Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro

Abstract

High molecular weight DNA was isolated from the yolk platelets of Xenopus laevis oocytes ovulated in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. The molecules are always linear. Cytochemical and biochemical controls preclude the possibility that the yolk DNA might be contaminated by nuclear or mitochondrial DNA. We conclude that the yolk DNA is an endogenous component of the yolk platelets.As described in a previous paper (1), DNA could be extracted from the yolk platelets isolated from whole ovaries of Xenopus laevis. Since it was likely that the yolk fraction was contaminated with nuclear DNA from the follicle cells in these earlier experiments, we now worked with oocytes that have been freed from their follicle cells in two differentways: (i) manual removal of follicle cells after a short treatment of the oocytes with chemical agents, such as Pronase (0.15%) or sodium dodecyl sulfate (0.3%); (ii) in vitro ovulation of the oocytes in the presence of hormones. In both cases, high molecular weight DNA could be extracted from the yolk platelets; the physicochemical properties of yolk DNA from oocytes ovulated in vitro will be described. Our experimental data exclude beyond reasonable doubt the possible contamination of yolk DNA by either nuclear or mitochondrial DNA arising from follicular or other ovarian contaminating cells. We shall discuss the extraction of yolk DNA from unfertilized eggs of Xenopus and Ambystoma mexicanum. MATERIALS AND METHODSIn Vitro Ovulation. Ovaries were removed from adult Xenopus laevis, and the membranes of the ovarian follicles were torn apart. (ViPyr). (pH 7.0); 10 ml of medium were used for each 1000 oocytes. The homogenate was layered on an equal volume of 1 M sucrose-5% (ViPyr). and centrifuged for 20 min at 500 X g. The pellet was suspended in 0.25 M sucrose-5% (ViPyr). and centrifuged again in the same way. Electron microscopic controls showed that contamination of the yolk platelets by pigment and mitochondria was negligible.Mitochondria were isolated by Dawid's method (4). Removal of the Jelly of Unfertilized Shed Eggs. For Xenopus, a cysteine-papain mixture at pH 7.8 was used (4). For Ambystoma, the jelly was removed with forceps without any chemical treatment. Both types of eggs were maintained in 0.1 X Holtfreter's medium (5).DNA Determination. Instead of the fluorometric method (6), we used a modification of Keck's indol method (7): 75 oocytes were homogenized in 2 ml of 0.25 M sucrose-0.03 M Tris HCl (pH 7.4)-0.1 M EDTA; 2 ml of cold 10% Cls-CCOOH were added; after 15 min, the pellet was centrifuged and washed with cold 5% Cl3CCOOH. Lipids were removed by single successive washings with 94% alcohol at 40, 94% alcohol at 500, n-butyl alcohol at 500, alcohol-ether (3: 1) at 500, and ether. The pellet was extracted for 20 min at 1000 with 600 ,l of 5% Cl3CCOOH and centri...

Biochemical characterization and developmental behavior of Artemia embryonic and nauplial deoxyribonucleases

“…(2) The processes involved may include the degradation and further utilization of the yolk DNA. Similar to earlier work in other systems (Hanocq et al, 1972), we have found a significant amount of DNA associated with the highly purified yolk fraction from dormant cysts (Marco et al, 1983); the fate and molecular characteristics of this DNA are being studied now in our laboratory. This second type of role is more likely associated with the type A-I/A-I* yolk granule associated enzyme.…”

Open questions on the functional biology of the yolk granules during embryo development

“…A similar but more thorough paper found high molecular weight DNA isolated from the yolk platelets of Xenopus oocytes. The yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded (Hanocq et al, 1972). The authors conclude that the yolk DNA is an endogenous component of the yolk platelets.…”

Deoxyribonucleic Acid Synthesis during Early Embryogenesis

“…Analyses of the DNA content of amphibian eggs by classical methods show that they contain a large excess of DNA over the expected 4C value ; part of this excess DNA is in the mitochondria, but studies on homogenates from oocytes show that more than 65% of cytoplasmic DNA is recovered in the yolk platelets. Studies on yolk DNA (Hanocq et al, 1972) have shown that it is made of linear double-stranded molécules somewhat smaller than those obtained by similar methods from Xenopus chromatin (respective mol.wts. 26x 10* and 40x 10*).…”