Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes

Obtaining karyotypes from human spermatozoa after microinjection intoSyrian golden hamster oocytes is difficult and the hitherto reportedresults are unsatisfactory. This may be related to the injection andculture technique or to the high susceptibility of the hamster oocytes toundergo parthenogenetic activation or both. Therefore, we investigated thehamster oocyte-human sperm microinjection model using the following twoapproaches: (i) application of contemporary techniques for injection(touching the sperm tail) and culture (hamster embryo culture medium,HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus,in the first series of experiments, 252 hamster oocytes were injected withhuman spermatozoa. Among the 219 (87%) oocytes that survived the injectionprocedure, the mean percentages of male pronucleus formation [two pronuclei(2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosomespreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocyteswhich failed to develop the male pronucleus following injection revealedthat most of them had developed only the hamster female PN while the spermnuclei were either intact or swollen (partially decondensed), indicatingthat failure of oocyte activation was not the likely reason for the failureof male PN formation in these oocytes. In the next series of experiments,sibling oocytes were alternately injected with spermatozoa suspended eitherin the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set2, n=278). A significant improvement was noted in the mean percentages ofoocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in theCa2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%,metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be oneof the contributing factors for the failure of male PN formation afterheterospecific hamster ICSI. From these experiments it can be concludedthat application of the advanced injection and culture techniques andomission of Ca2+ from the injection medium are promising for the routineapplication of the hamster oocyte microinjection for karyotyping of humanspermatozoa with poor fertilizing capacity.