development of mouse-bank vole interspecific...

/ . Embryol. exp. Morph. Vol. 33, 3, pp. 731-744, 1975 731Printed in Great Britain

Development of mouse-bank vole interspecificchimaeric embryos

By EWA T. MYSTKOWSKA1

From the Laboratory of Experimental Embryology, Institute of Obstetricsand Gynaecology, Warsaw, and the Department of Embryology,

Zoological Institute, University of Warsaw

SUMMARY

One bank vole {Clethrionomys glareolus) embryo and two mouse embryos were combinedat the 8- to 16-blastomere stage and cultured in vitro for 33-47 h. In 66% of cases singleregular blastocysts were formed. The chimaeric composition of blastocysts was confirmedkaryologically. Out of the 222 blastocysts transplanted to 49 pseudopregnant mouse recipients,a total of 52 implantations were found in 20 recipients. Among the 52 implantations, 14contained embryos and the remaining were resorptions. The majority of embryos wereabnormal and fell into two categories: (1) groups of cells surrounded by Reichert's membraneand lying freely in a cavity filled with giant trophoblastic cells, (2) small and retarded egg-cylinders usually composed of endoderm and ectoderm only, and containing a proamnioticcavity. The ectoplacental cone of these embryos was poorly developed or lacking altogether.

Two normal-looking embryos were recovered on the 9th and 10th day (4-somite and ca.12-somite stage). Chimaerism of the younger embryo was confirmed karyologically. Noevidence of chimaerism was available in the case of older embryo which was examined histo-logically. Thirteen implantations examined between 11th and 17th day contained onlyresorptions. It is suggested that the main cause of the heavy mortality of chimaeric embryosis the profound difference in the course of embryogenesis of these two species immediatelyfollowing implantation.

INTRODUCTION

Chimaeras are defined as individuals which contain cells derived from twoor more zygotes. In the mouse, chimaeras can be routinely produced by com-bining cleaving embryos at the 8- to 16-cell stage (Tarkowski, 1961; Mintz,1962). Recently reports have been published describing the combination ofmouse and rat cleaving eggs to form a single blastocyst (Mulnard, 1973; Stern,1973; Zeilmaker, 1973). Gardner & Johnson (1973) obtained chimaeric blasto-cysts by a different technique. They transplanted the whole inner cell mass ofthe rat blastocyst into the mouse blastocyst and succeeded in obtaining develop-ment of such interspecific chimaeric embryos up to the 10th day of pregnancy.

In the present study interspecific chimaeric embryos were produced by com-bining eggs of the bank vole {Clethrionomys glareolus) and of the mouse. These

1 Author's address: Laboratory of Experimental Embryology, Institute of Obstetrics andGynaecology, Medical Academy, 00-315 Warsaw, Karowa 2, Poland.

46 EM B 33

732 E. T. MYSTKOWSKA

two species are taxonomically distant as they belong to two different families,and they differ in the course of the early post-implantation development(Ozdzenski & Mystkowska, in preparation). It seemed interesting to find outwhether the cleaving embryos of these two taxonomically remote species couldintegrate to form a single blastocyst and, if so, how far such chimaeric embryoscould develop beyond implantation.

MATERIAL AND METHODS

Swiss albino mouse and bank vole females were induced to ovulate with 5 i.u.PMSG ('Gestyl', Organon) and 5 i.u. HCG ('Biogonadyl', Biomed) given atan interval of 35^48 h. Both mouse and vole eggs were collected at the 8- to16-cell stage. The procedure for combining the eggs and culturing them to theblastocyst stage was essentially similar to that used for obtaining mouse chim-aeras and was described in our earlier papers (Tarkowski, 1961; Mystkowska &Tarkowski, 1968; Mystkowska, 1974). The main difference was that three ratherthan two eggs were combined, namely one vole egg was placed centrally betweentwo mouse eggs. It was hoped that such an arrangement would increase thechance of the formation of trophoblast from the mouse blastomeres which, inturn, would facilitate implantation of the embryo in the mouse recipient. Theembryos were inspected 1-3 h after the start of culture, to make sure that theyhad remained stuck together. The embryos were cultured in a slightly modifiedMulnard's medium (personal communication, the full composition is describedin a paper by Mystkowska, 1974). They were kept in culture for 33-34 h or43-47 h. Chimaeric blastocysts were either examined in air-dried preparations(Tarkowski, 1966) or transplanted to the uterus of pseudopregnant Swiss albinofemales mated to vasectomized males. Transplantations were performed on theevening of the third day (20.00-22.00) or on the morning of the fourth day(09.00 to noon), counting the day on which the vaginal plug was found as the

FIGURES 1-13

Figs. 2, 5, 7, 9 and 4, 8, 10 represent successive stages of development of the sametriplets.

Figs. 1-2. Bank vole embryo (in the middle) joined with two mouse embryos, x 200.Figs. 3-5. Chimaeric morulae after 21 h of culture, x 200.Figs. 6-8. Chimaeric blastocysts after 44 h of culture, x 200.Figs. 9-10. Blastocysts shown in Figs. 7 and 8 after additional 5 h of culture, x 200.Fig. 11. Fragment of air-dried preparation made from chimaeric embryo no. 7,showing three mouse metaphase plates and one vole metaphase plate. Arrowsindicate plates shown in enlargement in Figs. 12 and 13: long arrow - mouse meta-phase plate, short arrow - vole plate, x 100.Fig. 12. Bank vole metaphase plate, x 1000.Fig. 13. Mouse metaphase plate, x 1000.

Mouse-bank vole chimaeric embryos 733

10

» A

13

46-2


first day. Recipients were sacrificed from 6th to 18th day of pregnancy. Uteriwith implantations (6th to 10th day) were fixed in a solution consisting of 14parts of 96 % ethyl alcohol, 5 parts of formalin and 1 part of glacial acetic acid.Sections were cut at 6 /tm and stained with haematoxylin and eosin. From oneembryo recovered on the 9th day karyological preparations were made by themethod of Evans, Burtenshaw & Ford (1972). Implantations found in femaleskilled between 11th and 18th day were torn open and inspected under dissectingmicroscope.

RESULTS

1. Preimplantation development in vitro

Altogether 784 mouse and 392 vole eggs were used. From 392 triplets 258single blastocysts (65-8%) were obtained. In 55 cases (14-0%) three or twoseparate blastocysts developed while in 79 cases (20-1 %) one, two or all threeeggs underwent degeneration. These results compare well with control experi-ments in which three mouse eggs were combined together. Out of 95 triplets79 single blastocysts (79%) were obtained. In two cases (2%) two separateblastocysts developed, and in 18 cases (19%) one, two or all three eggs degen-erated. Two females into which 11 blastocysts originating from three mouseeggs were transplanted gave birth to seven young.

Vole eggs differ in appearance from mouse eggs, in that they are smaller anddarker due to abundant granules present in the cytoplasm (Figs. 1, 2). Thedifference in the degree of granulation makes it possible to see in chimaericmorulae the boundaries between the contributing embryos even after 21-24 hof culture. At this moment in the compact and usually elongated morula, thedarker vole blastomeres are grouped centrally and the lighter mouse blasto-meres are on the sides (Fig. 5). As cell divisions progress and the morula roundsup the borders between the three components become obliterated. In somecases, however, already after 24 h of culture it is no longer possible to distinguishthe outlines of the three eggs (Figs. 3, 4). The blastocoel appears after about44 h of culture in the form of one (Fig. 8) or two (Figs. 6, 7) cavities. In thelatter case the two cavities merge together after a few hours and a typical blasto-cyst is formed (Figs. 9, 10). Chimaeric blastocysts derived from three eggs con-sisted on the average of 87-8 cells (including 10-1 metaphase plates; mean fromseven blastocysts). In air-dried preparations both mouse and vole metaphaseplates were found.

Only regular blastocysts in which all blastomeres had been incorporated wereselected for transplantation.

2. Post-implantation development

A total of 222 blastocysts were transplanted to 49 recipients. Altogether 52implantations were found in 20 recipients (Table 1).

Mouse-bank vole chimaeric embryos

Table 1. Post-implantation development of chimaeric embryos

735

Day ofdissection

of recipient

6789

10111214151718

Total

No. ofrecipients

3346

17354112

49

No. ofblastocysts

transplanted

98

192796142112349

222

No. of reci-pients with

implantations

122461

—211

—

20

No. ofimplantations

(no. oftransplantedblastocysts inparentheses)

3(3)6(6)5(9)8 (14)

17 (30)3(5)—

6(7)1(3)3(4)—

52 (81)

No. ofembryos

13253

——————

14

(a) Morphology of embryos recovered between the 6th and 10thday of development

Sixth day of development

Embryo no. 1 (Fig. 14). The egg-cylinder is elongated and consists of an outerlayer of flattened endodermal cells, and an inner layer of round or oval ecto-dermal cells. The ectoderm is subdivided into embryonic and extra-embryonicparts. The ectoplacental cone is not visible.

Seventh day of development

Embryo no. 2 (Fig. 15). The embryo is smaller than normal mouse and voleembryos of the same age and much less advanced in development. It is normallyimplanted and has a well-developed ectoplacental cone and a Reichert's mem-brane. Ectoderm and endoderm can be distinguished but there is no proamnioticcavity.

Embryo no. 4 (Figs. 16, 17). The egg-cylinder is composed of endoderm andectoderm which is subdivided into embryonic and extra-embryonic parts. Theupper part of the cylinder resembles that of a bank vole rather than a mousecylinder, in that instead of the ectoplacental cone there is a groove openinginto the lumen of the uterus (Fig. 16) (Ozdzeriski & Mystkowska, in preparation).The embryo is surrounded by a Reichert's membrane and a layer of tropho-blastic giant cells.

Embryo no. 3 (Fig. 18). This embryo was collected from the same recipientas embryo no. 2. It is represented by a group of embryonic cells situated in themiddle of a spongy structure of decidual origin filled with blood.

E. T. MYSTKOWSKA

tik;{ 23

Mouse-bank vole chimaeric embryos 111

Eighth day of development

Embryo no. 5 (Fig. 19). The embryo has reached the stage corresponding toa 7-day mouse egg-cylinder. The ectoderm is subdivided into embryonic andextra-embryonic parts and the proamniotic cavity is present. There is no ecto-placental cone. The embryo is surrounded by spongy decidual tissues con-taining giant trophoblastic cells in its meshes. The band of decidual tissuearound the embryo is degenerating and the trophoblastic layer together withthe Reichert's membrane is embedded in blood.

Embryo no. 6 (Fig. 20). This dead embryo was collected from the samerecipient as the previous one. It consists of a group of cells embedded in theReichert's membrane-like matrix and lies freely in the mesometrial part of thedecidual crypt. The crypt is surrounded by giant trophoblastic cells and isfilled with blood.

Ninth day of development

Embryo no. 7. This embryo was dissected from the uterus and examinedkaryologically. It had four pairs of somites and in its appearance and degree of

FIGURES 14-23

Chimaeric embryos, 6th-9th day of development (mesometrial pole orientedupwards).

Fig. 14. Embryo no. 1, 6th day of development. Ectoderm already subdivided intoembryonic and extra-embryonic part, x 270.Fig. 15. Embryo no. 2, 7th day of development. Embryonic and extra-embryonicparts of the cylinder and the ectoplacental cone are visible, x 300.Figs. 16,17. Two slightly oblique sections through embryo no. 4, 7th day of develop-ment. Left section shows groove in the upper part of the cylinder, characteristic forthe vole embryo. Right sectfon shows subdivision of ectoderm into embryonic andextra-embryonic parts, x 280.Fig. 18. Embryo no. 3, 7th day of development. Group of embryonic cells. x430.Fig. 19. Embryo no. 5, 8th day of development. Slightly oblique section showingembryonic and extra-embryonic parts. This egg-cylinder is deprived of an ecto-placental cone, x 230.Fig. 20. Embryo no. 6, 8th day of development. Group of embryonic cells embeddedin Reichert's membrane lie in a cavity occupied by giant trophoblastic cells andextravasated blood, x 80.Fig. 21. Two embryos, nos. 8 and 9, 9th day of development, implanted one abovethe other. The embryos are surrounded by a network formed by loose decidualtissue and giant trophoblastic cells, x 80.Fig. 22. Larger embryo (no. 8) from Fig. 21 under higher magnification. Beginningof mesoderm formation, x 180.Fig. 23. Embryo no. 11, 9th day of development. Degenerating two-layeied egg-cylinder. Loose network of decidual cells and giant trophoblastic cells surroundsthe cylinder, x 110.

Mouse-bank vole chimaeric embryos 739development it resembled a 9-day-old mouse embryo, with the exception thatthe ectoplacental cone was rather small. Its dimensions within the foetal mem-branes were 2x1-5 mm. Eighty-two mitotic plates were examined, 70 of whichhad a mouse karyotype and 12 a bank vole karyotype (56 chromosomes includinga pair of small metacentric chromosomes) (Figs. 11-12, 13).

Embryos nos. 8 and 9 (Figs. 21, 22). These two embryos were found in oneimplantation crypt, implanted close together. The embryos lie in a networkcomposed of decidual cells and giant trophoblastic cells. Each of them is sur-rounded by its own Reichert's membrane lined outside with trophoblast andinside with distal endoderm.

The embryo lying in the antimesometrial part of the crypt is larger and moreadvanced in development (Fig. 22). Both extra-embryonic endoderm (highlyvacuolated cells) and embryonic endoderm (flat cells) are present. The majorpart of the egg-cylinder is covered with extra-embryonic endoderm which isfolded in many places. The ectoderm is multilayered and subdivided intoembryonic and extra-embryonic parts. Mesoderm formation has just begun.The proamniotic cavity is very large. The ectoplacental cone is absent and theembryo is not directly attached to the uterus.

The second embryo is less advanced in development. Like the first one itlacks an ectoplacental cone and is surrounded by a network of giant tropho-blastic cells. The embryo consists of an egg-cylinder attached to a cellular massof unknown character.

Embryo no. 11 (Fig. 23). This embryo was a degenerating egg-cylinder sur-rounded by Reichert's membrane. The surrounding endometrium, which con-tained disseminated giant trophoblastic cells, shows signs of advanced degenera-tion. The embryo has no contact either with Reichert's membrane or theuterine tissues.

Embryo no. 10 (Fig. 24). The embryo was recovered from the same recipient

FIGURES 24-28Chimaeric embryos, 9th-10th day of development (mesometrial pole oriented

upwards).Fig. 24. Embryo no. 10, 9th day of development. Group of cells of embryonic originsurrounded by Reichert's membrane lying at the bottom of a large cavity filled withblood. Giant trophoblastic cells occupy peripheries of the cavity, x 130.Fig. 25. Normal-looking embryo no. 12, 10th day of development, x 16.Fig. 26. Abnormal embryo no. 13, 10th day of development. See text for detaileddescription, x 110.Fig. 27. Dead embryo no. 14, 10th day of development. Mass of degenerated cellssurrounded by Reichert's membrane. Decidual and giant trophoblastic cells havealso degenerated, x 100.Fig. 28. Giant trophoblastic cell characteristic for the bank vole, lying at the borderof endometrium and myometrium of the mouse uterus. This giant cell was found inthe implantation swelling containing embryo no. 14. x 130.


as embryos nos. 8 and 9. It is represented by a group of cells embedded in amatrix corresponding in character to Reichert's membrane. The abortive egg-cylinder lies in the antimesometrial part of a large crypt formed by loose deciduaand giant trophoblastic cells and filled with blood. It resembles the embryono. 6 described above.

Tenth day of development

Embryo no. 12 (Fig. 25). This was a healthy embryo at the stage of ca. 12somites. The embryo corresponds to a normal mouse embryo of the same stage.

Embryo no. 13 (Fig. 26). This was a litter-mate of embryo no. 12. It consistsof a small compact body with a fissure in the middle and a loose mass of cellspartly encompassing the compact body. Signs of degeneration are already visible.Adjacent to this structure is a mass of healthy cells, with large heavily stainednuclei; these cells are probably in the process of transformation into tropho-blastic giant cells. All these structures are surrounded by a network of gianttrophoblastic cells and are not directly connected with the decidua.

Embryo no. 14 (Fig. 27). In this case there was a mass of degenerated cellssurrounded by Reichert's membrane. Both the giant trophoblastic cells andthose of the decidua exhibit signs of advanced degeneration. Deep in the endo-metrium and close to myometrium, eight healthy looking giant cells were found(Fig. 28). By their size and location these cells correspond to giant cells charac-teristic of the bank vole (Brambell & Rowlands, 1936). Their dimensions rangedfrom 50 x 38 to 127 x 108 jum (mean 78 x 59 /mi). In the second implantationswelling from the same horn, the only traces of an embryo that could be detectedwere two giant trophoblastic cells (dimensions 85 x 46 and 96 x 77 /mi), againembedded deeply in the endometrium.

(b) Fate of chimaeric embryos in the second half of pregnancy

In order to find out how far chimaeric embryos can develop, seven femaleswere chosen in which pregnancy was recognized on the 9th day on the basis ofthe presence of abundant mucus in vaginal smears. The females were keptalive until the appearance of cornified cells in the smears, which was taken asa sign of termination of pregnancy and which occurred in all seven animalsbetween the 11th and 18th day (Table 1). Altogether 11 resorptions were col-lected and examined under a dissecting microscope. Five resorptions - onerecovered on the 1 lth, one on the 14th day and three recovered on the 17th day -contained large amounts of embryonic tissue, which suggested that the embryosmust have died in the second half of pregnancy.

DISCUSSION

The present experiments show that cleaving eggs of the mouse and of thebank vole can integrate to form single and regular blastocysts. Inspection

Mouse-bank vole chimaeric embryos 741during culture and karyological examination of a number of blastocysts pro-vided evidence that cells of both species were present at this stage and wereundergoing mitosis. However, it is not known whether the initial ratio of cells(2:1 in favour of the mouse) was maintained at the blastocyst stage, and whatthe spatial distribution of these two kinds of cells was within chimaeric blasto-cysts. One mouse embryo was placed on each side of a bank vole embryo inorder to increase the chances of trophoblast formation from the mouse cellsand thus to facilitate implantation in mouse recipients. The experiment wasdesigned in this way in view of the work of Tarkowski, who transplanted ratblastocysts to the mouse and vice versa (1962), and field vole (Microtus agrestis)blastocysts to the bank vole (personal communication), and who found thatthe blastocysts of different species, although they produced the decidual reaction,could not establish contact with the uterine mucosa and died shortly afterimplantation.

The implanted chimaeric embryos may be classified into the following groups:(1) Embryos consisting of a mass of cells lying in a cavity filled with giant

trophoblastic cells and showing no direct contact with the uterus (embryosnos. 3, 6, 10, 14).

(2) More or less normally developed egg-cylinders. The ectoderm, endodernand the proamniotic cavity (in the case of 7-day-old or older embryos) arealways distinguishable. Formation of mesoderm was observed in one embryo.Embryos either lack the ectoplacental cone or else the latter is poorly developed(embryos nos. 1, 2, 4, 5, 8, 9, 11, 13).

(3) Normally organized embryos (embryos nos. 7, 12).The embryos belonging to the second group, which is most numerous, present

a wide scale of variability, from egg-cylinders, which were almost normal butwith smaller ectoplacental cones than mouse embryos, up to irregular and smalltwo-layered egg-cylinders showing no direct contact with the endometrium.Frequently one horn contained embryos differing considerably in structure andstage of development (for example embryos nos. 2 and 3, 5 and 6, 12 and 13).These observations suggest that the distribution of both components in chimaericblastocysts as well as the contribution of vole cells to the trophoblast variedfrom case to case and that this factor was mainly responsible for the differentbehaviour of embryos in the post-implantation period.

The coexistence of vole and mouse cells in chimaeric blastocysts may lead toserious developmental disturbances as early as the time of implantation. Trans-formation of the blastocyst into an egg-cylinder takes a different course in thevole and in the mouse (Ozdzeriski & Mystkowska, in preparation). In the mousethe ectoplacental cone is large and well developed right from the beginning,whereas in the vole it is originally lacking and the lumen of the egg-cylinderopens into the uterine lumen. A solid ectoplacental cone arises much later andit is smaller than that of the mouse. Only in a few cases does this region ofchimaeric embryos take a form characteristic of the mouse (nos. 2, 7, 12) or the


vole (no. 4). In most cases the embryos lie loosely in the uterus without devel-oping direct connexion with the decidual tissue. Under these circumstancesearly death of the embryo and, consequently, degeneration of the surroundingdecidual tissue is inevitable. Disturbances due to the difference in the course ofdevelopment of the two species may be expected during the whole embryoniclife of an interspecific chimaera. However, the later the stage at which suchincompatabilities occur, the smaller is the chance that they would result inimmediate embryo death. In the case of the vole and the mouse profound differ-ences occur at the first stage of post-implantation development and this wasprobably the main cause of heavy mortality of chimaeric embryos.

Although the spatial arrangement of the bank vole and mouse eggs at themoment of fusion was always the same, the distribution of vole and mouse cellsin chimaeric blastocysts varied. Because of the position of the vole egg betweentwo mouse eggs, most of the vole cells in the majority of blastocysts must havecontributed to the inner cell mass. However, in some blastocysts part of the volecells must have remained in the trophoblast as indicated by the presence of volegiant trophoblastic cells in the mouse uterus. Development of the mesometrialpart of the chimaeric egg-cylinder in a way characteristic of the vole ratherthan of the mouse may be interpreted either as indicating the presence of volecells in the trophoblast covering the inner cell mass, or as a result of an inductiveinfluence of the vole inner cell mass on the mouse trophoblast. According toGardner & Johnson (1972), formation of the ectoplacental cone in the mousedepends on the influence of the inner cell mass.

The technique of producing interspecific chimaeras employed by Gardner &Johnson (1973), i.e. inoculation of the inner cell mass from the blastocyst ofone species (rat) into the blastocoel of a blastocyst of another species (mouse)is superior to the technique used in this study in that the inner mass becomeschimaeric, while the trophoblast remains homogenous. Rat-mouse chimaeraembryos develop normally up to the 10th day at least. It should, however, beborne in mind that embryonic development of the mouse and rat is much moresimilar than that of the mouse and bank vole and hence developmental dis-turbances are much less likely to occur in the former than in the latter com-bination.

Two normal-looking embryos found on the 9th and 10th day of developmentreached a rather advanced stage (four-somite embryo and ca. 12-somite embryo).Chimaerism of the 9th day embryo was confirmed karyologically. It had a smallectoplacental cone which in the course of further development might have causeddisturbances in the formation of the placenta. The 10-day-old embryo was quitenormal. Since it was only histologically examined no proof of its chimaerism isavailable. The conclusion that it consisted solely of mouse cells would requirethe assumption that all vole cells were eliminated in the post-implantationperiod. Among mice developed from two fused embryos there are always indi-viduals in which the presence of only one component can be detected (Myst-

Mouse-bank vole chimaeric embryos 743kowska & Tarkowski, 1968; Mintz, 1969; Mullen & Whitten, 1971). At leastin some of these cases the second component might have been included as awhole into the trophoblast or extra-embryonic membranes. As explained above(p. 741), in the case of mouse-bank vole chimaeras, the contribution ofall or the majority of bank vole cells to the trophoblast is likely to cause disordersin implantation and early death of the embryos. Such a situation could nottherefore have existed in the embryo in question. One cannot of course excludethe possibility that one component is overgrown by the other and either totallyeliminated or reduced to a negligible level. The author has studied a mousechimaera, in the bone marrow of which the ratio of metaphase plates of the twocomponents was 99:1 and in the cornea 20:2 (Mystkowska, unpublished results).The 10-day-old embryo referred to above (no. 12) may represent an example ofsuch an 'apparent' chimaera; that is, a chimaera in which one component isrepresented in a negligible and developmentally insignificant amount.

It is possible that in the case of combining eggs of taxonomically so distantspecies as mouse and vole, only such chimaeric embryos in which the cell linealien to the recipient is limited to the embryo itself and is represented in smallamount only have a chance of longer development.

The author wishes to express her thanks to Professor A. K. Tarkowski for his advice andguidance in the course of the present work. She is also indebted to Dr W. Ozdzenski forhelpful discussions and for preparing photographic documentation.

Bank voles used in the present study were obtained from the Mammals Research Institutein Biatowieza, Poland. The author wishes to express her thanks to Professor Z. Pucek andmgr A. Buchalczyk for making the animals available to her.

REFERENCESBRAMBELL, F. W. R. & ROWLANDS, 1. W. (1936). Reproduction of the bank vole (Evotomys

glareohis Schreber). Phil. Trans. R. Soc. Lond. B 226, 71-97.EVANS, E. P., BURTENSHAW, M. D. & FORD, C. E. (1972). Chromosomes of mouse embryos

and newborn young: preparations from membranes and tail lips. Stain Technol. 47, 229-234.

GARDNER, R. L. & JOHNSON, M. H. (1972). An investigation of inner cell mass and tropho-blast tissues following their isolation from the mouse blastocyst. / . Embryol. exp. Morph.28, 279-312.

GARDNER, R. L. & JOHNSON, M. H. (1973). Investigation of early mammalian developmentusing interspecific chimaeras between rat and mouse. Nature, New Biol. 246, 86-89.

MINTZ, B. (1962). Formation of genotypically mosaic mouse embryos. Am. Zool. 2, 432.MINTZ, B. (1969). Developmental mechanisms found in allophenic mice with sex chromo-

somal and pigmentary mosaicism. In First Conference on the Clinical Delineation of BirthDefects {Birth Defects Original Article Series) (ed. D. Bergsma & V. McKusick), pp. 11-22. New York: New York, National Foundation.

MULLEN, R. J. & WHITTEN, W. K. (1971). Relationship of genotype and degree of chimerismin coat color to sex ratios and gametogenesis in chimeric mice. / . exp. Zool. 178, 165-176.

MULNARD, J. (1973). Formation de blastocysts chimeriques par fusion d'embryons de rat etde souris au state VIII. C. r. hebd. Seanc. Acad. Sci., Paris 276, 379.

MYSTKOWSKA, E. T. & TARKOWSKI, A. K. (1968). Observations on CBA-p/CBA-T6T6 mousechimeras. /. Embryol. exp. Morph. 20, 33-52.


MYSTKOWSKA, E. T. (1974). Preimplantation development in vivo and in vitro in bank vole{Clethrionomys glareolus) treated with PMSG and HCG. / . Reprod. Fert. (in the Press).

STERN, S. (1973). Chimaeras obtained by aggregation of mouse eggs with rat eggs. Nature,Lond. 243, 472-473.

TARKOWSKI, A. K. (1961). Mouse chimaeras developed from fused eggs. Nature, Lond. 190,857-860.

TARKOWSKI, A. K. (.1962). Inter-specific transfer of eggs between rat and mouse. / . Embryol.exp. Morph. 10, 476-495.

TARKOWSKI, A. K. (1966). An air-drying method for chromosome preparations from mouseeggs. Cytogenetics 5, 394-400.

ZEILMAKER, G. H. (1973). Fusion of rat and mouse morulae and formation of chimaericblastocysts. Nature, Lond. 242, 115-116.

{Received 15 August 1974, revised 14 October 1974)

development of mouse-bank vole interspecific...

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