/ . Embryol. exp. Morph. Vol. 23, 3, pp. 729-737, 1970
729
Printed in Great Britain
Steroid control of
the normal development of glutamine synthetase
in the embryonic chick retina
By R. P I D D I N G T O N 1
From the Department of Histology and Embryology,
School of Dental Medicine, University of Pennsylvania
Glutamine synthetase (GS) activity in the neural retina of the chick embryo
is inducible with corticosteroids (Moscona & Piddington, 1966; Piddington,
1967; Moscona, Saenz & Moscona, 1967; Moscona, Moscona & Saenz, 1968;
Alescio & Moscona, 1969). Striking increases in retinal GS activity can be
elicited with hydrocortisone in ovo days in advance of the normal rapid rise of
this enzyme (Piddington & Moscona, 1967). Rapid increases in GS activity can
also be induced precociously with corticosteroids in organ cultures of young
retina (Piddington & Moscona, 1967; Reif-Lehrer & Amos, 1968). The natural
steroids most effective in stimulating retinal GS activities in vitro are the 1 \ßhydroxyl steroids hydrocortisone, corticosterone and aldosterone (Moscona &
Piddington, 1967; Reif-Lehrer, 1968).
The effectiveness of corticosteroids in promoting premature changes in retinal
GS suggests that the steroidogenic activity of the intact adrenals might control
the normal pattern of GS development in the retina. A previous study (Case,
1952) indicates that the normal growth and maturation of the chick adrenal
becomes dependent on endogenous corticotropin after the 15th day of embryonic
development. Retinal GS activities normally increase very rapidly after the
15th or 16th day. The work reported here asks whether a causal relationship
exists between these developmental events.
MATERIALS AND
METHODS
Single whole retinas from 12-day embryos were grown in organ culture in
3 ml of medium in 25 ml Erlenmeyer flasks. The medium consisted of 20% fetal
bovine serum (Microbiological Associates) in Tyrode's solution and 1 % penicillin-streptomycin mixture (5000 units of each per ml ; Microbiological Associates). Other additions (see below) were made at the expense of Tyrode's
1
Author''s address: Department of Histology and Embryology, School of Dental Medicine,
4001 Spruce Street, Philadelphia 19104, U.S.A.
47
E M B 23
730
R. P I D D I N G T O N
Solution. The cultures were gassed with 5 % C0 2 -air mixture and incubated at
37-5 °C on a rotary shaker operating at 70 rev/min. The retinas were harvested
at 24 h, washed twice in Tyrode's solution, frozen and lyophilized. The retina
powder was suspended in phosphate buffer (pH 7-1) and sonicated. Aliquots of
sonicate were assayed for GS activity by the method of Kirk & Moscona (1963)
and Kirk (1965). Protein was determined according to Lowry, Rosebrough,
Farr & Randall (1951). Specific activity was expressed as jamol of product
(glutamohydroxamate) formed per h per mg of protein.
Homogenates of adrenals, thyroids, testes and ovaries were prepared in
Tyrode's solution and added as a 20% fraction of the culture medium. Each
flask contained the homogenate of 2 adrenals, 2 thyroids, 2 testes or 1 left ovary.
Adrenal glands were also cut in half and cultured as pieces.
A steroid and a non-steroidal compound were tested in vitro. Hydrocortisone (Sigma Chemical Company) was used to promote GS activity in
cultured retina. Metopirone (2-methyl-l,2-bis-(3-pyridyl)-l-propanone, metyrapone, methopyrapone, SU 4885; Ciba Pharmaceutical Company) was used to
block the ll/?-hydroxylation of steroid molecules in cultured adrenals (Jenkins,
Meakin, Nelson & Thorn, 1958; Nagra, Sauers & Wittmaier, 1965; Frankel,
Garber & Nalbandov, 1967). Both compounds were prepared in Tyrode's
solution: Tween 80 (1 ml:0-04 ml) in concentrations stated in the text. Adrenocorticotropin (ACTH; Sigma Chemical Company) was used to stimulate adrenal
steroidogenesis in the embryo. It was dissolved in 0-025 N-HAC and injected in
concentrations of 15-30 i.u. ACTH/0-2 ml.
RESULTS
Effect of adrenal homogenates on retinal GS activities in vitro
Adrenal glands from 12-, 14-, 16-, 18- and 20-day embryos were collected and
homogenized in room temperature Tyrode's solution and cultured with 12-day
retina. The retinas were harvested at 24 h and assayed for GS activity. The
results (Fig. 1) show that 12- and 14-day adrenals have no significant effect on
control levels of retinal GS. 16-day adrenals double GS values and 18- and
20-day adrenals increase GS activities about 4 times under these conditions.
To test the specificity of the adrenal effect on retinal GS, thyroid glands, testes
and ovaries from 12- to 20-day embryos were prepared as above and cultured
for 24 h with 12-day retina. None of these glands had a significant effect on
retinal GS activities at any age (Fig. 1).
Effect of adrenal growth on adrenal activity
To determine the pattern of adrenal growth during embryonic development,
both adrenal glands from 12-, 14-, 16-, 18- and 20-day embryos were collected
and assayed for protein. As Fig. 2 indicates, the growth of the adrenal coincides
Steroid control of glutamine synthetase
731
closely with its effectiveness as a homogenate in promoting retinal GS activities
in vitro. The glands grow particularly rapidly between the 14th and 16th days
and reach maximal protein development by the 18th day.
5
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Mil titit iliii uiit
1214161820 1214161820 1214 161820
Adrenals
Thyroids
Testes
Age of glands (days)
1214161820
Ovaries
Fig. 1. Effects of homogenates of adrenals, thyroids, testes and ovaries on GS activities in 12-day retina cultured for 24 h. Numbers along abscissa indicate age of glands
at explantation; control cultures (C) were without glands. Vertical lines indicate
standard deviations of data in adjacent bars. Four to 31 (average of six) determinations per data point.
7 6
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12 14 16 18 20
Age of adrenals (days)
Fig. 2
4
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C
1214161820
Age of adrenals (days)
Fig. 3
Fig. 2. Protein content of both adrenal glands during the embryonic period.
Vertical lines indicate standard deviations of data in adjacent bars. Two to four
determinations per data point.
Fig. 3. Effect of adrenal organ cultures on GS activities in 12-day retina cultured
for 24 h. Numbers along abscissa indicate age of glands at explantation; control
cultures (C) were without glands. Vertical lines indicate standard deviations of data in
adjacent bars. Four to seven determinations per data point.
47-2
732
R. P I D D I N G T O N
Effect of adrenal organ cultures on retinal GS activities in vitro
The pattern of GS activities elicited by adrenal homogenates suggests that
adrenals become active relevant to the retinal GS system after the 14th day and
maximally effective after the 16th day. In view of this finding it became of
interest to determine whether the early adrenals are ineffective inducers of
retinal GS because they lack biosynthetic mechanisms essential for activity. To
examine this possibility adrenal glands were cultured under conditions presumed
to permit full expression of any inherent inductive properties. Both adrenals
from 12-, 14-, 16-, 18- and 20-day embryos were cut in half (0-5-1-0 mm3 pieces)
and grown in organ culture with 12-day retina for 24 h. The retinas were then
harvested and assayed for GS activity. The results (Fig. 3) show that, in organ
culture, adrenals of all ages induce striking increases in retinal GS activities.
Although the size of adrenals varied considerably, adrenal activity was remarkably similar under these conditions.
Inductive factors in adrenal organ cultures
The effectiveness of adrenal expiants in promoting retinal GS activities in
culture made it of interest to examine in more detail the adrenal factors responsible for these effects. Since 1 l/?-hydroxyl corticosteroids elicit striking
increases in retinal GS, the effects of these endogenous adrenal steroids on
retinal GS activities were examined in vitro. Adrenal pieces from 14-day embryos
were explanted with 12-day retina into normal medium containing metopirone.
This non-steroidal compound at low concentrations (Liddle, Island, Lance &
Harris, 1958; Dominguez & Samuels, 1963) affects adrenocortical secretions by
preferentially inhibiting the hydroxylation of the 1 Iß position on the steroid
molecule thereby preventing the synthesis of steroids such as hydrocortisone,
corticosterone and aldosterone. Control cultures were prepared in which hydrocortisone was substituted for the adrenal pieces to test the effects of metopirone on the retinal GS response to steroid induction. Appropriate control
cultures without metopirone were also examined. Retinas from all cultures were
harvested at 24 h and assayed for GS activity. The results (Table 1) show that,
in cultures with adrenal glands, metopirone (10~4-10-5 gm/ml) completely blocks
the rapid precocious rise in retinal GS activity. Metopirone clearly acts only on
the adrenals since it has no effect on GS levels elicited by hydrocortisone. While
individual steroids were not identified, these findings strongly suggest that
synthesis of 1 l/?-hydroxyl steroids in the adrenal pieces accounts for the effectiveness of adrenal organ cultures as inducers of retinal GS.
Steroid synthesis in adrenals at explantation
Differences between the effects of 14-day adrenal homogenates and organ
cultures on GS activities in vitro indicate that specific steroidogenic reactions are
activated in young adrenal expiants provided that some integrity of the gland
Steroid control of glutamine synthetase
733
tissue is maintained. This evidence suggests the possibility that homogenates of
older adrenals are uniquely effective in promoting GS activities in culture only
because the intact glands from older embryos are uniquely able to rapidly
synthesize 1 l/?-hydroxyl steroids during the short (5-15 min) period required
for their dissection. To test this possibility adrenal glands from 12-, 14-, 16-, 18and 20-day embryos were dissected, homogenized and cultured in the presence
Table 1. Effects of metopirone on retinal GS activities induced by adrenal
organ cultures and hydrocortisone
Specific activity
(12-day retina
cultured for
24 h)
14-day adrenal pieces
14-day adrenal pieces plus metopirone
(10- 4 -l(r 5 gm/ml)
Hydrocortisone (JO-6 gm/ml)
Hydrocortisone (10~6 gm/ml) plus
metopirone (10~ 4 -10 -5 gm/ml)
6-2+ 1-1 (4)*
1-3 ±0-5 (6)
7-3 ± 1 -0 (5)
7-3 ± 0-6 (5)
* Numbers in parentheses = number of assays.
of metopirone; controls were dissected and homogenized in room temperature
Tyrode's solution and then transferred to medium containing metopirone.
Twelve-day retinas were added to each flask and the cultures were incubated for
24 h. The retinas were then collected and assayed for GS activity. The results
(Fig. 4) show that early and continuous exposure of young (12-, 14- and 16-day)
adrenals to metopirone has little effect on control levels of retinal GS. On the
other hand similar treatment of 18- and 20-day adrenals with metopirone completely blocks the characteristic increases in GS activities indicating that 11/?hydroxyl steroids are normally synthesized rapidly in these glands at explantation.
When older adrenals were dissected and homogenized in cold (4 °C) Tyrode's
solution without metopirone, the rapid rise in retinal GS was also prevented
suggesting that these steroidogenic reactions are temperature sensitive. All of
these findings show clearly that the steroidogenic response in adrenals becomes
much more sensitive to environmental changes after the 16th day of embryonic
development.
Effect of ACT H on retinal GS activities in the embryo
The appearance of increased steroidogenic sensitivity to environmental factors
in adrenal expiants after the 16th day made it imperative to examine the pattern
of adrenal responsiveness to trophic influences in the embryo. Adrenocorticotropin (ACTH) or solvent (as a control) was injected on the chorioallantoic
membrane of 12-, 14- and 16-day embryos and reinjected after 24 h. Retinas
were then collected from 14-, 16- and 18-day embryos and assayed for GS
734
R. P I D D I N G T O N
activity. The results (Fig. 5) show that ACTH injected at the 14th and 15th days
precociously promoted GS activities at the 16th day. ACTH injected into earlier
embryos or during the final period of embryonic development had no effect on
control levels of retinal GS.
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12
14
16
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Age of adrenals at explantation (days)
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16
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Age of retinas (days)
Fig. 5
Fig. 4. GS activities in 12-day retina cultured for 24 h in medium containing
metopirone (10~ 4 -10 -5 gm/ml) and adrenal homogenates of various ages. Adrenals
were dissected and homogenized in the presence of metopirone (dark bars) or
Tyrode's solution (controls; light bars). Vertical lines indicate standard deviations
of data in adjacent bars. Three to five determinations per data point.
Fig. 5. Effect of ACTH (dark bars) or solvent (controls; light bars) on retinal GS
activities in embryos at various stages of development. Vertical lines indicate
standard deviations of data in adjacent bars. Five to nine determinations per data
point. Differences between the means of ACTH and control activities at the 16th day
are statistically significant (P < 001).
DISCUSSION
The evidence presented in this study indicates that adrenal glands from chick
embryos can elicit rapid precocious increases in GS activity in cultured retina.
This inductive influence is unique to adrenals and depends on the activation of
specific steroidogenic mechanisms in these glands; the induced rise in retinal GS
activities in culture requires adrenal synthesis of 1 l/?-hydroxyl steroids. Steroidogenesis in turn requires some integrity of the gland tissue under these conditions.
Homogenates of adrenals from early embryos are ineffective in retinal GS development whereas organ cultures of these glands can synthesize ll/?-hydroxyl
steroids in vitro and hence precociously advance GS activities in cultured retina.
Adrenals after the 16th day have special significance since only these older glands
Steroid control of glutamine synthetase
735
can rapidly synthesize 1 l/?-hydroxyl steroids as they are dissected from the
embryo. Thus, both homogenates and organ cultures of older adrenals are
effective inducers of retinal GS in vitro. The maturation of rapid corticosteroid
synthesis in adrenal expiants after the 16th day signals the development of
increased steroidogenic sensitivity to environmental changes. This period of
heightened sensitivity which appears as adrenal growth reaches a plateau,
follows closely the development of adrenal responsiveness to ACTH in the
embryo. It is of very special interest that these maturational events in adrenal
development coincide temporally with the appearance of the normal rapid phase
of increase in GS activity in the retina.
In view of the response of retinal GS to the inductive influence of specific
endogenous corticosteroids, it seems clear that the normal rapid rise in GS
activity in the embryo is controlled by increasing levels of 1 l/?-hydroxyl steroids
in late embryonic serum. These steroids very likely account for the maturational
change in 18-day chick embryo serum enabling it to rapidly advance GS activities in cultured retina (Piddington, 1967). While titers of all adrenal steroids
remain to be determined throughout the embryonic period, changes in the level
of corticosterone may be most significant to the development of retinal GS.
Corticosterone is the principal 11^-hydroxyl steroid known to be effective in the
GS system which is secreted by the embryonic (Bonhommet & Weniger, 1967)
and adult (Chester Jones, Phillips & Holmes, 1959; DeRoos, 1961; Sandor,
Lamoureux & Lanthier, 1963) chick adrenal.
The pattern of adrenal maturation presented here indicates that there is
enhanced secretion of steroids after the 16th day of development in response
to stimulation of adrenal steroidogenic mechanisms presumably by endogenous
corticotropin. This evidence implicates increased adrenal activity in the timing
of the normal rapid rise of retinal GS activities in the embryo and raises the
interesting possibility that pituitary regulation of adrenal steroidogenesis may
ultimately control the normal progression of GS development in the embryonic
retina.
SUMMARY
1. Adrenal ll/?-hydroxyl steroids are natural inducers of retinal glutamine
synthetase (GS) in the chick embryo.
2. The evidence presented in this study suggests that these steroids control
the characteristic pattern of changes in normal GS development as they are
rapidly secreted by the adrenals during the final embryonic period.
3. Endogenous ACTH very likely activates the synthesis and release of these
steroids into late embryonic serum.
736
R. P I D D I N G T O N
RÉSUMÉ
Contrôle par un Steroide du développement normal de la glutamine
synthetase rétinienne dans la rétine d'un embryon de poulet
1. Les ÏI/?-hydroxy-stéroïdes surrénaliens sont les inducteurs naturels de la
glutamine synthetase (GS) rétinienne chez l'embryon de poulet.
2. Les preuves expérimentales apportées dans cette étude suggèrent que ces
Steroides contrôlent la séquence caractéristique des changements au cours du
développement normal de la GS, puisqu'ils sont sécrétés rapidement par les
surrénales pendant la fin de la période embryonnaire.
3. L'ACTH endogène active très probablement la synthèse et l'émission de
ces Steroides dans le sérum d'embryon âgé.
I thank Dr Charles E. Wilde, jun. for his comments and criticisms and Miss Sheila Halpern
for her technical assistance. I am grateful to Ciba Pharmaceutical Company for the gift of
metopirone. This investigation was supported by General Research Support Grant 5 SO I
FR-05337-08 from the General Research Support Branch, Division of Research Facilities
and Resources, National Institutes of Health.
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(Manuscript
received 25 June 1969)