nuclear transplantation in mouse embryos

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 228~355-362 (1983)

Nuclear Transplantation in Mouse Embryos JAMES McGRATH AND DAVOR SOLTER The Wistar Institute of Anatomy and BLology, Philadelphia, Pennsylvania 19104

ABSTRACT The ability of foreign nuclei to support development in nuclear transplantation manipulations has proven an effective means to assess the consequences of nuclear differentiation. In addition, nuclear transplantation might serve to define the persistence and role of maternally inherited cytoplasmic constitutents during embryogenesis. We have extended the use of a technique that enables the efficient transfer of one-cell-stage pronuclei into the cytoplasm of enucleated mouse embryos, and have successfully transferred two-, four-, eight-cell-stage and inner cell mass (ICM) cell nuclei. We have also used this technique as a means to determining that the stage-specific embryonic antigen, SSEAS, is a cytoplasmic contribution of the unfertilized ovum. The potential value of this technique in determining the developmental capac- ity of nuclei from various embryonic stages, and in determining nuclear1 cytoplasmic origins of early embryonic gene products, is discussed. Key words nuclear transfer, mouse embryo, surface antigens

The transplantation of foreign nuclei into the amphibian embryo demonstrated the varied abilities of embryonic and adult nuclei to support development (Danielli and DiBerardino, '79). More recently, the ability of transplanted early embryonic nuclei from inner cell mass cells of the mouse blastocyst to support development has been reported (Illmensee and Hoppe, '81; Hoppe and 111- mensee, '82). These experiments have pro- vided valuable information regarding the status of nuclear differentiation at distinct stages of embryogenesis. A frequent difficulty encountered in microsurgical manipulations of the mammalian embryo, however, is the injury of a significant proportion of embryos due to micropipette penetration. To avoid this difficulty, we have devised a nuclear transplantation technique which does not require micropipette penetration of the embryo plasma membrane (McGrath and Solter, '83). This method allows the removal of the embryonic pronuclei within a membrane-bound vesicle. The subsequent introduction ofa donor nucleus into the enucleated embryo is achieved using a virus-mediated cell fusion technique. This procedure enables nuclear transplantation to be reproducibly performed, a t a high frequency of success, and with little or no effect on subsequent development. This report presents applica- tions of this procedure in defining nuclear/

cytoplasmic interactions during mammalian embryogenesis, specifically, in defining the nuclearlcytoplasmic origin of an early embryonic antigen.

MATERIALS AND METHODS Embryo isolation and culture

Embryos were obtained from spontaneous inter se matings of C57BL6lJ, C3H/HeJ and ICR (Swiss Albino) strain mice. One-cell- stage embryos were removed from mated females by puncturing the ampullary regions of excised oviducts with watchmaker forceps on the day of vaginal plug detection (day 1). Cumulus cells were removed with N-2- hydroxyethyl piperazine-N'-2-ethanesulfonic acid (Hepes)-buffered Whitten's medium (Whitten, '71) containing 500 NF unitdm1 bovine hyaluronidase (Sigma). Embryos were washed by transfer through successive drops (four) of modified Whitten's medium (Abram- czuk et al., '77) and cultured in this medium under silicone oil (Dow Corning 200 Fluid) as previously described (McGrath and Hillman, '80). Incubations were performed at 37°C in an atmosphere of 5% O2,5%CO2, and 90% Nz.

Two-cell-stage and four- to eight-cell-stage embryos were flushed from the oviducts of mated females on gestational days 2 and 3, respectively. Blastocyst-stage embryos were flushed from the uteri of mated females (day 4) and cultured overnight. Inner cell mass

0 1983 ALAN R. LISS. INC.

356 J. McGRATH AND D. SOLTER

TABLE 1. Preimplantation and postimplantation development of control and nuclear transplant embryos

Preimplantation . .. Postimplantation ~ _ _ _ _ _ AiControl 34/34 (100%)' 5/34 (i5%j2 BNuclear transulant 64167 196%) 1064 (16%)

'Number of embryos that successfullv develoDed to the morula-blastocvst starre after 5 davs of in vitro culture/total number of embryo"% '"umber of embryos born after transfer to the uteri of pseudopregant femaledtotal number of embryos transferred.

Fig. 1. Blastocysts obtained from control and nuclear transplant one-cell-stage embryos after 5 days of in vitro culture. A) Unmanipulated control ICR strain embryos.

B) C3WHeJ nucleus/C57BLG/J cytoplasm nuclear transplant embryos. x40.

cells were obtained from expanded blastocysts (day 5 ) immunosurgically (Solter and Knowles, '75). Single ICM cell suspensions were obtained by repeated pipetting of ICMs in 0.25% trypsin (Worthington) and 0.1% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline.

Microsurgery Microsurgical manipulations and the prep-

aration of microinstruments were as described previously (McGrath and Solter, '83). All manipulations were performed using Leitz micromanipulators and a fixed-stage Laborlux I1 microscope (Leitz). Embryos were incubated in cytochalasin B (5 pgiml; Sigma) (Hoppe and Illmensee, '77) and colcemid (0.1 pgiml; Sigma) for 15-60 min prior to microsurgery.

Detection of SSEA-3 Immunofluorescence tests were performed

as described by Shevinsky et al. ('82) to de- tect the expression of the stage-specific embryonic antigen, SSEA-3, on nuclear transplant and control embryos. This antigen is

present on unfertilized ova and preimplantation stage mouse embryos until the blastocyst stage. Briefly, all nuclear transplant and control embryos were incubated for 30 min at 37°C with anti-SSEA-3 monoclonal antibody used as either undiluted culture super- natant or nude mouse ascites (diluted 1:100, viv). Embryos were subsequently washed, incubated with fluorescein isothiocyanate-con- jugated rabbit anti-rat IgG, rewashed, and placed on a microscope slide under paraffin oil. Embryos were examined for the expression of SSEA-3 using a Leitz epi-illuminated fluoresence microscope.

RESULTS Interembryonic transfer of one-cell-stage

pronuclei A previous report (McGrath and Solter, '83)

described a nuclear transplantation technique in which the microsurgical removal of the pronuclei of the one-cell-stage embryo was combined with subsequent introduction of the pronuclei into a second embryo using a virus-mediated cell fusion technique (Fig. 2A, B). In that study, survival and incorpo-

NUCLEAR TRANSPLANTATION IN MOUSE EMBRYOS 357

Fig. 2. Light micrographs illustrating the formation of nuclear karyoplasts from preimplantation-stage mouse embryos and their introduction, along with inactivated Sendai virus, into the perivitelline space of previously enucleated one-cell-stage embryos. Contained within the

karyoplast are the pronuclei of the one-cell-stage embryo (A, B); the two-cell-stage nucleus (C, 0); the four-cell- stage nucleus (E, F) and the eight-cell-stage nucleus (G, HI. The transfer of an intact inner cell mass cell is also shown (I, J). X 250.

ration of the donor nuclei was greater than 90%. In addition, more than 90% of the nuclear transplant embryos successfully developed to the blastocyst stage after 5 days of in vitro culture (Fig. l), and approximately 15%

of these developed to term after transfer to the uteri of pseudopregnant females (Table 1). The proportion of nuclear transplant embryos that successfully developed to term was not significantly different from that of con-

358 J. McGRATH AND D. SOIATER

trol unmanipulated embryos. The pronuclei of the one-cell-stage embryo may therefore be efficiently transferred from one embryo to another with no significant effect on the ability of the embryo to undergo subsequent development.

Interembryonic transfer of two-, four-, eight- cell-stage and ICM nuclei

We have extended the use of our pronuclei transfer technique to transfer nuclei obtained from successive stages of preimplantation stage embryogenesis. Two-cell-, four- cell-, and eight-cell-stage embryos are affixed to a holding pipette and their zonae pelluci- dae penetrated by the enucleatiodinjection pipette. The pipette is subsequently ad- vanced so that its tip lies adjacent to the nucleus of one of the blastomeres. The nucleus is drawn into the pipette gently and the pipette is withdrawn, thereby surrounding the nucleus in the embryo plasma membrane with a small volume of cytoplasm (Fig. 2C, E, G). The karyoplast is then injected along with inactivated Sendai virus into the perivitelline space of a previously enucleated one-cell-stage embryo (Fig. 2D, F, H). The fusion of the plasma membrane of the karyoplast with the plasma membrane of the embryo introduces the donor nucleus into the recipient cytoplasm (Fig. 3B, C). The proportion of embryos that successfully incorporate the donor nucleus is typically observed to be greater than 90%. For example, of 72 two- cell karyoplasts that were introduced into the perivitelline space of enucleated one-cell- stage embryos, 70 (97%) successfully under- went fusion. The proportion of these nuclear transplant embryos that subsequently dis- play normal development is currently under investigation.

We have also used this technique to intro- duce successfully nuclei from ICM cells into

the cytoplasm of enucleated one-cell-stage embryos. In the latter experiments, a single intact ICM cell, with virus, is placed in the perivitelline space of enucleated embryos (Fig. 21, J).

Persistence of maternally inherited cytoplasmic constituents

The relatively high proportion of nuclear transplant embryos that undergo normal development after receiving one-cell-stage pronuclei enable experiments to be done that require analyses of entire populations of nuclear transplant embryos. In one such investigation, we have examined the nuclear/ cytoplasmic origin of a stage-specific embryonic antigen (SSEA-3) by performing reciprocal nuclear transplantations between genetically distinct one-cell-stage embryos that differ in their expression of this antigen. SSEA-3 is defined by a monoclonal antibody and is present on unfertilized ova and preimplantation stage mouse embryos until the early (day 4) blastocyst stage. In expanded blastocysts (day 5) SSEAS is no longer observed on trophectoderm but persists on ICM cells (Shevinsky et al., '82).

A strain difference exists in expression of SSEAS (Fig. 4A,B) wherein C57BL6/J embryos are antigen-positive (Ag+) and C3W HeJ embryos are antigen-negative (Ag-) (Shevinsky, Solter and Knowles unpublished results). In addition, a cytoplasmic inherit- ance of this antigen is implicated since C57BL6/J Q x C3HHeJ O" embryos are AgC, whereas C3HMeJ Q x C57BL6/J O" embryos are Ag-. Reciprocal pronuclear transplantation between C57BL6iJ and C3W HeJ one-cell-stage embryos were therefore performed and the resultant embryos cultured in vitro and assayed in immunofluorescence tests for their expression of SSEAS at

TABLE 2. Immunofluorescent staining of SSEA-3 on control and nuclear transplant embi-yos

Early Four-cell Eight-cell Morula blastocyst ____-____________________ Two-cell

C57 control 29/29' 20/20 17/17 5/5 212 C3H control 0/26 014 0115 0116 0119 C57n-C3H2 018 013 014 0/8 0/4 ..

c57",c3Hc; o/i9 O i l 1 017 019 017 C ~ H N " 2 5 7 ~ 17/17 10110 919 7/7 11/11

'Number of antigen-positive embryositotal number of embryos tested. 220-rm C57BLWJ cytoplasmic vesiclestnonenucleated C3H zygotes. "Nuclear transplant embryos: N refers to strain origin of nucleus; C refers to strain origin of cytoplasm


Fig. 3. Light micrographs of nuclear transplant embryos obtained shortly after the fusion of a nuclear karyoplast with the plasma membrane of an enucleated one- cell-stage embryo. The introduced nuclei were obtained from one-cell-state (A), two-cell-stage (B), and four-cell-

stage (C) donor embryos. The introduced nuclei are the same nuclei depicted in Figure 2A-F. Note the slight deformation of the embryo in the region of the incorporated karyoplast and the peripheral location of the nuclei.

successive stages of development. The data show (Table 2, Fig. 4) that nuclear transplant embryos with Ag- cytoplasm (C3HiHeJ) and Ag+ pronuclei (C57BL6iJ) did not express SSEA-3 at either the two-, four-, eight-cell or late morula-early blastocyst stages (Fig. 4D). Conversely, nuclear transplant embryos with Ag cytoplasm (C57BL6iJ) and Ag- pronuclei (C3HiHeJ) continued to expres SSEAS

through the early blastocyst stage (Fig. 4C). In control experiments, a small volume of membrane-bound cytoplasm, approximately equal to that present in the pronuclear karyoplast, from Agi C57BL6iJ one-cell embryo was transferred to the Ag- C3HiHeJ one-cell embryo; SSEA-3 could not be detected on the recipient embryos when tested during subsequent development (Table 2). We therefore


Fig. 4. Expression of SSEA-3 as determined by indi- rect immunofluorescence tests of two-cell-, four-cell-, and morula-stage control and nuclear transplant embryos. A) Control antigen-positive C57BL6iJ embryos. B) Con- trol antigen-negative C3HMeJ embryos. C) Antigen-

negative C3H/HeJ nucleusiantigen-positive C57BL6/J cytoplasm nuclear transplant embryos. D) Antigen-positive C57BL6iJ nucleus/antigen-negative C3H/HeJ cytoplasm nuclear transplant embryos.


conclude that a stage-specific embryonic antigen which is present at least until the blastocyst stage is inherited from the cytoplasm of the unfertilized ovum. Our inability to de- tect SSEA-3 when Ag' membrane-bound cytoplasm was incorporated into intact Ag- C3H/HeJ embryos suggests that the antigen may be either degraded or masked in the Ag - embryo.

DISCUSSION

Differentiation can be described as a pro- cess which results in sequential and ordered changes in cellular phenotype. The possible reversal of these changes at the level of the differentiating nucleus has been actively in- vestigated in the amphibian embryo in nuclear transplantation experiments. Only recently, however, have similar experiments been reported for the mammalian embryo (Illmensee and Hoppe, '81; Hoppe and 111- mensee, '82). In these experiments, inner cell mass cells obtained either from normal or parthenogenetic mouse blastocysts were shown to support complete development when introduced into the cytoplasm of one- cell-stage mouse embryos. Thus, it is now possible to define the extent of nuclear differentiation (and its possible reversal) during mammalian embryogenesis.

The ability to perform nuclear transplantation also permits analyses of the possible role of cytoplasmic constituents during development. The role of these maternally inherited constituents and their possible phenotypic effects on embryos and adults are largely unknown. An inherent difficulty in distinguishing maternally inherited cytoplasmic components from nuclear encoded gene products results from the presence of the maternal genome during both oogenesis and embryogenesis. Nuclear transplantation, however, offers a solution to this prob- lem since donor nuclei from genetically distinct cells may be used to replace the embryonic genome. Thereafter, the persistence of recipient-type gene products must be de- rived from the oocyte cytoplasm and the ap- pearance of donor-type gene products must be due to transcriptional activity by the embryonic genome.

We present in this report further applica- tions of a nuclear transplantation technique which enables the introduction of nuclei from many different stages of development. This procedure, however, is ideally suited for the

transfer of the pronuclei of the one-cell-stage embryo, nuclei which are prohibitively large for mechanical transfer. The developmental equivalence of the donor pronuclei and recipient cytoplasm, the protection of the transferred nuclei by the surrounding karyoplast membrane, and the lack of micropipette penetration in these experiments should provide optimal conditions for subsequent development. The relatively high rate of development exhibited by these nuclear transplant embryos (McGrath and Solter, '83) attests to the minimal effect of these manipulations on development. Thus, entire populations of synchronously developing nuclear transplant embryos can be readily produced and analyzed for nuclearicytoplasmic interactions. In one such investigation described here, we have defined the nuclearicytoplasmic source of an early embryonic antigen, SSEA-3, and have shown that this antigen, which persists until the blastocyst stage, is inherited from the cytoplasm of the unfertilized ovum. It will be of interest to apply this approach to other phenotypic traits of the mammalian embryo and adult in which possible maternallcytoplasmic inher- itance has been implicated (for reviews, see McLaren, '79, '81).

In addition to the transfer of nuclei, this procedure should also permit the controlled interembryonic transfer of membrane-bound cytoplasm. Thus, stage-specific properties of the embryonic cell surface membrance and its accompanying cytoplasm should be ame- nable to analysis.

ACKNOWLEDGMENTS

The authors wish to express their gratitude to Mariette Austin and Marina Hoffman for their critical reading of this manuscript. This work was supported by grants CA-25875, CA- 10815, and CA-27932 from the National Can- cer Institute, by grants HD-12487 and HD- 17720 from NICHD, and by grant PCM-81 18801 from the National Science Foundation. J.McG. was supported by grant T32-CA- 09171.

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nuclear transplantation in mouse embryos

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