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The Population Council and Rockefeller University, New York, New York 10021
Address all correspondence and requests for reprints to: Indrani C. Bagchi, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: Indrani{at}popcbr.rockefeller.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calcitonin, a 32-amino acid peptide hormone, has long been known to be synthesized and secreted primarily by the parafollicular C cells of the thyroid gland (6, 7, 8, 9). Its most well characterized physiological role is to regulate calcium levels in bone and kidney cells (6, 7, 8, 9). Our previous studies revealed that calcitonin synthesis is markedly induced in the pregnant rat uterus during implantation (5). The level of uterine calcitonin messenger RNA (mRNA) in cycling rats is low, i.e. less than 1% of the calcitonin mRNA present in the thyroid gland, the prinicipal site of calcitonin synthesis. The expression of calcitonin mRNA and protein rises dramatically after day 2 (postfertilization) of gestation and reaches a peak on day 4, the day before implantation. At this stage, the level of calcitonin within the uterus reaches about 10–20% of that synthesized by the thyroid gland. After day 5, the day implantation occurs, the expression of the gene starts to decline, and by day 6, when implantation is completed, the calcitonin level falls to below detection limits. The burst of calcitonin expression at the time of implantation is localized in the glandular epithelial cells of the endometrium by immunohistochemical analysis (5). The timing and location of its synthesis in the glandular epithelium of rat endometrium prompted us to speculate that calcitonin may regulate blastocyst implantation in an autocrine or paracrine manner.
A timely interplay of the maternal steroid hormones, estrogen and progesterone, is believed to orchestrate the pronounced morphological and biochemical alterations in the endometrium that prepare it to be receptive to the developing blastocyst (1, 2, 3). Our previous studies demonstrated that the expression of calcitonin in the endometrium is induced by progesterone (5). Although progesterone profoundly influences uterine functions during pregnancy, calcitonin is one of the few genes that have been identified as regulated by progesterone in the pregnant uterus (10, 11, 12, 13). Uterine expression of calcitonin could, therefore, be one of the links between steroid hormone action in the target cell nuclei and embryo-endometrial interactions during blastocyst implan-tation.
To gain insights into the functional role of calcitonin during implantation, we have employed an antisense oligodeoxynucleotide (ODN) methodology. The sequence-specific inhibition of gene expression by antisense ODNs relies on the ability of an ODN to bind specifically and efficiently to a complementary mRNA sequence (14, 15). Hybridization with antisense ODNs results in suppression of target mRNA levels by triggering degradation of the RNA strand of the RNA-DNA duplex. In addition, the ODNs are thought to prevent translation of target mRNAs (14, 15). Our approach involves suppression of calcitonin gene expression in the preimplantation phase uterus by using antisense ODNs targeted against calcitonin mRNA. Although there is mounting evidence from many laboratories that antisense oligomers targeted to cellular or viral mRNA sequences can produce specific biological effects in cultured cells or in intact tissue (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27), we report here, for the first time, that antisense ODNs can be successfully employed to regulate gene expression in the uterus. Our study shows that the administration of antisense calcitonin ODNs into the preimplantation phase uterus effectively reduced calcitonin mRNA and protein synthesis without affecting the expression of nontarget genes. Interestingly, the loss of calcitonin expression upon antisense ODN treatment was accompanied by a severe impairment in embryonic implantation. Based on these results, we propose that calcitonin plays a crucial role in the uterus during blastocyst implantation.
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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 adult males. 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. In certain experiments, animals were
ovariectomized and 14 days later were injected sc with estradiol (2
µg/kg BW). This was followed by an injection of progesterone (40
mg/kg BW) 24 h later.
Treatment of animals with ODNs
Sprague-Dawley rats were deeply anesthetized, and an incision
was made in the lower abdomen. The ODNs were mixed with DOTAP
(Boehringer Mannheim, Indianapolis, IN) and 20% F127 pluronic gel
(Sigma Chemical Co., St. Louis, MO). The solution was maintained in
liquid form at 4 C before injection. One hundred to 150 µl of this
ODN solution were taken in prechilled syringes and injected into each
uterine horn.
The injections were made into the luminal space of each horn. Each ODN sample was administered in two steps at two discrete locations along the length of the horn. We imagined each horn to be divided into three equal parts: the cervical one third, the central one third, and the ovarian one third. The needle was first inserted in the middle of the central one third portion of the horn with the tip pointing toward the ovarian side. Approximately 50% of the sample was delivered into the lumen while pulling the syringe out. The needle was then reinserted within the cervical one third of the horn, and the remaining sample was injected with the tip of the needle pointing toward the ovarian side. The solution turned into a transclucent gel as soon as it came in contact with the tissue. The incision was then closed, and the animals were returned to their cages. We noted that the gel slowly disappeared over several hours. We detected no gelled material in the injected horns 4–6 h after injection.
Detection of 33P-labeled ODNs in the
uterus
The antisense ODN-1 was labeled at the 5'-end using
[
-33P]ATP (SA, 1500 Ci/mmol) and polynucleotide
kinase. The labeled ODNs were administered into the uterus following
the procedure described above. The animals were killed after 2 h,
and the uteri were collected. Frozen uteri were sectioned at 8 µm.
The sections were obtained from both sides of the site of the first
injection within the middle one third portion of the uterine horn,
mounted on slides, fixed in 5% formaldehyde in PBS, and air-dried. The
sections were then coated with Kodak NTB2 emulsion (Eastman Kodak,
Rochester, NY) and stored in the dark at 4 C for 1 week. The slides
were developed in Kodak Dektol 19 at 20 C for 2 min, washed in
distilled water, and fixed with Kodak fixer for 5 min. The sections
were then counterstained with hematoxylin, mounted with coverslips, and
viewed under darkfield and brightfield illumination using a Nikon
microscope (Nikon Corp., Melville, NY).
Northern blot analysis
Uteri were isolated from ODN-treated animals and processed for
polyadenylated [poly(A)+] RNA isolation employing a fast
track mRNA isolation kit (Invitrogen, San Diego, CA). For Northern
analysis, 5–8 µg poly(A)+ mRNA were separated by
formaldehyde agarose gel electrophoresis and transferred to 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 (0.02% each of Ficoll,
polyvinylpyrrolidone, and BSA), 50% formamide, 0.1% SDS, and 100
µg/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 filters were stripped of the radioactive probe by
washing for 5 min in 0.1% SDS at 95 C and then reprobed with
32P-labeled glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) or ferritin light chain (FLC) probes.
Immunohistochemistry and image analysis
A polyclonal antibody against rat calcitonin (obtained from
Peninsula Laboratories, Belmont, CA) was diluted 1:1000 for
immunohistochemistry. A rabbit antibody against rat clusterin (obtained
from Dr. C. Y. Cheng, The Population Council, New York, NY) was
used at a dilution of 1:2000 (28). 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,
South San Francisco, CA). Sections were counterstained with
hematoxylin, mounted, and examined under brightfield. Red deposits
indicate the sites of immunostaining. Control experiments included
incubation of sections with 1) normal rabbit serum or preimmune serum
for clusterin, and 2) primary antibody that had been preabsorbed with
rat calcitonin (Peninsula Laboratories) or clusterin (C. Y. Cheng
Laboratory, The Population Council).
A quantitative analysis of the immunohistochemical data was performed by image analysis. The intensity of calcitonin-specific staining was determined using a Nikon Optiphot-2 microscope equipped with a Dage MTI video camera (CCD 72, Dage, Michigan City, IN). The video images of calcitonin protein signal were then digitized using a frame grabber (Quick Capture, Data Translation, Marlboro, MA) and displayed on an IPC (Sun Microsystems, Inc., Mountain View, CA). The stained cytoplasmic areas of the glandular epithelial 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 30 observations for each group.
Statistical analysis
Statistical evaluation of the data representing the effects of
ODN treatments on the number of implanted embryos in the uterus was
performed using Student’s t test. P < 0.05
was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies showed that calcitonin mRNA and protein are
synthesized predominantly in the glandular epithelial cells of the
uterus. To be an effective blocker of calcitonin gene expression, an
antisense ODN has to enter the glandular cells synthesizing this
hormone. We, therefore, needed to ascertain that the antisense
calcitonin ODNs injected into the uterine lumen were actually reaching
these glandular sites within the uterus. In the experiment described in
Fig. 1
, an antisense ODN, As-ODN-1, was
labeled at the 5'-end with 33P and injected into the
uterine lumen of pregnant rats (n = 4) on day 2 of gestation. Two
hours after injection, the animals were killed, and the uteri were
isolated and sectioned to monitor the presence of radioactivity. As
revealed by light microscope autoradiography (Fig. 1, right
panels), substantial amounts of 33P-labeled ODNs
accumulated in the glandular epithelial cells. Modest amounts of
radioactivity were also observed in the luminal epithelium and stroma.
These results showed that the ODNs injected into the uterine lumen were
indeed reaching the glandular sites of calcitonin synthesis.
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Two weeks after ovariectomy, the animals were primed with estrogen on day 15 and then injected with progesterone on day 16 to induce calcitonin gene expression. Three hours after progesterone treatment, the animals (n = 8) were subjected to a surgical operation, in which the lower abdominal cavity was opened. Both uterine horns of the same animal were injected with sense ODN, whereas both horns of a second animal were injected with antisense ODN. Twenty-four hours after this operation, the animals were killed, uteri were collected, and mRNAs were isolated for Northern blot analysis. The blot was hybridized with a 32P-labeled calcitonin cDNA (exon IV) probe as well as with FLC and GAPDH cDNA probes.
The results of these experiments are shown in Fig. 2. The uterine horns that were treated
with either 10 or 25 µg As-ODN-1 (upper panel, lanes 2 and
4) consistently exhibited drastically reduced (>90%) calcitonin mRNA
compared with the horns that were injected with the same doses of
S-ODN-1 (upper panel, lanes 1 and 3). Whereas treatment with
25 µg As-ODN-1 produced a slight decrease (
20%) in the intensity
of the FLC signal (lower panel, lane 2) compared with that
in the sense ODN-treated (lower panel, lane 1) animals,
treatment with 10 µg antisense or sense ODNs displayed FLC signals of
equal intensities (lower panel, lanes 3 and 4). The effects
of the ODNs on GAPDH expression were similar to those observed on FLC
expression (data not shown). Marked inhibition of both FLC and GAPDH
mRNA expression was noted when 40 µg or more of either sense or
antisense ODN were administered in the tissue (data not shown). These
results demonstrated that treatment with an optimal amount (10 µg) of
antisense ODNs could bring about specific and substantial reduction in
calcitonin gene expression in the uterus without significantly
affecting the expression of nontarget genes.
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To perform ODN treatments, animals (n = 6/ODN pair, 3 for
S-ODN-1 and 3 for As-ODN-1) on day 2 (afternoon) of pregnancy were
subjected to a surgical operation as described in Materials and
Methods. Both uterine horns were injected with 10 µg of one of
the antisense ODNs, As-ODN-1 or As-ODN-2, targeted against exon IV of
the calcitonin gene or the corresponding sense ODNs. Forty-eight or
72 h after the operation, the animals were killed, uteri were
collected, and mRNAs were isolated for Northern blot analysis. The blot
was hybridized with a 32P-labeled calcitonin cDNA (exon IV)
probe and control probes including FLC or GAPDH cDNAs. The results of
these experiments are shown in Fig. 3. As
shown in the top panel of the figure, the uterine horns that
were treated with antisense ODNs (right lane) exhibited
drastically reduced calcitonin mRNAs on day 4 compared with horns that
were injected with the same doses of sense ODNs (left lane).
Hybridization of the blot with FLC or GAPDH probes indicated no
reduction in the intensities of these signals in the antisense
ODN-treated uterus compared with those in the sense ODN-treated tissue
(Fig. 2
, middle and bottom panels). Due to a relative
overloading of RNA sample in the right lane (As), the FLC
and GAPDH signals in this lane appear to be slightly more intense than
those in the left lane (S). Upon quantitation and
normalization of the mRNA signals, we estimated that more than 90% of
the calcitonin expression on day 4 was suppressed by antisense ODN
treatment. A similar decline in calcitonin mRNA levels on day 5 was
observed upon antisense ODN treatment (data not shown). These results
demonstrated that antisense ODN intervention on the afternoon of day 2
of gestation effected a specific and drastic suppression of calcitonin
mRNA expression in the pregnant rat uterus immediately before (day 4)
and at the time (day 5) of implantation.
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. The
uteri of animals that were injected with either sense ODN, S-ODN-1 or
S-ODN-2, on day 2 of pregnancy and killed on day 4 exhibited an intense
immunostaining (upper panel a) that was mainly localized in
the glandular epithelial cells. This staining was calcitonin specific,
as control sections incubated with calcitonin antibody that was
preabsorbed with excess rat calcitonin showed no immunoreactivity (Fig. 4c). In contrast, the sections of uteri that were injected with the
corresponding antisense ODN, As-ODN-1 or As-ODN-2, showed strikingly
reduced calcitonin-specific staining (upper panel b).
Quantitation of protein staining by image analysis revealed that
greater than 80% of calcitonin immunoreactivity was lost upon
antisense ODN treatment (Fig. 4, middle panel).
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, lower panel b). Our
results show that treatment with either antisense ODN (Fig. 4, lower panel c) or sense ODN (data not shown) did not affect
clusterin protein expression in the glands. These results indicate that
antisense ODNs targeted against calcitonin mRNA can specifically
inhibit the synthesis of calcitonin protein in the glandular epithelial
cells of the uterus without affecting the expression of nontarget
proteins.
Administration of antisense oligonucleotides into rat uteri in the
early preimplantation phase reduces the number of the implanted embryos
We next investigated whether the suppression of
calcitonin mRNA and protein expression upon antisense ODN treatment
influenced embryonic implantation. As described above, we administered
either the antisense or the corresponding sense calcitonin ODNs into
both uterine horns of rats on the afternoon of day 2 of pregnancy.
After surgery, the animals were returned to their cages and killed on
day 9 of gestation. The uteri were opened, and the number of implanted
embryos in each uterine horn was counted.
On day 9 of pregnancy, control saline-injected rats contained
six to eight implanted embryos, on the average, in each uterine horn.
When the animals were injected with either S-ODN-1 (n = 8) or
S-ODN-2 (n = 10) on day 2 of gestation, no significant change in
the number of implanted embryos was observed compared with that in the
control animals (Fig. 5a, A). This
indicated that the dose of ODNs used in this study is not generally
toxic to the embryos. Moreover, the surgical perturbation and
the injection of vehicle without ODNs on day 2 did not have any harmful
effect on embryo implantation.
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, B. In this As-ODN-1-treated
uterus, there was no visible embryo in one horn, whereas the other horn
showed only three implanted embryos. The embryos also appeared to be
abnormal in that they were larger in the As-ODN-treated animals
compared with those in the S-treated animals. Isolated uteri from the
sense and antisense ODN-treated animals are compared in Fig. 5b, A and
B. As shown in Table 1, typically, a
50–80% reduction in the number of implanted embryos was evident in
each uterine horn in several independent sets of experiments performed
with two different sets of antisense ODNs as described in Materials and
Methods. The inhibition of implantation by the antisense ODNs was not
complete, presumably because a low level of calcitonin was still
expressed in the glands. Only minimal effects (<15%) on implantation
were observed in 1 of 8 animals treated with As-ODN-1 and 2 of 10
animals treated with As-ODN-2. The reasons for this weaker response in
10–20% of treated animals are not clear. A statistical test was
applied to the data obtained from several experiments to assess the
significance of the difference between the number of implanted embryos
obtained after treatment with either antisense or sense ODNs. Such
analysis produced a P < 0.05%, indicating that the difference in
the number of implantation sites in sense- and antisense-treated
animals is indeed significant. Taken together, these results indicate
that treatment of the uterine horns with antisense calcitonin ODNs, but
not with the complementary sense ODNs, can severely impair embryo
implantation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A major concern regarding the antisense methodology is the possibility of nonspecific inhibition of gene expression caused by ODN treatment. This issue has been tackled in the present study by the use of multiple sense and antisense ODNs. We have shown that treatment of pregnant uteri with two different antisense ODNs, As-ODN-1 and As-ODN-2, possessing different base compositions produced the same phenotype, but the corresponding sense ODNs of equal length had no effect. Moreover, the antisense, but not the sense, ODNs inhibited target mRNA expression, whereas neither inhibited nontarget mRNAs. Another concern is any general toxic effect the ODNs or the vehicle may exert on the embryo. In our studies, we have observed that administration of two different sense ODNs (10 µg) or the vehicle on day 2 of pregnancy did not have any significant effect on implantation or subsequent embryo development until day 9 of gestation. It is, therefore, highly unlikely that the administration of optimal levels of ODNs or vehicle will have any general deleterious effect on the embryo.
The mechanism of action of calcitonin in the uterus during implantation is unclear. Implantation is the culmination of a sequence of discrete functional events, such as successful attachment of the blastocysts to the appropriate sites on the luminal epithelium and proper embryonic development. If calcitonin is involved in the preparation of the endometrium for implantation, one would expect that the attenuation of calcitonin expression after treatment with antisense ODNs may prevent acquisition of the receptive state of the endometrium, leading to a failure of implantation. If, on the other hand, calcitonin has a role in early embryogenesis, attenuation of its expression during development by antisense ODN may hinder the proper growth and differentiation of the embryos, and these abnormal embryos might fail to implant.
During implantation in the rat, the initial interaction of the trophoblastic membrane with the endometrial epithelium is followed by the intrusion of the trophoblastic processes between the epithelial cells (1, 2, 3). The epithelial cells form tight junctions near their apical regions. These tight junctions need to be relaxed or dissociated before successful intrusion by trophoblastic processes. It has been reported previously that addition of calcium ions to a culture of polarized epithelial cells induces the formation of tight junctions, and removal of these ions leads to relaxation of tight junctions (29). Previous studies also demonstrated that treatment of isolated osteoclasts or cells stably transfected with cloned calcitonin receptor cDNA with calcitonin induces influx of extracellular calcium into these cells (30, 31, 32). It is, therefore, conceivable that calcitonin secreted by the glandular epithelium may act on the luminal epithelial cells to regulate embryo attachment. Lowering of extracellular calcium levels in the uterine fluid in the immediate surroundings of the implantation bed may induce junction disassembly, redistribution of junctional proteins, and opening of the tight junction barrier in epithelial cells to promote trophoblast invasion (33, 34). Consistent with this scenario, our recent studies detected the presence of a significant amount of calcitonin in the luminal secretions of pregnant rats only between days 3–5 of gestation (Bove, K., and I. C. Bagchi, unpublished observation).
One can also imagine that calcitonin secreted by the uterine epithelium may act on trophoblast cells to induce calcium uptake by these cells. This would also result in a lowering of the calcium ion concentration in the uterine fluid between the epithelial and the trophoblast cells, and this event, in turn, would trigger relaxation of the tight junctions between epithelial cells to facilitate implantation. Further studies leading to the localization of cells that are targets of uterine calcitonin will help us to decipher mechanism of action of this peptide hormone during implantation.
Calcitonin may also play a role in implantation by acting as a factor controlling early embryonic development. Calcium is known to regulate cell aggregation during early embryogenesis, and calcitonin, due to its hypocalcemic properties, may influence cell adhesion (35). In the rat after fertilization, the embryo enters the uterine lumen on day 4 of pregnancy and may potentially be influenced by maternal calcitonin for at least 24 h before implantation on day 5. The block in uterine calcitonin expression after antisense ODN injection might hinder the proper growth and differentiation of the embryos, and these abnormal embryos might fail to implant. A role of calcitonin in early differentiation of the vertebrate embryo has indeed been suggested by Burgess (36). Studies by this investigator have shown that treatment of Xenopus embryo during gastrulation with calcitonin leads to faulty induction of the neural plate and results in defects in the development of oral-facial and central nervous system architecture. One would also have to consider the possibility that antisense ODNs may directly act on the embryos to inhibit their development, perhaps by blocking early embryonic calcitonin synthesis. Although the target(s) of calcitonin action in the developing embryo remains to be identified, our current findings suggest that calcitonin, in addition to its regulatory role in calcium metabolism, may have a broader spectrum of activity at early stages of development. Such a role might be similar to the proposed functions of the cytokines, colony-stimulating factor-1, and leukemia inhibitory factor, in regulating trophoblast proliferation and differentiation (37, 38).
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Acknowledgments |
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Footnotes |
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Received August 14, 1997.
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