Satoshi Tsunoda

Peroxiredoxin 4 knockout results in elevated spermatogenic cell death via oxidative stress

Prx (peroxiredoxin) is a multifunctional redox protein with thioredoxin-dependent peroxidase activity. Prx4 is present as a secretory protein in most tissues, whereas in sexually mature testes it is anchored in the ER (endoplasmic reticulum) membrane of spermatogenic cells via an uncleaved N-terminal hydrophobic peptide. We generated a Prx4 knockout mouse to investigate the function of Prx4 in vivo. Prx4(-/y) mice lacking Prx4 expression in all cells were obtained by mating Prx4(flox/+) female mice with Cre-transgenic male mice that ubiquitously expressed Cre recombinase. The resulting Prx4(-/y) male mice were fertile, and most organs were nearly normal in size, except for testicular atrophy. The number of deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive spermatogenic cells was higher in Prx4(-/y) mice than in Prx4(+/y) mice and increased remarkably in response to warming the lower abdomen at 43 degrees C for 15 min. Cells reactive to antibodies against 4-hydroxynonenal and 8-hydroxyguanine were high in the Prx4(-/y) mice and concomitant with elevated oxidation of lipid and protein thiols. The cauda epididymis of Prx4(-/y) mice contained round spermatocytes, which were not found in Prx4(+/y) mice, and displayed oligozoospermia. However, mature spermatozoa from the epididymis of Prx4(-/y) mice exhibited normal fertilization In vitro. Taken together, these results indicate that spermatogenic cells lacking Prx4 are more susceptible to cell death via oxidative damage than their wild-type counterparts. Our results suggest that the presence of Prx4, most likely the membrane-bound form, is important for spermatogenesis, but not an absolute requisite.

Rescue of anaemia and autoimmune responses in SOD1-deficient mice by transgenic expression of human SOD1 in erythrocytes

Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.

Intrinsic oxidative stress causes either 2-cell arrest or cell death depending on developmental stage of the embryos from SOD1-deficient mice

Oxidative stress characterized by elevated reactive oxygen species is a well-known cause of developmental arrest and cellular fragmentation in the development of in vitro-produced embryos. To investigate the effects of intrinsic oxidative stress on the early development of embryos, oocytes from superoxide dismutase 1 (SOD1)-deficient mice resulting from in vitro fertilization, followed by culture for 4 days, were examined. Development of all embryos from SOD1-deficient oocytes was arrested at the 2-cell stage under conventional culture conditions with atmospheric oxygen (20% O(2)). Significantly higher levels of superoxide were detected in SOD1-deficinet embryos cultured under 20% O(2) using dihydroethidium. Among treatments with antioxidants, only hypoxic culture with 1% O(2) negated the 2-cell arrest and advanced the development of the embryos with efficacy similar to that in wild-type embryos. Mitochondrial function was investigated because its malfunction was a suspected cause of 2-cell arrest. However, respiratory activity, ATP content and mitochondrial membrane potential in the 2-cell embryos were not markedly affected by culture with 20% O(2). When embryos from SOD1-deficient oocytes were first developed to the 4-cell stage under 1% O(2) culture and were then transferred to 20% O(2), most of them developed to the morula stage but underwent total degeneration thereafter. Thus, oxidative stress was found to damage embryos differentially, depending on the developmental stage. These results suggest that embryos derived from SOD1-deficient mouse oocytes are an ideal model to investigate intrinsic oxidative stress-induced developmental abnormality.

Implication of oxidative stress as a cause of autoimmune hemolytic anemia in NZB mice

We have recently shown that deficiency of the superoxide dismutase 1 gene (SOD1) causes anemia and autoimmune responses against red blood cells (RBCs) and that transgenic expression of human SOD1 in erythroid cells rescues them. Because these phenotypes observed in SOD1-deficient mice are similar to autoimmune hemolytic anemia (AIHA), a causal connection between reactive oxygen species (ROS) and AIHA was examined using an AIHA-prone New Zealand Black (NZB) mouse. ROS levels in RBCs were high in young NZB mice, compared to control New Zealand White (NZW) mice, and increased during aging. Methemoglobin and lipid peroxidation products were elevated during aging, consistent with the elevated oxidative stress in RBCs of NZB mice. Severity of anemia and levels of intracellular ROS were positively correlated. Levels of antibodies against 4-hydroxynonenal and acrolein were also elevated in NZB mice. Transgenic expression of human SOD1 protein in RBCs of NZB mice suppressed ROS in RBCs and decreased the death rate. When RBCs from C57BL/6 mice were injected weekly into the same strain of mice, production of anti-RBC antibody was observed only in mice that had been injected with oxidized RBCs. Thus, oxidation-mediated autoantibody production may be a more general mechanism for AIHA and related autoimmune diseases.

Impaired Fertilizing Ability of Superoxide Dismutase 1-Deficient Mouse Sperm During In Vitro Fertilization

ABSTRACT The oxidative modification of gametes by a reactive oxygen species is a major deleterious factor that decreases the successful rate of in vitro fertilization. Superoxide dismutase 1 (SOD1) plays a pivotal role in antioxidation by scavenging the superoxide anion, and its deficiency causes infertility in female mice, but the significance of the enzyme in male mice remains unclear. In the present study, we characterized Sod1−/− (Sod1-KO) male reproductive organs and compiled the first report of the impaired fertilizing ability of Sod1-KO sperm in in vitro fertilization. Insemination of wild-type oocytes with Sod1-KO sperm exhibited lower rates of fertility compared with insemination by wild-type sperm. The low fertilizing ability found for Sod1-KO sperm was partially rescued by reductant 2-mercaptoethanol, which suggested the oxidative modification of sperm components. The numbers of motile and progressive sperm decreased during the in vitro fertilization process, and a decline in ATP content and elevation in lipid peroxidation occurred in the Sod1-KO sperm in an incubation time-dependent manner. Tyrosine phosphorylation, which is a hallmark for sperm capacitation, was also impaired in the Sod1-KO sperm. These results collectively suggest that machinery involved in sperm capacitation and motility are vulnerable to oxidative damage during the in vitro fertilization process, which could increase the rate of inefficient fertilization.

Redox regulation of fertilisation and the spermatogenic process.

Oxidative stress is one of the major causes of male infertility; it damages spermatogenic cells, the spermatogenic process and sperm function. Recent advances in redox biology have revealed the signalling role of reactive oxygen species (ROS) that are generated by cells. While highly reactive oxidants, such as the hydroxyl radical, exert largely deleterious effects, hydrogen peroxide can feasibly serve as a signal mediator because it is moderately reactive and membrane permeable and because it can oxidize only limited numbers of functional groups of biological molecules. The amino acid side chain most sensitive to oxidation is cysteine sulphydryl, which is commonly involved in the catalysis of some enzymes. Although the reactivity of cysteine sulphhydryl is not very high in ordinary proteins, some phosphatases possess a highly reactive sulphydryl group at their catalytic centre and are thereby oxidatively inactivated by transiently elevated hydrogen peroxide levels after extracellular stimuli and under certain environmental conditions. Peroxiredoxins, in turn, show moderate hydrogen peroxide-reducing activity, and their role in the modulation of ROS-mediated signal transduction in ordinary cells, mediated by protecting phosphatases from oxidative inactivation, has attracted much attention. Although knowledge of the signalling role of ROS in the male reproductive system is limited at present, its significance is becoming a focal issue. Here, we present a review of the emerging signalling role of hydrogen peroxide in testes.

A malfunction in triglyceride transfer from the intracellular lipid pool to apoB in enterocytes of SOD1-deficient mice

We compared lipid metabolism in the intestines of Sod1‐knockout mice with that found in wild‐type mice to elucidate the impact of oxidative stress in vivo. A high‐fat diet in wild‐type mice induced postprandial hypertriglyceridemia, but this adaptive response was impaired in Sod1‐knockout mice. While fewer triglycerides were secreted to the blood in the form of triglyceride‐rich lipoprotein, more lipid droplets accumulated in the enterocytes of Sod1‐knockout mice fed a high‐fat diet. These data collectively suggest that high‐fat diet induces oxidative stress, inhibits lipid secretion to the blood, and ultimately leads to dysfunctional lipid metabolism in enterocytes.

Deficiency of the cystine-transporter gene, xCT, does not exacerbate the deleterious phenotypic consequences of SOD1 knockout in mice

Because glutathione scavenges reactive oxygen species (ROS) and also donates electrons to antioxidative systems, it may compensate for the oxidative stress caused by SOD1 deficiency. The cystine/glutamate transporter, which consists of two proteins, xCT and 4F2hc, has been designated system xc−. This transporter system plays a role in the maintenance of glutathione levels in mammalian cells. In the present study, we created SOD1−/−; xCT−/− double-knockout mice by intercrossing xCT-knockout and SOD1-knockout animals. We determined if the double-knockout mice express the phenotypic characteristics unique to SOD1−/− mice—increased oxidative stress and the production of autoantibodies against erythrocytes. We also compared the phenotype of the double-knockout mice with those of the single-knockout and wild-type mice. Although two major antioxidative systems were found to be defective in the SOD1−/−; xCT−/− mice, relative to the SOD1−/− mice, no functional deficits were observed. Based on these results, it appears that defects in system xc− do not exacerbate the phenotypic consequences of SOD1 deficiency in postnatal mice under ordinary breeding conditions.

Oxidative stress and redox regulation of gametogenesis, fertilization, and embryonic development

Oxidative stress caused by elevated reactive oxygen species (ROS) is one of the predominant causes of both male and female infertility. Oxidative stress conditions cause either cell death or senescence by oxidation of cellular molecules including nucleic acid, proteins, and lipids. It is particularly important to minimize oxidative stress when in vitro fertilization is performed for the purpose of assisted reproduction. The problems associated with assisted reproductive technology are becoming evident, and it is now the time to clarify its mechanisms and cope with them. On the other hand, the beneficial roles of ROS, such as intracellular signaling, have become evident. The antithetical functions of ROS make it more difficult to overcome the problems caused by oxidative stress. Despite the difficulty in understanding mammalian reproduction, the mechanisms and problems can be gradually unveiled by advanced technology such as genetic modification of animals.

Reactive oxygen species exacerbate autoimmune hemolytic anemia in New Zealand Black mice

Elevated reactive oxygen species (ROS) and oxidative damage occur in the red blood cells (RBCs) of SOD1-deficient C57BL/6 mice. This leads to autoimmune responses against RBCs in aged mice that are similar to autoimmune hemolytic anemia (AIHA). We examined whether a SOD1 deficiency and/or the human SOD1 transgene (hSOD1) would affect phenotypes of AIHA-prone New Zealand Black (NZB) mice by establishing three congenic strains: those lacking SOD1, those expressing hSOD1 under a GATA-1 promoter, and those lacking mouse SOD1 but expressing hSOD1. Levels of intracellular ROS and oxidative stress markers increased, and the severity of the AIHA phenotype was aggravated by a SOD1 deficiency. In contrast, the transgenic expression of hSOD1 in an erythroid cell-specific manner averted most of the AIHA phenotype evident in the SOD1-deficient mice and also ameliorated the AIHA phenotype in the mice possessing intrinsic SOD1. These data suggest that oxidative stress in RBCs may be an underlying mechanism for autoimmune responses in NZB mice. These results were consistent with the hypothetical role of reactive oxygen species in triggering the autoimmune reaction in RBCs and may provide a novel approach to mitigating the progression of AIHA by reducing oxidative stress.