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Regular Article |
a Department of Reproductive Endocrinology,
b Department of Ultrasonography,
c Department of Family Planning, Affiliated Gynecological and Obstetric Hospital, The School of Medicine, Zhejiang University, Hangzhou 310006, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, trophoblast
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INTRODUCTION |
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Apoptosis serves as a sensitive indicator of environment insult and often progresses involving a family of cysteine proteases known as caspases. Caspase-3, an effector of apoptotic cascade, can be activated by upstream activators that are critical for the activation of apoptosis during development [1]. Activation of the executor caspase-3 is currently believed to commit the cell to apoptotic death [2]. Furthermore, there is indication for a link between differentiation and activation of apoptosis-related caspases in villous trophoblast [3]. Villous trophoblast provides elemental nutrients for embryonic growth. Its abnormality affects embryo development.
Activation of caspases is achieved via the extrinsic and intrinsic death pathways [4]. The intrinsic one is initiated at the mitochondria by cytochrome c release. When released, cytosolic cytochrome c binds together with dATP and the apoptosis-activating factor-1 to pro-caspase-9 to form the apoptosome, resulting in the activation of downstream caspases.
Recently, Stanton et al. reported that diagnostic ultrasound induces increased numbers of apoptotic bodies in the small intestines of mice [5]. To date, there is no report on exposure of human chorionic villi to transvaginal diagnostic ultrasound followed by activation of caspases. For the first time, we design an in vivo study to investigate the possible signaling pathway of apoptosis after long-duration exposure of chorionic villi to transvaginal ultrasound in the first trimester of pregnancy.
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MATERIALS AND METHODS |
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Twenty-four healthy women aged 22–28 yr were selected for an experimental protocol. They voluntarily demanded abortions at 7–8 wk of gestation. Informed consent for the use of human tissues in this study was obtained from all patients. According to different radiation times, they were randomly divided into four groups: a control group and 10-, 20-, and 30-min groups.
Transvaginal Ultrasound Exposure
Acuson128/HP10 B-mode diagnostic ultrasound equipment (frequency 5.0 MHz, Ispta = 13 mW/cm2, Isppa = 52 W/cm2) was used. The site of the attached embryo was exposed to a fixed beam as described previously [6]. The control group was sham exposed with the transducer switched off. Tissues were obtained through suction termination of pregnancy 4 h after exposure to ultrasound and were immediately rinsed in ice-cold PBS (pH 7.4) to remove blood. Once blood was washed away, tissues were floated in ice-cold PBS to facilitate identification of villi. Small bundles of villi were excised from freshly delivered first-trimester placentas. Pathologic examination of all samples showed typical structures of villi free of adjoining tissues. Samples were stored in liquid nitrogen.
Assessment of Apoptosis
Measurement of DNA fragmentation was carried out as described previously [7]. For the fragmentation assay, samples were lysed in buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM EDTA, 100 mM NaCl, 0.5% SDS, and 1 mg/ml proteinase K at 55°C overnight. After phenol/chloroform extraction, DNA was precipitated at -80°C, centrifuged at 4°C for 15 min, and resolved by 1.5% agarose gel electrophoresis. Then the fragmented DNA was visualized by ethidium bromide staining.
Extraction of Protein
Frozen tissues were sliced and thawed in lysis buffer containing 20 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml benzamidine, and further homogenized on ice. After centrifugation at 10 000 x g for 30 min at 4°C, the supernatants were saved. The protein content of the samples was determined by the method of Bradford [8].
Activation of Caspase-3 Measurement by Western Blotting
Caspase-3 activation was measured by Western blot as previously described [9], with minor modifications. The samples were added to an equal volume of 6x SDS sample buffer and boiled for 10 min. Protein extracts (50 µg/lane) were separated by 15% SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). The membrane was blocked with 10% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and then incubated with antibody against caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. The membrane was washed three times with TBST for 10 min each, followed by incubation with secondary antibody conjugated with peroxidase (Gibco BRL, Gaithersburg, MD). Immune complexes were visualized by the enhanced chemoluminescence (ECL) system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Equal protein loading was routinely confirmed by stripping the Ab off the membrane and probing with anti-ß-actin (Sigma, St. Louis, MO). The amounts of proteins recognized by the antibodies were quantified using densitometric analysis (OptiQuant software; Packard Instrument Co. Inc., Meriden, CT).
Cytochrome C Release Measurements
Cytochrome c release from mitochondria to cytosol was measured by Western blot as previously described [10] with minor modification. After lysis, lysates were centrifuged at 1000 x g for 10 min at 4°C to remove the cell nuclei. Supernatants were then centrifuged at 10 000 x g for 15 min at 4°C to obtain cytosolic extracts. The supernatants were loaded on a 15% SDS-PAGE and then transferred to nitrocellulose membranes (Schleicher and Schuell). Mouse monoclonal cytochrome c antibody (Neomarker, Fremont, CA) was used as the primary antibody. Peroxidase-labeled anti-mouse antibody (Gibco BRL) was used as a secondary antibody. Immune complexes were visualized by the ECL system (Amersham Pharmacia Biotech). The amounts of proteins recognized by the antibodies were quantified using densitometric analysis (OptiQuant software).
Statistical Analysis
Results was expressed as the mean ± SEM. Statistical significance was analyzed by one-way ANOVA, post hoc multiple comparisons (Student-Newman-Keuls test), and P < 0.05 was considered to be statistically significant.
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RESULTS |
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Activation of caspase-3 in human villi after exposure to transvaginal ultrasound was examined by Western blotting. In contrast with the control and 10-min groups, cleaved products of caspase-3 were significantly increased in the 20- and 30-min groups (P < 0.01). Intensities of active caspase-3 among different exposure groups occurred in a time-dependent manner (Fig. 2A). Among the four groups, activation of caspase-3 in the 30-min group was the most significant (P < 0.01). The cleaved products of precursor caspase-3 were detected in bands located at 17 and 10 kDa (Fig. 2B).
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Cytosolic extracts were obtained from villi in the four groups, and analysis of cytochrome c release from mitochondria to cytosol was performed. Enhanced cytochrome c appearance in the cytoplasm was clearly detectable in the 20- and 30-min groups (P < 0.01). As shown in Figure 3A, cytochrome c release further increased with elongation of the exposure to transvaginal ultrasound. The migration position of cytochrome c (15 kDa) is indicated in Figure 3B.
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DISCUSSION |
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Caspase-3, existing in the cytosolic fraction as an inactive 32-kDa precursor, can be proteolytically cleaved into active P17–20/P10–12 due to a variety of extracellular stimuli, such as radiation and chemical drugs [13]. To date, an association between exposure to transvaginal diagnostic ultrasound and activation of caspase-3 has not been studied. For the first time, this study has indicated that caspase-3 activation and subsequent DNA fragmentation are triggered by long-duration exposure of chorionic villi to transvaginal ultrasound in the first trimester of pregnancy. The length of time the ultrasound beam is fixed on a specific tissue target is an important component of thermal dosage [14]. It is suggested that long-duration exposure of transvaginal ultrasound induced caspase-3 activation of chorionic villi through a thermal mechanism. Caspase-3 cleaves cytoplasmic and nuclear proteins and thus initiates the irreversible stages of the apoptotic cascade [15].
Huppertz et al. [16] demonstrated activation of the initiator caspase-8 in villous cytotrophoblast, but activation of the execution caspase-3 could only be demonstrated in the syncytiotrophoblast after syncytial fusion, suggesting that the apoptosis cascade in villous trophoblast is regulated in parallel with trophoblast differentiation, syncytial fusion, and trophoblast turnover. The threshold at which excessive apoptosis induced by transvaginal ultrasound will affect chorionic villi and embryonic development needs additional study.
The precise pathways leading to caspase activation are not fully characterized. Nowadays, it is well known that there are at least two major mechanisms by which a caspase cascade may be initiated. 1) Several death receptors, including Fas and TNFR [17], induce the initiator caspase-8. Active caspase-8 cleaves and activates downstream caspases, initiating the caspase cascade in apoptosis. 2) Cytochrome c is released from the intermembrane space of mitochondria into the cytosol [18]. Oligomerization of Apaf-1/cytochrome c in complexes can activate procaspase-9. Activated caspase-9 in turn cleaves caspase-3, which functions as a downstream effector of the cell death program [19]. After a long dwell-time exposure to transvaginal ultrasound, villous trophoblast increases cytochrome c releases from mitochondria into the cytosol, presenting evidence for the involvement of the intrinsic pathway in transvaginal ultrasound-induced apoptosis.
In conclusion, our data suggest that long-duration exposure of the first-trimester villi to transvaginal ultrasound induces activation of caspase-3 through a mitochondrial pathway, which may be a response to DNA or mitochondria damage. These findings provide a molecular rationale for prudent use of ultrasound at the early stage of pregnancy. Apoptosis can be the predictor of biological effects of ultrasound. Care should be taken to minimize the duration of exposure to high-power transvaginal ultrasound.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: JiaYin Zhang, Hubin Campus 4170#, Zhejiang University, Hangzhou 310006, China. FAX: 86 0571 872 30480; jiayinzh{at}163.net or jiayinzh{at}hotmail.com 
Accepted: March 6, 2002.
Received: November 30, 2001.
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