METABOLISM OF ANDROGENS IN VITRO BY HUMAN FOETAL SKIN F. SHARP, J. B. HAY AND M. B. HODGINS Departments of Midwifery* and Dermatology, University of Glasgow, Glasgow Gli 6NU, Scotland
(Received 26 January 1976) SUMMARY
Fresh scalp, genital, chest and axillary skin from human foetuses of 12\p=n-\41weeks' maturity incubated in Krebs' improved Ringer I medium with [7\g=a\-3H]dehydroepiandrosterone, [7\g=a\-3H]testosterone and [7\g=a\-3H]androstenedione. The metabolites identified were
was
androstenedione, 5\g=a\-androstane-3,17-dione,androsterone, 3-epiandrosterone, 5\g=a\-dihydrotestosterone, 5\g=a\-androstane-3\g=a\,17\g=b\-diol,5\g=a\-androstane-3\g=b\,17\g=b\-diol,5-androstene-3\g=b\,17\g=b\\x=req-\ diol and testosterone. The results provide evidence for the presence of 3\g=b\-hydroxysteroid
dehydrogenase, \g=D\4\p=n-\5 isomerase, 17\g=b\-hydroxysteroid dehydrogenase, \g=D\4-3-oxosteroid-5\g=a\\x=req-\ reductase and 3\g=a\-hydroxysteroiddehydrogenase in human foetal skin. There were quantitative differences in the various enzyme activities between different body sites and skin specimens of different gestational age. 5\g=a\-Reductaseactivity was particularly high in genital skin. 3\g=b\-Hydroxysteroiddehydrogenase \g=D\4\p=n-\5isomerase activity was low in skin from a 12-week foetus, but high in skin specimens from 28-, 38- and 41-week foetuses. 17\g=b\-Hydroxysteroid dehydrogenase activity was already high in the skin of the 12-week foetus and remained so in the older foetuses. These results were correlated with the development of the foetal sebaceous glands, and were in general agreement with a parallel enzyme histochemical study. The role of androgen metabolism in human foetal skin is discussed. INTRODUCTION
Metabolism of
androgens by
human skin has
recently
been studied
intensively (Wilson
&
Walker, 1969; Hay & Hodgins, 1973). The control of sebaceous function and hair growth by testosterone may depend on the conversion, in the skin, of this steroid to 5a-dihydrotestosterone (Voigt & Hsia, 1973; Imperato-McGinley, Guerrero, Gautier & Peterson, 1974)
considered to be the active form of androgens (see review by King & Mainwaring, 1974). Other androgens, for example, dehydroepiandrosterone and androstenedione, may also be important in controlling sebaceous gland function and hair growth, and it has been shown that they also may be converted to 5a-dihydrotestosterone by the skin (Hay & Hodgins, 1973). Hydroxysteroid dehydrogenases have been demonstrated histochemically in sebaceous glands of human adult skin (Baillie, Caiman & Milne, 1965; Calman, Muir, Milne & Young, 1970). Similar histochemical studies using human foetal skin demonstrated the presence of 3/?- and 17/?-hydroxysteroid dehydrogenases (HSD) in the sebaceous glands (Sharp, Caiman, Milne & Young, 1970; Sharp, 1975, 1976). The appearance of histo¬ chemically detectable 17/7-HSD activity in foetal skin coincided with the appearance of
which is
*
t
now
Address for correspondence. Present address: Department of Dermatology, Victoria
Infirmary, Newcastle-upon-Tyne.
cells within the sebaceous glands, while 3/?-HSD activity appeared only from 22-24 weeks of gestation onwards. These studies have led to further investigation of the possible role of androgen metabolism in the development and function of the foetal pilo-sebaceous unit. In view of the limitations of the histochemical approach in the study of steroid metabolism, biochemical studies of the metabolism of [7a-3H]testosterone, [7a-3H]dehydroepiandrosterone and [7a-3H]androstenedione in the skin of human foetuses have been carried out in vitro. Metabolism has been related to differing body sites and gesta¬ tional ages. An earlier study (Flamigni, Collins, Koullapis, Craft, Dewhurst & Sommerville, 1971) reported the metabolism in vivo and in vitro of testosterone and androstenedione by genital and dorsal skin from foetuses between 12 and 24 weeks of gestation. Kelch, Lind¬ holm & Jaffe (1971) also described the metabolism in vitro of testosterone by perineal and thigh skin from two human foetuses of gestational age 14-18 weeks. However, no details were given in these studies of differences in metabolism related to specific gestational age, or other body sites.
lipid-containing
MATERIALS AND METHODS
Tissue
Full-thickness skin specimens were collected from freshly aborted or stillborn foetuses, the clinical details of which are summarized in Table 1. Where appropriate, permission for post mortem was obtained before dissection. In no case was there any doubt concerning gesta¬ tional age, and in three of the four foetuses this was confirmed by earlier ultrasonic scan¬ ning (Donald, 1969). In all cases the tissues were fresh, skin specimens being collected Table 1. Clinical details
Length of Foetus gestation no. (weeks) Sex
Ultrasonic confirmation of maturity Weight
performed
12 28
M M
Yes Yes
38
M
Yes
(kg)
Not known 1000 2830
offoetuses used
Other details
Skin sites examined
Hysterotomy Fresh stillbirth,
Scalp, axilla, perineum Scalp
premature labour Fresh cause
41
M
No
3410
Fresh cause
stillbirth, unknown
stillbirth, unknown
Scalp, anterior chest, scrotum
Scalp
within 30-45 min of death. The specimens were dissected free with minimal trauma, sub¬ was removed so far as possible, and each specimen was divided in half. One was portion rapidly frozen on carbon dioxide snow within a few minutes, and stored at 20 °C until histochemical examination for 3ß- and 17/?-HSD activity, using the method of Muir, Calman, Thomson, MacSween, Milne, Chakraborty & Grant (1968 a, b). The remaining portion was kept on crushed ice and transferred immediately to the laboratory for incubation as described below. Routinely a specimen of scalp skin was collected. Additional specimens were as follows: foetus No. 1, axillary and perineal tissue; foetus No. 3, skin from anterior chest wall and scrotal skin (Table 1). cutaneous fat
—
Radioactive steroids
[7a-3H]Testosterone (1-51 Ci/mmol), [7a-3H]androstenedione (5-88 Ci/mmol) and [7a-3H]dehydroepiandrosterone (0-5 Ci/mmol) were obtained from the Radiochemical Centre,
Amersham, England. Before use these were purified by thin-layer chromatography on gel (Merck HF 254+366), using chloroform:acetone (185:15, v/v) as solvent. Control
silica
experiments
showed these
purifications
to
be
satisfactory.
Incubations The fresh whole-thickness skin, collected as described above, was weighed and cut trans¬ versely into 0-5 mm thick slices using razor blades. These slices of fresh tissue were then incubated in 1-0 ml Krebs' improved Ringer I medium (Krebs, 1950) with either 3-5 nmol [7a-3H]testosterone (2-7 /.Ci), or 3-5 nmol [7a-3H]androstenedione (2-61 //Ci), or 3-5 nmol [7a-3H]dehydroepiandrosterone (1-76//Ci). The steroids were dried from methanol solution containing 5 µ\ propane-1,2-diol before addition of the Krebs' medium. Incubations were carried out with shaking under 95 % 02:5 °0 C02 at 37 °C for 2 h. Control incubations con¬ taining no skin were carried out for each radioactive steroid substrate. The incubations were stopped by the addition of 5 ml acetone and 250 pg each of the appropriate unlabelled carrier steroids in ethanol. Extraction and purification of steroids The extraction of steroids, removal of lipids and separation of neutral steroids and conju¬ gate fractions were carried out as described by Chakraborty, Thomson, MacSween, Muir, Calman, Grant & Milne (1970). The procedures for thin-layer and paper chromatography described by Hodgins (1971) were used to separate the following steroids: androstenedione,
5a-androstane-3,17-dione, 5/?-androstane-3,17-dione, androsterone, 3-epiandrosterone, aetiocholanolone, 5a-dihydrotestosterone, testosterone, dehydroepiandrosterone, and a polar neutral steroid fraction. Epitestosterone was separated as described by Hodgins & Hay
(1973).
The androstenediol-androstanediol fraction isolated by this procedure was also separated further as described by Hodgins & Hay (1973), the unlabelled carriers being 5-androstene-3/?,17/?-diol, 5a-androstane-3a,17/?-diol, and 5a-androstane-3/?, 17/i-diol. The polar neutral steroid fraction isolated as described above was not examined further.
Isolation and determination of steroids Samples of each isolated steroid were taken for estimation of radioactivity and carrier recovery. The procedures for this have been described by Chakraborty et al. (1970). A further sample was removed for determination of radiochemical purity by crystallization to constant specific activity; 10 mg carrier were used and the crystals were weighed on a Cahn Electrobalance (Cahn Instrument Co., Paramount, California, U.S.A.). RESULTS
Biochemical studies The metabolites formed on incubating the different steroid substrates with the various foetal skin samples are shown in Tables 2-4. The results are reported as percentage conversion of the substrate/2 h for the given weight of tissue, and are based on constant specific activities, examples of which are shown in Table 5. The metabolites produced in incubations with [7a-3H]testosterone were androstenedione, 5a-androstane-3,17-dione, androsterone, 3-epiandrosterone, 5a-dihydrotestosterone, 5a-androstane-3a,17/?-diol and 5a-androstane-3/?,17/?-diol. A similar spectrum of metabolites was isolated from the incubations with [7a-3H]androstenedione, except for the 5a-androstanediols, which were not formed in quantities sufficient for detection. Incubations with [7a-3H]dehydroepiandrosterone produced 5-androstene-3/?,17/?-diol, androstenedione, testosterone and a spectrum of 5a-reduced metabolites similar to that
found in incubations with [7a-3H]androstenedione. The percentage conversion of [7a-3H]testosterone to 17-oxosteroids greatly exceeded the conversion of [7a-3H]androstenedione and [7a-3H]dehydroepiandrosterone to 17/?-hydroxysteroids. The percentage conversion of the three substrates to neutral steroids more polar than 5-androstene-3/?,17/?-diol ranged from 0-02 to 1-5 %, with the exception of one incubation of scalp skin from the 12-week Table 2. Radiometabolites produced by various foetal skin samples obtained between the 12th and 41st weeks of gestation, when incubated with [7oc-3H]testosterone in Krebs' improved Ringer I medium (expressed as % conversion/100 mg tissue/2 h) Wet Skin site
Scalp Scalp Scalp Scalp
Length of weight of gestation tissue (weeks) (mg) 12 28 38 41
Metabolites* (% conversion/2 h)
Tt
1210 740
23-8
95-8 71-2 27-8 96-8 88-8 19-8
60-5 95-9 751 20-3
21-6
AE
5aAD
52-7 32-7 17-2 5-6 20
1-6 24-2 13-2 6-9
EA
5aT
01
0
1-7 2-7 40 03
2-3 0-6 0-5 005 20 0-4 0
01 07 3-4 3-2 16-7 35-7 9-7 0-2
3a, 17/?- 3/?, 17/?diol 0 0 01 0-3 0 2-4 06 0
diol
0 0 008 008 0 0-7 0-2 0
12 1-3 Perineum 38 0-7 Scrotum 10-9 13-7 54-4 2-2 7-7 41 Chest 38 5-4 0 87-4 Axilla 12 01 * T, testosterone; AE, androstenedione; 5aAD, 5a-androstane-3,17-dione; A, androsterone; EA,
3-epiandrosterone; 5aT, 5a-dihydrotestosterone; 3a,17/?-diol, 5a-androstane-3a,17/?-diol; 3/?,17/?-diol, 5a-androstane-3/?, 17/?-diol. t % incubated [7a-3H]testosterone recovered. Table 3. Radiometabolites produced by two samples offoetal scalp skin obtained during the third trimester when incubated with [7a-3H]androstenedione in Krebs' improved Ringer I medium Length of gestation (weeks) 38 41
Metabolites* ( %
Wet weight of tissue
5aAD
(mg)
AEt
53-4 93-8
57-7 0-3 141 66-9 0-3 8-2 See Table 2 for abbreviations.
*
conversion/2 h) EA
5aT
0-5 0-7
01 0-2
2-6 6-2
t % incubated [7a-3H]androstenedione recovered. Table 4. Radiometabolites produced by various foetal skin samples obtained between the 12th and 41st weeks of gestation, when incubated with [7a-3H]dehydroepiandrosterone in Krebs' improved Ringer I medium Wet Skin site
Scalp Scalp Scalp Scalp
Scrotum Chest
Length of weight of gestation tissue (weeks) (mg) Dt 12 28 38 41 38 38
1050 65-6 59-4 991 820 84-2
50-3 650 75-4 77-8 631 76-5
Metabolites*
001 007 004 002 002 002
(% conversion/2 h)
AE
5aAD
0-2 3-6 11 05 0-2 0-6
0 4-7 60 3-9 3-9 6-5
EA
0 0-2 11 1-3 2-7 1-9
0 1-6 10 0-4 1-3 1-4
5aT 0 005 006 0 06 008 0 10
A5diol 0 0 01 008 0-20 008
* D, dehydroepiandrosterone; A5diol, 5-androstene-3/?,17^-diol. See Table 2 for other abbreviations, t % incubated [7a-3H]dehydroepiandrosterone recovered.
Examples of constant specific activities obtained by recrystallization of metabolites produced from incubation of scalp skin, from 38-week male foetus, with the three steroid Table 5.
substrates
Specific activity of isolated metabolite (d.p.m./mg) Steroid isolated Testosterone
Androstenedione
Dehyd roepiandrosterone 5a-Androstane-3,17-dione Androsterone
3-Epiandrosterone 5a-Dihydrotestosterone
Crystallizations
Steroid substrate*
1 80640 835 93 46990 220000 4266 183500 26730 29510 142£0 3181 4091 1714
79580 811 86 48010 215100 4452 178900
536 757 771
507 775 770 3631 229 50 319 175 127
3827 243 54 295
268£0 29780 14040 3013 3916 1685
78230 832 93 49790 226900 4363 191300 26430 29290 13920
A DHA A DHA DHA A DHA
3077 4206 1732 521 796 803 3630 229 46
A DHA
800f
sot
310 5a-Androstene-3/?, 17/?-diol 173 181 5a-Androstane-3a, 17/?-diol 140 138 5a-Androstane-3/?,17/?-diol * DHA, A T, indicate the substrates dehydroepiandrosterone, androstenedione
respectively, t Crystals oxidized
to
A DHA A DHA DHA
and testosterone,
5a-androstane-3,17-dione before 4th crystallization.
[7a-3H]dehydroepiandrosterone, where there was a conversion of 8-7 %. From % of the incubated radioactivity was found in the aqueous phase after chloro¬ form/water partition of the incubates. No evidence was obtained for the transformation of any of the three substrates into 5/?-androstane-3,l 7-dione, aetiocholanolone or epitestosterone. The total activities of 3^-HSD 4~5 isomerase, 17/5-HSD and 5a-reductase, in relation to gestational age and body site of the skin, are shown in Figs 1-3. foetus with 0-02 to 2-2
Enzyme histochemical studies In skin from the 12-week foetus early hair pegs were present with no differentiated sebaceous glands, and histochemically demonstrable 3/?- or 17/?-HSD activity was absent. In all skin samples from the 28-, 38- and 41-week foetuses, fully differentiated sebaceous glands were present, and in all cases 3ß- and 17/?-HSD activity was demonstrable histochemically within the gland acini, but in no other structure.
15-
¬ o
(Received 26 January 1976) SUMMARY
Fresh scalp, genital, chest and axillary skin from human foetuses of 12\p=n-\41weeks' maturity incubated in Krebs' improved Ringer I medium with [7\g=a\-3H]dehydroepiandrosterone, [7\g=a\-3H]testosterone and [7\g=a\-3H]androstenedione. The metabolites identified were
was
androstenedione, 5\g=a\-androstane-3,17-dione,androsterone, 3-epiandrosterone, 5\g=a\-dihydrotestosterone, 5\g=a\-androstane-3\g=a\,17\g=b\-diol,5\g=a\-androstane-3\g=b\,17\g=b\-diol,5-androstene-3\g=b\,17\g=b\\x=req-\ diol and testosterone. The results provide evidence for the presence of 3\g=b\-hydroxysteroid
dehydrogenase, \g=D\4\p=n-\5 isomerase, 17\g=b\-hydroxysteroid dehydrogenase, \g=D\4-3-oxosteroid-5\g=a\\x=req-\ reductase and 3\g=a\-hydroxysteroiddehydrogenase in human foetal skin. There were quantitative differences in the various enzyme activities between different body sites and skin specimens of different gestational age. 5\g=a\-Reductaseactivity was particularly high in genital skin. 3\g=b\-Hydroxysteroiddehydrogenase \g=D\4\p=n-\5isomerase activity was low in skin from a 12-week foetus, but high in skin specimens from 28-, 38- and 41-week foetuses. 17\g=b\-Hydroxysteroid dehydrogenase activity was already high in the skin of the 12-week foetus and remained so in the older foetuses. These results were correlated with the development of the foetal sebaceous glands, and were in general agreement with a parallel enzyme histochemical study. The role of androgen metabolism in human foetal skin is discussed. INTRODUCTION
Metabolism of
androgens by
human skin has
recently
been studied
intensively (Wilson
&
Walker, 1969; Hay & Hodgins, 1973). The control of sebaceous function and hair growth by testosterone may depend on the conversion, in the skin, of this steroid to 5a-dihydrotestosterone (Voigt & Hsia, 1973; Imperato-McGinley, Guerrero, Gautier & Peterson, 1974)
considered to be the active form of androgens (see review by King & Mainwaring, 1974). Other androgens, for example, dehydroepiandrosterone and androstenedione, may also be important in controlling sebaceous gland function and hair growth, and it has been shown that they also may be converted to 5a-dihydrotestosterone by the skin (Hay & Hodgins, 1973). Hydroxysteroid dehydrogenases have been demonstrated histochemically in sebaceous glands of human adult skin (Baillie, Caiman & Milne, 1965; Calman, Muir, Milne & Young, 1970). Similar histochemical studies using human foetal skin demonstrated the presence of 3/?- and 17/?-hydroxysteroid dehydrogenases (HSD) in the sebaceous glands (Sharp, Caiman, Milne & Young, 1970; Sharp, 1975, 1976). The appearance of histo¬ chemically detectable 17/7-HSD activity in foetal skin coincided with the appearance of
which is
*
t
now
Address for correspondence. Present address: Department of Dermatology, Victoria
Infirmary, Newcastle-upon-Tyne.
cells within the sebaceous glands, while 3/?-HSD activity appeared only from 22-24 weeks of gestation onwards. These studies have led to further investigation of the possible role of androgen metabolism in the development and function of the foetal pilo-sebaceous unit. In view of the limitations of the histochemical approach in the study of steroid metabolism, biochemical studies of the metabolism of [7a-3H]testosterone, [7a-3H]dehydroepiandrosterone and [7a-3H]androstenedione in the skin of human foetuses have been carried out in vitro. Metabolism has been related to differing body sites and gesta¬ tional ages. An earlier study (Flamigni, Collins, Koullapis, Craft, Dewhurst & Sommerville, 1971) reported the metabolism in vivo and in vitro of testosterone and androstenedione by genital and dorsal skin from foetuses between 12 and 24 weeks of gestation. Kelch, Lind¬ holm & Jaffe (1971) also described the metabolism in vitro of testosterone by perineal and thigh skin from two human foetuses of gestational age 14-18 weeks. However, no details were given in these studies of differences in metabolism related to specific gestational age, or other body sites.
lipid-containing
MATERIALS AND METHODS
Tissue
Full-thickness skin specimens were collected from freshly aborted or stillborn foetuses, the clinical details of which are summarized in Table 1. Where appropriate, permission for post mortem was obtained before dissection. In no case was there any doubt concerning gesta¬ tional age, and in three of the four foetuses this was confirmed by earlier ultrasonic scan¬ ning (Donald, 1969). In all cases the tissues were fresh, skin specimens being collected Table 1. Clinical details
Length of Foetus gestation no. (weeks) Sex
Ultrasonic confirmation of maturity Weight
performed
12 28
M M
Yes Yes
38
M
Yes
(kg)
Not known 1000 2830
offoetuses used
Other details
Skin sites examined
Hysterotomy Fresh stillbirth,
Scalp, axilla, perineum Scalp
premature labour Fresh cause
41
M
No
3410
Fresh cause
stillbirth, unknown
stillbirth, unknown
Scalp, anterior chest, scrotum
Scalp
within 30-45 min of death. The specimens were dissected free with minimal trauma, sub¬ was removed so far as possible, and each specimen was divided in half. One was portion rapidly frozen on carbon dioxide snow within a few minutes, and stored at 20 °C until histochemical examination for 3ß- and 17/?-HSD activity, using the method of Muir, Calman, Thomson, MacSween, Milne, Chakraborty & Grant (1968 a, b). The remaining portion was kept on crushed ice and transferred immediately to the laboratory for incubation as described below. Routinely a specimen of scalp skin was collected. Additional specimens were as follows: foetus No. 1, axillary and perineal tissue; foetus No. 3, skin from anterior chest wall and scrotal skin (Table 1). cutaneous fat
—
Radioactive steroids
[7a-3H]Testosterone (1-51 Ci/mmol), [7a-3H]androstenedione (5-88 Ci/mmol) and [7a-3H]dehydroepiandrosterone (0-5 Ci/mmol) were obtained from the Radiochemical Centre,
Amersham, England. Before use these were purified by thin-layer chromatography on gel (Merck HF 254+366), using chloroform:acetone (185:15, v/v) as solvent. Control
silica
experiments
showed these
purifications
to
be
satisfactory.
Incubations The fresh whole-thickness skin, collected as described above, was weighed and cut trans¬ versely into 0-5 mm thick slices using razor blades. These slices of fresh tissue were then incubated in 1-0 ml Krebs' improved Ringer I medium (Krebs, 1950) with either 3-5 nmol [7a-3H]testosterone (2-7 /.Ci), or 3-5 nmol [7a-3H]androstenedione (2-61 //Ci), or 3-5 nmol [7a-3H]dehydroepiandrosterone (1-76//Ci). The steroids were dried from methanol solution containing 5 µ\ propane-1,2-diol before addition of the Krebs' medium. Incubations were carried out with shaking under 95 % 02:5 °0 C02 at 37 °C for 2 h. Control incubations con¬ taining no skin were carried out for each radioactive steroid substrate. The incubations were stopped by the addition of 5 ml acetone and 250 pg each of the appropriate unlabelled carrier steroids in ethanol. Extraction and purification of steroids The extraction of steroids, removal of lipids and separation of neutral steroids and conju¬ gate fractions were carried out as described by Chakraborty, Thomson, MacSween, Muir, Calman, Grant & Milne (1970). The procedures for thin-layer and paper chromatography described by Hodgins (1971) were used to separate the following steroids: androstenedione,
5a-androstane-3,17-dione, 5/?-androstane-3,17-dione, androsterone, 3-epiandrosterone, aetiocholanolone, 5a-dihydrotestosterone, testosterone, dehydroepiandrosterone, and a polar neutral steroid fraction. Epitestosterone was separated as described by Hodgins & Hay
(1973).
The androstenediol-androstanediol fraction isolated by this procedure was also separated further as described by Hodgins & Hay (1973), the unlabelled carriers being 5-androstene-3/?,17/?-diol, 5a-androstane-3a,17/?-diol, and 5a-androstane-3/?, 17/i-diol. The polar neutral steroid fraction isolated as described above was not examined further.
Isolation and determination of steroids Samples of each isolated steroid were taken for estimation of radioactivity and carrier recovery. The procedures for this have been described by Chakraborty et al. (1970). A further sample was removed for determination of radiochemical purity by crystallization to constant specific activity; 10 mg carrier were used and the crystals were weighed on a Cahn Electrobalance (Cahn Instrument Co., Paramount, California, U.S.A.). RESULTS
Biochemical studies The metabolites formed on incubating the different steroid substrates with the various foetal skin samples are shown in Tables 2-4. The results are reported as percentage conversion of the substrate/2 h for the given weight of tissue, and are based on constant specific activities, examples of which are shown in Table 5. The metabolites produced in incubations with [7a-3H]testosterone were androstenedione, 5a-androstane-3,17-dione, androsterone, 3-epiandrosterone, 5a-dihydrotestosterone, 5a-androstane-3a,17/?-diol and 5a-androstane-3/?,17/?-diol. A similar spectrum of metabolites was isolated from the incubations with [7a-3H]androstenedione, except for the 5a-androstanediols, which were not formed in quantities sufficient for detection. Incubations with [7a-3H]dehydroepiandrosterone produced 5-androstene-3/?,17/?-diol, androstenedione, testosterone and a spectrum of 5a-reduced metabolites similar to that
found in incubations with [7a-3H]androstenedione. The percentage conversion of [7a-3H]testosterone to 17-oxosteroids greatly exceeded the conversion of [7a-3H]androstenedione and [7a-3H]dehydroepiandrosterone to 17/?-hydroxysteroids. The percentage conversion of the three substrates to neutral steroids more polar than 5-androstene-3/?,17/?-diol ranged from 0-02 to 1-5 %, with the exception of one incubation of scalp skin from the 12-week Table 2. Radiometabolites produced by various foetal skin samples obtained between the 12th and 41st weeks of gestation, when incubated with [7oc-3H]testosterone in Krebs' improved Ringer I medium (expressed as % conversion/100 mg tissue/2 h) Wet Skin site
Scalp Scalp Scalp Scalp
Length of weight of gestation tissue (weeks) (mg) 12 28 38 41
Metabolites* (% conversion/2 h)
Tt
1210 740
23-8
95-8 71-2 27-8 96-8 88-8 19-8
60-5 95-9 751 20-3
21-6
AE
5aAD
52-7 32-7 17-2 5-6 20
1-6 24-2 13-2 6-9
EA
5aT
01
0
1-7 2-7 40 03
2-3 0-6 0-5 005 20 0-4 0
01 07 3-4 3-2 16-7 35-7 9-7 0-2
3a, 17/?- 3/?, 17/?diol 0 0 01 0-3 0 2-4 06 0
diol
0 0 008 008 0 0-7 0-2 0
12 1-3 Perineum 38 0-7 Scrotum 10-9 13-7 54-4 2-2 7-7 41 Chest 38 5-4 0 87-4 Axilla 12 01 * T, testosterone; AE, androstenedione; 5aAD, 5a-androstane-3,17-dione; A, androsterone; EA,
3-epiandrosterone; 5aT, 5a-dihydrotestosterone; 3a,17/?-diol, 5a-androstane-3a,17/?-diol; 3/?,17/?-diol, 5a-androstane-3/?, 17/?-diol. t % incubated [7a-3H]testosterone recovered. Table 3. Radiometabolites produced by two samples offoetal scalp skin obtained during the third trimester when incubated with [7a-3H]androstenedione in Krebs' improved Ringer I medium Length of gestation (weeks) 38 41
Metabolites* ( %
Wet weight of tissue
5aAD
(mg)
AEt
53-4 93-8
57-7 0-3 141 66-9 0-3 8-2 See Table 2 for abbreviations.
*
conversion/2 h) EA
5aT
0-5 0-7
01 0-2
2-6 6-2
t % incubated [7a-3H]androstenedione recovered. Table 4. Radiometabolites produced by various foetal skin samples obtained between the 12th and 41st weeks of gestation, when incubated with [7a-3H]dehydroepiandrosterone in Krebs' improved Ringer I medium Wet Skin site
Scalp Scalp Scalp Scalp
Scrotum Chest
Length of weight of gestation tissue (weeks) (mg) Dt 12 28 38 41 38 38
1050 65-6 59-4 991 820 84-2
50-3 650 75-4 77-8 631 76-5
Metabolites*
001 007 004 002 002 002
(% conversion/2 h)
AE
5aAD
0-2 3-6 11 05 0-2 0-6
0 4-7 60 3-9 3-9 6-5
EA
0 0-2 11 1-3 2-7 1-9
0 1-6 10 0-4 1-3 1-4
5aT 0 005 006 0 06 008 0 10
A5diol 0 0 01 008 0-20 008
* D, dehydroepiandrosterone; A5diol, 5-androstene-3/?,17^-diol. See Table 2 for other abbreviations, t % incubated [7a-3H]dehydroepiandrosterone recovered.
Examples of constant specific activities obtained by recrystallization of metabolites produced from incubation of scalp skin, from 38-week male foetus, with the three steroid Table 5.
substrates
Specific activity of isolated metabolite (d.p.m./mg) Steroid isolated Testosterone
Androstenedione
Dehyd roepiandrosterone 5a-Androstane-3,17-dione Androsterone
3-Epiandrosterone 5a-Dihydrotestosterone
Crystallizations
Steroid substrate*
1 80640 835 93 46990 220000 4266 183500 26730 29510 142£0 3181 4091 1714
79580 811 86 48010 215100 4452 178900
536 757 771
507 775 770 3631 229 50 319 175 127
3827 243 54 295
268£0 29780 14040 3013 3916 1685
78230 832 93 49790 226900 4363 191300 26430 29290 13920
A DHA A DHA DHA A DHA
3077 4206 1732 521 796 803 3630 229 46
A DHA
800f
sot
310 5a-Androstene-3/?, 17/?-diol 173 181 5a-Androstane-3a, 17/?-diol 140 138 5a-Androstane-3/?,17/?-diol * DHA, A T, indicate the substrates dehydroepiandrosterone, androstenedione
respectively, t Crystals oxidized
to
A DHA A DHA DHA
and testosterone,
5a-androstane-3,17-dione before 4th crystallization.
[7a-3H]dehydroepiandrosterone, where there was a conversion of 8-7 %. From % of the incubated radioactivity was found in the aqueous phase after chloro¬ form/water partition of the incubates. No evidence was obtained for the transformation of any of the three substrates into 5/?-androstane-3,l 7-dione, aetiocholanolone or epitestosterone. The total activities of 3^-HSD 4~5 isomerase, 17/5-HSD and 5a-reductase, in relation to gestational age and body site of the skin, are shown in Figs 1-3. foetus with 0-02 to 2-2
Enzyme histochemical studies In skin from the 12-week foetus early hair pegs were present with no differentiated sebaceous glands, and histochemically demonstrable 3/?- or 17/?-HSD activity was absent. In all skin samples from the 28-, 38- and 41-week foetuses, fully differentiated sebaceous glands were present, and in all cases 3ß- and 17/?-HSD activity was demonstrable histochemically within the gland acini, but in no other structure.
15-
¬ o
![Uptake, metabolism and release of androgens by human skin fibroblasts [proceedings].](/assets/img/hold.png)
