TERATOLOGY 42:635-642 (1990)
Whole Embryo Culture, Teratogenesis, and the Estimation of Teratologic Risk* D.A.T. NEW The Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG, England
ABSTRACT
Mammalian whole-embryo culture systems are now widely used and have proved useful in many studies of normal and abnormal development. The main advantages of these systems are that they allow precise control of experimental conditions and can often provide information unobtainable from in vivo studies; the main disadvantages are the rather short period of embryonic development that can be supported in culture and the present restriction of the techniques to very few species. The possibility of using whole-embryo culture systems for screening for new teratogenic agents remains controversial, but there are indications that the systems may have more potential in this area than has sometimes been supposed.
Recent years have seen a rapid increase in the use of mammalian culture systems for investigating problems of embryo physiology and teratogenesis. The systems include cell culture, tissue culture, organ culture, and embryo culture. Although several of these systems, and particularly those for cell culture (Flint and Orton, '84) have shown potential for screening for teratogenic agents, it is the application of wholeembryo culture to teratology that I wish to concentrate on here. Methods for whole-embryo culture, especially of postimplantation embryos, have for many years been a particular interest in my laboratory. We began to develop such methods during the 1960s, but i t was not until we had made a number of improvements in the 1970s that the techniques became good enough to be considered seriously for teratological work. They are now used in many different laboratories and have been the subject for several reviews (e.g., New '78, '83; Shepard et al., '87; Cockroft and Steele, '87). Technical details of explanting embryos, preparing media, and setting up cultures may be found in New ('71) and Cockroft ('89). Several variants of the culture systems are available (New, '83, '89), and the choice of system must depend on the requirements of a particular investigation. The system that we now use most often is shown in Figure 1.Culture bottles with embryos and medium are plugged with wide0 1990 WILEY-LISS, INC
bored stoppers, into a hollow drum. During incubation, the drum rotates and a n oxygenating gas with 5% CO, is passed continuously through it, ensuring that the embryos are exposed to a steady oxygen level and fairly steady pH. The bottles may be custom made in various sizes in glass or, as shown in Figure 1, may be standard disposable plastic bottles. (The rotating drum apparatus is obtainable from BTC Engineering, Milton, Cambridge, UK.) Rat and mouse embryos during early organogenesis show excellent development in such a culture system. Some years ago, we compared in detail the development during 48 h in culture of head-fold stage rat embryos removed from one horn of the uterus, with that of the embryos left to develop in vivo for the same period in the contralateral horn. Morphology, growth, degree of differentiation, and protein content were indistinguishable in the two groups of embryos (New et al., '76). Since then, a few histological and biochemical differences have been reported (e.g., Huber and Brown, '821, but most workers have confirmed that, during this period of development, overall growth of the rat or mouse embryo in vitro is almost identical to that in vivo. Received February 14, 1989; accepted July 10, 1990. *We have accepted this paper for publication in Teratology as part of the International Federation Teratology Society Symposium held in Kyoto, Japan, June 1988.
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Fig. 1. Rotator culture system providing continuous gassing of the culture bottles during incubation.
What can such culture techniques offer teratology and the estimation of teratologic risk? They undoubtedly meet some of the essential requirements; e.g., they reliably support embryonic development during organogenesis and they are relatively simple and cheap. But it has become increasingly clear that their value is much more evident in answering some types of questions than others. A few examples will illustrate this. NORMAL DEVELOPMENT
Although teratology, by definition, is concerned with abnormality, no investigation of abnormal mechanisms can proceed far without a prior or parallel study of normal development. And in the study of normal development, culture methods have much to contribute. They are particularly valuable where the minute size and inaccessibility of the organogenesis stage embryo in the uterus render in vivo experimentation im-
possible. For example, studies involving the transfer of cells to precisely located regions of the embryo, such as those made by Beddington ('81) and Morriss-Kay and Tan ('87) on ectodermal and neural crest derivatives, are possible only on explanted embryos. Similarly, the use of embryos in culture enabled Robkin et al. ('72, '76) to record with remarkable precision the embryonic heart beat and its response to cardioactive drugs (Fig. 2). Shepard et al. ('70) used cultured embryos to obtain the first reliable data on glucose consumption and changes in energy metabolism in the organogenesis embryo, work which has since been developed further by Ellington ('87), Hunter and Sadler ('881, and others. ABNORMAL DEVELOPMENT
Culture methods may also be of great value in analysing the mechanisms and
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causes of abnormal development. Because the experimenter has complete control of the environment of the embryo in vitro, the components of this environment can be varied at will and with great precision. An excellent example of this is the study by Cockroft ('88) of the effects of various vitamin deficiencies on development during organogenesis (Table 1). Culture serum was first made vitamin free by dialysis and then supplemented by glucose, amino acids, and different combinations of eight vitamins. The vitamins whose absence produced the most marked effect were pantothenic acid and riboflavin; protein content and crown-rump length of the embryos were significantly reduced, and there was a high frequency of
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malformations. Absence of folic acid also affected growth and differentiation, but to a lesser extent. Inositol was interesting in that its absence had little effect on overall growth but resulted in a high frequency of malformations, mostly of the brain and eyes. Younger embryos were more seriously affected than were older embryos. Cockroft's work may be particularly relevant to human development, in view of the reports (Smithells et al., '80; Seller, '85) that vitamin supplementation of the diet of women around the time of conception can reduce the incidence of neural tube defects in their children-findings that, for ethical reasons, have been very difficult to analyse clinically.
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TABLE 1. Protein contents and abnormalities in rat embryos cultured in dialysed rat serum supplemented with glucose, amino acids, and various combinations of vitamins' Pantothenic Choline Folic iPyridoxal Thiamine No. of Abnormal Protein acid chloride acid Inositol Nicotinamide HCl Riboflavin HCl embryos embryos (kg) + + + + + + + + 8 0 132 -t 12 8 8 23 t 3 + 8 8 37 t 5 ++ + + f + 8 1 114 9 + + + + + + + + + + + f + + 8 2 76 i- 7 + f + + + 8 6 124 2 9 + + + + + + 8 4 106 t 11 + f f f + f + 8 1 118 t 16 + f f + + + + + + +8 8 59 t 7 2 132 t 8 + + + + + f + 8 ~
*
'From Cockroft ('88).
When there is confusion about which of many possible factors in vivo is the source of a n abnormality, studies on cultured embryos can often be particularly effective in obtaining a clear-cut answer. I would like to illustrate this with two examples from our own laboratory. The first is a study that we made some years ago on hyperthermia. It was well known that epidemics of influenza and other fevers were associated with increased incidence of abnormalities in the newborn (Edwards, '72), but it was impossible to establish from the medical literature whether it was the raised temperature of the fever or some other factor in the mother that was the cause. Growing embryos at different temperatures in culture (Cockroft and New, '78; Mirkes, '85) has provided a simple way of resolving the problem and has shown that as little as a 2" rise in temperature (40°C) throughout the culture period causes abnormalities of development (Fig. 3), while even brief exposure to higher temperatures has severe effects. The second example is a study we made in response to a request from one of the drug companies to examine certain of their newly developed hypolipidaemic agents. This pharmaceutical company had found from their own in vivo tests that these agents were teratogenic when administered to pregnant rats. But they were unable to establish whether it was the drug itself that affected the embryos or-more worryingthe reduced lipid levels that were harmful. Our tests in culture (Steele et al., '83a) quickly showed that it was almost certainly the drugs and not the hypolipidaemia that caused the abnormalities (Table 2).
SCREENING
While the value of whole-embryo culture methods in studying the mechanisms of normal and abnormal development may be undisputed, their potential value in screening for teratogenic agents is much more problematical. Practical considerations One of the supposed problems-the labour and cost of culturing embryos-is often overstated. Setting up embryo culture for a drug test may not be a s simple as administering the drug to pregnant animals, although that has its problems too (Palmer, '741, but it is well within the bounds of feasibility. At Cambridge, we run a class on embryo culturing each year now for our undergraduate students and, without any previous experience of explanting these embryos, many of the students master the technique in a single afternoon. With a little practice, one person can easily explant and set up in culture 50 embryos a day. However, even if it be accepted that embryo culture would be feasible a s a screening method, there remain many questions concerning its relevance. Species variability First is the well-known problem of species differences in response to teratogenic agents and the uncertainty as to how far tests on one or a few species provide information of general validity. Although the same problem arises with in vivo tests, there is a t least a wide choice of species available for them; whereas culture methods for postimplantation embryos are a t present virtually
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Fig. 3. Rat embryos explanted at head-fold stage and grown in culture for 48 h a t different temperatures. Embryos cultured at 40°C were microcephalic but otherwise similar to the controls a t 38°C. Embryos cultured at 40.5" or 41°C were very severely affected (Cockroft and New, '78).
TABLE 2. Deueloument of embrvos in hvuoliuidaemic serum a n d in normal serum containing hypolipidaemic agent M&B 30,227' Total Blood Somites Group embryos circ. Turned (mean) Serum from treated rat 15 14 14 23 Treated 15 15 14 23 Control Serum with agent added 14 15 pgiml 15 0 0 22 14 5 pgiml 15 8 Control 15 14 15 23 " I
1
Protein (mean) (Pd 167 162 99 195 207
'Based on data from Steele et al. ('83a)
restricted to rats and mice. This would seem to put culture methods a t a serious disadvantage. But in one respect, culture methods may have an important contribution to make in trying to get round the problem of species variability. Variability of teratogenic response in vivo can be regarded as having three components: maternal variability, placental variability, and fetal variability. In vitro, two of these sources of variability are eliminated and the cultured isolated rat embryo may be a better model of, for example, human development than the rat embryo in the uterus. Furthermore, rat embryos may be cultured in serum from human patients after administration of drugs, thereby testing the response of the embryos to the products of the drugs and the human metabolism. This technique was first used with dramatic results by Norman
Klein and his colleagues at the University of Connecticut (Table 3). It has to be admitted that rat embryos often grow subnormally in whole human serum and that this sometimes caused problems with the controls in these earlier experiments. But more recent work (Beck et al., '85) has shown that the development of rat embryos in human serum can be much improved by the addition of as little as 10% rat serum. As a result, this approach would now seem to have considerable promise.
Duration of development in culture and assessment of results A serious problem of embryo culture in relation t o screening is the limited duration of development in culture. Development closely comparable to that in vivo, as regards both differentiation and growth rate, is obtained only up to early limb bud stages.
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TABLE 3. Growth of rat embryos in serum from patients receiving cancer chemotherapy or anticonuulsants' No. No. Protein Subject embryos abnormal (pgiembryo) Controls 1-19 107 20 76 Cancer chemotherapy lethal Vincristine etc. 21 3 9 0 82 (post-treatment) 21 3 0 22 3 (no treatment) 87 24 3 54 Methotrexate etc. 3 Tamoxifen 25 3 88 3 Anticonvulsants 26 3 3 64 Tegretol 27 3 3 61 Peganone etc. 28 3 3 14 Primidone etc. 3 ;33 Tegretol etc. 29 3 3 3 '79 Temetol etc. 30 'From data of Chatot et al. ('80).
TABLE 4. Results of an experiment to examine the effects o f c u l t u r i n ~on retinoic acid-treatc,d embryos'.' Anterior No. of Protein Turning Without limb neuropore Treatment embryos Somites (kg1 incomplete (%) buds (5%) open (5%) Control, vivo 64 24 262 3 5 2 160 15 36 7 Control, vivoivitro 61 22 159 37 61 63 RA, vivo 80 19 109 65 80 61 RA. vivoivitro 84 15 'Pregnant rats were injected a t 8-112 days p.c. with retinoic acid. The embryos from one uterine horn were explanted a t 9-112 days p.c. and grown in culture for 48 h; the embryos in the other horn were left to continue development for the same period in vivo. Control p u p s were treated similarly but without the retinoic acid injection. From data of Steele et al. ('83b).
TABLE 5. Data from the embryos of Table 4 analysed to assess ( A ) the frequency of abnormally open anterior neurooore and i B ) the freauencv o f abnormallv incomolete turning' Anterior neurouore ouen In embryos with 14 + somites Total no. Embryos with % of all 5% of all % of 1 4 + A Treatment of embryos 1 4 + somites embryos embryos embryos 64 62 2 0 0 Control, vivo 61 57 7 2 2 Control, vivohitro 80 68 63 55 65 RA, vivo 84 43 71 22 42 RA, vivoivitro
B Treatment Control, vivo Control, vivoivitro RA, vivo RA, vivoivitro
Toptal no. of embrvos 64 61 80 84
Embryos with 1 7 + somites 62 56 .~ 52 25
9% of all embrvos 3
15 37 65
Turning incomolete In embryos with 17 + somites % of all %of17+ embrvos embrvos 0 0 7 7 12 8 18 60
'Frequencies are expressed in three different ways
How far do abnormalities observed in culture during this period relate to those found in vivo at term? Beaudoin and Fisher ('81) treated pregnant rats with a number of teratogenic agents and then explanted the embryos into serum (from untreated animals) for culture and evaluation. The aim of this procedure was to expose the embryo to the
agent with the embryo-placenta-mother unit complete and then to add to this any additional effects of the culture environment itself. The agents tested were thiadiazole, cadmium sulphate, Dibrom, Dinoseb, lead nitrate, polybrominated biphenyls, sodium arsenate, and trypan blue. The investigators concluded that all these compounds
WHOLE EMBRYO CULTURE
TABLE 6. Results of a whole-embryo culture assay for teratogenic activity of various substances compared with the known activity in U ~ U O ' , ~ Response, both in vitro and in vivo Positive results Negative results Actinomycin D Azathioprine Cadmium chloride Colchizine Coumarin Cyclophosphamide Dexamethasone Diethylstilbestrol Doxorubicin Ethanol Fluorouracil Griseofulvin Hycanthone Hydrazinesulfate Meclizine Mercaptopurine Methotrexate Procarbazine Trypan blue Valproic acid Acetone Acrolein Allylamine derivative Aminopyrene Butylamine tert. Cyclosporin A Cysteine Dimethylsulfoxide EDTA Gentamycin Gelatin Glutamic acid Lysine Metronidazole Naftifine Olive oil Pleuromutilin derivative Saccharin Tween 80 'Data obtained from the literature of the same substances in vivo (Schmid et al., '83). 'Only 0-toluidine gave a different response: positive in vitro, negative in vivo.
inhibited the subsequent rate of development of the embryos in culture, but that the pattern of malformations observed bore little resemblance to that found in embryos allowed to continue development in the mother. We performed a similar experiment in our laboratory, but with excess vitamin A as the teratogen (Steele et al., '83b). Our conclusions were somewhat more optimistic that those of Beaudoin and Fisher. The abnormalities we obtained in vitro were broadly similar to those in vivo, including reduced protein synthesis, somite and limb bud formation, and increased neural tube defects, particularly microcephaly and abnormali-
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ties in the closure of the neuropores (Table 4). But the results illustrated well the problem of attempting to assess with any precision the frequency of specific malformations in culture. For example, Table 5 shows determinations of the frequency of abnormally open anterior neuropore. The anterior neuropore is normally open in rat embryos up to the 14-somite stage and closed at later stages. When embryos are explanted at 9.5 days (head-fold stage) and grown in culture in control medium, the anterior neuropore normally closes after about 30 h. Is an open neuropore therefore to be regarded as an abnormality, if it is found in any of the embryos of Table 4 (cultured for 48 hs), or only if present in an embryo with 14 + somites? If the latter, is the frequency to be expressed as a percentage of all the embryos or only of those with 14+ somites? The frequencies derived may vary widely depending on the answer t o this question (Table 5A); and similarly (Table 5B) with the assessment of incomplete turning (turning is normally complete at 17 somites) and other abnormalities. This is only one example of the kind of problem encountered in attempting to give numerical values to abnormalities in culture. In practice, different workers assess their embryos in many different ways, ranging from a description of a few of the embryonic organs to the calculation of a single figure expressing the development of the whole embryo, as in the useful morphological scoring system of Brown and Fabro ('81). Some of these methods are better than others, but none is entirely satisfactory, and often too little attention is paid to this problem both in evaluating individual experiments and in evaluating the potential of embryo culture for teratogenic screening. Current optimism Until a few years ago, the balance of the arguments appeared to be heavily against mammalian embryo culture as a practicable screening method (Wilson, '78). But recently, the picture has changed. Several studies, such as those of Schmid et al. ('831, have shown cultured embryos to have an impressively high predictive capacity for teratogens (Table 6). Sadler et al. ('88) and Webster ('86) and others have indicated the potential value of embryo culture used in conjunction with pharmacokinetic data in assessing teratogenic risk. And serious consideration is now being given by some com-
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mercial laboratories to the development of embryo culture as a screening procedure (Bechter and Schon, '88; Tesh, '88). The next few years should show how far this promise is realised in practice. LITERATURE CITED Beaudoin A.R., and D.L. Fisher (1981) An in vivoiin vitro evaluation of teratogenic action. Teratology 23: 57-61. Bechter, R., and H. Schon (1988) Use of the whole-embryo culture system in drug safety assessment. Toxicol. In Vitro 2t195-203. Beck, F., A.P. Gulamhusein, and I.M. Huxham (1985) The use of human serum for studying malformations in whole embryo cultures. In: Prevention of Physical and Mental Congenital Defects. Part C: Basic and Medical Science, Education, and Future Strategies. M. Marois, ed. Alan R. Liss, Inc., New York, pp. 265270. Beddington, R.S.P. (1981) An autoradiographic analysis of the potency of embryonic ectoderm in the 8th day postimplantation mouse embryo. J . Embryol. Exp. Morphol., 64:87-104. Brown, N.A., and S. Fabro (1981) Quantitation of rat embryonic development in vitro: A morphological scoring system. Teratology, 24:65-78. Chatot, C.L., N.W. Klein, J. Piatek, and L.J. Pierro (1980) Successful culture of rat embryos in human serum: Use in the detection of teratogens. Science 207t1471-1473. Cockroft, D.L. (1988) Changes with gestational age in the nutritional requirements of rat embryos in culture. Teratology, 38:281-290. Cockroft, D.L. (1990) Dissection and culture of postimplantation embryos. In: The Postimplantation Mammalian Embryo-A Practical Approach. D.L. Cockroft and A.J. Copp, eds. IRL Press, Oxford, pp. 15-39. Cockroft. D.L.. and D.A.T. New (1978) Abnormalities induced in cultured rat embryos by hyperthermia. Teratology, 17:277-284. Cockroft, D.L., and D.E. Steele (1987) Postimplantation embryo culture and its application to problems in teratology. In: In Vitro Methods in Toxicology. C.K. Atterwill, ed. Cambridge University Press, Cambridge, pp. 365-389. Edwards, M.J. (1972) Influenza, hyperthermia and congenital malformation. Lancet, 1:320. Ellington, S.K.L. (1987) In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos. Development, 1OOt431-439. Flint, O.P., and T.C. Orton (1984) An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol. Appl. Pharmacol., 76:383-395. Huber, B.E., and N.A. Brown (1982) Developmental patterns of ornithine decarboxylase activity in organogenesis phase rat embryos in culture and in utero. In Vitro, 18t599-605. Hunter, E.S. 111, and T.W. Sadler (1988) Embryonic metabolism of foetal fuels in whole-embryo culture. Toxicol. In Vitro, 2t163-167. Mirkes, P.E. (1985) Effects of acute exposures to elevated temperatures on rat embryo growth and development in vitro. Teratology, 32:259-266. Morriss-Kay, G., and S-S. Tan (1987) Mapping cranial neural crest cell migration pathways in mammalian embryos. Trends Genet 3.257-261. New, D.A.T. (1971) Methods for the culture of postimplantation embryos of rodents. In: Methods in Mammalian Embryology. J.C. Daniel, Jr., ed. W.H. Freeman & Co., San Francisco, pp. 305-319.
New, D.A.T. (1978) Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev., 53:81-122. New, D.A.T. (1983) The mammalian embryo and fetus in vitro. In: Methods for Assessing the Effects of Chemicals on Reproductive Functions. G.C. Butler, and N. Nelson, eds. John Wiley & Sons Ltd., New York, pp. 277-297. New, D.A.T. (1990) Introductory chapter. In: The Postimolantation Mammalian Embrvo-A Practical ADproach. D.L. Cockroft and A.L. dopp, eds. IRL Press, Oxford, pp. 1-13. New, D.A.T., P.T. Coppola, and D.L. Cockroft (1976) Comparison of growth in vitro and in vivo of postimplantation rat embryos. J. Embryol. Exp. Morphol., 36t133-144. Palmer, A.K. (1974) Problems associated with the screening of drugs for possible teratogenic activity. In: Experimental Embryology and Teratology. D.H.M. Woollam, and G.M. Morriss, eds. Paul Elek, London, Vol. 1, pp. 16-33. Robkin. M.A.. T.H. Sheuard. and T. Tanimura (1972) A new in vitro culture technique for rat embryos. Teratology, 5t367-376. Robkin, M.A., T.H. Shepard, and D.C. Dyer (1976) Autonomic receptors o f the early rat embryo heart: Growth and development. Proc. SOC. Exp. Biol. Med., 151:799-803. Sadler, T.W., L. Shum, C.W. Warner, and M. Kate Smith (1988) The role of pharmacokinetics in determining the response of rodent embryos to teratogens in whole-embryo culture. Toxicol. Vitro, 2t175-180. Schmid, B.P., A. Trippmacher, and A. Bianchi (1983) Validation of the whole-embryo culture method for in uitro teratogenicity testing. In: Developments in the Science and Practice of Toxicology. A.W. Hayes, R.C. Schnell, and T.S. Miya, eds. Elsevier Science Publishers B.V., Amsterdam, pp. 563-566. Seller, M.J. (1985) Periconceptual vitamin supplementation to prevent recurrence of neural tube defects. Lancet, lt1392-1393. Shepard, T.H., A.G. Fantel, and P.E. Mirkes (1987) Somite-stage mammalian embryo culture-Use in study of normal physiology and mechanisms of teratogenesis. Banbury Report 26: Developmental Toxicology: Mechanisms and Risk. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 29-44. Shepard, T.H., T. Tanimura, and M.A. Robkin (1970) Energy metabolism in early mammalian embryos. Dev. Biol. 4(Suppl.)t42-58. Smithells, R.W., S. Sbeppard, C.J. Schorah, M.J. Seller, N.C. Nevin, R. Harris, A.P. Read, and D.W. Fielding (1980) Possible prevention of neural-tube defects by periconceptual vitamin supplementation. Lancet, 1; 339-340. Steele, C.E., D.A.T. New, A. Ashford, and G.P. Copping (1983a) Teratogenic action of hypolipidaemic agents: An in vitro study with post-implantation rat embryos. Teratology, 28:229-236. Steele, C.E., D.G. Trader, and D.A.T. New (198313) An in vivoiin vitro evaluation of the teratogenic action of excess vitamin A. Teratology, 28r209-214. Tesh, J.M. (1988) The application of whole-embryo culture to new product development. Toxicol. In Vitro, 2t189-194. Webster, W.S., M.C. Johnston, E.J. Lammer, and K.K. Sulik (1986) Isoretinoin embryopathy and the cranial neural crest: An in vivo and in vitro study. J . Craniofac. Genet. Dev. Biol., 6.211-222. Wilson, J.G. (1978) Survey of in vitro systems: Their potential use in teratogenicity screening. In: Handbook of Teratology. J.G. Wilson and F.C. Fraser, eds. Plenum Press, New York, Vol. 4, pp. 135-154.
Whole Embryo Culture, Teratogenesis, and the Estimation of Teratologic Risk* D.A.T. NEW The Physiological Laboratory, University of Cambridge, Cambridge, CB2 3EG, England
ABSTRACT
Mammalian whole-embryo culture systems are now widely used and have proved useful in many studies of normal and abnormal development. The main advantages of these systems are that they allow precise control of experimental conditions and can often provide information unobtainable from in vivo studies; the main disadvantages are the rather short period of embryonic development that can be supported in culture and the present restriction of the techniques to very few species. The possibility of using whole-embryo culture systems for screening for new teratogenic agents remains controversial, but there are indications that the systems may have more potential in this area than has sometimes been supposed.
Recent years have seen a rapid increase in the use of mammalian culture systems for investigating problems of embryo physiology and teratogenesis. The systems include cell culture, tissue culture, organ culture, and embryo culture. Although several of these systems, and particularly those for cell culture (Flint and Orton, '84) have shown potential for screening for teratogenic agents, it is the application of wholeembryo culture to teratology that I wish to concentrate on here. Methods for whole-embryo culture, especially of postimplantation embryos, have for many years been a particular interest in my laboratory. We began to develop such methods during the 1960s, but i t was not until we had made a number of improvements in the 1970s that the techniques became good enough to be considered seriously for teratological work. They are now used in many different laboratories and have been the subject for several reviews (e.g., New '78, '83; Shepard et al., '87; Cockroft and Steele, '87). Technical details of explanting embryos, preparing media, and setting up cultures may be found in New ('71) and Cockroft ('89). Several variants of the culture systems are available (New, '83, '89), and the choice of system must depend on the requirements of a particular investigation. The system that we now use most often is shown in Figure 1.Culture bottles with embryos and medium are plugged with wide0 1990 WILEY-LISS, INC
bored stoppers, into a hollow drum. During incubation, the drum rotates and a n oxygenating gas with 5% CO, is passed continuously through it, ensuring that the embryos are exposed to a steady oxygen level and fairly steady pH. The bottles may be custom made in various sizes in glass or, as shown in Figure 1, may be standard disposable plastic bottles. (The rotating drum apparatus is obtainable from BTC Engineering, Milton, Cambridge, UK.) Rat and mouse embryos during early organogenesis show excellent development in such a culture system. Some years ago, we compared in detail the development during 48 h in culture of head-fold stage rat embryos removed from one horn of the uterus, with that of the embryos left to develop in vivo for the same period in the contralateral horn. Morphology, growth, degree of differentiation, and protein content were indistinguishable in the two groups of embryos (New et al., '76). Since then, a few histological and biochemical differences have been reported (e.g., Huber and Brown, '821, but most workers have confirmed that, during this period of development, overall growth of the rat or mouse embryo in vitro is almost identical to that in vivo. Received February 14, 1989; accepted July 10, 1990. *We have accepted this paper for publication in Teratology as part of the International Federation Teratology Society Symposium held in Kyoto, Japan, June 1988.
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Fig. 1. Rotator culture system providing continuous gassing of the culture bottles during incubation.
What can such culture techniques offer teratology and the estimation of teratologic risk? They undoubtedly meet some of the essential requirements; e.g., they reliably support embryonic development during organogenesis and they are relatively simple and cheap. But it has become increasingly clear that their value is much more evident in answering some types of questions than others. A few examples will illustrate this. NORMAL DEVELOPMENT
Although teratology, by definition, is concerned with abnormality, no investigation of abnormal mechanisms can proceed far without a prior or parallel study of normal development. And in the study of normal development, culture methods have much to contribute. They are particularly valuable where the minute size and inaccessibility of the organogenesis stage embryo in the uterus render in vivo experimentation im-
possible. For example, studies involving the transfer of cells to precisely located regions of the embryo, such as those made by Beddington ('81) and Morriss-Kay and Tan ('87) on ectodermal and neural crest derivatives, are possible only on explanted embryos. Similarly, the use of embryos in culture enabled Robkin et al. ('72, '76) to record with remarkable precision the embryonic heart beat and its response to cardioactive drugs (Fig. 2). Shepard et al. ('70) used cultured embryos to obtain the first reliable data on glucose consumption and changes in energy metabolism in the organogenesis embryo, work which has since been developed further by Ellington ('87), Hunter and Sadler ('881, and others. ABNORMAL DEVELOPMENT
Culture methods may also be of great value in analysing the mechanisms and
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causes of abnormal development. Because the experimenter has complete control of the environment of the embryo in vitro, the components of this environment can be varied at will and with great precision. An excellent example of this is the study by Cockroft ('88) of the effects of various vitamin deficiencies on development during organogenesis (Table 1). Culture serum was first made vitamin free by dialysis and then supplemented by glucose, amino acids, and different combinations of eight vitamins. The vitamins whose absence produced the most marked effect were pantothenic acid and riboflavin; protein content and crown-rump length of the embryos were significantly reduced, and there was a high frequency of
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malformations. Absence of folic acid also affected growth and differentiation, but to a lesser extent. Inositol was interesting in that its absence had little effect on overall growth but resulted in a high frequency of malformations, mostly of the brain and eyes. Younger embryos were more seriously affected than were older embryos. Cockroft's work may be particularly relevant to human development, in view of the reports (Smithells et al., '80; Seller, '85) that vitamin supplementation of the diet of women around the time of conception can reduce the incidence of neural tube defects in their children-findings that, for ethical reasons, have been very difficult to analyse clinically.
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TABLE 1. Protein contents and abnormalities in rat embryos cultured in dialysed rat serum supplemented with glucose, amino acids, and various combinations of vitamins' Pantothenic Choline Folic iPyridoxal Thiamine No. of Abnormal Protein acid chloride acid Inositol Nicotinamide HCl Riboflavin HCl embryos embryos (kg) + + + + + + + + 8 0 132 -t 12 8 8 23 t 3 + 8 8 37 t 5 ++ + + f + 8 1 114 9 + + + + + + + + + + + f + + 8 2 76 i- 7 + f + + + 8 6 124 2 9 + + + + + + 8 4 106 t 11 + f f f + f + 8 1 118 t 16 + f f + + + + + + +8 8 59 t 7 2 132 t 8 + + + + + f + 8 ~
*
'From Cockroft ('88).
When there is confusion about which of many possible factors in vivo is the source of a n abnormality, studies on cultured embryos can often be particularly effective in obtaining a clear-cut answer. I would like to illustrate this with two examples from our own laboratory. The first is a study that we made some years ago on hyperthermia. It was well known that epidemics of influenza and other fevers were associated with increased incidence of abnormalities in the newborn (Edwards, '72), but it was impossible to establish from the medical literature whether it was the raised temperature of the fever or some other factor in the mother that was the cause. Growing embryos at different temperatures in culture (Cockroft and New, '78; Mirkes, '85) has provided a simple way of resolving the problem and has shown that as little as a 2" rise in temperature (40°C) throughout the culture period causes abnormalities of development (Fig. 3), while even brief exposure to higher temperatures has severe effects. The second example is a study we made in response to a request from one of the drug companies to examine certain of their newly developed hypolipidaemic agents. This pharmaceutical company had found from their own in vivo tests that these agents were teratogenic when administered to pregnant rats. But they were unable to establish whether it was the drug itself that affected the embryos or-more worryingthe reduced lipid levels that were harmful. Our tests in culture (Steele et al., '83a) quickly showed that it was almost certainly the drugs and not the hypolipidaemia that caused the abnormalities (Table 2).
SCREENING
While the value of whole-embryo culture methods in studying the mechanisms of normal and abnormal development may be undisputed, their potential value in screening for teratogenic agents is much more problematical. Practical considerations One of the supposed problems-the labour and cost of culturing embryos-is often overstated. Setting up embryo culture for a drug test may not be a s simple as administering the drug to pregnant animals, although that has its problems too (Palmer, '741, but it is well within the bounds of feasibility. At Cambridge, we run a class on embryo culturing each year now for our undergraduate students and, without any previous experience of explanting these embryos, many of the students master the technique in a single afternoon. With a little practice, one person can easily explant and set up in culture 50 embryos a day. However, even if it be accepted that embryo culture would be feasible a s a screening method, there remain many questions concerning its relevance. Species variability First is the well-known problem of species differences in response to teratogenic agents and the uncertainty as to how far tests on one or a few species provide information of general validity. Although the same problem arises with in vivo tests, there is a t least a wide choice of species available for them; whereas culture methods for postimplantation embryos are a t present virtually
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WHOLE EMBRYO CULTURE
Fig. 3. Rat embryos explanted at head-fold stage and grown in culture for 48 h a t different temperatures. Embryos cultured at 40°C were microcephalic but otherwise similar to the controls a t 38°C. Embryos cultured at 40.5" or 41°C were very severely affected (Cockroft and New, '78).
TABLE 2. Deueloument of embrvos in hvuoliuidaemic serum a n d in normal serum containing hypolipidaemic agent M&B 30,227' Total Blood Somites Group embryos circ. Turned (mean) Serum from treated rat 15 14 14 23 Treated 15 15 14 23 Control Serum with agent added 14 15 pgiml 15 0 0 22 14 5 pgiml 15 8 Control 15 14 15 23 " I
1
Protein (mean) (Pd 167 162 99 195 207
'Based on data from Steele et al. ('83a)
restricted to rats and mice. This would seem to put culture methods a t a serious disadvantage. But in one respect, culture methods may have an important contribution to make in trying to get round the problem of species variability. Variability of teratogenic response in vivo can be regarded as having three components: maternal variability, placental variability, and fetal variability. In vitro, two of these sources of variability are eliminated and the cultured isolated rat embryo may be a better model of, for example, human development than the rat embryo in the uterus. Furthermore, rat embryos may be cultured in serum from human patients after administration of drugs, thereby testing the response of the embryos to the products of the drugs and the human metabolism. This technique was first used with dramatic results by Norman
Klein and his colleagues at the University of Connecticut (Table 3). It has to be admitted that rat embryos often grow subnormally in whole human serum and that this sometimes caused problems with the controls in these earlier experiments. But more recent work (Beck et al., '85) has shown that the development of rat embryos in human serum can be much improved by the addition of as little as 10% rat serum. As a result, this approach would now seem to have considerable promise.
Duration of development in culture and assessment of results A serious problem of embryo culture in relation t o screening is the limited duration of development in culture. Development closely comparable to that in vivo, as regards both differentiation and growth rate, is obtained only up to early limb bud stages.
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TABLE 3. Growth of rat embryos in serum from patients receiving cancer chemotherapy or anticonuulsants' No. No. Protein Subject embryos abnormal (pgiembryo) Controls 1-19 107 20 76 Cancer chemotherapy lethal Vincristine etc. 21 3 9 0 82 (post-treatment) 21 3 0 22 3 (no treatment) 87 24 3 54 Methotrexate etc. 3 Tamoxifen 25 3 88 3 Anticonvulsants 26 3 3 64 Tegretol 27 3 3 61 Peganone etc. 28 3 3 14 Primidone etc. 3 ;33 Tegretol etc. 29 3 3 3 '79 Temetol etc. 30 'From data of Chatot et al. ('80).
TABLE 4. Results of an experiment to examine the effects o f c u l t u r i n ~on retinoic acid-treatc,d embryos'.' Anterior No. of Protein Turning Without limb neuropore Treatment embryos Somites (kg1 incomplete (%) buds (5%) open (5%) Control, vivo 64 24 262 3 5 2 160 15 36 7 Control, vivoivitro 61 22 159 37 61 63 RA, vivo 80 19 109 65 80 61 RA. vivoivitro 84 15 'Pregnant rats were injected a t 8-112 days p.c. with retinoic acid. The embryos from one uterine horn were explanted a t 9-112 days p.c. and grown in culture for 48 h; the embryos in the other horn were left to continue development for the same period in vivo. Control p u p s were treated similarly but without the retinoic acid injection. From data of Steele et al. ('83b).
TABLE 5. Data from the embryos of Table 4 analysed to assess ( A ) the frequency of abnormally open anterior neurooore and i B ) the freauencv o f abnormallv incomolete turning' Anterior neurouore ouen In embryos with 14 + somites Total no. Embryos with % of all 5% of all % of 1 4 + A Treatment of embryos 1 4 + somites embryos embryos embryos 64 62 2 0 0 Control, vivo 61 57 7 2 2 Control, vivohitro 80 68 63 55 65 RA, vivo 84 43 71 22 42 RA, vivoivitro
B Treatment Control, vivo Control, vivoivitro RA, vivo RA, vivoivitro
Toptal no. of embrvos 64 61 80 84
Embryos with 1 7 + somites 62 56 .~ 52 25
9% of all embrvos 3
15 37 65
Turning incomolete In embryos with 17 + somites % of all %of17+ embrvos embrvos 0 0 7 7 12 8 18 60
'Frequencies are expressed in three different ways
How far do abnormalities observed in culture during this period relate to those found in vivo at term? Beaudoin and Fisher ('81) treated pregnant rats with a number of teratogenic agents and then explanted the embryos into serum (from untreated animals) for culture and evaluation. The aim of this procedure was to expose the embryo to the
agent with the embryo-placenta-mother unit complete and then to add to this any additional effects of the culture environment itself. The agents tested were thiadiazole, cadmium sulphate, Dibrom, Dinoseb, lead nitrate, polybrominated biphenyls, sodium arsenate, and trypan blue. The investigators concluded that all these compounds
WHOLE EMBRYO CULTURE
TABLE 6. Results of a whole-embryo culture assay for teratogenic activity of various substances compared with the known activity in U ~ U O ' , ~ Response, both in vitro and in vivo Positive results Negative results Actinomycin D Azathioprine Cadmium chloride Colchizine Coumarin Cyclophosphamide Dexamethasone Diethylstilbestrol Doxorubicin Ethanol Fluorouracil Griseofulvin Hycanthone Hydrazinesulfate Meclizine Mercaptopurine Methotrexate Procarbazine Trypan blue Valproic acid Acetone Acrolein Allylamine derivative Aminopyrene Butylamine tert. Cyclosporin A Cysteine Dimethylsulfoxide EDTA Gentamycin Gelatin Glutamic acid Lysine Metronidazole Naftifine Olive oil Pleuromutilin derivative Saccharin Tween 80 'Data obtained from the literature of the same substances in vivo (Schmid et al., '83). 'Only 0-toluidine gave a different response: positive in vitro, negative in vivo.
inhibited the subsequent rate of development of the embryos in culture, but that the pattern of malformations observed bore little resemblance to that found in embryos allowed to continue development in the mother. We performed a similar experiment in our laboratory, but with excess vitamin A as the teratogen (Steele et al., '83b). Our conclusions were somewhat more optimistic that those of Beaudoin and Fisher. The abnormalities we obtained in vitro were broadly similar to those in vivo, including reduced protein synthesis, somite and limb bud formation, and increased neural tube defects, particularly microcephaly and abnormali-
641
ties in the closure of the neuropores (Table 4). But the results illustrated well the problem of attempting to assess with any precision the frequency of specific malformations in culture. For example, Table 5 shows determinations of the frequency of abnormally open anterior neuropore. The anterior neuropore is normally open in rat embryos up to the 14-somite stage and closed at later stages. When embryos are explanted at 9.5 days (head-fold stage) and grown in culture in control medium, the anterior neuropore normally closes after about 30 h. Is an open neuropore therefore to be regarded as an abnormality, if it is found in any of the embryos of Table 4 (cultured for 48 hs), or only if present in an embryo with 14 + somites? If the latter, is the frequency to be expressed as a percentage of all the embryos or only of those with 14+ somites? The frequencies derived may vary widely depending on the answer t o this question (Table 5A); and similarly (Table 5B) with the assessment of incomplete turning (turning is normally complete at 17 somites) and other abnormalities. This is only one example of the kind of problem encountered in attempting to give numerical values to abnormalities in culture. In practice, different workers assess their embryos in many different ways, ranging from a description of a few of the embryonic organs to the calculation of a single figure expressing the development of the whole embryo, as in the useful morphological scoring system of Brown and Fabro ('81). Some of these methods are better than others, but none is entirely satisfactory, and often too little attention is paid to this problem both in evaluating individual experiments and in evaluating the potential of embryo culture for teratogenic screening. Current optimism Until a few years ago, the balance of the arguments appeared to be heavily against mammalian embryo culture as a practicable screening method (Wilson, '78). But recently, the picture has changed. Several studies, such as those of Schmid et al. ('831, have shown cultured embryos to have an impressively high predictive capacity for teratogens (Table 6). Sadler et al. ('88) and Webster ('86) and others have indicated the potential value of embryo culture used in conjunction with pharmacokinetic data in assessing teratogenic risk. And serious consideration is now being given by some com-
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mercial laboratories to the development of embryo culture as a screening procedure (Bechter and Schon, '88; Tesh, '88). The next few years should show how far this promise is realised in practice. LITERATURE CITED Beaudoin A.R., and D.L. Fisher (1981) An in vivoiin vitro evaluation of teratogenic action. Teratology 23: 57-61. Bechter, R., and H. Schon (1988) Use of the whole-embryo culture system in drug safety assessment. Toxicol. In Vitro 2t195-203. Beck, F., A.P. Gulamhusein, and I.M. Huxham (1985) The use of human serum for studying malformations in whole embryo cultures. In: Prevention of Physical and Mental Congenital Defects. Part C: Basic and Medical Science, Education, and Future Strategies. M. Marois, ed. Alan R. Liss, Inc., New York, pp. 265270. Beddington, R.S.P. (1981) An autoradiographic analysis of the potency of embryonic ectoderm in the 8th day postimplantation mouse embryo. J . Embryol. Exp. Morphol., 64:87-104. Brown, N.A., and S. Fabro (1981) Quantitation of rat embryonic development in vitro: A morphological scoring system. Teratology, 24:65-78. Chatot, C.L., N.W. Klein, J. Piatek, and L.J. Pierro (1980) Successful culture of rat embryos in human serum: Use in the detection of teratogens. Science 207t1471-1473. Cockroft, D.L. (1988) Changes with gestational age in the nutritional requirements of rat embryos in culture. Teratology, 38:281-290. Cockroft, D.L. (1990) Dissection and culture of postimplantation embryos. In: The Postimplantation Mammalian Embryo-A Practical Approach. D.L. Cockroft and A.J. Copp, eds. IRL Press, Oxford, pp. 15-39. Cockroft. D.L.. and D.A.T. New (1978) Abnormalities induced in cultured rat embryos by hyperthermia. Teratology, 17:277-284. Cockroft, D.L., and D.E. Steele (1987) Postimplantation embryo culture and its application to problems in teratology. In: In Vitro Methods in Toxicology. C.K. Atterwill, ed. Cambridge University Press, Cambridge, pp. 365-389. Edwards, M.J. (1972) Influenza, hyperthermia and congenital malformation. Lancet, 1:320. Ellington, S.K.L. (1987) In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos. Development, 1OOt431-439. Flint, O.P., and T.C. Orton (1984) An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol. Appl. Pharmacol., 76:383-395. Huber, B.E., and N.A. Brown (1982) Developmental patterns of ornithine decarboxylase activity in organogenesis phase rat embryos in culture and in utero. In Vitro, 18t599-605. Hunter, E.S. 111, and T.W. Sadler (1988) Embryonic metabolism of foetal fuels in whole-embryo culture. Toxicol. In Vitro, 2t163-167. Mirkes, P.E. (1985) Effects of acute exposures to elevated temperatures on rat embryo growth and development in vitro. Teratology, 32:259-266. Morriss-Kay, G., and S-S. Tan (1987) Mapping cranial neural crest cell migration pathways in mammalian embryos. Trends Genet 3.257-261. New, D.A.T. (1971) Methods for the culture of postimplantation embryos of rodents. In: Methods in Mammalian Embryology. J.C. Daniel, Jr., ed. W.H. Freeman & Co., San Francisco, pp. 305-319.
New, D.A.T. (1978) Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev., 53:81-122. New, D.A.T. (1983) The mammalian embryo and fetus in vitro. In: Methods for Assessing the Effects of Chemicals on Reproductive Functions. G.C. Butler, and N. Nelson, eds. John Wiley & Sons Ltd., New York, pp. 277-297. New, D.A.T. (1990) Introductory chapter. In: The Postimolantation Mammalian Embrvo-A Practical ADproach. D.L. Cockroft and A.L. dopp, eds. IRL Press, Oxford, pp. 1-13. New, D.A.T., P.T. Coppola, and D.L. Cockroft (1976) Comparison of growth in vitro and in vivo of postimplantation rat embryos. J. Embryol. Exp. Morphol., 36t133-144. Palmer, A.K. (1974) Problems associated with the screening of drugs for possible teratogenic activity. In: Experimental Embryology and Teratology. D.H.M. Woollam, and G.M. Morriss, eds. Paul Elek, London, Vol. 1, pp. 16-33. Robkin. M.A.. T.H. Sheuard. and T. Tanimura (1972) A new in vitro culture technique for rat embryos. Teratology, 5t367-376. Robkin, M.A., T.H. Shepard, and D.C. Dyer (1976) Autonomic receptors o f the early rat embryo heart: Growth and development. Proc. SOC. Exp. Biol. Med., 151:799-803. Sadler, T.W., L. Shum, C.W. Warner, and M. Kate Smith (1988) The role of pharmacokinetics in determining the response of rodent embryos to teratogens in whole-embryo culture. Toxicol. Vitro, 2t175-180. Schmid, B.P., A. Trippmacher, and A. Bianchi (1983) Validation of the whole-embryo culture method for in uitro teratogenicity testing. In: Developments in the Science and Practice of Toxicology. A.W. Hayes, R.C. Schnell, and T.S. Miya, eds. Elsevier Science Publishers B.V., Amsterdam, pp. 563-566. Seller, M.J. (1985) Periconceptual vitamin supplementation to prevent recurrence of neural tube defects. Lancet, lt1392-1393. Shepard, T.H., A.G. Fantel, and P.E. Mirkes (1987) Somite-stage mammalian embryo culture-Use in study of normal physiology and mechanisms of teratogenesis. Banbury Report 26: Developmental Toxicology: Mechanisms and Risk. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 29-44. Shepard, T.H., T. Tanimura, and M.A. Robkin (1970) Energy metabolism in early mammalian embryos. Dev. Biol. 4(Suppl.)t42-58. Smithells, R.W., S. Sbeppard, C.J. Schorah, M.J. Seller, N.C. Nevin, R. Harris, A.P. Read, and D.W. Fielding (1980) Possible prevention of neural-tube defects by periconceptual vitamin supplementation. Lancet, 1; 339-340. Steele, C.E., D.A.T. New, A. Ashford, and G.P. Copping (1983a) Teratogenic action of hypolipidaemic agents: An in vitro study with post-implantation rat embryos. Teratology, 28:229-236. Steele, C.E., D.G. Trader, and D.A.T. New (198313) An in vivoiin vitro evaluation of the teratogenic action of excess vitamin A. Teratology, 28r209-214. Tesh, J.M. (1988) The application of whole-embryo culture to new product development. Toxicol. In Vitro, 2t189-194. Webster, W.S., M.C. Johnston, E.J. Lammer, and K.K. Sulik (1986) Isoretinoin embryopathy and the cranial neural crest: An in vivo and in vitro study. J . Craniofac. Genet. Dev. Biol., 6.211-222. Wilson, J.G. (1978) Survey of in vitro systems: Their potential use in teratogenicity screening. In: Handbook of Teratology. J.G. Wilson and F.C. Fraser, eds. Plenum Press, New York, Vol. 4, pp. 135-154.
