l-carnitine supplementation during vitrification of mouse germinal vesicle stage–oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes
Advanced Access publication on August
L-carnitine supplementation during vitrification of mouse germinal vesicle stage - oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes
Adel R. Moawad 0 1 2 4
Baozeng Xu 0 1
Seang Lin Tan 0 2
Teruko Taketo 0 1 2 3
0 OriginElle Fertility Clinic and Women's Health Centre , Montreal, Quebec , Canada
1 Department of Surgery, McGill University , Montreal, Quebec , Canada
2 Department of Obstetrics and Gynecology, McGill University , Montreal, Quebec , Canada
3 Department of Biology, McGill University , Montreal, Quebec , Canada
4 Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University , Giza , Egypt
study question: How does L-carnitine (LC) supplementation during vitrification and in vitro maturation (IVM) of germinal vesicle stage (GV) - oocytes improve the developmental competence of the resultant metaphase II (MII) oocytes? summary answer: LC supplementation during both vitrification of GV - oocytes and their subsequent IVM improved nuclear maturation as well as meiotic spindle assembly and mitochondrial distribution in MII oocytes. what is known already: Vitrification of GV - oocytes results in a lower success rate of blastocyst development compared with nonvitrified oocytes. LC supplementation during both vitrification and IVM of mouse GV - oocytes significantly improves embryonic development after IVF. study design, size, duration: GV - oocytes were collected from (B6.DBA)F1 and B6 mouse strains and subjected to vitrification and warming with or without 3.72 mM LC supplementation. After IVM with or without LC supplementation, the rate of nuclear maturation and the quality of MII oocytes were evaluated. At least 20 oocytes/group were examined, and each experiment was repeated at least three times. All experiments were conducted during 2013 - 2014. participants/materials, setting, methods: Extrusion of the first polar body in IVM oocytes was observed as an indication of nuclear maturation. Spindle assembly and chromosomal alignment were examined by immunostaining of a-tubulin and nuclear staining with 4,6diamidino-2-phenylindole (DAPI). Mitochondrial distribution and oxidative activity were measured by staining with Mitotracker Green Fluorescence Mitochondria (Mitotracker Green FM) and chloromethyltetramethylrosamine (Mitotracker Orange CMTMRos), respectively. ATP levels were determined by using the Bioluminescent Somatic Cell Assay Kit. main results and the role of chance: LC supplementation during both vitrification and IVM of GV - oocytes significantly increased the proportions of oocytes with normal MII spindles to the levels comparable with those of non-vitrified oocytes in both mouse strains. While vitrification of GV - oocytes lowered the proportions of MII oocytes with peripherally concentrated mitochondrial distribution compared with non-vitrified oocytes, LC supplementation significantly increased the proportion of such oocytes in the (B6.DBA)F1 strain. LC supplementation decreased the proportion of oocytes with mitochondrial aggregates in both vitrified and non-vitrified oocytes in the B6 strain. The oxidative activity of mitochondria was mildly decreased by vitrification and drastically increased by LC supplementation irrespective of vitrification in both mouse strains. No change was found in ATP levels irrespective of vitrification or LC supplementation. Results were considered to be statistically significant at P , 0.05 by either x2- or t-test.
limitations, reasons for caution: It remains to be tested whether beneficial effect of LC supplementation during vitrification
and IVM of GV-oocytes leads to fetal development and birth of healthy offspring after embryo transfer to surrogate females.
wider implications of the findings: This protocol has the potential to improve the quality of vitrified human oocytes and
embryos during assisted reproduction treatment.
study funding/competing interest: Partially supported by the Natural Sciences and Engineering Research Council of Canada
(NSERC) Discovery Grant and Mitacs Elevate Postdoctoral Fellowship, Canada.
Infertility affects at least 15% of couples who anticipate having children
(Boivin et al., 2007)
. The development of assisted reproductive
technologies (ART) such as IVF and ICSI has been playing an important
role in the treatment of infertility caused by either female or male
factors. Embryo cryopreservation is another technique that has been
developed for infertility treatment; this technique became a routine
practise in clinical ART and has contributed to the delivery of many healthy
(Loutradi et al., 2008; Son and Tan, 2009; Abdelhafez et al.,
. However, embryo cryopreservation is not available for women
who will be subjected to cancer therapy and do not yet have male
(Huang et al., 2007; Chian et al., 2009)
. Furthermore, embryo
cryopreservation is forbidden due to ethical and legal issues in some countries
or for certain patients. Cryopreservation of unfertilized oocytes is an
alternative technology, giving flexibility in the time of IVF and a potential to
establish oocyte banking with oocyte donation
(Nagy et al., 2009; Noyes
et al., 2010a; Cobo et al., 2011)
Studies to date have mainly been concentrated on the
cryopreservation of mature oocytes at the metaphase II (MII)
Chen, 1986; Kuleshova et al., 1999; Huang et al., 2010)
. Of the two
main cryopreservation protocols, slow-rate freezing and vitrification,
vitrification became the favourite approach as it results in less damage to the
oocytes (Kuleshova and Lopata, 2002). However, only 7 – 8% of vitrified
MII oocytes have been reported to result in pregnancy and live birth,
much lower than 30 – 40% of non-cryopreserved oocytes
(Cobo et al.,
2008; Cobo and Diaz, 2011; Chang et al., 2013; Potdar et al., 2014)
Many characteristics of mature oocytes make them more vulnerable to
cryopreservation-inflicted damage than embryos, including their large
size, low surface area/volume ratio, high intracytoplasmic lipids and
high permeability of the ooplasmic membrane
(Leibo, 1980; Ruffing
et al., 1993; Agca et al., 1998; Isachenko et al., 1998)
cryopreservation-inflicted injuries include disruption of meiotic spindles
(Pickering et al., 1990; Mandelbaum et al., 2004)
and premature release
of cortical granules
(Johnson et al., 1988; Vincent et al., 1990; Ghetler
et al., 2006)
Cryopreservation of immature oocytes at the germinal vesicle stage
(GV) stage can be an alternative protocol to overcome the problems
associated with that of MII oocytes. GV – oocytes possess interphase
nuclei that have not yet formed metaphase spindles, hence rendering
them more tolerant to freezing-inflicted damage. Moreover, collection
of oocytes at the GV-stage has many clinical advantages; for example,
minimizing the risk of ovarian hyperstimulation syndrome and decreasing
the cost and duration of hormonal treatment
(Chian et al., 1999; Le Du
et al., 2005; Mikkelsen, 2005)
. Additionally, collection of GV – oocytes
does not require full ovarian stimulation, giving a benefit for certain
cancer patients to eliminate the risk of stimulating hormone-sensitive
tumours, such as breast cancer
(Rao et al., 2004; Kim et al., 2011)
Although GV – oocytes from many mammalian species have been
(Toth et al., 1994; Wu et al., 2001; Vieira et al., 2002;
Ruppert-Lingham et al., 2003; Kim et al., 2007; Tharasanit et al., 2009;
Somfai et al., 2010; Moawad et al., 2011)
and reportedly produced live
births in both human and mouse
(Tucker et al., 1998; Aono et al.,
, the rate of blastocyst development from cryopreserved GV –
oocytes remains low
(Aono et al., 2003, 2005)
. One major limitation is
the requirement of oocyte in vitro maturation (IVM) after freezing and
thawing of GV – oocytes in order to obtain mature oocytes that are
able to develop into embryos
(Ruppert-Lingham et al., 2003; Moawad
et al., 2013a,b)
. Unfortunately, no optimal IVM protocol for human
GV – oocytes has yet been established (Shahdei et al., 2013).
Improvement in the conditions for both cryopreservation of GV – oocytes and
their subsequent IVM would make many clinical applications possible.
We have previously reported that supplementation of the media for
vitrification, warming and IVM of mouse GV – oocytes with L-carnitine
(b-hydroxy-g-trimethylammonium-butyric acid, LC) improves the rate
of blastocyst development following IVF compared with those vitrified,
warmed and matured in vitro in basic media without LC supplementation
in both (B6.DBA)F1 (24 versus 42%) and B6 (0 versus 15%) mice
(Moawad et al., 2013c)
. Moreover, the genetic background of oocytes,
namely (B6.DBA)F1, (B6.C3H)F1, and C57BL/6J (B6), influences the
effects of vitrification and IVM, as well as LC supplementation, on
subsequent preimplantation development
(Moawad et al., 2013c)
. LC is a small
water soluble molecule, present in free and esterified forms, in most
tissues and body fluids. It has various metabolic functions, including
transfer of long-chain fatty acids inside mitochondria for b-oxidation,
transport of short- and medium-chain acyl groups from the peroxisome to
mitochondria, regulation of intracellular acyl-CoA/free CoA ratio and
removal of toxic acyl residues from mitochondria
(Melegh et al., 1993,
1994; Vaz et al., 2002)
. It also has antioxidant activity to protect the
cells from DNA damage
(Abdelrazik et al., 2009; Mansour et al.,
. Beneficial effects of LC supplementation on embryonic
development have previously been reported in many mammalian species.
However, we found that LC supplementation of all the media for
vitrification, warming and IVM has a greater beneficial effect on the
developmental competence of oocytes than LC supplementation of the IVM or
embryo culture medium alone
(Moawad et al., 2013c)
In the present study, we investigated the possible mechanisms by
which LC supplementation during vitrification/warming and IVM of
mouse GV – oocytes improves the developmental competence of the
resultant MII oocytes. We focused on MII-spindle assembly, mitochondrial
distribution and activity, and ATP levels in MII oocytes, all of which are
considered to be critical for successful embryonic development following
(Van Blerkom, 2004; Gomes et al., 2008; Noyes et al., 2010b)
Defects in the spindle configuration have been reported in the oocytes
from women at advanced ages
(Battaglia et al., 1996)
cryopreservation at GV
(Park et al., 1997)
. Mitochondria are one of the most
abundant organelles in oocyte and exert many cellular functions, including
(Van Blerkom, 2004)
. Localization of mitochondria
inside the oocyte is also associated with its developmental competence
(Nagai et al., 2006). Abnormal distribution and activity of mitochondria
has been shown after cryopreservation of MII oocytes in different
(Rho et al., 2002; Nagai et al., 2006; Shi et al., 2007; Yan et al.,
2010; Chen et al., 2012; Zander-Fox et al., 2013)
. We measured these
parameters in the MII oocytes after vitrification at the GV-stage followed
by IVM with or without LC supplementation. We also compared the
results in two mouse strains, (B6.DBA)F1 and B6, throughout our
studies, and discussed their association with the potential for embryonic
Materials and Methods
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless
All experiments were carried out according to the Guide to the Care and Use
of Experimental Animals released by the Canadian Council on Animal Care
with approval from the Animal Care Committee at McGill University. B6 mice
and DBA males were purchased from the Jackson Laboratory (Bar Harbor,
ME, USA) and Charles River (St. Constant, Quebec, Canada), respectively.
B6 females were crossed with DBA and B6 males to produce (B6.DBA)F1
and B6 progeny, respectively, in our mouse colony. All animals were
maintained under specific pathogen-free conditions with a humidity range of
30 – 60%, a temperature range of 21 – 248C, a light cycle of 12L:12D, and
ad libitum food and water.
Collection of cumulus – oocyte complexes, vitrification and IVM
Females (8 – 10-week-old) were i.p. injected with 5 IU equine chorionic
gonadotrophin (eCG) each and euthanized 45 – 47 h later. Cumulus –
oocyte complexes (COCs) were collected from ovaries and subjected
either to vitrification and warming or directly to IVM as previously described
(Moawad et al., 2013c)
. After vitrification/warming, only viable COCs
were selected and subjected to IVM. Vitrification, warming and IVM media
were supplemented with 3.72 mM (0.6 mg/ml) LC and compared with
Evaluation of meiotic spindle assembly and chromosomal alignment
After IVM for 16 h, oocytes were denuded of cumulus cells by using 300 IU/
ml hyaluronidase in the M2 medium. After fixation in a solution composed of
2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA)
and microtubule-stabilizing buffer
(Xu et al., 2014)
for 60 min at room
temperature, oocytes were washed three times in phosphate-buffered saline
(PBS) supplemented with 3% bovine serum albumin (BSA) (PBS – BSA),
transferred into blocking buffer (PBS supplemented with 5% FBS and 0.01% Triton
X-100), and kept in this solution overnight at 48C. Oocytes were then
incubated in anti-a-tubulin monoclonal antibody diluted 1:500 in the blocking
buffer for 60 min at room temperature, washed and incubated with a goat
anti-mouse IgG conjugated with fluorescein isothiocynate (FITC) (Jackson
ImmunoResearch Lab, West Grobe, PA, USA) diluted 1:500 in the blocking
buffer for 60 min at room temperature. After washing, the oocytes were
transferred to a small drop of Prolong Antifade mounting medium containing
4,6-diamidino-2-phenylindole (DAPI) (Molecular Probe, Eugene, OR, USA)
on a microscope slide (Thermo Fisher Scientific) and covered with a
coverslip. The slides were examined under an epi-fluorescence microscope
(Zeiss Axiophot, Germany). All images were captured with a digital
camera (Retiga 1300, QImaging, Burnaby, British Columbia, Canada),
processed with Northern Eclipse digital imaging software version 8.0 (Empix
Imaging, Mississauga, Ontario, Canada) and scored based on the criteria
Huang et al. (2008)
Evaluation of mitochondrial distribution
Denuded oocytes were fixed in 4% paraformaldehyde in PBS for 60 min in a
humidified chamber, washed and incubated in 100 nM Mitotracker
GreenFluorescence Mitochondria (Mitotracker Green FM; Molecular Probes,
Eugene, OR, USA) in PBS – BSA for 30 min in dark. After washing, the
oocytes were transferred into a drop of Prolong Antifade mounting
medium containing DAPI on a microscope slide and covered with a cover
slip. The slides were examined and fluorescence signals were captured and
processed under an automated fluorescence microscope system (Leica,
Evaluation of mitochondrial oxidative activity
Denuded oocytes were incubated in the M2 medium containing 100 nM
chloromethyltetramethylrosamine (Mitotracker Orange CMTMRos;
Molecular Probes, Eugene, OR, USA) and 5 mg/ml Hoechst 33342 for 20 min
at 378C in dark. After washing three times in the M2 medium, oocytes
were examined under an inverted fluorescence microscope (Leica,
DMIRB, Germany). Fluorescence signals were captured with a digital
camera and processed with Northern Eclipse digital imaging software as
Determination of ATP levels
ATP levels in 40 oocytes in a group were measured using Bioluminescent
Somatic Cell Assay Kit (FL-ASC, St Louis, MO) as described previously
(Xu et al., 2014)
Each experiment was repeated at least three times, and pooled data were
used for x2 test. Differences between experimental groups were analysed
by Student’s t-test. Results were considered to be statistically significant
at P , 0.05.
Effects of LC supplementation on oocyte nuclear maturation rates
GV – oocytes enclosed in COCs, collected from either (B6.DBA)F1 or
B6 females, were vitrified and warmed in the presence or absence of
3.72 mM LC, and viable oocytes (survival rates over 90%) were further
subjected to IVM with or without LC supplementation for 16 h.
Oocytes were then denuded of cumulus cells and examined as to the
extrusion of the first polar body, a characteristic of MII oocyte. As
shown in Fig. 1, the percentages of MII oocytes (nuclear maturation rates)
were significantly lower (P , 0.05, t-test) when COCs were vitrified,
warmed and matured without LC supplementation compared with
non-vitrified control groups in both (B6.DBA)F1 (65 versus 93%) and
B6 (69 versus 94%) strains. LC supplementation in the IVM medium
alone did not affect nuclear maturation rates, whereas LC
supplementation throughout vitrification/warming and IVM significantly increased
(P , 0.05) the maturation rates in both (B6.DBA)F1 (86%) and B6
(88%) strains to the levels comparable with those obtained from
nonvitrified control groups. In all experiments, when the vitrification
medium was supplemented with LC, the IVM medium was also
supplemented with LC to maximize the beneficial effects of LC
supplementation. The same experimental design was used for testing embryonic
development in our previous study
(Moawad et al., 2013c)
Effects of LC supplementation on spindle
assembly and chromosome alignment
in MII oocytes
shaped microtubule spindles with metaphase chromosomes aligned
along the equatorial metaphase plate were considered ‘normal’,
whereas asymmetrical or deformed spindles with misaligned metaphase
chromosomes were considered ‘abnormal’
(Huang et al., 2008)
shown in Fig. 2, the proportions of MII oocytes with normal spindles
were significantly lower in vitrified control groups than those in
nonvitrified control groups (P , 0.05, x2 test) in both mouse strains. LC
supplementation of the IVM medium alone did not affect the spindle
morphology in either non-vitrified or vitrified control groups, whereas
LC supplementation of all vitrification, warming and IVM media
significantly increased (P , 0.05) the proportions of MII oocytes with the
normal spindle configuration, compared with those in vitrified control
oocytes without LC supplementation, and to similar levels observed in
non-vitrified control groups in both mouse strains.
Effects of LC supplementation on mitochondrial distribution in MII oocytes
We examined the effects of LC supplementation during vitrification of
GV – oocytes and their subsequent IVM on spindle configuration and
chromosome alignment in the resultant MII oocytes. Symmetrical
barrelWe examined the effects of LC supplementation during vitrification/
warming and IVM of GV – oocytes on mitochondrial distribution in the
resultant MII oocytes by labelling with cell-permeable Mitotracker
Green FM probe. This probe contains a mildly thiol-reactive
chloromethyl moiety that becomes fluorescence once being accumulated in
the lipid environment of mitochondria regardless of the
mitochondrial membrane potential. Mitochondrial distribution was classified
as ‘peripherally concentrated’ when the scanning of fluorescence
signals showed a peak in the periphery compared with those in the
central area (Fig. 3A). As summarized in Fig. 3B (top), the majority
( 80%) of MII oocytes in non-vitrified control groups showed
peripherally concentrated mitochondrial distribution in both
(B6.DBA)F1 and B6 strains. In the (B6.DBA)F1 strain, the proportion
of such oocytes was significantly lower (P , 0.001, x2 test) in the
vitrified control group compared with the non-vitrified control group,
whereas LC supplementation of all vitrification/warming and IVM
media significantly increased (P , 0.001) the proportion of oocytes
with peripherally concentrated mitochondrial distribution. In the
B6 stain, however, no differences were observed between different
groups (Fig. 3B top).
Mitochondrial distribution was also categorized as ‘aggregated’ when
at least two large aggregates were seen in an MII oocyte or as
‘nonaggregated’ otherwise (Fig. 3A). As summarized in Fig. 3B (bottom), in
the (B6.DBA)F1 strain, 60% of MII oocytes showed non-aggregated
homogenous mitochondrial distribution and this percentage did not
change by either vitrification or LC supplementation. In contrast, in the
B6 strain, 60% of oocytes were seen with mitochondrial aggregates in
the non-vitrified control group; this percentage was higher than
that observed in the (B6.DBA)F1 strain (P , 0.05, x2 test). Vitrification
of GV – oocytes did not change the proportion of MII oocytes with
mitochondrial aggregates, whereas LC supplementation significantly
increased the proportion of oocytes with non-aggregated
mitochondrial distribution (P , 0.001, x2 test) in both vitrified and non-vitrified
Effects of LC supplementation on mitochondrial oxidative activity in MII oocytes
Mitochondrial distribution was examined above in the MII oocytes after
fixation. We also examined mitochondrial oxidative activity in the live MII
oocytes by labelling with Mitotracker Orange CMTMRos. This dye is
readily sequestrated only by actively respiring mitochondria depending
on the oxidative activity of the cells. Examples of fluorescence labelling
are shown in Fig. 4A. Fluorescence signals in the entire oocyte were
measured and normalized to the average in the non-vitrified control group in
each experiment. As summarized in Fig. 4B, vitrification of GV – oocytes
decreased the mitochondrial oxidative activity in the MII oocytes after
IVM from both mouse strains, but a significant difference was found
only in the (B6.DBA)F1 strain. LC supplementation of the IVM
medium alone significantly increased (P , 0.001, t-test) the
mitochondrial oxidative activity regardless of vitrification in both mouse strains.
Effects of LC supplementation on ATP levels in MII oocytes
Our results above indicated that LC supplementation during
vitrification/warming and IVM of GV – oocytes improved the MII-spindle
assembly and increased the mitochondrial oxidative activity in the
resultant MII oocytes. Since the MII-spindle assembly is known to depend on
ATP levels, we anticipated that ATP levels in MII oocytes may have
been increased by LC supplementation. Our results, however, showed
that ATP levels in MII oocytes were consistent irrespective of vitrification
or LC supplementation in both mouse strains (Fig. 5).
Cryopreservation of GV – oocytes would give women the opportunity to
have their biological children by circumventing the problems associated
with cancer treatment or aging. However, its clinical application is
currently limited because of the overall low pregnancy rate achieved with
these cryopreserved oocytes. A fundamental improvement in the
protocols for both GV – oocyte cryopreservation and IVM is needed
for its application in human and other mammalian species. We have
previously shown that LC supplementation during vitrification/
warming and IVM of mouse GV – oocytes increases the developmental
competence of the resultant MII oocytes
(Moawad et al., 2013c)
present results demonstrate that vitrification/warming and IVM of
GV – oocytes with LC supplementation improves their both nuclear
and cytoplasmic maturation as indicated by the rate of the first
polar body extrusion, MII-spindle morphology and mitochondrial
distribution. We propose that LC supplementation protects GV –
oocytes from cryopreservation-inflicted damage, particularly on
microtubule networks. However, LC supplementation during IVM
alone also caused changes in mitochondrial distribution and oxidative
activity in the resultant MII oocytes (present study), as well as their
developmental competence in the B6 strain
(Moawad et al., 2013c)
oocytes of the B6 strain are particularly vulnerable to in vitro
manipulations, and may be better benefited by LC supplementation. We suggest
that LC supplementation during both vitrification and IVM of GV –
oocytes has additive beneficial effects on the oocyte competence for
Beneficial effects of LC supplementation during vitrification of GV – oocytes on the oocyte nuclear maturation
Our results showed that the percentage of MII-oocytes after IVM (the
nuclear maturation rate) was significantly decreased when COCs had
been vitrified/warmed and matured in the basic media (without LC
supplementation) compared with non-vitrified control oocytes in both
(B6.DBA)F1 and B6 strains. A number of previous studies have also
reported lower maturation rates following cryopreservation of GV –
oocytes in humans
(Park et al., 1997; Isachenko et al., 2006; Shahedi
et al., 2013)
(Frydman et al., 1997; Cooper et al., 1998; Isachenko
and Nayudu 1999; Cha et al., 2011)
(Kim et al., 2007)
(Moawad et al., 2012)
(Kharche et al., 2005)
, horses (Tharasanit
et al., 2006) and cats
(Apparicio et al., 2013)
. The lower maturation
rate can be ascribed to the damage to certain cytoplasmic organelles
which are essential for oocyte maturation
(Huang et al., 2008;
Moawad et al., 2013b)
. Additionally, it has been reported that
cryopreservation of COCs severely disrupts physical communication
between the oocyte and its neighbouring cumulus cells, such as
(Vincent et al., 1990; Ruppert-Lingham et al., 2003;
Navarro-Costa et al., 2005; Vandevoort et al., 2008)
. In our study, LC
supplementation throughout vitrification, warming and IVM significantly
increased the nuclear maturation rates to the levels comparable with
those in non-vitrified control groups, while LC supplementation in the
IVM medium alone did not, in both mouse strains. However, as we
have previously reported
(Moawad et al., 2013c)
, the oocytes that
have reached MII after vitrification and IVM exhibit lower competence
for embryonic development than non-vitrified control oocytes, and LC
supplementation during vitrification/warming and IVM improves their
(Moawad et al., 2013c)
. We assume that
loss of developmental competence in the MII oocytes which have been
vitrified at GV is the major obstacle for the application of this technique
for ART, and hence our remaining studies were focused on the effects of
LC supplementation during vitrification/warming and IVM on the
features of MII oocytes that are relevant to their success in preimplantation
development. It remains to be determined whether beneficial effects of
LC supplementation on the oocyte nuclear maturation are shared with
those on its developmental competence.
Beneficial effects of LC supplementation during vitrification and IVM of GV – oocytes on the MII-spindle assembly
It is well documented that normal fertilization and subsequent embryonic
development from the MII oocyte requires proper MII-spindle assembly
and chromosome alignment, which is critical for correct sister
(Wang and Keefe, 2002; Keefe et al., 2003; Chen
et al., 2003)
. Our results showed that vitrification of GV – oocytes in
the basic medium decreased the proportion of oocytes with normal
spindle morphology, and LC supplementation throughout vitrification,
warming and IVM increased the proportion of such oocytes to
comparable levels with those in non-vitrified control groups in both mouse
strains. We assume that the improvement in MII-spindle morphology
and chromosomal alignment by LC supplementation during vitrification
and IVM of GV – oocytes may contribute to the higher rate of blastocyst
development following IVF as we have previously reported
et al., 2013c)
. Other laboratories have reported similar adverse effects
of GV – oocyte vitrification on the MII-spindle assembly and
(Park et al., 1997; Isachenko and Nayudu, 1999;
Rojas et al., 2004)
. However, no studies have previously reported the
beneficial effects of LC supplementation during vitrification/warming
and IVM of GV – oocytes on the MII-spindle assembly.
Beneficial effects of LC supplementation during vitrification and IVM of GV – oocytes on mitochondrial distribution in MII oocytes
Defects in meiotic spindle assembly have been associated with
impairment in mitochondrial distribution and activity in both human and
(Schon et al., 2000; Van Blerkom, 2004; Zhang et al.,
2006; Wang et al., 2009)
. Mitochondria, essential for respiration,
metabolism and apoptosis within a cell, play important roles in oocyte
maturation and competence for embryonic development
. It has been suggested that redistribution of mitochondria during
oocyte maturation is associated with redistribution of ATP, which is
required for spindle assembly and various molecular changes
and Squirrell, 2000; Van Blerkom, 2004)
. For example, mitochondria
are concentrated around the MI-spindle and retained in the ooplasm
after the first polar body extrusion (Dalton and Carroll, 2013).
Peripherally concentrated mitochondria may be required for proper segregation
of sister chromatids in oocytes upon fertilization
(Dalton and Carroll,
2013, Zander-Fox et al., 2013, Marei et al., 2012)
Our current results demonstrate that the majority ( 80%) of MII
oocytes in non-vitrified control groups showed peripherally
concentrated mitochondrial distribution in both (B6.DBA)F1 and B6 strains.
Vitrification of oocytes in the basic medium significantly decreased
the proportion of oocytes with peripherally concentrated
mitochondrial distribution while LC supplementation during vitrification/
warming and IVM increased this proportion to a comparable level
with that in the non-vitrified control in the (B6.DBA)F1 strain. Alterations
in the mitochondrial distribution after vitrification/warming of MII oocytes
have been previously reported in the (B6.CBA)F1 mouse strain
(ZanderFox et al., 2013) and this effect has been attributed to the dehydration of
oocytes during vitrification, which may also causes a damage to
(Eroglu et al., 1998; Rho et al., 2002)
. However, we
found no such changes in the oocytes of the B6 strain with or without
vitrification. This is rather surprising since the oocytes of B6 are generally
more vulnerable to manipulations in vitro than those of F1 hybrids
(Moawad et al., 2013c)
Our results, on the other hand, showed changes in the proportion of
oocytes containing mitochondrial aggregates with LC supplementation
in the B6 strain. It has been reported that an increase in the number
of mitochondrial aggregates in mouse oocytes is associated with
lower developmental competence
(Nagai et al., 2006; Wang et al.,
2009; Ou et al., 2012)
. In our study, 60% of MII oocytes in the
nonvitrified control group showed mitochondrial aggregates in the B6
strain; this proportion was significantly higher than that observed in
the (B6.DBA)F1 strain. These results may explain the lower rate of
blastocyst development from IVM oocytes (without vitrification) of
(Moawad et al., 2013c)
. In addition to species differences
(Bavister and Squirrell, 2000)
, mouse strain differences have been
reported to affect the mitochondrial distribution in fertilized oocytes
(Muggleton-Harris and Brown, 1988)
. In the present studies, we
found that vitrification/warming of GV – oocytes did not change the
proportion of MII oocytes with mitochondrial aggregates, whereas
LC supplementation during IVM significantly decreased the proportion
of MII oocytes with mitochondrial aggregates regardless of vitrification
in the B6 strain. Beneficial effects of LC supplementation on the
mitochondrial distribution in non-vitrified mouse and porcine oocytes
during IVM have previously been reported
(Somfai et al., 2011; Wu
et al., 2012)
. However, we found such effects only in the B6 strain.
We also previously found that LC supplementation during IVM of
nonvitrified oocytes improves embryonic development only in the B6 strain
(Moawad et al., 2013c)
. Thus, the mitochondrial distribution pattern
(with or without aggregates) appears to be well correlated with
developmental competence of oocytes.
Beneficial effects of LC supplementation
during vitrification and IVM of GV – oocytes
on the mitochondrial oxidative activity in
It has been reported that cryopreservation of human and mouse MII
oocytes significantly decreases the mitochondrial activity, indicated by
their membrane potential
(Jones et al., 2004; Yan et al., 2010; Chen
et al., 2012; Zander-Fox et al., 2013)
. Our results showed that
vitrification of GV – oocytes decreased the average mitochondrial oxidative
activity in the resultant MII oocytes in both mouse strains. LC
supplementation during IVM increased this activity to much higher levels than in
nonvitrified controls with or without vitrification. Higher levels of oxidation
may reflect higher mitochondrial functions on one hand, but it may result
in higher ROS production on the other. Since we did not find any adverse
effects of LC supplementation by all parameters that we have tested, we
speculate that the antioxidant property of LC
(Miyamoto et al., 2010;
Somfai et al., 2011)
may counterbalance the harmful effects of ROS to
favour the beneficial effects of LC supplementation. Further studies are
needed to clarify such possibility. These results may highlight the multiple
beneficial effects of LC supplementation during GV – oocyte vitrification
Effects of LC supplementation during vitrification and IVM of GV – oocytes on ATP levels in MII oocytes
It has been reported that redistribution of active mitochondria is
correlated with elevated ATP production during the MI – MII transition
et al., 2010)
. ATP is a major product of mitochondrial respiratory
oxidative phosphorylation and known to affect the MII-spindle assembly and
(Stojkovic et al., 2001; Wilding et al., 2001,
. However, contrary to our expectation, our results showed no
difference in ATP levels among different groups of MII oocytes irrespective
of vitrification or LC supplementation. Effects of oocyte
cryopreservation on ATP levels in relation to mitochondrial activity remain
controversial in the literature
(Jones et al., 2004; Manipalviratn et al., 2011;
Chankitisakul et al., 2013; Salehnia et al., 2013)
. One explanation for
the consistent ATP levels despite differences in mitochondrial activity
or distribution in MII oocytes in our study could be that bidirectional
communication between the oocyte and its neighbouring cumulus cells
has contributed to maintaining ATP levels (Xu et al., 2014). Alternatively,
the concentration of ATP in the periphery, rather than the total ATP
levels, may have been critical for proper spindle assembly in oocytes
during the MI – MII transition. We cannot exclude the possibility that
the mitochondrial oxidative activity that we measured may not have
directly contributed to ATP production.
LC supplementation during both vitrification and IVM of GV – oocytes
minimizes cryopreservation-inflicted damage on mitochondrial
distribution and the MII-spindle assembly, and improves their competence
for embryonic development. Since intracellular movement and
localization of mitochondria is controlled by cytoskeleton and kinesin-related
proteins integrated in microtubule networks
(Heggeness et al., 1978;
Nangaku et al., 1994; Tanaka et al., 1998; Van Blerkom, 2004)
antioxidant properties of LC
(Miyamoto et al., 2010)
may protect the
integrity of those components against oxidative stress that could occur during
oocyte vitrification/warming and IVM. In addition, as ATP is synthesized
by b-oxidation of fatty acids within mitochondria, which requires
(Dunning et al., 2010, 2011)
, LC supplementation may promote
local ATP production. If we can find similar beneficial effects of LC
supplementation in human oocytes, the use of human GV – oocytes for ART
may become more feasible.
A.R.M. conceived the research idea and experimental designs, executed
the experiments, analysed the data, and wrote the manuscript. B.X.
established the ATP assay, helped A.R.M. to perform the assay, and
reviewed the manuscript. S.L.T. contributed to the research idea and
experimental designs, and reviewed the manuscript. T.T. supervised the
research project, contributed to the research ideas, experimental designs
and data analyses, and edited the manuscript.
This work was partially supported by the Natural Sciences and
Engineering Research Council of Canada (NSERC) Discovery Grant to
T.T. A.R.M. is a recipient of Mitacs Elevate Postdoctoral Fellowship,
Conflict of interest
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