ADP-ribosylation of Rho proteins inhibits sperm motility.

The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins. They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function.

OBJECTIVE: To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. DESIGN: Prospectively designed, blinded, internally controlled study. SETTING: Tertiary care academic center. PATIENTS: Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. INTERVENTIONS: Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3-6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3-7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-antiperoxidase method and diaminobenzidine as color reagent. RESULTS: A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3-6-treated hemizonae than patients with sperm factor. CONCLUSIONS: These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting.

Direct comparison of skin physiology in children and adults with bioengineering methods.

The aim of the study was to investigate whether the physiologic skin parameters of transepidermal water loss (TEWL), stratum corneum hydration (capacitance and conductance), dynamic stratum corneum hydration parameters (hygroscopicity and water-holding capacity), skin color (a* and L* axes; chromameter), cutaneous blood perfusion [laser Doppler flowmetry (LDF)], and pH value differ between a sample of 44 children [C] (average age 3.5 years) and a directly comparable sample of 44 adults (their parents) [P] (average age 34.6 years). The results can be described as follows: TEWL C: 6.2 g/m2/h, P: 5.4 g/m2/h; stratum corneum hydration, capacitance C: 75.4 AU, P: 76.1 AU; conductance C: 27.1 microS, P: 19.2 microS; hygroscopicity C: 129.0 AU, P: 132.7 AU; water-holding capacity: C: 127.7 AU, P: 127.6 AU; redness (a*) C: 7.31, P: 8.21; lightness (L*) C: 67.63, P: 66.36; LDF (%) C: 24.6, P: 18.7; pH value C: 4.91, P: 5.07. In comparison to the skin of the adult sample we investigated (the parents of the 44 children), the skin of the small child can be characterized in the following way: it has a significantly lower hygroscopicity, a lighter (higher L* values) and less red color (lower a* values), and an increased cutaneous blood perfusion (LDF).

Identification of mouse ZP3 protein in mammalian oocytes with antisera against synthetic ZP3 peptides.

The mouse zona pellucida (ZP) protein ZP3 plays an important role in the process of fertilization by mediating sperm binding and the acrosome reaction. ZP3 primary structures are highly conserved, as revealed by cDNA cloning. We raised antisera against synthetic peptides that are either conserved in the structure of ZP3 from different mammalian species (AS ZP3-5 and AS ZP3-6) or specific for mouse ZP3 (AS ZP3-2). In ovary sections, AS ZP3-2 revealed immunoreactivity only to mouse ZP. AS ZP3-5 and AS ZP3-6 reacted with mouse, human, rat, hamster, porcine, and bovine ZP proteins. In porcine oocytes, immunoreactive material was highly abundant in the ooplasm. Immunoblots showed that antiserum AS ZP3-5 recognized the mouse ZP3 protein. In porcine ZP preparations, AS ZP3-5 recognized a 53-kDa ZP protein. No reaction was observed with purified porcine ZP3 alpha or with ZP3 beta. Immunofluorescence studies revealed that AS ZP3-5 and AS ZP3-6 antibodies react with ZP of isolated porcine and human oocytes. Our results show that antisera against synthetic mouse ZP3 peptides can be used as markers for the identification of ZP3-like proteins in mammalian oocytes and might be useful tools for the evaluation of ZP integrity and ZP3 function.

Anti-ZP3 antibodies binding to the human zona pellucida: effect of oocyte-storage conditions.

PROBLEM: The zona pellucida protein 3 (ZP3) is a zona pellucida (ZP) glycoprotein crucially involved in fertilization. ZP3 plays a major role in sperm binding and induction of the acrosome reaction. In different species, ZP3 proteins differ in their primary structure as derived from cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertilization outcome under in vitro conditions. METHOD: In these studies, we used antisera generated against synthetic ZP3 peptides to compare antibody binding patterns to ZP with sperm-ZP binding capacity under different HZA conditions. RESULTS: Analysis of antibody binding to hemizonae derived from metaphase II human oocytes that were used either after refrigeration at 4 degrees C or stored in a hyperosmotic salt solution revealed a strong reaction with human ZP3. However, treatment of human oocytes using a protocol to freeze embryos with the addition of 1,2 propanediol drastically reduced binding of ZP3 antibodies to the hemizonae. Nevertheless, no significant difference of sperm binding occurred under HZA conditions when oocytes were refrigerated, salt-stored, or frozen with 1,2 propanediol. CONCLUSIONS: Our results indicate that the ZP3 protein backbone might be altered by 1,2 propanediol-treatment while the glycoprotein-receptor remains intact. We conclude that antisera against ZP3 peptides can be used as markers for the ZP3 protein backbone in human oocytes and might be useful tools for the evaluation of ZP3 protein integrity.