Reagents and antibodies

All chemicals were obtained from Sigma (St. Louis, MO), unless otherwise stated. Goat polyclonal antibody against CRISP2 (aa77-89) (MBS422304) was obtained from MyBioso urce (San Diego, CA). Mouse monoclonal antibody against phosphotyrosine (pTyr) (clone 4G10) was purchased from Sigma. Rabbit polyclonal to alpha tubulin (ab15246) was obtained from Abcam. Mouse monoclonal antibody against GAPDH was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Boar spermatozoa preparation

Ethical review and approval were not required for the animal study because semen samples are delivered from a commercial breeder. A written informed consent was obtained from the owners for the participation of their animals in this study. Freshly ejaculated sperm cells from highly fertile boars were obtained from a commercial breeder (Cooperative Center for Artificial Insemination in Pigs, Veghel, the Netherlands). The collected semen was diluted to 20 million sperm/mL in a commercial diluter, shipped in 80-mL sealed insemination tubes in a cool box (17°C) until use. Diluted sperm from approximately two to three different ejaculates were pooled and washed through a discontinuous Percoll (GE Healthcare) gradient (35% v/v and 70% v/v) in HEPES-buffered saline (HBS; 20 mM HEPES, 137 mM NaCl, 10 mM glucose, 2.5 mM KCl, 0.1% kanamycin, pH 7.6) for 750× g for 15 min, room temperature (RT). Top and interface layers were removed, and sperm pellets were further washed in HBS at 750× g for 10 min at RT. All solutions were iso-osmotic (290–300 mOsm/kg) and at RT before use.

In vitro capacitation and calcium ionophore A23187-induced acrosome reaction

The capacitating medium (CM) used in this study is a modified Tyrode's medium containing 90 mM NaCl, 10.0 mM HEPES, 3.0 mM KCl, 0.4 mM MgCl₂, 2.0 mM CaCl₂·2H₂O, 0.3 mM Na₂HPO₄, 25 mM NaHCO₃, 2.0 mM Na-pyruvate, 5.0 mM D-glucose, 21.6 mM sodium DL-lactate, 3.0 mg/mL bovine serum albumin (BSA) (fatty acid free), 290–300 mOsm/kg, pH 7.4. Medium supplied with neither CaCl₂, NaHCO₃, nor BSA (300 mOsm/kg was achieved by compensatory NaCl addition) was defined as a non-capacitating medium (NCM). Na-pyruvate and BSA were added into the medium on the same day prior to use. Complete CM and NCM were brought to equilibrium in an incubator (38.5°C, 5% CO₂) with loose lids or in the water bath (38.5°C) with lids on for at least 2 h, respectively, before adding to sperms. Percoll-washed sperms were suspended in the CM (1 mL, 20 × 10⁶ sperm/mL) in open vials for 2.5 h at 38.5°C in the incubator with 5% CO₂ or incubated in NCM (1 mL, 20 × 10⁶ sperms/mL) in air-tight vials for 2.5 h at 38.5°C in a pre-warmed water bath.

Sperm cells were exposed to 5 µM calcium ionophore A23187 during the last 30 min of capacitation to induce an acrosome reaction. All sperm incubations were carried out in 5 mL polystyrene round bottom tubes (Falcon, 352,054; Life Sciences, Corning, NY). After incubation, sperm cells were spun down and washed twice with phosphate-buffered saline (PBS) (137 mM NaCl, 8.0 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 2.7 mM KCl, pH 7.4) at 750× g for 10 min at RT. Cells were either processed for immunofluorescence or stored at –80°C for later use.

Immunofluorescence staining of sperm

Non-capacitated (NC), capacitated (CAP), and AR sperm cells were stained using a LIVE/DEAD fixable blue kit (L23105, Thermo Scientific) to examine the cell viability following the manufacturer's instructions. Briefly, washed sperm cells (20 × 10⁶) were resuspended in 1 mL PBS and 2 µL fluorescent-reactive LIVE/DEAD dye was added. After mixing, cells were incubated in the dark with the dye for 30 min at RT. Then the cells were washed twice with PBS at 750× g for 10 min to remove excessive dye and fixed in 4% paraformaldehyde (PFA) for 15 min at RT. A suspension of 100 µL sperm (10⁵ sperm/mL) was deposited in the chamber built by imaging spacers (GBL654008, Merck) on Superfrost slides (Thermo Scientific). Sperm cells were settled down for 30 min at RT and further permeabilized using 0.5% (v/v) Triton X-100 for 15 min at RT. After rising with PBS, cells were blocked using 1% (w/v) BSA in PBS for 1 h at RT, incubated overnight at 4°C with goat anti-CRISP2 or/and mouse anti-pTyr. Wells were then washed three times for 30 min in PBS before incubation for 1 h at RT with Alexa Fluor 568-conjugated donkey anti-goat IgG [H + L] or/and Alexa Fluor 488-conjugated goat anti-mouse (Thermo Scientific) and counterstaining with Alexa Fluor 488-conjugated peanut agglutinin lectin (PNA) (Thermo Scientific) or/and Hoechst 33342 (1 µg/mL Sigma) at RT for 20 min. After extensive washing with PBS, slides were mounted with FluorSave reagent (Merck Millipore) and covered with coverslips. For negative controls, primary antibodies were omitted. Observations were performed on a Leica SPE-II confocal microscope using a 63× objective (NA 1.3, HCX PLANAPO oil). The scale bar was added by Image J software (bundled with 64-bit Java 1.8.0_172, National Institutes of Health, Bethesda, MD).

Porcine oocyte collection and in vitro maturation

Ovaries were collected from gilts from a local slaughterhouse within 2 h of slaughter. Cumulus–Oocyte complexes (COCs) were obtained by aspirating medium sized follicles (3–6 mm diameter) with an 18 g needle fixed to a vacuum pump via 50 mL conical tube and matured as previously described [46]. Briefly, COCs were recovered and washed in a modified Tyrode's lactate-HEPES medium (TL-HEPES, 114.0 mM NaCl, 3.2 mM KCl, 2.0 mM NaHCO₃, 0.25 mM Na-pyruvate, 0.4 mM NaH₂PO₄, 10.0 mM HEPES, 0.1% (w/v) polyvinylpyrrolidone (PVP), 10.0 mM Na-lactate, 0.5 mM MgCl₂·6H₂O, 2.0 mM CaCl₂·2H₂O). COCs were further washed twice in pre-warmed Medium 199 (Gibco) supplemented with 2.2 mg/mL NaHCO₃ (Medium C incomplete) and washed once in pre-equilibrated Medium C incomplete supplemented with 10% porcine serum, 1 mM Na-pyruvate, and 0.6 mM cysteine (oocyte maturation medium; OMM). COCs were transferred in groups of 40–50 per well in to a four-well dish containing 500 µL equilibrated OMM-I (OMM supplemented with 0.2 mM cysteamine, 0.05 IU/mL recombinant human follicle-stimulating hormone (rhFSH, Organon, Oss, The Netherlands), 100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco)) and incubated at 38.5°C with 5% CO₂ for 22 h. After 22 h, COCs were then washed twice in OMM- (OMM-I without rhFSH) and then placed in 500 µL OMM-II for an additional 20 h to reach the metaphase II stage.

Table 1.

The cleavage (≥2-cell) and blastocyst formation

In vitro fertilization

The expanded cumulus cells were removed by using a micropipette set at 150 µL and pipetting the contents of the dish in and out 30 times. Denuded oocytes were then washed twice in equilibrated IVF medium (113.1 mM NaCl, 3.0 mM KCl, 20.0 mM Tris, 11.0 mM glucose, 1.0 mM caffeine, 7.5 mM CaCl₂·2H₂O, 0.1% BSA (fatty acid free), 5 mM Na-pyruvate (add on the day of fertilization), 100 units/mL penicillin, and 100 µg/mL streptomycin, 275–290 mOsm/kg, equilibrated overnight in the incubator at 38.5°C in 5% CO₂) and transferred in groups of 40–50 oocytes into a four-well dish containing a 500 µL IVF medium and cultured at 38.5°C with 5% CO₂ until adding sperms. Freshly ejaculated sperm cells (from three different boars) were mixed and washed twice in an IVF medium at 700× g for 4 min at RT. Sperm pellets were resuspended in 3 mL of the IVF medium and pre-stained with 0.5 µM MitoTracker Red (Thermo Scientific) in 37°C water bath for 15 min. After twice washing with IVF medium, sperm suspensions were added into oocytes at a final concentration of 2.5 × 10⁵ sperm/mL and co-incubated at 38.5°C with 5% CO₂ for 6–8 or 24 h. At 24 h post-IVF, the presumptive zygotes were removed from the IVF wells, washed twice in pre-equilibrated synthetic oviductal fluid (SOF) medium [47] and placed in groups of 40–50 per 500 µL SOF medium for further development at 38.5°C with 5% CO₂ and 7% O₂. The cleavage rate (≥2-cell) and blastocyst formation of embryos were assessed 48 h and 7 days after IVF, respectively (Table 1).

Immunostaining of oocytes/zygotes

Immunostaining analysis of oocytes was carried out as previously described [20] with minor modifications. Before fixation, the ZP was removed by briefly incubating with 0.5% (w/v) protease in TL-HEPES. The zona-free zygotes were fixed in 4% PFA for 15 min at RT and stored in 0.1 M phosphate buffer (PB)-0.01% (w/v) PVP at 4°C, if not used immediately (no longer than 5 days). Oocytes were permeabilized in 0.1 M PB-0.01% (w/v) PVP with 0.1% Triton X-100 for 1 h at RT, then blocked in 0.1 M PB-0.01% PVP (w/v) with 1% (w/v) BSA for 45 min at RT. After this, zygotes were incubated with a CRISP2 antibody (1:100) diluted in 0.1-M PB-0.01% PVP (w/v) with 1% (w/v) BSA at 4°C, overnight. After three times washing in 0.1 M PB-0.01% PVP (w/v), oocytes were incubated with Alexa Fluor 488-conjugated or Alexa Fluor 568-conjugated donkey anti-goat IgG [H + L] (Thermo Scientific) for 1 h at RT in the dark. After extensive washing, oocytes were counterstained with 20 µg/mL Hoechst 33342 for 10 min at RT. Oocytes were mounted in imaging spacers (GBL654008, Merck) on Superfrost slides (Thermo Scientific) with FluorSave reagent (Merck Millipore) and covered with coverslips. Observations were performed on a Leica SPE-II confocal microscope using a 63× objective (NA 1.3, HCX PLANAPO oil).

Figure 1.

The distribution of porcine sperm CRISP2 during in vitro capacitation and the acrosome reaction. After incubation, NC, CAP, and AR sperm cells were washed and labelled with a live/dead blue dye before fixation and permeabilization. Sperm cells were then labelled with anti-CRISP2 and Alexa Fluor 568-conjugated secondary antibodies (red) followed by Alexa Fluor 488-conjugated PNA (green). The CRISP2 primary antibody was omitted and representative dead cells with bright blue signals were shown in control. Additional signals were observed on the apical ridge of sperm head and the EqS (arrows). Three ejaculates from different boars were mixed as one biological replicate and this experiment was replicated three times. Scale bar = 10 µm.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting

An equal number of sperm cells were lysed in the same volume of RIPA lysis buffer (Thermo Scientific) with freshly added protease inhibitors containing aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) (Thermo Scientific) on ice for 30 min with mixing. Cell debris was removed by centrifugation at 14000 g for 15 min, at 4°C. Supernatants were transferred to new tubes and denatured in a 4× SDS sample buffer (200 mM Tris–HCl, pH 6.8, 10% β-mercapto-ethanol, 8% SDS, 0.08% bromophenol blue, 40% glycerol) and boiled for 10 min. The same amount of each lysate was loaded on to an SDS-PAGE gel (5% stacking gel, 12% resolving gel) and blotted onto 0.2-µm nitrocellulose membranes (GE Healthcare, Piscataway, NJ) at 100 V for 1 h. After blocking for 3 h at RT in 5% (w/v) BSA in PBS with 0.05% (v/v) Tween-20 (PBST), membranes were incubated with primary antibodies (diluted in PBST with 1% BSA) overnight at 4°C. After three washes in PBST for 15 min, membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies (mouse anti-goat HRP IgG, sz-2354, Santa Cruz, CA; goat anti-rabbit and mouse HRP IgG, P0448, Agilent) for 1 h at RT. After rinsing four times in PBST for 20 min, membranes were developed using chemiluminescence (ECL-detection kit; Supersignal West Pico, Pierce, Rockford IL). The protein band size was determined using a PageRuler Plus pre-stained protein ladder, 10–250 kDa (Thermo Scientific).

Exposure of CRISP2 on the apical ridge and the EqS of the sperm head during capacitation

We have characterized the biochemical properties of CRISP2 in ejaculated boar spermatozoa in earlier work [44]. Here we explored the fate of CRISP2 during in vitro capacitation and during the calcium ionophore-induced acrosome reaction. To perform this study, we first focused on immunostaining of CRISP2 in NC, CAP, and AR porcine sperm. This was performed in the presence of PNA to assess the acrosome status. The results demonstrated that the fluorescent signals of CRISP2 in NC sperms were bright in the post-acrosomal region and the connecting piece, with weak signals in the sperm tail, as previously reported [44] (Figure 1,  Supplementary Figure S1A (supplementary_materials_ioac169.docx)). Interestingly, after incubation in the CM, a population (34.2 ± 3.8%; Table 2) of sperms with intact acrosomes showed strong CRISP2 labeling at the EqS region and the apical ridge of the sperm head (labeling type 2), and this CRISP2 labeling pattern was absent in NC sperms (Figure 1,  Supplementary Figure S1B (supplementary_materials_ioac169.docx)). While the majority possessed a staining pattern (labeling type1) that was consistent with NC sperms (Figure 1,  Supplementary Figure S1B (supplementary_materials_ioac169.docx)). To eliminate the possibility that staining pattern in type 2 was specific associated with cell death, a live/dead blue staining was used to assess sperm viability and revealed that type 2 stained sperm cells had no preference to deteriorated versus intact sperm cells. After the calcium ionophore-induced acrosome reaction, CRISP2 signals at the EqS and the apical ridge were not visible, but CRISP2 labeling was detected at the subdomain of EqS (EqSS) (Figure 1,  Supplementary Figure S1A (supplementary_materials_ioac169.docx)). To address the point that additional CRISP2 staining of capacitated sperms was likely an exposure, fresh spermatozoa was incubated with 0.01% saponin. Interestingly, the staining pattern of CRISP2 at the apical ridge and the EqS of the sperm head was mimicked. CRISP2 signal was present in the apical ridge when spermatozoa possessed a PNA-positive and swollen acrosome (Figure 2A). Further, immunoblotting analysis of CRISP2 showed that Saponin treatment did not cause visible loss of CRISP2 from sperm cells (Figure 2B).

Figure 2.

Saponin incubation with ejaculated spermatozoa mimics the exposure of CRISP2 at the apical ridge and the EqS of capacitated sperm. Percoll washed sperm cells were incubated in 0.01% Saponin supplied in HBS for 10 min at 37.5°C. After incubation, sperm cells were washed and sampled for immunostaining and immunoblotting analysis. (A) Sperm cells were fixed and permeabilized, probed with anti-CRISP2 followed by Alexa Fluor 568-conjugated (red) secondary antibodies, counterstained with Alexa Fluor 488-conjugated PNA (green) and Hoechst 33342 (blue). Scale bar = 10 µm. (B) Immunoblotting analysis of CRISP2 on the extracts from Saponin treated sperm probed with anti-CRISP2.

Table 2.

Type 2 labeling of CRISP2 in pig sperm after in vitro capacitation

Additional appearance of CRISP2 is specific for capacitated sperm cells that show protein tyrosine phosphorylation

To confirm that the translocation of CRISP2 to the EqS and apical region was dependent on capacitation, protein tyrosine phosphorylation was analyzed to confirm the capacitation status of the cells. Coomassie brilliant blue staining analysis of lysates from NC, CAP, and AR sperm was conducted to view total proteins loading and protein changes. As indicated in Figure 3A, the total protein amount among the conditions was comparable, with three protein bands that changed depending on capacitation and acrosome reaction treatments: (i) an ∼ 66-kDa protein band was absent in NC spermw and present in CAP and AR sperm. This likely corresponds to BSA, which was not present in the NCM. (ii) An ∼ 52-kDa protein band was present in NC and CAP sperms, but greatly decreased in AR sperms. This protein band likely represents proacrosin. Finally, (iii) the ∼32-kDa protein band associated with AR sperm is in line with the corresponding increased tyrosine phosphorylated protein detected by immunoblots in Figure 3B. Immunoblotting analysis on lysates from NC, CAP, and AR sperms showed that the intensity of ∼43, ∼40, and ∼36 kDa protein tyrosine-phosphorylation bands was increased when sperm were incubated in CM. Additional bands of ∼32 and ∼27 kDa also appeared after calcium ionophore-induced acrosomal exocytosis (Figure 3B). Previous studies have reported that protein tyrosine phosphorylation is enhanced at the acrosome in capacitated boar sperms, with consistently signals being present in the EqSS [48]. Similar results were observed in our study. In NC sperms, protein tyrosine phosphorylation was restricted to the EqSS (Figure 3D). After capacitation, a representative 25% sperms showed the tyrosine phosphorylation signal spreading over the acrosome. Among those, a representative 76% sperms showed additional CRISP2 labeling at the apical ridge and EqS (Figure 3D). After induction of the acrosome reaction, additional labeling of CRISP2 in the apical ridge was not observed despite protein tyrosine phosphorylation fluorescence labeling the acrosome region. Moreover, CRISP2 was colocalized with tyrosine phosphorylation fluorescence in the EqSS in AR sperms (Figure 3D). Immunoblot analysis of CRISP2 on lysates from NC, CAP, and AR sperm cells was shown in Figure 3C.

Distribution of CRISP2 of sperm bound to ZP

To further explore how CRISP2 is distributed post-fertilization, IVF was performed on matured porcine oocytes by coincubation with porcine sperms. Initial experiments focused on investigating the distribution of CRISP2 in the sperms that were bound to the oocyte ZP. Immunostaining of CRISP2 revealed CRISP2 signal at the apical ridge, the EqS, the post-acrosomal region of the sperm head and tail (Figure 4Aa). This distribution of CRISP2 was consistent with what we described above for CAP sperm that showed increased protein tyrosine phosphorylation. Sperms that were penetrating the ZP lost the signal on the apical ridge as well as on the EqS, but the immunofluorescence of CRISP2 in the post-acrosomal region was retained (Figure 4Aa). In Figure 4B, another capture of sperm penetrating the ZP showed that CRISP2 stayed condensed in the post-acrosomal region. A recent study has shown that there is no CRISP2 mRNA expression in porcine ovary [49]. In our study, mature oocytes were collected and processed for immunoblotting analysis. Ejaculated sperm cells were used as a positive control. We have described in our earlier study that 8 M urea treatments cause high molecular CRISP2 complexes disassociation into a monomer [44]. Here, denuded oocytes were also solubilized in 8 M urea to acquire an efficient denature of oocyte proteins. Our result revealed that no CRISP2 signal was detected on the immunoblot of the porcine oocytes lysates ( Supplementary Figure S2 (supplementary_materials_ioac169.docx)). Thus, the CRISP2 signal detected in Figure 4A/B was definitely coming from CRISP2 within boar spermatozoa.

Figure 3.

Exposure of CRISP2 at the apical ridge and the EqS of the sperm head during capacitation is an event associated with tyrosine phosphorylation. (A) Lysates from NC, CAP, and AR sperm cells were analyzed by SDS-PAGE and Coomassie blue staining. Immunoblotting analysis of tyrosine phosphorylation and CRISP2 on the extracts from NC, CAP, and AR sperm cells probed with anti-pTyr (B) and anti-CRISP2 (C) antibodies. (D) NC, CAP, and AR sperm cells were co-incubated with anti-CRISP2 and anti-pTyr, labelled with Alexa Fluor 568-conjugated (red) and Alexa Fluor 488-conjugated second antibodies (green) and counterstained with Hoechst 33342 (blue). This experiment was replicated three times. Scale bar = 10 µm.

Figure 4.

The distribution of sperm CRISP2 in zona-bound and zona-penetrated spermatozoa. Porcine oocytes and sperm were co-incubated for 6–8 h after insemination. Zygotes were fixed and permeabilized for immunochemistry analysis. (A) Zygotes were labelled with anti-CRISP2 (a) followed by Alexa Fluor 568-conjugated (red) secondary antibodies and counterstained with Hoechst 33342 (blue). (b) The anti-CRISP2 was omitted. (B) Immunolocalization of CRISP2 of sperm penetrated in zona detected by Alexa Fluor 488-conjugated (green) secondary antibodies. Scale bar = 10 µm.

Rapid dispersal of sperm CRISP2 prior to sperm nuclear decondensation

Intrigued by the findings that CRISP2 is a component of the PAS-PT and its important roles in early events of fertilization, we sought to investigate the fate of sperm CRISP2 in fertilized pig oocytes. Oocytes were co-incubated with fresh boar spermatozoa preloaded with fluorescent MitoTracker Red probe, cultured and sampled for CRISP2 detection. The dissociation of sperm CRISP2 from the PAS-PT was observed within the oolemma, while the sperm nucleus was still fully condensed and the entire mitochondrial sheath was still intact (Figure 5A). Remarkably, sperm CRISP2 was undetectable in zygotes that were in the two-pronuclear stage, while sperm mitochondria were still linear arranged (Figure 5B,  Supplementary Figure S3B (supplementary_materials_ioac169.docx)). Multilayer scanning (z-stack, step size 1 µm) was recorded to show the CRISP2 and sperm mitochondrial sheath originating from the fertilizing sperm cell ( Supplementary Files 1 and 2 (supplementary_materials_ioac169.docx)). Another fertilizing sperm was imaged and showed CRISP2 dispersal as well as a decreased intensity of CRISP2 immunofluorescence, while the sperm nucleus was still condensed ( Supplementary Figure S3A (supplementary_materials_ioac169.docx)). This indicates that the PT dissociation (as imaged with CRISP2 immunolabeling) and degradation events occur earlier than sperm chromatin decondensation and degradation of the sperm mitochondria.