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Abstract
In vitro maturation (IVM) is emerging as a popular technology at the forefront of
fertility treatment and preservation. However, standard in vitro culture (IVC) con-
ditions usually increase reactive oxygen species (ROS), which have been implicat-
ed as one of the major causes for reduced embryonic development. It is well-known
that higher than physiological levels of ROS trigger granulosa cell apoptosis and
thereby reduce the transfer of nutrients and survival factors to oocytes, which leads
to apoptosis. ROS are neutralized by an elaborate defense system that consists of
enzymatic and non-enzymatic antioxidants. The balance between ROS levels and
antioxidants within IVM media are important for maintenance of oocytes that de-
velop to the blastocyst stage. The effects of antioxidant supplementation of IVM
media have been studied in various mammalian species. Therefore, this article re-
views and summarizes the effects of ROS on oocyte quality and the use of antioxi-
dant supplementations for IVM, in addition to its effects on maturation rates and
further embryo development.
Citation: Khazaei M, Aghaz F. Reactive oxygen species generation and use of antioxidants during in vitro
maturation of oocytes. Int J Fertil Steril. 2017; 11(2): 63-70. doi: 10.22074/ijfs.2017.4995.
63
Khazaei and Aghaz
tonic generation of ROS triggers meiotic resump- converts H2O2 to O2 and H2O. The enzyme SOD
tion from diplotene as well as the MII arrest stage exists as three isoenzymes (49): SOD1, SOD2, and
in several mammalian species (44, 45). It has been SOD3. SOD1 contains Cu and zinc (Zn) as metal
reported that levels of ROS beyond the physiologi- co-factors in the cytosol. SOD2 is a mitochondrial
cal range could induce destabilization of matura- isoform that contains manganese (Mn), whereas
tion MPF, reduce survival factors, and trigger SOD3 encodes the extracellular form. Nutrients
mitochondria-mediated apoptosis of oocytes (15, such as Se, Cu, and Zn are required for the activi-
7KHELSKDVLFUROHRI526PXVWEHVXI¿FLHQWO\ ties of some antioxidant enzymes, although they
discussed in order to update OS and its impact on have no antioxidant actions. Non-enzymatic anti-
RRF\WH TXDOLW\ 7KH EHQH¿FLDO UROH RI 526 oxidants are composed of glutathione (GSH), vita-
comes from the observations that non-enzymatic min C, taurine, hypotaurine, vitamin E, Zn, seleni-
antioxidants, such as ascorbic acid and 3-tert-bu- um (Se), beta carotene, and carotene (47). GSH is
tyl-4-hydroxyanisole (BHA), inhibit spontaneous a tripeptide thiol compound with many important
meiotic resumption from diplotene arrest (47). functions in intracellular physiology and metabo-
7KHVH UHVXOWV VXJJHVW D EHQH¿FLDO WKUHVKROG OHYHO lism. One of the most important roles of GSH is
for ROS. to maintain the redox state in cells which protects
them against harmful effects effects caused by
$QWLR[LGDQWV oxidative injuries. The protective action of GSH
against ROS is facilitated by the interactions with
Antioxidants scavenge ROS, which helps main-
its associated enzymes, such as GPx and GSH re-
tain the cell oxidant/antioxidant balance. On the
ductase (Fig.2).
other hand, antioxidants are the compounds which
either suppress the formation of ROS or oppose Vitamin C (ascorbic acid) is a known redox
their actions. There are two types of antioxidants: FDWDO\VWWKDWFDQUHGXFHDQGQHXWUDOL]H526
enzymatic and non-enzymatic (Table 1). Based on its chemical structure, ascorbic acid is
an electron donor and therefore a reducing agent.
Table 1: >ŝƐƚ ŽĨ ƐƚƵĚŝĞƐ ƚŚĂƚ ƐŚŽǁ ƚŚĞ ĞīĞĐƚƐ ŽĨ ĂŶƟŽdžŝĚĂŶƚ
ƐƵƉƉůĞŵĞŶƚƐƚŚĂƚŝŵƉƌŽǀĞin vitroŵĂƚƵƌĂƟŽŶ It has two different biochemical roles-antioxidant
and enzymatic cofactor. Ascorbic acid is main-
$QWLR[LGDQW ([SHULPHQWDOPRGHO
tained through reactions with GSH and can be
Enzymatic antioxidants FDWDO\]HGE\SURWHLQGLVXO¿GHLVRPHUDVHDQGJOX-
Superoxide dismutase (SOD) Mouse taredoxins. Cysteamine is a low-molecular weight
Thioredoxin Porcine amino acid that contains thiol (51). The addition
Catalase (CAT) Bovine of cysteamine not only enhances the GSH content
Sericin Bovine
in MII oocytes but also protects the membrane li-
pids and proteins due to indirect radical scaveng-
Non-enzymatic antioxidants
ing properties (52). The concentrations of many
Glutathione (GSH) Hamster, pig, ovine, amino acids, including taurine and hypotaurine are
Bovine and equine non-enzymatic antioxidants that help maintain the
Cysteamine Canine, mice, goats, porcine redox status in oocytes (53).
Vitamin C (Ascorbic acid) Mouse 9LWDPLQ ( ĮWRFRSKHURO LV D OLSLG VROXEOH
Vitamin E and trolox - vitamin with antioxidant activity. It consists of
eight tocopherols and tocotrienols. Vitamin E
Enzymatic antioxidants neutralize excess ROS may directly destroy free radicals such as perox-
and prevent it from damaging the cellular struc- yl and alkoxyl (ROO•) generated during ferrous
ture. Enzymatic antioxidants are composed of ascorbate-induced lipid peroxidation (LPO),
superoxide dismutase (SOD), catalase (CAT), thus it is suggested as a major chain breaking
various peroxidases and peroxiredoxins (PRDXs), antioxidant (54). Hyaluronan, melatonin, tea
including glutathione peroxidases (GPXs), which and sericin are known to act as indispensable
can convert peroxides to water and alcohol (48). antioxidants in IVEP. They can block the release
SOD enzymes catalyze the dismutation of super- of pro-oxidant factors released as a result of OS
oxide anion (O2í) into O2 and H2O2 while CAT (12, 55, 56).
Hyaluronan, an essential component of the ex- (77). Although, other studies in goats (78), pigs
tracellular matrix and non-sulfated glycosami- KRUVHV EXIIDORV DQG FDWWOH
noglycan may play an important role in meiotic did not show any increase in nuclear maturation
resumption of oocytes (57). The hormone mela- rates. Addition of cysteamine to the IVM medium
tonin (N-acetyl-5-metoxy tryptamine) is an anti- improved embryo development to the blastocyst
oxidant that, unlike GSH and vitamins C and E, is stage in mammalian oocytes (82).
produced by mammals. In contrast to other anti-
Ascorbate is concentrated in granulosa cells,
oxidants, however, melatonin cannot undergo re-
theca cells, luteal cells, and oocytes (28). Choi
dox cycling. Once oxidized, it is unable to return
HW DO UHSRUWHG D EHQH¿FLDO UROH IRU YLWDPLQ
to its reduced state because of the formation of
C in protecting spindle structures of MII mouse
stable end-products after the reaction (14). As an
oocytes and chromosomal alignment against an
antioxidant, green tea has been shown to improve
oxidant (hydrogen peroxide)-induced damage. It
IVM and embryo development of sheep COCs to
is suggested that the effect of vitamin C is as-
the blastocyst stage in IVM medium (58). Sericin
sociated mainly with its capability to promote
a water-soluble globular protein (protein hydro-
RRSODVPLF PDWXUDWLRQ GXULQJ ,90 7KH EHQH¿-
lysate) is derived from the silkworm Bombyx
cial role of ROS comes from the observations
mori. This protein represents a family of proteins
that non-enzymatic antioxidants, such as ascor-
ZKRVHPROHFXODUPDVVUDQJHVIURPWRN'D
bic acid, inhibit spontaneous meiotic resumption
'DVK HW DO KDYH UHSRUWHG WKDW VHULFLQ
from diplotene arrest. We have presented a num-
PLJKW SURYLGH D SURWHFWLYH HIIHFW RQ ¿EUREODVWV
ber of these observations. Tatemoto et al. (84),
by promoting endogenous antioxidant enzymes
Kere et al. (85), and Córdova el al. (86) found
in vitro.
that the addition of vitamin C to the oocyte matu-
$QWLR[LGDQW VXSSOHPHQWV DQG LPSURYLQJ in ration medium exerted no effect on the matura-
vitroPDWXUDWLRQ tion rates of oocytes. Similarly, antioxidants such
as vitamin E and trolox had no effect on oocyte
The addition of enzymatic antioxidants such
maturation, but other antioxidants such as propyl
as SOD, CAT, and thioredoxin are effective for
gallate and 2,4,5-trihydroxybutrophenone inhib-
pre-embryo development as scavengers of ROS
ited the spontaneous resumption of meiosis (87).
and serving embryos a low OS condition in mice
7RJHWKHUWKHVHVWXGLHVHPSKDVL]HGWKHEHQH¿FLDO
(61, 62), porcine (63), and bovines (64). Sericin,
roles of ROS during IVM at certain concentra-
an antioxidant protein, improves embryo devel-
tions (low level).
RSPHQWDQGLVDFULWLFDOVXSSOHPHQWIRU
oocyte maturation (12, 56).
A series of non-enzymatic antioxidants protect
oocytes against ROS damage during oocyte mat-
uration. GSH is one of the naturally synthesized
antioxidants that protect cells from ROS toxic-
ity and regulate the intracellular redox balance
(66). The intracellular level of GSH increases
during oocyte maturation in hamsters (67), pigs
RYLQHERYLQHVDQGHTXLQHV
Recent reports have shown that addition of low
molecular weight thiol compounds, such as cy-
steamine and b-mercaptoethanol to IVM media
improved the cytoplasmic maturation of oocytes
and embryo development by increasing GSH
synthesis (66, 72, 73).
Cysteamine supplementation during IVM re- Fig.1: dŚĞƉŽƐƐŝďůĞĨĂĐƚŽƌƐƚŚĂƚŝŶĚƵĐĞŐĞŶĞƌĂƟŽŶŽĨƌĞĂĐƟǀĞŽdžLJŐĞŶ
ƐƉĞĐŝĞƐ;ZK^ͿŝŶƚŚĞŽŽĐLJƚĞ͘dŚĞŝŵďĂůĂŶĐĞďĞƚǁĞĞŶZK^ĂŶĚĂŶƟ-
portedly improved nuclear maturation rates in ŽdžŝĚĂŶƚƐ͕ƚŚĞŝŵƉĂĐƚŽĨŚŝŐŚůĞǀĞůƐŽĨZK^͕ĂŶĚƚŚĞƌĞƐƵůƟŶŐŽdžŝĚĂƟǀĞ
canines (74), mice (75), goats (76), and porcine ƐƚƌĞƐƐ;K^ͿŽŶŵĞŝŽƟĐĂƌƌĞƐƚĂŶĚĂƉŽƉƚŽƐŝƐŝŶŽŽĐLJƚĞƐ͘
Fig.2: dŚĞƉƌĞƐĞŶĐĞŽĨĂŶƟŽdžŝĚĂŶƚĞŶnjLJŵĞƐƐƵĐŚĂƐƐƵƉĞƌŽdžŝĚĞĚŝƐŵƵƚĂƐĞ;^KͿ͕ŐůƵƚĂƚŚŝŽŶĞƉĞƌŽdžŝĚĂƐĞƐ;'WdžͿ͕ĂŶĚĐĂƚĂůĂƐĞ;dͿĂƐǁĞůůĂƐŶŽŶͲ
ĞŶnjLJŵĂƟĐĂŶƟŽdžŝĚĂŶƚƐ͕ƐƵĐŚĂƐǀŝƚĂŵŝŶĂŶĚ;ĂƐĐŽƌďŝĐĂĐŝĚͿ͕ŐůƵƚĂƚŚŝŽŶĞ;'^,Ϳ͕ƵƌŝĐĂĐŝĚ͕ĂŶĚĂůďƵŵŝŶŝŶƚŚĞŽŽĐLJƚĞƐ͘džĐĞƐƐĂŵŽƵŶƚƐŽĨƌĞĂĐƟǀĞ
ŽdžLJŐĞŶƐƉĞĐŝĞƐ;ZK^ͿŵĂLJďĞŝŶǀŽůǀĞĚŝŶŽdžŝĚĂƟǀĞƐƚƌĞƐƐ;K^ͿŽĨŽŽĐLJƚĞƐĂŶĚŐƌĂŶƵůŽƐĂĐĞůůƐ͘
Conclusion Acknowledgements
It is well-known that high levels of ROS be- 7KLV VWXG\ ZDV VXSSRUWHG ¿QDQFLDOO\ E\
yond the physiological range could induce MPF Kermanshah University of Medical Sciences.
destabilization, reduce survival factors, and trig- 7KHUHLVQRFRQÀLFWRILQWHUHVWLQWKLVVWXG\
ger apoptosis in oocytes of several mammalian
species. Antioxidants are the main defense fac- References
tors against OS induced by ROS. Many reports 1. &DQLSDUL 5 2RF\WHJUDQXORVD FHOO LQWHUDFWLRQV +XP
suggest that antioxidant supplementation of IVM 5HSURG8SGDWH
2. (SSLJ--2RF\WHFRQWURORIRYDULDQIROOLFXODUGHYHORSPHQW
media improves cytoplasmic maturation by al- DQG IXQFWLRQ LQ PDPPDOV 5HSURGXFWLRQ
leviating OS during oocyte maturation via in- 829-838.
creasing GSH storage, and contributes to further 3. 7DQJKH69DQ6RRP$1DXZ\QFN+&RU\Q0GH.UXLI
$0LQLUHYLHZIXQFWLRQVRIWKHFXPXOXVRRSKRUXVGXULQJ
protect the embryo against oxidative aggressions RRF\WHPDWXUDWLRQRYXODWLRQDQGIHUWLOL]DWLRQ0RO5HSURG
during its early developmental stages. On the oth- 'HY
er hand, supplementation by antioxidants during 4. &RPEHOOHV &0 )LVVRUH 5$ $OEHUWLQL ') 5DFRZVN\ &
,Q YLWUR PDWXUDWLRQ RI KXPDQ RRF\WHV DQG FXPXOXV FHOOV
IVC improves oocyte quality by reducing ROS using a co-culture three-dimensional collagen gel system.
levels and apoptotic factors. However, some of +XP5HSURG
the non-antioxidants such as ascorbic acid and 2, 5. *LOFKULVW5%7KRPSVRQ-*2RF\WHPDWXUDWLRQHPHUJLQJ
FRQFHSWV DQG WHFKQRORJLHV WR LPSURYH GHYHORSPHQWDO
4, 5-trihydroxybutrophenone do not improve oo- SRWHQWLDOLQYLWUR7KHULRJHQRORJ\
cyte maturation; rather, they inhibit spontaneous 6. 6PLW] - 1RJXHLUD ' $OEDQR & &RUWYULQGW 5 'HYURH\
resumption of meiosis. Improvements to culture 3,PSURYLQJLQYLWURPDWXUDWLRQRIRRF\WHVLQWKHKXPDQ
WDNLQJ OHVVRQV IURP H[SHULHQFHV LQ DQLPDO VSHFLHV
conditions are complex challenges that depend 5HSURG'RPHVW$QLP
not only on the choice of an antioxidant but also 7. 7URXQVRQ$$QGHULHV]&-RQHV*0DWXUDWLRQRIKXPDQ
on its concentration, the medium and its compo- RRF\WHV LQ YLWUR DQG WKHLU GHYHORSPHQWDO FRPSHWHQFH
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ing to its developmental stage. Future efforts 437-446.
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should be placed on understanding the involve- R[\JHQVSHFLHVSHUVXDGHVSRVWRYXODWRU\DJLQJPHGLDWHG
ment of ROS in oocyte apoptosis and for guiding VSRQWDQHRXVHJJDFWLYDWLRQLQUDWHJJVFXOWXUHGLQYLWUR,Q
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