The Integrity Of The Inner Mitochondrial Membrane In Human Cumulus Cells In Relation To Oocyte Maturation And In Vitro Development

Objective: According to determine the mitochondrial membrane potential ( ΔΨ m ) in human cumulus cells removed from individual oocytes to assessment the inner mitochondrial membrane integrity and the relationship between the inner mitochondrial membrane integrity and the maturity of corresponding oocyte and in vitro embryo development.Methods: Cumulus cells were obtained from 23 patients undergoing intracytoplasmic sperm injections (ICSI) and undergoing oocyte collection. After egg collection, individual eggs were treated with hyaluronidase to remove the surrounding cumulus cells from the oocyte and prepare them for ICSI. The grade of each individual oocyte was noted. ICSI was performed on MII oocytes and the oocytes were incubated at 37℃, 5%CO2 in air. Cumulus cells from each oocyte were collected, washed in phosphate-buffered saline and counted the number of cumulus cells. Mitochondria can be isolated easily from cumulus cells by a simple method of homogenization followed by low and high speed centrifugation. The integrity of the inner mitochondrial membrane may be measured by observing the potential gradient over this membrane. This can be achieved by measuring the uptake of the cationiccarbocyanine dye JC-1 into the matrix. The mitochondrial membrane potential, across the inner membrane, determines the redistribution of this dye. Each of mitochondrial samples was added 2 ul of JC-1. The fluorescence of the sample was read in a spectrofluorometer. Calculate the fluorescence produced the same cell counts. According to the oocyte stage of maturity, fertilization and the development of embryo, divided the oocyte into five groups. Statistical analysis was carried out using SAS package (SAS 8.0) to determine the significance of any differences between the groups.Results: (1) The cumulus cells' fluorescence of the GV and MI oocytes was lower than MII oocytes. There was significant difference in the inner mitochondrial membrane integrity of cumulus cells between GV, MI or MII groups. (2) The cumulus cells' fluorescence of non-fertilized MII oocytes was lower than fertilized MII oocytes. There was significant difference of cumulus cells in the inner mitochondrial membrane integrity between non-fertilized MII oocytes and fertilized MII oocytes. (3) The cumulus cells' fluorescence of dead, arrested and with many fragments' embryos was lower than other embryos. There was significant difference in the inner mitochondrial membrane integrity of cumulus cells between dead, arrested, with many fragments' embryos and other embryos.Conclusions: The integrity of the inner mitochondrial in human cumulus cells can affect the oocyte maturation and in vitro development of corresponding oocyte after intracytoplasmic sperm injection.