Naoko Kimura

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.

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.

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.

Effect of Compound Exposure to Bisphenol A and Nonylphenol on the Development and Fertility of Fetal Mice

ABSTRACT Bisphenol A (BPA) and nonylphenol (NP) are endocrine disrupting chemicals (EDCs) which induce reproductive abnormalities. While single exposure studies have been reported, few investigations of the simultaneous administration of several chemicals to animals have been conducted. The purpose of this study was to estimate the effect of gestational exposure to BPA and NP simultaneously on reproductive function. Pregnant female ICR mice were given BPA, NP or BPA plus NP by subcutaneous injection from gestational day (GD) 5 to delivery. The daily doses of BPA and NP were 1/1,000 or 1/100 the median lethal doses (LD50; BPA: 2,500 mg/kg, NP: 1,231mg/kg). On postnatal day (PND) 1, the pups (F1) were thinned out to 8. On PND 42, the body weights of some F1 were recorded and they were sacrificed; the livers, testes, epididymes, ovaries, and uteri were then weighed. The remaining F1 mice were mated with non-treated heterosexual mice. On GD 17, female mice were dissected to count the total number of fetuses and dead fetuses. All NP-treated mice exhibited decreased body and testis weight on PND 42. The pregnancy rate was 100% in all treated female groups, although it declined in untreated female mice mated with male mice from some treatment groups. The average dead fetus rate changed significantly according to the dosage combination. This study shows that BPA and NP can enhance, suppress or be neutral for the effect of the other in combined exposure to BPA and NP.

Decreased reproductive performance in xCT-knockout male mice

Abstract Sulphoxidation occurs in protamines that are enriched in cysteine and supplies chromatin for packaging. The extracellular fluid contains higher levels of oxidised cysteine (cystine), and some cells utilise system xc−, a cystine transporter in which xCT is the main protein component, to fulfil the need for cysteine. We hypothesised that system xc− might ensure the supply of cysteine needed for spermatogenesis. The reproductive ability of xCT−/− male mice at 6- to 18-weeks of age appeared to be lower than xCT+/+ male mice. The courtship behaviour of the xCT−/− male mice was undynamic, which appeared to be associated with the low reproductive ability of xCT−/− male mice. xCT was found to be expressed in mouse testes, notably in Sertoli cells, as well as in the epididymis and the levels were increased at the time of sexual maturation. Despite the normal histological appearance of testicular tissues, the cauda epididymis of xCT−/− mice contained round, greater numbers of immature spermatogenic cells than that of xCT+/+ mice. However, there were no significant differences in the numbers of sperm stored in the cauda epididymis or in the concentrations of cysteine or glutathione in the testes. The resulting sperm had normal fertilising ability. Thus, system xc− appears to function as a backup system for supplying cysteine to testes and play a pivotal role in supplying cysteine for normal sexual behaviour by a mechanism that is different from that for the supply of cysteine in spermatogenesis.

Effect of In Utero and Lactational Exposure to Isoflavone on the Development and Fertility of Mouse Offspring

Abstract Soybean products contain high concentrations of phytoestrogens. Isoflavone is the major phytoestrogen in soy and has been shown to display estrogen-like activities. It is well known that the delivery of isoflavone components across the placenta and in milk occurs in fetuses and infants the effect of following maternal exposure to isoflavone. We examined the effect of in utero and lactational exposure to isoflavone on the development and fertility of mouse offspring over two generations (F1 and F2). Pregnant mice were given daily subcutaneous injections of isoflavone (5, 50, 100 mg/kg/day) during the gestational and lactational stages until 20 days after delivery. At postnatal day (PND) 42, the F1 pups were sacrificed, and their body masses and organs (liver, kidney, testis, epididymis, ovary and uterus) were weighed. The other F1 pups were mated with non-treated mice or F1 heterosexual mice to estimate F1 fertility and development of F2 pups. The body weights of F1 pups increased at dose of 5 mg/kg/day. The epididymis weights of F1 males increased at doses of 5 and 100 mg/kg/day. No significant differences were found in the uterus and ovary weights of F1 females in all treatments. In the F2 males, the body weights of the 50 mg/kg/day group were higher than those of the control group. The epididymis weights of the 5 and 100 mg/kg/day groups were higher than those of the control group. In the F2 females, the body weights of the 5 and 100 mg/kg/day groups and the ovary weights of the 5 and 50 mg/kg/day groups were higher than those of the control group. There was no significant difference in pregnancy rate or numbers of live or dead fetuses in any treatment for the F1 and F2 offspring. These results suggest that gestational and lactational exposure to isoflavone presumably affect the body weights and some reproductive organ weights of F1 and F2 offspring, but are not seriously harmful to potential fertility.

Significance of Mammalian Cumulus-Oocyte Complex Matrix in Oocyte Meiotic Maturation: Review of the Synthetic Control and Possible Roles of Hyaluronan (HA) and HA-binding Protein

ABSTRACT In most mammals, the growth and development of the oocyte and its surrounding somatic cell compartment in the follicle occur in a highly coordinated and mutually dependent manner. Oocytes acquire developmental competence sequentially during follicle growth, finally gaining the ability to undergo complete meiotic and cytoplasmic maturation at the final stage of the preovulatory follicle. Fully-grown immature oocytes are tightly surrounded by compact layers of specialized granulosa cells called cumulus cells that form the cumulus-oocyte complex (COC). After a preovulatory surge of gonadotrophin, the cumulus cells organize a special muco-elastic extracellular matrix (ECM) that requires synthesis and deposition of a large amount of hyaluronan (HA) and HA-binding matrix glycoproteins. Many studies have reported that the formation of the COC matrix mass plays important roles in a variety of reproductive phenomenons: oocyte meiotic maturation with changes of junctional communication and cytoskeletal modification in COC, ovulation, fertilization and early embryo development. Recently, we identified the expressions of HA synthases and the HA receptor, CD44, in the porcine COC matrix. The interaction of HA and CD44 appears to be closely related to gap-junctional communications and meiotic resumption during oocyte maturation. This review describes the recent findings on the regulation and the presumptive mechanism of COC matrix molecules, and physiological features in COC expansion.