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Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Dr. Stephen J. Winters, Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh, Montefiore N919, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213. E-mail: winters{at}med1.dept-med.pitt.edu
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Abstract |
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-subunit (P
< 0.01) mRNAs, whereas E increased -subunit (P
< 0.01), but did not alter LHß mRNA levels in rat cells. In monkey
cells, however, neither T nor E affected LHß or -subunit mRNA
levels significantly. Our results identify different regulatory
mechanisms by which testicular steroid hormones control LH secretion by
the pituitary in male primates and rodents. We propose that the primary
site of androgen negative feedback in the male primate is to restrain
GnRH pulsatile secretion, whereas in the male rat T also decreases
gonadotropin synthesis and secretion by directly affecting the
pituitary. E suppresses GnRH-stimulated LH secretion in the primate
pituitary, but amplifies the action of GnRH in the rat. Our data also
reveal that the action of T to suppress LH secretion and subunit mRNA
in male rats is not through decreased GnRH-R gene expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most of our knowledge about gonadal steroid regulation of
pituitary gonadotrophs at the cellular and molecular levels is from
studies in rats, in which castration increases (14) and T replacement
decreases (15) LH secretion, through changes in GnRH receptor (GnRH-R)
binding (16) and gene expression (17), as well as gonadotropin subunit
messenger RNAs (mRNAs) (18). The present experiments were performed to
begin to understand the molecular mechanisms subserving the hormonal
regulation of gonadotropin secretion in men using pituitary cells from
male rhesus monkeys, a representative nonhuman primate. We have
established an in vitro model of primary pituitary cultures
perifused with pulses of GnRH to clamp GnRH stimulation and eliminate
the effects of bioconversion of administered T into DHT and E in
peripheral tissues, nonsteroidal gonadal factors such as inhibin, and
hypothalamic modulators of GnRH action such as pituitary adenylate
cyclase-activating polypeptide (19). A constant flow of medium around
the cultured pituitary cells also reduces pituitary autocrine/paracrine
factors such as activin and follistatin (20). To determine whether
gonadal steroids affect the male primate pituitary directly, we
examined the LH response to pulsatile GnRH stimulation as well as
GnRH-R, LHß, and -subunit mRNA levels in dispersed, perifused
pituitary cells from intact adult male monkeys and compared the results
to those in adult male rat pituitary cells.
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Materials and Methods |
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Perifusion
Dispersed cells were allowed to attach to the surface of
preswollen Cytodex 3 beads (Pharmacia Biotech, Piscataway,
NJ) at a ratio of 5 x 106 cells:100 mg beads:30 ml
DMEM containing 10% dextran-charcoal-treated FCS (control medium) in
three 10-cm siliconized glass petri dishes at 37 C in 5%
CO2-95% air. After 48 h, in Exp 1 the three cultures
were treated with 10 nM T (Sigma Chemical Co.), 0.1 nM E (Sigma Chemical Co.), or
control medium, respectively. These are physiological steroid levels
for adult men. In Exp 2, after 48 h of preculture, one dish was
treated with 10 nM DHT (Sigma Chemical Co.),
and two dishes were treated with control medium. After an additional
48-h incubation, the cell/bead mixtures containing 2.8 ± 0.5
x 106 (monkey) or 5.6 ± 0.5 x 106
(rat) cells were packed into Acusyst-S 1.5-ml microchambers
(Endotronics, Inc., Coon Rapid, MN) and perifused at 0.3
ml/min with DMEM (pH 7.3) supplemented with 14.8 mm NaHCO3
and 0.25% BSA, and gassed with 10% CO2-90%
O2 using a computer-controlled perifusion system
(Endotronics, Inc.). Steroid hormone treatments
were continued during the perifusion. After 2-h perifusion with medium
alone, pulses of 2.5 nM GnRH (Sigma Chemical Co.) were introduced for 1 min every 60 min for 8 h. This
dose was selected because it produced a half-maximum LH secretory
response (21). In Exp 2, the control and DHT-treated chambers received
GnRH pulses, whereas the third chamber was perifused with control
medium only. Fractions of the column effluents were collected at 10-min
intervals and frozen at -20 C until determination of LH by RIA. At the
completion of the perifusions (45 min after the final GnRH pulse), the
cell/bead mixtures were suspended in 5 ml 4 M guanidinium
thiocyanate-20 mM ß-mercaptoethanol solution and stored
at -70 C for RNA extraction.
RIAs
The concentration of monkey LH in the perifusion effluent was
estimated using reagents supplied by the National Hormone and Pituitary
Program. The RIA system uses 125I-labeled recombinant
cynomolgus LH (AFP6936A), anti-recombinant cynomolgus LH (AFP342994),
and recombinant cynomolgus LH (rMoLH RP-1) as the standard. The average
sensitivity was 15 pg rMoLH RP-1/tube. The within- and between-assay
coefficients of variations were 7% and 15%, respectively. The
concentration of rat LH was estimated with reagents from the National
Hormone and Pituitary Program as described previously (22). Results are
expressed in terms of the LH RP-2 standard. The minimal detectable dose
was 0.07 ng/tube.
RNA extraction and Northern blot hybridizations
Total RNA was extracted by the guanidinium
thiocyanate-phenol-chloroform procedure (23). The concentration of
total RNA was estimated by measuring the OD at 260 nm using an
Ultrospec 2000 spectrophotometer (Pharmacia Biotech).
Sample purity was determined by calculating the ratio of OD at 260:280
nm, which was 1.7–2.1. GnRH-R, LHß, and -subunit mRNA levels were
determined by Northern analysis. Aliquots of each RNA preparation
(10–20 µg) were subjected to electrophoresis in 1.2%
agarose-formaldehyde gels, transferred to Nytran membranes
(Schleicher & Schuell, Inc., Keene, NH), and cross-linked
to the membranes by baking for 2 h at 80–90 C followed by UV
irradiation for 2 min.
RNA and DNA probe preparations
Human and murine GnRH-R RNA probes were synthesized using
MAXIscript In Vitro Transcription Kits (Ambion, Inc.,
Austin, TX) following the manufacturer’s protocol. The template
complementary DNAs for human and murine GnRH-R were gifts from Dr.
Stuart C. Sealfon (Mount Sinai Medical Center, New York, NY) (24) and
Dr. Kevin J. Catt (NICHHD) (25), respectively. RNA probes were labeled
with [-32P]CTP (
800 Ci/mmol; New England Nuclear
Research Products, Boston, MA) and added to the hybridization solutions
at a concentration of 106 cpm/ml. The purified
complementary DNAs for cynomolgus monkey LHß and (from Drs.
Christie Kelton and Scott Chappel), rat LHß (from Dr. James Roberts),
rat (from Dr. William Chin), and rat cyclophillin (from Dr. James
Douglass) were labeled by the random primer method with
[-32P]deoxy-CTP (3000 Ci/mmol; New England Nuclear
Research Products) to specific activities of 8–10 x
108 cpm/µg using an oligolabeling kit (Pharmacia Biotech) and were added to the hybridization solutions at a
concentration of approximately 5 ng/ml. The membranes were sequentially
hybridized for 48–72 h with the above probes without stripping and
were autoradiographed. Films were scanned with GS-700 Imaging
Densitometer (Bio-Rad Laboratories, Inc., Hercules, CA)
and analyzed using Molecular Analyst software (Bio-Rad Laboratories, Inc.).
Data analysis
Data are presented as the mean ± SEM. The LH
concentration in the perifusion fractions is expressed as the amount of
hormone secreted by 1.0 x 106 cells/10 min. LH
secretion was resolved into two components: interpulse secretion and
GnRH-induced pulsatile release. Interpulse secretion is the mean of the
three basal levels between successive GnRH pulses. Pulse amplitude was
calculated by subtracting the mean of two interpulse levels just before
and after a pulse from the average LH concentration in the three
fractions collected after delivery of the GnRH pulse. Because the
absolute LH levels differed among replicate experiments, the results
for each interval of interpulse secretion and for each GnRH-induced
pulse from T-, E-, or DHT-treated columns was expressed as a percentage
of the corresponding value for the simultaneous control chamber (cells
stimulated with GnRH but not treated with gonadal steroids). Then, a
mean value for pulse amplitude and interpulse secretion for each
perifusion was determined. Changes over time were analyzed by two-way
ANOVA with post-hoc Student’s t test.
Because of variation in the specific activities of the radiolabeled probes, mRNA results for each replicate experiment were also analyzed independently and then compiled for statistical analysis. In each Northern analysis, the mRNA value from cells stimulated by GnRH but not treated with gonadal steroids was set at 100%, and the value from cells treated with T, E, or DHT was expressed as a percentage of that control value. Two-tailed Student’s t test for unpaired data was used for comparison of results between two groups. Differences in mRNA levels in the steroid-treated cells were compared with those in control cells by ANOVA and post-hoc Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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illustrates representative LH secretory
profiles when pituitary cells from monkeys or rats were treated with 10
nM T or 0.1 nM E beginning 48 h before and
continuing during the perifusion. The results of replicate experiments,
normalized to permit between-experiment comparison, are summarized in
Table 1. Using rat cells, T suppressed
(P < 0.01) GnRH-stimulated LH pulse amplitude to
61 ± 10% of the control value with no effect on interpulse
secretion; consequently, significantly less LH (P <
0.05) was secreted during the perifusion by T-treated than by
vehicle-treated rat cells stimulated by pulses of GnRH. E, in contrast,
increased LH pulse amplitude more than 2-fold (P <
0.05) using rat cells. These findings confirm our previous results (4, 5). Interpulse secretion and total LH secretion were also increased by
E treatment, but these increases were not statistically significant
(P > 0.05).
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). Because reduced bioconversion of T to DHT in primate
pituitary cells is one explanation for the lack of effectiveness of T
compared with results in rat cultures, we next assessed whether 10
nM DHT would modify GnRH-induced LH secretion from monkey
pituitary cells. As shown in Fig. 2, DHT,
even at a 10-fold higher concentration than the physiological plasma
level (26), failed to suppress GnRH-induced LH secretion from monkey
pituitary cells.
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, a perfusion experiment extended to 10 h, and as summarized in Fig. 3, there was a period of initial
enhancement (P < 0.05) after which LH pulse amplitude
was significantly suppressed by E (P < 0.05). By
contrast, there was no significant change in LH pulse amplitude over
time in T-treated monkey cells. Moreover, as shown previously (3, 5),
T-suppressed and E-enhanced GnRH-induced LH pulsatile release from rat
pituitary cells was sustained over 8 h.
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Effects on GnRH-R, LHß, and -subunit mRNA levels
Androgen treatment was reported to decrease GnRH-R binding in rat
pituitary cell monolayer cultures (27). We therefore predicted that
GnRH-R gene expression would be decreased by T only in cells from rats
in which GnRH-stimulated LH secretion was suppressed, but not in
pituitary cells from monkeys. This hypothesis proved, however, to be
incorrect. As summarized in Fig. 4, GnRH-R mRNA was unchanged by T treatment in both rat and monkey
pituitary cells at the completion of the 8-h perifusions. Levels of
GnRH-R mRNA in rat pituitary cells were slightly increased by E
treatment to 118 ± 13% of control, but this increase was not
statistically significant (P > 0.05). E did not alter
GnRH-R mRNA levels in monkey pituitary cells.
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-subunit mRNA levels are
suppressed by T in rat pituitary cells stimulated with pulses of GnRH
(5). In agreement with those results, T suppressed LHß (83 ±
4% of the control value; P < 0.01) and -subunit
(43 ± 3% of the control value; P < 0.01) mRNA
levels in rat pituitary cells. E, on the other hand, increased
-subunit mRNA levels to 156 ± 5% of the control value
(P < 0.01), but did not alter LHß mRNA levels in rat
pituitary cells. In monkey pituitary cells, neither T nor E affected
LHß or -subunit mRNA levels significantly (Fig. 4). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We also confirmed that T down-regulates LHß and -subunit gene
expression in rat pituitary cells stimulated with pulsatile GnRH,
indicating that T directly suppresses LH secretion in that species in
part by decreasing LH biosynthesis. The action of GnRH to stimulate
LHß and -subunit gene expression in gonadotrophs appears to be via
intracellular calcium and mitogen-activated protein kinase,
respectively (32, 33), but whether these pathways are disturbed by
androgens remains to be determined.
In monkey pituitary cells, on the other hand, we found no evidence for
a direct suppressive effect of T on either GnRH-induced LH secretion or
on LHß or -subunit gene expression. In addition, DHT failed to
suppress GnRH-induced LH secretion, suggesting that the absence of a
direct androgen negative feedback action on the primate pituitary was
not due to T metabolism. As androgen receptors (ARs) are present in the
anterior pituitary of fetal (34) and adult male (35, 36) monkeys and
appear to colocalize with immunoreactive LH in the human pituitary
(37), it is likely that gonadotrophs in the monkey pituitary also
express ARs, but this has not yet been tested. We were surprised to
find that T did not down-regulate -subunit gene expression in monkey
pituitary cells because transcriptional repression of the human
-subunit gene by androgen was recently shown (38) to involve
interaction between the AR and other DNA-binding proteins (39). As
those findings were obtained using murine T3 cells transfected with
a human -subunit transgene, it is possible that the coactivator
proteins that are prerequisite for the AR-mediated repression of
-subunit gene transcription by T are expressed in rodent, but not in
normal, monkey gonadotrophs.
Both E secreted by the testis and that produced by peripheral and central aromatization of testicular T are thought to participate in LH feedback inhibition (18, 40, 41, 42, 43). The effect of E on rat pituitary cells is biphasic, with initial inhibition of GnRH-stimulated LH secretion followed by facilitation at 24 h (43). In the present study, E treatment for 48 h stimulated GnRH-induced LH secretion from rat pituitary cells, in agreement with the previous findings of others in static cultures (29, 44) and our prior experiments in perifusion (3, 4). Both inhibition and facilitation are partly linked to changes in GnRH-Rs (43), but other mechanisms are operative, in that E treatment of GnRH-deficient (hpg) mice doubled GnRH-R number, but when combined with GnRH treatment produced values identical to those in normal littermates (45). E treatment of hypothalamus-pituitary-disconnected ewes stimulated with GnRH also increases LH secretion and subunit mRNA levels in part by increasing GnRH-R (46). We observed a slight increase in GnRH-R mRNA levels in E-treated rat pituitary cell cultures, which might contribute to the increased responsiveness to GnRH. Taken together, these observations demonstrate that the pituitary effect of E in rodents and ewes is to up-regulate GnRH-R and consequently to enhance GnRH-induced LH secretion, but the overall inhibitory effect of E on LH secretion and subunit gene expression in vivo appears to reflect suppression of hypothalamic GnRH secretion, which predominates over the stimulatory effect on the pituitary.
We found that E increased -subunit, but not LHß, mRNA levels in
rat pituitary cells perifused with GnRH pulses. In previous studies, E
was found to stimulate the synthesis of LHß protein (29) as well as
the transcription of the rat LHß gene through estrogen receptors with
no effect on -subunit mRNA synthesis in cultured pituitary cells
from female rats (47). Discrepancies with our results may be explained
by the use of pulsatile GnRH stimulation in the present study, a lower
E dosage (0.1 nM in the present study vs. 1 or
20 nM), and/or the sex of the pituitary cell donors.
After an initial period of stimulation, E suppressed GnRH-induced LH secretion from monkey pituitary cells, by contrast, indicating a pituitary site of action for E negative feedback in the male primate. These results agree with studies in GnRH-deficient men receiving long term pulsatile GnRH replacement (13) in which mean plasma LH levels as well as LH pulse amplitude were decreased during E infusion. As a substantial amount of E is produced by peripheral aromatization of T in vivo, it follows from our results that infusion of T and E, but not of DHT, would suppress LH secretion in GnRH-deficient men replaced with exogenous pulsatile GnRH, as observed by Bagatell et al. (48). By studying steady state mRNA levels in perifused cells, we have to date been unable to explain E-mediated suppression of LH secretion in male primates by either a decrease in GnRH-R or gonadotropin subunit gene expression.
Notably, the inhibitory effect of E in perifused monkey pituitary cells was preceded by an initial enhancement of GnRH-stimulated LH secretion. These observations are consistent with experiments in which LH secretion from hypothalamus-lesioned female monkeys replaced with pulsatile GnRH was initially suppressed by E for approximately 2 days, followed by a massive discharge of LH for 1 day, and then finally suppressed again (49). The timing of our perifusion experiments corresponds to the second stimulatory and the third inhibitory phase of that in vivo observation. Moreover, in pituitary cell monolayer cultures from adult female monkeys, GnRH-induced LH secretion was initially suppressed by E administration, but then increased after 27-h exposure to E (44). Further, 72-h pretreatment with E increased LH secretion by cultured human fetal pituitary cells induced by a 3-h exposure to GnRH (50). That E induces LH surges after hypothalamic destruction in the female rhesus monkey (49, 51) further indicates a pituitary site of E action, but the precise mechanisms underlying the multiphasic effects of E on LH secretion remain unclear.
In summary, our results identify different regulatory mechanisms by
which testicular steroid hormones control LH secretion in male primates
and rodents as shown in Fig. 5. Taken
together with previous results in the literature, we propose that the
primary site of androgen negative feedback in the male primate is to
restrain GnRH pulsatile secretion, whereas in the male rat, T also
decreases gonadotropin synthesis and secretion by directly affecting
the pituitary. E suppression of gonadotropin secretion in the male
primate is partly due to suppression of GnRH-stimulated LH secretion,
whereas E amplifies the action of GnRH in the rat. Our data also reveal
that the action of T to suppress LH secretion and subunit mRNAs in male
rats is not through decreased GnRH-R gene expression. The potential
roles of GnRH binding and signal transduction through phospholipase C
activity in the differential responses of primate and rodent
gonadotrophs to T remain to be examined. Finally, the experiments
presented herein have established pituitary cell cultures from nonhuman
male primates stimulated with pulses of GnRH as a model for
understanding gonadotropin gene regulation in normal and hypogonadal
men.
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Acknowledgments |
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Footnotes |
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Received December 11, 1998.
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References |
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-reductase
activity in pituitaries of male rhesus macaques. J Clin Endocrinol
Metab 66:1251–1258 subunit gene by androgen may
involve direct binding of androgen receptor to the proximal promoter.
J Biol Chem 268:13556–13564-subunit gene by androgen receptor
occurs independently of DNA binding but requires the DNA-binding and
ligand-binding domains of the receptor. Mol Endocrinol 11:1497–1506This article has been cited by other articles:
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, LH secretion by cells stimulated by pulses of GnRH
without gonadal steroid; •, cells treated with GnRH and 10
nM T (A and C); 
, cells treated with GnRH and 0.1
nM E (B and D).
,
cells treated with GnRH and 10 nM DHT.
