Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model.

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Functionalized bridged silsesquioxane-based nanostructured microspheres: ultrasound-assisted synthesis and in vitro cytotoxicity characterization.

Different kinds of polymers have been employed in medicine as biomaterials for different purposes. In recent years, considerable attention has been focused on the development of new drug-delivery systems in order to increase bio-availability, sustain, localize and target drug action in the human body. The versatility of the sol-gel processing to synthesize nanostructured materials and the possibility of incorporating organic molecules into the matrix of the final hybrid material, represent a novel and attractive path to the synthesis of new functionalized hybrid biomaterials with advanced properties. In this work, acetylsalicylic acid (ASA)-functionalized hybrid microspheres based on bridged silsesquioxanes synthesized via ultrasound-assisted sol-gel processing, were characterized. An investigation concerning the cytotoxic response of these new microspheres on CHO-K1 cells was accomplished based on ISO 10993-5 standard (Biological Evaluation of Medical Devices). Microspheres incorporating ASA showed a cytotoxic effect when pure extracts of the microspheres were analyzed, however, they strongly diminished their cytotoxicity as the extracts were diluted. When a 10% concentration extract was employed, hybrid microspheres were shown to be non cytotoxic. These results are promising for considering these novel functionalized organic-inorganic microspheres as potential drug-carriers to be employed in drug delivery-related applications.

Antiacrosin antibodies and infertility. II. Gene immunization with human proacrosin to assess the effect of immunity toward proacrosin/acrosin upon protein activities and animal fertility.

OBJECTIVE: To assess the effect of antiacrosin antibodies upon proacrosin/acrosin activities and animal fertility. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): A gene immunization (GI) model was developed; mice were injected with the sequence encoding human proacrosin (h-proacrosin), cloned in an expression vector. INTERVENTION(S): Subcloning of h-proacrosin in a eukaryotic expression vector (promoter, CMV; leader sequence, alpha-1 antitrypsin; pSF2-Acro); GI of female mice with this plasmid. MAIN OUTCOME MEASURE(S): The following parameters were evaluated: [1] adequate conditions for GI protocols, [2] humoral response to GI with pSF2-Acro, [3] protein regions recognized by the antibodies, and [4] effect of antibodies upon proacrosin/acrosin-ZPA binding and amidase activity, and animal fertility. RESULT(S): Conditions of female mice GI with the proacrosin sequence were established (plasmid purification with anion exchange chromatography and 40 microg of pSF2-Acro per dose) to trigger an immune response, reaching maximum levels at week 9 after the first injection. Antibodies produced by GI recognized human and mouse sperm acrosin systems, inhibited human proacrosin/acrosin interaction with recombinant human ZPA and protease activity, and negatively affected mouse IVF and early embryonic development. In addition, mice immunized with SF2-Acro exhibited a significantly lower size of fetuses. CONCLUSION(S): Antiacrosin antibodies developed by using GI inhibit human proacrosin/acrosin activities and impair mouse fertility.

Further evidence on the role of heparan sulfate as protamine acceptor during the decondensation of human spermatozoa.

BACKGROUND: Human spermatozoa decondense in vitro upon exposure to heparin and glutathione. Glutathione is also the disulfide bond reducer in vivo, and heparan sulfate, a functional analogue of heparin, has been proposed as the protamine acceptor. The aim of this study was to evaluate the decondensing ability of chemically modified heparins and different glycosaminoglycans (GAGs) on isolated sperm nuclei in vitro, and to analyse the possible role of different GAGs as protamine acceptors. METHODS: Capacitated spermatozoa and isolated sperm nuclei from normospermic semen samples were decondensed in the presence of heparin (or its equivalent) and glutathione. After fixation with glutaraldehyde, the percentage of decondensed spermatozoa and nuclei was determined under phase-contrast. Proteins were extracted from sperm nuclei previously incubated in the presence of gluhathione and different GAGs by incubation with urea-beta-meracptoethanol-NaCl, and analysed by acid polyacrylamide gel electrophoresis. RESULTS: The ability of desulfated heparins and other GAGs to decondense isolated nuclei mirrored exactly the decondensation of capacitated spermatozoa, the only difference being the level of maximum decondensation achieved. Heparan sulfate and heparin, but not other GAGs, were able to release protamines from sperm chromatin. CONCLUSIONS: Heparan sulfate could be functioning as protamine acceptor in vivo during human sperm nuclear decondensation.

Exogenous interferon-gamma alters murine inner cell mass and trophoblast development. Effect on the expression of ErbB1, ErbB4 and heparan sulfate proteoglycan (perlecan).

Implantation is a crucial event in human pregnancy. The participation of cytokines in the implantation process has been widely documented, although the role of many of these molecules is still a matter of controversy. In a previous report from our laboratory, we demonstrated that addition of interferon-gamma to the culture medium produces deleterious effects on mouse embryo development. In this study we investigated the effect of this cytokine on outgrowing embryo morphology and on the expression of epidermal growth factor receptors (ErbBs) and heparan sulfate proteoglycan (perlecan) in mouse embryos cultured in vitro. Morphological assessment of inner cell mass and trophoblast development was carried on in-situ fixed and stained outgrowths. Localization of ErbB1, ErbB4 and perlecan on pre- and peri-implantation embryos was investigated by immunocytochemistry. Addition of interferon-gamma produced a deleterious effect on both inner cell mass and trophoblast morphology. Immunostaining demonstrated that ErbB1, ErbB4 and perlecan are present on pre-implantation embryos and blastocysts; interferon-gamma altered the expression of ErbB4 and Perlecan at the blastocyst stage. We propose that the effects produced by this cytokine could be related to the altered acquisition of adhesion competence and low implantation rates observed in certain reproductive immunological disorders.

Heparan sulphate: a putative decondensing agent for human spermatozoa in vivo.

BACKGROUND: Human sperm decondense in vitro upon exposure to heparin and glutathione. The aim of this study was to evaluate whether this decondensing ability of heparin in vitro is related to structural characteristics of the molecule and to test the in-vitro decondensing ability of other glycosaminoglycans. METHODS: Capacitated sperm obtained from normospermic semen samples were decondensed in the presence of heparin (or its equivalent) and glutathione. After fixation with glutaraldehyde, the percentage of decondensed sperm was determined under phase contrast. RESULTS: The decondensing ability of heparin was related to sulphation characteristics of the molecule: heparin, O-desulphated heparin and N-desulphated-N-acetylated heparin had similar decondensing abilities; N-desulphated was less active and O/N-desulphated-N-acetylated heparin was completely inactive. On the other hand, the decondensing ability of heparin was not affected by molecular weight, within the range 3000-18,000 kDa. When the decondensing ability of different glycosaminoglycans was tested, heparin and heparin sulphate were equally active, while chondroitin sulphate and hyaluronic acid were completely inactive and dermatan sulphate was slightly active. CONCLUSIONS: Our results indicate that heparin's decondensing ability in vitro is related to sulphation characteristics of the molecule and suggest that heparan sulphate, a structural analogue of heparin, could be a sperm-decondensing agent in vivo.