Akira Okano

Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay

The correlation of oxidative stress on development and DNA damage in bovine embryos was investigated by the comet assay (single-cell microgel electrophoresis), an effective technique for detecting single-strand DNA breakage. After in vitro maturation and fertilization, one-cell stage embryos without cumulus cells were cultured for 8 days in SOF medium containing amino acids plus 5% FCS under low (5%) and atmospheric (20% ) oxygen concentration. After 8 days of culture, the extent of blastocyst formation was significantly decreased (P<0.001) when embryos were cultured under 20% oxygen concentration (5.8 +/- 2.4%) when compared to embryos cultured under 5% oxygen concentration (35.1 +/- 6.7%). At the day 3 of development, DNA damage of individual embryos cultured under 5% or 20% oxygen concentration was measured by the comet assay, which entails microgel electrophoresis that can readily detect damaged DNA. After measuring the DNA damage in individual embryos by the comet assay, the length (149.9 +/- 15.3 microm) of the migrating DNA fragment that is indicative of damaged DNA was significantly increased (P<0.001) in the embryos cultured under 20% oxygen concentration when compared to embryos cultured in 5% oxygen concentration (42.3 +/- 7 microm). The length of damaged DNA in more than 50% of embryos was less than 50 microm. when embryos were cultured under 5% oxygen concentration. In contrast, the distribution of damaged DNA shifted to the more damaged extent when embryos were cultured under 20% oxygen concentration. These results demonstrate that the retardation in bovine embryo development than in likely due oxidative stress as a consequence of the higher atmospheric oxygen concentration is positively correlated with an increase in the extent of DNA damage. Moreover, these results demonstrate that the comet assay is a useful method to evaluate embryo culture conditions.

Promoting Effect of β-Mercaptoethanol on In Vitro Development under Oxidative Stress and Cystine Uptake of Bovine Embryos

Abstract The effects of β-mercaptoethanol (β-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O2 supplemented with β-ME. Addition of β-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O2, to almost the same rates when they were cultured in 5% O2. To investigate whether the growth-promoting effect of β-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without β-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without β-ME. In contrast, addition of β-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by β-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by β-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of β-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that β-ME has a protective effect in embryo development against oxidative stress and that the effect of β-ME is associated with the promotion of cystine uptake of low availability in embryos.

Assessment of DNA damage in individual hamster embryos by comet assay

DNA damage induced by either light exposure or oxidative stress likely contributes to the compromised development in vitro of cultured preimplantation embryos. Using the comet assay, which entails microgel electrophoresis that can readily detect single‐strand breaks in DNA, a significant increase in DNA damage was detected in individual one‐cell hamster embryos that were treated with either ultraviolet light or hydrogen peroxide. In addition, an increase in DNA damage also was observed following exposure of one‐cell embryos to visible light. When the embryos were placed in drops of culture medium that were covered with mineral oil and the dishes then placed in a portable incubator containing 5% CO2 in air at 37°C, visible and UV light irradiation for 30 min still induced extensive DNA damage when compared to control embryos that were kept in the dark. In contrast, infrared irradiation did not induce an increase in DNA damage. DNA damage also was measured in individual one‐ and two‐cell stage embryos developed in vivo or in vitro. The extent of DNA damage in the cultured embryos was significantly greater than in embryos that developed in vivo. These results highlight the usefulness of the comet assay to assess DNA damage in individual preimplantation embryos and how the assay can be used to monitor culture conditions in vitro. Mol. Reprod. Dev. 54:1–7, 1999.

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH92]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg7]-vasopressin, [Thr4,Gly7]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile3]-pressinoic acid (tocinoic acid)). The dissociation constant (Kd) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), Kd (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, Kd values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (Bmax) on D0 (910.0 ± 25.1 fmol mg−1 protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg−1 protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg−1 protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, Bmax values were constant and similar to that on D5 of the early luteal stage. Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.

Tumor necrosis factor-α and its receptor in the corpus luteum of pregnant cows.

The objective of this study was to investigate the presence of tumor necrosis factor (TNF)‐α mRNA and TNF‐α receptors in the bovine corpus luteum (CL) during the gestation period. TNF‐α mRNA and TNF‐α receptors were determined on bovine CL from pregnant cows at three stages: trimester I (fetal crown‐rump length; 6–20 cm), trimester II (25–45 cm) and trimester III (50–80 cm). TNF‐α mRNA was detected by an RT‐PCR analysis in the CL of all stages of gestation. A Scatchard analysis revealed the presence of a high‐affinity binding site (Kd; 5.1–6.9 nM) in the CL membranes collected at each stage of gestation. Furthermore, the concentrations of TNF‐α receptors in the CL of trimesters I (24.0 ± 1.95 pmol/mg protein) and III (21.6 ± 2.39 pmol/mg protein) of gestation were significantly higher than the concentration in trimester II (14.9 ± 2.07 pmol/mg protein) (P < 0.05). These results indicate that TNF‐α is locally produced and that TNF‐α receptors are present in bovine CL during the gestation period, and suggest that TNF‐α plays one or more roles as a paracrine factor in regulating bovine CL function during the entire gestation period. Mol. Reprod. Dev. 55:406–411, 2000.

Effect of b-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws

Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P < 0.05) when the embryos were transported in beta-ME supplemented medium (80 to 100%) than when transported without beta-ME (54 %). Blastocysts ranked as A or B after transportation in medium with or without 150 microM beta-ME were nonsurgically transferred to synchronous recipients; 60 d after embryo transfer, 21/36 and 19/35 cows, respectively, were diagnosed as pregnant by palpation per rectum. These results indicate that beta-ME maintains the quality of bovine blastocysts in plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

Biological Activity of Recombinant Bovine Interferon τ Produced by a Silkworm-Baculovirus Gene Expression System

ABSTRACT Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ.

Growth pattern of the genitalia from neonatal to pubertal heifer in the Japanese Black heifers

黒毛和種雌牛の0日齢から性成熟まで,及びそれ以後の生殖器各部位の重量を測定することにより,性成熟と生殖器発育の関係を検討して以下の結果をえた。1.供試牛のうち体重257kg以上の個体はすべて性成熟に達しており,子宮重量は,78.0~139.0g,頸管重量は,22.0~85.0gの範囲にあった。子宮は,出生時の約30倍となっていた。また,性成熟以後の子宮角の最大外径は,1.8~2.9cmの範囲にあった。性成熟に至るには少なくとも300日齢以上を要した。2. 性成熟時点で,左右卵巣重量の合計は4.9~10.3gの範囲にあり,出生時の2~5倍であった。3.性成熟時点で,左右卵管重量の合計は約2g,卵管の長さは約20cmとなって,出生時のおよそ2倍であった。4. 性成熟後,体重は増加し続けるのに対して,生殖器重量の増加は,子宮及び頸管でわずかに認められたが,卵管重量と長さには増加を認めなかった。

Development of mouse two-cell embryos to hatched blastocysts in a medium supplemented with bovine amniotic fluid

マウス胚の体外培養のための培養液へ,ウシ,ブタおよびニワトリ羊膜液添加の効果を明らかにしようとした.羊膜液および羊膜液を添加した培養液中で,マウス2細胞期胚を培養し,96時間後における透明帯脱出胚盤胞への発育率を検討した.3動物種からの羊膜液とも無処理のままでは,マウス胚の体外培養液として用いた場合は全く効果がなく,むしろ胚の発育を阻害する傾向を示した.しかしウシ血清アルブミンを含まないM16培養液にウシ羊膜液を10%添加して,マウス2細胞期胚を体外培養すると,ウシ血清アルブミンを含むM16培養液と同程度に,透明帯脱出胚盤胞へ発育させえた.マウス2細胞期胚を透明帯脱出胚盤胞へ発育させるのに有効なウシ羊膜中の因子は,100°Cで5分間の加熱処理で失活し,デキストランーチャコールで吸収されなかった.このことより,この因子は脂質やステロイド様の物質ではなく,分子量104以下のペプチドあるいは遊離アミノ酸と推定された.

Histological studies on uterine growth from neonatal to pubertal stage in Japanese Black heif-ers

3日齢から性成熟までの黒毛和種雌牛について,子宮の成長を組織学的に研究して以下の結果をえた。1. 子宮腺は,3日齢の子宮では発現しておらず,29日齢の子宮において機能層及び基底層の一部に発現していた。その後,日齢の進行とともに子宮腺は発達し,内膜機能層及び基底層において子宮腺の形質や分布状態が一定となるのは,性成熟時であった。2. 性成熟期以前の子宮では,内膜が筋層に比べて厚い。しかし日齢が進むのにつれて両層とも厚くなり,性成熟時には両層ともほぼ3mmを少し越えて等しい厚さを示した。3. 子宮内膜の表面上皮は,各日齢を通じて小丘領域より小丘間領域で高い傾向が認められ,性成熟期以後のもので,ほとんど円柱上皮であった。4. 性成熟期以後,子宮は妊娠が成立しない限り,組織学的にはほとんど変化しないでとどまることが示唆された。