C57CBAF1 female mice (7-8 weeks old) were superovulated by intraperitoneal injections of 5 IU of pregnant mare serum gonadotropin (PMSG, Folligon®, MSD Animal Health) and an equivalent dose of human chorionic gonadotropin (hCG, Sigma) at a 48-h interval.  

Cumulus-oocytes complexes (COCs) were recovered from the oviducts of superovulated female mice 14 h after hCG injection and placed in a 4-well dish with 400 µl of Human tubal fluid (HTF) medium. Cumulus cells were removed by incubation in 300 µg/ml hyaluronidase (Sigma) solution in M2 medium. Zona pellucida was removed by brief incubation in Acidic Tyrode´s medium. 

Sperm from WT, Hz or KO individuals was recovered from the cauda epididymis in HTF medium and placed in the bottom of a previously equilibrated 300 µl drop of HTF covered with mineral oil for 2 h prior to IVF. Following this pre-incubation time, the upper 150 µl of the drop were collected, and sperm concentration was analysed.

For the sperm penetration assay, zona-free mouse eggs were pre-incubated in HTF with Hoechst 33342 1 µg/ml for 10 min and washed before sperm addition. After 30 min of gametes co-incubation (at 1 million sperm/ml), the eggs were fixed in a 0.25 % glutaraldehyde solution in PBS.

For the sperm binding assay, eggs were also incubated for 30 min with sperm at a concentration of 1 million sperm/ml and stained with Hoechst after fixing; sperm from 3 KO and 3 WT males were tested on 12 eggs/male.