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Endocrinology Vol. 141, No. 10 3696-3702
Copyright © 2000 by The Endocrine Society

ARTICLES

Induction of Calcitonin and Calcitonin Receptor Expression in Rat Mammary Tissue during Pregnancy1

Lois A. Tverberg, Margaret F. Gustafson, Timothy L. Scott, Irina V. Arzumanova, Elayne R. Provost, Arthur W. Yan and Sonja A. Rawie

Department of Biology (L.A.T., M.F.G., T.L.S., I.V.A., S.A.R.), Hope College, Holland, Michigan 49423; Department of Pathology (E.R.P.), Yale University, New Haven, Connecticut 06510; and Amherst College (A.W.Y.), Amherst, Massachusetts 01002

Address all correspondence and requests for reprints to: Lois Tverberg, Ph.D., Department of Biology, Hope College, Holland, Michigan 49423. E-mail: tverberg{at}hope.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human breast milk samples at early time points after parturition contain high levels of calcitonin (CT) in both normal and thyroidectomized mothers, suggesting that mammary tissue produces CT. Using blot hybridization and reverse-transcriptase polymerase chain (RT-PCR) analysis of rat mammary RNA we found that CT messenger RNA is induced at midpregnancy (day 12), remains elevated through late pregnancy (day 19) but then decreases before the day of birth. RIA of mammary CT revealed that levels increase from 0.3 ng/g tissue in nonpregnant animals to peak at 1.6 ng/g on day 19 and then decline after that, paralleling messenger RNA expression. Dilution profiles for extracted mammary CT showed close parallelism with monomeric rat CT. Plasma samples from thyroparathyroidectomized rats contained 10–20 pg/ml CT that did not increase during pregnancy, suggesting that mammary CT is not released into plasma but functions locally. Consistent with this, RT-PCR detected that the CT receptor C1a isoform is expressed in rat mammary tissues during both pregnancy and lactation. This is the first report that mammary tissue expresses both CT and the CT receptor during pregnancy, suggesting that CT may have a paracrine regulatory role in the mammary gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN (CT) is secreted by thyroid C cells in response to elevated plasma calcium levels and acts to lower plasma Ca2+ (1, 2). Because it inhibits osteoclast resorption of bone, it is widely prescribed to treat osteoporosis and other degenerative bone diseases. It has been hypothesized that CT has a role in preserving the maternal skeleton during the calcium stress of pregnancy and lactation, as bone development occurs in the fetus and newborn. Supporting this, plasma CT levels are elevated during pregnancy and slightly elevated in lactating women (3). A similar pattern of plasma CT elevation is also found in rats (4, 5).

It has been known for several years that high levels of CT are present in early breast milk samples (6, 7). CT is present at equivalent levels in milk samples from both euthyroid and thyroidectomized women (8), suggesting that CT is produced in mammary tissues or another extrathyroidal source. The observations that CT is up to 90-fold more concentrated in breast milk than plasma (7) and that high-molecular-weight precursors are found in milk (6) imply that human mammary tissue may produce CT endogenously. Levels are highest in colostrum and drop rapidly within days of parturition (8), suggesting that CT expression is greatest during late pregnancy or at birth rather than during lactation.

Evidence also exists that mammary tissues express receptors for CT. The CT receptor (CTR) is expressed in T47D and MCF7 breast cancer cell lines (9), and CTR messenger RNA (mRNA) has been detected in human mammary tissue (10). Recently, transgenic mice containing the CTR promoter fused to ß-galactosidase, showed expression of CTR in mammary mesenchyme during embryonic development (11). Expression shut off before birth and was not detected in adult mouse mammary tissue, suggesting that CT may be involved in embryonic mammary gland development. Because mammary tissue resumes development and differentiation during pregnancy, it is possible that expression of the receptor may be induced again at this time.

Our laboratory has examined whether CT and CTR are expressed in rat mammary tissues during pregnancy and lactation. Previous investigators tested rat mammary tissue for CT mRNAs during lactation (14 days post partum) and did not detect expression of this transcript (12). However, we have found that expression of rat CT mRNA and peptide is induced during mid- to late pregnancy but then decreases before parturition. Interestingly, CTR mRNA expression is also induced in the mammary gland during pregnancy and lactation, suggesting that CT may have paracrine regulatory activity in the tissue during that time.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of mammary samples
For RNA samples, virgin female Sprague Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) between 8–10 weeks old were allowed free access to food (Formulab 5008 lab diet) and water and were housed in a 12-h light, 12-h dark cycle. For peptide extraction, thyroparathyroidectomized (TPTX) rats of the same age were obtained after surgery by the supplier and were fed normal lab diet and water containing 2% calcium lactate and 0.2 µg/ml T4 to maintain normal plasma Ca2+ and T4 levels. Each rat was caged with an adult male rat for insemination, and the date at which a seminal plug was found was denoted as day 1 of pregnancy. Virgin rats of the same age were used for nonpregnant (control) mammary samples. Rats were killed by sedation with ketamine (100 mg/kg ip) + acepromazine (1 mg/kg ip), followed by an intracardiac injection of Soccumb (1 ml/kg; Butler Co., Columbus, OH). The first, fourth, and fifth pairs of mammary glands were isolated on days 4, 8, 12, 15, 17, and 19 of pregnancy, on the day of birth (day 1 post partum), and on days 4 and 14 post partum. Involuted mammary tissues were taken 2 weeks after weaning at 21 days. Samples were individually isolated from three or four rats at each time point and stored at -70 for later extraction of RNA or peptide. All procedures were approved by the Hope College Animal Care and Use Committee.

RNA hybridization blots
Total RNA was purified from inguinal mammary glands by the guanidinium thiocyanate method (13). Poly-A selection was performed with oligo-dT Sepharose (Ambion, Inc.; Austin, TX), according to the manufacturer’s protocol, and the concentration of the poly-A-enriched RNA was measured by optical densitometry at 260 nm. RNA samples (10 µg each) were electrophoresed on a 1.2% formaldehyde-3-[N-morpholino)propanesulfonic acid gel for 1 h at 100 V and then transferred and UV-cross-linked to a positively-charged nylon membrane [Roche Molecular Biochemicals (RMB) Indianapolis, IN]. Antisense RNA probe was synthesized to the BglII fragment of rat CT exon 4 (14) with 1:2 digoxigenin-UTP:UTP mix using a strippable probe synthesis kit (Ambion, Inc.). This probe is specific to CT mRNA and does not detect CT gene-related peptide or other splicing products. The 250-bp KpnI-XbaI fragment of mouse tri-ß-actin (15) included with the kit was used for actin detection. Each blot was prehybridized for 1 h at 68 C in Ultrahybe hybridization buffer (Ambion, Inc.) with 100 µg/ml total yeast RNA before hybridizing in the same buffer with approximately 0.1 nM CT probe for 12–20 h. Blots were then washed and incubated with alkaline phosphatase-linked antidigoxigenin Fab fragments and detected with CDP-Star, according to manufacturer’s protocols (RMB). After stripping according to kit instructions, blots were rehybridized with actin RNA probe for 1–2 h, and the wash and detection steps were repeated as before. A Bioquant image analysis system was used to quantify CT and actin band density on autoradiograms.

Total RNA was isolated from thyroids using TRI reagent (Sigma, St. Louis, MO), according to the manufacturer’s protocol, and optical densitometry at 260 nm was used to determine concentration. Northern blots containing 2.5 µg of each sample were hybridized with CT and ß-actin probes consecutively, as described above, and the resulting autoradiograms were quantified for CT and actin band density.

RT-PCR
Primers for RT-PCR for CT were specific for the amplification of spliced rat CT mRNA and not other CT gene-splicing products. Oligonucleotides to exon 3 (5'-TAC TGG CTG CAC TGG TGC-3') and CT-specific exon 4 (5'-ACC CAT AAT AGC CCA GAG AA-3') were used in PCR reactions with an expected product size of 335 bp (14) These also distinguish complementary DNAs (cDNA)s amplified from spliced RNA from that of genomic DNA that would have an expected size of about 1 kb. All RNAs were verified for integrity before use in RT-PCR by observing ribosomal RNA bands by gel electrophoresis. For RT, 1 pmol of oligo dT primer was incubated with 5 µg total RNA, 2.5 mM deoxynucleotide 5'-triphosphates, 5 U ribonuclease inhibitor, and 1 U avian myeloblastosis (AMV) reverse transcriptase in a vol of 20 µl AMV buffer for 1 h at 42 C (reagents from Promega Corp., Madison, WI). Subsequent PCR reactions included 1 µl of the resulting cDNA in a 25-µl reaction containing 0.5 µM of each of the CT gene exon 3 and exon 4 primers, 200 µM deoxynucleotide triphosphates, 0.25 U Taq polymerase (Promega Corp.), and 1.5 mM MgCl2 in the supplied buffer. The PCR reaction was incubated for 5 min at 94 C, followed by 30 cycles of 30 sec at 94 C, 1.5 min at 55 C, 2 min at 72 C, and a final incubation at 72 C for 5 min.

PCR products were visualized and photographed under ethidium bromide fluorescence on a 1.5% agarose gel and then transferred and UV-cross-linked to a positively-charged membrane (RMB). For DNA blot hybridizations, a digoxigenin-labeled CT DNA probe was synthesized using a PCR synthesis kit (RMB), according to manufacturer’s directions. A subcloned BglII fragment of CT exon 4 was used as a template for a PCR reaction incorporating digoxigenin-11-deoxyuridine 5-triphosphate using polylinker primers. CT probe was incubated with the filter overnight at 45 C, and the probe was detected as described for RNA blots.

The one-tube Access RT-PCR reaction kit (Promega Corp.) was used in studies to detect CTR expression. We used primers outside the insertion site to distinguish the two species, with a predicted product size of 511 bp for C1a and 622 bp for C1b. An upstream primer beginning at + 742 of the cDNA with the sequence 5'-GGG ATC TTC TTG TTT TTC AAG AAC CT-3', and a downstream primer beginning at +1231 with the sequence 5'-ACA TGT AGG CCT CGG CTT CAT-3', were used to detect the reported rat CTR sequence (16). The upstream primer flanks the expected intron 7 splice site (17), to permit annealing only to spliced cDNAs. Each PCR reaction contained 1 µg total RNA, 1 mM of each primer, 1.5 U Tfl polymerase, and 3U AMV reverse transcriptase. The RT occurred at 48 C for 45 min and was followed by 40 cycles of: 30 sec at 94 C, 1 min at 62 C, and 2 min at 68 C; with a final extension step at 68 C for 7 min. All RNAs used in RT-PCR reactions to detect CT or CTR were also amplified in separate reactions with ß-actin primers to validate PCR conditions. As a control for contamination, a tube containing all reagents, but no cDNA, was included in each experiment.

RIA
For CT extraction, mammary tissues were homogenized in 10 vol of 0.1-M acetic acid, boiled for 10 min, and then incubated on ice for 10 min. The samples were centrifuged at 4 C for 20 min at 2,000 x g, followed by 30 min at 270,000 x g. The infranatant was removed and frozen at -70 C until the RIA was performed. Extraction efficiency was 40% using mammary tissues spiked with a known quantity of rat CT. From plasma samples, CT was extracted using a C18 SEP-COLUMN, according to manufacturer’s instructions (Peninsula Laboratories, Inc., Belmont, CA). Plasma CT extraction efficiency was also approximately 40%. The final eluate was lyophilized and resuspended in RIA buffer. Mammary tissue and plasma samples were then assayed using an RIA protocol, according to manufacturer’s instructions (Peninsula Laboratories, Inc.). The polyclonal rabbit antirat CT antiserum used in the assay showed no cross-reactivity with salmon, chicken, or eel CT, as well as CT gene-related peptide, CT C-terminal peptide, katacalcin (PDN-21), or amylin. The limit of detection of the RIA was 8 pg/tube. The intraassay variation was approximately 3–5% for samples from the same tissue processed independently but assayed at the same time. The interassay variation averaged 28% between tissues from different rats in separate assays.

DNA assay
Mammary tissue homogenates were also assayed for DNA content using a diphenylamine assay (18) that is specific for deoxyribose and does not detect ribonucleic acids. Trichloroacetic acid (2.5%) was added to a 2-ml aliquot of mammary homogenate from the peptide-extraction procedure, and samples were centrifuged at 2,000 x g for 20 min. Pellets were resuspended in 5% trichloroacetic acid and heated in a 90-C water bath for 15 min. After a second centrifugation (12,000 x g, 10 min), supernatants were assayed for DNA concentration immediately or stored at -20 C. For the assay, a standard curve of herring sperm DNA between 0–200 µg/ml was used. Diphenylamine reagent, composed of 1% diphenylamine (wt:vol) and 2.75% H2SO4 (vol:vol) in concentrated (17 M) glacial acetic acid, was added to the DNA samples in a 1:2 sample:reagent volume ratio. After boiling 10 min, absorption at 600 nm was measured in a spectrophotometer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CT mRNA detection by hybridization blot analysis
We used RNA hybridization to detect CT mRNA present in samples extracted from rat mammary tissues at various time points during pregnancy and lactation and from virgin animals (Fig. 1AGo). It is important to normalize expression to that of a nonregulated gene because mammary tissue increases in glandular content during pregnancy; and during lactation, high milk peptide gene expression dilutes all other mRNAs. We therefore determined the CT/ß-actin expression ratio at all time points. On hybridization blots, we saw that CT mRNA levels were induced between day 8 and day 12 of pregnancy and then decreased abruptly between day 19 and the day of birth (Fig. 1AGo, top panel). No CT expression was detectable before day 8 of pregnancy or after the day of birth. Rehybridization with actin probe showed that, when milk protein expression is fully induced during lactation (after day 4 post partum), actin and other transcripts are diluted until milk production ceases at involution (Fig. 1AGo, middle panel). However, CT mRNA levels dropped before birth, confirming that CT is shut off rather than diluted by other RNAs. CT normalization to actin mRNA level, from several blots, containing samples from 3–4 rats at each time point, revealed an approximately constant level of expression of CT in mammary tissue from day 12 to day 19 of pregnancy (Fig. 1BGo).



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  		Figure 1. Detection of CT mRNA in mammary tissues by RNA hybridization blot. A, Poly-A selected RNA (10 µg) from mammary samples from virgin rats (con) or the indicated days during pregnancy, birth, lactation, or involution were hybridized with a probe for CT (top panel). Thyroid total RNA (2 µg) was included as a positive control for CT. The same blot was stripped and rehybridized with an actin probe (middle panel). Ethidium bromide-stained 18 S ribosomal RNA from the same gel before transfer is shown in the bottom panel. B, Mammary CT mRNA expression normalized to actin. Autoradiograms were analyzed by densitometry, and CT/actin density ratio was calculated. One RNA sample (day 15 of pregnancy) was included on each blot to normalize for differences in detection sensitivity, and the CT:actin RNA ratio was arbitrarily set at 100. The error bar on day 15 was therefore calculated from its interassay variability. Means with SE bars for 3–4 rats at each time point are shown. n.d., No CT mRNA detectable; Invol, involution.

 
CT mRNA detection by RT-PCR
We used RT-PCR to examine expression of mammary CT because of the increased sensitivity of RT-PCR. Also, because mRNA makes up a small proportion of total RNA used in RT-PCR, transcript dilution effects should be minimized. Figure 2Go shows that RT-PCR with mammary RNA generates products of the expected size from tissues from mid- to late pregnancy (day 12 to day 19), but not in early pregnancy or after parturition, when visualized on ethidium bromide-stained gels. We then transferred the amplified DNAs to nylon filters and hybridized with a CT-specific DNA probe to confirm their identity (Fig. 2BGo). The increased sensitivity of hybridization could detect amplified cDNAs during early stages of pregnancy and up to the fourth day after birth but still did not detect CT expression later, at day 14 of lactation. This is consistent with the lack of expression reported by others at day 14 using less sensitive methods (12). In contrast, actin was detectable by RT-PCR at all times during pregnancy and lactation. These results confirm hybridization blot results and indicate that CT mRNA is mainly present in mammary tissue in late pregnancy.



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  		Figure 2. RT-PCR detection of CT mRNA in mammary tissues. A, Total RNA was isolated from rat mammary tissues at various days during pregnancy, on the day of birth, and during lactation. Reverse-transcribed cDNAs were amplified by PCR using primers specific for spliced CT mRNAs (upper panel) or ß-actin mRNA (lower panel). Resulting amplified DNAs were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. A 100-bp ladder is included as a reference marker (M). B, DNA from the gel in A was transferred to a nylon membrane and hybridized to a DNA probe to the CT gene exon 4.

 
CT peptide in mammary tissue and plasma
We used RIAs to measure CT peptide levels in mammary tissue at different time points during pregnancy and lactation. Rats were thyroparathyroidectomized to ensure that CT produced in thyroid or parathyroid would not be detected. At least 4 weeks elapsed between surgery and tissue harvest, to allow for residual CT to be eliminated. RIA of mammary tissue extracts revealed that CT levels rose from below 0.3 ng/g wet weight in nonpregnant tissues to peak at approximately 1.6 ng/g on day 19 of pregnancy (Fig. 3AGo, solid bars). Peptide levels declined by the day of birth and further decreased to approximately 0.6 ng/g by day 4 post partum. Because glandular differentiation during pregnancy increases cellular content of mammary tissue, DNA content was assayed to examine whether a correlation exists between CT expression and cellular proliferation. DNA content of the tissue was found to steadily increase through pregnancy and into lactation (Fig. 3AGo, white bars). In contrast, CT peptide levels peaked at day 19 and decreased thereafter, correlating with CT mRNA levels.



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  		Figure 3. CT peptide expression in mammary tissue. A, RIA quantification of mammary CT peptide during pregnancy and lactation. CT peptide content, after correcting for recovery inefficiency, is indicated by the solid bars. Tissue DNA content is indicated by the white bars. Means with SE bars for 3–4 rats at each time point are shown. B, Parallelism studies between monomeric rat CT and mammary CT. Increasing volumes of extract (10–100 µl) from mammary tissue at day 19 of pregnancy were compared, in an RIA, with self-competition by synthetic rat CT-monomer (diamonds). Two independent assays are shown by square and triangle symbols, each the average of duplicate samples.

 
A dilution assay was performed with CT peptide extracted from mammary tissue to assess the antigenic similarity between mammary CT and monomeric rat CT, since large-molecular-weight precursors were isolated from human milk samples (6). In two separate experiments, increasing volumes of extract from mammary tissues at day 19 of pregnancy were assayed for CT peptide and then evaluated for parallelism with a standard curve produced with synthetic rat CT-monomer. Figure 3BGo shows that both assays exhibited strong parallelism with monomeric rat CT. This implies that little to no binding of CT is occurring by factors present in the extract, and that the extracted CT peptide is recognized with nearly equal affinity by the RIA antibodies as monomeric CT.

Plasma from TPTX rats during pregnancy was also assayed for the presence of CT. In nonpregnant rats, low amounts of CT were detected (14 pg/ml), in comparison with 300–500 pg/ml typically seen in euthyroid female rats (5). During pregnancy, plasma CT levels remained low, between 10 and 20 pg/ml plasma (Fig. 4Go). No statistically significant increase (P < 0.05) was seen at any time point during pregnancy or lactation. The ratio of mammary CT peptide concentration to that of plasma in TPTX rats, therefore, ranged from between 20-fold in nonpregnant rats to 90-fold at day 19 of pregnancy.



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  		Figure 4. Plasma levels of CT during pregnancy. Plasma from rats before, during, and after pregnancy were assayed for CT content. Mean CT peptide concentration, after correcting for recovery inefficiency, is shown. Means with SE bars for 3–4 rats at each time point are shown. No statistically significant difference in CT level was detected by ANOVA where P < 0.05. conc., Concentration.

 
Thyroidal CT mRNA expression during pregnancy
We also used RNA hybridization analysis to examine the expression of CT mRNA in the thyroid during pregnancy, to determine if this correlated with the increased plasma peptide levels observed in rats and humans (Fig. 5AGo.) Surprisingly, thyroidal CT mRNA content did not change during pregnancy but was equivalent in tissues from nonpregnant and pregnant rats as well as during lactation (Fig. 5AGo, top panel). When CT:actin ratio of samples including day 4 of lactation from several blots were analyzed statistically, we saw no significant change in the CT:actin expression ratio between time points in the thyroid (Fig. 5BGo). Actin mRNA level correlated closely to ethidium bromide staining of the gel, suggesting that expression of neither transcript changes during pregnancy, and implying that if secretion of CT increases during pregnancy, it must be induced by a posttranscriptional mechanism. In comparing the CT:actin mRNA ratio of mammary tissues with that of thyroid tissues, we found that relative CT expression in mammary tissue during late pregnancy was approximately 3–5% of that in thyroid.



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  		Figure 5. Thyroidal CT mRNA expression during pregnancy. A, Total thyroid RNA (2.5 µg) from one rat at each indicated time point up until birth was blotted and hybridized with a probe for CT (top panel) or actin (middle panel). Ethidium bromide staining before transfer is shown in the bottom panel. An anomalous lower-molecular-weight band was detected by actin probe in some RNA samples. B, CT mRNA band density was normalized to that of actin from several blots containing thyroid RNA samples from 3–4 rats at each time point, including day 4 of lactation. The CT:actin RNA ratio of thyroid RNA from control (nonpregnant) rats was set at 100, and relative expression (Rel. Expr.) was normalized to the control on each blot. Means with SE bars at each time point except control are shown. No statistically significant difference in mRNA level was detected by ANOVA where P < 0.05.

 
Expression of CTR in mammary tissue
We next tested for expression of the CTR in mammary tissues at time points through pregnancy and lactation using RT-PCR. Rat CTR mRNA is alternatively spliced into two isoforms, C1a and C1b, which are identical except for a 111-bp insert in the C1b isoform (16). Whereas the C1b isoform is localized to brain, C1a has been found in most target organs of CT. To ensure that amplified products were from the CTR gene, PCR products were transferred and hybridized with a different, but overlapping, CTR fragment that had been identified by sequencing. Figure 6Go shows that RT-PCR using CTR primers amplifies a product of about 500 bp from RNA from pregnant and lactating mammary tissues, consistent with C1a CTR expression in the gland. No amplification occurred from two different RNA samples from nonpregnant rats, but samples from day 4 of pregnancy through day 14 of lactation contain detectable CTR mRNA. Actin mRNAs were amplified from all tissue samples. Although nonquantitative, these results indicate that the C1a isoform of the CTR RNA is induced in mammary tissues during early pregnancy and is still present during lactation.



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  		Figure 6. RT-PCR detection of CTR mRNAs. Total RNA was isolated from mammary tissue from nonpregnant rats or on the indicated day of pregnancy, on the day of birth, or at day 14 of lactation. Two micrograms of total RNA was used in an RT-PCR reaction with primers specific for CTR (upper panel) or ß-actin (lower panel). Kidney RNA was used as a positive control for C1a CTR expression. The negative control (-) contained all RT-PCR reagents but no RNA. A 100-bp DNA ladder is included as a reference marker.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have established that expression of CT and CTR are both induced in rat mammary tissue during pregnancy. Whereas the CTR is induced in early pregnancy and continues into lactation, transcription of CT mRNA is, for the most part, confined to late pregnancy. If the same expression pattern is present in humans, it is consistent with initially high, but rapidly decreasing, CT peptide levels seen in colostrum samples at early time points after birth (8, 19). The fact that CT gene expression is confined mainly to middle and late pregnancy suggests that CT has physiological activity before milk protein synthesis begins at the end of pregnancy. The CT found in colostrum may simply be what is present in the mammary tissues as the gland begins secretion, although it may also have a function in milk calcium sequestration initially or in the neonate. Progesterone, estradiol, and other maternal hormones that are present in mammary tissue during late pregnancy are released into colostrum at high concentrations initially that drop rapidly, in the same way that CT does (20).

We saw that CT peptide levels in mammary tissue rose during midpregnancy and then declined by birth. Though the decrease in mammary CT peptide levels after birth may be partially attributable to the fact that it is released into milk, the decreasing mRNA levels indicate that overall mammary expression declines at that time. Some CT was present even in nonpregnant mammary tissues, suggesting a very low basal level of CT expression in the gland. We did find that the CT peptide present in mammary tissue was immunologically very similar to monomeric rat CT, which contrasts with the observation that, in human milk, some of the CT peptide is present in large-molecular-weight forms (6). Whether rat mammary CT is processed differently from human CT or the peptide is later converted into larger forms in the milk is not known.

In mammary samples from TPTX rats, CT levels were approximately 90-fold higher than in plasma samples from the same rats, indicating that the peptide is produced in that tissue rather than being sequestered from circulating blood. We did not see increased CT levels in plasma samples from TPTX rats during late pregnancy, as would be expected if mammary tissue were contributing to systemic levels of the hormone. We also found that thyroparathyroidectomy almost entirely eliminated CT in plasma before, during, and after pregnancy, suggesting that thyroidal secretion is responsible for both basal secretion and elevated plasma CT levels during pregnancy (4). This, along with the fact that the concentration of CT in mammary tissue during late pregnancy is approximately 0.5% of that of the thyroid, suggests that CT produced in the mammary gland acts as a local paracrine factor rather than as a systemic regulator.

Coinduction of the CTR in mammary tissue during pregnancy also suggests that CT may have a local role in the mammary gland and is analogous to other tissues that have paracrine CT activity (21, 22, 23). In uterine endometrium, CT expression occurs briefly in glandular epithelial cells during implantation, and CT is secreted into the uterine lumen (24). Paracrine activity of the hormone is crucial for implantation to take place (25). CT is also expressed, along with its receptor, in the glandular endothelium of the prostate; and CT is found at appreciable levels in seminal fluid (26). Treatment of prostatic cell lines with CT stimulates cell proliferation (23). These observations suggest that CT may have paracrine activity that somehow regulates proliferation or other activity of secretory tissues.

Interestingly, expression of CT by mammary tissues is correlated to the rapid proliferation of alveolar secretory tissue, which in the rat begins at about day 12 and continues until birth. Mammary tissue undergoes extensive differentiation into a secretory tissue at this time and produces paracrine factors that regulate its own development (27). The hypothesis that CT plays a role in this process is supported by the fact that CTR-expressing breast cancer cell lines respond to CT with increased cAMP, Ca2+, and changes in proliferation (28). It is known that during pregnancy, intracellular cAMP levels in mammary tissue progressively increase (29). Also, exogenous cAMP and cholera toxin stimulate mammary growth and branching in virgin mice and in organ culture models (30, 31, 32). Induction of cAMP in mammary tissue during pregnancy may be one mechanism by which CT acts in the gland.

A suggestive parallel comes from PTH-related protein (PTHrP), another calcium-regulating hormone that is coexpressed in mammary tissue with its receptor during embryonic development, pregnancy, and lactation (33). PTHrP has been found to act in a paracrine manner to regulate development of several tissues; in PTHrP-knockout mice no mammary growth occurs, indicating a critical role in mammogenesis (34, 35). Intriguingly, the PTH/PTHrP receptor is a seven-transmembrane domain receptor homologous to that of CT. The fact that another calcitropic hormone acts as a developmental regulator suggests that CT may have a related function. The recent discovery that the CTR is expressed in mammary mesenchyme during embryonic development also supports this conclusion (11).

CT expression in mammary tissue may be induced by progesterone, which increases during pregnancy but drops immediately before birth (36). Progesterone induces CT expression in the uterus during implantation (24) and stimulates CT secretion from thyroid (37, 38), suggesting that it may stimulate mammary CT expression or secretion as well. Progesterone is known to regulate development of mammary tissue during pregnancy and also to inhibit onset of lactation (36). A role for CT in lactational inhibition is suggested by the fact that CT is known to oppose PRL activity by inhibiting pituitary secretion of the hormone (39, 40). Mammary tissue produces functional PRL endogenously (41), and it is possible that CT might inhibit mammary PRL activity also. Whether mammary CT is involved in some of progesterone’s regulatory effects or has some other activity in mammary tissue remains to be studied.

Paradoxically, we found that the CT mRNA level in the thyroid does not increase during pregnancy, even though several studies have shown that plasma CT peptide levels are elevated in both rats and humans during that time (3, 4, 5). Consistent with this, others have seen that elevated Ca2+ appears to stimulate CT posttranscriptionally, increasing translation and secretion of CT without affecting thyroid CT mRNA content (42). Segond et al. (43, 44) have found that hypercalcemia induces posttranscriptional modifications that cause a greater amount of CT RNA to be in a translatable form. Preliminary studies in our laboratory have seen that thyroid CT peptide content declines during pregnancy, suggesting a decrease in storage during that time. This result is also consistent with other reports that thyroid CT content decreases during times of demand (45) and suggests that increased thyroid secretion is the source of elevated plasma CT levels during pregnancy.

This report has established that the mammary gland produces CT during late pregnancy and suggests that CT may act locally through receptors coexpressed in the gland during that time. Paracrine factors have increasingly been seen to control proliferation and differentiation of both normal and cancerous tissues (27). Thus, understanding the possible paracrine role of CT in mammary tissue during pregnancy will be an important future goal.


   Acknowledgments
 
We gratefully acknowledge C. Newell and J. Roberts for their technical contributions. We also thank C. Barney and J. Roberts for their critical reviews and comments on the manuscript.


   Footnotes
 
1 This work was supported by funding from the Hope College Howard Hughes Medical Institute Grant, the National Science Foundation Research Experiences for Undergraduates Program (Grant DB-19322220), the Michigan Space Grant Consortium, and a National Institute of Health Academic Research Enhancement Award (HD-36497). Back

Received December 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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