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BACKGROUND: The channel-forming antimicrobial peptide, magainin-2-amide, interacts preferentially with negatively charged, non cholesterol-containing membranes, including those of sperm, oocytes and cells of pre-implantation embryos. Cyclodextrin and albumin remove membrane cholesterol and together with hydrogen peroxide (H2O2) are potential enhancers of embryotoxicity. METHODS: Two-cell murine embryos were cultured in vitro with magainin-2-amide at a high effective concentration (250 μg/ml) and at subthreshold concentrations (166 and 200 μg/ml). Embryos treated with sub-threshold concentrations of magainin were additionally treated with cyclodextrin, bovine serum albumin or H2O2 or were cultured under acidified conditions. Cell viability was verified with propidium iodide and fluorescein diacetate. RESULTS: The embryotoxic effect of magainin and H2O2 was dose- and time-dependent. Cyclodextrin, H2O2, acidification of the medium, and to a lesser extent albumin, enhanced the embryotoxicity of magainin at sub-threshold concentrations. CONCLUSION: Magainin on its own is highly embryotoxic. Its embryotoxicity is enhanced by cyclodextrin, albumin, H2O2 and acidification. Thus, magainin which has antibacterial, antifungal and antiprotozoal activity may also have a potential role as a contraceptive agent. The harmful effects of various concentrations of the exogenous H2O2 on 2-cell stage mouse embryos are reported here, to the best of our knowledge, for the first time.

Phorbol ester (PMA) in concentration 5 and 10 ng ml-1 blocks cytokinesis of the second maturation division in mouse oocytes. Karyokinesis is not impaired and digynic triploid oocytes are obtained which undergo first cleavage division. Effectiveness of blocking cytokinesis is dependent on the timing of exposure of oocytes to PMA action. When oocytes are subjected to PMA at the onset of the second maturation division only 14·5 % of eggs are triploid. PMA present during fertilization in vitro (about 1 h exposure to PMA) induces triploidy in 40 % eggs. Extending the time of exposure of oocytes to 2 h produces 76 % tripronucleate eggs. Applicability of PMA is compared with the use of cytochalasin B to induce triploidy in the mouse.

By subjecting A strain eggs at the time of fertilization and polar body extrusion to 5 μg/ml cytochalasin B, digynic triploidy was produced in 80 % fertilized eggs. Triplonucleate eggs were transplanted to recipients and examined between 9–11th day of pregnancy. Development of triploid mouse embryos up to day 7 is normal and most embryos form early egg cylinder. At day 8 the embryonic part of the cylinders is under-developed and later development fails to form an embryo. Development of foetal membranes is much less affected, CB-induced triploids survive to 10th-day of pregnancy.

Summary.The hardening of the zona pellucida as a result of the cortical reaction was estimated in triploid oocytes produced with cytochalasin B by two different procedures. Triploid oocytes resulting from fertilization in the presence of cytochalasin B did not reveal hardening of the zona pellucida unlike triploids obtained by inhibiting in-vitro extrusion of the second polar body in oocytes fertilized in vivo.

Summary.Mouse eggs and spermatozoa were treated in various ways with 5 or 10 μg cytochalasin B/ml. The fertilization rate in vitrowas reduced by treatment with the drug but 80–90% of the eggs fertilized were triploid. Many of the experimental eggs were penetrated by one or more spermatozoa but remained unfertilized (75% compared with 9% in control eggs). It is suggested that cytochalasin B weakens the zona reaction and interferes with fusion of gametes but does not prevent the block to polyspermy.

Mouse eggs fertilized in vivo were treated with cytochalasin B in vitro (5 µg/ml of culture medium) at the moment of extrusion of the second polar body (2·5, 3·0, 3·5 h after copulation). Cytochalasin B inhibits cytokinesis of the second maturation division, so that triploid digynic eggs are formed in over 50% of treated eggs. Triploid eggs were transplanted to the oviducts of recipients. On the 4th and 5th day of development 41·7% of transplanted eggs were recovered. All embryos recovered on the 4th day were morulae, while on the 5th day blastocysts predominated. Recovered embryos were studied for cell number and ploidy. Twenty-three of 27 embryos with analysable metaphase plates were triploid and four were diploid (the latter were found in females into which both triploid and control diploid eggs were transplanted). Sex chromosome constitution was determined in seven cases: four triploids were XX Y and three were XXX. Preliminary observations showed that triploid embryos obtained by the use of cytochalasin B can implant and survive at least to the 9th day.

The degree of chimaerism of the coat, bone marrow and cornea in mouse chimaeras obtained by aggregation of an albino inbred embryo of A strain with an A × CBA-T6T6 hybrid was investigated. Out of 21 individuals born, 18 were chimaeras. Statistically significant correlation has been demonstrated between chimaerism of the coat, bone marrow and the cornea. The contribution of the two components appears to be affected by two factors: first, variable participation of the two components in the embryonic ectoderm of the chimaeric egg cylinder, and second, the rate of proliferation of the two cell lines. A photometric method is described which permits the quantitative evaluation of coat chimaerism. The method is sensitive and reliable.