Vol. 91, No. 1, 1979 November
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14, 1979
Pages 284-288
STER0ID HORMONES MODULATE PROLACTIN BINDING BY CULTURED PORCINE GRANUL0SA CELLS James M. Hannnond and Elizabeth
Krall
Department of Medicine, Division of Endocrinology The Milton S. Hershey Medical Center The Pennsylvania State University Hershey, Pennsylvania 17033 Received
September
19,
1979
SUMMARY: We have studied the effects of the gonadal steroids - testosterone, 17&estradiol, progesterone, and Sol-dihydrotestosterone on the prolactinbinding activity of porcine granulosa cells maintained in monolayer culture. Testosterone, estradiol, and progesterone all significantly enhanced prolactin binding (55%, 107%, and 112% above control, respectively). In contrast, the non-aromatizable androgen, 5a-dihydrotestosterone, caused an insignificant suppression of prolactin binding. The anti-androgen, cyproterone acetate, did not influence prolactin binding when used alone, and did not inhibit the effects of testosterone. These data suggest that the stimulatory effects of testosterone may require aromatization to estradiol. INTRODUCTION Previous
studies
from
("receptors")
sites
and corpora ical
lutea
for
in vivo,
during
the
reproductive
have
examined activity
binding
to porcine
MATERIALS
since
binding
the
cycle role
by assessing granulosa
have
on freshly
This
a potential
receptor
laboratory
prolactin
(1,2).
control
this
isolated
activity
number
appears
(1,2).
for
ovarian
steroids
the
effect
of these
cells
porcine
of receptors/cell
and pregnancy
maintained
specific granulosa
to be under changes
In the current as regulators hormones
in monolayer
binding cells
physiolog-
substantially study,
we
of prolactin
on prolactin culture.
AND METHODS
Materials: Ovine the Hormone Distribution Schering AG, Berlin. Corp. Tissue culture bovine serum albumin, Schering Corporation; Na[l251] was purchased sterile solution from
prolactin (oPRL) (NIH-Sll and NIH-S12) was provided by Office, NIAMDD. Cyproterone acetate was a gift of All other steroids were purchased from Sigma Chemical medium and fetal calf serum were from Flow Laboratories: fraction V, from Miles Laboratories; gentamicin from the amphotericin B and mycostatin from Squibb. Carrier free from New England Nuclear. Lidocaine was obtained in Astra Pharmaceuticals.
Culture Techniques: Porcine granulosa cells (l-3 mm) porcine follicles as previously described cells were inoculated into 100 mm culture dishes 199 with Hanks salts, a bicarbonate buffer system, gentamicin (SOpg/ml), amphotericin B (2.5 us/ml), 0006-291X/79/210284-05$01.00/0 Copyright All rights
demonstrated
@ 1979 by Academic Press, Inc. in any form reserved.
of reproducti&
284
were isolated from small (1). Approximately lo7 (Falcon) containing Medium 10% fetal calf serum, and mycostatin (50 U/ml).
Vol. 91, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMIJNICATIONS
The cultures were maintained in monolayer for Co2 atmosphere. The medium was changed every added in absolute ethanol (0.5% v/v); control volume of ethanol.
8 days at 37 C in a 95% air-5% 48 hr. Steroid hormones were cultures received an equivalent
oPRL Binding Assay: The binding of oPRL to cultured qranulosa cells was assessed by the methods previously used with freshly isolated qranulosa cells (1) with a few exceptions. Initial studies showed that cells removed from culture plates by a 20-30 min incubation with the local anesthetic lidocaine binding characteristics which were superior to those (3.4 ti) (3), exhibited of cells assayed directly on culture plates or dispersed with trypsin, EDTA, or mechanical scraping. After dispersion with lidocaine, cells from 20-30 control and hormone-treated cultures were pooled, washed twice in medium 199, hepes buffer, pH 7.6, 1% bovine serum albumin, and aliquoted into plastic assay tubes containing the same medium and [ 1251]iodo-oPRL (50,000 cpm or 0.6 nq) in the presence or absence of saturating concentrations of oPPL (2 uq/ml). Preparation and validation of tracer hormone, incubation conditions and computation of specific binding have been described in detail (1). Four to six measurements of specific binding expressed per mq DNA (4) were made for control and hormone-treated cells in each experiment, and the significance of differences computed using Student's t test. The overall significance of a treatment effect was assessed using a chi-squared statistic derived from significance values for a series of individual experiments (5). For comparison, data from different experiments has been normalized as percent control (Table I). RESULTS Data from effect
all
of testosterone
qranulosa
cells
estradiol
appears
testosterone showed are
the experiments
and 17$-estradiol
were
observed
question,
differences
of aromatizing
or after
Sa-dihydrotestosterone.
the
a modest,
anti-androqen,
activity these
equivalent
quantitatively
(6),
the
with
that
exerts
to cultured Although
the
Since
qranulosa
with
testosterone
of binding. Further,
prolactin
testosterone. in this
this
androqen,
was suggested.
effects
could
To address
non-aromatizable
to alter
cells
effect
to show stimulation
failed
than
two hormones
testosterone
effect
17S-
binding
to estradiol. the
failed
acetate,
aromatization also
shown).
or in combination
suggest
after
not
inhibitory
cyproterone
experiments
Progesterone
steroid
insignificant,
when used alone
indirectly,
is
This
binding
comparing
conversion
two experiments
The stimulatory
of prolactin
directly (data
I.
experiments.
stimulator
testosterone
directly
we performed
Instead,
each of several
two experiments
no significant
have been exerted
in
in Table
on prolactin
to be a more potent
overall,
capable
are compiled
binding Collectively,
system
are
exerted
binding
which
to estradiol. a stimulatory to
that
encountered
285
effect
on prolactin with
testosterone
or
estradiol.
Vol. 91, No. 1, 1979
BIOCHEMICAL
TABLE I.
Effect of Steroid Treatment by Cultured Granulosa Cells Specific
Testosterone (500 nq/ml) Estradiol (500 ns/ml) Dihydrotestosterone (500 ns/d) Cyproterone acetate (5 vs/ml) Progesterone
on Prolactin
PRL binding f SEM)
(% control
(500
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Binding
No. of Experiments
*p (treatment vs. control)
155 f 42
10
c.01
207 _+ 87
3
e.01
61 * 27
2
>.5
103 + 64
4
>.9
212 f 73
6
c.01
108 f 73
4
>.9
nq/ml)
Testosterone CypZerone + Testosterone *
acetate
see Materials
and Methods
DISCUSSION Previous receptor tissue
studies
from
this
of
freshly
concentration varies
with
specific
prolactin
declines
with
corpora
lutea
binding
follicular
maturation
receptor
concentration
of luteal
hormonal
control
of prolactin
We have found
dihydrotestosterone, milieu
of granulosa (10)
of putative
In particular, of prolactin knowledge, the modulation
cells
estradiol, receptors the
tissue
high
current of ovarian
in rat studies
in
for
have been
prolactin
FSH, has been cells the
receptors
286
functioning
fluid,
seemed logical
or luteal
--in vivo first
testosterone,
(81,
(13).
--in vitro
by steroid
physiological
androgens
(9),
cells,
and a
to each
implicated
follicles,
suggested
the
assigned
of
in the prolactin-
steroids
estrogens
functions
constitute
in
of 178-estradiol,
in granulosa
granulosa
immature
observations
follicular
and luteal
from
increase
These
described
with
cells The number
again
and ovarian
Receptors
along
ovary.
a further (2).
the prolactin
granulosa
in cells
concentrations
(7).
regulatory
high
occasions
receptors,
have been
of the
and increases
and progesterone
and progestins number
is
shown that
porcine
state
sites/cell
Pregnancy
have
isolated
the physiological
(1,2).
candidates.
laboratory
(11,12).
in the control However,
to our
demonstration
hormones.
of
Vol. 91, No. 1, 1979
Such direct which
--in vitro
regulate
of hepatic
however,
receptors
induces
is
pertinent
it
themselves the
binding
after
explain
sane of the
for
(16),
estrogens
(17).
binding
Using
monolayer
binding
activity,
synergistically (veldhuis changes
suggests
Granulosa
have been
to those
and Hammond, unpublished). concentrations
thecal
exerts
These described
invoked
ovary
a direct
to
--in vivo
effect
to study
prolactin
interact
by porcine
here
prolactin
to synthesize
here
observations
Results
in 17ci-hydroxy-
and prolactin
secretion
alter
to be accounted
elements
employed
estradiol
progesterone
rat
are deficient
probably
cultures
to stimulate
in receptor
from
cell.
that
in the
are unlikely
by the granulosa
we have noted
administered
on the
of
cells,
pathways
cells
progesterone
similar
cultured
increases
testosterone
induction secretion
testosterone
Similar
derived
of the
steroids
of progesterone
androgens
estrogen
As discussed that
of exogenous
Consequently,
on prolactin
the
interactions
the
Even with
metabolites.
effects
hormonal
stimulation
(14).
whether
to estrogens.
and require
involves
to estradiol.
actions
complex
For example,
own receptor
evidence
the
by conversion
--in vivo
active
conversion
avoid
--in vivo.
to inquire
available
In contrast,
lase
its
or via
section,
(15).
binding
which
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
observations
prolactin
lactogen
prolactin
binding
BIOCHEMICAL
granulosa
suggest
that
cells the
may have physiological
relevance. ACKNOWLEDGEMENTS The authors appreciate the provision of ovine prolactin by the Hormone Distribution Program, NIAMDD, and of cyproterone acetate by Scheriny, AG. We also thank Dr. Marshall Jones, Department of Behavioral Science, The Milton S. Hershey Medical Center, for valuable statistical advice, Dr. Johannes Veldhuis for reviewing the manuscript, and Mrs. Marlene Thompson for secretarial assistance. This
work
was supported
by NIH Grant
No.HDlOl22.
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