Carolina Veaute

Acrosin antibodies and infertility. I. Detection of antibodies towards proacrosin/acrosin in women consulting for infertility and evaluation of their effects upon the sperm protease activities

OBJECTIVE To detect the presence of antibodies to the proacrosin/acrosin system and to evaluate their effect on the sperm acrosomal protein activities in women consulting for infertility. DESIGN Retrospective study. SETTING Basic research laboratory. PATIENT(S) Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10) and recombinant human zona pellucida (ZP) glycoprotein A( *) (rec-hZPA). INTERVENTION(S) Development of an ELISA-Acro to test for antiacrosin antibodies using Rec-40 and truncated acrosin proteins as antigens. MAIN OUTCOME MEASURE(S) Evaluation of: 1) the presence of antiacrosin antibodies; 2) the protein regions recognized by the antibodies; 3) the relationship between antiacrosin antibodies and surface antisperm antibodies (ASA) identified by the immunobead binding test (IBT); and 4) the effect of antiacrosin antibodies upon proacrosin/acrosin binding activity to ZPA and acrosin amidase activity. RESULT(S) Antiacrosin antibodies were detected in sera from 34 of 179 women (19%). Detection of ASA by the IBT resulted in a similar incidence (36 of 179, 20%), although only six of them showed correspondence between both assays; five of these six sera were IBT-positive IgGs to the sperm head. Antiacrosin antibodies directed toward different protein regions inhibited proacrosin binding activity to rec-hZPA as well as its activation and acrosin amidase activity in protein sperm extracts. CONCLUSION(S) Antiacrosin antibodies are present in sera of women consulting for infertility in both IBT-positive and IBT-negative samples, and they affect proacrosin/acrosin activities.

Antisperm Antibodies: Invaluable Tools Toward the Identification of Sperm Proteins Involved in Fertilization

The identification of sperm proteins involved in fertilization has been the subject of numerous investigations. Much interest has been dedicated to naturally occurring antisperm antibodies (ASA) and their impact in fertility. Their presence in men and women has been associated with 2–50% of infertility cases. ASA may impair pre‐ and post‐fertilization steps. Experimental models have been developed using sperm proteins as immunogens to evaluate their involvement in sperm function. Our team has pursued investigations to assess ASA presence in biological fluids from patients consulting for infertility and their effect on fertilization. We found ASA in follicular fluids with ability of inducing the acrosome reaction and blocking sperm–zona pellucida interaction and used them to identify sperm entities involved in these events. We generated and utilized antibodies against proacrosin/acrosin to characterize the sperm protease system. We implemented an ELISA to detect proacrosin/acrosin antibodies in human sera and evaluated their impact upon fertility by developing in vitro assays and a gene immunization model. This review presents a summary of ASA history, etiology, current approaches for detection and effects upon fertility. ASA (naturally occurring, generated by animal immunization and/or of commercial origin) are invaluable tools to understand the molecular basis of fertilization, better diagnose/treat immunoinfertility and develop immunocontraceptive methods.

Anti-human proacrosin antibody inhibits the zona pellucida (ZP)–induced acrosome reaction of ZP-bound spermatozoa

The anti-acrosin monoclonal antibody AcrC5F10 inhibited proacrosin activation, proacrosin-human zona pellucida glycoprotein A (ZPA) binding, and the zona pellucida (ZP)-induced acrosome reaction of the ZP-bound spermatozoa but had no significant effect on sperm-ZP binding. These results suggest that proacrosin-acrosin may play an important role in the ZP-induced acrosome reaction of spermatozoa after primary binding to the ZP.

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.

DNA Immunization Against Proacrosin Impairs Fertility in Male Mice

Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility.

In silico prediction of T- and B-cell epitopes in PmpD: First step towards to the design of a Chlamydia trachomatis vaccine

Background Chlamydia trachomatis is the most common sexually transmitted bacterial infection globally. Currently, there are no vaccines available despite the efforts made to develop a protective one. Polymorphic membrane protein D (PmpD) is an attractive immunogen candidate as it is conserved among strains and it is target of neutralizing antibodies. However, its high molecular weight and its complex structure make it difficult to handle by recombinant DNA techniques. Our aim is to predict B-cell and T-cell epitopes of PmpD. Method A sequence (Genbank AAK69391.2) having 99–100% identity with various serovars of C. trachomatis was used for predictions. NetMHC and NetMHCII were used for T-cell epitope linked to MHC I or MHC II alleles prediction, respectively. BepiPred predicted linear B-cell epitopes. For three dimensional epitopes, PmpD was homology-modeled by Raptor X. Surface epitopes were predicted on its globular structure using DiscoTope. Results NetMHC predicted 271 T-cell epitopes of 9-12aa with weak affinity, and 70 with strong affinity to MHC I molecules. NetMHCII predicted 2903 T-cell epitopes of 15aa with weak affinity, and 742 with strong affinity to MHC II molecules. Twenty four linear B-cell epitopes were predicted. Raptor X was able to model 91% of the three-dimensional structure whereas 57 residues of discontinuous epitopes were suggested by DiscoTope. Six regions containing B-cell and T-cell epitopes were identified by at least two predictors. Conclusions PmpD has potential B-cell and T-cell epitopes distributed throughout the sequence. Thus, several fragments were identified as valuable candidates for subunit vaccines against C. trachomatis.

Immune mediators associated to male infertility in a mouse model of DNA immunization with the sperm protease proacrosin

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-β1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.