In vitro maturation and fertilization of porcine oocytes after a 48 h culture in roscovitine, an inhibitor of p34cdc2/cyclin B kinase.
Maintaining oocytes at the germinal vesicle (GV) stage in vitro may permit enhanced acquisition of the developmental competence. The objective of the current study was to evaluate the nuclear and cytoplasmic maturation in vitro of porcine oocytes after pretreatment with S-roscovitine (ROS). Cumulus oocyte complexes (COC) were treated with 50 microM ROS for 48 h and then matured for various lengths of time in a conventional step-wise in vitro maturation (IVM) system by using dibutyryl cyclic AMP. The COC that were matured in the same system for 44 h without pretreatment with ROS were used as the control group. At various periods after the start of IVM, oocytes were assessed for the meiotic stages and subjected to in vitro fertilization (IVF) with fresh spermatozoa. The ROS treatment inhibited GV breakdown of 94.4% oocytes, with the majority arrested at the GV-I stage (67.4%). Maximum maturation rate to the metaphase-II stage after ROS treatment was achieved by 44 h of IVM (92.1%) and no differences were observed with control oocytes (95.0%). Penetration rate was correlated to the maturation rate. The duration of IVM had no effects on polyspermy and male pronuclear (MPN) formation rates at 8 h post insemination (hpi), whereas both rates increased at 22 hpi. Direct comparison with controls assessed at 22 hpi confirmed a lesser MPN formation in ROS-treated oocytes (73.7% compared with 53.6%). Glutathione (GSH) concentrations were less in oocytes treated with ROS than in control oocytes (5 compared with 7.7 pmol/oocyte) as well as blastocyst rate (22.0% compared with 38.1%, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pretreated with ROS for 48 h did not equal that of control oocytes in the current IVM system.
Romar
Raquel
R
Department of Animal Science and Technology, The Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Okayama 700-8530, Japan.
Funahashi
Hiroaki
H
D000818:Animals; D019926:Cyclin B / Q000037:antagonists & inhibitors / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development / Q000187:drug effects / Q000502:physiology; D004627:Embryonic Induction / Q000187:drug effects* / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005978:Glutathione / Q000378:metabolism; D008297:Male; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology*; D011897:Random Allocation; D000077546:Roscovitine; D013552:Swine / Q000502:physiology*; D013997:Time Factors; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D019926:Cyclin B; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D018844:Cyclin-Dependent Kinases; D005978:Glutathione; D000095043:Cyclin-Dependent Kinase-Activating Kinase