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Biology of Reproduction 62, 27-36 (2000)
©Copyright 2000 Society for the Study of Reproduction, Inc.

Articles

Complex Regulation of Calcium-Binding Protein D9k (Calbindin-D9k) in the Mouse Uterus During Early Pregnancy and at the Site of Embryo Implantation1

Gui-Ying Nie2,a, Ying Li Jian Wang3,a, Hiroyuki Minoura4,a, Jock K. Findlaya, and Lois A. Salamonsena

a Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Establishment of receptive endometrium is essential for implantation. Our aim was to identify and characterize genes uniquely regulated at the sites of implantation in mouse uterus by RNA differential display polymerase chain reaction (DDPCR). One of the gene fragments identified was 86% homologous to rat calcium-binding protein D9k (calbindin-D9k); the mouse counterpart had not then been cloned, but subsequently an mRNA sequence of mouse calbindin-D9k became available in GenBank (accession number: AF028071). This sequence is 99% homologous to the DDPCR-derived gene tag but has a shorter 3' end. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the sequence of 3' end of the DDPCR product and the 5' end of AF028071, and a full cDNA was obtained. This gene was primarily up-regulated by progesterone, but not by estrogen. It was further increased by the combination of the two steroids. Expression of calbindin-D9k was overall increased in the uterus during early pregnancy, but the level was significantly lower in implantation compared to interimplantation sites on Days 4.5 and 5.5 of pregnancy, becoming barely detectable in both sites after Day 6.5. In situ hybridization localized this mRNA predominantly in the luminal epithelium of the pregnant uterus. The complex regulation of calbindin-D9k in mouse uterus suggests an important role for this protein during pregnancy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryo implantation is a complex process involving active interactions between the blastocyst and the uterus. A successful implantation requires the development of the embryo to the blastocyst stage and its escape from the zona pellucida, and the establishment of a receptive uterus [1]. It has been well documented that only during the "window" of implantation, a limited time span when the uterine environment is suitable for blastocyst attachment and implantation, can a blastocyst successfully implant into the uterus and establish a pregnancy. During preparation for receptivity, the uterus undergoes proliferation and differentiation under the influence of ovarian hormones. Changes in growth factors, receptors, and other molecules, in association with the ovarian hormone-induced changes in the uterus during the periimplantation period have been reported [2–9]. However, given the complexity of the process, the exact molecular events during the uterine transition from the nonreceptive to receptive state are still far from clear.

The aim of this study was to use the mouse as a model to search for unrecognized molecules of importance for the very early stage of implantation. In the mouse, on Day 4.5 of pregnancy (vaginal plug = Day 0), the uterus undergoes dramatic morphological changes in association with cell proliferation and differentiation, leading to the acquisition of a receptive state. Uterine remodeling at this time is marked by an increase in vascular permeability at the implantation sites. We hypothesized that the proliferation and differentiation of endometrial cells at this time are associated with up- or down-regulation of a number of genes, many of which are still unknown. To identify these uterine genes potentially critical for uterine receptivity, we used the technique of RNA differential display polymerase chain reaction (DDPCR) [10,11] and compared the mRNA expression pattern of implantation and interimplantation sites on Day 4.5 of pregnancy.

One of the genes identified as being differently regulated between the two sites was calcium-binding-protein-D9k (calbindin-D9k), a molecule not previously well studied in the mouse uterus. In particular, its expression pattern and cellular distribution during the uterine preparation for receptivity and early pregnancy have not been examined. In the present investigation, we obtained the full-length cDNA sequence of mouse calbindin-D9k and examined its uterine expression during early pregnancy, in particular at implantation and interimplantation sites after Day 3 of pregnancy. Hormonal regulation of calbindin-D9k in the nonpregnant uterus was also investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Preparation

Swiss O/B mice were housed and handled according to the Monash University animal ethics guidelines on the care and use of laboratory animals. All experimentation was approved by the Institutional Animal Ethics Committee at the Monash Medical Centre. Adult female mice (6–8 wk old) were mated with fertile males of the same strain to produce normal pregnant animals or mated with vasectomized males to produce pseudopregnant mice. The morning of finding a vaginal plug was designated as Day 0 of pregnancy. Uterine tissues were collected from nonpregnant mice, pregnant mice on Days 3–11, and pseudopregnant mice on Days 3–5. A selection of other mouse organs was also collected from nonpregnant mice. Tissues were snap-frozen in liquid nitrogen for Northern analysis or fixed in 4% buffered formalin (pH 7.6) for in situ hybridization.

For nonpregnant, pseudopregnant, and 3-day pregnant mice, the entire uterus was collected. For 4.5-day pregnant mice, implantation sites were visualized by i.v. injections of a Chicago Blue dye solution (1% in saline, 0.1 ml/mouse) into the tail vein 5 min before the animals were killed, implantation sites were separated from interimplantation sites, and both sites were retained. For pregnant mice on Day 5.5 onwards, implantation and interimplantation sites were visualized without dye injection.

For nonpregnant mice, the uterus was also collected from different stages of the estrous cycle: metestrus, diestrus, proestrus, and estrus. The stages of the cycle were determined by analysis of vaginal smears [12]. For ovarian hormone treatments, the animals were first ovariectomized under anesthesia with tribromoethanol (Aldrich Chemical Company, Inc., Milwaukee, WI) without regard to the stage of the estrous cycle [12]. The animals were allowed to rest for two weeks, then treated with daily s.c. injections (0.1 ml per mouse) of steroid hormones for 3 days as follows: estradiol-17ß (100 ng), progesterone (1 mg), or a combination of both hormones. The steroids (Sigma Chemical Co., St. Louis, MO) were initially dissolved in minimal amounts of ethanol before dilution in peanut oil. Animals that received injection of oil alone served as controls. Mice were killed 24 h after the last injection.

DDPCR, Reamplification of cDNAs, and Subcloning

DDPCR was carried out as described by Liang and Pardee [10,11] by comparing the implantation and interimplantation sites of uterine tissue on Day 4.5 of pregnancy. To avoid embryonic contamination, the embryos were removed under the light microscope from the implantation sites. Total RNA was extracted from pools of implantation or interimplantation sites by acid guanidinium thiocyanate-phenol-chloroform extraction (GTC) [13]. The RNA was then treated with ribonuclease (RNase)-free deoxyribonuclease (DNase) in the presence of placental RNase inhibitor to remove any contaminating DNA. The amount of RNA in the final preparation was determined spectrophotometrically, and the RNA quality was evaluated by the ratio of optical density (OD)260:OD280 (> 1.8). DNA-free RNA (1 µg) from implantation and interimplantation sites was used as the template for the first-strand cDNA synthesis in a 20-µl reaction mixture in the presence of 20 µM dNTPs, 50 µM oligo(dT) anchored primers (one each of T12MG, T12MC, T12MA, and T12MT; M can be A, G, or C), 10 mM DTT, 10 U ribonuclease inhibitor (RNasin; Promega, Madison, WI), 25 U AMV reverse transcriptase (Boehringer Mannheim, Nunawading, Victoria, Australia), and the cDNA synthesis buffer. The resultant cDNA was then amplified by polymerase chain reaction (PCR) in 20 µl with the following components: 2 µl of cDNA, single-strength PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3), 10 µM dNTPs, 10 pmol of one random decamer (Operon 10-mer kit A; Operon, Alameda, CA), 50 pmol of one oligo(dT) anchored primer (as used in cDNA synthesis), 2 µCi of [33P]dATP (Du Pont [Australia] Ltd., North Sydney, Australia), and 1 U of Taq DNA polymerase (Boehringer). The PCR was performed in a Hybaid OmniGene PCR system (Hybaid Ltd., Teddington, Middlesex, UK) with the following conditions: initial denaturation at 94°C for 5 min; then 40 cycles of denaturation at 94°C for 30 sec, primer annealing at 39°C for 2 min, and extension at 72°C for 30 sec; and a final extension at 72°C for 10 min. The PCR products (4 µl) were run on a 6% high-resolution polyacrylamide/urea gel and visualized by autoradiography. The majority of amplified fragments were shared between the implantation and interimplantation sites. Bands unique to either implantation or interimplantation sites were identified as of interest. The candidate bands were excised from the dried sequencing gel, and the cDNA was eluted with H2O and reamplified by PCR using the conditions described above. The differential display pattern was further confirmed by Northern blotting analysis using the reamplified PCR products as probes, which were generated by random hexamer labeling. Those bands giving differential expression patterns on the Northern blots were subcloned into pGEM-T vector (Promega) via the T/A cloning procedure. The cDNAs were sequenced manually on both strands with T7 and SP6 primers by the fmol DNA sequencing system (Promega), and sequences were analyzed using the ANGIS (Australia National Genomic Information Service; The University of Sydney, Sydney, Australia) software package.

Northern Analysis

For Northern analysis, no attempt was made to separate the embryos from the implantation sites before Day 8 of pregnancy, but for 8- and 11-day pregnant mice, embryos were separated from the uterine tissue. Total RNA was extracted from whole uteri or pools of implantation or interimplantation sites by the GTC extraction method as described above. Total RNA (10–15 µg) was denatured at 50°C for 60 min in 50% dimethylsulfoxide (DMSO) and 1 M glyoxal, and the denatured RNA was fractionated by electrophoresis through a 1.2% agarose gel in 10 mM sodium phosphate buffer (pH 7.0) and transferred to positively charged nylon membranes (Hybond-N+; Amersham) by overnight capillary blotting in 5-strength SSPE (single-strength SSPE is 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4). Membranes were baked at 80°C for 2 h; this was followed by 3 min of UV cross-linking. Transcript size was estimated by comparison with RNA size standards (Gibco-BRL, Gaitherburg, MD).

A method of simplified filter paper sandwich blotting [14] was used for the hybridization process at 42°C overnight without a prehybridization step. The hybridization buffer contained 50% formamide, 6-strength SSPE, 5-strength Denhardt's solution (single-strength Denhardt's solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.5% SDS, 0.1 mg/ml sheared herring sperm DNA, and [32P]dCTP-labeled probes. The radiolabeled cDNA probes were generated by random primer labeling of 25 ng cDNA with [32P]dCTP (50 µCi/reaction). Unincorporated nucleotides were removed with a MicroSpin S-200 HR column (AMRAD Pharmacia Biotech, Melbourne, Australia). After hybridization, the blots were rinsed twice with 5-strength SSPE at 37°C, then twice for 15 min each at 37°C with double-strength SSC/0.1% SDS (w:v) (single-strength SSC is 150 mM NaCl, 15 mM Na3 citrate, pH 7.4). In some cases, additional washes were also performed with 0.5- or single-strength SSC/0.1% SDS for 15 min at 60°C. After autoradiography, the optical density of the hybridization signals was quantified using the Hewlett-Packard Scanjet IIp with Deskscan software (Hewlett-Packard, Palo Alto, CA) and NIH Image Version 1.54. To correct lane-to-lane loading variation, each blot was also probed with a mouse cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the quantitation was normalized against the GAPDH signal on the same membrane. Between hybridizations, blots were stripped by incubation at 80°C for 3 h in 1 mM EDTA/0.1% SDS and then rinsed in H2O.

Reverse Transcription (RT)-PCR and T/A Cloning

For RT-PCR, 1 µg DNA-free total RNA was reverse-transcribed at 46°C for 1–1.5 h in 20 µl reaction mixture using 100 ng random hexanucleotide primers and AMV reverse transcriptase (Boehringer) with the cDNA synthesis buffer. Negative controls were included in which either RNA or the reverse transcriptase was omitted. The resultant cDNA mixtures (RT reaction) were heated at 95°C for 3 min before storage at -20°C, or immediately used for PCR amplification. The following PCR was performed in a total volume of 40 µl with 1–1.5 µl of the RT reaction, single-strength PCR buffer, 20 µM dNTPs, 10 pmol forward and reverse primers, and 2.5 units of Taq DNA polymerase (Boehringer). The PCR was performed in 3 stages as follows: the first stage was one cycle of an incubation for 5 min at 95°C, 1 min at 50°C, and 2 min at 72°C; the second stage involved 30 cycles with a denaturation for 45 sec at 95°C, annealing at 52°C for 50 sec, and extension at 72°C for 1 min; finally the reaction mixture was incubated for 5 min at 72°C. PCR products were analyzed on a 1.5% agarose gel and stained with ethidium bromide. Bands of interest were cut out from the agarose gels, purified with Qiaquick gel extraction kit (Qiagen Pty Ltd., Clifton Hill, Victoria, Australia), and cloned into a pGEM-T easy vector (Promega) according to the instructions of the manufacturer.

In Situ Hybridization

Sense and antisense digoxigenin (DIG)-labeled RNA probes against clone 7.1 were generated using the DIG RNA Labeling kit (Boehringer), and the concentrations were determined according to the manufacturer's instructions. Five-micron sections of formalin-fixed paraffin-embedded tissues were subjected to in situ hybridization as described [15]. All sections were counterstained with Mayer's hematoxylin. Mouse kidney was used as positive control.

Detection of Apoptosis

Apoptosis was detected in sections (5-µm) of formalin-fixed tissues by the method of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) as described in detail [16] with minor modifications. Sections were pretreated with proteinase K (20 µg/ml) for 10 min at 37°C. Labeling of fragmented DNA was carried out with 7 µM DIG-labeled dUTP and 100 U/ml TdT, and the buffer supplied with the enzyme (all from Boehringer). The TdT was omitted on parallel negative control sections. Rat testis was used as positive control. Signals were visualized with the Liquid DAB-Plus Substrate Kit (Zymed Laboratories Inc., San Francisco, CA) for 5 min. The sections were lightly counterstained with Mayer's hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DDPCR and Confirmation of Clone 7.1 by Northern Analysis

To identify genes whose expression changes in mouse uterus during the initial implantation process, we compared the gene expression pattern of implantation and interimplantation sites on Day 4.5 of pregnancy using the DDPCR technique. One of the bands detected on the DDPCR gel, designated as band 7, was confirmed to be down-regulated in implantation sites compared to interimplantation sites (data not shown).

The cDNA products extracted from this band were reamplified and cloned into pGEM-T vector, and Northern blot analysis was repeated using the cloned inserts as probes. Clone 7.1 was confirmed to contain the cDNA representing the original expression pattern of band 7 (data not shown). Using cDNA of clone 7.1 as a probe, the mRNA expression pattern was determined by Northern analysis in the uterus of nonpregnant and Day 3 pregnant mice along with implantation and interimplantation sites of Days 4.5 and 5.5 pregnant uterus (Fig. 1A). A single band at around 600–700 basepairs (bp) was detected in all samples. Equal loading of RNA in all lanes was shown by probing the same membrane with a GAPDH cDNA. The mRNA detected by clone 7.1 was expressed in the uterus of nonpregnant as well as pregnant mouse. When the whole uteri were considered before and after pregnancy, it appeared that the expression level was much higher in the pregnant animals, in particular on Days 4.5 and 5.5 of pregnancy. Interestingly, the increased expression in the pregnant uterus was mainly in the interimplantation sites; the level in the implantation sites assessed by densitometric analysis was only 5–6% of that in the interimplantation sites on both days (Fig. 1B).



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  		FIG. 1. A) Northern blot analysis of mRNA detected by the DDPCR-derived 313-bp cDNA contained in clone 7.1 as a probe. Total RNA (15 µg) was isolated from whole uterus of nonpregnant mice at estrus (NP) and 3.5-day pregnant (d3.5) mice, from implantation sites (Imp) and interimplantation sites (Inter) of Days 4.5 (d4.5) and 5.5 (d5.5) pregnant mice. The top panel shows the 700-bp signal detected by the 313-bp cDNA; the lower panel shows that detected by the GAPDH probe on the same membrane as in the top panel. B) Relative expression of mRNA in implantation sites compared to interimplantation sites on 4.5 and 5.5 day of pregnancy. The means (± SEM) of 3 independent experiments are shown. For each experiment, the signal for each sample was analyzed by densitometry and corrected for loading according to the individual GAPDH level. The mRNA level in the implantation site was expressed as a percentage of that in the interimplantation site in the same animal (100%).

The precise percentage of increase in the expression of this gene during pregnancy in the whole uterus could not be determined accurately, because estrous cycle-related variation in the nonpregnant uterus was observed. Subsequently, nonpregnant mice were chosen at estrus, the stage at which mating occurs, representing the prepregnant state, to act as controls.

Cloning the Full cDNA Sequence of Calbindin-D9k

The nucleotide sequence contained in clone 7.1 (313 bp) was determined and is shown in Figure 2. When compared to the GenBank database, the cDNA sequences showing the highest homology with clone 7.1 were the vitamin D-dependent calcium-binding protein calbindin-D9k of the rat (86% identity in 259 nucleotides [nt]), chicken (92% in 150 nt), pig (76% in 172 nt), cow (76% in 172 nt), and human (72% in 189 nt). It was very likely, therefore, that clone 7.1 contained the partial cDNA sequence of mouse calbindin-D9k, a cDNA that had not been reported as being cloned at that time. During our efforts to clone mouse calbindin-D9k from a cDNA library of mouse uterus, a cDNA sequence (423 bp) of mouse calbindin-D9k became available in GenBank (accession number: AF028071). The first 290 nucleotides of clone 7.1 were 99% identical to this sequence (Fig. 2), but the remaining 23 nucleotides were different, with clone 7.1 having a longer 3' sequence (Fig. 2). To determine whether the sequence unique to clone 7.1 was authentic to mouse calbindin-D9k cDNA, RT-PCR of mouse uterine mRNA was performed using a forward primer based on the 5' end sequence of AF028071 and a reverse primer based on the 3' end sequence of clone 7.1 (Fig. 2). A cDNA of 441 bp (MCABP9k-A1) was obtained (Fig. 2); this cDNA contained most of AF028071 and the entire 3' end sequence unique to clone 7.1. Within this 441-bp sequence, two nucleotides at the 5' end were different from AF028071 (Fig. 2). RT-PCR was repeated two more times with different mice, and sequences the same as MCABP9k-A1 were obtained, confirming the difference from the sequence of AF028071. Subsequently the sequence of MCABP9k-A1 was deposited in GenBank (accession number: AF136283). These data indicated that the 441-bp sequence represented the full-length cDNA sequence of mouse calbindin-D9k, and that the sequence unique to clone 7.1 that was not found in AF028071 is part of the authentic 3' end. When the region between nucleotides 1–130 of MCABP9k-A1, which was not present in clone 7.1 and not previously used to probe mRNA on Northern blots, was excised with restriction enzyme ACCIII and tested as a probe, an expression pattern identical to the hybridization signal by clone 7.1 was obtained (data not shown). This confirmed that both clone 7.1 and the 130 bp of MCABP9k-A1 were from the same gene. Consequently, the full-length cDNA of 441 bp in clone MCABP9k-A1 was used in the subsequent Northern analysis. This 441-bp cDNA is 85% and 94% homologous to the rat counterpart at the cDNA and the protein level, respectively (data not shown). This cDNA was predicted to encode for a protein of 79 amino acids (Fig. 2). The last 77 amino acids of the deduced protein sequence were 100% identical to the amino acid sequence of mouse calbindin-D9k determined directly by tandem mass spectrometry of purified protein (the major form, consisting of 77 amino acids) [17]; the two amino acids from the N terminal of the deduced sequence were absent from the directly determined form (Fig. 2). It is very likely that the difference was caused by the difficulty in accurately determining the blocked terminal amino acids by mass spectrometry.



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  		FIG. 2. Comparison of cDNA sequences of clone 7.1 (313 bp), AF028071 (423 bp), and clone MCABP9K-A1 (441 bp). The nucleotides that were different in clone 7.1 and AF028071 compared with MCABP9K-A1 are highlighted in boxes. The predicted protein sequence consisting of 79 amino acids from both MCABP9K-A1 and AF028071 was the same and is shown. The stop codon is indicated (*). The forward and reverse primers are in bold and underlined on AF028071 and clone 7.1, respectively. The two amino N-terminal amino acids missing from direct measurement are underlined

Northern Analysis of Calbindin-D9k Expression in the Uterus During Early Pregnancy

To systematically determine the expression pattern of mouse calbindin-D9k in the uterus in relation to the time of implantation and early pregnancy, total RNA from the uterus of nonpregnant mice (at estrus) and pregnant mice at the initial stage of implantation (Day 4.5 of pregnancy) through to fully established placentation (Day 10.5 of pregnancy) was analyzed by Northern analysis using the full-length cDNA of 441 bp in clone MCABP9k-A1 (Fig. 3). Expression was detected in the uterus of nonpregnant mice and pregnant mice from Day 3.5 to Day 5.5 of pregnancy. It was increased in early pregnancy (as early as Day 3.5), but became barely detectable after Day 6.5 (Fig. 3). In addition, it was evident that at around the time of initial embryo attachment and during the actual implantation period (Days 4.5–5.5 of pregnancy), the mRNA level in the implantation sites was much lower than that in the interimplantation sites. This result confirmed the early observation that this gene was increased during early pregnancy, but the expression level was much lower in implantation sites compared to interimplantation sites, when the original DDPCR-derived clone 7.1 was used as probe (Fig. 1).



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  		FIG. 3. Northern blot analysis of mRNA detected by the full-length cDNA contained in clone MCABP9K-A1 (441 bp) in the mouse uterus across early pregnancy. Total RNA (15 µg) was isolated from whole uterus of nonpregnant at estrus (NP) and 3.5-day pregnant (d3.5) mice, and from implantation sites (Imp) and interimplantation sites (Inter) on Days (d) 4.5, 5.5, 6.5, 8.5, and 10.5 of pregnancy. On Days 8.5 and 10.5, three types of tissue were sampled: 1) the entire implantation unit containing the uterine implantation site, embryo and placenta (Imp [+]), 2) uterine implantation site tissue without embryo and placenta (Imp [-]), and 3) embryo and placenta sampled together (Emb +Pl). The top panel shows the 700-bp signal detected by this cDNA, and the lower panel shows the signal detected by the GAPDH probe on the same membrane

Effects of the Embryo on Uterine Expression of Calbindin-D9k

To determine whether the presence of embryos in the uterus was essential for the observed changes in calbindin-D9k during early pregnancy, total RNA was isolated from mice on Days 3.5 and 4.5 of pseudopregnancy, and the expression pattern was compared by Northern analysis with that in pregnant animals (Fig. 4). On Day 3.5, the expression level in the pseudopregnant animals was overall higher than that in the pregnant animals on the same day, but variations occurred because of the difficulties of determining the precise time of mating; on Day 4.5, the value in the pseudopregnant mice was equivalent to that in interimplantation sites of pregnant animals on the same day and was much greater than that at implantation sites (Fig. 4). These results implied that the observed increase in calbindin-D9k expression during early pregnancy is not embryo-dependent, but the down-regulation of calbindin-D9k at the implantation sites in the uterus required the presence of embryos.



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  		FIG. 4. Northern blot analysis of total RNA (15 µg) isolated from whole uterus of nonpregnant mice at estrus (NP) and Day (d) 3.5 pregnant (preg) mice, from implantation sites (Imp) and interimplantation sites (Inter) of Day 4.5 pregnant mice, and from whole uterus of Day 3.5 and 4.5 pseudopregnant (pseudo preg) mice. The top panel shows the 700-bp signal detected by the 414-bp cDNA; the lower panel shows that detected by the GAPDH probe on the same membrane

Calbindin-D9k mRNA Level During the Estrous Cycle and the Effects of Progesterone and Estradiol on its Expression in Ovariectomized Mice

The influence of the estrous cycle on the expression of calbindin-D9k in the nonpregnant uterus was examined by determining the level of its mRNA by Northern analysis. The study utilized 16 individual mice at different stages of the cycle (metestrus, diestrus, proestrus, and estrus), grouped to represent 4 cycles; one typical cycle is shown in Figure 5A. In all cases, the expression level was low at estrus and proestrus and became much higher during metestrus and diestrus (Fig. 5B). These results indicated a likely influence of the ovarian hormones estrogen and progesterone on the expression of calbindin-D9k in the uterus.



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  		FIG. 5. A) Northern blot analysis of total RNA (15 µg) isolated from whole uterus of nonpregnant mice at metestrus (met), diestrus (die), proestrus (pro), and estrus (est). One typical cycle is shown. The top panel shows the 700-bp signal detected by the 414-bp cDNA; the lower panel shows that detected by the GAPDH probe on the same membrane. B) Relative expression of mRNA in mouse uterus during the estrous cycle. The means (± SEM) of 4 independent experiments are shown. For each experiment, the signal for each sample was analyzed by densitometry and corrected for loading according to the individual GAPDH level. The mRNA level was expressed as a percentage of that in diestrus (100%)

To verify that the ovarian steroids can regulate the expression of calbindin-D9k in the uterus, estradiol and/or progesterone were administered to ovariectomized mice, and the expression level was determined by Northern analysis. Altogether, 16 animals consisting of 4 groups were used for this study. Very similar patterns of expression were observed in all 4 groups; results from one group are shown in Figure 6. The ovariectomized mice treated with vehicle (oil) showed very little expression of calbindin-D9k. Animals treated with estradiol alone had the same level of expression as the controls while those treated with progesterone had much higher expression compared to the control or to the estradiol-treated animals. Animals treated with both steroids had even higher expression than the progesterone-treated mice.



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  		FIG. 6. Northern blot analysis of total RNA (15 µg) isolated from whole uterus of ovariectomized mice treated with vehicle (oil), estradiol-17ß (E), progesterone (P), or E and P (E+P). The top panel shows the 700-bp signal detected by the 414-bp cDNA; the lower panel shows that detected by the GAPDH probe on the same membrane

Tissue Distribution of Calbindin-D9k mRNA

Multi-tissue Northern analysis was performed to investigate whether the expression of calbindin-D9k mRNA was specific to uterus (Fig. 7). Calbindin-D9k was not widely expressed; apart from the uterus, only kidney had a high level of expression while lung had a much lower signal of hybridization, among the 12 tissues tested. The lower expression of this gene in implantation sites compared to that in interimplantation sites in Day 4.5 pregnant uterus was demonstrated again on this blot (Fig. 7).



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  		FIG. 7. Northern blot analysis of the tissue specificity of the gene studied in Figures 3 and 4. RNA was isolated from mouse muscle, whole brain, kidney, spleen, heart, ovary, testis, liver, lung, intestine, and placenta. The relative expression level of this gene in the above tissues was compared to that at implantation sites (Imp) and interimplantation sites (Inter) of 4.5 day (d4.5) pregnant uterus; the mRNA level was lower in the implantation sites. Total RNA (15 µg) was loaded in each lane, and the full-length cDNA contained in clone MCABP9K-A1 was used as probe. The top panel shows the 700-bp signal detected by 441-bp cDNA, and the lower panel shows the signal detected by GAPDH probe on the same membrane

In Situ Hybridization of Calbindin-D9k mRNA in the Uterus of Early Pregnant Mice

The cell types that express calbindin-D9k mRNA in the uterus were determined by in situ hybridization using riboprobes generated against clone 7.1. Kidney was used as the positive control, and in this the mRNA was predominantly localized in some of the distal tubules (Fig. 8A). In nonpregnant uterus, no single cell type was distinctively positive. Signals in luminal epithelial cells appeared stronger than those in the glandular cells, and weak staining was present also in the myometrium (Fig. 8B). On Day 3.5 of pregnancy, a relatively higher level of expression was detected mainly in the luminal epithelial cells (Fig. 8C). Interestingly, on Day 4.5 of pregnancy, exceptionally strong signals were detected predominantly in the epithelial cells in the interimplantation site (Fig. 8, D and E); weaker signals were also present in the glandular epithelium and the stromal cells adjacent to the lumen on the same section. At this time, a very different pattern of staining was observed in the implantation site, in which a very strong signal was detected only in the uterine luminal epithelial cells where the embryo was not attached; no staining was present in the regions where the embryo was attached (Fig. 8, F and G). Very few glands were present on these sections because of the extensive decidualization of the stroma. This indicated that the lower expression of calbindin-D9k mRNA in implantation sites demonstrated by Northern analysis was mainly due to the absence of the signals in the luminal epithelial cells surrounding the embryo. To confirm this, the implantation sites were cut through to a region beyond the implanting embryo (Fig. 8, I and J); physically this region was away from the actual site of apposition by the embryo, but morphologically it was different from interimplantation sites because the decidual zone was still evident and the uterine lumen was more closed than at interimplantation sites. On these sections, very strong signals were detected in the entire luminal epithelium (Fig. 8I); glandular epithelial cells as well as stromal cells adjacent to the lumen also showed moderate staining on these sections (Fig. 8, I and J). This further demonstrated that only the uterine epithelial cells surrounding the embryo showed reduction in calbindin-D9k mRNA expression.



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  		FIG. 8. In situ hybridization of calbindin-D9k mRNA in the mouse kidney (A) and the uterus (B–L) across early pregnancy using DIG-labeled probes generated against clone 7.1. Uterine cross sections were hybridized with antisense probe and are shown for nonpregnant (B), pregnant Day 3.5 (C), interimplantation site (D and E) on Day 4.5 of pregnancy, implantation site where embryo is visible (F and G), and the end zone of the implantation site where no embryo is visible (I and J) on pregnant Day 4.5. The large arrowheads on I indicate the decidualized stromal cells that are also positive. H is the same as F, but sense-probe control was used. Sections are also shown for implantation (K) and interimplantation site (L) on Day 6.5 pregnancy. le, Luminal epithelium; ge, glandular epithelium; Imp, implantation site. Bar = 10 µm in each case

When the uteri of Day 5.5 pregnant mice were examined, signals were less intense, but an expression pattern similar to that in Day 4.5 pregnant uteri was observed (data not shown). On Day 6.5, the uterine lumen in the implantation sites was closed, and the implanting embryo occupied almost the entire uterine chamber (Fig. 8K). Very little staining was detected in these sections except for weak signal in remaining uterine epithelial cells that were not enclosing the embryo (Fig. 8K). At this time, the uterine lumen in the interimplantation sites was still relatively open (Fig. 8L), and moderate levels of staining were detected mainly in the luminal epithelial cells (Fig. 8L).

Detection of Apoptosis in the Uterus on Day 4.5 of Pregnancy

When uterine sections with blastocysts of different sizes and differently apposed to the lumen were examined by in situ hybridization, down-regulation of calbindin-D9k mRNA was observed in all cases (Fig. 9, A and C). Even those luminal epithelial cells surrounding the blastocyst but not directly in contact with the embryo were also devoid of calbindin-D9k mRNA (Fig. 9A). However, in such sections, only a few luminal epithelial cells were lightly stained positive for apoptosis, although morphologically they appeared to have normal nuclear shape (Fig. 9B); therefore, they may represent a early stage of apoptosis. On sections in which the blastocyst occupied the whole implantation chamber and was in close contact with the lumen, while only a few cells were positive for apoptosis, characteristic morphology such as increased nuclear density was seen (Fig. 9D). Some embryonic cells were also stained positive (Fig. 9B), because the TUNEL assay is designed to identify single-strand breaks of DNA, which occur in mitosis as well as in apoptosis [18]. Rat testis was used as control for the TUNEL assay [16].



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  		FIG. 9. In situ hybridization of calbindin-D9k mRNA and detection of apoptosis in parallel sections of mouse uterus on Day 4.5 of pregnancy. A and C are uterine sections for in situ hybridization and show variations in the extent of blastocyst development and apposition. Arrowheads indicate the sites at which mRNA disappears from luminal epithelial cells. B and D are stained for apoptosis and are serial sections of A and B, respectively. Luminal epithelial cells lightly stained for apoptosis are marked by small arrows, and the characteristic apoptotic cells are marked by arrowheads. Rat testis was used as a positive control for apoptosis (E); large arrowheads indicate the apoptotic region. F is negative for E. em, Embryo; le, luminal epithelium; ge, glandular epithelium; Imp, implantation site. Bar = 10 µm in each case


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We applied the technique of DDPCR to search for genes that are differentially regulated between implantation and interimplantation sites in the mouse uterus on Day 4.5 of pregnancy, when the uterus shows the first morphological changes associated with pregnancy. We reasoned that up- or down-regulation of these genes would be potentially important for conversion of the uterus from the nonreceptive to the receptive state. We detected several bands exhibiting different expression between the two sites, one of which we identified as mouse vitamin D-dependent calcium-binding calbindin-D9k.

The full cDNA sequence of mouse uterine calbindin-D9k was then determined. This sequence is longer than the published sequence of calbindin-D9k [19], and there are some differences at the 5' end, which is located in exon 1. The partial sequence of this exon [19] was exactly the same as that of MCABP9k-A1, which is different from the published cDNA sequence of calbindin-D9k [19]. The deduced protein sequence of mouse calbindin-D9k is in high agreement with the previously published amino acid sequence of this protein [17].

Vitamin D-dependent calcium-binding proteins (calbindins) are a family of intracellular proteins involved in regulating calcium metabolism by binding calcium with high affinity [20–22]. Two proteins, a 28-kDa (calbindin-D28k) and a 9-kDa (calbindin-D9k) form, which are the products of two distinct genes with limited sequence similarity, have been found in intestine, kidney, bone, uterus, and other tissues of birds [23–25] and mammals [26–29]. The importance of calbindins in reproductive function has been emphasized from studies involving vitamin D [30–34]. The role of vitamin D in fertility was mostly through its association with the calbindins that were expressed in the ovary, oviducts, testis, and other reproductive tissues [35–38].

While little is known about calbindin-D28k in pregnancy [39], a few studies have investigated calbindin-D9k in the rat uterus during early pregnancy. In pregnant rats, expression of calbindin-D9k mRNA is relatively high on Day 1 of pregnancy (P1); on P2, P3, and P5 the mRNA decreases to the detection limit of Northern analysis [40]; on P10, the transcripts begin to reappear at levels of about 30% of P1 [40]. In nonpregnant rats, calbindin-D9k was immunolocalized in the myometrium and endometrial stromal cells, but not in the luminal epithelial cells [41,42], whereas in pregnant rats, the expression was detected predominantly in luminal epithelial cells [42,43]. The dynamic nature of uterine expression of calbindin-D9k during pregnancy has also been reported in other species. In the porcine uterus, expression of endometrial calbindin-D9k mRNA was high on pregnancy Days 10–12 and below the detection limit on Days 15 and 18 (first attachment: Day 14). Later during gestation, the mRNA can be detected again at low levels from Day 60 [44]. In the bovine uterus, calbindin-D9k was localized to the maternal caruncular epithelium, and the expression level increased from the second trimester to term pregnancy, but no studies were performed at the time of implantation [45].

Apart from the recent report of Tatsumi et al. [19], no previous studies have investigated the expression pattern and the role of calbindin-D9k in the mouse uterus during early pregnancy. The only other data available on calbindin-D9k and pregnancy in the mouse concern its expression in the placenta during late gestation [46]. The present study confirms the study of Tatsumi et al. and more clearly illustrates cell-specific expression of calbindin-D9k in the mouse uterus during early pregnancy, and, in particular, its overall up-regulation during early pregnancy and differential pattern of expression between implantation and interimplantation sites. An increase in calbindin-D9k mRNA was also detected in the pseudopregnant mouse uterus, indicating that this increase does not require the presence of an embryo in the uterus. Thus, calbindin-D9k may play an important role in the control of uterine activity during early pregnancy in the mouse. On the other hand, the down-regulation of calbindin-D9k in implantation sites was dependent upon the presence of blastocysts because it did not occur in the pseudopregnant uterus in our study, and calbindin-D9k mRNA was suppressed by transfer of blastocysts to the pseudopregnant mice [19].

The observed increase in calbindin-D9k mRNA expression in early pregnancy followed by a decrease in the mouse uterus is in agreement with the previous reports in the rat [40] and mouse [19], in which a similar pattern was seen. However, the cellular origin of the increase immediately after pregnancy and the following decrease in calbindin-D9k mRNA in the uterus was not clear. This study demonstrates that, in the mouse, the dramatic increase of calbindin-D9k gene expression in early pregnancy was mainly due to the high expression of this gene in the luminal and to a lesser extent, in the glandular epithelial cells in the pregnant uterus; while in the prepregnant uterus, no single cell type highly expressed this gene.

The increased expression of calbindin-D9k mRNA in uterine epithelium in early pregnancy may be caused by the hormonal changes occurring at this time, but the exact mechanisms are not clear. In the rat, uterine expression of calbindin-D9k is dependent on critical levels of estradiol-17ß [41,47] and estrogen receptor [38,40], and it is also tightly regulated during the estrous cycle [48]. The present study is the first to demonstrate that, in the mouse, calbindin-D9k mRNA level likewise changes during the estrous cycle, indicating hormonal regulation. However, the expression pattern in the mouse is very different from that of the rat. In the rat, calbindin-D9k mRNA level was highest at proestrus and not detectable at diestrus, indicating positive regulation of this protein by estrogen [48]. By contrast, in the mouse, we detected high expression at diestrus and metestrus, and only basal levels of expression at proestrus and estrus. This very different expression pattern between the rat and mouse during the estrous cycle suggests quite different mechanisms of regulation. To pursue this, the effects of progesterone and estradiol on the expression of calbindin-D9k mRNA were examined in ovariectomized mice. Estrogen alone did not induce this mRNA, and progesterone alone moderately up-regulated the expression, while the highest expression was seen with a combination of both steroids, similar to the data reported by Tatsumi et al. [19]. Thus, the hormonal regulation of calbindin-D9k mRNA is quite different in the rat and mouse. In the rat, estrogen regulation of this protein was suggested to be mediated through an imperfect estrogen-responsive-like element (ERE) identified in intron A of the rat calbindin-D9k gene [49]. Recently, cloning of intron A of the mouse calbindin-D9k gene showed a single-base difference in the ERE compared to that of the rat [19]. This may partially explain the observed difference in the hormonal regulation between the rat and mouse. However, other unknown cell-, tissue-, and species-specific factors may also be very important. For example, regulation of calbindin-D9k expression in the rat by estrogen is only uterine-specific and does not occur in the intestine [41].

It was particularly interesting to observe that the regions where the uterine epithelium surrounded the implanting embryo were negative for calbindin-D9k mRNA, whereas uniform expression was detected in the entire luminal epithelium in the regions where no embryo was attached; this was true not only in the interimplantation site but also in the extremes of the decidualized zone, where the embryo was not directly in contact with the uterus. During progression of the pregnancy, the calbindin-D9k mRNA expression starts to diminish. On Day 6.5, expression was seen only in the little remaining epithelium not in contact with the embryo. Therefore, it is apparent that the absence of calbindin-D9k mRNA at implantation sites did not occur because the uterine epithelium never expressed this gene, but rather because expression was reduced at the site of blastocyst apposition. It is believed that during embryo implantation, in accordance with trophoblast growth, apoptosis occurs in the epithelial cells at the implantation sites [19,50]. However, the observed down-regulation of calbindin-D9k mRNA in the implantation site on Day 4.5 of pregnancy was not found to be associated with apoptosis of epithelial cells. All cells surrounding the blastocyst do not express calbindin-D9k mRNA on Day 4.5 of pregnancy, but only a few of these cells show characteristics of apoptosis such as condensed nuclei. Therefore, the calbindin-D9k mRNA must be actively down-regulated. The mechanisms by which the implanting blastocyst down-regulates the expression of calbindin-D9k mRNA are not clear. It will be particularly interesting to determine whether down-regulation of this gene at the blastocyst apposition site occurs before the blastocyst activation in the delayed implantation model.

The precise role of calbindin-D9k in the process of embryo implantation remains to be established. However, intracellular calcium flux is an important determinant of gene expression [51]. The observed complex regulation of calbindin-D9k mRNA in the mouse uterus and its changing expression pattern during early pregnancy, not only in the mouse but also in species with different types of implantation, suggests that it may play a crucial role in this process.


   ACKNOWLEDGMENTS
 
Hiroyuki Minoura was on sabbatical leave from School of Medicine, Mie University, Japan. We are grateful to Jin Zhang, Anne Hampton, and Leigh Batten for their technical assistance, and thank Sue Panckridge for assistance with the illustrations.


   FOOTNOTES
 
First decision: 5 May 1999.

1 Research supported by the Rockefeller Foundation Contraceptive 21 Initiative Project, the Wellcome Trust (grant 52666), and the NH&MRC of Australia (grant 971297). Back

2 Correspondence: Gui-Ying Nie, Prince Henry's Institute of Medical Research, PO Box 5152, Block E, Level 4, Monash Medical Centre, 246 Clayton Rd, Clayton, Vic 3168, Australia. FAX: 61 3 9594 6125; guiying.nie{at}med.monash.edu.au Back

3 Current address: Shanghai Institute of Planned Parenthood Research, 2140 Xie Tu Road, Shanghai 200032, China. Back

4 Current address: Department of Obstetrics and Gynecology, Mie University School of Medicine, 2-174 Edobashi Tsu, Mie, 514 Japan. Back

Accepted: August 24, 1999.

Received: April 1, 1999.


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