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BOR - Papers in Press, published online ahead of print April 13, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037168
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BIOLOGY OF REPRODUCTION 73, 382–388 (2005)
DOI: 10.1095/biolreprod.104.037168
© 2005 by the Society for the Study of Reproduction, Inc.

Nonoxynol-9 Induces Apoptosis of Endometrial Explants by Both Caspase-Dependent and -Independent Apoptotic Pathways1

John K. Jain 2 3, Aimin Li 3, Deborah L. Nucatola 3, Parviz Minoo 4, and Juan C. Felix 3,5 

Departments of Obstetrics and Gynecology,3 Pediatrics,4 Pathology,5 Keck School of Medicine, University of Southern California, Los Angeles, California 90033


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Contraceptive microbicides formulated as vaginal gels offer the possibility of women-controlled contraception and prevention of HIV infection. The effects of these gels on the upper reproductive tract are largely unknown. The purpose of this study was to determine whether nonoxynol-9 (N-9) induces apoptosis in human endometrium using endometrial explant as a model. 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. The ability of the broad-spectrum caspase inhibitor and CASP3-specific inhibitor to prevent N-9-induced cell death was measured. Expression of apoptosis-related genes such as BCL2, BAX, Fas receptor (FAS), and Fas ligand (FASLG) was quantified using real-time polymerase chain reaction (PCR) analysis. This study demonstrated that N-9 induced DNA fragmentation and caspase activity in endometrial explants in a dose- and time-dependent manner. Caspase inhibitors did not fully prevent the N-9-induced DNA fragmentation. Real-time PCR analysis revealed that FAS and FASLG were largely increased following N-9 treatment. Together, these results suggested that apoptosis triggered by N-9 in endometrial explants is mediated upstream via FAS and FASLG, followed by CASP3 activation leading to final cell death. It appears that other factors besides caspases are also involved in the N-9-induced apoptosis.

apoptosis, caspase activation, endometrial explants, female reproductive tract, gene expression, gene regulation, nonoxynol-9, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Contraceptive microbicides offer the possibility of women-controlled contraception and prevention of HIV infection. Nonoxynol-9 (N-9) is a surfactant microbicide that has been used as a spermicide for more than a half a century. It acts by disrupting the cell membrane of sperm as well as those of some sexually transmitted viral and bacterial pathogens [1]. Unfortunately, serious public health concerns were raised following a study of N-9 in African sex workers that reported an increased rate of HIV infection when N-9 was used frequently [2–4]. It has been suggested that vaginal epithelial disruption, induction of cytokines, and recruitment of HIV host cells to the damaged area may underlie this observation [5, 6].

The effect of microbicides on the upper reproductive tract is largely unknown. Several studies have demonstrated that upper reproductive tract epithelial cell populations are susceptible to HIV transmission in a menstrual cycle-dependent manner [7–9]. Columnar epithelium of the upper reproductive tract is more susceptible to sexually transmitted bacterial diseases and may be more susceptible to HIV as well [10].

Recent studies have demonstrated normal physiologic movement of vaginal gels into the upper reproductive tract. Using magnetic resonance imaging, Barnhart demonstrated movement of 5 ml of Gadolinium-labeled N-9 into the upper female reproductive tract within 10 min of vaginal insertion [11, 12]. In another study, vaginal fluids, semen, and microbicides labeled with a sonographic contrast agent (Optison) were seen to move into the upper reproductive tract by uterine peristalsis. Finally, using a mouse model, Dayal demonstrated loss of uterine mucosa by 24 h following intravaginal or intrauterine placement of N-9 [10].

Apoptosis describes a process of programmed cell death. It is an essential process to remove excess, unwanted, and harmful cells and maintain homeostasis in all body tissues, including the endometrium [13]. In mammalian cells, the onset of apoptosis correlates with the activation of a family of cysteine proteases called caspases, which are constitutively expressed as inactive zymogens in the cytosol. Caspases constitute a potent machinery that cleaves crucial proteins of the nucleus and the cytoskeleton, inducing the phenotypic changes of apoptosis, including advanced chromatin condensation and internucleosomal DNA fragmentation [14]. Typically, two major pathways, death receptor-mediated signal and mitochondria-dependent signal, are involved in the process of caspase activation and apoptosis [15, 16]. Death receptor-mediated pathway for apoptosis involves ligation of the death receptor (FAS) to its ligand (FASLG) leading to the cleavage of procaspase-8 [17]. Members of the BCL2 family of proteins play a major role in governing the mitochondria-dependent apoptotic pathway, with proteins such as BAX functioning as inducers of apoptosis and proteins such as BCL2 as suppressors of cell death [18]. Both pathways converge on CASP3 and other executioner caspases and nucleases that drive the terminal events of programmed cell death [19].

Apoptosis can be induced in susceptible cells by a broad spectrum of events and agents such as withdrawal of growth factors [20], anticancer drugs [21], and detergents [22]. We recently demonstrated that N-9 can interrupt the functional barrier provided by the endometrium and thus facilitate infection with HIV and other pathogens [23]. The purpose of this study was to determine whether N-9, a detergent-type spermicide, could induce apoptosis in endometrial explants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Explant Culture and Reagents

This study was approved by the Institutional Review Board of the University of Southern California and Los Angeles County Medical Center. Informed consent was obtained from women participating in a different IRB-approved contraceptive study to use a portion of their initial biopsy for additional contraceptive-related studies. These subjects (n = 12) were healthy, regularly menstruating between 18 and 45 yr of age without contraindications to hormonal contraceptives. Endometrial biopsies were obtained during the midsecretory phase (Cycle Days 21–23) using a Pipelle endometrial biopsy instrument. Part of each biopsy was processed for routine histological evaluation. A portion of each biopsy was cultured under sterile conditions similar to the methods of Marbaix et al. [24] and Osteen et al. [25] with some modifications. Briefly, each specimen was washed two to three times with DMEM/F-12 medium (Gibco, Grand Islands, New York, NY) and cut into 1–2-mm3 uniform explants with a sterile scalpel blade. The tissue pieces were then washed once more, placed in multiple separate tissue culture inserts (Millicell-CM, Fisher Scientific Co., Springfield, NJ), and cultured in DMEM/F-12 medium devoid of phenol red and serum, supplemented with estradiol (10 nM) and progesterone (100 nM) with or without N-9 (0.03%, 0.3%, and 3.0%; Spectrum Chemical Mfg., Gardena, CA). Explants were incubated for either 6 or 24 h. Explants cultured from 6 of the 12 biopsies were incubated with caspase inhibitors, Z-VAD-FMK (100 µM), and Z-DEMD-FMK (100 µM) (R&D Systems, Minneapolis, MN).

Histology and Immunohistochemistry

All samples, including treated and untreated explants generated from the 12 biopsies, were fixed in 10% neutral buffered formaldehyde solution for 12–24 h, processed into paraffin blocks, stained with hematoxalin and eosin, and analyzed histologically by an expert pathologist. Because of a smaller amount of tissue in the biopsies of some patients, not all biopsies generated a sufficient number of explants to perform all the assays. Explants from 8 of the 12 biopsies were used to perform immunohistochemical staining using Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) as previously described [23]. Briefly, after routine deparaffinization and rehydration, sections were microwaved in citrate buffer (pH 6.0) for 8 min. After blocking endogenous peroxidase activity, sections were incubated at 4°C overnight with either M30 CytoDEATH (1:250; Roche Applied Science, Indianapolis, IN), a mouse monoclonal antibody that detects the caspase cleavage product, cytokeratin 18 [26], or anticleaved caspase-3 rabbit polyclonal antibody (1:50; Oncogene, San Diego, CA). 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 streptoavidin solution was applied for 50 min and visualized by 0.05% 3'-3' diaminobenzidine. Counterstaining was performed lightly with hematoxylin. The sections were then dehydrated and coverslipped with mounting medium (Richard-Allan Scientific, Kalamazoo, MI). Examination and photography was performed using an Olympus BX-50 light microscope equipped with a Canon EOS D30 digital camera.

Detection of DNA Fragmentation

DNA fragmentation was examined by agarose gel electrophoresis. DNA from the 12 original biopsies and correspondingly treated or untreated endometrial explants was extracted using a DNeasy Tissue Kit (Qiagen, Valencia, CA). One microgram of each DNA sample was loaded onto a 1.2% agarose gel, and electrophoresis was performed at 75 V for 90 min. DNA was visualized under UV light after staining with ethidium bromide. As a nuclear weight standard, a 100-bp ladder was run in parallel with the DNA samples. Apoptosis was detected by visualizing a typical ladder pattern representing multiple small DNA fragments of 180–200 bp (size of one nuclesome) [27].

Real-Time Polymerase Chain Reaction

Endometrial explants from four of the 12 biopsies were harvested individually and processed for real-time polymerase chain reaction (PCR) analysis as previously described [23, 28]. Briefly, total RNA was extracted from treated or untreated endometrial explants using TRIzol Reagent (Invitrogen, Carlsbad, CA). Two micrograms of total RNA were reverse transcribed with Superscript II RNase H- reverse transcriptase (Invitrogen) using random primers (Invitrogen) according to the manufacturer's instructions. The quantification of apoptosis-related genes was carried out in triplicate for each gene using LightCycler Real Time PCR system (Roche Diagnostics Corporation, Indianapolis, IN). Oligonucleotide primers were designed for BCL2, BAX, FAS, FASLG, and GAPD using LightCycler Probe Design Software. The nucleotide sequences of the primers are shown in Table 1. The optimal PCR reaction was established using the LightCycler Fast Start DNA MasterPLUS SYBR Green I Kit (Roche), according to the manufacturer's instructions. Annealing temperature and MgCl2 concentration were optimized to create a one-peak melting curve. Following PCR analysis, amplicons were recovered and checked by agarose gel electrophoresis for a single band of the expected size.


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  		TABLE 1. Primers for amplification of targeted messages with real-time PCR

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 (GAPD) in samples from cultured explants treated with N-9 to the ratio of the same two sequences in samples from untreated explants, which therefore served as a "calibrator" (ratio = 1) for the experiment. The results are expressed as a normalized ratio.

Statistical Analysis

Descriptive terms were used for nonquantitative methods such as gel electrophoresis and immunohistochemistry. Real-time PCR results were expressed as mean ± SD for the number of experiments performed (repeated three times for each gene, of each condition from four biopsies) and analyzed using an unpaired two-tailed Student t-test. Differences were considered significant at a level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
N-9 Induces Apoptosis in Endometrial Explants

Gel electrophoresis of extracted DNA from samples of N-9-treated endometrial explants demonstrates the presence of fragmented DNA, a hallmark of apoptosis. As shown in Figure 1A, N-9 induced a time and dose-dependent DNA laddering in endometrial explants after 24 h of treatment. In addition, examination of N-9-treated endometrium by light microscopy revealed morphological changes characteristic of apoptosis, including nuclear condensation, DNA fragmentation, and apoptotic body formation (Fig. 1B). Taken together, these data provide strong evidence in support of N-9-induced apoptosis in endometrial explants.



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  		FIG. 1. Nonoxynol-9 (N-9)-induced apoptosis in endometrial explants. A) Representative photographs showing internucleosomal DNA fragmentation after 6 and 24 h of N-9 exposure. L, 100-bp ladder; BC, before culture. B) Representative images showing histological analysis of endometrial explants following N-9 treatments. a) Untreated control at 24 h; (b) 3% N-9 at 24 h (b insert). Arrows in b indicate condensed nuclei, DNA fragment, and apoptotic bodies. Arrows in b insert show typical apoptotic bodies. Original magnification a and b x40; insert in b x100

N-9 Induces Caspase Activation in Endometrial Explants

To investigate possible mechanisms of N-9-induced endometrial apoptosis, we tested for caspase activation in explants with and without N-9. Using M30, a monoclonal antibody for the detection of a caspase cleavage product of cytokeratin 18, we demonstrated an increase in the number of M30-positive cells in glandular epithelium in a dose-dependent manner after only 6 h of N-9 exposure (Fig. 2). After 24 h, the labeling with M30 decreased compared with 6 h but was still higher than untreated controls (data not shown).



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  		FIG. 2. Nonoxynol-9 (N-9)-induced caspase activation in endometrial explants after 6 h of treatment. The image presents immunohistochemical staining for M30 antigen, a caspase-cleaved epitope of cytokeratin 18 protein. A) Untreated control at 6 h. B) 0.03% of N-9. C 0.3% of N-9. D) 3% of N-9. Original magnification x20

To determine if CASP3, one of the central effector caspases of apoptosis, was involved in N-9-induced apoptosis, we used an anti-caspase-3 polyclonal antibody, which only detects cleaved CASP3 product. As shown in Figure 3, cleaved CASP3 immunoreactivity was observed at both 6 and 24 h, with more pronounced effect at 24 h. The effect was present but less prominent at lower doses of N-9. These data suggest that the apoptosis observed following N-9 exposure is likely caspase dependent.



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  		FIG. 3. Nonoxynol-9 (N-9)-induced CASP3 activation in endometrial explants. The image presents immunohistochemical staining for cleaved CASP3 antigen. A) Untreated control at 6 h. B) Cultured with 3% N-9 for 6 h. C) Untreated control at 24 h. D) Cultured with 3% N-9 for 24 h. Original magnification x40

N-9-Induced Apoptosis Is Partially Inhibited by Caspase Inhibitors

To further explore the role of caspases in N-9-induced endometrial apoptosis, the explants from six women were pretreated with either a broad caspase inhibitor (Z-VAD-FMK) or a CASP3-specific inhibitor (Z-DEVD-FMK) for 2 h and then treated with N-9 for another 24 h. As shown in Figure 4, treatment of endometrial explants with both inhibitors only partially inhibited the DNA fragmentation induced by N-9, suggesting that a caspase-independent pathway may also be involved in N-9-induced endometrial cell death. To determine whether the partial inhibition of DNA fragmentation is accompanied by complete, partial, or no inhibition of caspase activity, we performed the immunohistochemical staining with anti-CAP3 polyclonal antibody to detect caspase activity on samples subjected to N-9 and caspase inhibitor treatment. As shown in Figure 5, caspase activity in endometrial explants increased strongly after 24 h of N-9 treatment but was dramatically inhibited in the presence of either inhibitor from five samples of the six biopsies. The positive reactivity to CASP3 antibody labeling of some cells after caspase inhibitor treatment would be insufficient to be detected by gel electrophoresis, confirming our suspicion that a caspase-independent pathway of apoptosis may be involved.



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  		FIG. 4. Representative photographs showing DNA fragmentation after cotreatment of nonoxynol-9 (N-9) and caspase inhibitors in endometrial explants. Endometrial tissues were preincubated with either Z-VAD-FMK or Z-DEVD-FMK for 2 h and incubated with or without 0.3% N-9 combined with caspase inhibitors for another 24 h. VAD, Z-VAD-FMK, a broad caspase inhibitor; DEVD, Z-DEVD-FMK, a CASP3-specific inhibitor; L, DNA ladder; BC, before culture



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  		FIG. 5. Immunohistochemical staining of cleaved CASP3 in endometrial explants after cotreatment of nonoxynol-9 (N-9) and caspase inhibitors. Endometrial tissues were preincubated with either VAD or DEVD for 2 h and incubated with or without 0.3% N-9 combined with caspase inhibitors for another 24 h. A) Untreated control at 24 h. B) Cultured with 0.3% N-9 for 24 h. C) Cultured with 0.3% N-9 and a broad caspase inhibitor for 24 h. D) Cultured with 0.3% N-9 and a CASP3-specific inhibitor for 24 h. Original magnification x40

N-9 Induces the Expression of Fas Receptor and Fas Ligand

Given that CASP3 activity can be regulated by both antiapoptotic genes such as BCL2 and proapoptotic genes such as BAX, FAS, and FASLG, we examined the gene expressions of BCL2, BAX, FAS, and FASLG in endometrial explants (from four women) with and without N-9 treatment by real-time PCR analysis using specific primers (Table 1). Although wide variations of gene expression levels were present between the explants from different individuals, the pattern of the gene responses for BCL2, BAX, FAS, and FASLG to N-9 treatment in all samples analyzed show similar trends. The results with relative quantification analysis revealed that the level of both FAS and FASLG mRNAs were significantly increased by N-9 treatment (P < 0.05; Table 2). Both FAS and FASLG mRNAs increased at 6 h after the addition of N-9 and continued to increase significantly (P < 0.01) in an apparent dose-dependent manner with time. The maximal effect (58.65-fold increase) was achieved with the highest dose of N-9 (3%) at 24 h (Table 2). In contrast, the effect of N-9 on BCL2 and BAX expression was variable and not significant (P > 0.05) except Bcl-2 mRNA with 0.03% of N-9 at 24 h (significant decrease, P < 0.01; Table 2). Figure 6 demonstrates a single band of the expected molecular size of all mRNAs detected by real-time PCR. Taken together, these data suggested that the apoptosis triggered by N-9 in endometrial explants is mediated upstream via increased expression of FAS and FASLG, followed by CASP3 activation leading to final cell death.


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  		TABLE 2. Relative quantification (mean ± SD) of apoptosis-related gene expression



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  		FIG. 6. Gel electrophoresis of apoptosis-related mRNA expression in endometrial explants after 6 and 24 h of N-9 treatments. Untreated explants (C) served as a control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates for the first time that N-9 induces apoptosis in human endometrial explants. As a membrane-active surfactant spermicide, N-9 induces membrane permeability and membrane fusion [29–31]. It is well known that cell membrane integrity is essential to maintain intracellular homeostasis, viability, and function. It was recently reported that detergents such as Triton X-100, Nonidet P-40 (NP-40), n-octylglucoside, and the bile salt sodium deoxycholate similarly induce apoptosis [22], indicating that apoptosis induction in the setting of this study may be not a feature peculiar to N-9 but a more general effect of the interaction of detergents with tissue. Furthermore, the irreversible effects of other spermicides such as vanadocene have also been attributed to apoptosis [32]. These findings are relevant to the development of future detergent microbicides and suggested that vaginally applied agents should be tested for potential deleterious effects to the upper reproductive tract. Such damage could facilitate infection with bacterial or viral pathogens such as HIV.

There are several pathways involved in apoptosis including caspase-dependent and caspase-independent mechanisms [33, 34]. The current study provides evidence of caspase activation in N-9-treated endometrial explants by demonstrating an increase of M30 CytoDEATH and anticleaved CASP3 immunoreactivity. M30 recognizes a peptide in epithelial cells generated after caspase-directed cleavage within the cytokeratin 18 molecule. Cytokeratin 18 is cleaved by caspase even before disruption of membrane asymmetry and DNA strand break occurs. The M30 antibody thus becomes a useful marker for detecting early apoptosis [34]. We observed that M30 immunoreactivity decreased with time from the 6-h to the 24-h samples, probably as a result of progressive degradation of CK18 [35, 36]. For this reason and also to observe possible apoptosis in stromal cells (which do not express cytokeratins), we tested the same samples with anticleaved CASP3 antibody. The increased cleaved CASP3 expression with N-9 treatment confirms that seen with the M30 antibody and supports activation of caspase in the mechanism of apoptosis.

We demonstrated that although N-9 activates a caspase-dependent apoptotic pathway, it appears also to induce apoptosis of endometrial explants through caspase-independent pathways. This is suggested by only a partial inhibition of N-9-induced DNA fragmentation with either broad caspase inhibitor or a CASP3-specific inhibitor. This observation was further confirmed by demonstrating that although apoptosis was occurring in the presence of caspase inhibitors seen in DNA fragmentation assay, there was an almost complete absence of caspase activity on N-9 treatment, as demonstrated by very low reactivity to CASP3 antibody in the presence of caspase inhibitors (Fig. 5). The partial inhibition of apoptosis using caspase inhibitors has been suggested as evidence of caspase-independent apoptosis [37, 38]. Since there are several components present in endometrial explants, it is possible that certain cell types within the endometrial tissue underwent caspase-dependent while the others underwent caspase-independent apoptosis in response to N-9. Although the partial inhibition by both inhibitors demonstrated by DNA fragmentation assay may be due to incomplete tissue penetration and thus failure to accomplish inadequate intracellular concentrations in the relevant cells undergoing apoptosis, no evidence of such partial penetration, such as central sparing of caspase activity, could be demonstrated in our samples. These data therefore suggest that a caspase-independent pathway may be involved in N-9-induced endometrial apoptosis.

Two caspase-dependent pathways leading to CASP3 activation have been described, including the death receptor pathway (FAS/FASLG) and a mitochondria-dependent pathway [39–41]. The molecular mechanism underlying the N-9-induced apoptosis in endometrial explants is not known. In the present study, we demonstrated that mRNA for both FAS and FASLG was increased by the treatment of N-9 in endometrial explants and tightly associated with the event of apoptosis. This increase in FAS and FASLG would suggest these genes as mediators of apoptosis in the N-9-treated endometrial explants system. In contrast, we did not observe expected changes in either BCL2 or BAX expression, supporting a proapoptotic environment and suggesting that these two gene products may not directly mediate N-9-induced apoptosis in endometrial explants.

The current study provides initial evidence that N-9 can induce apoptosis in endometrial explants. These findings were confirmed to be a drug effect by using parallel, untreated cultured explants that exhibited little apoptosis over 6–24 h and in many cases a dose response to the drug. Explant viability was maintained by supplementing with estrogen and progesterone to prevent tissue breakdown. Furthermore, expression of GAPD and increased expression of FAS/FASLG are evidence that the explants were viable and capable of responding to N-9 and not the result of massive tissue destruction. Finally, in drawing conclusions from the results of our studies, important consideration should be given to the inherent limitations of an in vitro culture system as well as cycle variations between individuals from whom biopsies were obtained.

In conclusion, our findings suggest that N-9 induced apoptotic cell death in endometrial explants through both caspase-dependent and possibly caspase-independent pathways. The N-9-triggered apoptosis of endometrial explants appears to be mediated through involvement of FAS/ FASLG mechanism, followed by CASP3 activation leading to final cell death. These findings may have important practical implications to future microbicide development by demonstrating that vaginally administered agents may cause upper reproductive tract toxicity and possibly facilitate HIV infection.


   FOOTNOTES
 
1 Supported by a grant from the National Institute of Child Health and Human Development (RO-1 HD 43189). Back

2 Correspondence: John K. Jain, Department of Obstetrics and Gynecology, The Keck School of Medicine of the University of Southern California, 1240 Mission St., Room 1M20, Los Angeles, CA 90033. FAX: 323 226 2850; jjain{at}usc.edu Back

Received: 4 November 2004.

First decision: 15 December 2004.

Accepted: 12 April 2005.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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