In vitro development of the mammalian embryo
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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 228:235-251(1983) 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. 236 M.K. SANYAL AND F. NAFTOLIN 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 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). SPRECIFIC MEDIUM REQUIREMENTS FOR PREIMPLANTATION EMBRYOS 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). CHARACTERISTICS OF PREIMPLANTATION EMBRYOGENESIS IN VITRO 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- IN VITRO EMBRYO DEVELOPMENT 237 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- ment with proteolytic enzymes. Culture of blastocysts denuded by pronase in minimum essential medium supplemented with 10% fe- tal calf serum produced more advanced de- velopment into egg cylinder stage embryos. Spindle ('80) has attempted to culture blas- tocysts in a defined medium modified from Eagle's basal medium containing essential amino acids, uridine M), and P-mercap- toethanol M). In this medium, most of the blastocysts hatched and formed tropho- blastic outgrowths, but 62% developed two- layer egg cylinders. The most advanced growth and differentia- tion of blastocysts in culture have been achieved by Hsu and colleagues ('79). They have been able to obtain growth of a two-cell mouse embryo into an early somite embryo with beating heart, and, more recently, a blastocyst into limb-bud stage embryo (Hsu and Baskar, '74; Chen and Hsu, '82). The sequence of in vitro development and differ- entiation under the conditions utilized ap- pears to be synchronous and comparable to that in utero. In our laboratory, approxi- mately half the mouse blastocysts cultured with frequent changes of the medium begin to grow to advanced stages (unpublished). The blastocyst (Witschi stage 6) develops into early somite embryo (stage 15) in 8 days of culture in CMRL 1066 medium containing different volumes of fetal calf serum (FCS) and human cord serum (HCS). Fetal calf serum is required for growth from stage 6 to 11, and human placental cord serum from 11 to 15 (Hsu, '80). Thus, these sera seem to have different factors which allow growth and differentiation at successive stages. These factors are distinguishable by separa- tion with Amicon DIAFLO membrane fil- ters, and have been named as embryo growth and differentiation factors (EGDF). The FCS has at least two factions: EGDF-1 with higher molecular weight required for development of stages 6-8, and EGDF-2 with lower molec- ular weight needed for stages 8-11. The HCS has EGDF-3, which allows further develop- ment from stage 8 to 15 (Hsu, '79). POSTIMPLANTATION EMBRYONIC DEVELOPMENT In utero, the blastocyst gradually develops its contacts with the maternal system by dif- ferentiation of extraembryonic membranes and a highly specialized organ, the placenta. These adaptations provide nutritional and hormonal requirements for initial as well as continued growth and differentiation of the embryo into fetus. The embryonic develop- ment a t this period is associated with process of cellular commitment, differentiation, and specialization for organogenesis. The period of organogenesis is characterized by a fast rate of cell division, various cellular interac- tions, migration of cells from one region to another, and loss of some unwanted and no longer needed cells. Embryos are also highly susceptible to anomalous development dur- ing this phase, and major structural anom- alies begin to appear a t this period by alteration of the course of cellular differen- tiation or disorganization of the cellular functions (Wilson, '73). Although attempts a t in vitro culture of whole embryos have been made since the 1930s, progress in this area has been slow, owing to lack of knowledge of nutritional needs and complexities of growth processes. In rodents, such as rats and mice, the vis- ceral yolk sac is inverted and highly vascular (Witschi, '62; Thieler, '72). By removing the uterine decidual tissues, it is possible to ob- tain a "conceptus preparation" composed of a presomite primitive streak stage or early so- 238 M.K. SANYAL AND F. NAFTOLIN mite neurula embryo (pregnancy days 9/10) associated with the extra embryonic mem- branes. After surgical rupturing of the Reich- ert membrane, this conceptus preparation can be cultured in homologous rat sera (New, '78; Buckley et al., '78; Sanyal, '80; Sanyal and Wiebke, '79). The exposed vascular visceral yolk sac allows transport of gases, electro- lytes, and nutrients for continuous, synchron- ous, and autonomous growth of the embryo and associated membranes. There are three methods for culture of postimplantation rat or mouse conceptuses: 1) in static medium on watch glasses; 2) in circulating medium pro- pelled by air or by mechanical devices; and 3) in roller bottles. The roller bottle method was initially described by New, and appears to be the most convenient (New et al., '73, '76a,b, '77, '79). The culture medium promoting opti- mal growth is homologous serum collected by immediate centrifugation method (Steele and New, '74). Rat conceptuses can also be grown in human or monkey serum and mouse con- ceptuses in rat serum (Chatot et al., '80; Klein et al., '81; Clarkson et al., '69; Sadler, '79). In a standardized regime of sera (2-5 ml per con- ceptus) with gradual increase in concentra- tions of oxygen 5% to 95% in rotating bottle atmosphere, the presomite or early somite embryos develop considerably with increases of DNA and protein contents and extensive organogenesis (New, '78). Fig. 1. Comparison of rat embryonic growth in utero (upper row) and in vitro (lower row). Upper left preg- nancy day 11; middle, day 12; and right, day 13 embryos. Lower left, initial day 11 embryo; middle, cultured for 24 h; and right, cultured for 48 h. Embryonic growth in vitro closely resembles those in vivo. Magnification, x50. IN VITRO EMBRYO DEVELOPMENT 239 REQULREMENTS OF POSTIMPLANTATION EMBRYO DEVELOPMENT Embryonic differentiation in vitro appears to be comparable to that in utero in roller bottle cultures utilizing homologous serum (Fig. 1). Organogenesis is extensive in these embryos even during the 2-day culture periods and more like that occurring in vivo. Allantoic placental differentiation and growth is practically absent (Figs. 2, 3). Em- bryonic development assessed by DNA and protein content measurements in roller bot- tle culture and relative development of em- bryos at identical stages of development in utero is given in Figures 4 and 5 . Pregnancy day 10 embryo growth in 10% 02, 5% C02 and N2 and immediately centrifuged male rat serum is almost identical to that in utero during the 2-day culture period. There is pro- gressive reduction of the embryonic growth when pregnancy day 11 and 12 conceptuses were cultured by similar methods for 2 days in 20 and 40% 0 2 concentrations, respectively, with 5% C02 and Nz in bottle gas atmospheres. Pregnancy day 11 embryos showed 50%, and day 12 embryos only 20% of in utero growth. Growth of embryos is not improved in pregnancy sera collected on days 12, 15-16, or 19-20 (Figs. 6, 7; Sanyal and Biggers, '79). No apparent effect on DNA/ protein content was also observed by adding Fig. 2. Comparison of rat placental growth in utero (upper row) and in vitro (lower row). Upper left preg- nancy day 11; middle, day 12; and right, day 13 placenta. Lower left, initial day 11 placenta; middle, cultured for 24 h; and right, cultured for 48 h. Preplacental tissues were allowed to remain attached to these pregnancy day 11 conceptus preparations. These preplacental tissues do not differentiate further concurrent with the embryonic growth in culture. Magnification, x50. 240 M.K. SANYAL AND F. NAFTOLIN estrogen and progesterone to rat serum from castrated rats (Table 1). Embryogenesis in sealed roller bottle cul- tures is affected by three processes (Sanyal, '80): 1) rapid depletion of nutrients (e.g., glu- cose) and decrease of pO2; 2) accumulation of C02 shown by higher pC02, thereby creating disturbance of the medium buffer system; and 3) absence of allantoic placental growth and differentiation (Fig. 3). This points to the area that requires further effort-i.e., devel- opment of culture methods for growth of al- lantoic placenta and trophoblast cells in concert with the embryonic compartment for continued growth of the embryo. In current culture procedures, the trophoblast cells do not multiply, and eventually begin to degen- erate along with other placental chorionic tissues when preplacental tissues were al- lowed to remain attached to the conceptus preparations. It is noteworthy, however, that in conceptus cultures of pregnancy day 10 for 2 days, there is an initial appearance of cir- culation in the ectoplacental cone and for- mation of functional umbilical vessels. How- ever, this circulation disappears, and placen- tal tissues remain undifferentiated during prolongation of the culture period (New, '78). To remedy some of the drawbacks of sealed culture bottles, we have developed a modified method of culture of rat postimplantation conceptuses utilizing 100-ml capacity roller bottles capped by New Brunswick swivel caps (Fig. 8; Tarlatzis et al., '83). These caps have five inlets which allow infusion of humidified gases continuously into bottle atmospheres and removal of the excess C02. In a series of experiments with pregnancy day 10 concep- tuses, we have been able to grow embryos at pre- or early somite stage continuously for 4 consecutive days in immediately centrifuged rat sera with increasing regimens of 0 2 con- centration (5 , 20, 40, and 95%) during each 24-h period. The initial gas mixture for the Fig. 3. Sections of rat allantoic placental tissues. Left: in utero control showing giant trophoblast cells and cho- rionic tissues with numerous blood sinusoids and capil- laries. Right placental tissues after 48 h culture of conceptus preparation. Progressive necrosis of placental chorionic elements are observed. Magnification, x 200. IN VITRO EMBRYO DEVELOPMENT DAY 12 CONCEPTUS 48 HR CULTURE - 241 I IZI) EMBRYO EBl YOLK SAC 6oo t v) Y DAY II CONCEPTUS a 48 HR CULTURE 0 2 1 5 w 200 . I DAY 10 CONCEPTUS 48 HR CULTURE 5. 1 612 i i s INITIAL IN VlTRO IN UTERO INITIAL IN VITTRO IN UTERO INITIAL IN VITRO IN UTERO OAY 10 48 HR DAY 12 DAY II 48 HR DAY I3 DAY 12 48 HR DAY 14 Fig. 4. Comparison of rat conceptus growth in utero and in vitro for 48 h in homologous male rat serum. DNA contents of embryo and yolk sac tissues of preg- nancy day 10 conceptus cultured for 48 h are near 95% of those in utero. DNA contents of day 11 embryo and yolk sac when conceptus cultured for 48 h are approxi- mately 50% and day 12,20% of those in utero. 0 EMBRYO fZl YOLK SAC 0 8 1500 3 ,,,- 48HRCULTURE DAY I I CONCEPTUS 48 HR CULTURE 4742 * 148 DAY 12 CONCEPTUS 48 HR CULTURE INITIAL INVITRO INUTERO INITIAL IN VITRO INUTERO INITIAL IN VITRO 1 UTERO DAY 10 48 HR DAY 12 DAY II 4 8 HR DAY I3 DAY 12 4 8 H R DAY14 Fig. 5. Comparison of rat conceptus growth in utero and in vitro for 48 h in homologous male rat serum, assessed by protein measurement. Growth pattern is identical to that shown in DNA measurement (Fig. 4). 242 M.K. SANYAL AND F. NAFTOLIN 250 200 u 3 J Y -1 150 0 0 > u m E 100 \ z 0 a ul li 50 DAY 12 CONCEPTUS 0 EMBRYO 48 HR CULTURE a YOLK SAC DAY I I CONCEPTUS 48 HR CULTURE DAY 10 CONCEPTUS 48 HR CULTURE DAY12 15-16 20-21 DAY12 15-16 20-21 DAY 12 15-16 20-21 PREGNANCYSERA PREGNANCY SERA PREGNANCY SERA Fig. 6. Rat pregnancy day 10, 11, and 12 conceptuses cultured for 48 h in pregnancy sera obtained from preg- nant rats on days 12, 15-16, and 20-21. Embryo and yolk sac DNA contents are identical in these cultures, although sera at different days of gestation have differ- ent hormonal composition. 2500 0 EMBRYO 1 rZa YOLK SAC DAY 12 CONCEPTUS 48 HR CULTURE [L 1500 V v) Y _I a 9 0 >- (L m ' 1000 z E (5 W t 0 500 DAY 10 CONCEPTUS 48 HR CULTURE DAY II CONCEPTUS 48 HR CULTURE T DAY 12 15-16 20-21 DAY 12 15-16 20-21 DAY12 15-16 20-21 PREGNANCY SERA PREGNANCY SERA PREGNANCY SERA Fig. 7. Protein contents of embryos and yolk sac of the same experiment described in Figure 6. Embryo protein contents of pregnancy day 10,11, and 12 concep- tuses after culture for 48 h in three different pregnancy sera are similar. However, yolk sac protein is increased considerably in day 12 conceptuses cultured with preg- nancy sera, and these values are higher than those cul- tured with homologous male rat sera. IN VITRO EMBRYO DEVELOPMENT 243 TABLE I . Effects ofprogesterone and estradiol-17fl on in witro development of rat embryos ofpregnancy day 11 i n serum from castrated male rats pg/Embryo ____. pgiYolk Sac ___ _ _ _ _ _ _ _ _ _ ~ pg/Placenta Progesterone Estradiol-170 d m l pglml medium medium Protein - DNA Protein DNA Protein DNA _- 127 i 9 872 i 71 38 i 1 627 f 38 12 i 1 193 7 127 i 8 953 i 62 39 i 2 585 f 46 11 i 1 170 i 13 10 - 10 0.1 130 i 11 953 I 92 34 i 1 562 i 45 11 i 0 170 2 13 10 1.0 126 i 10 932 i 64 39 i 2 576 i 38 12 1 196 & 15 Mean i S.E.M.; n = 6-8; culture period = 46-48 h - - first 24 h was 5% 0 2 , 5% COz and Nz; the second 20% O,, 5% COZ and N,; the third 40% 0 2 , 5 % COZ and N,; and the fourth 95% 0 2 , 5% COZ, and Nz. Infusion of these gas mixtures maintains average partial pres- sures of oxygen during the respective 24 h periods at 69,131,213, and 409 mm Hg. The pCO2 values remained stable a t 25-35 mm Hg, and pH at 7.5-7.6 throughout the entire culture period. In addition, to minimize defi- ciencies of nutrients in homologous rat serum culture medium, during the first 2-day cul- ture periods six conceptuses were grown per 24-ml volume, and during the second 2-day periods two conceptuses were in each 24-ml volume of fresh sera. In this modified culture method, the em- bryonic growth is approximately 200-fold when assessed by DNA and protein contents. The egg cylinder embryos without any pri- mary organs grow into embryos with various organs. For example, cultured embryos de- velop closed neural tubes with distinct seg- mentation of the brain into anterior tele- cephalon and diencephalon, middle mesen- W I GAS IN FUSION cephalon, and posterior metencephalon and myelencephalon. The development of sen- sory organs-olfactory, optic, and otic-is also considerable. Embryonic heart differentia- tion with distinct chambers and rhythmic beating, and the formation of networks of vessels and capillaries extending into the yolk blood islands, are prominent. The struc- tural differentiation of approximately 50 somites and both anterior and posterior limb buds are also prominent in these embryos cultured continuously for 4 days. It is as- sumed the embryogenesis in vitro is orderly and that cellular processes of differentiation occur as in normal embryos, but little actual study has yet tested that assumption (Tar- latzis et al., '831. INDUCTION OF ANOMALOUS EMBRYOGENESIS Although effects of toxic agents usually lead to embryo death rather than to struc- tural fetal malformations in the preimplan- tation embryos, they are susceptible to mutations and chromosomal aneuploidy. Donahue ('72) estimated nearly 4% of mouse J SAMPLE THDRAWAL U Fig. 8. Roller bottle to which New Brunswick swivel cap has been attached. The cap has five inlets which allow continuous flushing of bottle gas atmosphere with humidified mixtures o f gases 0 2 , COa, and Nz. The cap also allows withdrawal and supplementation of culture medium with nutrients. 244 M.K. SANYAL AND F. NAFTOIJN preimplantation embryos carry chromo- somal anomalies. The expression of Ah locus in the mouse, regulating mixed function oxi- dase activity and cytochrome P-450, occurs during the preimplantation period. Mouse embryos with Ah-positive locus may at this period metabolize polycyclic hydrocarbons as indicated by higher sister chromatic ex- change compared to A h-negative embryos (Shum et al., '79; Galloway, '81; Filler and Lew, '81). Embryogenesis during this period is affected considerably by exposure to var- ious other toxins, such as microtubule inhib- itors or metallic ions (Spielmann and Eibs, '77; Eibs and Spielmann, '77; Izquierdo and Becker, '82; Chang, '74; Siracusa et al., '80; Storens and Jonsen, '80). These processes perhaps account for a significant portion of embryonic loss during the preimplantation period. During the period of organogenesis after implantation the embryos are highly vulner- able to structural malformations. Innumer- able direct and indirect experimental data indicate that deleterious exposure of mothers during this period produces malformation in the offspring (Wilson, '73). The relative con- tributions of the mother, placenta, and fetus in this teratogenic process are unclear. Therefore, the availability of controlled cul- ture methods for whole embryos during or- ganogenesis which are free from the mater- nal barriers would be of considerable interest and value to studies on the mechanisms of normal and anomalous embryonic differ- entiation. Utilizing the postimplantation embryo cul- ture system, effects of individual teratogens and their propensity toward anomalous or- ganogenesis are being studied in a number of laboratories (Agnish and Kochhar, '76; Sanyal et al., '81; Kitchin et al., '81a,b; Allen et al., '81; Schmid et al., '81, '82; Kitchin and Ebron, in press; Kitchin and Ebron, '82; Carey and Klein, '82; Klein et al., '80; Chatot and Klein, '82; Cockroft, '80; Brown et al., '79; Sadler and Kochhar, '76; Webb and Cop- pola,, '76; Fantel et al., '79, '81; Greenaway et al., '82a,b; Mirkes et al. '81, '82; Gulam- husein et al., '82). There are four basic ap- proaches of such research utilizing postim- plantation conceptus culture: 1) teratogens directly added to the culture medium; 2) te- ratogens added in combination with a micro- soma1 activating system; 3) whole-embryo culture in the presence of teratogen com- bined with specific organ culture; and 4) con- ceptuses cultured with serum from human or animal sera exposed to teratogens. In these in vitro studies utilizing different research approaches, information on the mode of ac- tion of toxic agent on embryos, toxic effects mediated by the mother, and relative suscep- tibility of different embryonic organs toward toxic agents are made available which one cannot obtain conveniently in in vivo studies. MECHANISM OF TERATOGEN ACTION AND DETECTION OF HUMAN TERATOGENS Time and dose effects Addition of model teratogens to the concep- tus culture medium induces stage-dependent structural embryo anomalies proportional to the dose. In our studies with alkylating agent 2,4,6-triethylimino-1,3,5-triazine, (TEM, 50- 100 ngiml medium) produce considerable re- duction in growth of pregnancy day 10 rat em- bryos in culture, with degenerative changes in the neuroepithelial tissues (Fig. 9; Sanyal and El-Nahass unpublished). These changes were not prominent when older embryos of preg- nancy day 12 were utilized. Similarly bromo- deoxyuridine (BrdU) treatment (5-50 pg/ml) to day 10 conceptuses in cultures produced dose- dependent retardation of embryo develop- ment with open neural tubes (Fig. 10; Biggers and Sanyal, '83). The neurite outgrowth from embryos treated with BrdU was markedly re- duced when isolated fragments of neural tubes were cultured over collagen-plated Petri dishes in the minimum essential medium containing horse and calf sera (Fig. 11). The alteration of the differentiation process is pre- sumably related to the incorporation of BrdU into DNA. BrdU at these doses is known to af- fect cell division; its influence on neurite out- growth from neural tube fragments is, however, unlikely to be dependent upon cell division. Although nonspecific effects of agents on embryogenesis in whole-embryo cultures require careful consideration, it is noteworthy that compounds such as amino- pyrine, which are nonteratogenic in in vivo tests, do not produce anomalous embryogene- sis upon direct addition to the conceptus cul- ture medium. Bioactivation of teratogens and xenobiotics Despite impressive potency of some terato- genic agents, not all model teratogens pro- duce anomalous in vitro embryogenesis when added to the culture medium (Sanyal et al., '79, '81): some teratogens require bioactiva- tion into toxic products. Therefore, it appears that this step is carried out in vivo by mater- nal or nonembryonic tissues, since the capa- IN VITRO EMBRYO DEVELOPMENT 245 Fig. 9. Section of embryos cultured with alkylating agent TEM (lower). Necrosis of neuroepithelium is pro- duced by treatment with this compound. A section from control embryo shows deeply stained neuroepithelium (upper). Magnification, X 200. 246 M.K. SANYAL AND F. NAFTOLIN Fig. 10. Anomalous embryos produced by treatment of pregnancy day 10 conceptuses with BrdU M) for 48 h. These embryos are frequently a t reverse position and retarded compared to controls. Morphogenesis is significantly retarded and anomalous. Neural and sen- sory organ differentiation are markedly affected. Mag- nification, ~ 5 0 . bility of bioactivation of xenobiotics by the embryonic tissue at organogenesis stage is very small, and may not be sufficient to in- duce structural malformation by numerous teratogens (Hales, '81; Nebert, '80). Preg- nancy day 11 rat conceptuses, when cultured with a teratogen (cyclophosphamide) and a microsomal activating system composed of rat hepatic microsomes and NADPH cofactor (Kitchin et al., '81a,b; Mirkes et al., '81, '821, produce deleterious effects on embryogene- sis. The embryonic growth and differentia- tion were markedly anomalous and retarded in such cultures, whereas cyclophosphamide alone or microsomes and cofactors added to cultures separately did not produce deleteri- ous effects. Furthermore, use of microsomes treated with cumene hydroperoxide in the activating system was completely ineffective in inducing deleterious effects (Kitchin et al., '81a). Since cumene hydroperoxide is known to specifically inhibit cytochrome P-450 ac- tivities in microsomes, these studies indicate possible involvement of cytochrome P-450 in biotransformation processes of the teratogen. This capability of coculture of conceptuses with a teratogen-activating system utilizing microsomes from various soures is an inva- luable method which will allow in-depth in- vestigation of mechanisms of teratogen bio- activation. Transplacental transport of activated tox- ins from maternal and placental compart- ments may also induce deleterious mutations in the embryo. The contribution of maternal tissue in producing mutagenic substances by bioactivation processes and mutagenic sus- ceptibility of embryonic cells during the pos- timplantation period can be assessed in whole-embryo cultures by sister chromatid exchange analysis (Allen et al., '81). In such experiments, chromatids of rat yolk sac and embryonic cells of pregnancy days loill are labeled with BrdU pellets implanted in vivo in the mother, and estimates of sister chro- matid exchanges (SEC) are made in cells of conceptuses cultured in the presence of tera- togens. SCE index was low and identical to controls in the presence of cyclophosphamide in the culture media. In contrast, phosphor- amide mustard, a metabolite of cyclophos- phamide produced by bioactivation processes when added to the culture, induced 8-fold increase in SCE indices in yolk sac cells over controls of cyclophosphamide-treated cultures. Endogenous teratogens Another approach utilizing rat conceptus culture has been the detection of teratogens in human or animal sera. Klein and associ- ates have shown higher anomalous rat em- bryogenesis rates in cultures with human sera from patients with a history of sponta- neous abortions and patients treated with anticancer drugs or in sera from monkeys that have the history of spontaneous abor- tions, compared to those cultured in normal control serum (Chatot et al., '80; Klein et al, '81). Since human serum has low levels of glucose, by adding exogenous glucose to hu- man serum medium, pregnancy day 10 con- ceptuses can be grown for longer periods with considerable embryonic differentiation, and different types of rat embryo anomalies are induced by culture in human sera containing teratogens. Therefore, it seems that a rat whole-embryo culture method could be used IN VITRO EMBRYO DEVELOPMENT 247 Fig. 11. Neurite outgrowth from neural tube frag- ments of BrdU M) treated pregnancy day 11 em- bryos is highly retarded during 3-day post embryo culture in collagen-coated petri dishes (right). Untreated controls under identical conditions produce vigorous neurite out- growths (left). Magnification, X200. as a biological assay system for assessment of drug metabolites which may be injurious to embryos and teratological potential of other deleterious substances in the human sera. MICROADMINISTRATION OF TERATOGENS AND CELLS INTO EWfRRYOS The postimplantation conceptuses are au- tonomous as they develop a circulatory sys- tem with numerous blood vessels and a network of capillaries while growing in vitro. Teratogens in the medium must cross the capillary barriers of the yolk sac to reach the embryonic cells. Therefore, we have recently utilized microinjection techniques in which the effects of teratogens on embryonic devel- opment are determined more specifically by direct administration into embryonic com- partments (Satish et al., '83). Anomalous ern- bryogenesis is induced following injection of teratogens directly into pregnancy day 11 conceptus Compartments or into various re- gions of the embryo, during the subsequent 2-day culture of treated and control concep- tuses. In our initial experiments with pon- tarnine blue dye, we determined that approximately 10-50 nl volumes of fluid could be conveniently injected into different compartments of pregnancy day 11 concep- tuses (Fig. 12). Utilizing these micromethods, marked anomalous development of the em- bryo can be produced by microinjection of approximately 100 ng quantity of the phos- phoramide mustard in 50 nl sterile distilled water into yolk sac compartment of such con- ceptuses. In contrast, the same quantity of phosphoramide mustard when injected into amniotic sac produced no apparent effect de- tectable in gross and histological examina- tion (Fig. 13). In similar experiments, when phosphoramide mustard is injected into the anterior neural tube region, the embryos be- come anencephalic with substantial post- cephalic differentiation (Satish et al., '83). The effects of phosphoramide mustard on the 248 M.K. SANYAL AND F. NAFTOLIN Fig. 12. Microinjection procedure into conceptus com- partments is illustrated by pontamine blue dye injec- tions. Left, the micromanipulator pipette (marked by an arrow) is being inserted into amniotic sac. Right, the dye solution is distributed uniformly within the cavity in 2- 3 min. Magnification, x50. IN VITRO EMBRYO DEVELOPMENT 249 posterior somite region are minimal in com- parison to its effects on the neural tube under identical conditions of treatment. Explora- tory microinjection studies also show that isolated cells can also be placed into various embryonic regions; however, behavior of such cells in cultured conceptuses remains to be determined. Thus, the in vitro culture methods allow a more precise elucidation of process associ- ated with differentiation, nutritional re- quirements, and mechanisms that promote anomalous embryogenesis during the pre- and postimplantation periods. In these in vi- tro procedures, various experimental manip- ulations are possible to explore the specificity of teratogens, maternal contribution in the production of active teratogens, and influ- ences of genomic alteration on organogenesis of the embryo. ACKNOWLEDGMENTS We acknowledge participation of Drs. J. Sa- tish, B. Pratt, B. Tarlatzis, E. El-Nahass, and K.T. Kitchin, and Mr. W. Biggers in some of the unpublished studies presented. We thank Miss Jacki Fitzpatrick for her assistance in preparing the manuscript. These studies have been supported by the National Insti- tutes of Health (HD 14587) and Rockefeller Foundation grants. F.N. is supported by a Fogarty Senior International Fellowship (F06-TW00706) and a John Simon Guggen- heim Memorial Foundation Fellowship. LITERATURE CITED Agnish, N.D., and D.M. Kochhar (1976) Direct exposure of postimplantation mouse embryos to 5-bromodeoxy- uridine in vitro and its effect on subsequent chondro- genesis in the limbs. J . Embryol. Exp. 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