In vitro development of the mammalian embryo

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    In Vitro Development of the Mammalian Embryo MRINAL K. SANYAL AND FREDERICK NAFTOLIN Department of Obstetrics and Gynecology, Yale University School of Medicine, New Hauen, Connecticut 06510

    ABSTRACT Normal growth and differentiation of mammalian embryos in vitro during the preimplantation period appear to be dependent upon the availability of appropriate metabolic substrates. For preimplantation embryos, defined conditions of culture have been achieved only in a few laboratory species. There is now evidence that differentiation factors isolated from fetal calf serum and human placental cord serum may promote further development of blastocysts. Postimplantation rat and mouse embryos can be cultured during the organogenesis period with rat or human sera in roller bottles. The embry- onic differentiation of the rat at this stage of development is progressively retarded in such cultures with male rat serum. The embryonic development is not improved, even in sera obtained from rats a t different days of gestation (12, 15-16, and 20-21). Inability to grow placental tissues simultaneously with embryos, accumulation of unfavorable substances, and rapid depletion of nu- trients contribute to the retardation of embryonic growth. To improve growth and differentiation of conceptuses, a continuous culture system with the possi- bility of infusion of increasing concentrations of oxygen in the roller bottle gas atmosphere has been developed. This improved method allows considerable continuous growth and differentiation from the neurula stage with develop- ment of numerous primary organs.

    Utilizing these in vitro culture methods during pre- and postimplantation periods, it is now possible to assess embryotoxic or teratogenic potential of drugs and chemical agents. The postimplantation culture procedure allows a more precise assessment of mechanisms associated with anomalous embryonic differentiation. Bioactivation of teratogens and effects of active toxic metabo- lites on organ primordium differentiation have been shown by combining embryo culture with a hepatic microsomal activating system. Microinjection of teratogens and cells into conceptus compartments is being used to elucidate specific anomalous differentiation processes. Key words in vitro, embryo, tera- togen, organogenesis, development

    Early mammalian embryos at different stages of development are now cultured suc- cessfully for short periods in specific media. The embryonic development in vitro is close to normal; growth and differentiation are often comparable to intrauterine gestation. While development processes and patterns are by and large programmed intrinsically, problems with in vitro embryo culture mainly relate, as might be expected, to the availability of appropriate nutrients and clearance of waste. The mammalian embryo is not sufficiently provided with nutritional and growth-promoting substances within it- self; development occurs by drawing these resources from the mother. During the course

    of development and in successive steps, the embryo has specific needs which are pro- vided by the secretions, cellular interactions, and transport mechanisms regulated by com- plex physiological and immunological pro- cess in the mother. The free-floating zygote within ampulla or oviduct differentiates in follicular fluid and oviduct secretions. As it enters into the uterine cavity, the preimplan- tation-stage embryo faces a different set of secretory products closely regulated by the stimulus of hormones. Finally, nidation of the embryo promotes a more intimate asso- ciation between the maternal system and the developing embryo. Active transport pro- cesses between the embryo and maternal sys-

    0 1983 ALAN R. LISS, INC.


    tems are established for further growth and development.

    The elucidation of components of the in vivo embryonic environment appears to be a rational approach of research for devising culture conditions optimally supporting de- velopment in vitro. If growth and develop- ment of the embryo outside the maternal environment are dependent upon favorable conditions and appropriate substrates, such a system should provide investigators with the means to determine important elements of the maternal environment and effects of changes in this environment on develop- ment. Viewed from another standpoint, the study of embryonic development under de- fined culture conditions permits elucidation of basic processes of embryonic differentia- tion, and assessment of metabolic require- ments crucial to embryo survival and normal differentiation. There is no assurance, how- ever, that in vitro conditions are equivalent to in vivo conditions.

    In this paper, the current capabilities and limitations for growth and differentiation in vitro of pre- and postimplantation laboratory animal embryos will be reviewed. Various chemical and physical manipulations in these culture systems, and the effects of such ma- nipulations on growth and development of processes of embryos will also be described.


    Preimplantation embryonic development in vitro from the fertilized ovum to blastocyst stage has been attempted in numerous labo- ratory species with varying degrees of suc- cess. The low rate of embryogenesis in vitro in some species is due to as yet unknown gaseous and metabolic requirements for in vitro development. Of all the laboratory spe- cies, these requirements have been studied most extensively in the mouse (Biggers, '79; Brinster and Troike, '79; Brackett, '81). Pre- implantation mouse embryos can be grown to blastocyst stage under defined culture con- ditions, since metabolic requirements for growth and differentiation at this stage of development are known for this species. The stages of in vitro development are identical to those in utero and such embryos grown in vitro when transferred to foster mothers de- velop into normal neonates. A completely de- fined culture condition for rabbit embryo development is also available (Seidel et al., '76; Binkerd and Anderson, '79; Kane and Headon, '80). Only a few studies have been

    made in rats and hamsters during this period of development (Yamamura and Markert, '81; Whittingham and Bavister, '74; Leibfried et al., '82). In general, some success with domes- tic species has been achieved, but often un- defined media containing supplements of sera are, used (Wright, '77; Davis and Day, '78; Linder and Wright, '78; Tervit and Rowson, '74; Peters et al., '77 and Wright, et al., '76a,b,c; Boone et a1,'78; Brackett et al, '80).


    The discovery that substrates for energy metabolism change during the stages of early embryonic development in the mouse was important in formulating a defined culture medium for development of fertilized em- bryos to blastocysts. The preimplantation mouse embryo can now be grown from the one-cell to the blastocyst stage in a medium similar to Krebs-Ringer bicarbonate salt so- lution containing pyruvate and lactate (Whitten and Biggers, '68; Biggers et al., '65, '67). Glucose, the most common energy source for mammalian cells, is unable to support development of the one- or two-cell stage em- bryo; pryuvate is an essential requirement in the culture medium (Biggers, '71; Brin- ster, '71; Hoppe and Pitts, '73). These mouse embryos apparently do not have specific es- sential amino acid requirements. Mouse pre- blastocyst embryos can be grown to blastocyst stage with single amino acids as the amino- nitrogen source, and crystallized serum al- bumin appears to be adequate in supporting normal differentiation (Brinster, '65, '68). The preimplantation embryo does not require nu- cleic acid precursors, since the embryo is able synthesize the required nucleic acids from simple exogenous carbon and nitrogen sources. The embryo can also utilize exoge- nous nucleotide precursors for nucleic acid synthesis when available in the culture me- dium (Sanyal and Meyer, '70).


    A remarkable variability in embryonic growth in vitro is also found among mice of different genetic strains (McLaren and Bow- man, '73; Chapman et al., '78; Biggers, '71). It is known that strain differences exist in the fertilizing efficiency of mouse spermato- zoa in various media (Parkening and Chang, '76; Kaleta, '77). Recently, Shire and Whit- ten ('80a, b) and Niwa et al. ('80) have iden-


    tified influences of maternal and paternal genotypes on the first cleavage after fertili- zation. The progressive development of em- bryos beyond the two-cell stage in mice is often blocked owing to metabolic deficiencies in glycolytic pathways (Barbehen et al., '74). Genetic characterization of such deficiencies has not been pursued in depth; however, it is clear in a number of mouse inbred strains that the developmental capabilities of the zygote are more restricted than in F1 hybrids under identical conditions of cultures. In cul- ture media designed for maintenance of the one-cell stage, zygotes of inbred strains fre- quently do not grow beyond the two-cell stage, while zygotes of random bred or F1 hybrid mice strains may grow into blasto- cysts (Biggers '71).

    Requirements for embryonic development become more complex beyond the blastocyst stage (articles in Glasser and Bullock, '79). Blastocysts do not grow into egg cylinder stage embryos without the addition of essen- tial amino acids or serum to the culture medium (Gwatkin, '66). Hatching of the blastocyst is associated with dissolution of the zona pellucida. Studies of Konwinski et al. ('78) indicate that the in vitro differentia- tion of the blastocyst is facilitated by treat


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