How many blastomeres of the 4-cell embryo contribute cells to the mouse body?

The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body.

Mouse singletons and twins developed from isolated diploid blastomeres supported with tetraploid blastomeres.

The aim of this study was to obtain mice, hopefully identical multiplets, from single diploid blastomeres isolated at the 4-cell stage, or from pairs of sister blastomeres isolated at the 8-cell stage. To this end isolated blastomeres were aggregated with one or two tetraploid carrier embryos produced by electrofusion of 2-cell embryos. Diploid embryos were albino and homozygous for the "a" allele of glucose-phosphate isomerase (GPI-1a1a) and tetraploid embryos were pigmented and GPI-1b1b. The aggregates were cultured in vitro up to the blastocyst stage. Each quartet (occasionally triplet or doublet) of chimaeric blastocysts was transplanted to the oviduct of a separate pseudopregnant recipient. Altogether 62 blastocysts were transplanted to 17 recipients. Eight full-term foetuses (two singletons and three pairs of twins) were rescued by Caesarian section on day 19, 20 or 21 of pregnancy. Three young (one singleton and twins) were successfully reared by foster mothers and proved to be normal and fertile females. All foetuses and animals were albino. In five individuals only the 1-A form of GPI (characteristic for 2n blastomere) was found. In one adult female traces of the 1-B form of GPI (characteristic for 4n carrier blastomeres) were detected in the heart and the lungs while 4 other organs contained only the 1-A form. These observations strongly suggest that the majority of foetuses/animals produced according to our experimental system are 'pure' diploids rather than 2n/4n chimaeras, and that the described method can be used in future to produce twins, triplets and quadruplets in the mouse. Our study confirms earlier work by Kelly (1975, 1977) that 'quarter' blastomeres of the mouse are still totipotent.

Postimplantation development of mouse blastocysts with two separate inner cell masses.

Blastocysts with double inner cell masses (ICMs) were produced by electrofusion of two blastocysts and transplanted to pseudopregnant recipients. The implanted embryos were either examined histologically (8th day of pregnancy) or dissected and inspected in toto (11th or 12th day). In most cases both ICMs of experimental blastocysts developed into separate egg cylinders. Both cylinders were located in the common yolk sac cavity. Some cylinders were quite normal, but most of them were small and deprived of embryonic membranes. Ectoplacental cones of these cylinders were often oriented laterally or even antimesometrially. The development of cylinders seems to depend upon the position of their ectoplacental cones--cylinders with cones situated antimesometrially were handicapped in development. Among four sets of twin embryos examined on the 11th or 12th day, in one set each of the twins were equally developed, and in the three others one of the twins was more advanced.

Hermaphroditism in young mouse chimeras.

Among 23 5-day old mouse chimeras, 11 were sex chimeras. Four of them were hermaphrodites, six were males and one was female. In the gonads of three hermaphrodites fragments of ovarian tissue were very small. Growing oocytes were observed not only in ovotestes but also in testes. The course of transformation of ovotestes into testes is discussed.

Precocious initiation of meiosis by male germ cells of the mouse.

12 1/2-15 1/2 day embryonic mouse testes of 129/terSv and CBA/T6T6 strains were transplanted under the kidney capsule of adult hosts. After 3-5 days in 41% of CBA/T6T6 transplants and in 82% of 129/terSv transplants a limit number of germ cells began meiosis. The percentage of meiotic germ cells was inversely related to the total number of gonocytes and the organization of seminiferous cords. The presented evidence indicates that the ability of the germ cells to begin meiosis precociously depends on: 1) genotype of donor embryos; 2) age of transplanted testis, and 3) using whole of half of gonad for transplantation. After 10-15 days in two out of 46 129/terSv testes (4%) growing oocytes were observed.

Factors regulating the degree and extent of experimental chimaerism in the mouse.

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 X 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 inthe embryonic ectoderm of the chimaeric egg cyclinder, 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.

Implantation and early postimplantation development of the bank vole Clethrionomys glareolus, Schreber.

The development of the bank vole Clethrionomys glareolus is described from implantation to the formation of the foetal membranes. The embryonic development of this species combines features of primitive rodent species, for example Geomys bursarius and highly specialized ones, for examples Mus musculus. The egg-cylinder is formed by invagination into the blastocoelic cavity of the inner cell mass and polar trophoblast overlying it; this resembles in many respects the early stages of development of primitive species. The fully formed egg-cylinder, however, resembles that of the mouse and the formation of foetal membranes is also similar to that in Muridae. It is concluded that in the bank vole and also in other rodents, the extra-embryonic ectoderm of the egg-cylinder is derived from the polar trophoblast rather than from the inner cell mass.

Influence of embryonic and adult testis on the differentiation of embryonic ovary in the mouse.

The development of 11 1/2-13 1/2-day embryonic mouse ovaries subjected to the influence of adult and embryonic testes was investigated. The environment of adult testis caused severe restriction of ovarian growth, but did not produce any effects which might be considered as masculinization. The presence of embryonic testis was distinctly unfavourable to the embryonic ovary, resulting in restriction of the growth of the latter and degeneration of oocytes. Reversal of the course of differentiation of genetically female germ cells has never been observed.