Evaluation of contraceptive potential of a novel epididymal sperm protein SFP2 in a mouse model.

PROBLEM: Sperm flagellar protein 2 (SFP2), which was earlier identified using a novel combinatorial approach, was evaluated for its contraceptive potential in mice. METHOD OF STUDY: Male mice were actively immunized with two synthetic peptides of SFP2. Antipeptide antibody was characterized by Western blot and indirect immunofluorescence. Immune response was monitored, and mating studies were performed 6 and 22 weeks post-immunization. RESULT: Antibodies to the SFP2 peptide 1 recognized a doublet at 220- to 230-kDa region only in the epididymal protein extract. Peptide 1 antibody recognized the cognate protein on spermatozoa from mouse, rat, and human. Histological analysis of testis and epididymis of the immunized mice indicated no deleterious effect. Incubation of sperm with the immune sera of peptide 1 caused significant reduction in motility and viability but did not agglutinate sperm. Only synthetic peptide 1 gave rise to high-level antibodies in all the immunized mice, which on mating resulted in reduced fertility rate (20%) when compared with PBS control animals (100%). The antibody levels in the immunized males declined by 22 weeks post-immunization, resulting in 100% reinstatement of fertility. CONCLUSION: These data provide an experimental basis for the development of effective contraceptive vaccine based on new epididymal target.

Diagnostic and prognostic impact of serum HE4 detection in endometrial carcinoma patients.

BACKGROUND: To date, no good marker for screening or disease monitoring of endometrial cancer (EC) is available. The aims of this study were to investigate HE4 gene, protein expression and serum HE4 (sHE4) levels in a panel of ECs and normal endometria (NEs) and to correlate sHE4 with patient clinicopathological characteristics and prognosis. METHODS: Using quantitative real-time PCR we tested 46 ECs and 20 NEs for HE4 gene expression. Protein expression was analysed by immunohistochemistry on tissue microarrays in 153 ECs and 33 NEs. Pre-operative serum samples from 138 EC and 76 NE patients were analysed with HE4-EIA assay. Association between sHE4 and patient clinicopathological characteristics or outcome was evaluated. RESULTS: Protein and HE4 gene were significantly upregulated in EC tissues and sera, compared with controls. High sHE4 levels were significantly associated with worse EC clinical characteristics. By univariate survival analysis, high sHE4 levels significantly correlated with decreased overall survival, progression-free survival and disease-free survival, retaining their independent prognostic value on the poorly differentiated EC cohort. CONCLUSION: We demonstrate, for the first time, that high sHE4 levels correlates with an aggressive EC phenotype and may constitute an independent prognostic factor for poorly differentiated-ECs. Determination of sHE4 could be clinically useful in identifying high-risk EC patients for a more aggressive adjuvant therapy.

Use of yeast-secreted in vivo biotinylated recombinant antibodies (Biobodies) in bead-based ELISA.

PURPOSE: To measure circulating antigens, sandwich ELISA assays require two complementary affinity reagents. Mouse monoclonal antibodies (mAb) and polyclonal antibodies (pAb) are commonly used, but because their production is lengthy and costly, recombinant antibodies are emerging as an attractive alternative. EXPERIMENTAL DESIGN: We developed a new class of recombinant antibodies called biobodies (Bb) and compared them to mAb for use in serodiagnosis. Bbs were secreted biotinylated in vivo by diploid yeast and used as affinity reagents after Ni purification. Bead-based assays for HE4 and mesothelin were developed using Bbs in combination with pAbs (Bb/pAb assays). To assess precision, reproducibility studies were done using four runs of 16 replicates at six analyte levels for each marker. Pearson correlations and receiver-operator characteristic analyses were done in 214 patient serum samples to directly compare the Bb/pAb assays to mAb assays. Diagnostic performance of the Bb/pAb assay was further assessed in an expanded set of 336 ovarian cancer cases and controls. RESULTS: On average across analyte levels, Bb/pAb assays yielded within-run and between-run coefficients of variations of 11.7 and 23.8, respectively, for HE4 and 14.0 and 14.5, respectively, for mesothelin. In the subset (n = 214), Pearson correlations of 0.95 for HE4 and 0.92 for mesothelin were observed between mAb and Bb/pAb assays. The area under the curves for the mAb and Bb/pAb assays were not significantly different for HE4 (0.88 and 0.84, respectively; P = 0.20) or mesothelin (0.74 and 0.72, respectively; P = 0.38). CONCLUSION: Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead-based assays for use in large validation studies.

Molecular and functional characterization of the rabbit epididymal secretory protein 52, REP52.

As part of a systematic study of rabbit epididymal proteins involved in sperm maturation, we have identified and characterized a novel glycoprotein (rabbit epididymal secretory protein 52 [REP52]) of 52 kDa. REP52 is synthesized and secreted in a tissue-specific manner by the mid (region 6) and distal (region 7) corpus epididymidis and associates weakly with the sperm surface overlying the principal piece of the tail. Sequencing of cloned REP52 cDNA demonstrated that this protein represents a novel member of the highly conserved fibronectin type II (FN2) module protein family. The protein appears related but not homologous to ungulate seminal plasma proteins and is the first known example to be identified as a rabbit epididymal secretory protein. Consistent with other members of this protein family, REP52 possessed a high level of sequence identity within the FN2 module-encoding domains, but a highly variable N-terminal sequence that failed to show significant homology with published sequences. By analogy with evidence from studies of the ungulate seminal plasma proteins it is hypothesized that the tandemly arranged FN2 modules could facilitate the association of REP52 with sperm phosphatidylcholine residues on the outer leaflet of the sperm tail. It is also considered likely that these domains represent key elements for the function of this novel protein, a conclusion supported by the fact that antisera raised against the REP52 protein blocked in vitro fertilization in a concentration-dependent fashion.

Immunocontraceptive properties of recombinant sperm protein DE: implications for the development of novel contraceptives.

OBJECTIVE: To evaluate the immunocontraceptive properties of recombinant DE, a sperm epididymal protein involved in fertilization, via an experimental study in rats as a critical step toward the development of a human immunocontraceptive. DESIGN: In vivo study in rats. SETTING: Animal care facility of an academic research center. ANIMAL(S): Seventy-four 90-day-old Wistar male and female rats distributed into three groups. INTERVENTION(S): Animals received five injections (intramuscular and subcutaneous) of recombinant DE (recDE), native DE (nDE), or MBP (maltose-binding protein). At various times, animals were anesthetized and bled. MAIN OUTCOME MEASURE(S): Anti-DE levels and tissue specificity of sera were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot, respectively. Fertility was analyzed by natural mating. The testes and epididymides were analyzed by histology. RESULT(S): Recombinant DE raised an immune response with the same kinetics and higher anti-DE levels than that elicited by nDE. Sera against recDE recognized epitopes of DE that were different from those recognized by anti-nDE sera but specifically reacted with DE in epididymis and sperm without cross-reacting with other tissues tested. Male and female recDE-injected animals presented a statistically significant reduction in their fertility with no evidence of pathologic effects. CONCLUSION(S): Recombinant DE is able to both elicit a specific immune response and inhibit male and female fertility, supporting the use of this sperm epididymal protein for the development of an immunocontraceptive approach.

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating.

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K(d)=4.8 x 10(-9) M and K(d)=5.1 x 10(-9) M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

Beta-defensin 126 on the cell surface protects sperm from immunorecognition and binding of anti-sperm antibodies.

Beta-defensin 126 (DEFB126), formerly known as epididymal secretory protein 13.2 (ESP13.2), coats the entire primate sperm surface until completion of capacitation, and it is a candidate for providing immune protection in the female reproductive tract. To further examine the potential role of DEFB126 as a means of protection from immune recognition, cynomolgus macaque sperm were exposed to a number of treatments that are known to alter sperm surface coats, including capacitation. We used a novel in vivo assay to determine immune recognition: aldehyde-fixed whole sperm injections into rabbits. Following booster injections, immunoblot analyses of whole sperm prepared in various manners was conducted. On Days 60 and 80 post-initial immunization, the antisera showed a remarkably strong reaction to a single 34-36 kDa protein, which was shown to be DEFB126. Sera from rabbits that were immunized with sperm washed more rigorously using Percoll gradients showed an increase in the number and intensity of proteins recognized on whole sperm Western blots, although DEFB126 was still the major immune response. When capacitated sperm, from which most DEFB126 had been released, were used as the immunogen, there was a dramatic increase in the immune recognition to a variety of protein bands. Sperm treated with neuraminidase to remove sialic acid on DEFB126 before fixation were shown to still possess DEFB126, but lacked the sialic acid component of the glycoprotein. These sperm were as immunogenic as capacitated sperm even though the desialylated DEFB126 still covered the entire cell surface. These sperm lost their highly negative charge (the isoelectric point of DEFB126 shifted from pI 3.0 to pI 6.4). Experiments using different sperm plasma membrane protein-specific Igs showed that recognition did not occur when DEFB126 was present, but following capacitation these Igs readily recognized the exposed sperm membrane. Our data suggest that DEFB126 protects the entire primate sperm surface from immune recognition and that the sialic acid moieties are responsible for the cloaking characteristic of this unique glycoprotein.

Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization.

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.