Bovine testicular hyaluronidase

HA distribution in RSFs after treatment with HAoligos was visualized by staining with a biotinylated HA binding protein (b-HABP; Seikagaku Biobusiness Co., Tokyo, Japan) that has a high affinity for the decasaccharide unit of HA. RSFs were seeded into chamber slides (BD Biosciences) for 48 hours and treated with or without 250 μg/ml HAoligos for one or three hours. Cells were then fixed with 4% paraformaldehyde, buffered with PBS at room temperature for one hour, treated with 0.3% H₂O₂ in PBS for 30 minutes at room temperature to block internal peroxidase activity, and incubated with 1% bovine serum albumin in PBS for one hour at room temperature. Cells were incubated with 2.0 μg/ml b-HABP for one hour at room temperature, and bound b-HABP was detected by the addition of streptavidin-peroxidase reagents and diaminobenzidine (DAB)-containing substrate solution (Nichirei Biosciences Inc., Tokyo, Japan). As a negative control, cells were pretreated with 100 units/ml bovine testicular hyaluronidase (Sigma-Aldrich) for three hours before incubation with b-HABP.

Two hundred microliters of native SF prior to further processing and 200 μl of PB supernatants after density gradient centrifugation (see below) were harvested for MACSPlex analysis (Miltenyi Biotec, Germany) and deep-frozen at − 80 °C with a maximum time between sample collection and cryopreservation of 30 min. The remaining volume of SF samples was treated with bovine testicular hyaluronidase (10 mg/ml, Sigma-Aldrich, USA) for 30 min at 37 °C and washed twice with PBS. SM samples were rinsed twice with phosphate-buffered saline (PBS), minced finely with sterilized scissors, and digested with collagenase B (1 mg/ml; Roche, USA) and bovine testicular hyaluronidase IV (2 mg/ml; Sigma-Aldrich, USA) at 37 °C for 2 h in RPMI 1640 culture medium (Invitrogen, USA) supplemented with 10 μg/ml penicillin-streptomycin (Invitrogen, USA) and 5% FCS (Biochrom AG, Germany). The cell suspension was filtered through a 100-μm (BD Biosciences, USA) and a 40-μm pore-size cell strainer (EMD Millipore, USA) to remove any undigested tissue. The filtered cell suspension was washed twice with PBS. Mononuclear cells were isolated from heparin anti-coagulated whole blood, SF, and SM cell suspensions using Ficoll-PaqueTM PLUS (GE Healthcare, USA) density gradient centrifugation. T cells were isolated from PB, SF, and SM mononuclear cells by CD3 MACS bead separation (Miltenyi Biotec, Germany).

Both oocytes and cumulus cells were prepared by the superovulation of 8‐ to 10‐week‐old B6D2F1 female mice. Superovulation was induced by sequential injection 5 IU of pregnant mare serum gonadotropin (Sigma‐Aldrich), followed by 5 IU of human chorionic gonadotropin (hCG, Sigma‐Aldrich) with an interval of 48 hours. Cumulus‐oocyte complexes (COCs) were collected from oviducts in M2 medium (Sigma‐Aldrich) at 14 hours after hCG injection and were treated with M2 containing 0.1% bovine testicular hyaluronidase (Sigma‐Aldrich) to obtain dissociated cumulus cells and oocytes. The cumulus‐free oocytes were then cultured in potassium simplex optimized medium (KSOM; Millipore) at 37°C under 5% CO₂ until further use. Dispersed cumulus cells were dissociated by hyaluronidase treatment, diluted in M2 medium and collected. The pellet was then resuspended in a small volume of polyvinylpyrrolidone (PVP) in M2 medium in a manipulator chamber.

Adult female M. fascicularis monkeys were super-ovulated via intramuscular injection of 15 IU of rhFSH (recombinant human follitropin alfa, GONAL-F, Merck Serono, Germany) twice daily for 8 days and then with 1000 IU of hCG (Sigma, USA) on day 9. Oocytes were collected by laparoscopic follicular aspiration 36 h after hCG injection. Cumulus cells were removed by adding hyaluronidase (2 mg/ml) to the follicle and gently pipetting. Cumulus-free high-quality oocytes (MII) were visually inspected using a microscope and then cultured in HECM-9 medium at 37 °C in an atmosphere of 5% CO₂ for approximately 2 h. All oocytes and embryos were washed thrice in cold Dulbecco’s phosphate-buffered saline containing 0.01% polyvinylalcohol, immediately transferred to PCR tubes containing 3 µl of DEPC-treated water with 6 U of RiboLock RNase inhibitor (Thermo Fisher Scientific, Massachusetts, USA), and quickly frozen on dry ice. The samples were stored at −80 °C before use. Female C57BL/6 mice (3–4 weeks old) were super-ovulated via intraperitoneal injection of 10 IU of pregnant mare serum gonadotropin and 10 IU of hCG at 43- to 44-h intervals. Cumulus–oocyte complexes were collected from the oviducts 13 to 15 h later and treated with bovine testicular hyaluronidase (1 mg/ml, Sigma-Aldrich) for 1 to 2 min. Cumulus cells were removed and high-quality oocytes (MII) were then collected.

The sperm ZP-binding assay was performed as described previously (69 (link)). In brief, cumulus cells of oocytes were removed by treatment with bovine testicular hyaluronidase (175 U/mL; Sigma-Aldrich) for 5 min. In the TYH medium, cumulus-free oocytes were mixed and incubated for 2 h with spermatozoa of the indicated genotypes and fixed with 0.25% glutaraldehyde for 30 min. The bound spermatozoa were observed with an Olympus IX73 microscope (Olympus) (Fig. 3A) or an IX-70 fluorescent microscope (Olympus) (Fig. 3B and SI Appendix, Fig. S6 A and B). IZUMO1 was used as a marker of the acrosome reaction in spermatozoa bound to the ZP. After fixation, the oocytes were incubated with rat anti-IZUMO1 antibody (KS64-125) for 30 min, followed by incubation with goat anti-rat immunoglobulin G (IgG) Alexa Fluor 488 for 30 min. The AR spermatozoa were observed with a BZ-X710 fluorescent microscope (Keyence) (Fig. 3B and SI Appendix, Fig. S6 A and B).