Douglas E. Chandler

Allurin, a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins

Previously, we demonstrated that a protein from Xenopus egg jelly exhibits sperm chemoattractant activity when assayed by either video microscopy or by sperm passage across a porous filter. Here we describe the isolation and purification of allurin, the protein responsible for this activity. Freshly oviposited jellied eggs were soaked in buffer, and the conditioned medium was loaded onto an anion exchange column and eluted with an NaCl gradient. The active fraction was purified further by RP-HPLC, the chemoattractant protein appearing as a single sharp peak. The amino acid sequence of the protein, determined by direct sequencing and cloning of cDNAs coding for the protein, consisted of 184 amino acids having a molecular mass of 21,073 Da. The protein shares homology with the mammalian cysteine-rich secretory protein (CRISP) family that includes testes-specific spermatocyte protein 1, a cell adhesion protein which links spermatocytes to Seritoli cells, and acidic epididymal glycoproteins that bind to sperm and have been implicated in sperm–egg fusion. Phylogenetic analysis suggests that allurin evolved from the ancestral protein that gave rise to the mammalian CRISP family. Addition of allurin to this family portends that the CRISP family represents a group of “sperm escort” proteins, which bind to sperm at various steps in their life history, facilitating passage from one functional stage to the next. Allurin stands out in this regard, representing both the first vertebrate sperm chemoattractant to be purified and sequenced and the first member of the CRISP family to be found in the female reproductive tract.

Exocytotic fusion pores exhibit semi-stable states

SummaryRapid-freezing/freeze-fracture electron microscopy and whole-cell capacitance techniques were used to study degranulation in peritoneal mast cells of the rat and the mutant beige mouse. These studies allowed us to create a time-resolved picture for fusion pore formation. After stimulation, a dimple in the plasma membrane formed a small contact area with the secretory granule membrane. Within this zone of apposition no ordered proteinaceous specializations were seen. Electrophysiological technique measured a small fusion pore which widened rapidly to 1 nS. Thereafter, the fusion pore remained at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15,000 msec. These conductances correspond to pore diameters 25–36 nm. Ultrastructural data confirmed small pores of hourglass morphology, composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increased in conductance, consistent with the observed morphology of omega-figures. The hallmarks of channel-like behavior, instantaneous jumps in pore conductance between defined levels, and sharp peaks in histograms of conductance dwell-time, were not seen. Since the morphology of small pores shows contiguous fracture planes, the electrical data represent pores that contain lipid. These combined morphological and electrophysiological data are consistent with a lipid/protein complex mediating both the initial and later stages of membrane fusion.

Postfertilization growth of microvilli in the sea urchin egg: new views from eggs that have been quick-frozen, freeze-fractured, and deeply etched.

Abstract We have used quick-freezing, freeze-fracture, and deep etching to study postfertilization growth of microvilli in the sea urchin, Strongylocentrotus purpuratus . Between 1 and 2 min postinsemination, we observed rapid growth of numerous finger-like microvilli to about 0.7 μm in length. Subsequently, groups of two to four microvilli became raised off the egg surface on broad cytoplasmic bases. Finally, by 5 min postinsemination, these bases had become interconnected by thin sheets of cytoplasm to form a continuous network of ruffles across the egg surface. Corresponding thin sections of quick-frozen and freeze-substituted eggs showed that these microvilli contained networks of microfilaments. The finger-like processes had bundles of parallel microfilaments that ran their entire length, while the ruffles of cytoplasm contained sheets of crisscrossed microfilaments. These observations support the hypothesis that microvillar growth results from formation of new microfilament networks.

Exocytosis in vitro: Ultrastructure of the isolated sea urchin egg cortex as seen in platinum replicas

Sea urchin egg cortices, isolated on polylysine-coated cover glasses by the method of V.D. Vacquier (1975, Dev. Biol. 43, 62-74), have been fixed with 2% glutaraldehyde, critical point-dried, and rotary shadowed with platinum-carbon. The replicas produced show that the isolated cortex consists of a plasma membrane having a regular array of microvillar evaginations each containing a filamentous core. Attached to the plasma membrane is a densely packed layer of cortical granules; each granule is linked to the plasma membrane and to four to six adjacent granules by fine, 6-nm-diameter filaments. Between the plasma membrane and cortical granule layer runs a tubular endoplasmic reticulum consisting of bulb-like varicosities joined in chains that are arranged in a network of polygons. Interspersed among the cortical granules are occasional spherical organelles which correspond in size to osmiophilic granules seen in the intact egg cortex. Addition of 1000 microM or 50 microM Ca2+ to the cortex initiates fusion of individual granules with the plasma membrane, as well as extensive fusion between granules, producing extended, branched exocytic pockets. Frequently, fusion patterns are observed that suggest a propagation of granule fusion outward from one or more foci within a single cortex.

A sperm chemoattractant is released from Xenopus egg jelly during spawning

Although the jelly layers surrounding amphibian eggs are known to be essential to fertilization, no biological activities have been reported for jelly macromolecules in any vertebrate. Here we provide evidence that the jelly surrounding Xenopus laevis eggs releases a small diffusible protein into the surrounding media that serve as sperm chemoattractant. Using video microscopy we find that Xenopus sperm will preferentially turn toward and contact a glass capillary filled with egg jelly extract. In experiments using a two-chamber bioassay device we find that egg jelly extracts are capable of stimulating sperm movement across a membrane barrier sixfold over controls. This activity is not observed in materials unrelated to egg jelly and the response of sperm to egg jelly extract is clearly chemotactic rather than chemokinetic. A concentration gradient of the extract is absolutely necessary, and the chemotactic activity of the extract exhibits a biphasic dose dependence similar to that of chemotactic agents in other systems. We have been able to characterize the factor as being a heat-stable protein about 10 kDa in size. This study, therefore, provides the first clear evidence for a diffusible sperm chemoattractant in a nonhuman vertebrate, as well as the first demonstration of a physiological role for egg jelly macromolecules in Xenopus fertilization.

Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis.

SummaryDuring meiotic maturation, the cortex of oocytes of Xenopus laevis undergoes structural reorganization, visualized in this study by freeze-fracture electron microscopy. In the full-grown but immature oocyte, annulate lamellae are dispersed throughout the subcortex of the egg, 5 to 20 μm from the plasma membrane. The annulate lamellae consist of well-organized stacks of membrane with visible pores. Stimulation of meiotic maturation by progesterone leads to disruption of the annulate lamellae and formation of an elaborate cortical endoplasmic reticulum which surrounds the cortical granules and intertwines throughout the cortex of the mature egg. Pore-like structures similar to those previously observed in the subcortical annulate lamellae are observed in the mature cortical endoplasmic reticulum. The cortical endoplasmic reticulum is often in close apposition with the plasma membrane and with membranes of cortical granules, but no junctions are visualized. This study provides further evidence that the cortical endoplasmic reticulum develops during progesterone-stimulated meiotic maturation in vitro, and that the annulate lamellae are precursors to the cortical endoplasmic reticulum.

Calcium signals in neutrophils can be divided into three distinct phases

Rabbit neutrophils were loaded with the fluorescence probe indo-1 and cytosolic free calcium levels were monitored during chemotactic peptide (fMet-Leu-Phe) stimulation. The fMet-Leu-Phe-induced calcium signal consisted of three consecutive phases: (1) an initial peak that was independent of extracellular calcium, (2) a secondary shoulder that required extracellular calcium but was totally blocked by hyperosmolality and (3) a final plateau of elevated calcium that was dependent on extracellular calcium but insensitive to hyperosmolality.

Xenopus laevis fertilisation: analysis of sperm motility in egg jelly using video light microscopy

Xenopus laevis eggs are surrounded by an extracellular matrix consisting of a vitelline envelope, and three jelly layers, J1, J2, and J3 (from egg surface outward). The jelly layers vary in thickness (about 150, 15 and 200 microns for J1, J2 and J3 respectively) but all are translucent allowing observation of sperm penetration. Video microscopy demonstrated that sperm are able to penetrate and traverse J3 at velocities approaching 30 microns/s. Sperm swim through jelly in a corkscrew-like manner with their rotational and forward velocities being tightly coupled at about 30 degrees/micron forward travel. They are propelled by whip-like power strokes involving hairpin bends in the flagellum that are generated every 180 degrees of rotation and which are propagated from base to tip. The overall trajectories of individual sperm are quite variable. Many sperm head directly for J2 but some do not, these swimming circumferentially, or even away from the egg surface. Most sperm (over 97%) that enter the jelly do not get to the egg surface but are stopped at a variety of positions within J3 or at the outer surface of J2. Efficient sperm penetration and passage through the jelly layers requires a low electrolyte concentration in the surrounding medium, and is inhibited by the lectin wheat germ agglutin (WGA) in a dose-dependent manner. WGA does not block sperm penetration of J3 but does block further progression towards the egg surface. This observation suggests that sperm motility within the jelly is dependent on the carbohydrate moieties of the large glycoconjugates present, and that their alteration by WGA binding accounts for the inability of sperm to reach the egg surface and fertilise the egg.

Egg jelly layers of Xenopus laevis are unique in ultrastructure and sugar distribution.

Jelly coats surrounding the eggs of the South African clawed toad, Xenopus laevis, consist of three transparent, gelatinous layers: the innermost layer (J1), the middle layer (J2), and the outer layer (J3). The distribution of N‐acetylglucosamine within these jelly coats, as probed with FITC‐conjugated wheat germ agglutinin (WGA‐FITC), and the matrix ultrastructure of each layer, as visualized in platinum replicas produced by the quick‐freeze, deep‐etch, and rotary‐shadowing technique, suggests that each layer has a unique fiber and glycoprotein composition. J1 extends nearly 200 μm from the egg surface and exhibits no WGA‐FITC staining. Stereo images of platinum replicas indicates that J1 consists of a tightly knit network of 5–10 nm fibers decorated with 10–20 nm particulate components. In contrast, J2 is a relatively thin layer, extending only 25–40 μm from the outer aspect of J1. When visualized by confocal microscopy, J2 displays a multilayered WGA‐FITC staining pattern. The ultrastructure of J2 consists of sheets of fine fibers that run parallel to one another and that can be identified by their ability to bind WGA‐colloidal gold. The fibers of each sheet run at an oblique angle to fibers in neighboring layers. J3 extends 100 μm or more from J2. The WGA‐FITC staining pattern shows high intensity in its outer region and less intensity in regions closer to J2. Like J1, the J3 ultrastructure consists of a network of 5–10 nm fibers, decorated with 10–20 nm particulate components. The results of these studies add to a growing body of information that suggests the jelly coats surrounding the eggs of many animals consist of a fibrous glycoprotein superstructure that acts as a scaffold to which globular glycoproteins are bound.

The coelomic envelope of Xenopus laevis eggs: A quick-freeze, deep-etch analysis

The extracellular matrix of Xenopus laevis oocytes was analyzed before and after meiotic maturation using quick-freeze, deep-etch, rotary-shadow electron microscopy. The perivitelline space (PS) of the meiotically immature oocyte contains a filamentous network which connects microvilli (MV) and follicle cell macrovilli to the folded oocyte surface below. The envelope overlying the PS is composed of bundles of large fibers which course between the tips of the MV. Spaces between these bundles contain smaller fibrils which secure the egg envelope to the microvillar tips. Meiotic maturation is accompanied by flattening of the oocyte plasma membrane, formation of an orderly array of MV, and elevation of the egg envelope. In the coelomic eggs, the reorganized envelope is composed of loosely bundled large fibers which course above the microvillar tips rather than between them. The spaces between these bundles contain small fibers similar to those seen in the meiotically immature oocyte. This reorganized envelope, however, will not bind sperm; further modifications must transpire during passage through the oviduct to render it sperm receptive.