Thomas G. Honegger

Fertilization in ascidians: Studies on the egg envelope, sperm and gamete interactions in Phallusia mammillata☆

Abstract Eggs and sperm of the ascidian Phallusia mammillata were studied by light microscopy and scanning and transmission electron microscopy. The egg is enclosed in a rigid acellular envelope, the vitelline coat (vc). The vc consists of a fibrous surface layer, a homogeneous electron dense central layer and a thick inner layer with thin fibers and globular elements. Vacuolated follicle cells are attached to the outer, test cells to the inner surface of the vc. Upon staining with phalloidine-rhodamin both cell types exhibit a distinct pattern of actin filaments. Studies of gamete interactions during fertilization reveal that sperm binding is established between the tip of the sperm and the fibrous layer of the vc at sites which are not covered by follicle cells. Freeze fractures of quick frozen sperm show a cap-like structure at the sperm apex. This structure contains a number of vesicles as detected in sperm prepared for TEM by quick freezing and freeze substitution. In sperm just about to penetrate the vc apical vesicles which may contain the vitelline coat lysins have been found. Wheat germ agglutinin (WGA), which binds to the vc but not to follicle cells and sperm, inhibits fertilization by interfering with sperm-vitelline coat binding. This inhibition was completely abolished by competitive sugars such as N,N′,N″-triacetylchitotriose (1 mM) or N-acetylglucosamine. However, there are indications that WGA binding sites on the vc may not only be N-acetylglucosamine but also sialic acid residues. Preliminary studies reveal similarities between ascidian and mammalian fertilization concerning the phagocytosis-like gamete fusion process.

Oogenesis in Hydra carnea: A new model based on light and electron microscopic analyses of oocyte and nurse cell differentiation

Oogenesis in Hydra carnea starts with an accumulation of a great number of I-cells in the interstitial spaces of the ectoderm of the body column. One centrally located I-cell becomes the future oocyte, the others differentiate into nurse cells. Presumptive oocyte and nurse cells are not easily distinguishable at that time. The earliest stage of an oocyte we could identify on ultrastructural criteria was in prophase of its first meiotic division. Only at this stage autosynthesis of nutritive substances predominates, the following rapid increase of the oocyte volume relies on the successive adoption of cytoplasmic fragments from nurse cells. Extending fingerlike processes between the epitheliomuscular cells, the oocyte then starts to phagocytose apoptotic nurse cells. Nurse cell differentiation is indicated by the appearance of lipid vesicles in I-cells. As differentiation proceeds glycogen, rEr and Golgi complexes appear and the cells increase due to a continuous production and accumulation of lipid, glycogen and yolk-like electron dense material. Then the loss of cytoplasmic fragments and degenerative changes typical of apoptosis, a morphologically defined form of cell death, converts the nurse cells into apoptotic bodies. The bulk of nurse cells becomes phagocytosed by the oocyte at late stages of their transformation into apoptotic bodies. At the end of oogenesis which in Hydra carnea takes about 4 days, the egg consists for the largest part of apoptotic nurse cells which persist in the developing embryo until hatching.

Glycoprotein constituents of the vitelline coat of Phallusia mammillata (Ascidiacea) with fertilization inhibiting activity

Vitelline coats (VCs) of Phallusia mammillata were isolated and purified following homogenization of live eggs in order to investigate the molecular basis of sperm-egg recognition. Clean VCs were partly solubilized by sonication in H2O and the soluble fraction (SFVC), derived from the outer surface of VCs, was used for further characterization. Electrophoretic analyses of radioiodinated VCs revealed that SFVC consists of two major glycoprotein components with apparent average Mrs of 450,000 and 180,000, respectively. The 450,000 Mr component is composed of several charge isomers, whereas the 180,000 Mr component is supposed to consist of two oligomers, both with acidic pI, held together by a disulfide linkage(s). Each of the two components possesses WGA-binding sites as shown by transblotting followed by WGA-peroxidase treatment. The amino acid composition of SFVC was determined after acid hydrolysis and its carbohydrate composition was analyzed and quantified by GLC. GlcNAc and GalNAc were found to predominate with 86% by weight of total sugar content and fucose, mannose, and glucose accounted for the remaining 14%. The susceptibility of SFVC to enzymatic (N-glycosidase F) and chemical (TFMS) deglycosylation as well as to protease (trypsin and chymotrypsin) digestion was investigated. Furthermore, sperm receptor activity of SFVC was tested in a fertilization assay. The fertilization rate decreased in a concentration-dependent manner when sperm were preincubated with SFVC. Additionally, sperm treated with SFVC showed binding for FITC-WGA or WGA-gold at the apical portion of the sperm head. Therefore, we strongly assume that one or both of the identified glycoprotein macromolecules of SFVC are involved in sperm-egg recognition.

Molecular cloning and sequence analysis of an ascidian egg β‐N‐acetylhexosaminidase with a potential role in fertilization

β‐N‐Acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate‐based transient sperm–egg coat binding. In ascidians and mammals, β‐hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata, N‐acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of β‐hexosaminidase. In the present study, P. mammillataβ‐hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known β‐hexosaminidases; however, P. mammillataβ‐hexosaminidase had a unique potential N‐glycosylation site. A phylogenetic analysis suggested that P. mammillataβ‐hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage‐specific expression of β‐hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg β‐hexosaminidase forms are specific for the oocyte, test cells and follicle cells.

Ultrastructural and experimental investigations of sperm-egg interactions in fertilization ofHydra carnea

SummaryFertilization in the freshwater hydrozoanHydra carnea has been examined by light, scanning and transmission electron microscopy. Sperm penetrate the jelly coat which covers the entire egg surface only at the site of the emission of the polar bodies. The egg surface exhibits a small depression, the so called fertilization pit at this site. Sperm-egg fusion takes place only at the bottom of the fertilization pit.Hydra sperm lack a structurally distinct acrosome and in most of the observed cases, fusion was initiated by contact between the membrane of the lateral part of the sperm head and the egg surfacce. Neither microvilli nor a fertilization cone are formed at the site of gamete fusion. The process of membrane fusion takes only a few seconds and within 1 to 2 min sperm head and midpiece are incorporated in the egg.Electron dense material is released by the egg upon insemination but cortical granule exocytosis does not occur and a fertilization envelope is not formed. The possible polyspermy-preventing mechanisms in hydrozoans are discussed. Hydra eggs can be cut into halves whereupon the egg membranes reseal at the cut edges and the fragments assume a spherical shape. Fragments containing the female pronucleus can be inseminated and exhibit normal cleavage and development. The observation that in such isolated parts the jelly coat will not fuse along the cut edges was used to determine its role in site-specific gamete fusion. These experiments indicate that site-specificity of gamete fusion can be attributed to special membrane properties at the fertilization pit.

A single layer of microtubules is part of a complex cytoskeleton in mature nematocytes of hydra

The architecture of microtubules in mature nematocytes (stinging cells) of Hydra attenuata was investigated in detail by an indirect immunofluorescence study and by scanning and transmission electron microscopy in order to comprehend the function of the cytoskeleton in this extremely complex cell type. Microtubules were detected in all types of nematocytes in the tentacles and were found to be arranged in parallel arrays forming a highly organized basket-like structure around the nematocysts.

The ascidian egg envelope in fertilization: structural and molecular features

In this report, unpublished and recent findings concerning the structure and function of the ascidian egg coat are compiled in context with fertilization. In the initial stage of ascidian fertilization, sperm interact with a complex egg investment that consists of a layer of follicle cells attached to an acellular vitelline coat. Increasing evidence exists that ascidian sperm are activated at their encounter with the follicle cells. The molecular basis of sperm-follicle cell interactions is discussed in context with sperm binding, membrane proteins and sperm bound glycosidase. The model that suggests a block to polyspermy established by glycosidase released from the follicle cells on fertilization is evaluated and compared with assured facts. Although a number of questions remain to be answered, our recent findings that a cloned beta-hexosaminidase from P. mammillata binds exclusively to the follicle cells of unfertilized but not fertilized eggs, indicates that the follicle cells participate in the block to polyspermy. A dual function, mediating sperm activation and a block to polyspermy attributes to the ascidian follicle cells a key position in fertilization.

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species‐specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio‐iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non‐radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration‐dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg‐binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species‐specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species‐specific recognition in P. mammillata.

Structural and Molecular Investigations on the Egg Coat in Phallusia mammillata

Sperm that approach the ascidian egg encounter first the follicle cells (FCS) and increasing evidence points to a crucial role of these cells for several fertilization steps. The main objective of our study was to investigate the particular surface structure of the follicle cells as potential sperm receptor site. SEM and TEM observations reveal the free upper surface and the filopodia of the FCS covered with an extracellular matrix (ECM) exhibiting a regular surface ornamentation consisting of small ring-like structures, the so-called microannuli (MA). To investigate the ECM properties we used lectin binding to characterize the carbohydrate moieties. From the lectins tested only WGA showed affinity for the FCS surface by specifically binding to the MA. Fluorescence and electron microscopy using solubilized biotinylated sperm surface proteins (SSP) was used to evaluate the ability of the FCS to act as sperm receptors. Biotinylated SSP was found to bind exclusively to MA. These findings are discussed in context with the suggested multiple role of follicle cells in fertilization and previously characterized components of the egg vitelline coat that exhibit sperm-binding activity.

Identification of Phallusia mammillata Egg β-N-Acetylhexosaminidase with a Potential Role in Prevention of Polyspermy

Sperm-egg binding in ascidian fertilization is mediated by the interaction of a sperm-bound glycosidase and the corresponding glycan of the egg coat. Polyspermy is thought to be prevented by the release of egg-associated β-N-acetylhexosaminidase in response to monospermic fertilization, thus blocking supernumerary sperm-binding sites. In the present study β-N-acetylhexosaminidase was isolated and partially purified from unfertilized eggs of Phallusia mammillata by liquid chromatography. Zymographic analyses of enriched enzyme preparations resulted in the identification of a glycoprotein with an apparent molecular weight of 69 kDa under non-reducing and 61 kDa under reducing conditions. Database searches with amino acid sequences of three tryptic gp69 fragments suggest a homology to mammalian β-N-acetylhexosaminidases.