Inner cell masses (ICMs) dissected from -day rat blastocysts were aggregated with -day mouse morulae. Successful aggregates formed blastocysts in vitro and morphologically normal -day conceptuses in the mouse uterus. Immunofluorescent analysis of these conceptuses revealed that rat cells were only present in the embryonic ectoderm and endoderm and never in the trophectoderm derivatives, although rat trophoblast did develop in the mouse uterus in various control experiments. The single-cell resolution of this technique extends the results obtained from aggregating mouse ICMs with mouse morulae and provides strong evidence that ICM cells, although not overtly differentiated, are determined by the blastocyst stage.

The inner cell mass (ICM) isolated from the mouse blastocyst, although not an overtly differentiated tissue (Gardner, 1972) is apparently determined by days post coitum, as judged by its inability to contribute to post-implantation trophoblast after aggregation with morulae or after transfer to the oviduct and later injection into blastocysts (Rossant, 1975 a, b). In these experiments, electrophoretic variants of glucose phosphate isomerase (GPI) were used as genetic markers. However, the low sensitivity of this technique means that a minor contribution of ICM cells to the proliferating trophoblast or a contribution to the non-dividing mural trophoblast giant cells cannot be excluded (Gardner, Papaioannou & Barton, 1973). A marker that can detect individual cells is required to eliminate this possibility. No such markers are yet available for the mouse (Gardner & Johnson, 1975), but immunofluorescent analysis of rat/ mouse chimaeras permits identification of single rat or mouse cells (Gardner & Johnson, 1973, 1975). Thus, an attempt was made to extend the results of experiments using mouse ICM/mouse morula aggregates by aggregating rat ICMs with mouse morulae. Immunofluorescent analysis can then be used to discover whether any rat ICM cells contribute to the trophoblast of resulting conceptuses. Analysis was performed at days of pregnancy in the mouse when all three presumptive derivatives of the trophectoderm -ectoplacental cone, primary giant cells and extra-embryonic ectoderm -should be present (Gardner & Johnson, 1975; Gardner & Papaioannou, 1975).

Recovery of embryos from donor females

Mouse morulae (8–16 cell) were flushed from the oviducts of CFLP females (Anglia Laboratory Animals Ltd) on the afternoon of the 3rd day after mating. Rat morulae at similar cleavage stages were flushed from the uterotubal junctions of CFHB random-bred females (Anglia Laboratory Animals Ltd) on the afternoon of the 4th day after mating, while rat blastocysts were obtained from the uterus on the afternoon of the 5th day. It is assumed throughout that rat early development is 24 h behind that of the mouse.

PB1 medium +10 % foetal calf serum (FCS) was used for recovery, storage, microsurgery and transfer of embryos (Whittingham & Wales, 1969) and M16 medium ± 10 % FCS was used for culture (Whittingham, 1971).

Aggregation of culture and embryos

1. Rat ICM/mouse morula aggregates

ICMs were dissected from -day rat blastocysts and aggregated with mouse morulae as described previously for mouse ICM/mouse morula aggregates (Rossant, 1975a). All aggregates were cultured in Ml6. A few pairs were cultured to the late blastocyst stage to observe and photograph the process of aggregation. The remainder were cultured for either 2–4 h or 24 h (late morula/early blastocyst) before transfer to pseudopregnant mice.

2. Rat blastocysts

Some -day rat blastocysts were not dissected but were stored briefly in PB1 +10 % FCS before transfer to mice.

3. Rat morulae

Rat morulae ( days) were cultured for 24 h in M16 or M16 + 10 % FCS before transfer to recipient rats or mice. In most cases the zonae were removed by Pronase (Mintz, 1962) before culture.

4. Rat morula/mouse morula aggregates

The zonae were removed from both rat and mouse morulae by Pronase and rat/mouse pairs were brought into contact in drops of M16 or M16 +10 % FCS. The cultures were then transferred to a 37 °C incubator. Contact between the morulae was checked after 1 h. After 24 h in culture, aggregated embryos were transferred to pseudopregnant mice.

Transfer to recipient animals

All four types of embryos listed above were transferred to recipient mice on the 3rd day of pseudopregnancy. Rat recipients were used for uterine transfer on the 4th day of pregnancy after the oviducts had been ligated on day 2. Rat embryos cultured for 24 h were transferred unilaterally so that the contralateral horn provided a control for effective ligation of the oviduct.

Analysis of post-implantation development

Histology

Recipient mice which had received non-cultured or cultured rat embryos, and one recipient which contained rat morula/mouse morula aggregates, were killed on the 6th day after mating (-day embryos). Uteri containing implants were fixed in AFA, processed and embedded in wax (Orsini, 1962). Serial sections were cut at 6–7 μm, stained with haemalum and eosin and examined for embryonic derivatives.

Recipient rats which had received cultured rat embryos were killed on the 7th day after mating and uteri containing implants were processed as above.

Immunofluorescent analysis

Mice which had received rat ICM/mouse morula aggregates and rat morula/ mouse morula aggregates were killed on the sixth day of pregnancy. Decidua were processed and embedded in wax (Sainte-Marie, 1962) and sectioned at 6 μm. The sections were treated with antisera specific for mouse and rat antigens as described previously (Gardner & Johnson, 1973) and examined by epifluorescent illumination in a Zeiss fluorescence microscope (Primary filter, barrier filter HBO 200). Camera lucida drawings were made of all sections, marking rat and mouse cells.

In vitro observations

1. Rat ICM/mouse morula aggregates

Successful aggregation of rat ICMs with mouse morulae was achieved in vitro in Ml6 (Table 1). The rate of aggregation was lower than for mouse ICM/ morula combinations (Rossant, 1975a) but a large proportion of successful aggregates formed blastocysts after 24 h in culture (Table 1).

Table 1.

Rates of va vitro aggregation and blastocyst formation of rat ICM/mouse morula and rat morula/mouse morula combinations

Rates of va vitro aggregation and blastocyst formation of rat ICM/mouse morula and rat morula/mouse morula combinations
Rates of va vitro aggregation and blastocyst formation of rat ICM/mouse morula and rat morula/mouse morula combinations

2. Rat morulae

Rat morulae were cultured to the blastocyst stage in both M16 and M16 + 10 % FCS. However, success was rather variable and only 32/66 and 10/28 morulae respectively underwent further cleavage.

3. Rat morula/mouse morula aggregates

Successful aggregation of rat and mouse morulae was achieved in culture in both M16 and M16 +10 % FCS (Table 1). The rather low rate of aggregation was chiefly due to the fairly frequent failure of rat morulae to develop in culture, as reported above. A large proportion of the successful aggregates formed blastocysts after about 24 h in culture (Table 1), as reported previously by other workers (Mulnard, 1973; Stern, 1973; Zeilmaker, 1973).

Analysis of post-implantation development

1. Rat ICM/mouse morula aggregates

The implantation rate of these aggregates was rather low (Table 2; see Rossant, 1975a). However, seven embryos were obtained and examined by immunofluorescence. Five proved to be interspecific chimaeras; their patterns of chimaerism are summarized in Table 3. Conceptus 90a was obtained from an aggregate transferred after 2– 4 h in culture, whereas the others were cultured for 24 h before transfer. All five were morphologically normal although embryos 90a and 95b were retarded slightly and lacked extra-embryonic ectoderm. None showed contribution of rat cells to the mural trophoblast, ectoplacental cone or extra-embryonic ectoderm.

Table 2.

Implantation rates (only pregnant recipients considered)

Implantation rates (only pregnant recipients considered)
Implantation rates (only pregnant recipients considered)
Table 3.

Estimation by immunofluorescence of the proportion of rat cells in 512-day conceptuses derived from rat ICM/mouse morula aggregates

Estimation by immunofluorescence of the proportion of rat cells in 512-day conceptuses derived from rat ICM/mouse morula aggregates
Estimation by immunofluorescence of the proportion of rat cells in 512-day conceptuses derived from rat ICM/mouse morula aggregates

2. Rat blastocysts

As a control for survival of rat trophoblast in the mouse uterus, non-cultured rat blastocysts were transferred to recipient mice. A high proportion were capable of inducing decidual formation (Table 2). Histological examination of the -day decidua revealed that most contained embryonic cells. Six out of eight contained a definite egg-cylinder but this was never well organized (Fig. 1). Ectoderm and endoderm were generally delineated but no division into embryonic and extra-embryonic ectoderm was apparent and the orientation of the embryos was often abnormal. All conceptuses contained trophoblast cells and in six out of eight definite mural giant cells could be identified (Fig. 1). Pycnotic cells were present to a greater or lesser extent in all conceptuses.

Fig. 1.

Section of 512 -day conceptus developed from non-cultured rat blastocyst transferred to the mouse uterus. Arrows indicate definite trophoblast giant cells.

Fig. 1.

Section of 512 -day conceptus developed from non-cultured rat blastocyst transferred to the mouse uterus. Arrows indicate definite trophoblast giant cells.

3. Rat morulae

To control for the ability of rat trophoblast to survive in the mouse after development in culture, blastocysts developed from rat morulae cultured for 24 h in M16 + 10%FCS were transferred to pseudopregnant mice. Decidual formation was induced (Table 2) and 50 % of the embryos transferred produced embryonic structures. Four such structures consisted solely of groups of trophoblast cells with some giant cell formation (Fig. 2). The other four consisted of well-expanded trophoblastic vesicles, containing only a few dispersed cells on their inner surface (Fig. 3). No organized ICM was present and the inner cells were presumably endoderm. The beginning of giant cell formation was also apparent in these structures.

Fig. 2.

Group of trophoblast cells produced 512 days after transfer of cultured rat blastocyst to the mouse uterus. Arrows indicate definite trophoblast giant cells.

Fig. 2.

Group of trophoblast cells produced 512 days after transfer of cultured rat blastocyst to the mouse uterus. Arrows indicate definite trophoblast giant cells.

Fig. 3.

Section of 512-day trophoblast vesicle containing only a few endoderm cells after transfer of cultured rat blastocyst to the mouse uterus.

Fig. 3.

Section of 512-day trophoblast vesicle containing only a few endoderm cells after transfer of cultured rat blastocyst to the mouse uterus.

When rat blastocysts cultured in M16 alone were transferred to rats, the implantation rate was low (Table 2). However, considering the fairly high implantation rate in transfers to mice, this may be largely due to lack of practice in performing rat uterine transfers. Of the eight decidua formed, four had normal rat egg-cylinders and two had trophoblast cells only.

4. Rat morula/mouse morula aggregates

To control for the development of rat trophoblast in competition with mouse cells, rat morula/mouse morula aggregates were transferred to mouse uteri. The number of post-implantation embryos formed from these aggregates was rather low (Table 2). The first three embryos were examined histologically and were found to be egg-cylinder-like but not well organized (Fig. 4). Trophoblast, ectoderm and endoderm were present in all three, but the expected division into embryonic and extra-embryonic ectoderm was not found. However, since trophoblast cells were found, a further 11 conceptuses were analysed by immunofluorescence to see if any of the trophoblast cells were rat.

Fig. 4.

Section of 512 -day conceptus derived from rat morula/mouse morula aggregate.

Fig. 4.

Section of 512 -day conceptus derived from rat morula/mouse morula aggregate.

The patterns of chimaerism in these embryos are summarized in Table 4. None was a completely normal -day embryo. Some resembled retarded blastocysts (e.g. R/M 19.3-4) while others were more advanced but more disorganized (e.g. R/M 19.3-3). The orientation of some was abnormal (e.g. R/M 28.3-2). However, distal and proximal endoderm, mural trophoblast, ectoplacental cone and embryonic ectoderm were present in all embryos. There was a definite tendency for the rat cells to colonize the endoderm in preference to other tissues (Table 4) but 7 out of 11 contained rat ectoderm cells. Also, 10 out of 11 contained rat giant trophoblast cells, and 9 out of 11 rat polar trophoblast.

Table 4.

Estimation by immunofluorescence of the proportion of rat cells in 512 -day conceptuses derived from rat morula/mouse morula aggregates

Estimation by immunofluorescence of the proportion of rat cells in 512 -day conceptuses derived from rat morula/mouse morula aggregates
Estimation by immunofluorescence of the proportion of rat cells in 512 -day conceptuses derived from rat morula/mouse morula aggregates

Conceptuses 92a74 and 96b74 and the three conceptuses examined histologically were derived from aggregates cultured in M16 while the rest had been cultured in M16 + 10%FCS. No obvious differences in morphology were apparent between the two groups of embryos.

Aggregation of -day rat ICMs with -day mouse morulae has been achieved in vitro and morphologically normal blastocysts were produced. After transfer to pseudopregnant mice, these blastocysts formed apparently normal day conceptuses, of which a high proportion (5/7) were inter-specific chimaeras. However, immunofluorescent analysis revealed rat cells only in the presumptive ICM derivatives, i.e. embryonic ectoderm, distal and proximal endoderm and never in the trophectoderm derivatives, i.e. ectoplacental cone, extra-embryonic ectoderm and mural trophoblast (Gardner & Johnson, 1975 ; Gardner & Papaio-annou, 1975). Because of the single-cell resolution of the analysis these results completely exclude the possibility of a contribution, however minor, of ICM cells to the trophoblast. Thus they extend the conclusions drawn from GPI analysis of mouse ICM/mouse morula aggregates reported previously (Rossant, 1975a).

However, since interspecific chimaeras were used, the validity of the present results could be queried, unless it can be shown that rat trophoblast can develop in the mouse until at least days of pregnancy. Non-cultured rat blastocysts produced both proliferating trophoblast and giant cells at days in the mouse, although the embryonic regions were rather disorganized (Fig. 1).

Culture of rat morulae to the blastocyst stage before transfer to mice also did not prevent trophoblast formation although no conceptus contained an organized ICM (Figs. 2, 3). Finally, rat trophoblast, both proliferating and giant, could even be formed in competition with mouse cells in rat morula/mouse morula aggregates (Table 4, Fig. 4). Development of rat trophoblast in the mouse under such a variety of conditions means that the absence of rat trophoblast in conceptuses derived from rat ICM/morula aggregates can be considered as a valid result. Thus, the present experiments provide strong evidence that ICM cells, although not overtly differentiated, are determined by the blastocyst stage. This is supported by recent evidence of molecular differentiation between ICM and trophectoderm (Van Blerkom, Barton & Johnson, 1976).

The present experiments also provide some new information on the morphology of rat/mouse chimaeras. Chimaeras derived from rat ICM/mouse morula aggregates are morphologically normal at least in early stages, as are those produced by injection of rat ICMs into mouse blastocysts (Gardner & Johnson, 1973, 1975). However, none of the 14 -day conceptuses derived from rat morula/mouse morula aggregates could be considered normal (Fig. 4). The reasons for this difference are not clear, but at least two possible explanations can be suggested.

Firstly, culture may produce deleterious effects on rat morulae and not on rat ICMs, since consistent success in culturing rat morulae to the blastocyst stage was not achieved. In particular, the predominance of endodermal chimaerism in rat morula/mouse morula aggregates (Table 4) might be related to the effects of culture. Cultured rat blastocysts never produced egg-cylinder structures when transferred to the mouse, but four out of eight produced trophoblast vesicles containing only a few dispersed endoderm cells. However, six out of eight noncultured rat blastocysts did produce egg-cylinders containing ectoderm cells. Thus there seems to be a correlation between culture of rat embryos and lack of ectoderm formation, although Tarkowski has observed similar trophoblast vesicles containing only endoderm cells after transfer of rat eggs to the mouse oviduct (Tarkowski, 1962).

The second factor exerting a deleterious effect on the development of rat morula/mouse morula chimaeras may be the formation and continued presence of rat trophoblast, since neither rat ICM/mouse morula nor rat ICM/mouse blastocyst chimaeras contain this tissue. Tarkowski (1962) has suggested that the abnormal development of rat eggs after implantation in the mouse may be due to defective interaction of the rat trophoblast with the mouse uterine epithelium. Similar effects could be occurring in some rat morula/mouse morula aggregates. Mystkowska (1975) has reported similar poor postimplantation development from bank vole morula/mouse morula aggregates, and she suggested that the wide taxonomic gap and differences in the course of embryogenesis between the two species were the main causes. However, the present experiments indicate that even morulae of more closely related species like the mouse and the rat are rarely able to integrate and form normal conceptuses.

I should like to thank Dr R. L. Gardner, Dr M. H. Johnson and Mr A. Copp for valuable discussion. Special thanks are due to Dr Johnson, who provided the specific antisera and laboratory facilities for performing the immunofluorescent analysis. Mrs L. Ofer and Mrs S. Clutterbuck-Jackson provided technical assistance. The author was supported by a Medical Research Council Research Studentship and a Beit Memorial Junior Research Fellowship. The work was supported by Medical Research Council project grants to Dr Gardner and Dr Johnson.

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