10 Dec

Segregation of the Pathways Leading to Cortical Reaction: RESULTS(5)

Loading eggs with BAPTA-AM resulted in a concentration-dependent inhibition of both the CR and resumption of meiosis (Fig. 7A). However, inhibition of meiotic resumption was more sensitive to Ca2+ levels than was inhibition of the CR. When 0.5 |xM BAPTA was used, only 14% of the eggs resumed meiosis, whereas more than 50% underwent a CR. Only at 5 and 10 (xM BAPTA-AM were both aspects of egg activation inhibited to a similar level.

Assessment of the CR at different concentrations of BAPTA-AM displayed a concentration-dependent decrease in strong labeling (Fig. 7B). At low concentrations of BAPTA-AM, most of the activated eggs presented strong labeling of CG exudate, whereas at a higher concentration the majority demonstrated medium labeling. ventolin inhaler

The amplitude of the Ca2+ transient in each treatment was measured, and the results revealed a concentration-dependent decrease, correlated with the increasing concentrations of BAPTA-AM (Fig. 8). In contrast to a mean Ca2+ rise of 440 nM in eggs treated with OAG, when increasing concentrations of BAPTA-AM were used, the mean Ca2+ transient decreased from 250 to 95 nM. No [Ca2~], response to OAG was observed when 10 |xM BAPTA-AM was used.
Fig8Segregation of the pathways leading
FIG. 8. Amplitude of [Ca2+] transients in the presence of BAPTA-AM (dotted bars). Eggs were loaded for 30 min simultaneously with fura-2-AM and increasing concentrations of BAPTA-AM, washed, and allowed to attach to a coverslip in TH medium containing 20 n.g/ml OAG. [Са2+]| changes were followed for 15 min. The amplitude values are given after subtraction of the basal [Са2 + ]; levels. The results are expressed as means ± SEM. The percentage of eggs that responded by a Ca2 + rise is indicated (diamonds). Each data point represents 24-55 eggs from at least 3 experiments.

Fig9Segregation of the pathways leading
FIG. 9. Schematic model for early egg activation. Sperm-egg interaction leads to activation of PLC, resulting in hydrolysis of PIP2. One cleavage product, IP3, leads to the release of Ca2+ from the endoplasmic reticulum (ER). The other cleavage product, DAG, activates PKC. This PKC activation is Ca2+-dependent and is also activated by the initial Ca2+ rise. Resumption of meiosis results directly from the Ca2+ elevation, while CR is triggered by PKC activation. It is not clear yet whether the CR can be triggered directly by calcium elevation, bypassing the PKC channel.

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