Peanut Agglutinin

To image the endothelial glycocalyx, an Alexa Fluor 594-conjugated wheat germ agglutinin (WGA, Thermo Fischer Scientific, W11262, dilution 2 μg/ml), anti-heparan sulfate antibody (Abcam, Cambridge, UK, ab23418, 1:100), and peanut agglutinin (PNA, Vector Labs, Ontario, CA, FL-1071-5, 1:200) were used. Cells were cultured to confluence on coverslips and exposed to cyclosporine as described. Cells exposed to 500 mU/mL neuraminidase for 1 h were used as a positive control in WGA and PNA experiments. Cells exposed to 0.5 U/mL Heparinase III (H8891-5UN, Sigma-Aldrich, St. Louis, MO) for 30 min were used as a positive control in heparan sulfate experiments. Cells were incubated with Alexa Fluor 594-conjugated WGA for 5 min on ice and washed two times with ice-cold HBSS, and the coverslips were mounted in a Chamlide magnetic chamber (Life Cell Instrument, Seoul, Korea) and overlaid with media. Confocal microscopy was performed as detailed in Supplementary material, and total fluorescence intensity was measured using ImageJ software. For experiments using anti-heparan sulfate and PNA, cells were washed and fixed with 2% paraformaldehyde, followed by incubation with mouse anti-heparan sulfate (1:100) and anti-PNA (1:100) for 1 h. Alexa Fluor 488-conjugated species-specific secondary antibodies were used at a dilution of 1:1,000. Nuclei of cells were stained with 0.12 μg/ml Hoechst stain (Thermo Fisher Scientific, Waltham, MA) for 5 min.

Eight semen samples collected from each of the three male monkeys were performed for cryopreservation and evaluation. The TRIS-egg yolk-based sperm freezing medium (TTE) containing 0.2% Tris, 1.2% TES, 2% glucose, 2% lactose, 0.2% raffinose, and 20% (v/v) fresh egg yolk was prepared as previously described [32 (link),33 (link)]. Before an experiment, the medium was thawed in a 37 ℃ water bath. Semen was collected from the male monkeys by penile electroejaculation [34 (link)]. The volume of the remaining semen was measured, and TTE solution of equal volume containing 20% glycerol was prepared and placed at 4 °C. After 2 h, a TTE solution containing 20% glycerol was slowly added into the semen sample [33 (link)]. The diluted semen was sealed in 0.25 mL cryostraws and at 4 cm above liquid nitrogen with vapor for 10 min before being placed in liquid nitrogen and was stored at −196 °C until use. The cryopreserved semen were thawed in a 37 °C water bath.
Sperm motility, sperm acrosome integrity and mitochondrial membrane potential of fresh and frozen–thawed sperm were evaluated as in our previous description [20 (link)]. Fresh sperm and thawed sperm samples (10 μL) were placed on a pre-warmed Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) under a microscope for motility assessment. At least 200 sperm of each sample were evaluated for motility. The sperm acrosome integrity for fresh and thawed samples was determined using the Alexa Fluor-488-peanut agglutinin conjugate assay (Eugene, OR, USA). Briefly, 10 μL fresh semen or frozen–thawed semen was evenly coated on a slide, and then was dried at room temperature and fixed for 30 min (200 μL anhydrous ethanol). Then 50 μL of 10 μg/mL Alexa Flu-488-peanut lectin was added, and the slides were incubated in a 37 °C dark box. Thirty minutes later, they were observed using a microscope (emission ratio of 530 nm, wavelength of 488 nm). Sperm with uniform green fluorescence in the acrosome region of the head were considered to have an intact acrosome, while sperm with little or no green fluorescent staining in the front of the head were considered to have acrosomal sperm impairment. At least 200 sperm per semen sample were evaluated for this staining. We used a JC-1 assay kit (Solarbio, Beijing, China) to detect mitochondrial membrane potential in 2 × 10⁵ fresh sperm and thawed resuscitated sperm. JC-1 reagent was incubated with semen in a 37 °C water bath for 20 min according to the instructions, and fluorescence detection was performed (emission ratio of 530 nm, wavelength of 488 nm). Sperm with intact mitochondria fluoresce in orange and yellow. In contrast, sperm with damaged mitochondria emit green fluorescence. At least 200 sperm in each sample were evaluated for mitochondrial potential using a fluorescent staining procedure.