| Abstrakt: |
EQUATORIN is localized in mammalian sperm acrosomes and specifically recognized by MN9 monoclonal antibody (MN9). The purified MN9 effectively inhibited sperm-egg fusion under in vitroand in vivofertilization condition. These previous findings suggest EQUATORIN is an important molecule for sperm-egg fusion. Our recent biochemical data show EQUATORIN is a 41-49kDa N-,O-sialoglycoprotein (poster presented by K Yamatoya). Here we present the nature of mouse EQUATORIN analyzed by two anti-EQUATORIN antibodies, MN9 and EQ70-83. Material and Methods: Antibodies; Primary antibodies were used MN9 and polyclonal EQ70-83. MN9 was used at a 1/2,000 dilution for Western blot (WB), 1/30,000 dilution for indirect immunofluorescence (IIF). EQ70-83was produced in rabbit by immunizing with 14 amino acid sequences conserved widely in mammals as immunogens. Purified EQ70-83was used at a 1/2,000 dilution for WB, 1/5,000 dilution for IIF. Secondary antibodies, Alexa 488-conjugated anti-mouse Ig (H+L) and Alexa 546-conjugated anti-rabbit Ig (H+L) were used at a dilution 1/4,000 dilution for IIF. HRP conjugated anti-mouse IgG and anti-rabbit IgG were used at a 1/10,000 dilution for WB. WB; Spermatozoa (1 x 107) collected from cauda epididymides were lysed in SDS sample buffer and boiled for 5 min at 98°C. After centrifugation, the supernatants were subjected to WB. IIF; Spermatozoa collected from cauda epididymides were incubated in TYH medium at 37°C until use. Spermatozoa that had been treated with 0.1% Triton X-100 (Triton) in PBS were incubated in primary antibody for 4°C overnight. After washing in PBS, the samples were incubated with secondary antibodies at room temperature for 30 min. Nuclei were stained with Hoechst (1/400 dilution). Results: Both MN9 and EQ70-83recognized the same bands mainly at 41kDa and 49kDa. IIF showed EQ70-83strongly stained equatorial segment (ES) of mature spermatozoa, while MN9 strongly stained both the anterior acrosome (AA) and ES. Such a staining pattern was attenuated in Triton treatment. In testes, both MN9 and EQ70-83stained whole acrosomes of developing spermatids in a similar fashion; no significant difference was found. By contrast, marked difference was found in the later stage of elongating spermatids; the staining was confined to ES as similarly shown in mature spermatozoa. During acrosome reaction, EQ70-83weakly stained AA in the initial stage, but the later strongly stained ES. By contrast, MN9 strongly stained AA in the initial stage of acrosome reaction, and the staining signals extended from AA to ES as acrosome reaction proceeded. As approaching to the end of acrosome reaction, both antibodies strongly stained ES. Discussion: The epitope of EQ70-83in AA is hardly detectable, but consistently detectable in ES in mature spermatozoa and developing spermatids. This staining tendency of the epitope of EQ70-83during spermatogenesis is basically same to that found in acrosome reaction. The reason is unclear at present, but the epitope region in AA may be modified during acrosome reaction. By contrast, the epitope of MN9 is consistently exposed in AA and ES with specific localization on ES. The difference in these antibodies is presumed to be due to different condition of EQUATORIN in the AA and ES. Therefore, MN9 and EQ70-83is useful for further analysis of molecular mechanism. This research was supported by grant from the Japan Society for the Promotion of Science (KT). |
