Floriana Rosati

Studies on fertilization in the ascidans: I. Self-sterility and specific recognition between gametes of Ciona intestinalis☆

Abstract In Ciona intestinalis of the Gulf of Naples, self-sterility is not absolute, 15% of the animals being self-fertile. Self-sterility and self-fertility are specific properties of the gametes; eggs from self-sterile animals are not fertilized even at very high sperm concentrations. Observations on the eggs with their envelopes and on eggs deprived of their follicle cells point to the chorion as the site at which the specific sperm-egg recognition occurs and as the primary site of one of the most stringent requirements of fertilization, namely the self-not-self recognition between gametes. Recognition results in the formation of a firm binding between the sperm receptors of the chorion and the plasma membrane of the unreacted spermatozoa.

A study of the chorion and the follicle cells in relation to the sperm-egg interaction in the ascidian, Ciona intestinalis☆

Abstract We present ultrastructural observations on the processes underlying interaction between the spermatozoon and the egg envelopes in Ciona intestinalis. The morphological and cytochemical basis of sperm binding to the chorion and the subsequent steps such as the acrosome reaction and penetration of the spermatozoon through the chorion are described. Some speculations are also presented on the role of the follicle cells in fertilization.

Glycoproteomics: Past, present and future

This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O‐glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi‐automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.

Studies on fertilization in the ascidians. II. Lectin binding to the gametes of Ciona intestinalis.

Abstract In the present work we have compared the binding of fluorescein-conjugated lectins (concanavalin A (ConA), wheat germ agglutinin (WGA), fucose binding protein (FBP) and soybean agglutinin (SBA)) to the sperm surface and to the egg and its envelopes of Ciona intestinalis. Only WGA is bound to the follicle cells: yet this lectin has no binding sites on the sperm surface. Both ConA and FBP are bound by the chorion, the oolemma and the sperm surface. However, while ConA reacts only with the sperm head, FBP is bound both to the head and to the flagellum. Experiments on the effect of ConA and FBP on the fertilization reaction have been carried out. The role of the lectin-binding sites that are shared by the surfaces of both gametes is discussed in connection with the nature of the sperm-binding sites.

Protein profile of capacitated versus ejaculated human sperm.

Freshly ejaculated sperm acquire the fertilizing potential by a continuing process that occurs during sperm transport through the female genital tract, and it is physiologically not complete until the spermatozoon reaches the oocyte. The process termed capacitation can be mimicked in vitro by using appropriate capacitation media. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. This work deals with a proteomic approach to the analysis of protein profile variations in human normospermic samples as a consequence of three hours in vitro capacitation. 2DE gels were produced per freshly ejaculated sperm and per capacitated sperm and several quantitative and qualitative significant variations were found. Among the MS obtained identifications, proteins with a significant decrease after capacitation were found to be involved in protein fate, metabolism, and flagellar organization; on the contrary, increasing proteins were found to be related to cellular stress. Interestingly, the detected flagellar organization proteins decreased during capacitation whereas their corresponding fragments increased. A swim-up selected and three-hour capacitated sperm subpopulation has also been resolved by 2DE, and its synthetic gel has been analyzed for the variations observed in the entire sperm population. An immunofluorescence analysis of this sperm typology was carried out with antiactin and antitubulin antibodies.

Further observations on the morphogenesis of the round headed human spermatozoa.

Sperm and testicular biopsies of an infertile human patient have been investigated using histochemical and electron microscopical technique. Spermiogram revealed a head defect, characterized with lacking acrosome and round and immature nucleoplasm, occuring in practically all cells and coiled tails in about a half of spermatozoa. EM study of spermatids has shown an abortive development of acrosome, whose primordium failed to attach the nucleus and expand, instead regressed. The nucleus failed in shaping and retarded in maturing, a disturbance apparently associated with the aplasia of hypoplasia of caudal manchette. The role of zinc in nuclear differentiation and the shape of head in the movement pattern are discussed.

A fucosyl glycoprotein component with sperm receptor and sperm-activating activities from the vitelline coat of ciona intestinalis eggs☆

In this paper we describe a mild procedure which results in the extraction of a glycoprotein fraction from the vitelline coat (VC) of Ciona intestinalis while leaving behind the bulk of the VC components. When acting upon the spermatozoa this fraction inhibits sperm binding to the VC and fertilization and elicits sperm activation including the acrosome reaction. SDS-PAGE shows that it contains the same (fucosyl) glycoprotein components previously recognized in the total extracts of VC. It is suggested that this material contains the sperm receptors or those components of the receptors that are essential for their Chinese function.

Electron microscopy on tardigrades. III. The integument

Electron microscopic studies on the body wall of Tardigrada show the presence of three layers from the outside, (1) a compact acellular cuticle, (2) a one cell-thick epidermis, and (3) an exceedingly thin collagen coat. Histochemical studies performed at a submicroscopic level resolve in the cuticle a set of nonchitinous layers (epicuticle, intracuticle, and wax layer) and a thicker chitinous procuticle. Within the epicuticle three layers were recognized: (a) a mucous coat consisting of an acid mucopolysaccharide arising from particular craters of the cuticle (“pearls” of AA), (b) a triple-layered tanned lipoprotein cuticulin, and (c) a dense homogeneous epicuticle containing an untanned lipoprotein and a neutral polysaccharide. The intracuticle is composed of (a) a tanned outer membrane; (b) a dense untanned glycoprotein and lipid layer; (c) a tanned glycoprotein and a lipid inner flake. Between all these layers and the procuticle, a discontinuous wax layer is present. The procuticle is always untanned and corresponds to the arthropods endocuticle. Pore canals and ecdysial membrane are absent. The epidermal cells are devoid of microvilli, contain lipids, polysaccharides, proteins, a rich ergastoplasm, and enzymes.

Studies on fertilization in the ascidians: Fucosyl sites on vitelline coat of Ciona intestinalis

Abstract The vitelline coat (originally called chorion) of the ascidian egg is the site where species-specific recognition and binding of spermatozoa occurs. Recent findings from this laboratory have suggested that fucosyl residues are present on the vitelline coat of Ciona intestinalis eggs and play an important role in the process of fertilization. The results reported in this paper confirm and extend those findings. With Fucose Binding Protein (FBP) and Fucosyl-Ferritin as markers, fucosyl sites have been localized on the fibrillar tufts emerging from the outer surface of the vitelline coat, both on glycerol-treated eggs and on living eggs deprived of follicle cells at pH 5. Observations on isolated vitelline coats have shown that the inner surface does not contain fucosyl sites. Studies performed with 125I-FBP on glycerol-treated eggs have indicated that two distinct classes of fucosyl sites are present on the vitelline coat. The association constants for FBP of the high affinity and of the low affinity class are 2.3×106 and 4.1×105 respectively. The gel electrophoresis of the proteins extracted in sodium dodecyl sulfate (SDS) from sonicated vitelline coats has shown three fucosyl-containing polypeptide bands.

The spermatozoon of arthropoda XIII. The cell surface

In this paper the authors observe that the limiting membrane of the insect spermatozoon is asymmetrical. There is a “unit membrane” of the classical type, displaying an intense phosphatase activity, on the outer surface of which is a glycoprotein coat occurring in three structural patterns exhibiting progressive structural complexity. In Ceratitis and Drosophila (“fruit-fly model”), this coat is very thin and apparently amorphous; in Ctenocephalus (“flea” model), it is thicker and composed of transverse fibres; in Pezotettix and Aiolopus (the “locust” model), the coat is very thick and made up of short filaments or rodlets which are usually oriented normal to the sperm surface and grouped in tetrads with a rhomboidal arrangement.