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

Human Reproduction, Oct 2014

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?

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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. Introduction 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 babies (Loutradi et al., 2008; Son and Tan, 2009; Abdelhafez et al., 2010) . However, embryo cryopreservation is not available for women who will be subjected to cancer therapy and do not yet have male partners (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) (Whittingham 1977; 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) . Reported 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 cryopreserved (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., 2005) , 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., 2009) . 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 IVF (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) or after cryopreservation at GV (Park et al., 1997) . Mitochondria are one of the most abundant organelles in oocyte and exert many cellular functions, including ATP production (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 species (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 development. Materials and Methods Reagents Animals All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise stated. 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 non-supplemented media. 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 by 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, DMR6000B, Germany). 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 described above. 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) . Statistical analysis 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. Results 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) . As 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 groups. 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). Discussion 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) . Our 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) . The 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 embryonic development. 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) , mice (Frydman et al., 1997; Cooper et al., 1998; Isachenko and Nayudu 1999; Cha et al., 2011) , cattle (Kim et al., 2007) , sheep (Moawad et al., 2012) , goats (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 transzonal processes (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 developmental competence (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 chromatids segregation (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 (Moawad et al., 2013c) . Other laboratories have reported similar adverse effects of GV – oocyte vitrification on the MII-spindle assembly and chromosome alignment (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 mouse oocytes (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 (Van Blerkom, 2004) . 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 (Bavister 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 microtubule networks (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 B6 mice (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 MII oocytes 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 and IVM. 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 (Yu et al., 2010) . ATP is a major product of mitochondrial respiratory oxidative phosphorylation and known to affect the MII-spindle assembly and embryonic development (Stojkovic et al., 2001; Wilding et al., 2001, 2009) . 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. Conclusion 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) , the 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 carnitine (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. Authors’ roles A.R.M. conceived the research idea and experimental designs, executed the experiments, analysed the data, and wrote the manuscript. 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Adel R. Moawad, Baozeng Xu, Seang Lin Tan, Teruko Taketo. 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, Human Reproduction, 2014, 2256-2268, DOI: 10.1093/humrep/deu201