Birth of piglets after transferring of in vitro-produced embryos pre-matured with R-roscovitine.
The objectives of this study were to evaluate: (1) the nuclear maturation, (2) the intracellular glutathione (GSH) content, (3) the normality of fertilization and (4) full development after transplantation of embryos derived from porcine oocytes pre-cultured with 50 micromol/l roscovitine (an inhibitor of p34cdc2/cyclin B kinase) for 22 h. After treatment with roscovitine, the nuclear configuration of oocytes (Hoechst staining) was comparable with those examined just after collection: the majority of oocytes were arrested at the germinal vesicle (GV) 1 stage (63.2%). Roscovitine-treated oocytes progressed through meiosis to the metaphase II stage in a conventional step-wise in vitro maturation (IVM) program for 44 h in a proportion similar to control ones (>85.0%). When roscovitine-treated oocytes and non-treated oocytes were matured for 44 h and then co-cultured with fresh spermatozoa for 18 h, no differences were observed in oocyte penetrability, proportion of monospermic penetration and male pronuclear formation (>87%). Roscovitine increased the GSH synthesis in oocytes at 22 h, whereas, after 44 h, roscovitine-treated oocytes had similar amounts of GSH to non-treated oocytes. Finally, surgical transfer of zygotes at 22-24 h post-insemination, derived from roscovitine-treated oocytes, resulted in one pregnancy with 12 piglets born; control non-treated zygotes resulted in one pregnancy and 10 piglets born. The full-term developmental ability of mammalian oocytes pre-cultured with roscovitine prior to IVM is thereby demonstrated. This validation is important before the introduction of roscovitine into routine procedures.
Coy
Pilar
P
Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia 30071, Spain. pcoy@um.es
Romar
Raquel
R
Ruiz
Salvador
S
Cánovas
Sebastián
S
Gadea
Joaquín
J
García Vázquez
Francisco
F
Matás
Carmen
C
D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors; D018920:Coculture Techniques; D046149:Embryo Culture Techniques; D004624:Embryo Transfer / Q000662:veterinary*; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D005978:Glutathione / Q000378:metabolism; D008098:Litter Size; D008297:Male; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000378:metabolism; D009866:Oogenesis / Q000187:drug effects; D011247:Pregnancy; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D013194:Staining and Labeling; D013552:Swine; D015053:Zygote
D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D016203:CDC2 Protein Kinase; D005978:Glutathione