti-mouse rERp29 antibodies or with normal rabbit IgG at the concentrations of 20, 50, 100, 200 μg/ml for 40 minutes. After sperm/antibody incubation, sperm motility (including movement parameters: velocity average path (VAP), velocity curvilinear (VCL) and velocity straight line (VSL)) was assessed using computer-assisted semen analysis (CASA; Hamilton-Thorn Research, Beverly, MA, USA) with parameters optimized for detection of mouse sperm.
AR assays were conducted essentially as described . Sperm suspensions were fixed with 4% paraformaldehyde solution (pH 7.4) for 10 minutes, and then centrifuged and washed twice with 100 mM ammonium acetate (pH 9.0). The final sperm pellet was resuspended in 100 mM ammonium acetate, and 50 μl of the sperm suspension was smeared on a glass slide and incubated with Coomassie Blue (0.22% Coomassie Blue G-250, 50% methanol, 10% glacial acetic acid, 40% water) for 2 minutes. Finally, the percentage of acrosome-reacted cells was calculated by evaluating at least 200 spermatozoa and every experiment was repeated three times.
Data are presented as mean ± SEM and analyzed using the SAS 8.2 statistical software. Fertilization rate was analyzed by chi-square test. Student-Newman-Kuel's test was performed to assess the significance of individual variations among the treated groups. Differences were considered statistically significantly different when P < 0.05.