Immunofluorescence (IF) and F-actin staining

Briefly, 30 oocytes from each group were fixed in 4% paraformaldehyde for 15 to 20 min at room temperature. Oocytes were then transferred to a membrane permeabilization solution (1% Triton X-100 in PBS) for 8 to 12 h at 4 °C. After 1 h in blocking buffer (1% bovine serum albumin in PBS), oocytes were subsequently incubated overnight at 4 °C with rabbit anti-connexin 45 antibody (marker protein of GJs, BS3470, Bioworld, 1:100), mouse anti-β-actin antibody (AP0060, Bioworld, 1:200), mouse anti-γ-actin antibody (ab123034, Abcam, 1:200) or rabbit anti-tubulin α (ab52866, Abcam, 1:250) antibody. After three washes (5 min each) in wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were incubated with Alexa Fluor 488 (Molecular Probes, A11008, 1:500) or Alexa Fluor Plus 555 (Molecular Probes, A32727, 1:1000) secondary antibody for 1 h at 38.5 °C under 5% CO₂. For nuclear and F-actin staining, oocytes were sequentially washed for three times (5 min each) after the blocking or secondary antibody incubation operations, and transferred into the staining solution containing the fluorescent dye Hoechst 33342 (Invitrogen, USA, Cat# H3570) or TRITC Phalloidin (Yeasenbio Co., Ltd., China, Cat# 40734ES75) at a final concentration of 10 μmol/L or 200 nmol/L, respectively, and then incubated for another 15 to 30 min at 38.5 °C under 5% CO₂. Finally, after three washes in wash buffer, oocytes were mounted on glass slides, and examined with a confocal laser-scanning microscope (Zeiss LSM 700 META, Germany). TZPs numbers and the colocalization of TZPs and monomeric actins in the zona pellucida areas were calculated manually. The fluorescence intensity of F-actin co-localized with connexin 45 in oolemma areas were quantified by ImageJ software.