A comparative study of the isolation of the cortex and the role of the calcium-insoluble protein in several species of sea urchin egg.

A comparative study was made of the isolation of the cortex in the eggs of several sea urchin species. Since the isolation method developed by Sakai depends on the presence of magnesium in the medium, the protein composition of the cortex was investigated to determine whether the protein component of the egg described by Kane and Hersh which is gelled by divalent ions, is present in these cortices. Isolation of the cortex was found to require the same divalent ions at the same concentrations as protein gelation, and in the eggs of some species much of the gel protein of the cell was found in the isolated cortical material. In the eggs of other species a smaller fraction of this protein was found in the isolated cortex, although it was more concentrated there than in the endoplasm, and in one species this protein appeared to be uniformly distributed throughout the cell. These results indicate that this protein is localized in the cortical region of the eggs of some species of sea urchin, possibly in the cortical granules, but also point up the fact that results from one species cannot be uncritically extrapolated to others.

Preparation and purification of polymerized actin from sea urchin egg extracts.

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits sperm-egg binding and fertilization.

The role of cell surface glycoproteins of the sea urchin egg in binding sperm has been examined by studying the biological activity of glycopeptides derived from these glycoproteins. Glycopeptides were produced from egg surface glycoproteins by Pronase digestion. After fractionation by gel filtration the glycopeptides were tested for their ability to inhibit the binding of sperm to eggs, presumably by competing with the egg surface glycoproteins for binding sites on the sperm. One glycopeptide fraction with an apparent molecular weight of approximately 6,000 was found to be a potent inhibitor of sperm-egg binding, as well as fertilization, even at nanomolar concentrations. This activity was heat stable and exerted its effect against the sperm and not the egg. Experiments with a radiolabeled form of the glycopeptide fraction directly demonstrated that at least one component of it bound to sperm. Specific binding of the radiolabeled glycopeptide occurred only to acrosome-reacted sperm. Because the isolated glycopeptide fraction has many of the characteristics that one would expect of a biologically active fragment of an egg surface receptor for sperm, these findings are consistent with the idea that one or more glycoconjugates on the surface of the egg are involved in sperm binding.

Some properties of hyalin: the calcium-insoluble protein of the hyaline layer of the sea urchin egg.

The principal protein component of the hyaline layer of sea urchin eggs is the calcium-insoluble protein first described by Kane and Hersh. The protein hyalin is abnormally high in acidic amino acids, almost devoid of basic amino acids, and characteristically rich in valine and proline. Essentially all of the cysteine present is found in the disulfide form; no evidence points to intermolecular disulfide linkages. Hyalin from several species has a minimal subunit weight of about 100,000, though evidence exists for a particle three times this weight in urea or guanidine hydrochloride from one species. Optical rotatory dispersion measurements indicate no alpha-helix content, though the dispersion has unique characteristic features. Addition of small quantities of calcium causes hyalin to gel to a birefringent fibrous form. The fibrous, birefringent form of hyalin is rendered isotropic upon addition of EDTA, but the birefringence is restored with re-addition of divalent cation.

Intramolecular heterogeneity of mitochondrial DNA of Drosophila melanogaster.

DNA from purified mitochondria of Drosophila melanogaster can be isolated as supercoiled molecules which when nicked have a contour length of 5.9 micron. Partial denaturation mapping shows regional heterogeneity of base composition with one early denaturing region, with a calculated GC content close to zero, extending over 20% of the genome. DNA isolated from unfertilized eggs shows nuclear and mitochondrial DNA in equal proportions; we found no evidence of other cytoplasmic species.

Image analysis of chromatin in cells of reimplantation mouse embryos.

Mouse two-celled embryos and blastulae were Feulgen stained and the DNA content of their nuclei was measured with an integrating microdensitometer. The cells considered on the basis of their nuclear DNA content to be in G(1), S, and G(2) phases of the cell cycle were selected and their total chromatin area and chromatin areas at different gray levels were measured by the image analyzing computer, Quantimet. The measurements were aimed at quantitation of several features of the chromatin morphology of cells in different functional states. The total area of chromatin was found to increase, and the mean density of chromatin to decrease, from the G(1) to the G(2) phase of the cell cycle in both two-celled embryos and blastulae. The area of chromatin decreased, and the mean density of chromatin increased, as embryos developed from two-celled to blastula stage. It was concluded that nuclear morphology in preimplantation mouse embryos depends on both the phase of the cell cycle and the stage of development. The method of image analysis described was found to be useful for quantitation of changes in chromatin morphology.

Histones synthesized for use in early development of Xenopus laevis are stored as a complex with antigenic properties similar to those of the octamer core of nucleosomes.

Serum of patients with systemic lupus erythematosus (SLE) contains crossreacting autoantibodies which recognize histones in nucleosomes or when they are induced to form octamers in solution in the presence of 2 M NaCl, but not when they are dissociated free in solution at physiological ionic strength. We have found that histones stored in eggs of Xenopus laevis for use in rapid nuclear synthesis during early development react with this antibody. This reaction has been observed by radioimmunoassay, inhibition of chromatin assembly by the extracts in the presence of antibody, and, in a preliminary result, by identification of a histone-antibody complex bound to protein A-sepharose. Further evidence that the extract antigen corresponds to the stored histone pool comes from sedimentation and charge fractionation experiments where the chromatin assembly activity and antigen (measured by radioimmunoassay) were found to cofractionate. BEcause the extract histones are not bound to DNA, our results suggest that they are stored as a soluble complex in a conformation similar or identical to the octameric core of the nucleosome. Our data suggest that the histones in this complex are bound to an anionic factor or factors which presumably replaces the DNA in shielding the positive charges on the histones.

Immunochemical studies of 22S protein from isolated mitotic apparatus.

Evidence is presented that the "22S protein" of mitotic apparatus isolated from sea urchin eggs is not microtubule protein. An antibody preparation active against 22S protein is described, and immunochemical studies of the distribution of 22S protein in various cellular fractions and among morphological features of mitotic apparatus are reported. The protein is ubiquitous in the metaphase egg fractions that were tested but is not found in sperm flagella. It is immunologically distinct from proposed microtubule protein isolated from mitotic apparatus by the method of Sakai, and from proposed microtubule protein obtained after extraction with mild acid. It exists in nontubule material of isolated mitotic apparatus but is not detectable in microtubules.

Localization of ribosomal DNA within oocytes of the house cricket, Acheta domesticus (Orthoptera: Gryllidae).

A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.

Isolation and characterization of mitochondrial DNA from Drosophila melanogaster.

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm(3) has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 microm; it reassociates with a low C(0)t(1/2) after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm(3). MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm(3), slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.

Actin in Xenopus oocytes.

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.

Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata.

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.

Actin in Xenopus oocytes. II. Intracellular distribution and polymerizability.

The largest oocytes of Xenopus Laevis were broken open in the absence of shearing forces which might transfer actin from particulate to supernatant fractions. Particulate and postmitochondrial supernatant fractions were prepared by centrifugation. SDS-electrophoretic fractionation on polyacrylamide gels and quantitative scanning techniques were used to separate actin and to assay its amount in cellular fractions. The actin has been identified in electrophoretograms by its molecular weight and its binding to DNase I. oocytes contain 1.4-1.7 {um}g of actin per cell, of which up to 88 percent is recovered in the postmitochondrial supernate under a variety of conditions. In the soluble fraction, it represents about 8.8 percent of the total protein. Its concentration in native cytoplasm was directly assayed at 4.1 mg/ml. There is no detectable actin that can be transferred from the particulate to the soluble phase by neutral detergents or ionic conditions that would depolymerize muscle actin. Centrifugation of the soluble oocyte fractions showed that 75-95 percent of the actin can not be sedimented under forces that would pellet filamentous actin. Addition of potassium and magnesium to the cytoplasm, to concentrations that would polymerize muscle actin, does not increase the amount of sedimentable actin. Roughly one-third of the soluble actin is recovered from Sephadex columns at about the position of monomer. About two- thirds is in complexes of 100,000 daltons or greater.

Specific protein synthesis in cellular differentiation. Production of eggshell proteins by silkmoth follicular cells.

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3-1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30-37 codons per ribosome) is similar to that encountered in other eukaryotic systems.

Receptor function of mouse sperm surface galactosyltransferase during fertilization.

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.

Metabolic and ultrastructural changes in the frog ovarian follicle in response to pituitary stimulation.

Frog ovarian fragments were prevented from ovulating in vitro by the addition of actinomycin D up to 3 hr following pituitary stimulation; but addition of Actinomycin D 6 hr after stimulation was far less effective. Puromycin, on the other hand, effectively inhibited ovulation when added as late as 6 hr after pituitary stimulation. Although actinomycin D reduced uptake of uridine-(3)H, and puromycin reduced uptake of leucine-(3)H and lysine-(14) by pituitary-stimulated ovarian tissue minus oocytes (OTMO) in vitro, it was found that pituitary stimulation did not significantly increase uptake of these compounds by OTMO. Radioautographs of ovarian follicles fixed 6 hr after the addition of pituitary extract and uridine-(3)H in vitro revealed increased RNA synthesis in the peritoneal surface epithelium, compared with unstimulated controls, while the ovarian sac epithelium showed no increase. Gross ultrastructural changes occurred in the peritoneal area of ovarian follicles following pituitary stimulation in vivo, including loss of collagen fibrils, and general disorganization of the connective tissue theca. Changes in the rough endoplasmic reticulum of the peritoneal epithelial cells, while frequently encountered, were less pronounced. None of these changes was observed in the ovarian sac area, or in the interfollicular region. The above data are consistent with the hypothesis that pituitary stimulation of the frog ovary results in increased synthesis of RNA and protein by the peritoneal epithelial cells, and that the protein may be collagenase.

Parthenogenesis in Xenopus eggs requires centrosomal integrity.

Xenopus eggs are laid arrested at second metaphase of meiosis lacking a functional centrosome. Upon fertilization, the sperm provides the active centrosome that is required for cleavage to occur. The injection of purified centrosomes mimics fertilization and leads to tadpole formation (parthenogenesis). In this work we show that the parthenogenetic activity of centrosomes is inactivated by urea concentrations higher than 2 M. The loss of activity is correlated with a progressive destruction of the centriolar cylinder and extraction of proteins. This shows that centrosomes are relatively sensitive to urea since complete protein unfolding and solubilization of proteins normally occurs at urea concentrations as high as 8-10 M. When present, the parthenogenetic activity is always associated with a pelletable fraction showing that it cannot be solubilized by urea. The parthenogenetic activity is progressively inactivated by salt concentrations higher than 2 M (NaCl or KCl). However, only a few proteins are extracted by these treatments and the centrosome ultrastructure is not affected. This shows that both parthenogenetic activity and centrosomal structure are resistant to relatively high ionic strength. Indeed, most protein structures held by electrostatic forces are dissociated by 2 M salt. The loss of parthenogenetic activity produced at higher salt concentrations, while the structure of the centrosome is unaffected, is an apparent paradox. We interpret this result as meaning that the native state of centrosomes is held together by forces that favor functional denaturation by high ionic strength. The respective effects of urea and salts on centrosomal structure and activity suggest that the centrosome is mainly held together by hydrogen and hydrophobic bonds. The in vitro microtubule nucleating activity of centrosomes can be inactivated at salt or urea concentrations that do not affect the parthenogenetic activity. Since egg cleavage requires the formation of microtubule asters, we conclude that the extracted or denatured microtubule nucleating activity of centrosomes can be complemented by components present in the egg cytoplasm. Both parthenogenetic and microtubule nucleating activities are abolished by protease treatments but resist nuclease action. Since we find no RNA in centrosomes treated by RNase, they probably do not contain a protected RNA. Taken together, these results are consistent with the idea that the whole or part of the centrosome structure acts as a seed to start the centrosome duplication cycle in Xenopus eggs.

Yolk pigments of the Mexican leaf frog.

Eggs of the Mexican leaf frog contain blue and yellow pigments identified as biliverdin and lutein, respectively. Both pigments are bound to proteins that occur in crystalline form in the yolk platelet. The major blue pigment is biliverdin IX alpha. The eggs vary in color from brilliant blue to pale yellow-green depending on the amount of each pigment. These pigments may provide protective coloration to the eggs.

Deoxyribonucleic acid in germinal vesicles of oocytes of Rana pipiens.

The amount of DNA isolated from germinal vesicles of Rana pipiens oocytes is similar to that isolated from whole unfertilized eggs, and this suggests that oocyte nuclei are the source ofmuch of the DNA obtained from whole eggs. Fluorometric determinations of the isolated DNA show the presence of 0.004 microgram of DNA equivalents per germinal vesicle. Similar values were obtained by estimations from comparisons of the cesium chloride densitygradient profiles of sample DNA and a known amount of reference DNA from Pseudomonas aeruginosa. The buoyant density of DNA prepared from isolated germinal vesicles is the same as that of frog-liver DNA, 1.706 grams per cubic centimeter, as determined by equilibrium sedimentation in a cesium chloride density gradient.

2-Deoxy-alpha-ecdysone from ovaries and eggs of the silkworm, Bombyx mori.

From ovaries dissected from developing and emerged adults of the silkworm, Bombyx mori, two substances having high molting hormone activity were isolated. One of these was identified as 2-deoxy-alpha-ecdysone by means of high-pressure liquid chromatography and mass spectrometry. Although this compound had previously been isolated from the fern Blechnum minus and postulated to be the precursor of alpha-ecdysone, it had not been obtained from insect material. The compound is also contained in the form of a conjugate in ovaries as well as in diapausing silkworm embryos.