M. Takahashi

National Institute of Animal Health, Tsukuba, Ibaraki, Japan

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Publications (15)14.66 Total impact

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    ABSTRACT: Fibroblast growth factor 4 (FGF4) is a crucial growth factor for the development of mammalian embryos. We previously produced hexahistidine-tagged, bovine and porcine FGF4 (Pro32 to Leu206) proteins without a secretory signal peptide at the amino-terminus in Escherichia coli. Here, we found that these were unstable; site-specific cleavage between Ser54 and Leu55 in both FGF4 derivatives was identified. In order to generate stable FGF4 derivatives and to investigate their biological activities, aminoterminally truncated and hexahistidine-tagged bovine and porcine FGF4 (Leu55 to Leu206) proteins, termed HisbFGF4L and HispFGF4L, respectively, were produced in E. coli. These FGF4 derivatives were sufficiently stable and exerted mitogenic activities in fibroblasts. Treatment with the FGF4 derivatives promoted the phosphorylation of ERK1/2, which are crucial kinases in the FGF signaling pathway. In the presence of PD173074, an FGF receptor inhibitor, the phosphorylation of ERK1/2 was inhibited and resulted in abolition of the growth-promoting activity of FGF4 derivatives. Taken together, we demonstrate that HisbFGF4L and HispFGF4L are capable of promoting the proliferation of bovine- and porcine-derived cells, respectively, via an authentic FGF signaling pathway. These FGF4 derivatives may be applicable for dissecting the roles of FGF4 during embryogenesis in cattle and pigs.This article is protected by copyright. All rights reserved
    Biotechnology and Applied Biochemistry 01/2014; in press. · 1.35 Impact Factor
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    ABSTRACT: The goal was to understand the role of heat shock at the zygote stage in causing infertility. Culture at 40°C reduced the percentage of inseminated oocytes that became a morula or blastocyst by d 6 or that were a blastocyst at d 8. An additional experiment was done to test whether effects of heat shock occur early in development or at the time of morula formation. Exposure to 40°C for 24 h decreased development to the blastocyst stage if exposure was at the zygote stage [8 to 32 h postinsemination (hpi)] but not if exposure occurred at the morula stage (116 to 140 hpi). To test effect of oxygen concentration, inseminated oocytes were cultured at 40°C for 12 or 24 h in either air (20.95% O₂; high oxygen) or a 5% (vol/vol) O₂ environment (low oxygen) that approximates the partial oxygen pressure of the reproductive tract. Blastocyst development was reduced by 40°C for 12 or 24 h under both atmospheres and was higher for embryos cultured in low oxygen than for embryos cultured in high oxygen. Examination of cell numbers at 72 hpi indicated that heat shock reduced developmental potential of embryos by reducing competence to complete cleavage divisions after first cleavage. Changes in expression of genes involved in heat shock and oxidative stress were measured to determine whether zygotes are more susceptible to heat shock because of reduced capacity for transcription. Heat shock was performed for 24 h at the 1-cell stage (expression examined in 2-cell embryos) or at d 5 (examined in morulae). Heat shock increased amounts of steady-state mRNA for HSPA1A but not for HSP90AA, SOD1, or CAT. We observed a tendency for a stage × temperature interaction for HSPA1A because the difference in expression between 38.5 and 40°C was greater for morulae than for 2-cell embryos. The amount of HSPA1A mRNA was less for morulae that were heat shocked than for 2-cell embryos cultured at 38.5°C. Heat shock at a temperature and oxygen tension similar to those seen in vivo can disrupt developmental competence of bovine zygotes. Increased susceptibility of the early embryo compared with the morula to heat shock was not due to reduced HSPA1A mRNA because amounts were higher for 2-cell embryos than for morulae.
    Journal of Dairy Science 06/2012; 95(6):3080-91. · 2.57 Impact Factor
  • Animal Science Journal 01/2012; in press. · 1.04 Impact Factor
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    ABSTRACT: A serious decline in the reproductive performance of dairy cows occurs in southern Japan in the summer period, when the total number of hot days ≥35°C numbers more than 20 days annually. Previous reports have mentioned the effectiveness of embryo transfer (ET) at 7 days after AI (AI/ET) under heat-stressed conditions. In the present study, we investigated the effect of AI/ET on conception rate (CR) under heat-stressed conditions in the summer period. Artificial insemination was performed at 13 commercial dairies in this study from August through September in 2007 and 2008. Seven days after AI, a single embryo was transferred into the uterine horn contralateral to the ovary with a corpus luteum (AI/ET, n=82). Artificial insemination at oestrus without further treatment was assigned as the control group (AI, n=367). In 2007, frozen-thawed embryos of Japanese Black cattle were transferred, and the same cattle were used for ET of fresh embryos in 2008. The temperature-humidity index [0.8×temperature+0.01×relative humidity (temperature -14.4)+46.4], rectal temperature, and diurnal highest or lowest and average ambient temperatures were measured at the time of AI and ET. Cows were diagnosed for pregnancy at 42 days after AI by palpation per rectum and were reexamined by transrectal ultrasonography at 60 days after AI. The CR was calculated as the number of cows diagnosed as pregnant 60 days after AI divided by the number of cows inseminated. Fetal loss was calculated as the number of cows that did not deliver calves after term divided by the number of cows diagnosed as pregnant. The CR, number of AI, fetal loss, and type of newborn (Holsteins, AI origin; Japanese Black, ET origin) were confirmed retrospectively. For statistical analysis, Fisher's exact test and Student's t-test were used for comparison of the CR, fetal loss, and body temperature by using a statistical software program for PC (Excel Statistics 2006). The CR for AI/ET was 30.4% and for AI was 13.8% in 2007 (P<0.01), and the CR for AI/ET was 30.8% and for AI was 21.5% in 2008 (P=0.294). The average diurnal temperature was 31.1°C in 2007 and 30.1°C in 2008, and the temperature-humidity index was 81.8 and 80.8, respectively. On Day 8, the pregnant cows had a lower rectal temperature than the open cows in 2007, but not in 2008 (38.9 v. 39.4°C in 2007; P<0.05; and 39.1 v. 38.9°C in 2008; P>0.05). The fetal loss was 38.1% in AI/ET v. 7.4% in AI in 2007 (P<0.05) and 12.5% v. 0% in 2008 (P<0.05), respectively. The AI/ET procedure could improve CR in dairy cows during the summer period in southern Japan. However, other problems may accompany AI/ET, such as higher fetal losses.
    Reproduction Fertility and Development 01/2011; 23(1):179. · 2.58 Impact Factor
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    ABSTRACT: Recently, the activity of cathepsins B was found to be correlated inversely with the developmental competence of bovine oocytes. In this study, we investigated (1) the role of intracellular cathepsin B expression and developmental competence as well as the quality of bovine preimplantation embryos, and (2) the effect of cathepsin B inhibitor (E-64) during in vitro culture (IVC) on the development and quality of bovine embryos. After in vitro fertilization (IVF) followed by IVC for 7 days, good and poor quality embryos classified by morphology and developmental rate on days 2, 4, and 7 were assessed for cathepsin B expression and activity. To investigate the effect of cathepsin B inhibition on embryonic development, putative zygotes were cultured with or without E-64, followed by evaluation of cleavage and blastocyst rates on days 2 and 7, respectively. Embryonic quality was evaluated by both TUNEL staining and total cell number in day-7 blastocysts. In each developmental stage, cathepsin B expression and activity were significantly higher in poor quality embryos than good quality ones. Moreover, addition of E-64 during IVC significantly increased both the blastocyst rate and the total cell number. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in day-7 blastocysts. These results indicate that cathepsin B activity can be useful as a marker for inferior quality embryos. Moreover, inhibition of cathepsin B greatly improves the developmental competence of preimplantation embryos and increases the number of good quality embryos.
    Molecular Reproduction and Development 12/2010; 77(12):1031-9. · 2.81 Impact Factor
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    ABSTRACT: Recently, the quantity of cathepsin transcripts in cumulus cells was found to be associated with low-developmental competence of bovine oocytes. In the present study, we investigated (1) the relation between cathepsin B activity and the quality of in vitro-matured cumulus-oocyte complexes (IVM COCs) and denuded oocytes and (2) the effect of a cathepsin B inhibitor (E-64) on embryo development and quality. The activity of cathepsin B was evaluated in IVM COCs and denuded oocytes. After maturation of COCs with or without E-64, followed by in vitro fertilization, zygotes were cultured for 8 days. Cleavage and blastocyst rates were evaluated on days 2 and 8, respectively. Quality of embryos was evaluated by differential staining of day 8 blastocysts. TUNEL staining was conducted on IVM COCs and blastocysts. Cathepsin B activity was clearly detected in the low-quality oocytes, and in the cumulus cells of both high- and low-quality oocytes. This latter activity was diminished by addition of E-64. The presence of E-64 during IVM also significantly increased both the blastocyst rate and the total cell number, and improved blastocyst quality associated with a significant increase of trophoectoderm cells. TUNEL staining revealed that inhibition of cathepsin B significantly decreased the number of apoptotic nuclei in both the cumulus cell layer of matured oocytes and blastocysts. These results indicate that cathepsin B activity can be a useful marker of oocyte quality. Furthermore, inhibition of cathepsin B greatly improves the developmental competence of bovine oocytes and increases the number of high-quality embryos.
    Molecular Reproduction and Development 03/2010; 77(5):439-48. · 2.81 Impact Factor
  • K. Yamanaka, M. Sakatani, M. Takahashi
    Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2009; 21(1).
  • M. Sakatani, K. Yamanaka, M. Takahashi
    Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2009; 21(1).
  • C Suzuki, K Yoshioka, M Sakatani, M Takahashi
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    ABSTRACT: We previously developed an in vitro-production system for porcine embryos and reported that the addition of glutamine (Gln) and hypotaurine (HT) during in vitro culture improved embryo development. This study examined the effects of Gln and HT on in vitro development, intracellular oxidative status and DNA damage of porcine preimplantation embryos. Porcine zygotes produced by in vitro maturation (IVM) and in vitro fertilization (IVF) were cultured until day 2 (day 0 = day of IVF) in porcine zygote medium (PZM) including 2 mM Gln and 5 mM HT, namely PZM-5. On day 2, the cleaved embryos were selected and cultured for 24 h in PZM-5 to which one of the following substances was added: (1) none (control); (2) Gln; (3) HT; or (4) Gln + HT. After 24 h of culture in each medium, the embryos were then returned to PZM-5 and cultured until day 5. Day-5 blastocyst yield was significantly higher in the Gln and Gln + HT groups (p < 0.05) than in the control and HT groups. In addition, Gln + HT significantly increased the total number of cells in blastocysts (p < 0.05) compared with the control. Although the number of cells and the intracellular GSH levels in day-3 cleaved embryos did not differ among treatments, addition of Gln, HT or Gln + HT significantly (p < 0.05) reduced the intracellular H2O2 content and the extent of DNA damage compared with the control. These results indicate that the presence of Gln and HT in PZM-5 from day 2 to day 3 promotes the development of porcine embryos by improvement of intracellular oxidative status.
    Zygote 12/2007; 15(4):317-24. · 1.50 Impact Factor
  • Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2007; 19(1).
  • Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2007; 19(1).
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    ABSTRACT: We investigated the antioxidative effect of brown algae phlorotannins on reactive oxygen species (ROS) generation and embryo development of parthenogenetically activated porcine embryos under oxidative and heat-stressed conditions. Cumulus–oocyte complexes (COCs) were aspirated from follicles on the surface of porcine ovaries collected from an abbattoir. COCs were matured in NCSU-23 containing 10% (v/v) porcine follicular fluid and hCG during the first 22 h, followed by an extra 22 h of culture in hormone-free NCSU-23. After 44 h of maturation, oocytes were denuded of cumulus cells and used for parthenogenetic activation. Oocytes were activated by single 100-µs pulse of 1.5 kV cm-1 DC in 1-mm electrodes. Activated oocytes were cultured for 5 h in NCSU-23 containing BSA, EGF, and 5 µg mL-1 cytochalasin B. Embryos were then cultured for 7 days in PZM-5 medium that was a slightly modified version of the PZM-4 medium reported by Yoshioka et al. (2002 Biol. Reprod. 60, 112–119). In Experiment 1, after parthenogenetic activation, embryos were cultured for 7 days at 38.5°C under 5% O2, 5% CO 2, and 90% N2 (defined as 5% O2) as a control. Embryos were also cultured under 5% CO2 in air (defined as 20% O2) with or without 100 ng mL-1 brown algae phlorotannins extracted from Ecklonia kurome. The number of embryos developed to the blastocyst stage was observed on Day 6. The total cell number of Day 7 blastocysts was counted by DAPI staining of nuclei. On Day 2, intracellular ROS levels of individual embryos were measured with fluorescent dyes (222,722-dichlorodihydrofluorescein diacetate). In Experiment 2, on Day 1 or 2, embryos cultured in 5% O2 concentration at 38.5°C were exposed to 41.5°C for 6 h with or without 100 ng mL-1 phlorotannins and cultured at 38.5°C until Day 7. After 6 h of heat-shock on Day 1 or Day 2, intracellular ROS levels were measured as described in Experiment 1. Statistical analysis was carried out by ANOVA. In Experiment 1, the rate of blastocyst formation and the total cell number were significantly decreased (P < 0.05) when embryos were cultured under 20% O2 compared to 5% O2. In contrast, addition of phlorotannins significantly increased the rate of blastocyst formation under high O2 concentration. ROS levels were also significantly increased by higher O2 concentration. In contrast, addition of phlorotannins significantly reduced the ROS levels. In Experiment 2, heat-shock to embryos on Days 1 and 2 significantly (P < 0.05) decreased the rate of blastocyst formation compared to the control. In contrast, addition of phlorotannins significantly (P < 0.05) increased embryo development and decreased the intracellular ROS levels of heat-stressed embryos. These results indicate that oxidative and heat stress conditions decrease embryo development and increase the level of intracellular ROS. However, addition of phlorotannins promotes embryo development by decreasing the oxidative stress.
    Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2007; 19(1).
  • S. Kobayashi, M. Sakatani, M. Takahashi
    Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2006; 18(2).
  • Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2006; 18(2).
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    ABSTRACT: Development of cleavage-stage pre-implantation embryos is disrupted by exposure to heat shock. Heat shock also increases intracellular reactive oxygen species (ROS) in pre-implantation embryos. Therefore, reduction of intracellular ROS levels might improve the development of heat-shocked embryos. Recently the antioxidative activities of polyphenols have been widely reported to reduce the oxidative stress. In this study, we investigated the effect of purple sweet potato anthocyanin, a kind of polyphenol that is a strong ROS scavenger, on development and intracellular redox status of bovine pre-implantation embryos exposed to heat shock. Experiment 1: In vitro-produced 8-16-cell-stage embryos on Day 2 after fertilization were exposed to 41.5°C for 6 h in CR1aa containing 0, 0.1, 1, and 10 ¼g/mL anthocyanin at 5% CO2, 5% O2, and 90% N2. After heat shock, embryos were cultured at 38.5°C at 5% CO2, 5% O2 until Day 8. On Day 8, the proportion of embryos developing to the blastocyst stage was evaluated. Blastocyst total cell number and the ratio between inner cell mass and tropheoderm were evaluated by differential staining. The experiment was replicated five times with more than 70 embryos used in each treatment. Experiment 2: Heat shock treatment of in vitro-produced 8-16-cell-stage embryos was carried out as described in experiment 1. After heat shock, intracellular ROS and glutathione (GSH) levels were measured in individual 8-16 cell stage embryos with fluorescent probes (22,72-dichlorodihydrofluorescein diacetate for ROS and CellTracker" Blue (Invitrogen Japan K. K., Tokyo, Japan) for GSH). The fluorescence emissions of each treatment were normalized to those of 8-16 cell stage embryos cultured at 38.5°C without anthocyanin to obtain the relative fluorescence emission. This experiment was replicated four times. Embryos treated with heat stress without anthocyanin (0 ¼g/mL) showed low development (14.6 ± 3.6%) and blastocyst total cell number (88.2 ± 9.4). However, embryos treated with 0.1 ¼g/mL anthocyanin improved development (31.7 ± 4.5%, P < 0.05) and increased the total cell number (96.5 ± 11.3). The higher concentrations of anthocyanin (1 and 10 ¼g/mL) did not affect development and cell number. The intracellular ROS levels in heat-shocked embryos were significantly reduced by all concentrations of anthocyanin (P < 0.05). In addition, anthocyanin increased GSH levels at all doses tested (P < 0.05). These results indicate that an appropriate concentration of anthocyanin improves development by regulating intracellular redox balance in bovine embryos exposed to heat shock.
    Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2006; 18(2).