Human Sperm EQUATORIN Analyzed by Anti-EQUATORIN Antibodies, MN9 and EQ70-83. (2008)

EQUATORIN is localized in mamalian sperm acrosomes and specifically recognized by monoclonal antibody MN9. The purified MN9 inhibited sperm-egg fusion, suggesting EQUATORIN plays an important role during fertilization. Our recent biochemical data show EQUATORIN is a 41-50kDa N-, O-sialoglycoprotein (Yamatoya et al; poster in this meeting). Here, we present the nature of human EQUATORIN analyzed by anti-EQUATORIN antibodies, MN9 and EQ70-83. Materials and methods: Antibodies. MN9 (IgG2a) and purified EQ70-83 were used as primary antibodies at 1/2,000 and 1/4,000 (dilution) for Western blot (WB), 1/25,000 and 1/5,000 for indirect immunofluorescence (IIF), and 1/20,000 and 1/20,000 for immunohistochemistry (IHC), respectively. EQ70-83 was produced in rabbit by immunizing with 14 amino acid sequnces (70-84) conserved in mammals. Alexa 488-conjugated anti-mouse immunoglobulin (H+L) and Alexa 546-conjugated anti-rabbit immunoglobulin (H+L) were used at 1/4,000 for IIF. HRP conjugated anti-mouse IgG and anti-rabbit IgG were used at 1/10,000 for WB, and 1/400 for IHC. WB: Ejaculated spermatozoa were lysed with SDS sample buffer and boiled for 5 min at 98°C. After centrifugation, the supernatant was subjected to SDS-PAGE and WB. IIF: Spermatozoa incubated in HTF (human tubal fluid) at 37°C were treated with 0.1% Triton X-100 (Triton) in PBS and incubated in primary antibodies for 4°C overnight. The samples were then incubated with secondary antibodies at RT for 30 min. Nuclei were stained with Hoechst at 1/400 dilution. IHC: Castrated human testis was donated from volunteer (53 years old patient) who had suffered from prostate cancer without hormonal treatment in Chiba University Hospital. The testis was routinely fixed, embedded in paraffin and processed for IHC. The samples were treated in primary antibodies at 4°C overnight and in secondary antibodies for 30 min to 1 hr. The reaction was visualized by diaminobenzidine-peroxidase. Result and Discussion: Human EQUATORIN migrated at broad region from approximately 40kDa and 64kDa with strong intensity on 50kDa on WB. This size was a bit bigger than that of mouse EQUATORIN. In IIF for mature spermatozoa, MN9 strongly stained both anterior acrosome (AA) and equatorial segment (ES), while 70-83 weakly stained AA, but strongly stained ES. This staining pattern was same to that found in mouse spermatozoa. IHC showed both MN9 and EQ70-83 recognized whole acrosomes of developing spermatids, with slight difference when compared to mouse. In IIF for spontaneously induced acrosome reaction, a marked difference was found in the staining pattern; MN9 strongly stained AA in the initial stage, and the stained area extended from AA to ES as acrosome reaction proceeded, evntually confining to ES. By contrast, EQ70-83 weakly stained AA throughout the acrosome reaction, but strongly stained ES as acrosome reaction proceeded. This tendency was same to that observed in mouse spermatozoa. Conclusion: Human EQUATORIN is a 40-64kDa N-, O-sialoglycoprotein. EQUATORIN at AA is well recognized by MN9, and EQUATORIN at ES is well recognized by both MN9 and EQ70-83 antibodies. The difference is presumed to be due to the different conditions of the epitope; carbohydrate moiety is required for the MN9 epitope, while peptide per se is the epitope of EQ70-83. This research was supported by grant from the Japan Society for the Promotion of Science (KT).