Differentiation of a calsequestrin-containing endoplasmic reticulum during sea urchin oogenesis.

We have used light and electron microscopic immunolocalization to study the distribution of a sea urchin calsequestrin-like protein (SCS) during sea urchin oogenesis. SCS was localized exclusively in the lumen of the endoplasmic reticulum (ER) and in the nuclear envelope of oocytes of all maturation stages. Immunoelectron microscopy also revealed that SCS is not present in golgi complexes of oocytes. Double label immunofluorescent staining of frozen sections of ovary with the SCS antiserum and an antibody to the cortical granule protein hyalin indicated a dramatic morphogenesis of the SCS-containing ER (SCS-ER) coincident with oocyte maturation. This differentiation included an apparent increase in the amount and complexity of the cytoplasmic SCS-ER network, the transient appearance of stacks of SCS-ER cisternae in synthetically active vitellogenic oocytes, and the restructuring of the SCS-ER in the cortex. Immunofluorescence of isolated oocyte cortices showed a plasma membrane-associated SCS-ER which was much less dense and regular than that found surrounding the cortical granules in the mature unfertilized egg cortex. Cytoplasmic and cortical microtubule arrays are present in oocytes and may provide the basis for the SCS-ER distributional dynamics. The results of this study underscore the dynamic nature of ER and how it's organization reflects cellular functions. We suggest that the establishment during oogenesis of the dense SCS-ER tubuloreticulum provides the egg with the calcium sequestration and release apparatus that regulates calcium fluxes during egg activation and early development.

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo.

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.