Expression and immunolocalization of the calpain-calpastatin system during parthenogenetic activation and fertilization in the rat egg.
Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca(2+)-dependent thiol protease. The calpain-calpastatin system constitutes one functional proteolytic unit whose presence and function has already been investigated in various cell types, but not in the egg. We have previously shown that calpain is expressed in rat eggs and is activated upon egg activation. The present study was designed to investigate the calpain-calpastatin interplay throughout the process. Western blot analysis revealed two main calpastatin isoforms, the erythrocyte type (77 kDa) and the muscle tissue type (110 kDa). By immunohistochemistry and confocal laser scanning microscopy, we demonstrated that the 110 kDa calpastatin was localized at the membrane area and highly abundant at the meiotic spindle in eggs at the first and second meiotic divisions. The 77 kDa calpastatin isoform appeared to be localized as a cortical sphere of clusters. The 110 kDa calpastatin and beta-tubulin have both been localized to the spindle of metaphase II eggs, both being scattered all through the cytoplasm following spindle disruption by nocodazole treatment, implying a dynamic interaction between calpastatin and microtubule elements. Upon egg activation, membranous calpastatin translocated to the cortex whereas cortical millimolar (m)-calpain shifted towards the membrane. Spindle calpastatin and calpain remained static. We suggest that calpastatin serves as a regulator of m-calpain. The counter translocation of m-calpain and calpastatin could serve as a means of calpain escape from calpastatin inhibition and may reflect a step in the process of calpain activation, throughout egg activation, that is required for calpain to exert its proteolytic activity.
Haim
K
K
Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel.
Ben-Aharon
I
I
Shalgi
R
R
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western / Q000379:methods; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D002154:Calpain / Q000032:analysis* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D005306:Fertilization / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D018613:Microscopy, Confocal; D008970:Molecular Weight; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D010312:Parthenogenesis / Q000502:physiology*; D020033:Protein Isoforms / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D002135:Calcium-Binding Proteins; D007476:Ionophores; D020033:Protein Isoforms; D015759:Ionomycin; C033668:calpastatin; D002154:Calpain