Chi-Yu Gregory Lee

Monoclonal antibodies to human sperm antigens -- II

As part of our continuous effort to elucidate the biochemical and immunological nature of human sperm surface antigens, monoclonal antibodies to human spermatozoa were generated by improved hybridoma techniques. Following immunizations with the membrane fraction of human spermatozoa and cell fusions, hybrid cells were cultured in a semi-solid HAT-selection medium to maximize the number of monoclones recovered. Subcultures were made in liquid phase 7 to 10 days after cell fusions by removing colonies from the initial medium. Based on the results of screening by microplate enzyme-linked immunoassay, 143 of 552 initial clones were found to secrete antibodies to human sperm antigens. More than one-hundred independently derived hybrid cell lines were established. Using indirect immunofluorescent procedures, 62 cell lines were shown to produce antibodies to surface antigens of human spermatozoa. Unique sperm antigens that react with monoclonal antibodies were identified by the SDS gel/protein blot radioimmunobinding method. Sperm agglutinating and immobilizing antibodies were exhibited by 4 and 15 hybrid cell lines, respectively. Fourteen of the monoclonal antibodies also exhibited cross-reactivity with methanol-fixed sperm cells of the rabbit or mouse or both whereas a reaction was not seen with viable sperm of these species. Generation of monoclonal antibodies against a wide spectrum of human sperm antigens should facilitate future investigations regarding immunologic-associated human infertility and fertility control.

Inhibitory effects of monoclonal sperm antibodies on the fertilization of mouse oocytes in vitro and in vivo

The inhibitory effects of monoclonal antibodies on the fertilization of mouse oocytes were evaluated in vitro and in vivo. Among the 40 sperm-specific monoclonal antibodies that had been examined, nine showed significant inhibition of the fertilization of mouse oocytes in vitro. MS 204 was shown to cause a high incidence of penetration of the zona pellucida of mouse eggs by multiple sperm, when the sperm concentration for insemination exceeds 1 X 10(5)/ml. This antibody prevented further penetration of the vitelline membrane by sperm. On the other hand, sperm penetration to zona was inhibited in the presence of MS 207. However, neither MS 204 nor MS 207 caused significant inhibition of penetration of zona-free mouse or hamster eggs by sperm. MS 204 and MS 207 were also found to inhibit the fertilization of mouse oocytes in vivo and embryo development in vitro, when superovulated mice were injected intraperitoneally with given doses of antibodies prior to the mating. Further in vitro culture of the recovered 2-cell oocytes revealed little or no further embryo development beyond two- to four-cell stages. In the controls, greater than 80% of the retrieved oocytes were fertilized and successively developed to the blastocyst stages in vitro when the ascites fluid from NS-1 cells was administered. Two of the monoclonal antibodies generated against human sperm antigens, HS 11 and HS 63, were shown to cross-react specifically with mouse sperm acrosomal antigens and also inhibited the fertilization of mouse oocytes in vitro and in vivo. The results of this study suggest that some monoclonal antibodies to sperm acrosomal antigens exhibit strongly inhibitory effects on the in vitro and in vivo fertilization of mouse oocytes as well as subsequent development of early embryos. As a comparative control, rabbit antisera against sperm-specific enzymes, lactate dehydrogenase-X and 3-phosphoglycerate kinase-2, showed little or no inhibition on the fertilization of mouse oocytes in vitro or in vivo or the subsequent embryo development.

Generation of mouse oocyte monoclonal isoantibodies: their effects and those of antisperm monoclonal antibodies on in vitro fertilization

Monoclonal isoantibodies to mouse oocyte antigens were generated by modified hybridoma techniques similar to those described for mouse sperm monoclonals. Following isoimmunization with mouse oocytes and cell fusion, hybrid cells were cultured initially in a semi-solid medium containing methylcellulose. Seven to ten days after cell fusion about 350 hybrid clones were recovered for subculture. By an indirect immunofluorescence assay using frozen or fresh mouse oocytes, twenty hybridomas were shown to produce antibodies that bind to various oocyte components including antigens of the zona pellucida. However, they did not cross-react with mouse spermatozoa or lymphocytes. A system was established to evaluate whether monoclonal antibodies to gamete-specific antigens have any inhibitory effects on the fertilization of mouse oocytes in vitro. A monoclonal antibody against zona antigen(s), ME 56, was shown to block fertilization of mouse oocytes via the inhibition of sperm binding to the zona pellucida. On the other hand, three out of four antibodies reacting with mouse sperm acrosomes were also inhibitory to mouse in vitro fertilization, perhaps mainly due to the inhibition of sperm acrosomal reactions. Using a sodium dodecylsulfate gel/protein blot radioimmunobinding method, the molecular weight of zona antigen(s) that react with ME 56 was determined to be in the range of 95,000, whereas that of the acrosomal antigen(s) reacting with the fertilization-inhibiting antibody, MS 207, was about 30.000. The results of this preliminary study suggest that monoclonal antibodies to certain gamete antigens can be a valuable tool for the analysis of sperm-egg interactions during the fertilization processes.

Monoclonal antibodies affecting sperm-zona binding and/or zona-induced acrosome reaction

The majority of anti-sperm monoclonal antibodies which were shown to inhibit in vitro fertilization in our laboratory react with antigens in the acrosomal region of spermatozoa. To elucidate the mechanism of human and/or mouse fertilization inhibition, the effects of these antibodies on mouse sperm-zona binding and mouse zona-induced acrosome reaction in vitro were studied. Among these antibodies, MS-4 and MS-7 were shown to react partially with uncapacitated mouse sperm, and to react more with capacitated mouse sperm, whereas HS-9, HS-11 and HS-63 react only with capacitated and acrosome-intact human or mouse sperm. HS-63 and MS-4 were shown to inhibit zona-induced acrosome reaction significantly, but not the sperm-zona binding. On the other hand, HS-9, HS-11 and MS-7 were shown to inhibit both sperm-zona binding and zona-induced acrosome reaction. The results of this study suggest that fertilization inhibition caused by these antibodies could result from that of the initial sperm-zona binding and/or zona-induced acrosome reaction.

Developmental studies of sperm surface antigens using sperm-specific monoclonal antibodies

Sperm-specific monoclonal antibodies generated against mouse sperm isoantigens were used to analyze the developmental expressions of sperm surface antigens during different stages of spermatogenesis. Indirect immunofluorescent assays using the freshly isolated testicular cells from mature and immature mice revealed that a number of monoclonal antibodies did not stain the surface of spermatogenic cells in testis. Instead these antibodies reacted with the sperm surface antigens in testis and/or epididymis. Further analysis was performed using frozen testicular sections from mice of day 14 to day 30 after birth. It was generally observed that a significant number of these antibodies reacted with the cytoplasmic components of spermatogenic cells in testis at the postmeiotic stages (e.g. day 22 after birth). After meiosis, the percentage of seminiferous tubules that were stained by immunofluorescence was found to increase with the ages of the mice. The results of this study suggest that some cytoplasmic components (especially acrosomal) of spermatogenic cells are expressed postmeiotically in testis, but later translocated to the sperm surface during very late stages of spermatogenesis.

Analysis of mouse sperm isoantigens using specific monoclonal antibodies

ABSTRACT: Female mice were isoimmunized with homologous spermatozoa of the same strain. Hybrid cells that secrete monoclonal antibodies to mouse sperm isoantigens were generated by modified hybridoma techniques using a semi‐solid Iscoves modified Dulbeccos medium containing methylcellulose for the initial cloning. Out of more than 1,000 colonies that were initially recovered for subculture, 246 were shown to produce antibodies reacting with various cytological regions of mouse spermatozoa, when methanol‐fixed sperm were employed in an indirect immunofluorescent assay. More than 75% of the generated monoclonal isoantibodies were shown to bind the acrosomal regions of mouse spermatozoa. Some were found to cross‐react with spermatozoa from other mammalian species including those of human, rabbit, rat, and guinea pig. However, none were shown to cross‐react with mouse lymphocytes. Two‐thirds of the generated monoclonal antibodies can also bind live mouse spermatozoa. By an immunohistochemical technique using testicular sections, some of these monoclonal antibodies were shown to react with specific antigens expressed during different stages of spermatogenesis. It is concluded that these mouse sperm isoantigens are sperm‐specific and appear uniquely during spermatogenesis. Monoclonal isoantibodies produced in the present study may have potential applications regarding the investigations of sperm iso‐ or autoimmunity, spermatogenesis, and fertility control.

Studies of a sperm/placenta cross-reacting antigen, STX-10

A monoclonal antibody, HSA-10 initially produced against acrosome-reacted human sperm was also shown to cross-react with human placenta/trophoblast. Transmission electron microscopy, as well as indirect immunofluorescent assay, demonstrated that HSA-10 was found to react with antigen on the inner acrosome of human sperm. The cognate antigen, designated as STX-10, was found to exist as an aggregate in the native form when analyzed by Sephacryl S-300 gel filtration chromatography. When purified by HSA-10-immunoaffinity chromatography from human placenta extract, STX-10 was found to be predominantly a group of glycoproteins with a subunit molecular mass in the range of 75 +/- 5 kDa, whereas an additional group of three proteins with subunit molecular mass less than 20 kDa were copurified from human sperm extract. A sandwich enzyme immunoassay was designed to quantitatively determine the immunoactivity of STX-10 in solution, using HSA-10 monoclonal antibody for coating and for signal detection via enzyme conjugation. Based on this assay, it was found that STX-10 could be detected only in human sperm and placenta extract, but not in any other human somatic tissues, such as serum, brain, heart, muscle, kidney and liver. The immunoactivity of STX-10 was found to be sensitive to proteolytic digestion, low pH, in the presence of reducing agent, but resistant to treatment with sodium periodate. This observation suggests that HSA-10 specific epitope is a peptide in nature and not a carbohydrate moiety. Results of antifertility studies revealed that HSA-10 significantly inhibited human sperm penetration to zona-free hamster ova. Thus, the results of this study are consistent with those of WHO Workshop evaluations that seem to suggest that STX-10 is a highly gamete-specific antigen localized on the inner acrosome of human sperm and in human trophoblast/placenta. Therefore, it may play an important role during human fertilization and embryo development.

Purification and characterization of a sperm antigen recognized by HSA-5 monoclonal antibody.

Among the monoclonal antibodies generated against acrosome-reacted human sperm, HSA-5 was shown to react with a sperm antigen localized predominantly to the equatorial region of the acrosome of human sperm and to the head and tail of mouse sperm. This antibody reacted with the methanol-fixed sperm, but not with fresh live sperm. When purified by immunoaffinity column, a major protein band with a molecular mass of approximately 100 kDa on SDS gel was isolated from fresh human sperm extract. The immunospecificity of isolated human sperm protein to this monoclonal antibody was verified by enzyme-linked immunosorbent assay and Western blot analysis. This antigen, designated as HSA-5, was susceptible to proteolytic degradation and revealed multiple immunoreactive bands in Western blot analyses of some preparations. Mouse sperm homogenates showed a similar polymorphic pattern to that of human samples. The tissue specificity of this antigen was examined immunohistochemically using various mouse and human tissues. HSA-5 did not cross-react with any other tissues except for sperm in adult testes and epididymis. This antibody also showed no binding activity to testicular tissue sections from mice of 13 and 21 days of age. The results of our study suggest that the sperm antigen recognized by HSA-5 monoclonal antibody is a differentiation antigen, which is expressed postmeiotically in testicular sperm but not in any somatic tissues.

Monoclonal antibodies as direct probes for human sperm acrosome reaction.

PROBLEM: To develop simple and rapid assay procedures for determining human sperm acrosome reaction under various experimental conditions.

Molecular nature of a sperm acrosomal antigen recognized by HS-13 monoclonal antibody

Among the numerous monoclonal antibodies generated against human sperm antigens, HS-13 monoclonal antibody was shown to react with an intra-acrosomal antigen from human, mouse and rat. In this study, HS-13 was used as the affinity ligand for the purification of the cognate antigen from human sperm by immunoaffinity chromatography. The purified cognate antigen from human sperm, designated as HSAg-13, was found to be a protein with a molecular weight of approximately 80 kDa on SDS-PAGE in the presence of reducing reagents. This monoclonal antibody was used as the probe to study the tissue distributions and developmental expression of the cognate antigen from human, mouse and rat by immunohistochemical assays. It was concluded that the antigen recognized by HS-13 antibody is highly sperm specific and found only in sperm and mature testis, but not in any other somatic tissues examined in human and mouse. The antigen was shown to be expressed at the postmeiotic stages of spermatogenesis in mouse and rat. By using indirect immunofluorescent staining assay, HS-13 was shown to react only with the methanol-fixed acrosome-intact sperm but not with the live sperm. Following calcium ionophore A23187 treatment, acrosome-reacted sperm showed either negative staining or residual staining in the equatorial region, suggesting the intra-acrosomal location of HSAg-13. The spontaneous acrosome reaction following overnight incubation in BWW medium resulted in a statistically significant decrease of antibody-stained human sperm. In view of excellent correlations for the scoring of acrosome-intact sperm with that of fluorescence-labeled Pisum sativum agglutinin (PSA) probe, HS-13 monoclonal antibody can be routinely used for monitoring sperm capacitation and acrosome reaction.