Nuclear Transfer in Mammalian Embryos

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 120

Nuclear Transfer in Mammalian Embryos RANDALLSCOTTPRATHER* AND NEALL. FIRST Department of Meat and Animal Science, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706

I. Introduction

Transfer of nuclei from one cell to another provides a powerful method to study the interactions of the cytoplasm of one cell with the nuclei of another. Nuclei from various differentiated states can be transferred to nondifferentiated cytoplasm and the effect on the nucleus can be monitored (e.g., nuclear swelling, DNA replication, RNA production, developmental competence). Such an experiment was proposed by Spemann in 1938 and completed by Briggs and King and 1952. The original experiment was in Ranu pipiens, and it was not until the present decade that similar types of nuclear transfers were successfully completed in mammalian eggs (McGrath and Solter, 1983a,b;Willadsen, 1986;Prather et al., 1987,1989a; Stice and Robl, 1988; Kono et al., 1988). Therefore, the history of nuclear transfer experiments in mammalian eggs is very short. It is the purpose of this review to collate the limited information in mammals and draw comparisons with the amphibians for which nuclear transfer has been well characterized. It is anticipated that this review will provide stimulation and direction to a further understanding of mammalian development. 11. Differentiation Events in Early Mammalian Development

Early mammalian development is characterized by an initial period of zygotic inactivity (little or no RNA synthesis) followed by a transition period during which development is directed by both maternally stored message RNA and newly synthesized RNA. The period when the zygote begins producing its own RNA, but is still translating RNA that was stored in the egg during oogenesis, has been termed the maternal-to-zygotic transition. The zygotic transition appears to occur at a species-specific cell stage. One method for determining the onset of zygotically produced transcripts is to block their appearance and subsequent protein production * Present address: Department of Animal Science, University of Missouri, Columbia, Missouri 6521 1. 169

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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with a-amanitin. a-Amanitin blocks mRNA synthesis by binding to a subunit of polymerase 11, thus blocking chain elongation (Cochet-Meilhac et al., 1974). It is less effective on polymerase I11 and ineffective on polymerase I (Weinmann and Roeder, 1974). In the mouse the major onset of zygotically derived RNA production and subsequent translation occurs at the two-cell stage (Flach et ai., 1982; Bolton et a)., 1984). The two-cell stage is characterized by the production of a-amanitin-sensitive heat shock-like proteins (hsp) in G I (Bensaude et al., 1983) and a set of nine a-amanitin-sensitive proteins in Gz (Bolton et al., 1984). In the cow embryo the first a-amanitin-sensitive proteins are produced at the late 4-cell stage (Barnes, 1988). However, the major change in protein profiles occurs at the 8- to 16-cell stage (Barnes, 1988). The major a-amanitin-sensitive protein change in rabbits (Van Blerkom and McGaughey, 1978) and sheep (Crosby et al., 1988) also occurs at the 8- to 16-cell stage. It is interesting to note that in three species a minor protein change precedes the major shift. This occurs at the pronuclear stage in the mouse (Clegg and Piko, 1982), 4-cell stage in the rabbit (Kanka and Flechon, 1987), and 4-cell stage in the cow (Barnes, 1988). Total RNA production can also be assayed by evaluating uridine incorporation. Uridine is incorporated into 2-cell mouse embryos (Mintz, 1964) and at the 8-cell stage in the cow (Camous et al., 1986). A morphological change of nucleoli from fibrillar to reticular and silver staining of nucleolar organizing regions on metaphase chromosomes has been correlated with an initiation of rRNA synthesis. These changes suggest that rRNA is first transcribed at the 2-cell stage in the mouse (Hansmann et ul., 1978) and goat (Chartrain et ul., 19871, the 4-cell stage in the pig (Norberg, 1970, 1973) and rat (Szollosi, 1966), and the 8-cell stage in the cow (Camous et al., 1986; King et al., 1988), and the 16-cell stage in the sheep (Calarco and McLaren, 1976). Thus there appears to be a period during early development when little or no RNA is produced. This period is characterized by short cell cycles with short or no G I or Gz periods (see the review by First and Barnes, 1989). Subsequent cell cycles are lengthened by the addition of RNAsynthetically active GI and G2 periods. The first period of sufficient length to initiate transcription may actually signal the first genomic differentiation event. After activation of the genome, a variety of events occur that are sensitive to a-amanitin and thus thought to be under the direction of zygotically produced transcripts. These include compaction, gap junction formation, and blastomere polarization (reviewed by Prather and First, 1988).

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111. Nuclear Transfer

As seen in the previous section, the mammalian genome is quiescent during early cleavage stages. However, at a species-specific cleavage stage, the embryonic genome begins to transcribe RNA and thus may signal the first differentiation event at the level of the genome. Therefore, by assessing the resulting gene activity and development, nuclear transfer can be used to evaluate whether or not nuclei that are undergoing active transcription at progressively differentiated states are different from nuclei at the earlier quiescent stages. In amphibians the developmental potential of nucli gradually decreases as the stage of the donor nucleus is advanced (reviewed by Gurdon, 1986; DiBerardino, 1987). Whether this decreased developmental rate is a result of progressive differentiation events or increased asynchronies of the length of the cell cycles remains to be fully determined. As we shall see later, a similar developmental result is expected to occur in certain mammals as well. Nuclear transfer procedures in mammals generally incorporate a method of cell fusion between a cytoplast and karyoplast to complete the nuclear transfer (McGrath and Solter, 1983a), whereas nuclear transfer in amphibians utilizes a procedure that penetrates the plasma membranes of the donor cell and the recipient cell (Gurdon and Laskey, 1970). This difference, though superficially trivial, should be borne in mind when making comparisons between nuclear transfer in mammals and amphibians. The method of nuclear transfer used in mammals usually results in the transfer of a relatively large amount of cytoplasm as compared to the methods used in amphibians, in which adult cytoplasmic proteins can bring about abnormalities in normal fertilized Rana eggs (Markert and Ursprung, 1963). It should also be note that the use of the term clone in the following discussion does not necessarily imply that the embryos or offspring are identical, but simply that they are the result of the transfer of an advanced-stage nucleus to an enucleated oocyte. Achievement of genetically identical status requires that a larger number of criteria be fulfilled, as outlined by Seidel (1983), including identical cytoplasmic inheritance, epigenetic phenomena, uterine environment, neonatal environment, and later environment. A. MICEAND RATS For economic reasons, the majority of mammalian nuclear transfer studies have been conducted with mouse eggs. The results obtained have

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generally been applied to the rest of the mammalian animal kingdom; however, as we shall see, this is not a prudent application. I. Nuclear Transfer Successful nuclear transfer in mammals requires the eggs to be resilient to the conditions employed for the nuclear transfer procedure. These conditions may include those described in the following paragraphs. a. Treutment with Cytochalasins. Cytochalasins, mold metabolites, inhibit the function of microfilaments by binding to one end of the actin filament and preventing further polymerization. This disruption of microfilaments makes the plasma membrane less rigid and more elastic, thus permitting nuclei or cytoplasts to be removed from cells with a minimal amount of tearing of the microfilaments, thereby maintaining the integrity of the plasma membrane (McGrath and Solter, 1983a). Undesired effects of cytochalasins include decreased polysaccharide synthesis and inhibition of sugar transport (Granholm and Brenner, 1976). b. Treatment with Colchicine or Colcemid. These compounds inhibit the polymerization of microtubules by displacing guanosine triphosphate. The importance of the disruption of the microtubules on micromanipulation is dependent on the stage at which the egg is manipulated. Unfertilized oocytes, apart from the meiotic spindle, have a minimal microtublar structure, whereas after activation the microtubules form a large, highly organized intracellular meshwork (Schatten et al., 1985). This internal network, in pronuclear-stage eggs, must be destabilized before successful micromanipulation can take place. An undesirable side effect of this drug is inhibition of nucleoside transport systems, which thereby prevents chromosome replication (Mizel and Wilson, 1972). If unfertilized oocytes are used as recipient cells, colchicine use may be omitted. c. Exposure to Cell Fusion Conditions. Cell fusion can be facilitated electrically (Berg, 1982), chemically (Fisher and Goodall, 1981), or by a virus (Graham, 1969). All three fusion methods result in a destabilization of the plasma membrane. If the membranes of two adjacent cells are destabilized, small channels form between the two cells upon restabilization. These small channels are thermodynamically unstable, thus they open larger and larger until the two cells become one. Since the conditions described are less than ideal for normal in uitro development, it must first be demonstrated that these conditions are compatible with development to term.

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2 . Within-Stage Nuclear Transfer Within-stage nuclear transfers were first achieved by McGrath and Solter (1983a) when they exchanged pronuclei between two mouse zygotes. This relatively simple manipulation demonstrated that the mouse zygote is remarkably resilient to the insults inflicted during nuclear transfer and can still direct development to term. Similar results have been obtained with rat zygotes (Kono et al., 1988). The exchange of pronuclei between zygotes provides a method to study both the nuclear and cytoplasmic contributions to early development. Examples are described in the following. a. Pronuclear Exhanges. The hairpin tail (Thp)mutation is a maternally derived lethal mutation, whereas viable offspring result when the mutation is paternally inherited (Johnson, 1975). Pronuclear exchanges between normal zygotes and maternal ThPzygotes show the maternal ThP cytoplasm receiving normal pronuclei will result in normal offspring, whereas normal zygote cytoplasm receiving either ThPpronuclei or normal pronuclei result in the production of both normal-tailed and short-tailed offspring (McGrath and Solter, 1984a). This also illustrates the nonequivalence of the maternally and paternally derived genomes, which will be discussed further in a later section. Stage-specific embryonic antigen 3 (SSEA-3) is an antigen present on unfertilized ova, cleavage stage eggs, and the cells of the inner cell mass of blastocyst stage embryos of certain strains of mice (McGrath and Solter, 1983b; Prather and First, 1987). Stage-specific embryonic antigen 3 is present on eggs resulting from the transfer of SSEA-3- pronuclei into the cytoplasm of enucleated SSEA-3' zygotes. However, the antigen does not appear on cells resulting from the transfer of SSEA-3' pronuclei to enucleated SSEA-3- zygotes (McGrath and Solter, 1983b).This suggests that the presence of the antigen is due to a cytoplasmic component stored in the ooctye during oogenesis. Female DDK mice, when mated to non-DDK males, produce small litters as a result of the death of many embryos between the morula and implantation stages. This incompatibility can be overcome if a DDK maternally derived pronucleus, along with the non-DDK paternally derived pronucleus, is transferred to a non-DDK enucleated zygote. A high proportion of these eggs develop (Renard and Babinet, 1986) and result in offspring (Mann, 1986). This deficiency also appears to be cytoplasmically inherited, in that the transfer of DDK cytoplasm to non-DDK zygotes reduces the developmental potential of these eggs (Renard et al., 1988). Thus the egg cytoplasm can have an effect on the paternally derived

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pronucleus, but the exposure of the alien paternal pronucleus results in the paternally derived pronucleus affecting the cytoplasms such that development is reduced even when normally developing pronuclei are transferred to the affected cytoplasm (Renard and Babinet, 1986). The lethal component may also affect the proteins responsible for gap junction communication in the early embryo (Buehr et al., 1987). The exchange of pronuclei interspecifically between Mus musculus and Mus caroli zygotes results in only a few cleavage divisions (Solter et al., 1985; McGrath and Solter, 1986). This incompatibility is likely due to insufficient communication between the pronuclei and the cytoplasm. This result as such should not discourage attempts at other combinations of interspecific nuclear transfer.

b. Two-Cell Nuclear Exchange. Within-phase nuclear transfers are not limited to pronuclear exchanges, in that two-cell nuclei can be transferred to enucleated two-cell blastomeres and result in high rates of development (Robl et a / . , 1986; Tsunoda et al., 1987). In uitro development in a variety of mammalian eggs results in a cessation of cleavage at a species-specific cell stage. In the mouse this block in development occurs at the two-cell stage (Goddard and Pratt, 1983). Reciprocal nuclear transfer, at either the pronuclear or two-cell stage, between eggs that do not exhibit this block to development and eggs that block, demonstrate that both the nucleus and the cytoplasm regulate in uitro development from the one-cell stage, but that the nucleus alone is responsible for strain differences in in uitro development beyond the twocell stage (Robl et al., 1988). Therefore, within-stage nuclear transfer has provided a method to evaluate early mouse devlopment and elucidate both developmentally regulated events (two-cell block) and the route of inheritance, either cytoplasmic or nuclear, of specific characteristics of the egg. 3. Cloning The genomes of all of the nuclei of an early mouse embryo are thought to be identical, therefore the procedure of nuclear transfer also potentially provides a method for creating large numbers of clones. After the first series of nuclear transfers is carried out, the resulting embryos could be recloned and/or frozen for later use, thus providing a potentially unlimited supply of genetic maternal. However, as we shall see, the usefulness of this tenchique in mice is currently limited.

a. Developmental Potential. The successful results of nuclear transfer in amphibians (Briggs and King, 1952; Fischberg et al., 1958; McKinnel,

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1962; Gurdon, 1962) have provided in incentive for its application in mammals. Initial studies by Modlinski (1978, 1981) using the T6 chromosomal marker showed the nuclei from cleavage stage cells and inner cell mass cells, but not trophectodermal cells, can participate in the development of a tetraploid blastocyst. This is supported by the observation that the inner cell mass can contribute to the trophectoderm but not vice versa (Rossant and Vijh, 1980), suggesting that trophectoderm is more differentiated than is inner cell mass. A similar result can be seen when an eight-cell blastomere is transferred to a haploid zygote; that is, a blastocyst develops but is triploid (Howlett er al., 1987). However, when cleavage stage nuclei are transferred to enucleated zygotes they rarely support development to the blastocyst stage (pronuclear to enucleated zygote, 95%; two-cell to enucleated zygote, 13%; four-cell to enucleated zygote, 0%; McGrath and Solter, 1984b). Similar results have resulted from nuclear transfers in rats, even though pronuclear exchange can result in development to term (Kono et al., 1988). Attempts to evaluate development after transfer of nuclei at different stages of the cell cycle in mice have yielded conflicting results (Howlett et al., 1987; Smith et al., 1988). A major developmental transition occurs at the two-cell stage in the mouse (Johnson, 1981). Therefore, the low development resulting from the transfer of nuclei across this stage may be a result of differentiation events that occur at the two-cell stage that the enucleated zygote is unable to reprogram. After the two-cell stage there are few morphological or protein synthetic changes until the late eight-cell stage. Since these stages appear to be functionally similar, the transfer of nuclei from the eight-cell stage to the two-cell stage may permit extended development. This type of transfer results in 58% of the eggs developing to the blastocyst stage (Rob1 et al., 1986) and up to 63% forming implantation sites by day 10 of gestation. Further experiments that incorporate cooling of the eggs prior to nuclear transfer have resulted in the production of offspring (Tsunoda er al., 1987). Nuclear transfer experiments that use enucleated zygotes as recipients, however, do not directly mimic those of the amphibian experiments. It has been postulated that removal of interphase nuclei removes components that are responsible for genomic reprogramming, because they likely have an affinity for nuclei (Rather, 1989). The amphibian experiments use an oocyte in metaphase as a recipient for nuclear transfer. Coincident with nuclear transfer and enucleation is activation. This results in both a remodeling and a genetic reprogramming of the transferred nucleus (reviewed by Gurdon, 1986; DiBerardino, 1987; Rather, 1989). In the mouse remodeling occurs only in nuclei transferred within a 90-minute window around activation of the ooctye (Czolowska et al., 1984). The occurrence

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of the remodeling of sperm chromatin is also dependent on the timing of activation in relation to fertilization (Usui and Yanagimachi, 1976; Komar, 1982), and factor(s) responsible for this remodeling appear to be quantitatively limiting (CzoYowska et al., 1984; Witkowska, 1981). In order to stimulate more closely the amphibian experiments, 8- to 16-cell nuclei were transferred to oocytes within 3 hours of activation. One of the 198 resulting eggs developed to the blastocyst stage (McGrath and Soiter, 1986). The nuclear modifications responsible for the observed development may have been a result of an extended period of exposure to developmentally early cytoplasm or greater accessibility to a possibly limiting and labile supply of cytoplasmic components. As we have seen, the developmental potential of cleavage stage nuclei in mouse embryos is very limited. The discussion that follows describes nuclear changes occurring after nuclear transfer and may provide an explanation for this limited development. b. Nuclear Changes after Nuclear Transfer. A variety of nuclear modifications occur after transfer of nuclei to amphibian oocytes (Gurdon, 1986; DiBerardino, 1987). Similar nuclear modifications in mammals have been less well characterized, but are presumed to include changes in DNA synthesis and gene regulation. In amphibian eggs, nuclei in G I ,but not Gz, prior to the nuclear transfer undergo DNA synthesis (DeRoeper et al., 1977). One mammalian study has shown that nuclei transferred to hamster oocytes also undergo DNA synthesis (Naish et a / . , 1987). Gene expression has been closely monitored in the mouse. During early mouse development, zygotically derived proteins first appear in GI of the two-cell stage with the transient appearnace of hsp (Bensuade et al., 1983). When eightcell nuclei are transferred to enucleated zygotes, they have abnormally low levels of methionine uptake (Barnes et a f . , 1987) but exhibit normal production of the hsp (Barnes et al., 1987; Howlett et al., 1987). However, when an eight-cell nucleus is transferred to an enucleated two-cell egg, the resulting embryo has normal methionine uptake, but continues producing polypeptides partially with its own developmental program (Barnes et al., 1987). One of the first events following nuclear transfer indicative of reprogramming is a swelling of the nucleus and a redistribution of the nucleoli (Gurdon, 1964). However, eight-cell nuclei transferred to either enucleated zygotes or enucleated two-cell blastomers show no signs of nuclear swelling (Barnes et al., 1987). If nuclei are transferred to intact two-cell blastomeres they swell only slightly (Graham, 1969), but if transferred to oocytes and then activated, they swell considerably (Graham, 1969; Tarkowski and BaYakier, 1980). Reprogramming of the timing of the occurrence of developmental events

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such as compaction and blastocoele formation is not clear. Some results suggest incomplete reprogramming of eight-cell nuclei transferred to enucleated two-cell blastomeres because compaction (Tsunoda et al., 1987; Howlett et al., 1987) and blastocyst formation (Tsunoda et al., 1987) occurred earlier than for controls, whereas Barnes et al. (1987) showed the timing of blastulation occurs at the same time as for control two-cell blastomeres. Others have failed to report this important parameter (Tsunoda and Shioda, 1988; Kono and Tsunoda, 1988; Smith et al., 1988). Certainly additional studies are essential to evaluate the many possible parameters that change in mouse embryos during early development and how these are affected by nuclear transfer. 4 . Developmental Potential of Androgenones and Gynogenones Nuclear transfer can be used to study the development of embryos that lack a maternal genomic contribution (androgenones) or lack of paternal genomic contribution (gynogenones). These can be either haploid, by removal of the appropriate pronucleus, or diploid, by either subsequent transfer of a suitable (maternally or paternally derived) pronucleus, or by culture in the presence of cytochalasin. Regardless of the method employed, development to term is inhibited (reviewed by McGrath and Solter, 1986; Surani e? al., 1987). Androgenones and gynogenones stop development at characteristic stages. Diploid eggs develop better than haploid eggs; however, haploid development can be improved by reducing the cytoplasmic volume, which may make the ratio of DNA content to cytoplasmic volume closer to normal (McGrath and Solter, 1986; Howlett et al., 1987). Androgenones can develop to implantation (day 10-1 1) and have relatively normal extraembryonic membranes but lack a well-developed fetus and yolk sac (Barton e? al., 1984). When adrogenetic blastomeres are chimerized with normal cleavage stage blastomeres they can make a contribution to the trophoblast- and trophectoderm-derived tissues but not the fetus, extraembryonic mesoderm, or extraembryonic endoderm (Surani et al., 1988; Thomson and Solter, 1988). This is in contrast to the limit of development achieved by gynogenones. These embryos develop similarly to parthenogenones and can reach to 25-somite stage, but have relatively small extraembryonic membranes and yolk sac (Surani and Barton, 1983; Surani et al., 1984). The development of a gynogenetic or parthenogenetic inner cell mass can be enhanced by chimerizing the inner cell mass with normal trophoblastic cells (Barton et al., 1985). The inability of parthenogenetically activated eggs to develop is not due to a cytoplasmic defect, since artificially activated eggs receiving a normal paternally derived pronucleus from other fertilized eggs develop to term (Mann and Lovell-Badge, 1984; Surani et al., 1984). Parthenogenetic cells can also contribute to the germ,

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somatic, and extraembryonic mesodermal cells of a chimera (a normal egg chimerized with a parthenogenone: Surani et al., 1977; Stevens, 1978; Andregg and Markert, 1986; Thomson and Solter, 1988), but parthenogenetic cells disappear from trophectodermal cells by day 6.5 and from primitive endoderm by day 9.5, and decrease in primitive ectodermal cells between day 9.5 and birth (Nagy et al., 1987; Clark et al., 1988). The disappearance of parthenogenetic cells in uiuo parallels that found in uitro (McGrath and Solter, 1986). The above results, in combination with the results of studies with the ThPmutation, suggest that imprinting of both the sex chromosomes and autosomes may occur during gametogenesis (Mann and Lovell-Badge, 1987, 1988). Therefore nuclear material would be modified differentially depending on whether or not it went through spermatogenesis or oogenesis. Indeed, differential methylation patterns have been detected in transgenes, and the degree of methylation is dependent on whether the transgene is maternally or paternally inherited (Reik et al., 1987; Sapienza et al., 1987; Swain et al., 1987; Hadchouel et al., 1987). The fact that both the male and female contribution must be present in the egg to facilitate development to term, but that limited development can be obtained with eggs that lack one parental gene expression contribution, raises the question of when both maternal and paternal gene expression are necessary to achieve development. Barra and Renard (1988) have attempted to answer this by creating haploid eggs, culturing them to different stages, and diploidizing them by cell fusion. These eggs are then allowed to develop in uiuo. They have found that complementarity is not required by the pronuclear or two-cell stage (Barra and Renard, 1988); when eggs are combined at the four-cell stage they proceed to develop until at least 15 days of gestation ( J.-P. Renard, personal communication). Another approach to study possible cloning in mice is to transfer just half of the genome (Surani et al., 1986). This can be accomplished by creating a haploid, culturing it to a multicell stage, and then transferring these haploid nuclei to a haploid I-cell egg. This reestablishes the normal ploidy and the maternal-paternal complementarity necessary for complete development. Nuclei from 4-cell haploid androgenones can be transferred to haploid gynogenetic zygotes, and development will proceed to term. In comparison, nuclei from 16-cell haploid gynogenones can be transferred to haploid androgenetic zygotes and development can proceed to term. An analysis of the protein-synthetic profiles suggests that these advanced haploid nuclei are reprogrammed, but the eggs exhibit compaction earlier than controls (Surani et al., 1986). Therefore, we can see that a large amount of research has been conducted in the mouse. This research has been very rewarding by elucidating the mechanisms of murine genetics and early murine development. This, by extrapolation, has in turn led to many theories about mammalian devel-

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opment in general. However, as we shall see later, the direct application of theories regarding murine embryo development to other mammals should not be completed without careful evaluation. AND PIGS B. SHEEP,Cows, RABBITS,

Studies using nuclear transfer in mammals other than mice are very few. The reasons for this modest data base are multiple, but mainly involve the great financial costs and the disappointing result of McGrath and Solter (1984b) in the mouse. However, as we shall see, the mouse is a very poor model for nuclear transfer in mammals. So far the mouse appears to be the mammalian exception to the models for cloning as first developed in the amphibians (Briggs and King, 1952). This is suggested because cloned offspring have been born in sheep (Willadsen, 1986; Smith and Wilmut, 1988), cattle (Prather et al., 1987), rabbits (Stice and Robl, 1988),and pigs (Prather et al., 1989a),but not mice (McGrath and Solter, 1984b). Because of the limited data base and similarities in response to nuclear transfer between these four species, they will be discussed together. 1. Within Stage Within-stage nuclear transfer has only been reported in the cow (Robl et al., 1987) and pig (Prather et al. 1989a). An initial obstacle to conducting these experiments was the invisibility of pronuclei through the opaque cytoplasm. This difficulty was overcome by centrifuging the embryos at low speeds (Wall et al., 1985). The dense cytosol within the cytoplasm is translucent, while the less dense lipid granules are opaque (Fig. 1). Centrifugation causes these layers to become stratified within the egg. Centrifuged pig and cow eggs develop at a similar rate in uiuo as noncentrifuged eggs (Wall et al., 1985; Wall and Hawk, 1988). The method used for nuclear transfer in these species is a modification of that of McGrath and Solter (1983a), using electrically induced cell-cell fusion instead of viralmediated cell fusion. Pronuclear exchange in the bovine egg results in development to morula or blastocyst stage of -50% of the eggs as compared with unmanipulated controls (Robl et al., 1987), suggesting that the conditions employed, though effective, are less than ideal. The rate of fusion in porcine eggs was 76% (89/117: Prather et al., 1989a)as compared with 79% (15/19) in bovine eggs (Robl et al., 1987). Normal offspring have been produced in both species, thus illustrating that the conditions employed are compatible with complete and normal development (Robl et al., 1987; Prather et al., 1989a).

2. Cloning The first report in the literature of attempted cloning by nuclear transfer in nonmurine mammals was in the pig (Robl and First, 1983, followed by

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FIG. I . Centrifuged bovine zygote. The two pronuclei can be seen in the center of the egg (arrowheads). Note the stratification of the cytoplasmic contents. Bar = 10 pm.

sheep (Willadsen, 1986) and cows (Prather et al., 1986) and then rabbits (Stice et al., 1987).The common feature among the studies that resulted in development to term is the use of metaphase oocyte as a recipient (Willadsen, 1986; Prather et al., 1987, 1989a; Stice and Robl, 1988; Smith and Wilmut, 1988) as opposed to an enucleated pronuclear-stage egg as in the mouse (McGrath and Solter, 1984b) and rat (Kono et a/., 1988). Interestingly, the use of an enucleated zygote as a recipient for early cleavage stage eggs rarely results in cleavage in the bovine (Robl et al., 1987).As we shall see, very little research has described the developmental potential of nuclei or actual reprogramming in the domestic species. a. Efjciency of Nuclear Transfer. Since this field has received little study, it may be useful to review the basics and efficiencies of the micromanipulation procedures. When using meiotic metaphase I1 ooctyes as recipients, three major factors will limit subsequent development of the clone: enucleation of the oocyte, activation of the oocyte, and fusion of the enucleated ooctye with the transferred karyoplast. The first step in the nuclear transfer process is enucleation of the metaphase I1 oocyte. This is more readily accomplished in some species than others. In the cow the first polar body breaks down soon after extrusion

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and the enclosed chromatin is released into the perivitelline space. This is in contrast to the pig where the first polar body remains intact and attached to the plasma membrane overlying the metaphase chromosomes for as long as 48 hours after onset of estrus (Prather et al., 1988). Since the first polar body is used as a landmark to estimate the location of the metaphase I1 chromosomes, these characteristics may explain the enucleation efficiencies: cow 60%(Prather et al., 1987) and pig 74%(Prather et al., 1989a). In these species the metaphase I1 chromosomes are difficult to visualize even when in the pipette. However, in the rabbit the metaphase I1 chromosomes can be visualized in the pipette, and therefore even higher enucleation rates have been obtained (92%: Stice and Robl, 1988). In sheep this efficiency is 75% to 67% (Willadsen, 1986; Smith and Wilmut, 1988, respectively). The next requirement for success is activation of the oocyte. If the oocyte is not activated, a transferred nucleus will break down and the chromosomes will condense into a metaphaselike structure and enter a “meioticlike” arrest (Czolowska er al., 1984). In species where electrically induced activation has been evaluated, activation appears to be dependent on oocyte age and species. In the cow and pig, electrically induced activation can be >90% (Ware et al., 1989; Prather et al., 1988). However, the percentage activation in the rabbit currently appears to be much lower (52%: Stice and Robl, 1988). Fusion for cloning can be accomplished with Sendai virus or electrofusion. The percentage electrofusion is generally high for the four species in which it has been evaluated (cow, 75%: Prather et al., 1987; pig, 87%: Prather et al., 1989a; rabbit, 84%: Stice and Robl, 1988: Sheep, 90%: Willadsen, 1986; Smith and Wilmut, 1988). Sendai virus causes fusion in sheep (Willadsen, 1986) but is less effective in rabbit (Bromhall, 1975), rat (Kono er al., 1988), and cow embryos (Robl et al., 1987). Virus-mediated cell fusion has an advantage over electrically induced cell fusion, in that small cells can be fused to large cells virally, but large and small cells do not fuse as readily with electrofusion (Prather et al., 1987). The ability to transfer smaller cells provides the opportunity to study nuclear reprogramming and developmental potential in developmentally more advanced cells. The overall potential efficiencies are presented in Table I and show that on a per nuclear transfer basis, the cow, pig and rabbit have an equal likelihood of developing. This comparison, however, may be misleading, since the eggs in which fusion occurs may be the same eggs that are activated. If this is the case the overall efficiency may be 45% for the cow, 68% for the sheep, 62% for the pig, and 48% for the rabbit. These figures would not be taken as the maximum potential, but as a basal level when establishing a nuclear transfer program.

I82

RANDALL SCOTT PRATHER AND NEAL L. FIRST TABLE 1. PERCENTAGE ENUCLEATION, ACTIVATION, A N D FUSIONUSED IN THE CLONING PROCEDURE B Y NUCLEAR TRANSFER TO METAPHASE 11 OOCYTES'

Soecies

Enucleation

Activation

60 (1) 75 (2) 74 (3)

90 ( 5 ) NEb 81 (3) 52 (4)

-~

cow

Sheep Plg Rabbit

92 (4)

Fusion

Overall

75 (1)

41 NE 52 40

90 (6) 87 (3) 84 (4)

" Numbers in parentheses refer to references 1. Prather et ul (1987);2 Willadsen (1986), 3 Prather er cri I1989a). 4 Stice and Robl (1988).5 Ware p f nl 0989). 6 Smith and Wilmut (1988) Not evaluated

'

b. Developmental Potential. The developmental potential of nuclear transfer embryos as reported in the literature is very limited. Most reports of offspring to date have used donor nuclei derived from embryos that are not more than two cleavages beyond the major onset of zygotic transcription. In the cow the major transition occurs at the 8- to 16-cell stage (Barnes, 1988), and calves have resulted from 9- to 16- and 32-cell stage nuclei (Prather et al., 1987; N. L. First, unpublished). In the sheep the major transition occurs at the 8- to 16-cell stage (Crosby et al., 1988), and lambs have resulted from transfer of 16-cell donor nuclei (Willadsen, 1986; Smith and Wilmut, 1988). In the rabbit the major transition occurs at the 8-cell stage (Van Blerkom and McGaughey, 1978; Cotton et al., 1980) and %cell nuclei can promote development to term (Stice and Robl, 1988). In the pig the transition appears to occur at the 4-cell stage (Norberg, 1970, 1973), and 4-cell nuclei can direct the development of piglets (Prather et al., 1989a). Since the rat also appears to begin zygotic control of development at the 4-cell stage (Szollosi, 1966), it is interesting to speculate that if an enucleated oocyte is used as a recipient rather than an enucleated zygote (Kono et al., 1988), development may continue to term. In the instances just mentioned it would be difficult to state that much actual reprogramming had occurred, since few differentiation events had been detected by the time of the transfer. The only overt indication of reprogramming is the delay in the timing of blastocoele formation, such that blastocoele formation occurs on the temporal schedule of a pronuclearstage egg (cow: Prather et al., 1987; rabbit: Stice and Robl, 1988; pig: Prather et al., 1989a). Using more advanced-stage donor nuclei in both the sheep and the cow has resulted in at least limited development. In the sheep, inner cell mass nuclei can promote in uiuo development to the blastocyst stage and subsequently to term (Smith and Wilmut, 1988).

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Although data regarding development of nuclear transfer embryos from the blastocyst stage to term are few, a progressive decrease in pregnancy maintenance is observed. Nuclear transfer in cattle (Prather et al., 1987) can result in 20% morula-blastocyst formation. Of the morula-blastocyst embryos transferred, only 37% (7/ 19) established pregnancies, as confirmed by ultrasound between day 21 and day 30. Among the confirmed pregnancies, 71% were aborted and the recipients cycled by day 88. This resulted in two calves born (ll%, 2/19) as compared to an expected birth rate of 5 0 4 0 % for normal morula-blastocyst stage embryos. A similar result, of aborted conceptuses, is inferred from nuclear transfer in the pig. Three gilts, receiving 10 or more nuclear transfer embryos, had extended cycles of 28,52, and 72 days (Prather et al., 1989a).In Xenopus the rate of development of nuclei from late blastula and late gastrula stage embryos transferred to oocytes, to the blastula stage, tailbud tadpole, swimming tadpole, or young frog is 62%, 48%, 38%, and 35%, respectively (Gurdon, 1964). Of those that do not develop to young frogs, their developmental restriction point is stably inherited, as shown with serial nuclear transfer, and is likely due to karyotypic abnormalities (reviewed by DiBerardino, 1987). We hypothesize that the same mechanism is responsible for the limits of development attained in amphibians and mammals.

c . Nuclear Changes after Transfer. The changes that occur after transfer of a nucleus to an enucleated oocyte, both morphological and biochemical, are well characterized in amphibians (reviewed by Gurdon, 1986; DiBerardino, 1987; Prather, 1989). These changes include an increase in nuclear volume (Gurdon, I964), and exchange of proteins between the nucleus and the cytoplasm (Merriam, 1969; DiBerardino and Hoffner, 1971, 1975), highly specific gene regulation (Wakefield and Gurdon, 1983), and DNA synthesis in GI nuclei (DeRoeper et al., 1977). The extent of our knowledge in mammals is more limited. We do know that hamster nuclei transferred to metaphase I1 oocytes initiate DNA synthesis (Naish et al., 1987),and that nuclear transfer in rabbits and pigs results in a swelling of the transferred nucleus (Stice and Robl, 1988; Prather et al., 1988). There is also an acquisition by the transferred nucleus of A/C-like nuclear lamin proteins from the cytoplasm of the oocyte by the transferred nucleus. The A/C antigen is lost from the nuclei of the developing pig egg by the 8-cell stage. However, transfer of a 16-cell-stage nucleus to an enucleated metaphase I1 oocyte results in a positive A/C staining of the transferred nucleus (Prather et at., 1989b: Fig. 2). The same result is seen in the mouse; however, if 16-cell nuclei are transferred to enucleated zygotes, the transferred nuclei do not readily acquire the A/C antigen (Prather et al., 1989b).

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FIG. 2. Nuclear lamin epitope after transfer of a 16-cell-stage porcine nucleus to an activated, enucleated meiotic metaphase I1 oocyte. DNA stain Hoechst 33258 on (A) cells from a 16-cell stage egg; (C) pronuclear stage egg; and (E) the nuclear transfer egg; and corresponding lamin AIC reactivity (B. D, and F, respectively). Note the absence of lamin AIC reactivity in the 16-cell-stage blastomeres, whereas after transfer to an activated, enucleated metaphase I1 oocyte the nucleus acquires the antigen. (B, D, and E) photographed and developed under identical conditions. Bar = 10 pM.From Rather ef at. (1989b).

As can be deduced from the foregoing discussion, a very limited amount of research has been conducted on nuclear transfer in nonmurine mammals. It would be of great benefit, both scientifically and practically, of the efficiencies of the various nuclear transfer procedures to be increased and for a more thorough evaluation of various donor nuclei and the nuclear changes that are associated with each type of nuclear transfer to be carried out.

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IV. Model for Programming From this review it is anticipated that the reader will have a grasp of the types of nuclear transfers that result in sufficient chromatin remodeling and reprogramming for complete development to continue. Therefore in this section we propose a model that not only may be applicable to chromatin remodeling after nuclear transfer, but also will be suitable for describing events in early development. This model will require a few basic assumptions, the validity of which is uncertain: (1) the components responsible for nuclear modifications after transfer have an affinity for interphase nuclei. (2) The components responsible for the chromatin modifications disperse evenly into the cytoplasm during metaphase and have no affinity for metaphase chromosomes. Examples of proteins that exhibit this type of behavior but may or may not influence gene expression include nuclear lamins, which line the inner nuclear envelope and polymerize and depolymerize with the cell cycle (Gerace and Blobel, 1980), and small nuclear ribonuclear proteins, which are localized in the nucleus during interphase and are dispersed into the cytoplasm during mitosis (Lobo et al., 1987). A. METAPHASE VERSUS INTERPHASE RECIPIENT CELLS

Based on the foregoing assumptions, if a cell in interphase is used as a recipient and the interphase nucleus is removed during the nuclear transfer, then the components responsible for the chromatin remodeling are also removed. It is not suggested that the nuclear envelope serves as an impermeable container for these proteins (Feldher and Pomerantz, 1978), but as a scaffold containing selective protein-binding sites. Nuclear transfer to these enucleated interphase cells would not be expected to result in development to term. Examples include the cow (Robl et al., 1987), mouse (Robl e f al., 1986; McGrath and Solter, 1984b), and rat (Kono et al., 1988). However, if a cell in metaphase is used as a recipient, removal of the chromosomes would not result in removal of the remodeling components. Examples include the amphibian (Gurdon, 1986), the sheep (Willadsen, 1986; Smith and Wilmut, 1988), the cow (Prather et al., 1987), the rabbit (Stice and Robl, 1988), and the pig (Prather et al., 1989a). A notable exclusion here is the mouse, where nuclear transfer to an enucleated metaphase oocyte rarely results in cleavage J. M. Robl, personal communication); moreover, if the transfer is completed shortly after activation (4 hours postactivation), followed by removal of the maternal pronucleus (7 hours postactivation), then development can proceed to the blastocyst, but rarely does (McGrath and Solter, 1986). A modification of the nuclear

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transfer scheme requires transferring just half of the genome, either maternal or paternal (Surani et al., 1986). In this instance the resident pronucleus may retain sufficient remodeling components that after the first metaphase and completion of syngamy there is sufficient reprogramming for complete development. However, the resulting embryos compact early, suggesting incomplete chromatin remodeling. Finally, to explain the results of Tsunoda et al., (1987) we suggest that the cooling process employed somehow disassociated the remodeling components from the interphase nuclei before enucleation, and therefore these components were present in the recipient cell to act on the transferred nucleus and subsequently permit a continuation of development. This is suggested because Rob1 et al. (1986), using procedures similar in all respects except for the cooling, did not obtain development to term. The effects of cooling donor nuclei in some amphibians (Rana: Hennen, 1970) indicate that it would be of interest to test directly these effects on development after nuclear transfer in mammals. Therefore we hypothesize that there are components in the cytoplasm of a metaphase stage cell that are in a limiting and labile state. Upon formation of the nucleus, these components associate with and modify the chromatin such that specific gene expression is altered to be consistent with nuclei that are normally found in the cytoplasm of the metaphase stage cell.

B. DIFFERENTIATIVE STATEOF THE DONORNUCLEUS The state of differentiation of the donor nucleus has only begun to be evaluated in mammals. In amphibians the degree of differentiation and the length of the cell cycle are confounded, as more differentiated cells have longer cell cycles. Many highly differentiated nuclei, such as those of erythrocytes (Orr et al., 1986) and keratinized epithelial cells (Gurdon et af., 1975), are capable of promoting limited cleavage after transfer. Since the length of the cell cycle is early amphibian eggs is so very short (35 minutes: Newport and Kirschner, 1982) as compared with mammals 2 1 5 hours: reviewed by First and Barnes, 1989), an answer may come from mammalian studies and may give direction for answering the same question in amphibians. OF THE NUCLEAR COMPONENTS C. MODIFICATIONS

Nuclei that swell after nuclear transfer are considered to be reprogrammed (Gurdon, 1964). Swelling may be a result of the exchange of proteins between the cytoplasm and the nucleus (Merriam, 1969; DiBerardino and

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Hoffner, 1971, 1975; Prather el al., 1989b), resulting in specific gene regulation (reviewed by Gurdon, 1986; DiBerardino, 1987; Prather, 1989). But how is this gene regulation accomplished? In a certain amphibians the addition of spermine (Hennen, 1970)or protamines (Brothers, 1985)during nuclear transfer enhances the developmental potential of nuclei. Spermine interacts with chromatin, affects the spatial conformation of DNA and RNA, and stimulates both RNA chain initiation and chain elongation (Karpetsky et al., 1977).Interestingly, disruption of the synthetic pathway of ornithine to spermine affects differentiation in both plants and animals (Malmberg et al., 1985; Alexandre and Gueskins, 1984; Lane and Davis, 1984).

It will be important to test other compounds that affect differentiation. Then we may be able to begin to develop comprehensive hypotheses that will adequately describe differentiation at the molecular level.

V. Conclusions and Future Prospects This review illustrates the importance of species comparisons and the danger of extrapolating results from one species to another. Nuclear transfer for cloning has been successful in a variety of mammals, excluding the mouse. This suggests that careful between-species comparisons should be made before any overall hypothesis regarding nuclear transfer is made. The comparisons need to include an evaluation of the most advanced stage of donor nuclei capable of promoting development and the length of the cell cycle of these nuclei, what components aid in promoting development (cooling, spermine, protamines), and evaluation of nonnuclear inheritance, and the possibility of using nondifferentiated embryonal carcinoma or embryonic stemlike cells as a source of donor nuclei. ACKNOWLEDGMENTS The authors would like to acknowledge funding by the United States Department of AgricuIture and W. R. Grace and Company. Helpful critiques of all or portions of the manuscript were provided by Dan Hagen, J. P. Renard, Jim Robl, and the laboratory group.

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