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Population Council and The Rockefeller University, New York, New York 10021
Address all correspondence and requests for reprints to: Dr. Indrani C. Bagchi, Population Council, 1230 York Avenue, New York, New York 10021. E-mail: indrani{at}popcbr.rockefeller.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A timely interplay of the ovarian steroids, estrogen and progesterone, orchestrates the entry of the fertilized ova into the uterus followed by pronounced morphological and physiological alteration of the endometrium leading to the acquisition of the receptive state of the uterus (1, 2, 3). Estrogen initiates hypertrophy and hyperplasia of endometrial epithelia. Progesterone transforms this prepared endometrium into a secretory tissue and creates an environment within the uterine milieu that is conducive to embryo attachment. Steroid hormones act through their intracellular receptors, which are ligand-inducible gene regulatory factors (4, 5, 6). It is therefore likely that steroids trigger the expression of a unique set of genes during the early stages of pregnancy and that these eventually lead to the synthesis of new proteins that prepare the uterus to accept the invading blastocyst. It has been previously proposed that steroid hormones transform uterine epithelial cells from a polarized or nonadhesive phenotype to a nonpolarized or adhesive state, which then allows attachment of the trophoblast (7, 8, 9). The loss of apical-basal polarity of epithelial cells is believed to be due to hormone-induced expression or redistribution of adhesion molecules. Indeed, a number of recent studies have shown that expression of adhesion molecules such as integrins, trophinin, and H-type carbohydrates or loss of nonadhesive molecules such as MUC-1 from uterine surface epithelium coincides with the putative window of receptivity (10, 11, 12, 13, 14, 15, 16).
To gain further insights into the molecular basis of uterine receptivity and identify steroid-regulated markers of this crucial physiological state, we previously employed a gene expression screen technique (17) to isolate a number of putative implantation stage-specific genes. Nucleotide sequence analysis identified one of these genes as that encoding the peptide hormone calcitonin (18). Our previous studies revealed that the level of uterine calcitonin messenger RNA (mRNA) or protein rises dramatically during the implantation phase of gestation. The expression of calcitonin increases by day 2 (postfertilization) of gestation and reaches a peak on day 4, the day before implantation. On day 5, the day implantation occurs, the expression of the gene starts to decline, and by day 6, when the embryo has attached to the endometrium, the calcitonin level falls to below detection limits (18). The transient expression of calcitonin at the time of implantation is restricted to the glandular epithelial cells of the endometrium (18). In immunocytochemical experiments, no significant calcitonin protein signal has been detected in the stromal cells or the myometrium. Our studies also indicated that the expression of calcitonin in the uterus is regulated by progesterone (18). However, it was not clear whether this gene regulatory effect of progesterone is exerted at the transcriptional or the posttranscriptional level.
In the present study, we reexamined in more detail the steroid hormone regulation of calcitonin gene expression in rat uterus and cultured endometrial epithelial cells. Our study confirmed that progesterone induces calcitonin mRNA synthesis in the glandular epithelium of uteri of pregnant as well as pseudopregnant rats. The action of progesterone is mediated through intracellular progesterone receptors (PRs), which are present in the glandular epithelial cells and enhance transcription from the calcitonin promoter. We also observed that calcitonin is secreted into the uterine lumen immediately preceding implantation. Calcitonin is, therefore, a useful marker of the progesterone-primed preimplantation endometrium, which is poised to enter the receptive state for blastocyst attachment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
All experiments involving animals were conducted according to
NIH guidelines for the care and use of experimental animals. Virgin
female rats (Sprague-Dawley, from Charles River, Wilmington, MA; 60–75
days of age) in proestrus were mated with fertile or vasectomized males
of the same strain to induce pregnancy or pseudopregnancy,
respectively. The different stages of the cycle in the nonpregnant rats
were ascertained by examining vaginal smears. The presence of a vaginal
plug after mating was designated day 1 of pregnancy or pseudopregnancy.
In some experiments, animals were injected sc with either mifepristone
(8 mg/kg BW) or vehicle (sesame oil) as described in
Results. The rats were killed 16 h after final
injection.
To induce and maintain delayed implantation, rats were ovariectomized on day 4 of pregnancy and injected daily with progesterone (19) from days 5–8. To terminate delayed implantation and induce blastocyst activation, the progesterone-primed delayed implanting rats were given an injection of estrogen (19) on the third day of the delay (day 8). Rats were killed 24 h after an estrogen injection.
Northern blot analysis
For Northern analysis, 5–10 µg polyadenylated
[poly(A)+] mRNA were separated by formaldehyde agarose
gel electrophoresis and transferred to a Duralon membrane (Stratagene,
La Jolla, CA). After transfer, the membranes were baked at 80 C for
2 h. Blots were prehybridized in 50 mM
NaPO4 (pH 6.5), 5 x SSC (standard saline citrate),
5 x Denhardt’s solution, 50% formamide, 0.1% SDS, and 100
mg/ml salmon sperm DNA for 4 h at 42 C. Hybridization was carried
out overnight in the same buffer containing 106 cpm/ml
32P-labeled calcitonin complementary DNA (cDNA) fragment.
The filters were washed twice for 15 min each time in 1 x
SSC-0.1% SDS at room temperature, then twice for 20 min each time in
0.2 x SSC-0.1% SDS at 55 C, and the filters were exposed to
x-ray films for 24–72 h. The intensities of signals on the
autoradiogram were estimated by densitometric scanning. To correct for
RNA loading, the signals obtained were normalized with respect to the
ferritin light chain (FLC) signal in the same blot. For this, the
filters were stripped of the radioactive probe by washing for 5 min in
0.1% SDS at 95 C. The blots were then reprobed with a
32P-labeled FLC probe as described above.
In situ hybridization
Uterine tissue from nonpregnant and pregnant animals was
collected and frozen. Tissues were fixed in 4% paraformaldehyde at 4
C. Cryostat sections were cut at 8 µm and attached to 3-aminopropyl
triethyl silane (Sigma)-coated slides. In situ hybridization
was performed with digoxygenin (DIG)-labeled sense or antisense RNA
probes complimentary to nucleotides 2600–3000 of the rat calcitonin
gene. DIG-labeled RNA probes were synthesized from calcitonin cDNA
using T3 or T7 RNA polymerase and DIG-labeled nucleotides according to
the manufacturer’s specifications (Boehringer Mannheim).
Prehybridization was carried out in a damp chamber at 37 C for 60 min
in hybridization buffer (50% formamide, 5 x SSC, 2% blocking
reagent, 0.02% SDS, and 0.1% N-laurylsarcosine).
Hybridization was carried out at 42 C overnight in a damp humidified
chamber. To develop the substrate, sections were sequentially washed in
2 x SSC, 1 x SSC, and 0.1 x SSC for 15 min in each
buffer at 37 C. Sections were then incubated with anti-DIG alkaline
phosphatase-conjugated antibody. Excess antibody was washed away, and
the color substrate (nitro blue tetrazolium salt and
5-bromo-4-chloro-3-indoylphosphate) was added. Slides were allowed to
develop in the dark, and the color was visualized under light
microscopy until maximum levels of staining were achieved. The reaction
was stopped, and the slides were counterstained in Nuclear Fast Red for
5 min. The slides were washed in water, dehydrated, and coverslipped.
Control incubations used a DIG-labeled RNA sense strand and were
performed under identical conditions.
Immunohistochemistry and image analysis
Polyclonal antibodies against rat calcitonin (Peninsula
Laboratory, Belmont, CA) and PR (Zymed, Burlingame, CA) were diluted
1:1000 for immunohistochemistry. Frozen uteri were sectioned at 7 µm,
mounted on slides, and then fixed in 5% formaldehyde in PBS. Sections
were washed in PBS for 20 min and then incubated in a blocking solution
containing 10% normal goat serum for 10 min before incubation in
primary antibody overnight at 4 C. Immunostaining was performed using a
streptavidin-biotin kit for rabbit primary antibody (Zymed, Burlingame,
CA). Sections were counterstained with hematoxylin, mounted, and
examined under brightfield microscopy. Red deposits indicate the sites
of immunostaining.
A quantitative analysis of the immunohistochemical data was performed by image analysis. The intensity of PR-specific staining was determined using a Nikon Optiphot-2 microscope (Nikon, Melville, NY) equipped with a Dage MTI video camera (CCD 72, Dage, Michigan City, IN). The video images of PR protein signal were then digitized using a frame grabber (Quick Capture, Data Translation, Marlboro, MA) and were displayed on a Sun IPC work station (Mountain View, CA). The stained cytoplasmic areas of the glandular epithelial cells, luminal epithelial cells, and stromal cells were traced. The integrated pixel intensity was determined for the traced areas using image analysis software (Image-Pro, Media Cybernetics, Silver Spring, MD). The intensities were normalized by dividing the integrated pixel intensity by the cytoplasmic area (which equaled the total number of pixels within the traced boundary). The background intensities were determined for each group by tracing an unlabeled area adjacent to the labeled cells. The background was subtracted from the values obtained for the labeled cells, and the adjusted values are referred to as the relative signal intensities. There were 25 observations for each group.
Transient transfection experiments
HEC-1B (American Type Culture Collection, Rockville, MD) cells
were maintained in MEM in Earle’s salt solution (Life Technologies,
Grand Island, NY) supplemented with 5% FBS (HyClone Laboratories,
Logan, UT). Semiconfluent cells were transiently transfected using the
calcium phosphate coprecipitation procedure as described previously
(20). Cells (5 x 105) were plated on 10-cm tissue
culture dishes in phenol red-free MEM medium containing 5%
charcoal-stripped serum and after 24–48 h were transfected with
plasmid DNAs. Typically, cells received 10 µg chloramphenicol
acetyltransferase (CAT) reporter plasmid and 2 µg of an internal
control plasmid pSV-ßgal (Promega Corp., Madison, WI), which contains
the gene for ß-galactosidase enzyme. After 12–14 h of exposure to
the calcium phosphate precipitate, the cells were washed with PBS and
incubated in fresh phenol red-free medium with 10-8
M progesterone, 10-8 M
mifepristone, or solvent. Cells were harvested after 24 h for
determination of ß-galactosidase and CAT activities. The
ß-galactosidase assay was performed according to the method of
Herbomel et al. (21). The amount of cell extract used per
CAT assay was determined after normalization with respect to the
ß-galactosidase activity. Quantitation of the CAT activities was
performed by liquid scintillation analysis of the acetylated
[14C]chloramphenicol product and the remaining
unacetylated substrate. Each experiment was repeated at least three
times.
RIA
Animals were killed at different stages of pregnancy. The uteri
were removed, and each horn was isolated. PBS (200 µl) was flushed
through each horn and collected. For the mifepristone experiments,
animals received either vehicle or 1.5 mg mifepristone on day 3 of
pregnancy and were killed on day 4. RIA (Peninsula Laboratories) was
performed with the uterine flushings using rabbit anticalcitonin serum
and 125I-labeled calcitonin according to the
manufacturer’s specification.
Statistical analyses
Statistical evaluations of the data representing the levels of
PR in different uterine compartments at the estrous stage and on day 4
of pregnancy were performed using ANOVA and Fisher’s least significant
difference (LSD) test. The data representing the level of calcitonin in
the uterine flushings of rats are presented as the mean ±
SEM. For statistical evaluation of the data representing
the level of calcitonin in the uterine flushings of rats on different
days of pregnancy, ANOVA and Fisher’s LSD test were used. To evaluate
the data depicting the level of calcitonin in the uterine flushings of
day 4 pregnant rats before and after treatment of mifepristone,
Student’s t test was employed. P < 0.05
was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a transient expression of
calcitonin was observed during days 3–5 of pseudopregnancy (lanes
2–4). The level of calcitonin mRNA was barely detectable on day 1. It
rose sharply on day 3 and then increased further to reach a peak on day
4. Calcitonin mRNA declined on day 5 and was undetectable on day 7. The
temporal profile of calcitonin expression in the uterus of the
pseudopregnant rat was therefore essentially similar to that observed
previously in the normal pregnant rat. By comparing the normalized
levels of calcitonin mRNA on days 1 and 4 of pseudopregnancy, we
estimated a 25-fold induction in calcitonin gene expression. The
magnitude of this induction was therefore comparable to that observed
previously in the pregnant animals. The induction of calcitonin in the
endometrium during pseudopregnancy suggested that its expression in the
pregnant rat uterus at the time of implantation is primarily under
maternal control and does not require the presence of embryos in the
uterine lumen.
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We observed a strong hybridization signal in the glandular epithelial
cells of the uterine sections obtained from day 4 pregnant animals
(Fig. 2B). In contrast, little, if any, signal was detected in the
glandular epithelial cells of the uteri of nonpregnant animals in the
estrus stage (Fig. 2A). Control uterine sections (nonpregnant, estrus
stage, or pregnant, day 4) hybridized with the corresponding sense RNA
probe of equal length did not exhibit any significant signal (Fig. 2, A' and B', respectively). We did not detect any calcitonin mRNA signal
in the luminal epithelium, stroma, or myometrium. These results
demonstrated that the glandular epithelial cells are the actual sites
of synthesis of calcitonin mRNA in the uteri of pregnant rats.
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, treatment with
mifepristone caused a remarkable decline in the level of calcitonin
mRNA in the glandular cells relative to that in the uteri of untreated
day 4 pregnant animals (Fig. 3A). This observation confirmed the role
of progesterone in regulating calcitonin mRNA expression in the
glandular epithelial cells of pregnant rats.
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, A and A', an intense
calcitonin-specific staining was observed in the glandular epithelial
cells of uterine sections from animals on day 4 of pseudopregnancy.
However, administration of mifepristone markedly suppressed the level
of calcitonin protein in the uterus of pseudopregnant rats within
16 h of treatment (Fig. 4, B and B'). The observation that
mifepristone effectively reduced calcitonin synthesis in the glandular
epithelial cells of pseudopregnant rats confirmed the role of
progesterone in regulating uterine calcitonin gene expression during
pseudopregnancy.
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. The uteri of nonpregnant
(estrus) animals exhibited PR-specific staining in both glandular and
luminal epithelium and stromal cells (Fig. 5A). A 1.5- to 2-fold
(P < 0.05) increase in the level of PR at each of
these sites was noted (Fig. 5B) in the uterine sections of day 4
pregnant animals and was considered statistically significant (Fig. 6). Among the different cellular
compartments, the maximal increase (>2-fold) in the level of PR was
observed in the glandular epithelial cells on day 4 of pregnancy.
Sections of uteri (estrus or day 4) incubated with control serum did
not exhibit any specific immunostaining (Fig. 5, B and D,
respectively). These results showed that sufficient quantities of PRs
are localized in the glandular epithelial cells where calcitonin mRNA
is induced on day 4 of gestation. These findings are consistent with a
direct regulatory role for PR in calcitonin gene expression during
implantation.
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(lanes 1, 3, and 4), a low level of
basal promoter activity was observed when the calcitonin promoter
construct was transfected alone into HEC-1B cells in the presence or
absence of progesterone or mifepristone. This basal promoter activity
did not change significantly when the promoter construct was
cotransfected with a vector expressing the PR-B isoform in the absence
of hormone (lane 2). However, when the promoter construct and the PR-B
expression vector were cotransfected in the presence of 100
nM progesterone, a 6- to 8-fold induction of CAT expression
was observed (lane 5). This PR-mediated induction of calcitonin
promoter was dependent on hormone agonist, as PR-B complexed with the
antagonist mifepristone failed to promote CAT expression (lane 6).
These results indicated that calcitonin gene induction by progesterone
is a transcriptional event mediated by the PR at the calcitonin
promoter.
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Rats were ovariectomized on day 4 of pregnancy and subsequently
injected with progesterone (2 mg) through days 5–8 of pregnancy. On
day 9 of gestation, rats were treated with either progesterone or an
implantation-initiating dose of estrogen (0.2 µg). Twenty-four hours
after the last steroid hormone treatment, animals were killed, and
uteri were isolated to monitor the calcitonin mRNA level by Northern
blot analysis. As shown in Fig. 8, the
level of calcitonin mRNA in the uteri of delayed rats during
progesterone treatment (equivalent to day 10 postconception) was
elevated similarly to that of day 4 pregnant rats (compare lanes 1 and
2). Interestingly, although the expression of calcitonin declined on
day 5 in pregnant animals and virtually disappeared after day 6, the
level of the peptide hormone in the ovariectomized pregnant animals was
elevated for several days beyond day 4 in the presence of progesterone
alone. Administration of estrogen to the delayed animals initiated
implantation, but also diminished the level of calcitonin mRNA within
24 h of treatment. Quantitation of the signals by densitometric
scanning followed by normalization to FLC mRNA signals showed that the
estrogen treatment suppressed calcitonin levels to about 50% of that
found in the delayed rats treated with progesterone alone (compare
lanes 2 and 3). Taken together, these results suggest that progesterone
is necessary and sufficient to induce and maintain calcitonin
expression in the preimplantation uteri, and this steroid is the
primary positive regulator of calcitonin synthesis in the endometrium.
Estrogen, on the other hand, functions as an antagonist of
progesterone-mediated uterine calcitonin expression.
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, only low levels of calcitonin were
detectable in the uterine flushings from nonpregnant and day 3 pregnant
rats. However, on day 4 of gestation, a marked rise (
8-fold compared
with nonpregnant state) in the level of calcitonin was detected in the
uterine flushings. This rise in calcitonin secretion declined by day 6
of pregnancy (D6). These results demonstrated that a significant amount
of calcitonin is secreted in the uterine lumen of pregnant rats on day
4 of gestation immediately preceding the receptive state on day 5.
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Calcitonin is therefore a progesterone-regulated measurable indicator
of the state of the endometrium that immediately precedes the receptive
state during embryo implantation. |
Discussion |
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There has been a long standing effort to identify molecules that will
correctly predict or indicate the receptive state of human endometrium
and therefore may serve as markers of the fertile endometrium. The
putative window of uterine receptivity for implantation in the human is
believed to exist between days 19–24 of the secretory phase of the
menstrual cycle. A number of human endometrial proteins that appear to
undergo changes in expression around the time of implantation have been
identified (for a review, see Ref. 24). These include the
progesterone-associated endometrial protein, insulin-like growth
factor-binding protein-1, leukemia inhibitory factor, and several
extracellular binding proteins and their receptors (10, 11, 25, 26, 27, 28, 29, 30, 31, 32, 33).
The most promising candidate marker among these is the
vß3 vitronectin receptor, an integrin that
plays a role in cell-cell adhesion. This integrin appears on the
epithelial cells of human endometrium at a time that closely overlaps
the putative window of implantation (10, 11). We recently observed that
calcitonin is expressed in human endometrium in the midsecretory phase
(day 20) of the menstrual cycle (Kumar, S., and I. Bagchi, unpublished
observation). Interestingly, no calcitonin expression is detected in
the endometrium in the proliferative phase (day 8 or 11). These
findings raise the possibility that the appearance of calcitonin in
secretory endometrium may serve as a potential marker of uterine
receptivity for embryo implantation in the human. Most importantly, we
report in this paper that calcitonin is secreted into rat uterine lumen
on day 4 of gestation and, therefore, is a measurable marker. This
finding, if found valid for human endometrium in future studies, may
permit the development of sensitive methods for detection (such as RIA)
of this hormone in uterine secretions or other body fluids of the
human. This will give calcitonin a clear advantage over other potential
markers of uterine receptivity such as integrins, which are not
secretory proteins.
Calcitonin expression is regulated by progesterone, one of the principal pregnancy hormones. In previous studies we observed that treatment of ovariectomized rats with progesterone led to a 20-fold increase in the synthesis of calcitonin (18). In the present study, we observed that in pseudopregnant rats, progesterone regulates calcitonin mRNA synthesis. The fact that progesterone is indeed an inducer of calcitonin mRNA expression in the uterus during pregnancy or pseudopregnancy is confirmed by experiments employing the antiprogestin drug mifepristone (RU486). We observed that treatment of rats on day 3 of pregnancy or pseudopregnancy with a single dose of mifepristone suppressed calcitonin mRNA synthesis in the glandular epithelium to undetectable levels within 16 h. It is likely that RU486 exerts its inhibitory effects by impairing the gene regulatory activity of the PR (34, 35). Consistent with this prediction, we observed that 1) PRs are present in the glandular cells during calcitonin mRNA induction; and 2) cotransfection of PR and a reporter gene linked to a 1.3-kb fragment of calcitonin promoter into a human endometrial cell line led to a significant progesterone-dependent enhancement of reporter gene expression. These results strongly suggest that the regulatory effects of progesterone are indeed exerted at the level of transcription of the calcitonin gene.
Although progesterone profoundly influences uterine functions during pregnancy, to date only a few genes have been identified as being regulated by progesterone in the pregnant uterus. Uteroglobin, one of the first progesterone-regulated genes to be identified, is reported to be expressed in the rabbit endometrium between days 3 and 9 of pregnancy (36, 37). Uteroferrin, a secretory glycoprotein implicated in transplacental iron transport, is induced by progesterone in pregnant pig uterus (38). A recent report indicated that progesterone stimulated the expression of amphiregulin in mouse uterine epithelial cells (39). In the present study the development of a reconstituted progesterone response system in endometrial cells in which calcitonin promoter is regulated by PR in a ligand-dependent fashion offers an opportunity to study the mechanism of progesterone regulation of a cellular gene. Progesterone may modulate calcitonin promoter activity through direct interactions of its nuclear receptor with specific progesterone response elements of the gene or through interactions of the receptor with other transcription factors that bind to the promoter. Further functional analyses using various calcitonin promoter mutants in the transient cotransfection system would help us to distinguish between these mechanisms.
Our previous studies in ovariectomized animals have shown that administration of estrogen together with progesterone inhibited the progesterone-mediated calcitonin gene induction (18). Such antagonistic interactions between estrogen and progesterone pathways have been documented previously in breast and uterine cells (40, 41, 42). It has been proposed that these phenomena reflect transcriptional cross-talk occurring between estrogen and PRs coexpressed in the same target tissue (40, 41, 42). During pregnancy in the rat, the circulating levels of estrogen remain unchanged on day 2 after fertilization, increase sharply on the evening of day 4, decline again by day 5 of pregnancy, and remain low throughout gestation until term (43). Previous studies established that the surge of estrogen on the evening of day 4 is essential for implantation on day 5 (1). It is interesting to note that the transient surge of estrogen on the evening of day 4 of pregnancy is quickly followed by the decline in calcitonin expression on day 5.
To functionally dissect the regulatory effects of progesterone and estrogen on the calcitonin gene, we analyzed the expression of this gene in pregnant rats that had undergone bilateral ovariectomy on day 4 of gestation. In these rats, the implantation is blocked in the absence of the ovarian steroids. We found that in the absence of estrogen, continued administration of progesterone to these ovariectomized animals maintained calcitonin mRNA synthesis in the glands for several days beyond day 5 while the embryo remained free floating but viable. This is in contrast to the sharp decline in calcitonin expression that is observed on day 5 in normal pregnancy. Administration of estrogen to the ovariectomized pregnant rats triggered implantation, but also rapidly reduced the progesterone-mediated expression of calcitonin. The effect of estrogen in the delayed rats, therefore, mimicked the physiological shut off of calcitonin expression at the time of implantation. This observation raises the interesting possibility that the decline in calcitonin expression may be a prerequisite for embryonic implantation and may therefore serve as a signal for this process.
By definition, a true marker of uterine receptivity must have a relevant functional role during the implantation process. The most well characterized physiological role of calcitonin is to regulate calcium levels in bone and kidney cells (44, 45, 46, 47, 48). Calcitonin is synthesized and secreted primarily by the parafollicular C cells of the thyroid gland (44, 45, 46, 47, 48). In response to hypercalcemia, the C cells release calcitonin rapidly, which, in turn, lowers blood calcium by inhibiting osteoclast activity and thereby reducing bone resorption and remodeling (44, 45, 46, 47, 48). The hormone is also present in small amounts in tissues such as lung, liver, intestine, pituitary, and the central nervous system (49). Although the precise functional role of calcitonin in these tissues remains unknown, its wide distribution throughout the body, including the central nervous system, and its presence in animals that have no bony skeleton suggest that calcitonin may have other effects in addition to its action in bones. The common denominator in the various physiological actions of calcitonin could well be the modulation of calcium flux across the membranes of a number of different types of cells and thus of the intracellular-extracellular distribution of calcium in various systems (50). Although the creation of the receptive endometrium during implantation undoubtedly involves the actions of multiple effector molecules, we speculate that calcitonin might regulate uterine receptivity for blastocyst implantation by controlling calcium homeostasis within the uterus in an autocrine or paracrine manner.
Previous studies hinted at a role for calcium efflux in maternal recognition signals, although the precise function of calcium remains unclear (51, 52, 53). During implantation, the initial interaction of the trophoblastic membrane with the endometrial epithelium is followed by the intrusion of the trophoblastic processes between the epithelial cells, which form tight junctions near their apical regions. These tight junctions of the surface epithelial cells in the uterus need to be relaxed or dissociated before successful adhesion and subsequent intrusion by trophoblastic processes (1, 2, 3). At the time of implantation, uterine epithelial cells are thought to undergo reprogramming from a polarized to a nonpolarized phenotype, which prepares the apical pole toward adhesiveness for trophoblast (7, 8, 9). It has been reported previously that addition of Ca2+ ions to a culture of polarized epithelial cells induces the formation of tight junctions and removal of these ions leads to a relaxation of tight junctions (54, 55). It is conceivable that secretion of calcitonin by the glands at the time of implantation may alter calcium homeostasis in the surface epithelial cells in the immediate surroundings of the implantation bed. The change in calcium signaling by calcitonin may trigger the redistribution or expression of critical cell adhesion molecules or junctional complexes that control polarized epithelial phenotype and can prepare the apical cell pole for contact with the trophoblast.
To assess the functional role of calcitonin during implantation, we recently employed an antisense technology to regulate calcitonin gene expression in rat uterus (56). Administration of two different antisense oligodeoxynucleotides (ODNs), targeted specifically against calcitonin mRNAs, into the lumen of the preimplantation phase uterus resulted in a dramatic reduction in the number of implanted embryos (56). Similar treatment with the corresponding sense ODNs had no effect on implantation. We also observed that treatment with the antisense calcitonin ODNs, but not with the sense ODNs, markedly suppressed the steady state level of the targeted mRNA in the uterus. These data collectively suggest that calcitonin performs critical functions in the endometrium to regulate uterine receptivity before implantation.
Taken together, our studies demonstrate that calcitonin fulfills a large number of criteria to be an appropriate marker that forecasts uterine receptivity for implantation in the rat. There is an urgent need to identify biochemical markers that are faithful and sensitive predictors or indicators of the receptive state of the human endometrium during blastocyst implantation. This knowledge in the long run will assist in the diagnosis and treatment of female infertility caused by lack of uterine receptivity. Future studies involving an extensive analysis of spatio-temporal expression of calcitonin in human endometrium will determine whether this peptide hormone would emerge as a bonafide marker of uterine receptivity in the human.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this report.
3 Supported by NIH Grants R01-DK-50257 and HD-13541-18.
Received December 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v are apically distributed in
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J.-W. Jeong, K. Y. Lee, I. Kwak, L. D. White, S. G. Hilsenbeck, J. P. Lydon, and F. J. DeMayo Identification of Murine Uterine Genes Regulated in a Ligand-Dependent Manner by the Progesterone Receptor Endocrinology, August 1, 2005; 146(8): 3490 - 3505. [Abstract] [Full Text] [PDF]
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J. R. A. Sherwin, T. C. Freeman, R. J. Stephens, S. Kimber, A. G. Smith, I. Chambers, S. K. Smith, and A. M. Sharkey Identification of Genes Regulated by Leukemia-Inhibitory Factor in the Mouse Uterus at the Time of Implantation Mol. Endocrinol., September 1, 2004; 18(9): 2185 - 2195. [Abstract] [Full Text] [PDF]
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 M. S. Jansen, S. C. Nagel, P. J. Miranda, E. K. Lobenhofer, C. A. Afshari, and D. P. McDonnell Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition PNAS, May 4, 2004; 101(18): 7199 - 7204. [Abstract] [Full Text] [PDF]
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 Q. Li, Y.-P. Cheon, A. Kannan, S. Shanker, I. C. Bagchi, and M. K. Bagchi A Novel Pathway Involving Progesterone Receptor, 12/15-Lipoxygenase-derived Eicosanoids, and Peroxisome Proliferator-activated Receptor {gamma} Regulates Implantation in Mice J. Biol. Chem., March 19, 2004; 279(12): 11570 - 11581. [Abstract] [Full Text] [PDF]
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Y.-P. Cheon, X. Xu, M. K. Bagchi, and I. C. Bagchi Immune-Responsive Gene 1 Is a Novel Target of Progesterone Receptor and Plays a Critical Role during Implantation in the Mouse Endocrinology, December 1, 2003; 144(12): 5623 - 5630. [Abstract] [Full Text] [PDF]
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B. Chen, D. Zhang, and J. W. Pollard Progesterone Regulation of the Mammalian Ortholog of Methylcitrate Dehydratase (Immune Response Gene 1) in the Uterine Epithelium during Implantation through the Protein Kinase C Pathway Mol. Endocrinol., November 1, 2003; 17(11): 2340 - 2354. [Abstract] [Full Text] [PDF]
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S. Kumar, A. Brudney, Y.-P. Cheon, A. T. Fazleabas, and I. C. Bagchi Progesterone Induces Calcitonin Expression in the Baboon Endometrium Within the Window of Uterine Receptivity Biol Reprod, April 1, 2003; 68(4): 1318 - 1323. [Abstract] [Full Text] [PDF]
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Y.-P. Cheon, Q. Li, X. Xu, F. J. DeMayo, I. C. Bagchi, and M. K. Bagchi A Genomic Approach to Identify Novel Progesterone Receptor Regulated Pathways in the Uterus during Implantation Mol. Endocrinol., December 1, 2002; 16(12): 2853 - 2871. [Abstract] [Full Text] [PDF]
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Q. Li, J. Wang, D. R. Armant, M. K. Bagchi, and I. C. Bagchi Calcitonin Down-regulates E-cadherin Expression in Rodent Uterine Epithelium during Implantation J. Biol. Chem., November 22, 2002; 277(48): 46447 - 46455. [Abstract] [Full Text] [PDF]
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Y.-P. Sun, T. J. Lee, and G. V. Shah Calcitonin Expression in Rat Anterior Pituitary Gland Is Regulated by Ovarian Steroid Hormones Endocrinology, October 1, 2002; 143(10): 4056 - 4064. [Abstract] [Full Text] [PDF]
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N. Takamoto, B. Zhao, S. Y. Tsai, and F. J. DeMayo Identification of Indian Hedgehog as a Progesterone-Responsive Gene in the Murine Uterus Mol. Endocrinol., October 1, 2002; 16(10): 2338 - 2348. [Abstract] [Full Text] [PDF]
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 O. M. Conneely, B. Mulac-Jericevic, F. DeMayo, J. P. Lydon, and B. W. O'Malley Reproductive Functions of Progesterone Receptors Recent Prog. Horm. Res., January 1, 2002; 57(1): 339 - 355. [Abstract] [Full Text] [PDF]
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Q. Li, M. Zhang, S. Kumar, L.-J. Zhu, D. Chen, M. K. Bagchi, and I. C. Bagchi Identification and Implantation Stage-Specific Expression of an Interferon-{{alpha}}-Regulated Gene in Human and Rat Endometrium Endocrinology, June 1, 2001; 142(6): 2390 - 2400. [Abstract] [Full Text] [PDF]
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K. Pavelock, K. M. Braas, L'H. Ouafik, G. Osol, and V. May Differential Expression and Regulation of the Vascular Endothelial Growth Factor Receptors Neuropilin-1 and Neuropilin-2 in Rat Uterus Endocrinology, February 1, 2001; 142(2): 613 - 622. [Abstract] [Full Text] [PDF]
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 G.-Y. Nie, Y. Li, L. Batten, B. Griffiths, J. Wang, J. K. Findlay, and L. A. Salamonsen Uterine expression of alternatively spliced mRNAs of mouse splicing factor SC35 during early pregnancy Mol. Hum. Reprod., December 1, 2000; 6(12): 1131 - 1139. [Abstract] [Full Text] [PDF]
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 B. Mulac-Jericevic, R. A. Mullinax, F. J. DeMayo, J. P. Lydon, and O. M. Conneely Subgroup of Reproductive Functions of Progesterone Mediated by Progesterone Receptor-B Isoform Science, September 8, 2000; 289(5485): 1751 - 1754. [Abstract] [Full Text]
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G. A. Johnson, T. E. Spencer, R. C. Burghardt, K. M. Taylor, C. A. Gray, and F. W. Bazer Progesterone Modulation of Osteopontin Gene Expression in the Ovine Uterus Biol Reprod, May 1, 2000; 62(5): 1315 - 1321. [Abstract] [Full Text]
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G.-Y. Nie, Y. L. Jian Wang, H. Minoura, J. K. Findlay, and L. A. Salamonsen Complex Regulation of Calcium-Binding Protein D9k (Calbindin-D9k) in the Mouse Uterus During Early Pregnancy and at the Site of Embryo Implantation Biol Reprod, January 1, 2000; 62(1): 27 - 36. [Abstract] [Full Text]
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