estrogen on the pituitary growth hormone response to hypo- thalamic growth ..... over the. 15-mm sampling period, in male rats treated with testosterone was.
BIOLOGY
OF
REPRODUCTION
The Effects
32,
369-375
(1985)
of Testosterone and Estrogen on the Pituitary Growth Response to Growth Hormone-Releasing Factor
WILLIAM
B. WEHRENBERG,’
ANDREW
and Laboratories The
for
Salk
BAIRD,
NICHOLAS
YING
LING
Neuroendocrinology
Institute
for
La Jolla,
SHAO-YAO
Hormone
Biological
California
Studies
92037
ABSTRACT The
effects
testosterone and estrogen on the pituitary growth hormone response to hypohormone-releasing factor (GRF) were evaluated in vivo using male and female rats and in vitro using a pituitary cell monolayer culture system. In vivo the increase in plasma growth hormone (GH) concentration in response to a 500 ng/kg dose of GRF was similar in gonadectomized male and female rats. Pretreatment of intact and gonadectomized male rats with testosterone caused significant enhancement of the pituitary GH response to GRF, whereas pretreatment of gonadectomized female rats with 17j3-estradiol did not alter the response. The GH response to GRF was not different between prepubertal (i.e., 30-day-old) male and female rats. However, following puberty (i.e., by 60 days of age), the response in male rats was significantly greater than that observed in female rats. The in vitro preincubation of anterior pituitary cells with either testosterone or 1713-estradiol did not cause any shift in the dose-response curve between GRF and GH. These results demonstrated that androgens play an active role in modulating the pituitary response to GRF in vivo.
thalamic
of
growth
INTRODUCTION
Males
of
humans than
and their
most laboratory
female
in weight are potent anabolic olic processes 1972; Perry
species,
rodents,
are
counterparts. known effects
et
It
vertebrate
to
Sex
be due to of androgens
role the of normal
pathways
(see
review,
example,
female
rats
during the tion weigh (Swanson Bell and medial more
critical more and Van Zucker,
Accepted Received
than
McEwen, injected
direct and on metab-
in the system For
androgens
Ten Bosch, Following
lesions, rats
female (Cox
et al.,
The Salk CA 92138.
to
1966;
Illig
stimuli and rats
that
release humans
growth
to have (Shupnik
1984)
or on the
GH
response
et
al.,
1979).
growth been
investigated
the pituitary
in rats (Somana pituitary GH GH
through
(Deller
et al.,
1970; Eastman et a!., (Jansson et al., 1982). Estrogens little or no effect on GH synet al., 1979; Copeland et al.,
and
the
gonadal
on
Prader,
1971)
has
the
in both
appear thesis
on
that
effects
GH content enhances
pathways
thalamic (GRF)
to indirect Because
the
hormone-releasing isolated and characterized, actions GH
of
the
response
gonadal
stimuli hypofactor we steroids
to GRF.
gain
1969).
MATERIALS
AND
METHODS
Sprague-Dawley rats were used in these experiments. Animals were housed in a temperature (2122#{176}C)- and humidity-controlled vivarium with a 14L: 1OD lighting schedule (lights on at 0600 h). Food and water were freely available.
October 15, 1984. June 25, 1984.
‘Reprint requests: 85800, San Diego, 453-4100, Ext. 394.
their
and
(Negro-Vilar
1963; ventrorats
1978)
indirect
in growth through an
suggested
pituitary
aI.,
response
for sexual differentiathan control females
der Werff 1971). male
et
been
mediate
regulating growth hormone (GH) and release. Androgen treatment
increases
et al., the rat
1976).
also
might
through synthesis
differences
with
has
steroids
larger
sex steroids have central nervous
period as adults
hypothalamic weight
much
in target tissues (Schemmel al., 1979). Evidence in
also suggests that the difference between males and females is organizational development
including
Institute,
Telephone:
P.O.
In
Box (619)
Vivo
Methods
Animals
with 369
ether
were
at least
gonadectomized 2 wk prior
to
while anesthetized any further experi-
370
WEHRENBERT
mentation.
Steroid
at the time daily between rats received
replacement
therapy
was
initiated
of
surgery and subsequently performed 0800 and 1000 h. Testosterone-treated 1.0 mg of testosterone propionate per day s.c. in 0.1 ml sesame oil, estrogen-treated rats received 10 g of estradiol benzoate per day s.c. in 0.1 ml sesame oil, and control rats received 0.1 ml sesame oil per day s.c. The pituitary GH response to GRF was evaluated in sodium pentobarbital-treated rats (50 mg/kg, i.p.)
by quantitating plasma GH concentrations, as previously described (Wehrenberg et al., 1982b). Experiments began at 0900 h and were completed in approximately 2 h. The design was as follows. Five to 10 mm after pentobarbital administration, the animals were fitted with a polyethylene catheter (0.28 mm i.d. X 0.61 mm o.d.; Clay Adams, Parsippany, N.J.) that was placed in an external jugular vein. One of heparin were then injected i.v. A
hundred blood
units sample
was drawn 15 mm after pentobarbital administration. This was immediately followed by administration of 500 ngfkg GRF i.v. In the initial experiments, 8 blood samples were drawn over the next 15 mm to monitor the pituitary GH response to GRF. This was reduced to
5
blood
samples
in
later
experiments
since
the
pituitary response could be accurately monitored with fewer samples. The GRF used in these experiments was the synthetic replicate of the 44-amino acid peptide isolated from human hypothalamic tissue (Ling et al., 1984). The peptide was dissolved in water and diluted to a working concentration of 500 ng/ml with sterile saline immediately before use. It has been shown previously that the GH response in rats to human Ling,
and 1983).
rat
GRF
is not
different
(Wehrenberg
and
ET
AL.
determined by analysis of variance, with consideration given for repeated measures since multiple observations were made in each animal (Winer, 1971). RESULTS
Figure
1 illustrates
(panel
testosterone The mean the
period, was
significantly response
greater observed
(345
± 24
the
Vitro
The anterior bioassay where estradiol
exact procedures for the preparation of rat pituitary cells in monolayer culture and the for GRF have been described in detail else(Brazeau et al., 1982). Testosterone and 173at 1 MM or 0.01 MM concentrations were
added to following serum-free incubation immunoassay
Radio
the cells on the third day of culture. The day, the cells were washed twice with medium and treated with GRF for 3 h. The medium was then collected for the radioof GH.
immunoassay
Methods
Plasma concentrations of GM were determined by radioimmunoassay using a double antibody method with reagents provided by the National Institutes of Health, with the exception of the first antiserum, which was provided by Dr. V. Sinha (Sinha et al., 1972). The within-assay variation was 7% and the between-assay variation 10%. Data are presented as mean ± SEM. In vitro treatments were performed in triplicate and in vivo treatments were carried out in groups of at least 5 animals. Multiple dose-response curves in the in vitro bioassay were analyzed by regression analysis and their relative potencies calculated using the BIOPROG procedure described by Rodbard (1974). Since the variances of the data from the in vivo studies were not homogeneous, the data were subjected to logarithmic transformation prior to statistical analysis. Significant treatment effects were
800
± (P
