M. Weiss

Corticosteroid secretion in the Australian phalanger (Trichosurus vulpecula)

Abstract Corticosterone (B) as well as cortisol (F) is secreted by the adrenal gland of the marsupial Trichosurus vulpecula . The F:B ratio in controls varied from 3.5 to 12.8; and in all animals (including ACTH-treated) from 1.6–16.0. Both steroids were present in the erythrocytes as well as the plasma of adrenal venous blood. Their secretion was stimulated by intravenous ovine corticotrophin; and maximum cortisol secretion rate was 20.8–22.3 μg/hour per 100 mg adrenal, or 10–25 μg/hour per kg body weight. Cortisol concentration in peripheral blood plasma did not exceed 2.5 μg/100 ml. Aldosterone was sought, but not detected in excess of 0.1 μg in adrenal venous plasma, corresponding to a 2-hour collection period; or in a 48-hour urine sample. Urinary excretion of free cortisol, cortisone, their tetrahydro derivatives, and 17-ketosteroids was demonstrated; but their excretion rates were all very low, compared with eutherians.

Sex differences in steroidogenesis by adrenal homogenates of adult possum (Trichosurus vulpecula) attributable to the steroids formed by the adrenocortical special zone of the female

A marked sex difference was found in the nature of steroid conversion products formed from [14C]progesterone by whole adrenal homogenates of adult possums. The major conversion products from adrenals of males were: cortisol in yields of 46 ± 2% and corticosterone, 25 ± 2% (mean ± SEM, n = 35). The total 5β-reduced products were 80% of the yield, the predominant steroids being 5β-pregnane-3α,17α-diol-20-one, 25 ± 4% and 5β-pregnane-3α-o1-20-one, 21 ± 3% (mean ± SEM, n = 39). The yields of cortisol was 70% and reduced 5α- and 5β-androstan derivates, <15%. The products of major yield were: 5β-pregnan-3α-o1-20-one, 5β-pregnane-3α,17α-diol-20-one, and 5β-pregnane-3α,17α,20α-triol. Similarly from [17α-14C]hydroxyprogesterone and [11-14C]deoxycortisol substrates the conversion products by the special zone were mainly 5β-reduced pregnane derivates, the C19 steroids being <10%. Dissected medullary tissue had no steroidogenic activity. The addition of medullary tissue to cortex proper had no effect on the synthetic activity of the cortex. With mixtures of cortex proper and special zone the conversion products were similar to those obtained by whole adrenal homogenates. It is concluded that the dissimilarity in the adrenal steroid formation was due to the activity of the 5β-reductase, which prevailed in the female adrenal and was very low in the male adrenal. It was found that the location of the reducing enzymes was specific to the unique adrenocortical special zone which is present only in the female possum.

Differences in steroidogenesis by the subcellular fractions of adrenocortical special zone and cortex proper of the female possum (Trichosurus vulpecula).

Steroidogenesis by subcellular fractions of adrenal cortex proper (C.P.) and special zone (S.Z.) of female possum (Trichosurus vulpecula) was studied. Mitochondrial, microsomal and cytosol cell fractions were incubated with appropriate substrates in the presence of an NADPH-generating system. The major products formed from [3H]progesterone and [3H]17 alpha-hydroxyprogesterone by the microsomal fraction of the C.P. were 11-deoxycortisol and 11-deoxycorticosterone and 3 alpha (beta)-hydroxy-5 alpha-androstan-17-one by the S.Z. The mitochondrial fraction converted [3H]11-deoxycortisol to cortisol in yields twenty times higher by the C.P. than by the S.Z. and to 17 alpha, 20 beta,21-trihydroxy-4-pregn-3-one thirty times higher by the S.Z. The conversion of [3H]androstenedione to 11 beta-hydroxyandrostenedione by the C.P. was approximately double that of the S.Z., while 18-hydroxyandrostenedione (tentatively identified) formed the highest yield in both zones. Incubation of the same substrates with cytosol formed two 5 beta-pregnane and two 5 beta-androstane derivatives in total yields less than 5% by C.P. and greater than 60% by S.Z. Aromatase activity, estimated by the release of [3H2O] from [1 beta 3H]testosterone, in the adrenals of 8 possums, was in each experiment negligibly low. Determination of total enzyme activities in the two zones revealed that 11 beta, 18 and 21-hydroxylases were higher in the C.P., while 17 alpha-hydroxylase was higher in the S.Z. Similar results were obtained when the rates of formation of hydroxylated products were estimated in the presence of saturating amounts of substrates. Active 5 alpha- and 5 beta-reductases, C17-20-lyase and 3 alpha (beta) and 20 beta-hydroxysteroid dehydrogenases were found almost exclusively in the S.Z. We conclude that the S.Z. at lower levels of activity than the C.P. could contribute to the basal secretion of corticosteroids. In addition, the S.Z. has a high capacity to form C19 steroids and 5 alpha- and 5 beta-reduced steroids. The possible role of the S.Z. in possum is discussed.

A study of steroid 11β-hydroxylation by adrenal mitochondria of marsupials—Part I. A comparison of 11β-hydroxylase activity and specificity for different steroid substrates by possum (Trichosurus vulpecula), kangaroo (Macropus major) and beef☆

A kinetic approach was used to investigate the properties of adrenal mitochondrial 11β-hydroxylation in two marsupials, possum (Trichosurus vulpecula) and kangaroo (Macropus major), as compared to beef under similar experimental conditions. For possum, kangaroo and beef, the KM values of 11β-hydroxylation of 11-deoxycortisol (S) were 86, 714 and 38 μM respectively and for 17α-OH-progesterone (17OHP) 147, 294 and 118 μM respectively. The V values for S were in the respective species, 36, 32 and 2 270 pmol/mg mitochondrial protein/min and for 17OHP59, 101 and 11 pmol/mg/min. The KM values for 11β-hydroxylation of progesterone and androstenedione by kangaroo mitochondria were 769 and 87 μM and the V values were 6.3 and 39 pmol/mg/min respectively. With possum and kangaroo simultaneous additions of S and 17OHP acting as alternate inhibitors, showed a mutual competitive inhibition of 11β-hydroxylation in the range of 24–52%, indicating a single 11β-hydroxylase enzyme system. No evidence of competitive inhibition was found with beef, indicating two, substrate-specific 11β-hydroxylases. The findings are discussed in terms of possible evolutionary processes in adrenal steroidogenesis.

The effects of ACTH on adrenal steroidogenesis and blood corticosteroid levels in the echidna (Tachyglossus aculeatus)

1. The effects of short-term (S.T., 30 min) and long-term (L.T., 4 days) administration of ACTH on peripheral blood corticosteroid levels and on in vitro steroidogenesis were investigated. 2. Control levels of cortisol, corticosterone and aldosterone were 58 +/- 12, 130 +/- 26 and 10 +/- 6 (SEM) ng/100 ml respectively. 3. Corticosterone was 70% higher after S.T. and 150% higher after L.T., when cortisol was 800% higher. 4. Adrenal homogenates from control echidnas converted [14C]progesterone predominantly to 11-deoxycorticosterone (45%) and 11-deoxycortisol (12%). 5. After L.T. the principal product was corticosterone (25%), but S.T. had no effect. 6. In control echidnas the Km and V for 11 beta-hydroxylation of 11-deoxycorticosterone were 20 microM and 2.8 rho mol/min/mg respectively. After L.T. V increased to 10 rho mol/min/mg.

Changes in steroid biosynthesis by adrenal homogenates of the possum (Trichosurus vulpecula) at various stages of sexual maturation with special reference to 5β-reductase activity☆

Abstract 1. 1. A marked difference was found in the yields of 11β-hydroxylated and 5β-reduced products from 14C progesterone by adrenal homogenates of immature and adult possums. 2. 2. From adrenals of immature possums and possums in early stages of gonadal activity (ages 5–18 months) the yields of conversion products were; cortisol 52.4 ± 2.4% , corticosterone 16.3 ± 2.2% , total 5β-reduced products 9.6 ± 3.4% (mean ± S.E.M.) . In yields aldosterone, 21-deoxycortisol, 11β- and 17α-hydroxyprogesterone. 3. 3. The yields of products from adrenals of young sexually mature possums (ages 15–24 months) were variable. From five the levels were similar to that of immature animals. From four there was an increase in the yields of 5β reduced products (of varying degrees) with a concomitant fall in unreduced products, the average yields being; cortisol 12%, corticosterone 3% and total 5β-reduced metabolites 60%. 4. 4. The yields of conversion products from adrenals of adults ( > 2 1 2 years old) were cortisol 1.3 ± 0.8% and total 5β-reduced-11-deoxy-steroids 71 ± 8% (mean ± S.E.M.) . 5. 5. The similarities of the changes in adrenal 5β-reductase activity in the possum and guinea pigs at different stages of development are discussed.

The effects of age, gonadectomy and testosterone treatment on adrenal 5β-reductase activity in guinea pigs☆

Abstract 1. 1. The major conversion products formed by adult guinea pig adrenal homogenates incubated with 14 C pregnenolone or 14 C progesterone were; 17α, 21-dihydroxy-5β-pregnane-3,20-dione and 17α, 20β,21-trihydroxy-50-pregnane-3-one. Foetuses (30–55 days gestation) and immature animals (1–20 days old) formed cortisol. 2. 2. The amount of 5β-reductase activity increased with age. The total yields of unreduced products were 4 -3-ketosteroids there was a concomitant increase in 5β reduced products. 3. 3. Gonadectomized animals of pre- or post-puberal age produced steroids of similar yields as controls from the same litters. 4. 4. Treatment of gonadectomized animals with testosterone propionate for periods of 10–14 days increased the weights of seminal vesicles, ventral prostate and uterus by 200%, 80% and 150% respectively. No difference in the yields of conversion products from those of control animals of similar ages was observed. 5. 5. The possibility that the controlling mechanisms for adrenal 5β-reductase activity may differ from those for 5α reduction are discussed.

Steroid formation by the adrenal tissue of the possum (Trichosurus vulpecula).

Abstract 1. 1. Possum adrenal homogenates incubated with 14C-progesterone yielded A-ring reduced metabolites, the major ones being 5β-pregnane-3α-ol-20-one, 5β-pregnane-3α,17α,20α-triol, 5β-pregnane-3α,17α,21-triol-20-one and 5β-androstane-3α-ol-17-one. 2. 2. Similar experiments with adrenal slices yielded 17α-hydroxyprogesterone, cortisol, corticosterone and androstenedione. 3. 3. A time-yield study indicated that reduction followed the usual synthetic pathways.

A study of steroid 11β-hydroxylation by adrenal mitochondria of marsupials—Part II. The effect of corticotrophin, metopirone and pH on 11β-hydroxylation of 11-deoxycortisol and 17α-OH-progesterone by adrenal mitochondria of possum (Trichosurus vulpecula)

Abstract ACTH administration to possum (Trichosurus vulpecula) caused an increase in adrenal weight and in mitoehondrial protein. Compared with untreated animals, the KM for 11β-hydroxylation of 11-deoxycortisol (S) and 17α-OH-progesterone (170HP) after ACTH treatment decreased for both substrates, by approximately 2.6 times and V increased by approximately 3.6 times. Simultaneous addition of S and 170HP acting as alternate inhibitors, showed a mutual competitive inhibition of 11β-hydroxylation, the per cent inhibition being the same before and after ACTH treatment. The effect of pH on 11β-hydroxylation of S and 170HP over a range, of 6.2 to 9.5 indicated a similar pattern of change with optima at approximately pH 8 for both substrates. Addition of metopirone competitively inhibited the 11β-hydroxylation of S and 170HP, which at equimolar concentrations, was for both substrates 38%. It was concluded, in conjunction with results in Part I, that a single non-substrate-specific 11β-hydroxylase enzyme operates in marsupials.

21-Hydroxylation of 21-deoxycortisone by adrenal glands of a marsupial and two eutherian species.

Abstract 1. 1. 3H cortisone was produced by possum and rabbit adrenals infused with 3H 21-deoxycortisone in vivo. 2. 2. The 21-hydroxylation was unaffected by the simultaneous infusion of 14C 11-deoxycorticosterone, 14C 11-deoxycortisol or 14C 17αOH-progesterone. 3. 3. Possum adrenal tissue incubated with 3H 21-deoxycortisone and 14C 11-deoxycortisol produced more 3H-labelled than 14C-labelled cortisol-cortisone. 4. 4. Similar results were obtained with rabbit and with guinea-pig adrenal tissue, 14C 17αOH-progesterone being tested as well in the guinea-pig. 5. 5. In both eutherian species more cortisol-cortisone was produced from mixtures than from 3H 21-deoxycortisone alone. 6. 6. Rabbit adrenals incubated with 230 μg of 21-deoxycortisol yielded 89 μg cortisol and 19 μg cortisone.