ABSTRACT
The presence of acidic phosivitins phosvettes in Xenopus laevis yolk platelets and their purification by (NH4)2SO4 precipitation of associated lipovitellin were documented by polyacrylamide gel electrophoresis followed by staining with Stains-all. Procedures were further developed to resolve the various entities present in the crude phosvitin/phosvette fraction by size-exclusion, anion-exchange, and hydrophobic interaction chromatography, using a Pharmacia FPLC system, and their resolution was documented by both electrophoresis and two-dimensional chromatography. Four major entities (phosvitins 1 and 2; phosvettes 1 and 2) were observed, but microheterogeneity was also apparent, particularly by hydrophobic interaction chromatography. The new separation procedures require min/h rather than h days.
Subject(s)
Chromatography, High Pressure Liquid/methods , Egg Proteins/analysis , Phosphoproteins/analysis , Phosvitin/analysis , Animals , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Electrophoresis, Polyacrylamide Gel , Female , Xenopus laevisABSTRACT
Full grown Xenopus oocytes were incubated with trifluoperazine (TFP) or injected with TFP. Incubation of oocytes in TFP resulted in normal-appearing meiotic maturation, as judged by the presence of the white spot and the absence of the germinal vesicle. Cortical granule breakdown in TFP-incubated oocytes was not normal. Abnormal cortical granule breakdown was also observed when progesterone-maturated oocytes were activated in the presence of TFP. Oocytes microinjected with TFP and incubated with progesterone appeared to mature in a normal manner, as judged by the absence of the germinal vesicle; these underwent cortical granule breakdown following activation, but frequently lacked the white spot. Oocytes microinjected with TFP did not mature in the absence of progesterone. We conclude that incubation, although not microinjection, of oocytes with TFP induces essentially normal resumption of meiotic maturation.
Subject(s)
Oocytes/drug effects , Trifluoperazine/pharmacology , Animals , Calcium/metabolism , Calmodulin/metabolism , Cytoplasmic Granules/drug effects , Female , Kinetics , Meiosis/drug effects , Oocytes/growth & development , Progesterone/pharmacology , Sulfonamides/pharmacology , Trifluoperazine/metabolism , Xenopus laevisABSTRACT
Xenopus oocytes were exposed to trifluoperazine, a drug that binds to the calcium-regulating protein, calmodulin. The drug induced meiotic maturation even in the absence of progesterone. When trifluoperazine was microinjected directly into oocytes the maturation of the cells was partially inhibited in that the white spot, indicative of germinal vesicle breakdown, did not appear even though the germinal itself was absent in dissected oocytes.
Subject(s)
Meiosis/drug effects , Oocytes/growth & development , Ovum/growth & development , Trifluoperazine/pharmacology , Xenopus laevis/physiology , Animals , Female , Kinetics , Microinjections , Oocytes/ultrastructure , Progesterone/pharmacology , Trifluoperazine/administration & dosageABSTRACT
Microinjection of 0.1 microgram of Ca++ into Xenopus laevis oocytes induces breakdown of the cortical granules. The cortical granules disappeared in both full grown (Stage VI) and small growing (Stage IV) oocytes. Microinjection of Mg++, K+, or Na+ had no effect on cortical granules in either Stage IV or Stage VI oocytes. Small quantities (0.03 microgram) of Ca++ induced dehiscence of the cortical granules only in proximity to the injection site.
Subject(s)
Calcium/pharmacology , Oocytes/drug effects , Ovum/drug effects , Animals , Female , Microinjections , Oocytes/physiology , Oocytes/ultrastructure , Oogenesis , XenopusSubject(s)
Lipoproteins/metabolism , Oocytes/metabolism , Oogenesis , Ovum/metabolism , Vitellogenins/metabolism , Animals , Anura , Cytoplasm/analysis , Female , Progesterone/pharmacology , Rana pipiensABSTRACT
Microinjection of approximately 0.3 mug of calcium into maturing oocytes of Rana pipiens after nuclear dissolution resulted in cleavage-like constrictions, cortical granule breakdown, and formation of a structure resembling a two-cell embryo. Mg2+, Na+, or K+ did not induce any of these reactions. Larger amounts of Ca2+-induced contraction over the entire surface of oocytes or eggs, but did not induce cleavage-like constrictions; smaller amounts of Ca2+ produced either a local cortical granule reaction of the formation of one large and one small "blastomere." Furrow formation was not observed during normally induced maturation until after germinal vesicle breakdown. The location of microinjected Ca2+ determined the orientation of the resulting furrow. Ca2+-induced cortical granule breakdown occurred in full-grown nonmaturing oocytes near the site of injection. Cortical granule breakdown also occurred in maturing oocytes (after germinal vesicle breakdown but before second meiotic metaphase), but only in the blastomere containing the infected Ca2+. As expected, in mature oocytes (at second meiotic metaphase) cortical granule breakdown occurred over the entire oocyte surface, including both blastomeres. The results indicate that furrow formation and cleavage-like constrictions may be directly influenced by Ca2+, and that functional contractile elements are present near all areas of the oocyte surface. Furthermore, Ca2+ injection initiates localized cortical granule breakdown in full-grown immature and maturing oocytes.
