Ventolin Inhalers

- Sperm Factor Induces Intracellular Free Calcium: MATERIALS AND METHODS(4)


The activity of column fractions was arbitrarily classified as follows: — = absence of activity; + = fractions that initiated a first spike within 10-20 sec after the injection but [Ca2+]i rises did not last for 10 min; ++ = fractions that initiated oscillations that lasted for at least 10 min and showed four or fewer rises per 10 min; +++ = fractions whose Ca2+ responses lasted more than 20 min and four or more rises occurred per 10-min interval; + + + + = fractions that induced one rise per minute in the first 10 min of monitoring.

In the last group, the decline in activity was minimal for the first 20 min of monitoring. Assessment of the frequency of [Ca2+]i rises for activity determination was done 5 min postinjection and lasted for 10 min. In those cases in which the first rise lasted for more than 5 min, activity was monitored starting as soon as the baseline was reestablished and continued for 10 min.

IP3 Concentration Determination in Xenopus Oocytes

The concentration of IP3 was measured in small regions of single Xenopus oocytes as described by Luzzi et al. with some modifications. Boar SF or SF buffer alone (50 nl, 30 ^g/^l) was microinjected into Xenopus oocytes. Five to 8 min after injection, a small sample of cytoplasm (10 nl) at an average distance of 200 ^m from the injection site was aspirated into a capillary tube, and electrophoresis was initiated. The elapsed time between the initiation of cytoplasmic sampling and the onset of electrophoresis was 250 msec. The effluent from the capillary tube was directed onto a single permeabilized detector cell, a baby hamster kidney (BHK 21) cell, which had been previously loaded with mag-fura-2, a Ca2+-sensitive fluorophore contained only in the ER. After elution onto the detector cell, the sample containing IP3 opened the IP3R channel, releasing Ca2+ from the ER and increasing the fluorescence of the mag-fura-2. Mag-fura-2 fluorescence was monitored and quantified as previously described. The IP3 concentrations were obtained from a previously constructed calibration curve.

September 17, 2013 Sperm
Tags: calcium developmental biology fertilization