Effect of monoclonal antibody to bull sperm sp18 on murine fertilization and embryos development

Abstract

Western blot analysis revealed that one IgG1 monoclonal antibody (mAb) to sp18 family membrane proteins (Mr. 14, 16 and 18 ku) of bovine sperm reacted faintly with protein bands of 14, 18, 22, 30 and 60 ku (reducing) in samples of mouse sperm. The mAb also reacted to protein of egg lysozyme. Using a laser confocal microscope, indirect immunofluorescence (IIF) showed that the sp18 antigens were present in the posterior head of murine sperm. In murinein vitro fertilization (IVF) and embryo development trails, a total of 426 oocytes from C57,BL/6 and F1 hybrid strain (CD1 × C57BL/6 cross) of 12 female mice were used in 3 independent trails. After preincubating capacitated sperm with 182 μ/mL of sp18 mAb in the modified TYH IVF medium for 15–20 min, cumulus-oocyte-complexes were introduced. The fertilization rate in sp18 rnAb groups was 77.1 %, which was not significantly (P > 0.05) different from the nonspecific mouse IgG (79.2 %) and non-lgG (80.3 %) control groups. Fertilized oocytes had been continuously cultured in modifed CZB medium. 100%, 100% and 97.9% of 1cell embryos developed to 2-cell stage in sp18 mAb, nonspecific mouse IgG and non-lgG group 30 h after the start of fertilization, respectively. In the nonspecific mouse IgG and nonlgG groups, 64.1% and 64.3% of embryos developed to the 4-cell stage, respectively, but all developing eggs in sp18 mAb groups arrested developmentin vitro at 2-cell stage. After zonae of 2-cell blocked embryos were enzymatically removed with 0.5% pronase, detection of sp18 antigens by IIF indicated that the fluorescence scattered on two embryonic cells. For embryos fertilizedin vivo and co-cultured with 182 μg/mL sp18 mAb, the numbers of 1-cell embryos reaching the 2-cell and 4-cell stage were 95.2% and 70.5%, which were not significantly (P> 0.05) different from the control group (92.9% and 77.9%). These results indicate that the sp18 antigens on posterior head of mouse sperm were incorporated into the egg plasma membrane during fertilization, and played an active role in development of murine preimplantation embryo.