Calcitonin Expression in Rat Anterior Pituitary Gland Is Regulated by ...

0013-7227/02/$15.00/0 Printed in U.S.A.

Endocrinology 143(10):4056 – 4064 Copyright © 2002 by The Endocrine Society doi: 10.1210/en.2001-210908

Calcitonin Expression in Rat Anterior Pituitary Gland Is Regulated by Ovarian Steroid Hormones YA-PING SUN, TAE JIN LEE,

AND

GIRISH V. SHAH

Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, Texas 79106 Gonadotroph-derived calcitonin-like peptide (pit-CT) is a potent inhibitor of lactotroph function. We investigated the effect of ovarian hormones on pit-CT mRNA expression in the anterior pituitary (AP) gland of cycling female rats. Levels of mRNAs for pit-CT, CT receptor, prolactin (PRL), and ␤-LH during 4-d estrous cycle were determined. In a second study, the effects of estrogens and progesterone on pit-CT and PRL mRNA levels were investigated. In a third group, the effect of estrogen or progesterone depletion on pit-CT mRNA expression was studied. In a fourth group, the effect of passive pit-CT immunization on PRL and LH mRNA expression was examined. Pit-CT mRNA levels varied during estrous cycle. They were

C

ALCITONINS (CTs) ARE a group of polypeptide hormones containing 32 amino acid residues (1– 4). In addition to the thyroid gland, CTs are widely distributed in the central nervous system, the pituitary gland, and several neuroendocrine organs such as lungs, uterus, and the prostate (5–11). Receptors for CT have also been detected in these organs (12–15). CT exerts significant effects on secretion and production of neurotransmitters, and also alters the growth and function of various target organs (16 –19). These include uterine cells, prostate basal epithelial cells, tuberoinfundibular dopaminergic neurones, as well as anterior pituitary (AP) lactotrophs. The diversity of sites of CT production as well as its actions suggests a variety of paracrine and autocrine roles for CTs in addition to their originally described function of regulation of serum calcium. CT-like pituitary peptide (Pit-CT) has been reported to be synthesized and released by gonadotrophs of rat AP gland (9, 20, 21). Pit-CT has been suggested as a negative regulator of lactotroph function in rat AP gland because of its selective, potent inhibition of prolactin (PRL) biosynthesis secretion and lactotroph cell proliferation (22–24). There is evidence to suggest that ovarian steroid hormones significantly affect lactotroph function (25–28). Specifically, estrogens directly stimulate PRL gene expression, biosynthesis, and also increase lactotroph cell proliferation (26, 28, 29). In addition, estrogens have also been shown to induce the synthesis of autocrine stimulators of lactotroph function such as galanin and vasoactive intestinal peptide (30 –33). Progesterone may not have a direct role in regulation of lactotrophs but may

Abbreviations: AP, Anterior pituitary; CT, calcitonin; CT-R, CT receptor; DAPI, 4,6-diamino-2-phenylindole; FITC, fluorescein isothiocyanate; IHC, immunohistochemistry; OVX, ovariectomized; PAM, proestrous morning; Pit-CT, CT-like pituitary peptide; PN, proestrous early afternoon; PPM, proestrous late afternoon; PRL, prolactin.

highest in diestrus, but lowest in the evening of proestrus. CT-receptor mRNA levels displayed smaller fluctuations. Estrogen repletion caused a decline in pit-CT mRNA expression in ovariectomized rats, but progesterone produced a marked increase. ICI 182,780 prevented the decline of pit-CT mRNA levels during late proestrus-estrus, but RU 486 attenuated pit-CT mRNA levels. Passive CT immunization in diestrus altered PRL and LH mRNA expression, and advanced the estrus cycle. These results suggest that pit-CT mRNA expression is regulated by ovarian hormones, and depletion of pit-CT advances their estrous cycle. (Endocrinology 143: 4056 – 4064, 2002)

indirectly affect it by modulating the actions of estrogen at hypothalamic and/or pituitary level (34). Because pit-CT is a gonadotroph-derived paracrine inhibitor of lactotroph function (21), it can significantly influence pituitary function by reducing lactotroph cell populations as well as by attenuating PRL biosynthesis and secretion. The primary objective of the present study was to investigate the role of ovarian steroid hormones in regulation of pit-CT mRNA expression in gonadally intact as well as ovariectomized adult female rats. Materials and Methods Experimental animals Sixty-day-old Fisher 344 rats were purchased from Harlan Sprague Dawley, Inc. (Milwaukee, WI) and housed two per cage. The animals were maintained under the conditions of 12 h of light and 12 h of darkness (lights on at 0600 h) with ad libitum access to tap water and Purina rat chow (Ralston Purina Co., St. Louis, MO). Euthanasia was performed by decapitation under ketamine anesthesia. All animal procedures were conducted according to the protocol approved by the Institutional Animal Care and Use Committee of the Texas Tech University Health Sciences Center.

Reagents 32 P-Uridine triphosphate was purchased from NEN Life Science Products (Boston, MA). ICI 182,780 was purchased from Tocris (Bristol, UK). Mifeprestone (RU 486) and all other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Experimental procedures 1. Estrous cycle. After a 4-d period of acclimatization, estrous cycle of adult female rats was monitored by vaginal cytology. Only rats showing at least three consecutive 4-d estrous cycles were used in the study. After following four consecutive regular 4-d cycles, the rats were killed either at proestrus [at 0900 h PAM (proestrous morning), 1300 h PN (proestrous early afternoon), or 1800 h], estrus, metestrus, or diestrus. At least four rats were used for each data point of the cycle. The AP glands were

4056

Sun et al. • Ovarian Steroids and Pituitary Calcitonin

rapidly collected, and total RNA was extracted under ribonuclease-free conditions. The experiment was repeated three separate times. 2. Ovariectomy and ovarian steroid hormone administration. In some experiments, adult female rats were bilaterally ovariectomized (OVX) under ketamine anesthesia. After a recovery period of 10 d, the OVX rats were divided into four groups, where each group had five rats. The first group received only vehicle injection (0.2 ml sesame oil, sc). The second group received estrogens (E2-2 mg estradiol propionate in 0.2 ml sesame oil, sc). The third group received progesterone (P4, 5 mg in 0.2 ml sesame oil, sc), and the last group received E2 as well as P4. P4 injections in third and fourth group were given 15 h after the E2 (or vehicle) injection. The rats were killed 12 h after the P4 (or vehicle) injection, and the AP glands were collected for RNA preparation. The experiment was repeated three separate times. 3. Treatment with antiestrogen ICI 182,780 compound. Regularly cycling female rats were injected with either ICI 182,780 (1.25 mg in 0.2 ml sesame oil, sc) or vehicle (control group) at 0900 h of proestrus. The dose of the compound was derived from preliminary experiments, which determined a dose-response effect of the compound on PRL mRNA abundance. The animals were then killed on either proestrus [PN or late afternoon (PPM)], estrus, metestrus, or diestrus (four animals per group), and the AP glands were collected for RNA preparation. The experiment was repeated three separate times. 4. Treatment with antiprogestin RU 468 compound. As described in the previous experiment, regularly cycling rats were treated with either RU 486 (4 mg in 0.2 ml sesame oil, sc) or vehicle at 0900 h of proestrus. The dose of the compound was derived from a dose-response effect of the compound on ␤-LH mRNA abundance. The animals were then killed either at proestrus (PN or PPM), estrus, metestrus, or diestrus. The AP glands were harvested in ribonuclease-free condition. 5. Passive immunization with anti-CT serum. The animals for this experiment were monitored for cyclicity by vaginal cytology for at least four 4-d cycles, and those displaying three consecutive regular cycles were included in the experiment. The rats were treated with 75 ␮l rabbit antiserum (iv) in the afternoon of diestrus (1300 h). Control rats received an equivalent amount of nonimmune serum. Our previous results have shown that this treatment caused a large, rapid but long-lasting increase in serum PRL levels in conscious OVX rats (35). The animals were then either monitored for estrous cycle by vaginal cytology, or were killed on the following day (proestrus) at three different times: PAM, PN, or PPM. The AP glands were harvested and total mRNA was extracted as described before. The experiment was repeated one more time.

Plasmid constructs and riboprobe preparation Partial cDNA probes for pit-CT (86 –580 bp; Ref. 21) and CT-receptor (CT-R, 381–739 bp; Ref. 15) were cloned in pGem-T vector, and linearized with ApaI or PstI. A 504-bp fragment of ␤-LH cDNA was cloned into pBluescript SK(⫹/⫺), the plasmid was linearized with BamH1 or HindIII. A 416-bp fragment of rat PRL cDNA (115–531 bp) was cloned into pBluescript SK (⫹/⫺), the plasmid was linearized with SacII or HindIII. The pTRI-␤-actin mouse antisense control template contained a 245-bp fragment of mouse cytoplasmic ␤-actin (220 –303 bp, Ambion, Inc., Austin, TX). 32P-labeled antisense riboprobes for ␤-LH, PRL, CT, CT-R, ␤-actin were transcribed using either SP6, T7, or T3 RNA polymerase. After transcription, the reaction mixtures were digested with ribonuclease-free deoxyribonuclease (Roche Molecular Biochemicals, Indianapolis, IN), the riboprobes were extracted with phenol/chloroform, and precipitated in ethanol. The quality of riboprobes was tested by running a small aliquot in 8 m urea and 5% polyacrylamide gel.

RNA isolation and S1 nuclease protection assay Total RNA from AP glands was extracted by the modified method of Chomczynski and Sacchi as described by Xie and Rothblum (36). In brief, the AP glands were homogenized with prechilled tissue grinder, and lysed using a single step acid-guanidinium thiocyanate-phenol-chloroform extraction. Total RNA was precipitated in isopropanol. The precipitates were washed in 70% ethanol, and dissolved in diethyl pyrocarbonate-treated water.

Endocrinology, October 2002, 143(10):4056 – 4064 4057

Total RNA samples from the AP glands were analyzed for the abundance of CT, ␤-LH, PRL, ␤-actin, and in some cases CT-R mRNA by S1 nuclease protection assays. The S1-nuclease assay kit was purchased from Ambion, Inc., and the manufacturer’s instructions were followed. In brief, 20 ␮g of total RNA were incubated with appropriate antisense riboprobes (approximately 500,000 cpm) for 18 h at 42 C. Sense riboprobes served as negative controls. The samples were then digested with 50 U of S1-nuclease for 30 min at 37 C. The protected RNA was fractionated on 8 m Urea 5% polyacrylamide gel. The gel was then dried and autoradiographed. The autoradiograms were scanned on a GS-700 imaging densitometer (Bio-Rad Laboratories, Inc., Hercules, CA). Each experiment was repeated three separate times.

CT immunohistochemistry (IHC) The AP glands from OVX or steroid hormone (either E2, P4, or E2 ⫹ P4)-treated rats were fixed in Zamboni’s solution, and frozen by submersion in isopentane-dry CO2 bath after mounting in the embedding medium (OCT compound, Tissu-Tek, Miles Laboratories, Elkhart, IN) as previously described (37). The frozen tissues were sliced to 5- to 10␮m-thick sections and thaw-mounted on Superfrost plus glass slides (Fisher Scientific, Pittsburgh, PA). The sections were processed for pit-CT IHC as previously described except that the second antibody (antirabbit IgG) was conjugated to fluorescein isothiocyanate (FITC) (20). The sections were then counterstained in the DNA dye (DAPI solution containing 4,6-diamino-2-phenylindole). The negative controls were treated with primary antiserum that was preincubated with 1 ␮m salmon CT at 37 C for 1 h. The experiments were repeated two more times. Three animals per group were used for these experiments. Sections from all animals were processed simultaneously. Two researchers independently evaluated the slides under Nikon Optiphot microscope (Nikon Instruments, Melville, NY) with epifluorescence attachment, scoring all slides at the same time to avoid comparing preparations that had been stored or exposed to UV-light for different periods of time. Separate digital images for FITC and DAPI were captured on a G3 Power PC computer by a Spot camera attached to the microscope, and number of cells displaying FITC immunofluorescence and DAPI per high power field (⫻400) were determined.

Statistical analysis The results were statistically evaluated by one way ANOVA, and significance was derived using Newman-Keuls test.

Results Modulation of CT and CT-R mRNA expression during estrous cycle

Abundance of CT mRNA in the AP gland varied significantly during the course of estrous cycle (Fig. 1). The mRNA levels were highest during diestrus, and declined to almost 60% in proestrus. A further dramatic decline (almost 3-fold) was observed in estrus where CT mRNA levels were the lowest. The mRNA levels increased markedly in metestrus but were still lower than those seen in diestrus. In contrast, CT-R mRNA levels showed only moderate changes from met- to diestrus (Fig. 1). Therefore, the subsequent experiments focused on regulation of CT, and not CT-R, mRNA expression. In the next series of experiments, we examined the relationship between CT, PRL, (target gene) and ␤-LH mRNAs (because CT originates from gonadotrophs) during estrous cycle. Moreover, proestrous phase of the cycle was examined in a greater detail by obtaining samples at three different times during proestrus: 0900 h (PAM), 1300 h (PN), and 1800 h (PPM). As presented in Fig. 2, CT mRNA levels were higher on the morning of proestrus but declined steadily through the day and reached a nadir at PPM. They remained very low during estrus but increased in metestrus and

4058

Endocrinology, October 2002, 143(10):4056 – 4064


FIG. 1. Changes in pit-CT and CT-R mRNA levels in the AP gland of rats during estrous cycle. Groups of animals (n ⫽ 4 per group) were decapitated in early afternoons of proestrus, estrus, metestrus, and diestrus. The results from S1-nuclease protection assays were digitized, normalized with ␤-actin mRNA levels, pooled and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean ⫾ SEM (n ⫽ 12). Representative autoradiogram has been represented. Level of significance: a ⬍ 0.01, b ⬍ 0.01, c ⬍ 0.001 when compared with E (estrus).

showed a further increase in diestrus. The profile of PRL mRNA expression was completely opposite of CT mRNA expression profile. PRL mRNA levels were highest during PPM when CT mRNA levels were at their lowest. Similarly, PRL mRNA levels were lowest during diestrus when CT mRNA levels displayed their highest abundance. Interestingly, ␤-LH mRNA profile was more similar to PRL mRNA than CT mRNA during early phases of estrous cycle. However, both CT and ␤-LH mRNA levels increased significantly in diestrus. Modulation of CT and CT-R mRNA by ovarian hormones

To examine individual as well as combined effects of estrogen and progesterone on CT mRNA levels, we tested the effects of administration of these hormones in OVX rats. The results presented in Fig. 3 depict the changes in CT and PRL mRNA abundance in the AP glands of OVX animals treated with either the vehicle, E2, P4, or a sequential treatment of E2 followed by P4. The results demonstrate that the treatment of OVX rats with E2 caused a significant, greater than 3-fold,

decline in CT mRNA abundance. As expected, this treatment also resulted in a large, almost 4-fold increase in PRL mRNA abundance. Treatment of OVX rats with P4 resulted in a smaller, nonsignificant, decline in CT mRNA levels but an increase in PRL mRNA abundance. However, P4 significantly increased CT mRNA abundance when given subsequent to the E2 treatment. In contrast, P4 reversed E2-induced increase in PRL mRNA expression. We next examined whether the actions of steroid hormones on CT mRNA abundance translate into alterations in immunoreactive CT in the AP gland (pit-CTI). This was tested by CT IHC of AP glands obtained from OVX rats administered with steroid hormones. The results presented in Fig. 4 demonstrate that AP glands from vehicle treatedOVX and E2 ⫹ P4-treated OVX rats displayed significantly greater and more intense pit-CTI cell populations compared with those treated with either E2 or P4. Pit-CTI cell populations in E2-treated rats were fewer, weaker, but hyperplastic. These results are consistent with the results of Fig. 3 on pit-CT mRNA expression.


FIG. 2. Changes in pit-CT, PRL, and ␤-LH mRNA in the AP gland of rats during estrous cycle. Various groups of rats (n ⫽ 4 per group) were decapitated in the morning, early afternoon, and late afternoon of proestrus, and afternoons of estrus, metestrus, and diestrus. The results from S1-nuclease protection assays were digitized, normalized with ␤-actin mRNA levels, pooled and statistically evaluated by oneway ANOVA and Newman-Keuls test. The results are expressed as mean ⫾ SEM (n ⫽ 12). A representative autoradiogram has been represented. 1, Proestrus 0900 h; 2, proestrus 1300 h; 3, proestrus 1800 h; 4, estrus; 5, metestrus; 6, diestrus. *, P ⬍ 0.05 when compared with 4 (estrus).

Effect of antiestrogen ICI 182,780 on CT mRNA expression during estrous cycle

To further examine the role of estrogens on CT mRNA expression in normal cycling rats, the animals were treated with ICI 182,780 during early proestrous phase. The administration of this antiestrogenic compound prevented the steep decline in CT mRNA abundance during late proestrous and estrous phases (Fig. 5). Similarly, the treatment also prevented a large increase in PRL mRNA abundance during proestrous phase. The treatment did not significantly alter ␤-LH mRNA expression during early phases of estrous cycle but caused an increase in ␤-LH mRNA during met- and di-estrus (Fig. 5). Effect of antiprogestin RU 486 on CT mRNA expression during estrous cycle

To further examine the role of progesterone in CT mRNA expression in cycling rats, we treated cycling rats with RU 486 during proestrus. This treatment caused a decrease in CT


FIG. 3. Effect of ovarian hormone manipulations on pit-CT and PRL mRNA abundance in the AP glands of rats. OVX rats were treated with either vehicle (OVX), E2 (E), P4 (P), or E2⫹P4 (E⫹P) as described in Materials and Methods, and the AP glands were harvested for RNA preparation. Pit-CT, PRL and ␤-actin mRNA levels were determined by S1-nuclease protection assays. The experiment was repeated two more times. The results were digitized, normalized with ␤-actin mRNA levels, pooled, and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean ⫾ SEM (n ⫽ 12) of normalized mRNA abundance. A representative autoradiogram has been represented. *, P ⬍ 0.05 when compared with OVX.

mRNA abundance during all phases of estrous cycle compared with vehicle-treated rats (Fig. 6). However, a decline in CT mRNA abundance in estrous and met-estrous phases was statistically significant. There were no significant changes in PRL mRNA abundance in early phases but the mRNA levels significantly increased during met- and diestrus compared with vehicle-treated rats (Fig. 6). This treatment did not significantly alter ␤-LH mRNA abundance except in met-estrus. Effect of passive pit-CT immunization on PRL mRNA expression

Because CT expression is highest during diestrus, we passively immunized rats with anti-CT serum at 1300 h on that day and examined its effect on PRL and LH mRNA abundance on the day of proestrus. The results presented in Fig. 7 show that the profile of PRL and LH mRNA expression on proestrus in NIS-treated rats is very similar to untreated cycling rats in proestrus as depicted in Fig. 2. The PRL and LH mRNA levels were low at PAM and PN but dramatically higher at PPM. However, passively immunized rats showed a completely different profile. These animals displayed higher PRL mRNA expression at PAM, which declined at PN, and increased slightly at PPM (Fig. 7). Similarly, LH mRNA levels were markedly higher at PAM and PN com-

4060



FIG. 4. Effect of ovarian hormone manipulations on pit-CTI cell populations in AP glands of rats. OVX rats were treated with either vehicle (OVX), E2 (E), P4 (P), or E2⫹P4 (E⫹P) as described in Materials and Methods. The AP glands were obtained, and pit-CT IHC was performed. Number of Pit-CTI-positive cell populations per field (⫻400) were counted, and the percentage pit-CTI cell population was calculated. At least 20 fields per section, and four sections per AP gland, were counted. The results are presented as mean percent pit-CTI cells ⫾ SEM (n ⫽ 3). The results were statistically evaluated by one-way ANOVA and Newman-Keuls test. Representative fluoromicrographs have been presented. *, P ⬍ 0.05 when compared with OVX.

pared with NIS-treated rats and showed much smaller increase at PPM compared with NIS-treated rats (Fig. 7). Most notable change was the complete absence of a dramatic increase in PRL as well as LH mRNA levels. Passive pit-CT immunization may have advanced the estrous cycle because a majority of these rats (8 of 12) displayed estrus as indicated by the presence of large populations of cornified cells in their vaginal smear (examined at PAM). Although, a significant percentage (less than 40%) of nucleated cells were also present. In contrast, almost all NIS-treated rats displayed proestrus phase of the cycle (10 of 11). Discussion

The present results demonstrate that the expression of pit-CT mRNA varied significantly during estrous cycle in rats. However, the changes in CTR mRNA expression were not as marked. This suggests that the expression of CT, but not CTR, is primarily targeted by ovarian steroid hormones. However, these results do not rule out the possibility that ovarian hormones may affect other CT-R actions such as binding characteristics or their ability to activate signal transduction. The results further indicate that CT mRNA levels were significantly lower in follicular phase of the cycle compared with luteal phase and raise a possibility that estrogens may be inhibiting pit-CT mRNA expression where as progesterone may act as a stimulatory hormone. Because pit-CT has been shown to be paracrine inhibitor of PRL gene expression (9), we attempted to examine a relationship between PRL and pit-CT mRNA levels. The pat-

tern of PRL mRNA abundance during estrous cycle was consistent with previous reports, which demonstrated that PRL mRNA levels peaked in the evening of proestrus (38 – 40). Interestingly, PRL mRNA levels were higher during late proestrus and estrus, and pit-CT mRNA levels were almost undetectable during these phases of the cycle. The PRL mRNA levels declined to their lowest during diestrus when pit-CT mRNA abundance increased significantly. These results suggest an inverse relationship between pit-CT and PRL mRNA levels in the AP gland during different phases of estrous cycle and are consistent with the inhibitory action of CT on PRL gene transcription (23). Because pit-CT is synthesized in gonadotrophs, we also examined the relationship between pit-CT mRNA and ␤-LH mRNA abundance during estrous cycle. Present results that ␤-LH mRNA levels begin to increase in early afternoon of proestrus and peak in the late afternoon are in agreement with a previous report (41). ␤-LH mRNA levels were very low in estrus and metestrus, but a large increase was observed in diestrus. When comparing the mRNA abundance profiles of pit-CT and ␤-LH, it appears that the changes in pit-CT mRNA precede those in ␤-LH mRNA. For example, a decline in CT mRNA begins in the noon of proestrus. In contrast, the same in ␤-LH mRNA begins in the evening of proestrus. Similarly, a rise in pit-CT mRNA levels in metestrus is followed by a rise in ␤-LH mRNA levels in diestrus. Although ␤-LH and pit-CT are both expressed in gonadotrophs, these results indicate that they are differentially regulated.



FIG. 5. Effect of ICI 182,780 on pit-CT, PRL, and ␤-LH mRNA levels in AP glands of cycling rats. Normal, cycling female rats were treated with ICI 182,780 or vehicle in the morning of proestrus as described in the Materials and Methods section. The AP glands were collected on proestrus afternoon (P), estrus (E), metestrus (M), and diestrus (D). The mRNA levels for CT, PRL, ␤-LH, and ␤-actin were determined by S1-nuclease protection assay. The experiment was repeated two more times. The results were digitized, normalized with ␤-actin mRNA levels, pooled, and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean normalized mRNA abundance ⫾ SEM (n ⫽ 12) of normalized mRNA abundance. A representative autoradiogram has been presented. *, P ⬍ 0.05 when compared with C (vehicle treated).

Because estrous cycle is predominantly regulated by the timely interplay of ovarian steroids, estrogen and progesterone, we attempted to delineate the specific roles of estrogen and progesterone in regulation of pit-CT mRNA expression. We examined pit-CT mRNA abundance in pituitaries obtained from OVX rats as well as those treated with estrogen alone, progesterone alone, and with estrogen followed by progesterone. These results have demonstrated that estrogen is a potent inhibitor of pit-CT gene expression. In contrast, progesterone has a stimulatory effect in estrogenprimed AP gland. These results suggest a direct action of these two hormones on pit-CT mRNA expression at the level of AP gland. However, an indirect action through the hypothalamus cannot be ruled out. The actions of estrogen and progesterone on pit-CT mRNA expression could be further explained by the current evidence that lactotrophs contain estrogen receptors, but gonadotrophs contain both estrogen and progesterone receptors (42, 43). Moreover, a prior estrogen treatment is necessary for the induction of progesterone receptors, and thereby, for the effectiveness of progesterone treatment (34, 42, 44). Results of pit-CTI immunohistochemistry are consistent with the results of pit-CT mRNA abundance and suggest that pit-CT biosynthesis in

gonadotrophs is regulated at the transcriptional, and not protein, level. Again, these results suggest that ovarian steroid hormones are potent regulators of pit-CT mRNA expression, and confirm our earlier studies that estrogen is a potent inhibitor of pit-CT biosynthesis and secretion (37), whereas progesterone has stimulatory effects. Similar stimulatory effect of progesterone on CT mRNA expression has been reported in the uterus (45). To elucidate the role of estrogens and progesterone in regulation of pit-CT mRNA expression in cycling female rats, we selectively but individually blocked estrogen and progesterone actions by injecting either true antiestrogen ICI 182,780 compound or antiprogestin receptor RU 486 compound during proestrous phase of the cycle. In consistence with the results of experiment 2, administration of ICI 182,780 prevented the decline of pit-CT mRNA levels during proestrous PM and estrous phases of the cycle. A concurrent decline in PRL mRNA levels was also observed at these time points. Interestingly, this treatment caused a marked increase in ␤-LH mRNA abundance during metestrus and diestrus. This may have been due to a possible shift in the estrous cycle caused by the decline in estrogen action at the level of hypothalamus and the AP gland, and subsequent

4062



FIG. 6. Effect of RU 486 on pit-CT, PRL, and ␤-LH mRNA levels in AP glands of cycling rats. Normal, cycling female rats were treated with RU 486 or vehicle in the morning of proestrus as described in the Materials and Methods section. The AP glands were collected on PN, PPM, estrus (E), metestrus (M), and diestrus (D). The mRNA levels for CT, PRL, ␤-LH, and ␤-actin were determined by S1-nuclease protection assay. The experiment was repeated two more times. The results were digitized, normalized with ␤-actin mRNA levels, pooled, and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean normalized mRNA abundance ⫾ SEM (n ⫽ 12). A representative autoradiogram has been presented. *, P ⬍ 0.05 when compared with C (vehicle treated).

shift in GnRH secretion (46, 47). Likewise, the treatment with RU 486 during proestrus resulted in the decline of pit-CT mRNA abundance during estrus, metestrus and diestrus. There was a concomitant increase in PRL mRNA abundance at these time points. However, this affect cannot be exclusively linked to antagonism of progesterone alone because RU 486 also inhibits glucocorticoid receptor. These results again suggest that pit-CT expression by gonadotrophs is inhibited by estrogens and stimulated by progesterone, and reinforce the possibility that the modulation of pit-CT expression in the AP gland could alter the expression of PRL gene. While these results demonstrate tight regulation pit-CT gene expression by steroid hormones, they do not reveal the contribution of pit-CT on PRL gene expression, and its impact on estrous cycle. There, passive pit-CT immunization approach was attempted to deplete endogenous pit-CT. The results show that the depletion of pit-CT during diestrus dramatically alters the proestrus profile of PRL and LH mRNA expression, and prematurely advances the cycle to estrus phase as indicated by PRL and LH mRNA expression as well as vaginal cytology. Specifically, we see premature

increase in LH mRNA expression at PAM and PN, and abolition of evening peaks in PRL and LH mRNAs. The profile of PRL and LH mRNA levels in anti-CT-treated rats looks similar to estrus rather than proestrus, and raise a possibility that the deprival of CT during diestrus may have caused premature increase in PRL, and possibly LH expression (48). These changes could accelerate the process follicular development, ovulation and luteinization (49). Alternatively developing follicle(s) may differentiate without undergoing ovulation (49). Specifically, luteolytic actions of PRL on older corpora lutea, but luteotropic actions on a developing follicle are well documented (35, 50, 51). Additionally, these changes in the estrous cycle can also be caused by direct or indirect (through modulation of LH) actions of CT on ovarian function. Although present results demonstrate observable changes in PRL and LH mRNA expression in response to passive pit-CT immunization, they do not identify the precise chain of events initiated by anti-CT serum. Additional studies will be necessary to systematically evaluate all possibilities, and delineate the chain of events. The AP gland, as indicated by relative proportion of various cell populations as well as their responsiveness to hy-



FIG. 7. Effect of anti-CT serum on PRL and LH mRNA expression. Regularly cycling female rats were treated with 75 ␮l rabbit anti-CT serum (iv) at 1300 h of diestrus. Concurrent controls received equivalent amount of NIS. The AP glands were collected on proestrous 0900 h (PAM), proestrous 1300 h (PN), and proestrous 1800 h (PPM). The mRNA levels for PRL, ␤-LH, and ␤-actin were determined by S1-nuclease protection assay. The experiment was repeated one more time. The results were digitized, normalized with ␤-actin mRNA levels, pooled, and statistically evaluated by one-way ANOVA and Newman-Keuls test. The results are expressed as mean normalized mRNA abundance ⫾ SEM (n ⫽ 12). A representative autoradiogram has been presented. *, P ⬍ 0.05 when compared with respective NIS-controls. NS, Not significant.

pothalamic and gonadal hormones, is continuously remodeled by dynamic changes in the hormonal environment (52, 53). It has been suggested that paracrine/autocrine factors within the AP gland may be responsible for both these changes. For example, estrogen induces vasoactive intestinal peptide and galanin synthesis in the AP gland, which stimulate PRL secretion and induce lactotroph proliferation (30 – 32, 54 –56). Similarly, our studies suggest that pit-CT inhibits PRL gene transcription as well as lactotroph cell proliferation (19, 22–24, 57). Considering the secretion of CT by gonadotrophs and antagonistic functional relationships between gonadotrophs and lactotrophs, these results raise a strong possibility that ovarian hormones may remodel the AP gland by direct actions as well as by modulating the expression of CT and other paracrine/autocrine factors. In conclusion, we have shown that pit-CT expression in the AP gland is attenuated by estrogens but stimulated by progesterone in estrogen-primed rats. Therefore, the timespecific interplay of estrogen and progesterone secretion can significantly modulate pit-CT secretion, and consequently affect lactotroph function. The results from CT passive immunization studies demonstrate that an increase in pit-CT

expression during diestrus may be necessary for the build-up of preovulatory surges in PRL and LH expression. Acknowledgments

The authors gratefully acknowledge Dr. A. F. Parlow and National Hormone and Peptide Program for the gift of rat anti-PRL serum (AFP 425-10-91). Received August 7, 2001. Accepted June 6, 2002. Address all correspondence and requests for reprints to: Girish V. Shah, Ph.D., Texas Tech University Health Sciences Center, Department of Pharmaceutical Sciences, 1300 Coulter, Amarillo, Texas 79106. E-mail: [email protected]. This work was supported by Grant DK-45044 from NIDDK, NIH.

References 1. Azria M 1989 The calcitonins. Basel, Switzerland: Karger 2. Fischer JA, Born W 1985 Novel peptides from the calcitonin gene: expression, receptors and biological function. Peptides 6(Suppl 3):265–271 3. Potts Jr JT 1976 Chemistry of calcitonins. In: Aurbach GD, ed. Handbook of physiology. Washington, DC: American Physiological Society; 423– 437 4. Stevenson JC 1980 The structure and function of calcitonin. Invest Cell Pathol 3:187–193 5. Fischer JA, Tobler PH, Kaufmann M, Born W, Henke H, Cooper PE, Sagar

4064

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30. 31.


SM, Martin JB 1981 Calcitonin: regional distribution of the hormone and its binding sites in the human brain and pituitary. Proc Natl Acad Sci USA 78:7801–7805 Flynn JJ, Margules DL, Cooper CW 1981 Presence of immunoreactive calcitonin in the hypothalamus and pituitary lobes of rats. Brain Res Bull 6:547–549 Sexton PM, Hilton JM 1992 Biologically active salmon calcitonin-like peptide is present in rat brain. Brain Res 596:279 –284 Hilton JM, Mitchelhill KI, Pozvek G, Dowton M, Quiza M, Sexton PM 1998 Purification of calcitonin-like peptides from rat brain and pituitary. Endocrinology 139:982–992 Shah GV, Deftos LJ, Crowley WR 1993 Synthesis and release of calcitonin-like immunoreactivity by anterior pituitary cells: evidence for a role in paracrine regulation of prolactin secretion. Endocrinology 132:1367–1372 Gropp C, Luster W, Havemann K 1985 Salmon and human calcitonin like material in lung cancer. Br J Cancer 51:897–901 Zhu LJ, Cullinan-Bove K, Polihronis M, Bagchi MK, Bagchi IC 1998 Calcitonin is a progesterone-regulated marker that forecasts the receptive state of endometrium during implantation. Endocrinology 139:3923–3934 Rizzo AJ, Goltzman D 1981 Calcitonin receptors in the central nervous system of the rat. Endocrinology 108:1672–1677 Wang J, Rout UK, Bagchi IC, Armant DR 1998 Expression of calcitonin receptors in mouse preimplantation embryos and their function in the regulation of blastocyst differentiation by calcitonin. Development 125:4293–302 Chien J, Ren Y, Wang YQ, Bordelon W, Thompson E, Davis R, Rayford W, Shah GV 2001 Calcitonin is a prostate epithelium-derived growth stimulatory peptide. Mol Cell Endocrinol 181:69 –79 Albrandt K, Mull E, Brady EM, Herich J, Moore CX, Beaumont K 1993 Molecular cloning of two receptors from rat brain with high affinity for salmon calcitonin. FEBS Lett 325:225–232 Clementi G, Nicoletti F, Patacchioli F, Prato A, Patti F, Fiore CE, Matera M, Scapagnini U 1983 Hypoprolactinemic action of calcitonin and the tuberoinfundibular dopaminergic system. J Neurochem 40:885– 886 Franceschini R, Cataldi A, Cianciosi P, Garibaldi A, Corsini G, Barreca T, Rolandi E 1993 Calcitonin and ␤-endorphin secretion. Biomed Pharmacother 47:305–309 Freed WJ, Perlow MJ, Wyatt RJ 1979 Calcitonin: inhibitory effect on eating in rats. Science 206:850 – 852 Judd AM, Kubota T, Kuan SI, Jarvis WD, Spangelo BL, MacLeod RM 1990 Calcitonin decreases thyrotropin-releasing hormone-stimulated prolactin release through a mechanism that involves inhibition of inositol phophates production. Endocrinology 127:191–199 Chronwall BM, Sands SA, Li Z, Shah GV 1996 Calcitonin-like peptide containing gonadotrophs are juxtaposed to cup-shaped lactotrophs. Endocr J 4:27–33 Ren Y, Chien J, Sun Y, Shah GV 2001 Calcitonin is expressed in gonadotropes of the anterior pituitary gland: its possible role in paracrine regulation of lactotrope function. J Endocrinol 171:217–228 Shah GV, Wang W, Grosvenor CE, Crowley WR 1990 Calcitonin inhibits basal and thyrotropin-releasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretogogueinduced increases in cytosolic Ca2⫹. Endocrinology 132:621– 628 Zhang Q, Stanley SM, Shah GV 1995 Calcitonin inhibits prolactin gene transcription in rat pituitary cells. Endocr J 3:445– 451 Shah GV, Chien J, Sun YP, Puri S, Ravindra R 1999 Calcitonin inhibits anterior pituitary cell proliferation in the adult female rats. Endocrinology 140:4281– 4291 Platia MP, Catt KJ, Aguilera G 1986 Effects of 17 ␤-estradiol on angiotensin II receptors and prolactin release in cultured pituitary cells. Endocrinology 119:2768 –2772 Amara JF, Van Itallie C, Dannies PS 1987 Regulation of prolactin production and cell growth by estradiol: difference in sensitivity to estradiol occurs at level of messenger ribonucleic acid accumulation. Endocrinology 120:264 –271 Wiklund J, Wertz W, Gorski J 1981 A comparison of estrogen effects on uterine and pituitary growth and PRL synthesis in F344 and Hotzman rats. Endocrinology 108:1700 –1707 Spady TJ, McComb RD, Shull JD 1999 Estrogen action in the regulation of cell proliferation, cell survival, and tumorigenesis in the rat anterior pituitary gland. Endocrine 11:217–233 Perez RL, Machiavelli GA, Romano MI, Burdman JA 1986 Prolactin release, oestrogens and proliferation of prolactin-secreting cells in the anterior pituitary gland of adult male rats. J Endocrinol 108:399 – 403 Hsu DW, el Azouzi M, Black PM, Chin WW, Hedley Whyte ET, Kaplan LM 1990 Estrogen increases galanin immunoreactivity in hyperplastic prolactinsecreting cells in Fisher 344 rats. Endocrinology 126:3159 –3167 Wynik D, Hammond PJ, Akinsaya KO, Bloom SR 1993 Galanin regulates basal and estrogen-stimulated lactotroph function. Nature 364:529 –532


32. Carretero J, Rubio M, Sanchez F, Vazquez RJ, Santos M, Blanco J, Vazquez R 1995 In vitro morphometric and proliferative variations in VIP-immunoreactive pituitary cells induced by estradiol. Neuroendocrinology 62:277–282 33. Montagne MN, Dussaillant M, Chew LJ, Berod A, Lamberts SJ, Carter DA, Rostene W 1995 Estradiol induces vasoactive intestinal peptide and prolactin gene expression in the rat anterior pituitary independently of plasma prolactin levels. J Neuroendocrinol 7:225–231 34. Brann DW, Rao IM, Mahesh VB 1988 Antagonism of estrogen-induced prolactin release by progesterone. Biol Reprod 39:1067–1073 35. Shah GV, Pedchenko, V, Stanley S, Li Z, Samson WK 1996 Calcitonin is a physiological inhibitor of prolactin secretion in ovariectomized female rats. Endocrinology 137:1814 –1822 36. Xie WQ, Rothblum LI 1991 Rapid, small-scale RNA isolation from tissue culture cells. Biotechniques 11:324, 326, 327 37. Li Z, Shah GV 1995 Estrogen attenuates expression of calcitonin-like immunoreactivity in the anterior pituitary gland. Endocr J 3:452– 459 38. Haisenleder DJ, Ortolano GA, Landefeld TD, Zmeili SM, Marshall JC 1989 Prolactin messenger ribonucleic acid concentrations in 4-day cycling rats and during the prolactin surge. Endocrinology 124:2023–2028 39. Lee BJ, Kim JH, Lee CK, Kang HM, Kim HC, Kang SG 1995 Changes in mRNA levels of a pituitary-specific trans-acting factor, Pit- 1, and prolactin during the rat estrous cycle. Eur J Endocrinol 132:771–776 40. Shimokawa N, Kato Y, Imai K, Wakabayashi K 1990 Changes in content of prolactin mRNA during the rat estrous cycle. Exp Clin Endocrinol 96:8 –14 41. Zmeili SM, Papavasiliou SS, Thorner MO, Evans WS, Marshall JC, Landefeld TD 1986 ␣ and luteinizing hormone ␤ subunit messenger ribonucleic acids during the rat estrous cycle. Endocrinology 119:1867–1869 42. Park D, Cheon M, Kim C, Kim K, Ryu K 1996 Progesterone together with estradiol promotes luteinizing hormone ␤-subunit mRNA stability in rat pituitary cells cultured in vitro. Eur J Endocrinol 134:236 –242 43. Sakabe K, Seiki K, Fujii-Hanamoto H, Haruki Y, Ogawa H 1986 Immunohistochemical evidence of progestin target cells in the pituitary gland of ovariectomized rat. Endocrinol Jpn 33:777–785 44. Genazzani AR, Petraglia F, Silferi M, Lattesa AM, Coukos G, Genazzani AD, Artini P, Nappi C, Volpe A 1990 Progestins modulate the action of estrogen on gonadotropin-releasing hormone, luteinizing hormone and prolactin in the rat. Gynecol Obstet Invest 29:197–202 45. Ding YQ, Zhu LJ, Bagchi MK, Bagchi IC 1994 Progesterone stimulates calcitonin gene expression in the uterus during implantation. Endocrinology 135:2265–2274 46. Tebar M, Ruiz A, Sanchez-Criado JE 1998 Involvement of estrogen and follicle-stimulating hormone on basal and luteinizing hormone (LH)-releasing hormone-stimulated LH secretion in RU-486-induced 3-day estrous cycle in the rat. Biol Reprod 58:615– 619 47. Tebar M, Bellido C, Aguilar R, Sanchez-Criado JE 1994 Inappropriate ovarian feedback in basal gonadotropin secretion in 4-day cyclic rat treated with mifepristone: role of endogenous estradiol. J Endocrinol Invest 17:425– 430 48. Moro M, Inada Y, Miyata H, Komatsu H, Kojima M, Tsujii H 2001 Effects of dopamine D2 receptor agonists in a pituitary transplantation-induced hyperprolactinemia/anovulation model in rats. Clin Exp Pharcol Ther 28:651– 658 49. Lindner HR, Tsafriri A, Lieberman ME, Zor U, Koch Y, Bauminger S, Barnea A 1974 Gonadotropin action on cultured Graafian follicles: induction of maturation division of the mammalian oocyte and differentiation of the luteal cell. Recent Prog Horm Res 30:79 –138 50. Bowen JM, Keyes PL 2000 Repeated exposure to prolactin is required to induce luteal regression in the hypophysectomized rat. Biol Reprod 63:1179 –1184 51. Gaytan F, Bellido C, Morales C, Sanchez-Criado JE 2001 Luteolytic effect of prolactin is dependent on the degree of differentiation of luteal cells in the rat. Biol Reprod 65:433– 441 52. Orgnero de Gaisan E, Maldonado C, Diaz Gavier MF, Aoki A 1997 Diversity of pituitary cells in primary cell culture. An immunocytochemical study. Anat Anz 179:453– 460 53. Pasolli HA, Torres AI, Aoki A 1992 Influence of lactotroph cell density on prolactin secretion in rats. J Endocrinol 134:241–246 54. Cai A, Bowers RC, Moore Jr JP, Hyde JF 1998 Function of galanin in the anterior pituitary of estrogen-treated Fischer 344 rats: autocrine and paracrine regulation of prolactin secretion. Endocrinology 139:2452–2458 55. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408 –7412 56. Balsa JA, Cacicedo L, Lara JI, Lorenzo MJ, Pazos F, Sanchez-Franco F 1996 Autocrine and/or paracrine action of vasoactive intestinal peptide on thyrotropin-releasing hormone induced prolactin release. Endocrinology 137: 144 –150 57. Shah GV, Epand RM, Orlowsky RC 1988 Calcitonin inhibits prolactin secretion in isolated rat pituitary cells. J Endocrinol 116:279 –286