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 11β-hydroxyl 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.

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 solution. The cultures were gassed with 5 % CO2-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 μmol 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 11 β-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. Adreno-corticotropin (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.

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.

Fig. 1.

Effects of homogenates of adrenals, thyroids, testes and ovaries on GS activities 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.

Fig. 1.

Effects of homogenates of adrenals, thyroids, testes and ovaries on GS activities 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.

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 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.

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. 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.

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.

Fig. 3.

Effect of adrenal organ cultures on GS activities in 12-day retina cultured adjacent bars. Four to seven determinations per data point. cultures (C) were without glands. Vertical lines indicate standard deviations of data in adjacent bars. Four to seven determinations per data point

Fig. 3.

Effect of adrenal organ cultures on GS activities in 12-day retina cultured adjacent bars. Four to seven determinations per data point. cultures (C) were without glands. Vertical lines indicate standard deviations of data in adjacent bars. Four to seven determinations per data point

Inductive factors in adrenal organ cultures

The effectiveness of adrenal explants in promoting retinal GS activities in culture made it of interest to examine in more detail the adrenal factors responsible for these effects. Since 11β-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 11β 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 meto-pirone 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 11β-hydroxyl steroids in the adrenal pieces accounts for the effectiveness of adrenal organ cultures as inducers of retinal GS.

Table 1.

Effects of metopirone on retinal GS activities induced by adrenal organ cultures and hydrocortisone

Effects of metopirone on retinal GS activities induced by adrenal organ cultures and hydrocortisone
Effects of metopirone on retinal GS activities induced by adrenal organ cultures and hydrocortisone

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 explants provided that some integrity of the gland 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-, 18- and 20-day embryos were dissected, homogenized and cultured in the presence 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 Wff 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.

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. 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.

Effect of ACTH 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 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.

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 < 0·01).

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 < 0·01).

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 11β-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 11β-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 can rapidly synthesize 11β-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 explants 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 lβ-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.

  1. Adrenal 11β-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.

Contrôle par un stéroïde du développement normal de la glutamine synthétase rétinienne dans la rétine d’un embryon de poulet

  1. Les 11β-hydroxy-stéroïdes surrénaliens sont les inducteurs naturels de la glutamine synthétase (GS) rétinienne chez l’embryon de poulet.

  2. Les preuves expérimentales apportées dans cette étude suggèrent que ces stéroïdes 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 stéroïdes 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 1 FR-05337-08 from the General Research Support Branch, Division of Research Facilities and Resources, National Institutes of Health.

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