Electrophoretic and immunocytochemical analysis of Hsp72 and Hsp73 expression in heat-stressed mouse testis and epididymis.

OBJECTIVE: One of the most profound events in stressed cells is the synthesis of a highly conserved family of proteins, the 'heat shock proteins' (Hsp). The Hsp70 family is the most diverse, and includes constitutive as well as stress-inducible proteins with overlapping or unique functions in different cell compartments. Elucidation of Hsp70 expression during different stages of spermatogenesis and maturation of germ cells is of particular interest due to their high sensitivity to heat treatment. STUDY DESIGN: Expression of the main isoforms of the Hsp70 family (constitutive Hsp73 and stress-inducible Hsp72) was determined in normal and heat-stressed mouse testes and epididymis from sexually mature (60-day-old) mice during spermatogenesis and maturation of germ cells. Immunocytochemical analysis and one- and two-dimensional gel electrophoresis were used to separate mouse testicular and epididymal proteins from saline extracts, followed by Western blotting. RESULTS: Using a polyclonal anti-Hsp70 antibody that recognizes both isoforms, inducible Hsp72 expression, was demonstrated immunocytochemically only in heat-stressed tissues, while a high level of constitutive Hsp73 isoform expression was found in both normal and heat-stressed mouse male reproductive tissues. Morphological studies have shown that round and elongated spermatids in the testes, as well as all segments of the epididymis, are most sensitive to heat stress. In the epididymis, the reaction was localized in different cell compartments. CONCLUSION: In heat stress conditions, Hsp73 is mobilized to prevent apoptosis in the testes and epididymis, and assists Hsp72 in the repair of stress-altered protein conformations.

Identification and isolation of boar sperm specific antigens with potential role in sperm-egg interaction.

Investigations on specific and functionally active sperm antigens would bring about the elucidation of the mechanisms of gamete interaction and help the search to new approaches for prognosis and regulation of fertility. Previously, we have produced a polyclonal rabbit anti-boar spermatozoa antibody (RABSA) that might affect the fertilizing capacity of boar spermatozoa. The sperm specificity of RABSA was demonstrated by double immunodiffusion, immunoelectrophoresis and ELISA against boar spermatozoa, as well as against saline extracts of boar reproductive and somatic organs. Using indirect immunofluorescence (IIF) test, here we provide evidence that RABSA stained the acrosomes of ejaculated and capacitated boar and human spermatozoa, the fluorescence being intensified on the equatorial region after the acrosome reaction. The RABSA cognate antigen/s is a subject of interest because of their specific localization in sperm structures, which is shown to be a binding and/or fusion competence region. Using ion-exchange (Heparin-Sepharose) chromatography, we eluted an antigen with molecular mass 60 kDa (Ag60) in SDS-PAGE from NP40 extracts of capacitated boar spermatozoa. In Western blot, RABSA recognized specifically this antigen. The Ag60 did not affect the sperm-ligand activity of zona pellucida in a porcine sperm-zona binding assay. IIF experiments showed that zona-free porcine oocytes preincubated with Ag60 and RABSA presented fluorescent labeling over the entire egg surface. The biological and IIF experiments provide evidence supporting the involvement of Ag60 in functional steps required for sperm-egg binding and/or fusion, but not sperm-zona pellucida binding.

Role of a capacitation-related protein on some sperm functional parameters.

In previous studies a series of Mabs against boar capacitated sperm have been produced. One of these Mabs--4B12--was found to recognize a surface membrane-associated protein located in the acrosome portion of the spermatozoa that became accessible to antibody after capacitation. In biological experiments it was shown that Mab 4B12 significantly inhibited boar sperm-porcine ZP binding. In attempts to investigate the mechanisms by which Mab 4B12 affected sperm-ZP binding, the role of the cognate protein on some functional parameters such as sperm motility and ability of the capacitated spermatozoa to undergo AR was studied. Experimental models of premature AR and AR physiologically induced with ZP were applied to study the effect of Mab 4B12 on boar sperm AR using PSA staining to estimate the acrosome-reacted state of spermatozoa. Sperm motility characteristics were determined by the time-exposure photokinesigraphic method. The results obtained in the present study, together with previously established inhibition of sperm-ZP binding by Mab 4B12, documented the participation of the 4B12 protein in primary sperm-ZP binding. The protein is not connected with sperm motility and secondary sperm-ZP binding.

Monoclonal antibodies to intra-acrosomal proteins inhibit gamete binding in vitro.

The sperm-zona pellucida-binding assay in vitro was used as a functional test for zona pellucida-binding ability of boar spermatozoa after co-incubation with monoclonal antibodies against intra-acrosomal proteins. The effect of monoclonal antibodies ACR.2 against boar acrosin (55, 53, 45 and 38 kDa), and Hs-8 against boar intra-acrosomal protein (230, 110, 88, 60, 48 kDa) on boar spermatozoa-porcine oocyte binding was examined. The sperm-zona pellucida-binding was reduced when medium was supplemented with monoclonal antibodies during sperm-oocyte co-incubation, but not when capacitated spermatozoa were pretreated with monoclonal antibodies before incubation with oocytes. Our results show that the monoclonal antibodies (ACR.2, Hs-8) against intra-acrosomal proteins reduce the secondary sperm-zona pellucida-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization.

Isolation and biological characterization of boar sperm capacitation-related antigen.

PROBLEM: Monoclonal anti-capacitated sperm antibody has been used as a probe to identify, isolate, and characterize specific, fertilization-related antigen with some characteristic features that point to its possible significance in immunocontraception. METHOD OF STUDY: Fast protein liquid chromatography (FPLC), enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectrofocusing were used for isolation, immunochemical and physicochemical characterization of a monoclonal antibody (mAb) 1F10 cognate antigen. Sperm-zona pellucida binding and hemizona assay were used for testing the biological roles of mAb 1F10 and Ag 1F10 in boar and human fertilization processes. RESULTS: The Ag 1F10 was found to be eluted in the eighth protein peak of FPLC-fractionated Nonidet P40 (NP40) extracts of capacitated boar spermatozoa. The SDS-PAGE and immunoelectrofocusing experiments showed that Ag 1F10 is a protein composed of a single peptide chain with a relative molecular mass of 68/70 kDa and an isoelectric point of 3.5. It was demonstrated that the zona binding activity of spermatozoa preincubated in the presence of mAb 1F10 was significantly inhibited both in porcine and human in vitro fertilization (IVF) systems. A dose-dependent manner of inhibition of the sperm/ligand activities of porcine and human zona pellucida was observed when the effect of purified Ag 1F10 was investigated by its preincubation with zona pellucida. CONCLUSIONS: It is assumed that the protein bearing the epitope recognized by mAb 1F10 may be accepted as one of the molecules with receptor function in sperm-zona pellucida interaction during fertilization.

Zona pellucida autoantibodies in women undergoing ART.

The presence of ZP autoantibodies in serum and Ffl samples of 109 women attending the ART Center in Kiev was investigated using IIF and ELISA. Positive serum and Ffl samples examined by both methods were found in 20 (18.34%) and in 19 (17.43%) patients respectively; 31 (28.44%) serum samples and 33 (30.27 %) Ffl samples analyzed by IIF were positive; of the samples analyzed by ELISA 21 (19.26%) and 20 (18.34%), respectively, were positive. No relationship was found between ZP autoantibody incidence and the type and cause of infertility. A significant prevalence of ZP autoantibodies detected by ELISA in Ffl was found in patients with fertilization failure (39.13%) and with low fertilization rate (42.85%) when compared to patients with middle fertilization rate (5.71%) and high fertilization rate (8.1%). The clinical significance of ZP autoantibodies in Ffls for in vitro fertilization outcome was suggested.

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro.

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.

Human sperm surface glycoprotein involved in sperm-zona pellucida interaction.

To identify peptide-specific antibodies which define sperm surface antigens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited proteolytic cleavage under reducing conditions prior to immunization. A total of 13.7% of the hybrid clones secreted antibodies which reacted with deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-dependent manner, and totally blocked sperm penetration in vitro. This inhibition did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome reaction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and binding of lectins, did not abrogate the reactivity of mAb 4A8 to the cognate epitope whereas antibody binding was precluded upon digestion with proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polypeptides, a major glycoprotein of M(r) 78 kDa and less prominent bands of M(r) 56 and 44 kDa. Immunocytochemical staining of a number of human reproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human spermatozoa was demonstrated by immunofluorescence, although on undigested spermatozoa the antigen epitope was less accessible. Upon capacitation the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced peripheral immunostaining of the sperm head was accentuated after DTT/trypsin treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that the protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and interaction with the human zona pellucida.

Monoclonal antibodies reacting against capacitated but not with freshly ejaculated boar spermatozoa.

PROBLEM: The involvement of individual sperm proteins in differentiation of antigenically specific and functionally defined regions on sperm membrane has not yet been completely elucidated. METHOD: BALB/c mice were immunized with live capacitated boar spermatozoa and used for production of monoclonal antibodies (MAbs). ELISA, IIF, SDS-PAGE, IVF, and cytologic methods were used for selection and biological characterization of MAbs as well as for identification of corresponding antigens. RESULTS: MAb1F10, MAb2E2, and MAb4B12 react with antigens in the acrosome portion of live capacitated spermatozoa, MAb 1F10 reacted with human sperm cells along with those from bull, ram, mouse, dog, whereas MAb2E2--with mouse's spermatozoa and MAB4B12-with bull's, mouse's, and dog's spermatozoa. Some glycolytic enzymes seemed to reduce mildly the reactions of the MAbs with enzyme treated sperm cells; proteolytic enzymes eliminated the binding of MAbs to the sperm acrosome. These MAbs have no sperm agglutinating and/or sperm-immobilizing activities and reduced the number of spermatozoa binding to zona pellucida. CONCLUSIONS: MAb1F10, MAb2E2, and MAb4B12 seemed to recognize membrane associated antigens with potential role in the initial stages of fertilization, specific for capacitated but not for freshly ejaculated spermatozoa.

Contraceptive potential of porcine zona pellucida in cats.

The contraceptive potential of solubilized porcine zona pellucida (spZP) was studied in 2 groups of cats after active immunization using slightly different protocols. Cats from Group 1 (n = 3) were immunized with a total of 300 8g spZP divided in 4 s.c. multisite injections (each of 37.5 8g) given at 10 day intervals followed by a booster 150 days after the initial immunization. Cats from Group 2 (n = 5) were immunized with a total of 400 8g spZP divided in 4 i.m. injections (each of 50 8g) given at 2 wk intervals followed by a booster 92 days after initial immunization. Immunogen was emulsified in Complete Freund Adjuvant for the first dose and in Incomplete Freund Adjuvant for the following 3 doses. The respective controls were immunized in the same manner using only adjuvant and PBS. Immunofluorescence studies showed intense fluorescence on the zona pellucida (ZP) of the oocytes isolated from immunized cats, as well as on the ZP of porcine and cat oocytes preincubated in immune sera. Sera from cats immunized with spZP inhibited in vitro binding was demonstrated in oocytes isolated from immunized group 1 cats. In vivo fertility data in Group 2 cats revealed that only 1 of 5 cats became pregnant, the one with the lowest anti-spZP titer. The results from the experiments reported above, suggest that in this preliminary study spZP can elicit antibodies with contraceptive potential in actively immunized female cats.

Zona-penetration in vitro test for evaluating boar sperm fertility.

We investigated the capacity of porcine sperm-zona binding and penetration by using bioassay to differentiate between spermatozoa from fertile and subfertile boars. Semen was collected from Large White boars grouped into categories of fertile and subfertile (n=5 per each group) according to the results of artificial insemination. Boars in both groups showed similarly hyperactivated sperm motility at insemination (44.72 and 43.03% respectively) regardless of the lower percentage of progressive motility observed in the ejaculates of subfertile boars. At in vitro insemination, a high proportion of the sperm population (43.76%) in the subfertile boars was without acrosomes, while in the fertile boars this proportion was only 24.35%. The sperm penetration rate of fertile boars reached 66.03% while that of subfertile boars was only 25.08%. In conclusion, the results of our study showed that the penetration rate by boar spermatozoa of the zona pellucida can be used to predict fertility and/or as an in vitro standard for describing porcine semen characteristics.

Identification and characterization of human acrosomal antigen defined by a monoclonal antibody with blocking effect on in vitro fertilization.

A monoclonal anti-human sperm antibody (Mab 1A1) has been produced by fusion of myeloma cells with splenocytes from a BALB/c mouse immunized with in vitro capacitated human spermatozoa. Immunofluorescence studies with Mab 1A1 show that it recognizes an antigen(s) (Ag 1A1) which is located in the acrosome of human spermatozoa. As shown by Western blotting experiments, 1A1 antigen represents a family of proteins with Mr ranging from 20 kDa to 34 kDa. Immunofluorescence observations on epitope exposure and location suggest that during in vitro capacitation of human spermatozoa, Mab 1A1 epitope-bearing molecules are concentrated in regularly arranged granules in the acrosome. After long-term incubation the epitope is exposed on the apical acrosome surface exhibiting a spot-like arrangement. The 1A1 epitope is widely distributed among mammalian species: boar, ram, mouse and rat acrosome is intensively stained by Mab 1A1. The antibody inhibits in vitro fertilization mainly by blocking sperm attachment to and penetration through the zona pellucida when included in the medium for the in vitro fertilization of mouse, porcine and human oocytes.