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Search for other papers by Elena Ibáñez in
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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. C57BL/6 oocytes also showed slower release from MII arrest but faster progression to telophase (TII) after ethanol exposure, and they exhibited the most rapid exit from TII under both activation treatments. Strontium caused delayed meiotic resumption, spindle rotation and PB2 extrusion, but rapid TII exit, in B6D2F1, CF-1 and NU/+ oocytes when compared with ethanol. Compared with all other strains, NU/+ oocytes were significantly slower in completing spindle rotation and PB2 extrusion, irrespective of the activating stimulus, and a significant decrease in activation rates and pace of meiotic progression was observed after strontium exposure. Thus, our findings demonstrated that the kinetics of meiosis resumption and completion, spindle rotation and PB2 extrusion following parthenogenetic activation depends on both genotype-specific factors and on the activation treatment applied.
Department of Molecular and Integrative Physiology, Unidade de Biologia da Reprodução, Marine Biological Laboratories, Center for Reproductive Sciences, 3088 Kansas Life Science Innovations Center, University of Kansas Medical Center, KLSIC Room 3016, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA
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Department of Molecular and Integrative Physiology, Unidade de Biologia da Reprodução, Marine Biological Laboratories, Center for Reproductive Sciences, 3088 Kansas Life Science Innovations Center, University of Kansas Medical Center, KLSIC Room 3016, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA
Search for other papers by David F Albertini in
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In the perinatal ovary of most mammals, external and internal factors establish a primordial follicle reserve that specifies the duration of the reproductive lifespan of a given species. We analyzed the mechanism of follicle loss and survival in C57BI/6 mice using static and dynamic assays of apoptosis, autophagy, and ovarian morphogenesis. We confirm an initial loss soon after birth, when about 44% of the germ cells detectable at the end of the fetal period abruptly disappear. The observations that (1) few germ or somatic cells were apoptotic in newborn ovaries, (2) vitally stained organ cultures exhibit active extrusion of non-apoptotic germ cells and (3) germ-cell lysosome amplification occurs at birth suggested that additional mechanisms are involved in perinatal germ cell loss. Newborn mouse ovaries cultured in the pH sensitive dye lysotracker red exhibit an increased incidence of acidified non-apoptotic germ cells when maintained in the absence but not in the presence of serum, implying a role for autophagy in germ cell attrition. Inhibitors of autophagy, but not apoptosis, reduce germ cell acidification induced by serum starvation in ovary organ cultures and protein mediators of both autophagy and apoptosis are expressed at birth. From these findings we suggest that multiple perinatal mechanisms establish the primordial follicle reserve in mice.
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Cloning mammalians by somatic cell nuclear transfer (SCNT) remains inefficient. A majority of clones produced by SCNT fail to develop properly and of those which do survive, some exhibit early aging, premature death, tumors, and other pathologies associated with aneuploidy. Alterations of centrosomes are linked to aberrant cell cycle progression, aneuploidy, and tumorigenesis in many cell types. It remains to be determined how centrosomes are remodeled in cloned bovine embryos. We show that abnormalities in either distribution and/or number of centrosomes were evident in approximately 50% of reconstructed embryos following SCNT. Moreover, centrosome abnormalities and failed ‘pronuclear’ migration which manifested during the first cell cycle coincided with errors in spindle morphogenesis, chromosome alignment, and cytokinesis. By contrast, nuclear mitotic apparatus protein (NuMA) exhibited normal expression patterns at metaphase spindle poles and in ‘pronucleus’ during interphase. The defects in centrosome remodeling and ‘pronuclear’ migration could lead to chromosome instability and developmental failures associated with embryo production by SCNT. Addressing these fundamental problems may enhance production of normal clones.
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Search for other papers by David F Albertini in
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Department of Psychology, University of Toronto Mississauga, Mississauga, Ontario, Canada
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The naked mole-rat (NMR, Heterocephalus glaber) is renowned for its eusociality and exceptionally long lifespan (> 30 y) relative to its small body size (35–40 g). A NMR phenomenon that has received far less attention is that females show no decline in fertility or fecundity into their third decade of life. The age of onset of reproductive decline in many mammalian species is closely associated with the number of germ cells remaining at the age of sexual maturity. We quantified ovarian reserve size in NMRs at the youngest age (6 months) when subordinate females can begin to ovulate after removal from the queen’s suppression. We then compared the NMR ovarian reserve size to values for 19 other mammalian species that were previously reported. The NMR ovarian reserve at 6 months of age is exceptionally large at 108,588 ± 69,890 primordial follicles, which is more than 10-fold larger than in mammals of a comparable size. We also observed germ cell nests in ovaries from 6-month-old NMRs, which is highly unusual since breakdown of germ cell nests and the formation of primordial follicles is generally complete by early postnatal life in other mammals. Additionally, we found germ cell nests in young adult NMRs between 1.25 and 3.75 years of age, in both reproductively activated and suppressed females. The unusually large NMR ovarian reserve provides one mechanism to account for this species’ protracted fertility. Whether germ cell nests in adult ovaries contribute to the NMR’s long reproductive lifespan remains to be determined.
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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. When oocytes were matured in culture, meiotic spindle and cytoplasmic phenotypic properties of M-II oocytes were affected relative to in vivo conditions and between strains. Specifically, measures of meiotic spindle size, shape, polar pericentrin distribution and cytoplasmic MTOC number all revealed characteristic variations. Interestingly, the overall reduction in cytoplasmic MTOC number noted upon IVM was concomitant with an overall increase in spindle and polar body size. Maturation under IVM + conditions resulted in a further decrease in cytoplasmic MTOC number, but spindle and polar body characteristics were intermediate between IVO and IVM. How these oocyte phenotypic properties of maternal origin may be linked to predictive assessments of fecundity remains to be established.
