Long Dwell-Time Exposure of Human Chorionic Villi to Transvaginal

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					BIOLOGY OF REPRODUCTION 67, 580–583 (2002)

Long Dwell-Time Exposure of Human Chorionic Villi to Transvaginal Ultrasound
in the First Trimester of Pregnancy Induces Activation of Caspase-3
and Cytochrome C Release

JiaYin Zhang,1,2 FuZhen Zhou,2 YiLi Song,3 WeiWen Ying,3 and Ying Zhang4
Department of Reproductive Endocrinology,2 Department of Ultrasonography,3 Department of Family Planning,4
Affiliated Gynecological and Obstetric Hospital, The School of Medicine, Zhejiang University,
Hangzhou 310006, China

ABSTRACT                                                                    nutrients for embryonic growth. Its abnormality affects em-
                                                                            bryo development.
   Bioeffects after exposure to ultrasound are correlated to its               Activation of caspases is achieved via the extrinsic and
duration. Although diagnostic ultrasound has been suggested to              intrinsic death pathways [4]. The intrinsic one is initiated
induce apoptosis, the underlying signal transduction pathway re-
mains elusive. In this study, women in the first trimester of preg-
                                                                            at the mitochondria by cytochrome c release. When re-
nancy were exposed to transvaginal diagnostic ultrasound with
                                                                            leased, cytosolic cytochrome c binds together with dATP
5.0-MHz frequency for 0, 10, 20, or 30 min. The chorionic villi             and the apoptosis-activating factor-1 to pro-caspase-9 to
were obtained 4 h after exposure and activation of caspase-3                form the apoptosome, resulting in the activation of down-
and cytochrome c release were analyzed by Western blotting.                 stream caspases.
In contrast with the 0- and 10-min groups, cleavage products of                Recently, Stanton et al. reported that diagnostic ultra-
active caspase-3 and cytochrome c release significantly in-                  sound induces increased numbers of apoptotic bodies in the
creased in 20- and 30-min groups in a time-dependent manner.                small intestines of mice [5]. To date, there is no report on
We show that long-duration exposure to transvaginal ultrasound              exposure of human chorionic villi to transvaginal diagnostic
activates effector caspase-3-mediated apoptotic cascade of cho-             ultrasound followed by activation of caspases. For the first
rionic villi in the first trimester of pregnancy. This occurs                time, we design an in vivo study to investigate the possible
through the intrinsic death pathway involved in cytochrome c                signaling pathway of apoptosis after long-duration exposure
release. Our findings provide a molecular rationale for discrim-             of chorionic villi to transvaginal ultrasound in the first tri-
inant use of transvaginal ultrasound at the early stage of preg-            mester of pregnancy.
apoptosis, trophoblast                                                      MATERIALS AND METHODS
INTRODUCTION                                                                    Twenty-four healthy women aged 22–28 yr were selected for an ex-
   In assisted reproductive technologies, high-power trans-                 perimental protocol. They voluntarily demanded abortions at 7–8 wk of
                                                                            gestation. Informed consent for the use of human tissues in this study was
vaginal ultrasonography is increasingly used to observe or                  obtained from all patients. According to different radiation times, they
transfer embryos in the early stages of genesis. Its potential              were randomly divided into four groups: a control group and 10-, 20-, and
bioeffects for conceptus should be given more attention. An                 30-min groups.
understanding of the mechanism by which the bioeffects of
ultrasound are produced is indispensable for prudent use,                   Transvaginal Ultrasound Exposure
especially in the first trimester of pregnancy. However, little
is known about the bioeffects of transvaginal ultrasound on                     Acuson128/HP10 B-mode diagnostic ultrasound equipment (frequency
                                                                            5.0 MHz, Ispta 13 mW/cm2, Isppa 52 W/cm2) was used. The site of
early conceptus and the mechanism by which biological                       the attached embryo was exposed to a fixed beam as described previously
effects are produced.                                                       [6]. The control group was sham exposed with the transducer switched
   Apoptosis serves as a sensitive indicator of environment                 off. Tissues were obtained through suction termination of pregnancy 4 h
insult and often progresses involving a family of cysteine                  after exposure to ultrasound and were immediately rinsed in ice-cold PBS
proteases known as caspases. Caspase-3, an effector of ap-                  (pH 7.4) to remove blood. Once blood was washed away, tissues were
optotic cascade, can be activated by upstream activators                    floated in ice-cold PBS to facilitate identification of villi. Small bundles
                                                                            of villi were excised from freshly delivered first-trimester placentas. Path-
that are critical for the activation of apoptosis during de-                ologic examination of all samples showed typical structures of villi free
velopment [1]. Activation of the executor caspase-3 is cur-                 of adjoining tissues. Samples were stored in liquid nitrogen.
rently believed to commit the cell to apoptotic death [2].
Furthermore, there is indication for a link between differ-                 Assessment of Apoptosis
entiation and activation of apoptosis-related caspases in vil-
lous trophoblast [3]. Villous trophoblast provides elemental                   Measurement of DNA fragmentation was carried out as described pre-
                                                                            viously [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%
 Correspondence: JiaYin Zhang, Hubin Campus 4170#, Zhejiang Univer-         SDS, and 1 mg/ml proteinase K at 55 C overnight. After phenol/chloro-
sity, Hangzhou 310006, China. FAX: 86 0571 872 30480;                       form extraction, DNA was precipitated at 80 C, centrifuged at 4 C for
e-mail: or                            15 min, and resolved by 1.5% agarose gel electrophoresis. Then the frag-
                                                                            mented DNA was visualized by ethidium bromide staining.
Received: 30 November 2001.
First decision: 2 January 2002.                                             Extraction of Protein
Accepted: 6 March 2002.
   2002 by the Society for the Study of Reproduction, Inc.                    Frozen tissues were sliced and thawed in lysis buffer containing 20
ISSN: 0006-3363.                                  mM Tris-HCl, 1 mM EDTA, 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml

                                            APOPTOSIS OF VILLI INDUCED BY ULTRASOUND                                                                 581

FIG. 1. DNA fragmentation in four groups. The DNA was isolated as
described in Experimental Procedures and was analyzed with 1.5% aga-
rose gel electrophoresis. The DNA was visualized by ethidium bromide

benzamidine, and further homogenized on ice. After centrifugation at
10 000    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 de-
scribed [9], with minor modifications. The samples were added to an equal
volume of 6 SDS sample buffer and boiled for 10 min. Protein extracts
(50 g/lane) were separated by 15% SDS-PAGE. Proteins were electro-
transferred 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
                                                                             FIG. 2. Caspase-3 activation after exposure to transvaginal ultrasound
for 10 min each, followed by incubation with secondary antibody conju-
                                                                             (A); temporal pattern of activation of caspase-3 in the first-trimester villi.
gated with peroxidase (Gibco BRL, Gaithersburg, MD). Immune com-
                                                                             *P 0.05; **P 0.01. B) The Western blot was analyzed with antibodies
plexes were visualized by the enhanced chemoluminescence (ECL) system
                                                                             against caspase-3. The migration position of precursor forms (32 kDa) and
(Amersham Pharmacia Biotech, Buckinghamshire, UK). Equal protein
                                                                             the cleavage products (p17/p10) are indicated. Actin was used as the
loading was routinely confirmed by stripping the Ab off the membrane
                                                                             internal control.
and probing with anti- -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).
                                                                             Newman-Keuls test), and P       0.05 was considered to be statistically sig-
Cytochrome C Release Measurements
    Cytochrome c release from mitochondria to cytosol was measured by        RESULTS
Western blot as previously described [10] with minor modification. After
lysis, lysates were centrifuged at 1000   g for 10 min at 4 C to remove         The fragmented DNA of villi following exposure to
the cell nuclei. Supernatants were then centrifuged at 10 000    g for 15    transvaginal ultrasound was isolated as described in the ex-
min at 4 C to obtain cytosolic extracts. The supernatants were loaded on     perimental procedures and analyzed with 1.5% agarose gel
a 15% SDS-PAGE and then transferred to nitrocellulose membranes              electrophoresis. The DNA was visualized by ethidium bro-
(Schleicher and Schuell). Mouse monoclonal cytochrome c antibody (Neo-
marker, Fremont, CA) was used as the primary antibody. Peroxidase-la-
                                                                             mide staining. Clean bands represented DNA fragmenta-
beled anti-mouse antibody (Gibco BRL) was used as a secondary anti-          tion, suggesting apoptosis throughout the villi after ultra-
body. Immune complexes were visualized by the ECL system (Amersham           sound exposure in the 10-, 20-, and 30-min groups (Fig. 1).
Pharmacia Biotech). The amounts of proteins recognized by the antibodies        Activation of caspase-3 in human villi after exposure to
were quantified using densitometric analysis (OptiQuant software).            transvaginal ultrasound was examined by Western blotting.
                                                                             In contrast with the control and 10-min groups, cleaved
Statistical Analysis                                                         products of caspase-3 were significantly increased in the
   Results was expressed as the mean SEM. Statistical significance was        20- and 30-min groups (P        0.01). Intensities of active
analyzed by one-way ANOVA, post hoc multiple comparisons (Student-           caspase-3 among different exposure groups occurred in a
582                                                                 ZHANG ET AL.

                                                                               This family of proteases is divided into two major groups:
                                                                               initiator caspases (caspase-8 and -9), which are involved in
                                                                               triggering the apoptotic cascade, and effector caspases (cas-
                                                                               pase-3 and -6), which catalyze the disassembly of the cell
                                                                               structure when activated by initiator caspases [12].
                                                                                   Caspase-3, existing in the cytosolic fraction as an inac-
                                                                               tive 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 ultra-
                                                                               sound induced caspase-3 activation of chorionic villi
                                                                               through a thermal mechanism. Caspase-3 cleaves cytoplas-
                                                                               mic and nuclear proteins and thus initiates the irreversible
                                                                               stages of the apoptotic cascade [15].
                                                                                   Huppertz et al. [16] demonstrated activation of the ini-
                                                                               tiator 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 addition-
                                                                               al 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, in-
                                                                               cluding Fas and TNFR [17], induce the initiator caspase-8.
                                                                               Active caspase-8 cleaves and activates downstream caspas-
                                                                               es, initiating the caspase cascade in apoptosis. 2) Cyto-
FIG. 3. Cytochrome c release induced by long-duration exposure of the          chrome c is released from the intermembrane space of mi-
first-trimester villi to transvaginal ultrasound (A); the level of cytochrome   tochondria into the cytosol [18]. Oligomerization of Apaf-
c released from mitochondria into the cytosol among different groups. *P       1/cytochrome c in complexes can activate procaspase-9.
   0.05; **P      0.01. B) Cytosolic fractions (15 kDa) were obtained by
Western blot. These were typical results from three independent experi-
                                                                               Activated caspase-9 in turn cleaves caspase-3, which func-
ments. Actin was used as the loading control.                                  tions as a downstream effector of the cell death program
                                                                               [19]. After a long dwell-time exposure to transvaginal ul-
                                                                               trasound, villous trophoblast increases cytochrome c re-
                                                                               leases from mitochondria into the cytosol, presenting evi-
time-dependent manner (Fig. 2A). Among the four groups,                        dence for the involvement of the intrinsic pathway in trans-
activation of caspase-3 in the 30-min group was the most                       vaginal ultrasound-induced apoptosis.
significant (P     0.01). The cleaved products of precursor                         In conclusion, our data suggest that long-duration ex-
caspase-3 were detected in bands located at 17 and 10 kDa                      posure of the first-trimester villi to transvaginal ultrasound
(Fig. 2B).                                                                     induces activation of caspase-3 through a mitochondrial
   Cytosolic extracts were obtained from villi in the four
                                                                               pathway, which may be a response to DNA or mitochondria
groups, and analysis of cytochrome c release from mi-
tochondria to cytosol was performed. Enhanced cyto-                            damage. These findings provide a molecular rationale for
chrome c appearance in the cytoplasm was clearly de-                           prudent use of ultrasound at the early stage of pregnancy.
tectable in the 20- and 30-min groups (P        0.01). As                      Apoptosis can be the predictor of biological effects of ul-
shown in Figure 3A, cytochrome c release further in-                           trasound. Care should be taken to minimize the duration of
creased with elongation of the exposure to transvaginal                        exposure to high-power transvaginal ultrasound.
ultrasound. The migration position of cytochrome c (15
kDa) is indicated in Figure 3B.
                                                                                    We thank Dr. Jiali Lee from the National Laboratory of Neurobiology,
   The caspase family of cysteine proteases plays a central                    Fudan University School of Medicine, for skillful assistance in these stud-
role in initiating and executing the apoptosis cascade [11].                   ies.
                                            APOPTOSIS OF VILLI INDUCED BY ULTRASOUND                                                               583

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