This work was approved by the Ethics Committee on Animal Experimentation of South China Agricultural University. The license number is SYXK (Guangdong) 2019-0136.

Thirty-three Yorkshire semen samples and the fertility data for each boar were supplied by Shuitai Pig Farm (Guangdong, China). The protein samples of sperm were extracted immediately after semen collection. The sperm proteins were extracted with a whole protein extraction kit (KeyGEN, Jiangsu, China) according to the manufacturer's instructions. Briefly, the sperm samples were centrifuged at 10,000 rpm for 5 min. Supernatant was discarded and sperm pellet was washed thrice with ice-cold DPBS. The sperm pellet was incubated with ice-cold lysis buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF), and the tubes were incubated on ice for 4 min and vortexed for 30 s for 5 times. After incubation, the tubes were centrifuged at 14,000 rpm at 4°C for 4 min. After centrifugation, supernatant was retained as the sperm protein samples.

The reproductive tissues (at least 3 samples) of immature (3 months old) and adult (24 months old) male and female pigs from Shuitai Farm were collected immediately after slaughter and stored in liquid nitrogen or fixed in 4% paraformaldehyde. Testis, epididymis (distal caput), bulbourethral gland (BUG), prostate, and seminal vesicle gland (SVG) were collected from the males. Ovaries, oviduct, uterine horn, uterine body, and cervix were collected from females. The oocytes and granulosa cells were collected from the ovaries in a local slaughterhouse, and the isolation of granulosa cells was performed according to previous study (18). The total RNA and protein of each sample were extracted by using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and a whole protein extraction kit (KeyGEN), respectively, according to the manufacturer's instructions. Purity and concentration of RNA were measured using a NanoDrop ND-1000 instrument (Thermo Fisher Scientific, Waltham, USA). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent, San Jose, USA). For protein extraction from tissue samples, the tissues were homologized in lysis buffer containing 1 mM PMSF and were then subjected to centrifugation (14,000 rpm) for 5 min at 4°C. The supernatant was collected as the protein samples.

The protein levels of samples were measured and diluted to an appropriate concentration with a BCA protein assay kit (KeyGEN) according to the manufacturer's instructions. Briefly, a standard curve (range 0–2,000 μg/mL) was derived with nine points of serial dilution with bovine serum albumin (BSA) and a working reagent. All samples and standard points were replicated three times. The samples (100 μL each) were mixed with 2.0 mL of working reagent and incubated at 37°C for 30 min. After cooling to room temperature, each absorbance difference, which was subtracted by averaged absorbance of blank standard replicates at 562 nm, was measured by a spectrometer, and the absorbance differences were converted to μg/mL via the standard curve. If a protein concentration exceeded the upper limit of the standard curve of 2,000 μg/mL, the sample was diluted until it could be measured within the standard range, and the final concentrate was calibrated considering the dilution factor.

The protein levels of CRISP2 of each sample were quantified with a porcine CRISP2 ELISA kit (PG1898, TSZ, USA). The assay range was 18–1,450 pg/mL according to the kit instructions. The relative expression level of CRISP2 protein was obtained by dividing the protein levels of CRISP2 by the total protein content. The correlation between the relative content of CRISP2 and the fertility data was conducted using Pearson correlation analysis.

The IVF experiment was conducted as previously reported (19). Briefly, porcine ovaries were obtained from a slaughterhouse and transported to the laboratory in sterile 0.9% NaCl at 38.5°C within 2 h of slaughter. Oocytes were aspirated from follicles (3–6 mm in diameter) with an 18-gauge needle attached to a disposable syringe. Oocytes covered with multilayers of cumulus cells were selected. Oocytes collected were cultured for 44–6 h and denuded in 1 mg/ml hyaluronidase in DPBS by mechanically pipetting; then, 10–15 oocytes were grouped and transferred to the 50 μl mTBM fertilization medium containing 2.5 mM caffeine and 2 mg/ml bovine serum albumin (BSA; fraction V) covered with mineral oil. The fresh semen provided by the Shuitai Farm was washed three times by centrifugation with DPBS supplemented with 0.1% BSA at 1,500 rpm for 4 min. The spermatozoa pellets were resuspended and diluted to 1 × 10⁶ sperm/ml with mTBM for capacitation in the CO₂ incubator for 30 min. Then, the capacitated sperm were added to the drop containing oocytes with a final sperm concentration of 1 × 10⁵ sperm/ml and co-incubated for 6 h at 39°C in an atmosphere of 5% CO₂ in air. After fertilization, the oocytes were washed 3 times and cultured with PZM3 medium at 39°C, 5% O₂, 5% CO₂, 90% N₂, and 100% humidity. The cleavage rate was determined after culturing for 48 h.

The effect of CRISP2 on fertilization was tested by adding the anti-CRISP2 antibody (SAB2501635, Sigma, USA) to the fertilization medium mTBM. Briefly, 2 μl of the antibody was added to 500 μl fertilization medium to a final concentration of 2 μg/ml of anti-CRISP2 antibody. The same volume of dilution medium (20 mM Tris (pH 7.3) + 150 mM NaCl + 0.02% sodium azide + 0.5% BSA) or the IgG were added as controls.

The gene expression of CRISP2 in different reproductive organs of different aged male and female pigs was examined by reverse transcriptase PCR. The primers used in the analysis are presented in Table 1. The PCR conditions were as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 40 s, and a final extension at 72°C for 7 min.

The relative expression levels of CRISP2 in the reproductive organs of male and female pigs at different ages were further verified by qRT-PCR using a SYBR-Green RT-PCR Kit (Thermo Fisher Scientific) in an Applied Biosystems 7900HT Real-time PCR Thermal Cycler (Applied Biosystems, Foster City, USA). GAPDH was employed as an internal control, and each sample was analyzed three times. The mean values were calculated using the ΔΔCt method as previously reported (20). The PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s and extension at 72°C for 30 s. The qRT-PCR primers are listed in Table 1.

The proteins (20 μg) of adult boar reproductive tissues and sperm were separated by SDS-PAGE using 12% (v/v) gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies against CRISP2 (1:1,000; SAB2501635, Sigma, USA) or β-actin (1:1,000; HC201, TransGen Biotech, China) overnight at 4°C. The membranes were washed 3 times for 10 min each with TBST (0.1% Tween 20, 20 mM Tris/HCl, 150 mM NaCl; pH 8.0) and incubated for 1 h with horseradish peroxidase (HRP)-conjugated rabbit anti-goat (1:3,000; E030130-02; Earthox, San Francisco, USA) or goat-anti-mouse (1:3,000; HS201, TransGen Biotech, China) secondary antibodies at room temperature for 1 h. The membranes were incubated for 5 min with the enhanced chemiluminescence (ECL) detection reagent in the dark and then exposed with a Tanon-5200 Imaging System (Tanon, Shanghai, China). β-actin was used as the internal control, and the relative protein expression levels of CRISP2 were analyzed by using ImageJ software (https://imagej.nih.gov/ij/index.html).

Immunohistochemical detection of CRISP2 in the adult tissues of testis was carried out on 5 μm tissue sections mounted onto siliconized slides. Briefly, paraffin sections were dewaxed with xylene, rehydrated in a graded series of ethanol, and antigen retrieval was performed by heating at 95°C in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase was quenched with 0.3% H₂O₂ in methanol for 15 min at room temperature. After 3 washes in PBS (pH 7.4), the slides were incubated in a blocking solution containing 3% BSA for 30 min at room temperature. Sections were incubated overnight at 4°C with antibodies against CRISP2 (1:150; SAB2501635; Sigma, USA), and the primary antibody replaced with normal IgG diluted was served as a negative control. After washing 3 times in PBS, sections were incubated with HRP-conjugated secondary antibodies for 50 min at room temperature. Then, the sections developed with a DAB chromogenic solution and counterstained with a hematoxylin solution. Sections were dehydrated, cleared, covered with Permount solution (Fisher, NH, USA) and viewed under an Olympus BX53F microscope (Olympus, Japan).

To evaluate the distribution of CRISP2 in the sperm before and after capacitation, immunofluorescence detection was performed as previously described (21). The sperm before and after in vitro capacitation were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, permeabilized with 0.5% Triton-100 for 10 min, and blocked in 1% BSA (Sigma) for 30 min. The sperm were incubated with a goat anti-CRISP2 antibody (1:200; SAB2501635, Sigma, USA) at 4°C overnight and washed three times in PBS. After that, the sperm were incubated with Alexa Fluor 568-donkey anti-goat IgG (1:100; A-11057, Thermo Fisher) 1 h at 37°C in the dark. The samples were coated onto slides and observed under a fluorescence microscope (BX53F, Olympus).

All the data analysis was performed using the SPSS 18.0 software (IBM, USA). All the data were expressed as mean ± standard deviation. The correlation between the relative content of CRISP2 and the fertility data was conducted using Pearson correlation analysis. The unpaired Student's t-test was performed to assess the significant differences between treatment groups. P < 0.05 was considered statistically significant.

The relative content of CRISP2 in sperm of 33 boars was detected by ELISA, and effective data was obtained for 29 boars, because a few ELISA wells showed null data. The sperm CRISP2 protein levels and boar reproductive parameters of 29 boars were shown in Supplementary Table 1. The 29 boars were divided into low CRISP2 and high CRISP2 group based on the median values of the sperm CRISP2 protein levels. The correlation analysis listed in Table 2 showed that the protein levels of sperm CRISP2 were significant positive correlation with the boar breeding parameters of litter size (r = 0.412, p = 0.026), live-born piglets per litter (r = 0.421, p = 0.023) or qualified piglets per litter (r = 0.381, p = 0.042), but not with parturition rate (r = 0.029, p = 0.880) or boar fecundity (r = 0.315, p = 0.096).

To further analyze the correlation between sperm CRISP2 protein levels and the reproductive capacity of boars and to even explore their feasibility as biomarkers for screening boars with high fertility, we ranked the boars corresponding to their protein levels of sperm CRISP2 and divided them into 2 groups: the high CRISP2 group (n = 14) and the low CRISP2 group (n = 15). The reproductive parameters of the boars are shown in Table 3. There is no significant difference in the number of sows bred (low CRISP2 group: 977 vs. high CRISP2 group: 865) and parturition rate (low CRISP2 group: 92.95 ± 1.51% vs. high CRISP2 group: 93.26 ± 1.22%) between low CRISP2 group and high CRISP2 group. The litter size (low CRISP2 group: 12.18 ± 0.26 vs. high CRISP2 group: 13.08 ± 0.17), live-born piglets per litter (low CRISP2 group: 11.57 ± 0.27 vs. high CRISP2 group: 12.61 ± 0.20), qualified piglets per litter (low CRISP2 group: 10.39 ± 0.26 vs. high CRISP2 group: 11.18 ± 0.17) and boar fecundity (low CRISP2 group: 11.32 ± 0.33 vs. high CRISP2 group: 12.21 ± 0.28) in the CRISP2 group were significantly higher than that in the low CRISP2 group. These results indicated that CRISP2 might play critical roles in the sperm fertilizing ability or boar fertility, and might have the potential to serve as a biomarker for selecting high fertility boars.

The effect of the CRISP2 protein on the cleavage rate of in vitro fertilization was indirectly investigated by adding the anti-CRISP2 antibody to the fertilization medium during in vitro fertilization. As shown in Table 4, the cleavage rate in anti-CRISP2 group (50.37 ± 1.94%) was significantly lower than that of the control group (59.53 ± 2.54%) and the IgG group (57.81 ± 2.19%), which suggests that CRISP2 plays a critical role in the process of fertilization.

To elucidate the potential role of CRISP2 in the sperm fertilizing ability and boar fertility, the mRNA expression of CRISP2 in the reproductive organs of male and female pigs with different ages was detected by reverse transcriptase PCR. As shown in Figure 1, CRISP2 was expressed specifically in the testis and epididymis of adult boars, and no expression was detected in the reproductive tissues examined from the female pigs (Figure 1A).

To verify the CRISP2 expression patterns detected by reverse transcriptase PCR, qRT-PCR was conducted on the expression levels of CRISP2 in the testis, epididymis, SVG, prostate, bulbourethral gland, sperm, ovary and oviduct of adult, and 3-month-old pigs. The results showed that CRISP2 mRNA was highly expressed in adult testis and sperm (Figure 1B).

The protein level expression of CRISP2 in the boar reproductive organs and semen were further detected by Western blot. The results showed that CRISP2 was mainly expressed in the testis and sperm (Figure 1C).

The distribution of CRISP2 in the testis tissues was detected by immunohistochemical analysis. The results showed that the CRISP2 protein was expressed in the cytoplasm of spermatogonia, spermatocytes, and sperm cells in the seminiferous tubules (Figure 2).

To gain further insight into the function of CRISP2 in fertilization, immunofluorescence detection of CRISP2 before and after sperm capacitation was carried out. As shown in Figure 3, CRISP2 is mainly distributed in the post-acrosomal region, neck, and tail of sperm before capacitation and relocated to the apical segment and posterior of the acrosome and to the middle piece of the tail after sperm capacitation. The number of CRISP2-staining sperm in the apical segment and posterior of the acrosome and middle piece of the tail after capacitation was significantly higher than that before capacitation (before capacitation: 20.63 ± 2.22% vs. after capacitation: 70.23 ± 2.15%, P < 0.001).