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
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Abstract |
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Key words: apoptosis/BCL2:BAX ratio/caspase activity/endometrial explant/menstrual breakdown
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Introduction |
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Apoptosis has been suggested to play a pivotal role in the reproductive physiology of the endometrium (Kokawa et al., 1996; Nakano and Shikone, 1996
; Shikone et al., 1997
; Vaskivuo et al., 2000
, 2002
; Castro et al., 2002
). In the endometrium, apoptosis has been shown to be regulated by steroid hormones in several mammalian species including rabbits (Rotello et al., 1989
), hamsters (Chen et al., 2001
), and monkeys (Sengupta et al., 2003
). Hopwood and Levinson (1976)
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., 1996
; Nakano and Shikone, 1996
; Shikone et al., 1997
; Vaskivuo et al., 2000
, 2002
; Castro et al., 2002
). 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., 1996
).
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, 1998). The BCL2 proto-oncogene promotes cell survival by blocking the apoptosis induced by different stimuli (Adams and Cory, 2001
), 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., 1999
).
Several investigators have successfully maintained human endometrial explants in tissue culture (Csermely et al., 1969; Abel and Baird, 1980
; Marbaix et al., 1992
, 1996
; Illouz et al., 2000
). 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., 1996
). The purpose of this study was to assess the role of apoptosis in menstrual-like breakdown in human endometrial explants.
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Materials and methods |
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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., 1997; Leers et al., 1999
; Michael-Robinson et al., 2001
) (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 310) and corresponding explants were labelled by fluorescein-conjugated TUNEL assay (Gavrieli et al., 1992) 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 TrisHCl (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., 2000
). 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 310) 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 180200 bp (size of one nucleosome) when apoptosis occurred (Bortner et al., 1995).
Immunohistochemistry
Immunohistochemical staining was performed on eight original biopsies (post-ovulation days 310) and correspondingly treated and untreated explants using Vectastain Elite ABC Kit (Vector Laboratories, USA) as reported previously with a few modifications (Weiss et al., 2001; Zhu et al., 2003
). 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., 1995
; Dong et al., 1997
; Mommers et al., 1999
) 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 310) 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 -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.
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Results |
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Discussion |
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The observation that menstrual-like breakdown occurs in cultured endometrial explants was first noted by Marbaix et al. (1996). 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)
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., 1999). 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., 1996
). 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., 2000; Fadeel, 2001
, 2003
; Mor et al., 2001
). Although apoptosis can be seen in senescence of embryological structures and other destructive events (Jacobson et al., 1997
; Todaro et al., 2004
), 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)
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)
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., 1997; Fadeel et al., 1999
). In contrast, we observed a variable expression of BCL2 in the endometrial explants. Predominantly, the explants from POD 910 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., 1999
; Marone et al., 2000
; Vaskivuo et al., 2000
). Cyclic BCL2 gene expression in human endometrium during menstrual cycle has been reported (Gompel et al., 1994
; Otsuki et al., 1994
). 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., 1994
; Dahmoun et al., 1999
). 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., 1999
). 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., 2000
, 2002
). 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, 2001
; Opferman and Korsmeyer, 2003
), 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.
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Acknowledgements |
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Submitted on May 14, 2004; resubmitted on August 23, 2004; accepted on January 28, 2005.