1 Department of Physiology, In the present study, the roles of ovarian
steroid hormones and thyroxine
(T4) in regulating the secretion
of calcitonin (CT) in pregnant rats were examined. The levels of plasma
progesterone, pre- and post-CaCl2
plasma CT, and recovery time of plasma CT and calcium after calcium
challenge were greatest in midterm pregnant rats. The levels of basal
plasma progesterone, CT, calcium, and recovery time of plasma CT after
calcium challenge were less in late pregnant rats, but basal plasma
estradiol was highest in late pregnancy. The concentrations of plasma
T4 were gradually decreased in
rats during pregnancy. Regardless of the presence of estradiol,
administration of progesterone in ovariectomized (Ovx) rats resulted in
an increase of plasma T4 as well
as the basal and calcium-induced secretion of CT. Administration of
estradiol alone did not alter the
CaCl2-induced levels but decreased
the post-CaCl2 levels of plasma
calcium in Ovx rats. The basal levels of plasma CT were decreased in
Ovx rats treated with T4. These results suggest that the hypercalcitoninemia in midterm pregnant rats
is due to an increased secretion of progesterone. Hypocalcitoninemia in
late pregnant rats, however, is due in part to lower plasma calcium.
estradiol; progesterone
DURING PREGNANCY, the metabolism of maternal calcium is
influenced by fetal requirements (14). This adaptive process depends on
the interrelationship between parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3],
which shows quantitative rather than qualitative changes from the
nonpregnant state (14). In addition, the metabolism of calcium is also
regulated by calcitonin (CT) and
1,25-(OH)2D3.
Changes in the secretion of CT and
1,25-(OH)2D3 in pregnancy are well characterized. For example, the level of CT in
rat plasma has been shown to increase up to 19.5 days and to decrease
subsequent to 21.5 days of gestation (10). Halloran et al. (13) found
that plasma
1,25-(OH)2D3
levels in female rats increased threefold during the latter stages of
pregnancy. In humans, plasma levels of CT and
1,25-(OH)2D3
have also been shown to increase during pregnancy (24). A significant
increase in plasma vitamin D concentration has been noted in pregnant
rats from day 19 to
day 21 (17a). A linear reduction of
bone density has been observed during the third trimester (17), when
the greatest calcium transfer occurs between the mother and the fetus (13). Thus the simultaneous rise in CT and
1,25-(OH)2D3
during pregnancy reduces the bone-resorbing activities at this critical stage and thereby maintains the integrity of the maternal skeleton and
protects against osteoporosis (13). However, the mechanism by which CT
increases during pregnancy remains unknown.
It is well documented that the level of plasma CT is influenced by
gonadal steroid hormones. Androgen deficiency per se has been shown to
play an influential role in the pathogenesis of osteoporosis in
hypogonadal subjects and may influence bone metabolism by regulating CT
secretion (7). Whitehead et al. (23) found that estrogen increases CT
secretion in humans. Clinical studies have indicated that
postmenopausal estrogen replacement therapy is effective in the
prevention of rapid bone loss (1). Because estrogen regulates CT
secretion in postmenopausal women, CT may mediate estrogen action on
bone (1).
In both humans (20) and rats (11), circulating plasma progesterone
concentrations are higher in pregnant subjects than in nonpregnant
controls, and estradiol concentrations are increased in pregnant
subjects approaching full term. Furthermore, ovariectomy causes a
decrease in serum CT and calcium levels (15). In addition, both
estradiol and progesterone cause an increase of in vitro CT release
from the thyroid C cells of 8-day-old rats (12). These findings suggest
that ovarian steroid hormones play a prominent role in regulating the
secretion of CT during pregnancy.
In addition to the changes of gonadal steroid hormones, the
concentration of plasma thyroxine
(T4) is gradually decreased during pregnancy (8). Compared with the hormonal profile of early
pregnancy, the concentration of plasma 3,5,3'-triiodothyronine (or T3) is lower and that of
plasma thyrotropin is higher in late gestation (8). Because the
skeletal density (4) and the concentration of plasma CT (2) decrease in
hypothyroid patients, CT secretion may also be regulated by
T4.
This investigation was designed to study the role of ovarian steroid
hormones and that of T4 in
regulating CT secretion in rats during pregnancy. To study the effects
of steroid hormones and T4 on CT
release, ovariectomized (Ovx) rats were treated with ovarian steroid
hormones or T4 to characterize the
hormonal effects on CT secretion.
Animals.
Female Sprague-Dawley rats weighing 220-270 g were housed in a
temperature-controlled (22 ± 1°C) room with 14 h of artificial illumination daily (0600-2000) and were given food and water ad libitum.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Experiments. Rats were catheterized via the right jugular vein and left femoral vein under ether anesthesia (22). Twenty hours later, CaCl2 (10 mg/ml) was infused (1 ml/30 min) via the femoral catheter connected to a peristaltic pump (21). Blood samples (0.6 ml each) were collected from the right jugular vein at 0, 30, 60, and 120 min after CaCl2 challenge (21).
Plasma was separated by centrifugation at 10,000 g for 1 min and stored atRIA of CT.
Concentrations of plasma and medium CT were measured by a human CT RIA
kit purchased from Nichols Institute Diagnostics (San Juan Capistrano,
CA) (21). Inhibition curves of rat thyroid medium, rat plasma, and
human plasma paralleled those of human CT standards (21). The antisera
for CT showed negligible or no cross-reactivity against bovine
PTH-(184), human PTH-(1
34), insulin, prolactin, human growth
hormone, thyroid-stimulating hormone, and adrenocorticotropic hormone.
Salmon CT up to 40 ng/ml did not cross-react with the antisera. The
sensitivity of the RIA was 4 pg/ml. The recovery of CT from human serum
pools was 86-94%. Intra- and interassay coefficients of variation
were 6.7% (n = 10) and 8.3%
(n = 10), respectively.
RIAs of estradiol and progesterone. Concentrations of plasma progesterone were determined by RIA as described previously (16). With antiprogesterone serum no. W5, the sensitivity of the progesterone RIA was 5 pg/assay tube. Intra- and interassay coefficients of variation were 4.8% (n = 5) and 9.5% (n = 4), respectively.
The antiserum against estradiol (no. W1) was generated by immunizing the rabbit with 1,3,5(10)-estratrien-3,17RIA of T4. The concentration of total T4 in plasma and media samples was measured by an Amerlex-M T4 RIA kit provided from Johnson & Johnson Clinical Diagnostics (Amersham International). The sensitivity of this assay was 3 ng/ml, and the intra- and interassay coefficients of variation were 3.3% (n = 10) and 4.7% (n = 10), respectively.
Statistical analysis. Treatment means were tested for homogeneity with analysis of variance, and the difference between specific means was tested for significance by use of Duncan's multiple range test (19). P < 0.05 was taken to indicate statistical significance.
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RESULTS |
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Concentrations of plasma total T4,
estradiol, progesterone, calcium, and CT in rats during pregnancy.
Plasma levels of total T4 on
days 7, 14, and
21 of gestation were lower than
T4 levels in diestrous rats (Fig.
1, top).
The plasma total T4 was negatively
correlated with the day of gestation (correlation coefficient = 0.87, P < 0.01) and decreased
gradually during gestation.
|
Response of CT to CaCl2 challenges in pregnant rats. The percent changes of plasma calcium and CT levels in response to intravenous infusion of CaCl2 are illustrated in Fig. 2. After 30 min of CaCl2 infusion, plasma calcium levels increased in all groups (Fig. 2, top). Thereafter (at 60 and 120 min), calcium levels either returned to basal levels (diestrous, and days 7 and 14 of gestation) or were further reduced in 21-day-pregnant rats (60 min; Fig. 2, top). In addition, plasma calcium levels were lower on day 21 of gestation than for other groups at 60 min. Before and after CaCl2 challenge, the calcium levels were not different among diestrous rats and pregnant rats at days 7 and 14 of gestation at 0-60 min. Ninety minutes after termination of CaCl2 challenge, plasma calcium levels were lower on day 7 of gestation than for diestrous rats and pregnant rats at day 14 of gestation.
|
Concentrations of plasma T4, calcium, and CT in steroid-treated Ovx rats. Concentrations of plasma estradiol and progesterone in EB- and/or progesterone-injected Ovx rats ranged from 17 to 30 pg/ml and from 19 to 47 ng/ml, respectively (data not shown).
The concentration of plasma total T4 was higher in Ovx rats treated with progesterone (P < 0.01) or with EB plus progesterone (P < 0.05) than in Ovx rats treated with oil (Fig. 3, top). The levels of plasma total T4 in Ovx rats were not different between oil and EB treatments, between EB and EB plus progesterone treatments, or between progesterone and EB plus progesterone treatments.
|
Response of CT to CaCl2 challenge in Ovx rats treated without or with ovarian steroids. Replacement of EB or progesterone resulted in a lower (P < 0.01 and P < 0.05, respectively) plasma calcium at 60 min and a lowest (P < 0.01) plasma calcium at 120 min after CaCl2 infusion in EB-treated Ovx rats (Fig. 4, top). Meanwhile, the levels of plasma calcium at 60 min in Ovx rats were different between those EB and oil treated, or between those progesterone and oil treated. At 120 min, the levels of plasma calcium in Ovx rats were different between EB- and oil-treated, between progesterone- and EB-treated, or between EB plus progesterone- and EB-treated animals.
|
Concentrations of plasma T4, calcium, and CT in T4-treated Ovx rats. The concentration of plasma total T4 was higher in Ovx rats treated with T4 (P < 0.01) than in Ovx rats treated with saline (Fig. 5, top). Basal levels of plasma calcium in Ovx rats remained unaltered by the treatments of T4 (Fig. 5, middle). In contrast, concentrations of plasma CT were lower in Ovx rats treated with T4 than in Ovx rats treated with saline (Fig. 5, bottom).
|
Response of CT to CaCl2 challenge in Ovx rats treated with or without T4. After CaCl2 infusion (at 30 min), plasma calcium levels were increased in all animals. Thereafter (at 60 and 90 min), calcium levels returned to basal (Fig. 6, top).
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DISCUSSION |
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In the present study we found that 1) the midterm pregnant rats exhibited highest levels of plasma progesterone and CT and a long recovery time of plasma CT after calcium challenge; 2) the late pregnant rats exhibited lower basal plasma calcium and CT and a short recovery time of plasma calcium and CT after calcium challenge; 3) regardless of the presence of estradiol, administration of progesterone increased the levels of plasma CT and T4; 4) administration of progesterone alone or progesterone plus estradiol increased the basal level of plasma CT and the response of CT to calcium; 5) administration of estradiol increased the clearance of plasma calcium; and 6) administration of T4 decreased the basal levels of plasma CT but did not alter CT secretion induced by calcium challenge.
A previous study has shown that the circulating progesterone concentration is higher in rats during pregnancy, except at term, than in virgin controls (11). However, the level of plasma estradiol markedly increases during late pregnancy in rats (11). These profiles of ovarian steroid hormone levels correspond with our observations in the pregnant rats. In this study, both the basal and calcium-induced levels of plasma CT and plasma progesterone are markedly higher in midterm pregnant rats. Apparently, a higher secretion of CT is correlated with progesterone production.
Garel and Jullienne (10) found that the levels of CT in the plasma after 17.5 days of gestation were already higher than the level in control rats, increased further up to 19.5 days, and subsequently decreased at 21.5 days. Moreover, an increased plasma CT level during pregnancy has also been reported in humans (5, 25). Collectively, these data suggest that the midterm enhancement of circulating CT may be due in part to the higher secretion of progesterone. This is supported by our observation that progesterone alone increased the basal and calcium-induced plasma CT in Ovx rats. Because no increase of plasma estradiol level was observed in midterm pregnant rats, and neither basal nor calcium-induced plasma CT levels were enhanced by the administration of estradiol in Ovx rats, we suggest that the greater secretion of CT in midterm pregnant rats is due to the stimulatory effect of progesterone rather than the invalid effect of estradiol.
Despite the increase in CT secretion, plasma calcium levels were not altered in midterm pregnant rats compared with nonpregnant rats. These results suggest that hypercalcitoninemia in pregnant rats is calcium independent; they compare favorably with the data reported by Quan-Sheng and Miller (17a). However, in addition to the reduction of CT secretion, this study shows that hypocalcemia always occurs in rats during late pregnancy (i.e., day 21). Administration of estradiol did not affect basal, calcium-induced, and postcalcium levels of plasma CT but decreased postcalcium levels of plasma calcium in Ovx rats. The reason for lower post-CaCl2 levels of plasma calcium caused by estradiol remains unknown but may be an overcompensation as the body returns plasma calcium levels to normal. Because estrogen depresses serum calcium levels in postmenopausal women (23), the decreased basal level of plasma calcium and short recovery time of plasma calcium after CaCl2 challenge in late pregnancy might be due to a high level of plasma estradiol.
On the basis of our results of a lower basal level of plasma calcium and a short recovery time of plasma calcium after CaCl2 challenge, hypocalcemia may be one of the main factors for the occurrence of hypocalcitoninemia in late pregnancy. The observation that hypocalcemia occurs in late pregnancy has been noted not only in rats (10, 17a) but also in women (5) and has been suggested to result from the increased demand of fetal growth (17a). Because administration of progesterone alone or progesterone plus estradiol increased the levels of plasma T4 in Ovx rats (Fig. 3), the decrease of plasma T4 levels in rats during pregnancy was independent of the levels of plasma progesterone and/or estradiol but might be due to the increased metabolic clearance rate of T4 in pregnant rats (9). Although it has been shown that CT deficiency is present in primary hypothyroidism (2), we found that administration of T4 decreased the basal levels of plasma CT in Ovx rats. Because we found a lower level of plasma T4 in midterm pregnant rats but a higher level of plasma T4 in progesterone-treated Ovx rats, there seems to be no correlation between T4 and CT levels in the plasma of female rats. There is no evidence at the present time to indicate that T4 is a physiological regulator for CT secretion in pregnant rats.
In women, the occurrence of osteoporosis is due either to menopause or to aging (3, 18). Administration of estrogen and/or CT is facilitative in relieving the syndromes of osteoporosis (1). Our observations in Ovx rats indicate that progesterone alone or in combination with estradiol increases the secretion of CT. However, more studies are needed before progesterone can be considered as a promising therapeutic reagent for treating osteoporosis.
In summary, results of this study demonstrate that abundant production of progesterone is associated with the increase of both spontaneous and calcium-induced levels of plasma CT in midterm pregnant rats. Because administration of progesterone increased rather than decreased the levels of plasma T4 and CT in Ovx rats, the hypercalcitonemia in midterm pregnant rats is dependent on progesterone rather than T4. The hyposecretion of CT in late pregnant rats is at least partially due to the hypocalcemia.
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ACKNOWLEDGEMENTS |
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The authors greatly appreciate Dr. C. Weaver's English editing.
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FOOTNOTES |
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This study was supported by Grant NRICM-84104 from the National Research Institute of Chinese Medicine, Grant NSC 86-2314-B-010-074 from the National Science Council, and awards from the Medical Research and Advancement Foundation in memory of Dr. Chi-Shuen Tsou, ROC, to P. S. Wang.
Address for reprint requests: P. S. Wang, Dept. of Physiology, National Yang-Ming Univ., Shih-Pai, Taipei, Taiwan, Republic of China.
Received 19 May 1997; accepted in final form 29 October 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Agnusdei, D.,
R. Civitelli,
A. Camporeale,
and
C. Gennari.
Calcitonin and estrogens.
J. Endocrinol. Invest.
13:
625-630,
1990[Medline].
2.
Body, J. J.,
N. Demeester-Mirkine,
A. Borkowski,
S. Suciu,
and
J. Corvilain.
Calcitonin deficiency in primary hypothyroidism.
J. Clin. Endocrinol. Metab.
62:
700-703,
1986[Abstract].
3.
Christiansen, C.
Estrogen therapy of osteoporosis.
In: New Horizons in Osteoporosis, edited by C. Christiansen. Carnforth, UK: Parthenon, 1988, p. 13-19.
4.
Coindre, J. M.,
J. P. David,
L. Riviere,
J. F. Goussot,
P. Roger,
A. de Mascarel,
and
P. J. Meunier.
Bone loss in hypothyroidism with hormone replacement.
Arch. Intern. Med.
146:
48-53,
1986[Abstract].
5.
Dahlman, T.,
H. E. Sjoberg,
and
E. Bucht.
Calcium homeostasis in normal pregnancy and puerperium: a longitudinal study.
Acta Obstet. Gynecol. Scand.
73:
393-398,
1994[Medline].
7.
Foresta, C.,
G. P. Zanatta,
B. Busnardo,
G. Scanelli,
and
C. Scandellari.
Testosterone and calcitonin plasma levels in hypogonadal osteoporotic young men.
J. Endocrinol. Invest.
8:
377-379,
1985[Medline].
8.
Fukuda, H.,
K. Ohshima,
M. Mori,
I. Kobayashi,
and
M. A. Greer.
Sequential changes in the pituitary-thyroid axis during pregnancy and lactation in the rat.
Endocrinology
107:
1711-1716,
1980[Medline].
9.
Galton, V. A.
Thyroxine metabolism and thyroid function in the pregnant rat.
Endocrinology
82:
282-290,
1968[Medline].
10.
Garel, J. M.,
and
A. Jullienne.
Plasma calcitonin levels in pregnant and newborn rats.
J. Endocrinol.
75:
373-382,
1977[Medline].
11.
Garland, H. O.,
J. C. Atherton,
C. Baylis,
M. R. A. Morgan,
and
C. M. Milne.
Hormone profiles for progesterone, oestradiol, prolactin, plasma renin activity, aldosterone and corticosterone during pregnancy and pseudopregnancy in two strains of rat: correlation with renal studies.
J. Endocrinol.
113:
435-444,
1986.
12.
Greenberg, C.,
S. C. Kukreja,
E. N. Bowser,
K. Hargis,
W. J. Henderson,
and
G. A. Williams.
Effects of estradiol and progesterone on calcitonin secretion.
Endocrinology
118:
2594-2598,
1986[Abstract].
13.
Halloran, B. P.,
E. N. Barthell,
and
H. F. DeLuca.
Vitamin D metabolism during pregnancy and lactation in the rat.
Proc. Natl. Acad. Sci. USA
76:
5549-5553,
1979[Abstract].
14.
Hosking, D. J.
Calcium homeostasis in pregnancy.
Clin. Endocrinol.
45:
1-6,
1996[Medline].
15.
Kalu, D. N.,
R. R. Hardin,
and
R. Cockerham.
Evaluation of the pathogenesis of skeletal change in ovariectomized rats.
Endocrinology
115:
507-512,
1984[Abstract].
16.
Lu, S.-S.,
C.-P. Lau,
Y.-F. Tung,
S.-W. Huang,
Y.-H. Chen,
H.-C. Shih,
S.-C. Tsai,
C.-C. Lu,
S.-W. Wang,
J.-J. Chen,
E. J. Chien,
C.-H. Chien,
and
P. S. Wang.
Lactate stimulates progesterone secretion via an increase in cAMP production in exercised female rats.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E910-E915,
1996
17.
Paparella, P.,
R. Giorgino,
A. Maglione,
D. Lorusso,
P. Scirpa,
A. Del Bosco,
and
S. Mancuso.
Maternal ultrasound bone density in normal pregnancy.
Clin. Exp. Obstet. Gynecol.
22:
268-278,
1995[Medline].
17a.
Quan-Sheng, D.,
and
S. C. Miller.
Calciotrophic hormone levels and calcium absorption during pregnancy in rats.
Am. J. Physiol.
257 (Endocrinol. Metab. 20):
E118-E123,
1989
18.
Rubin, C. D.
Southwestern internal medicine conference: age-related osteoporosis.
Am. J. Med. Sci.
301:
281-298,
1991[Medline].
19.
Steel, R. D.,
and
J. H. Torrie.
Principles and Procedures of Statistics. New York: McGraw-Hill, 1960.
20.
Tovanabutra, S. P.,
J. Illingworth,
W. L. Ledger,
A. F. Glasier,
and
D. T. Baird.
The relationship between peripheral immunoactive inhibin, human chorionic gonadotropin, oestradiol and progesterone during human pregnancy.
Clin. Endocrinol.
38:
101-107,
1993[Medline].
21.
Tsai, C.-L.,
H.-F. Pu,
C.-P. Lau,
P. S. Wang,
and
T.-K. Liu.
Age-related differences in basal and calcium-stimulated plasmacalcitonin levels in female rats.
Am. J. Physiol.
262 (Endocrinol. Metab. 25):
E557-E560,
1992
22.
Wang, P. S.,
J. Y. Liu,
C. Y. Hwang,
C. Hwang,
C. H. Day,
C. H. Chang,
H. F. Pu,
and
J. T. Pan.
Age-related differences in the spontaneous and thyrotropin-releasing hormone-stimulated release of prolactin and thyrotropin in ovariectomized rats.
Neuroendocrinology
49:
592-596,
1989[Medline].
23.
Whitehead, M. I.,
G. Lane,
P. T. Townsend,
G. Abeyasekera,
C. J. Hillyard,
and
J. C. Stevenson.
Effects in postmenopausal women of natural and synthetic estrogens on calcitonin and calcium-regulating hormone secretion. Relevance to postmenopausal osteoporosis.
Acta Obstet. Gynecol. Scand. Suppl.
106:
27-32,
1981[Medline].
24.
Whitehead, M. I.,
G. Lane,
O. Young,
S. Campbell,
G. Abeyasekera,
C. J. Hillyard,
I. MacIntyre,
K. G. Phang,
and
J. C. Stevenson.
Interrelations of calcium-regulating hormones during normal pregnancy.
Br. Med. J.
283:
10-12,
1981[Medline].
25.
Woloszczuk, W.,
J. Kovarik,
and
R. Pavelka.
Calcitonin in pregnant women and in cord blood.
Gynecol. Obstet. Invest.
12:
272-276,
1981[Medline].