I treated the mature cumulus-free oocytes with pronase 0.3% (w/v in PBS), the zona pellucida was digested within 60-90 seconds however when the oocytes were first electrically activated (parthenogenesis) the digestion time became more longer (120-180 seconds or even more). Is there any explanation?
there could be more porous membrane after activated with electric pulse (BTX electrophoresis unit), which might be affect the activity of pronase.
You should be seeing the effect of zona hardening, which is a byproduct of fertilization but might be brought about my the Ca++ transient elicited by activation, depending on the species you are working with.
I support the Rita Vassena explanation. The activation causes besides other things the cortical reaction (efflux of cortical granules) with these consequences.
As already explained very clearly by Rita Vassena and Pavel Trávník, the longer digestion time of the zona pellucida you are observing after oocyte activation is due to the zona hardening caused by the cortical reaction, the slow mechanism of polyspermy prevention normally elicited upon fertilization.
Cross linking of zona pellucida through sulfide linkages have been shown to reduce the pronase activity. Please see the paper
http://www.reproduction-online.org/content/135/1/19.full
Try the same experiment with fertilized and unfertilized eggs. This will peg it down to the role of egg activation. This might eliminate the possible electrical mystery aspect of your observation.
Hi Dr.Islam.
I will answer you after discussing with my colleagues .
Dear Islam,
I agree with the comments made by the previous people. This hardening is mediated by the cortical granule content released after oocyte activation. A similar observation was reported after freezing oocytes and thawing. I can add that if you want to reduce this hardening then you can use an inhibitor of protease. Very recently Jurrien Dean lab in the discovery of the ovastacin, a protease involved in the ZP2 proteolysis.
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