Comp. Biochent Physiol., 1977,VoL 57B,pp. 15 to 18. Pergamon Press. Printed in Great Britain
CHANGES IN STEROID BIOSYNTHESIS BY ADRENAL HOMOGENATES OF THE POSSUM (TRICHOSURUS VULPECULA) AT VARIOUS STAGES OF SEXUAL MATURATION WITH SPECIAL REFERENCE TO 5fl-REDUCTASE ACTIVITY* MAGDA WEISS AND VALERIEL. FORD Department of Physiology, Monash University, Clayton, Victoria, Australia (Received 27 September 1976)
Abstract--1. A marked difference was found in the yields of llfl-hydroxylated and 5fl-reduced products from 14C progesterone by adrenal homogenates of immature and adult possums. 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~o, corticosterone 16.3 + 2.270, total 5fl-reduced products 9.6 _+ 3.4~o (mean + S.E.M.). In yields < 4 ~ aldosterone, 21-deoxycortisol, llfland 17~-hydroxyprogesterone. 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 5fl reduced products (of varying degrees) with a concomitant fall in unreduced products, the average yields being; cortisol 1270, corticosterone 3~o and total 5fl-reduced metabolites 60~o. 4. The yields of conversion products from adrenals of adults ( > 2½ years old) were cortisol 1.3 + 0.870 and total 5fl-reduced-ll-deoxy-steroids 71 ___8~o (mean ___S.E.M.). 5. The similarities of the changes in adrenal 5fl-reductase activity in the possum and guinea pigs at different stages of development are discussed.
found in the guinea pig (Weiss & Ford, 1975). However, we have shown previously (Weiss & Ford, 1975) that in guinea pigs a gradual rise in 5fl-reductase activity occurs from birth to puberty. Adrenal homogenates from prenatal animals and up to I 18 days postnatally, formed predominantly cortisol, whereas the major conversion products from glands of animals over 25 days old were 5fl-reduced products. The observations that adult possums possess an equally high 5fl-reductase activity (Weiss, 1975), prompted us to investigate whether a similar age induced change in this activity occurs in the possum. In addition the aim was to find out whether the increase in activity is in any way related to stages of gonadal development.
INTRODUCTION
The ability of the adrenal gland to reduce A4-3-oxosteroids to 5~ and 5fl metabolites has now been well established and a species-difference exists with regard to the preferential formation of either metabolite. For example, human (Charreau et al., 1968) beef (Lantos et al., 1965; Levy & Saito, 1966) and rat (Kitay et al., 1971 ; Maynard & Cameron, 1972) adrenal glands produce prevalently 5or-reduced steroid metabolites, whereas in the guinea pig (Brown-Grant et al., 1960; Spatz & Hofmann, 1966), the mouse (Ertel & Ungar, 1964; Maynard & Cameron, 1972) and the possum (Weiss, 1975) 5fl-reduced products predominate. The respective reductases have different cellular locations (Brown-Grant et al., 1960; Kitay et al., 1971; Maynard & Cameron, 1973a; Collins & Cameron, 1975) and for each reductase multiple substrate specific enzymes differing in activity are postulated (Brown-Grant et al., 1960; Maynard & Cameron, 1973a; Frgacic-Cala et al., 1975). The physiological role of reductases in the adrenal remains to be established. Extensive studies in rats by several workers (Witorsch & Kitay, 1972; Colby & Kitay, 1972; Maynard & Cameron, 1973b) have indicated a gonadalpituitary involvement in the control of 5ct reductase activity, but no evidence of a similar inter-relationship controlling 5fl-reductase activity could be
MATERIALS AND METHODS
Possums (Trichosurus vulpecula) of both sexes; 12 males and 10 females ranging in ages from 5 to 24 months a n d 5 adults > 30 months old were used. Some animals were born in captivity and were of known ages while others were captured with their mothers as pouch-young and age determination was made from growth curve tables based on body weights and measurements of linear dimensions of various body parts (Lyne & Verhagen, 1957). Experiments were carried out during the peak breeding season (Pilton & Sharman, 1962; Gilmore, 1969). The testes and ventral prostate in males and the ovaries, uterus and vaginae in females were dissected free and weighed. The testes and ovaries were placed in Bouin's
* Supported by the Australian Research Grants Committee Grant No. D68/16837. 15
16
MAGDA WEISS AND VALERIE L. FORD
fixative and microscopic sections cut at 8#m were stained with Ehrlich's haematoxylin and eosin. The adrenals were removed under ether anaesthesia, freed of adhering tissue and weighed. They were then immediately homogenized and incubated in the presence of 1 #C ~4C progesterone (sp. act. 53 mCi/m-mole) substrate for 30 min. The procedures for incubation, extraction and characterization of steroids were the same as described in detail elsewhere (Weiss, 1973; Weiss & Ford, 1975). Final identifications were based on the ability of the labelled conversion product to form crystals of constant specific activity when mixed with authentic inert carriers. The specific activities of the last 3 crystallizations (from different solvent mixtures) did not differ by > 3~o. After correction for losses (Weiss & Ford, 1975), the results were expressed in terms of yields of labelled conversion products as percentages of added substrate. RESULTS
Determination of sexual development Based on the examination of histological sections of gonads and the weights and appearance of reproductive organs, the animals were subdivided into 3 groups: a, sexually immature; b, early gonadal activity; c, sexually mature. The criteria for males were the same as described by Tyndale-Biscoe (1955) and Gilmore (1969) and for females as described by Tyndale-Biscoe (1955) and Pilton & Sharman (1962). Histological examination of testes showed in animals of group a, no evidence of gonadal activity, group b, numerous stages of spermatogenic activity but mature spermatozoa were absent, group c, mature spermatozoa present in tubular lumen. Histological examination of ovaries showed in a, no evidence of follicular growth b, follicles in various stages of development and growth, the largest being 2-3 mm in diameter, corpus luteum absent, c, the presence of one or more corpoa lutea. In a large scale study of several hundred possums (Tyndale-Biseoe, 1955; Pilton & Sharman, 1962; Gilmore, 1969) it was found that puberty is reached between 1 and 2 years of age and our observations were in agreement with these findings.
Adrenal weight to body weight ratios The adrenals of immature animals were morphologically well established. The combined adrenal weights relative to body weight were, in immature animals (ages 5-15 months) 10.8___ 1.06mg/100g mean +_ S.E.M. (7 animals), in animals with early gonadal activity (ages 12-18 months) 7.9_ 0.72mg/ 100g (6 animals) and, in sexually mature animals (ages 15-24 months) 9.1 _+ 0.73 mg/100 g (6 animals).
No apparent sex difference in the ratios was found in either of the two sexually immature groups. In sexually mature animals the adrenals of the females were 30-50~o larger than those of males and fell into the range of values as published for adult possums (Weiss & McDonald, 1966).
Conversion products formed from 14C progesterone by adrenal homogenates of male and female possums at various stages of sexual development Immature animals and animals in early gonadal activity (ages 5-18 months) Table 1. tn both groups cortisol and corticosterone formed the major conversion products in total yields of > 70~o. Appreciable conversion to aldosterone and 21-deoxycortisol also occurred. The compounds sought, which were either not detected, or present in yields
CHANGES IN STEROID BIOSYNTHESIS BY ADRENAL HOMOGENATES OF THE POSSUM (TRICHOSURUS VULPECULA) AT VARIOUS STAGES OF SEXUAL MATURATION WITH SPECIAL REFERENCE TO 5fl-REDUCTASE ACTIVITY* MAGDA WEISS AND VALERIEL. FORD Department of Physiology, Monash University, Clayton, Victoria, Australia (Received 27 September 1976)
Abstract--1. A marked difference was found in the yields of llfl-hydroxylated and 5fl-reduced products from 14C progesterone by adrenal homogenates of immature and adult possums. 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~o, corticosterone 16.3 + 2.270, total 5fl-reduced products 9.6 _+ 3.4~o (mean + S.E.M.). In yields < 4 ~ aldosterone, 21-deoxycortisol, llfland 17~-hydroxyprogesterone. 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 5fl reduced products (of varying degrees) with a concomitant fall in unreduced products, the average yields being; cortisol 1270, corticosterone 3~o and total 5fl-reduced metabolites 60~o. 4. The yields of conversion products from adrenals of adults ( > 2½ years old) were cortisol 1.3 + 0.870 and total 5fl-reduced-ll-deoxy-steroids 71 ___8~o (mean ___S.E.M.). 5. The similarities of the changes in adrenal 5fl-reductase activity in the possum and guinea pigs at different stages of development are discussed.
found in the guinea pig (Weiss & Ford, 1975). However, we have shown previously (Weiss & Ford, 1975) that in guinea pigs a gradual rise in 5fl-reductase activity occurs from birth to puberty. Adrenal homogenates from prenatal animals and up to I 18 days postnatally, formed predominantly cortisol, whereas the major conversion products from glands of animals over 25 days old were 5fl-reduced products. The observations that adult possums possess an equally high 5fl-reductase activity (Weiss, 1975), prompted us to investigate whether a similar age induced change in this activity occurs in the possum. In addition the aim was to find out whether the increase in activity is in any way related to stages of gonadal development.
INTRODUCTION
The ability of the adrenal gland to reduce A4-3-oxosteroids to 5~ and 5fl metabolites has now been well established and a species-difference exists with regard to the preferential formation of either metabolite. For example, human (Charreau et al., 1968) beef (Lantos et al., 1965; Levy & Saito, 1966) and rat (Kitay et al., 1971 ; Maynard & Cameron, 1972) adrenal glands produce prevalently 5or-reduced steroid metabolites, whereas in the guinea pig (Brown-Grant et al., 1960; Spatz & Hofmann, 1966), the mouse (Ertel & Ungar, 1964; Maynard & Cameron, 1972) and the possum (Weiss, 1975) 5fl-reduced products predominate. The respective reductases have different cellular locations (Brown-Grant et al., 1960; Kitay et al., 1971; Maynard & Cameron, 1973a; Collins & Cameron, 1975) and for each reductase multiple substrate specific enzymes differing in activity are postulated (Brown-Grant et al., 1960; Maynard & Cameron, 1973a; Frgacic-Cala et al., 1975). The physiological role of reductases in the adrenal remains to be established. Extensive studies in rats by several workers (Witorsch & Kitay, 1972; Colby & Kitay, 1972; Maynard & Cameron, 1973b) have indicated a gonadalpituitary involvement in the control of 5ct reductase activity, but no evidence of a similar inter-relationship controlling 5fl-reductase activity could be
MATERIALS AND METHODS
Possums (Trichosurus vulpecula) of both sexes; 12 males and 10 females ranging in ages from 5 to 24 months a n d 5 adults > 30 months old were used. Some animals were born in captivity and were of known ages while others were captured with their mothers as pouch-young and age determination was made from growth curve tables based on body weights and measurements of linear dimensions of various body parts (Lyne & Verhagen, 1957). Experiments were carried out during the peak breeding season (Pilton & Sharman, 1962; Gilmore, 1969). The testes and ventral prostate in males and the ovaries, uterus and vaginae in females were dissected free and weighed. The testes and ovaries were placed in Bouin's
* Supported by the Australian Research Grants Committee Grant No. D68/16837. 15
16
MAGDA WEISS AND VALERIE L. FORD
fixative and microscopic sections cut at 8#m were stained with Ehrlich's haematoxylin and eosin. The adrenals were removed under ether anaesthesia, freed of adhering tissue and weighed. They were then immediately homogenized and incubated in the presence of 1 #C ~4C progesterone (sp. act. 53 mCi/m-mole) substrate for 30 min. The procedures for incubation, extraction and characterization of steroids were the same as described in detail elsewhere (Weiss, 1973; Weiss & Ford, 1975). Final identifications were based on the ability of the labelled conversion product to form crystals of constant specific activity when mixed with authentic inert carriers. The specific activities of the last 3 crystallizations (from different solvent mixtures) did not differ by > 3~o. After correction for losses (Weiss & Ford, 1975), the results were expressed in terms of yields of labelled conversion products as percentages of added substrate. RESULTS
Determination of sexual development Based on the examination of histological sections of gonads and the weights and appearance of reproductive organs, the animals were subdivided into 3 groups: a, sexually immature; b, early gonadal activity; c, sexually mature. The criteria for males were the same as described by Tyndale-Biscoe (1955) and Gilmore (1969) and for females as described by Tyndale-Biscoe (1955) and Pilton & Sharman (1962). Histological examination of testes showed in animals of group a, no evidence of gonadal activity, group b, numerous stages of spermatogenic activity but mature spermatozoa were absent, group c, mature spermatozoa present in tubular lumen. Histological examination of ovaries showed in a, no evidence of follicular growth b, follicles in various stages of development and growth, the largest being 2-3 mm in diameter, corpus luteum absent, c, the presence of one or more corpoa lutea. In a large scale study of several hundred possums (Tyndale-Biseoe, 1955; Pilton & Sharman, 1962; Gilmore, 1969) it was found that puberty is reached between 1 and 2 years of age and our observations were in agreement with these findings.
Adrenal weight to body weight ratios The adrenals of immature animals were morphologically well established. The combined adrenal weights relative to body weight were, in immature animals (ages 5-15 months) 10.8___ 1.06mg/100g mean +_ S.E.M. (7 animals), in animals with early gonadal activity (ages 12-18 months) 7.9_ 0.72mg/ 100g (6 animals) and, in sexually mature animals (ages 15-24 months) 9.1 _+ 0.73 mg/100 g (6 animals).
No apparent sex difference in the ratios was found in either of the two sexually immature groups. In sexually mature animals the adrenals of the females were 30-50~o larger than those of males and fell into the range of values as published for adult possums (Weiss & McDonald, 1966).
Conversion products formed from 14C progesterone by adrenal homogenates of male and female possums at various stages of sexual development Immature animals and animals in early gonadal activity (ages 5-18 months) Table 1. tn both groups cortisol and corticosterone formed the major conversion products in total yields of > 70~o. Appreciable conversion to aldosterone and 21-deoxycortisol also occurred. The compounds sought, which were either not detected, or present in yields
