MANIPULATION OF THE MAMMALIAN EMBRYO 1

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1 MANIPULATION OF THE MAMMALIAN EMBRYO 1 G. B. Anderson 2 University of California, Davis Summary Technological advances in manipulation of mammalian embryos outside the maternal environment have resulted in opportunities for study of preimplantation embryo development, identification of developmental phenomena that are unique to mammals, and further improvement of technology. Mammalian embryos may be cultured in vitro at 37 C for up to several days or they may be stored at 196 C indefinitely. The mammalian embryo possesses the unique capacity to regulate its development and differentiate into a normal individual after being stimulated to incorporate foreign cells or after a portion of its cells are removed. This regulatory ability has proven useful in research dealing with the production of chimeras. It allows genetic copies of an embryo to be produced by dissociation of an early cleavage-stage embryo into its component blastomeres or by bisection of a morula. Production of large sets of identical animals may be possible in the future by serial transplantation of nuclei from one embryo into enucleated ova. Progress has been made in producing unique genetic combinations by manipulation of the pronuclei of fertilized ova. It is also possible in some cases to identify the sex of a living cleavage-stage embryo. Some of these manipulations have been carried out primarily in laboratory mice, but as animal scientists identify beneficial uses in farm animals, these procedures are being extended to embryos of the large domestic species. (Key Words: Embryo, Embryo Manipulation, Mammalian, Biotechnology.) Introduction Because of difficulty with manipulation of lthe assistance of Susan Donahue, Mary Horton, Suzanne Jones and Shelle Winkler in preparation of this manuscript is gratefully acknowledged. 2 Dept. of Anim. Sci. mammalian embryos in the laboratory, less is known about early development of mammals than of invertebrates and nonmammalian vertebrates. Research contributing to the understanding of requirements for viability of mammalian embryos outside the maternal environment has enabled progress to be made in manipulation of mammalian embryos. Laboratory mice have been the most commonly used animal in studies of mammalian embryo development, but establishment of the commercial embryo transfer industry has provided the impetus for extending studies of embryo development to large domestic animals and to primates. In keeping with the purpose of this symposium, I will present an overview of procedures dealing with manipulation of mammalian embryos and describe potential applications of these procedures to large animal production. The areas covered include storage in vitro, identification of sex, manipulations that exploit unique abilities of mammalian embryos to regulate their development, and surgical procedures that may be applied to embryos. Exciting new developments in maturation of oocytes in vitro, fertilization in vitro and gene transfer are covered elsewhere in this symposium. Storage of Embryos Removal of embryos from the reproductive tract requires conditions that maintain viability, or the ability of the embryo to develop normally when transferred to the female tract. Optimum conditions for maintenance of embryo viability depend on a number of factors. One of the more important of these is the duration of storage. Storage for only a few hours may require different conditions than storage for several days. For example, phosphate buffered saline (PBS) supplemented with serum is commonly used to store bovine embryos during the time between collection from a donor and transfer to recipients. Often, because of the stability of this medium, embryos are held at RNAL OF ANIMAL SCIENCE, Vol. 61, Suppl. 3, 1985

2 2 ANDERSON ambient temperature and with no special control of the gaseous atmosphere. While use of PBS yields acceptable results when embryos are held for short periods (i.e., less than 1 d), support of continued development for longer periods requires different culture media and stricter control of the conditions under which the embryos are incubated. Transfer of embryos to the reproductive tract of heterologous species may also provide adequate short-term storage and continued development. If it is desired that embryos be held for several days at a specific stage of development, they may be refrigerated. This method requires a medium different than that used for either storage for several hours or for culture at body temperature. Long-term storage at 196 C requires yet another set of conditions. No system has yet been devised for maintaining embryos of farm animals at body temperature with development arrested, as can be accomplished in those mammals where delayed implantation may be induced or may occur spontaneously (e.g., mouse and rat). Storage at Body Temperature. Embryos may be stored by transfer to the reproductive tract of another female, which serves as an incubator in which development may continue. The host female and embryo may be of either the same or different species. In some cases it is desirable that the transfer be made to a female of the same species, especially if some criticial developmental process is to occur. For example, bovine oocytes that are removed from their follicles and complete meiosis I in vitro may be transferred to the oviducts of previously inseminated heifers, where they are fertilized and begin early development (Newcomb et al., 1978). Normal fertilization of oocytes matured in vitro occurs infrequently either in vitro or in the oviducts of a different species (DeMayo et al., 1980). In many cases, however, the reproductive tract of one of the laboratory species provides a convenient and acceptable incubator for embryos. Oviducts of rabbits have been used to store embryos of pigs (Polge et al., 1972), sheep (Lawson et al., 1972a), cows (Lawson et al., 1972b; Trounson et al., 1976; Boland et al., 1978) and mares (Allen et al., 1976). The ability of embryos to develop into viable young may be retained in rabbit oviducts for as long as 3 to 4 d, making this system useful for long-distance transport of embryos (Hunter et al., 1962; Allen et al, 1976). The usefulness of temporary culture in the oviduct of another animal has been overshadowed by development of systems for storing embryos in vitro, which often provide equivalent survival with greater convenience. For some types of manipulations, however, development of the embryo proceeds more successfully in vivo than in vitro, and the oviducts of an intermediate host remains an important step in the manipulation procedure. For example, culture of individual blastomeres of a cleavage-stage embryo may be performed more successfully in vivo than in vitro (Willadsen, 1982). Embryos may be cultured in vitro in a variety of culture systems and under a variety of environmental conditions (reviewed by Maurer, 1976; Brackett, 1981; Wright and Bondioli, 1981; Anderson, 1983). The first successful culture systems to employ chemically defined media were developed for mice (Brinster, 1963; Whitten and Biggers, 1968) and rabbits (Kane and Foote, 1970). By virtue of employing chemically defined media, these culture systems provided reasonably consistent results, which are not always possible using undefined biological media. Even synthetic media usually contain bovine serum albumin (BSA), a component that is not strictly chemically defined but is necessary for development in vitro. A number of hypotheses have been proposed to explain the critical need for BSA in culture media (see Brackett, 1981, for further discussion). Principles learned from culturing embryos of laboratory species have been applied to establishment of culture systems that support development of embryos from farm animals and, more recently, primates. In most cases, development in vitro proceeds to the blastocyst stage, at which time implantation occurs in most rodents and primates. A method has recently been described for culturing mouse embryos from preimplantation blastocysts to the limb bud stage where embryos showed blood circulation in the yolk sac and primordia of liver, pancreas and lungs (Chen and Hsu, 1982), development consistent with approximately the first half of gestation in this species. Cultured blastocysts of some farm animal species also have been shown to hatch from the zona pellucida, attach themselves to a substratum and exhibit outgrowth of trophoblast cells (Shaffer and Wright, 1978; Kuzan and Wright, 1982). Requirements for culture of embryos vary with animal species, and in some cases with genetic strain within a species. Culture systems

3 EMBRYO MANIPULATION 3 that support development in vitro of mouse embryos are quite different than those for rabbit embryos. Sheep (Peters et al., 1977) and cattle (Wright et al., 1976) have been shown to develop in vitro from one-celled zygotes into blastocysts, but substantially different culture media are required. Culture systems that support development of cleavage-stage goat (Wright and Bondioli, 1981) and horse (Imel et al., 1981) embryos to the blastocyst stage are not yet available. The basis of these species differences is unknown. Stage of embryo development at which culture is initiated is also an important variable affecting success of culture. In general, early cleavage-stage embryos cannot be cultured to the blastocyst stage as successfully as later stage embryos. This may be due in part to the length of time required to reach the blastocyst stage, but is also related to greater difficulty in promoting early cleavages in vitro. References were cited above for experiments in which one-celled zygotes developed in vitro to the blastocyst stage, but success in culture from the one-cell stage is relatively low. Even for mice and rabbits, zygotes from some strains may be cultured more successfully than those of other strains. As nonsurgical techniques were developed for flushing embryos from cattle and horses, interest waned in culturing early stage embryos, because nonsurgical collection produces morulae and blastocysts that will readily continue to develop in vitro. Questions still remain about how requirements for development of premorula-stage embryos differ from those of later embryos. Brackett (1981) has discussed the shifts in metabolism that occur during preimplantation development and how they may affect requirements for successful culture. Previous treatment that an embryo has received may also affect survival in vitro. For example, embryos that have been frozen and thawed may not survive well in a culture system that is adequate for fresh embryos, but may develop if transferred into recipients (Tervit and Elsden, 1981). In spite of widespread use of cultured embryos for studying developmental processes and in applications of embryo transfer technology, there is still question regarding how closely development in vitro parallels development in vivo. Ultrastructural features and embryonic protein profiles have been reported to be similar for embryos that developed in vitro and in vivo (VanBlerkom et al., 1973; VanBlerkom and Manes, 1974). In contrast, differences in size and in synthesis of macromolecules have been described between embryos cultured in vitro and in vivo (Anderson and Foote, 1975a,b; Beier and Maurer, 1975). Qualitative and subjective appraisals of rates of embryonic development in specific culture systems often indicate that development in vitro and in vivo are equivalent. From controlled studies in mice (Bowman and McLaren, 1970), rabbits (Binkerd and Anderson, 1979) and monkeys (Kreitmann and Hodgen, 1981), however, it has been concluded that cleavage rates are generally slower in vitro than in vivo. Slower in vitro development has also been reported in studies with sheep embryos (Tervit and Rowson, 1974). Perhaps most disturbing in terms of differences between in vitro and in vivo development are reports that embryos fail to maintain normal viability in vitro, even though development continues and gross morphology is normal. The magnitude of loss of viability tends to be a function of time; embryos cultured for short periods (<24 h) are reasonably viable, but survival after transfer decreases as embryos are cultured for longer periods (Hahn and Schneider, 1982). Bowman and McLaren (1970) transferred mouse blastocysts cultured from the eight-cell stage and reported reduced survival and fetal weights compared with noncultured embryos. Rabbit blastocysts cultured from early cleavage stages have also been reported to have dramatically reduced viability (Schneider, 1977; Maurer, 1978; Binkerd and Anderson, 1979). Adjustment of the luteal age of the recipient to compensate for slower rates of development in vitro has been shown to affect survival, but not completely reverse the negative effects of long-term culture (Binkerd and Anderson, 1979). Reduced survival of cultured embryos has also been reported for the horse (Imel et al., 1981), pig (Pope and Day, 1977), sheep (Peters et al., 1977) and cow (Hahn et al., 1978; Renard et al., 1978, 1980). While these reports for cow embryos indicated reduced viability after 24-h culture, other researchers have reported normal survival after culture of cow embryos for 24 h (Peters et al., 1978; Schneider et al., 1980). In spite of results indicating that survival after transfer of cultured embryos decreases as length of culture period increases, pregnancies have been established with bovine embryos cultured as long as 4 d (Tervit et al., 1972) and with sheep embryos cultured as long

4 4 ANDERSON as 6 d (Tervit and Rowson, 1974). While these reports demonstrate that embryos can survive long-term culture, they probably represent exceptional cases and viability that cannot be routinely expected with our current knowledge of requirements for in vitro development. Renard et al. (1980) have reported that higher than usual embryonic losses occur between d 21 and 60 of pregnancy in bovine embryos that were cultured for 24 h. Additional research is needed to determine whether this represents an isolated or a recurrent event. Storage at Refrigeration Temperatures. When mammalian embryos are stored at body temperature, or even room temperature, they continue to develop. When they are cooled to below 20 C, however, mitosis is halted and they remain at the stage of development at which they were cooled. This may be desirable when there is an inadequate supply of synchronized recipients; as additional recipients reach the appropriate stage of the estrous cycle, embryos may be warmed and transferred. As early as 1947, Chang demonstrated that mammalian embryos can survive refrigeration for several days. Viable lambs have been obtained after refrigeration of sheep embryos for as long as 10 d (Kardymowicz, 1972), but, generally, refrigeration of embryos is considered to be a means of short-term storage, with viability maintained for only a few days. Considerable variability exists among species in embryo survival after cold storage. Rabbit embryos stored at 4 C for 7 d retain viability similar to nonstored embryos, as measured by development to term after transfer to recipients (Hughes and Anderson, 1982). Bovine embryos stored under the same conditions survive only 2 d (BonDurant et al., 1982; Lindner et al., 1983). Pig embryos do not survive cooling to refrigeration temperatures for even short periods (Wilmut, 1972). There is some evidence that cells from the inner cell mass (ICM) may be more susceptible to damage by prolonged exposure to refrigeration temperatures than are cells of the trophoblast (Lindner et al., 1983). Freezing of Embryos. For long-term storage, embryos may be frozen to 196 C in liquid nitrogen and held indefinitely. In farm animals, freezing of embryos facilitates transport and sale of embryos, as well as preservation of excess embryos. Little advantage in terms of genetic improvement may be realized through embryo freezing, however, because the genotype of the embryo is established, but the population is improving. For inbred laboratory animals, freezing provides the opportunity to create embryo banks and a method of preserving important genotypes for later reestablishment. The first successful method for freezing and thawing of mammalian embryos was reported for mice (Whittingham et al., 1972), and techniques for other mammals followed soon thereafter. Evolution of this freeze-thaw technique resulted from the collaborative efforts of cryobiologists and embryologists. The large size of ova, their low surface to volume ratio and their low permeability to water at low temperatures required unusually slow freezing and thawing rates. In general, freezing rates that are too fast do not provide sufficient time for the cells to dehydrate, allowing intracellular crystallization. Death of the embryo is not likely to occur at 196 C, but rather at intermediate temperatures during cooling and warming (Mazur, 1978). The crybiological theory that supports development of embryo freezing and thawing technology is available elsewhere (Mazur, 1977; Leibo, 1981). Early procedures for freezing of mouse embryos relied upon slow cooling (^.5 C/min) to approximately 80 C, after which embryos were immersed in liquid nitrogen. They were later thawed by slow warming (Whittingham et al., 1972). Willadsen etal. (1977) reported that bovine embryos could be plunged into liquid nitrogen (i.e., rapidly cooled) from 33 C, but subsequent survival depended upon rapid thawing. If such embryos are warmed slowly, intracellular recrystallization (crystal growth) occurs during thawing and may be associated, perhaps indirectly, with failure of the embryo to survive (Rail, 1981; Lehn-Jensen and Rail, 1983). These protocols require careful control of temperature changes. Recently, a new procedure has been introduced that may significantly reduce the time and equipment required, thus making it more attractive for on-the-farm freezing of embryos (Kasai et al., 1980; Wood and Farrant, 1981). Called the two-step method, it involves rapid cooling of embryos to an intermediate temperature (^ 20 C) at which they are temporarily held before immersion in liquid nitrogen. A modification includes rapid cooling first to -v-20 C, then to ^ 100 C before immersion at 196 C (Kasai et al., 1980). Pregnancy rates achieved with frozen embryos sometimes approach those with fresh embryos (Niemann et al., 1982), but in many

5 EMBRYO MANIPULATION 5 studies, selection of embryos results in transfer of only those embryos that are morphologically normal after freezing and thawing. With current technology, some loss of embryos is to be expected during the freeze-thaw process. Considering the short history of embryo freezing, based on research with mice embryos, survival after freezing and thawing has been reported for a remarkable number of species, including cattle (Wilmut and Rowson, 1973), sheep (Willadsen et al, 1976), horses (Griffin et al., 1981), goats (Bilton and Moore, 1976), rabbits (Bank and Maurer, 1974) and rats (Whittingham, 1975). Interest is particularly strong in improving the success rate and practicality of freezing bovine embryos. Embryos may be frozen in plastic straws used for nonsurgical embryo transfer. Because cryoprotectant must be diluted from the embryo after thawing, the embryo must be removed, moved through a series of diluents and then replaced in a straw for nonsurgical transfer. Efforts are being made to devise a successful means of diluting the cryoprotectant without removing the embryo from the freezing straw (Renard et al, 1982, 1983). In some trials in which this procedure was used, pregnancy rates approached those for other dilution procedures. Another simplification of the freeze-thaw procedure being tested is freezing embryos in plastic straws by a modified two-step process (Bouyssou and Chupin, 1982; Massip et al., 1982). Problems that still exist include the loss of embryos during the freezethaw process, accuracy in determining embryo survival after freezing and thawing, and assessment of the role of embryo quality in the survival of frozen embryos (Kennedy et al., 1983). Identification of Sex of Preimplantation Embryos One of the uncertainties associated with using embryo transfer procedures in farm animals is the sex of the resulting offspring. This is of particular concern when the economic value of one sex is greater than that of the other, as may be the case with both beef and dairy cattle. A reliable test is needed for sexing embryos without reducing viability. Currently, no consistently successful method exists, but identification of sex chromosomes and immunological detection of a male-specific antigen are each useful under some conditions. Hare et al. (1976) described a technique for identifying the sex of d 12 to 15 bovine embryos by chromosome analysis of a biopsy. Results using this technique were encouraging, but several drawbacks prevented its widespread use. Approximtely 68% of the attempts resulted in successful sexing, but a pregnancy rate of only approximately 33% could be achieved with these later stage embryos (Betteridge et al., 1981). Furthermore, embryos at this stage do not survive freezing and thawing, requiring immediate transfer after chromosomal analysis. Efforts to identify the sex of d 6 to 7 bovine embryos have yielded variable results (Moustafa et al., 1978; Singh and Hare, 1980). In one report (Moustafa et al., 1978), sex could be determined in 59% of the embryos and an acceptable pregnancy rate was achieved after a small number of transfers. In another report (Singh and Hare, 1980), in only 33% of the embryos could sex be correctly identified. These authors indicated that the low mitotic index of bovine morulae would likely prevent consistently acceptable results. They also reported difficulty removing cells from bovine blastocysts. The relative complexity associated with identifying sex chromosomes of species other than cattle reduces the overall usefulness of chromosomal analysis to sex embryos. A recent approach to sexing preimplantation mammalian embryos is immunological detection of male-specific antigen, referred to here as H-Y antigen (Wachtel et al., 1981; Ohno, 1982), although others have used the term SDM antigen (serologically-detectable male-specific antigen; Silvers et al., 1982). Krco and Goldberg (1976) reported the detection of H-Y antigen on eight-cell mouse embryos. From a developmental standpoint, this was an interesting observation because it provided additional evidence for early expression of the embryonic genome. Because H-Y antigen is thought to be coded on the Y-chromosome, its presence demonstrates expression of a paternal allele at the eight-cell stage. [Recent evidence supports expression of the paternal genome as early as the two-cell stage (Sawicki et al., 1981).] Epstein et al. (1980) cultured mouse embryos with H-Y antiserum and complement, and karyotyped unaffected embryos, 92% of which were found to be female. White et al. (1982) transferred unaffected mouse embryos to recipients and obtained 86% females from embryos cultured in H-Y antiserum and guinea pig complement, and approximately 50%

6 6 ANDERSON females in a variety of control media. A shortcoming of using complement-mediated cytolysis to detect H-Y antigen is the destruction of male embryos. Use of monoclonal H-Y antibody and fluorescent second antibody has been shown to determine sex of mouse embryos with approximately 80% accuracy and to maintain viability of both male and female embryos (White et al, 1983). Mouse H-Y antiserum has been used to detect H-Y antigen on cells of a number of other mammalian species (Wachtel et al., 1975). Because this antigen appears to lack species-specificity, its detection may provide a method of identifying sex of embryos from species other than the mouse. Use of mouse H-Y antiserum and an indirect immunofluorescent assay to identify sex of bovine embryos has yielded results similar to those obtained with mouse embryos (White et al., 1984). Examples of Unique Regulatory Ability During Mammalian Embryo Development Production of Chimeras. The unique ability of the mammalian embryo to regulate its early development is demonstrated by the production of chimeras. A chimera is a composite animal (or plant) in which the cell population is derived from more than one fertilized egg, or the union of more than two gametes. These composite animals are also termed allophenic or quadriparental. Chimeras are usually produced experimentally by one of two techniques, aggregation of two cleavage-stage embryos (aggregation chimeras) or injection of one or more cells from one embryo into the blastocoele of another embryo (injection chimeras; McLaren, 1976). The majority of chimeras produced in the laboratory have been mice, although they have also been produced in sheep (Tucker et al., 1974), rabbits (Gardner and Munro, 1974; Babinet and Bordenave, 1980) and rats (Mayer and Fritz, 1974). Viable interspecific chimeras have also been reported between two species of mice (Rossant and Frels, 1980) and between sheep and goats (cited by Seidel, 1983). Experimentally produced chimeras have been used in a variety of studies in developmental biology. Intra- and interspecific chimeras are useful for investigating cell lineage and clonal growth, as well as other aspects of differentiation, because growth and movement of cells may be observed. Chimeras are also useful for studying certain types of abnormal development by observing the individual produced from combining an abnormal with a normal embryo. The examples that follow illustrate the types of studies that are possible. Parthenogenetic mouse embryos often undergo apparently normal cleavage and blastocyst formation, appear to have a normal diploid chromosome complement, and may even implant in the uterus; however, all die by midgestation. When parthenogenetically activated embryos are aggregated with normally fertilized embryos, cells from the parthenogenome are able to contribute to development of a normal chimeric mouse (Surani et al., 1977; Stevens, 1978), demonstrating their ability to undergo normal development in certain environments. Germ cell chimerism has been observed in these chimeric individuals, and fully functional totipotent ova of parthenogenetic origin have been produced. Chimeras can be used to examine genetic abnormalities as shown by studies involving the sex-reversed (Sxr) mutation in mice, where individuals of the genotype X/X, Sxr/+ are sterile phenotypic males. Bradbury (1983) produced a female chimeric mouse by aggregating a normal embryo with one carrying the sex-reversed aberration. The germ line of this animal was entirely composed of X/X, Sxr/+ cells capable of producing fully functional oocytes. Because X/X, Sxr/+ germ cells are usually found only in testes where they are incapable of forming gametes, the importance of the environment to normal development is clearly demonstrated. One of the more extreme examples of the use of chimeras to redirect abnormal development is the reversal of embryonal carcinoma cells from the malignant state. Embryonal carcinoma cells are malignant cells derived from a type of tumor called a teratocarcinoma (see Stevens, 1981; Martin, 1978 for detailed descriptions). Chimeras can be formed when teratocarcinoma cells are injected into the blastocoeles of normal blastocysts (Brinster, 1974; Mintz and Illmensee, 1975; Papaioannou et al., 1975) or by aggregation of teratocarcinoma cells with normal embryos (Stewart, 1982). Not only are normal chimeric mice produced that show no signs of malignancy, but germ cells may be derived from the teratocarcinoma line, producing normal offspring whose genetic mother was a tumor cell. Interspecific chimeras have proven useful for studying differentiation in the early embryo, because the chromosomes of each species uni-

7 EMBRYO MANIPULATION 7 quely mark each cell. Interspecific chimeras are also useful for studying hybridization of species. Chimeras between two species of mice may be chimeric in their germ cell populations and produce hybrid offspring when mated to one of the two parental types (Rossant et al., 1982). It has been concluded from this observation that failure of a hybrid fetus to develop to term in a cross of two species may be due to immunological incompatibility between the mother and fetus. Production of Sets of Identical Offspring, In addition to the mammalian embryo's unique ability to incorporate foreign cells, the embryo may also continue normal development when a portion is destroyed or removed. This discovery has been utilized in studying retention of totipotency by individual blastomeres or groups of embryonic cells. The ability of a complete mammalian embryo to develop from a fraction of its original cells has recently been applied to producing multiplets from a single embryo. Two approaches to producing multiplets are bisection of a morula into halves or quarters and separation of an early cleavage-embryo into its component blastomeres. The technology for bisection of morulae to produce monozygotic twins is developing rapidly. This procedure is finding immediate application in the commercial bovine embryo transfer industry where twins are produced from particularly valuable embryos may be desirable. Pregnancy rates after nonsurgical transfer of half-embryos approximate those with whole embryos and the technique may be used to simply increase the number of calves produced from embryo of a superovulated donor cow (Ozil et al., 1982; G. Seidel, personal communication). Techniques vary considerably and skill is required for successful production of half-embryos (Williams and Seidel, 1983; Lambeth et al., 1983). Splitting bovine morulae into quarters does not increase the number of calves produced compared with splitting the embryo into halves, probably because of the greatly reduced cytoplasm available in each quarter-embryo (Willadsen et al., 1981). A second approach to producing identical animals is separation of early cleavage-stage embryos into their component blastomeres. Early research with mouse embryos indicated that single blastomeres from two-cell embryos will readily develop to the blastocyst stage and subsequently into viable young, but development of blastomeres from four- and eight-cell embryos is less likely (Tarkowski and Wroblewska, 1967; Fiser and Macpherson, 1976; Rossant, 1976). It appears this lack of development is not due to restricted developmental potential of individual cells, but rather to the low number of cells present at the time of blastocyst formation. This problem may be peculiar to mouse embryos, which form blastocysts after fewer cleavage divisions than other species. Viable young have been produced from single blastomeres taken from four- and eight-cell embryos of rabbits (Moore et al, 1968), sheep (Willadsen, 1981), cows, pigs and horses (Willadsen, 1982). The greatest success to date has been achieved with sheep embryos using a method described by Willadsen (1979, 1980), whereby each isolated blastomere is transferred into a foreign empty zona pellucida, embedded in agar and transferred for several days to the oviduct of a temporary recipient ewe. This intermediate culture in vivo is used because of the difficulty in achieving normal development of early cleavage-stage embryos in vitro. Embryos are recollected at approximately 5 to 7 d of age, removed from the agar and transferred to suitable recipients. Genetically identical triplet and quadruplet lambs have been produced with single blastomeres from a four-cell embryo and pairs of blastomeres from an eight-cell embryo, respectively (Willadsen, 1981). Attempts to culture isolated blastomeres from early porcine embryos have also been reported (Menino and Wright, 1983), but the resulting embryos were not transferred. Totipotency of individual blastomeres of eight-cell sheep embryos, as well as production of up to five genetically identical lambs from a single embryo, have been reported by Willadsen and Fehilly (1983). These researchers incorporated into the procedure previously described for producing lambs from single blastomeres a step that allowed isolation of single blastomeres from an eight-cell embryo without the drastic reduction in cytoplasm that would otherwise accompany such a procedure. Their modification was based on observations by Kelly et al. (1978) and Spindle (1982) that presumptive ICM cells can be identified in the early cleavagestage embryo. In general, mitotically advanced cells (i.e., the first to undergo mitosis at each cleavage division) are likely to be incorporated into ICM. Later cleaving cells, or less advanced cells, tend to form trophectoderm or cells of the trophoblast. Because the embryo is derived

8 8 ANDERSON from ICM, it was correctly reasoned that the combination of a single blastomere from a fourcell embryo with a single blastomere from an eight-cell embryo would result in an individual with a genotype derived exclusively from the eight-cell embryo. Cells derived from the fourcell embryo contributed primarily to trophectoderm and, therefore, placental membranes. The techniques for producing identical offspring may be combined with other manipulations. One such combination is the freezing of half- and quarter-embryos. This has been performed successfully with embryos from cattle (Lehn-Jensen and Willadsen, 1983), sheep (Willadsen, 1980), rats and rabbits (Hiroshi and Ogawa, 1981), and mice and rabbits (Hiroshi et al., 1982). In some of these reports, embryos appeared to be morphologically normal after freezing and thawing, but failed to develop after transfer; in others viable offspring were produced. The applications of combining these techniques are many and as yet not thoroughly exploited. If the number of identical blastocysts from a single embryo can be increased, it may be possible to transfer some immediately and freeze the rest for long-term storage. After performance or progeny testing, decisions can be made regarding the desirability of producing young from the embryos in storage. Freezing identical embryos also allows the birth of genetically identical young into different environments and at different times. A female may even serve as recipient of an embryo that is her identical sibling. Genetically identical animals are useful as research animals. If multiplets are assigned to various treatments of an experiment, individual variation will be reduced and treatment effects more accurately measured. Production of genetically identical animals has stimulated interest in research concerning phenotypic differences in such individuals. It is known that genetically identical individuals are not exact phenotypic copies of one another. Expression of certain traits (e.g., some coat-color spotting patterns) is not solely controlled by genotype. Two genetically identical animals may show slight variations in spotting patterns. It is not known at this time how many production traits (e.g., milk production, growth rate, fertility) are affected in a similar fashion. The corollary is that offspring that are genetically identical may, for some traits, be more similar to one another than can be accounted for by genetics alone. Gartner and Baunack (1981) reported that monozygotic twins produced from highly inbred mice were more similar to one another for certain traits than could be accounted for by residual heterozygosity in the line. They suggested this may have been due to nongenetic influences that occur as early as the third cleavage division. These interesting questions may be studied now that production of sets of genetically identical individuals is feasible. Other Micromanipulation Procedures The removal and transplantation of pronuclei from zygotes and nuclei from embryos provide new approaches to studying development and may have applications to animal agriculture. Amphibian nuclear transplantation has been performed for a number of years (Gurdon, 1974; McKinnell, 1981; Etkin, 1982). The usefulness of this technique in amphibia is related only indirectly to principles of animal breeding; it has been used primarily to study the ability of nuclei from more or less differentiated tissue to support normal development when transplanted into an activated egg. Illmensee and Hoppe (1981) reported the first successful nuclear transplantations in mammals that resulted in the birth of viable young. In this research, nuclei were transplanted from ICM into activated zygotes whose pronuclei had been removed. These same researchers (Hoppe and Illmensee, 1982) also reported full-term development of mice after transplantation of parthenogenetic embryonic nuclei into fertilized eggs. Neither of these accomplishments has been duplicated by other researchers, which may reflect the skill required to successfully perform nuclear transplantation in mammals. Another technique using microsurgery and virus-mediated cell fusion for transplantation of pronuclei into mammalian zygotes has recently been described (McGrath and Solter, 1983). This technique offers exciting possibilities for measuring the effects of cytoplasmic inheritance by allowing exchange of pronuclei between zygotes. Nucleo-cytoplasmic interactions have been studied in the past by virus-mediated cell fusion between oocytes and other embryonic or differentiated cells (Soupart et al., 1978; Tarkowski, 1982). It has been demonstrated by transfer of cytoplasm between embryos that the genetic origin of the cytoplasm can have profound effects on preimplantation development (Muggleton-Harris et al., 1982). Removal of pronuclei from developing mam-

9 EMBRYO MANIPULATION 9 malian zygotes has also been reported as a means of producing completely homozygous offspring (Markert and Petters, 1977; Hoppe and Illmensee, 1977). This was accomplished in mice by microsurgically removing one pronucleus, either male or female, from a fertilized egg. The activated, haploid egg could be diploidized by incubation in cytochalasin B and cultured to the blastocyst stage in vitro. Development to term after transfer at the blastocyst stage to the reproductive tract of a recipient female was reported by Hoppe and Illmensee (1977). This procedure may be useful for rapidly producing highly inbred strains of animals. It may be more difficult to accomplish in livestock because of greater difficulty in visualizing the pronuclei. Another complication is the existence in livestock of undesirable recessive genes, which have been eliminated from many inbred strains of laboratory species. One intriguing aspect of this procedure is that either pronucleus is able to support subsequent development. It would seem possible then to remove one pronucleus and replace it with another pronucleus from the same sex, thereby producing offspring with two male or two female parents rather than one of each. Because of our ability to assess genetic superiority more accurately in the male, it may be particularly useful in animal agriculture to produce offspring from two males. For example, the use in artificial insemination of semen from a bull produced from two of the top proven dairy sires may increase the rate of genetic gain. Conclusion The livestock and dairy industries can benefit from adopting new technologies arising from research with early embryos. A good example is the Widespread use of embryo transfer in farm animals, which in turn has stimulated efforts to refine techniques for manipulating embryos and to expand technologies for future applications. 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