Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine.
Cyclin dependent kinase inhibitors (CDKIs) may be used for pre-maturation culture, but can accelerate nuclear maturation. The aim of the present research was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at lesser than typically used concentrations on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 microM BLI (B) or 6.25 microM BLI+12.5 microM ROS (BR) in TCM-199 for 24 h. Oocytes were subsequently submitted to in vitro maturation (IVM) in TCM-199+0.5 microg/ml FSH, 50 microg/ml LH and 10% FCS for another 24 h, during which oocytes were fixed every 3 h. In Experiment 2, oocytes were submitted to 24h pre-maturation treatments, with the inhibitors being diluted in TCM-199 or DMEM. IVM lasted 21 h in the culture media DMEM+0.5 microg/ml FSH, 50 microg/ml LH, 5% FCS and 50 ng/ml EGF. After IVM, oocytes from all groups were fertilized in vitro. Oocytes and sperm (2x10(6) sperm cells/ml) were co-cultured for 18 h. Embryos were co-cultured with granulosa cells in CR2aa for 8 days. All cultures were in droplets under oil, at 38.5 degrees C and 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. In Experiment 1, at 0 h, C and B oocytes were all (100%) at the germinal vesicle stage (GV) of development. BR had fewer GV oocytes (89%, P<0.05). After 3 h IVM, B and BR had fewer oocytes in GV (84.7 and 79.6%, P>0.05) than C (100%, P<0.05). At 12 h, most oocytes were at intermediate stages (metaphase to telophase I) in all groups (approximately 80%, P>0.05). After 21 (77-89%) and 24 h (85-95%), all groups had similar metaphase II (MII) rates of development (P>0.05). In Experiment 2, cleavage (79-84%, P>0.05) and Day 7 blastocyst rates (26-36%, P>0.05) were similar. After 8 days, the group pre-matured with BR in DMEM had lesser blastocyst rates of development (32.3%) lower than C (40.1%, P<0.05). The other groups were similar to C (35-38%, P>0.05). Hatching rates were similar (10-15%, P>0.05) as were total cell numbers (141-170). In conclusion, BR is less effective in maintaining meiosis block; B and BR accelerate meiosis resumption; and use of pre-maturation medium may affect developmental rates.
Adona
P R
PR
ZAB, FZEA, USP, Pirassununga, SP, Brazil.
Pires
P R L
PR
Quetglas
M D
MD
Schwarz
K R L
KR
Leal
C L V
CL
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002417:Cattle / Q000196:embryology*; D018929:Cell Culture Techniques / Q000662:veterinary; D003470:Culture Media; D047108:Embryonic Development / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D008297:Male; D009865:Oocytes / Q000502:physiology*; D011247:Pregnancy; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine
D003470:Culture Media; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; C082868:butyrolactone I; D015107:4-Butyrolactone