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J. J. BASS
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J. B. G. BELL
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D. LACY
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The presence of cholesterol side-chain cleavage enzymes in the rat testis was established by Shimizu, Hayano, Gut & Dorfman (1961). More recently Hall, Irby & DeKretser (1969) separated interstitial cells from seminiferous tubules in an attempt to compare the relative abilities of interstitium and seminiferous tubules to convert cholesterol to testosterone in incubations in vitro. They reported that the interstitial cells represent the major and probably only source of testicular androgens. This conclusion failed to explain the significance of the findings by Bell, Vinson & Lacy (1968, 1971) that the seminiferous tubules can convert pregnenolone to androgens. Therefore, the cholesterol side-chain cleavage activity of both the interstitium and seminiferous tubules of rat testis was examined by extracting and measuring the labelled isocaproic acid produced by the tissues when incubated with [26-14C]cholesterol.

Mature male Wistar rats (body weights 240–360 g) were killed with coal gas. The testes were removed

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P. J. Hyatt
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J. B. G. Bell
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K. Bhatt
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J. F. Tait
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Mixtures of zona fasciculata (ZF) and zona reticularis (ZR) cells, obtained by enzyme dispersion of decapsulated guinea-pig adrenal glands, were separated either by unit gravity sedimentation or by equilibrium density sedimentation. There was no evidence of deleterious effects on ultrastructural integrity or the ability of cells to respond to (1–24)ACTH (Synacthen) after either separation technique. Unit gravity sedimentation gave one fraction in which 90% of the cells were from the ZR and another fraction in which 70% of the cells were from the ZF. Equilibrium density sedimentation of cell mixtures on Percoll gradients gave fractions containing either 90% pure ZR or 95% pure ZF cells.

Cortisol, 11-deoxycortisol, corticosterone, deoxycorticosterone, 11 β-hydroxyandrostenedione and androstenedione were all formed from [14C]pregnenolone on incubation with purified preparations of both types of cell. No product was seen to be unique to either cell type although ZR cells appeared deficient in 11β-hydroxylase activity relative to ZF cells. The ratio of androstenedione to cortisol (formed either from labelled pregnenolone or from endogenous precursors) was higher for ZR cells than for ZF cells. When the purer cells obtained by equilibrium density sedimentation were studied, it was found that (1–24)ACTH stimulated greater steroid production (both androstenedione and cortisol) by the ZF cells compared with the ZR cells.

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E. M. Wintour
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M. B. Smith
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R. J. Bell
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J. G. McDougall
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M. N. Cauchi
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ABSTRACT

The switch from γ (fetal) to β (adult) globin production was studied by the analysis of globin synthesis in chronically cannulated ovine fetuses and newborn lambs. The γ/α globin synthesis ratio decreased from 0·98 ± 0·11 (s.d.) (n = 4 samples) at 100–120 days of gestation to 0·15± 0·07 (n = 4) in lambs of 150–156 days post-conception, and the β/α synthesis ratio increased from 0·04 ± 0·06 (n = 4) to 1·13 ± 0·21 (n = 4) over the same period. In bilaterally adrenalectomized fetuses, which survived in utero until 151–156 days, the γ/α and β/α synthesis ratios were 0·64 ± 0·14 (n = 3) and 0·25 ± 0·07 (n = 3) respectively in the 150- to 156-day period. Bilateral adrenalectomy did not affect the time of onset of β globin synthesis, but significantly decreased the rate. In one bilaterally adrenalectomized fetus the infusion of increasing concentrations of cortisol restored the rate of β globin synthesis to normal. Treatment of three intact fetuses with 100 μg cortisol/h for 3 weeks, from 100 to 121 days, did not affect the timing or rate of switch from γ to β globin synthesis. Thus fetal adrenal secretions, probably cortisol, affected the rate of change of γ to β globin synthesis but other factors must have been involved in the initiation of the switch.

J. Endocr. (1985) 104, 165–170

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P. J. Hyatt
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J. B. G. Bell
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K. Bhatt
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F. W. Chu
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J. F. Tait
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S. A. S. Tait
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G. St J. Whitley
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ABSTRACT

Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bis-phosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0·25 nmol/l to 0·25 μmol/l) highly significantly (P < 0·01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by α-MSH (above 0·1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 μmol α-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P > 0·05) by any dose of α-MSH (10 nmol/l, 1 μmol/l and 0·1 mmol/l) used. Therefore, it can be concluded that α-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol α-MSH/l (highly significant above 10 nmol α-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0·1 to 1 μmol α-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata–reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that α-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, α-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells.

J. Endocr. (1986) 110, 405–416

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J. B. G. BELL
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J. F. TAIT
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S. A. S. TAIT
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G. D. BARNES
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B. L. BROWN
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The effects of pure [Asp1, Val5]- and [Asn1, Val5]-angiotensin II and also [des-Asp1, Ile5]-angiotensin II (angiotensin III) on cyclic AMP and steroid outputs by dispersed rat capsular cells, comprising 95% zona glomerulosa and 5% zona fasciculata cells, have been studied.

The results showed that [Asp1, Val5]- and [Asn1, Val5]-angiotensin II, at doses between 2·5 × 10−1 1 and 2 × 10−4 mol/l, which produced typical increases in steroidogenesis, failed to increase output of cyclic AMP. This lack of effect was observed whether the nucleotide was measured by radioimmunoassay or by adrenal binding protein and under the same conditions in which 8·4 mm-K+ consistently increased the output of cyclic AMP. Instead the results showed a small but significant decrease in cyclic AMP output with angiotensin II. Similar results were obtained with incubations for 60 rather than 120 min and with medium containing a concentration of 5 or 40 g bovine serum albumin/l. Although the levels of cyclic AMP were generally higher in the presence of the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, the same decrease relative to basal outputs was observed with angiotensin II which increased steroidogenesis. Angiotensin III also failed to increase output of cyclic AMP at doses (2·5×10−9 to 2·5×10−6 mol/l) which produced increases in steroid output equivalent to those obtained with angiotensin II.

These results indicate that angiotensin II and III can act through a cyclic AMP- independent mechanism.

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J. F. TAIT
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S. A. S. TAIT
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J. B. G. BELL
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P. J. HYATT
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B. C. WILLIAMS
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Preparations of capsular rat adrenal cells consisting mainly of zona glomerulosa with less than 5% zona fasciculata contamination are described. The responses of the aldosterone and corticosterone outputs of these preparations to various stimuli were of four types. (1) Variations in K+ concentration gave a maximum aldosterone response at 5·9–8·4 mm-K+, about sixfold greater than the control output at 3·6 mmol/l. At higher K+ concentrations, such as 13 mmol/l, the response decreased. (2) Serotonin (at a concentration of about 10−4 mol/l) gave only a slightly lower maximal aldosterone response than did K+ but this did not decrease significantly at higher concentrations. Serotonin gave significant steroidogenic response at 10−8 mol/l. (3) [Asp1,Val5]-Angiotensin II (10−10 mol/l) with 3·6 mm-K+ gave a significant response and a constant maximal response at 2·5 × 10−8 mol/l. This maximum response was about half that found for both aldosterone and corticosterone when stimulated maximally by K+ or serotonin: [des-Asp1,Ile5]- and [des-Asp1,Val5]-angiotensin II (angiotensin III) gave similar response characteristics but had a lower potency in this cell preparation. The initial maximum response could be further increased at a higher concentration (from 2·5 × 10−5 mol/l) of a preparation of [Asn1,Val5]-amide angiotensin II (Hypertensin-Ciba) and might eventually be greater than with K+. This additional response was, to a major extent, due to stimulation of the contaminating zona fasciculata cells and was not seen with high concentrations of the free acid, angiotensin II. It was also not seen in two experiments with pure [Asn1]-amide angiotensin II and therefore it could have been due to some impurity in Hypertensin-Ciba. (4) Adrenocorticotrophin (Synacthen) at 3 × 10−11 mol/l gave a significant steroidogenic response. Higher concentrations (3 × 10−10 to 7·5 × 10−9 mol/l) gave no constant maximum but the response could be much greater than for other stimuli such as K+, serotonin and [Asp1]-angiotensin II. This additional response was again due to steroid precursors, e.g. deoxycorticosterone and corticosterone from contaminating zona fasciculata cells. Similar results were obtained with ACTH (ACTHAR) in three experiments. Threshold sensitivity (a significant increase in steroidogenesis) for ACTH (Synacthen) was, in two experiments, greater for zona fasciculata-reticularis cells (3 × 10−12 mol/l) than for zona glomerulosa cells (3 × 10−11 mol/l).

The data show that aldosterone output was approximately a function of the square of the corresponding corticosterone value. Specific effects on this pathway can be shown by values of aldosterone/corticosterone2 greater than one. Of all stimuli used, only K+ concentrations of 5·3, 5·9 and 13 mmol/l gave such effects. However, because of several considerations, only positive results with other stimuli may be meaningful. Calculation of this parameter might be useful as a screening test in bioassays for substances with aldosterone-stimulating activity.

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J. B. G. BELL
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R. P. GOULD
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P. J. HYATT
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J. F. TAIT
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S. A. S. TAIT
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An enriched fraction of zona reticularis cells was obtained by unit gravity sedimentation of decapsulated adrenal glands from female rats. From light microscopic and ultrastructural studies of the whole gland and the isolated cell fractions, the zona reticularis cells of the adrenal gland can be classified mainly on the bases of size, position and mitochondrial morphology. This cell population consists of two types of cell, the 'true' zona reticularis cells (Type I, modal diameter 9 μm), which usually constitute 90% of the isolated reticularis fraction and 80% of the intact reticularis tissue, and cells (Type II, modal diameter 13 μm) with fasciculata-like properties (rich in lipid and spherical mitochondria with vesicular cristae). Staining of the cell preparation for 3β-hydroxysteroid dehydrogenase activity also demonstrates the existence of two types of cell in the zona reticularis.

The zona reticularis cell fraction, like the zona fasciculata cell fraction, was capable of producing the subsequent steroids from radioactive pregnenolone: corticosterone, deoxycorticosterone, 18-hydroxydeoxycorticosterone, 11-dehydrocorticosterone, progesterone and androstenedione. However, the pattern of steroid production differed markedly between the zona reticularis and zona fasciculata cells, particularly with respect to the production of deoxycorticosterone and corticosterone (and its correlated steroids, 11-dehydrocorticosterone and 18-hydroxydeoxycorticosterone). When R (the ratio of deoxycorticosterone: corticosterone plus 11-dehydrocorticosterone) for the purest preparation of reticularis cells was compared with R for the corresponding preparation of fasciculata cells, the normalized ratio was found to be 6·4, 16·4 and 20·1 in three experiments. The pattern of production of androstenedione per cell was similar in the reticularis and fasciculata cell fractions. The exact mechanism for the altered pattern of steroid metabolism remains to be elucidated. However, these results establish that the corticosteroids produced by the cells of the zona reticularis may be quantitatively, if not qualitatively, different from those produced by the zona fasciculata cells.

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J. B. G. BELL
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R. P. GOULD
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P. J. HYATT
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J. F. TAIT
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S. A. S. TAIT
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The outputs of corticosterone, deoxycorticosterone and androstenedione from dispersed, purified rat adrenal zona reticularis and zona fasciculata cells have been measured by radioimmunoassay.

Preferential production of deoxycorticosterone by zona reticularis cells was demonstrated by their higher basal deoxycorticosterone: corticosterone ratio when compared with zona fasciculata cells.

Adrenocorticotrophin (ACTH) stimulated corticosterone output by all cell pools prepared by unit gravity (1 g) sedimentation, zona fasciculata cells being stimulated 130-fold compared with 20-fold for the zona reticularis cells in relation to their basal corticosterone output.

In every cell pool, ACTH stimulated the output of corticosterone more than it stimulated the output of deoxycorticosterone. In parallel cell preparations, it was shown that ACTH increased the conversion of tracer amounts of radioactive deoxycorticosterone to corticosterone and decreased the conversion of radioactive corticosterone to 11-dehydrocorticosterone. Adrenocorticotrophin did not increase the conversion of radioactive deoxycorticosterone to total 11-oxygenated steroids (corticosterone+ 11-dehydrocorticosterone). It is unlikely therefore that ACTH stimulates 11 β-hydroxylation.

Data indicate that the ratio of deoxycorticosterone to total 11-oxygenated steroids (corticosterone +11-dehydrocorticosterone) is characteristic for each cell type, and that this ratio will be relatively independent of ACTH stimulation or the amount of pregnenolone substrate available.

Basal androstenedione outputs were similar for both types of cell, and ACTH stimulation was very small, being slightly greater for zona fasciculata than for zona reticularis cells.

The contribution of the zona reticularis cells to the basal output of any steroid by the cells of the inner two zones of the adrenal cortex of the rat was relatively small (20% for deoxycorticosterone and 10% for corticosterone) and was even less after stimulation by ACTH. Unless a specific stimulus can be found, therefore, a significant role for the zona reticularis cannot yet be established.

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P. J. HYATT
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L. W. WALE
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J. B. G. BELL
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J. F. TAIT
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S. A. S. TAIT
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Cyclic AMP levels were measured in combined cells and supernatant fraction from incubations of dispersed rat adrenal zona fasciculata and zona reticularis cell preparations purified by unit gravity sedimentation. These measurements were correlated with deoxycorticosterone (DOC) and corticosterone outputs from the cells in the presence or absence of ACTH.

Similar measurements of cyclic AMP outputs were made for unpurified dispersed, decapsulated rat adrenal cell preparations and they were found to correspond to previously reported measurements made by other workers on such preparations.

The response of the purest zona reticularis cells to ACTH in terms of cyclic AMP output was 28-fold lower than that of the purest zona fasciculata cells (compared with a fivefold lower DOC output and a 20-fold lower corticosterone output) and the response to ACTH of the mixed-cell preparations was related to the number of zona fasciculata cells in the preparation, i.e. the greater the proportion of zona fasciculata cells in the preparation the greater the response in terms of both outputs of cyclic AMP and of either of the two steroids measured. This correlation is in accordance with the theory that cyclic AMP may be the secondary messenger for both zona fasciculata and zona reticularis cells of the rat adrenal cortex in mediating the response to an ACTH stimulus.

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