ABSTRACT
Abnormalities of the ocular system which appear in organ culture in Way-mouth’s medium with freshly added glutamine (Armstrong & Elias, 1968) resemble those caused by transitory pteryolglutamic acid (PGA or folic acid) deficiency in vivo (Armstrong & Monie, 1966). The configurations of such malformations as lens herniations, retinal diverticula, and rosette-like formations of the retina are remarkably similar in both cases. The experiments reported in this paper were undertaken in an effort to understand the mechanisms involved in the production of similar abnormalities by two very different experimental conditions: the addition of glutamine in vitro and the transitory deficiency of PGA in vivo.
One series of experiments involved the effects of manipulation of the PGA and glutamine content of the culture medium on eye development in vitro. Parallel studies on PGA-deficiency in vivo were undertaken in conjunction with organ-culture experiments in order to compare the effects on abnormal eye morphogenesis.
MATERIALS AND METHODS
1. Culture experiments
Eyes from normal day-11 Long-Evans rat embryos were used as well as eye tissues from embryos of equivalent age whose mothers had been subjected to transitory PGA-deficiency in vivo from days 9-11 of gestation (Armstrong & Monie, 1966). Waymouth’s MB 752/1 culture medium was employed in both experiments, the content of glutamine and PGA being altered as required. In addition, the effect of the PGA antimetabolite 9-methyl PGA, used to induce PGA-deficiency in vivo, was tested in the culture system by adding it directly to Waymouth’s medium both with and without PGA; only day-11 eyes were cultured under the latter condition. In all cases the culture and histological procedures were the same as those previously described by Arm-strong & Elias (1968), and freshly prepared glutamine was always added just before use.
2. PGA deficiency in vivo
The earlier experiments on congenital eye defects and PGA deficiency in vivo (Armstrong & Monie, 1966) were extended for the purposes of this study. Thus, the period of deficiency was prolonged for an additional 1-4 days, from days 9-12 through days 9-15 of pregnancy, as com-pared to the 9-to 11-day transitory deficiency reported previously. A few animals were kept on the PGA-deficient regimen for the entire length of the experi-ment (i.e. days 9-15). Eye tissues from these two groups of experimental em-bryos were compared histologically.
RESULTS
1. In vitro experiments (Table 1)
A. Eyes of normal day 11 embryos in Way mouth’s medium without PGA
Expiants in Waymouth’s medium lacking PGA, but containing glutamine, showed a greater degree of degeneration as compared to cultures in normal Waymouth’s medium. The marked increase in growth rate observed when both glutamine and PGA were present in the medium (i.e. normal Waymouth’s medium) was greatly reduced. A few minor anomalies such as irregularities in the lenses were observed but the majority of the eyes were only slightly larger than those cultured in glutamine-deficient medium (Armstrong & Elias, 1968) and did not show malformations.
B. Eyes of day 11 embryos from PGA-deficient mothers cultured in normal and in glutamine-deficient medium
At the time of explanation the eyes of 11-day-old embryos from PGA-deficient (days 9-11) mothers are not abnormal, but retardation of growth and develop-ment is apparent. However, it is probable that the eyes would have become abnormal if the embryos had remained in vivo until day 12 and the mother had been placed on a normal diet from day 11 onwards (Armstrong & Monie, 1966). When these day-11 eyes were cultured in normal Waymouth’s medium they showed marked increase in size, but to a somewhat lesser degree than normal eye tissues. Furthermore, all of the abnormal patterns of eye development ob-served in normal eyes under these conditions were present in PGA-deficient expiants (Plate 1, figs. A, B). When such PGA-deficient tissues were cultured in glutamine-deficient medium, however, they remained normal but were small and retarded (Plate 1, fig. C). Thus, it appears that tissues retarded by previous ex-posure to PGA deficiency in vivo are still capable of responding to glutamine in culture.
C. Comparison of normal eye tissues cultured in Waymouth’s medium containing 9-methyl PGA in the presence and absence of PGA
The PGA antimetabolite 9-methyl PGA was first tested in standard Way-mouth’s medium which contains 0·40 mg/1. PGA and 350 mg/1. glutamine. Eyes cultured in the presence of this amount of PGA with concentrations of 0·1,1,10, 100 and 1000 μg/ml of 9-methyl PGA exhibited a slight increase in size as com-pared with medium deficient in both PGA and glutamine. They did not, how-ever, become abnormal or as large as eyes grown in medium containing both PGA and glutamine. Since tissues exposed to the 1000 μg/ml concentration of 9-methyl PGA showed increased degeneration indicative of a generalized toxic effect, the 100 μg/ml level was selected as a test concentration in Waymouth’s medium deficient in PGA.
Normal eye tissues cultured in PGA-deficient Waymouth’s medium with 100 μg/ml of 9-methyl PGA behaved just as those cultured in standard Way-mouth’s medium (i.e. they became large and abnormal). It should be noted that the vitreous body did not develop under any of the previously described culture conditions. The failure of a vitreous body to appear in vitro and some possible consequences of its absence are discussed elsewhere (Armstrong & Elias, 1968).
2. In vivo experiments
Further studies on PGA deficiency in vivo revealed some interesting and un-expected findings. In these experiments the deficiency was continuous, and not transitory as had been the case in previous experiments (Armstrong & Monie, 1966).
Continuous deficiency from days 9-11 or 9-12 led to arrested growth and differentiation. The optic vesicles did not invaginate to form optic cups, and although the surface ectoderm had been in direct contact with the vesicles since day 11, lens formation did not occur (Plate 1, fig. D). Malformations of the eye were not evident.
The walls of the optic vesicles were extremely thin and the overlying surface ectoderm was squamous in type in day-13 embryos whose mothers were on the deficient regimen from days 9-13. Only rarely did the vesicles invaginate to form cups; in most instances, however, a small lens placode had formed in relation to their center (Plate 1, figs. E, F). The lens placode remained attached to the sur-face ectoderm and did not form a lens vesicle. Thus evidence of lens differentia-tion was manifested although the optic cups did not form and growth was vir-tually at a standstill. The embryo resorption rate was near 50 % in this group of experimental animals.
Approximately 90 % of the embryos from mothers continuously deficient from days 9-14 and 9-15 were resorbed at autopsy on the 14th and 15th days of pregnancy but a few surviving embryos seemed to have ‘escaped’ the inhibi-tory effects of PGA-deficiency after this prolonged exposure. Although the eyes of these embryos were microphthahnic and retarded as compared with normal eyes, they had begun to grow and differentiate again and were becoming ab-normal. They appeared to be more comparable to those observed after transitory PGA-deficiency from days 9-11 followed by a normal diet, rather than the picture observed following a continuous deficiency from days 9-13 of pregnancy where both growth and differentiation had seemingly come to a standstill.
In another series of experiments embryos were examined from mothers which had been on a PGA-deficient diet starting on the 9th day of pregnancy and sub-sequently returned to a PGA-supplemented diet 24 h prior to autopsy. The transitory PGA deficiency periods lasted from days 9-12, 9-13 and 9-14 of pregnancy. These embryos exhibited the typical increase in growth rate and resulting abnormalities described previously for transitory PGA deficiency from days 9-11 of pregnancy (Armstrong & Monie, 1966). Thus, in all cases, transi-tory PGA deficiency followed by replacement on a normal diet led to abnormal eye development. In contrast, as noted above, short-term continuous PGA-deficiency per se did not cause eye malformations.
DISCUSSION
When glutamine was continuously present in the culture medium eye expiants exposed to a PGA-deficient medium remained relatively small and did not become abnormal, suggesting that the ‘glutamine response’ described previously (Armstrong & Elias, 1968) may have been dependent on the presence of PGA in the medium. In contrast, those exposed to a PGA-deficient medium con-taining 9-methyl PGA became large and abnormal. In the latter condition the tissues behaved the same as those in a medium containing PGA. The fact that abnormalities occur in PGA-deficient medium containing 9-methyl PGA and glutamine may indicate that 9-methyl PGA is altered in some manner in vitro so that it can substitute for PGA in the absence of the latter. On the other hand, when both PGA and 9-methyl PGA were present in the medium, abnormalities did not occur even when the level of the antimetabolite had an over-all toxic effect on the tissues. These latter findings suggest the existence of a competitive mechanism between the metabolite and the antimetabolite, so that when both are present neither can elicit the response in the tissue which each alone can do in the presence of fresh glutamine. Abnormalities observed under these condi-tions would thus be attributable mainly to the presence of a functional form of PGA plus glutamine in the medium, both of which are required for rapid growth in culture.
Embryonic eye tissues previously exposed to 9-methyl PGA in vivo did not become abnormal in culture unless the growth factors PGA and glutamine were present in the medium. This indicates that 9-methyl PGA does not produce ocular malformations by altering the tissue irreversibly into an abnormal pattern of development. Rather, it would appear that such tissues when provided with a growth-promoting environment may ‘recover’ from the temporary inhibition and continue to grow, but that growth no longer follows normal pathways. A few earlier studies on PGA deficiency tend to support this contention. Thus, certain cell cultures exposed to the PGA-antimetabolite, aminopterin, for long periods in vitro also overcame the inhibitory effects of the antimetabolite (Jacobson, 1954), and these cells were found to contain a factor which could temporarily inhibit or inactivate the antimetabolite. Observations by Monie, Nelson & Evans (1954) on kidney malformations following long-term PGA deficiency in vivo (induced with 9-methyl PGA) from days 11-21 of pregnancy showed that fetal urethral atresia usually resulted from retarded growth and differentiation ; however, in approximately 33 % of the cases the abnormality was apparently due to hyperplasia. It is possible that tissues exposed to an antimetabolite in the absence of the essential metabolite may succeed in utilizing, normally or abnor-mally, the antimetabolite to some degree. That this process may result in mal-formation should be explored further by direct experimental means. Asling et al. (1960) and Asling (1961) found that long-term PGA deficiencies (days 11-21) in vivo produced only slight anomalies of the palate resulting mainly from retardation, as compared to the marked abnormalities observed after transitory PGA deficiency (days 9-11) during early stages of development. In the present study on PGA deficiency in vivo, eyes exposed to a continuous deficiency had less tendency to become grossly abnormal than those exposed to a transitory deficiency. However, if the deficiency was extended for periods up to 5 or 6 days eye tissues in the small percentage of embryos which survived ‘overcame’ the initial inhibitory effects, began to grow again and became abnormal.
A collation of results of experiments on PGA deficiency in vitro and in vivo as well as on glutamine in vitro indicates that ocular malformations observed under these conditions may have resulted from the growth response of the tissue following an initial transitory lag in the normal growth rate and pattern. If the growth-inhibitor influences were overcome either in vitro or in vivo, growth was resumed but seemed to follow an aberrant course. The temporary delay in growth and differentiation was produced in vivo by transitory PGA deficiency, and in vitro by the ‘initial period of adaptation’ to culture conditions (Arm-strong & Elias, 1968). In each case ocular abnormalities appeared only after this initial period of temporary retardation. In the case of PGA deficiency in vivo, eye defects were observed only when a transitory type of deficiency was instituted or in the few embryos who apparently ‘escaped’ the inhibitory effects of long-term deficiency. In both instances rapid growth and differentiation followed the initial period of inhibition in vivo. Similarly, ocular malformations were never observed in the presence of glutamine and PGA during the first two days of culture (‘the initial period of adaptation’) when growth and differentia-tion were relatively slow. Abnormalities did occur, however, in the subsequent period of rapid growth when both glutamine and a PGA source were available; if this rapid growth period was prevented by either glutamine deficiency or PGA deficiency, then the eyes showed normal development but were small in size.
On the basis of these data it is proposed that a ‘rebound’ hypothesis of tera-togenesis may explain both the in vitro and the in vivo eye responses observed. It is postulated that neither PGA and glutamine in the culture medium, nor transitory PGA deficiency during pregnancy, acts on the tissues as a specific teratogen. Rather, the initial period of adaptation, in the in vitro system, and the transient dietary deficiency, in the in vivo system, are comparable in their early inhibition of tissue growth and differentiation. Thus, following adaptation in culture or dietary repletion of the mother the tissue appears to respond by under-going compensatory growth and/or differentiative phenomena.
During this ‘recovery’ period mitotic figures are more numerous in tem-porarily inhibited tissues than in control tissues at the same developmental stage. Also, rapid differentiation is occurring simultaneously with a very active growth phase, so that morphologically the degree of differentiation achieved in a temporarily inhibited tissue during later stages of development may be com-parable to that of a control tissue of equivalent age.
It is suggested that gross morphological abnormalities produced in other experimental conditions be studied in order to determine whether they develop by means of this compensatory ‘rebound’ growth mechanism.
SUMMARY
Normal and PGA-deficient embryonic ocular tissues have been cultured in Waymouth’s medium in which the amounts of glutamine and PGA were varied. In addition, normal eye tissues were cultured in the presence of the anti-metabolite 9-methyl PGA (used to induce PGA deficiency in vivo) which had been added directly to Waymouth’s medium.
Similarities between ocular malformations observed in vitro following the addition of fresh glutamine to Waymouth’s-MB 752/1 medium and those resulting from transitory PGA deficiency in vivo are discussed.
Parallel in vivo studies on PGA deficiency for both continuous and transi-tory periods were also undertaken.
To account for the results obtained, a ‘rebound’ hypothesis of teratogenesis is proposed and discussed.
RÉSUMÉ
Développement des yeux d’embryons de Rat, en culture d’organe. II. Une approche in vitro, des mécanismes de la tératogénèse
Des tissus oculaires embryonnaires, normaux et déficients en PGA, sont cultivés sur le milieu de Waymouth, dans lequel les taux de glutamine et de PGA sont modifiés. De plus, des tissus des yeux normaux sont cultivés en présence de l’antimétabolite 9-méthyl PGA (utilisé pour induire in vivo la déficience en PGA), ajouté directement au milieu de Waymouth.
Les analogies entre les malformations oculaires observées in vitro après l’addition de glutamine fraîche au milieu MB/752/1 de Waymouth et les mal-formations oculaires résultant d’une déficience transitoire en PGA in vivo sont discutées.
Des recherches parallèles sur la déficience transitoire ou continue en PGA, sont aussi entreprises in vitro.
Pour expliquer les résultants obtenus, une hypothèse ayant un rebondisse-ment sur la tératogénèse est proposée et discutée.
ACKNOWLEDGEMENTS
The authors are indebted to Dr Ian W. Monie for his valuable advice and to Mr Dave Akers for technical assistance. This investigation was supported by U.S.P.H.S. grants HD-00142 and HD-00149.
REFERENCES
Plate 1
Abbreviations: ae, anterior epithelium of lens; bv, brain vesicle; l, lens; lf, lens fibers; lp, lens placode; nl, neural layer of retina; ov, optic vesicle; pl, pigmented layer of retina; rd, retinal diverticulum ; se, surface ectoderm.
Fig. A. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Way-mouth’s medium. The lens is irregular in shape and the anterior epithelium is ill defined. × 177.
Fig. B. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Way-mouth’s medium. Note the extensive diverticulum lying between pigmented and neural epithelia of the retina. × 177.
Fig. C. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Way-mouth medium deficient in glutamine. The lens is small and normal. The neural and pig-mented epithelia of the retina have also differentiated normally. × 177.
Fig. D. Day-12 embryonic eye from a mother which had been on a continuous PGA deficiency from days 9-12 of pregnancy. The optic vesicle has not invaginated and remains in direct contact with the surface ectoderm; the lens placode also failed to differentiate. × 177.
Fig. E. Day-13 embryonic eye from a mother which had been on a continuous PGS defi-ciency from days 9-13 of pregnancy, showing the beginning formation of a lens placode, although the optic vesicle has not yet invaginated. × 177.
Fig. F. Day-13 embryonic eye from a mother which had been on continuous PGA-deficiency from days 9-13 of pregnancy, showing a more advanced stage of lens placode formation than observed in Fig. E. The optic vesicle, however, has still not invaginated. × 177.
Fig. A. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Way-mouth’s medium. The lens is irregular in shape and the anterior epithelium is ill defined. × 177.
Fig. B. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Way-mouth’s medium. Note the extensive diverticulum lying between pigmented and neural epithelia of the retina. × 177.
Fig. C. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Way-mouth medium deficient in glutamine. The lens is small and normal. The neural and pig-mented epithelia of the retina have also differentiated normally. × 177.
Fig. D. Day-12 embryonic eye from a mother which had been on a continuous PGA deficiency from days 9-12 of pregnancy. The optic vesicle has not invaginated and remains in direct contact with the surface ectoderm; the lens placode also failed to differentiate. × 177.
Fig. E. Day-13 embryonic eye from a mother which had been on a continuous PGS defi-ciency from days 9-13 of pregnancy, showing the beginning formation of a lens placode, although the optic vesicle has not yet invaginated. × 177.
Fig. F. Day-13 embryonic eye from a mother which had been on continuous PGA-deficiency from days 9-13 of pregnancy, showing a more advanced stage of lens placode formation than observed in Fig. E. The optic vesicle, however, has still not invaginated. × 177.