dihydrotestosterone from testosterone in organs from 2M mice. No significant differences were found in tes- ticular steroidogenic enzymes between. 2M and.
BIOLOGY
OF
REPRODUCTION
47,
Intrauterine
723-729
(1992)
Position
Effects
on Steroid
of Reproductive Dj.
NONNEMAN,3
of Veterinary
Departments
Biomedical University
Organs
GANJAM,2’3
V.K.
Metabolism
W.V.
Sciences3
in Male
of Missouri-Columbia,
and
F.S.
Sciences,4
Columbia,
Steroid
Receptors
Mice’
WELSHONS,3
Biological
and
and
VOM
SAAL4
College
Missouri
of
Medicine
Veterinary
65211
ABSTRACT Mice
differ
in their
adult
fetuses
(2M
males),
which
levels.
The
present
study
of testicular P4w),
were
tions,
which
vesicle
and to
ticular for
form
OM
of
between no
exerts
organs
of adult
male
in
organs
receptors)
(occupied
on subsequent
In mice
and
the
rats,
among
contiguity sex
during
urally
occurring
rangement
animal
of
fetuses
greater
than (OM);
that
male
develop
have
between
distance
than
females;
i.e.,
females
2M
age
Intrauterine opment; Gonadally
are
intact
June
Received
December was
2Correspondence.
ar-
intrauterine
of
has
OM males paired
FAX:
males
in OM
of
Su-
concentra-
For both
males,
suggest
findings
androgen
seminal
indicating
greater
were
found in teshigher for 2M males had three times the that intrauterine fetal
responsiveness
in utero slightly
also exhibit with
fefe-
in reproductive
a larger
an-
between
masculinized
two
influences
longer young
male
extensively more
females
mounts [6]. When
devel-
male
physiological on
to eliminate treated with were
differences
later
found
any postT by imto be
more
present
in
fetal
hormone
con-
development.
study,
we
investigated
allow position
certain
biochemical
us to further characterize the in adult male mice. We mea-
important potential. sex tissues
sex organs to androgens
MATERIALS
testicular enzymes to 5a-Reductase activity (i.e., seminal vesicles
AND
to assess the ability and estrogens.
of
METHODS
Materials
described. and
2M males
ceptors in the accessory these tissues to respond
[21. As
[4-6].
less
the
at birth and then
and prostate) to determine the ability of 2M and OM mice to convert T to dihydrotestosterone (DHT), a more potent androgen. In addition, we measured steroid hormone re-
Females
have
develop
were castrated exposure,
sured activities of three monitor the steroidogenic was measured in accessory
of estwo on
small In the
have
(2M)
extensively.
that
been
by NSF grant (314)
Our
Activity
receptor
organs.
sex
than
parameters that would effects of intrauterine
Steroid standards Co. (St. Louis, MO)
more 2M
were purchased and recrystallized
prior to use. New England source for [1 ,2,6,7,16,17-3H]T 3HIDHT (148 Ci/mmol),
27, 1991.
supported
accessory
in adulthood,
centrations
22, 1992.
Accepted work
the
two
OM females
position
when
nat-
random
are more aggressive, have cycles, and cease producing
this
products.
Activities C,7.20-lyase
aggressive towards other males than were the OM males [5, 6]. For these reasons, the naturally occurring phenomenon of random intrauterine positioning of male and female fetuses affords a unique model for studying the effects
or op-
concentrations
studied
males
do
fetal
intromissions
‘This
than
however,
as
to
plants
to
elevated until birth at [2]. Amniotic fluid and
lesser
females
2M female mice vs. 4-day) estrous
at an earlier
same
that develop between fetal position effects
been two
ogenital adults, (6-day
to
between
of animals Intrauterine
development
that
develop
of T, but
those [4-61.
is due
[2]. Fetal mouse testes differentiate 13 of gestation and begin to secrete
concentrations
tradiol, males
due
which remains 19 in CF-i mice
Day
of animals
the
and
androgen
and
males
and
metabolism
and OM mice natal androgen
[1, 2]. This
variation referred
sex
of the
development
[31,
(T)
in
in 2M
to 2M prostates.
androgen
of phenotypic same
to siblings
is
position phenomenon at approximately Day
blood
of the
intrauterine phenotypic
testosterone approximately
component
individuals
of an
posite
of substrate Estrogen
and
male
estradiol
mice.
a significant
adult
17u-hydroxylase,
from 2M mice. No significant differences the trend for all three activities was receptor concentrations, OM prostates
compared
adult
INTRODUCTION
variation
fractionation techniques.
greater
two
fetuses
also examined
60%
OM animals, whereas found in estrogen
and
were
influence
HPLC
were
approximately
testosterone
2M
differences
receptors
androgen a significant
position
from
and
by similar
hormones,
were
female
dehydrogenase/isomerase,
measured
to steroid
they developed in utero between (OM males), which have higher parameters of 2M and OM adult male mice.
of whether two
biochemical
assays
was
activities
enzymes While
characterize
prostate
to respond
or between
-3(3-hydroxysteroid
of radiometric
and
dihydrotestosterone
males.
concentration
vesicle
as a function
levels,
further
to
namely
5a-reductase
steroidogenic
than
designed
capacity
prostate,
characteristics testosterone
by means
seminal
indicate
higher
enzymes,
measured
in both
capacity
was
steroidogenic
reductase
reproductive
have
mmol), [1 ,2,6,7-3H]progesterone hydroxyprogesterone (42.3
DCB8518094.
882-2950.
723
from from
Sigma aqueous
Nuclear (Boston, (168 Ci/mmol), [2,4,6,7-3Hlestradiol (115 Ci/mmol), Ci /mmol), and
Chemical ethanol
MA) was the [1,2,4,5,6,7(106.9 Ci! [1 ,2-3H]17a[7-3H ]preg-
NONNEMAN
724
nenolone Acetonitrile,
(10.5
Ci/mmol); methanol,
purity
their benzene,
and
was checked by HPLC. hexane were HPLC-
ET AL.
After
the incubation, the incubation
placing
reaction
the vials
on ice.
was
The
terminated
sample
by
preparation
grade from Burdick and Jackson (Muskegan, MI). Ethanol (HPLC-grade) was purchased from J.T. Baker Chemical Company (Glen Ellyn, IL). -NADPH, NAD, and other re-
procedure, using C18 solid-phase extraction (SPE) columns, was conducted as described and analyzed in detail elsewhere [8]. Briefly, the C18 SPE columns were activated by
agents
discharging 2.0 ml of methanol followed by 5.0 ml of water through the column with positive air pressure. Incubation vial contents were transferred to the column and washed
were
Animals
purchased
and
(Wilmington,
Sigma.
of Intrauterine
Iden4/ication
An outbred colony domesticu.s), originally oratories
from
of CF-i
Fetal
purchased MA), was
mice (Mus musculus from Charles River Labused to take advantage of
the large litter size (12 pups). The mice were on a 12L: 12D cycle. Adult female mice were and checked for the presence of vaginal plugs [)ay
19 of gestation,
the
Position
house
mothers
were
maintained time-mated (Day 0). On
killed
by
cervical
dislocation, and the pups were delivered by Caesarean section wIthin 2 miii of maternal death. Fetal sex was assessed by the length of the anogenital space, the intrauterine frtal I)051ti011
of each
pup by toe clipping
was
determined,
and
each
pup
WilS
marked Was between
for positive identification, A 2M pup tWo males in the uterus, while a OM pup was l)etWeen IWO female fetuses. Pups between a male and female fetus in a uterine horn were not used In these cx-
l)CrlmeIitS, The male pups, fostered to females
in groups of five OM and five 2M, were that had delivered normally within the preceding 24 Ii. The animals were weaned at 23 days and liotised individually at 3S days of age. OM and 2M males were matched for I)ody weight when assigned to the experiments. In adulthood, after 10-Il were killed by cervical dislocation, collected and stored at 70#{176}C until
/‘cstlcular Testes Krebs-Rlnger previously
Sieroido,en1c
Ifnzme
5 days of age, animals and the tissues were further analyses
Aa
individually homogenized at 10 mug in phosphate buffer (KRPB, phI 6.9) prepared as described 7) and were held at 0-4#{176}C at all times
were
prior to Incubation. Ten milligrams placed into I ncu bat ion flasks
of testicular containing
tissue 0.5
8.0 ml
hexane,
methanol/water
Steroids
of benzene orated, and
were
eluted
into glass conical the conical tube
(1:3
v/v)
from
the
incubation metahohites
2.0 ml of nwith
was calculated initially
and
in nanomoles
T. Data
of product
4.0 ml
by using
present
in each
the percentage of the total i.e., I 7a-hydroxy-progestcrone,
flask and formed,
drostene-3,17-dione, activity
and column
The benzene was evapwere concentrated to
tubes. contents
the tip. The activity of l7ct-hydroxylase the value of 5 pM progesterone
radioactive 4-an-
are expressed formed
as specific
in 30 minutes
per
milligram of protein, Lyase or side-chain cleavage activity was determined in exactly the same manner except that 17a[11 ljhiydroxyprogesterone was used as substrate. The ,er centage of conversioli to 4-androscene-3,17-dione and T was used to calculate specific activity. The enzymatic activity of -3-hiydroxysteroid dehydrogenase/isornerase was similarly determined except that [4Hjpregnenolone was used as substrate and 0,5 mM -NAl)4 was used as the cofactor, The of conversion
percentage
culate
specific
Quantification
to progesterone
was
used
to cal-
activity.
of Steroid
Metaholites
by Isocratlc
HPLC
We employed the use of an Isocratic ternary solvent system and a narrow-bore reversed-phase IIPLC column to accomplish the separation of pregnenolone, progesterone, 17ahydroxyprogesterone, 4-androstene-3,1 7-dione, and T, Steroids were judged to he completely separated from each other on the basis of the resolution of adjacent radioactive
was
peak
Ci
than
I]progesterone and enough nonradiouctive progesterone final progesterone concentration equalled S In a 1.0-mi incubation volume. This mixture was preincubated for 2 mlii at 37#{176}C before initiation of the reaction via addition of 0.5 mM -NAl)Pi=I. The reaction was terminated after 30 mlii. Assay conditions were specifically designed (for each enzyme) to maintain a linear reaction velocity with respect to time and protein concentration. Product accumLllatIon for all three enzymes was linear through 60 mlii and 2 mg protein, which were the maximum parameters examined. Control incubations were processed In exactly the same manner with boiled testicular homogenate from each group used. The background activity was below the limits of detection for all control incubations. All incubadons were performed In duplicate. [4j
so that the
with
pairs. The resolution between peaks was always greater 1.25, Indicating complete separation of compounds [71.
A Perkin-Elmer
(Norwalk,
CT)
Series
lO liquid
chroma-
tograph equipped with a Rainin (Woburn, MA) ltheodyne model 7125 Injector, a i00-.ti sample loop, a Beckman-Alccx (Fullerton, CA) 2 x 250-mm IJltrasphere ODS (5 m) reversed-phase C18 column protected by an Upchurch (Oak I larbor, WA) 2-ni precolumn filter, was used for the chromatography. The elutlon of 4-ene-3-ketosteroids was monitored through liv absorhance detection at 240 nm; the elution of pregnenolone (a 5-ene-3-hydroxysteroid) was monitored at 219 nrn with a Perkin-Elmer model LC-95 t// VIS spectrophiotonietric detector equipped with an 18-fJ.l flow cell. Detection and quantifIcation of 311-isotope elution was accomplished by use of a Beckman Model 171 on-lIne radiochromatogr.iiliIc detector equipped with a 300-pA flow cell. An isocratic acetonitrlle:methanol:water (35:24:40, v:v:v)
INTRAUTERINE
ternary solvent system psi) was employed for
at a flow rate of 0.3 the 20-mm analysis.
POSITION
mI/mm
EFFECTS
Radiometric
Prostate
and
seminal
polypropylene in an amount weight.
Homogenization
(Brinkman ice
vesicle
so
tissues
was
Westbury,
into
transferred
and
to 10 ml/g a type
remained
placed
were
cylinders with
Instruments, that
tissue
tissues
homogenizing of KRPB equal
homogenized
tissue
original
PT 10/35
NY)
was
wet
Polytron
performed
at 0-4#{176}C.Ten
separate
to
incubation
on
milligrams vials
of
containing
0.05 pCi [3H}T (100 000 dpm) and enough recrystallized nonradioactive T so that the final T concentration was 5 x i09
M in a 1.0-mi
incubation
tion
of 3-NADPH, was 0.5 mM.
prepared Incubations
vial,
1 h [8]. Assay signed
conditions
to give
volume.
The
for the
enzyme
rate
than 15% to ensure to time and tissue
der
any
for
homogenates,
same We
did
control
endogenous tissues
tissue
homogenate.
periments
using
boiled
failed
to detect
tane-3a-diol the
that above
cubation period. used to isolate as described
to aid
mm
X 25-cm
in the
seminal
the
reduction conditions
ex-
vesicular (e.g.,
tissue
5a-andros5a-DHT), the
un1-h
steroid elution procedure from the biological
in-
was matrix,
chromatographic
in 30 pA HPLC-grade ethanol standards T, 3j3, 3a, and
UV visualization ODS
at 210
a Brownlee C18 guard
(Rainin, column
conditions
(45:55
A Rainin
4.6-
C18 column
(5-
Woburn, MA) 3.2-mm were used. All other
were
acetonitrile/water
nm.
reverse-phase
the v/v)
same
as above.
An
system
at a
solvent
Assay
receptors were measured by the hydroxylapamethod of Williams and Gorski [10]. Briefly,
and
and placed immediately in 10 mM Tris-HC1,
1 mM
dithiothreitol
was
determined
radiolabeled bound from a 60%
by using
steroid in free hormone,
slurry
of HAP
pH 7.3. After
the
final
wash,
the pellet scintillation
with 2 ml cocktail.
Androgen
Receptor
(Scintiverse
LC; Fisher
with
HPLC
effluent
The
formation
use of the incubation and
DHT
equipped
efficiency was 20% Scientific, (3:1
ratio)
of 5a-reduced value of 5 X iOand the percentage
formed.
with
a 1.0-mi
flow
cell
of on-line radiochromatowhen scintillation cocktaii Pittsburgh, in
PA) was
a high-efficiency
androgens
was
calculated
M T initially present of total radioactive
mixed mixer. with in the
313, 3ct,
at a
100-fold
Tissues
were
1.5 mM
Tris,
dithiothreitol, molybdate;
Tris
+
ml buffer) was
mogenized buffer
used
every
as the
15 mm
used were
that
and
low-salt
in
from 10 ml
DHT
(10
1 mM
each
extract [12].
centrifuged
and
The
of
hormone removed
mM of
sodium on ice The This
contained
was ho-
pellet buffer
(TEDG
as before.
high-salt extract by endogenous
by competitive
This
su-
and contained hormone [12]. for the binding from the low-
charcoal
(DCC; After
of extract).
by centrifugation at 10000 of [3H]DHT by the extracts inhibition
using
nonlabeled
(550 fii) was incubated with 0.5tubes contained 100-fold excess
to determine
incubated
buffer
and
1 h in high-salt
removed binding
DHT. An aiiquot of extract 20.0 nM [3H]DHT. Parallel were
TEDG
with dextran-coated 1.25 mg dextran/mI
1 h at 0-4#{176}C,DCC was x g for 10 mm. Specific
nonradioactive
and
extracted
fluoride, and was homogenized
with labeled steroids were
salt extract by incubation 12.5 mg charcoal plus
estimated
K1-l2PO4
5-sec bursts of a poiytron. at 30000 X g for 30 mm.
for
as the occupied
Before incubation assay, endogenous
was
was
[v/v],
receptors
0.4 M KCI)
was
pernatant receptors
with
cytosolic
with
was
counted
in ice-cold
10% glycerol
was centrifuged
unoccupied
separate added to
mixings for 30 mm. in 50 mM Tris-HCI,
and
phenylmethylsulfonyl pH 7.4) [11]. Tissue
supernatant
of non-
To
1 mM
radioactivity
immersed
EDTA,
(1 g tissue/S homogenate
excess
Assay
to the occupied receptors roid was separated from
detector
tis-
ice. Tissues mM sodium-
25#{176}C) (TED)
incubations. pA of cytosol
of ethanol
line
counting detection
1.5
7.3,
allowed to stand on ice with several The HAP pellet was washed five times
extracts
radioisotope
parallel 200
in 50 mM
flow rate of 1.0 mI/mm (1800 psi) was used for the 20-mm analysis. Detection and quantification of 3H-isotope elution was accomplished by use of the Beckman Model 171 on[9]. The graphic
(pH
on
of 1:20 (w/v). Aliquots of the cytosol (100 000 X g for containing 0.5-2.0 mg/mi of protein were incubated 1-10 nM [3H]estradiol overnight at 4#{176}C. Nonspecific
binding
the
of T occurs through
ratio 1 h) with
725
MICE
were excised homogenized
EDTA,
in the
control
[313], and
spontaneous
Microsorb
packing) and 1.5-cm HPLC
isocratic
than
in exactly
preliminary
and
were reconstituted authentic recrystallized
DHT
X
less
of the reIn or-
ability
T metabolites
A C18 SPE the androgens
de-
above.
Samples containing
pm
prostatic
experimental
were
did not contain 13-NADPH. endogenous reducing abil-
5a-androstane-3j3-diol little
of not
processed
Our
5a-reduced [3a],
suggesting der
were
incubation at 22#{176}C for
linearity concentration. reducing
manner except that they not see any measurable
ity in the
concentra-
reactions
conversion
1% and not more action with respect to account
final
with KRPB in each were conducted
a substrate
Estrogen (I-lAP)
sues were
Assay
MALE
Receptor
Estrogen
(2000
tite
5a-Reductase
IN
for
nonspecific
binding.
24 h at 4#{176}C, during
which
The la-
beled
DHT bound to the unoccupied receptors in the lowsalt extract or exchanged with endogenous hormone bound
plicate, 0.5 ml several
in the bound
high-salt steroid
200 pA of extract to 0.3-0.35 ml buffer and incubating this mixture mixings. The samples were then
extract. Free steby adding, in duof packed HAP in for 20 mm with centrifuged for 2
mm at 600 X g and 4#{176}C. The supernatant was aspirated, packed I-LAP was washed four times with 2 ml ice-cold mM tracted
Tris
buffer with
ethanol
(pH
7.3),
and
as described
bound above.
hormone
was
the 10 ex-
726
NONNEMAN
TABLE
1.
enzyme
Effects
of
intrauterine
position
on testicular
El
AL.
steroidogenic
Enzyme
OM
i-3-Hydroxysteroid Dehydrogenase/isomerase 17a-Hydroxyiase C,7-Lyase
ESTROGEN
PROSTATE
activities.#{176}
BINDING
2M
7.68
±
0.64
7.89
±
1.34 2.54
± ±
0.89
0.11
1.60
±
0.20
0.13
2.62
±
0.31
0
E
Data are expressed as nmol product formed/30 mm/mg protein (mean ± SEM, n = 7 testes/group). The designations OM and 2M represent pups neighbored in utero by two females or two males, respectively.
0
z
0
Protein
Determination
Protein
determinations
ried were
out according digested with
Tris
buffer
(pH
concentration
of tissue to Bradford 0.1 N NaOH
7.5),
and
against
homogenates
were
[13]. Briefly, at 60#{176}C for
assayed
in duplicate
a standard
curve
car-
homogenates 1 h, diluted
in
FREE
for protein
of 5-50
g/ml
ence
BSA.
Data
Analysis
Data
tradiol.
were
expressed
as
enzyme
activity
number per and receptor
milligram numbers
protein. Data were subjected
For all organ
weights,
body
iate
in covariance
atp
analysis.
weight The
or
for enzyme to one-way
was
level
used
DHT
receptor
and
activities
Total
(0),
total
of significance
was
differences
set
ble
We
Steroidogenic examined
The
the
activities
of
three
diometrically moles the
and
are
of product testis
weights
adult
mice
total
activity
expressed
formed
enzymes,
were
(123.3
±
not 5.3
paralleled
as specific
per vs.
the
milligram
different 121.2
were
values
namely
progesterone
activity
homogenates
3.0
seen
2M
mg,
for
Since and
With tor,
and
C17 20-Lyase
2M and
OM adult
mice
OM
to androstenedione
with
about
oc-
1.5 nmol
throughout 17a-hydroxylase incubation, C17 20-lyase activity yielded from
[14]. hofrom
as substrate and activity, whole of progesterone
and androstenedione 17a-hydroxyprogesterone
nating
activity.
(Ta-
Activity
(Table 1). With progesterone cofactor for 17a-hydroxylase converted
of C19-steroids
5-313-Hydroxysteroid
between
of progesterone
1 7a-hydroxyprogesterone gram of protein,
respectively)
specific
as the
NADPH
in nano-
of protein.
seen
conversion
17a-hydetermined ra-
between ±
of
curs through two activities of one testicular enzyme Both activities were measured in OM and 2M testicular mogenates to evaluate their ability to form androgens
Enzynes
5-313-hydroxysteroid dehydrogenase/isomerase, droxylase, and C1., 20-lyase. Activities were
nonspecific)
1).
I 7a-Hydroxylase RESULTS Testicular
(A),
nonspecific
and
ANOVA.
as the covar-
0.05.
15.77 group).
Receptor
Androgen 0
vs.
7 per
=
performed
with 2 nM [3H]estradiol 10 nM DHT. Prostate
concentrations expressed nificantly different (p
z
were
in of
in the (occupied
PROSTATIC
than
prostate,
androgen
receptors)
ANDROGEN
receptors
were
=
significantly
RECEPTORS
30
2M prostates. 25
Receptor
Estrogen
Specific estrogen pared from mouse seminal vesicles [3H]estradiol. tate cytosois Scatchard
binding duced
binding saturable
labeled lower-affinity
found
in cytosols
cytosols low
studies binding
prepared
binding
estrogen
of
prefrom 1713-
20
z Do
androgen
component.
The
0
10
E 9-
a high-affinity
and a lower-affinity DHT (to reduce to
15
OE
using mouse prosby 2 nM estradiol.
two components:
revealed
with low capacity addition of 10 nM of
the
G.)
receptors were prostate, whereas exhibited very
Estrogen showed analysis
component nent. The
Studies
receptors) main
5
compoany cross-
2M
re-
component
showed high-affinity binding and low capacity: K. = 0.15 nM and Bmw, = 30 fmol/mg protein (Fig. 1). Competition studies carried out as previously described [15], i.e. using
FIG. OM and cubated
3. Androgen binding 2M mouse prostates. in duplicate overnight
free
hormone
2M
prostates).
were
separated
OM by high-salt extracts (occupied receptor) of Extracts from individual prostates were inat 4#{176}C with 20 nM (3HIDHT, and bound and by use of HAP ( p < 0.01; n = 7 OM and
728
NONNEMAN
higher (p The values protein
