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5α-steroid reductase activity in tissues from rats with end organ insensitivity to testosterone
Vol. 9, Part I, pp. 701-705, 1970. Life Sciences Printed in Great Britain
Pergamon Press
5a-STEROID RSDUCTASS ACTIVITY IN TISSUES FROM RATS WITH SND ORGAN INSENSITIVITY TO TESTOSTERONE Leslie Bullock, George Schneider, C. Wayna Bardia Endocrinology Branch, National Cancer Institute,
Bethesda, liaryland
(Received 28 October 1989; in final form 2 April 1970) Stanley~umbreck pseudohermaphrodite (Pe) rate are phenotypic females but genetic and goaadal melee (1) .
The lack of androgen dependent dif-
ferentiation ie due to sad organ insensitivity to testosteronel
(2) and
these animals represent the rodent counterpart to a syndrome is maa-testicular feminization (3) .
Recent studies of patients with testicular
feminization have demonstrated decreased Sa-steroid reductase activity and, ae a result, reduced metabolism of testosterone to 5a-dihydrotestoeterone (DHT)(4,5,6), a proposed active intracellular product of testosterone (7,8) .
These observations suggested that the androgen insensitivity in
patients with testicular fe~inization was due to defective DST formation . It was thus pertinent to study 5a-steroid reductase activity and DHT formation in the tissues of the Stanley-Gumbreck pseudohermaphrodite rat in an attempt to eaplain the testosterone insensitivity in these animals. Materials and Methods Duplicate samples of dorsal akin and preputial glands from male, female and Pa rate were studied.
One gram of akin from a single animal or
120-230 mg preputial gland from several animals were minced with iris scissors and added to incubation flasks containing 5 ml Rrebs-Ringer phosphate buffer, pH7 .4, 12 yMol . glucose-6-phosphate,
2 Roraberg unite
The following trivial vanes are used : testosterone ~ androst-4-en-17ß01-3-one ; dihydrotaetosterone - 5a-androetan-l7ß-ol-3-one ; androstenadione aadroet-4-en-3, 17-thons ; androsterone ~ 5a-androetan-3a-o1-17-one. 701
702
5 d-BTEROtD REDUCTASE ACTIVITY
glucose-6-phosphate debydrogenaae, (specific activity 50 mCi/mKol .)
Vol . 9, No. 12
3 uMnl . NADP, 8 muMol . testosterone-4- 14 0,
500 u panicillia and 500 ug streptomycin .
Tissues were incubated under 02-002
(95 :5) for 4 hours.
Reactions were ter-
urinated by addition of 12 ml methanol and Frozen until analyzed .
Prior to
analysis 50 ug of each of the following steroids were added to the individual flasks :
testosterone, DHT, and androaterone .
flask were thawed and filtered through glass wool .
The contents of each The filtrates were di
luted to 50 ml with water a~ eztracted twice with 2 volumes of ether .
The
eatracte were washed with 0 .1 N Na0H,.6x acetic acid and water, dried, and chromatographed on Whatman 1 urn paper in Ligroin :Mathanol :Water
(10:7 :3) for
18 hours eaparatiag testosterone and 5a-androstan-3a,17ß-diol from other metabolites.
The rva off . from this chromatography was collected,
dried, and
rechramatographed in the same egstem for 9 hours resolving dihydrotestoeterone and aadrostenedione .
The associated rue off was rerun for 4 hours to isolate
androeterone and Sa-androatan-3,17-dione.
Standard reference eteroide were
located with UV light, alcoholic sulfuric acid, or m-dinitro beazene in alcoholic benzyltrimethyl ammonium hydrozide. Gated with a Nuclear Chicago Actigraph III.
Radioactive peaks were 1oThe eteroide listed in Table I
were further identified by thin layer chromatography and crystallization to constaat specific activity .
Radioactivity in testosterone, DHT and andros-
teroae was corrected for losses by marker steroid recovery with gee-liquid chromatography .
Recovery of other steroids was estimated from the radio-
active peaks on the paper chromatograms . Results Testosterone metabolism to DHT and other 5a-steroids was similar in normal and Ps akin and preputial glands . given in Table I.
Results of a typical study are
Ia addition, androstenedione was tentatively identified
as a metabolite, but was not crystallized .
Two other studies, is which akin
and preputial gland were incubated under slightly different conditions, gave
Vol. 9, No. 12
5 aC-3TEROII~ REDUCTA3E ACTIVITY
qualitatively similar results to those in Table I.
709
Thus, in the Ps rat
a
defective 5a-steroid reductase was not detected which would distinguish these animals from normal litter mates. TABLE I 5a-Metabolites of Testosterone after Incubation with Tiesues from lisle, Female a~ Pseudohermaphrodite (Ps) Rats Steroid
Male
Skin Female
Ps
Testosterone+
49 47
46 46
5a-Androstan-l7ß-ol-3-one+ (Dihydrotestosterone)
10 7
5a-Androstan-3a-o1-17-one+ (pndrosterone)
z*
x
x
x
55 31
22 15
18 9
19 26
8 7
5 6
12 21
12 13
18 10
4 2
2 4
4 4
++
5 8
6 3
5a-Androstan-3a,17ß-diol
5 5
10 5
10 5
19 23
20 25
20 15
5a-Androstan-3,17dione
2 2
2 4
4 4
++
3 6
5 3
*
x
x
Preputial Gland Male Female Ps
Percent of initial testosterone-4- l4 C radioactivity recovered as testosterone and its metabolites iri duplicate 4 hr . incubations . The skin i.n each flask came from a different animal . Preputial glands from 2-3 animals were pooled for each flask. Details of the incubations are given in the teat . Corrected for losses by marker steroids . Not isolatedDiscussion There is considerable evidence which suggests that steroids act on the
nucleus of the call .
Ia studies of androgen responsive tissues such as
prostate (7,9) and duck preen gland (10), radioactivity localized in nuclei following 3H-testosterone administration .
Most of the proetatic intra-
nuclear radioactivity was not the injected steroid but DriT, a potéat androgen, which may be the effector of testosterone action (7,8,11) . Recent in vivo studies in patients with testicular feminization demonetrated reduced conversion of testosterone to several 5a-metabolites
(12) .
~al
5o(-STEROID RBDUCTA3E ACTIVITY
Vol. 9, No. 12
These observations, suggesting decreased 5a-reductaee activity, were corroborated by is vitro studies showing decreased metabolism of testosterone to DHT by akin slices (5,6,13) .
As a result of these studies, defective
DHT formation from testosterone was proposed as the basis for the androgen insensitivity in testicular femlaizAtion
(5,6,12) .
In the present study, mincae of skin and preputial gland from androgen insensitive Ps rate metabolized testosterone to DHT and 3 other 5a~metabolitea in the same proportions as tissues from normal rata .
On the basis of these
observations, a decrease in 5a-steroid reductase activity of the whole cell is probably not the ezplanation for the a~rogen insensitivity in Ps .
Hw-
ever, since 5a-steroid reductase ie normally present in 'both cytoplasm and nucleus,
it is possible that a deficiency of int~anuclear Sa-steroid reduc-
tale wsa undetected .
Aa alternative e~lanation of the androgen insensiti-
vity could be that intranualear binding of androgens ie decreased. In studies of praltata tissue culture radioactive testosterone was metabolized to two aadrogeaic metabolites:
Dâf wLich vas predominantly in
the nuc %i, and 5a-aldroatan-3ß,17ßdiol whic4 vea in the cytoplasm (14) . When these metabolites were added to prostatic culture, DHT stimulated cell growth and the dial promoted secretion (14) .
Recently, we haws isolated
5a-a~rostaa-3ß,17ßdiol froze both normal and Ps preputial gland incubations . Ackaarledgaent The authors ezprase appreciation to Amel French for technical assistance and to Dre. J. S. Allison, A. J. Stanley, and L. G. Gumbreck (Departments of Anatomy and Physiôlogy, IIaiveraity of Oklahoma liedical Center) for supplying the rats . References 1.
J . S. Allison, A. J . Stanley and L. G. Gumbreck, Gnat . Rec. 153, 85 (1965) .
Voi . 9, No. 12
706
6o(-gTERO1D REDUCTA3E ACTIVITY
2.
C. W. Bardia, L . Bullock and G. Schneider,
Science 167, 1136
(1970) .
3.
H. H. Suer, R. J. Pion and W. J . Dignam, Testicular Feaiaization . Thomas, Springfield, I11 . (1965) .
4.
P. Mauvais-Jarvie, J. P. Bercovici and F . Gauthier, J. Clin . Endo . 29, 417 (1969) .
5.
R. C. Northcutt, D . P. Island and G. W. Liddle, J . Clin . Endo . 29 , 422, (1969) .
6.
L. R. Heinrichs, Clin . Research 17, 143 (1969) . a
7.
N . Bruchovsky a~ J. D . Wilson, J . Biol . Chew . 243, 2012 (1968) .
8.
N. Bruchovsky and J. D . Wilson, J . Biol . Chem . 2~, 5953
9.
K. J. lbeter and A. Attramadal, Endo . _85, 350 (1969) .
(1968) .
10 .
J. D. Wilson and P . M. Loeb in Developmental and Metabolic Control Mechanisms , p . 375, M. D. Anderson Hospital and Tumor Institute (Fde) The Williams and Wilkins Co ., Baltimore, Md . (1965) .
11 .
&. M. Anderson and S . Lino, Nature 219, 277 (1968) .
12 .
P. Mauvais- Jarvie, H. H. Floch and J. P. Bercovici, J . Clin . Endo . 28, 460 (1968) .
13 .
J. D. Wilson and J. D. Wallcar, J. Clin . Invest . 48 , 371 (1969) .
14 .
E. Haulieu, Le Concours Medical, 91, 2181 (1969) .
Pergamon Press
5a-STEROID RSDUCTASS ACTIVITY IN TISSUES FROM RATS WITH SND ORGAN INSENSITIVITY TO TESTOSTERONE Leslie Bullock, George Schneider, C. Wayna Bardia Endocrinology Branch, National Cancer Institute,
Bethesda, liaryland
(Received 28 October 1989; in final form 2 April 1970) Stanley~umbreck pseudohermaphrodite (Pe) rate are phenotypic females but genetic and goaadal melee (1) .
The lack of androgen dependent dif-
ferentiation ie due to sad organ insensitivity to testosteronel
(2) and
these animals represent the rodent counterpart to a syndrome is maa-testicular feminization (3) .
Recent studies of patients with testicular
feminization have demonstrated decreased Sa-steroid reductase activity and, ae a result, reduced metabolism of testosterone to 5a-dihydrotestoeterone (DHT)(4,5,6), a proposed active intracellular product of testosterone (7,8) .
These observations suggested that the androgen insensitivity in
patients with testicular fe~inization was due to defective DST formation . It was thus pertinent to study 5a-steroid reductase activity and DHT formation in the tissues of the Stanley-Gumbreck pseudohermaphrodite rat in an attempt to eaplain the testosterone insensitivity in these animals. Materials and Methods Duplicate samples of dorsal akin and preputial glands from male, female and Pa rate were studied.
One gram of akin from a single animal or
120-230 mg preputial gland from several animals were minced with iris scissors and added to incubation flasks containing 5 ml Rrebs-Ringer phosphate buffer, pH7 .4, 12 yMol . glucose-6-phosphate,
2 Roraberg unite
The following trivial vanes are used : testosterone ~ androst-4-en-17ß01-3-one ; dihydrotaetosterone - 5a-androetan-l7ß-ol-3-one ; androstenadione aadroet-4-en-3, 17-thons ; androsterone ~ 5a-androetan-3a-o1-17-one. 701
702
5 d-BTEROtD REDUCTASE ACTIVITY
glucose-6-phosphate debydrogenaae, (specific activity 50 mCi/mKol .)
Vol . 9, No. 12
3 uMnl . NADP, 8 muMol . testosterone-4- 14 0,
500 u panicillia and 500 ug streptomycin .
Tissues were incubated under 02-002
(95 :5) for 4 hours.
Reactions were ter-
urinated by addition of 12 ml methanol and Frozen until analyzed .
Prior to
analysis 50 ug of each of the following steroids were added to the individual flasks :
testosterone, DHT, and androaterone .
flask were thawed and filtered through glass wool .
The contents of each The filtrates were di
luted to 50 ml with water a~ eztracted twice with 2 volumes of ether .
The
eatracte were washed with 0 .1 N Na0H,.6x acetic acid and water, dried, and chromatographed on Whatman 1 urn paper in Ligroin :Mathanol :Water
(10:7 :3) for
18 hours eaparatiag testosterone and 5a-androstan-3a,17ß-diol from other metabolites.
The rva off . from this chromatography was collected,
dried, and
rechramatographed in the same egstem for 9 hours resolving dihydrotestoeterone and aadrostenedione .
The associated rue off was rerun for 4 hours to isolate
androeterone and Sa-androatan-3,17-dione.
Standard reference eteroide were
located with UV light, alcoholic sulfuric acid, or m-dinitro beazene in alcoholic benzyltrimethyl ammonium hydrozide. Gated with a Nuclear Chicago Actigraph III.
Radioactive peaks were 1oThe eteroide listed in Table I
were further identified by thin layer chromatography and crystallization to constaat specific activity .
Radioactivity in testosterone, DHT and andros-
teroae was corrected for losses by marker steroid recovery with gee-liquid chromatography .
Recovery of other steroids was estimated from the radio-
active peaks on the paper chromatograms . Results Testosterone metabolism to DHT and other 5a-steroids was similar in normal and Ps akin and preputial glands . given in Table I.
Results of a typical study are
Ia addition, androstenedione was tentatively identified
as a metabolite, but was not crystallized .
Two other studies, is which akin
and preputial gland were incubated under slightly different conditions, gave
Vol. 9, No. 12
5 aC-3TEROII~ REDUCTA3E ACTIVITY
qualitatively similar results to those in Table I.
709
Thus, in the Ps rat
a
defective 5a-steroid reductase was not detected which would distinguish these animals from normal litter mates. TABLE I 5a-Metabolites of Testosterone after Incubation with Tiesues from lisle, Female a~ Pseudohermaphrodite (Ps) Rats Steroid
Male
Skin Female
Ps
Testosterone+
49 47
46 46
5a-Androstan-l7ß-ol-3-one+ (Dihydrotestosterone)
10 7
5a-Androstan-3a-o1-17-one+ (pndrosterone)
z*
x
x
x
55 31
22 15
18 9
19 26
8 7
5 6
12 21
12 13
18 10
4 2
2 4
4 4
++
5 8
6 3
5a-Androstan-3a,17ß-diol
5 5
10 5
10 5
19 23
20 25
20 15
5a-Androstan-3,17dione
2 2
2 4
4 4
++
3 6
5 3
*
x
x
Preputial Gland Male Female Ps
Percent of initial testosterone-4- l4 C radioactivity recovered as testosterone and its metabolites iri duplicate 4 hr . incubations . The skin i.n each flask came from a different animal . Preputial glands from 2-3 animals were pooled for each flask. Details of the incubations are given in the teat . Corrected for losses by marker steroids . Not isolatedDiscussion There is considerable evidence which suggests that steroids act on the
nucleus of the call .
Ia studies of androgen responsive tissues such as
prostate (7,9) and duck preen gland (10), radioactivity localized in nuclei following 3H-testosterone administration .
Most of the proetatic intra-
nuclear radioactivity was not the injected steroid but DriT, a potéat androgen, which may be the effector of testosterone action (7,8,11) . Recent in vivo studies in patients with testicular feminization demonetrated reduced conversion of testosterone to several 5a-metabolites
(12) .
~al
5o(-STEROID RBDUCTA3E ACTIVITY
Vol. 9, No. 12
These observations, suggesting decreased 5a-reductaee activity, were corroborated by is vitro studies showing decreased metabolism of testosterone to DHT by akin slices (5,6,13) .
As a result of these studies, defective
DHT formation from testosterone was proposed as the basis for the androgen insensitivity in testicular femlaizAtion
(5,6,12) .
In the present study, mincae of skin and preputial gland from androgen insensitive Ps rate metabolized testosterone to DHT and 3 other 5a~metabolitea in the same proportions as tissues from normal rata .
On the basis of these
observations, a decrease in 5a-steroid reductase activity of the whole cell is probably not the ezplanation for the a~rogen insensitivity in Ps .
Hw-
ever, since 5a-steroid reductase ie normally present in 'both cytoplasm and nucleus,
it is possible that a deficiency of int~anuclear Sa-steroid reduc-
tale wsa undetected .
Aa alternative e~lanation of the androgen insensiti-
vity could be that intranualear binding of androgens ie decreased. In studies of praltata tissue culture radioactive testosterone was metabolized to two aadrogeaic metabolites:
Dâf wLich vas predominantly in
the nuc %i, and 5a-aldroatan-3ß,17ßdiol whic4 vea in the cytoplasm (14) . When these metabolites were added to prostatic culture, DHT stimulated cell growth and the dial promoted secretion (14) .
Recently, we haws isolated
5a-a~rostaa-3ß,17ßdiol froze both normal and Ps preputial gland incubations . Ackaarledgaent The authors ezprase appreciation to Amel French for technical assistance and to Dre. J. S. Allison, A. J. Stanley, and L. G. Gumbreck (Departments of Anatomy and Physiôlogy, IIaiveraity of Oklahoma liedical Center) for supplying the rats . References 1.
J . S. Allison, A. J . Stanley and L. G. Gumbreck, Gnat . Rec. 153, 85 (1965) .
Voi . 9, No. 12
706
6o(-gTERO1D REDUCTA3E ACTIVITY
2.
C. W. Bardia, L . Bullock and G. Schneider,
Science 167, 1136
(1970) .
3.
H. H. Suer, R. J. Pion and W. J . Dignam, Testicular Feaiaization . Thomas, Springfield, I11 . (1965) .
4.
P. Mauvais-Jarvie, J. P. Bercovici and F . Gauthier, J. Clin . Endo . 29, 417 (1969) .
5.
R. C. Northcutt, D . P. Island and G. W. Liddle, J . Clin . Endo . 29 , 422, (1969) .
6.
L. R. Heinrichs, Clin . Research 17, 143 (1969) . a
7.
N . Bruchovsky a~ J. D . Wilson, J . Biol . Chew . 243, 2012 (1968) .
8.
N. Bruchovsky and J. D . Wilson, J . Biol . Chem . 2~, 5953
9.
K. J. lbeter and A. Attramadal, Endo . _85, 350 (1969) .
(1968) .
10 .
J. D. Wilson and P . M. Loeb in Developmental and Metabolic Control Mechanisms , p . 375, M. D. Anderson Hospital and Tumor Institute (Fde) The Williams and Wilkins Co ., Baltimore, Md . (1965) .
11 .
&. M. Anderson and S . Lino, Nature 219, 277 (1968) .
12 .
P. Mauvais- Jarvie, H. H. Floch and J. P. Bercovici, J . Clin . Endo . 28, 460 (1968) .
13 .
J. D. Wilson and J. D. Wallcar, J. Clin . Invest . 48 , 371 (1969) .
14 .
E. Haulieu, Le Concours Medical, 91, 2181 (1969) .