HUMAN SPERM CYTOSOLIC FACTOR TRIGGERS CA2+ OSCILLATIONS AND OVERCOMESACTIVATION FAILURE OF MAMMALIAN OOCYTES

Among the possible mechanisms of oocyte activation after sperm penetra tion, it appears most likely that a protein released by the spermatozo on elicits a calcium elevation in the ooplasm. To further test this id ea, cytosolic factors obtained from human spermatozoa by two different methods, freezing-thawing and sonication, were injected into mouse oo cytes following which intracellular calcium release was measured. Of a total of 42 mouse oocytes, a pattern of calcium oscillations was obse rved in nine out of 16 oocytes injected with sonicated fraction, in al l of eight oocytes with the frozen-thawed fraction and in none of 18 c ontrol oocytes. Injection of the frozen-thawed fraction also produced regular calcium oscillations in all of five in-vitro matured human ooc ytes. To assess the putative factor's ability to support fertilization , human oocytes that were not activated by prior intracytoplasmic inje ction of spermatozoa (ICSI) and round spermatids were reinjected with the frozen-thawed sperm fraction. Of 23 human oocytes which remained u nfertilized after ICSI, 19 became activated after injection with sperm cytosolic factor; eight showed two pronuclei, three one pronucleus an d eight showed three or more pronuclei. Of 11 oocytes unfertilized aft er prior round spermatid injection, two developed two pronuclei, four developed one pronucleus and two had three or more pronuclei. Cytogene tic analysis by fluorescence in-situ hybridization confirmed the exist ence of a male pronucleus in eight out of nine such zygotes displaying two or more pronuclei. Thus, human sperm extracts activated mouse and human oocytes after injection, as judged by calcium flux patterns in conjunction with male pronucleus formation.