BIOLOGY
OF
9, 330-337
REPRODUCTION
Biological
(1973)
Evidence
Fetal
YOUNG
S.
MOON,
for Androgen
Pig Testes
MARGARET
Secretion
in Organ
H.
Accepted
Culture’
HARDY,
Department
of Biomedical
University
of Guelph,
Guelph,
April
by the Early
AND
JAMES
I.
RAESIDE’
Sciences,
Ontario,
Canad.a
13,
1973
A bioindicator
method
was used to detect
the presence
of androgens
in fetal
organs;
114 portions
of the androgen-sensitive
ventral
prostate
of the 21- to 28-day-old
rat were
grown
in organ
culture
with entire
gonads
from fetal pigs of 2.0 cm C-R
(crown-rump),
or portions
of gonads
from
fetal
pigs of 2.5,
3.0, 3.5, 4.0, and 4.5 cm C-R
lengths.
After
6 days
in medium
199
containing
10% bovine
serum,
isolated
explants
of prostate
glands
remained
healthy,
but showed
slight
regressive
changes
in the acinar
epithelitun.
Testes
from
2.5 and 3.0 cm C-R
fetuses
developed
normally
in vitro,
and adjacent
rat
prostate
gland explants
responded
with a secretory
epithelium,
enlarged
acini containing
some secretion
in the lumen, and with reduction
in the stroma.
From 2.0 cm C-R fetuses,
the gonads which differentiated
into testes during
cultivation
for 6 days in vitro, stimulated
explants
of prostate
to a lesser degreee.
It was concluded
that androgens
are secreted
by male gonads
of the fetal pig and, whereas
their production
is of limited
duration
in the early stages
of pregnancy,
it commences
perhaps
even before
sex can be distinguished
histologically.
Ovaries
from
pig fetuses
at similar
stages
did not affect adjacent
prostate
explants.
Adrenal
glands
from male and female
fetuses
similarly
had no androgenic
action
on prostate
explants.
When
testosterone
was added
to the medium
for explants
of rat
prostate,
for comparison,
the responses
in the tissues
were
largely
similar
to those
seen
where
fetal pig testes were the source
of the androgenic
stimulation
during
cultivation.
It is now
malian
fetal
bility
well
testes
in early
especially
Woiffian
and
(Jost,
been
demonstrated
development
in
1965;
Price
its
male
time
On the
influence
tract
1
the
development
et a!.,
in press),
genihas
studies
species
control
reproductive
of gonadal
other
hand,
the
female
sex,
it
culture
several
and
Our histological
ation
of interstitial
of
the
Mullerian
of external
Moreover,
in organ
testes
of
androgens,
of the
after
entiation.
do not
of somatic
the
maintenance
regression
of
masculinization
1965,
1970).
that
the fetal
rodents
secrete
shortly
that
the mammain
responsi-
differentiation
for
ducts,
ducts,
talia
the
accepted
have
the
of
the
tract
sex
differ-
the ovaries
reproductive
(Price
and
Ortiz,
1969).
Supported
in part by a Grant
Medical
Research
Council,
Send reprint
requests
to J.I.R.
(MA-3566)
Canada.
#{174}
1973
of
by
reproduction
The
Society
in any
for
form
histochemical
studies
of hy-
dehydrogenase
and
Raeside,
activity
1972)
of the
during
fetal
the
life
of
in
pig,
Reproduction.
strongly
fetal
pig testes
thesize
steroidal
have
the
hormones
stage
development.
in
their
report
fetal
describes
pig
gonads
ventral
prostate
gland
test
has
for
the
testes
her
associates
fetal
suggest
The
(Price
in media
of
for
been
of rodents
containing
rats
in
secretion.
with
success
by
Ortiz,
prostate
present
of early
from
the
young
androgen
used
and
that
capacity
to synat a very
early
the
cultivation
with
explants
bioindicator
The
method
cultivated
the Study
reserved.
on the differenti(Moon
and Hardy,
droxysteroid
testis
(Moon
et a!., 1969).
Explants
of rat
from
330
Copyright
All rights
and
studies
cells
gland
Price
1965;
were
testosterone,
and
Price
also
a
ANDROGEN
in
to
order
to
testosterone
compare
with
developing
the
the
gonads.
prostate
tissue
androgenic
studied
by
1965;
of rodent
the
in
has
been
(Lasnitzki,
1968;
a!.,
of
presence
other
steroids
investigators
et
response
to the
behavior
vitro
and
several
Baulieu
type
of
response
The
in
SECRETION
Robel
et
a!.,
OF
FETAL
to
5% CO
in
of
culture
was
were
the
during
explants
the
culture
AND
METHODS
fetuses
from
sows
used
in
a
slaughter
at
these
selected
were
Yorkshire
Fetuses
ranged
from
(C-R)
length.
Fetal
examining
external
C-R
lengths,
studies
fluid
containing
and
stained
house,
or
2.0
to
sex
4.5
genitalia
and
of
from
4.0
testis
animals
crown-rump
be
organ,
judged
and
by
4.5
morphology
of prostatic
the
present
Rats
where
into
its
explants
as
glands
size
and
was
for
teased,
of
were
cut
soluapart,
appropriate
and
bioindicator
the
medium
test.
mixture
and
tract
(from
seemed
tissue
to
one
improve
one
of
50%
with
part
chicken
embryos).
the
cultivated
studies
of
part
11-day
when
on lens
For
This
maintenance
alone.
tissue
paper
which
medium.
The
culture
Explants
rested
dishes
testosterone
of
the
above
embryo
ex-
organs
of pig
were
fe-
culti-
and
adrenals
from
The
normal
differentia-
in
placed
surface
1 mm
organ
the
on
pig
cultures
has
highly
of
of
prostatic
tissue
organs
and
of
columnar
of
et
al.
were
(b)
acini,
with
tissue,
amounts
folded
of
squamous
low
Where
response,
a
as
fetal
a
occurred
were
reflection
of
ethanol
in-
attempt
for
of
medium
199
in
rat
was
the
overall
production
for
6
days
prostate
with
were
activity,
for
m
were
grown
ethanol
made
and
principally
proliferation
comparison
made
bovine
0.2%
with
con-
dissolved
dilutions
10%
total-
medium
was
serial
containing
contahiing
prostate,
Testosterone
and
niedia
media
ventral
tain the desired
concentrations.
of ethanol
in all culture
media
epithelium,
with
androgen
grown
cultured
absolute
secretory
(d)
acini,
greatly
an
subjectively
Explants
testosterone.
examinations
tissue;
small
tissue.
score
explants
of
or
large
organ.
taining
in
cuboidal
epithelium;
connective
stimulation
give
with
relatively
ally
cuboidal
interacinous
with
connective
genei
large
incompletely
acini,
interacinous
or
to
connective
epithelium;
regressed-
to
connective
some
(c)
large
with
supranuclear
secretions,
sparse;
columnar,
creased
rarely,
interacinous
relatively
or
acini
large
epithelium;
(a)
with
interacinous
without
gressed-moderately
from
acini
stimulated-moderately
columnar
grading
follows:
many,
and,
sparse
or
for
as
or
fetal
prostatic
modified
(1966),
all,
of
the
used
large
present;
tissue;
65,
114
characteristics
criteria
explants
epithelium
zones
the
and
were
stage
of
histological
1. The
secretions;
made
alone,
gonads
cultivated,
classification
basis
Zaaijer
without
ing
cultivated
stimulated-generally
low
contact
experiment,
numbers
with
in Table
of
clear
in
every
were
total
appearance
those
thickness,
In
prostate
the
shown
the
in
fragment.
numbers
explants
are
one
prostatic
exposed
were
eosin.
prostate
gonads
development,
explants
were
in para-
sections
and
with
of
Zenker’s
respectively.
The
pig
of
were
in
-R.
cultivated
14,
modification
on the
embedded
The
end
ovaries,
cm
The
Prostate
testosterone.
selected
microscope.
consisted
of
controls.
and
by
Falcon
plastic
organ
culture
dishes
were
used
with
medium
TC
199
(Difco)
containing
10%
bovine
serum
(Grand
Island
Biological
Co.)
for
the
fixed
ion.
ventral
of
the
acid,
grown
prostate
adrenals
aseptisalt
gently
Pieces
explants
dissecting
and
were
removed
were
6
rat
isolated
completely
glands
balanced
subdivisions.
shape
the
were
Hanks’
gland
1.
dislocation
prostate
appearance
At
hemaluin
exceeding
the
cm
cultivation
Fig.
cervical
to
each
natural
for
in
ventral
transferred
tion
under
The
taken
shown
by
the
free.
and
is
killed
containing
dissected
tissue
study
were
tissues
the
at
testes,
not
with
for
fetuses
from 2.5 to 3.5 Clfl C-R. In all cases additional
confirmation
was made
for each fetus from
the histology
of the
contralateral
gonad
which
was not cultivated.
With
2.0 cm C-R
fetuses
the
sex was recorded
after the gonad
had differentiated
into an ovary or testis in culture.
Ventral
prostate
glands
of 21- to 28-day-old
postnatal
rats of the Fischer
strain
were
chosen
as the androgen
responsive
tissues.
A detailed
description
of the histology
of the rat ventral
prostate
gland
has been given
by Price
and Williams-Ashman (1961).
Briefly,
peripheral
acini of rat prostate glands
at 21-28
days are usually
small
and
have
a high epithelium.
In some
acini which
arc
distended
with
secretion
the epithelium
is lower.
Acini of the inner
area of the gland
are generally
smaller,
with an epithelial
height
slightly
less than
in those
of undistended
peripheral
acini.
The ap-
pearance
the
Duration
experiments.
been
demonstrated
(Moon
and
Hardy,
in press).
Each
culture
dish
contained
a whole
organ
(from
2.0 cm
C-R
fetuses)
or part
of an
crossbreds.
cm
could
of
already
obtained
where
Yorkshire
on
explants
of 2.0-4.5
the
series
cultivation.
Mayer’s
with
at 35CC.
all
3% acetic
with
of
in
made
sectioned
fetuses
were
maintained
period,
ffin,
tion
All
of
and
6 days
Observations
vated
MATERIALS
cally
air,
331
Prostate
explants
tuses.
Explants
of
1971).
in
TESTES
serum,
The
was
in
using
to
ob-
concentration
0.2%. Control
without
also.
testosterHistological
on
of
the
cell
the
height,
prostatic
findings
of
332
MOON,
HARDY,
EXPLANTS
OF
TABLE
RESPONSES
OF
IN
Mean
C-it
length
(cm)’
and
CONTACT
WITH
type
PROSTATE
ORGANS
OF
No.
number
of
1
RAT
Total
of organ
RAESIDE
AND
GLAND
Pio
of explants
in wh ich prostate
Highly’
explantsb
CULTURED
FETUSES
gland
was
Incompletely
Completely
stimulated
Stimulated”
regressed
regressed
Testes
2.0
9
3
4
2
0
2.5
17
10
7
0
0
3.0
17
15
2
0
0
3.5
6
0
5
1
0
4.0
6
0
6
0
0
4.5
7
1
2
4
0
2.0
12’
0
0
3
9
2.5
9
0
0
1
3.0
12
0
0
1
8
11
3.5
5
0
0
0
5
4.0
8
0
0
0
8
4.5
6
0
0
0
6
2.0
7’
0
0
0
7
2.5
7
0
0
0
7
Ovaries
Adrenals
‘Mean
Only
C-R length
± 0.25
one organ
from each
‘Whole
organs
d Subjective
were
cm.
fetus
was
used
for explants.
cultivated.
scores
for
the
overall
response
rose
to
highest
values
for
fetuses
of
than
in
2.5
and
3.0
cm
C-R
length.
Robel
were
et a!. (1971).
graded
subjectively.
Responses
these
in
criteria
RESULTS
Prostate
explants
pig
fetuses.
which
with
rat
fetal
Results
to
are
tissues
grown
to 4.5
or highly
plants.
It
activity
the
for
of
with
organs
with
the
C-R
length
stimulated,
was
noted
was
greater
in
were
grown
adrenals
for
test the androgenicity
shown
in Table
1. The
cm
of
experiments
prostate
explants
testes,
ovaries,
and
6 days,
organs,
2.0
grown
fetal
of these
prostatic
testes
were
from
fetal
testes
from
2.5 and 3.0 cm C-R
stages
and declined
older
fetuses.
The
acini
in prostate
ex-
plants
cultivated
with
larged
geneous
and most
secretions.
of them
These
occupied
a much
greater
fetal
testes
were
en-
contained
distended
homoacini
proportion
of the
tissue
and,
tissue
consequently,
was sparse
were
cuboidal,
fetal
or columnar
stages
cultures
control
interacinous
connective
(Fig.
2). Epithelial
cells
sionally
with
supranuclear
few
large
explants
of tests
showed
(Fig.
3),
clear
from
zones.
different
degeneration
occa-
in
A
the
center
of the explant,
but this did not seem
to be reflected
in the prostatic
response.
Ovaries
from
2.0 to 4.5 cm C-R
fetuses
which
grew
stimulated,
in 55 out of 62 exthat
the
androgenic
for
prostatic
were
well
stimulate,
explanted
but
the
with
prostatic
did not maintain,
rat prostate.
In
tissue
let alone
all sections
of such
explants
the acinar
epithelium
markedly
regressed.
Most
lumina
were
row,
usually
with
a folded
epithelium
had
naror
some
cell
loss
debris.
The
considerably
4,
of cytoplasm,
and
connective
throughout
tissue
the
contained
had increased
explant
(Figs.
5).
Adrenal
glands
were
well
maintained
as
ANDROCEN
explants
from
while
the
separately,
the
fetuses
of 2.0
gonads
to
were
to 2.5
being
determine
cultivation
SECRETION
No
C-R
at the
end
evidence
drogen
glands
secretion
was
from
male
or
shown
female
judged
explants
from
the
accompanying
which
had
completely
(Table
After
1).
6 days
explants
grown
alone
dergone
slight
regressive
of
of an-
by adrenal
fetuses,
as
prostatic
regressed
of cultivation,
The
as
the
had
changes
the
either
containing
were
filled
philic
material
thelial
a
and
7),
or
with
eosino-
a marked
the
epi-
high
doses
DISCUSSION
cause,
ventral
prostate
androgenic
for
while
it
was
is normally
chosen
activity
as
be-
maintained
in
vivo by testicular
hormones,
it can be stimulated
under
in vitro
conditions
by androgenic
hormones
glands
cal
(Zaaijer
of
The
present
a very
1966).
and
prostate
of
fact,
early
evidence
testis
from
was
test
in
its
have
development.
provided
gonad.
et
1968).
from
the
secretion
in vitro
A similar
study
Black
the
domestic
occur,
rat
the
served
that
early
pig.
appar-
laboratory
anicondensed
time
A decline
in
in
the
in vitro
testes
has
and
Tuilner,
been
1965;
Steinberger
observations
and
raise
chemical
of
testes.
identity
early
and
androgenic
in fetal
Christensen
guinea
(1969)
undifferentiated
days,
possessed
a role
in the
the
or be
studies
and
points,
for
however,
formation
significance
of the fetal
ultrastructural
in
decline
slightly
pattern
1966;
These
about
pigs,
testes
structures
biosynthesis
ob-
at 22-24
consistent
of steroid
with
hor-
mones.
A steroid
ular
secretions
nature
for the fetal
testicmight
be implied,
at least
for
stages,
later
fetal
radioactive
rabbit,
and
steroid
rhesus
from
measured
day
ture
from
of
fetal
30
of
of
gestation,
the onset
evidence
genases
in
fetus,
as
fetal
11-day
(Baillie
et
1964).
In
(Moon
and
genital
and
gnenolone
a!.,
a
as
which
is
early
5
days
of sexual
differentiation.
about
the chemical
the
the
androstene-
sheep,
of early
secretions
is
histochemical
studies.
and 17$-hydroxysteroid
human
rat,
et
1965;
Resko,
(1969)
has
and
gonads
with
in the
(Noumura
Tullner,
Attal
testosterone
in
studies
precursors
monkey
a!., 1966; Lipsett
and
1970).
More
directly,
rats
testis
of the
hormones
that
androgen
the
differentiation
indifferent
al.,
before
Indirect
results
that
the
androgenic
stage
Noumura
Ficher,
was
present
not
and
(Lipsett
cytologiand
may
re-
the inhighly
subsequent
in the
in
in the
rabbit
demonstrated
as
most sensiof andro1961).
the
been
differentiating
the
postnatal
period,
amount
of testosterone
dione
adrenal
The
experimental
clearly
secrete
can
bioindicator
occurs
during
of
the
testes
is known
to be one of the
indicators
for the presence
(Price
and Williams-Ashman,
demonstrated
pig fetus
In
the
et a!.,
structure
mice
tive
gens
at
of
a pattern
In
8).
The
rat
a bioindicator
in
has
which
days,
was
secretion
biological
products
acini
it
to a biphasic
questions
most
in
ent, prenatally
in the small
mals
which
have
a more
scale
in their
development.
showed
homogeneous,
(Fig.
hyperplasia
(Fig.
fetuses
con-
the
pigs
(1970)
at 22-23
when
sumgrown
in which
columnar
with
are
testosterone
condition,
was
bethan
fetal
in medium
The findings
2. The
explants
a stimulated
epithelium
cells
abundant
with
the
guinea
culture
as a testis.
The
in androgen
production
Such
un-
in
tissue
grown
testosterone.
in Table
in medium
Price
gonad,
androgenic
older
prostatic
controls
connective
explants
tai fling
marized
fetal
by
androgen
tween
the acini
were
more
in those
explants
grown
testes.
Prostate
with
ported
different
333
TESTES
however,
acinar
epithelium
(Fig.
6). Serial
sections
showed
that
the
area
occupied
by acini
was
greatly
reduced
in many
parts
of the
explants.
FETAL
study
cultivated,
sex
period.
cm
OF
also
The
ridge
a weak
of
na-
provided
presence
dehydroa 1.4
cm
reaction
with
substrate
in
gonad
mouse
have
been
reported
Baillie
and
Griffiths,
1966;
recent
Raeside,
the
histochemical
1972),
preof
study
some
53f3
334
MOON,
FIG.
acinar
1.
size,
Portion
folded
gland. x656. All
Fic. 2. Part of
portion
of
prostate
of
prostatic
epithelium,
figures
testis
gland
are
tissue
as
and
abundant
of 6 m
explanted
of
AND
HARDY,
sections
from
25-day-old
a
control
RAESIDE
from
stained
a 3.0
rat.
cm
with
C-R
Stimulation
rat
a
interacinous
26
days
connective
hemalum
Mayer’s
fetus
and
of
of
the
age.
tissue
cultivated
prostate
of
and
Note
an
small
immature
eosin.
for
is
6 days
with
shown
by
the
ANDROGEN
Fic.
small
6.
A
acini
Fic.
7.
An
showing
g
of
8.
An
from
columnar
explant
cultivated
x
25-day-old
epithelium
enlarged
tissue.
Fic.
explant
low
parts
connective
25
prostate
with
for
acini
OF
SECRETION
rat,
and
6
with
days
on
columnar
explant
of
rat
3.
gland
marked
epithelial
of
low
of
prostate
cuboidal
loss of cytoplasm.
Fic. 5. Higher
epithelium,
(homogeneous,
magnification
columnar
4. Part
for
containing
6
days,
showing
x 656.
tissue.
6.25
g
testosterone/mI,
secretion,
and
sparse
6
on
medium
interacinous
cultivated
finely
for
were
not
(Allen,
days
x
hyperplasia.
yet
intensity
granular,
reactions
2.5
and
histologically
were
at maxi3.0
cm
C-H
and
interacinous
recognizable
The
1904).
mum
containing
656.
at
eosinophilic),
sparse
x 76.
Higher
acini,
Fic.
portion
with
secretions
tissue.
Fic.
of two
medium
epithelium,
prostate
showing
with
acini
alone
connective
1200.
testosterone/mI,
connective
335
TESTES
cultivated
abundant
and 17$-hydroxysteroid
dehydrogenase
activity
was found
in gonads
of 1.5 and 2.0
cm G-R pig fetuses,
that is, when
the testes
large
FETAL
glandular
of
epithelium,
ovary
explanted
gland
of
epithelium,
Note
abundant
magnification
material
portion
of
and secretion.
from a 3.0
25-day-old
and
rat.
no
secretion
prostate
gland
shown
in
Fig.
2,
showing
parts
x 1200.
cm
C-R
The
fetus
and
considerably
but
interacinous
connective
of portion
of prostate
in lumen,
and abundant
some
contain
cultivated
for
6 days
regressed
acini
are
cell
tissue.
Compare
gland
shown
in
connective
tissue.
debris
from
with
small,
partial
with Fig. 2. x 78.
Fig. 4, with
cuboidal
x
1200.
336
MOON,
ILAIWY,
AND
TABLE
EFFECTS
OF
TESTOSTERONE
IN
ON
ORGAN
RAESIDE
2
RAT
PROSTATE
CULTURE
EPITHELIUM
6
FOR
GROWN
DAYS
Concentration
in
medium
No.
(ug/ml)
25.0
12.5
6.25
3.125
‘Number
and
5.0
levels
at
vide
a very
in
by
declined
C-R.
These
of
the
bioindicator
test
early
fetal
the
to reflect
androgen
for this
No evidence
of
adrenal
glands
experiments
to the
activity
studies
appear
pattern
of
(20)’
± (14)
± (15)
which
low
Baulieu
pro-
changes
as
mea-
(Table
1).
the same
secretion
biin
pig
secretion
by
in the present
fetuses,
from
(Table
1). In contrast,
Ortiz
cultivated
fetal
adrenal
glands
of male
and female
guinea
pigs with
rat prostate
explants
and
found
prostatic
fetal
adrenal
not seem
to
all stages
according
glands
secrete
tested
to Jost
of rabbits
important
androgenic
hormones
logical
conditions.
under
(22-30
(1953),
and rats
amounts
normal
cultivation
physio-
in the bioindicaearly
fetal
ovaries
Steroid
malian
period
hormone
fetal ovary
formation
has not
strated
biochemically
and, in histochemical
xysteroid
seen
early
1967).
The
results
with testosterone
fetal
of
yet
1970).
in the
mambeen
demon-
in early
pregnancy;
studies,
5-3$-hydro-
dehydrogenase
in
(Price,
pig
activity
was
ovaries
(Dufour,
the
present
are similar
this
at.
(1968)
by the
A more
ity
of fetal
Robel
et
a!.
fetal
pig testes
answer
cannot
are
yet
factors
activ-
testes.
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the
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No androgenic
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by
of
of
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the
earlier
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et
a!.
(1966)
maintenance
at
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-
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++
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± (14)
of explants
species.
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was seen
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to
findings
androgenic
Both
phasic
life
number
Secretion
-
++
rapidly
parallel
height
+++
indicates
striking
amount
sured
then
cm
Hyperplasia
4
28
16
17
in parentheses
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SECRETION
ACKNOWLEDGMENTS
The authors
thank
J. M. Schneider,
Ltd., Kitchener,
Ontario,
for providing
the facilities
for the
collection
of the pig fetuses.