Endometrial Cell Death During Early Pregnancy in the ... - Springer Link

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Wendy Tassell, Michael Slater∗, Julian A. Barden & Christopher R. Murphy. Institute for Biomedical Research and Department of Anatomy and Histology, The ...
The Histochemical Journal 32: 373–379, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

Endometrial cell death during early pregnancy in the rat Wendy Tassell, Michael Slater∗ , Julian A. Barden & Christopher R. Murphy Institute for Biomedical Research and Department of Anatomy and Histology, The University of Sydney, NSW 2006, Australia ∗

Author for correspondence

Received 8 December 1999 and in revised form 29 March 2000

Summary In a study of early pregnancy in the rat, a high proportion of morphologically apoptotic, TUNEL and P2X7 positive cells were found to be present in the luminal epithelium and stroma prior to implantation. At the time of implantation on Day 6, apoptosis as measured by these indicators was reduced up to 4-fold in the non-implantation uterine epithelium but was markedly increased adjacent to the implanting blastocyst. It is proposed that apoptotic cell death is an important regulatory factor involved in uterine remodelling prior to and during implantation.

Introduction Apoptosis is a genetically-controlled process of selective cell deletion that is characterized by a series of morphological, biochemical and molecular changes prior to actual cell death (Arends & Wyllie 1991). These changes include the disruption of cellular metabolism (Sen 1992, Kerr et al. 1995), condensation and margination of chromatin, degradation of the nucleus (Wiley et al. 1980), destruction of functional ion-gated channels (Wiley et al. 1980) and degradation of DNA into oligonucleosomal-sized fragments. The latter process does not occur in non-specific cellular necrosis. Other notable characteristics of apoptosis include the production of apoptotic by-products, the formation of nuclear and cytoplasmic apoptotic bodies, cytoplasmic vacuoles and membrane ‘blebbing’ (Wiley et al. 1980, Sen 1992). In the rat, the non-pregnant endometrium acts as a barrier to implantation (Murphy 1998). Complex molecular and morphological changes to the endometrium during early pregnancy are necessary for the implantation of a fertilized ovum into the wall of the uterus and subsequent formation of a definitive placenta (Enders & Schlafke 1975, Weitlauf 1994). The purpose of implantation in the rat, as in humans, is to establish contact between the blastocyst and the maternal blood, thus providing the blastocyst with nutrients for growth and development (Welsh & Enders 1991, Welsh 1993). In the course of early pregnancy, the sequence of apoptotic events and ultrastructural appearance of the uterine cells vary in different regions of the uterus. This suggests that a complex series of apoptotic events contributes to the preparation of the endometrium for implantation and pregnancy (Welsh 1993). Uterine stromal cells displaying apoptotic features have been localized both in pregnant tissue and during the oestrous cycle. This finding suggests that apoptotic processes not only allow implantation to proceed, but act as a

proliferation-regulating mechanism throughout the reproductive cycle (Welsh 1993, Sato et al. 1997). The events leading up to implantation of the rat blastocyst occur during the first 6 days of pregnancy. Day 1 of pregnancy, defined as the day of mating, is considered representative of oestrous (Murphy 1993). Day 3 represents the peri-implantation (intermediate) period. Apposition of the blastocyst against the uterine wall occurs on Day 5 of pregnancy. Implantation, defined as irreversible adhesion of the blastocyst to the luminal epithelium, is completed on Day 6. On Day 7, penetration of the blastocyst through the luminal epithelium to the maternal stroma is completed by displacement penetration. Implantation is characterized by a localized interaction between trophoblast and luminal epithelial cells (Murphy 1995). During this process, the basal lamina of the latter is destroyed and removed by phagocytic macrophages from the maternal blood supply (Enders & Schlafke 1967, Weitlauf 1994). The purinergic (P2X1–7 ) receptors constitute a group of newly defined fast-response, membrane-bound, ligand-gated, calcium-permeable, cation-selective channels that are activated by extracellular ATP from nerve terminals or a local tissue source (Brake et al. 1994, Valera et al. 1994). They are predominately permeable to calcium ions but also admit other cations, such as potassium and sodium (Filipovic et al. 1998) and are found in a wide variety of mammalian tissues (Abbracchio & Burnstock 1998). Binding of ATP to P2X receptors on the cell membrane triggers an influx of cytosolic Ca2+ (Gong et al. 1995) that is characteristic of apoptotic cells of all types (Kyprianou et al. 1998). Following calcium ion influx, the apoptotic process continues. Fragmentation of the genomic DNA (nuclear or mitochondrial) occurs by activation of Ca2+ /Mg2+ -dependent endonucleases, which selectively hydrolyse DNA at sites located between nucleosomal units. This DNA cleavage precipitates cell death

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(Kyprianou et al. 1998, Fernando et al. 1999). Ca2+ influx through the P2X7 receptor also activates Ca2+ -dependent phospholipase D (Gargett et al. 1996). The expression of P2X receptors leads to the death of many different cell types either through necrosis or apoptosis (di Virgilio et al. 1998). The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions has been suggested (di Virgilio et al. 1998). The main P2X subtype associated with apoptotic change is P2X7 which has been shown to up-regulate in association with the process of apoptosis in a variety of cell types (Pizzo et al. 1992, Ferrari et al. 1997, Wiley et al. 1998, Freedman et al. 1999). Ion flow through the P2X7 channel has been shown to stimulate both Ca2+ dependent and -independent phospholipase D (Gargett et al. 1996, Gu et al. 1998). After a delay of several hours, apoptotic cell death is seen in these cells (Pizzo et al. 1991, Zheng et al. 1991, Fernando et al. 1999). P2X7 activation also stimulates the activity of intracellular caspases immediately before ATP-induced apoptosis occurs (Ferrari et al. 1999). In view of the role of cell death in implantation, our objective was to study the relationship between P2X7 expression and apoptosis in the luminal epithelium in early pregnancy.

Worthington et al. 1999b). The serum was tested with an ELISA assay for specific antibodies against the peptide epitope (Hansen et al. 1998, Dutton et al. 1999, Worthington et al. 1999b). The antibody titre, defined as the reciprocal of the serum dilution resulting in an absorbance of 1.0 above background in the ELISA assay, fell in the range 75,000 ± 4000 compared with 225 ± 25 for pre-immune samples. The P2X7 antibody, like all the other P2X subtype-specific antibodies produced, bound selectively and specifically as shown by Western blots in a range of tissues (Worthington et al. 1999a). Specificity of the antibodies had previously been demonstrated using immunolocalization (Dutton et al. 1999, Hansen et al. 1999). The P2X7 antiserum was raised against the non-homologous extracellular epitope conjugated to a diphtheria toxin domain. The epitope was chosen in part to ensure no cross-reaction with any of the other P2X subtypes. Individual antibody specificity was checked using a specific rat P2X cDNA transfected into Xenopus oocytes. Specific P2X antibody was added to transfected cells in the presence and absence of either a 10 µM concentration of the specific P2X epitope or a 10 µM concentration of a non-specific P2X epitope. Only the specific antibody reacted with the specific epitope (Dutton et al. 1999).

Materials and methods

Confocal microscopy

Experimental animals

Using a Reichert Jung 2800 Frigocut cryotome, 16 µm sections were collected at −21 ◦ C. The sections were immediately transferred to Coplin jars containing 4% paraformaldehyde for 20 min. Sections were then immersed in a permeabilizing solution consisting of 2% normal horse serum (NHS), 1% bovine serum albumin (BSA), 0.1% dimethylsulphoxide, 0.1% Triton X-100 in phosphatebuffered saline (PBS) for 30 min, followed by three 10 min PBS washes. This was followed by immersion in 20% NHS in PBS for 1 h to block non-specific binding sites. Incubation of sections with 1 : 100 dilutions of rabbit P2X7 antibody in PBS for 1 h at 37 ◦ C followed. The tissue was then rinsed using four 10 min PBS washes. Sections were then incubated in a 1 : 200 dilution of secondary fluorescent antibodies (antirabbit Cy2, Jackson Immunoresearch) in PBS for 2 h. They were then washed four times for 10 min in PBS, mounted in a gelatin/glycerol mix, coverslipped, sealed and examined in a Leica TCS NT laser confocal microscope.

Female virgin Wistar rats used for these experiments were kept on a light cycle of 12 h light and 12 h dark. Randomly chosen male Wistar rats were placed overnight in cages containing females displaying vaginal smears appropriate for mating. The following morning, the male rats were removed from the cages and females displaying spermatazoa in their vaginal smears were designated as being at Day 1 of pregnancy. Twenty rats were used in each experimental group of Days 1, 3, or 6 of pregnancy. Animals were sacrificed at 9 am on Days 1, 3, 6 and 7 of pregnancy respectively. Pregnant females were anaesthetized with 0.6 ml sodium pentabarbitone (Nembutal) and their uterine horns surgically excised. Following this procedure, they were sacrificed by exposing the diaphragm to air. The uterine horns were excised, coated in OCT, quenched in nitrogen-chilled isopentane and then immersed in liquid nitrogen for storage. Ethical approval was previously granted. At the time of uterine horn excision, a section of kidney was simultaneously removed. Fixation and processing of the kidney tissue was performed according to the histochemical protocol outlined below. This tissue was used as a positive control for both the TUNEL and P2X receptor assays. Antibody production A suitable epitope from the extracellular domain of rat P2X7 was synthesized using standard t-BOC chemistry (Barden et al. 1997) on an ABI synthesizer (Hansen et al. 1997,

Labelling intensity quantitation The method of Slater (Slater 1999, Slater & Murphy 1999) was used for image quantitation. In short, photographs were taken using the 25× objective of a Leica ‘Diaplan’ microscope with Ilford Pan F film used at 50 ASA. The microscope settings remained constant for each exposure. A contact sheet (Agfa multigrade) of suitable density was prepared from the negatives. This ensured all images experienced the same photographic variables. Each contact sheet was scanned using an Agfa Duoscan scanner. The image file was then transferred

Endometrial cell death during early pregnancy to NIH Image 1.6, resulting in a computer image 10 times the size of the contact print. Using the freehand selection tool, the labelled epithelium on each section was selected and analysed for image density and standard deviation. Data were analysed using a two-tailed modified Wilcoxon test. Reproducibility was tested by outlining a single defined area of reaction product ten times, resulting in a variation coefficient of 1.0%.

375 binding of biotinylated-deoxyuridine (b-dUTP), uterine sections taken from Days 1, 3 and 6 were incubated in media free from TdT or b-dUTP, resulting in a significant loss of label. Previous studies have shown TUNEL of apoptotic cells to be successful in the rat kidney. This tissue was, therefore, chosen as the positive control tissue to detect TUNEL-positive cells (Zhang & Xiao 1998). Methylene blue staining

TUNEL labelling TUNEL (terminal deoxynucleotidyl transferase [TdT]mediated dUTP nick end labelling) was performed as follows. Frozen sections (8 µm) were collected at −21 ◦ C using a Reichert Jung 2800 Frigocut cryotome, left on gelatin chrome alum-coated glass slides to air dry for 1 h and fixed in 4% paraformaldehyde for 12 h. After fixation, the slides were immersed in 70% ethanol and washed twice in distilled water. They were then equilibrated with 1× TdT buffer 0.03 M Tris base (Sigma) with added 0.001 M cobalt chloride (Sigma) and 0.14 M sodium-cacodylate (Alltec) at pH 7.2 for 10 min. The reaction mix was prepared by adding the following reagents added to eppendorf tubes: 1120 µl water, 125 µl 10× TdT buffer, 5 µl biotin-16dUTP (Boehringer Mannheim, Germany), and 1.5 µl terminal transferase enzyme (Boehringer Mannheim). Sections were incubated in this mix at 37 ◦ C for 60 min. The reaction was terminated by transferring the slides into SSC (0.03 M sodium citrate, 0.4 M sodium chloride, at pH 7.4) for 15 min, followed by a 10 min wash in 1% BSA–PBS to prevent non-specific binding. Sections were then incubated with horseradish peroxidase (HRP)-conjugated avidin (Zymed, USA). Avidin– HRP sections were incubated in a 1 : 500 dilution with PBS for 60 min at 37 ◦ C. The avidin–HRP complex was then visualized in 3,30 -diaminobenzidine (DAB) for 10 min as follows: the slides were transferred to Coplin jars containing 20 ml Tris (0.05 M, pH 7.6), 20 mg DAB, 200 µl sodium azide, and 11.2 µl H2 O2 (30%) to start the reaction. After this procedure, the slides were rinsed in water for 5 min, air-dried, and mounted in DPX.

Sections were stained for 15 s in a solution of 100 mg methylene blue in 100 ml of distilled water. This was used for assessment of epithelial cell proliferation. Electron microscopy Immediately after excision, uterine horns from Day 3 of pregnancy were pre-fixed in 2.5% glutaraldehyde for 15 min. They were then cut into 4–5 mm pieces, fixed for a further 10 min, cut into blocks 1–2 mm in length, followed by fixation for a further 40–50 min. Following fixation, the tissue was rinsed in 0.1 M phosphate buffer and post-fixed in 2% osmium tetroxide (in 0.1 M phosphate buffer, pH 7.2), for 1 h. Tissue was then dehydrated through graded alcohols and embedded in Spurr’s resin. Sections (100 nm) were cut on a Reichart-Jung Ultracut Microtome for transmission electron microscopy, collected on copper grids and stained with uranyl acetate and lead citrate. Positive electron micrographs were prepared from negatives taken with a Phillips 400 CX electron microscope. Results Using brightfield microscopy, the uterine epithelium was continuous and relatively thick (Figure 1a). By Day 3, the uterine epithelium was of similar thickness (Figure 1b) but on Day 6 it was considerably reduced (Figure 1c). These observations were consistent with our previous experience with this tissue. P2X7 distribution

Controls P2X7 antibodies were affinity-purified, tested on positive and negative tissue and the working dilutions determined. Negative controls in which both pre-immune serum and PBS was substituted for the primary antibody, did not label. These sections were completely blank and were, therefore, not photographed. Sections of pregnant rat endometrium were reacted with and without TdT (as a control) and assessed during early pregnancy to define changes in the distribution of apoptotic cells in the endometrium. Non-apoptotic cells remained cuboidal in shape and maintained a regular morphology. TUNEL cells displayed the characteristic pyknotic nuclei and irregular morphology of apoptotic cells. To detect non-specific

The pattern of P2X7 distribution changed over time in tissue taken from Days 1, 3, and 6 of pregnancy (Figure 1). The apical epithelium (Figure 1a, inset-A), basement membrane (Figure 1a, inset-B) and lateral plasma membranes between each cell, were examined using several procedures including immunolabelling with an antibody to the rat P2X7 subtype, transmission electron microscopy, as well as analysis by immuno-confocal microscopy and the results quantified. Day 1 tissue exhibited an abundance of P2X7 labelling in the apical epithelium (Figure 1d, A), lateral plasma membranes (Figure 1d, L) and basement membrane (Figure 1d, B). Day 3 tissue showed a similar labelling distribution and intensity (Figure 1e). There was no significant difference between the labelling intensity of Day 1 and Day 3 tissue.

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Figure 1. (a) Methylene blue-stained section of Day 1 of pregnancy (oestrous). Inset – outline of the area of the uterine epithelium depicted in d, e and f, incorporating the apical (A) and basal (B) compartments, stroma (S). (b) Methylene blue-stained section of Day 3 of pregnancy. (c) Methylene blue-stained section of Day 6 of pregnancy, the time of implantation. a–c, bar = 40 µm. (d) Immuno-confocal microscopy for the purinergic receptor P2X7 in the non-implantation epithelium on Day 1 of pregnancy. Labelling is seen in the apical (A), lateral (L) and basal (B) compartments. The area shown is outlined by the inset in a. (e) Immuno-confocal microscopy for P2X7 on Day 3 of pregnancy. Labelling is not significantly reduced from that of the intensity of the Day 1 label. (f) Immuno-confocal microscopy for P2X7 on Day 6 of pregnancy, the time of implantation. Labelling is (significantly) reduced, by 3.9-fold (p < 0.0089). d–f, bar = 6 µm. Table 1. Statistical mean and standard error for TUNEL cell counts in non-implantation tissue. Group n Central mean (lumen) SE Peripheral mean (stromal) SE

1

3

20 148.6

20 179.1

6 20 67.6

7 20 14

30.3 129.7

23.4 151.2

10.2 79.8

3.9 20.8

16

11.2

5

3.1

SE = Standard error, n = number of groups in parameter series.

In non-implantation tissue on Day 6, the P2X7 signal was reduced 3.9-fold (p < 0.0089) compared to Day 1 tissue. The lateral plasma membrane label also disappeared at this time (Figure 1f), (Table 1). TUNEL assay Sections of tissue were reacted with and without TdT (for control tests) and assessed over the days of early pregnancy to define temporal changes in the distribution of apoptotic cells in the endometrium. Non-apoptotic cells remained cuboidal in shape and maintained a regular morphology, whilst labelled

cells displayed the characteristic pyknotic nuclei and irregular morphology of apoptotic cells. In peroxidase-stained sections, the proportion of TUNEL-stained cells corresponded precisely to the proportion of pyknotic nuclei observed. Temporal changes in the pattern of distribution of TUNELpositive cells were observed microscopically in tissue taken from Days 1, 3, 6 and 7 of pregnancy. Day 1 (Oestrous) Peroxidase labelling of TUNEL-positive cells appeared to be both more intense and more sensitive than the complementary fluorescence labelling of these cells. The greatest intensity of peroxidase-mediated signalling was observed in tissue taken from Days 1 and 3. Day 1 tissue exhibited an abundance of peroxidase-labelled TUNEL-positive cells in the luminal epithelium. To a lesser extent, peroxidase-labelled TUNELpositive cells were also evident in the stroma of Day 1 tissue. The use of FITC-conjugated avidin to visualize the location of b-dUTP was used to confirm results obtained with avidin– HRP. Again, TUNEL-positive cells were visualized in the luminal epithelium. Labelled stromal cells surrounded the (stromal) blood vessels. FITC-labelled cells were also stained with Hoechst 33258 to distinguish between pyknotic and normal nuclei.

Endometrial cell death during early pregnancy Day 3 (Peri-implantation) With respect to the proportion of TUNEL-positive cells, Day 3 tissue did not differ significantly from Day 1 tissue. The use of FITC-conjugated avidin to detect b-dUTP confirmed the presence of cells labelled with peroxidase. TUNELpositive cells were also visualized in the luminal epithelium. Days 6 and 7 (non-implantation sites) Tissue taken from non-implantation sites on Days 6 and 7 of pregnancy failed to yield significant numbers of TUNELpositive cells. Days 6 and 7 (implantation sites) In the implantation chamber on Day 6 however, a strong TUNEL signal was recorded in both the uterine epithelium and the immediately adjacent stroma (Figure 2). TUNEL positive cells (Figure 2, T+) surrounded the implantation site to a distance of approximately 100 µm. The cells of the blastocyst within the implantation chamber were not TUNEL positive (Figure 2, IC). Control sections from Days 1, 3, and 6 were negative. Controls To detect non-specific binding of b-dUTP, sections taken from Days 1, 3, 6 and 7 were incubated in media free from TdT or b-dUTP. When both exogenous terminal transferase or b-dUTP were excluded from the incubation mixture, there was a significant loss in intensity of both peroxidase- and fluorescence-mediated colour signals. Substantial numbers of peroxidase-labelled TUNEL-positive cells were observed in the positive control (rat kidney).

377 Table 2. Results of the Tukey–Kramer post hoc tests for the TUNEL series at the 5% level of significance. The groups that are not significantly different to each other are in brackets (Days 1 and 3). Parameter

Results

Central (luminal) Peripheral (stromal)

(3; 1) 6; 7 (3; 1) 6; 7

Table 3. Results of alternate Welch t-test on combined means for P2X7 labelling. Day 1

Day 3

Day 6

431 ± 70

370 ± 78

110 ± 22

Units are arbitrary NIH Image 1.62 units. Days 1 and 3 data were not significantly different. The Day 6 P2X7 label was reduced 3.9-fold (p < 0.0089) compared to Day 1 tissue.

Cell counts and statistical analysis Data are presented as mean, standard deviation and standard error. Analyses of equal variance were not necessary to perform a one-way analysis of variance (ANOVA), provided a constant number of scores appeared in all groups tested. The two parameters measured for statistical analyses were luminal (central) and stromal (peripheral) cell counts. Table 2 shows the descriptive statistical mean and standard error for the two parameters in the series. Table 3 shows the results from the Tukey–Kramer post hoc tests. The results of the ANOVA between groups for the two parameters indicate that the following days are statistically significant at the 5% level of significance (p < 0.0001) in each of the ensuing cases: 1 and 6; 1 and 7; 3 and 6; and 3 and 7 (Tables 2 and 3). Observations that the cells described in this study were apoptotic were confirmed by electron microscopy. Figure 3 shows a cell within the basement membrane in Day 1 tissue. This cell displays the ultrastructural characteristics of apoptosis such as cell shrinkage, margination of nuclear chromatin, pinopods (Figure 3, arrow) and membrane blebbing. An adjacent (normal) nucleus is included for comparison (Figure 3, N). Figure 4 shows additional characteristics of apoptosis such as degradation of the nucleus, condensation (Figure 4, arrowhead) and cytoplasmic apoptotic bodies (Figure 4, short arrows). The size of the P2X7 receptor clusters was approximately 150 nm, as measured by immunoelectron microscopy (not shown). Discussion

Figure 2. TUNEL-positive cells (T+) in the epithelium and stroma of an implantation chamber (IC) were observed to a distance of approximately 100 µm. The blastocyst within the implantation chamber is not significantly labelled. Bar = 100 µm.

In this study there was a significant decrease in the number of endometrial cells displaying apoptotic features in the nonimplantation epithelium but an increase immediately adjacent to the implantation chamber, at the time of implantation on Day 6. During implantation, the shape and volume of the uterus alters dramatically during its change from a fluid-filled cavity

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Figure 3. Transmission electron microscopy of apoptotic cells within the basement membrane of Day 1 tissue. Apoptotic features include cell shrinkage, margination of nuclear chromatin, pinopods (arrow) and membrane blebbing. Uranyl acetate and lead staining. Bar = 3 µm.

Figure 4. Transmission electron microscopy of apoptotic cells within the basement membrane of Day 1 tissue showing additional characteristics of apoptosis – degradation of the nucleus, condensation (arrowhead) and cytoplasmic apoptotic bodies (short arrows). Uranyl acetate and lead staining. Bar = 2 µm.

for transporting sperm to a vessel for blastocyst attachment and implantation (Finn & Publicover 1981). This study confirms and extends results from several studies that noted apoptotic-like features in endometrial and myometrial cells at various stages of the oestrous cycle and during early pregnancy (Finn & Publicover 1981, Murdoch 1995). Cell death in the implantation chamber is a well-documented phenomenon (Parr et al. 1987, Abrahamsohn & Zorn 1993, Welsh 1993) and is a necessary procedure for the removal of epithelial cells prior to implantation. In the current study, the endometrium displayed a high degree of apoptotic activity prior to implantation on Day 6. This may be a response to the oestrogen-induced mitotic surge that occurs during

W. Tassell et al. late proestrous (Psychoyos 1973). This study also established that apoptosis occurs in the uterine epithelium during the tissue remodelling phase prior to implantation. As pregnancy proceeds, the endometrium is required to supply the developing conceptus with mechanical support, protection and nutrition. This necessitates a shift from apoptotic cell death to an increase in protein synthesis and proliferation from the time of implantation (Weitlauf 1994). Distribution of the P2X7 receptor was also studied. P2X activation occurs when ATP, possibly produced by the uterine epithelium itself, binds to these receptors. A high proportion of P2X7 immunoreactivity was found in tissue taken from Days 1 and 3, followed by a sudden decrease from the time of implantation on Day 6 of pregnancy. This finding correlated with the TUNEL and electron microscopy results. Several previous electron microscopy studies have shown that many endometrial cells display features characteristic of apoptosis throughout the oestrous cycle and pregnancy in the rat (Sato et al. 1997). The majority of these studies have focussed on apoptosis as a prominent mechanism for the removal of maternal tissues, primarily luminal epithelial cells, during formation of the implantation chamber (Schlafke et al. 1985, Pollard et al. 1987, Welsh & Enders 1991, Abrahamsohn & Zorn 1993). In the current study we used electron microscopy to confirm the presence of apoptotic-like morphological features since TUNEL does not differentiate between apoptosis and necrosis. Studies have suggested that the P2X7 receptor plays an important role in the physiological turnover of continuously regenerating cells (Groschel-Stewart et al. 1999a) and that P2X7 receptor immunoreactivity is seen in the membranes of enterocytes and goblet cells in the rat duodenal villus, where cells are exfoliated into the lumen. This observation is consistent with earlier findings that P2X7 is involved in apoptotic events (Groschel-Stewart et al. 1999b). In summary, using several different approaches to examine apoptosis, the results of the current study suggest that apoptosis is an important part of the process of uterine remodelling prior to implantation of the blastocyst. It further demonstrates that apoptosis in the uterine epithelium decreases significantly at the time of implantation except in the uterine epithelium and stroma in the immediate vicinity of the implantation chamber.

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