Menstrual-like breakdown and apoptosis in human endometrial explants

A. Li1, J.C. Felix1,2, J. Hao1, P. Minoo3 and J.K. Jain1,4

Department of 1Obstetrics and Gynecology, 2 Pathology and 3 Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Keck School of Medicine of the University of Southern California, 1240 Mission Street, Room 1M20, Los Angeles, CA 90033, USA. Email: jjain{at}usc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Apoptosis occurs in late secretory and menstrual human endometrium and is thought to play an important role in endometrial physiology. Menstrual-like breakdown has been observed in vitro in endometrial explants. The purpose of this study was to assess the role of apoptosis in menstrual-like breakdown in human endometrial explants. METHODS: Human endometrial tissue was obtained during the mid-secretory phase and cultured with or without estrogen and progesterone. The occurrence of breakdown was assessed by histology. Apoptosis was determined by gel electrophoresis for the detection of DNA fragmentation and by immunohistochemistry using the M30 CytoDEATH and anti-cleaved caspase-3 (CASP3) antibodies for the detection of caspase activity. Expression of BCL2 and BAX was quantified using real-time PCR analysis. RESULTS: Apoptosis occurred in human endometrial explants at all time-points studied. Cleaved CASP3 and M30 antigen expression increased in all explants, suggesting the involvement of CASP3 in the apoptosis. Low BCL2:BAX ratios were observed in all samples when compared with pre-culture controls. Estradiol and progesterone supplementation of the culture media reduced or eliminated menstrual-like breakdown but did not affect the degree of apoptosis observed. CONCLUSIONS: The apoptosis observed in endometrium during the late secretory phase and menstrual phase does not appear to be mechanistically related to the tissue breakdown but rather may be involved in the impending remodelling that occurs in the endometrium in the transition from secretory to proliferative phase following the menses.

Key words: apoptosis/BCL2:BAX ratio/caspase activity/endometrial explant/menstrual breakdown


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
During every reproductive cycle, the human endometrium undergoes extensive tissue remodelling in response to cyclic hormonal changes. Estrogen stimulates proliferation and re-establishment of the stromal and vascular components of the tissue during the proliferative phase, whereas the influence of progesterone results in glandular differentiation and stromal decidualization during the secretory phase. In the absence of pregnancy, the decreased serum levels of ovarian steroid hormones in the late secretory phase provides a critical trigger that leads to tissue breakdown, loss of the functional layer and menstruation.

Apoptosis has been suggested to play a pivotal role in the reproductive physiology of the endometrium (Kokawa et al., 1996Go; Nakano and Shikone, 1996Go; Shikone et al., 1997Go; Vaskivuo et al., 2000Go, 2002Go; Castro et al., 2002Go). In the endometrium, apoptosis has been shown to be regulated by steroid hormones in several mammalian species including rabbits (Rotello et al., 1989Go), hamsters (Chen et al., 2001Go), and monkeys (Sengupta et al., 2003Go). Hopwood and Levinson (1976)Go showed for the first time changes in apoptosis of the glandular epithelium in the human endometrium throughout the menstrual cycle. Several studies in humans have reported the presence of endometrial apoptotic cells appearing mainly at the beginning of the secretory phase during the receptive period, becoming more numerous during the late secretory phase, and finally peaking during the menstrual phase (Kokawa et al., 1996Go; Nakano and Shikone, 1996Go; Shikone et al., 1997Go; Vaskivuo et al., 2000Go, 2002Go; Castro et al., 2002Go). The temporal association of apoptosis and menstruation has prompted hypotheses suggesting a mechanistic role of apoptosis and the tissue breakdown seen in menstruation (Kokawa et al., 1996Go).

Apoptosis is generally controlled by three major components: the BCL2 family proteins; the caspases; and the APAF1/CED4 protein that relays the signals between BCL2 proteins and caspases (Adams and Cory, 1998Go). The BCL2 proto-oncogene promotes cell survival by blocking the apoptosis induced by different stimuli (Adams and Cory, 2001Go), whereas another member of the family, BAX, promotes apoptosis. There are currently two well-characterized caspase-activating cascades that regulate apoptosis: one is initiated from the cell surface death receptor (FAS/FASLG) and the other is triggered by changes in mitochondrial integrity (Budihardjo et al., 1999Go).

Several investigators have successfully maintained human endometrial explants in tissue culture (Csermely et al., 1969Go; Abel and Baird, 1980Go; Marbaix et al., 1992Go, 1996Go; Illouz et al., 2000Go). Menstrual-like breakdown has been observed in these cultured endometrial explants, a process that can be experimentally prevented or minimized by supplementation with sex steroid hormones (Marbaix et al., 1996Go). The purpose of this study was to assess the role of apoptosis in menstrual-like breakdown in human endometrial explants.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Endometrial histology, explant culture and antibodies
This study was approved by the Institutional Review Board of the University of Southern California and Los Angeles County Medical Center. Endometrial biopsies were obtained from 29 regular menstruating women during the mid-secretory phase (cycle days 21–23) using a PipelleTM endometrial biopsy instrument. Part of each biopsy was processed for routine histological evaluation with haematoxylin and eosin after standard formalin fixation and paraffin embedding. Original endometrial samples were evaluated for normalcy as well as for determination of phase using the criteria of Noyes et al. (1975)Go. Of the 29 samples, 24 biopsies were obtained from endometrium post-ovulation days 3–9 and five biopsies were obtained from endometrium post-ovulation days 10–12. A potion of each biopsy was cultured under sterile conditions similar to the methods of Marbaix et al. (1992)Go and Osteen et al. (1994)Go with some modifications. Briefly, human endometrium from biopsies during the secretory phase were washed two or three times with Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with 1% antibiotic/antimycotic solution (penicillin/streptomycin/amphotericin; Life Technologies, USA), and cut into 1–2 mm3 uniform explants with a sterile scalpel blade. The tissue pieces were then washed once more, placed in tissue culture inserts (Millicell-CM inserts; Fisher Scientific Co., USA), and cultured in DMEM/F-12 medium, devoid of Phenol Red and serum with and without supplementation of 17{beta}-estradiol (E2, 10 nM/l), progesterone (100 nM/l), and E2 + progesterone. Explants were incubated for 6, 24 and 48 h as indicated. The medium was renewed daily (2 ml/insert).

All samples including treated and untreated explants generated from the 29 biopsies were processed for haematoxylin and eosin staining and analysed histologically by an expert pathologist. Due to a smaller amount of tissue in the biopsies of some patients, not all biopsies generated a sufficient number of explants to perform all of the assays. Twelve of the 29 biopsies and corresponding explants were processed for TdT (terminal deoxynucleotidyl transferase) mediated dUDP nick-end labelling (TUNEL) staining; eight for detection of DNA fragmentation; eight for M30 and cleaved CASP3 immunostaining; and six for real-time PCR analysis.

The primary antibodies used were: M30 CytoDEATH (Roche Applied Science, USA), a mouse monoclonal antibody (clone M30) for the detection of a caspase cleavage product cytokeratin 18 (Caulin et al., 1997Go; Leers et al., 1999Go; Michael-Robinson et al., 2001Go) (1:250); and anti-cleaved CASP3 (ab-2) rabbit polyclonal antibody (1:50; Oncogene, USA).

TUNEL assay
To detect apoptosis in individual cells, DNA strand breaks from 12 biopsies (post-ovulation days 3–10) and corresponding explants were labelled by fluorescein-conjugated TUNEL assay (Gavrieli et al., 1992Go) with In Situ Cell Death Detection Kit (Roche, USA) following the manufacturer's instructions. Briefly, after deparaffinization, sections were microwaved in 0.1 mol/l citrate buffer (pH 6.0) for 5 min at 350 W and washed and immersed in 0.1 mol/l Tris–HCl (pH 7.5) containing 3% bovine serum albumin, and 20% normal bovine serum. Sections were incubated with TUNEL reaction mixture for 60 min at 37 °C, and propidium iodide (PI) was added to stain all cells. The sections were then mounted and photographed with a Zeiss confocal microscope (Carl Zeiss, Inc., Germany) (Cheng et al., 2000Go). The number of fluorescein-dUTP end-labelled cells was quantified by counting the number of labelled cells per 1000 total cells. Two consecutive, randomly selected x 200 microscopic fields were selected for each specimen processed.

Detection of DNA fragmentation
DNA fragmentation was detected by agarose gel electrophoresis. DNA from eight biopsies (post-ovulation days 3–10) and correspondingly treated or untreated explants was extracted using a DNeasy Tissue Kit (Qiagen, USA). One microgram of each DNA sample was loaded on to a 1.2% agarose gel and electrophoresis was performed at 75 V for 90 min. DNA fluorescence was visualized by UV transillumination after staining with ethidium bromide. As a marker, a 100 bp ladder was run in parallel with the DNA samples. We expected to see a typical ladder pattern representing multiple small DNA fragments of 180–200 bp (size of one nucleosome) when apoptosis occurred (Bortner et al., 1995Go).

Immunohistochemistry
Immunohistochemical staining was performed on eight original biopsies (post-ovulation days 3–10) and correspondingly treated and untreated explants using Vectastain Elite ABC Kit (Vector Laboratories, USA) as reported previously with a few modifications (Weiss et al., 2001Go; Zhu et al., 2003Go). Briefly, after routine deparaffinization and rehydration, sections were treated with microwaves in citrate buffer (pH 6.0; Zymed Laboratories Inc.) for 15 min. After blocking of endogenous peroxidase activity, the sections were then incubated with a primary antibody (McGuckin et al., 1995Go; Dong et al., 1997Go; Mommers et al., 1999Go) at 4 °C overnight. After washing with phosphate-buffered saline (PBS), biotinylated anti-mouse or anti-rabbit IgG was applied for 30 min at room temperature. After washing with PBS, peroxidase-conjugated streptavidin solution was applied for 50 min and visualized by 0.05% 3',3'-diaminobenzidine (DAB). Counterstaining was performed lightly with haematoxylin. The sections were then dehydrated and coverslipped with mounting medium (Richard-Allan Scientific, USA). Examination and photography were performed using a Nikon light microscope equipped with a digital camera.

Real-time PCR
Six original biopsies (post-ovulation days 3–10) and correspondingly treated or untreated explants were processed for real-time PCR analysis. Total RNA was extracted using TRIzol Reagent (Invitrogen). Two micrograms of total RNA were reverse-transcribed with SuperscriptTM II RNase H reverse transcriptase (Invitrogen) using random primers (Invitrogen), according to the manufacturer's instructions. The quantification of the selected genes by real-time PCR was performed using a LightCycler (Roche, Germany). Oligonucleotide primers were designed using LightCycler Probe Design Software. The nucleotide sequences of the primers used are: BCL2: sense, 5'-GCCTTCTTTGAGTTCGG-3', antisense, 5'-GGGTGATGCAAGCTCC-3', 286 bp; BAX: sense, 5'-GCATCGGGGACGAACTGG-3', antisense, 5'-GTCCCAAAGTAGGAGAGGA-3', 306 bp; and {beta}-actin (ACTB): sense, 5'-CTTCCCCTCCATCGTGGG-3', antisense, 5'-GTGGTACGGCCAGAGGCG-3', 255 bp. The optimal PCR reactions for all investigated genes were established using the LightCycler Fast Start DNA Master SYBR Green I Kit (Roche), according to the manufacturer's instructions; annealing temperatures and MgCl2 concentrations were optimized to create a one-peak melting curve. Additionally, the PCR reactions were recovered after each PCR analysis and amplicons were checked by agarose gel electrophoresis for a single band of the expected size. The running protocol was programmed on the LightCycler software, version 3.5. Each reaction had a total volume of 20 µl including 2 µl of cDNA and 18 µl of a reaction mixture. The program consisted of the following four steps: (i) denaturation program, 94 °C, 5 min; (ii) amplification and quantification program, 10 s at 95 °C, 5 s at 60 °C, and 15 s at 72 °C; (iii) melting curve program: the reaction temperature was rapidly increased to 95 °C, then decreased to 60 °C for 15 s, and finally slowly increased to 98 °C at a rate of 0.1 °C/s, with continuous fluorescence monitoring); (iv) cooling program down to 40 °C.

A relative quantification analysis on a single channel experiment was carried out with the LightCycler software, version 4 (Roche). The analysis uses the sample's crossing point, the efficiency of the reaction (specified an efficiency value of 2), the number of cycles completed, and other values to compare the samples and generate the ratios. Two ratios were compared: the ratio of a target DNA sequence to a reference DNA sequence (ACTB) in samples from cultured explants with or without treatment, and the ratio of the same two sequences in samples from original biopsies serving as ‘Calibrator’. The results are expressed as a normalized ratio.

Statistical analysis
Results are expressed as means ± SD for the number of experiments indicated. Statistical analysis was performed using unpaired two-tailed Student's t-test for continuous variables and Fisher's exact test for configured variables. Differences were considered significant at P<0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Menstrual-like breakdown in cultured endometrial explants
Menstrual-like endometrial breakdown as defined by stromal breakdown, glandular collapse and architectural fragmentation was identified in all endometrial explants (n=29) that were cultured in the absence of estradiol (E2) and progesterone. In most instances the degree of breakdown was significant, affecting large portions of the explant (Figure 1B, E). Addition of E2 and progesterone alone or in combination substantially reduced or completely prevented the menstrual-like breakdown seen in the explants. Subjectively, the reduction of the menstrual-like changes appeared to be greatest when utilizing the combination of E2 and progesterone and least when treated with E2 (data not shown). In some tissue samples treated with the combination of E2 and progesterone, no tissue breakdown changes were detected (Figure 1C). The morphological changes observed in this in vitro system appear highly associated with the post-ovulation day of the harvested human endometrial sample. When the endometrial biopsy sample was harvested from women who were 3–9 days post-ovulation, the explant cultured in the absence of hormone supplementation exhibited a large extent of menstrual-like breakdown, a change that was minimized or did not occur at all when the same sample was cultured in the presence of E2 and/or progesterone (Figure 1AC). In contrast, if the explant was obtained from women who were 10–12 days post-ovulation, the degree of menstrual-like breakdown was greater and more extensive than that seen in samples of earlier endometrium and could not be reversed by hormonal supplementation (Figure 1DF). So although in most instances menstrual-like breakdown could be prevented in endometrial explants by the addition of sex steroids to the culture media, very late secretory endometrium did not seem to respond in the same manner.



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Figure 1. Histological analysis of menstrual-like breakdown in endometrial explants. (AC) An endometrial biopsy sample harvested from a woman who was 4 days post-ovulation. (A) Before culture; (B) after culture for 48 h; (C) endometrium from same patient cultured 48 h with estradiol (E2) 10 nM/l and progesterone 100 nM/l supplementation. Note that there is less tissue breakdown. (DF) An endometrial biopsy sample harvested from a woman who was 11 days post-ovulation. (D) Before culture; (E) after culture for 48 h; (F) culture 48 h with E2 10 nM/l and progesterone 100 nM/l supplementation. Note that breakdown could not be reversed by hormone supplementation.

 
Apoptosis in cultured endometrial explants
TUNEL analysis was performed to identify the cells involved in the apoptotic DNA fragmentation in endometrial explants (n=12: six hormone-supplemented explants, six non-supplemented with hormones). As shown in Figure 2A, in situ labelling revealed a marked increase in the number of apoptotic cells (133 ± 33 cells per 1000) in cultured endometrial explants when compared to non-cultured endometrium (7.4 ± 4.1 cells per 1000) (P<0.05). Identical or similar results were observed in all endometrial explants determined. To verify that apoptosis, not necrosis, had occurred, we further examined DNA ladder formation, which is regarded as another indicator of apoptosis. Figure 2B shows that the DNA of endometrium from women who were either 3 days or 10 days post-ovulation was markedly fragmented, and demonstrated a distinctive ladder pattern of multiple 200 bp fragments after incubation of 24 and 48 h. In contrast, control samples extracted from the original endometrial tissue biopsy lacked this pattern.



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Figure 2. Detection of apoptosis in cultured endometrial explants. (A) Representative sample of TUNEL assay. The endometrial biopsy sample was harvested from a woman 8 days post-ovulation and cultured for 48 h. The fragmented DNA of apoptotic cells was detected by catalytically incorporating fluorescein-12-dUTP at the 3'-OH DNA ends using the principle of TUNEL assay. The DNA strand breaks of apoptotic cells were labelled with fluorescein-12-dUTP (b, e and h, green), and cell nuclei were stained with propidium iodide (a, d and g, red). (ac) The endometrium before culture; (df) the endometrium after culture for 48 h. (gi) DNAase-treated endometrium (before culture) serves as a positive control. (c) Overlay of a and b; (f) overlay of d and e; (i) overlay of g and h. (B) Agarose gel analysis of DNA fragmentation. The endometrial biopsy samples were harvested from women 3 and 10 days post-ovulation and cultured for 24 and 48 h. Genomic DNA was extracted and loaded onto agarose gel. POD = post-ovulation; L = 100 bp ladder.

 
Importantly, although supplementation of endometrial explants was noted to decrease or eliminated menstrual-like breakdown in virtually all samples, there was no appreciable difference in the amount of apoptosis seen in any of the endometrial explant samples whether or not they had received supplementation with E2 (192 ± 26 cells per 1000), progesterone (151 ± 42 cells per 1000) or E2 + progesterone (185 ± 54 cells per 1000) when compared to untreated control samples (133 + 33 cells per 1000) (P=0.07, P=0.60, P=0.23 respectively) (Figure 3A). The amount of apoptosis for E2 or E2 + progesterone-supplemented explants were higher than for non-supplemented or progesterone-supplemented explants, but the differences were not statistically significant. Figure 3B shows the DNA of endometrium as well as endometrial explants from a woman 8 days post-ovulation. The DNA ladder pattern remained unchanged after culture with or without hormone supplementation. This dissociation of apoptotic activity from the presence or absence of menstrual-like breakdown strongly suggests that apoptosis and breakdown are not mechanistically related.



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Figure 3. Detection of apoptosis in cultured endometrial explants with estradiol + progesterone (EP) supplementation. (A) Representative TUNEL assay for endometrial explants with or without EP supplementation. The endometrial biopsy sample was harvested from a woman 8 days post-ovulation and cultured for 48 h. PI = propidium iodide; C = cultured explants without treatment; E = 10 nM/l; progesterone = 100 nM/l. Scale bar = 20 µm. (B) Representative agarose gel analysis of DNA fragmentation for endometrial explants with or without EP supplementation. The endometrial biopsy sample was harvested from a woman 4 days post-ovulation and cultured for 48 h. L = 100 bp ladder; BC = before culture; C = cultured explants without treatment; E1 = 1 nM/l; E2 = 10 nM/l; P1 = 10 nM/l; P2 = 100 nM/l; EP1 = E1 + P1; EP2 = E2 + P2.

 
Caspase activation in cultured endometrial explants
In order to investigate possible mechanisms of apoptosis during explant culture, we tested for the presence of caspase activation (n = 8). Using M30, a monoclonal antibody for the detection of a caspase cleavage product of cytokeratin 18, we noted a significant increase in the expression of M30-positive cells in glandular cells of endometrial explants following 6 h (50.25 ± 11.84 cells per 1000) and 24 h (6 ± 2.45 cells per 1000) of culture when compared to endometrium prior to culture (1.25 ± 1.26 cells per 1000) (P<0.001, P<0.05 respectively) (Figure 4AD). Since the labelling with M30 decreases in the later stages of apoptosis due to progressive degradation of CK18 (Bantel et al., 2001Go; Duan et al., 2003Go), we next applied an anti-cleaved CASP3 antibody to the tissues. Our data demonstrated that activated CASP3 immunoreactivity was mostly found in the cytoplasm of apoptotic cells and increased remarkably in both glandular and stromal cells after culture (Figure 4EH). In support of the data demonstrating the degree of apoptosis identified in our samples using both the TUNEL and DNA fragmentation assays, no significant decrease in expression of CASP3 activation was identified in endometrial explants supplemented with E2 and/or progesterone compared to those without supplementation (Figure 4IL and Table I). These data seem further to dissociate the occurrence of apoptosis and menstrual-like breakdown by demonstrating that the enzymatic mediators of apoptosis are not modulated by interventions that prevent breakdown.



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Figure 4. Detection of caspase activation in cultured endometrial explants. (AD) Representative immunohistochemical staining for M30 antigen, a caspase-cleaved epitope of cytokeratin 18 protein. The endometrial biopsy sample was harvested from a woman 7 days post-ovulation and cultured for 6 and 24 h. (A) Before culture; (B) negative control (the same section as in A), stained in the absence of primary antibody; (C) culture 6 h; (D) culture 24 h. (EH) Representative immunohistochemical staining for cleaved CASP3 antigen. The endometrial biopsy sample was harvested from a woman 7 days post-ovulation and cultured for 6 and 24 h. (E) Before culture; (F) negative control (the same section as in E), stained in the absence of primary antibody; (G) culture 6 h; (H) culture 24 h. (IL) Representative immunohistochemical staining for cleaved CASP3 antigen in endometrial explants with estradiol + progesterone supplementation. The endometrial biopsy sample was harvested from a woman 9 days post-ovulation and cultured for 48 h. (I) Untreated control; (J) E, 10 nM/l; (K) P, 100 nM/l; (L) estradiol + progesterone.

 

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Table I. Determination of CASP3 activation in human endometrium and endometrial explants with estradiol (E2) and progesterone (P) supplementation at 48 h by immunohistochemistry

 
Apoptosis-related gene expression in cultured endometrial explants
To detect further which genes were involved in the regulation of apoptosis in cultured endometrial explants, apoptosis-related gene mRNA levels for BCL2 and BAX were quantified by real-time PCR analysis (n=6). Agarose gel electrophoresis of PCR products demonstrated a single band of the expected molecular weight (Figure 5). Relative quantification analysis revealed that BAX mRNA levels increased after culture in all samples analysed (Table II). In contrast, BCL2 mRNA varied greatly in the patients evaluated. BCL2 decreased after culture in samples obtained in the mid-secretory phase whereas those obtained in the early secretory phase demonstrated an increase. However, when the BCL2:BAX ratio was calculated, a consistently low ratio was present in all samples. The low BCL2:BAX ratio was also observed in explants with or without steroid supplementation when compared to non-cultured tissue samples in both early and mid-secretory phase (Table III). The levels of these two molecular determinants of apoptotic activity support the increased apoptotic activity observed in our explants. Persistently low BCL2:BAX ratios, in the face of hormone supplementation, also supports the notion that apoptosis and menstrual-like breakdown are dissociated in endometrial explants.



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Figure 5. Agarose gel electrophoresis of real-time PCR products. A single band of the expected molecular weight confirms the specificity of the LightCycler PCR. ACTB was used as a normalization control for real-time PCR reaction. (A) Gene expressions in original endometrial biopsies and endometrial explants without hormone supplementation. (B) Gene expressions in original endometrial biopsies and endometrial explants with or without hormone supplementation for 48 h. POD = post-ovulation day; BC = before culture; C = cultured explants without hormone supplementation; E = estradiol; P = progesterone.

 

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Table II. Quantification of BCL2 and BAX mRNA in human endometrium and endometrial explants by real-time PCR

 

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Table III. Quantification of BCL2 and BAX mRNA in human endometrium and endometrial explants with estradiol (E2) and progesterone (P) supplementation at 48 h by real-time PCR

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The results of the studies described here demonstrate the occurrence of apoptosis in human endometrial tissue explants in culture. We have also identified evidence that caspase activation is involved in this process. To our knowledge, this is the first report to demonstrate that apoptosis occurs in a human in vitro model of endometrium. The current study also shows that cultured endometrial explants demonstrate morphological changes analogous to those seen in physiological menstrual endometrium in vivo. Finally, the menstrual-like changes seen in the explant model can be reduced or prevented, in most instances, by the addition of hormonal supplementation with E2 and progesterone.

The observation that menstrual-like breakdown occurs in cultured endometrial explants was first noted by Marbaix et al. (1996)Go. As in our endometrial explant model, Marbaix et al. showed that the addition of physiological levels of sex steroid hormones was able to delay or prevent the menstrual-like changes seen in explants cultured in media lacking these hormones. Our findings parallel those seen by Marbaix et al. (1995)Go in most instances; we also demonstrated that the ability of supplemental hormones to reduce or prevent tissue breakdown in the implant depends, in large part, on the number of days post-ovulation at which the original endometrial sample is obtained. In our studies, if the endometrium had been harvested very late in the secretory phase (e.g. post-ovulation day 12), the process of menstrual-like breakdown was irreversible. This apparent irreversibility seen in the late secretory period may represent a point of no return at which molecular and biochemical changes in the endometrium will inevitably lead to a breakdown of tissue. This finding is important in the selection of endometrial tissue for the establishment of explants in culture. If selected too late in the secretory phase, hormonal and molecular manipulations may not accurately represent the normal physiological response of endometrium in the secretory phase but rather that of the menstrual phase.

In normal human endometrium in vivo, apoptosis has been reported to appear in the mid-secretory phase, to increase in the late secretory phase, and be maximal during the menstrual phase (Dahmoun et al., 1999Go). The association between apoptosis and the tissue breakdown that occurs at the time of menstruation has led some investigators to postulate a mechanistic role of apoptosis in the process of endometrial tissue breakdown (Kokawa et al., 1996Go). Our findings confirm that apoptosis occurs in vivo in samples of secretory endometrium examined immediately after biopsy. We also observed a significant increase in apoptosis in vitro in endometrial explants that were undergoing menstrual-like breakdown. The degree of apoptosis in our explants did not appreciably decrease when tissue breakdown was minimized or prevented by the addition of physiological levels of both estradiol and progesterone to the culture media. The dissociation between tissue breakdown and apoptosis shown in our samples suggests that, although menstrual-like breakdown and apoptosis can occur at the same time in an endometrial sample, the two processes may not be linked and are probably not mechanistically related.

Apoptosis is most commonly associated with processes that require tissue remodelling (Fadeel et al., 2000Go; Fadeel, 2001Go, 2003Go; Mor et al., 2001Go). Although apoptosis can be seen in senescence of embryological structures and other destructive events (Jacobson et al., 1997Go; Todaro et al., 2004Go), it is seldom, if ever, seen in massive tissue destruction. We speculate that the apoptosis seen in endometrium during the late secretory phase and menstrual phase is not responsible for the tissue breakdown but rather is involved in the impending remodelling that will occur in the endometrium during or following the secretory phase. The two possible outcomes of the endometrium, after the secretory phase, are either implantation, in the event that the ovum is fertilized, or menstruation, in the case when no fertilization occurs. Both of these functions require massive remodelling of the endometrium. With fertilization, the zygote will implant into the endometrium during the process of nidation, triggering changes that will result in the decidua of pregnancy. If fertilization fails, the endometrium will need to reorganize to form the new functional layer for the following cycle. Von Rango et al. (1998)Go previously noted that the occurrence of apoptosis during the mid-secretory phase coincided with the window of endometrial receptivity for implantation of a fertilized ovum. They postulated that endometrial apoptosis was probably involved in the process of endometrial preparation for implantation and pregnancy. We propose that the increase in apoptosis during the late secretory and menstrual phase is not linked to the process of tissue breakdown but rather is present in preparation for the endometrial remodelling that needs to occur to restore normal endometrial architecture following menstruation. Based on their findings demonstrating the discrepancy between glandular and stromal apoptosis observed at various points in the menstrual cycle, Dahmoun et al. (1999)Go postulated a similar remodelling role for apoptosis in the endometrium. Although the mechanism of this remodelling is unknown, one can speculate that after shedding of the endometrial functionalis, residual basal functionalis and upper basalis must coordinate cell death and cell growth in concert to provide rapid and architecturally appropriate repair of the bleeding endometrium.

The increased expression of BAX mRNA detected in our endometrial explants is consistent with previous observations correlating elevated levels of BAX with apoptosis (Tao et al., 1997Go; Fadeel et al., 1999Go). In contrast, we observed a variable expression of BCL2 in the endometrial explants. Predominantly, the explants from POD 9–10 demonstrated the expected decrease of BCL2 expression that is usually associated with apoptosis. BCL2 has been shown to be an inhibitor of apoptosis in numerous experimental and naturally occurring models (Fadeel et al., 1999Go; Marone et al., 2000Go; Vaskivuo et al., 2000Go). Cyclic BCL2 gene expression in human endometrium during menstrual cycle has been reported (Gompel et al., 1994Go; Otsuki et al., 1994Go). The decreased expression seen in some of our explants is consistent with a permissive molecular environment for apoptosis to occur. Several of our samples exhibited an increase in the expression of BCL2 when apoptosis increased. This discrepancy has also been demonstrated in vivo (Gompel et al., 1994Go; Dahmoun et al., 1999Go). This phenomenon has been explained by a difference of behaviour between glandular and stromal BCL2 immunostaining as well as the timing of sampling in the pre-menstrual days (Dahmoun et al., 1999Go). In our study, in all cases examined, the ratio of BCL2 to BAX was low. It has been proposed that the cellular BCL2:BAX ratio may be more indicative of the regulation of apoptosis than one or the other factor independently (Vaskivuo et al., 2000Go, 2002Go). A high BCL2:BAX ratio makes cells resistant to apoptotic stimuli, whereas a low ratio induces cell death. Given that not only BCL2 family members but also FAS/FASLG can regulate CASP3 activity (Goyal, 2001Go; Opferman and Korsmeyer, 2003Go), the role of FAS and FASLG in endometrial explants should also be taken into consideration.

Finally, our experimental explant system has the inherent limitations of in vitro culture systems as well as limitations of tissue obtained from different subjects. Variations between individuals can be seen within similar post-ovulation day samples. In addition, samples of different histologically confirmed post-ovulation days are difficult to obtain due to variation in follicular phase duration between individuals. However, we feel that the benefits of having an in vitro system in which endometrium with a preserved glandular and stromal architecture can be manipulated experimentally greatly outweigh these limitations. It should be borne in mind that the apoptosis seen in the endometrial explants could have occurred as a result of the preparation and culture process. We have performed the experiments to compare the explants cultured in serum-containing medium to explants in serum-free medium, and have found that there is no difference between these two conditions (data not shown). It appears that serum starvation is not the cause of apoptosis in our system. We have also examined apoptosis using the TUNEL assay of post-ovulation day 10 endometrium and compared it to endometrium explants obtained from post-ovulation day 8 endometrium and then cultured with EP for 48 h (data not shown). Under these conditions, we found much higher apoptotic rates in the endometrium cultured for 48 h when compared to endometrium of the same post ovulation period without culture. These data support an increase in apoptosis of endometrium when cultured in vitro. Although the process of tissue culture may be responsible for increased apoptosis, we show that endometrial breakdown can be prevented with the addition of hormones, again suggesting dissociation between apoptosis and breakdown.

In summary, we have confirmed the occurrence of menstrual like breakdown in cultured endometrial explants and showed that this breakdown can be reduced or prevented by supplementation with E2 + progesterone. This is the first report describing the presence of apoptosis in an in vitro model of human endometrium similar to what is seen in human endometrium in vivo. Importantly, our results show that in vitro menstrual-like breakdown and apoptosis may not be functionally linked. Rather, apoptosis is more likely to be involved in the impending remodelling of the endometrium following menstruation. The parallels we have found between normal human endometrium and our in vitro endometrial explant system support our use of this easily manipulated system to study the physiology of normal human endometrium.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Dr Richard Paulson for his guidance and support in the preparation of this manuscript. This work was supported by a grant from the National Institute of Child Health and Human Development (RO-1 HD 43189).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Abel MH and Baird DT (1980) The effect of 17 beta-estradiol and progesterone on prostaglandin production by human endometrium maintained in organ culture. Endocrinology 106, 1599–1606.[Abstract]

Adams JM and Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326.[Abstract/Free Full Text]

Adams JM and Cory S (2001) Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 26, 61–66.[CrossRef][ISI][Medline]

Bantel H, Ruck P, Gregor M and Schulze-Osthoff K (2001) Detection of elevated caspase activation and early apoptosis in liver diseases. Eur J Cell Biol 80, 230–239.[ISI][Medline]

Bortner CD, Oldenburg NB and Cidlowski JA (1995) The role of DNA fragmentation in apoptosis. Trends Cell Biol 5, 21–26.[CrossRef][ISI][Medline]

Budihardjo I, Oliver H, Lutter M, Luo X and Wang X (1999) Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15, 269–290.[CrossRef][ISI][Medline]

Castro A, Johnson MC, Anido M, Cortinez A, Gabler F and Vega M (2002) Role of nitric oxide and bcl-2 family genes in the regulation of human endometrial apoptosis. Fertil Steril 78, 587–595.[CrossRef][ISI][Medline]

Caulin C, Salvesen GS and Oshima RG (1997) Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 138, 1379–1394.[Abstract/Free Full Text]

Chen JC, Lin JH, Jow GM, Peng YI, Su TH, Tsai YF and Chen TJ (2001) Involvement of apoptosis during deciduomal regression in pseudopregnant hamsters effect of progesterone. Life Sci 68, 815–825.[CrossRef][ISI][Medline]

Cheng L, Zhu H, Wang A, Ren F, Chen J and Glasier A (2000) Once a month administration of mifepristone improves bleeding patterns in women using subdermal contraceptive implants releasing levonorgestrel. Hum Reprod 15, 1969–1972.[Abstract/Free Full Text]

Csermely T, Demers LM and Hughes EC (1969) Organ culture of human endometrium. Effects of progesterone. Obstet Gynecol 34, 252–259.[ISI][Medline]

Dahmoun M, Boman K, Cajander S, Westin P and Backstrom T (1999) Apoptosis, proliferation and sex hormone receptors in superficial parts of human endometrium at the end of the secretory phase. J Clin Endocrinol Metab 84, 1737–1743.[Abstract/Free Full Text]

Dong Y, Walsh MD, Cummings MC, Wright RG, Khoo SK, Parsons PG and McGuckin MA (1997) Expression of MUC1 and MUC2 mucins in epithelial ovarian tumours. J Pathol 183, 311–317.[CrossRef][ISI][Medline]

Duan WR, Garner DS, Williams SD, Funckes-Shippy CL, Spath IS and Blomme EA (2003) Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol 199, 221–228.[CrossRef][ISI][Medline]

Fadeel B (2001) [Apoptosis—emperor's new clothes?]. Lakartidningen 98, 4613–4614.[Medline]

Fadeel B (2003) Programmed cell clearance. Cell Mol Life Sci 60, 2575–2585.[CrossRef][ISI][Medline]

Fadeel B, Zhivotovsky B and Orrenius S (1999) All along the watchtower: on the regulation of apoptosis regulators. FASEB J 13, 1647–1657.[Abstract/Free Full Text]

Fadeel B, Henter JI and Orrenius S (2000) [Apoptosis required for maintenance of homeostasis: familial hemophagocytic lymphohistiocytosis caused by too little cell death.] Lakartidningen 97, 1395–1400. 1402.[Medline]

Gavrieli Y, Sherman Y and Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119, 493–501.[Abstract]

Gompel A, Sabourin JC, Martin A, Yaneva H, Audouin J, Decroix Y and Poitout P (1994) Bcl-2 expression in normal endometrium during the menstrual cycle. Am J Pathol 144, 1195–1202.[Abstract]

Goyal L (2001) Cell death inhibition: keeping caspases in check. Cell 104, 805–808.[CrossRef][ISI][Medline]

Hopwood D and Levison DA (1976) Atrophy and apoptosis in the cyclical human endometrium. J Pathol 119, 159–166.[ISI][Medline]

Illouz S, Boubli L, Lavaut MN, Allasia C and Charpin C (2000) Endometrial response to sexual steroids as assessed by prostaglandin F (2alpha) output in explant culture and hormone receptor expression. Gynecol Obstet Invest 50, 43–49.[CrossRef][ISI][Medline]

Jacobson MD, Weil M and Raff MC (1997) Programmed cell death in animal development. Cell 88, 347–354.[CrossRef][ISI][Medline]

Kokawa K, Shikone T and Nakano R (1996) Apoptosis in the human uterine endometrium during the menstrual cycle. J Clin Endocrinol Metab 81, 4144–4147.[Abstract]

Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B, Bjorklund P, Ramaekers FC, Bjorklund B, Nap M et al. (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187, 567–572.[CrossRef][ISI][Medline]

Marbaix E, Donnez J, Courtoy PJ and Eeckhout Y (1992) Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants. Proc Natl Acad Sci USA 89, 11789–11793.[Abstract/Free Full Text]

Marbaix E, Kokorine I, Henriet P, Donnez J, Courtoy PJ and Eeckhout Y (1995) The expression of interstitial collagenase in human endometrium is controlled by progesterone and by oestradiol and is related to menstruation. Biochem J 305(Pt 3), 1027–1030.[ISI][Medline]

Marbaix E, Kokorine I, Moulin P, Donnez J, Eeckhout Y and Courtoy PJ (1996) Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases. Proc Natl Acad Sci USA 93, 9120–9125.[Abstract/Free Full Text]

Marone M, Ferrandina G, Macchia G, Mozzetti S, de Pasqua A, Benedetti-Panici P, Mancuso S and Scambia G (2000) Bcl-2, Bax, Bcl-x (L) and Bcl-x (S) expression in neoplastic and normal endometrium. Oncology 58, 161–168.[CrossRef][ISI][Medline]

McGuckin MA, Walsh MD, Hohn BG, Ward BG and Wright RG (1995) Prognostic significance of MUC1 epithelial mucin expression in breast cancer. Hum Pathol 26, 432–439.[CrossRef][ISI][Medline]

Michael-Robinson JM, Biemer-Huttmann A, Purdie DM, Walsh MD, Simms LA, Biden KG, Young JP, Leggett BA, Jass JR and Radford-Smith GL (2001) Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status. Gut 48, 360–366.[Abstract/Free Full Text]

Mommers EC, Leonhart AM, von Mensdorff-Pouilly S, Schol DJ, Hilgers J, Meijer CJ, Baak JP and van Diest PJ (1999) Aberrant expression of MUC1 mucin in ductal hyperplasia and ductal carcinoma in situ of the breast. Int J Cancer 84, 466–469.[CrossRef][ISI][Medline]

Mor G, Aschkenazi S and Song J (2001) Sex hormones, apoptosis and the fas/fas ligand system in normal endometrial tissue remodeling. Sci World J 1, 99.

Nakano R and Shikone T (1996) [Apoptosis in the reproductive system.] Nippon Sanka Fujinka Gakkai Zasshi 48, 721–732.[Medline]

Noyes RW, Hertig AT and Rock J (1975) Dating the endometrial biopsy. Am J Obstet Gynecol 122, 262–263.[Medline]

Opferman JT and Korsmeyer SJ (2003) Apoptosis in the development and maintenance of the immune system. Nat Immunol 4, 410–415.[CrossRef][ISI][Medline]

Osteen KG, Rodgers WH, Gaire M, Hargrove JT, Gorstein F and Matrisian LM (1994) Stromal-epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium. Proc Natl Acad Sci USA 91, 10129–10133.[Abstract/Free Full Text]

Otsuki Y, Misaki O, Sugimoto O, Ito Y, Tsujimoto Y and Akao Y (1994) Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle. Lancet 344, 28–29.[ISI][Medline]

Rotello RJ, Hocker MB and Gerschenson LE (1989) Biochemical evidence for programmed cell death in rabbit uterine epithelium. Am J Pathol 134, 491–495.[Abstract]

Sengupta J, Dhawan L, Lalitkumar PG and Ghosh D (2003) A multiparametric study of the action of mifepristone used in emergency contraception using the Rhesus monkey as a primate model. Contraception 68, 453–469.[CrossRef][ISI][Medline]

Shikone T, Kokawa K, Yamoto M and Nakano R (1997) Apoptosis of human ovary and uterine endometrium during the menstrual cycle. Horm Res 48(Suppl 3), 27–34.[ISI][Medline]

Tao XJ, Tilly KI, Maravei DV, Shifren JL, Krajewski S, Reed JC, Tilly JL and Isaacson KB (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.[Abstract/Free Full Text]

Todaro M, Zeuner A and Stassi G (2004) Role of apoptosis in autoimmunity. J Clin Immunol 24, 1–11.[CrossRef][ISI][Medline]

Vaskivuo TE, Stenback F, Karhumaa P, Risteli J, Dunkel L and Tapanainen JS (2000) Apoptosis and apoptosis-related proteins in human endometrium. Mol Cell Endocrinol 165, 75–83.[CrossRef][ISI][Medline]

Vaskivuo TE, Stenback F and Tapanainen JS (2002) Apoptosis and apoptosis-related factors Bcl-2, Bax, tumor necrosis factor-alpha and NF-kappaB in human endometrial hyperplasia and carcinoma. Cancer 95, 1463–1471.[CrossRef][ISI][Medline]

von Rango U, Classen-Linke I, Krusche CA and Beier HM (1998) The receptive endometrium is characterized by apoptosis in the glands. Hum Reprod 13, 3177–3189.[Abstract]

Weiss ER, Ducceschi MH, Horner TJ, Li A, Craft CM and Osawa S (2001) Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction. J Neurosci 21, 9175–9184.[Abstract/Free Full Text]

Zhu X, Brown B, Li A, Mears AJ, Swaroop A and Craft CM (2003) GRK1-dependent phosphorylation of S and M opsins and their binding to cone arrestin during cone phototransduction in the mouse retina. J Neurosci 23, 6152–6160.[Abstract/Free Full Text]

Submitted on May 14, 2004; resubmitted on August 23, 2004; accepted on January 28, 2005.