Acknowledgements. We wish to acknowledge the collaboration by Professor Christian ... Dhein, J., Walczak, H., Bäumler, C. et al. (1995) Autocrine T-cell suicide.
Human Reproduction vol.13 no.11 pp.3177–3189, 1998
The receptive endometrium is characterized by apoptosis in the glands
Ulrike von Rango1, Irmgard Classen-Linke, Claudia A.Krusche and Henning M.Beier Department of Anatomy and Reproductive Biology, Medical Faculty, RWTH University of Aachen, Wendlingweg 2, D-52057 Aachen, Germany 1To
whom correspondence should be addressed
Apoptosis in the human endometrium up to now has been detected during the mid to late luteal phase and therefore connected to the onset of the menstrual shedding. However, there is increasing evidence that regulated apoptosis may be important during decidualization and implantation. To investigate a possible role for apoptosis in the human endometrium and its regulation, we correlated the immunolocalization of the apoptosis regulatory protein bcl-2 and the proliferation marker Ki67 to the in-situ nuclear DNA fragmentation — a key feature of apoptosis — detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) method during the menstrual cycle. Whereas proliferation and bcl-2-expression were predominantly detected in the glandular compartment during the proliferative phase, only single apoptotic cells could be shown during this period. During the transformation of the endometrium (days 15–19) proliferation and bcl-2 expression decreased markedly and there was no sign of apoptosis. At the beginning of the implantation window (days 19–20) we could detect the first signs of apoptosis in the glandular epithelia in the basalis, which extended to the functionalis during the luteal phase. Proliferation and bcl-2 expression are limited to the stromal compartment — comprising the large granular lymphocytes — during this time, and extend in parallel with apoptosis from the basal to the functional layers. Apoptosis therefore may be related to the loss of the protective effect of bcl-2 and may have significance for the establishment of an endometrium adequately prepared for successful implantation. Key words: apoptosis/bcl-2/endometrium/implantation/proliferation
Introduction Apoptosis is a process by which single cells undergo programmed cell death and are deleted from the tissue. The first description of this phenomenon was published 100 years ago, when Weismann (1863) described cellular loss in the embryonic development of Diptera and called it histolysis. The apoptotic programme is characterized by cell shrinkage, reorganization of the nucleus, membrane blebbing and finally fragmentation © European Society of Human Reproduction and Embryology
of the nucleus into apoptotic bodies (Kerr et al., 1972; Wyllie 1980). These apoptotic bodies are phagocytosed by neighbouring cells or macrophages. In a single-layered epithelium, they are shed into the lumen (Kerr et al., 1994). Apoptosis occurs not only in embryonic development, but plays a significant role in the maintenance of cellular balance in differentiated tissues which show high turnover such as the intestine (Merrit et al., 1994; Krammer, 1996), in the involution of the thymus, and the mammary gland after weaning (Cohen and Duke 1994; Quarrie et al., 1995; Lund et al., 1996), and in the regulation of the immune system (Dhein et al., 1995; Nagata and Golstein 1995; Mitra et al., 1996). Hormonally regulated dynamic tissue reorganization, for example in the prostate and in the ovary, also seems to involve apoptotic cell death (Tilly et al., 1991; Colombel and Buttyan, 1995). The late key event of apoptosis is the internucleosomal fragmentation of the DNA, which was first detected in apoptotic thymocytes (Wyllie, 1980) and demonstrated by the DNAladdering in agarose gel electrophoresis. Since fragmentation of the DNA in apoptotic cells is the common feature of all apoptotic programs (Bortner et al., 1995), it is the best way to prove apoptosis in intact fixed tissue sections. We performed the detection of apoptosis within histological sections using the terminal deoxynucleotidyl transferase-mediated dUTP nickend-labelling (TUNEL) method, according to Gavrieli et al. (1992) with slight modifications. The regulation of apoptosis is very complex and involves the expression of various genes which display either positive or negative regulatory effects. One of the most accepted inhibitors of apoptosis is the proto-oncogene bcl-2 (Tsujimoto et al., 1985), which is localized in the inner and outer mitochondrial membrane (Hockenbery et al., 1991; Krajewski et al., 1993). Today, bcl-2 is known as the prototype of a family of genes regulating the susceptibility of a cell to commit apoptosis in response to various stimuli and by various pathways (Oltvai et al., 1993; Reed, 1994; Wyllie 1994; Nunez and Clarke, 1994). Immunoreactivity for bcl-2 protein has been identified in the human endometrium (Gompel et al., 1994; Otsuki et al., 1994, Koh et al., 1995). Recently it was shown that the onset of decidualization was associated with increased expression of bax and decreased expression of bcl2 (Akcali et al., 1996), which could induce apoptosis in the glands. Because of its translational and post-translational regulation (Chleq-Deschamps et al., 1993; Reed, 1996) we decided to compare the expression of bcl-2 protein with the detection of the apoptotic cells in the human endometrium. The relationship between steroid hormone concentrations and endometrial apoptosis is already proven in several animal models. In the hamster (Sandow et al., 1979) and the mouse 3177
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(Pollard et al., 1987), apoptosis of glandular epithelial cells was shown to depend on oestradiol. In the rabbit, decreasing progesterone concentrations or progesterone antagonists, such as RU486 or Onapristone, are responsible for the apoptotic degeneration of glandular epithelia (Nawaz et al., 1987; Hegele-Hartung et al., 1992; Rotello et al., 1992). The appearance and regulation of apoptosis in the human endometrium have until now not been examined. Earlier morphological studies (Hopwood and Levison, 1976) and recently performed exemplary assessment of fragmented DNA (Tabibzadeh et al., 1994) showed the first signs of apoptosis during the mid to late luteal phase. Consequently this phenomenon was hypothetically associated with the onset of menstruation. Taking into account that menstruation comprises shedding of the greatest part of the mucosa, apoptosis — a process of single cells – may not account for its regulation. Consequently, we investigated apoptosis in relation to endometrial proliferation and protein expression of bcl-2 for a better understanding of the onset, localization and the potential significance of endometrial apoptosis for implantation and early pregnancy.
Materials and methods Uterine tissues Human endometrial tissues were obtained from normal cycling women undergoing hysterectomy for myoma, descensus uteri or hypermenorrhoea. The use of these specimens was approved by the Ethics Committee of the Medical Faculty of the University of Aachen. At the time of hysterectomy serum concentrations of 17β-oestradiol, progesterone and luteinizing hormone (LH) were measured. For histological dating (according to Noyes et al., 1950 and DallenbachHellwig and Poulsen, 1985) tissue sections were stained with haematoxylin and eosin. If there was any disagreement between the hormone concentrations, the histological dating and the announced day of the last menstrual bleeding, the specimen was excluded from our study. Women who had received any exogenous hormones within the last 3 months prior to hysterectomy or had used an intrauterine device were also excluded. A total of 59 specimens were obtained ranging with ages between 29 and 52 years. The endometria were dated to the proliferative (days 4–14; n 5 26), early secretory (days 15–19; n 5 10), mid secretory (days 20–25; n 5 18) and late sectretory (or premenstrual) phase (day 26–28; n 5 5) of the menstrual cycle. Specimens obtained from the corpus of the uterus contained myometrium and endometrium. They were either cryoconserved using liquid nitrogen and stored at –40°C until use, or fixed with buffered 4% formalin overnight and embedded in paraffin using nuclease-free solutions to avoid false positive DNA fragmentation signals when using the TUNEL method to detect apoptosis in the histological sections. Pretreatment of the sections for immunohistochemistry The 4–6 µm thick sections were mounted on 3-amino-propyl-triethoxy-silane (APES)-coated glass slides, deparaffinized with xylol and rehydrated. For antigen retrieval the sections were heated four times (each 5 min) in citric acid buffer (1.8 mM citric acid, 8.2 mM tri-sodium-citrate-dihydrate, pH 6.0) using a microwave oven (600 W). After they were allowed to cool down for about 30 min, the sections were placed in 0.3% H2O2/methanol for 30 min to block endogenous
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peroxidase activity. After a washing in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA), the sections were blocked for non-specific binding of the secondary antibody by an incubation for 20 min with normal rabbit serum (1:20) in the case of Mib1 or normal goat serum (using HistostainTM Kit; Zymed, San Francisco, CA, USA) in the case of bcl-2. Immunolocalization of Ki-67 Tissue sections were incubated with the monoclonal antibody Mib1 (Dianova, Hamburg, Germany) diluted 1:40 with PBS/0.1% BSA for 1 h at room temperature (RT). After a wash with PBS/0.1% BSA and the incubation with the biotinylated secondary antibody (goat anti-mouse 1:300; Dianova) in PBS/0.1% BSA for 30 min at RT, immunohistochemical detection was performed using the biotin– streptavidin–peroxidase method. The sections were placed in streptavidin–peroxidase conjugate (1:300) for 30 min at RT. Subsequently antibody-binding was detected by immersing the sections for 5– 10 min in a solution of the chromogen amino-ethyl-carbazole (AEC; Genzyme, Cambridge, MA, USA). The slides were washed with PBS and deionized H2O and embedded with glycerol gelatine without previous counterstaining. Negative controls were performed by using normal rabbit serum at a 1:20 dilution instead of the primary antibody. Immunolocalization of bcl-2 Section were incubated with the mouse monoclonal anti-human bcl2 antibody (Dako, Hamburg, Germany) in a dilution of 1:4 in PBS/ 0.1% BSA for 90 min at 37°C. After an additional wash with PBS/ 0.1% BSA the sections were incubated with the biotinylated secondary antibody (goat anti-mouse; HistostainTM Kit). The antibody binding was detected by the biotin–strepavidin–peroxidase method using a streptavidin–peroxidase solution (HistostainTM Kit) for 20 min at RT twice to intensify the immunohistochemical signal. The further treatment of the sections was performed as described for the Ki-67 immunohistochemistry. Detection of the apoptotic cell death in situ using the TUNEL method Fragmentation of the DNA in the nucleus is one of the first morphological changes of the apoptotic process and can be detected in histological sections using the TUNEL method according to Gavrieli et al. (1992). The method was adapted to the conditions required for the use in the human endometrium. The 4–6 µm thick sections were mounted on APES-coated glass slides, deparaffinized with xylol and rehydrated. Endogenous peroxidase activity was blocked by incubating the sections for 30 min at RT in 0.3% H2O2/methanol. Sections were rinsed in PBS and heated for 1 min in citric acid buffer using a microwave oven (600 W). This pretreatment enhances the sensitivity of the TUNEL reaction without loss of specificity (see Figure 6) (Stra¨ter et al., 1995; Negoescu et al., 1996). After another wash in PBS the slides were treated with proteinase K (2 µg/ml Boehringer Mannheim, Germany; in 10 mM Tris, 5 mM EDTA, pH 7.5) for 15 min at 37°C. After washing the sections in PBS they were preincubated in 13TdT buffer (MBI Fermentas, Vilnius, Lithuania: 200 mM potassium cacodylate, 25 mM Tris, 1 mM CoCl2, 0.5 mM dithiothreitol, pH 7.5) for 30 min at 37°C in a humidified chamber. The labelling reaction was carried out in 13TdT buffer containing 35 pmol/µl digoxigenin (Dig)-dUTP (Boehringer Mannheim), 350 pmol/µl dATP and 0.3 U/µl terminal deoxynucleotidyl transferase (TdT; MBI Fermentas) for 60 min at 37°C in a humid atmosphere. The dATP was added to allow the tailing with more than one Dig-dUTP by avoiding the steric barrier of the Dig to render the method more sensitive. After two washes in 23 standard saline citrate (SSC: 0.3 M NaCl, 0.03 M Na citrate) and a preincubation in blocking buffer (100 mM Tris–HCl pH 7.5,
Endometrial apoptosis and implantation
Table I. Proliferation and apoptosis during the menstrual cycle
Proliferative phase Early luteal phase Mid luteal phase Late luteal phase
Cell proliferation (marker Ki-67)
Protein expression bcl-2
In-situ detection of apoptosis (TUNEL)
Basal layer
Functional layer
Basal layer
Functional layer
Basal layer
Functional layer
S
GE (%)
S
GE (%)
LE (%)
S
GE (%)
S
GE (%)
LE (%)
S
S
4 4 9 16
14.7 11.4 0.4 0.0
29 20 16 26
34.9 24.4 0.5 0.0
29.4 4.5 1.6 0.8
1a 2a 5a 8a
84.0 35.0 0.0 0.0
6 6 11 11
55.0 51.7 5.0 7.5
39.0 48.3 28.3 35.0
,1b 0.2b ,1 ,0.1 ,1 3.5 ,1c 2.9
GE (%)
GE (%)
,1b 0.3b ,1 ,0.1 ,1 2.3 ,1d 3.4
LE (%) 0.7b ,0.1 ,0.1 0.2
TUNEL 5 terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling; S 5 stroma; GE 5 glandular epithelium; LE 5 luminal epithelium. Summary of the semiquantitative image analysis of the histological data concerning the cell proliferation, protein expression of the apoptosis-inhibiting protooncogene bcl-2 and the in-situ analysis of apoptosis in the human endometrium. Because of inter- and intraspecimen differences in the signal intensity, deviations using this semiquantitative method were obtained. Deviations were not uniform for all phases of the menstrual cycle. The largest deviations were obtained for the proliferative phase, which was not subdivided into early, mid and late phases. Even specimens from the early luteal phase were not uniform for all markers used. The best fitting values were obtained for the mid and late luteal phase. For the epithelia, the percentage value represents the positive cells out of all counted epithelial cells for Ki-67 and TUNEL and a relative estimated intensity of the immunological detection of bcl-2. For the stromal compartment, the values are numbers of positive cells within a randomly posititioned frame (3–5 times) using a magnification of 3400. aThe predominantly basal localized lymphoid follicles were excluded. bThere were occasionally single cells committing apoptotic suicide probably because they could not pass successfully through the cell cycle. cIn the lymphoid follicles of the basal layer we observed apoptotic nuclei between the mantle zone and germinal centre in some specimens; these cells were excluded in this semiquantitative analysis. dIn some very exceptional cases we observed single apoptotic cells in the predecidua of the late secretory phase (data not shown).
150 mM NaCl) the slides were blocked for 30 min at RT using 1% (w/v) blocking reagent (Boehringer Mannheim) in blocking buffer. Detection of the biotinylated deoxynucleotides was carried out using a 1:300 dilution (in blocking buffer) of an anti-Dig antibody conjugated with peroxidase (Boehringer Mannheim) for 45 min at RT in a humidified chamber. After the removal of the unbound antibody by washing the slides with blocking buffer containing 0.1% Tween (w/ v) and a short rinsing with deionized water, the tailing reaction was detected by the chromogen AEC (Genzyme). It is important to use only nuclease-free solutions during the whole procedure, to avoid artificial DNA fragmentation. As positive control the slides were treated with 3 U DNase I/µl (Boehringer Mannheim) prior to the tailing reaction. As negative control either the Dig-dUTP or TdT was omitted during the tailing reaction (see Figure 6). It was also shown (see Figure 6) that there were no artificial TUNEL-positive reactions because of the microwave pretreatment. The myometrium and the samples from the proliferative phase could be used as an additional internal control. Image analysis As well as the sections for immunhistochemistry the TUNEL slides were assessed and evaluated using an Axiophot (Zeiss). Our special interest focused on the patterns of proliferation, apoptosis and bcl-2 protein expression. For a semiquantitative analysis the sections were analysed as follows. For each marker and each menstrual cycle phase 5–11 tissue sections were analysed. In the case of the nuclear detections of Ki-67 and apoptosis the percentage of positive cells within the glandular and luminal epithelia was counted in 3–5 randomly selected glands and in the whole luminal epithelium of the section. Stromal analysis was performed by counting the positive cells within a randomly positioned frame (3–5 times) using a magnification of 3400. This stromal counting was also applied to the bcl-2 immunohistochemical detection. The epithelial staining was estimated taking into account the extent and intensity of the immunohistochemical signal. The data are summarized in Table I.
Results Immunolocalization of the proliferation antigen Ki-67 Cell proliferation was determined by detecting the nuclear proliferation-associated antigen Ki-67 (Gerdes et al., 1983).
Ki-67 is visible in all phases of the cell cycle, with exception of the G0-phase and the early G1-phase of the first cell-cycle after a resting phase. It is predominantly expressed in the G2and M-phase of the cell cycle. Fifty-three uterine tissue samples were examined by immunolocalization of Ki-67 (follicular phase n 5 23, early luteal phase n 5 10, mid luteal phase n 5 15, late luteal phase n 5 5). Immunoreactivity for Ki-67 was observed exclusively in the nucleus. The follicular phase was characterized by strong immunoreactivity in the glandular and surface epithelia as well as in the stromal compartment. During the whole follicular phase the functional layer showed stronger immunoreactivity than the basal layer (Figure 1). At the beginning of the luteal phase, the proliferation in the glandular epithelia showing the first characteristics of transformation ceased. After a decrease of stromal staining during the early luteal phase a second wave of proliferation was detected beginning in the mid luteal phase on day 22–23 of the menstrual cycle (Figure 2) and persisting to the end of the menstrual cycle. Immunohistochemical staining for bcl-2 The proto-oncogene bcl-2 is widely accepted to promote cell survival by blocking apoptosis. The membranes of the endoplasmic reticulum, the mitochondria and the outer nuclear membrane (Krajewski et al., 1993) account for the exclusively cytoplasmic immunostaining for bcl-2. A set of 31 endometria were examined for expression of bcl-2 protein (follicular phase n 5 11, early luteal phase n 5 6, mid luteal phase n 5 10, late luteal phase n 5 4). During the follicular phase, positive immunoreactivity was observed predominantly in the glandular epithelium of the basal and lower functional layers (Figure 3). Stromal immunoreactivity was detected in lymphoid follicles of the basal layer and in isolated cells of the upper functionalis, particular within the stromal compartment underlying the surface epithelium. The luminal epithelium was negative for bcl-2 protein expres3179
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Figure 1. Immunohistochemical staining for Ki-67 in the follicular (a and b, days 8–10 of the menstrual cycle), the early luteal phase (c and d, days 17–18) and the beginning of implantation (e and f, days 19–20). For each phase, the staining in the functional (a, c, e) and basal (b, d, f) layer is shown. Bar 5 100 µm.
sion during the whole follicular phase. Immunoreactivity was most marked during the mid and late follicular phase. During the transformation of the endometrium in the early luteal phase, this signal decreased rapidly (Figure 4). From day 21 of the menstrual cycle the immunoreactivity in the deeper glands was absent, but in the mid to late luteal phase the luminal epithelium and the glandular epithelia of the upper functionalis showed weak bcl-2 immunoreactivity. During the luteal phase the number of bcl-2 positive fibroblasts increased in the whole endometrium. In particular we detected bcl-2 expression within lymphoid follicles and proliferating immune competent cells within the basalis and functionalis layer. There was a strong immunoreactivity within the myometrium in all phases of the menstrual cycle. In situ detection of apoptosis The TUNEL method detects the fragmentation of the DNA taking place during the process of apoptosis. Therefore only nuclear staining will be observed. As we show in Figure 5, the morphological appearance of the apoptotic bodies detected 3180
using our TUNEL method matches very well with the schematic drawing of the events known to occur during apoptotic cell death. Therefore we conclude that we detected only apoptotic and no necrotic cell death, and that our method was suitable even to distinguish certain phases of the nuclear degradation following the onset of the DNA fragmentation. Figure 6 clearly shows that even the microwave pretreatment did not produce artificial DNA fragmentation. For this study 47 specimens were included (follicular phase n 5 20, early luteal phase n 5 7, mid luteal phase n 5 15, late luteal phase n 5 5). During the follicular phase of the menstrual cycle, only occasional isolated apoptotic cells could be detected within all compartments (Figure 7). At the time of transformation (early luteal phase) no sign of apoptosis could be seen. From days 19–21 of the menstrual cycle there is a strong increase in apoptotic activity (Figure 8). Beginning in the basal glands, the epithelium shows marked DNA fragmentation. During the later luteal phase, this activity expands further into the
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Figure 2. Immunohistochemical staining for Ki-67 in mid and late luteal phase. (a and b) Negative control with serial sections of the mid secretory specimen shown in (c) and (d). The primary antibody was replaced by using normal rabbit serum. (c and d) Staining in the basalis (d) and functionalis (c) layer at days 22–24 of the cycle. (e and f) Ki-67 staining in the late secretory phase endometrium at days 26–28 of the cycle; basalis (f) and functionalis (e) layer. Bar 5 100 µm.
functionalis layer (Figure 8). Apoptotic activity seems to be more marked in glandular epithelial cells localized near spiral arteries or lymphoid follicles. The luminal epithelium and the glandular epithelium of the upper one-third of the functionalis never showed signs of apoptosis. During the late luteal phase, we could detect apoptosis even in the stromal compartment in the mantle zone of secondary lymphoid follicles predominantly in the basalis layer of the endometrium (Figure 9). All data are summarized in Table I. Discussion The present data illustrate the apoptotic processes in the human endometrium during the menstrual cycle. Apoptosis is correlated with endometrial proliferation and expression of the proto-oncogene bcl-2 protein, which blocks apoptosis in various cell types (Vaux et al., 1992; Reed, 1994). During the follicular phase, we could show some apoptotic cells within all compartments of the proliferating functional
layer of the endometrium. During transformation of the endometrium in the early luteal phase, a decrease of proliferation and expression of bcl-2 protein was noticed. In addition, there were no apoptotic bodies detected. The luteal phase was characterized by a second wave of proliferation, located solely in the stromal compartment, representing large granular lymphocytes (LGL) (Bulmer et al., 1988; King et al., 1988; King and Loke, 1991), accompanied by strong bcl-2 expression in scattered stromal cells and lymphoid follicles. From days 19–20 of the luteal phase, there was an increasing number of apoptotic cells in the glandular epithelium of the basalis, and later on in the functionalis of the endometrium. At this time there was no sign of proliferation or bcl-2 expression in these compartments. The luminal epithelium did not show any apoptotic bodies during this phase, but displayed a weak bcl2 expression towards the end of cycle. During the luteal phase, we observed apoptotic, cell death between the proliferating centre and the bcl-2 expression mantle zone of secondary lymphoid follicles. The qualitative detection of endometrial 3181
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Figure 3. Immunohistochemical detection of bcl-2 protein expression during the follicular phase of the cycle. (a and b) Negative control sections (primary antibody was replaced by normal goat serum). Immunohistochemical detection of bcl-2 day 5 (c and d) and days 11–12 (e and f) of the menstrual cycle in the basal and functional layers of the endometrium. Bar 5 50 µm.
glandular apoptosis together with the semiquantitative analysis of our data, show the onset of apoptosis at days 19–20 and its extension to all endometrial layers. Detection of apoptosis using the TUNEL method Apoptosis is a sequence of cellular events beginning in the cytoplasm through the activation of caspases, the inactivation of cell survival proteins and the disruption of mitochondrial transmembrane potential. The following morphological changes, shown in Figure 5, include membrane blebbing, chromatin condensation and the fragmentation of cell and nucleus into apoptotic bodies with maintained membrane integrity. To determine apoptotic activity in situ, it is possible to localize caspases by immunohistochemistry or to show the DNA content of the nucleus. However, the localization of caspases is no proof of an active enzyme, and DNA content does not only vary during apoptosis. Every pathway to apoptosis converges in fragmentation of the DNA (Bortner et al., 1995). Therefore the TUNEL method used in our study is the best way to show apoptotic activity in histological sections. Even 3182
during the process of necrosis there is DNA fragmentation, but as Figure 5 shows, it is accompanied by an uncontrolled fragmentation of the cell without maintainance of membrane integrity. Additionally apoptosis is a single cell event which can be correlated to an inducing stimulus, whereas necrosis represents the destruction of a large part of the tissue. Comparing the morphology of the apoptotic bodies (see Figure 5) with the schematic drawing, and by detecting single or clustered cells undergoing apoptosis, we can be confident we have shown apoptosis using the TUNEL method. The appearance of apoptotic bodies is restricted to a time window between the beginning of DNA fragmentation and the phagocytosis of cellular shrinkage products, which occurs within ~24 h (Kerr et al., 1972; Earnshaw 1995). Therefore our results can be interpreted as snap-shots of the investigated day of the cycle. In consequence the apoptotic score of up to 3.5% (see Table I) represents a score per cycle day and should be amplified during the ongoing luteal phase. The proliferation marker Ki-67 is expressed during all phases of the cell cycle with the exception of the early
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Figure 4. Bcl-2 expression in the luteal phase of the cycle. (a and b) Weak expression in the glands and stronger signals in the stromal lymphocytes are obtained during early luteal phase (days 15–17). During the mid (c and d, days 19–20) to late (e and f, day 26) luteal phase bcl-2 expression was predominantly localized in the stromal compartment. (a, c, e functional layer; b, d, f basal layer). Bar 5 50 µm.
G1-phase of resting cells entering their first cell cycle, or differentiated cells (in G0-phase; Gerdes et al., 1984; Landberg et al., 1990). Because of its short half-life (Schlu¨ter et al., 1993), Ki-67 indicates the growth fraction of the examined tissue (Gown et al., 1996). The expression of cell cycle regulatory proteins during the apoptotic pathway (Grassilli et al., 1992; Evan et al., 1992) and the evidence of apoptosis predominantly in tissues with a high proliferation index has led to the proposal that the apoptotic pathway represents an abortive attempt to pass through the cell proliferation cycle. However, recent studies, particularly on hormone-induced apoptosis, show that resting cells can also enter the apoptotic pathway (Galand and Martelaer 1992; Kroemer et al., 1995; Lindenboim et al., 1995). The use of the proliferation marker Ki-67 seems to be a useful tool in defining the association of apoptosis with proliferation (Coates et al., 1996). Our studies on the correlation of apoptosis and proliferation indicate three types of apoptosis in the human endometrium: (i) occasional apoptotic cells in proliferating compartments (stroma and epithelia) during the first half of the menstrual cycle, (ii)
apoptosis of lymphocytes in secondary lymphoid follicles at the end of their proliferation and the beginning of their differentiation, (iii) apoptosis in resting differentiated nonproliferating glandular cells from the beginning of the midluteal phase. Correlation of the expression of the proto-oncogene bcl-2 should allow insights into the regulation of apoptosis. Our data concerning the immunohistochemical staining of bcl-2 protein generally support the findings of previous investigators (Gompel et al., 1994; Otsuki et al., 1994, Koh et al., 1995; Tabibzadeh et al., 1995). Bcl-2 is expressed during the whole menstrual cycle, and its localization shifts from predominantly glandular expression during the follicular phase to a more stromal expression during the luteal phase, probably respresenting scattered LGL. We observed that glandular staining was confined to the early luteal phase (according to Gompel et al., 1994) and decreased just after the LH peak, but occasionally we found some bcl-2 expression in the luminal and the upper functionalis glandular epithelia during the mid to late luteal phase. These patterns support the hypothesis, by several 3183
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Figure 5. Schematic diagram of the morphological processes observed during the apoptotic program compared with images of single apoptotic cells and nuclei detected by the TUNEL method during our study. This figure illustrates that we have detected apoptotic cell death and no necrosis, and that it is even possible to distinguish certain stages of the apoptotic death program using the 39 labelling method presented in this paper. Bar 5 10 µm. [Schematic drawing taken from: Kerr, F.K. et al. (1994) Cell Biology: A Laboratory Handbook. Academic Press, New York.]
Figure 6. TUNEL reaction with various pretreatments of the sections, and negative and positive controls for a typical specimen from days 22–24 of the menstrual cycle. (a) Negative control by omitting the enzyme terminal transferase (TdT) during the labelling reaction. In a second negative control the Dig-dUTP was omitted, which gave the same results (data not shown). (b) Positive control using a DNase digestion (3 U DNase I/µl for 10 min at 37°C) prior to the labelling reaction. (c) TUNEL reaction without microwave pretreatment. (d) TUNEL reaction with 1 min microwave pretreatment. It is clear from (c) and (d) that using the microwave pretreatment causes no artificial DNA fragmentation. Bar 5 100 µm.
investigators, that bcl-2 expression is hormonally regulated by oestradiol and down-regulated during cell differentiation (Sabourin et al., 1994; Marthinuss et al., 1995; Furuchi et al., 1996). Our observations match very well with the appearance of the first apoptotic glandular cells during the mid luteal 3184
phase in the basal layer of the endometrium, and with the fact that we did not find any apoptoses in the luminal epithelium during the whole luteal phase. Therefore, we conclude that bcl-2 must play an important role in regulating endometrial cell death. Koh et al. (1995) only found weak immunoreactivity
Endometrial apoptosis and implantation
Figure 7. TUNEL reaction to detect single apoptotic cells in the follicular phase. (a and b) Functional and basal layer of an endometrial specimen from day 5 of the menstrual cycle. (c and d) Functional and basal layer of an endometrial specimen from days 8–10 of the menstrual cycle (serial sections to Figure 1a and b). Bar 5 100 µm.
for bcl-2 in endocrinologically rescued endometria whereas during early pregnancy — after successful implantation — bcl-2 was shown to be expressed in the glandular epithelia (Lea et al., 1997). These data are in accord to our current opinion that there is a need for glandular apoptosis during the time of implantation. Recent studies on other members of the bcl-2 family show that the bcl-2/bax protein ratio can influence the cell’s susceptibility to apoptotic stimuli (Oltvai et al., 1993; Oltvai and Korsmeyer, 1994; Reed, 1996; Farrow and Braun, 1996). In the human endometrium, bax protein was found to persist exclusively in the glandular epithelium through the whole menstrual cycle (McLaren et al., 1997) and may be upregulated during the luteal phase (Tao et al., 1997). This persistence and the dramatic down-regulation of bcl-2 in the glandular epithelium in the mid luteal phase (beginning at day 19–20 of the cycle) may predispose these cells to undergo apoptosis during the period when the earliest signs of apoptosis appear. Consequently we conclude that glandular apoptosis, evident at the time of implantation, is hormonally regulated by oestradiol and some variation of the bcl-2/bax ratio.
As mentioned above, we could show three different occurrences of apoptosis in the human endometrium. The first is scattered apoptosis in proliferating compartments with positive immunostaining for bcl-2. These occasionally appearing apoptotic bodies probably represent cells unable to pass successfully through the cell cycle, perhaps due to DNA damage. Concerning this type of apoptosis, the effect of p53 as a guardian of the genome is discussed (Lane, 1992) which perhaps is mediated by the down-regulation of bcl-2 and the up-regulation of bax (Haldar et al., 1994; Miyashita et al., 1994; Miyashita and Reed, 1995). This phenomenon was recently described for the secretory epithelia of the lactating mammary gland (Quarrie et al., 1995) and seems to be a mechanism of cell removal, not exclusively related to the endometrium. The second occurence of apoptosis detected was the apoptosis of lymphocytes at the end of their proliferation and just before the beginning of differentiation in lymphoid follicles of the human endometrium, which to our knowledge we have described for the first time in this paper. These immunecompetent cells could be either activated T-lymphocytes, removed by apoptosis to terminate an immunological reaction, or negatively selected B-lymphocytes from the germinal centre of a secondary lymphoid follicle. Both types of cell death in the immune system are mediated through the apoptotic pathway (Chleq-Deschamps 1993; Debatin, 1994; Krammer, 1996). The most essential third occurrence of apoptosis shown in our study is the cell death of differentiated glandular epithelial cells during the beginning of implantation (days 19–20). Our data confirm the findings of previous studies of morphological detection of apoptosis (Hopwood and Levison, 1976; Tabibzadeh et al., 1994) concerning the localization of this phenomenon to the glandular epithelium, and the beginning of apoptosis in the basal layer of the endometrium. But the time-course of the apoptosis is different. Hopwood and Levison (1976), Tabibzadeh et al. (1994), and recently Watanabe et al. (1997) detected the first apoptotic cell death in the mid to late luteal phase, and therefore associated these processes exclusively with the onset of menstruation (Tabibzadeh, 1996). We could detect apoptotic cells just after the transformation of the endometrium during the early to mid luteal phase. These differences may be explained by the different detection methods. Hopwood and Levison detected morphologically apoptotic bodies, the latest sign during the apoptotic program. Tabibzadeh and his co-workers used a TUNEL method without microwave pretreatment. Our data show many early apoptotic stages at day 19–20 of the menstrual cycle. At this time, it is not yet determined if the endometrium will be shed at the end of the cycle, or if a pregnancy will be established. The first known hormonal signal of the conceptus is human chorionic gonadotropin (HCG), dated to days 7–9 after LH (Lenton and Woodward, 1988). This would correspond to days 19–21 of the menstrual cycle. Hence we could determine the first signals of DNA fragmentation at the time when implantation is determined. The execution phase of the apoptotic pathway is preceded by a ‘decision’ phase of several hours up to 1–2 days (Lazebnik et al., 1993; Earnshaw, 1995; Kroemer et al., 1995) and DNA fragmentation detected by the TUNEL method is a late event in this process 3185
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Figure 8. TUNEL reaction showing glandular apoptosis beginning at days 19–20 of the cycle. (a and b) Days 17–18 (serial sections to Figure 1c and d), (c and d) days 19–20 (serial sections to Figure 1e and f), (e and f) days 26–28 (serial sections to Figure 2e and f) of the menstrual cycle. For each specimen the pattern in the functional (a, c, e) and basal (b, d) or lower functional (f) layer of the endometrium are given. Bar 5 100 µm.
(Collins et al., 1997). Therefore the cell death seen at days 19–20 could be initiated on days 17–18 of the menstrual cycle. It seems unreasonable to prepare the endometrium for shedding at this early point of the cycle. Hence, other molecular pathways, mentioned by Tabibzadeh (1996), rather than apoptosis seem to regulate the onset of menstruation. There is increasing evidence that apoptosis may be important for implantation and very early pregnancy. In the rat, glandular apoptosis during implantation and in oestrus and metoestrus has been shown (Parr et al., 1987; Sato et al., 1997) and seems to be regulated by changing concentrations of bcl-2 and bax proteins (Akcali et al., 1996). This apoptosis seems to be an intrinsic program, and the conceptus may play only a minor role in regulating the death of the endometrial cells in this species (Welsh, 1993; Gu et al., 1994). Significance of the apoptotic processes in the human endometrium In contrast to the former idea of an immunologically tolerant endometrium at the time of implantation, current opinion deals 3186
with the concept of limiting trophoblast invasion by the decidua (Bulmer, 1992; Loke and King, 1996). During the first trimester of pregancy 70–80% of the decidual stromal immunocompetent cells are uterine natural killer (NK)-cells (Hunt, 1994; Loke et al., 1995; Whitelaw and Croy, 1996). These members of the innate immune system have been shown to be able to lyse trophoblast cells after the activation by interferon (IFN)-γ or interleukin-2 (Billington, 1987; King and Loke, 1990; Loke et al., 1995). A paracrine function of IFNγ is also proposed in the endometrium (Tabibzadeh, 1990, 1991). The secretion of IFNγ by activated T-lymphocytes at the beginning of the luteal phase can induce proliferation and cytotoxicity of the NK-cells even against glandular epithelia. This may explain the occurence of glandular apoptosis in bcl-2-negative epithelia expanding in parallel with the appearance of NK-cells from the basal layer in the functionalis during the luteal phase. Glandular apoptosis may serve several mechanisms to prepare the endometrium for implantation. (i) Glandular apoptosis
Endometrial apoptosis and implantation
Figure 9. The detection of cell proliferation, apoptosis and bcl-2 protein expression in serial sections of a lymphoid follicle localized in the basal layer during the late luteal phase (day 26). (a) Cell proliferation was predominantly found in the germinal centre of the lymphoid follicle (probably proliferating B-lymphocytes or activated T-lymphocytes). (b) Apoptosis is located in the border of the germinal centre to the mantle zone of the follicle (probably B-lymphocytes which did not pass through the B-cell selection successfully) and in the epithelia of the surrounding glands. (c) Bcl-2 expression is confined to the mantle zone lymphocytes (probably successfully passed through the B-cell selection) and the scattered stromal cells (probably large granular lymphocytes). Bar 5 100 µm.
with subsequent shedding of the apoptotic bodies into the glandular lumen may serve as a secretory mechanism of the luteal glands whose secretory activity is maximal on days 19– 21 of the cycle (Loke and King, 1995). Nuclear components (e.g. histones) may be extruded into the uterine secretion material. Our recently published identification of histones in this compartment (Hilmes et al., 1993; Beier-Hellwig et al., 1994) supports this hypothesis. (ii) The establishment of the characteristic immunological situation of the mid to late luteal phase and the first trimester of pregnancy comprises large amounts of immune-competent cells, particularly LGL. Their proliferation may be supported by the release of histones during apoptosis, a known stimulus of lymphocyte proliferation (Bell and Morrison, 1991; Laurent-Crawford et al., 1991). This effect additionally may explain the parallel extension of glandular apoptosis and lymphocyte proliferation from the basal layer to the functional layer. (iii) Predominantly in the functional layer, apoptosis may serve for tissue remodelling (in particular glandular reduction) during the decidualization beginning at days 23–24 of the menstrual cycle, when glandular apoptosis is even seen in the epithelia of the functional layer. At the end of this process the upper layer (decidua compacta) contains decidualized stroma with attenuated non-secretory glands (Loke and King, 1995). In the rodent model a role for glandular apoptosis in tissue remodelling during decidualization has recently been suggested (Akcali et al., 1996; Sato et al., 1997). During the first trimester of pregnancy lymphoid aggregates are found in the basal layer of the decidua (Loke and King, 1995). These immune-competent cells may serve to regulate and to limit the invasion of the extravillous trophoblast cells in the decidual tissue. The perhaps activation-induced apoptotic cell death or clonal selection of lymphocytes in the basal lymphoid aggregates described in this paper may be the first observation of an immunological preparation of the maternal
endometrium for implantation. Apoptosis therefore will not serve to facilitate the adhesion of the blastocyst, but rather seems to help to regulate the invasion of the extravillous trophoblast. In conclusion, the comparison of proliferation and bcl-2 expression with the detection of apoptosis seems to be a useful tool to distinguish different forms of apoptosis. For the first time, we have demonstrated apoptotic cell death in the glandular epithelia of the human endometrium at the onset of implantation. This process probably depends on the periovulatory decrease of oestradiol and it involves the down-regulation of bcl-2. Its possible significance for tissue remodeling and immunological preparation of the endometrium for implantation has been discussed. In consequence, the apoptotic cell death we detected during mid luteal phase could be assessed as a cell biological marker of an adequately prepared endometrium for implantation. Further examination of apoptotic cell death in the decidua of the first trimester pregnancy, and studies on the mechanism of the induction of glandular epithelial apoptosis during the menstrual cycle, will serve to elucidate the significance of apoptotic cell death for the receptive endometrium and implantation. Acknowledgements We wish to acknowledge the collaboration by Professor Christian Karl, Department of Gynecology and Obstetrics, St Antonius Hospital Eschweiler. Thanks are due to Diana Behrend, Elisabeth Bru¨gmann and Sabina Hennes-Mades for excellent technical assistance and to Gaby Bock for elaborate photographic work. Financial support by the Deutsche Forschungsgemeinschaft (Grant Cl 88/3-1/3-2) is gratefully acknowledged.
References Akcali, K.C., Khan, S.A. and Moulton, B.C. (1996) Effect of decidualization on the expression of bax and bcl-2 in the rat uterine endometrium. Endocrinology, 137, 3123–3131.
3187
U.von Rango et al. Beier-Hellwig, K., Sterzik, K., Bonn, B. et al. (1994) Hormone regulation and hormone antagonistic effects on protein patterns of human endometrial secretion during receptivity. In The Human Endometrium, Ann. NY Acad. Sci., 734, 143–156. Bell, D.A. and Morrison, B. (1991) The spontaneous apoptotic cell death of normal human lymphocytes in vitro: the release of and immunoproliferative response to nucleosomes in vitro. Clin. Immunol. Immunopathol., 63, 13–26. Billington, W.D. (1987) Immunological aspects of implantation and fetal survival: the central role of the trophoblast. Curr. Top. Dev. Biol., 23, 209–232. Bortner, C.D., Oldenburg, N.B.E. and Cidlowsky, J.A. (1995) The role of DNA fragmentation in apoptosis. Trends Cell Biol., 5, 21–26. Bulmer, J.N. (1992) Immune aspects of pathology of the placental bed contributing to pregnany pathology. Baillie`re’s Clin. Obstset. Gynecol., 6, 461–488. Bulmer, J.N., Lunney, D.P. and Hagin, S.V. (1988) Immunohistochemical characterization of stromal leucocytes in nonpregnant human endometrium. Am. J. Reprod. Immunol. Microbiol., 17, 83–90. Chleq-Deschamps, C.M., LeBrun, D.P., Huie, P. et al. (1993) Topographical dissociation of bcl-2 m-RNA and protein expression in human lymphoid tissues. Blood, 81, 293–298. Coates, P.J., Hales, S.A. and Hall, P.A. (1996) The association between cell proliferation and apoptosis: studies using the cell cycle associated proteins Ki67 and DNA-polymerase. J. Pathol., 178, 71–77. Cohen, J.C. and Duke, R.C. (1994) Glucocorticoid activation of calclium dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol., 132, 38–42. Collins, J.A., Schandl, C.A., Young, K.K. et al. (1997) Major DNA fragmentation is a late event in apoptosis. Histochem. Cytochem., 45, 923–934. Colombel, M.C. and Buttyan, R. (1995) Hormonal control of apoptosis: the rat prostate as a model system. Methods Cell. Biol., 46, 369–385. Dallenbach-Hellweg, G. and Poulson, H. (eds) (1985) Atlas der Histopathologie des Endometriums. Springer, Berlin. Debatim, K.M. (1994) Apo-1 (CD95) and bcl-2: determinants of cell death in the human thymus. Res. Immunol., 145, 146–151. Dhein, J., Walczak, H., Ba¨umler, C. et al. (1995) Autocrine T-cell suicide mediated by Apo-1 (Fas/CD95). Nature, 373, 438–441. Earnshaw, W.C. (1995) Nuclear changes in apoptosis. Curr. Opin. Cell Biol., 7, 337–343. Evan, G.I., Wyllie, A.H. and Gilbert, C.S. (1992) Induction of apoptosis in fibroblasts by c-myc protein. Cell, 69, 119–128. Fadok, V.A., Voelker, D.R., Campbell, P.A. et al. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol., 148, 2207–2216. Farrow, S.N. and Brown, R. (1996) New members of bcl-2 family and their protein partners. Curr. Opin. Genet. Dev., 6, 45–49. Furuchi, T., Masuko, K., Nishimune, Y. et al. (1996) Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing bcl-2 in spermatogonia. Development, 122, 1703–1709. Galand, P. and de Maertelaer, V. (1992) Models of oestrogen action: a cell kineticist’s view. Epith. Cell Biol., 1, 177–188. Gavrieli, Y., Sherman, Y. and Ben-Sasson, A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNAfragmentation. J. Cell Biol., 119, 493–501. Gerdes, J., Schwab, U., Lemke, H. and Stein, H. (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer, 31, 13–20. Gerdes, J., Lemke, H., Baisch, H. et al. (1984) Cell cycle analysis of a cell proliferation associated human nuclear antigen defined by the monoclonal antibody Ki-67. J. Immunol., 133, 1710–1715. Gompel, A., Sabourin, J.C., Martin, A. et al. (1994) Bcl-2 expression in normal endometrium during the menstrual cycle. Am. J. Pathol., 144, 1195–1202. Gown, A.M., Jiang, J.J., Matles, H. et al. (1996) Validation of the S-phase specifity of histone (H3) in situ hybridization in normal and malignant cells. J. Histochem. Cyctochem., 44, 221–226. Grassilli, E., Carcereri de Prati, A., Monti, D. et al. (1992) Studies of the relationship between cell proliferation and cell death. Biochem. Biophys. Res. Commun., 188, 1261–1266. Gu, Y., Jow, G.M., Moulton, B.C. et al. (1994) Apoptosis in decidual tissue regression and reorganization. Endocrinology, 135, 1272–1279. Haldar, S., Negrini, M., Monne, M. et al. (1994) Down-regulation of bcl-2 by p53 in breast cancer cells. Cancer Res., 54, 2095–2097.
3188
Hegele-Hartung, C., Mootz, U. and Beier, H.M. (1992) Luteal control of endometrial receptivity and its modification by progesterone antagonists. Endocrinology, 131, 2446–2460. Hilmes, U., Beier-Hellwig, K., Sterzik, K. et al. (1993) Identification of histones in human uterine secretion samples. J. Reprod. Fertil. Abstr. Ser. 12, 43. Hockenbery, D.M., Zutter, M., Hickey, W. et al. (1991) Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc. Natl. Acad. Sci. USA, 88, 6961–6965. Hopwood, D. and Levison, D.A. (1976) Atrophy and apoptosis in the cyclical human endometrium. J. Pathol., 119, 159–166. Hunt, J.S. (1994) Immunologically relevant cells in the uterus. Biol. Reprod., 50, 461–466. Janeway, C.A. and Travers, P. (1997) Immunobiology: the Immune System in Health and Disease. Churchill Livingstone, Edinburgh, Vol. 3. Kerr, J.F.R., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer, 26, 239–257. Kerr, J.F.R., Winterford, C.M. and Harmon, B.V. (1994) Morpholigical criteria for identifying apoptosis. In A Laboratory Handbook, Academic Press, New York, pp. 319–328. King, A., and Loke, Y.W. (1990) Human trophoblast and JEG chorioncarcinoma cells are sensitive to lysis by Il-2 stimulated decidual NK cells. Cell. Immunol., 129, 435–448. King, A. and Loke, Y.W (1991) On the nature and function of human uterine granular lymphocytes. Immunol. Today, 12, 432–435. King, A., Wellings, V., Gardner, L. and Loke, Y.W. (1988) Immunocytochemical characterization of the unusual large granular lymphocytes in the human endometrium throughout the menstrual cycle. Hum. Immunol., 24, 195–205. Koh, E.A.T., Illingworth, P.J., Duncan, W.C. and Critchley, H.O.D. (1995) Immunolocalization of bcl-2 protein in human endometrium in the menstrual cycle and simulated early pregnancy. Mol. Hum. Reprod., 10, 1557–1562. Krajewski, S., Tanaka, S., Takayama, S. et al. (1993) Investigation of the subcellular distribution of bcl-2 oncoprotein: Residence in the nuclear envelope, ER, and outer mitochondrial membranes. Cancer Res., 53, 4701–4714. Krammer, P.H. (1996) Apoptose im Immunsystem: Mord oder Selbstmord. Robert Koch Mitt., 20, 32–43. Kroemer, G., Petit, P., Zamazmi, N. et al. (1995) The biochemistry of programmed cell death. FASEB J., 9, 1277–1287. Landberg, G., Tan, E.M. and Roos, G. (1990) Flow cytometric multiparameter analysis of proliferating cell nuclear antigen (cyclin) and Ki67 antigen: a new view of the cell cycle. Exp. Cell Res., 187, 111–118. Lane, D.P. (1992) p53 the guardian of the genome. Nature, 358, 15–16. Laurent-Crawford, A.G., Krust, B., Muller, S. et al. (1991) The cytolytic effect of HIV is associated with apoptosis. Virology, 185, 829–839. Lazebnik, Y.A., Cole, S., Cooke, W.G. et al. (1993) Nuclear events of apoptosis in vitro in cell free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol., 123, 7–22. Lea, R.G., Al.Sharekh, N., Tulppala, M. and Critchley, H.O.D. (1997) The immunolocalization of bcl-2 at the maternal–fetal interface in healthy and failing pregnancies. Hum. Reprod., 12, 153–158. Lenton, E.A. and Woodward, A.J. (1988) The endocrinology of conception and implantation in women. J. Reprod. Fertil., 36 (Suppl.), 2–15. Lindenboim, L., Diamond, R., Rothenberg, E. and Stein, R. (1995) Apoptosis induced by serum deprivation of PC12 cells is not preceded by growth arrest and can occur at each phase of the cell cycle. Cancer Res., 55, 1242–1247. Loke, Y.W. and King, A. (1995) Uterine mucosal leucocytes. In Loke Y.W. and King A. (eds), Human Implantation; Cell Biology and Immunology. Cambridge University Press, Cambridge, p. 102. Loke, Y.W. and King, A. (1996) Immunology of human implantation an evolutionary perspective. Hum. Reprod., 11, 283–286. Loke, Y.W., King, A. and Burrows, T.D. (1995) Decidua in human implantation. Hum. Reprod., 10 (Suppl. 2), 14–21. Lund, L.R., Romer, J., Thomasset, N. et al. (1996) Two distinct phases of apoptosis in mammary gland involution: proteinase-independant and dependent pathways. Development, 122, 181–193. Marthinuss, J., Lawrence, L. and Seiberg, M. (1995) Apoptosis in Pan 212, an epidermal keratinocyte cell line: a possible role for bcl-2 in epidermal differentiation. Cell Growth Diff., 6, 239–250. McLaren, J., Prentice, A., Charnock-Jones, D.S. et al. (1997)
Endometrial apoptosis and implantation Immunolocalization of the apoptosis regulating proteins Bcl-2 and Bax in human endometrium and endometriosis. Hum. Reprod., 12, 146–152. Merrit, A.J., Potten, C.S., Kemp, C.J. et al. (1994) The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice. Cancer Res., 54, 614–617. Miyashita, T. Krajewski, S., Krajewska, M. et al. (1994) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene, 9, 1799–1805. Miyashita, T. and Reed, J.C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell, 80, 293–299. Mitra, D., Steiner, M., Lynch, D.H. et al. (1996) HIV-1 upregulated Fasligand expression in CD41 – T cells in vitro and in vivo; association with Fas-mediated apoptosis and modulation by aurintricarboxylic acid. Immunology, 87, 518–585. Nagata, S. and Golstein, P. (1995) The Fas death factor. Science, 267, 1449–1456. Nawaz, S., Lynch, M.P., Galand, P. and Gerschenson, L.E. (1987) Hormonal regulation of cell death in rabbit uterine epithelium. Am. J. Pathol., 127, 51–59. Negoescu, A., Lorimier, P., Labat-Moleur, F. et al. (1996) In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations. J. Histochem. Cytochem., 44, 959–968. Noyes, R.W. and Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25. Nunez, G. and Clarke, M.F. (1994) The Bcl-2 family of proteins: regulators of cell death and survival. Trends Cell Biol., 4, 399–403. Oltvai, Z.N. and Korsmeyer, S.J. (1994) Checkpoints of dueling dimers foil death wishes. Cell, 79, 189–192. Oltvai, Z.N., Milliman, C.L. and Korsmeyer, S.T. (1993) Bcl-2 heterodimerises in vivo with a conserved homologue Bax that accelerates programmed cell death. Cell, 74, 609–619. Otsuki, Y., Misaki, O., Sugimoto, O. et al. (1994) Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle. Lancet, 344, 28–29. Parr, E.L., Tung, H.N. and Parr, M.B. (1987) Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rats. Biol Reprod., 36, 211–225. Pollard, J.W., Pacey, J., Cheng, S.V.Y. and Jordan, E.G. (1987) Estrogens and cell death in murine uterine luminal epithelium. Cell Tissue Res., 249, 533–540. Quarrie, L.H., Addey, C.V.P and Wilde, C.J. (1995) Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end-labeling. Cell Tiss. Res., 281, 413–419. Reed, J.C. (1994) Bcl-2 and the regulation of programmed cell death. J. Cell Biol., 124, 1–6. Reed, J.C. (1996) A day in the life of the bcl-2 protein: does the turnover rate of bcl-2 serve as a biological clock for cellular lifespan regulation? Leukemia Res., 20, 109–111. Rotello, R.J., Lieberman, R.C., Lepoff, R.B. and Gerschenson, L.E. (1992) Characterization of uterine epithelium apoptotic cell death kinetics and regulation by progesterone and RU486. Am. J. Pathol., 140, 449–456. Sabourin, J.C., Martin, A., Baruch, J. et al. (1994) Bcl-2 expression in normal breast tissue during the menstrual cycle. Int. J. Cancer, 59, 1–6. Sandow, B.A., West, N.B., Norman, R.L. and Brenner, R.M. (1979) Hormonal control of apoptosis in hamster uterine luminal epithelium. Am. J. Anat., 156, 15–36. Sato, T., Fukazawa, Y., Kojima, H. et al. (1997) Apoptotic cell death during the estrous cycle in the rat uterus and vagina. Anat. Rec., 248, 76–83. Schlu¨ter, C., Duchrow, M., Wohlenberg, C. et al. (1993) The cell proliferationassociated antigen of antibody Ki67: a very large ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell-cycle maintaining proteins. J. Cell Biol., 123, 313–522. Stra¨ter, J., Gu¨nthert, A.R., Bru¨derlein, S. and Mo¨ller, P. (1995) Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells. Histochemistry, 103, 157–160. Tabibzadeh, S. (1990) Evidence of T-cell activation and potential cytokine activation in human endometrium. J. Clin. Endocrinol. Metab., 71, 740–747. Tabibzadeh, S. (1991) Induction of HLA-DR expression in endometrial epithlial cells by endometrial T-cells: potential regulatory role of endometrial T-cells in vivo. J. Clin. Endocrinol. Metab., 71, 1352–1359. Tabibzadeh, S. (1996) The signs and molecular pathways involved in human menstruation, a unique process of tissue destruction and remodelling. Mol. Hum. Reprod., 2, 77–92. Tabibzadeh, S., Kong, O.F., Satyaswaroop, P.G. et al. (1994) Distinct regional and menstrual cycle dependent distribution of apoptosis in human
endometrium. Potential regulatory role of T-cells and TNFα. Endocr. J., 2, 87–95. Tabibzadeh, S., Zupi, E., Babaknia, A. et al. (1995) Site and menstrual cycledependent expression of proteins of the tumor necrosis factor (TNF) receptor family, and Bcl-2 oncoprotein and phase-specific production of TNFα in human endometrium. Hum. Reprod., 10, 277–286. Tao, X.J., Tilly, K.I., Maravei, D.V. et al. (1997) Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax. J. Clin. Endocrinol. Metab., 82, 2738–2746. Tilly, J.L., Kowalski, K.I., Johnson, A.L. and Hsueh, A.J. (1991) Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology, 129, 2799–2801. Tsujimoto, Y., Cossman, J., Jaffe, E. and Croce, C.M. (1985) Involvement of the bcl-2 gene in human follicular lymphoma. Science, 228, 1440–1443. Vaux, D.L., Aguila, H.L. and Weissman, I.L. (1992) Bcl-2 prevents death of factor-deprived cells but fails to prevent apoptosis in targets of cell mediated killing. Int. Immunol., 4, 821–824. Watanabe, H., Kanzaki, H., Narukawa, S. et al. (1997) Bcl-2 and Fas expression in eutopic and ectopic human endometrium during menstrual cycle in relation to endometrial cell apoptosis. Am. J. Obstet. Gynecol., 176, 360–368. Weismann, A. (1863) Die Entwicklung der Dipteren im Ei nach Beabachtungen an Chironomus spec., Musca vomitoria and Pulex carnaria. Z. Wiss. Zool., 14, 187–336. Welsh, A.O. (1993) Uterine cell death during implantation and early placentation. Microsc. Res. Tech., 25, 223–245. Whitelaw, P.F. and Croy, B.A. (1996) Granulated lymphocytes in pregnancy. Placenta, 17, 533–543. Wyllie, A.H. (1980) Glucocortocoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 284, 555–556. Wyllie, A.H. (1994) Death gets a break. Nature, 369, 272–273. Received on January 9, 1998; accepted on July 31, 1998
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