Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models

Accepted Manuscript Title: Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models Author: Takamasa Ishii Kayo Yasuda Masaki Miyazawa Junji Mitsushita Thomas E. Johnson Phil S. Hartman Naoaki Ishii PII: DOI: Reference:

S0047-6374(16)30018-5 http://dx.doi.org/doi:10.1016/j.mad.2016.02.013 MAD 10822

To appear in:

Mechanisms of Ageing and Development

Received date: Revised date: Accepted date:

17-12-2015 16-2-2016 28-2-2016

Please cite this article as: Ishii, Takamasa, Yasuda, Kayo, Miyazawa, Masaki, Mitsushita, Junji, Johnson, Thomas E., Hartman, Phil S., Ishii, Naoaki, Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models.Mechanisms of Ageing and Development http://dx.doi.org/10.1016/j.mad.2016.02.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Infertility

and

recurrent

miscarriage

with

complex

II

deficiency-dependent mitochondrial oxidative stress in animal models Takamasa Ishiia, b*, Kayo Yasudaa, c, Masaki Miyazawad, Junji Mitsushitae, Thomas E. Johnsonf, Phil S. Hartmang and Naoaki Ishiia a

Department of Molecular Life Science, Tokai University School of Medicine, 143

Shimokasuya, Isehara, Kanagawa 259-1193, Japan. b

Institute of Medical Sciences, Tokai University, 143 Shimokasuya, Isehara, Kanagawa

259-1193, Japan. c

Support Center for Medical Research and Education, Tokai University, 143 Shimokasuya,

Isehara, Kanagawa 259-1193, Japan. d

Department of Biological Sciences, North Carolina State University, Box 7633, Raleigh, NC

27695, USA. e

Department of Obstetrics and Gynecology, Saitama Medical Center, Jichi Medical University,

1-847 Amanuma-cho, Omiya, Saitama 330-8503, Japan. f

Institute for Behavioral Genetics, University of Colorado, Box 447, Boulder, CO 80303, USA.

g

Department of Biology, Texas Christian University, Fort Worth, TX 76129, USA.

*Corresponding Author: Tel: +81-463-93-1121 (ext. 2650); Fax: +81-463-94-8884; E-mail: [email protected]; [email protected] (T.I.)

Highlights · SDHC mutation causes complex II deficiency-dependent oxidative stress in female reproductive organs, not male. · The oxidative stress causes ovarian hemangiomas leading to abnormal follicle maturation. · The oxidative stress does not affect spermatogenesis and early embryogenesis. · The oxidative stress causes placental angiodysplasia and inflammation. · The oxidative stress causes embryopathy and developmental arrest with excessive apoptosis.

Abstract Oxidative stress is associated with male and female infertility. However, there is insufficient evidence relating to the influence of oxidative stress on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. There are a number of animal models for understanding age-dependent decreasing reproductive ability and diabetic embryopathy, especially abnormal spermatogenesis, oogenesis and embryogenesis with mitochondrial dysfunctions. Several important processes occur in mitochondria, including ATP synthesis, calcium ion storage, apoptosis induction and reactive oxygen species (ROS) production. These events have different effects on the several aspects of reproductive function. Tet-mev-1 conditional transgenic mice, developed after studies with the mev-1 mutant of the nematode C. elegans, offer the ability to carefully regulate expression of doxycycline-induced mutated SDHCV69E levels and hence modulate endogenous oxidative stress. The mev-1 models have served to illuminate the effects of complex II deficiency-dependent mitochondrial ROS production, although interestingly they maintain the normal mitochondrial and intracellular ATP levels. In this review, the reproductive dysfunctions are presented focusing on fertility potentials in each gamete, early embryogenesis, maternal conditions with placental function and neonatal development.

Abbreviations: ROS: reactive oxygen species, TCA: tricarboxylic acid SDH: succinate dehydrogease, OXPHOS:

mitochondrial oxidative phosphorylation, ETC: electron transport chain,

ANT: adenine nucleotide transporter, SDHC: succinate dehydrogenase C subunit, ETF: electron transferring flavoprotein, CoQ: ubiquinone, CAD: caspase activated DNase, ICAD: inhibitor of caspase activated DNase, TNF: tumor necrosis factor, TNFR1: TNF receptor 1, TRAIL: TNF-related apoptosis-inducible ligand, DISC: death-inducing signaling complex, AIF: apoptosis-inducing factor, TRADD: TNFR1-associated death domain, Apaf-1: apoptotic protease-activating factor-1, MPTP: mitochondrial permeability transition pore, cypD: cyclophilin D, VDAC: voltage-dependent anion channel, JNK: c-Jun-N-terminal kinase, ASK1: apoptosis signal-regulating kinase 1, 8-OHdG: 8-hydroxydeoxyguanosine, TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling, NGF: non-growing follicles, HIF: hypoxia inducible factors, VEGF: vascular endothelial growth factors, VEGFR-1: VEGF receptor-1, PLGF: placenta growth factor, SOD: superoxide dismutase, Dox: doxycycline, HE: hematoxilin-eosin, ICM: inner cell mass, TE: trophectoderm, PKC: protein kinase C, DAG: diacylglycerol, PGE2: Prostaglandin E, AA: arachidonic acid, PGF2: prostagladin F2, PGLs: paragangliomas, PHEOs: phaeochromocytomas

Keywords: Mitochondria, Oxidative stress, SDHC, Infertility, Miscarriage, Embryopathy 1. Introduction Mitochondrial morphologies and mitochondrial physiological functions encompass a wide variety of characteristics depending on the cell type and tissue. As a consequence, their dysfunctions are implicated in several diseases. Mitochondrial dysfunctions that lead to fundamental defects in cell viability contribute to many pathophysiological phenomena, neuronal degenerative diseases, cancer, diabetes, cardiovascular diseases and infertility (Wallace, 1999; Dröge, 2002; Wallace et al., 2009). Here we review the current understanding of the reproductive disorders and mitochondrial oxidative stress, male and female infertilities with abnormal spermatogenesis, oogenesis and embryogenesis, and describe the complex II succinate dehydrogenase (SDH) activity-dependent oxidative stress-specific reproductive pathologies from our experimental results using mev-1 animal models with a SDHC mutation. We have isolated an ROS-generating chemical methyl viologen and oxygen hypersensitive short-lived mutant, mev-1, from the nematode Caenorhabditis elegans (Ishii et al., 1990; Honda et al., 1993). The mev-1(kn-1) mutation results in an amino acid substitution at the 71st position from glycine to glutamate (G71E) in C. elegans and

has been identified as residing in the putative gene cyt-1 (a human SDHC gene homologue), which is homologous to the succinate dehydrogenase (SDH) cytochrome b large subunit in complex II (Ishii et al., 1998). Complex II catalyzes electron transport from succinate to CoQ and contains the citric acid (tricarboxylic acid: TCA) cycle enzyme succinate dehydrogenase (SDH). The SDHC

mutation dramatically

compromises the ability of complex II to participate in electron transport. The electron transport system of C. elegans is composed of about 70 nuclear and 12 mitochondrial gene products. It closely parallels its mammalian counterpart in its metabolism and structure, and C. elegans mitochondrial DNA (mtDNA) is similar in size and gene content to that of mammals (Murfitt et al., 1976; Okimoto et al., 1992). We then have established mouse fibroblast mev-1 (SDHC E69) cell lines and conditional transgenic Tet-mev-1 mice with the equivalent mutation, changing a neutral amino acid (valine) to an acidic amino acid (glutamate) at the 69th position (V69E) in SDHC as is the case with C. elegans mev-1 (Ishii et al., 2005; Ishii et al., 2011a and 2011b; Ishii et al., 2013). It is thought that the serine, histidine and cysteine residues in the close mutation site, which are located within the CoQ binding region, constitute an active center of oxidoreductase and act as nucleophilic amino acids (Ishii et al., 2005; Ishii et al., 2013). In the mouse fibroblast mev-1 cells, the succinate-dependent reduced

cytochrome c level through complex II-III electron transport activity, including succinate-CoQ oxidoreductase, was decreased to 40% of wild-type levels (Ishii et al., 2005). As in C. elegans, the mutation results in an excess electron leakage from electron transport system with decreasing the affinity between complex II and ubiquinone and thereby uncouples electron transfer or reverses electron transport from succinate dehydrogenase to complex I. Interestingly, ATP level was not affected, suggesting that this mutation does not directly compromise cell survival through reduced respiration per se such as is also the case in the C. elegans mev-1mutant.

2. Mitochondrial ROS production from electron transport system with flavoenzymes The electron transport system is the major endogenous source of reactive oxygen species (ROS) such as superoxide anion (O2•-), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH) (Turrens, et al., 2003). It is known that oxygen is initially converted to O2•- by electrons leaked from complexes I and mainly complex III (Turrens, 1997; Lenaz, 1998; Finkel et al., 2000; Raha et al., 2000). Complex III generates O2•- by the auto-oxidation of ubisemiquinone (QHo), formed during the Q cycle (Sun et al., 2003). O2•- can be generated at two different sites, Qo and Q1, within complex III. The Qo site

releases O2•- into the intermembrane space, whereas the Q1 site releases O2•- into the matrix. Most of the O2•- generated by complex III is generated at the Qo site (St-Pierre et al., 2002). In contrast, O2•- generated by complex I is probably released into the matrix. The mechanisms of O2•- generation in complex I are poorly understood, controversial and dependent on experimental conditions. Several studies demonstrated that O2•production from complex I was shown to occur through both forward electron transfer, involving electrons originating from NADH, and reverse electron transfer, involving electrons derived from succinate (Turrens et al., 2003; Liu et al., 2002; Tahara et al., 2009). Another study revealed a much higher rate of O2•- production during reverse electron transport from succinate to NAD+ than during forward electron transport (Lenaz et al., 2006). Tahara and colleagues have demonstrated that reverse electron transfer from succinate dehydrogenase (SDH) to complex I leads to very substantial levels of ROS. Their findings indicate an important role played by complex II flavoenzymes for reverse electron transport to complex I as ROS sources, and that succinate is an important substrate for mitochondrial O2•- production leading to hydrogen peroxide (H2O2) in brain, heart, kidney, and skeletal muscle (Tahara et al., 2009). For reference, the mitochondrial fatty acid β-oxidation primarily generates FADH2

through the activity of mitochondrial acyl-CoA dehydrogenase. The electrons from mitochondrial fatty acid β-oxidation are then transported to the electron transferring flavoprotein (ETF) and reduce ubiquinone (CoQ) to enter the mitochondrial electron transport chain (ETC) through the activity of ETF-ubiquinone oxidoreductase (Ghisla et al., 2004; Tahara et al., 2009; Cornelius et al. 2013; Cornelius et al., 2014). More experimental supports the involvement of other mitochondrial enzymes, in particular flavoenzymes

such

as

α-ketoglutarate

dehydrogenase,

glycerol

phosphate

dehydrogenase, monoamine oxidases and dihydroorotate dehydrogenase, as important sources of O2•- production (Lenaz, 2001; Miwa et al., 2003; Starkov et al., 2004; Tahara et al., 2007; Tretter et al., 2004; Hauptmann et al., 1996; Forman et al., 1975).

3. Mitochondrial ROS production with SDHC mutation Indeed, the C. elegans mev-1 mutants enhance O2•- levels in intact mitochondria and sub-mitochondrial particles and are approximately two times greater, leading to decreased reduced glutathione as compared to wild type (Senoo-Matsuda et al., 2001). This means that the mev-1 mutation either exacerbates O2•- production at this domain or, increases O2•- production at another point in reverse electron transfer from succinate dehydrogenase (SDH) to complex I, perhaps even at complex II (Turrens et al., 2003;

Liu et al., 2002; Tahara et al., 2009). The ATP level is identical between wild type and mev-1. This was initially surprising but may suggest that mev-1 relies more heavily on glycolysis for energy acquisition, thus explaining the elevated lactate levels, which could lead to reverse electron transport from complex II to complex I. It is also possible that ATP consumption is decreased in mev-1 because of some sort of global decrease in the metabolic rate that acts to counterbalance the compromised ATP synthesis in mev-1. Either way, mev-1 mutants accumulate fluorescent materials (lipofuscin) and protein-carbonyl derivatives, specific indicators of oxidized lipid and protein. These are formed in vivo as a result of metal-catalyzed oxidation at significantly and age-dependent higher rates compared to wild-type N2 cohorts (Honda et al., 1993; Strehler et al., 1959; Spoerri et al., 1974; Stadman et al., 1991; Stadman 1992; Hosokawa et al., 1994). In addition, the oxidative stress by hyperoxia in C. elegans mev-1 animals renders them hypermutable to nuclear mutations (Hartman et al., 2001). In mouse fibroblast mev-1 cells, O2•- levels were significantly higher in intact mitochondria depending on the culture time (Ishii et al., 2005). Consequently, the mev-1 cells accumulated cytoplasmic carbonyl proteins, a marker of oxidative stress, at a faster rate. They also accumulated 8-hydroxydeoxyguanosine (8-OHdG), a DNA marker of oxidative stress, at two-fold higher than control cells. This presumably resulted in

hypermutation in the 6-thioguanine tolerance test as an indicator of mutations in the nuclear HPRT gene (Ishii et al., 2005). In addition, we have comprehensively examined age-related changes in the levels of oxidative damage, mitochondrial ROS production, mitochondrial antioxidant enzyme activity and apoptosis induction in key organs of an inbred mouse strain (C57BL/6j). Mitochondrial O2•- production levels increased with aging in the brain, oculus and kidney, and did not significantly increase in the heart and muscle. In contrast to O2•production, mitochondrial SOD activities increased in heart and muscle, and remained unchanged in the brain, oculus and kidney with aging. Oxidative damage accumulated and excess apoptosis occurred in the brain, oculus and kidney with aging, but comparatively little occurred in the heart and muscle. These levels were correlated with mitochondrial O2•- levels. These results suggest that mitochondrial O2•- production and oxidative stress-dependent dysfunctions has high organ specificity, and can lead to a large variety of physiological dysfunctions with age (Miyazawa et al., 2009). The experimental results demonstrated that the oculus was especially sensitive to age-dependent oxidative stress by mitochondrial O2•- production (Miyazawa et al., 2009; Onouchi et al., 2012; Uchino et al., 2012). Tahara and colleagues have demonstrated an important participation of

flavoenzymes for reverse electron transport from complex II; SDH to complex I as ROS sources. In addition, they showed that succinate is an important substrate for mitochondrial hydrogen peroxide (H2O2) production in brain, heart, kidney, and skeletal muscle, particularly brain and heart (Tahara et al., 2009). They also showed high H2O2 release promoted by palmitate as a substrate of the fatty acid β-oxidation with acyl-CoA dehydrogenase in kidney and liver. Increased respiratory rates promoted by oxidative phosphorylation or uncoupling significantly prevented H2O2 production in brain and heart, and, to a lesser extent, kidney and skeletal muscle (Tahara et al., 2009). Collectively these data indicate that mitochondrial ROS production has tissue specificity that depends on energy metabolism, species of ROS and antioxidants redox ability or balance. In the case of succinate dehydrogenase (SDH)-dependent mitochondrial ROS production, it has been hypothesized that high succinate concentrations outside the normal physiological range in mitochondrial or cytosolic energy metabolism might be important for O2•- production during reverse electron transport (Gottlieb et al., 2005; Echtay et al., 2007). The mev-1-mimic conditional transgenic Tet-mev-1 mouse (C57BL/6j mouse with the SDHCV69E mutation) have been established using our modified Tet-On/Off system (Gossen et al., 1992; Gossen et al., 1995; Freundlieb et al., 1999; Uchida et al., 2006;

Ishii et al., 2011a). The mutated SDHCV69E coding gene expression was induced by doxycycline treatment, and total SDHC protein levels were less than twice compared to only endogenous levels in wild-type C57BL/6j mice or non-doxycycline treated Tet-mev-1 mice (Ishii et al., 2011a and Fig. 1A). The 3-month old Tet-mev-1 mice induce mitochondrial O2•- in energy-intensive organs; brain, lung, liver, kidney, spleen and pancreas, excepting heart and muscle same as mentioned above in aged C57BL/6j non-transgenic mice (Miyazawa et al., 2009 and Fig. 1B).

4. Ovarian hemangioma and decreasing ovulation with SDHC mutation Several studies report that exposing cells or tissues to hypoxia leads to an increase in mitochondrial ROS production, and that it is required for the cellular response to hypoxia (Chandel et al., 1998; Guzy et al., 2005; Emerling et al., 2009). Gimenez-Roqueplo and colleagues studied the biological effects of a loss-of-function SDHD germline mutation (p.Arg22X) in an extra-adrenal PGL and a missense SDHB germline mutation (p.Arg46Gln) in a malignant PHEO with a somatic terminal deletion of 1p (Gimenez-Roqueplo et al., 2001; Gimenez-Roqueplo et al., 2002). In tumor tissues, the inactivation of SDH activity and succinate accumulation inhibited prolyl-hydroxylation of hypoxia inducible factors (HIF) 1alpha and 2alpha, which is an

essential step for its degradation through the complex VHL-ElonginD-C-Cul2 (Selak et al., 2005). In addition, expression of vascular endothelial growth factors (VEGFs) associated with VEGF-R1 and VEGF-R2 was increased in endothelial cells. This is in agreement with the high vascularization of this endocrine tumor (Pollard et al., 2005; Favier et al., 2009). From these aspects, SDH deficiency appears to trigger hypoxic conditions, resulting in increased activity of HIFs. Based on this, we have speculated that the SDHC mutation could be associated with the high vascularization in ovaries like SDH-mutated endocrine tumors. In fact, the Tet-mev-1 mice with SDHCV69E mutation have an excessive vascularization in swollen and vacuolated ovaries with increasing mitochondrial O2•production, carbonylated protein levels and excessive apoptosis (Ishii et al., 2014 and Fig. 2). In Tet-mev-1 mice, mitochondrial oxidative stress with SDHC mutation possibly causes a mimic hypoxic condition in ovary, as mentioned above. As a result, Tet-mev-1 females develop symptoms such as ovarian hemangioma leading to endocrine-related disorders and show decreased ovulations with unsynchronously developed follicle maturation (Ishii et al., 2014 and Fig. 2). These results are consistent with the fact that the number of non-growing follicles (NGF) declines under the endocrine defects with age as in humans. It is estimated that only 12% of the maximum pre-birth NGF population is

present in 30-year old women and by the age of 40 years only 3% remain (Uppal et al., 2004; Wallace et al., 2010). Oogenesis occurs within a follicular microenviroment that is cooperating between theca cells, granulosa cells, cumulus cells and oocyte (Eppig 2001). Oocyte maturation and developmental competence is regulated by cumulus cell-dependent mechanisms (Paula-Lopes et al., 2007). During oocyte development, glucose is used by the cumulus cells leading to the production of pyruvate that is utilized by the oocytes, although both pyruvate and glucose are used by in primordial follicles (Biggers et al., 1967; Boland et al., 1993; Jansen et al., 2004; Wycherley et al., 2005). During oogenesis there is an amplification in mitochondrial number in parallel with the cytoplasmic volume increase. The increase in mitochondrial number during oocyte growth is accompanied by changes in their ultrastructure (Wassarman et al., 1978; Motta et al., 2000; Au et al., 2005). In periovulatory oocytes, the mitochondria appear oval or spheroid with dense matrix and few cristae. At ovulation, mitochondria still have a spherical immature structure, are highly vacuolated, with a dense matrix and only few cristae (Wassarman et al., 1978; Motta et al., 2000). Given this situation, it has been suggesting that mitochondria might be barely involved in the periovulatory oogenesis. In fact, the morphology of oocyte at ovulation is completely normal in Tet-mev-1 mice.

5. Decreasing sperm motility with SDHC mutation In testes, the different germ cell types differ in their preferred substrates for energy metabolism, including glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) pathways, because of the blood-testis barrier that alters the condition of surrounding substrates for energy metabolism (Robinson and Fritz, 1981; Grootegoed et al., 1984; Nakamura et al., 1984; Bajpai et al., 1998; Meinhardt et al., 1999). In spermatogonia on the basal compartment of seminiferous tubules, substrates are exclusively supplied and easy access to oxygen is provided through adjacent blood vessels for energy metabolism. However, spermatocytes, spermatids and spermatozoa in the luminal compartment of seminiferous tubule rely on the breakdown of pyruvate or lactate provided by Sertoli cells, or lactate in seminiferous tubule fluid (Bajpai et al., 1998; Boussouar et al., 2004). Therefore, it has been suspected that succinate dehydrogenase-dependent complex II-III electron transport activity, which causes the mitochondrial O2•- leading to oxidative stress, has a smaller effect on spermatogenesis in Tet-mev-1 mice. Death via apoptosis seems to be a survival strategy for some male germ cells, allowing survival of remaining healthy spermatozoa under various stresses (Narisawa et

al., 2002; Sinha-Hikim et al., 2003). The increased activation of caspase-2 in the mitochondria of germ cells plays an important role in apoptosis induction through cytochrome c release from mitochondria during the process of spermatogenesis (Zheng et al., 2006). Bax, one of Bcl-2 family members is also crucial factor for the apoptosis induction through the mitochondrial pathway in dividing spermatogonia (Rodriguez et al., 1997; Knudson et al., 1995; Russell et al., 2002). Mitochondrial ROS might have a part in the mitochondria-dependent apoptosis induction pathway in dividing spermatogonia. In fact, Tet-mev-1 mice induce excessive apoptosis in only spermatogonia on the basal compartment of seminiferous tubules (Ishii et al., 2014). Not surprisingly, the apoptosis induction in spermatogonia does not affect the total number of spermatozoa in epididymis of Tet-mev-1 mice, indicating that spermatogenesis is successfully normal after the meiotic phase. The spermatozoal morphology is also completely normal in Tet-mev-1 mice. Spermatozoal mitochondria are rearranged in elongated tubular structures and are helically arranged around the anterior portion of the flagellum (Otani et al., 1988; Olson et al., 1990; Ho et al., 2007). The outer membranes of sperm mitochondria are enclosed in a keratinous structure and formed by disulfide bonds between cysteine- and proline-rich selenoproteins, including the sperm-specific glutathione peroxidase,

catalases and superoxide dismutases (SOD) (Ursini et al., 1999; Tremellen, 2008). These formations contribute to protect the spermatozoon against ROS generated by surrounding defective spermatozoa in the ejaculate (Aitken et al., 1996; Koppers et al., 2008). Mitochondria supply sperm with energy for motility (Mundy et al., 1995; Ruiz-Pesini et al., 1998; Ruiz-Pesini et al., 2000b; Amaral et al., 2007). Mito-mice models with pathogenic mtDNA-derived electron transport chain (ETC) from complex I to III demonstrate male infertility with low motility and abnormal morphology of spermatozoa (Nakada et al., 2006). It has been reported that low quality and motility sperm possess abnormal mtDNA leading to mitochondrial ETC deficiency (May-Panloup et al., 2003; Amaral et al., 2007; Trifunovic et al., 2004). This supports the notion that mitochondria-generated ATP depending on ETC activity from complex I to III closely correlates with sperm motility and fertilization. In addition, it has been reported that the activation of caspase-3 and -9 decrease motility in sperm at ejaculation (Weng et al., 2002; Paasch et al., 2004; Grunewald et al., 2005; Varum et al., 2007). The sperm motility of Tet-mev-1 mice is slightly decreased to 50-80% of wild type depending on culture time in an in vitro fertilization assay (Ishii et al., 2014). The reasons for this infertile phenomenon have not been identified, but could be related to

compromised

energy

or

apoptotic

protease

activation

with

complex

II

deficiency-dependent mitochondrial ROS production. Future studies would be needed to determine the triggers of decreasing sperm motility and the role played by mitochondria with complex II deficiency as well as oxidative stress.

6. Low in vitro fertilization rate with SDHC mutation Using an in vitro fertilization assay, fertilized eggs found to be significantly decreased using either Tet-mev-1 mouse sperm, eggs or both. In this assay, the effect of cross-species fertilization in each gamete between C57BL/6j and Tet-mev-1 mice more significantly affects the in vitro fertilization rate as compared to that of both gametes in Tet-mev-1 or C57BL/6j mice (Ishii et al., 2014). Interestingly, the percentage of abnormally fertilized eggs, e.g., polyspermy eggs and developmentally arrested eggs before the 2-cell stage is normal in Tet-mev-1mice. Similarly, early embryogenesis through the blastocyst stage was no different between Tet-mev-1 and wild-type mice (Ishii et al., 2014). The mtDNA bottleneck theory (Hauswirth and Laipis, 1982) suggests that the number of homoplastic mtDNA molecules are transmitted to progeny are restricted in order to select high quality oocytes with healthy mitochondria (Cummins 2001;

May-Panloup et al., 2007; Cao et al., 2007; Cree et al., 2008; Khrapko, 2008). The ATP levels increase during oocyte maturation (Nagano et al., 2006; Duran et al., 2011) and oocytes with higher mtDNA copy number and higher ATP levels have greater fertilization rates and embryo development than those with lower ATP levels (Stojkovic et al., 2001; Santos et al., 2006; Nagano et al., 2006; Ge et al., 2012). In mature oocytes, each of the 105-108 mitochondria possesses a single copy of mtDNA (Chen et al., 1995; Jansen and de Boer, 1998). The mitochondria propagate with changes in their ultrastructure during oocyte growth (Wassarman and Josefowicz, 1978; Au et al., 2005). It has been reported that the mitochondria of zygotes are barely active through the blastocyst stage before implantation. The mitochondrial shapes are spherical and vacuolated with a dense matrix and few cristae before ovulation (Hewitson and Leese, 1993; Houghton, 2006; Motta et al., 2000), and then become elongated dumb-bell shapes with decreases in matrix density before changing to numerous transversally-supported cristae from concentrically located cristae after fertilization through implantation (Dvorak et al., 1982). This is logical given that energy metabolism is mainly from glycolysis in the enclosed environment during the blastomere stage (Leese et al., 1984). After that, the blastocyst stage oocyte, which starts cell division for the involved differentiation in both

inner cell mass (ICM) and trophectoderm (TE), prepares an increase in oxidative phosphorylation (OXPHOS) in mitochondria with glycolysis and oxygen consumption. Given this, it is logical that the mitochondrial complex II SDHCV69E mutation could not affect the fertilization and early embryogenesis but rather exert its effects later in development.

7. Placental angiodysplasia and inflammation with SDHC mutation Under natural conditions, 75% of 30-year old women attempting to conceive will have a conception ending in a live birth within 1 year, 66% at age 35 years, and only 44% at age 40 (Leridon 2004). Miscarriage refers to the unintentional termination of a pregnancy before fetal viability at 20 weeks of gestation or when fetal weight is < 500 g. The incidence of such miscarriages is 12-24% of all pregnancies (Jurkovic et al., 2013). The risk of a miscarriage was 8.9% in women aged 20-24 years and 74.7% in those aged 45 years or older (Nybo Andersen et al., 2000). Unexplained infertility is defined as the inability to conceive after 12 months of unprotected intercourse in couples where known causes of infertility have been ruled out. Up to 22.3% of infertile couples have unexplained infertility (Collins et al., 1989). Indeed, in Tet-mev-1mice, the natural pregnancy and safe delivery rates are decreased to about 55% and 65%, respectively,

before they first experience a successful delivery (primiparity). In addition, the fetal loss and placental inflammation resulting in occasional maternal death were frequently observed in 13.5-day pregnant Tet-mev-1 mice (Ishii et al., 2014). The hermaphrodite C. elegans short-lived mev-1 mutant is also characterized by low fertility and progeny (Ishii et al., 1990). Based on this, we are posit that the C. elegans short-lived mev-1-mimics Tet-mev-1 mice and therefore would be a suitable model for demonstrating the phenomena of age-related infertility with oxidative stress such as it occurs in humans. The total number of mitochondria in a normal human blastocyst is about 14,000, and the average number of mitochondria per cell is about 150 (Van Blerkom, 2008). The average number of mitochondria per cell is higher in the trophectoderm (TE) than in the inner cell mass (ICM) of mouse blastocyst (Barnett et al., 1996; Van Blerkom, 2008). The existence of two types of mitochondria in the mouse blastocyst has been reported: spherical mitochondria in the ICM and elongated mitochondria in the TE (Sathananthan et al., 2000). In both types mitochondrial cristae are transversely oriented and their matrix is less dense than the mitochondrial matrix found in earlier developmental stages (Stern et al., 1971). It has been anticipated with those mitochondrial shape’s characterizations that the TE cells become the placenta and extraembryonic tissues, are

highly polarized and very active at producing ATP and oxygen consumption (Hewitson et al., 1993; Barnett et al., 1996; Houghton, 2006; Van Blerkom et al., 2006). The precise role mitochondria play in placental development remains unknown. Tet-mev-1 mice exhibit thrombocytosis and splenomegaly with megakaryocytic differentiation by intracellular oxidative stress-activated Nrf-2 signaling (Motohashi et al., 2010; Ishii et al., 2014 and Fig. 3). This causes placental angiodysplasia with decreasing Flt-1/VEGF receptor-1 (VEGFR-1) protein, occasionally leading to placental inflammation (Ishii et al., 2014). Vascular endothelial growth factors (VEGF) signaling is important in regulating vascular cell recruitment and proliferation for placental formation (Vuorela et al., 2000; De Falco 2012). Flt-1, also known as VEGFR-1, is well known as the most abundant and active member of the VEGF receptor family. This binds to VEGF-A and PLGF-1 as a key factor promoting angiogenesis in the placenta. Disruptions contribute to the pathogenesis of female infertilities such as preeclampsia (Kita et al., 2008). We expected that these placental phenotypes would lead to placental inflammation and hypoxic conditions in embryos. Given this, it is not surprising that the embryos of Tet-mev-1mice frequently have a developmental arrest that mimics human embryopathy and a randomly abnormal angiogenesis leading to decreasing progeny (Ishii et al., 2014). In future studies, the complex II-deficiency dependent oxidative

stress and placental development or maintenance machinery in Tet-mev-1 mice could be clarified.

8. Recurrent miscarriage under excessive apoptosis induction with SDHC mutation 8.1. Apoptosis induction with mitochondrial ROS Apoptosis induction pathways are classified largely into the extrinsic and intrinsic pathways activated by receptor-ligand binding signals through caspase-8 activation and mitochondria-dependent machinery through caspase-9 activation (Sinha et al., 2013). The extrinsic apoptotic pathway induces apoptosis mediated through the receptor-ligand binding signals of the tumor necrosis factor (TNF) receptor superfamily: FasL (Fas ligand)/Fas (also called CD95 or APO-1), TNF-α/TNFR1 (TNF receptor 1), and TRAIL (TNF-related apoptosis-inducible ligand)/TRAILR-R1 and 2 (TRAIL-receptor 1 and 2; also called DR4 and 5) (Ashkenazi et al., 1998; Ashkenazi et al., 1999; Circu et al., 2010; Elmore 2007). The FasL/Fas binding signal requires Fas-associated DD (FADD) and procaspase-8 resulting in activation of the initiator caspase-8 with the endocytosed death-inducing signaling complex (DISC) (Watanabe et al., 1988; Lee et al., 2006; Huang et al., 2007). The caspase-8 activation exists at the DISC and determines Type 1 or Type 2 mechanisms; significant caspase-8 activation directly activates caspase-3

(Type 1), whereas low caspase-8 activation mediates caspase-3 activation through an amplification loop in mitochondria (Type 2) (Barnhart et al., 2003). In Type 2, the activated caspase-8 cleaves pro-apoptotic Bid leading to outer mitochondrial membrane permeabilization through the interactions of cleaved Bid (tBid) with Bax/Bak, resulting in apoptogenic cytochrome c release from mitochondria, involved in intrinsic pathway as described below. On the other hand, TNFα/TNFR1 binding signal requires the TNFR1-associated death domain (TRADD)-composed complex I and II formation that activates distinct downstream survival and apoptotic signaling pathways, respectively (Jiang et al., 1999; Hsu et al., 1995). Apoptosis-inducible complex II is composed of TRADD, FADD and caspase-8, and is formed after TNFR1 receptosome endocytosis leading to caspase-8 activation as mentioned below (Schneider-Brachert et al., 2004; Micheau et al., 2003; Lin et al., 1999). Given this, the both TNFα/TNFR1 and FasL/Fas -mediated apoptosis are similarly induced by the caspase-8-dependent modulations. The intrinsic apoptotic pathway, which is activated by intracellular stimuli such as mitochondrial oxidative stress, depolarization, DNA mutation is mediated by promoting mitochondrial outer membrane permeabilization and cytoplasmic translocation of mitochondrial apoptogenic or pro-apoptotic proteins: cytochrome c, Smac/DIABLO and HtrA2/Omi, which are caspase-dependent factors, and apoptosis-inducing factor (AIF),

EndoG and CAD, which are released from mitochondria to directly go to the nucleus for induction of nuclear chromatin condensation and DNA fragmentation (Susin et al., 1999; Verhagen et al., 2000; Ryter et al., 2007; Orrenius et al., 2007). Cytochrome c normally resides in the intermembrane space of mitochondria for electron transport chain reaction. The released cytochrome c from mitochondria forms an apoptosome complex with apoptotic protease-activating factor-1 (Apaf-1) and procaspase-9, producing active caspase-9 which is a crucial factor to induce apoptosis by directly activating caspase-3, whereas Smac/DIABLO and HtrA2/Omi inhibit inhibitors of apoptosis (IAPs) and thereby indirectly activate caspase-3 (Hirsch et al., 1997; Verhagen et al., 2000; Elmore 2007). The mitochondrial permeability transition pore (MPTP) is composed of cyclophilin D (cypD), voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT). It plays a key role in mitochondrial permeabilization and release of mitochondrial pro-apoptotic proteins (Baines et al., 2007; Kokoszka et al., 2004; Schinzel et al., 2005; Rasola et al., 2007; Circu et al., 2010). The anti-apoptotic (Bcl-2, Bcl-XL, and Bcl-w) and pro-apoptotic (Bax, Bak, Bad, Bim, and Bid) Bcl-2 superfamily are major players to modulate mitochondrial outer membrane permeabilization (Kelekar et al., 1998; Cory et al., 2002). Upon receiving apoptotic

stimuli, Bad is activated by phosphorylation and inhibits Bcl-2 and Bcl-XL that maintains Bax/Bak inactivation (Yang et al., 1995; Pastorino et al., 1998; Orrenius et al., 2011). Activated Bax and Bak then directly interact with VDAC leading to cytochrome c release from mitochondria (Shimizu et al. 1999). In addition, the tBid converted from Bid is degraded by caspase-8. This promotes Bax and/or Bak oligomerization resulting in megapore formation, a highly orchestrated and active process (Desagher et al., 1999; Eskes et al., 2000; Wei et al. 2000). The contribution of pro-apoptotic tBid in both mitochondria and the death receptor-ligand binding signals has a crucial role for cross talk between the intrinsic and extrinsic apoptotic pathways. In addition, it has been reported that oxidative stress directly plays an important role in the mitochondrial permeability transition (MPT) modifications through ROS-mediated modification of the thiol group of ANT or ROS-mediated oxidation of cardiolipin for binding between tBid and VDAC (Bustamante et al., 2005; Orrenius et al., 2011). Another interesting report indicated that ROS-activated c-Jun-N-terminal kinase (JNK) can be induced by extrinsic or intrinsic apoptotic signaling (Dhanasekaran et al., 2008). TNFα is a potent activator of the MAPK cascade, and the apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway plays an important role in TNFR1-mediated apoptotic signaling in various cell types (Matsuzawa et al., 2008).

TNFα induces pro-apoptotic effects via ASK1 signaling with a prolonged and robust JNK activation by ROS; however, a transient and modest JNK activation mediates cell survival via NF-κB-induced antiapoptotic gene expression (Kamata et al., 2005; Liu et al., 2002; Deng et al., 2003). Furthermore, the mitochondrial ASK1-dependent apoptotic signaling pathway reportedly activated both JNK-dependent and JNK-independent apoptosis (Zhang et al., 2004). Nuclear translocation of activated JNK promoted activator protein-1 (AP-1)-mediated expression of pro-apoptotic TNFα, FasL, and Bak (Fan et al., 2001), whereas mitochondrial JNK translocation promoted cytochrome c release (Kharbanda et al., 2000). The above-mentioned cross talk between intrinsic and extrinsic apoptotic pathways via caspase-8 or JNK activated by intracellular and extracellular stimuli with oxidative stress has not been adequately clarified. More studies will be needed to fully identify the mechanisms, control and consequences of this cross talk.

8.2. Developmental arrest, embryopathy and growth retardation with SDHC mutation Indeed, the oxidative stress-sensitive, short-life mev-1 mutant of C. elegans that exhibits growth retardation and small body size has supernumerary embryonic apoptosis

mediated by the oxidative stress-stimulated ced-9 (Bcl-2 family)/ced-3 (caspase)/ced-4 (Apaf-1) apoptotic pathway (Sulston et al., 1983; Ishii et al., 1990; Senoo-Matsuda et al., 2003). They show a complex II-deficiency which presumably leads to oxidative stress and excessive apoptosis which results in occasional developmental arrest and a reduction in the number of progeny (Fig. 4). C. elegans individuals are composed exclusively of post-mitotic cells, excepting reproductive organs. Therefore the random excessive apoptosis induction might affect the decreased progeny with decreasing zygotes and the small body size with decreasing number of somatic cells as compared to that of control N2 worms (Fig. 4). The mev-1-mimic SDHCI71E mutation also causes very similar pathophysiological phenotypes in Drosophila (Tsuda et al., 2007). In mouse embryonic fibroblast mev-1-mimic cells (NIH3T3 cells with SDHCV69E mutation, SDHC E69 cells), the activity of the apoptotic effector caspase-3 activity through initiator caspase-8 and -9 activations leading to cytochrome c release from mitochondria was 1.3 to 1.8 times higher than as compared to control mouse embryonic fibroblast cells (NIH3T3 cells) (Ishii et al., 2005; Miyazawa et al., 2008; Fig. 5). Interestingly, mev-1-mimic cells maintained caspase-3 activity through caspase-9 activation of both caspase-9-dependent intrinsic and caspase-8 and -9-dependent extrinsic pathways as shown by the observation that caspase-8 inhibition had less effect

on caspase-3 inhibition, whereas caspase-8 inhibition completely affected caspase-3 inhibition in control cells (Fig. 5A). Interestingly, the inhibition of apoptosis induction (yellow arrows) with caspase-8 inhibitor did not affect the survival rate in mev-1-mimic cells, although that increased survival rate in control cells and transformed mouse embryonic fibroblast mev-1-mimic cells (transformed mev-1-mimic cells). On the other hand, the inhibition of apoptosis with caspase-9 inhibitor decreased survival rate with necrotic cell death induction (red arrows) in both control and mev-1-mimic cells (Fig. 5B). From these results, it has been suggested that the caspase-9 is affecting the caspase-8-mediated extrinsic apoptotic pathway and mitochondrial deficiency-mediated intrinsic pathway plays a crucial role in survival in both control and mev-1-mimic cells excepting transformed mev-1-mimic cells. Moreover JNK activation patterns were dramatically changed in each cell type: phosphorylated JNK pattern was completely changed between control and mev-1-mimic cells and the JNK activity was robustly enhanced in transformed mev-1-mimic cells (Fig. 5C). This JNK functional change might affect or at least reflect different apoptosis induction machinery via in each intrinsic and extrinsic pathway or both, as mentioned above. Tet-mev-1 mice, which like the C. elegans mev-1 mutant, have a mitochondrial complex II SDHCV69E mutation, exhibit fetal and infant growth retardation. When

exposed continuously to doxycycline for induction of SDHCV69E mutated protein before fertilization through fetal development, these animals show a low birthweight and growth retardation through the weaning age. Two separate measures (TUNEL staining and immunodetection of caspase-3) reveal excessive apoptosis in several tissues: brain, lung, liver, kidney (especially in the adrenal region), salivary grand, nasal sinus tissue (especially secretory cells and mucosal cells) and muscles. In Tet-mev-1mice, the neonatal unexplained mortality is increased, thus the number of weaning mice is dramatically decreased at first delivery (Ishii et al., 2011a; Ishii et al., 2014). Also interestingly, complex II deficiency-dependent excessive apoptosis in several tissues was not observed after the period of growth of body length at about 12-week age. This indicates that the excessive apoptosis induction might be caused by mitochondrial electron transport chain (ETC) defect-enhanced intracellular oxidative stress with cell differentiation and proliferation. These phenomena, including decreasing electron transport ratio, increase in O2•- production and accumulation levels, excessive apoptosis induction, low-birth-weight infants and growth retardation in Tet-mev-1 mice, are completely recovered by CoQ supplementation to that of the wild-type C57BL/6j (Ishii et al., 2011a). This strongly suggests that the excessive apoptosis induction caused by the electron leakage from mitochondria with complex II-CoQ oxidoreductase deficiency

leading to O2•- overproduction during cell proliferation stage.

9. Conclusions and future perspectives Complex II activity has a crucial role for energy metabolisms of both the citric acid (tricarboxylic acid: TCA) cycle and electron transport chain (ETC) in mitochondria. Complex II contains the citric cycle enzyme succinate dehydrogenase (SDH), which is composed of the flavin protein (Fp, SDHA), the iron-sulfur protein (Ip, SDHB) and two other subunits (a large subunit of cytochrome b encoded by cyt-1, SDHC and a small subunit of cytochrome b, SDHD) and catalyzes electron transport from succinate to ubiquinone (CoQ). The crystal structure of mitochondrial respiratory membrane protein complex II has been elucidated in porcines (Sun et al., 2005). In vivo, the SDHC and SDHD subunits containing a heme are anchored to mitochondrial inner membrane and involved in catalyzing FADH2 produced by SDH activity. In humans, it has been reported that homozygous germline mutations affecting the SDHA gene cause Leigh syndrome, a sub-acute necrotizing encephalomyelopathy during infancy (Bourgeron et al., 1995; Ackrell 2002). SDHB, SDHC and SDHD heterozygous

mutations

cause

a

genetic

predisposition

to

non-chromaffin

palagamgliomas (PGLs) and adrenal ⁄extra-adrenal phaeochromocytomas (PHEOs)

called ‘PGL/PHEO syndrome’ (Baysal et al., 2000; Niemann et al., 2000; Astuti et al., 2001). In the lower animal model C. elegans , the SDHCG71E homozygous germline mutation causes an oxygen-hypersensitive precocious aging with low fertility, developmental arrest and small body size. In contrast, in mice, the overexpression of mutated SDHCV69E coding transgene that causes a dominant negative effect results in widespread cellular lethality. Mice with mutated SDHCV69E expression equal to that of the endogenous SDHC gene are born with growth retardation, low fertility and accelerating age-dependent phenotypes. The relationship between oxidative stress and infertility has been widely reported, although low fertility and the effects of SDHC mutations remain comparatively unexplored. SDHC mutations can cause intracellular oxidative stress with mitochondrial electron transport chain dysfunction from complex II to ubiquinone (CoQ) (Fig. 6). Thus, the low-fertility phenotypes in Tet-mev-1 mice demonstrate a relationship between mitochondrial ROS and infertility (Fig. 6). Future studies using Tet-mev-1 mice may help clarify the influence of oxidative stress from mitochondria with complex II-III electron transport deficiency on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. Collectively these data could demonstrate that chronic oxidative stress caused by complex II-III electron transport

deficiency without mitochondrial energy metabolic rate changes provoke spontaneous abortions and recurrent miscarriage resulting in age-related female infertility (Fig. 6). Recent studies have shown a diabetic embryopathy resulting in birth defects and miscarriage caused by oxidative stress-induced apoptosis (reviewed in Yang et al., 2015). The maternal diabetes-induced birth defects, most commonly neural tube defects and heart defects, occur in 6-10% of babies born to mothers with pre-gestational diabetes. It has been demonstrated that embryos under hyperglycemic conditions exhibit high levels of oxidative stress in animal models. The oxidative stress is a trigger of apoptosis induction by activated ASK1, JNK1/2 and caspases through the intrinsic and extrinsic pathways as mentioned in section 3. It has been also revealed that maternal diabetes activates protein kinase C (PKC) isoforms: PKCα, βII and δ. These signaling intermediates exert their effects through the extrinsic pathway. The PKC family of serine/threonine protein kinases consists of 12 members, which can be divided into the following 3 groups based on their activation mechanisms: (1) PKCα, βI, βII, and γ require calcium and diacylglycerol (DAG) for activation; (2) PKCδ, ε, η, ν, and θ require only DAG; and (3) PKCμ, ξ, and ι/λ do not require calcium or DAG, but instead require distinct lipid cofactors (eg, ceramide and phosphatidylinositol-4-phosphate) (Dempsey et al., 2000; Shirai et al., 2002). Specific PKC isoforms (α, βII, and δ) are

up-regulated and are activated for prolonged periods, while others (ε and ξ) are down-regulated in diabetic embryopathy (Curtis et al., 2004; Srivastava 2002; Way et al., 2001; Park et al., 2000). The maternal diabetes-induced oxidative stress is a major contributor to PKCα/βII and δ activation causes a positive feedback loop and induces lipid peroxidation in diabetic embryopathy (Li et al., 2011; von Ruecker et al., 1989). Prostaglandin E2 (PGE2), a product of arachidonic acid (AA) metabolism catalyzed by cyclooxygenase-2, aids in preventing malformations. PGE2 has been detected in embryos under maternal hyperglycemic conditions (Goldman et al., 1985; Pinter et al., 1986; Kudo et al., 2003; Claria 2003; Goto et al., 1992). In diabetic patients, AA is converted into PGE2-like isoprostanes such as 8-iso-prostagladin F2 (PGF2) by noncyclooxygenase catalyzed peroxidation involving free radicals that have damaging effects on embryos (Morrow et al., 1990; Morrow 2000; Wentzel et al., 2002). It has been also suggest that the metabolic shift from AA/PGE2 to AA/isoprostanes is caused by the level of 8-iso-PGF2-elevated embryos under diabetic conditions (Gopaul et al., 1995; Decsi et al., 2002; Wentzel et al., 1999). Based upon these observations, it has been postulated that diabetic oxidative stress contributes to the proapoptotic kinase signaling ASK1/JNK activation and the positive feedback loop of PKC activation and lipid peroxidation activates apoptosis induction through extrinsic and intrinsic pathways

in embryos. Indeed, in the corneal epithelium of Tet-mev-1 mice, the proliferation of epithelial basal cells was decreased, resulting in delayed epithelialization with keratitis relative to wild-type C57BL/6j mice, particularly as animals aged (Onouchi et al., 2012). It has been reported that keratitis and delayed epithelialization might be caused by hyperglycemia and high oxidative stress conditions (Kim et al., 2011). From these observations, it is also anticipated that the study of Tet-mev-1 mice could clarify the contributions between hyperglycemia and oxidative stress leading to placental dysfunctions. These could be associated with abnormal angiogenesis and diabetic embryopathy. Acknowledgements Our research in this review was supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and Japan Society for the Promotion of Science (JSPS), and the Tokai University School of Medicine Research Aid.

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Figure legends Fig. 1. The SDHC/β-actin protein ratios with SDHCV69E-mutated protein (A) and mitochondrial O2•- levels (B) in energy-intensive key organs at 3-month old complex II-deficiency mev-1-mimic animal model, Tet-mev-1 mice. Results are expressed as mean ± SD; *P < 0.01; ** P < 0.05; n = 4 in each group.

Fig. 2. Micrographs of hematoxylin-eosin stained ovaries of non-pregnant and pregnant Tet-mev-1 mice. Yellow arrows indicate different developmental stages of follicle. Black and red arrows indicate blood vessels and corpus luteums, respectively.

Fig. 3. Micrographs of hematoxylin-eosin stained spleens of Tet-mev-1 mice. Black arrows are indicating megakaryocytes.

Fig. 4. C. elegans control N2 and mev-1 growth rates through the gravid adult (GA) stage. N >= 20 in each group. Body length was used as a proxy of growth rate.

Fig. 5. Alterations in caspase-3 activity in the presence of inhibitors of caspase-8, -9 or both (A), JNK activities (B), micrographs of cultured cells subjected to caspase-8 or -9

inhibition (C) in mev-1-mimic cells and control NIH3T3 cells (modified Miyazawa et al. 2008). Results are expressed as mean ± SD; *P < 0.01; n = 3 in each group. Yellow and red arrows are indicating apoptotic cell bodies and necrotic cell death, respectively.

Fig. 6. Graphical summary of reproductive dysfunctions caused by complex II-deficiency dependent oxidative stress in the mev-1 mouse model. The SDHCV69E mutation decreases the affinity between complex II and ubiquinone (CoQ) resulting in electron leakage that leads to ROS production. Consequently, the oxidative stress-activated intrinsic apoptosis pathway induces an excessive apoptosis resulting in developmental arrest and growth retardation. On the other hand, oxidative stress induced a mimic-hypoxic condition, which caused ovarian hemangiomas leading to decreasing ovulation and placental angiodysplasia resulting in inflammation. The oxidative stress also decreases sperm motility and survival. These aspects could associate with low fertility and recurrent miscarriage with occasional maternal death.

**

SDHC/β-actine protein ratios

A - Doxycycline + Doxycycline

*

2.5 2.0

*

*

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*

*

** **

**

1.0 0.5 0

B

H

Lu

Li

Ki

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3-month aged Tet-mev-1 mice

B O2•- accumulation levels in mitochondria

*

*

3-month aged C57BL/6j

1200

3-month aged Tet-mev-1 mice

1000 800

*

*

**

*

*

600

*

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B

H

Lu

Li

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Sp

Pa

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B: Brain, H: Heart, Lu: Lung, Li: Liver, Ki: Kidney, Sp: Spleen, Pa: Pancreas, St: Stomach, M: Muscle, T: Testis, O: Ovary

Figure 1

I Non-pregnant Tet-mev-1 mouse

II I

Pregnant Tet-mev-1 mouse

II

I

I

II II

Figure 2

C57BL/6j mouse

Tet-mev-1 mouse

Figure 3

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Figure 4

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NIH3T3 cells Non-transformed mev-1-mimic cells Transformed mev-1-mimic cells

Relative caspase3 activities

2.0

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mev-1-mimic cells NIH3T3 cells

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Non-transformed Transformed PhosphoJNK/SAPK

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DMSO

Caspas-8 inhibitor

Caspase-9 Caspase-8 and -9 inhibitors inhibitor

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g

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Caspase-8 inhibitor

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Figure 5

Decreasing ovulations with ovarian hemangioma

Decreasing sperm motility

Low fertility

SDHA (Fp)

ROS

Mitochondria

SDHB (Ip)

eCoQ SDHD SDHC

Flt-1

JNK/SAPK Cytochrome c

V69E

Caspase-9 Placental angiodysplasia and inflammation

Miscarriage and maternal death

Figure 6

Caspase-3 Apoptosis

Fetal developmental arrest and Infant growth retardation