Nathan H. Hart

Fertilization in Teleost Fishes: Mechanisms of Sperm-Egg Interactions

Publisher Summary This chapter describes some of the principal morphological and cellular features of fertilization in fishes, from the initial encounter between sperm and egg to their fusion to form the zygote. The pathway leading to the fusion between a single sperm and a previously quiescent egg and the subsequent union of male and female pronuclei consists of a predictable, highly ordered sequence of events. The chapter describes the events and changes that take place during the sperm–egg interaction and their temporal relationships to each other. The eggs of fishes, particularly those of teleosts, appear to be particularly useful and advantageous for investigations of fertilization. Teleost eggs are generally large and possess the built-in advantage of having the location of sperm entry topographically restricted to a predetermined, identifiable site in the animal pole. The direct interaction of sperm and egg is mediated by complementary receptors located on their plasma membranes. Such receptors may function in sperm–egg recognition, binding, fusion, and even the activation of the egg in some species. An important functional component of these receptors is carbohydrate based on studies of the mouse sperm receptor (ZP3) and the sea urchin egg-binding protein (bindin).

Comparative analysis of tissue esterases of the zebra danio (Brachydanio rerio) and the pearl danio (B. albolineatus) by disc gel electrophoresis

Abstract 1. 1. The multiple forms of soluble esterases from liver, ovary, skeletal muscle and intestine of Brachydanio rerio and Brachydanio albolineatus have been analyzed by polyacrylamide (disc) gel electrophoresis. 2. 2. Nineteen separable zones of soluble esterase activity were observed in B. rerio and 10 separable zones in B. albolineatus. 3. 3. Although each tissue had its own unique esterase profile, different tissues within a species shared several esterase isozymes in common. The same tissues of the two species had fewer esterases in common. 4. 4. Esterase isozymes were identified in liver and intestinal tissues that showed unusual patterns of inhibition with DFP, eserine sulfate and sulfhdryl-group inhibitors.

The cortical actin cytoskeleton of unactivated zebrafish eggs: Spatial organization and distribution of filamentous actin, nonfilamentous actin, and myosin-II

Actin and nonmuscle myosin heavy chain (myosin‐II) have been identified and localized in the cortex of unfertilized zebrafish eggs using techniques of SDS‐polyacrylamide gel electrophoresis, immunoblotting, and fluorescence microscopy. Whole egg mounts, egg fragments, cryosections, and cortical membrane patches probed with rhodamine phalloidin, fluorescent DNase‐I, or anti‐actin antibody showed the cortical cytoskeleton to contain two domains of actin: filamentous and nonfilamentous. Filamentous actin was restricted to microplicae and the cytoplasmic face of the plasma membrane where it was organized as an extensive meshwork of interconnecting filaments. The cortical cytoplasm deep to the plasma membrane contained cortical granules and sequestered actin in nonfilamentous form. The cytoplasmic surface (membrane?) of cortical granules displayed an enrichment of nonfilamentous actin. An antibody against human platelet myosin was used to detect myosin‐II in whole mounts and egg fragments. Myosin‐II colocalized with both filamentous and nonfilamentous actin domains of the cortical cytoskeleton. It was not determined if egg myosin was organized into filaments. Similar to nonfilamentous actin, myosin‐II appeared to be concentrated over the surface of cortical granules where staining was in the form of patches and punctate foci. The identification of organized and interconnected domains of filamentous actin, nonfilamentous actin, and myosin‐II provides insight into possible functions of these proteins before and after fertilization.

11 Cytoskeleton in Teleost Eggs and Early Embryos: Contributions to Cytoarchitecture and Motile Events

Publisher Summary Successful interaction between gametes initiates a cascade of pathways that lead to cleavage, cellular differentiation, morphogenesis, and eventual development of an adult organism. Shortly after fertilization of the teleost egg, the egg cortex undergoes a predictable sequence of structural changes in response to intracellular signals. Early transformation of the cortex includes formation of the fertilization cone and second polar body, sperm entry into the egg, cortical granule exocytosis, and surface membrane retrieval by endocytosis. Subsequently, cytoplasm moves toward the animal pole where it accumulates in preparation for cell division and cellular rearrangement. By analogy to other cell systems that show secretion, mitosis, intracellular cytoplasmic flow, and cell migration, it is predicted that similar events in eggs are mediated by action of the cytoskeleton. Most of the knowledge about the egg cytoskeleton has been obtained from analyses of sea urchin, amphibian, and mammalian eggs. By comparison, the composition, organization, and function of the egg cytoskeleton in teleost fishes have received considerably less attention. The eggs of teleost fish have long provided favorable material for the study of vertebrate development. Unlike mammalian and amphibian eggs, teleost eggs can be obtained in large numbers, are optically transparent, and are easily accessible for experimental manipulation. Furthermore, teleosts now also offer the possibility of employing genetic and molecular approaches to investigate the role of the cytoskeleton in early development. This chapter surveys the teleost egg cytoskeleton and evaluates its proposed role(s) in events of fertilization, ooplasmic segregation, and gastrulation.

Effects of A23187 upon cortical granule exocytosis in eggs of Brachydanio

SummaryThe effects of the divalent ionophore A23187 upon unfertilized eggs of the freshwater teleost fish, Brachydanio rerio, have been examined by light, scanning (SEM) and transmission (TEM) electron microscopy. Treatment of eggs with micromolar amounts (1 μM, 10 μM) of A23187 triggers cortical granule exocytosis and elevation of the chorion. However, the exocytosis of cortical granules in ionophore-activated eggs is explosive and occurs more rapidly than in eggs naturally activated in conditioned tap water. Eggs treated with A23187 in a medium lacking extra-cellular calcium also show cortical granule exocytosis, suggesting strongly that egg activation in Brachydanio results from release of calcium primarily from intracellular stores; however, there is a distinct delay in the onset of cortical granule breakdown. Unfertilized eggs exposed to A23187 for 1–5 min show noticeable disturbances in cell surface topography, including loss of microplicae and the appearance of prominent membrane-limited blebs.To determine if cortical granule exocytosis is self-propagating once initiated, A23187 was applied to a localized portion of the unfertilized egg surface, using either a G-50 sephadex gel bead or a 1 mm glass capillary tube. Eggs placed in continuous contact for 15 min with a bead coated with 10 μM A23187 show neither exocytosis of cortical granules nor elevation of the chorion. All eggs exhibit exocytosis when positioned against a glass rod coated with 1 μM A23187. The cortical granule breakdown is partial and restricted to less than 50% of the egg surface in most cells. The complete exocytosis of cortical granules in the zebra danio egg appears to require the stimulation and release of calcium from multiple sites over the cortex.

Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg

The responses of the egg to insemination in a modified Fish Ringers solution (FRS) were examined in eggs of the zebrafish (Brachydanio rerio) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5–8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3–4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish.

LSD: Teratogenic Action in Chick Blastoderms

Summary Chick blastoderms at H & H stage 8-8+ were cultured in vitro on 1 ml aliquots of medium containing either 50 or 100 μg of lysergic acid diethylamide. Following 20 hr incubation, drug-treated embryos failed to demonstrate any increase in mortality or growth-depressing effects. At both dose levels, however, disturbances were observed in fusion of the neural folds. A significant reduction in the mean number of somites per embryo was observed in embryos exposed to 50 μg of LSD.

Ontogeny of glucose-6-phosphate and phosphogluconate dehydrogenases in Brachydanio

Abstract 1. 1. The tissue distributions and developmental progressions of glucose-6-phosphate dehydrogenase (GPD) and phosphogluconate dehydrogenase (PGD) in Brachydanio rerio (zebra danio) and B. albolineatus (pearl danio) are reported. 2. 2. The five GPD isozymes separated from adult and embryonic tissues consisted of two electrophoretically distinct components: a hexose-6-phosphate dehydrogenase (HPD) and a glacose-6-phosphate specific GPD. 3. 3. HPD isozymes were expressed continuously throughout development. 4. 4. Phosphogluconate dehydrogenase existed as a single form in all tissues and was continuously expressed in embryos. 5. 5. Spectrophotometric analyses of GPD and PGD profiles showed that activities of both enzymes were high between fertilization and blastulation and prior to hatching at 4 days.