Bovine somatic cell nuclear transfer using mitomycin C-mediated chemical oocyte enucleation

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml−1MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.</jats:p

Ultrastructural characteristics of sheep oocytes during in vitro maturation (IVM)

AbstractThe aim of this study was to describe the ultrastructure of sheep oocytes during 24h of in vitro maturation (IVM). Ovaries were collected from a slaughterhouse and the follicles aspirated. The selected cumulus oocyte complexes (COC's) were divided into groups and transferred to maturation medium, where they were maintained for 6, 12, 18 or 24h. Control immature oocytes were also collected (0h). After different maturation intervals the structures were removed and processed for transmission electron microscopy. Immature oocytes (0h) demonstrated pleomorphic mitochondria and presumptive lipid droplets as normally expected, and a moderate number of vesicles scattered throughout the cytoplasm. After 6h of IVM, there was a migration of the mitochondria and presumptive lipid droplets to the cytoplasm periphery and the onset of cumulus cell expansion. Between 12 and 18h of IVM, cytoplasmic rearrangements of the organelles were also observed e.g. aligning and increasing of cortical granules under the plasma membrane. Hooded mitochondria were observed during oocyte maturation. After 24h of IVM, the oocytes showed signs of degeneration, which can be related to cellular aging – because of the excessive time in the maturation medium, after acquisition of developmental competence. This work described the ultrastructural aspects of sheep oocytes during IVM and provided insight into oocyte maturation in the ovine species