Isabel A. Lea

Autoimmunogenicity of the human sperm protein Sp17 in vasectomized men and identification of linear B cell epitopes.

OBJECTIVE To assess whether human sera positive for antisperm antibodies have detectable levels of Sp17 autoantibodies and to determine the linear B cell epitopes to which these are raised for both native and recombinant Sp17. DESIGN Enzyme-linked immunoaborbent assays were performed against recombinant HSp17 on 15 serum samples from prevasovasostomy and postvasovasostomy patients. Positive sera then were used in mimotope analyses to determine HSp17 immunodominant linear B cell epitopes. These were compared with the linear B cell epitopes of recombinant HSp17. SETTING University research laboratory. PATIENT(S) Fifteen vasectomized or vasovasostomized men. MAIN OUTCOME MEASURE(S) Serum antibody reactivity to human Sp17. RESULT(S) Sera from vasectomized and vasovasostomized men exhibit Sp17 antibodies raised predominantly to two immunodominant linear B cell epitopes (amino acids 4 to 19 and amino acids 118 to 127), which differed from those of recombinant HSp17 (amino acids 52 to 79 and amino acids 124 to 136). CONCLUSION(S) The results show that Sp17 is an antigen to which vasectomized men raise autoantibodies. Two linear B cell epitopes predominate in native Sp17 and these differ from (but overlap with) those of the bacterially expressed recombinant protein.

Zonadhesin: Characterization, Localization, and Zona Pellucida Binding

Abstract Zonadhesin is a multiple-domain transmembrane protein that is believed to function as a sperm-zona pellucida binding protein. In this study we sequenced zonadhesin from rabbit testis and analyzed its processing, expression, localization, and zona pellucida binding. We show that the precursor protein occurs exclusively in the testis and that proteolytic processing results in the formation of three fragments: p43 (D1 domain), p97 (D2–D4 domains), and p58 (D4 domain-C-terminal). In mature spermatozoa the p43 and p97 fragments exist as disulfide-bonded dimers. During spermatogenesis, synthesis of zonadhesin mRNA chiefly occurs in primary spermatocytes, whereas the protein is abundant in both Sertoli cells and spermatids. In spermatozoa the protein is localized exclusively to the anterior acrosome but is not available for binding antibody on live spermatozoa. Once the acrosome reaction is induced, zonadhesin is lost from the spermatozoon, but remains with the acrosomal shroud. We show that recombinant D4 domain can bind zona pellucida, and we propose that zonadhesin functions after the acrosome reaction has been initiated to bind the acrosomal shroud to the zona pellucida.

Association of sperm protein 17 with A-kinase anchoring protein 3 in flagella

BackgroundSperm protein 17 (Sp17) is a three-domain protein that contains: 1) a highly conserved N-terminal domain that is 45% identical to the human type II alpha regulatory subunit (RII alpha) of protein kinase A (PKA); 2) a central sulphated carbohydrate-binding domain; and 3) a C-terminal Ca++/calmodulin (CaM) binding domain. Although Sp17 was originally discovered and characterized in spermatozoa, its mRNA has now been found in a variety of normal mouse and human tissues. However, Sp17 protein is found predominantly in spermatozoa, cilia and human neoplastic cell lines. This study demonstrates that Sp17 from spermatozoa binds A-kinase anchoring protein 3 (AKAP3), confirming the functionality of the N-terminal domain.MethodsIn this study in vitro precipitation and immunolocalization demonstrate that Sp17 binds to AKAP3 (AKAP110) in spermatozoa.ResultsSp17 is present in the head and tail of spermatozoa, in the tail it is in the fibrous sheath, which contains AKAP3 and AKAP4. Recombinant AKAP3 and AKAP4 RII binding domains were synthesized as glutathione S-transferase (GST) fusion proteins immobilized on glutathione-agarose resin and added to CHAPS extracts of human spermatozoa. Western blots of bound and eluted proteins probed with anti-Sp17 revealed that AKAP3 bound and precipitated a significant level of Sp17 while AKAP4 did not. AKAP4 binds AKAP3 and expression of AKAP3 is reduced in AKAP4 knockout sperm, therefore we tested AKAP4 knockout spermatozoa for Sp17 and found that there was a reduction in the amount of Sp17 expressed when compared to wild type spermatozoa. Co-localization of AKAP3 and Sp17 by immunofluorescence was demonstrated along the length of the principal piece of the flagella.ConclusionsAs predicted by its N-terminal domain that is 45% identical to the human RIIα of PKA, Sp17 from spermatozoa binds the RII binding domain of AKAP3 along the length of the flagella.

Analysis of recombinant mouse zona pellucida protein 2 (ZP2) constructs for immunocontraception

In this study we have examined the potential of recombinant mouse zona pellucida glycoprotein 2 (ZP2) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in outbred Swiss-Webster mice using four ZP2 constructs: Val(35)-Gly(200) (ZP2(V35-G200)), Val(35)-Leu(331) (ZP2(V35-L331)), Pro(325)-Ala(637) (ZP2(P325-A637)), and Val(35)-Ala(637) (ZP2(V35-A637)). A significant antibody response occurred to three of the four immunogens, however antibodies capable of recognizing native ZP occurred only after immunization with ZP2(V35-A637) and ZP2(P325-A637). Only immunization with ZP2(V35-A637) correlated with a reduction in fertility. Examination of the physiological basis for infertility revealed that: (1) passive transfer of ZP2 antiserum induced infertility in non-immune mice; (2) ovaries of infertile mice appeared histologically normal; (3) infertile mice produced normal numbers of eggs and (4) ZP of ovulated eggs from infertile mice demonstrated a significant reduction in the number of sperm bound compared to eggs from adjuvant controls. Infertility can be caused entirely by ZP2 antibodies without the incidence of significant ovarian pathology. This study also demonstrated that immunization with the bioactive (sperm binding) region of ZP2, recombinant ZP2(V35-G200), did not result in a significant immune response that recognized native ZP or inhibited fertility. Consequently we designed a ZP2-sperm antigen construct, replacing the C-terminal region of ZP2 with Sp17. This construct proved to be immunogenic and reduce fertility while directing the immune response to the Val(35)-Gly(200) region of ZP2.

Designing an effective immunocontraceptive1

Abstract The immunological inhibition of fertilization is the goal of gamete immunocontraception. To achieve this goal a gamete-specific antigen target must be defined and the presentation of the immunogen to the immune system must be clearly understood in order to elicit a defined immune response which will target the native gamete molecule. Almost 20 years ago C.B. Metz suggested six studies which would answer the questions necessary for the development of a successful immunocontraceptive, and although much work has gone into answering each of these questions, none has been completed. Hyaluronidase is an example of a well studied sperm antigen whose native, membrane bound form (PH-20) is a successful immunocontraceptive in female guinea pigs. However, it remains to be demonstrated that a successful native antigen can be a successful synthetic or recombinant gamete immunocontraceptive (GAMICON). The problem of converting a successful native contraceptive antigen into an effective synthetic or recombinant GAMICON is at the heart of the problem of GAMICON design. If the epitopes of native and synthetic immunogens are not the same, then can a conversion ever be made? One approach to understanding how to make this conversion is to use defined, synethetic B- T-cell epitopes as specific B-cell epitopes on native antigens affect fertility.