title: Development of a reliable in vitro maturation system for zebrafish oocytes.
abstract: In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, these in vitro maturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliable in vitro maturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50-100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5-9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16-29%; hatching rate, 8-21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism of in vitro maturation in zebrafish oocytes and for the practical use of immature oocytes.
authors.affiliation: Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Seki
authors.forename: Toshimitsu
authors.initials: T
authors.lastname: Kouya
authors.forename: Ryoma
authors.initials: R
authors.lastname: Tsuchiya
authors.forename: Delgado M
authors.initials: DM
authors.lastname: Valdez
authors.forename: Bo
authors.initials: B
authors.lastname: Jin
authors.forename: Takao
authors.initials: T
authors.lastname: Hara
authors.forename: Naoya
authors.initials: N
authors.lastname: Saida
authors.forename: Magosaburo
authors.initials: M
authors.lastname: Kasai
authors.forename: Keisuke
authors.initials: K
authors.lastname: Edashige
mesh_terms: D000818:Animals; D003470:Culture Media; D003593:Cytoplasm / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods; D006863:Hydrogen-Ion Concentration; D006908:Hydroxyprogesterones; D009865:Oocytes / Q000502:physiology*; D009866:Oogenesis / Q000502:physiology*; D009930:Organic Chemicals; D009994:Osmolar Concentration; D012710:Serum Albumin, Bovine; D013997:Time Factors; D046509:Tissue Culture Techniques; D015027:Zebrafish / Q000502:physiology*
chemical_list: D003470:Culture Media; D006908:Hydroxyprogesterones; C095240:L15 culture medium; D009930:Organic Chemicals; C007433:17,20-dihydroxy-4-pregnen-3-one; D012710:Serum Albumin, Bovine