Akiko Hasegawa

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Delineation of a Conserved B Cell Epitope on Bonnet Monkey (Macaca radiata) and Human Zona Pellucida Glycoprotein-B by Monoclonal Antibodies Demonstrating Inhibition of Sperm-Egg Binding

Abstract To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136–147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136–147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136–147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.

Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters

Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine.

Monoclonal antibodies to porcine zona pellucida antigens and their inhibitory effects on fertilization

Five hybridomas which produce monoclonal antibodies (Mabs) to porcine zona pellucida (ZP) were established. The immunoglobulin classes were IgG2a from hybridoma B11C8 and IgM from the other four. Using immunofluorescent staining, the Mab from B11C8 stained only porcine oocytes, the Mab from G10G5 stained oocytes of pigs and hamsters but Mabs from C6H1, D3H4, G10F9 stained oocytes of pigs, humans, hamsters, rats and mice. Mabs from B11C8 and G10F9 strongly blocked boar sperm binding to porcine ZP but other Mabs produced only slight blocking. However, no Mabs could block sperm penetration during in vitro fertilization (IVF) of hamster oocytes. When a goat antibody (IgG) to mouse serum gamma-globulin was applied after each Mab as a second antibody, only Mab from G10G5 impaired IVF of hamster oocytes.

Production and characterization of monoclonal antibodies to cross-reactive antigens of human and porcine zonae pellucidae.

Three monoclonal antibodies (Mabs), 3A4-2G1, 1D5-2B7 and 1F2-1B8 were produced against heat-solubilized porcine zona pellucida (ZP). Each Mab stained intact ZP but no other pig tissues using immunofluorescence staining. All three Mabs stained selectively zonae pellucidae (ZPe) from pigs and humans but not from hamsters, rats or mice, and showed no inhibitory effect on sperm binding to human oocytes. When goat antiserum to mouse gamma-globulin was added to human oocytes pre-treated with 3A4-2G1 or 1D5-2B7, sperm binding to oocytes was completely blocked with formation of immune precipitates around them. SDS-PAGE analysis of the immune precipitates of 125I-labeled porcine zona proteins and Mab showed that the antigen binding 3A4-2G1 was mainly composed of components with approximate molecular weights of 92,000, 65,000 and 23,000 and the antigen binding 1D5-2B7 contained two components with approximate molecular weights of 57,000 and 49,000, respectively. The epitope of ZP antigen, corresponding to 3A4-2G1, was found to be present in the molecule of 92,000 daltons as demonstrated by enzyme immunostaining of the proteins after blotting to nitrocellulose membrane from SDS-PAGE gels.

Contraceptive potential of synthetic peptides of zona pellucida protein (ZPA)

Previously, we produced a fertilization-blocking monoclonal antibody (MAb-5H4) to find a candidate peptide for a contraceptive vaccine. MAb-5H4 recognized a linear amino acid sequence of ZPA (No. 50-67) in pigs, humans and rabbits. In the present study, 18mer peptides corresponding to the sequence were conjugated with diphtheria toxoid as a carrier protein before immunization in rabbits. All three antisera recognized human zona pellucida on testing by immunofluorescent staining method. The two produced against human and rabbit peptides effectively inhibited human sperm binding to the zona pellucida, but the antiserum against the pig peptide did not. The former two peptides include an identical sequence (LDPEKLTL) of the minimum binding motif for MAb-5H4, but the latter peptide includes one amino acid replacement (K to N) in the sequence. It is thus concluded that a synthetic peptide including the sequence of LDPEKLTL could be a feasible candidate for developing a contraceptive vaccine for humans.

Gene expression profile during ovarian folliculogenesis

Assisted reproductive technologies have progressed significantly and have provided successful treatment for many infertile couples. However, more advanced technologies are required for severe infertility such as premature ovarian failure and ovarian impairment due to adjuvant therapy for cancer. Ovarian tissue cryopreservation followed by in vitro growth of isolated follicles is a feasible proposition for such patients. Close coordination of communication among follicle cells including oocytes, granulosa and theca cells is required for follicle growth. Crucial factors may regulate the gonadotropin-independent and -dependent follicle growth stages. To facilitate development of a culture system for early growing follicles, DNA microarray analysis of mouse ovaries recovered at 7, 10, 13, 16 and 19 days of age was performed to identify factors required for the growth of early-stage follicles. These studies showed strong intensity of zona pellucida glycoproteins, bone morphogenic protein-15 (BMP-15) and growth differentiation factor (GDF-9) in 7 days old mice, which gradually declined in 19 days old mice. KIT, KIT ligand, anti-müllerian hormone (AMH) and platelet-derived growth factor (PDGF), known as granulosa cell secreted factors, also showed relatively high expression. These studies will facilitate our understanding of the regulatory factors involved in folliculogenesis and thereby enable establishment of in vitro culture system for ovarian follicles.

Sperm immobilizing and fertilization-blocking monoclonal antibody 2C6 to human seminal plasma antigen and characterization of the antigen epitope corresponding to the monoclonal antibody

A monoclonal antibody (Mab 2C6) with strong sperm immobilizing and agglutinating activities was generated by cell fusion between spleen cells from a mouse immunized with human seminal plasma (HSP) and mouse myeloma cells. It also showed a strong inhibitory effect on human sperm-egg interaction. The corresponding antigen was present on the whole surface of ejaculated spermatozoa. In male genital organs, immunostaining with Mab 2C6 was observed in epididymis and seminal vesicle but not in testis. By Western blotting, immunostaining with Mab 2C6 was detected around the 15-25 kDa region under both reducing and non-reducing conditions. The antigen corresponding to Mab 2C6 was susceptible to treatment with periodate or trifluoromethanesulfonic acid. The antigenic activities were slightly increased by treatment with neuraminidase but reduced by further treatment with glycosidases. Enzymatic digestions with pronase and papain also reduced the antigenic activities. The antigen molecules exhibited a strong binding affinity to RCA lectin. These results indicated that Mab 2C6 recognized one of the components which might be secreted from epididymis or seminal vesicle and bind to ejaculated spermatozoa as a sperm coating antigen. The corresponding antigen seems to be a glycoprotein and its carbohydrate moiety has an important role in the conformation of the antigen epitope.

Functional aspects of CD52 in reproduction

CD52 is a GPI-anchored protein present in lymphocytes and male reproductive tissues (mrt) including mature sperm and seminal plasma. It has been shown that mrt-CD52 is synthesized in epithelial cells of the epididymis and vas deferens, but not in the testis. The mrt-CD52 is transported to mature sperm during sperm transition in the male reproductive tract. Lymphocyte CD52 functions to stimulate suppressor T cell induction, while mrt-CD52 is associated with seminogelin and involved in clot formation and liquefaction of semen. In a landmark study, a monoclonal antibody (Mab H6-3C4) from peripheral B lymphocytes of a patient with complement-dependent sperm-immobilizing antibody in the serum has been generated. Using Mab H6-3C4, the carbohydrate moiety of CD52 as an epitope for infertility-related antigen was identified. Mab H6-3C4 also shows strong complement-dependent sperm-immobilizing activity. This suggests CD52 may have a function in protecting sperm from complement activation. Indeed, purified mrt-CD52 from human sperm interferes with the classical pathway, but not lectin-binding or alternative pathways, of the complement systems. Recently, we found CD52 in ovulated cumulus cells from the female reproductive tissues (frt). The frt-CD52 is not recognized by Mab H6-3C4, suggesting that it harbors distinct carbohydrate antigenicity. Further studies are necessary to determine the molecular features and biological functions of CD52 in male and female reproductive tissues.

Harmful effects of anti-zona pellucida antibodies in folliculogenesis, oogenesis, and fertilization

The zona pellucida (ZP) is an extracellular matrix that surrounds the mammalian oocyte and plays an important role in normal folliculogenesis and fertilization. Because of its strong immunogenicity and its possible relation with premature ovarian failure, we conducted the present study to examine whether or not anti-ZP antibodies impaired folliculogenesis. Mouse preantral follicles were cultured with anti-ZP antibodies to evaluate the effects on follicle growth and antral formation. The cultured follicles were also examined by electron microscope and assessed for oocyte maturation, fertilization capacity, and embryo development. The results showed that follicles cultured with anti-ZP antibodies had a smaller diameter than the controls. Also, these antibodies reduced antral formation, mucification, maturation of oocytes (metaphase II), and fertilization rates. Morphologically, ZP thickness was lower in the anti-ZP antibody groups. The quantity of granulosa cell microvillous processes that transverse the ZP was diminished in follicles cultured with anti-ZP antibodies. In conclusion, anti-ZP antibodies were harmful to the normal development of mouse follicles and oocytes in vitro. These antibodies may be a cause of premature ovarian failure syndrome because they disrupt the gap junctions between the oocyte and granulosa cells and, as a consequence, damage the bidirectional communication necessary for normal folliculogenesis.