/. Embryol. exp. Morph. Vol. 19, 3, pp. 407-14, May 1968
With 1 plate
Printed in Great Britain
407
Development of embryonic rat eyes
in organ culture
II. An in vitro approach to teratogenic mechanisms
By R. CHRISTY ARMSTRONG 1 & JOEL J. ELIAS 1
From the Department of Anatomy, University of California
Medical Center, San Francisco
Abnormalities of the ocular system which appear in organ culture in Waymouth'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 Arm1
Authors' address: Department of Anatomy, University of California Medical Center,
Third and Parnassus, San Francisco, California 94122. U.S.A.
408
R. C. ARMSTRONG & J. J. ELIAS
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 compared 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 experiment (i.e. days 9-15). Eye tissues from these two groups of experimental embryos were compared histologically.
RESULTS
1. In vitro experiments (Table 1)
A. Eyes of normal day 11 embryos in Waymouth 's medium without PGA
Explants 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.
Table 1. Summary of organ culture conditions and effects of eyes
Source of embryonic eye tissues
11-Day normal
Waymouth's medium (MB 752/1)
containing 350 mg/1. freshly
prepared glutamine +
PGA and glutamine
PGA
Glutamine
9-Methyl PGA,* PGA
and glutamine+
9-Methyl PGA* and
glutamine
11-Day; mother PGAdeficient from days 9-11
of pregnancy
Very large; many
abnormalities
Very small;
normal
Moderately small;
mostly normal
Large; normal
Large; many
abnormalities
Very small;
normal
—
Very large; many
abnormalities
—
* 9-Methyl PGA added at lOO^g/ml.
—
/. Embryol. exp. Morph., Vol. 19, Part 3
R. C. ARMSTRONG & J. J. ELIAS
PLATE 1
facing p. 409
Embryonic rat eyes in culture. II
409
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 development 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 observed in normal eyes under these conditions were present in PGA-deficient
explants (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 exposure 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 Waymouth'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,
PLATE 1
Abbreviations: ae, anterior epithelium of lens; bv, brain vesicle; /, lens; If, lens fibers; Ip, lens
placode; nl, neural layer of retina; ov, optic vesicle; pi, 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 Waymouth's medium. The lens is irregular in shape and the anterior epithelium is ill denned.
xl77.
Fig. B. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Waymouth's medium. Note the extensive diverticulum lying between pigmented and neural
epithelia of the retina, x 177.
Fig. C. Day-11 embryonic eye from a PGA-deficient mother after 5 days of culture in Waymouth medium deficient in glutamine. The lens is small and normal. The neural and pigmented epithelia of the retina have also differentiated normally, x 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, x 177.
Fig. E. Day-13 embryonic eye from a mother which had been on a continuous PGS deficiency from days 9-13 of pregnancy, showing the beginning formation of a lens placode,
although the optic vesicle has not yet invaginated. x 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. x 177.
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R. C. ARMSTRONG & J. J. ELIAS
100 and 1000 /*g/ml of 9-methyl PGA exhibited a slight increase in size as compared with medium deficient in both PGA and glutamine. They did not, however, become abnormal or as large as eyes grown in medium containing both
PGA and glutamine. Since tissues exposed to the 1000 /^g/ml concentration of
9-methyl PGA showed increased degeneration indicative of a generalized toxic
effect, the 100 /*g/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 /*g/ml of 9-methyl PGA behaved just as those cultured in standard Waymouth'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 unexpected 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 surface ectoderm and did not form a lens vesicle. Thus evidence of lens differentiation was manifested although the optic cups did not form and growth was virtually 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 inhibitory effects of PGA-deficiency after this prolonged exposure. Although the eyes
of these embryos were microphthalmic and retarded as compared with normal
eyes, they had begun to grow and differentiate again and were becoming abnormal. 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.
Embryonic rat eyes in culture. II
411
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 subsequently 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, transitory PGA deficiency followed by replacement on a normal diet led to abnormal
eye development. In contrast, as noted above, short-term continuous PGAdeficiency per se did not cause eye malformations.
DISCUSSION
When glutamine was continuously present in the culture medium eye explants
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 containing 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 conditions 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
27
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R. C. ARMSTRONG & J. J. ELIAS
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 abnormally, the antimetabolite to some degree. That this process may result in malformation 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 (Armstrong & 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 differentiation 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 teratogenesis 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
Embryonic rat eyes in culture. II
413
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 undergoing compensatory growth and/or differentiative phenomena.
During this 'recovery' period mitotic figures are more numerous in temporarily 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 comparable 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
1. 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 antimetabolite 9-methyl PGA (used to induce PGA deficiency in vivo) which had
been added directly to Waymouth's medium.
2. 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.
3. Parallel in vivo studies on PGA deficiency for both continuous and transitory periods were also undertaken.
4. To account for the results obtained, a' rebound' hypothesis of teratogenesis
is proposed and discussed.
RESUME
Developpement des yeux d'embryons de Rat, en culture d'organe. II. Une
approche in vitro, des mecanismes de la teratogenese
1. Des tissus oculaires embryonnaires, normaux et deficients en PGA, sont
cultives sur le milieu de Waymouth, dans lequel les taux de glutamine et de
PGA sont modifies. De plus, des tissus des yeux normaux sont cultives en presence
de l'antimetabolite 9-methyl PGA (utilise pour induire in vivo la deficience en
PGA), ajoute directement au milieu de Waymouth.
2. Les analogies entre les malformations oculaires observees in vitro apres
l'addition de glutamine fraiche au milieu MB/752/1 de Waymouth et les malformations oculaires resultant d'une deficience transitoire en PGA in vivo sont
discutees.
3. Des recherches paralleles sur la deficience transitoire ou continue en PGA,
sont aussi entreprises in vitro.
27-2
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R. C. ARMSTRONG & J. J. ELIAS
4. Pour expliquer les resultants obtenus, une hypothese ayant un rebondissement sur la teratogenese est proposee et discutee.
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
R. C. & ELIAS, J. J. (1968). Development of embryonic rat eyes in organ culture.
I. Effect of glutamine on normal and abnormal development in a chemically defined
medium. /. Embryol. exp. Morph. 19, 397-405.
ARMSTRONG, R. C. & MONIE, I. W. (1966). Congenital eye defects in rat fetuses following
maternal petroyglutamic acid deficiency. J. Embryol. exp. Morph. 16, 531-42.
ASLING, C. W. (1961). Congenital defects of face and palate in rats following maternal
deficiency of pteroylglutamic acid. In Congenital Anomalies of the Face and Associated
Structures (ed. Samuel Pruzansky), pp. 173-87. Springfield: Charles C. Thomas.
ARMSTRONG,
ASLING, C. W., NELSON, M. M.,
DOUGHERTY, M. D.,
WRIGHT, H. V. & EVANS, H. M.
(1960). The development of cleft palate resulting from maternal pteroylglutamic (folic) acid
deficiency during the latter half of gestation in rats. Surgery, Gynec. Obstet. Ill, 19-28.
JACOBSON, W. (1954). Mode of action of folic acid antagonists and the function of the Leuconostic Citrovorum factor. In CIBA Foundation Symposium on Chemistry and Biology of
Pteridines (ed. G. E. W. Wolstenholme and M. P. Cameron), pp. 329-55. Boston: Little
Brown.
MONIE, I. W., NELSON, M. M. & EVANS, H. M. (1954). Abnormalities of the urinary system
of rat embryos resulting from maternal pteroylglutamic acid deficiency. Anat. Rec. 120,
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(Manuscript received 7 September 1967)