Discrimination of maternally or paternally derived pronuclei by epigenetic divergence in human zygotes

Abstract

Oocyte fertilization is generally judged by the number of pronuclei (PN) after insemination. However, whether the PN are maternally or paternally derived remains uncertain. This study focused on the epigenetic divergence of male and female PN, with respect to differences in DNA methylation status, to establish a method for PN discrimination using immunostaining in abnormally fertilized human oocytes. Research study. Donated frozen normally (n=3) and abnormally (1PN: n=2, 3PN: n=5) fertilized oocytes were used. After thawing, oocytes were fixed in 4% paraformaldehyde in PBS for 1h, washed with 0.05% Tween 20 in PBS (PBST), and then permeabilized with 0.1% Triton-X 100 for 30min. Oocytes were denatured with 2 M HCl for 30min and neutralized with 100 mM Tris-HCl (pH 8.5) for 15min, and then blocked overnight at 4° in 5% BSA in PBS. After blocking, oocytes were incubated for 1h with the primary antibodies, anti-H3K9me2 or anti-5mC to detect fPN and anti-5hmC to detect mPN. After washing in PBST, oocytes were incubated with secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG for 1h. Images were obtained on an FV1000 confocal microscope. We discerned male PN (mPN) and female PN (fPN) in all 2PN oocytes by using antibody. The average size of fPN was 24.6±2.1μm, while for mPN it was 29.8±0.6μm. In 3PN oocytes, four oocytes showed one fPN and two mPNs, but one showed three mPNs and small two female signals (≤4.5μm). The average size of fPN was 21.3±0.9μm, while mPN measured 22.1±1.0μm. Two of the 1PN oocytes showed one mPN and a small maternal-specific signal (≤5.0μm). This study established a novel method to discern PN origin in human zygotes using immunochemical analysis. Our data showed that mPN were slightly larger than fPN. The 1PN and 3PN results suggested the possibility of PN formation failure and extrusion failure of the second polar body that might lead to 1PN and/or ≥3PN formation.