Kiyoshi Totsukawa

Identification, Cloning, and Initial Characterization of a Novel Mouse Testicular Germ Cell-Specific Antigen

Abstract A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.

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.

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.

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.