Genetic strain variations in the metaphase-II phenotype of mouse oocytes matured in vivo or in vitro.

The interplay between genetic and epigenetic factors plays a central role in mammalian embryo production strategies that superimpose ex vivo or in vivo manipulations upon strain background characteristics. In this study, we examined the relationship between genetic background and the phenotypic properties of mouse metaphase-II (M-II) oocytes that were matured under in vivo (IVO) or in vitro conditions, either in a basal (IVM) or a supplemented (IVM + ) medium. Differences existed amongst inbred (C57BL/6), outbred (CF-1, Black Swiss, NU/NU) and hybrid lines (B6D2F1) induced to superovulate with regard to cytoplasmic microtubule organizing center (MTOC) number but not spindle size or shape, except for larger and asymmetrical spindles in Black Swiss oocytes.

Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes.

With the aim of investigating the effects of oocyte genotype and activating stimulus on the timing of nuclear events after activation, oocytes collected from hybrid B6D2F1, inbred C57BL/6 and outbred CF-1 and immunodeficient nude (NU/+) females were activated using ethanol or strontium and fixed at various time-points. Meiotic status, spindle rotation and second polar body (PB2) extrusion were monitored by fluorescence microscopy using DNA-, microtubule- and microfilament-selective probes. Although activation efficiency was similar in all groups of oocytes, a significant percentage of CF-1 and NU/+ oocytes treated with ethanol and of C57BL/6 oocytes treated either with ethanol or strontium failed to complete activation and became arrested at a new metaphase stage (MIII) after PB2 extrusion.

Meiotic spindle morphogenesis in in vivo and in vitro matured mouse oocytes: insights into the relationship between nuclear and cytoplasmic quality.

Background: This work addresses the hypothesis that events occurring within the follicle soon after the LH surge are essential for coordinating morphogenesis of the spindle and cytoplasm in mouse oocytes matured in vivo (IVO); we further tested whether in vitro maturation (IVM) fails to support these events.

Methods: Oocytes collected at 1, 2, 3, 4 and 5 h post-hCG or after IVM were analyzed for chromatin, nuclear lamina, microtubules (MTs) and centrosomal proteins by conventional fluorescence and confocal microscopy. In addition, these parameters were monitored in oocytes maintained in 50 microM roscovitine, followed by IVM, or in oocytes retrieved at 1.

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes.

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules.

Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion.

Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes.