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Endocrinology Vol. 140, No. 9 4281-4291
Copyright © 1999 by The Endocrine Society

ARTICLES

Calcitonin Inhibits Anterior Pituitary Cell Proliferation in the Adult Female Rats1

Girish V. Shah, Jeremy Chien, Ya Ping Sun, Sanjeev Puri and R. Ravindra

Departments of Surgery (S.P., G.V.S., R.R.) and Physiology (J.C., C.V.S.), University of Kansas Medical Center, Kansas City, Kansas 66160; and Department of Pharmaceutical Sciences (J.C., Y.P.S., G.V.S.), Texas Tech University Health Sciences Center, Amarillo, Texas 79106

Address all correspondence and requests for reprints to: Girish V. Shah, Ph.D., Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, 1300 South Coulter Drive, Amarillo, Texas 79106. E-mail: girish{at}cortex.ama.ttuhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that CT-like immunoreactive peptide(s) (pit-CT) is synthesized by the anterior pituitary (AP) gland, and exogenously added salmon(s) CT inhibits PRL release and PRL gene transcription in cultured AP cells. Anti-sCT serum, which immunoreacts with pit-CT, stimulates PRL secretion, suggesting pit-CT is a physiologically relevant PRL-inhibiting hormone. Using proliferating cell nuclear antigen (PCNA) staining and 5-bromo-2'deoxyuridine (BrdU) incorporation into newly replicated DNA, the effect of calcitonin (CT) on cellular proliferation in the rat anterior pituitary gland (AP) was examined. CT significantly attenuated PCNA-immunopositive as well as BrdU-positive AP cell populations in dispersed rat AP cells.

A second series of experiments tested the effects of CT on AP cell proliferation in vivo. OVX + E2 rats were injected with 200 µg CT (iv), the rats killed at various time points, and the APs were processed for BrdU staining. CT inhibited BrdU incorporation at all time points up to 15 h after the injection, and this inhibitory effect was reversed at later time points. The effect of CT was concentration dependent, and a maximal inhibition was observed 10 h after the CT injection. Subsequent experiments identified CT-responsive AP cell populations using double immunofluorescence for BrdU and either PRL or FSH. The number of BrdU-labeled lactotropes in the AP gland declined by 74% in the CT-treated rats. Neutralization of endogenous pit-CT by passive immunization with anti-sCT serum caused a 2-fold increase in AP cell proliferation. These results suggest an important role for the endogenous pit-CT in regulation of lactotrope population of the AP gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ADDITION TO the thyroid gland, calcitonin (CT) and CT-like peptides are present in regions of human and rat brain including the hypothalamus and the anterior pituitary (AP) gland (1, 2, 3), suggesting a physiological role for CT-like products in these tissues. Recent evidence suggests that endogenous CT-like peptide(s) may play an important role in regulation of PRL secretion in the AP gland. For example, serum PRL levels in rats and humans decline significantly after iv injections of salmon (s) CT (4, 5); sCT attenuates baseline and TRH-stimulated PRL secretion by dispersed AP cells from the rat (6, 7, 8), and also inhibits PRL gene transcription in primary AP cell cultures from the rat as well as GH3 cells (9).

Supporting the physiological relevance of sCT actions on lactotrope function are the findings of the presence of sCT-like immunoreactivity in the hypothalamus and the pituitary gland by several investigators (1, 3, 10). It has been further demonstrated that CT-like immunoreactive peptide (pit-CT) is synthesized and secreted by primary cultures of the rat AP gland (11). Pit-CT may share antigenic sites with human CT (hCT) and sCT because antisera raised against these peptides immunoprecipitate molecules of similar size from AP cell lysates (11). Anti-sCT serum stimulates PRL release from primary AP cells and causes a dramatic increase in serum PRL levels when administered iv to ovariectomized conscious rats (12). These results strongly suggest that pit-CT is a physiologically relevant PRL-inhibiting peptide of pituitary origin.

Receptors recognizing sCT have been detected in specific regions of rat brain and the AP gland (13, 14), and complementary DNAs for two such receptors have recently been cloned from a rat brain complementary DNA library (15). Therefore, it is conceivable that pit-CT, which shares antigenic epitopes with sCT and hCT, may serve as an endogenous ligand for sCT receptors in the AP gland; and in concert with other paracrine and hormonal factors, regulates PRL gene expression and modulates responsiveness of lactotropes to neurohormones during different physiological conditions.

Several investigators have shown that cell populations of the AP gland are not static but constantly dynamic. Cell populations of the AP gland are shown to proliferate consistently at a low rate in a normal adult rat, and a dramatic increase in the number of mitotic lactotropes has been shown to occur during the afternoon of estrus (16). There is evidence to suggest a direct correlation between PRL gene expression and lactotrope proliferation. For example, blockers of DNA synthesis such as nicotinamide inhibit PRL gene expression (17). On the other hand, dopamine, a PRL-inhibiting hormone, inhibits lactotrope cell proliferation (18). Because sCT is a potent inhibitor of PRL release and gene expression, the present study was undertaken to investigate the effect of sCT on cellular proliferation in the rat AP gland. Using 5-bromo-2'deoxyuridine (BrdU) incorporation into newly replicated DNA as an index of cellular proliferation (19), we report here that CT has a profound inhibitory effect on proliferation of AP cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Antimouse PCNA (proliferating cell nuclear antigen) antibody and 5-bromo-2'deoxyuridine (BrdU)-staining kit were purchased from Zymed Laboratories, Inc. (South San Francisco, CA). Salmon calcitonin (sCT) was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Culture media and other tissue culture reagents such as Trypsin-EDTA solution, Penicillin G-Streptomycin mixture, horse and fetal calf sera were obtained from Life Technologies, Inc. (Grand Island, NY). All other chemicals were purchased from Sigma (St. Louis, MO).

Animals
Sixty-day-old adult female Fisher (or Holtzman in some experiments) 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 Pharmaceuticals, Inc. water and Purina rat chow (Ralston Purina Co., St. Louis, MO). After a 4-day period of acclimatization, the rats were bilaterally ovariectomized under ketamine anesthesia. Ovariectomized (OVX) rats were allowed to recover for 10 days and then implanted with a silastic tube (30 mm long, id 1.57 mm, od 3.18 mm) containing either a vehicle (sesame oil) or 500 µg/ml of 17-ß estradiol propionate (E2) dissolved in sesame oil under the skin of the back. In some experiments, ovariectomized rats were implanted with E2 and progesterone (P4; 1 mg/ml) or P4 alone. Three days after implantation, age-matched steroid-treated rats were killed, and the APs were removed. This E2 treatment has previously been demonstrated to produce serum E2 concentrations similar to those observed in the afternoon of proestrus (20, 21).

Euthanasia was performed by decapitation under ketamine anesthesia. Protocols for the surgery as well as euthanasia were approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center and the Texas Tech University Health Sciences Center.

Preparation of primary cultures of anterior pituitary cells
The anterior pituitary (AP) glands obtained from OVX + E2 rats were transferred to serum-free culture medium, minced to very small pieces, trypsinized, and filtered through a stainless steel mesh as described before (11). The filtrate was centrifuged at 400 x g for 10 min at room temperature, and the cell pellet was resuspended in the culture medium (DMEM supplemented with 10 mM HEPES, 10% horse serum, 5% FCS, 280 µg/ml bacitracin, 100 U/ml penicillin G-sodium, and 100 µg streptomycin sulfate). After trituration, approximately 5000 cells were seeded onto each well of 10-well microscopic slides (Fisher Scientific, Pittsburgh, PA), and incubated at 37 C in 95% air-5% CO2.

Proliferating cell nuclear antigen (PCNA) staining of dispersed AP cells
Dispersed AP cells were allowed to attach for 24 h, and then washed three times with serum-free DMEM supplemented with 10 mM HEPES, 280 µg/ml bacitracin, 100 U/ml penicillin G-sodium and 100 µg streptomycin sulfate. The cells were then treated with various concentrations of sCT and incubated in the serum-free DMEM for an additional period of 24 h. At the end of this period, the cells were fixed in Zamboni’s solution for 1 h, washed three times with PBS, and incubated with blocking solution (10% goat serum in 0.4% Triton X-100-PBS for 10 min. This was followed by an overnight incubation with anti-PCNA antibody (1:5 dilution of the working solution in 0.4% Triton X-100-PBS). Subsequently, the cells were washed three times with PBS, incubated with biotinilated antimouse second antibody (1:200) for 3 h, and processed for horse radish peroxidase assay according to the manufacturer’s instructions (Zymed Laboratories, Inc.). The brown color, indicating PCNA staining, was observed using a Nikon (San Francisco, CA) Optiphot microscope equipped with a Sony video camera. The total numbers of cells as well as PCNA-labeled cells were counted in 5–10 different fields (at 400x magnification) of each well; 500–800 cells per each treatment group were counted. Two researchers independently made the measurements on all slides. The data were expressed as number of cells per field = total number of cells (or immunopositive cells) counted/number of fields examined.

BrdU staining of dispersed AP cells
Twenty-four hours after plating, the AP cells were washed three times with serum-free DMEM, various concentrations of sCT and 1 µM BrdU added, and the cell cultures incubated in serum-free condition for an additional 24-h period. At the end of the incubation period, the cells were fixed in Zamboni’s solution for 1 h, washed three times with PBS, and incubated with the blocking solution for 10 min at room temperature. This was followed by an overnight incubation with anti-BrdU antibody. The cells were then processed for BrdU staining according to the manufacturer’s instructions (Zymed Laboratories, Inc.). The brown color, indicating BrdU staining, was observed using a Nikon Optiphot microscope equipped with a Sony video camera. The total numbers of cells as well as BrdU-labeled cells were counted in 5–10 different fields of each well (as described in PCNA ICC).

Immunocytochemistry (ICC) of the AP tissue
Normal cyclic, ovariectomized (OVX), or OVX rats implanted with E2, P4, or E2 and P4 were injected with sCT (200 µg/rat, iv), and the rats were killed after 10 h; BrdU (10 mg/rat, ip) was administered 3 h before rats were killed. The AP glands obtained from these rats were rapidly fixed in Zamboni’s solution for 2 h, rinsed in PBS, and frozen by submersion in isopentane-dry CO2 bath after mounting in the embedding medium (OCT compound, Tissue-Tek, Miles Laboratories, Elkhart, IN). The frozen tissues were sliced to 5–10 µm thick sections and thaw-mounted on Superfrost plus glass slides (Fisher Scientific, Pittsburgh, PA). The sections were stored frozen at -70 C until ICC analysis. The tissue sections were stained for BrdU using BrdU-staining kit and the manufacturer’s protocol was followed (Zymed Laboratories, Inc.). Each section was examined under a Nikon optiphot microscope and the total numbers of cells as well as BrdU-labeled cells were counted in 10 randomly selected fields. At least four sections per AP gland were examined, and each experimental group had a minimum of three AP glands.

Double immunofluorescence for BrdU and PRL
To identify lactotropes, BrdU-PRL double immunofluorescence was performed. The AP sections from OVX + E2 rats were incubated with biotinilated mouse BrdU antibody and rat anti-PRL serum (1:250, NIH, Bethesda, MD); the secondary antibody for the PRL antiserum was conjugated to rhodamine, and was used at a dilution of 1:50 (Jackson ImmunoResearch Laboratories, Inc. Westgrove, PA). BrdU-labeled cells were identified by incubating with streptavidin-FITC (1:20, Zymed Laboratories, Inc.). In preliminary experiments, it was determined that the best double immunostaining was obtained when the sections were first incubated with antibodies to BrdU and PRL. Subsequent incubation was with a secondary antibody to anti-PRL serum, followed by streptavidin-FITC (for biotinilated BrdU antiserum). Each section was examined under a Nikon microscope equipped with filters XF23 (for fluorescein), XF 32 (for rhodamine), XF 101 (for dual wavelengths, Omega Optical, Brattleboro, VT) and a 100W high pressure mercury lamp. The total number of cells and BrdU + PRL labeled cells were identified and counted in 10 randomly selected fields (as described before).

Modulation of CT action by ovarian hormones
In the female rat, E2 is secreted from the ovaries during the period between the afternoon of diestrous II and the morning of proestrous. E2 influences the vaginal smears, increases uterine weight (23), and alters the pituitary responsiveness to hypothalamic hormones at proestrous (24). E2 has also been shown to attenuate expression pit-CT and increase proliferation of lactotropes (10, 25). In addition, E2 is known to stimulate endocrine functions of lactotropes including the synthesis and secretion of PRL in vivo (26) and in vitro (27, 28). The E2 actions on the preovulatory gonadotropin and PRL surges in cycling female rats can be duplicated by administering E2 to OVX rats (25). The present study examined whether the action of CT on AP cell proliferation is altered by either 1) administration of E2, P4 or the combination; and 2) by physiological changes in the hormonal status of rats during estrous cycle.

Statistical analysis of data
Each experiment was conducted using a minimum of three rats per group. Statistical significance of the data were examined by ANOVA and Fisher tests. Means were deemed significantly different at a P value of less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of CT on proliferating activity of dispersed AP cells
Proliferative activity of AP cells obtained from OVX + E2 rats was investigated by PCNA ICC. The results from three independent experiments (using AP cells from three different sets of rats) suggest that approximately 22% of total AP cells were PCNA immunopositive. These results were obtained after counting at least eight wells per each experiment (total 500–800 cells); and 5–10 fields per well were counted. Incubation with CT caused a marked reduction in the number of PCNA-immunopositive AP cells. Although the inhibition by CT appeared to be concentration dependent, CT did not induce statistically significant inhibition at concentrations of 1 nM or lower; the inhibition ranged from 50 and 59% at 10 nM and 1 µM CT (Fig. 1AGo; P < 0.05). The number of PCNA-immunopositive cells per field was 19.8 ± 2.1, 9.9 ± 0.7 and 8.6 ± 0.37 (mean ± SEM) with 0, 10 nM, and 100 nM, respectively. A representative photomicrograph of PCNA-positive cells is shown in Fig. 2Go.



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  		Figure 1. Effect of CT on proliferative activity of dispersed AP cells. A, Primary cell cultures of AP from OVX + E2 rats were incubated with various concentrations of CT at 37 C for 24 h. Subsequently, the cells were incubated with anti-PCNA antibody (1:5 dilution) and processed to identify PCNA-immunopositive cells as described in Materials and Methods. Each point represents mean number of cells per field ± SEM (n = 30); ten fields were counted in each of the three wells. This is a representative of three similar experiments. *, Value significantly different from the control value (P < 0.05). B, Primary cell cultures of AP from OVX + E2 rats were incubated with various concentrations of CT at 37 C for 24 h. Subsequently, BrdU incorporation by these cells was carried out as described inMaterials and Methods. Each point represents mean number of cells per field ± SEM (n = 30); ten fields were counted in each of the three wells. This is a representative of three similar experiments. *, Value significantly different from the control value (P < 0.05).

 


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  		Figure 2. Photomicrograph of PCNA-immunopositive AP cells. Primary cell cultures of AP from OVX + E2 rats were incubated 1 nM CT at 37 C for 24 h. Subsequently, the cells were incubated with anti-PCNA antibody (1:5 dilution) and processed to identify PCNA-immunopositive cells as described inMaterials and Methods. Upper panel, Control cells: dark staining indicates PCNA-immunopositive AP cells (magnification, 400x). Lower panel, CT-treated cells: light staining indicates hematoxylin-stained PCNA-negative AP cells (magnification, 400x).

 
We conducted a similar experiment in the AP cells obtained from Holtzman rats. This is because the Fisher rats are known to be highly responsive to estrogens and form pituitary tumors when exposed to high concentrations of estrogens for a longer period of time (22). In contrast, Holtzman rats are resistant to tumorigenic actions of estrogens on the AP gland. AP cells were obtained from OVX + E2 Holtzmann rats (as described in the Materials and Methods section), and treated with 100 nM CT (the most effective concentration in Fig. 1Go) for 24 h. The cells were then processed for PCNA-ICC. Consistent with the previous results, CT also attenuated PCNA-immunopositive AP cells from Holtzman rats. The treatment with 100 nM CT caused a 78% decrease in PCNA-immunopositive cell populations. The numbers of PCNA-immunopositive cells were 11.54 ± 3.86 per field (for control) and 2.25 ± 1.28 per field for CT-treated cells.

Effect of CT on BrdU incorporation of dispersed AP cells
In another experiment, proliferative activity of AP cells obtained from OVX + E2 Fisher rats was investigated by immunostaining of 5-bromo-2'deoxyuridine (BrdU). The AP cell cultures were incubated with BrdU (1 µM/well) for 24 h before BrdU staining. Approximately 18% of the total AP cells incorporated BrdU in control primary cultures, and the presence of CT caused a significant decrease in BrdU incorporation. The number of BrdU-labeled cells per field was 17.5 ± 2.6, 14.7 ± 0.7, 9.8 ± 0.24, 8.7 ± 0.6, 8.7 ± 3.1, and 7.1 ± 0.66 with 0, 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 µM CT, respectively. The inhibition of BrdU-labeled cells by CT was concentration dependent; the inhibition was 16, 44, 50, 50, and 59% at 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 µM CT, respectively (Fig. 1BGo; P < 0.05).

Effect of CT on BrdU incorporation: in vivo studies
Time course of CT action. OVX + E2 rats were injected with 200 µg CT (iv), the rats killed at different time points, and the APs were processed for BrdU staining as described in Materials and Methods. BrdU-labeled cells comprised of 14.7 ± 0.34% of total AP cells (mean ± SEM, n = 3 rats; from each AP gland, four sections were analyzed, and in each section ten fields were counted). The inhibition of cell proliferation by CT appeared to be time dependent. The number of BrdU-labeled cells per field were 43.7 ± 1.9, 24.7 ± 2.2, 11.5 ± 1.0, and 14.4 ± 1.0 at 0, 5, 10, and 15 h, respectively. The inhibition of cell proliferation by 200 µg CT was 43, 74, and 67% at 5, 10, and 15 h, respectively (Fig. 3Go; P < 0.05). A maximal inhibition of 74% occurred at 10 h.



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  		Figure 3. Time course effect of CT on in vivo BrdU incorporation by AP cells. OVX + E2 rats were injected (iv) with 200 µg CT, and the rats killed at the indicated time points, and the AP sections were processed for BrdU labeling as described in Materials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before the rats were killed. Each point represents the mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. *, Value significantly different from the control value (P < 0.05).

 
Dose response
CT inhibited AP cell proliferation in a dose-dependent fashion. At 0, 50, 100, and 200 µg of CT, the number of BrdU-labeled cells per field were 42.2 ± 2.1, 19.9 ± 1.1, 13.8 ± 1.1, and 10.7 ± 1.6, respectively; the inhibition was 53, 67, and 75% at 50, 100, and 200 µg CT, respectively (Fig. 4Go; P < 0.05). Although 50 µg CT produced a statistically significant inhibition of cell proliferation in this particular experiment, subsequent experiments used a higher dose of 200 µg of CT. This was in consideration for possible individual variations in transport of the injected CT to target tissues as well as in biological responses by these organs.



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  		Figure 4. Dose response of CT on in vivo BrdU incorporation by AP cells. OVX + E2 rats were injected (iv) with various doses of CT, and the rats killed after 10 h, and the AP sections were processed for BrdU labeling as described inMaterials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before the rats were killed. Each point represents the mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. *, Value significantly different from the control value (P < 0.05).

 
Reversibility of CT action
To examine whether the effect of CT is transient, reversible, and not cytotoxic, OVX + E2 rats were injected with CT (200 µg iv) or vehicle (saline). The rats were then killed at 10 h, 24 h, and 48 h after the injection, and the APs were processed for BrdU ICC. Similar to the previous experiments, the APs from CT-treated rats (10 h after the injection) displayed dramatically lower number of BrdU-positive cells compared with the concurrent controls. However, 24 h after the CT injection, BrdU-labeled cells in the APs from CT-treated rats were slightly higher than those in concurrent controls. This may have been due to a possible rebound effect following the removal of CT suppression. Forty-eight hours after the CT injection, numbers of BrdU-labeled cells in the controls as well as CT-treated rats were similar (Fig. 5Go). The numbers of BrdU-labeled cells per field were 17 ± 1.1, 67 ± 3.4 and 58 ± 2.9 at 10 h, 24 h, and 48 h after the CT injection. Concurrent control APs showed 53 ± 2.4, 57 ± 2.9 and 58± 3.1 BrdU-labeled cells per field. CT caused 68% inhibition in BrdU incorporation at the 10-hour data point. However, the APs of CT-treated rats showed a 17% increase in BrdU-labeled cell populations at the 24-h data point. The control APs did not show any significant variations in BrdU-labeled AP cell populations during the experimental period.



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  		Figure 5. Inhibitory effect of CT on AP cell proliferation is reversible. OVX + E2 rats were injected (iv) with 200 µg CT, and the rats killed at the indicated time points, AP tissue dissected out and processed for BrdU labeling cells as described in Materials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before the rats were killed. Each point represents the mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. *, Value significantly different from the control value (P < 0.05).

 
Influence of E2 and P4 on the effect of CT on AP cell proliferation in OVX rats
Present results suggest that CT inhibited AP cell proliferation by about 75% in E2-treated OVX rats (Fig. 6AGo; P < 0.05). The average numbers of BrdU-labeled cells per field were 42.2 ± 2.1 in OVX + E2 + vehicle rats compared with 10.7 ± 1.6 in OVX + E2 + CT rats. A representative photomicrograph of BrdU-labeling of AP sections in OVX + E2 rats is shown in Fig. 7Go.



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  		Figure 6. Influence of ovarian steroids on the effect of CT on AP cell proliferation in OVX rats. OVX rats were implanted with 17ß-estradiol (E2), E2 and progesterone (P4), or P4 alone for 3 days and injected (iv) with 200 µg CT, and the rats killed after 10 h, and the AP sections were processed for BrdU labeling as described inMaterials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before the rats were killed. Each point represents the mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. *, Value significantly different from the control value (P < 0.05).

 


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  		Figure 7. A typical photomicrograph of BrdU-labeled cells from OVX + E2 rats. OVX + E2 rats were injected (iv) with 200 µg CT, and the rats killed after 10 h, AP tissues were obtained and processed for BrdU labeling as described inMaterials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before rats were killed. Upper panel, A typical AP section of control rats (magnification, 200x). Lower panel, A typical AP section of CT-treated rats (magnification, 200x).

 
It is well established that P4 dampens the effect of E2 (29), and therefore the number of BrdU-labeled cells is expected to be less in OVX + E2 + P4 rats. BrdU-labeled cells per field were 21.3 ± 1.15 in OVX + E2 + P4, which is significantly lower than 42.2 ± 2 in the case of OVX + E2 rats. Thus, the decrease in the BrdU-labeled cell number in E2 and P4 treated rats again appears to corroborate the observation that P4 negates the effect of E2 (29). In OVX + E2 + P4 rats, the inhibition of BrdU labeling by CT was 42%; the cell numbers per field were 21.3 ± 1.15 and 12.4 ± 0.64 in control and CT-injected rats respectively (Fig. 6BGo; P < 0.05).

In another experiment, OVX rats were implanted with P4 alone and the effect of CT was studied. In these rats also, the inhibition of cell proliferation by CT was 43%. The cell numbers per field were 19.1 ± 1.1 and 10.9 ± 0.52 in control and CT-injected rats, respectively (Fig. 6CGo; P < 0.05). Again, BrdU-labeled cells in P4-treated APs were about 50% lower than those from OVX + E2 rats.

Effect of anti-sCT serum on AP cell proliferation in OVX rats
We have previously demonstrated that pituitary content of pit-CT in the ovariectomized (OVX) rats was significantly higher than sham-operated controls (10). Therefore, the injection of CT to OVX rats should not inhibit AP cell proliferation as the AP gland is already exposed to high concentrations of endogenous pit-CT. This hypothesis is validated by the observation that CT did not affect BrdU incorporation in OVX AP glands; the cell numbers per field were 20.7 ± 1.7 and 19.3 ± 1 in OVX rats and OVX rats injected with CT, respectively (200 µg for 10 h; Fig. 8AGo). It was previously shown that anti-sCT serum neutralizes endogenous pit-CT under in vivo as well as in vitro conditions (12). To examine if anti-CT antibodies might also remove inhibitory effect of endogenous pit-CT on AP cell proliferation, we injected two doses of anti-sCT serum to OVX rats (0.35 ml x 2; the first one at 1100 h and the second injection at 2300 h; the concurrent controls received equivalent amounts of nonimmune serum), and the rats were killed at 1100 h on the subsequent day. BrdU was administered 3 h before killing. In rats treated with the antiserum, a dramatic increase in BrdU labeling of the AP cells was observed. The cell number per field was 22.9 ± 0.9 and 11.1 ± 0.7 in antiserum- and nonimmune serum-treated rats, respectively; anti-sCT serum stimulated the AP cell proliferation by about 100% (Fig. 8BGo; P < 0.05).



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  		Figure 8. AP cell proliferation in OVX rats. A, OVX rats were injected (iv) with 200 µg CT and the rats killed after 10 h, and AP sections were processed for BrdU labeling as described in Materials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before rats were killed. Each point represents the mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. B, OVX rats were injected (iv) with anti-sCT serum. Two doses (0.35 ml x 2) of the antibody were administered, the first one at 1100 h and the second injection at 2300 h, and the rats were killed at 1100 h on the subsequent day. The controls received a similar treatment with nonimmune serum. BrdU was administered 3 h before rats were killed. The AP glands were sectioned and processed for BrdU labeling cells as described in Materials and Methods. Each point represents the mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. *, Value significantly different from the control value (P < 0.05).

 
Effect of CT on lactotrope proliferation
To identify the type of AP cells affected by CT treatment, tissue sections obtained from OVX + E2 rats were processed for double labeling with BrdU and PRL. The number of BrdU-labeled lactotropes per field was 17. ± 1.06 and 4.6 ± 0.35 in control and sCT-treated rats, respectively (Fig. 9AGo; P < 0.05); CT inhibited lactotrope proliferation by 74%. A representative photomicrograph of BrdU + PRL labeling of the AP cells is shown in Fig. 9BGo.



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  		Figure 9. Effect of CT on lactotrope proliferation. A, OVX + E2 rats were injected (iv) with 200 µg CT, and the rats killed after 10 h, AP tissue dissected out and processed for double-labeling with BrdU and PRL as described in Materials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before rats were killed. Each point represents mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. *, Indicates the value significantly different from the control value (P < 0.05). B, A typical section of OVX + E2 AP gland shows that a majority of proliferating cells are lactotropes. Upper panel, PRL-rhodamine labeled secondary antiserum in cytoplasm (magnification, 200x). Middle panel, BrdU- fluorescein label in nucleus (magnification 200x). Lower panel, Overlay of the middle panel over the upper panel.

 
Effect of CT on AP cell proliferation in cycling rats
The last series of experiments investigated the effect of CT on AP cell proliferation in different stages of estrous cycle. The three stages of estrous cycle: proestrus, estrus, and diestrus were determined by vaginal histology every afternoon [around 1600 h, (23)]. The rats were then treated with CT on the following morning; the AP glands were obtained and processed for BrdU immunohistochemistry. The number of BrdU-labeled cells per field was 18.05 ± 1.02, 16 ± 1.1, and 23.7 ± 1.1 in diestrous, proestrous, and estrous rats, respectively; (diestrous vs. estrous P < 0.05, proestrous vs. estrous P < 0.05). In proestrous rats, the administration of CT had no effect on the AP cell proliferation (Fig. 10AGo). However, in estrous and diestrous rats, CT inhibited cell proliferation by 43 and 24%, respectively (P < 0.05). In estrous rats, BrdU-labeled cells per field were 23.7 ± 1.1 and 13.4 ± 0.7 in control and CT-treated rats, respectively; in diestrous rats, BrdU-labeled cells per field were 18.0 ± 1.0 and 13.2 ± 0.7 in control and CT-treated rats, respectively (Fig. 10Go, B and C). A representative photomicrograph of BrdU-labeled AP sections from estrous rats is shown in Fig. 11Go.



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  		Figure 10. Effect of CT on AP cell proliferation in cycling rats. Groups of rats were injected (iv) with 200 µg CT and the rats killed after 10 h; the AP glands were sectioned and processed for BrdU labeling as described in Materials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before rats were killed. Each point represents the mean number of cells per field ± SEM from three rats; from each AP gland four sections were analyzed, and in each section ten fields were counted. *, Value significantly different from the control value (P < 0.05).

 


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  		Figure 11. Photomicrograph of BrdU-labeled cells from estrous rats. Estrous rats were injected (iv) with 200 µg CT, and the rats killed after 10 h, the AP glands were sectioned and processed for BrdU labeling as described in Materials and Methods. BrdU (10 mg/rat, ip) was administered 3 h before rats were killed. Upper panel, A typical AP section from the control rats (magnification 200x). Lower panel, A typical AP section from the CT-treated rats (magnification 200x).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is generally accepted that fully differentiated cells rarely undergo mitotic division. Interestingly, the AP cells synthesizing various hormones and considered fully differentiated were observed to undergo mitotic division, albeit at a low rate except under certain physiological conditions (30). In fact, recent reports suggest that the AP cell proliferation is significantly altered by a variety of gonadal and neuroendocrine hormones. In vivo treatment of rats with TRH was shown to increase BrdU labeling by AP cells (31) as well as cells from the intermediate lobe (32). In the adult male rat AP gland, angiotensin II stimulated proliferation of lactotropes (33, 34). The proliferation of mouse lactotropes and corticotropes was stimulated by IGF-I (35).

Several techniques have been described to analyze the rate of cell proliferation in various organs. It appears that the nonradioactive BrdU labeling technique is the method of choice to investigate cellular proliferation. BrdU is a thymidine analog and its incorporation into DNA of S phase cells is detected by anti-BrdU serum (36). This technique has been used extensively to characterize cellular proliferation in the AP tissue sections from rats treated with BrdU in vivo (19, 36, 37). Alternatively, PCNA expression has also been used as an index of cell proliferation. PCNA (36 kDa) is produced in G1 and S phases of the cell cycle (38). Because it is an auxiliary protein of DNA polymersase {delta}, PCNA is required for DNA replication (38). Several studies indicate that the PCNA labeling index represents a reliable approach to investigate cellular proliferation in tissues from various species (37). However, PCNA values were reportedly very high in histological sections obtained from human AP tissue, indicating that PCNA may be unsuitable as a proliferation marker for human AP adenomas (39). In the present study, PCNA proliferation indices correlated well with BrdU incorporation in vitro. The percentages of PCNA positive and BrdU-labeled cells in untreated primary AP cultures were similar, and were 22 and 18 respectively. Using both the methods, we observed that CT maximally inhibited AP cell proliferation by about 50% in AP cells obtained from OVX Fisher 344 rats receiving short-term term estrogen treatment. However, the peptide was more potent in inhibiting AP cell proliferation of Holtzman rats. It has been shown that the AP glands of Holtzman rats are resistant to the tumorigenic actions of estrogens (22). These results, when combined with the previous findings that immunoreactive CT is synthesized and released by gonadotropes, suggest that CT may be a physiological regulator of lactotrope proliferation.

The data presented in this study suggest that the inhibitory effect of CT on AP cell proliferation was more dramatic when the peptide was administered in vivo to the rats than when it was added to the cultured AP cells in vitro. The percentages of BrdU-labeled cells in control AP sections as well as in dispersed AP cells were comparable (15% in vivo vs. 18% in vitro). However, the inhibitory effect of CT on cell proliferation was more dramatic under in vivo conditions (75% in vivo vs. 50% in vitro). This may have been due to a combination of reasons such as: 1) enzymatic dispersal of the AP gland may disrupt cellular cytoskeleton and could also alter cell-to-cell interactions within the AP gland; 2) secretion and/or effectiveness of endogenous pit-CT may be less than optimal under in vitro conditions. It has been shown that pit-CT originates from gonadotropes (41), and gonadotropes show close apposition with lactotropes (42); and 3) in addition to its direct effect on lactotropes, in vivo administration of CT may also influence lactotrope proliferation by other extrapituitary mechanisms such as an increase in the hypothalamic dopamine synthesis (43). The inhibitory action of CT was dose dependent, was relatively rapid, and the maximal inhibition occurred 10 h after the injection. The inhibitory action of CT was reversible and a small but significant rebound in cell proliferation was observed after 24 h.

Previous findings from this laboratory have shown that ovariectomy (OVX) induced a large increase in pit-CT secretion (10). Therefore, in the presence of high endogenous CT, infusion of additional exogenous sCT should not significantly influence AP cell proliferation. Consistent with this assumption, CT did not influence AP cell proliferation in OVX rats. In contrast, immunoneutralization of endogenous pit-CT in OVX rats with anti-sCT serum caused a 2-fold increase in AP cell proliferation. These results strongly suggest a role for the endogenous pit-CT in the AP cell proliferation and corroborate our previous observations of stimulation of PRL secretion by passive immunization with anti-sCT serum (12).

The present results that E2 treatment of OVX rats significantly increases AP cell proliferation are consistent with the current evidence (44, 45, 46). BrdU-labeled cell population in E2-treated rats was at least 2-fold greater than that seen in OVX rats. E2 has also been shown to induce PRL gene expression and lactotrope cell proliferation by influencing multiple events such as the attenuation of dopamine secretion, an increase in TRH secretion and a direct effect at the lactotrope level (47, 48, 49, 50). Recent evidence suggests that at least some of E2 actions involve various paracrine peptides such as VIP, galanin, neurotensin, and pit-CT (10, 51, 52). Thus, E2-primed rats should have minimal endogenous pit-CT levels, and therefore, the AP gland should be most responsive to exogenously administered sCT. Consistent with this hypothesis, sCT was most effective in inhibiting AP cell proliferation in E2-treated OVX rats. Taken together, these results raise a possibility that estrogens stimulate lactotrope proliferation, at least in part, by removing the inhibitory influence of endogenous pit-CT.

To determine the specific AP cell type that is affected by CT, AP tissue sections from OVX + E2 rats were processed for double immunostaining for FSH/PRL and BrdU. Although a small population of AP cells from OVX rats stained positively for the gonadotropin, CT did not affect the proliferation of gonadotropes (data not shown). This is consistent with the observation that, in OVX rats, CT did not affect the proliferation of AP cell population in general. E2 treatment of OVX rats was observed to increase lactotrope proliferation as determined by double immunostaining for PRL and BrdU (16). Using this paradigm, we report here that CT specifically inhibited lactotrope proliferation by 74%. These results further support the paracrine inhibitory role for pit-CT in regulation of lactotrope function.

Our results that rat AP cells proliferate at a lower rate during estrous cycle and that a marked increase in the rate of cell proliferation occurs at the estrus are in agreement with the previous reports (25, 53). The results have shown that CT affected proliferation of AP cells in normal cycling rats. Although a modest inhibition of AP cell proliferation during diestrus and estrus by CT was observed, CT did not influence cell proliferation during proestrus. This discrepancy with the hypothesis, that E2 surge (known to occur at proestrus) is expected to inhibit endogenous production of CT and thus make the AP cells more receptive to the actions of exogenous CT, could possibly be explained by the fact that proestrous phase was determined on the basis of vaginal smears on the previous afternoon, and either the time of CT administration or the time of killing of proestrous rats may not have coincided with the E2 surge. It is also conceivable that neuroendocrine and/or hormonal secretions during E2 surge may have antagonized the CT action (16, 25). Additional studies will be necessary to explain this phenomenon.

In conclusion, using PCNA and BrdU methods, we have demonstrated that CT inhibited AP cell proliferation in vitro as well as in vivo. A modest inhibition of AP cell proliferation by CT in cycling rats was observed, whereas in cells from OVX + E2 rats a dramatic effect of the peptide was demonstrated. In addition, a 2-fold stimulation of proliferation by anti-CT antibodies in AP cells from OVX rats indicates a role for the endogenous CT in modulation of this important function. Finally, using double immunostaining for PRL and BrdU, it was demonstrated that CT specifically inhibits lactotrope proliferation. These results suggest that pit-CT may play an important role in remodeling of the pituitary tissue.


   Acknowledgments
 
The authors gratefully acknowledge the National Hormone and Pituitary Program, NIDDK for anti-rPRL and anti-rFSH sera for immunocytochemistry.


   Footnotes
 
1 This work was supported by NIH Grant DK-45004 (to G.V.S.) Back

Received November 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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