Reproduction Abstracts

Introduction: Mature mouse oocytes, arrested at meiotic metaphase II (MII), are activated by a series of Ca²⁺ oscillations caused by sperm-specific phospholipase C zeta (PLCz) which has been shown to localize in cytoplasmic vesicles. The substrate for PLCz, phosphatidylinositol 4,5-bisphosphate (PIP₂), is also present in cytoplasmic vesicles in MII oocytes. Throughout oocyte maturation there is reported to be a gradual increase in sensitivity to PLCz and InsP₃ -Ca²⁺ oscillations, which has been attributed to increased Ca²⁺ store content or InsP₃receptor sensitivity. Here, we have examined the sensitivity of maturing oocytes to PLCz-induced Ca²⁺ oscillations in relation to the appearance of PIP₂ vesicles.

Methods: Mouse oocytes were collected from culled female mice and maintained in M2 media with, or without, IBMX. Oocytes were microinjected with PLCz-luciferase cRNA and a dextran-linked fluorescent Ca²⁺ dye. Chemiluminescence and fluorescence were measured in oocytes maintained on the heated stage of an epifluorescence microscope equipped with an intensified CCD camera. Immunostaining was carried out on similarly-treated, fixed and permeabilized oocytes.

Results and Discussion: Injection of luciferase-tagged PLCz RNA showed that cytoplasmic PLCz expression of up to ~35-fold that which is effective in MII oocytes, does not trigger Ca²⁺ oscillations in 41/49 GV oocytes. However, after germinal vesicle breakdown (GVBD), 10/10 oocytes expressing similar PLCz levels to those active in MII oocytes displayed Ca²⁺ oscillations of reduced amplitude. This marked increase in sensitivity to PLCz correlated with the appearance of PIP₂-positive cytoplasmic vesicles that were evident after GVBD. Immunostaining of MII oocytes suggested that PLCz co-localized with markers of Golgi, which is known to fragment during M-phase. These data suggest that the appearance of PIP₂ in cytoplasmic vesicles after GVBD may also sensitize mature MII oocytes to sperm-induced Ca²⁺ oscillations at fertilization.