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A. A. WRIGHT

SUMMARY

1. Attempts have been made to estimate oestrogens in residues from sheep faeces using two methods of purification.

2. Low recoveries of oestrogens added to the faeces were recorded with both methods.

3. Some batches of faeces contain contaminants which remain in the final fractions and interfere markedly in colour development.

4. The analysis of faeces, including the effect of diet on the contaminants, is discussed in relation to these methods.

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N. A. WRIGHT

Corticotrophin (ACTH) has been shown to increase adrenocortical DNA content (Bransome, 1968) and to stimulate adrenal DNA polymerase and thymidine kinase activity (Masui & Garren, 1970). Bransome (1968) has shown that dexamethasone also produces a significant decrease in adrenocortical DNA. It is therefore surprising that a recent report by Ueberberg, Stöcker & Städtler (1970) concluded that cell proliferation is not influenced by dexamethasone treatment. The present results indicate that in prepubertal rats dexamethasone has a profound inhibitory effect or adrenocortical cell proliferation by acting on a particular point in the cell cycle.

Male Wistar rats aged 14 days were given a single i.p. injection of 3 μg dexamethasone phosphate (Merck, Sharp and Dohme)/g body weight. Controls received normal saline. Animals were killed serially thereafter. Tritiated thymidine ([3H]Tdr) was administered 1 h before death, and autoradiographs of the adrenal glands prepared as previously described (Wright, 1971). The adrenal cortex was

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N. A. WRIGHT

SUMMARY

On the basis of labelling indices measured with tritiated thymidine at intervals throughout its thickness, the adrenal cortex of prepubertal male rats has been divided into four compartments. These are called the glomerular, proliferative, fascicular and reticular compartments, respectively. Labelling indices measured for each compartment showed highest values in the glomerular and proliferative compartments, with values of 6·73% and 7·09% respectively. The fascicular compartment showed a lower index of 3·16% while the reticular compartment gave the lowest value of 1·15%. These differences are further reflected in measurements of the mitotic indices for each compartment.

The phases of the cell cycle have been measured by pulse-chase analysis in each compartment, and all phases estimated showed an increase in duration as the inner compartments were approached. The duration of interphase DNA synthesis (ts) was found to be shortest in the glomerular and proliferative compartments, with values of 7·45 and 7·73 h, respectively. The fascicular compartment showed lengthening of ts to 8·56 h, and the reticular compartment gave the highest value of 9·21 h. Similarly, the values obtained for G 2 (the post-DNA synthetic interval) and tm (the duration of mitosis), and a calculated value of the cell cycle time all showed a general increase in duration from the outer to the inner compartments. The relation of these results to theories of adrenocortical cytogenesis is discussed, and it is suggested that the differences in cell cycle components can best be explained by the inward migration of cortical cells from the outer compartments.

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W. KLYNE and A. A. WRIGHT

SUMMARY

1. Pregnant cow's urine was hydrolysed with acid, and the lipid material obtained was submitted to the fractionation procedures customary in the study of urinary steroids.

2. Oestrone (0·3 mg/1.) and oestradiol-17α (0·1 mg/1.) were isolated from the phenolic fraction. Oestradiol-17β could not be detected.

3. The heterocyclic phenol equol isoflavan-7:4′-diol) was also isolated (6 mg/1.).

4. The neutral non-ketonic fraction contained 5β-androstane-3α: 17α-diol (0·2 mg/1.) and 5α-androstane-3β:17α-diol. A very small quantity of material resembling 5β-pregnane-3α:20α-diol (the common 'pregnanediol' of human pregnancy urine) was isolated but not satisfactorily identified.

5. The volatile part of this fraction contained two tetrahydroionanediols A and B.

6. The neutral non-volatile ketonic fraction was small (0·6 mg/1.). Three α-17-oxosteroids were tentatively identified.

7. The results are discussed in relation to the routes of steroid excretion in cows, and in relation to the determination of steroids in animal urines.

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I. CHESTER JONES and A. WRIGHT

SUMMARY

Male adult rats, with established drinking patterns, were given the choice of saline or tap water to drink, immediately after adrenal enucleation. Both saline and water were taken, but by the 6th day after operation the rats had returned to drinking predominantly tap water. The adrenals at this stage showed a small compact cortex, no distinguishable zona glomerulosa, and they appeared to be composed for the most part of cells in 'fascicles'. Adrenalectomized animals chose saline, drinking more and more pari passu with time. Other short-term enucleated animals were injected with ACTH, and the tendency for the regenerating cortex to form in 'fascicles' was very pronounced.

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I. CHESTER JONES and A. WRIGHT

SUMMARY

The adrenal of male and female rats with persistent diabetes insipidus showed a prominent zona fasciculata and zona reticularis. The zona glomerulosa was narrow or absent.

The results from this and the preceding three papers are here reviewed together. It is concluded that control of salt-electrolyte metabolism cannot be ascribed to the zona glomerulosa. It is probable that the zona fasciculata is reponsible for most of the adrenocortical secretions. The zona glomerulosa is a vegetative back-water of cells, which is able to produce minimal amounts of adrenocortical secretions without stimulation by pituitary hormones, but is only of significance when the latter are absent. Rising amounts of circulating adrenocorticotrophic hormone (ACTH) can transform the zona glomerulosa into actively secreting cells of the zona fasciculata type. After cessation of such activity the zona glomerulosa re-forms, as the amount of ACTH will maintain only a certain volume of zona fasciculata (and zona reticularis) against the rigid limiting inner circumference formed by the medulla; some of the cells derived from the chief area of cell division in the outer part of the zona fasciculata do not mature to cells of the zona fasciculata type, but form zona glomerulosa cells. It is thought that cell migration occurs from the cells of the outer region of the zona fasciculata to the zona reticularis and that this is, normally, a slow process.

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A. WRIGHT and I. CHESTER JONES

SUMMARY

The anatomy and histology of the adrenal glands of the West African lizard (Agama agama L.) and of the common grass snake (Natrix natrix L.) are described.

In lizards, 30 days after hypophysectomy, there was considerable degeneration, with a 60% loss in weight, of the adrenal. The chromaffin cells underwent no histological change. Some cortical cells contained large sudanophilic, Schultz-positive lipid droplets, some nuclei remaining normal, others showing intense basophilia and shrinkage. In other cells, the cytoplasm was reduced to a small acidophilic patch, with pyknotic nuclei. The sodium and potassium contents of the blood and muscle of these hypophysectomized animals were within the normal range.

In snakes, the successive histological stages in the degeneration of the adrenal cortex 9–11, 20 and 30–39 days after hypophysectomy are described. Injection of mammalian ACTH allowed the adrenal to maintain a normal histological appearance in hypophysectomized snakes. In addition, these animals, and normal animals similarly injected, showed degenerative areas in the cortex, attributable to over-stimulation. Injections of cortisone and DCA into unoperated animals were followed by degeneration of the adrenal cortex. After unilateral adrenalectomy, the contralateral adrenal was hypertrophic.

The sodium and potassium contents of blood and muscle, together with the water content of the latter, were obtained in all groups of animals. Despite the histological changes induced in the adrenal cortex by the various experimental procedures, there were no profound changes in the distribution of salt-electrolytes in any of the animals. The sodium content of muscle of normal snakes was about twice the value found in vertebrate muscle in general.

The results are discussed in the light of present-day knowledge of the adrenal cortex in vertebrates, especially of the gland in mammals.

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A. R. MORLEY and N. A. WRIGHT

SUMMARY

Castrated male mice were injected with 250 μg testosterone propionate on 3 consecutive days. With the third injection, 1 μCi tritiated thymidine/g body weight was given intraperitoneally. A pulse-chase analysis in the seminal vesicle and coagulating gland was performed during the next 50 h and labelling and mitotic indices were obtained.

One hour after the injection of tritiated thymidine the labelling index was 19% in the seminal vesicle and 13% in the coagulating gland. The mitotic index was 1·7% in the seminal vesicle and 1·4% in the coagulating gland.

The observed cell cycle (T c) duration in the seminal vesicle was 25 h, and the duration of DNA synthesis (t s) was 9 h. The calculated duration of mitosis (t m) was 0·6 h, the presynthetic duration (t G1) was 14·2 h, and the postsynthetic duration (t G2) 1·2 h. Forty-eight hours after testosterone propionate the growth fraction was 0·62 and a decycling ratio of 0·8 was calculated. In the coagulating gland T c was 52·0 h, t s 9·0 h, t m 1·0 h, t g1 41·0 h and t G2 1·0 h.

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N. A. WRIGHT and A. R. MORLEY

Testosterone influences cell proliferation in a number of tissues (Tuohimaa & Niemi, 1968). We have produced evidence that testosterone affects the position of the 'critical decision phase' (Quastler & Sherman, 1959), or 'cut-off position' (Cairnie, Lamerton & Steel, 1965) of the small intestine of the castrated male mouse. This is the point in the crypt where the decision is made to differentiate. Any change in the cut-off position will lead to an alteration in the growth fraction (GF) or proportion of the cell population progressing round the cell cycle (Cleaver, 1967).

Male Balb/c mice aged 3 months were castrated. Fourteen days later animals were given 250 μg testosterone propionate s.c. in sesame oil for 3 consecutive days. Castrated controls were given sesame oil only. Tritiated thymidine ([3H]Tdr) was administered at the same time as the third injection of testosterone or sesame oil. Autoradiographs were prepared from samples of upper

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M. R. ALISON and N. A. WRIGHT

The effects of estramustine phosphate (EMP) have been measured during the proliferative response of the seminal vesicle and coagulating gland of the castrated mouse after continuous treatment with testosterone. Daily subcutaneous injections of 60 μg testosterone were begun 14 days after castration. Three daily injections of EMP (100 mg/kg body wt) were begun on either day 11 or day 13 after castration. Cell proliferation was monitored by [3H]thymidine-labelling techniques combined with autoradiography, and by determinations of mitotic index.

Treatment with EMP on days 11, 12 and 13 after castration almost completely blocked the testosterone-induced proliferative response in the coagulating gland. Treatment with EMP commencing on day 13 after castration was less effective. Estramustine phosphate had a small stimulatory effect on DNA synthesis and mitosis in the seminal vesicle. Measurements of 5α-reductase activity in vitro did not reflect the dynamic cell kinetic changes observed in both tissues in vivo. We have concluded that previous assay systems using the Coffey model are unsuitable for the assessment of antiandrogens and prostatic anti-cancer drugs.