Reactive oxygen and nitrogen species in reproductive biology

Abstract

The aim of this review is to analyze the increasing number of data regarding the relationship between reactive oxygen and nitrogen species (ROS/RNS) and the physiology of reproduction. ROS/RNS have been found to be implicated in the etiology of a high number of male and female infertility cases. Excessive ROS levels, which can be attained by either an increased ROS production or by low levels of antioxidants, lead to a number of defects especially in membranes and DNA of germinal cells and gametes that severely compromise maturation and fertilization processes. Embryo early development is also affected by the imbalance of the redox status, which is associated with cell fragmentation and blocking of development, usually at the two-cell stage. We will focus on the sources and the participation of ROS, mainly superoxide anion, hydrogen peroxide, and nitric oxide in sperm capacitation, ovary function and, more interestingly, in the fertilization process. The role of NADPH-oxidoreductases, which initiate the early oxidative burst during sea urchin egg fertilization and new data linking ROS/RNS with cell signaling pathways involved in early development, will be also covered. Finally, special attention will be given to the oxidative stress induced during in vitro manipulation of gametes and embryos in assisted reproduction techniques, which can be minimized in order to accomplish successful in vitro fertilization.

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Free Radicals in Biology and Medicine, 2008: 43-66 ISBN: 978-81-308-0267-1
Editors: Carlos Gutiérrez-Merino and Christiaan Leeuwenburgh
3
Role of reactive oxygen and
nitrogen species in reproductive
biology


Francisco Javier Martin-Romero1* and Ignacio S. Alvarez2*
*Reproductive Biology and Development Group (ReDes), 1Departamento de
Bioquimica y Biologia Molecular and 2Departamento de Biologia Celular
Universidad de Extremadura, 06071-Badajoz, Spain




Abstract
The aim of this review is to analyze the increasing
number of data regarding the relationship between
reactive oxygen and nitrogen species (ROS/RNS) and
the physiology of reproduction. ROS/RNS have been
found to be implicated in the etiology of a high
number of male and female infertility cases. Excessive
ROS levels, which can be attained by either an
increased ROS production or by low levels of
antioxidants, lead to a number of defects especially in
membranes and DNA of germinal cells and gametes
that severely compromise maturation and fertilization

Correspondence/Reprint request: Dr. Francisco Javier Martín-Romero, Departamento de Bioquimica y
Biologia Molecular, Facultad de Ciencias, Universidad de Extremadura, 06071-Badajoz. Spain
E-mail: fjmartin@unex.es

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Francisco Javier Martin-Romero & Ignacio S. Alvarez
44
processes. Embryo early development is also affected by the imbalance of the
redox status, which is associated with cell fragmentation and blocking of
development, usually at the two-cell stage. We will focus on the sources and the
participation of ROS, mainly superoxide anion, hydrogen peroxide, and nitric
oxide in sperm capacitation, ovary function and, more interestingly, in the
fertilization process. The role of NADPH-oxidoreductases, which initiate the
early oxidative burst during sea urchin egg fertilization and new data linking
ROS/RNS with cell signaling pathways involved in early development, will be
also covered. Finally, special attention will be given to the oxidative stress
induced during in vitro manipulation of gametes and embryos in assisted
reproduction techniques, which can be minimized in order to accomplish
successful in vitro fertilization.

Abbreviations
ART, assisted reproductive techniques; [Ca2+]i, intracellular free calcium
concentration; CaMKII, calmodulin-dependent kinase II; cAMP, 3',5'
adenosine monophosphate; DAG, diacylglycerol; DPI, diphenylene iodonium;
GPx, glutathione peroxidase; GSH, reduced glutathione; GVDB, germinal
vesicle breakdown; hCG, human chorionic gonadotropin; IP3, inositol 1,4,5-
trisphosphate; IVF, in vitro fertilization; NEM, N-ethyl maleimide; L-NMMA,
NG-monomethyl-L-arginine; NOS, Nitric oxidase synthase; Ovothiol, 1-
methyl-αN,αN-dimethyl-4-thiohistidine; PHGPx, phospholipid hydroperoxide
glutathione peroxidase; pHi, intracellular pH; PKA, cyclic AMP-dependent
protein kinase; RNS, reactive nitrogen species; ROS, reactive oxygen species;
SIN-1, 3-morpholinosydnonimine; SNP, sodium nitroprusside; SOD,
superoxide dismutase; TAC, total antioxidant capacity; WST, 1, 2-(4-
iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium.

1. Relevance of reactive oxygen species in sperm
function
1.a. Role of superoxide and hydrogen peroxide
Sperm maturation is a process that takes place during the transport of
spermatozoa through the epididymus, where these cells acquire progressive
motility, complete nuclear condensation and modify expression and
distribution of surface proteins. It has been proposed that reactive oxygen
species (ROS) play a key role in the regulation of sperm maturation, and the
seminiferous epithelium and mature sperm require high protection against
oxidative stress. Phospholipid hydroperoxide glutathione peroxidase (PHGPx
or GPx4) is a selenoprotein enriched in the mitochondrial capsule of the
spermatozoa, a keratinous matrix of protein surrounding mitochondria in these

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cells [1]. Together with this selenoprotein, another glutathione peroxidase,
GPx1 (cytosolic GPx or cGPx), has been implicated in antioxidant defense in
Leydig cells that are assumed to produce H2O2 during steroid hormone
synthesis [2]. This latter glutathione peroxidase is expressed ubiquitously in all
types of cells. However, the expression of cGPx and the glutathione peroxidase
activity are very low in testis [3] and cGPx knock-out mice reproduce and
develop normally [4]. On the contrary, expression of PHGPx (GPx4) is very
high in rat testis [5], and is expressed almost exclusively in round spermatids
[3], but the expression decays with elongation of spermatids and is not present
in mature spermatozoa, where the PHGPx activity is almost undetectable [6].
Although the mRNA expression is absent, the protein PHGPx is still present in
spermatozoa. This phenomenon is explained in terms of functional
transformation of the protein during spermatogenesis. The soluble PHGPx that
is active as peroxidase in spermatids, switches to an inactive and structural
protein in mature spermatozoa [6] by an oxidative process that is still under
study. PHGPx in the mitochondrial capsule complex can be depolymerized
using reductive treatment, while oxidant treatment with H2O2 leads to
polymerization [6]. Basically, PHGPx is oxidized by hydroperoxides in the
absence of GSH, and then reacts with thiol groups of proteins to become
crosslinked by Se-S bonds [1]. Interestingly, the decrease in the GSH content,
which is required for this alternative reaction, takes places in parallel to the
maturation from spermatids to spermatozoa [7], increasing the probability to
occur this kind of crosslinking reactions in late spermatogenesis.
On the other hand, ejaculated sperm can not fertilize until spermatozoa
acquire the ability to increase motility and develop the acrosomal reaction.
These modifications are induced by different substances present in semen, by
the female reproductive tract, or by progesterone secreted by the oocyte
cumulus complex [8-10], in a process termed capacitation. Sperm capacitation
is associated with an increase in protein tyrosine phosphorylation levels [11]
and it is known that ROS trigger capacitation, being this effect mediated by the
cAMP-dependent transduction pathway. Two proteins have been identified as
major targets of tyrosine phosphorylation during sperm capacitation (mol. wt.
of 105 and 81 kDa) [12]. It has been proposed that ROS (mainly superoxide
and H2O2), at physiological levels, induce sperm capacitation by enhancing
adenylate cyclase activity that leads to the increase of cAMP and stimulates
tyrosine kinase activity, and by inhibiting tyrosine phosphatases at the same
time. This stimulation is similar to that found with the addition of NADPH, or
with the addition of dibutyryl-cAMP [13]. The convergence of the redox- and
cAMP-regulated pathways has been demonstrated by the ability of the PKA
inhibitor, H89, to block both routes of signaling. In addition, catalase can
inhibit the redox pathway, while it has no effect on the signaling stimulated by
cAMP, indicating that ROS generation is upstream from cAMP-regulation

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Francisco Javier Martin-Romero & Ignacio S. Alvarez
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reaction [13]. This conclusion is supported with the inhibition of ROS
generation with 2-mercaptoethanol, 2-deoxyglucose or diphenylene iodonium
(DPI), that leads to the failure of activation mediated by cAMP [13].
On the contrary, other authors have reported that capacitation process is
mediated by extracellular Ca2+ uptake and subsequent elevation in cAMP
levels. This cAMP stimulates the generation of superoxide anion, by a not still
defined mechanism that involves serine/threonine protein phosphorylation,
which causes the increase in p105/81 phosphotyrosine content [14]. On the
other hand, Lewis and Aitken reported that production of H2O2 by caudal
epididymal cells was stimulated by NADPH, and this H2O2 generation induces
an increase in tyrosine phosphorylation [15]. Taken together all results, it is
proposed that tyrosine phosphorylation in rat spermatozoa is mediated by ROS
by two complementary mechanisms; (1) superoxide stimulates tyrosine kinase
activity indirectly through the elevation of intracellular cAMP while (2) H2O2
acts directly stimulating the kinase and inhibiting the phosphatase activity.
There are plenty of evidences that demonstrate the production of ROS by
sperm. The most important pathway for the intracellular generation of ROS is
the mitochondrial electron transport chain. Nevertheless, many other metabolic
processes have been added as sources of ROS, like intracellular oxidases (e.g.
xanthine oxidase), cytochrome P450, nitric oxide synthase, NAD(P)H oxidases
or the plasma membrane redox system. The production of ROS in human
sperm has been demonstrated with the chemiluminescence probe luminol, after
activation with the ionophore A23187 [16]. This rapid production of ROS is
not affected by mitochondrial inhibitors, but cytochrome c decreased in a 50%
the response to activation by A23187, suggesting that superoxide is a major
product of human activated spermatozoa. These results, together with other
reports [17-20] indicate that this ROS production is in fact associated to the
degree of defectiveness in sperm function. The source of these ROS has been a
central point of discussion, in part due to the low specificity of methods used
for ROS detection in spermatozoa. However, early reports confirmed that
extracellular NADPH triggers a rapid dose-dependent increase in the rate of
superoxide generation. This superoxide production can not be inhibited by
inhibitors of the mitochondrial electron transport chain (antimycin A, rotenone,
carbonyl cyanide m-chlorophenylhydrazone [CCCP], and sodium azide).
Dicoumarol, allopurinol or sodium oxamate do not inhibit this oxidative burst,
but the involvement of a flavoprotein in the electron transfer process was
indicated by the high sensitivity of the oxidase to inhibition by diphenylene
iodonium (DPI) and quinacrine [21]. Both NADH and NADPH were active
electron donors in this system, and this stimulation of ROS production was
accompanied by tyrosine phosphorylation and capacitation in the same degree
[22]. All these data are consistent with the existence of a NAD(P)H oxidase in
the plasma membrane of spermatozoa. Actually, the gene for the NADPH

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ROS in reproductive biology
47
oxidase family member NOX5 is expressed in human immature sperm cells
[23]. NOX5 is similar to the gp91(phox) subunit of the phagocyte NADPH
oxidase. However, NOX5 contains three EF hand motifs that are associated
with the response to elevations of the cytosolic Ca2+ concentration. Upon Ca2+
activation, NOX5 acts not only as superoxide source but also it functions as a
proton channel, presumably to compensate charge and pH imbalance due to
electron export. Aitken et al. [24] have recently shown that human
spermatozoa are capable to reduce 1, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-
(2,4-disulphophenyl)-2H-tetrazolium (WST-1), in the presence of extracellular
NADH. This activity resembles the described trans-membrane NADH oxidase,
which is inhibited by capsaicin, superoxide dismutase (SOD) and N-ethyl
maleimide (NEM) [25,26], but in spermatozoa the reduction of WST-1
induced by NAD(P)H was not suppressed by capsaicin. In sum, human
spermatozoa have diverse plasma membrane redox systems that are involved
in the physiological control of sperm function, although their pharmacological
sensitivity is still under study.

1.b. Contribution of the nitric oxide pathway
Nitric oxide synthase (NOS) isoforms are present in mouse and human
spermatozoa [27,28]. Localization and intensity of immunoreactivity is
dependent on the specie. In human spermatozoa NOS expression is localized
on postacrosomal and equatorial segments, and it has been shown to be intense
in normozoospermic patients, whereas asthenozoospermic patients show no
immunoreactivity against NOS [28]. In fact, NOS pattern of expression is
assumed to be under the molecular basis of the sperm motility classification,
indicating that NO· is involved in normal sperm physiology [29,30]. Western
blot analysis have confirmed the expression of NOS in human [31], mouse
[32] and sea urchin sperm [33]. Using this latter animal model of study, NOS
have been detected at high levels in spermatozoa, being an active protein as
determined with the citrulline assay, determination of nitrites and by
fluorescence changes after nitrosation of the NO· indicator diaminofluorescein
(DAF) [33]. However, there are controversial data using human spermatozoa,
in part due to the small amount of NO· produced by these cells. Electron
paramagnetic resonance (EPR) has been used to address this question and
these measurements indicated that NO· is synthesized by human motile
spermatozoa, and that this synthesis is associated with capacitation (8-fold
increase compared to non-capacitated spermatozoa) [34].
NO·-donors have been used to better know the involvement of NO· in the
normal physiology of spermatozoa and motility, although with diverse result.
Early reports showed that sodium nitroprusside (SNP) at low concentrations
was beneficial in preserving viability and motility of thawed human sperm
[35], as well as for improving motility in hamster sperm [36]. On the contrary

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48
high concentrations of NO·-donors, i.e., 1 mM SNP, 100-125 µM 3-
morpholinosydnonimine (SIN-1) and 25-125 µM pure NO· gas dissolved in
buffer, inhibited motility in a dose-dependent fashion, due to the inhibition of
sperm respiration, as measured by the tetrazolium-formazan assay, but without a
significant elevation of intracellular cGMP [37]. In this regard high levels of NO·
have been found in patients with low motility spermatozoa associated to reduced
fertility [38]. Consistently, inhibition of NO· synthesis with NG-monomethyl-L-
arginine (L-NMMA), prevents sperm motility decay after 24 hours of incubation
at 37ºC, and prevents loss of viability, indicating that the NO· pathway plays a
role in modulating sperm motility and survival [39].
In addition to motility, NO· has been related to its ability to accelerate as
well as to increase the percentage of sperm capacitation to undergo acrosomal
reaction [40], although the effect was dependent on the NO-releasing agent,
due to differences in the kinetics of NO· formation (SNP vs. diethylamine-
NONOate). During NO·-induced capacitation of human sperm, two proteins
(p81 and p105) resulted phosphorylated in tyrosine residues, whereas the
inhibition of capacitation with L-NAME was accompanied by a significant
decrease in tyrosine phosphorylation of these two proteins [40], which indeed
are also modulated by superoxide and H2O2, as we stated above [12]. More
recently Thundathil et al. showed that the double phosphorylation of the
threonine-glutamine-tyrosine motif (P-Thr-Glu-Tyr-P) in human sperm
proteins of 81 and 105 kDa during capacitation is modulated by nitric oxide
[41]. Superoxide dismutase and catalase do not prevent this phosphorylation
and exogenous addition of superoxide or H2O2 does not trigger the increase in
P-Thr-Glu-Tyr-P. However, L-NAME prevents the increase in P-Thr-Glu-Tyr-
P related to sperm capacitation, whereas L-arginine reverses the inhibitory
effect of L-NAME. Therefore, the regulation of P-Thr-Glu-Tyr-P seems to be
specific to nitric oxide and not to superoxide anion or H2O2. Thus, it is now
accepted that nitric oxide regulates the level of P-Thr-Glu-Tyr-P in sperm
proteins of 81 and 105 kDa during capacitation [41].

2. Role of ROS/RNS in oogenesis and ovary function
ROS are related with many aspects of female reproduction, including
folliculogenesis, oocyte maturation and atresia, embryo implantation and
parturition; however and due to space restrictions we just will focus on ovary
physiology in this chapter.

2.a. Involvement of ROS in follicular development and ovary
physiology
Regarding oogenesis and ovary function, Tilly et al. [42] have reported
that members of the Bcl-2 gene family are expressed in ovarian granulosa cells

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49
during follicular maturation and atresia, and that this expression is
gonadotropin-regulated. Because of the antioxidant properties ascribed to Bcl-
2 it is reasonable to hypothesize that ROS play a role in the apoptosis of
granulosa cells during follicular atresia in the immature rat ovary. Extracellular
addition of SOD, ascorbic acid, N-acetyl-L-cysteine prevents apoptosis in
granulosa cells in vitro, in the same extent as the blockade by treatment with
FSH. These data correlates with the higher expression of extracellular SOD
isoforms (ecSOD) and MnSOD in granulosa cells from rats primed with
equine chorionic gonadotropin (eCG) which promotes antral follicular growth
and survival. However, gonadotropin priming does not increase Cu/Zn-SOD,
GPx or catalase [42]. Thus, the induction of ecSOD and MnSOD expression by
eCG provides evidence that gonadotropins promote granulosa cell survival in
developing antral follicles via activation of an oxidative stress response to
protect granulosa cells from the damaging effects of reactive oxygen species.
Oestradiol is one of the primary luteotropic hormones and the withdrawal
of oestradiol results in a rapid decline in serum progesterone and corpus
luteum regression. Depletion of oestradiol levels in vivo leads to an increase in
bax mRNA and a decrease in bcl-x mRNA levels, synchronized with luteal cell
apoptosis, demonstrating that the expression of bcl-2 gene family members are
one of the mechanisms by which oestradiol exerts its luteotropic effect in the
corpus luteum [43]. ROS are well-established modulators of luteal cell
apoptosis during corpus luteum regression in the estrous cycle. One example of
this modulation is found on the activity of superoxide as inhibitor of the
progesterone production by luteal cells. The suppression of Cu, Zn-SOD
activity with antisense oligonucleotides reduces significantly the progesterone
production by rat luteal cells, an effect that is completely blocked by the
simultaneous addition of N-acetyl-L-cysteine, suggesting that superoxide
radicals and intracellular Cu,Zn-SOD play important roles in the regulation of
luteal function [44]. In addition to SOD and N-acetyl-L-cysteine, and similarly
to those results obtained with granulosa cells during folliculogenesis and
follicular atresia, ascorbic acid or catalase delay apoptosis of corpus luteum, as
well as they reduce levels of bax mRNA, a prooxidant member of the Bcl-2
protein family, although do not modify bcl-x expression [45]. Finally, human
chorionic gonadotropin (hCG) significantly increases expression of
mitochondrial MnSOD, suggesting that Mn-SOD is responsible for the
gonadotropin-mediated inhibition of apoptosis [45].
On the other hand, stimulation of bovine luteal cells by H2O2 resulted in
the induction of apoptotic nuclear condensation and caspase-3 activation.
Accumulation of 8-hydroxy-2'-deoxyguanosine, a marker of DNA damage
induced by oxidative stress, was also detected in luteal cells from the late
estrous stage [46], further confirming the relationship between oxidative stress
and the apoptosis in this phase of the follicle cycle. Hydrogen peroxide

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treatment induces up-regulation of cyclooxygenase (COX-2), p53, and Bax in
vitro, while in vivo histology confirmed higher expression of these messengers
in late luteal cells, suggesting that the enhancement of ROS in the bovine
corpus luteum induces expression of COX-2, p53, and Bax mRNAs, resulting
in activation of the signaling pathway for luteal-cell apoptosis [46].
Finally, reactive oxygen and nitrogen species play a physiological role
during ovulation, as supported by the finding that perfusion of in vitro cultured
ovaries with SOD or catalase, delays ovulation [47]. In fact, it is well
established that the ovary and uterus of cycling and pregnant mice generate
NADPH-dependent superoxide [48], and both ovarian and uterine NADPH-
dependent superoxide production are likely to be luteinizing hormone (LH)-
inducible [49,50].

2.b. Nitric oxide signaling in ovary function
The presence of the constitutive and the inducible isoforms of nitric oxide
synthase in both male and female reproductive organs suggest that local
synthesis of NO· is required for the reproductive physiology. In the female
reproductive tract the three isoforms of NOS (iNOS, nNOS and eNOS) are
expressed in rat cervix, whereas iNOS and eNOS are found in the uterus [51],
and it is believed that NO· signaling have a role in the cascade of events
involved in preparing the uterus and cervix for parturition.
Being more specific in the description of the role of NO· on the ovary
function, it is well known that the three isoforms of the NOS are expressed in
the ovary [52-54]. Immunolocalization of NOS has been reported for thecal
and stromal cells of the ovary during follicular development, ovulation and
luteal formation [53]. Thus, NO· must be considered as an important endocrine
regulator of the ovulatory process. The contribution of NO· in ovulation is
supported by the effects induced by the NOS inhibitors aminoguanidine and L-
NMMA, in vivo, which significantly reduced the rate of folliculogenesis and
the production of mature oocytes [55]. On the contrary, intraperitoneal
injection of the NO-donor sodium nitroprusside completely reverses the effects
of the NOS inhibitors. From these experiments it is concluded that the ovarian
NO·/NOS system is required for the follicle development during oogenesis.
Roselli et al. reported that NO· synthesis, measured as circulating
nitrite/nitrate, increases during follicular development [56], as well as with the
nitrite/nitrate concentration in the follicular fluid [57]. This raised NO·
synthesis correlates well with oestradiol concentration, further supporting for a
role of NO· during the folliculogenesis process.
Human chorionic gonadotropin (hCG) induces germinal vesicle
breakdown (GVBD) in oocytes of preovulatory follicles, and promotes meiotic
progress; however, the nitrate/nitrite concentrations in preovulatory follicles
significantly decreases after hCG injection [58]. In accordance, NO· donors

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51
prevented GVBD and inhibitors of iNOS induced GVBD as well as hCG.
Moreover, it is known that high concentrations of NO· inhibits progesterone
production [59,60] and induces apoptosis in rat granulosa cells [59]. In sum,
concentrations of NO· metabolites (nitrate/nitrite) in preovulatory follicles are
at high levels before hCG injection but decrease after hCG stimulation.
Expression of eNOS, mainly localized in the thecal layer, significantly
increased after hCG injection [52,54,59,61]. However, the direct contribution
of eNOS-derived NO· to oocyte maturation is unclear, because no change of
eNOS expression can be seen in the oocyte during the ovulatory process. On
the contrary, iNOS expression, mainly localized in granulosa cells,
significantly decreased after hCG injection, which induced a decrease of NO·
concentrations in preovulatory follicular fluid. Thus, the reported changes in
NO· concentrations reflect changes of iNOS expression, but not of eNOS, in
granulosa cells of preovulatory follicles [59]. These results suggest that NO·
produced by iNOS is acting as inhibitor of the oocyte maturation. An
intrafollicular high-concentration of NO· likely plays a role in the meiotic
arrest, and the subsequent decrease of NO· concentration after hCG injection
triggers oocyte maturation [58].
The involvement of iNOS in ovary physiology was later confirmed with
experiments showing that inhibition of iNOS decreased cGMP production in
preovulatory follicles and that the addition of an NO· donor blocked this
suppression [58]. Nitric oxide derived from iNOS induces an increase in
cGMP concentration in preovulatory follicles, and then cGMP is transported
via gap junctions into the oocyte, where it has a key role in the meiotic arrest
of oocytes [62]. In fact, levels of both cGMP and cAMP decrease in oocytes in
parallel to spontaneous meiosis and the microinjection of both signaling
molecules into oocytes caused a delay in oocyte maturation [62,63]. It is
believed that cGMP maintains the meiotic arrest of preovulatory oocytes via
two pathways: (1) increasing cAMP levels by the inhibition of oocyte cAMP
phosphodiesterase and (2) activating cGMP-dependent protein kinase in
oocytes [62]. Interestingly, mitogen-activated protein kinase (MAPK)
modulates the initiation of the mammalian oocyte maturation [64], but NO·
attenuates MAPK activity via generation of cGMP [65], and directly via
activation of tyrosine kinase [66]. In conclusion, iNOS inhibition as well as
hCG induce oocyte meiotic maturation, while NO-donors prevents this
process, suggesting that the iNOS-NO·-cGMP pathway modulates the oocyte
meiotic maturation during the ovulatory process.
Further support to the key role of NO· in ovulation came from the
experiments reported by Vega et al. [67], that show that incubation of corpus
luteum with L-arginine elicits an inhibitory action on the production of
oestradiol, and increases apoptosis and the number of positive cells for the
expression of iNOS, while decreases bcl-2 expression. Opposite effects were

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found with L-NMMA and hCG, that are mediated by the upregulation of bcl-2
expression.

3. Reactive oxygen species at fertilization
3.a. Involvement of NADPH oxidases in early signaling at
fertilization
It is well documented that a “respiratory burst” takes place at fertilization
of the egg of the sea urchin and other echinoderm species [68-74]. The oxygen
consumption of zygotes reaches a peak 2 minutes after insemination and then
decreases to a plateau of one-third maximal rate. Although this final rate of O2
consumption is cyanide-sensitive and mitochondrial, the initial burst is CN--
insensitive and develops concomitantly with H2O2 production [75]. All these
events are firstly initiated by an intracellular Ca2+ rise that leads to the
exocytosis of cortical granules [76]. This is a conserved strategy to avoid
multiple insemination, since secreted proteins during exocytosis interact with
the vitelline envelope in a divalent cation manner that allow the sea urchin
eggs alter their extracellular protein envelope at fertilization. Ovoperoxidase, a
70 kDa heme protein, is one of the proteins secreted by the exocytosis of
cortical granules and this protein binds to extracellular proteoliaisin [77]. The
primarily synthesized H2O2 is subsequently used by the secreted
ovoperoxidase to drive the cross-linking of the extracellular protein matrix by
the formation of dityrosil bonds between the ortho carbon atoms of adjacent
tyrosyl groups [78], hardening the fertilization envelope and leading to the
prevention of polyspermy. The assembly of ovoperoxidase into the fertilization
envelope and crosslinking reaction take place within 10 min after gamete
fusion [79].
The enzyme responsible for the initial oxidative burst and H2O2
production was initially discovered by Bennett M. Shapiro in 1985 from egg
cortices (consisting of extracellular vitelline layer, plasma membrane, and
cortical granules) as a Ca2+-dependent NAD(P)H oxidoreductase activity
resistant to inhibition by cyanide [68], distinguishing this oxidase activity from
the NAD(P)H oxidase activity of many peroxidases, which is CN--sensitive.
The active cofactor of this cortical NAD(P)H oxidoreductase activity was later
identified as 1-methyl-αN,αN-dimethyl-4-thiohistidine, or ovothiol, and was
originally described as the cofactor that confers this oxidoreductase activity on
the ovoperoxidase [69]. However, this oxidoreductase activity was later
attributed to other protein fraction purified from egg cortex. The
characterization of this fraction revealed that synthesis of H2O2 at fertilization
of the sea urchin eggs was stimulated by micromolar Ca2+ (Km ~ 4 µM) and
MgATP2- [70]. Nonhydrolyzable ATP analogs and other nucleotides can not
replace ATP [70]. The activated oxidase was found to utilize NADPH as

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53
substrate with Km value of 40 µM (Km for NADH >400 µM), and it is not
affected by cyanide, azide, and aminotrizole [73]. On the contrary this NADPH
oxidoreductase is sensitive to protein kinase inhibitors such as promethazine
and trifluoperazine, N-ethylmaleimide, H-7 and HA1004 in the micromolar
range [70], and by nanomolar concentrations of staurosporine, suggesting a
role for Ca2+-calmodulin and protein kinase C in early signaling and oxidative
burst at fertilization. The absolute requirement of protein kinase C in the
activation of the oxidase was later demonstrated with isolated rat brain PKC,
that could replace the soluble factor as an activator of the egg membrane
oxidase [72]. In addition, the [Ca2+]i required for activation of oxidase is
decreased from 40 to 0.8 µM by phorbol ester, and neither CaMKII, PKA,
casein kinase II, nor myosin light chain kinase activate the oxidase [72].
Moreover, the [Ca2+]i rise at fertilization activates the calmodulin-
dependent stimulation of NAD+ kinase [80], increasing the NADP+/NAD+
ratio. Thereafter, hexose monophosphate shunt activity reduces NADP+ to
NADPH, providing a source of electron donor to the NADPH oxidoreductase
as an early event in fertilization. The postfertilization increase in NADPH in
sea urchin eggs could be involved in subsequent biosynthetic reactions like the
reductive synthesis of deoxyribonucleotides or fatty acids required during the
metabolic onset after fertilization [71]. Additionally redox changes may also
be involved in the reinitiation of DNA synthesis following fertilization [81],
and NADPH might enhance the protein translation rate through its modulation
on the initiation factor eIF-2B in echinoid eggs [82]. In lysates from sea urchin
eggs eIF-2B activity is sensitive to NADPH [83], and eIF-2B affects the
binding of the initiator tRNA to the small ribosomal subunit. Thus, it has been
hypothesized that NADPH generation at fertilization may be one of the factors
responsible for the 5- to 15-fold increase in protein synthesis after fertilization
[82].
On the contrary to close related oxidases like neutrophil oxidase, the
NADPH oxidase found in sea urchin eggs does not generate ·O2
by the superoxide dismutase-inhibitable reduction of cytochrome c, or
nitroblue tetrazolium [73], leading to the conclusion that egg oxidase reduces
O2 directly to H2O2 by a two-electron transfer reaction. Direct measurement on
a single-cell level using luminol and an imaging photon detector system, Wong
et al. found that 60-65 nM H2O2 accumulates in the perivitelline space [84], the
region between the plasma membrane and the fertilization envelope, where
active ovoperoxidase is present. When peroxidase-dependent oxidation of
Amplex Red to resorufin was used to track the kinetics of H2O2 generation it
was found a significant difference between the oxidase activities from
fertilized zygotes compared to A23187-activated eggs. The activation induced
by the Ca2+ ionophore evoked a linear generation of H2O2, while fertilization
induced a biphasic response [84]. A simple explanation for this difference is
-, as assessed

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Francisco Javier Martin-Romero & Ignacio S. Alvarez
54
that ionophore acts uniformly over the egg whereas sperm initiates a polarized
wave of activation.
Another early event after gamete fusion induced by the calcium rise is the
Na+-dependent intracellular alkalinization (from pH 6.9-7.0 to 7.2-7.4 [85]).
Calcium stimulates the Na+/H+ antiporter activation leading to the increase of
the intracellular pH (pHi). This alkalinization enhances the synthesis of H2O2,
since the replacement of Na+ by choline inhibits the O2 consumption and the
H2O2 synthesis by ionophore-stimulated eggs, an effect that is reversed by
treatment with NH4Cl which directly alkalinizes the cytoplasm [86]. In
addition, phorbol ester-treated eggs show an increase in pHi that is Na+-
dependent, like the effect reported at fertilization, suggesting a role for protein
kinase C (PKC) in the regulation of pHi upon fertilization of sea urchin eggs
[87]. For this reason it is assumed that cytoplasmic alkalinization may act as
modulator of the NADPH oxidoreductase activity. A proposed model for
regulation of the oxidative burst at fertilization is shown in Figure 1. This
model suggests that the gamete fusion activates phospholipase C and the
phosphoinositide pathway, releasing diacylglycerol (DAG) and inositol 1,4,5-
trisphosphate (IP3). Increased IP3 activates the release of Ca2+ from
intracellular stores [88], which together with DAG activates PKC. The
respiratory burst could have a negative regulation by the subsequent decrease
in [Ca2+]i after the first propagated calcium wave [88], and following
completion of dityrosine cross-linking [75].
The long half-life time of H2O2, and the capability to diffuse through
plasma membrane, makes critical the existence of a mechanism that controls
sea urchin eggs exposure to potentially lethal ROS. These eggs are highly
enriched in an aminoacid that scavenges H2O2, ovothiol, which is present at
millimolar concentrations [89,90]. Ovothiol is a mercaptohistidine with a low
thiol pKa, which is present as thiolate at physiological pH, making ovothiol
particularly efficient in the reduction of H2O2 [89,91]. Ovothiol is more
effective than catalase in eliminating H2O2 at concentrations attained during
fertilization and is a major mechanism for preventing oxidative damage at
fertilization. Oxidized ovothiol is subsequently reduced by glutathione, which
can be regenerated by NADPH-dependent glutathione reductase, present in
high levels in eggs [90]. Thus, increased NADPH/NADP+ ratio following
fertilization serves both to generate extracellular H2O2 and to protect egg
cytoplasm from H2O2 back diffusion into the cell.
Although the calcium rise that follows fertilization is ubiquitous and is
considered a major signaling event in early development, it has been shown
that activation of NAD+ kinase is not a universal feature of egg activation in
marine invertebrates, and it was found only in clam and sea urchin eggs.
However, postfertilization redox ratio ([NADPH]/ [NADP+]) is 2 or higher in

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ROS in reproductive biology
55
all studied species, suggesting that there is a restoration of a normal
metabolism in fertilized eggs [71].
More recently, a sea urchin ortholog of the thyroid oxidase has been found
to be expressed in zygotes. This new oxidase is a member of the dual oxidase
family of transmembrane proteins, with a N-terminal peroxidase domain and a
C-terminal reductase domain linked by a cytoplasmic bridge with two EF
hands [84], suggesting a role of calcium in the regulation of this enzyme. This
urchin dual oxidase 1 (Udx1) expresses a 6 kb udx1 transcript that accumulates
in developing oocytes, as well as the final product, a 185 kDa protein present
at the egg cell surface. The incubation of eggs with a polyclonal antibody
raised against the dual oxidase inhibits 30% the H2O2 synthesis, a value close
to the one found with DPI, a potent inhibitor of both the neutrophil oxidase
burst and endothelial nitric oxide synthase [92,93]. However microinjection of
this antibody into eggs resulted in a larger inhibition at lower concentration of
the antibody [84], further supporting the hypothesis that Udx1 is responsible
for the respiratory burst at fertilization.

Gamete fusion
NAD+
NADP+
NADPH
[Ca2+]i
NAD+
kinase
Protein
Kinase C
Protein
Kinase C
Na+/H+
antiporter
pHi
NADP+
oxidase activation
O2
H2O2
activated PLC
IP3 + DAGPIP2

Figure 1. Model for regulation of the oxidative burst at fertilization in the sea urchin
egg [70,73].

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Francisco Javier Martin-Romero & Ignacio S. Alvarez
56
Udx1 was found to use NADPH as preferential substrate, in accordance to
previous studies (as stated above). The oxidase activity of Udx1 is sensitive to
PKC inhibitors, such as rottlerin or staurosporine, although this sensitivity is
species-specific [84]. In addition Udx1 shows a pH-dependent activity that
overlaps with the described transient rise in pHi at fertilization (from 7.1 to 7.5
in Strongylocentrus purpuratus), once again indicating that Udx1 is the
activated oxidase at fertilization. Finally, the inhibition of Udx1 with DPI or 3-
aminotriazole, directly affects ovoperoxidase activity, due to the inhibited
production of its substrate H2O2, depleting the crosslinking level of the
proteins at the fertilization envelope [84].
In sum, after insemination, a rise in [Ca2+]i takes place in the zygote,
similar to other species. This initial calcium wave induces the secretion of the
cortical granules content to the perivitelline space. In addition there is calcium-
dependent activation of PKC that slightly increase intracellular pH through the
activation of the Na+/H+ antiporter. Both, the PKC activation and the increase
in pHi are required for the subsequent activation of Udx1, a plasma membrane
protein with NADPH oxidase activity that produces H2O2. Secreted
ovoperoxidase consumes this H2O2 in the formation of dityrosyl bonds to
harden the fertilization envelope and block polyspermy. At the same time, eggs
have developed a system to protect the intracellular environment against
oxidative damage. Increased NADPH synthesis, which is stimulated by the
initial rise of [Ca2+]i, serves as electron donor for the glutathione reductase that
recycles the glutathione/ovothiol system within the cell.

3.b. Nitric oxide in early signaling following fertilization
In addition to H2O2, nitric oxide comes out as a key regulator in early
steps of fertilization and embryo development. Nitric oxidase synthase is
present at high levels in the sea urchin sperm, which can be immunolocalized
primarily at the head and the acrosome region. NOS is active in spermatozoa,
as revealed by the NADPH-dependent diaphorase assay, and the citrulline
assay, whereas this activity is sensitive to NG-nitro-L-arginine, supporting
previous findings. Eggs were found positive to this NOS activity, which is
enhanced after activation, although not at the high levels detected in sperm,
most probably due to the high content in ovothiol in eggs, which may scavenge
this NO· generation [33]. It is well known that NO· elicits an increase in [Ca2+]i
in many mammalian tissues, and it has been described also that NO· triggers
the release of calcium from intracellular stores in sea urchin eggs [94]. This
effect is mediated through cGMP and cADPr pathway, since the NO·-
stimulated Ca2+ release is decreased with inhibitors of the cGMP-dependent
protein kinase or with competitors, such as 8-amino-cADPr. Moreover,
activation of eggs with ionomycin does not increase cGMP concentration

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ROS in reproductive biology
57
within eggs, suggesting that NO· production and subsequent activation of the
cGMP pathway precedes Ca2+ rise at fertilization [95].
Using diaminofluorescein (DAF) it has been monitored the kinetics of NO·
production in sea urchin eggs, and found that an initial transient takes place
within seconds after insemination, followed by a sustained increase during
minutes. The kinetics of NO· production was initially found to precede the first
calcium pulse of fertilization, and the microinjection of NO· scavengers, like
oxyhaemoglobin, prevents the egg activation stimulated by insemination.
Microinjection of NO·-donors, like SNAP, or recombinant nNOS with
calmodulin, activate the eggs [33]. However Kuo and co-workers did not
measured NO· and Ca2+ simultaneously, and the temporal relationship between
the NO· rise and the Ca2+ transient was indirectly concluded. Indeed, the results
reported by this group could be affected by the initial rise in pHi that occurs in
sea urchin eggs, since the fluorescent product of the reaction of DAF-2 with NO·
exhibits pH-dependent fluorescence intensity. Therefore, the involvement of the
NO· pathway at fertilization is still a matter of intense debate. A more recent
study with mouse oocytes and with the ascidian Ascidiella aspersa, studied
intracellular NO· and Ca2+ levels simultaneously, and found that sperm-induced
Ca2+ rises were not associated with any global or local change in intracellular
NO· [96]. Furthermore, NG-nitro-L-arginine methyl ester had no effect on sperm-
induced Ca2+ release but did block completely ionomycin-induced NO· synthase
activation [96], as described earlier for the sea urchin egg [33].
Finally, a recent study has monitored NO· and Ca2+ levels at fertilization
using fluorescence indicators of NO· (DAF and DAF-FM), and it has been
established that NO· levels rise after, not before, the initiation of the Ca2+ wave
[97]. Inhibition of the increase in NO· at fertilization does not abolish the initial
Ca2+ transient, although its duration is reduced. Similar results were obtained
when cGMP and cADPR signaling was inhibited, leading to the conclusion
that cADPR is generated at fertilization after the initial rise of Ca2+, similarly
to NO·. Thus, NO· is not a primary activator in sea urchin eggs, but it
modulates the duration of the Ca2+ transient [97].
In conclusion, the precise molecular mechanism underlying the role of
NO· as egg activator remains unknown, and it is believed that is specie-
specific. In addition to its role on the cGMP and cADPR pathway, NO· may
induce calcium release from ryanodine receptor-stores, which have been
described to be sensitive to poly-S-nitrosylation [98], and nitrosylation may
trigger signaling molecules such as src [99] which in turn could elicit
phospholipase C activity [100].

4. Redox metabolism in early embryo development
Early development, i.e. preimplantation development between 2-cell and
blastocyst stage has been described as highly sensitive to oxidative stress [101-