Hypertonicity-Induced Projections Reflect Cell Polarity in Mouse Metaphase II Oocytes: Involvement of Microtubules, Microfilaments, and Chromosomes1

Abstract

A previous study showed that with hypertonic sucrose treatment, a projection is formed in mouse metaphase II (MII) oocytes in proximity to the spindle and chromosomes, where a polarized cortical domain is located. However, little is known about the mechanisms involved in this process. Here, we designed a series of experiments to test the hypothesis that hypertonicity is the induction factor for the formation of projections in mouse MII oocytes. Our hypothesis was supported by the following evidence: 1) different concentrations of sucrose affected the formation and shape of projections, whereas serum or basic media had little effect; 2) other hypertonic sugar solutions could also induce projection formation; and 3) projections could also be induced by hypertonic NaCl solution. We then tested the hypothesis that the cytoskeleton was involved in the formation of hypertonicity-induced projections. This was investigated by culturing MII- and germinal vesicle-stage mouse oocytes in the presence or absence of cytoskeletal inhibitors, including cytochalasin B (disruption of actin filaments), nocodazole (disruption of microtubules), and taxol (polymerization of tubulin molecules). We found that none of the cytoskeletal inhibitors alone could prevent hypertonicity-induced projection formation, whereas the combination of cytochalasin B with nocodazole or with taxol blocked the formation of these projections in most matured oocytes. When immature oocytes were incubated in cytochalasin B, but not in nocodazole or taxol, the formation of an actin-rich domain and the peripheral positioning of the spindle were blocked during maturation; hence, no projections were formed, even after hypertonic sucrose treatment. Based on these observations, we propose that three components are necessary for projection formation: 1) a polarized cortical patch (e.g., an actin-rich domain), 2) rigid submembrane structures (e.g., a spindle and/or chromosomes), and 3) solid connections between the above. Any disturbance of one of these factors will affect the hypertonicity-induced projection formation. Hypertonicity-induced projection in mouse oocytes thus provides an experimental model for studies regarding cell polarity and the interaction between membrane and submembrane components.

Demecolcine-Assisted Enucleation of Goat Oocytes: Protocol Optimization, Mechanism Investigation, and Application to Improve the Developmental Potential of Cloned Embryos

“…The mitotic spindle of porcine oocytes also became disorganized after demecolcine treatment (Kawakami et al, 2003). Mouse oocytes are different from goat and bovine oocytes in that the actin-rich domains formed independent of demecolcine during oocyte maturation (Liu et al, 2002). In the present study, however, when goat oocytes were treated with demecolcine and CB in combination, the number of oocytes forming projections decreased significantly.…”

Effects of Demecolcine and Sucrose on the Incidence of Cytoplasmic Protrusions Containing Chromosomes in Pig Oocytes Matured In Vitro

“…It has been reported that the basic medium is not involved in induction of a cytoplasmic protrusion containing chromosomes in mouse oocytes using sucrose [22]. However, the effects of the basic medium on the phenomenon induced by demecolcine or in other species are unclear.…”

“…The cytoplasmic protrusion containing chromosomes in the mouse oocyte is considered to be induced by hypertonicity, the common role for sucrose in cryopreservation [24][25][26][27], because hypertonic solutions containing high concentrations of other sugars, such as maltose, trehalose, sorbitol, mannitol and glucose, or NaCl can also induce the same phenomenon [22]. Sucrose, which is unable to permeate through the plasma membrane, induces water molecules to move out of oocytes and, thus, effectively causes them to shrink.…”

“…Sucrose, which is unable to permeate through the plasma membrane, induces water molecules to move out of oocytes and, thus, effectively causes them to shrink. Liu et al [22] estimated that shrinkage of mouse oocytes induced by hypertonicity of more than 470 mOsm brings the spindle-chromosome complex, where both poles of the spindle are anchored to the cortex by microtubule and non-microtubule elements, closer to the plasma membrane. The chromosomes are then attached to the plasma membrane, and cytoplasmic protrusions are formed due to bulging of the rigid spindle-chromosome complex through the plasma membrane.…”

“…In the presence of sucrose, the concentration of demecolcine affects the cytoplasmic protrusion of pig oocytes [19]. On the other hand, it has been reported that hypertonic treatment with sucrose induces formation of a protrusion around the chromosome and spindle area in mouse [21,22] and bovine [23] oocytes, suggesting that the hypertonicity induced by sucrose might also be essential for the cytoplasmic protrusion in pig oocytes [19]. However, there are no reports that have tested this hypothesis.…”