Human sperm cytosolic factor triggers Ca2+ oscillations and overcomes activation failure of mammalian oocytes

Among the possible mechanisms of oocyte activation after sperm penetration,it appears most likely that a protein released by the spermatozoon elicitsa calcium elevation in the ooplasm. To further test this idea, cytosolicfactors obtained from human spermatozoa by two different methods,freezing-thawing and sonication, were injected into mouse oocytes followingwhich intracellular calcium release was measured. Of a total of 42 mouseoocytes, a pattern of calcium oscillations was observed in nine out of 16oocytes injected with sonicated fraction, in all of eight oocytes with thefrozen-thawed fraction and in none of 18 control oocytes. Injection of thefrozen- thawed fraction also produced regular calcium oscillations in allof five in-vitro matured human oocytes. To assess the putative factor'sability to support fertilization, human oocytes that were not activated byprior intracytoplasmic injection of spermatozoa (ICSI) and round spermatidswere reinjected with the frozen-thawed sperm fraction. Of 23 human oocyteswhich remained unfertilized after ICSI, 19 became activated after injectionwith sperm cytosolic factor; eight showed two pronuclei, three onepronucleus and eight showed three or more pronuclei. Of 11 oocytesunfertilized after prior round spermatid injection, two developed twopronuclei, four developed one pronucleus and two had three or morepronuclei. Cytogenetic analysis by fluorescence in-situ hybridizationconfirmed the existence of a male pronucleus in eight out of nine suchzygotes displaying two or more pronuclei. Thus, human sperm extractsactivated mouse and human oocytes after injection, as judged by calciumflux patterns in conjunction with male pronucleus formation.