Detection of Bovine Early Pregnancy Factor in the Culture Medium of Sperm-Injected or Plasmamembrane-Perforated Oocytes.

Abstract

This study was undertaken to determine the mechanism of early pregnancy factor (EPF) secretion from ova. EPF released into the culture medium from in vitro matured bovine oocytes that were sperm-injected and plasmamembrane-perforated was detected. Bovine follicular oocytes were matured in vitro for 19 to 22 h in 5% CO2 in air at 39 C. A capacitated spermatozoa was injected into each of the three parts of the matured oocyte, namely, the zona pellucida, the perivitelline space and the ooplasm. The plasmamembrane of matured oocytes were also perforated with a glass micropipette into the ooplasm. After sperm-injection or perforation, these ova were cultured separately in TCM199 containing 2% fetal calf serum (FCS) for 24 h in 5% CO2 in air at 39 C. Then EPF activity in these culture media were assayed by rosette inhibition test. A rosette inhibition titer (RIT) above 4 was considered as a positive expression of EPF activity. Of the medium samples from the sperm-injected oocytes, 4 out of 5 of those injected in the perivitelline space and 4 out of 5 of those injected in the ooplasm showed positive EPF activity. Further, all 7 medium samples of oocytes perforated in the plasmamembrane with a glass micropipette also showed positive EPF activity. However, all 4 medium samples containing ova injected with sperm into the zona pellucida showed negative EPF activity. Consideration of these data led to a remarkable discription of EPF release from oocytes micromanipulated with sperm and inorganic matter, and provided the evidence to show that EPF release is triggered by sperm penetrating through the zona pellucida.