Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: An analysis of individual oocytes

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.