Study on the regulation of sulfated zona glycoproteins associated with the reduction of polyspermy during in vitro fertilization of porcine oocytes

• Project/Area Number: 16K07992
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Animal production science
• Research Institution: University of the Ryukyus
• Principal Investigator: TATEMOTO Hideki 琉球大学, 農学部, 教授 (70227114)
• Project Period (FY): 2016-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2019: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2018: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: ブタ体外受精 / 多精子受精 / 透明帯 / 糖タンパク質 / 硫酸化糖鎖 / arylsulfatase A / 脱硫酸化処理 / 脱硫酸化 / 先体反応 / 透明帯硬化 / sulfatase / dextran sulfate / 畜産学 / 体外受精 / ブタ卵胞卵 / 透明帯構成糖タンパク質 / 精子－透明帯間相互作用

Outline of Final Research Achievements

This study was conducted to elucidate the reduction of polyspermy during IVF without adverse effect on embryo developmental competence through the regulation of sulfated glycoprotein of porcine zona pellucida (ZP).
Treatment of oocytes with 5 mU/mL sulfatase for 10 min before the onset of insemination or 5 ng/ml ARSA blocking peptide during IVF effectively protected polyspermic fertilization under conditions that maintained the high sperm penetration rate and developmental potency. The number of sperm bound to ZP and the incidence of inducing acrosome reaction were significantly decreased by these treatments; however, it should be noted that the treatments had no effect against the time course of changes in ZP glycoproteins with egg activation.
These findings indicate the possibility of reduction of polyspermy during porcine IVF but not the decrease of development to blastocyst stage in keeping with a role for sulfated glycoprotein of ZP and ARSA of spermatozoa.

Academic Significance and Societal Importance of the Research Achievements

ブタ卵体外受精(IVF)時には他の家畜種に比べて多精子受精が高頻度に起こることが知られている。そこで，本研究では，透明帯構成糖タンパク質の硫酸化糖鎖に着目し，媒精前のブタ卵透明帯に脱硫酸化処理を行うことで，効果的に多精子受精を抑制する新しいIVF法の確立が可能か否かを検討した。
本研究結果から，精子arylsulfatase Aと透明帯の硫酸化糖鎖との結合を処理することで，透明帯への結合精子数と透明帯結合精子の先体反応誘起率を有意に減少させ，精子侵入や胚発生能に悪影響を及ぼさずに多精子受精を効果的に抑制できることが明らかになった。

Research Products

• [Journal Article] Role of endoplasmic reticulum stress on developmental competency and cryo-tolerance in bovine embryos (2020)
  • Author(s): Khatun H, Ihara Y, Takakura K, Egashira J, Wada Y, Konno K, Tatemoto H, Yamanaka K
  • Journal Title: Theriogenology Volume: 142 Pages: 131-137
  • DOI: 10.1016/j.theriogenology.2019.09.042
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Efficient <i>in vitro</i> embryo production using <i>in vivo</i>-matured oocytes from superstimulated Japanese Black cows (2019)
  • Author(s): Junki Egashira, Yusuke Ihara, Hafiza Khatun, Yasuhiko Wada, Toshihiro Konno, Hideki Tatemoto, Ken-ichi Yamanak
  • Journal Title: Journal of Reproduction and Development Volume: 65 Issue: 2 Pages: 183-190
  • DOI: 10.1262/jrd.2018-155
  • NAID: 130007630934
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Heat-shock-induced cathepsin B activity during IVF and culture compromises the developmental competence of bovine embryos (2018)
  • Author(s): Ken-ichi Yamanaka, Hafiza Khatun, Junki Egashira, AhmedZ Balboula, Hideki Tatemoto, Miki Sakatani, Naoki Takenouchi, Yasuhiko Wada, Masashi Takahashi
  • Journal Title: Theriogenology Volume: 114 Pages: 293-300
  • DOI: 10.1016/j.theriogenology.2018.04.005
  • NAID: 120006650532
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Sericin enhances the developmental competence of heat-stressed bovine embryos (2018)
  • Author(s): Khatun H, Egashira J, Sakatani M, Takenouchi N, Tatemoto H, Wada Y, Yamanaka K
  • Journal Title: Molecular Reproduction and Development Volume: 85 Issue: 8-9 Pages: 696-708
  • DOI: 10.1002/mrd.23038
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Effect of Heat Stress on the Quality of Cumulus-Oocyte Complex Obtained by Ovum Pick-Up in Japanese Black Cows (2018)
  • Author(s): 江頭潤将, 建本秀樹, 和田康彦, 山中賢一
  • Journal Title: Nihon Danchi Chikusan Gakkaihou Volume: 61 Issue: 2 Pages: 111-119
  • DOI: 10.11461/jwaras.61.111
  • NAID: 40021697288
  • ISSN: 2185-081X, 2185-1670
  • Peer Reviewed / Open Access
• [Journal Article] Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos (2018)
  • Author(s): Yamanaka K, Yamashita K, Khatun H, Wada Y, Tatemoto H, Sakatani M, Takenouchi N, Takahashi M, Watanabe S
  • Journal Title: Animal Science Journal Volume: 89 Issue: 10 Pages: 1406-1414
  • DOI: 10.1111/asj.13086
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Improvement of the post-thaw qualities of Okinawan native Agu pig spermatozoa treated with α-tocopherol and ascorbic acid 2-O-α-glucoside during semen transportation and sperm freezing procedure (2017)
  • Author(s): 建本 秀樹、舩曳 美和、佐渡山 祐希、上原 みなみ、金野 俊洋、山中 賢一
  • Journal Title: Nihon Danchi Chikusan Gakkaihou Volume: 60 Issue: 2 Pages: 111-120
  • DOI: 10.11461/jwaras.60.111
  • NAID: 130006301348
  • ISSN: 2185-081X, 2185-1670
  • Peer Reviewed / Open Access
• [Presentation] 沖縄在来豚アグー精子凍結時におけるL-カルニチン処理が融解後の精子性状に及ぼす影響 (2019)
  • Author(s): 獺越真土，本岡愛美，平井萌，上原みなみ，建本秀樹
  • Organizer: 日本暖地畜産学会
• [Presentation] ブタ卵体外受精時における精子arylsulfatase A阻害による多精子受精抑制効果 (2018)
  • Author(s): 上原みなみ，佐渡山佑希，建本秀樹
  • Organizer: 日本畜産学会第124回大会
• [Presentation] Mural trophectoderm specific activation of adhesion competence in peri-implantation murine blastocyst (2017)
  • Author(s): Nishi I, Kawase F, Kurane T, Ohta K, Tatemoto H, Konno T
  • Organizer: 4th World Congress of Reproductive Biology
  • Int'l Joint Research
• [Presentation] ブタ卵体外成熟時の透明帯構成糖タンパク質における硫酸化阻害と多精子受精抑制効果との関係 (2017)
  • Author(s): 上原みなみ，佐渡山佑希，建本秀樹
  • Organizer: 第10回日本暖地畜産学会佐賀大会
• [Presentation] Improvement in the post-thaw qualities of Okinawan native Agu pig spermatozoa treated with skim milk and Trolox prior to cryopreservation (2016)
  • Author(s): Tatemoto H, Mitsube R, Tokeshi I, Uehara M, Sadoyama Y, Funabiki M, Konno T
  • Organizer: The 17th AAAP Animal Science Congress
  • Place of Presentation: Kyushu Sangyo University, Fukuoka
  • Year and Date: 2016-08-22
  • Int'l Joint Research