Sperm functional parameters assays

Human normozoospermic A- and B-SPZ collected from CTRL (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) were used for in vitro AR induction and capacitation state assessment as previously reported ³⁵. In brief, for AR induction SPZ treated with CYTO-D were incubated with 10 μM of Ca²⁺ ionophore A23187 (C7522; Sigma-Aldrich, Milano, Italy) for 1 h at RT. After incubation, SPZ were dried on slides and stored for following PNA and IZUMO1 immunofluorescence analyses.

For capacitation assessment, human normozoospermic A- and B-SPZ fractions from CTRL (pre-A; pre-B) and following in vitro CYTO-D treatment (post-A; post-B) were stained with a chlortetracycline (CTC) solution contained 750 μmol of CTC I-1 (26430; Sigma-Aldrich, Milano, Italy) in a buffer containing 130 mmol NaCl, 5 mmol cysteine and 20 mmol Tris-HCl. After incubation, sperm cells were fixed in PFA 4%, dried on slides and analyzed under an optical microscope (Leica DM 5000 B + CTR 5000).