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 into Syrian golden hamster oocytes is difficult and the hitherto reported r esults are unsatisfactory. This may be related to the injection and cu lture technique or to the high susceptibility of the hamster oocytes t o undergo parthenogenetic activation or both. Therefore, we investigat ed the hamster oocyte-human sperm microinjection model using the follo wing two approaches: (i) application of contemporary techniques for in jection (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 with human spermatozoa, Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronuc leus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic m etaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had develop ed only the hamster female PN while the sperm nuclei were either intac t or swollen (partially decondensed), indicating that failure of oocyt e activation was not the likely reason for the failure of male PN form ation in these oocytes, In the next series of experiments, sibling ooc ytes were alternately injected with spermatozoa suspended either in th e regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n = 278), A significant improvement was noted in the mean percentag es of oocytes,vith 2PN, 2PB, metaphase entry and sperm chromosome spre ads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 vers us 36.6 %, metaphase entry: 36.3 versus 26.9 % and sperm chromosome sp reads: 28 versus 20.4%; all P < 0.04), Thus, parthenogenetic activatio n appears to be one of the contributing factors for the failure of mal e PN formation after heterospecific hamster ICSI, From these experimen ts it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjec tion for karyotyping of human spermatozoa with poor fertilizing capaci ty.