In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model.

Abstract

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.

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In Vitro Acute Exposure to DEHP Affects Oocyte Meiotic
Maturation, Energy and Oxidative Stress Parameters in a
Large Animal Model
Barbara Ambruosi1., Manuel Filioli Uranio1*., Anna Maria Sardanelli2, Paola Pocar3, Nicola Antonio
Martino1, Maria Stefania Paternoster2, Francesca Amati2, Maria Elena Dell’Aquila1
1Department of Animal Production, University of Bari Aldo Moro, Valenzano, Bari, Italy, 2Department of Medical Biochemistry, Biology and Physics, University of Bari Aldo
Moro, Bari, Italy, 3Dipartimento di Patologia Animale, Igiene e Sanita ` Pubblica Veterinaria, University of Milan, Milan, Italy
Abstract
Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer
products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an
endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte
maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status
were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence
of 0.12, 12 and 1200 mM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological
assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin
configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative
parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 mM; P,0.05). This effect was
related to increased CC apoptosis (P,0.001) and reduced ROS levels (P,0.0001). At higher doses (12 and 1200 mM), DEHP
induced apoptosis (P,0.0001) and ROS increase (P,0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed
MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular
ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content
(P,0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently
scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure
to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured
oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to
confirm this possibility.
Citation: Ambruosi B, Filioli Uranio M, Sardanelli AM, Pocar P, Martino NA, et al. (2011) In Vitro Acute Exposure to DEHP Affects Oocyte Meiotic Maturation,
Energy and Oxidative Stress Parameters in a Large Animal Model. PLoS ONE 6(11): e27452. doi:10.1371/journal.pone.0027452
Editor: Fernando Rodrigues-Lima, University Paris Diderot-Paris 7, France
Received March 1, 2011; Accepted October 17, 2011; Published November 4, 2011
Copyright: ? 2011 Ambruosi, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Progetto COFIN PRIN 2007 (2007S75KSE_003 Contributo d’Ateneo) University of Bari ‘‘Aldo Moro’’ – Italy. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: m.filioliuranio@gmail.com
. These authors contributed equally to this work.
Introduction
Phtalates are a family of industrial compounds used as
plasticizers in the manufactures of many products such as infant
toys, building and food packaging materials, and biomedical
devices [1]. These plasticizers are not covalently bound to the
polymer and leach out into the environment, thus becoming
ubiquitous environmental contaminants [2]. Humans are exposed
to these compounds through ingestion, inhalation, and dermal
exposure for their whole lifetime, since the intrauterine life [3,4].
Among phtalates, the di-(2-ethylhexyl) phthalate (DEHP) is the
most widely used [5,6]. This agent is rapidly hydrolyzed to
produce its major metabolite mono (2-ethylhexyl) phthalate
(MEHP). Both DEHP and MEHP are reported as potent
reproductive toxicant and they impair fertility by acting as
endocrine disruptors, thus causing gonadal mophological or
functional alterations in both sexes [7]. Despite experimental data
provide good evidence that MEHP is highly active in mediating
many of the effects of DEHP, in vitro studies have recently
demonstrated that monoesters (such as MEHP) did not enter the
cells as readily as did the diesters (DEHP), possibly because the
charged molecules cannot pass the plasma membrane [8].
Furthermore, in vitro studies, largely conducted in cell lines or
primary cell cultures, have demonstrated that DEHP is active at a
cellular level, indicating either that DEHP itself has some intrinsic
activity in mediating the observed effects, or that cells have some
capacity for conversion of DEHP to MEHP [9].
In studies in rats, DEHP [10] has been shown to suppress
granulosa cell estradiolproduction with consequent alterationof the
gonadic-hypothalamus feedback, modifications of follicle stimulat-
ing hormone (FSH) and luteininzing hormone (LH) levels,
prolonged estrous cycles, absence of ovulation and corpus luteum
formation and ovarian degeneration. Biological action mechanisms
of phthalates are not clearly understood besides their known ability
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to activate the PPAR nuclear receptors which are known to be
expressed in granulosa and theca cells [11]. Until now, few studies
focusing on the impact of phthalates on meiotic maturation have
been reported. The first study, performed in bovine oocytes,
demonstrated that the addition of MEHP, during in vitro
maturation (IVM), inhibits meiotic maturation in a dose-dependent
manner [1]. This result was confirmed in a subsequent study
performed in mouse oocytes [12] whereas no effect was noticed by
adding DEHP in IVM culture of pig oocytes [13]. Eimani et al.,
2005 [14] reported inhibition of meiotic maturation in the mouse
after in vivo oral DEHP administration. A very recent study in
zebrafish [15] firstly reported deleterious effects of DEHP on
molecular biomarkers of oocyte growth, maturation and ovulation.
It has been reported that oxidative stress (OS) may be an
important mechanism underlying the toxic effects of DEHP [16-
18]. Oxidative stress occurs if disequilibrium between reactive
oxygen species (ROS) production and antioxidative capacity of the
cell takes place [19] and it has also been implicated in the etiology
of some forms of female infertility [20]. Mitochondria represent
the major source of ROS, in which they are produced in a
stepwise process with a final reduction of O2 to H2O during
oxidative phosphorylation, in particular at the level of complex I
and III [21]. Under physiological conditions, ROS are neutralized
by an elaborate defence system consisting of enzymes such as
catalase, superoxide dismutase, glutathione peroxidase or reduc-
tase and numerous non enzymatic antioxidants such as vitamin C,
E, A, pyruvate, glutathione, ubiquinone, taurine and hypotaurine
[22]. Thus, any perturbation in mitochondrial or in the activity of
scavenger systems can lead to profound implications in ROS
production, OS induction, and mitochondrial cytochrome c
release, which is an important step for apoptosis [23]. Oocytes,
as other aerobic cells, produce ATP and ROS by means of
mitochondrial oxidative phosphorylation. Recent studies evi-
denced that, during functional maturation of the oocyte or in
pathological conditions, mitochondria display dynamic tubular
networks undergoing fission, fusion, organization in granules and
tubules and intracellular movements [24-26].
The aim of the present study was to analyze the in vitro effects
of DEHP on oocyte maturation, energy and oxidative status in the
horse, a large animal model. In order to assess the role of cumulus
cells in mediating or counteracting the effects of DEHP, cumulus
cell (CC) apoptosis and oxidative status were also assessed.
Results
The study was conducted in Southern Italy (41stNorth parallel)
during three subsequent breeding seasons. No statistically significant
differences were found in all examined parameters as related to
cumulus morphologyat retrieval,whethercompact(Cp) orexpanded
(Exp), so that data of all cumulus-enclosed oocytes were pooled.
Experiment 1
In Experiment 1, cytological assessment of CC apoptosis and
evaluation of oocyte nuclear maturation rate, mt and ROS
distribution pattern, fluorescent intensities and colocalization were
performed. DEHP was used at the concentrations of 0.12, 12 and
1200 mM and control oocytes were cultured in the absence of
DEHP. Cumulus cell apoptosis was analyzed by Hoechst 33258
staining in order to follow nuclear chromatin fragmentation and
condensation as indicators of late-stage chromatin damage. The
ovaries of 39 mares were processed. One hundred and twenty five
oocytes were recovered (1.6 oocytes/ovary), 67 surrounded by a
Cp cumulus and 58 with an Exp cumulus and cultured for IVM in
five consecutive trials.
DEHP induces chromatin fragmentation and
condensation in CCs
A clear harmful effect of DEHP on CC nuclear chromatin
fragmentation and condensation was visible at all tested
concentration (P,0.001; Figure 1A). At higher doses (12 and
1200 mM), DEPH induced a significant increase of late stage
apoptotic morphologies (Type D=apoptotic bodies; P,0.01;
Figure 1B). Moreover, DEHP was effective at all tested
concentrationsirrespectively of
(P,0.01; Figure 1 C).
oocyte nuclearmaturation
DEHP at low doses affects oocyte nuclear maturation
A statistically significant reduction of the maturation rate was
observed at 0.12 mM DEHP compared with controls (9/32, 28%
vs 16/28, 57%; P,0.05; Table 1). No effects were noticed at the
other tested concentrations. In order to evaluate the effects of
DEHP on oocyte developmental potential, a set of fluorescent
labelling confocal microscopy energy/redox ooplasmic parame-
ters, such as mt distribution pattern, apparent energy status,
intracellular ROS localization and levels, were examined in single
MII oocytes obtained after IVM in presence of DEHP.
DEHP does not affect mitochondrial distribution pattern
and ROS intracellular localization in MII oocytes
Mitochondrial distribution pattern did not vary in DEHP-
treated oocytes compared with controls (Table 2). Oocytes were
found as showing either heterogeneous (pericortical/perinuclear,
P/P) or homogeneous or abnormal distribution pattern. Intracel-
lular ROS localization also did not vary upon DEHP exposure.
Data concerning ROS localization corresponded to data of mt
distribution pattern presented in Table 2. Figure 2 shows matured
equine oocytes representative of heterogeneous (A), homogeneous
(B) and abnormal (C) mt distribution patterns with corresponding
intracellular ROS localization and merge.
DEHP increased intracellular ROS levels without affecting
apparent energy status and mt/ROS colocalization in MII
oocytes
Mitotracker Orange CMTM Ros fluorescence intensity,
indicating oocyte apparent energy status, did not vary upon
DEHP exposure (Figure 3A). On the contrary, oocytes treated
with DEHP at all tested concentrations showed significantly higher
values of DCF fluorescence intensity, indicative of intracellular
ROS levels, than controls (Figure 3B; P,0.05). At examined
concentrations, DEHP did not affect mt/ROS co-localization in
MII oocytes (Figure 3C).
Experiment 2
In Experiment 2, based on the observation that DEHP
increased intracellular ROS levels, the effects of DEHP on total
ATP content and total SOD activity in single MII oocytes were
investigated. DEHP was used at the concentration of 12 mM
which was reported to be within the range of real environmental
exposure levels [27]. For the evaluation of ATP content, the
ovaries of 49 mares were processed. One hundred and seventy six
oocytes were recovered (1.8 oocytes/ovary), 84 surrounded by a
Cp cumulus and 92 with an Exp cumulus and cultured for IVM,
in six trials. For total SOD activity, the ovaries of 23 mares were
processed. One hundred and fifteen oocytes were recovered (2.5
oocytes/ovary), 57 surrounded by Cp cumulus and 58 with an Exp
cumulus and cultured for IVM in five trials.
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DEHP increases ATP content but does not affect total
SOD activity in single MII oocytes
The ATP content was significantly higher in oocytes treated
with DEHP compared with controls (Figure 4A; P,0.05). The
total SOD activity did not vary between DEHP-treated and
control oocytes (Figure 4B; NS).
Experiment 3
Experiment 3 was performed to clarify whether and how the
effects of DEHP on CC apoptosis are related to oocyte
maturation. To this aim, co-treatments between DEHP and the
antioxidant N-Acetyl-Cysteine (NAC) were performed at all tested
DEHP concentrations and, after culture, CCs of each COC were
Figure 1. Effects of DEHP on cumulus cell chromatin fragmentation and condensation. At all tested concentration, DEHP increased
cumulus cells chromatin fragmentation and condensation in equine COCs (A). In separate evaluation of morphological features of apoptosis, such as
marginated chromatin (Type A), single small densely stained nucleus (Type B), multiple densely nuclear fragments (Type C), apoptotic bodies (Type
D), a significant increase of apoptotic bodies could be observed after IVM in presence of DEHP (B). Cumulus cell chromatin morphology was affected
irrespectively to oocyte maturation stage (C). Numbers of analyzed cumuli oophori per group, from which cells analyzed where obtained, are
indicated on the top of each histogram. One-way ANOVA: a, b P,0.001; c,d P,0.01.
doi:10.1371/journal.pone.0027452.g001
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simultaneously analyzed for apoptosis and intracellular ROS levels
and oocytes were analyzed for nuclear maturation rate and
energy/oxidative parameters, as in Experiment 1. In order to
evaluate the effect of DEHP on late-stage molecular DNA
damage, CC apoptosis was analyzed by Terminal Deoxynucleo-
tidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL).
The ovaries of 58 mares were processed. Two hundred and eighty
seven oocytes were recovered (2.5 oocytes/ovary), 131 surrounded
by a Cp cumulus and 156 with an Exp cumulus and cultured for
IVM in three trials.
NAC reversed DEHP-induced apoptosis in CCs
The addition of 12 and 1200 mM DEHP increased CC
apoptosis (P,0.0001; Figure 5A,C), thus confirming morpholog-
ical observations in Experiment 1. At any DEHP tested
concentration, the apoptotic index was higher in CCs from
oocytes found as non matured (NM) after IVM compared with
CCs from MII oocytes (46622% vs 2468%) and this difference
attained statistical significance at 0.12 mM (P,0.0001; File S1,a).
NAC co-treatment was able to counteract DEHP-induced CC
apoptosis observed at 12 mM (P,0.0001; Figure 5A) and, at this
concentration, the apoptotic index was significantly higher in CCs
from NM oocytes compared with CCs from MII oocytes
(46614% vs 29610%; P,0.0001; File S1,b).
NAC counteracts intracellular ROS increase in
DEHP-exposed CCs
The exposure to 0.12 mM DEHP significantly reduced
intracellular ROS levels (P,0.0001) in CCs whereas it increased
ROS levels when used at 12 and 1200 mM (P,0.0001;
Figure 5B,D). The ROS reduction observed at 0.12 mM DEHP
was not related to nuclear maturation of corresponding oocytes
(reduced ROS levels were found in CCs from either NM or MII
oocytes) whereas the increase observed at 12 and 1200 mM DEHP
was significantly higher in CCs from MII oocytes (P,0.0001; File
S1,c). Co-treatment with NAC efficiently scavenged excessive
ROS caused by DEHP at 1200 mM (P,0.0001) whereas it had no
effects at 12 mM (Figure 5B). At this DEHP concentration, ROS
levels were significantly higher in CCs from MII oocytes compared
with CCs from NM oocytes (P,0.0001; File S1,d).
Overall assessment of DEHP-induced oocyte damage
Concerning DEHP-induced oocyte damage, overall data
(Experiment 1+Experiment 3) are provided. DEHP was shown
to inhibit oocyte nuclear maturation only when used at low doses
(0.12 mM; 19/61, 31% vs 29/57, 51% for DEHP-treated and
control oocytes, respectively; P,0.05) thus confirming the results
of experiment 1. At any tested concentration, it had no significant
effect on examined microscopy ooplasmic energy/redox param-
eters (File S2).
NAC does not improve the maturation rate of
DEHP-exposed oocytes
NAC co-treatment tended to reverse the inhibitory effect of
DEHP on oocyte maturation (14/31, 45% vs 10/29, 34% for
DEHP/NAC- and DEHP-treated oocytes, respectively) but this
effect did not attain statistical significance. Interestingly, the
addition of NAC ‘‘per se ´’’ had a stimulatory effect on oocyte
maturation (23/31, 74% vs 13/29, 45% for NAC-treated and
control oocytes, respectively; P,0.05).
Discussion
To our knowledge, this is the first study which analyzes the
effects of in vitro acute exposure to DEHP on energy and oxidative
parameters of single cumulus-oocyte complexes by using a large
Table 1. In vitro effects of DEHP on oocyte meiotic maturation.
DEHP concentration
(m mM)
N. of evaluated
oocytes Nuclear chromatin configuration:
Germinal vesicle
Metaphase I to
Telophase I
Metaphase II and
1stPolar Body Abnormal
0 280 (0) 7 (25) 16 (57)a
5 (18)
0.12322 (6) 8 (25)9 (28)b
13 (41)
12343 (9)3 (9) 18 (53)10 (29)
1200313 (10)6 (19) 13 (42)9 (29)
Chi-square Test: a,b P,0.05.
doi:10.1371/journal.pone.0027452.t001
Table 2. In vitro effects of DEHP on oocyte mitochondrial distribution pattern.
DEHP concentration (m mM)
N. of oocytes found at the MII
stage and evaluated
Mitochondrial distribution pattern
Pericortical/Perinuclear Small aggregates Abnormal
014 7 (50) 5 (36)2 (14)
0.129 6 (67)3 (33) 0 (0)
12 18 9 (50)4 (22)5 (28)
1200117 (64)2 (18) 2 (18)
Chi-square Test: NS.
doi:10.1371/journal.pone.0027452.t002
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animal model. The mare represents a valid model to investigate
oocyte physiology due to its particularly large ovarian follicle size,
which allows the possibility to relate oocyte meiotic and
developmental competence with biochemical and molecular
features of corresponding follicle cells as reported in the studies
performed by several groups [28-35].
As the viability/quality of CCs has been indicated as a crucial
factor influencing the outcome of oocyte maturation and
subsequent developmental competence, the incidence of apoptotic
chromatin modification in CCs after exposure to DEHP has been
investigated first. DEHP, at any tested concentration, induced
chromatin damage in CCs which was displayed in all character-
istics morphological features of apoptosis. On the other hand, the
TUNEL assay revealed DEHP-induced DNA fragmentation only
at higher tested concentrations (12 and 1200 mM). Thus,
chromatin condensation and fragmentation observed after expo-
sure at low DEHP doses (0.12 mM) may not be associated with
DNA apoptotic cleavage. In addition, chromatin breakage was
observed irrespectively of oocyte maturation, whereas DNA
cleavage was higher in CCs from NM oocytes. These observations
indicate that these two methods provide complementary informa-
tion. Our data confirmed previous observations obtained with
MEHP. The inhibitory effect of MEHP on maturation was
reported to be more severe in denuded oocytes compared to intact
COCs, indicating that cumulus cells are primarily affected by
MEHP and are able to reduce the harmful effect of MEHP on
oocyte maturation [1].
The presence of DEHP at low doses (0.12 mM) in the
maturation medium negatively influenced the ability of equine
oocytes to reach meiotic maturation at the MII stage. One of the
main question involving phthalates, is whether the level of
exposure is sufficient to adversely affect female reproductive
health. Recently, several studies reported that treatment of rat with
active phthalates may result in non-monotonic response curves
and low-dose effects [36-38]. This is in agreement with results
reported in the present study, showing major adverse effects on
oocyte maturation at the lowest dose investigated. It is noteworthy
to observe that the current no observed adverse effect level
(NOAEL) adopted by the European Food Safety Authority for
DEHP is 5 mg/Kg/day, based on a multigeneration study using
alterations in male reproductive organs as endpoint [39].
However, it is also important to note that the significance of
low-dose changes, as observed in the present study, is still largely
unknown and, due to difficulties in in vitro to in vivo extrapolation,
cannot be taken as clear evidence for concern about real-life
exposure. To address this question, further analysis in animal
models are necessary in order to better understand the cellular and
molecular mechanisms at the basis of the observed effects. At the
moment, few studies have been published regarding the in vitro
effects of phthalates on oocyte maturation. To our knowledge,
only one study reported the in vitro effects of DEHP on meiotic
maturation [13]. These authors found that DEHP at the
concentrations of 0.0001, 0.01, 1 and 100 mM did not affect
meiotic stage of porcine oocytes. Another study [1] reported that,
in bovine oocytes, MEHP at concentrations between 25 and
100 mM blocked oocytes from reaching the MII stage of
maturation. A subsequent study on mouse oocytes [12] confirmed
these results, as the proportion of oocytes that progressed to the
MII stage was significantly reduced by adding MEHP in a dose-
related manner. Mlynarcı ´kova ´ et al. in their study (2009) [13]
supposed that the different results obtained in their study may be a
consequence of the related but still dissimilar chemical structure of
MEHP and DEHP. Our results lead us to hypothesize that
different sensitivity to DEHP may be observed in different species.
How phthalates influenced oocyte maturation is not clearly
understood. It was reported that MEHP inhibited FSH-stimulated
Figure 2. Mitochondrial distribution pattern and ROS localization in equine matured oocytes exposed to DEHP. For each oocyte,
corresponding bright-field (A,B,C), UV light (A1,B1,C1) and confocal laser scanning images showing mt distribution pattern (A2,B2,C2), intracellular
ROS localization (A3,B3,C3) and mt/ROS merge (A4,B4,C4) are shown. Oocytes are representative of heterogeneous (pericortical/perinuclear; A),
homogeneous (B) and abnormal (C) mitochondrial distribution pattern, respectively. Scale bar represents 60 mm.
doi:10.1371/journal.pone.0027452.g002
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cAMP production in cultured granulosa cells [40] considering that
intracellular build up of cAMP is necessary for optimum
developmental competence [41,42]. DEHP/MEHP probably
mimic the effects of fatty acids on granulosa cells [6] because
they are ligands for fatty acid binding proteins [43], and it has
been shown that fatty acids adversely affect in vitro maturation
[44,45]. Phthalates have been involved in PPARs activation, that
lead to a decrease in aromatase and estradiol levels [46] and
several reports [14] indicate that lower estradiol secretion from
granulosa cells is responsible for impaired oocyte maturation. A
more recent study [27] indicated that MEHP is a specific inhibitor
of estradiol production in human granulosa cells with a post-
Figure 3. Effects of DEHP on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in matured oocytes. Dose-
response curve of the in vitro effects of DEHP on mitochondrial activity and intracellular ROS levels in single equine metaphase II stage oocytes
expressed as Mitotracker Orange CMTM Ros (A) and DCF (B) fluorescence intensities. Oocytes treated with DEHP showed significantly higher ROS
levels compared to controls. Values are expressed as arbitrary densitometric units (ADU). Pearson’s correlation coefficents of Mitotracker Orange
CMTM Ros and DCF fluorescent labelling in oocytes cultured in presence of DEHP (C). Numbers of analyzed oocytes per group are indicated on the
top of each histogram. Student’s t-Test: a, b P,0.05.
doi:10.1371/journal.pone.0027452.g003
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cAMP site of action. These authors observed the inhibition of
estradiol production resulting from a reduction of aromatase
activity at the transcript level.
In order to evaluate whether DEHP could affect cytoplasmic
maturation, thus developmental potential of exposed oocytes, a set
of cytoplasmic energy/redox parameters was investigated in
oocytes found at the MII stage after culture in presence of DEHP.
In Experiment 1, the addition of DEHP at any used concentration
did not affect mt distribution pattern, intracellular ROS
localization, apparent energy status and mt/ROS colocalization
whereas it increased ROS levels. This observation prompted us to
hypothesize that the DEHP-induced ooplasmic ROS levels
increase could be responsible for altered oocyte maturation, also
in agreement with previous studies reporting that oxidative stress
may be an important mechanism underlying the toxic effects of
DEHP [18-20]. However, 0.12 mM was the only dose that
inhibited oocyte maturation while all tested doses increased
ooplasmic ROS levels.
The increase of intracellular ROS was supposed to be due to a
lack of one or more scavenging enzyme activities or to a loss of
function of the mitochondrial respiratory chain proteins, as
reported by Bonilla and del Mazo (2010) [47] who showed a
deregulation of genes encoding Cu-Zn superoxide dismutase
(SOD1) and Nd1 mitochondrial protein upon in vitro exposure to
MEHP. In addition, our finding that DEHP did not affect mt/
ROS colocalization lead us to hypothesize that ROS produced in
excess in DEHP-treated oocytes could be located at mitochondrial
level. In order to explore whether increased ROS levels could be
due to unefficient scavenging enzyme activity, our subsequent aim
(Experiment 2) was to investigate total SOD activity in oocytes
found as matured after culture in presence of 12 mM DEHP. To
our knowledge, this is the first study reporting total SOD activity
in single equine oocytes matured in vitro. The results of our study
indicated that SOD activity was not affected in DEHP-exposed
oocytes compared with controls. Thus, the intracellular ROS
increase observed in DEHP-exposed oocytes could be due to
altered efficiency of other antioxidant systems.
Moreover, ATP content analysis showed an increase of ATP
levels in MII oocytes obtained after incubation with 12 mM DEHP
compared to controls. These data apparently disagree with the
Figure 4. Effects of DEHP on total ATP content and total SOD activity in single equine matured oocytes. ATP content and total SOD
activity in single equine MII oocytes cultured in presence of 12 mM DEHP. Control oocytes were cultured in absence of DEHP. Numbers of analyzed
oocytes per group are indicated on the top of each histogram. Oocytes treated with 12 mM DEHP showed significantly higher ATP content compared
to controls. Values are expressed as pmol/oocyte (A). Total SOD activity did not vary upon 12 mM DEHP exposure in matured equine oocytes
compared with controls. Values are expressed as IU/mg protein (mean6sd of 9 evaluations per oocyte; B). Student’s t-Test: a, b P,0.05.
doi:10.1371/journal.pone.0027452.g004
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results of confocal analysis in which no differences in mt apparent
energy status (Mitotracker Orange CMTM Ros fluorescence
intensity) were found between treated and control samples.
However in the performed test, total ATP levels were measured
and it could be possible that the observed ATP increase could be
of cytosolic (or glycolitic) origin. To our knowledge the only study
about DEHP effects on glycolisis was reported by Martinelli et al.
(2006) [48] who found increased pyruvate and lactate content in
skeletal muscle after in vivo DEHP administration. The reported
increase of pyruvate and lactate, which are responsible for
increased cytosolic ATP, could be considered in agreement with
our findings.
In order to assess whether the observed inhibitory effect of
DEHP on oocyte maturation was related to oxidative stress and
apoptosis in the COC, the potential reversibility of DEHP-induced
COC damage by the antioxidant NAC was examined. NAC was
shown to counteract the damaging effects of the environmental
toxicant arsenic trioxide on mitochondrial function in mouse
matured oocytes [49]. Apoptotic index and ROS genreation were
also reversed by NAC in human ovarian cancer cells treated with
WP 631, an anticancer anthracycline analog, and with doxoru-
bicin, the best known first-generation anthracycline [50]. Another
recent study [51] reported no differences in nuclear maturation,
fertilization and cleavage rate, but higher blastocysts formation
rate in presence of NAC compared with controls in porcine
oocytes.
It came out that:
N at low doses, DEHP increased CC apoptosis, observed as
chromatin condensation/fragmentation but not as DNA
cleavage, and reduced CC intracellular ROS levels, thus
causing loss of CC viability with consequent inhibitory effect
on oocyte nuclear maturation;
N at higher doses, DEHP increased CC apoptosis, at chromatin
and DNA level, and increased CC ROS levels but it does not
affect oocyte maturation.
In conclusion, our data indicate that DEHP exerts a dose-
dependent effect on CC apoptosis and oocyte meiotic maturation
which was more deleterious at low doses. This effect is not
mediated by cellular stress but rather by loss of CC viability. CC
acts as a protection barrier against the harmful effects of DEHP.
Energy/redox parameters of MII DEHP-exposed oocytes are
consistent with the possibility that they can sustain fertilization and
embryo development. These findings means that DEHP-exposed
oocytes can be used, with apparent lack of effects on female
fertility, but could also represent a potential risk of generating
abnormal embryos and this topic needs further investigations.
Recognition that the toxic effects of DEHP may originate in
disruption of mitochondrial biology, at CCs or ooplasmic level,
could represent important findings for the future development of
therapeutical procedures to phthalates exposure-derived infertility.
Figure 5. Effects of DEHP and DEHP/NAC co-treatment on cumulus cell apoptosis and intracellular ROS levels. At the concentration of
12 and 1200 mM, DEHP increased cumulus cell apoptosis in equine COCs. NAC reversed DEHP-induced apoptosis observed at 12 mM (A). At the
concentrations of 12 and 1200 mM, DEHP increased CC intracellular ROS levels and co-treatment with NAC reduced the DEHP-induced ROS increase
observed at 1200 mM (B). Numbers of analyzed cumuli oophori per group (from MII+NM oocytes), from which analyzed cells where obtained, are
indicated on the top of each histogram. Representative images of equine CCs after IVM in presence of DEHP subjected to TUNEL analysis (C) to
determine apoptosis and DCF staining (D) to evaluate intracellular ROS levels. In C, corresponding bright-field and UV light images are also provided.
One-way ANOVA: comparisons between used DEHP doses: a,b,c P,0.0001; comparisons between DEHP and DEHP/NAC:*,**P,0.0001.
doi:10.1371/journal.pone.0027452.g005
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The risk of infertility may be reduced by optimizing mitochondrial
energy metabolism within the ‘‘contaminated oocyte’’ by new
therapeutic skills of nutritional, pharmacological or technological
means, such as antioxidants-enriched diets, antioxidants added to
culture media for assisted reproductive technologies and homol-
ogous mitochondrial ooplasmic transfer.
Materials and Methods
Chemicals and culture media
All chemicals were purchased from Sigma–Aldrich (Milano,
Italy) unless otherwise indicated.
Medium TCM-199 with Earle’s salts, buffered with 4.43 mM
HEPES and 33.9 mM sodium bicarbonate and supplemented
with 0.1 g/L L-glutamine, 2 mM sodium pyruvate, 2.92 mM
calcium-L-lactate pentahydrate (Fluka 21175 Serva Feinbiochem
GmbH & Co. Heidelberg, Germany No. 29760) and 50 mg/mL
gentamicin was used. After preparation, pH was adjusted to 7.18
and the medium was filtered through 0.22-mm filters (No. 5003-6,
Lida Manufacturing Corp., Kenosha WI, USA) and further
supplemented with 20% (v/v) Fetal Calf Serum (FCS). Then,
gonadotrophins (10 mg/mL ovine FSH and 20 mg/mL ovine LH)
and 1 mg/mL 17b Estradiol were added. The medium was filtered
again and allowed to equilibrate for 1 h under 5% CO2in air
before being used.
Collection and culture of cumulus-oocyte complexes
Ovaries from mares of unknown reproductive history, obtained
at two local abattoirs (Fin.Sud. Import s.r.l. and Maselli Carni Per
Te s.r.l.) located at a maximum distance of 30 km (30 min) from
the laboratory, were transported and processed for the scraping
procedure as previously described [32]. Cumulus-oocyte complex-
es (COCs) were recovered from medium size follicles (0.5–2.5 cm
in diameter), identified in the collected mural granulosa cells by
using a dissection microscope and only healthy COCs, classified as
having an intact Cp or Exp cumulus investment [30,32] were
selected for culture; degenerating oocytes (having shrunken, dense
or fragmented cytoplasm) were recorded and discarded. The time
between follicle scraping and beginning of oocyte culture was less
than 1 h. Total time between slaughter and culture ranged
between 2 and 4 h.
In vitro maturation was performed following the procedure by
Dell’Aquila et al. (2003) [32]. Cp and Exp COCs were washed
three times in the culture medium and groups of COCs with the
same cumulus morphology were placed in 400 mL of medium/well
of a four-well dish (Nunc Intermed, Roskilde, Denmark), covered
with pre-equilibrated lightweight paraffin oil and cultured for 28–
30 h at 38.5uC under 5% CO2in air. DEHP (Sigma Supelco, Cat
# 47994) stock solution (1 g/1 ml methanol) was prepared
following manufacturers instructions and dilution 1:20 (v/v) in
pure ethanol was performed. The effects of DEHP for the whole
duration of IVM culture were tested at final concentrations of
0.12, 12 and 1200 mM. Due to unknown phtalates concentration
in equine follicular fluid, the minimum dose has been calculated
on the basis of observations on daily dose exposure in humans
[52,53] divided by a factor of 1000. The dose range was then
obtained by multiplying the calculated dose by a factor of 100, up
to the highest dose which showed reproductive toxicity in previous
in vitro studies [1]. Control oocytes were cultured in the absence of
DEHP. The N-Acetyl Cysteine was used at the concentration of
5 mM, reported as being effective in counteracting energy and
oxidative damage in mouse oocytes [49]. The effects of NAC were
analyzed at all DEHP tested concentrations. After IVM, oocytes
underwent cumulus and corona cells removal by incubation in
TCM 199 with 20% FCS containing 80 IU hyaluronidase/mL
and aspiration in and out of finely drawn glass pipettes. Oocyte
maturation was initially assessed after denuding by observation
under a Nikon SMZ 1500 stereomicroscope (60–110x magnifica-
tion) evaluating the extrusion of the first polar body (PB) in the
perivitelline space and was confirmed by nuclear chromatin
evaluation as described below.
Morphological assessment of cumulus cell apoptosis
Cumulus cells in experiment 1 were fixed overnight at 4uC in
3.8% (v/v) buffered formaldehyde solution (J T Baker; No. 7385)
in PBS, then stained with 2.5 mg/ml Hoechst 33258 in 3:1 (v/v)
glycerol/PBS and observed under an E-600 Nikon fluorescent
microscope equipped with a 365 nm excitation filter. Morpholog-
ical criteria for apoptotic cell and bodies described previously were
used [54]. Cells were classified in 4 different categories: A, B, C
and D. Type A: cells with nuclei containing marginated
chromatin; type B: cells with a single small nucleus with densely
stained chromatin; type C: cells containing multiple nuclear
fragments and type D: membrane-bound structures containing
variable amount of chromatin and/or cytoplasm (apoptotic
bodies). Evaluation of apoptotic cells and apoptotic bodies was
performed on a mean of 10 to 15 COCs per culture condition. For
each COC, two fields with 50 cumulus cells each were countered.
Terminal Deoxynucleotidyl Transferase-mediated dUTP
Nick-End Labeling (TUNEL)
To assess the rate of apoptotic cells, CCs were separated in
groups according to the treatments and oocyte nuclear matura-
tion. Briefly, CCs were fixed in 2% PBS-buffered paraformalde-
hyde over night at 4uC. An in situ cell death detection kit (Roche
Molecular Biochemicals, code: 11684795910; Mannheim, Ger-
many) was used for labeling apoptotic cells. CCs were washed
three times in PBS and then permeabilized with 0.5% Triton X-
100, 0.1% sodium citrate in PBS for 10 min. CCs were washed
twice with PBS before labeling. The TUNEL reagent was
prepared immediately before use and kept on ice. CCs were
placed in 50 ml drops of TUNEL reagent and incubated in the
dark for 1 h at 37uC in a humidified chamber. After incubation,
CCs were washed three times with PBS. Total cell nuclei were
stained with 10 mg/ml Hoechst 33258, 2.3% Na-citrate in 3:1 (v/
v) glycerol/PBS, mounted on microscope slides, covered with
cover-up micro slides, sailed with nail polish and kept at 4uC in the
dark until observation. CCs were observed under an E-600 Nikon
fluorescent microscope equipped with a 365 nm excitation filter.
Positive and negative controls were performed following the
manufacturers instructions. Apoptosis was determined as the
percentage of labeled cells to the total cell number. For each
culture condition, a minimum of 1000 randomly chosen cells was
examined.
Mitochondrial and ROS staining
Oocytes were washed three times in PBS with 3% bovine serum
albumin (BSA) and incubated for 30 min in the same medium
containing 280 nM MitoTracker Orange CMTM Ros (Molecular
Probes M-7510, Oregon, USA) at 38.5uC under 5% CO2. The
cell-permeant probe contain a thiol-reactive chloromethyl moiety.
Once the MitoTracker probe accumulates in the mitochondria, it
can react with accessible thiol groups on peptides and proteins to
form an aldehyde-fixable conjugate. This cell-permeant probe is
readily sequestered only by actively respiring organelles depending
on their oxidative activity [55,56]. After incubation with
MitoTracker Orange CMTM Ros, oocytes were washed three
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times in PBS with 0.3% BSA and incubated for 15 min in the
same media containing 10 mM 29,79-dichlorodihydrofluorescein
diacetate (DCDHF DA). The non-ionized DCDHF DA is
membrane permeant and therefore is able to diffuse readily into
cells. Once within the cell, the acetate groups are hydrolysed by
intracellular esterase activity forming 29,79-dichlorodihydrofluor-
escein (DCDHF) which is polar and thus trapped within the cell.
DCHF fluoresces when it is oxidized by H2O2or lipid peroxides to
yield 29,79-dichlorofluorescein (DCF). The level of DCF produced
within the cells is linearly related to that of peroxides present and
thus its fluorescent emission provides a measure of the peroxide
levels [57]. After incubation, oocytes were washed three times in
prewarmed PBS without BSA and fixed overnight at 4uC with 2%
paraformaldehyde solution in PBS. The organelle-specificity of the
probe was assessed, as reported by Valentini et al., (2010) [58], in
control samples which were imaged after incubation in Mito-
Tracker Orange and further incubation for 5 min in the presence
of 5 mM of the mt membrane potential (Delta Psi)-collapsing
uncoupler carbonyl cianide 3-chloro phenylhydrazone (CCCP;
Molecular Probes), which inhibits mt respiratory activity thus
reducing fluorescence intensity. Cumulus cells were stained with
DCDHF DA in order to evaluate intracellular ROS levels.
Particular attention was paid to avoid sample exposure to the light
during staining and fixing procedures in order to reduce
photobleaching.
Assessment of oocyte nuclear maturation
To evaluate nuclear chromatin, oocytes were stained with
2.5 mg/ml Hoechst 33258 in 3:1 (v/v) glycerol/PBS, mounted on
microscope slides, covered with cover-up micro slides, sailed with
nail polish and kept at 4uC in the dark until observation. Nuclear
chromatin status was observed under a Nikon Eclipse 600
fluorescent microscope equipped with B2A (346 nm excitation/
460 nm emission) filter and classified as follows: GV including
those oocytes with fluorescent nucleus and those with a condensed
chromatin, metaphase to telophase I (MI to TI) and complete
maturation at metaphase II with the first polar body extruded
(MII+PB) [59]. Oocytes with irregular chromatin distribution or
non detected chromatin were considered as abnormal.
Assessment of mitochondrial distribution pattern and
intracellular ROS localization in matured oocytes
For mt distribution pattern evaluation, oocytes were selected
among those having regular ooplasmic size and texture (no
vacuoles). Oocytes were observed at 600 x magnification in oil
immersion with Nikon C1/TE2000-U laser scanning confocal
microscope. A helium/neon laser ray at 543 nm and the G-2 A
filter (551 nm exposure/576 nm emission) was used to point out
the MitoTracker Orange CMTM Ros. An argon ions laser ray at
488 nm and the B-2 A filter (495 nm exposure/519 nm emission)
was used to point out the DCF. Scanning was conducted with 25
optical series from the top to the bottom of the oocyte with a step
size of 0.45 mM to allow three-dimensional distribution analysis.
General criteria for mt pattern definition were adopted on the
basis of previous studies in equine oocytes as well as in other
species [59]. Thus, homogeneous/even distribution of small mt
granules throughout the cytoplasm was considered as an
indication of immature cytoplasmic condition. Heterogeneous/
uneven distribution of small and/or large mt granules indicated
metabolically active ooplasm. In particular, accumulation of active
mitochondria in the peripheral cytoplasm (pericortical mt pattern)
and/or around the nucleus (perinuclear and pericortical/perinu-
clear mt pattern, P/P) was considered as characteristic of full
cytoplasmic maturation. Oocytes showing irregular distribution of
large mt clusters unrelated to the specific cell compartments were
classified as abnormal. To our knowledge, few studies are reported
to date on intracellular ROS localization in mammalian oocytes. A
recent study performed in mouse oocytes reported that regions
producing high levels of ROS colocalized with the active
mitochondria in the majority of in vivo matured ovulated oocytes
[60]. All oocytes found at the MII stage were analyzed.
Quantification of Mitotracker Orange CMTM Ros and DCF
fluorescence intensity in matured oocytes
Measurements of fluorescence intensities were performed in
oocytes having either heterogeneous (pericortical/perinuclear) or
homogeneous (small granules) mt distribution pattern. Oocytes
showing abnormal mt distribution pattern were excluded from this
analysis. In each individual oocyte, the fluorescence intensity was
measured at the equatorial plane (plane no. 13), with the aid of the
EZ-C1 Gold Version 3.70 software platform for Nikon C1
confocal microscope. A circle of an area (arbitrary value=100 in
diameter) was drawn in order to measure only the cytoplasmic
area. Fluorescence intensity encountered within the programmed
scan area was recorded and plotted against the conventional pixel
unit scale (0–255). Parameters related to fluorescence intensity
were maintained at constant values for all evaluations. In detail,
images were taken under fixed scanning conditions with respect to
laser energy, signal detection (gain) and pinhole size. Based on the
observation that co-localization of actively respiring mitochondria
and ROS is considered as indication of healthy cell [60,61],
Pearson’s correlation coefficient, which describes the correlation of
the intensity distribution between channels [62], was used to
quantify mt/ROS colocalization as related to DEHP treatment.
Quantification of DCF fluorescence intensity in CCs
Measurements of DCF fluorescence intensity were performed in
CCs isolated from matured and non matured COCs as assessed by
PB extrusion evaluation. For each COC, fluorescence intensity
was measured on a minimum number of 50 individual randomly
chosen cells, with the aid of the EZ-C1 software. A circle was
drawn in order to measure the cytoplasmic area of 10 cells and
measures were repeated for five fields. Fluorescence intensity
encountered within the programmed scan area was recorded and
plotted against the conventional pixel unit scale (0–255).
Parameters related to fluorescence intensity were maintained at
constant values for all evaluations as reported for oocyte
evaluations.
Measurement of the ATP content of single matured
oocytes
The ATP content of denuded matured oocytes, cultured with 0
and 12 mM DEHP, was measured using a commercial assay (based
on the luciferin-luciferase reaction, ATP lite Perkin Elmer, Monza,
Italy). Briefly, after IVM samples were rinsed in TCM 199
supplemented with 20% FCS and then transferred individually in
100 ml of the same medium into plastic tubes. Then, 50 ml of
mammalian cell lysis solution was added, and the tubes were kept
in the darkness for 5 minutes at room temperature in an orbital
shaker. Subsequently, 50 ml of substrate solution was added and
after 5 minutes the measurement of the luminescence was
performed. The ATP content of the samples was measured using
a luminometer (VictorTMX, Perkin Elmer) with high sensitivity
(0.01 pmol). A seven point standard curve (0-6 pmol/tube) was
routinely included in each assay. The ATP content was
determined from the formula for the standard curve (linear
regression) [25].
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Measurement of the total superoxide dismutase (SOD)
activity in single matured oocytes
The SOD activity was determined on MII oocytes, cultured in
presence of 12 mM DEHP. Control oocytes were cultured in
absence of DEHP. Single oocytes were previously treated with SB
buffer (TRIS/HCl 60 mM pH 6.8, Glycerol 40%) and solubilized
for one hour at 4uC in the presence of 1.0% Triton X-100. The
protein concentration was assessed by the method of Bradford
[63]. Each test was performed on 7 mg of proteins of a single
solubilized oocytes. The superoxide dismutase activity was
determined with the Fluka analytical assay kit using a spectropho-
tometer VictorTMX, Perkin Elmer at l=440 nm. Total SOD
activity (SOD, EC 1.15.1.1) was assayed by its ability to inhibit the
reduction of a novel tetrazolium salt, WST-1 [2-(4-Iodophenyl)-3-
(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium
salt] by superoxide anions generated with the xanthine/xanthine
oxidase method [64,65]. One unit of SOD activity was defined as
the amount of the enzyme causing half maximum inhibition of
WST-1 reduction. It was measured with high sensitivity
(0.01 pmol) and expressed as U/mg proteins. A nine point
standard curve was routinely included in each assay.
Statistical analysis
The apoptotic index and intracellular ROS levels in CCs
surrounding MII and immature oocytes were analyzed by using
GraphPad Prism software (GraphPad Software 5.03, San Diego
CA). The percent number of apoptotic cells per cumulus was
calculated using oocytes from at least 5 different trials for
morphological analysis and 3 trials for TUNEL test. Differences
between the means were evaluated by one-way ANOVA, with
statistical significance assigned at P 0.05. When a significant P
value was obtained with ANOVA, the Bonferroni test was used in
the post hoc analysis. Oocyte nuclear maturation rates and the
rates of oocytes showing the different mt distribution patterns and
ROS intracellular localization were compared between treated
and control groups by x2-analysis with the Yates correction for
continuity. Fisher’s exact test was used when a value of , 5 was
expected in any cell. For confocal quantitative analysis of mt and
ROS fluorescence intensity, the least-square means of the
dependent variables (mt and ROS fluorescence intensity) were
calculated in examined samples and the statistical significance of
the least-square means between control and treated groups was
calculated by the Student’s t-test. Mean values of Pearson’s
correlation coefficient, ATP content and SOD activity were
compared between treated and control groups by the Student’s t-
test. Differences with P,0.05 were considered statistically
significant.
Supporting Information
File S1
(MII) and non matured (NM) oocytes after IVM in
presence of DEHP (a) and DEHP+ +NAC (b), as assessed
by TUNEL test. Intracellular ROS levels of CCs isolated from
MII and NM oocytes after IVM in presence of DEHP (c) and
DEHP+NAC (d), as assessed by DCF fluorescence intensity.
(XLS)
Overall assessment (Experiment 1+ +Experiment
3) of DEHP effects on oocyte apparent energy status,
intracellular ROS levels and mt/ROS colocalization as
assessed at ooplasmic level in individual MII oocytes.
Number of examined oocytes are reported at the top of each
histogram.
(XLS)
Apoptotic index of CCs isolated from matured
File S2
Author Contributions
Conceived and designed the experiments: MED PP. Performed the
experiments: BA MFU NAM MSP FA. Analyzed the data: BA MFU AMS
PP NAM MSP FA. Contributed reagents/materials/analysis tools: AMS
PP MED. Wrote the paper: BA MFU MED.
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DEHP Affects Oocyte Energy and Oxidative Status
PLoS ONE | www.plosone.org12 November 2011 | Volume 6 | Issue 11 | e27452