Archives for
Arch. Derm. Res. 256, 265-273 (1976)
e e s e r m a t o l o g i c a l a r c n
© by Springer-Verlag 1976
Metabolism of TestOsterone 4- 4C in Skin of Castrated Rats* E. Rampini, P. Cardo, Carla Cipriani, and G. Moretti Department of Dermatology (Head: Prof. Dr. G. Moretti) University of Genoa, I- 16132 Genoa, Italy
Summary.Testosterone metabolism was studied in the skin and preputial glands of normal and prepuberally castrated male rats during the 2nd hair cycle. In catagen-telogen the 17-beta-OHSDH of dorsal skin was higher in castrated than in control animals; 5-alpha-reductase instead, remained unchanged through out the hair cycle. Also in the preputial glands of castrated rats 17-beta-OHSDH was higher than in normal rats. So was 5-alpha-reductase. A possible direct control of a substrate, like Testosterone, and/or of a hypophyseal tropin, like prolactin, on enzymes that direct the metabolism of steroids in target tissues is conceivable. Zusammenfassung. Die Autoren studierten den Testosteron-Metabolismus der Haut und der Prfiputialdrfisen yon normalen m/innlichen Ratten, die in der Vorpubertfit wiihrend des zweiten Haarzyklus kastriert wurden. Im Katagen- und im Telogen-Stadium war die 17-beta-Hydroxysteroid-Dehydrogenase der Riickhaut in den kastrierten Ratten h6her als in den normalen Ratten; dagegen blieb die 5-alpha-Reductase w~ihrend des ganzen Haarzyklus unver~indert. Auch in den Pr~iputialdrfisen der kastrierten Ratten waren die 17-beta-Hydroxysteroid-Dehydrogenase und die 5-alpha-Reductase h6her als in den normalen Ratten. Man daft annehmen, dab eine direkte Kontrolle eines Substrates - Testosteron und/oder eines pituitfiren Tropin als Prolactin - fiber Enzyme, die ffir den Metabolismus der Steroide im ,,Target"-Zielgewebe verantwortlich sind, denkbar ist. Introduction Estradiol and testosterone metabolization in hairy dorsal rat skin is cyclic; 17-betahydroxysteroid dehydrogenase activity (17-beta-OHSDH is maximal in anagen; * Presented at the Joint Meeting of the Society for Investigative Dermatology and of the European Society for Dermatological Research, Amsterdam, June 1975.
266
E. Rampini et al.
5-alpha-steroid reductase activity increases remarkably in telogen [8, 9]. The activity of these enzymes, however, is probably conditioned by a variety of factors, apart from the hair cycle. 1 7 - b e t a - O H S D H and 5-alpha-reductase activity in rats f.i. is influenced by age since during the period b e t w e e n birth and 90 days of age, (puberty occurs in rats within the second m o n t h of life) both enzymes tend to diminish with progression of cycles [10]. Hypophysectomy, furthermore, markedly increases the activity of 17-betaO H S D H in the rat's skin whereas that of 5-alpha-reductase remains unchanged [11]. Does this m e a n that in the absence of the pituitary a directly repressive effect on 1 7 - b e t a - O H S D H may be lacking as already shown for prolactin on other enzymes of steroid metabolfsm [13, 14]? 1 7 - b e t a - O H S D H and 5-alpha-reductase activities of skin and preputial glands were consequently studied in castrated rats, since castration substantially reduces the production of prolactin a m o n g tropins [5,7].
Materials and Methods
Chemicals. NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and reference steroids were from Sigma Chemical Co. Testosterone-4-~C (specific activity 40 ~tC/p.mole) was purchased from Amersham and a stock solution in ethanol was prepared and stored in a refrigerator. Solvents and chemicals were of reagent grade. Chloroform for chromatography was redistilled before use and stabilized by 0.75 % of ethanol. Silica Gel H was obtained from Merck AG Co. Skin Specimens Specimens of hairy (dorsal) and glabrous (plantar) skin and the preputial glands were obtained from Wistar 36 male rats, which were either castrated before puberty on the 18th day of life or were intact litter mate controls. Dorsal skin, in particular, was taken on several days of the second hair cycle induced by plucking i.e.: on the 32nd, 37th, 42th day of life (anagen) when hairs were in anagen and animals in the puberal period, which in male rats ends around the 45th day [3]; on the 50th, 55th and 60th days of life when hairs are in Catagen-telogen and rats have reached adulthood [3]. The length of hair cycle was controlled in the two groups of animals by histology (H.E. stain of dorsal skin) and the general bodily effect of castration by recording the weight of the animals. On each of the specified days, three castrated and three normal rats were killed by decapitation and skin specimens were obtained from the supracaudal region. The panniculus carnosous was removed with a scalpel and 300 mg of skin were minced for incubation with testosterone-4-a4C. On the 37th and 55th day 150 mg of plantar skin and pairs of preputial glands were also taken from the same controls and castrated animals and incubated with testosterone-4-14C.
Incuba~on Each tissue sample (300 mg dorsal skin; 150 mg plantar skin; 40-80 mg preputial glands) was finely minced with scissors and suspended in 5 ml of KrebsrRinger phosphate buffer pH 7.4 containing a NADPH generating system (1 ~tmoleNADP; 25 ~tmoles glucose-6-phosphate, 1 unit of glucose-6phosphate dehydrogenase). Incubations were initiated with the addition of 5 x 105 dpm of testosterone-4-14C (5.6 m~moles) in 0.1ml of ethanol and carried out at 37 ° C in a shaking incubator.
Extraction and Separation of Steroids Incubation time was 30 rain since previous determinations of the progress of testosterone metabolism in dorsal skin during anagen, catagen and telogen had shown that at that time reaction was still linear [9].
Metabolism of Testosterone 4-14Cin Skin
267
Incubations were terminated by adding 20 ml methanol/dichlorometane 2 : 1. This single-phase mixture was filtered and the filtrate evaporated under vacuum. The acqueous residue was transferred to a separatory funnel with an additional 10 ml of water and extracted three times with 20 ml dichloromethane. Extracts were dried by passing through anhydrous sodium sulfate. Radioactivity in the extracts was assayed by counting an aliquot in a Packard Tricarb liquid scintillation spectometer model 3320 with 80 % counting efficiency. The scintillation fluid contained 14 g PPO and 0.7 g dimethyl POPOP in 2 1 of toluene. I4C recovery was always more than 85 %. Extracts were concentrated under nitrogen flow and residues were applied to thin layer plates (5 x 40 cm) prepared with silica gel H 0.3 mm thick and activated at 80 ° C for 1 h. The plates were developed by ascending chromatography in system chloroform-methanol 99 : 1 [4]. Distribution of 14C on the plates was located by a Packard 7201 autoscanner. Each of the five radioactive bands was scraped off the plates, eluted with methanol, and an aliquot was radio assayed by scintillation counting. Radioactive metabolites in each band were identified by co-chromatography with reference steroids, chromatography in different systems and isotopic dilution already employed in previous work [9] and in current use in our laboratories. The 17-beta-hydroxysteroid dehydrogenase activity was evaluated as a percentage of total radioactivity on plates by summing up the 14C in the 17 keto products i.e. androstanedione (band I) and androstenedione (band II). Values given as P moles of metabolites produced/mg f.w. 5-alpha-reductase activity was obtained in a similar way, by summing up the radioactivity present in androstanedione (band I). 5-alpha dihydrotestosterone (band III) and androstanediols (band V). Also in this case activity was expressed as pmoles of products/mg f.w. Statistical evaluation of results was made with the analysis of variance, "F'" test and "Tukey's" test.
Results H i s t o l o g i c e x a m i n a t i o n of dorsal skin of the 2 g r o u p s of s t u d i e d rats did n o t reveal, in a g r e e m e n t w i t h p r e v i o u s o b s e r v a t i o n s [2] a p p r e c i a b l e d i f f e r e n c e in the l e n g t h of h a i r - c y c l e phases. B o d y - w e i g h t of t h e two g r o u p s of animals was n o t statistically d i f f e r e n t until p u b e r t y after w h i c h the smaller w e i g h t of c a s t r a t e d rats b e c a m e highly significant ( T a b l e 1). T h e activity of 1 7 - b e t a - O H S D H and 5 - a l p h a - r e d u c t a s e in dorsal skin, p l a n t a r skin and p r e p u t i a l glands was as follows:
Table 1. Body weight Puberty Days of life Normal rats
32nd 113.3
Adulthood 37th 103.3
+_1.17
_+50.20
Castrated 95.0 rats +_12.42
+_31.26
101.7
42nd
50th
181.7
226.7
+_31.26 _+68.42
171.7
180.0
+__25.86 +-24.84
Statistical analysis "E" test 55th
60th
221.7
256.7
+_14.34
+_37.95
210.0
226.7
+-21.52
+-37.95
Groups of animals P 0.050
Days of life
< 0.001
Interaction
> 0.050
32nd vs 37th 37th vs 42nd 32nd vs 42nd 42nd vs 50th 50th vs 55th 55th vs 60th
Probability
Slope
< < < < > >
1'
0.001 0.001 0.020 0.001 0.050 0.050
~,
]" = Ascending values; + = Descending values; ~ = Stable values
Table 7. 17-beta-OHSDH activity in plantar skin
Days of life
Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
2.22 +_2.108
1.22 +_0.382
Groups of animals Days of life
P > 0.05 P > 0.05
Castrated rats
1.91 +_2.224
1.77 +_0.220
Interaction
P < 0.05
Values are given as pmoles of 17 keto-metabolites of T/mg f.w. __+95% confidence interval
b) P l a n t a r S k i n I n b o t h g r o u p s of a n i m a l s 1 7 - b e t a - O H S D H a n d 5 - a l p h a - r e d u c t a s e - a c t i v i t i e s d e creased with time though only significantly over 5-alpha-reductase. T a b l e 7 a n d 8 give t h e m e a n s of v a l u e s of t h e s e e n z y m e s , t h e i r c o n f i d e n c e i n t e r v a l s a n d t h e s t a t i s t i c a l analysis.
Metabolism of Testosterone 4-i4C in Skin
271
Table 8.5-alpha-reductase activity in plantar skin Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
12.73 +_2.001
10.86 +_1.483
Groups of animals Days of life
P > 0.05 P < 0.05
Castrated rats
12.80 +_4.399
11.80 +_0.756
Interaction
P > 0.05
Days of life
Values are given as pmoles of 5-alpha-reduced metabolites of T/mg f.w. + 95 % confidence interval
Table 9, 17-beta-OHSDH activity in preputial glands
Days of life
Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
7.84 +_7.136
5.04 +_2.608
Groups of animals Days of life
P < 0.10 P > 0.05
Castrated rats
9.08 +-6.276
9.32 +-8.105
Interaction
P > 0.05
Values are given as pmoles of 17 keto-metabolites of T/rag f.w. + 95 % confidence interval
Table 10. 5-alpha-reductase activity in preputial glands Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
114.86 +_56.380
74.76 +_11.048
Groups of animals Days of life
P < 0.01 P < 0.05
Castrated rats
135.75 +_11.195
125.75 +__53.019
Interaction
P > 0.05
Days of life
Values are given as pmoles of 5-alpha-reduced metabolites of T/mg f.w. _+95 % confidence interval
c) Preputial Glands
In these sebaceous glands the activity of 1 7 - b e t a - O H S D H was constant with time though always higher in castrated than in normal rats. 5-alpha-reductase activity significantly decreased with time in both groups; on days of puberty and adulthood, however, it increased, to a higly significant level in castrated rats only. Table 9 and 10 report the means of values of 1 7 - b e t a - O H S D H and 5-alpha-reductase, their confidence intervals and the statistical analysis.
Discussion Factors regulating the peripheral metabolism of steroid hormones are complex. A factor probably controlling the periphery enzymatic processes of steroid metabolism
272
E. Rampini et al.
might be constituted by testosterone itself, which as an enzymatic substrate could act as a stimulator as well as an inhibitor of its metabolizing enzymes. The lack of testosterone in castrated rats could, thus, be responsible for the increase of 17-beta-OHSDH-activity in the preputian glands (Table 10), liver [1], adrenals [6] and contemporaneously for its reduction in the prostate [17]. A n o t h e r factor is hypophysis. Prolactin, f. i. a hypophyseal increation, notoriously inhibits steroid enzymes in target-tissue of rats 5-alpha-reductase activity in adrenals [14] and the reductase of the 20 ketone of progesterone in the corpus luteum [13]. Our results have demonstrated that two conditions, characterized by an absence or scarsity of prolactin in the blood, prepuberal castration and prepuberal hypophysectomy, in rat-dorsal skin habe the same enzymatic behaviour i. e the activity of 1 7 - b e t a - O H S D H increases notably; that of 5-alpha-reductase remains unchanged (Fig. 1 and 2) [11]. The absence of scarsity of prolactin in the blood of these endocrine mutilated animals can, therefore, be responsible for the lack of inhibition on 1 7 - b e t a - O H D H activities. A further indication of the possibly regulatory role of pituitarian incretions on the skin enzymes involved in the metabolism of steroid hormones is given by puberty: this m o m e n t in life, in fact is crucial in establishing differences in the activity levels of 1 7 - b e t a - O H S D H (Fig. 1). In conclusion, our own and literature's data suggest that, besides the stimulatory or inhibitory effects of a substrate such as testosterone on its metabolizing enzymes, the role of hypophysis may go well beyond the control of steroidogenesis in adrenals and gonads to extend to the extreme body periphery, the skin f.i. and there condition the course of steroid metabolism.
References 1. Bullock, L. P., Bardin, C. W., Gram, T. E., Schroeder, D. H., Gillette, J. R.: Hepatic ethylmorphine demethylase and A4-Steroid Reductase in the androgen-insensitive pseudoherm aphroditic rat. Endocrinology 88, 1521-1523 (1971) 2. Dieke, S. H.: The effect of removing various endocrine glands on the hair cycles of black rats. Endocrinology 42, 315-319 (1948) 3. Farris, E. J., Griffith, J. Q.: The rat in laboratory investigation, p. 3. New York and London: Hafner Publishing Company 1967 4. Gomez, E. C., Hsia, S. L.: In vitro metabolism of testosterone-4-14C and A4-androstene-3, 17-dione-4D4C in human skin. Biochemistry 7, 24-32 (1968) 5. Grindeland, R. E., McCulloch, W. A., Ellis, S.: Meeting of the Endocrine Society, 1969, Abstract, p. 44 6. Kitay, J. I.: Functions of the adrenal cortex. (K. W. McKerns, ed.) Vol. 2, p. 755. New York: Appleton 1968 7. Lierena, L. A., Molina, A., Pearson, O. H.: Meeting of the Endocrine Society, 1969, Abstract, p. 45 8. Rampini, E., Davis, B. P., Moretti, G., Hsia, S. L.: Cyclic changes in the metabolism of estradiol by rat skin during the hair cycle. J. invest. Derm. 57, 75-80 (1971) 9. Rampini, E., Voigt, W., Davis, B. P., Moretti, G., Hsia, S. L.: Metabolism of testosterone-4-14C by rat skin: Variations during the hair cycle. Endocrinology 89, 1506-1514 (1971) 10. Rampini, E., Moretti, G.: Cutaneous metabolism of estradiol and testosterone in the newborn rat till puberty. Minerva Dermat. 1, 49-53 (1973)
Metabolism of Testosterone 4-14C in Skin
273
11. Rampini, E., Gambini, C., Moretti, G.0 Cardo, P., Cipriani, C.: L'attivit/i della 17-beta-OH steroido deidrogenasi e della 5-alpha-steroido reduttasi nella cute del ratto ipofisectomizzato. Min. Dermat. 111, 74-82 (1976) 12. Shimazaki, J., Sato, J., Nagai, H., Shida, K.: Effects of inhibitors of nucleic acids and protein biosynthesis on the rate of 5-alpha-reduction of testosterone, the activity of DNA polymerase and nucleic acid contents of testosterone-stimulated prostate of rats. Endocrin. (Jap.) 17, 175-179 (1970) 13. Wiest, W. G., Kidwell, W. R.: In: K. W. McKerns, The Gonads, p. 295. New York: AppletonCentury-Crofts 1969 14. Witorsch, R. J., Kitay, J. I.: Pituitary hormones affecting adrenal 5-alpha-reductase activity: ACTH, growth hormone and prolactin. Endocrinology 91, 764-769 (1972) Received June 22, 1976
Arch. Derm. Res. 256, 265-273 (1976)
e e s e r m a t o l o g i c a l a r c n
© by Springer-Verlag 1976
Metabolism of TestOsterone 4- 4C in Skin of Castrated Rats* E. Rampini, P. Cardo, Carla Cipriani, and G. Moretti Department of Dermatology (Head: Prof. Dr. G. Moretti) University of Genoa, I- 16132 Genoa, Italy
Summary.Testosterone metabolism was studied in the skin and preputial glands of normal and prepuberally castrated male rats during the 2nd hair cycle. In catagen-telogen the 17-beta-OHSDH of dorsal skin was higher in castrated than in control animals; 5-alpha-reductase instead, remained unchanged through out the hair cycle. Also in the preputial glands of castrated rats 17-beta-OHSDH was higher than in normal rats. So was 5-alpha-reductase. A possible direct control of a substrate, like Testosterone, and/or of a hypophyseal tropin, like prolactin, on enzymes that direct the metabolism of steroids in target tissues is conceivable. Zusammenfassung. Die Autoren studierten den Testosteron-Metabolismus der Haut und der Prfiputialdrfisen yon normalen m/innlichen Ratten, die in der Vorpubertfit wiihrend des zweiten Haarzyklus kastriert wurden. Im Katagen- und im Telogen-Stadium war die 17-beta-Hydroxysteroid-Dehydrogenase der Riickhaut in den kastrierten Ratten h6her als in den normalen Ratten; dagegen blieb die 5-alpha-Reductase w~ihrend des ganzen Haarzyklus unver~indert. Auch in den Pr~iputialdrfisen der kastrierten Ratten waren die 17-beta-Hydroxysteroid-Dehydrogenase und die 5-alpha-Reductase h6her als in den normalen Ratten. Man daft annehmen, dab eine direkte Kontrolle eines Substrates - Testosteron und/oder eines pituitfiren Tropin als Prolactin - fiber Enzyme, die ffir den Metabolismus der Steroide im ,,Target"-Zielgewebe verantwortlich sind, denkbar ist. Introduction Estradiol and testosterone metabolization in hairy dorsal rat skin is cyclic; 17-betahydroxysteroid dehydrogenase activity (17-beta-OHSDH is maximal in anagen; * Presented at the Joint Meeting of the Society for Investigative Dermatology and of the European Society for Dermatological Research, Amsterdam, June 1975.
266
E. Rampini et al.
5-alpha-steroid reductase activity increases remarkably in telogen [8, 9]. The activity of these enzymes, however, is probably conditioned by a variety of factors, apart from the hair cycle. 1 7 - b e t a - O H S D H and 5-alpha-reductase activity in rats f.i. is influenced by age since during the period b e t w e e n birth and 90 days of age, (puberty occurs in rats within the second m o n t h of life) both enzymes tend to diminish with progression of cycles [10]. Hypophysectomy, furthermore, markedly increases the activity of 17-betaO H S D H in the rat's skin whereas that of 5-alpha-reductase remains unchanged [11]. Does this m e a n that in the absence of the pituitary a directly repressive effect on 1 7 - b e t a - O H S D H may be lacking as already shown for prolactin on other enzymes of steroid metabolfsm [13, 14]? 1 7 - b e t a - O H S D H and 5-alpha-reductase activities of skin and preputial glands were consequently studied in castrated rats, since castration substantially reduces the production of prolactin a m o n g tropins [5,7].
Materials and Methods
Chemicals. NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and reference steroids were from Sigma Chemical Co. Testosterone-4-~C (specific activity 40 ~tC/p.mole) was purchased from Amersham and a stock solution in ethanol was prepared and stored in a refrigerator. Solvents and chemicals were of reagent grade. Chloroform for chromatography was redistilled before use and stabilized by 0.75 % of ethanol. Silica Gel H was obtained from Merck AG Co. Skin Specimens Specimens of hairy (dorsal) and glabrous (plantar) skin and the preputial glands were obtained from Wistar 36 male rats, which were either castrated before puberty on the 18th day of life or were intact litter mate controls. Dorsal skin, in particular, was taken on several days of the second hair cycle induced by plucking i.e.: on the 32nd, 37th, 42th day of life (anagen) when hairs were in anagen and animals in the puberal period, which in male rats ends around the 45th day [3]; on the 50th, 55th and 60th days of life when hairs are in Catagen-telogen and rats have reached adulthood [3]. The length of hair cycle was controlled in the two groups of animals by histology (H.E. stain of dorsal skin) and the general bodily effect of castration by recording the weight of the animals. On each of the specified days, three castrated and three normal rats were killed by decapitation and skin specimens were obtained from the supracaudal region. The panniculus carnosous was removed with a scalpel and 300 mg of skin were minced for incubation with testosterone-4-a4C. On the 37th and 55th day 150 mg of plantar skin and pairs of preputial glands were also taken from the same controls and castrated animals and incubated with testosterone-4-14C.
Incuba~on Each tissue sample (300 mg dorsal skin; 150 mg plantar skin; 40-80 mg preputial glands) was finely minced with scissors and suspended in 5 ml of KrebsrRinger phosphate buffer pH 7.4 containing a NADPH generating system (1 ~tmoleNADP; 25 ~tmoles glucose-6-phosphate, 1 unit of glucose-6phosphate dehydrogenase). Incubations were initiated with the addition of 5 x 105 dpm of testosterone-4-14C (5.6 m~moles) in 0.1ml of ethanol and carried out at 37 ° C in a shaking incubator.
Extraction and Separation of Steroids Incubation time was 30 rain since previous determinations of the progress of testosterone metabolism in dorsal skin during anagen, catagen and telogen had shown that at that time reaction was still linear [9].
Metabolism of Testosterone 4-14Cin Skin
267
Incubations were terminated by adding 20 ml methanol/dichlorometane 2 : 1. This single-phase mixture was filtered and the filtrate evaporated under vacuum. The acqueous residue was transferred to a separatory funnel with an additional 10 ml of water and extracted three times with 20 ml dichloromethane. Extracts were dried by passing through anhydrous sodium sulfate. Radioactivity in the extracts was assayed by counting an aliquot in a Packard Tricarb liquid scintillation spectometer model 3320 with 80 % counting efficiency. The scintillation fluid contained 14 g PPO and 0.7 g dimethyl POPOP in 2 1 of toluene. I4C recovery was always more than 85 %. Extracts were concentrated under nitrogen flow and residues were applied to thin layer plates (5 x 40 cm) prepared with silica gel H 0.3 mm thick and activated at 80 ° C for 1 h. The plates were developed by ascending chromatography in system chloroform-methanol 99 : 1 [4]. Distribution of 14C on the plates was located by a Packard 7201 autoscanner. Each of the five radioactive bands was scraped off the plates, eluted with methanol, and an aliquot was radio assayed by scintillation counting. Radioactive metabolites in each band were identified by co-chromatography with reference steroids, chromatography in different systems and isotopic dilution already employed in previous work [9] and in current use in our laboratories. The 17-beta-hydroxysteroid dehydrogenase activity was evaluated as a percentage of total radioactivity on plates by summing up the 14C in the 17 keto products i.e. androstanedione (band I) and androstenedione (band II). Values given as P moles of metabolites produced/mg f.w. 5-alpha-reductase activity was obtained in a similar way, by summing up the radioactivity present in androstanedione (band I). 5-alpha dihydrotestosterone (band III) and androstanediols (band V). Also in this case activity was expressed as pmoles of products/mg f.w. Statistical evaluation of results was made with the analysis of variance, "F'" test and "Tukey's" test.
Results H i s t o l o g i c e x a m i n a t i o n of dorsal skin of the 2 g r o u p s of s t u d i e d rats did n o t reveal, in a g r e e m e n t w i t h p r e v i o u s o b s e r v a t i o n s [2] a p p r e c i a b l e d i f f e r e n c e in the l e n g t h of h a i r - c y c l e phases. B o d y - w e i g h t of t h e two g r o u p s of animals was n o t statistically d i f f e r e n t until p u b e r t y after w h i c h the smaller w e i g h t of c a s t r a t e d rats b e c a m e highly significant ( T a b l e 1). T h e activity of 1 7 - b e t a - O H S D H and 5 - a l p h a - r e d u c t a s e in dorsal skin, p l a n t a r skin and p r e p u t i a l glands was as follows:
Table 1. Body weight Puberty Days of life Normal rats
32nd 113.3
Adulthood 37th 103.3
+_1.17
_+50.20
Castrated 95.0 rats +_12.42
+_31.26
101.7
42nd
50th
181.7
226.7
+_31.26 _+68.42
171.7
180.0
+__25.86 +-24.84
Statistical analysis "E" test 55th
60th
221.7
256.7
+_14.34
+_37.95
210.0
226.7
+-21.52
+-37.95
Groups of animals P 0.050
Days of life
< 0.001
Interaction
> 0.050
32nd vs 37th 37th vs 42nd 32nd vs 42nd 42nd vs 50th 50th vs 55th 55th vs 60th
Probability
Slope
< < < < > >
1'
0.001 0.001 0.020 0.001 0.050 0.050
~,
]" = Ascending values; + = Descending values; ~ = Stable values
Table 7. 17-beta-OHSDH activity in plantar skin
Days of life
Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
2.22 +_2.108
1.22 +_0.382
Groups of animals Days of life
P > 0.05 P > 0.05
Castrated rats
1.91 +_2.224
1.77 +_0.220
Interaction
P < 0.05
Values are given as pmoles of 17 keto-metabolites of T/mg f.w. __+95% confidence interval
b) P l a n t a r S k i n I n b o t h g r o u p s of a n i m a l s 1 7 - b e t a - O H S D H a n d 5 - a l p h a - r e d u c t a s e - a c t i v i t i e s d e creased with time though only significantly over 5-alpha-reductase. T a b l e 7 a n d 8 give t h e m e a n s of v a l u e s of t h e s e e n z y m e s , t h e i r c o n f i d e n c e i n t e r v a l s a n d t h e s t a t i s t i c a l analysis.
Metabolism of Testosterone 4-i4C in Skin
271
Table 8.5-alpha-reductase activity in plantar skin Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
12.73 +_2.001
10.86 +_1.483
Groups of animals Days of life
P > 0.05 P < 0.05
Castrated rats
12.80 +_4.399
11.80 +_0.756
Interaction
P > 0.05
Days of life
Values are given as pmoles of 5-alpha-reduced metabolites of T/mg f.w. + 95 % confidence interval
Table 9, 17-beta-OHSDH activity in preputial glands
Days of life
Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
7.84 +_7.136
5.04 +_2.608
Groups of animals Days of life
P < 0.10 P > 0.05
Castrated rats
9.08 +-6.276
9.32 +-8.105
Interaction
P > 0.05
Values are given as pmoles of 17 keto-metabolites of T/rag f.w. + 95 % confidence interval
Table 10. 5-alpha-reductase activity in preputial glands Puberty 37th
Adulthood 55th
Statistical analysis "F" test
Normal rats
114.86 +_56.380
74.76 +_11.048
Groups of animals Days of life
P < 0.01 P < 0.05
Castrated rats
135.75 +_11.195
125.75 +__53.019
Interaction
P > 0.05
Days of life
Values are given as pmoles of 5-alpha-reduced metabolites of T/mg f.w. _+95 % confidence interval
c) Preputial Glands
In these sebaceous glands the activity of 1 7 - b e t a - O H S D H was constant with time though always higher in castrated than in normal rats. 5-alpha-reductase activity significantly decreased with time in both groups; on days of puberty and adulthood, however, it increased, to a higly significant level in castrated rats only. Table 9 and 10 report the means of values of 1 7 - b e t a - O H S D H and 5-alpha-reductase, their confidence intervals and the statistical analysis.
Discussion Factors regulating the peripheral metabolism of steroid hormones are complex. A factor probably controlling the periphery enzymatic processes of steroid metabolism
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might be constituted by testosterone itself, which as an enzymatic substrate could act as a stimulator as well as an inhibitor of its metabolizing enzymes. The lack of testosterone in castrated rats could, thus, be responsible for the increase of 17-beta-OHSDH-activity in the preputian glands (Table 10), liver [1], adrenals [6] and contemporaneously for its reduction in the prostate [17]. A n o t h e r factor is hypophysis. Prolactin, f. i. a hypophyseal increation, notoriously inhibits steroid enzymes in target-tissue of rats 5-alpha-reductase activity in adrenals [14] and the reductase of the 20 ketone of progesterone in the corpus luteum [13]. Our results have demonstrated that two conditions, characterized by an absence or scarsity of prolactin in the blood, prepuberal castration and prepuberal hypophysectomy, in rat-dorsal skin habe the same enzymatic behaviour i. e the activity of 1 7 - b e t a - O H S D H increases notably; that of 5-alpha-reductase remains unchanged (Fig. 1 and 2) [11]. The absence of scarsity of prolactin in the blood of these endocrine mutilated animals can, therefore, be responsible for the lack of inhibition on 1 7 - b e t a - O H D H activities. A further indication of the possibly regulatory role of pituitarian incretions on the skin enzymes involved in the metabolism of steroid hormones is given by puberty: this m o m e n t in life, in fact is crucial in establishing differences in the activity levels of 1 7 - b e t a - O H S D H (Fig. 1). In conclusion, our own and literature's data suggest that, besides the stimulatory or inhibitory effects of a substrate such as testosterone on its metabolizing enzymes, the role of hypophysis may go well beyond the control of steroidogenesis in adrenals and gonads to extend to the extreme body periphery, the skin f.i. and there condition the course of steroid metabolism.
References 1. Bullock, L. P., Bardin, C. W., Gram, T. E., Schroeder, D. H., Gillette, J. R.: Hepatic ethylmorphine demethylase and A4-Steroid Reductase in the androgen-insensitive pseudoherm aphroditic rat. Endocrinology 88, 1521-1523 (1971) 2. Dieke, S. H.: The effect of removing various endocrine glands on the hair cycles of black rats. Endocrinology 42, 315-319 (1948) 3. Farris, E. J., Griffith, J. Q.: The rat in laboratory investigation, p. 3. New York and London: Hafner Publishing Company 1967 4. Gomez, E. C., Hsia, S. L.: In vitro metabolism of testosterone-4-14C and A4-androstene-3, 17-dione-4D4C in human skin. Biochemistry 7, 24-32 (1968) 5. Grindeland, R. E., McCulloch, W. A., Ellis, S.: Meeting of the Endocrine Society, 1969, Abstract, p. 44 6. Kitay, J. I.: Functions of the adrenal cortex. (K. W. McKerns, ed.) Vol. 2, p. 755. New York: Appleton 1968 7. Lierena, L. A., Molina, A., Pearson, O. H.: Meeting of the Endocrine Society, 1969, Abstract, p. 45 8. Rampini, E., Davis, B. P., Moretti, G., Hsia, S. L.: Cyclic changes in the metabolism of estradiol by rat skin during the hair cycle. J. invest. Derm. 57, 75-80 (1971) 9. Rampini, E., Voigt, W., Davis, B. P., Moretti, G., Hsia, S. L.: Metabolism of testosterone-4-14C by rat skin: Variations during the hair cycle. Endocrinology 89, 1506-1514 (1971) 10. Rampini, E., Moretti, G.: Cutaneous metabolism of estradiol and testosterone in the newborn rat till puberty. Minerva Dermat. 1, 49-53 (1973)
Metabolism of Testosterone 4-14C in Skin
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11. Rampini, E., Gambini, C., Moretti, G.0 Cardo, P., Cipriani, C.: L'attivit/i della 17-beta-OH steroido deidrogenasi e della 5-alpha-steroido reduttasi nella cute del ratto ipofisectomizzato. Min. Dermat. 111, 74-82 (1976) 12. Shimazaki, J., Sato, J., Nagai, H., Shida, K.: Effects of inhibitors of nucleic acids and protein biosynthesis on the rate of 5-alpha-reduction of testosterone, the activity of DNA polymerase and nucleic acid contents of testosterone-stimulated prostate of rats. Endocrin. (Jap.) 17, 175-179 (1970) 13. Wiest, W. G., Kidwell, W. R.: In: K. W. McKerns, The Gonads, p. 295. New York: AppletonCentury-Crofts 1969 14. Witorsch, R. J., Kitay, J. I.: Pituitary hormones affecting adrenal 5-alpha-reductase activity: ACTH, growth hormone and prolactin. Endocrinology 91, 764-769 (1972) Received June 22, 1976
