Jerry L. Hedrick

Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis

Abstract A method has been found which distinguishes between a size isomer family of proteins (e.g. bovine serum albumin polymers) and a charge isomer family of proteins (e.g. lactate dehydrogenase isoenzymes), utilizing disc gel electrophoresis. When the log of protein mobility relative to the dye front was plotted versus acrylamide gel concentration, size isomeric proteins gave a family of nonparallel lines extrapolating to a common point in the vicinity of 0% gel concentration; charge isomeric proteins gave a parallel family of lines. Proteins differing in both charge and size gave non-parallel lines intersecting at gel concentrations other than 0% gel concentration. The slope of such a plot is related to molecular weight. The molecular weight-slope relation was established utilizing 17 well-characterized proteins as standards. From this relation, it is possible to determine the molecular weight of a protein with an average precision of ±4%. This molecular weight method can be applied to a single protein in a mixture of proteins provided a specific detection test is available. This method should find uses in distinguishing between size and charge isomer families of proteins, for the rapid, easy, and accurate determination of protein molecular weights, and as a valuable aid in indicating the procedures to be used in enzyme purifications.

Formation and structure of the fertilization envelope in Xenopus laevis

Abstract This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro .

A molecular approach to fertilization: II. Viability and artificial fertilization of Xenopus laevis gametes

Abstract Conditions necessary for procuring, handling, and storing Xenopus laevis gametes have been investigated in order to develop a simple and reliable method for artificial fertilization. Temperature, sperm concentration, and ionic strength influence sperm and/or egg viability. Extension of the fertilizing capacity of sperm suspensions could be correlated with reversible repression of sperm motility. In the case of various salt solutions used for testes maceration, inhibition of sperm motility was primarily a function of the ionic strength. However, there was some specificity in the salt employed as KCl and CaCl 2 solutions gave anomalous results in comparison with those obtained with other salt solutions. The fertilizability of Xenopus laevis eggs could be extended by shedding the eggs in DeBoers solution. Using this information a simple reliable method for artificial fertilization of Xenopus laevis gametes has been described.

Evidence that the fertilization envelope blocks sperm entry in eggs of Xenopus laevis: Interaction of sperm with isolated envelopes☆

The cortical reaction in eggs of Xenopus laevis results in conversion of the vitelline envelope (VE) into the fertilization envelope (FE). The FE is composed of the former VE plus a new component, the fertilization layer, that forms as a consequence of the cortical reaction. An in vitro test system was developed to compare the VE and FE in terms of their penetrability by sperm. VEs and FEs were isolated from unfertilized eggs and cleaving embryos, respectively. Although they swell during the isolation procedure, the isolated VEs and FEs retain the morphological characteristics by which they can be distinguished in situ. Penetrability of isolated envelopes was tested by exposing suspensions of envelopes to sperm in a saline solution to which solubilized components from the jellycoat of the egg were added. Isolated VEs were penetrated by sperm from both sides. By contrast, isolated FEs proved impenetrable: In no instance did sperm penetrate either the fertilization layer or the VE component of the FE. We concluded that the fertilization envelope blocks sperm entry in Xenopus laevis.

On the macromolecular composition of the zona pellucida from porcine oocytes

The chemical and immunological relation between the glycoprotein components of the pig oocyte zona pellucida resolved by two-dimensional polyacrylamide gel electrophoresis was investigated. After disulfide bond reduction, four microheterogeneous glycoprotein components with apparent molecular weights of 25K, 55K, 65K, and 90K were resolved. When disulfide bonds were left unreduced, two microheterogeneous glycoprotein components were resolved with apparent molecular weights of 55K and 90K. Actin was present, but as a contaminant of the zona pellucida rather than as a true component. The structural relation of these components was investigated using deglycosylation with trifluoromethane-sulfonic acid, limited proteolysis with Staphylococcus aureus V8 protease, amino acid and carbohydrate composition analyses, sequence analysis, and monoclonal antibodies. The 25K and 65K components comigrated with the 90K component when disulfide bonds were not reduced. When the intermolecular disulfide bonds crosslinking the two components were reduced, the 25K and 65K components behaved independently. The 25K and 65K components were derived from the 90K glycoprotein family by proteolysis. The 25K component originated from the C-terminal end, and the 65K component from the N-terminal end of the 90K glycoprotein. The 55K component was composed of two chemically and antigenically distinct glycoproteins, termed 55K alpha and 55K beta, that electrophoretically comigrated. The N-terminal amino acid of the 55K alpha family was blocked. The 55K beta family had an N-terminal amino acid sequence of Asp-Val-Pro-Thr-Ile-Gly-Leu-Ser-X-Ala-Pro-Thr. Thus, the two to four electrophoretic components of the zona pellucida observed on gel electrophoresis are derived from three glycoprotein families.

A molecular approach to fertilization: I. Disulfide bonds in Xenopus laevis jelly coat and a molecular hypothesis for fertilization

Abstract The jelly coat surrounding the egg of Xenopus laevis is dependent on the integrity of disulfide bonds for maintenance of its structure. This was proved by the jelly coat dissolving action of various disulfide bond-breaking reagents, by the rate of jelly coat solubilization as a function of pH, and by the appearance of free sulfhydryl groups due to ultraviolet photolysis of the jelly coat. From the presence of disulfide bonds in the jelly coat, it is postulated that sulfhydryl-disulfide bonds may be involved in the process of fertilization. Sperm penetration through egg integuments (jelly coat and vitelline membrane) may involve a lysin which utilizes a disulfide bond as a substrate. In addition, gamete fusion is visualized as occurring by a sulfhydryl-disulfide bond interchange. In view of the occurrence of disulfide bonds in egg integuments of many different organisms, this molecular hypothesis for fertilization may be generally applicable. The observation of jelly coat solubilization by mercaptans now makes possible a study of the macro-molecular composition of jelly coats and their role in fertilization.

Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes

The isolation of the porcine zona pellucida, the glycoprotein envelope surrounding the mammalian oocyte, and the purification of its glycoprotein families is described. The zona pellucida was prepared from oocytes isolated from pig ovaries using a razor blade device and sieving procedures with Teflon or nylon screens. In 6-7 man h, the zona pellucida from 5 X 10(5) oocytes was obtained yielding 12 mg protein and 2.2 mg carbohydrate. The absorptivity of heat solubilized and filtered zona pellucida was A1%280 nm, 1 cm = 10.8. The four glycoprotein families of the zona pellucida were purified by two-dimensional polyacrylamide gel electrophoresis and electrophoretic elution. The electrophoretic purity of these families was greater than 90%. The protein and carbohydrate content and the amino acid and monosaccharide compositions of each of the glycoprotein families were determined.

Hatching in the toad Xenopus laevis: Morphological events and evidence for a hatching enzyme☆

Abstract The hatching process in embryos of the toad Xenopus laevis consists of two temporally distinct phases. In phase 1, the embryo escapes sequentially from the two outermost jelly layers, J3 and J2, and during phase 2 the embryo hatches from the last remaining jelly coat layer J1 and the fertilization envelope. Phase 1 hatching appears to be a physical process caused by water inbibition of jelly coat layer J1 and dynamic changes in the volume enclosed by the fertilization envelope. The combined turgor pressure ruptures jelly coat layers J3 and J2. The subsequent phase 2 hatching is a result of both physical and chemical processes. Phase 1 hatching exposes layer J1 to the medium which, in contrast to jelly layers J2 and J3 is partially soluble, and permits its gradual dissolution during Phase 2. The embryo secretes a proteolytic enzyme from the frontal region which partially digests the fertilization envelope; subsequent embryo movement ruptures the weakened envelope and completes the hatching process.

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N‐linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin‐like (convertase) site and a C‐terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure‐fertilization function predictions for this phylogenetically conserved family of glycoproteins.

Proteases released from Xenopus laevis eggs at activation and their role in envelope conversion

During fertilization of the Xenopus laevis egg, the egg envelope is converted so that further sperm contact with the egg is prevented. In this study two envelope conversion reactions were investigated, envelope hardening and limited hydrolysis of two structurally related envelope glycoproteins. Both of these reactions were shown to be sensitive to protease inhibitors. In an attempt to identify egg proteases involved in envelope conversion, the medium around activated dejellied eggs was collected and analyzed. The exudate was able to convert isolated envelopes and, when the exudate was analyzed using peptide substrates, two major activities were found, one with a preference for cleavage after argininyl peptide bonds and one with a preference for phenylalaninyl peptide bonds. Analysis of exudate using SDS-polyacrylamide gel electrophoresis with gelatin cast into the gel showed two bands of proteolytic activity, one at Mr 45,000 that was identified as the trypsin-like activity and one at Mr 30,000 that was identified as the chymotrypsin-like activity. When cortical granule exocytosis was suppressed using ammonium chloride, release of the two exudate proteases was also suppressed. Studies of the envelope conversion reactions using protease inhibitors indicated that the chymotrysin-like protease was involved in envelope conversion once it had been activated by the trypsin-like protease.