THE JounNAL m: I NVEST IC.ATJVE Ot::HMATOLOGY
Copy ri ght © 1972 "-"' Th e William s & Wil kins Co.
\ 'o\. 59. No. 4
Print ed in L'.S .A .
A POSSIBLE MECHANISM OF ACTION OF ESTROGEN AT THE CELL ULAR
LEVEL IN A MODEL SEBACEOUS GLAND *
GAIL SANSONE-BAZZ ANO, Ph.D., RONALD M. REISNER, M.D.
Ph .D. j"
AND
GAETANO BAZZANO, M .D.,
ABSTRA CT
The effect of estradiol a dministration a nd castra tio n on testosterone (T) up ta ke a nd
meta bolism in vitro in t wo sebaceous gland ana logues has been investigated . Uptake ofT
was dec reased in the preputial glands of both castrated and norma l male rats a nd cast rated
mice by estradiol adm inis trati on. Conversion ofT to dihydrotestoster one (O HT) was also
decrease d in the prepu t ia l glands of norma l rats by estradiol admin istrat ion .
Castrat ion a lone caused increased upta ke ofT and decreased convers ion ofT to DHT
when ca lculate d as percent of total uptake co mpa red to percentage conversion in gla nds of
normal anima ls. The demonst rat ion that estradiol suppresses T upta ke a nd metabolis m to
DHT in norma l male rats sugge ts a mechanism at the cellular level to expla in how est ra diol mi ght inhibit sebu~. production .
Current ev idence indicates that dihydrotestoste rone (OHT) may be t he cellula rly active a n drogen in severa l t issues, includin g t he prostate
an d such sebaceous gland homologues as the
preen gland (1) of the duck a nd the preput ial
gla nd of t h e rat (2) and poss ibly in huma n s kin
(3) . W e have recentl y demonstrated that the production of DHT from testoste ron e (T) is greate r
in acne bearing s kin t han in comparable no n-ac ne
bearing s kin (4). The enzym e 5-a lpha reductase
which converts T to DHT is a lso present in the
preputial glands of rodents whi ch ca n thus be
us ed as model sebaceou structures to study the
possib le influence of various age nts on T to DHT
convers ion.
Systemicall y administered t herape ut ic agents
used in the treatment of ac ne migh t poss ibl y act,
at least in part, by modifyin g the upta ke or metabolism of testosterone wit'hin the gland. Such
suppress ion of T upta ke or of T metabolis m
within the gland might in turn be a facto r respon sible for decreasing sebum production.
Since estrogen is the prototy pe agent fo r sup pression of sebum production and further, si nce
its mechanis m of action rema ins unknown , it
seemed worthwhil e to investigate the effect of
estrogen on T upta ke and metabolis m in the rat
preputial gland. The preputial gla nd is s imila r to
human sebaceous glands in its lipidizing responses to T and estrogen a nd vefY similar to
Received December 27, 1971 ; accepted for publication
May 6, 1972.
Supported in part by Grant AM 11164-04 from the
National Institute of Arthritis and Metabolic Diseases
a nd in part by a gra nt from Th e Elliot and Ru t h Han dler Foundat ion , and in part by a grant from The Max
Factor Memorial Fund.
* From the Division of Dermatology, Depa rtment of
Medicin e, Harbor General Hos pita l, T orrance, Ca li fornia 90509. Reprint requests to Docto r Sansone-Baz za no .
i" Section of Nutrition and Meta bolis m, Departm ent
of Internal Medicine a nd Biochemistry, St. Louis Un iversity, St. Louis, Missouri 63ll2.
299
human sebum m co mpos ition of the lipid it ex c re tes (5).
MATEHIALS AND METHODS
All rats (S prague -Dawley, 3 to 4 months old) were
purchased from Charl es Rive rs and were cast rated 1
wee k prior to initiat ion of the expe rim ent. All mi ce used
were between 5 and 6 weeks old ( Ha/1 R strai n) a nd
were castrated at 3 to 4 weeks of age.
Cast rated a nd norma l rats were divided into gro ups
of 5. Each group rece ived one of t he following regimes
for a 2-week period.
1. Estradiol (P rogy non)-0.25 mg per day sub cutaneously, or
2. Testosterone-0.3 mg every other day intravenously .
The normal and castrated control groups were in jected wit h sa li ne.
The castrated mice were also divided into groups of
5. Each group received 1 of t he fo ll ow in g t herapeuti c
regimes for a 2-week period:
1. Estradiol (Progynon)-100 mg daily sub cutaneousl y, or
2. T estoste rone-0.1 mg dail y subcuta neously.
The norm al and cas ! rated cont rol groups were in jected with sa line.
At the termination of the treatment period , each
group of anima ls was sac rifi ced and the preputial glands
were removed. weighed and immediately chill ed in
Krebs-Ringer buffer. The in cubation med ia co nsisted of
10 p c of testosterone- ! , 2'H (New England Nucl ear.
specific activity 1 mCi/6 mi cro gra ms) Krebs-Rin ge r
phosphate buffer, pH 7.4 and approxima tely 150 mg of
ra t preputia l gland t issue (7 - 10 mg of protein) per in cubation or app roxi mately 120 mg of mouse preputial
gland tiss ue (6- 8 mg of protein) per incubation.
The in cubation s were te rminated afte r 45 minut e .
the glands homoge ni zed. and a sma ll portion of the
homoge nate saved for protein determination. The remaind er wa s extracted by t he method of Bligh and Dyer
(6). The chl oroform ext ract ion co ntai nin g t he I ritiated
ste roid fra ction was ana lyzed and quantitated by thin layer and gas- liquid chromatographY: The co mpl ete
deta ils of methodol ogy for identifi cation and quantitat ion of steroids have been previously reported (4).
The amou nt of testoste rone taken up by the gland
TH E J OURNA L OF I NV ESTI GAT I VE D ERMATOLOGY
300
a nd t he a mou nt of DHT produ ced was ca lcul aled in Lh e
foll ow in g manner:
Afte r in cubati on, t he whole gla nd tiss ue was removed
from the in cubat ion medi um a nd both t iss ue an d med ium we re ext racted se pa rate ly a nd a na lyzed for rad io ac ti ve s t e roid meta bolites. T estoste rone within t he
gla nd a nd the metabo li tes form ed from testoste rone
wit hin the gland and th ose whi ch ha d diffused in to t he
medium were totaled a nd t hi s fi gure wa s repo rted as
testoste rone uptak e. Th e DHT prod uced was ca lculated
from that found within t he gla nd a nd that whi ch had
d iffused out and was in the in cubation media . Co nve rs ion ratio was th us ca lculated as DHT/ DHT + in t ragla ndu la r T + T meta boli tes other tha n DHT intra - a nd
extrag la ndul a r.
RESULT S
Resul ts of t he t hin -laye r a nd gas chromatogra phy were in close agreeme nt. Alt hough DHT
cou ld not be sepa rated from a ndrosterone by t hin layer chromatography, t he amo unt of ra dioac tivity in t he a ndroste ron e pea k was found to be
negligib le by gas chroma togra phi c a na lys is a nd it
was concl ud ed t hat th in -layer chromatog raphy
provided a dequate mea ns for routin e assays .
F igures 1 a nd 2 re prese nt zo na l sca n profi les of
t hin-laye r chromatograms of th e testosterone
1, 2 3 H metabo li tes found in huma n s kin a nd rat
preputi a l gland respectively.
Figure 3 prese nts data on t he effect of castrat io n a nd est ra diol a dmini s trat ion on testoste rone
l , 2 3 H upta ke a nd meta bolis m in the rat pre putia l
gla nd. The open bar re presents mea n upta ke a nd
t he shaded ba r represe nts mea n DHT production
in cpm/mg prote in. T he dotted li nes represent
sta nda rd dev ia tion.
In t he norma l (no n-cas trated) ma le ra t prepu -
tial gla nd th e mea n T upta ke was :2500 c pm/ m g
protein a nd th e mea n DHT production was 800
c pm/ mg prote in rep rese nt in g a mea n co nve rs ion
of 32% of t he T ta ken up to DHT.
Th e castrated no n-trea ted ra t preputia l gla nds
de monstrated mea n T upta ke of 5000 c pm/ mg
prote in . M ea n DHT production was 600 cp m/ mg
prote in , re present in g a mea n co nve rs ion of 12% of
the T taken up to DHT. It is im po rtant to note
that castra tion in c reased by a lmost two-fo ld t he
T uptake with no conco mi ta nt in c rease in DHT
produ ct ion. T to DHT convers ion in the castrated
mal e ra t was onl y 12% as co mpa red to 32% in the
normal non-cast ra ted rat pre putia l gla nd.
ln t he prep uti a l gla nds of non-castrated estradiol -trea ted ma le ra ts, T upta ke was suppressed
from 2500 cpm/ mg prote in to 1500 c pm / mg prote in , a nd t he mean DHT producti on was dec reased from 500 cpm/mg protein to a pproximatel y 170 c pm / mg prote in , re present in g a mean
co nv ers ion of 11 % of th e T tak en up to DHT .
lt is ev iden t that both upta ke a nd convers ion
were s ignifi ca ntl y dec reased by estra di ol a dministrat ion in t he norma l non-castrated ma le rat
preputia l gla nd . Preputia l gla nds of castrated
ma le rats t rea ted with estradiol a lso d emonstrated a decreased upta ke ofT wh en compared
with cas trated ma le rats not treated with estradiol. However, DHT prod uction was not s ignifi ca ntl y a lte red .
Fi gure 4 presents data on th e effec t ofT a nd
estrad iol a dminis tration on T up ta ke a nd T to
DHT co nvers ion in t he preputi a l gla nds of castrated ma le mi ce. Results s imila r to those foun d
in the preputi a l gla nds of castrated ma le rats
were obta in ed. Th e major diffe rence obse rved is
T ESTOSTERONE METABOLITES in HUMAN SKIN
w
40, 000
z
1') ,000
0
w
f(f)w
[t
9 .000
B,O OO
oz
f-O
7.0 0'J
(/)[t
ww
6,('00 ·
f- t-
w
f-
::>
z
::';'
ll::
4,000
w
n.
3,000
(/)
2 ,0 (JU
z
::>
0
(.)
0
w
0
z
~(f)
1,0 00
z
({)
0
({)
[t
0
0
1-
0
<!
w
<!
0
[t
0
({)
z
<!
f-
z
0
w
({)
[t
600
z
~
f-
f-
0
BOO
Q
~J
0 <!
[t
z
i5
0
>-o
Jz
z
0
f-
cr: o
Ocr:
w
...J
w
z
w
0({)
~.ooo
z
400
200
100
0
A.__,..J'
/""..._
... ....r""""""..,................,--
..,......,-..-..,.,.,--rry~~.-.-.T"
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
BO
85
90
~5
NUMB ER OF VIIILS
FI G.
l. Zona l sca n profil e of t:1in -laye r chromatogram of testoste rone- ! , 2' H metabol ites in huma n s kin .
ACTIO N OF ESTROGEN I N A MODEL SEBACEOUS G LAND
301
TESTOSTERONE METABOLITES in RAT PREPUTIAL GLAND
nooo
2000 ·
2GOO 2400 --
w
z
0
w 2200
a::
w
~ 2000
z
~
f(f)W
oz
1800
0:: 1600 - 1
LLI 1400 -
f-
0
0
~
w
0
0
0:
z
(f)
120 0 -
u::
a::o
Ou::
w
:Z
0
?::o
Q<{
w
0
f(f)
0
1(f)
z
z IGOO ::J
ww
f- f0(f)
z
0...
(f)
f- 0
(f)Q::
-'
0
0
<{
300 ·GOO -
w
f-
z
0
z
~
(f)
0
a::
0
z
<{
400 ·-200 TTTrT Tr
t}
rrrr·t
10 15 20 25 30 35
NUMBER OF V IALS
40
FIG. 2. Zonal scan profile of thin-layer chromatogram of testosterone- 1, 2' H metaboli tes in rat preputial gland .
EFFECT of CASTRATION and ESTROGEN ADMINISTRATION
on TESTOSTERONE METABOLISM
in the RAT PREPUTIAL GLAND
6000
CASTRATED
~ 400 0
6a:
a.
CASTRATED
+
ESTROGEN
"'
E
l2000
F IG. 3. Effect of castration and est rogen administrat ion on testos terone metabolis m in th e rat preputial
gland-testosterone- 1, 2' H up take (o pen bars) and dihyd rotes toste rone production (s haded ba rs). Dotted
lin es represent one standa rd deviation. T uptake = lntragla ndular T + lntraglandu\ar T metabolites + T
metaboli tes in medium.
t hat the preput ia l gla nd of t he castra ted ma le
mouse is a bl e to ta ke up 4 t im es as much T per
mg o f protein as the pre putia l gla nd of th e cast rated mal e rat. M ea n DHT p roduction in t he
p reput ia l gland of castrated male mice ave rages
2500 cp m/ mg protein represe ntin g a mea n con ve rsio n of 6% of t he T ta ken up to DHT. Th e
a dmini strat io n of est ra di o l resul ted in a de creased T up ta ke, but no signifi ca nt a lte ration in
DHT production in the preputia l gla nds of cas-
trated mice-findin gs s imila r to t hose obse rved in
th e preputia l glands of cas trated ma le rats .
As mi ght be expected, T adm in ist ration ca used
a decrease in uptake of tritiated T.
It furth er see med impo rta nt , espec ia lly s in ce
DHT product ion in seve ra l of the rat experiments
proved lo be in t he ra nge of 500 cpm / mg of protein , to d ete rmin e wh ether max imum DHT pro ductio n was in fa ct a funct ion of tota l T upta ke or
was limi ted by enzy me saturation . To dete rmin e
t hi s, con centrations of tritiated T ra ngin g from 5
!J. C (. 11 1111 mo les) to 50 !J.C (1 .1 1111 moles) were
used in t he in cuba t ion media ma inta inin g con stant s pec ifi c activit y.
T he resu lts of th ese experiments are summari zed in F igu re 5. Gla ndul a r up ta ke ofT per mg of
protein ra n ge d from 2000 c pm/ m g of protein to
27.000 c pm/ mg of protei n . Co rrespo ndin g DHT
production levels ra nged from 350 cpm / mg of
prote in to 9000 cpm / m g of protein. Co rres pondin g T to DHT conve rsi on rates ra nge d from
30 to 38%. These resul ts clea rl y de monstrate t hat
T to DHT co nve rs ion in t he rat preput ia l gla nd is
not limi ted by saturation of t he 5-a lpha red uctase
enzy me wi t hin t he 10-fold ra nge ofT concentratio ns tested , but rather t hat it is a fun ction of
uptake wit h conve rsion ra tes re ma in ing relativel y
co nsta nt wi t hin a ra nge of 30 -38 "~ . Thus , dec reased production of OHT by t he preputia l
gla nd of t he castrated ra t is a resu lt of dec reased
5-a lpha reductase activ ity. T his is equa lly true of
est rad iol ad ministration. whi ch reduced conversion of from 30% to 11 % of t he T take n up, a decrease of a lmost 1 :t .
THE JO URNAL OF I N VE STI GA TIVE DERMATOLOG Y
302
Th e pre puti a l gla nd of t he castrated ra t demo n strates a n inc reased up ta ke ofT a nd a dec reased
produ ct ion of DHT as a resul t of dec reased con vers ion . E st radiol a d mini stration results in dec reased upta ke of T wi t h a dis proport ionately
great decrease in DHT prod uction (11% vs 3038% in t he untreated a nim a ls).
DI SCUSS ION
We ha ve demonstrated that hi gh close estra diol
administratio n suppresses th e up take of T by
the preputia l gla nds of both castrated and norma l
male rats a nd castrated mi ce a nd that it suppresses th e co nvers ion of T to DHT in the preput ia l glands of norma l ma le rats. It is poss ibl e
that estradiol acts by replacin g or displac in g T
fro m a ndrogen rece ptor prote in in th e pre putia l
gla nd, thereby co mpetin g with T for uptake by
t he gla nd cell s. Fa ng and Liao (7) hav e recently
reported that in prostatic cells estra diol is a bl e to
fo rm a co mplex with a ndrogen protein receptor
within t he cell, a nd it is poss ibl e t hat estradi ol
could dis pl ace from 10 to 30% of t he T co mpl exed
to th e androgen r.ece ptor.
We pro pose that a n an drogen recepto r protei n
formin g a s imila r co mpl ex mi ght be present in
the preputia l gla nd a nd t hat estra di ol mi ght di spla ce androgen on thi s rece pto r protein , t hereby
dec reas ing t he up ta ke. Our data a lso d emonstrate
that es tradi ol treat ment ca n decrease t he conve rs ion of T to DHT. This mecha ni s m has not yet
been in ves ti gated but it could be due to a direct
effect of est ra di ol on t he 5-a lpha reductase en zy me, s uch as subst rate co mpetition. In li ght of
recent ev id ence, howeve r, a noth er mecha ni s m is
more lik ely . We ha ve previously shown that rat
a nd mouse preputi a l gla nds conta in ac id phospha tase and beta-glu curo nidase act ivi ty associated with th e lysoso ma l fraction (8). T hi activity ca n be in creas ed by T admini st rat ion (9).
The in crease may be so lely within t he lysoso me
a nd is not la bil e or released unl ess t he lysoso mes
a re rupt ured. Recent findings by Smith et al. (1 0)
d e monstrate t hat in t he ra t prepu t ia l gla nd , e trogen activates lysoso ma l enzy me release in to
t he cytoplasm and in to t he nuclea r envelope. Intra nu c lea r and nu c leo la r pen et rat io n of fra gmented lysoso ma l co mponents is ev id ent by elect ron mi c rosco py within 1.5 minutes afte r estrogen
a dmini st ration to ovari ectomi zed rats .
Th ese findin gs show that estroge n a dministrat io n ca n ca use lysoso ma l enzy me release . It is
EFFECT of CASTRATION and HORMONE ADM!NISTRATION
on TESTOSTE RONE METABOLISM
in the MOUSE PREPUTIAL GLAND
35,000l
-1
3o,ooo-=l
CASTRATED
c:-.:~
CASTRATED
+
ESTROGEN
25,000
z
w
I-
0
0::
20,000
Q._
0">
E
.......
15,000
E
CASTRATED
TESTOSTERONE:
TREATED
a.
u
10,000
5,000
6%
23%
10%
% CONVERSION
ial
FI G. 4. E ffect of castration and hormone a dminist ra tion on testosterone metabolis m in the mouse preput
gla nd -testosterone -!, 2' H uptak e (open bars) a nd dihydrotestost erone produ ction (shaded bars). Dotted lines
represent one s ta nd a rd deviati on. T upta ke = ln t raglandul a r T + lntraglandu la r T metabolites + T metabolites in
medium .
ACTION OF ESTROGEN IN A MODEL SEBACEOUS GLAND
DHT FORMATION AS A FUNCTION
OF TESTOSTERONE UPTAKE IN
PREPUTIAL GLANDS
OF NORMAL MALE RATS
26,000 -
CJ TEST UPTAKE
~ DHT
z
w
b
g:
14,000 -
Q"'
'EE
1o,ooo
0..
(..)
5. Dihydrotestosterone formation as a function
of testosterone-! , 2'H uptake in preputial glands of
norma l male rats. T upta ke ~ ln traglandular T + ln tragland ular T metabolites + T metabolites in medium.
FIG.
possible that the resul ta nt release of ac id phosphatase, beta-glucuronidase a nd oth er cata lytic
enzymes within t he gla ndul a r cell s results in
premature intracellula r destruction, in creas in g
the t urn over of cell s, res ul t in g in d ec reased
sebum production a nd t ri gge rin g rap id mi tot ic
activ ity. Thi s th en is a poss ibl e exp la nat ion for
dec r eased DHT production in the estra di olt reated a nima ls. Supp rt for t he above is a lso
found in the work of Eb lin g (11), who describ ed
the effect of lo ng-ter m est rogen adm inistrat ion on
the rat preputial gla nd . He showed t hat estroge n
adm inistrat ion causes just such a n in cr eased m itotic act ivi ty a nd that the indi vidua l cell s of t he
gland re ma in much sma ll er a nd undi fferentiated
when com pa red to t he ce ll s of testostero netreate d a nima ls.
Cast ratio n res ults in in c reased T up take a nd
decr ease d convers ion ofT to DHT in the prepu t ial g la nds of both rats an d mice. T his dec reased
conversion ofT to DHT may be a con sequence of
decrease d en zy m e present or decreased enzyme
activity, poss ibly du e to decreased co factor activity. NADPH is a n ecessa ry co facto r for t h e
convers ion of T to DHT by t he en zy m e 5-a lpha
redu ctase. In prep utia l gla nds of castrated r· ts,
decreased con ve rs io n or T to DHT may resu lt
from lac k of s uffi c ien t NADP H. We have pre vio us ly shown (12) the glu cose-1- "C ox idation via
303
t he pen tose ph osphate pat hway in t he mouse preputia l gland is decreased by at least 50% by cast rati on and it is poss ibl e t hat t h e resul ta nt decrease in NADPH is in part responsible for decreased T to DHT conversion in the preputial
gla nds of castrated rats as opposed to testosterone-treated a nd norma l rats. The compa rative
lac k of glucose-6-phos phate dehydrogenase activity a nd of NADPH production in preputial
glands of castrated rats may result in decrease of
5-a lpha redu ctase en zyme activity due to t he lack
of this n ecessary cofactor, t herefore, decreasing
conve rs ion ofT to DHT.
The mec ha nis m by which estrogen results in
d ec reased se bum produ ct io n has hi t he rto remained un expla in ed .
These stud ies, demon strat in g estrad iol suppressio n ofT uptake a nd its conve rs ion to DHT in a
sebaceous gla nd ana logue, point to a poss ib le
m echa ni s m at t he ce llul ar level by which est radiol
could inhi bit seb um production.
S in ce t he pentose phosphate pathway is t he
major source of NAOP H , castration would result
in decreased
ADPH a nd T a dmini st rat ion
would res ul t in in creased levels in previous ly cast rated a nima ls .
The authors gratefully acknowledge the superb techni cal assistance of Miss Badryeh Cum min gs and Miss
Ru th Chang.
REFERENCES
1. Wilson , J.D. and Loeb, P.M. (Eds.) : D evelopm ent
and M etabolic Con t rol M echanisms and Neoplasia. Anderson Hospita l and Tumor Institute,
Willia ms and Wi lkins Co., Baltimo re. 1965.
2. Bruchovsky, . and Wilson, J. D.: The conversion
of testosterone to 5 androstan-17b ol-3one by rat
prostate in vivo and in vitro. J. Bioi. Chem.. 243:
2012, 1968 .
3. Voigt, W. , Fernandez. E. and Hsia, S. L. : Transformation of testoste ron e in 17b-hydroxy-5a-a ndrostan -3 -one by microso ma l preparati ons of
human skin . J. Bioi. Chem. , 245: 5594, 1970
4. Sansone, G. an d Reisner, R. M.: Differentia l ra tes
of conver ion of testosterone to di hydrotestosterone in acne and in normal human skin-A
possible pathogeni c factor in acne. J. In vest.
Derm., 56: 366, 197 1.
5. Nicolaides, N.: Skin lipids ll-Lipid class co mpositio n of sa mpl es from various species and anatomicalnotcs . J. Ame r. Oil Chern. Soc., -12: 69 1. 1964 .
6. Bligh, F. G. and Dyer, W. J.: A rapid method of
tota l li pid extraction and purificat ion. Canad. J.
Biochem. Physio l. , 37: 911 , 1959.
7. Fang, S. and Liao, S.: Androgen receptors. J. Bioi.
Chern. , 240: 16, 197l.
8. Sansone, G.: M etabo lism of Gly cerol Eth ers and
Wax Esters in The Preputial Gland of th e Mous e.
Ph.D. Dissertation, Tu lane University, ew Orleans, 1968 .
9. Patterson, J. F. , Cheney. M. and Fisma n.
W. L. H. L.: Preputial gland b-glucuronidase response to testoste rone and to two anabolic
steroids. Endocrinology, 75: 273, 196<1.
10. S mi th, E. and Szego, C. M.: Direct photomicroscopic evidence for rapid nu clear penetrat ion of
lysosomal produ cts in steroid targets after es troge n in v ivo . Endocrinology, 88: Supplement
XRA-151: A-151, 1971.
304
THE JO URNAL OF I N VE ST I GAT IV E DERMATOLOG Y
ll . E b ling, F. J .: Horm ona l co ntrol of sebaceous gla nds
in expe rim e nta l a n ima ls, A dvances in Biology of
th e Shin , Vo l. 4. E ds., M ontagna, W ., E ll is, R. A.
a nd S ilve r, A. F ., M ac M ill a n Pub li sh ing Co.,
1963.
12. Sa nso ne, G ., Da vidso n, W ., C umm ings, B. a nd
Re is ne r, R. M .: Sebaceous gla nd lipoge nes is in du ced by tes toste ro ne: E a rly meta boli c eve nts J.
Tn vesl. De rm ., 57: 144, 197 1.