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Biology of Reproduction 63, 1764-1773 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Desmosomes Are Reduced in the Mouse Uterine Luminal Epithelium During the Preimplantation Period of Pregnancy: A Mechanism for Facilitation of Implantation1

Ian M. Illingwortha, Irena Kiszka3,a, Steven Bagley4,a, Grenham W. Irelanda, David R. Garroda, and Susan J. Kimber2,a

a School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

ABSTRACT

Dynamic regulation of intercellular junctions is an essential aspect of many developmental, reproductive, and physiological processes. We have shown that expression of the desmosomal protein desmoplakin decreases in the luminal uterine epithelium during the preimplantation period of pregnancy in mice. By the time of implantation (between Days 4.5 and 5 of pregnancy), desmoplakin protein can barely be detected by SDS-PAGE and Western blotting, and by immunocytochemistry, it is restricted to well-spaced, punctate dots at the apicolateral junction. Using confocal XZ series and electron microscope quantitation, both the density and distribution of desmosomes along the lateral cell surfaces of luminal epithelial cells were observed to change during early pregnancy. On Day 1 of pregnancy, desmosomes were found at high density in the apicolateral junctional complex, being present here in 79% of ultrathin sections examined, whereas on Day 5, the density was much reduced (present in only 18% of ultrathin sections examined). Desmosomes were found along the lateral surfaces, at or below the level of the nucleus, in 15% of ultrathin sections examined on Day 1 of pregnancy but in only 1% on Day 5. Desmoplakin mRNA declined during the first 4–5 days of pregnancy, along with the protein, suggesting that these changes are controlled at the level of mRNA. This study shows that desmosomes are regulated during early pregnancy, and we propose that a reduction in desmosome adhesion facilitates penetration of the luminal epithelium by trophoblast cells at implantation.

female reproductive tract, uterus

INTRODUCTION

Trophoblast invasion in the mouse occurs via a "displacement" mechanism in which the trophoblast penetrates between the intact lining epithelial cells of the uterus [1–3]. Implantation of the blastocyst is initiated by adhesion between the apical surfaces of the abembryonic trophectoderm [4] and the lining cells of the uterine endometrium [2]. After invasion, cells of the lining epithelium are displaced and appear to undergo apoptosis [5]. This exposes the basal lamina to the trophoblast, which penetrates into the stroma after stroma-induced basement membrane degradation [6, 7].

Implantation of the embryo occurs on Days 4.5–5 postcoitum (pc) in the mouse. Implantation is controlled by ovarian steroids, and it requires both the uterine endometrial epithelium and the stroma to undergo a process of maturation. Mice require a brief "nidatory" surge of estrogen for the endometrium to become receptive to the implanting blastocyst [8]. Nidatory estrogen induces a "window of implantation", or a receptive period, lasting approximately 24 h in rodents, after which the uterus becomes nonreceptive or "refractory" by 36 h post-nidatory estrogen [9, 10]. Although we have considerable knowledge regarding the ovarian steroid environment required for implantation, we know much less concerning the cell biological changes in the epithelium that render it receptive.

Clearly, implantation requires fundamental changes in the endometrial epithelium [2, 11–13]. Murine uterine epithelial cells exhibit characteristics that are typical of a simple epithelium, including the polar organization of surface membrane components into apical and basolateral domains. At the time of implantation, rapid changes in the epithelial cells result in flattening and a less distinctly polar organization of the cells [2, 3, 11, 12, 14–18].

Changes in the junctional complexes between luminal epithelial cells likely facilitate trophoblast invasion. In the rat, tight junctions change from a compact, apicolateral pattern to a highly branched, lateral distribution by Day 6 pc [19]. In mouse luminal uterine epithelium, a pronounced decrease in E-cadherin staining intensity is seen from Day 3 to 6 pc, as is redistribution from basal and lateral regions to a more apicolateral pattern [20]. E-Cadherin mRNA levels increase under the control of progesterone and 17ß-estradiol [21]. Changes in the distribution of desmosomal proteins and glycoproteins have also been reported in the rabbit [22], but these have not been rigorously documented. To our knowledge, desmosomal changes have not been examined in the mouse, in which trophoblast invasion occurs by a different mechanism from that in the rabbit (termed displacement, rather than by cell fusion).

In this study, we focus on the changes in desmosomes of the uterine epithelium during early pregnancy and implantation. Desmosomes are multimolecular, adhesive junctions of approximately 0.5 µm in diameter. These junctions are present in nearly all types of epithelial cells [23–25]. They confer structural integrity and organization to the tissues in which they occur [26, 27]. Desmosomes appear very early during murine development, at the early blastocyst stage, suggesting a critical role in developmental processes [28]. The principal desmosomal transmembrane glycoproteins are desmogleins (Dsg) and desmocollins (Dsc), both of which exist as three distinct isoforms. Each Dsg and Dsc isoform is the product of a different gene of the cadherin superfamily of calcium-dependent, cell adhesion molecules [29, 30]. Cytoplasmic structural proteins associated with desmosomes [27] include plakoglobin, plakophilin, and desmoplakins I and II. The desmoplakins link desmosomes to the intermediate filament cytoskeleton. Several recent reviews of desmosomes have appeared [31–33].

At implantation, the integrity of the receptive epithelium clearly is breached as the trophoblast penetrates between the epithelial cells. We show that changes in desmosomes occur during the preimplantation period that may contribute to changes in cell-cell adhesion of luminal epithelium, thus facilitating trophoblast invasion.

MATERIALS AND METHODS

Animals and Mating

Six-wk-old MF1 female mice (Harlan Olac Ltd., Bicester, UK) were maintained in a 12L:12D cycle with water and food provided ad libitum. Individual female mice were paired overnight with MF1 male mice (Harlan Olac Ltd.) and checked each morning for a vaginal plug. The day of plug observation was termed Day 1 pc. Mated female mice were removed to individual holding cages. At the required stage of pregnancy, female mice were killed by cervical dislocation. Uterine horns were dissected and trimmed of excess fat and mesentery tissue before transfer into Hanks Balanced Salt Solution (HBSS; Gibco, Paisley, UK) on ice. Pregnancy was confirmed by either flushing of fertilized embryos or the observation of decidualized regions in the uterine horn.

Antibodies

For both immunocytochemistry and Western blotting, monoclonal antibody 11-5F [34] was used at a dilution of 1:10 v:v. This mouse IgG recognizes bovine desmoplakins I and II and cross-reacts with the murine forms of these proteins. Additionally, a rabbit anti-desmoplakin polyclonal and a mouse monoclonal antibody to desmoglein were used (data not shown), and a guinea pig anti-desmoplakin polyclonal antibody at a dilution of 1:200 v:v was also used. Normal goat serum (NGS; Sigma, Dorset, UK) was used at a dilution of 1:20 v:v as a negative control in both procedures. For immunocytochemistry, goat anti-mouse IgG-fluorescein isothiocyanate (FITC) conjugate (Sigma) was used at dilution of 1:125 v:v. For Western blotting procedures, a peroxidase-conjugated goat anti-mouse IgG (Sigma) was used at a dilution of 1:500 v:v. For double staining, LE-41, a mouse monoclonal antibody recognizing cytokeratin 8 (gift of Prof. Birgit Lane, University of Dundee, UK) [35] was used in conjunction with guinea pig anti-desmoplakin (O70), both at a dilution of 1:10 v:v. In this case, secondary antibodies were a FITC-labeled donkey anti-mouse and lisamine-rhodamine-labeled donkey anti-guinea pig (Jackson Laboratories, West Grove, PA) used together, both at a dilution of 1:100 v:v.

Immunocytochemistry

Dissected uterine horns were cut into lengths of approximately 0.5 cm, which were then rapidly frozen in OCT cryoembedding compound (Tissue Tek, Bayer Corp., Tarrytown, NY) in aluminum foil boxes using liquid nitrogen. Seven-micrometer, transverse uterine cryosections were cut using a Leitz cryostat at -20°C (Leica UK Ltd., Milton Keynes, UK). Two sections at least 100 µm apart in the tissue were mounted on each glass microscope slide. Sections were dried for 1 h at room temperature, fixed for 10 min in acetone at 4°C, and air-dried for 5 min. Immunocytochemistry was performed as described elsewhere [36] with a 45-min NGS blocking step preceding the primary (overnight at 4°C) and secondary (30 min at room temperature) antibody incubations. Slides were examined using confocal microscopy as described later.

Scoring

Uterine luminal and glandular epithelium were scored independently for staining intensity at the apicolateral border and the lateral, basal, and cytoplasmic regions. A five-point scoring system, ranging from no signal to bright, was used to score each region. The scorer was blind to the day of pregnancy of the section. The actual scores are not included, because they are not truly quantitative and give only information concerning relative levels of staining. However, scoring is consistent between trained observers.

Isolation and Immunocytochemical Staining of Endometrial Epithelial Cell Sheets

Dissected uterine horns were placed in a 0.5% dispase II (Boehringer Mannheim, Lewes, UK) in HBSS and incubated at room temperature for 2 h and 20 min. Using a Wild dissecting microscope (Leica), tubes of luminal endometrial cells were then squeezed from the horn by running a fine glass rod along its length. Epithelial tubes were opened out using vanass scissors and slid onto 8-mm diameter, circular coverslips. Excess buffer was removed from the epithelial sheets, and time was allowed for minimally hydrated specimens to attach to the slide. The sheets were fixed in acetone:methanol (1:1) at -20°C for 10 min. Fixed epithelial cell sheets were stained as described elsewhere [36] in a volume of 100 µl. After removal of secondary antibody, sheets were incubated with 100 µl of propidium iodide solution (40 µg/ml; Sigma) for 30 min at room temperature. Sheets were incubated with 1 µg/ml of RNase A for 30 min to reduce cytoplasmic RNA staining. They were then washed in six changes of PBS before mounting in Gelvatol. Staining was observed using confocal microscopy as described later.

Confocal Microscopy

A BioRad MRC 600 laser scanning attachment (BioRad, Hemel-Hemstead, UK) was linked to a 90-MHz Pentium Compaq personal computer running COMOS version 6 control software. The laser scanning attachment was attached to a Zeiss photomicroscope II fluorescence microscope equipped with a 40x plan neofluor multi-immersion objective lens (Zeiss, Wehryn Garden City, UK; numerical apperture = 0.9). The 488-nm line of an argon-ion laser was used to excite FITC, employing 30% transmission with neutral-density filters. Minimal pinhole size was used, with a Kalman average of seven scans forming each image. Other settings remained constant for each immunocytochemistry series, with no further contrast correction or image processing being performed.

For observation of epithelial cell sheets, a 100x Neofluor (Zeiss; NA = 1.3) oil-immersion objective was used. Krypton/argon laser illumination providing 488/568 lines was used to excite FITC and propidium iodide, respectively. Emitted light was separated into two channels by T1 and GR2 filters. Minimal pinhole size was used, with a Kalman average of three scans forming each image. The Z series were collected with a Z-step of 2 µm. The XZ (vertical) sections were also collected from epithelial cell sheets using a 63x Plan-Neofluor (Zeiss; NA = 1.4) oil-immersion objective. Images for the Z series were collected starting from the apical cell surface.

Cytokeratin/desmoplakin colocalization was observed using a Zeiss LSM 510 confocal microscope with a 63x Plan-Apochromat oil-immersion objective (NA = 1.4). Line scan averaging of two scans was used to collect single images, with a pinhole aperture of 100 µm.

All image files were converted to uncompressed TIFF files before output using CorelDRAW version 8 and dye sublimation printing using a Hewlett-Packard DeskJet 720C printer (Hewlett-Packard, Palo Alto, CA).

Electron Microscopy

Segments of uterus were dissected into 1-mm3 pieces and fixed in 2.5% gluteraldehyde/1% paraformaldehyde in 100 mM sodium cacodylate and 2 mM CaCl2 buffer at pH 7.4. After fixation, the pieces were washed in 100 mM sodium cacodylate and 2 mM CaCl2 and then postfixed in 2% OsO4 for 1 h. Next, the tissue was dehydrated in a stepped ethanol series before embedding in Spurr resin (Agar Scientific, Starsted). Sections of 150–200 nm were cut using an ultramicrotome (Reichert, Austria) and mounted on 200-mesh copper grids. Grids were stained with 2% uranyl acetate in 70% ethanol and 0.3% lead citrate in 100 mM NaOH before observation using a Philips 200 electron microscope at 80 kV. In total, 16 sections from Day 1 and 12 sections from Day 5 were examined. Sections were taken from two animals for each day of pregnancy examined. Each individual cell assessed for distribution of desmosomes was fully visible from the apical to the basal surface. Any cell for which this was not true was not included in the analysis.

Endometrial Epithelial Protein Extraction

Epithelial cell sheets (prepared as described earlier) were squeezed from the uteri into HBSS on ice. Cells were washed twice in HBSS at 4°C, then lysed in Laemmli sample buffer [37] without reducing agent or bromophenol blue. The preparation was boiled for 5 min, which was followed by sonication and removal of insoluble material by centrifugation at 15 000 x g.

SDS-PAGE/Immunoblot Analysis

Endometrial epithelial cell extracts from each of Days 1 to 6 of pregnancy were assayed for total protein content using bicinchonic acid protein assay reagent (Pierce, Warriner Chester, UK). ß-Mercaptoethanol (Sigma) and bromophenol blue were added to each sample before boiling for 5 min. Equal quantities of protein from each stage were loaded into sequential lanes of a SDS-PAGE gel (usually 20 µg/lane). Two identical, 7.5% gels [37] were run in parallel. One gel was blotted onto nitrocellulose (BioRad Microscience), whereas the other was silver stained to check protein loading. The nitrocellulose membrane was transferred to a solution of 4% dried skimmed milk (Tesco, Manchester, UK) in TBST (0.1 M Tris-buffered saline [pH 7.5] with 0.1% Tween-20 [BioRad Microscience, UK]) and incubated overnight at 4°C. The blot was then washed in TBST before being transferred into primary antibody for 1.5 h, which was followed by further washes in TBST and incubation in secondary antibody for 1.5 h. The membrane was washed extensively before incubation in enhanced chemiluminescence reagent (Nycomed-Amersham, Amersham, UK) for 1 min and exposure to autoradiographic film (Hyperfilm-ECL; Nycomed-Amersham, Amersham, UK) for 5 min.

RNA Extraction

Epithelial cell tubes were prepared as described earlier. The tubes were centrifuged at 3000 x g and the HBSS removed. The epithelial tubes were snap frozen in liquid nitrogen and stored at -80°C. Tissue from each of Days 1 to 6 of pregnancy was prepared in this manner, using two or three animals in each instance. The complete analysis was repeated three times. Total RNA was extracted from each cell pellet using an RNAeasy extraction kit (Quiagen, Crawley, UK). Tissue homogenization was achieved by use of Quiashredder columns (Quiagen). Purified total RNA was eluted in 30 µl of RNase-free water. The RNA preparations from each day of pregnancy were quantified by absorbance at 260 nm using a Gene Quant UV spectrophotometer (Pharmacia Biotech, St. Albans, UK). Purity of RNA was assessed by comparing the ratio of absorbance at 260 nm with that at 280 nm. From 300 to 1000 µg of RNA of between 90% and 100% purity was obtained from two animals on each day of pregnancy.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction

Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to examine relative changes in desmoplakin mRNA levels from luminal epithelium on Days 1–6 of pregnancy. Changes in PCR product for desmoplakin were compared with an endogenous gene ß-actin (as an internal standard), the expression of which was fairly constant in the luminal epithelium between Days 1 and 6 of pregnancy.

First Strand Synthesis

From each RNA sample, 2 µg were incubated with 1–5 U of RNase free DNase (Boehringer Mannheim) at 37°C for 20 min before denaturing DNase at 94°C for 10 min. Next, 1 µl of oligo(dT)12–18 (Life Technologies Ltd., Paisley, UK) was added to each sample, and the total volume was made up to 10 µl with RNase free H2O before incubation at 70°C for 10 min. Samples were then placed on ice before addition of 4 µl of 5x RT buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, and 15 mM MgCl2), 2 µl of 0.1 M 1,4-dithiothreitol, 1 µl of deoxynucleoside 5'-triphosphate (dNTP) mix (10 mM each dNTP), 1 µl of Moloney murine leukemia virus reverse transcriptase (all from Life Technologies Ltd.), and 2 µl of RNase free H2O. The cDNA synthesis mix was incubated at 37°C for 1 h, which was followed by RT inactivation through heating to 95° (5 min) and cooling on ice. Subsequently, 1 µl of this cDNA was used in each PCR reaction. The RT negative controls consisted of 1 µl of RNA subsequent to treatment with RNase free DNase.

PCR Primers

The PCR primers were designed using GCG primer design software (HMGP, Hinxton, UK). Desmoplakin primers were designed to a published cDNA sequence [38]. This short cDNA fragment was derived from murine ectoplacental cone cells and is highly homologous to the published sequence of human desmoplakin cDNA [39]. ß-Actin primers were designed to the published cDNA sequence of murine ß-actin [40]. Specific sequences were as follows:

Desmoplakin: CTC TGG AGG AGT CAA GCC C CCT TTT ACC CCT TCA AAG CC Product length: 274 base pairs (bp)ß-Actin: AAA CTG GAA CGG TGA AGG C CCT GGG CCA TTC AGA AAT TAProduct length: 344 bp

PCR Protocol

The PCR master mix consisted of 5 µl of 10x PCR buffer (Boehringer Mannheim), 0.5 µl of both forward and reverse primers (100 pM; Life Technologies, Ltd., custom oligonucleotide synthesis services, UK), 0.5 µl Taq DNA polymerase (2.5 U; Boehringer Mannheim), 0.4 µl of dNTP (25 mM) and 42.1 µl of RNase and DNase free H2O (Sigma). This master mix was added to 1 µl of template cDNA and then mixed briefly before centrifugation at 15 000 x g and thermal cycling.

The PCR thermal cycling was performed using an Eppendorf multicycler gradient PCR machine (Eppendorf, Hamburg, Germany) under the following conditions for desmoplakin: initial denaturation at 94°C for 2 min, annealing at 60°C for 30 sec, elongation at 72°C for 40 sec, and denaturation at 94°C for 30 sec. The cycle number to be used was determined by performing the described reaction with cDNA from Day 1 of pregnancy using increasing cycle number. The cycle number to be used was defined as being the last cycle number at which the PCR product appeared to double in quantity, which was 40 cycles. ß-Actin cycling conditions were initial denaturation at 94°C for 2 min, annealing at 60°C for 30 sec, elongation at 72°C for 30 sec, and denaturation at 94°C for 30 sec. The cycle number to be used was determined as being that at which the PCR product could be first visualized (20 cycles).

Differing cycle numbers were used for ß-actin and desmoplakin amplification. The cycle number at which ß-actin is first detected was considered to be the most accurate method for ensuring equal cDNA quantities for each day of pregnancy [41]. The desmoplakin reaction was optimized such that a cycle number was used at which all days of pregnancy were in the logarithmic phase of amplification.

The PCR products were separated on a 1.5% (v:v) agarose gel containing 0.05 µg/ml of ethidium bromide using a 100-bp DNA ladder (Life Technologies Ltd.) for reference. The PCR products were verified by sequencing on an automated ABI DNA sequencer (ABI Analytical, Ramsey, NJ).

RESULTS

Desmoplakin Protein Expression Decreases in Uterine Epithelium Between Days 1 and 6 pc

To determine whether a change occurs in desmosome expression in the uterine epithelium during the preimplantation period, immunoblot analysis of the major desmosomal protein desmoplakin was performed on endometrial extracts taken at 1-day intervals using monoclonal antibody 11-5F. This antibody recognized two polypeptide bands of 230 and 215 kDa, which correspond to desmoplakins I and II (Fig. 1A). Immunoblots of equal amounts of protein extracted on successive days showed a progressive decrease in desmoplakin expression, so that it became barely detectable from Days 4 to 6 (Fig. 1, A and B).



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  		FIG. 1. Western blot detection of desmoplakins I and II in murine luminal epithelial cell extracts. A) Western blot detection using monoclonal antibody 11-5F, of luminal epithelial cell extracts from Days 1 to 6 pc. On Day 1, two bands of 230 and 215 kDa, corresponding with the known sizes of desmoplakins I and II, can be clearly identified. On Day 2 pc, the quantity of protein detected is significantly reduced, and a further reduction is observed by Day 3 pc. On Days 4, 5, and 6 pc, little or no protein can be seen. No desmoplakin bands are observed in the NGS control. B) Silver-stained gel illustrating equal protein loading using identical cell extract quantities to those used for Western Blotting

Desmoplakin Immunofluorescence Decreases and Changes in Distribution Between Days 1 and 6 pc

To provide more detailed evidence for the decline in desmoplakin expression during the preimplantation period, cryostat sections of uterine endometrium were examined by immunofluorescence with monoclonal antibody 11-5F on successive days of pregnancy. The results showed a striking, progressive decrease in desmoplakin immunofluorescence, from very bright staining on Day 1 to much weaker staining on Day 4 to very weak staining on Days 5 and 6 (Fig. 2, A–D). Thus, the immunofluorescence results showed a corresponding decrease in desmoplakin expression to that found by immunoblotting.



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  		FIG. 2. Immunolocalization of desmoplakin in mouse uterine luminal epithelium. Transverse sections of mouse uterine horns stained using either monoclonal antibody 11-5F (recognizing desmoplakins I and II; A–D) or control NGS (E–H) are shown. A) Day 1 pc mouse uterus shows a clear "chicken wire" pattern of desmoplakin immunostaining around the apicolateral region of the luminal epithelial (LE) cells. Some punctate lateral staining can also be observed. Staining visible in the stroma (S) is thought to be endogenous IgG recognized by the secondary antibody. This is confirmed by observation of the immunological control NGS (E), in which only stromal staining can be identified. B) Day 4 pc LE cells show a more punctate and faint pattern of apicolateral staining, with little or no lateral staining visible. Stromal staining has also decreased, an observation that is mirrored in the NGS controls (F). C and D) Days 5 and 6 pc, respectively. Mouse uterus shows little or no staining on either the apicolateral or lateral cell surfaces. No stromal staining is visible, as reflected in the NGS controls (G and H). Ap, Apical cell surface; Ba, basal cell surface; L, lumen; LE, luminal epithelium; S, stroma. Bars = 50 µm

As well as showing a marked quantitative decrease, desmoplakin immunofluorescence changed in distribution during the preimplantation period. On Day 1 pc, the distribution was typical of that seen in other simple columnar epithelia, with a bright punctate ring of staining around the apicolateral cell margins and weaker punctate staining along the entire lateral cell borders (Fig. 2A). Staining was absent from the apical and basal cell surfaces. By Day 4, the apicolateral rings had decreased in intensity, and lateral staining was barely detectable (Fig. 2B). On Days 5 and 6, only very weak apicolateral staining was present (Fig. 2, C and D). At the implantation site at early Day 5, desmosomes appeared to be reduced to a similar extent to that seen elsewhere in the luminal epithelium (Fig. 3A).



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  		FIG. 3. Immunolocalization of desmoplakin at the implantation site and in mouse uterine glandular epithelium. Transverse sections of mouse uterine horns stained with monoclonal antibody (mAb) 11-5F (recognizing desmoplakins I and II) are shown. A) Day 6 pc. Section through an implantation site showing very low staining with mAb 11-5F in the luminal epithelium, whereas desmosomal staining between trophoblast cells is visible. B) Day 1 pc. The apicolateral region of the glandular epithelial cells (GE) shows the clear "chicken wire" pattern of desmoplakin immunostaining. Some punctate lateral cell staining can also be observed. Staining visible in the stroma (s) is thought to be endogenous IgG recognized by the secondary antibody (confirmed by observation of the immunological control NGS; data not shown). C) Day 6 pc. GE cells show poor morphology at this stage but bright, diffuse staining in the apicolateral region. Ap, Apical cell surface; Ba, basal cell surface; EC, egg cylinder; EPC, ecoplacental cone; GE, glandular epithelium; LE, luminal epithelium; S, stroma. Bars = 50 µm

Some bright diffuse staining was present in the uterine stroma on Day 1 pc (Fig. 2A). By staining with secondary antibody alone, this was shown to indicate the presence of endogenous mouse IgG (Fig. 2E). This stromal staining decreased progressively during the preimplantation period (Fig. 2F), becoming undetectable on Days 5 and 6 pc (Fig. 2, G and H). Such staining represents a complication of using mouse monoclonal antibodies for detecting antigens in mouse tissue. However, the results also indicate a decrease in the presence of endogenous IgG in the uterine stroma during the preimplantation period. This was confirmed by the decrease, between Days 1 to 2 and later days of pregnancy, in the IgG light-chain band in Western blots stained with the secondary antibody (data not shown).

The uterine glandular epithelium showed a less marked decrease in desmoplakin immunofluorescence during the preimplantation period. On Day 1, staining was similar to that seen in the luminal epithelium (Fig. 3B). By Day 6, the morphology of glands was poor, reflecting their degeneration at implantation; however, bright, diffuse, apically distributed staining was still evident (Fig. 3C).

Double labeling with guinea pig antiserum to desmoplakin together with a monoclonal antibody to cytokeratin 8 showed a similar decrease in desmosomal staining, with restriction to the apicolateral luminal epithelial domain, during the preimplantation period (Fig. 4, A–C), with a parallel decrease in cytokeratin immunofluorescence (Fig. 4, D–F). Examination of desmoglein immunofluorescence again indicated a similar decrease in bound antibody (data not shown).



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  		FIG. 4. Immunolocalization of desmoplakin and intermediate filaments in luminal epithelium. Transverse sections of uterine horns were simultaneously double labeled with guinea pig anti-desmoplakin (A–C) and monoclonal antibody LE-41 to cytokeratin 8 (D–F). The same section is illustrated in the following pairs of micrographs: A and D, B and E, and C and F. A and D) Day 2 pc. Lateral desmosome staining is abundant (A) and associated with strong staining of the cytokeratin network throughout the cell (D). B and E) Day 3 pc. Desmoplakin staining is now predominantly localized to the apicolateral junctional complex, although lateral desmosomes are still evident (B). The cytokeratin staining is much reduced (E). C and F) Day 5 pc. Desmoplakin staining is almost entirely restricted to the apicolateral junctional complex (C), and staining of the cytokeratin network is low throughout the cell, except for moderate staining near the apical and basal cell surfaces (F). Ap, Apical cell surface; Ba, basal cell surface; L, lumen; LE, luminal epithelium; S, stroma. Bars = 20 µm

Confocal Microscopy of Isolated Cell Sheets Confirms Desmoplakin Changes

To provide further support for the changes found by immunoblotting and conventional immunofluorescence, isolated endometrial cell sheets were analyzed by confocal microscopy after staining with monoclonal antibody 11-5F and propidium iodide. Figure 5, G–J, shows immunofluorescence images at the level of the most intense apicolateral staining on Days 1, 3, 5, and 6 pc. Note that the staining becomes progressively less concentrated here as the punctae become more widely spaced. Figure 5, A–F, shows XZ sections of epithelia isolated on successive days of pregnancy. The figure also shows a progressive decrease in the intensity of immunofluorescence and a progressive loss of the subapical lateral staining between Days 1 and 5–6 of pregnancy.



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  		FIG. 5. Confocal analysis of desmoplakin localization in mouse uterine epithelial cells. Sheets of uterine luminal epithelial cells were stained using monoclonal antibody 11-5F (recognizing desmoplakins I & II) and the nuclear stain propidium iodide. Regions of most intense apicolateral staining are illustrated (A–D), as are confocal XZ sections from each of Days 1 to 6 pc (E–J). Confocal settings are optimized for each day of pregnancy. A) Day 1 pc. Desmoplakin immunostaining (green) can be seen in a continuous ring surrounding most cells. (Unstained areas result from the slightly undulating nature of epithelial cell sheets on this day of pregnancy.) Most staining is located in the region apical with respect to the nucleus (red). B) Day 3 pc. Distribution of desmoplakin is similar to that seen on Day 1, although the undulated nature of the cell sheets is less pronounced. C) Day 5 pc. Desmoplakin staining has become significantly more punctate in nature. The cells are more cuboidal at this stage; hence, the apical part of the nucleus is also visible. D) Day 6 pc. By this stage, the desmoplakin staining is highly punctate in nature and the density of the stain reduced. E) Day 1 pc. Desmoplakin immunostaining (green) is concentrated in the apicolateral region, but additional staining extends further down the lateral cell surfaces in regions adjacent to the nucleus (red). F) Day 2 pc. Desmoplakin immunostaining has a similar distribution to that observed at Day 1, although staining in the apicolateral region is somewhat fainter. G and H) Days 3 and 4 pc, respectively. Desmoplakin staining is beginning to become localized to the apicolateral region. I and J) Days 5 and 6 pc, respectively. Desmoplakin immunostaining is now significantly reduced in intensity and mainly located in the region apical with respect to the nucleus. Ap, Apical cell surface. Bars = 25 µm (A–D) and 20 µm (E–J).

Electron Microscopy Shows Fewer Desmosomes in Endometrium on Day 5 than on Day 1 pc

To provide direct, quantitative evidence for loss of desmosomes during the preimplantation period, luminal epithelial cells were examined by electron microscopy. Desmosomes had similar morphology on Days 1 and 5 (Fig. 6). To quantify desmosome expression, the lateral cell membranes were divided onto two compartments: membrane above the nucleus, and membrane level with or below the nucleus (Fig. 7, top). Desmosomes in each compartment were counted on epithelium from Days 1 and 5 pc. One hundred and forty-four lateral cell surfaces between epithelial cells were examined from Day 1 pc, and 114 borders (79%) had at least one desmosome on the membrane apical to the nucleus (Fig. 7, top). Most of these desmosomes were in the apicolateral junctional complex. Lateral membrane adjacent to or below the nucleus showed a desmosome in only 22 instances (15%). For Day 5 pc uterus, 159 epithelial cell borders were examined. Twenty-nine (18%) had desmosomes apical to the nucleus in the apicolateral junctional complex. Only two desmosomes were observed adjacent to or below the nucleus (1%). These data are summarized in Figure 7, bottom. The results confirm that desmosomes are reduced during the preimplantation period, as indicated by immunoblotting and immunofluorescence for desmoplakin.



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  		FIG. 6. Ultrastructural analysis of desmosomal distribution in murine luminal epithelium. Sections through the apical region of murine luminal epithelial cells are illustrated. A) Day 1 pc. Two adjacent epithelial cells are shown. A morphologically distinct desmosome can be clearly seen, along with the attached intermediate filaments. This appearance is typical of most apicolateral regions of adjacent cells at this stage of pregnancy. B) Day 5 pc. Two adjacent epithelial cells are illustrated. In this instance, no desmosomes are visible, although the apical region of the lateral cell borders appears to have a significant region of close membrane apposition. Again, this appearance is the most common for cells at this stage of pregnancy. Bi) Day 5 pc. Appearance of a desmosome between two adjacent epithelial cells. D, Desmosome; E, epithelial cell; L, lumen; MA, region of tight membrane apposition. Bars = 0.3 µm



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  		FIG. 7. Top: Illustration of defined "apical" and "basal" regions of lateral cell membrane. Bottom: Quantitative analysis of desmosomal distribution in murine luminal epithelium. On Day 1 pc, 79% of the cell-cell borders observed had at least one desmosome visible in the apical region of the lateral cell membrane. This compares with only 18% of the cell-cell borders in the similar region on Day 5 pc. In the basal region, 15% of the cell-cell borders observed on Day 1 pc had one or more desmosomes present, compared with 1% on Day 5.

Additionally, little close membrane apposition was found by electron microscopy in the apicolateral junctional region on Day 1 pc (Fig. 6A). By contrast, close membrane apposition appeared much more extensively on Day 5 pc (Fig. 6B). This observation was not quantified.

Desmoplakin Expression Is Regulated at the Level of mRNA

To determine whether desmoplakin downregulation during the preimplantation period is regulated at the mRNA level, desmoplakin mRNA was studied by semiquantitative RT-PCR using ß-actin mRNA as a standard. ß-Actin primers yielded a single RT-PCR product of 344 bp, and desmoplakin primers yielded a single product of 274 bp. These products were sequenced to confirm their identities. The level of ß-actin mRNA expression was relatively consistent throughout the preimplantation period, and more so than that of an alternative control, glyceraldehyde phosphate dehydrogenase. By contrast, desmoplakin mRNA decreased between Days 1 and 4–5 of pregnancy (Fig. 8). These results indicate that desmoplakin expression during this period is regulated at the level of mRNA.



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  		FIG. 8. Semiquantitative RT-PCR analysis of desmoplakin mRNA expression in murine luminal epithelium. The expression of ß-actin RT-PCR product (344 bp) was on all but Day 1 of pregnancy. A low level of expression is observed, because the reaction is optimized for the lowest number of cycles at which a product is visible. In contrast, desmoplakin RT-PCR product was highest on Day 1 pc and decreased steadily until Day 5, when it reaches its lowest level. A slight increase was observed on Day 6 pc

DISCUSSION

Our results show that desmosome expression decreases during the pre- and peri-implantation phases of pregnancy in the luminal epithelium of the murine uterus. This suggests a change in lateral adhesiveness between these cells consistent with facilitation of implantation: the trophectoderm cells of the embryo need to penetrate between the luminal epithelial cells. Interestingly, these changes were not seen in the glandular epithelium, which is continuous with the luminal epithelium. Furthermore, the level of desmoplakin mRNA in the luminal epithelium decreases during this period in parallel with downregulation of desmosome expression. This suggests regulation at the transcriptional level, although changes in the mRNA stability and additional regulation of translation cannot be ruled out.

The fundamental alteration in luminal epithelial cell phenotype during transition from the prereceptive to the receptive uterus (described earlier) are accompanied by several other changes in intercellular junctions and adhesion mechanisms. Thus, tight junction distribution also appears to be regulated in the endometrial epithelium in a species-specific manner. The complexity (human) or both the distribution and complexity (rabbit and rat) of tight junction particle networks changes in the lateral membrane [19, 42, 43]. In rats, the beaded strands seen in freeze fractures of tight junctions extend further down the lateral membrane from the junctional complex and show complex interconnections on Day 5 of pregnancy (the day of implantation) compared with Day 1 [19]. Our own preliminary results suggest that uterine endometrial tight junctions also increase in complexity in the mouse during this period. This suggests that tight junctions may provide a better barrier to transepithelial movement of molecules at the time of implantation. Compromised epithelial tight junction integrity does not prevent implantation in humans [44], and that tight junctions play a role in the structural integrity of the luminal epithelium is equally possible. Considering our results, we suggest that the downregulation of desmosomes accompanied by the change in distribution of tight junctions produces a stable, but more plastic, epithelium, in which the intercellular space can be penetrated more easily by invading trophoblast cells. Furthermore, a similar reduction in desmosomes to that described here is indicated between proliferative and midluteal phase in human endometrial epithelium based on quantitation of desmosomes [45].

Additional adhesive changes occur in the interactions between luminal epithelial cells at the time of implantation. Specific gap junctional proteins are expressed in the implantation chamber epithelium [46]. In the mouse, apical epithelial staining for syndecan was demonstrated on Days 3.5–4.5 of pregnancy. Syndecan is a membrane heparan sulfate proteoglycan that usually is found basolaterally in epithelia, including that of the endometrium [47]. Endocytosis of syndecan at the basolateral luminal epithelium surface is induced by estrogen [48, 49], so estrogen-induced changes in syndecan presumably contribute to relaxation of the typical simple epithelial phenotype. In human endometrial epithelium, {alpha}6-integrin redistributes from basal to both lateral and basal surfaces during the secretory phase of the menstrual cycle [50]. Estrogen stimulates murine uterine E-cadherin mRNA levels [21] but also induces E-cadherin degradation, leading to reduced lateral epithelial expression on Day 4.5 of pregnancy [20]. Cadherins on the lateral membranes have also been reported to relocate to the apical luminal epithelial surface at the time of implantation in the rat [51]. However, in the rabbit, basal E-cadherin expression with concurrent loss of apicolateral expression has been reported at this time [52], perhaps reflecting species differences in hormonal control and different mechanisms of implantation. Loss of E-cadherin from luminal epithelium has been ascribed to a number of possible mechanisms, including protease degradation, dissociation from the cytoskeleton, and increased endocytosis [20]. Such mechanisms may also account for changes in desmosomes.

After trophoblast penetration between the lateral surfaces of the epithelium, the cells are displaced from contact with the underlying stroma, and the epithelial cells adjacent to the embryo undergo apoptosis [4]. Cell death may be facilitated by the reduced cell adhesion, because cell-cell contact may attenuate survival signals. Reduction of desmosomal contacts may be a contributory factor in cell death along with other changes in cell-cell adhesion, particularly the reduction in interaction with basal extracellular matrix. Such changes in desmosomes, however, occur throughout the epithelium, whereas apoptosis is restricted to the cells adjacent to the embryo. Thus, the loss of cell-cell contact caused by trophectoderm penetration and displacement of the epithelium may be the crucial death-inducing signal.

To our knowledge, no systematic studies of adhesive changes in human endometrium have been performed, though a partial epithelial to mesenchymal transition of luminal epithelial cells during pregnancy has been hypothesized [16]. Such a transition would necessarily involve loss of epithelial cell-cell adhesion mechanisms. Two human endometrial carcinoma cell lines, HEC-1-A and RL-95, appear to model the phenotypic changes of luminal cells in the mouse. Thus, HEC-1-A cells exhibit a polarized phenotype, whereas RL-95 cells are rounded and unpolarized. Furthermore, RL-95 cells are adhesive to competent blastocysts, whereas HEC-1-A cells are not [15]. Interestingly, HEC-1-A cells have typical desmosomes as well as a normal polarized distribution of tight junction proteins, E-cadherin and catenins, whereas RL-95 cells have abnormally distributed E-cadherin and catenins and only a few desmosomes. These differences in desmosomes between HEC-1-A and RL-95 cells thus resemble the differences between mouse luminal epithelial cells on Days 1–2 and 4.5–6 of pregnancy, respectively. This suggests that such phenotypic changes may be instrumental in the control of luminal epithelial receptivity.

In the light of these findings, we are now investigating whether desmosome protein/glycoprotein synthesis or assembly is under the control of ovarian steroids, and whether a relationship exists between decreased desmosome density and redistribution and increased tight junction complexity in the mouse.

ACKNOWLEDGMENTS

We thank Emma Millican for facilitating the initiation of this project.

FOOTNOTES

First decision: 30 September 1999.

1 Supported by a Wellcome Trust grant to S.J.K., G.W.I., and D.R.G. Back

2 Correspondence: Susan J. Kimber, School of Biological Sciences, University of Manchester, 3.239 Stopford Building, Oxford Rd., Manchester M13 9PT, UK. FAX: 161 275 3915; skimber{at}fs1.scg.man.ac.uk Back

3 Current address: Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107. Back

4 Current address: CRC Structural Cell Biology Group, Paterson Institute for Cancer Research, Christie Hopital, Manchester M20 9BX, UK. Back

Accepted: July 12, 2000.

Received: August 10, 1999.

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