Inhibition of salmon calcitonin on secretion of progesterone
and GnRH-stimulated pituitary luteinizing hormone
Shiow-Chwen
Tsai1,
Chien-Chen
Lu1,
Jiann-Jong
Chen1,
Yu-Chung
Chiao1,
Shyi-Wu
Wang2,
Jiuan-Jiuan
Hwang1, and
Paulus S.
Wang1
1 Department of Physiology,
Schools of Medicine and Life Science, National Yang-Ming
University, Taipei 11221; and
2 Department of Physiology,
College of Medicine, Chang-Gung University, Taoyuan 33333, Taiwan,
Republic of China
 |
ABSTRACT |
The effects of salmon
calcitonin (sCT) on the production of progesterone and secretion of
luteinizing hormone (LH) were examined in female rats. Diestrous rats
were intravenously injected with saline, sCT, human chorionic
gonadotropin (hCG), or hCG plus sCT. Ovariectomized (Ovx) rats were
injected with saline or sCT. In the in vitro experiments, granulosa
cells and anterior pituitary glands (APs) were incubated with the
tested drugs. Plasma LH levels of Ovx rats were reduced by sCT
injection. Administration of sCT decreased the basal and hCG-stimulated
progesterone release in vivo and in vitro. 8-Bromo-cAMP dose
dependently increased progesterone production but did not alter the
inhibitory effect of sCT. H-89 did not potentiate the inhibitory effect
of sCT. Higher doses of 25-hydroxycholesterol and pregnenolone
stimulated progesterone production and diminished the inhibitory
effects of sCT. sCT did not decrease basal release of LH by APs, but
pretreatment of sCT decreased gonadotropin-releasing hormone
(GnRH)-stimulated LH secretion. These results suggested that sCT
inhibits progesterone production in rats by preventing the stimulatory
effect of GnRH on LH release in rat APs and acting directly on ovarian
granulosa cells to decrease the activities of post-cAMP pathway and
steroidogenic enzymes.
cytochrome P-450 side-chain
cleavage; 3
-hydroxysteroid dehydrogenase
 |
INTRODUCTION |
CALCITONIN (CT) is a peptide hormone produced mainly by
the parafollicular C cells of the thyroid gland in mammals, and classic concepts of CT function have focused on the effects of CT on calcium homeostasis (2). Recently, CT actions on the brain, pituitary, and
gonads have been investigated. In addition to thyroid, CT-like immunoreactivity has been found in the pituitary gland of humans and
rats (7, 15). Specific CT-binding sites have been found in the brain
and pituitary gland (14, 27), testicular Leydig cells (3), and ovarian
cells (16). These observations suggest an endocrine role of endogenous
CT at cerebral, pituitary, and gonadal levels.
Both basal release and thyrotropin-releasing hormone (TRH)-stimulated
release of prolactin in isolated rat pituitary cells are inhibited by
salmon CT (sCT; Ref. 37). We found that CT peptides, including human CT
(hCT), sCT, and CT gene-related peptide (CGRP), inhibit the spontaneous
and gonadotropin-stimulated secretion of testosterone by acting
directly at testes and reducing the release of pituitary luteinizing
hormone (LH) through a mechanism involving an increase in cAMP
production (44). In rats, CT exhibited a peak concentration in plasma
on the day of diestrus and dropped to the lowest on the day of estrus
(8). These observations indicate an endocrine role of sCT at ovarian level.
It has been well-known that the LH-increased productions of
progesterone (11, 22, 25) are correlated with the increased generation
of cAMP (11, 22). An increased expression of the cytochrome
P-450 side-chain cleavage
(P-450scc;
Refs. 22, 23) and 3
-hydroxysteroid dehydrogenase
(3
-HSD; Ref. 17) has also been demonstrated. The
conversion of cholesterol to pregnenolone is the rate-limiting step in
the final formation of progesterone, and this step is regulated by
mitochondria enzyme
P-450scc
(12, 41, 46). The interconversion of pregnenolone to progesterone is
catalyzed by microsomal enzyme 3
-HSD. Progesterone is the main
secretory product of granulosa cells and diffuses into theca cells to
serve as a substrate for biosynthesis of androgens (19, 25). The theca
cells provide androgens, whereas granulosa cells convert androgens to
estrogens by 17
-HSD and cytochrome
P-450 aromatase.
In the present study, sCT was employed instead of rat CT, because the
ultimobranchial CT is biologically more active and stable than
mammalian CT (18, 32). The effects of sCT on the basal and human
chorionic gonadotropin (hCG)-stimulated in release of progesterone in
rats and the release of LH from anterior pituitary glands (APs) were
examined. We found that sCT inhibits production of progesterone both in
vivo and in vitro through the mechanisms involving decreased
gonadotropin-releasing hormone (GnRH)-stimulated pituitary LH release
and the activities of
P-450scc
and 3
-HSD in granulosa cells. Furthermore, we suggested that the
inhibitory effect of sCT on progesterone release may be related to the
post-cAMP pathway.
 |
MATERIALS AND METHODS |
Animals. Mature diestrous female
(250-300 g) and immature female (30-40 g) Spraque-Dawley rats
were housed in a temperature-controlled room (22 ± 1°C) with 14 h of artificial illumination daily (0600-2000) and were given food
and water ad libitum.
Effect of sCT on progesterone release in
vivo. Diestrous rats were catheterized via the right
jugular vein (44). Twenty hours later, they were injected with saline
(1 ml/kg), sCT (3.4 ng · ml
1 · kg
1),
hCG (5 IU · ml
1 · kg
1),
or hCG plus sCT via the jugular catheter. Blood samples (0.5 ml each)
were collected at 0, 30, 60, and 120 min after the challenge. Plasma
was separated by centrifugation of blood samples at 10,000 g for 1 min. The concentration of
progesterone in plasma was measured by RIA (6).
Effect of sCT on LH release in vivo.
Some rats were ovariectomized (Ovx) 2 wk before being catheterized and
were injected with saline or sCT (3.4 ng · ml
1 · kg
1)
via the jugular catheter. Blood samples were collected at 0, 15, 30, 60, and 120 min after the challenge. Plasma was separated by
centrifugation at 10,000 g for 1 min.
The concentration of LH in plasma was measured by RIA (44).
Dispersion and preparation of rat granulosa
cells. The preparation of granulosa cells was modified
from the method described by Too et al. (42). The immature female rats
at 25-27 days of age were subcutaneously injected with pregnant
mares' serum gonadotropin (PMSG; 15 IU/rat). Forty-eight hours later,
rats were killed by cervical dislocation. Ovaries were excised and
transferred into the sterile DMEM/Ham's F-12 (1:1) medium, containing
0.1% bovine serum albumin (BSA, Sigma, St. Louis, MO), 20 mM HEPES,
100 U/ml penicillin-G, and 50 µg/ml streptomycin sulfate. After the
fat and connective tissues were trimmed free, the large and
medium-sized follicles were punctured with a 26-gauge needle to release
granulosa cells. The harvested cells were pelleted and resuspended in
growth medium (DMEM/Ham's F-12 containing 10% fetal calf serum, 2 µg/ml insulin, 100 IU/ml penicillin, and 50 µg/ml streptomycin
sulfate). Cell viability was >90% as determined with a hemacytometer
and the trypan blue method. Granulosa cells were plated in 24-well plates at ~1 × 105 viable
cells per well and were incubated at 37°C with 5%
CO2-95% air for 2 days.
Morphologically, the cultured granulosa cells appeared nearly round (or
polygonal) in shape, not like fibroblasts, throughout our culture
conditions. Total cell proteins were determined by the method of Lowry
et al. (26).
Incubation of granulosa cells with hCG and/or
sCT. The granulosa cells were washed twice by
serum-free BSA-M199 medium (medium 199, 0.3% BSA, 100 IU/ml
penicillin, 50 µg/ml streptomycin sulfate) and then incubated with
sCT (0-10
8 M), hCG
(0.5 IU/ml), or hCG plus sCT for 2 h. To further evaluate the role of
intracellular cAMP in regulation of progesterone release by sCT, the
effects of 8-bromo-cAMP, a cAMP analog to mimic increase of
intracellular cAMP, 10
4 or
10
3 M) and H-89 (an
inhibitor of protein kinase A catalytic subunit, 5 × 10
9 or 5 × 10
8 M; Refs. 10, 30, 34) on
the action of sCT in granulosa cells were examined. After incubation at
37°C with 5% CO2-95% air for
2 h, samples of medium were then collected, cleared by centrifugation,
and stored at
20°C until analyzed for progesterone by RIA.
Effect of sCT on steroidogenesis. The
activities of
P-450scc
and 3
-HSD were determined by measuring the conversion of
[3H]pregnenolone to
17
-[3H]hydroxyprogesterone
and [3H]progesterone
as described previously (43). Granulosa cells were incubated with
[3H]pregnenolone
(10,000 counts/min, 0.2 pmol) in the absence or presence of sCT
(10
8 or
10
6 M) for 2 h. The medium
was collected and extracted by vigorous agitation in 1 ml diethyl ether
and then quick-frozen in a mixture of acetone and dry ice. The diethyl
ether layer was collected, dried, and reconstituted in 100 µl of
100% ethanol containing 5 µg of each of the unlabeled carriers,
including pregnenolone, progesterone, and 17
-hydroxyprogesterone.
Aliquots (50 µl) were applied to a TLC plate and developed in a
mixture of carbon tetrachloride and acetone (4:1, vol/vol). The sheets
were dried, and the location of steroid-containing spots was indicated
under ultraviolet light. The migration rates
(Rf) were 0.55 for pregnenolone,
0.71 for progesterone, and 0.50 for 17
-hydroxyprogesterone. The
spots were cut off and transferred into vials containing 1 ml of liquid scintillation fluid (Ready Safe, Beckman, Fullerton, CA) before the
radioactivity was counted in an automatic beta counter (Wallac 1409, Pharmacia, Turku, Finland).
To ascertain the activities of
P-450scc
and 3
-HSD separately, the precursors, including
25-hydroxycholesterol (25-OH-cholesterol, which acts as a substrate of
P-450scc
that readily passes through cell and mitochondrial membranes; Refs. 4,
13) and pregnenolone (which acts as substrate of 3
-HSD), were added.
The granulosa cells were washed and incubated with fresh BSA-M199
medium containing 25-OH-cholesterol
(10
7-10
5
M) or pregnenolone
(10
9-10
5
M) in the absence or presence of sCT
(10
12-10
8
M). Two hours later, medium was collected and analyzed for progesterone by RIA.
Effect of sCT on LH release in vitro.
The diestrous rats were killed by decapitation. The
anterior pituitary glands (APs) were excised, bisected, preincubated,
and then incubated for 30 min with Locke's medium containing 10 mM
glucose, 0.003% bacitracin, and 0.05% HEPES at 37°C. One hemi-AP
was assigned to a flask containing 1 ml of medium. APs were then
incubated with sCT (0, 10
12,
10
10,
10
8 M) for 30 min before
being incubated with GnRH
(10
9 M). After further
incubation of APs with GnRH, AP tissues were weighed. The media were
collected and stored at
20°C until analyzed for LH by RIA
(44). The effect of sCT on GnRH-stimulated LH release was compared with
that of controls (non-sCT pretreated).
RIA of progesterone. The concentration
of progesterone in plasma and media was determined by RIA as previously
described (6). The sensitivity of progesterone RIA was 5 pg/assay tube.
The intra- and interassay coefficients of variability were 4.8%
(n = 5) and 9.5%
(n = 4), respectively.
RIA of LH. The concentration of LH in
plasma and media was determined by RIA as previously described (44).
The rat LH-I-6 used for iodination and rat LH-RP-3 serving as standard
preparation were provided by the National Hormone and Pituitary
Program, the National Institute of Diabetes and Digestive and Kidney
Diseases, the National Institute of Child Health and Human Development, and the United States Department of Agriculture. The
sensitivity was 0.1 ng for LH. The intra- and interassay coefficients
of variability were 3.8% (n = 4) and
6.6% (n = 5), respectively.
Statistical analysis. All data were expressed
as means ± SE. Treatment means were tested for homogeneity with
ANOVA, and the differences between the specific means were tested for
significance by means of Duncan's multiple range test or Student's
t-test (39). The level of significance
chosen was P < 0.05.
 |
RESULTS |
Plasma calcium and progesterone in response to hCG
and/or sCT. The mean levels of plasma calcium of
diestrous rats at all time points were 10.28 ± 0.14 mg/dl for the
saline-injected group, 10.21 ± 0.11 mg/dl for the hCG-injected
group, 10.66 ± 0.12 mg/dl for the sCT-injected group, and 10.30 ± 0.12 mg/dl for the animals injected with both hCG and sCT. There
were no significant differences in plasma calcium levels among these
four groups. The mean levels of plasma calcium of Ovx rats at all time
points were 10.0 ± 0.16 mg/dl.
Intravenous injection of saline did not alter the level of plasma
progesterone (Fig. 1,
top). Thirty to 120 min after sCT injection, the mean concentrations of plasma progesterone dropped by
37-49%. Injection of hCG stimulated progesterone secretion (Fig.
1, bottom). Injection of hCG plus
sCT resulted in a significantly lower level of plasma progesterone at
30 min after challenge compared with that induced by hCG alone
(P < 0.01).

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Fig. 1.
Effects of human chorionic gonadotropin (hCG; 5 IU · ml 1 · kg 1),
salmon calcitonin (sCT; 3.4 ng · ml 1 · kg 1),
and hCG + sCT on concentration of plasma progesterone in
diestrous rat. Rats were given a single intravenous injection of hCG,
sCT, or hCG + sCT via right jugular vein. Blood samples were collected
via jugular catheter at time indicated after hormone injection. Plasma
progesterone was extracted by ether before measuring by a RIA. Each
value is mean ± SE. * P < 0.05 and ** P < 0.01 vs.
saline-injected (top) or
hCG-injected (bottom) animals. + P < 0.05 and
++ P < 0.01 vs. value at 0 min.
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Plasma LH in response to sCT.
Intravenous injection of saline did not alter the level of plasma LH
(Fig. 2), but a single injection of sCT
decreased plasma LH at 15, 30, and 60 min after challenge
(P <0.05). A maximal reduction in
the plasma LH level was observed at 1 h after injection of sCT. After 2 h, the plasma LH concentration rose to the basal level.

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Fig. 2.
Change of rat plasma luteinizing hormone (LH) in response to sCT.
Female rats were ovariectomized (Ovx) 2 wk before a single intravenous
injection of saline or sCT (3.4 ng · ml 1 · kg 1)
via right jugular vein. Each value is mean ± SE.
** P < 0.01 vs.
saline-injected animals. + P < 0.05 and ++ P < 0.01 vs.
value at 0 min.
|
|
Effect of sCT on progesterone in granulosa cells in
vitro. The effect of sCT ranging from
10
12 to
10
8 M on progesterone
release by granulosa cells of PMSG-primed rats is illustrated in Fig.
3. During a 2-h incubation, sCT at
10
8 M caused a significant
inhibition of progesterone release (P < 0.05). Incubation with hCG for 2 h caused a significant increase of
progesterone release from granulosa cells
(P < 0.01). The hCG-induced release
of progesterone was significantly (P < 0.05 or P < 0.01) inhibited by
sCT ranging from 10
10 to
10
8 M.

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Fig. 3.
Dose-response effects of sCT on basal (hatched bar) and hCG-stimulated
(solid bar) release of progesterone by rat granulosa cells in vitro.
Each value is mean ± SE. * P < 0.05 and ** P < 0.01 vs.
sCT at 0 M. ++ P < 0.01 vs.
hCG at 0 IU/ml.
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8-BrcAMP at 10
3 M
stimulated the release of progesterone either in the absence or
presence of sCT (P < 0.01), but
8-BrcAMP could not fully reverse the inhibitory effect of sCT (Fig.
4). Administration of 5 × 10
8 M of H-89 resulted in a
decrease in the release of progesterone (Fig.
5). In the presence of H-89, no further
inhibition of progesterone release by sCT was observed.

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Fig. 4.
Effects of sCT on release of progesterone in rat granulosa cells in
absence (hatched bar) or presence of 8-bromo-cAMP [8-BrcAMP;
10 4 M (crosshatched bar) or
10 3 M (solid bar)].
Each column is mean ± SE.
* P < 0.05 vs. sCT at 0 M. ++ P < 0.01 vs.
corresponding control group.
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Fig. 5.
Effects of sCT (10 8 M) on
release of progesterone in rat granulosa cells in absence (hatched bar)
or presence of H-89 [5 × 10 9 M (crosshatched bar) or
5 × 10 8 M (solid
bar)]. Each column is mean ± SE.
* P < 0.05 vs. sCT at 0 M. ++ P < 0.01 vs. H-89 = 0 M.
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Effect of sCT on the activities of
P-450scc and
3
-HSD. With unlabeled steroids as carriers,
[3H]progesterone,
17
-[3H]hydroxyprogesterone,
and
[3H]androstenedione
(<100 counts/min, data not shown) were produced after incubation of
granulosa cells with
[3H]pregnenolone (Fig.
6). sCT did not alter the accumulation of [3H]pregnenolone and
production of 17
-[3H]hydroxyprogesterone but decreased the
production of
[3H]progesterone,
indicating that the 3
-HSD activity (conversion of
[3H]pregnenolone to
[3H]progesterone) was
inhibited.

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Fig. 6.
Effects of sCT (10 8 and
10 6 M) on activities of
3 -hydroxysteroid dehydrogenase in rat granulosa cells. Rat granulosa
cells were incubated with
[3H]pregnenolone
[10,000 counts/min (cpm)] and different doses of sCT at
37°C for 2 h. Medium was extracted by ether and dried and then
reconstituted in ethanol before analysis by TLC.
[3H]pregnenolone,
[3H]progesterone, and
17 -[3H]hydroxyprogesterone
levels were measured. Each symbol is mean ± SE. Administration of
sCT significantly decreased production of
[3H]progesterone by
rat granulosa cells (470 ± 25 cpm · 1 × 105
cells 1 · 2 h 1 for sCT at
10 8 M and 354 ± 34 cpm · 1 × 105
cells 1 · 2 h 1 for sCT at
10 6 M vs. 660 ± 39 cpm · 1 × 105
cells 1 · 2 h 1 for sCT
at 0 M, P < 0.01).
** P < 0.01 vs. sCT
at 0 M.
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Administration of 25-OH-cholesterol
(10
7-10
5
M; Fig. 7) and pregnenolone
(10
9-10
5
M; Fig. 8) increased progesterone release
(P < 0.05 or
P < 0.01). sCT at
10
8 M decreased not only
the basal release of progesterone but also the progesterone response to
the addition of lower doses of 25-OH-cholesterol (10
7 M,
P < 0.05; Fig. 7) or
pregnenolone
(10
9-10
7
M, P < 0.05; Fig. 8).
25-OH-cholesterol and pregnenolone at
10
5 M reversed the
inhibitory effect of sCT on progesterone release (Figs. 7 and 8).

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Fig. 7.
Effects of sCT on release of progesterone after incubation of rat
granulosa cells with 25-hydroxycholesterol (25-OH-cholesterol) at
different doses
(10 7-10 5
M). Each symbol is mean ± SE.
* P < 0.05 vs. sCT at 0 M.
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Fig. 8.
Effects of sCT on release of progesterone after incubation of rat
granulosa cells with pregnenolone at different doses
(10 9-10 5
M). Each symbol is mean ± SE. Administration of sCT at
10 8 M resulted in a
significantly lower level of progesterone release vs. group treated
with 0 M of sCT in presence of pregnenolone at 0 M (20.86 ± 2.81 vs. 33.85 ± 3.82 pg · µg
protein 1 · 2 h 1,
P < 0.05),
10 9 M (27.22 ± 3.40 vs.
48.95 ± 9.29 pg · µg
protein 1 · 2 h 1,
P < 0.05),
10 8 M (51.19 ± 3.95 vs.
64.34 ± 5.50 pg · µg
protein 1 · 2 h 1,
P < 0.05), or
10 7 M (232.43 ± 5.25 vs. 475.18 ± 17.41 pg · µg
protein 1 · 2 h 1,
P < 0.05).
* P < 0.05 vs. sCT at 0 M.
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Effect of sCT on LH release in vitro.
The mean pre-sCT levels of LH release from AP ranged between 11.1 and
11.8 ng/mg of AP (Fig. 9). Incubation of
sCT did not alter the release of LH. Although GnRH
(10
9 M) increased LH
release significantly (Fig. 9, P < 0.01), pretreatment with sCT dose dependently diminished the
stimulatory effect of GnRH on LH release (Figs. 9 and
10).

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Fig. 9.
In vitro release of LH by rat anterior pituitary gland (AP) before and
after incubation with sCT at 37°C for 30 min. After basal
incubation (open bar) and incubation with sCT (hatched bar), APs were
then incubated with GnRH (solid bar;
10 9 M) for 30 min. One
hemi-AP was assigned to each flask. Each value is mean ± SE.
** P < 0.01 vs. sCT
pretreatment at 0 M. ++ P < 0.01 vs. basal level.
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Fig. 10.
Effects of sCT on increment of LH release stimulated by
gonadotropin-releasing hormone in rat APs in vitro. Each value is mean ± SE. ** P < 0.01 vs. sCT
at 0 M.
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 |
DISCUSSION |
In the present study, we found that administration of sCT in rats
significantly 1) inhibited the
spontaneous and hCG-stimulated secretion of progesterone in vivo and in
vitro, 2) inhibited the GnRH-stimulated release of LH by rat anterior pituitary glands, and
3) decreased the activities of
P-450scc
and 3
-HSD. Furthermore, we suggested that the inhibitory effect of
sCT on progesterone release was through the post-cAMP pathway in
granulosa cells.
The presence of CT in the brain and pituitary glands has been
demonstrated in many mammalian species, including humans (7, 27). In
humans, intravenous infusion of sCT caused a serum calcium-independent reduction in thyrotropin and LH secretion in response to
hypothalamic-releasing hormone (24). Administration of CT inhibits the
release of rat growth hormone (28), thyrotropin (29), LH (44), and
prolactin (9), as well as the secretion of LH from APs (44), the
secretion of prolactin in isolated rat pituitary cells (37), and the
rise of rat prolactin after stress (9). The CT receptors designated C1a
and C1b receptors have been identified in rat brain (1, 35). Both C1a
and C1b-transfected cells responded to increasing concentrations of sCT
with increases of cAMP (1, 36).
The present data provide evidence that sCT diminishes the
GnRH-stimulated release of rat LH by acting directly on the APs (Fig.
9, 10). Because of the sample size and RIA sensitivity, we measured the
concentration plasma LH in Ovx rather than in intact rats.
After injection of sCT, the levels of plasma calcium were not altered
(data not shown), but the release of plasma progesterone and LH was
significantly decreased (Figs. 1 and 2). We concluded that the
decreases of progesterone and LH were independent of the
calcium-decreased effect of sCT. After pretreatment with sCT, the
reduction of LH secretion in response to GnRH in the anterior pituitary
glands (Fig. 10) might be one of the reasons for diminishing the level
of plasma LH and progesterone after a single injection of sCT.
According to our in vitro observation,
10
8 M sCT peptides are
effective in reducing both spontaneous and hCG-stimulated release of
progesterone. Another reason for sCT-induced diminution in plasma
progesterone is that sCT inhibits progesterone production by acting
directly on granulosa cells in ovarian follicles of PMSG-treated
immature female rats. At the present time, the inhibitory effect of sCT
on progesterone release was in part due to
1) decreased progesterone release
directly, 2) decreased progesterone release in response to gonadotropin (LH or hCG), and
3) decreased LH release in response
to GnRH. A link between sexual hormones and CT in female rat CT had
been suggested because of an increased level of plasma CT in the
afternoon during diestrus (8). Because the secretion of both LH and
progesterone is decreased during diestrus in rats, it seems that CT
plays a significant physiological role in regulating the production of
pituitary gonadotropins and ovarian steroid hormones.
It has been well established that hCG stimulates progesterone secretion
both in vivo (5) and in vitro (38) and increases granulosa cAMP content
(38). In the present study, we found that both the stimulatory effects
of hCG on plasma progesterone and the progesterone production in vitro
were diminished by sCT. The increased production of cAMP caused by CT
has been demonstrated in perfused rat bone (40), rat osteoblast-like
cell line (20), rat osteoclasts (33), atria (45), and aortic smooth
muscle cells (21). An increase of the conversion of
3H-cAMP in TM3 mouse testis cell
line by sCT has been described (31). We have found that sCT, hCT, or
CGRP induces an increase of cAMP production in both testicular tissues
and anterior pituitary glands in rats (44). These observations reflect
the presence of a different post-cAMP event between hCG and CT actions.
Our results indicated that administration of 8-BrcAMP did not alter the
inhibitory effect of sCT and that H-89 did not potentiate the
inhibitory effect of sCT. We suggested that sCT-inhibited progesterone
release might be through a post-cAMP pathway, and the interaction
between sCT and hCG on the signal transduction in rat granulosa cells
is still open to elucidate.
The accumulation of
[3H]pregnenolone was
not altered and the amount of
[3H]progesterone was
decreased by sCT, indicating that the activity of 3
-HSD was
decreased by administration of sCT. It has been demonstrated that
25-OH-cholesterol at 2.5 × 10
5 M stimulates
testosterone release by Leydig cells in rat testes (4). In the
adrenocortical cell culture, a significant effect of 25-OH-cholesterol
(1.86 × 10
5 M) on
progesterone release has been observed (13). In the present study, the
attenuation of a higher dose of 25-OH-cholesterol on the decrease of
progesterone release by sCT suggests an inhibition of sCT on the
activity of
P-450scc,
the rate-limiting enzyme in progesterone biosynthesis, for the
conversion of cholesterol to pregnenolone. The final step in
progesterone biosynthesis is the conversion of pregnenolone to
progesterone under the activity of the microsomal enzyme 3
-HSD.
Administration of pregnenolone produced an increase in the release of
progesterone and attenuated the inhibitory effect of sCT. Although the
inhibitory effects caused by sCT might be mediated by decreasing the
expression of steroidogenic enzymes and/or decreasing the cholesterol
transport, the inhibition of the activity of
P-450scc
and 3
-HSD by sCT may account for the reduction of progesterone
secretion in rat granulosa cells.
In summary, our present findings suggest that sCT inhibits progesterone
production in rats by 1) acting
directly on granulosa cells in the ovary,
2) inhibiting LH release and
preventing LH response to GnRH, and
3) preventing progesterone response
to gonadotropin, without altering plasma calcium level. The inhibitory
effect of sCT on progesterone release is associated with a decrease of
P-450scc and 3
-HSD activities in granulosa cells. The effective dose of sCT
in reducing progesterone and LH secretion in vitro is 1-10 nM,
which is greater than the normal level of rat plasma CT (<100 pg/ml,
data not shown). We therefore conclude that CT expresses mainly a
pharmacological rather than a physiological effect on progesterone and
LH secretion. The inhibitory effects of CT on LH and progesterone
secretion may be interesting in the therapy of hypogonadotropic or
hypergonadotropic hypergonadism.
 |
ACKNOWLEDGEMENTS |
We greatly appreciate A. L. Vendouris for English editorial
assistance. The rat LH RIA kit was kindly supplied by the
National Hormone and Pituitary Program, the National Institute of
Diabetes and Digestive and Kidney Diseases, the National Institute of
Child Health and Human Development, and the United States Department of Agriculture.
 |
FOOTNOTES |
This study was supported by the Grant No. NSC85-2331-B010-53
from the National Science Council of the Republic of China and awards
from the Medical Research and Advancement Foundation in memory of Dr.
Chi-Shuen Tsou, Republic of China, to P. S. Wang.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. S. Wang,
Dept. of Physiology, National Yang-Ming Univ., Shih-Pai, Taipei, Taiwan
11221, Republic of China (E-mail: pswang{at}ym.edu.tw).
Received 25 August 1998; accepted in final form 25 March 1999.
 |
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