Ultrastructural modifications in bovine oocytes maintained in meiotic arrest in vitro using roscovitine or butyrolactone.
Butyrolactone-I (BL-I) and roscovitine (ROSC) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes for 24 hr without having a negative affect on subsequent development to the blastocyst stage. The aim of the present study was to describe the morphological changes occurring in fully grown immature and in vitro matured bovine oocytes following exposure to either BL-I or ROSC for 24 hr at concentrations known to be consistent with normal development. Immature bovine cumulus oocyte complexes, recovered from the ovaries of slaughtered heifers, were incubated for 24 hr in the presence of one of the inhibitors. They were then either fixed immediately and processed for transmission electron microscopy (TEM), or cultured for a further 24 hr in the absence of the inhibitor, in conditions permissive to maturation, and subsequently processed for TEM. A control group of oocytes were processed for TEM immediately upon recovery (0 hr) or following in vitro maturation (IVM) for 24 hr. In general, incubation with either inhibitor disrupted the integrity of the surrounding cumulus cells and affected their subsequent expansion during IVM. Within the oocyte cytoplasm, swelling of the mitochondrial cristae was immediately noticeable following meiotic inhibition in the presence of ROSC, while an increased population of pleomorphic mitochondria and mitochondria with electron lucent matrices following BL-I treatment was not observed until after the subsequent IVM period. Both inhibitors caused degeneration of the cortical granules, effectively reducing the population, most noticeably following IVM. At the level of the nucleus, both inhibitory treatments caused convolution of the nuclear membrane, furthermore, aberrant structures were observed within the nucleoplasm of ROSC-treated cumulus oocyte complexes (COCs). In conclusion, while it has been shown that inhibition of meiotic resumption using specific cdk inhibitors is possible and that such oocytes are capable of undergoing maturation, fertilization, and early embryo development, there is as yet no definitive proof that oocytes treated in this way can ultimately give rise to normal offspring. We have shown here that some modifications are induced in the oocytes at the ultrastructural level. Whether or not these modifications are compatible with normal gestation and the birth of a live calf remain to be elucidated.
Lonergan
Patrick
P
Department of Animal Science, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland.
Faerge
Inger
I
Hyttel
Poul Maddox
PM
Boland
Maurice
M
Fair
Trudee
T
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology*; D008540:Meiosis / Q000187:drug effects; D009865:Oocytes / Q000378:metabolism / Q000648:ultrastructure*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D015044:Zona Pellucida / Q000648:ultrastructure
D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D011687:Purines; D000077546:Roscovitine; C082868:butyrolactone I; D015107:4-Butyrolactone