1993 Fiscal Year Annual Research Report

ほ乳動物の精子の受精能の獲得の分子機構

Research Project

Project/Area Number	05044121
Research Institution	University of Tsukuba
Principal Investigator	岡村直道筑波大学, 基礎医学系, 講師 (30134224)
Co-Investigator(Kenkyū-buntansha)	HARRISON RAP AFRC, 動物生理学・遺伝学研究所 (英国), 主任科学職員 DACHEUX JL CNRS, 国立農学研究所 (フランス), 主任研究官永井卓農水省, 東北農試・家畜繁殖研究室, 室長
Keywords	精子成熟 / 運動能 / 受精能 / 精巣上体 / カルシウム / Capacitation / フォルスコリン / 重炭酸イオン
Research Abstract	1.精巣上体における精子成熟の分子機構(岡村・Dacheux) ほ乳動物の精子は、精巣で形成されてから受精に至るまで、常に、体細胞の影響下にある。特に、精巣上体における精子成熟は、上皮細胞から分泌される種々のタンパク質により精子表層が修飾され、初めて精子が運動能と受精能を獲得する非常に重要なステップである。本年は、それらの分泌タンパク質の分離と同定を試みた。精巣上体尾部分泌液から12種類のタンパク質を分離し、それらのN-末端アミノ酸配列を決定した。Northern法によって、それらのタンパク質をコードしているmRNAの発現を調べたところ、135,65,40,23,19kDaのタンパク質が、それぞれ精巣上体の異なる部位で合成されていることが判明し、精巣上体の部位による機能分化を直接証明する事ができた。これらのうち、135,65,23kDaタンパク質については、cDNAをクローニングし、全塩基配列を決定した。現在、抗体を用いて、これらのタンパク質の精子成熟における役割を検討している。 2.カルシウムイオンの精子受精能に対する作用の解析(永井・Harrison・岡村) 精子のCapacitationや先体反応、受精にカルシウムイオンが重要な調節因子として働いている事はよく知られている。我々は、既に、精子表層に電位非依存性でベラパミルによって阻害されるカルシウムイオン輸送系の存在を報告している。本年、更に、フォルスコリン、カフェイン、及び重炭酸イオンが、精子のcapacitationの際に、精子内へのカルシウムイオンの取り込みを高める事を見いだした。又、これらは、体外受精系における精子の受精活性も著しく高める作用を持つ事が判明した。その厳密な作用機構は、更なる検討が必要であるが、カルシウムイオンの取り込みを促進する事によって受精能を高めていると思われる。畜産や不妊症治療における体外受精へのこれらの試薬の応用を検討中である。

Research Products
(22 results)

All Other

All Publications (22 results)

[Publications] Okamura,N.(Nagai,T): "Forskolin stimulates porcine sperm capacitation by increasing calcium uptake." FEBS LETTERS. 316. 283-286 (1993)
[Publications] Okamura,N.: "精巣上体におけるほ乳類精子の成熟" Cell Science. 9. 19-29 (1993)
[Publications] Okamura,N.: "精巣上体における精子成熟" Perinatal Care. 12. 62-67 (1993)
[Publications] Takahashi,M.(Nagai,T.,Okamura,N.): "Effect of thiol compounds on in vitro fertilization of pig oocytes matured in vitro." Biol.Reprod.49. 228-232 (1993)
[Publications] Nagai,T(Okamura,N): "Effects of caffeine on invitro fertilization of pig follicular oocytes." J.Reprod.Develop.39. 347-352 (1993)
[Publications] Nagai,T.: "Current status and perspectives in IVM-IVF of porcine oocytes." Theriogenology. 41. 73-78 (1994)
[Publications] Nagai,T.: "Effect of follicle cells and steroidgenesis on maturation and fertilization in vitro of pig oocytes." J.Exp.Zool.266. 146-151 (1993)
[Publications] Kikuchi,K.(Nagai,T.): "Effect of follicle cells on in vitro fertilization of pig follicular oocytes." Theriogenology. 39. 593-599 (1993)
[Publications] Yoshida,M(Nagai,T.): "Glutathione concentration during maturation and after fertilization in pig oocytes - Relevance to the ability of oocytes to perform male pronucleus." Biol.Reprod.49. 89-94 (1993)
[Publications] Yoshida,M(Nagai,T.): "Birth of piglets derived from in vitro fertilization of pig oocytes matured in vitro." Theriogenology. 39. 1303-1311 (1993)
[Publications] Gatti,JL(Dacheux,JL): "External ionic conditions,internal pH and motility of ram and boar spermatozoa." J.Reprod.Fert.98. 439-449 (1993)
[Publications] Harrison,RAP: "Flow cytometric studies of bicarbonate-mediated Ca^<2+> influx in boar sperm populations." Mol.Reprod.Develop.35. 197-208 (1993)
[Publications] Okamura,N.(Dacheux,JL): "Cloning and characterization of complementary DNA encoding 135kDa protein,a possible marker for the functional differentiation of pocine epididymis." J.Biol.Chem.(in press). (1994)
[Publications] Okamura,N.(Dacheux,JL): "Direct evidences for the functional differentiation among the regions of porcine epididymis." FEBS LETTERS. (in press). (1994)
[Publications] Nagai,T.(Okamura,N.): "Forskolin promotes sperm penetration of pig oocytes in vitro without increasing cyclic AMP levels in sperm." Anim.Reprod.Sci.(in press). (1994)
[Publications] Suzuki,K.(Nagai,T.,Harrison,RAP): "Bicarbonate/CO^2 is required for pig fertilization in vitro." Reprod.Fertil.Develop.(in press). (1994)
[Publications] Nagai,T.: "Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture." Mol.Reprod.Develop.(in press). (1994)
[Publications] Hirao,Y.(Nagai,T.): "In vitro growth and maturation of pig oocytes." J.Reprod.Fert.(in press). (1994)
[Publications] Yoshizawa,M.(Nagai,T.): "Native zona pellucida structure is required for completion of sperm acrosome reaction in porcine fertilization." Theriogenology. (in press). (1994)
[Publications] Takada,M.(Nagai,T.): "pH-Sensitive dissociation and association of -N-acetyl-hexoaminidase from boar sperm acrosome." Biol.Reprod.(in press). (1994)
[Publications] Okamura,N.(Dacheux,JL): "Cloning and characterization of complementary DNA encoding 23kDa protein secreted from caput epididymis of porcine." J.Biol.Chem.(投稿中).
[Publications] OKAMURA,N.(Nagai,T.): "“Assisted reproductive technology/andrology" Glutathione in mammalian embryos:effects of thiol compounds." Archives of Andrology ed.Hafez,E.S.E. (in press), (1994)

1993 Fiscal Year Annual Research Report

ほ乳動物の精子の受精能の獲得の分子機構

Principal Investigator

岡村 直道 筑波大学, 基礎医学系, 講師 (30134224)

Research Products

[Publications] Okamura,N.(Nagai,T): "Forskolin stimulates porcine sperm capacitation by increasing calcium uptake." FEBS LETTERS. 316. 283-286 (1993)

[Publications] Okamura,N.: "精巣上体におけるほ乳類精子の成熟" Cell Science. 9. 19-29 (1993)

[Publications] Okamura,N.: "精巣上体における精子成熟" Perinatal Care. 12. 62-67 (1993)

[Publications] Takahashi,M.(Nagai,T.,Okamura,N.): "Effect of thiol compounds on in vitro fertilization of pig oocytes matured in vitro." Biol.Reprod.49. 228-232 (1993)

[Publications] Nagai,T(Okamura,N): "Effects of caffeine on invitro fertilization of pig follicular oocytes." J.Reprod.Develop.39. 347-352 (1993)

[Publications] Nagai,T.: "Current status and perspectives in IVM-IVF of porcine oocytes." Theriogenology. 41. 73-78 (1994)

[Publications] Nagai,T.: "Effect of follicle cells and steroidgenesis on maturation and fertilization in vitro of pig oocytes." J.Exp.Zool.266. 146-151 (1993)

[Publications] Kikuchi,K.(Nagai,T.): "Effect of follicle cells on in vitro fertilization of pig follicular oocytes." Theriogenology. 39. 593-599 (1993)

[Publications] Yoshida,M(Nagai,T.): "Glutathione concentration during maturation and after fertilization in pig oocytes - Relevance to the ability of oocytes to perform male pronucleus." Biol.Reprod.49. 89-94 (1993)

[Publications] Yoshida,M(Nagai,T.): "Birth of piglets derived from in vitro fertilization of pig oocytes matured in vitro." Theriogenology. 39. 1303-1311 (1993)

[Publications] Gatti,JL(Dacheux,JL): "External ionic conditions,internal pH and motility of ram and boar spermatozoa." J.Reprod.Fert.98. 439-449 (1993)

[Publications] Harrison,RAP: "Flow cytometric studies of bicarbonate-mediated Ca^<2+> influx in boar sperm populations." Mol.Reprod.Develop.35. 197-208 (1993)

[Publications] Okamura,N.(Dacheux,JL): "Cloning and characterization of complementary DNA encoding 135kDa protein,a possible marker for the functional differentiation of pocine epididymis." J.Biol.Chem.(in press). (1994)

[Publications] Okamura,N.(Dacheux,JL): "Direct evidences for the functional differentiation among the regions of porcine epididymis." FEBS LETTERS. (in press). (1994)

[Publications] Nagai,T.(Okamura,N.): "Forskolin promotes sperm penetration of pig oocytes in vitro without increasing cyclic AMP levels in sperm." Anim.Reprod.Sci.(in press). (1994)

[Publications] Suzuki,K.(Nagai,T.,Harrison,RAP): "Bicarbonate/CO^2 is required for pig fertilization in vitro." Reprod.Fertil.Develop.(in press). (1994)

[Publications] Nagai,T.: "Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture." Mol.Reprod.Develop.(in press). (1994)

[Publications] Hirao,Y.(Nagai,T.): "In vitro growth and maturation of pig oocytes." J.Reprod.Fert.(in press). (1994)

[Publications] Yoshizawa,M.(Nagai,T.): "Native zona pellucida structure is required for completion of sperm acrosome reaction in porcine fertilization." Theriogenology. (in press). (1994)

[Publications] Takada,M.(Nagai,T.): "pH-Sensitive dissociation and association of -N-acetyl-hexoaminidase from boar sperm acrosome." Biol.Reprod.(in press). (1994)

[Publications] Okamura,N.(Dacheux,JL): "Cloning and characterization of complementary DNA encoding 23kDa protein secreted from caput epididymis of porcine." J.Biol.Chem.(投稿中).

[Publications] OKAMURA,N.(Nagai,T.): "“Assisted reproductive technology/andrology" Glutathione in mammalian embryos:effects of thiol compounds." Archives of Andrology ed.Hafez,E.S.E. (in press), (1994)

岡村直道筑波大学, 基礎医学系, 講師 (30134224)