120 EMBRYONIC AND POSTNATAL DEVELOPMENT OF DIPLOID-TRIPLOID MOUSE CHIMAERAS

Abstract

A chimaera is an organism composed of cells derived from two (or more) zygotes. Spontaneously originated diploid-triploid (2n-3n) chimaeric embryos and adults have been described in many species of mammals. In man, between 1960 and 2002 over 30 cases of chimaerism were discovered (van de Laar I et al. 2002 Clin. Genet. 62(5), 376–382). A deeper insight into the developmental consequences of this rare and odd phenomenon requires experimental production of 2n-3n embryos and animals. The present study is the first and successful attempt to produce diploid-triploid chimaeric embryos, fetuses, and postnatal animals in the mouse. Diploid embryos originated from BAMIZ females crossed with BAMIZ males. The zygotes that were the source of triploid embryos were obtained from females F1 (C57Bl/6 × CBA/H) crossed with F1 males as a result of “delayed mating.” The triploidy was induced by suppression of the extrusion of the second polar body with cytochalasin D (1 μg mL⁻¹, 5 h). Diploid-triploid chimaeric embryos were created by aggregation of diploid embryos with triploid embryos at 4–8 cell stage. In chimaeras created according to this procedure, the triploid component was agouti and produced the 1B1B isoform of glucose phosphate isomerase (GPI) and the diploid component was albino and produced the GPI-1A1A isoform. Electrophoresis of GPI was performed in order to determine the contribution of both populations of cells in tissues of embryos and individuals. Over a thousand oocytes were subjected to triploidization. A total number of 201 diploid-triploid aggregates developed into blastocysts and were transplanted to the oviducts of 30 recipients. Our experiment yielded 23 living and 6 dead embryos (age: 8th–19th day) out of which 22 proved to be chimaeric and 3 were adults. Two of these animals were albino but had the triploid component in several internal tissues; both were fertile. The third animal, a male, was an overt chimaera. It turned out to be infertile (no sperm in the ejaculate; testes small and deprived of germ cells). The infertility of this individual is puzzling because the FISH studies with the help of X and Y chromosome painting probes proved that the diploid component was XY and the triploid component was XXX. The results of our study indicate that the rate of postimplantation development of 2n-3n chimaeric embryos is normal or only slightly retarded. Developmental stage of chimaeric embryos was assessed by comparison of their external morphology with normal diploid embryos of equivalent post-coital age according to the descriptions given by Theiler K (1972 The House Mouse, Springer-Verlag, Berlin). With the exception of one embryo lacking both eyes (but otherwise looking quite normal) no other morphological abnormalities were observed. Comparison of the contribution of both components to the fetal and extra-embryonic tissues at the consecutive foetal stages has shown that participation of triploid cells slightly but steadily decreased in all tissues examined. However, the presence of triploid cells in mouse chimaeras was compatible with their normal postnatal development to adulthood.