Ts4, an autosperm-monoclonal antibody (mAb), reacts with a specific oligosaccharide (OS) of glycoproteins containing bisecting N-acetylglucosamine residues. Ts4 reactivity was observed against epididymal spermatozoa, testicular germ cells, and the early embryo, but not against major organs in adult mice. In mature testis, Ts4 exhibits immunoreactivity with a germ cell-specific glycoprotein, TEX101, whereas the mAb immunoreacts with alpha-N-acetylglucosaminidase in the acrosomal region of cauda epididymal spermatozoa. Thus, Ts4 seems to react against different molecules throughout spermiogenesis via binding to its OS epitope. Since the Ts4-epitope OS is observed only in reproduction-related regions, the Ts4-reactive OS may play a role in the reproductive process. The aim of this study is to investigate the characteristics of the Ts4-reactive molecule(s) during testicular development. Ts4 reactivity was observed in testes from the prenatal period; however, its distribution changed according to the stage of maturation and was identical to that of the adult testes after 29-day-postpartum (dpp). Ts4 immunoreactivity was detected against a protein with 63 kDa in testis from 1 to 29 dpp. In contrast, Ts4 showed reactivity against some other glycoproteins after 29 dpp, including TEX101 at the 5-week-old stage and onward. To identify the Ts4-reactive 63 kDa molecule, we identified NUP62 as the target of Ts4 in 22 dpp testis using liquid chromatography-tandem mass spectrometry analysis. Because NUP62 has been known to play active roles in a variety of cellular processes including mitosis and cell migration, the bisecting GlcNAc recognized by Ts4 on NUP62 may play a role in regulating the early development of germ cells in male gonadal organs.
Supplementary Table 1. Information of primary antibodies used in this study
Supplementary Table 2. LC-MS/MS analysis for confirmation of molecules immunoprecipitated with Ts4 excised from equivalent area showing in Figure 2D-(b)
Supplementary Figure 1. Characterization of DPEP3 in the testis from 22-dpp mice. Immunohistochemical studies of the Ts4-reactive molecule and DPEP3 in the 22-dpp mice testis (A-C). The specimens were treated with these Abs, and then an Alexa Fluor 488- or 594-conjugated secondary antibody was used for detection of the primary antibodies. Green: Ts4 (A), red: anti-DPEP3 pAb (αDPEP3) (B). Overlay image of A and B (C). The samples were counterstained with TO-PRO3 (blue). Bars: 20 µm. White dotted lines indicate the borders of seminiferous tubule. Western blot analysis of Ts4-immunoprecipitant using the anti-DPEP3 pAb (D). The testicular WP fraction of 2.5 µg (p.c.) and immunoprecipitants with either Ts4 or RP-3 using the same fraction (lanes 1 and 2) were applied and separated by a 7.5% SDS-PAGE gel under reducing conditions. Control experiments were conducted under the same conditions, except for the absence of the tissue extract (-) (lanes 3 and 4).
Supplementary Figure 2. Expression of CD73 in the testes from 22-dpp mice. Immunolocalization of Ts4 and the anti-CD73 pAb (αCD73) in the 22-dpp mice testis (A-C). The specimens were treated with these Abs, and then an Alexa Fluor 488- or 594-conjugated secondary antibody was used for detection of the primary Abs. Green: Ts4 (A), red: αCD73 (B). Overlay image of A and B (C). The sample was counterstained with TO-PRO3 (blue). Bars: 20 µm. Dotted lines: the border of seminiferous tubule. Arrowheads: plasma membranes of germ cells in the seminiferous tubule. Western blot analysis of Ts4-immunoprecipitant detected by αCD73 (D). Testicular WP fraction (20 µg) (p.c.) and immunoprecipitants (IP) using 200 µg of WP fraction with either Ts4 or RP-3 (n.c.) (each 15 µg) (lane 1 and 2) were applied and separated by a 7.5% SDS-PAGE gel under reducing conditions. Control experiments were conducted under the same conditions, except for the absence of the tissue extract (-) (lane 3 and 4). Western blot analysis of the αCD73-immunoprecipitant detected by the same pAb (E) or Ts4 (F). Testicular WP fraction (20 µg) (p.c.) and immunoprecipitants (IP) using 200 µg of WP fraction with either αCD73 or negative control sheep Ig (n.c.) (each 10 µg) were applied to each lane of a 7.5% SDS-PAGE gel (lane 1 and 2) and separated under reducing conditions. Control experiments were conducted under the same conditions, except for the absence of the tissue extract (-) (lane 3 and 4).
Supplementary Figure 3. Western blot analysis of the 22-dpp testicular proteins immunoprecipitated with anti-TEX101 mAb. Immunoprecipitants (IP) using 100 µg of WP fraction with either TES101 (an anti-TEX101 mAb, compatible for IP) or 3H9 (n.c.) (each 15 µg) were applied to each lane of a 7.5% SDS-PAGE gel under reducing conditions, electroblotted onto PVDF membrane, and then detected with 6035 (the anti-TEX101 mAb; αTEX101) (lanes 1, 2), Ts4 (lane 3, 4), the anti-CD73 pAb (αCD73, lanes 5, 6),　the anti-NUP62 (αNUP62, lanes 7, 8), or the anti-DPEP3 pAb (αDPEP3, lanes 9, 10). Arrowheads indicate the specific immunoreactive bands observed.
Supplementary Figure 4. Western blot analysis after 2-D electrophoresis of ES cells lysate immunoprecipited with Ts4. The cell pellet of mouse ES cells (a kind gift from Dr. Yoichi Shinkai (RIKEN, Saitama, Japan)) were suspended in buffer A and extracted by Sonifier ultrasound homogenizer for 10 sec, six times. After Triton X-100 was added to the final concentration of 1%, the suspension was incubated for 20 min on ice, and then centrifuged at 10,000 x g for 5 min. The resultant supernatant (ES cell lysate) (200 µg) were immunoprecipitated using Ts4 (30 µg), then applied to an IPGphor strip (7cm; GE Healthcare Life Sciences), and IEF was performed at pH range from 3 to 10. EF and the following Western blot analysis with anti-NUP62 pAb (αNUP62) or Ts4 were performed as described in the legend for Figure. 3. ES cell lysate (50 µg) was applied to the left lane of the IPGphor strip for SDS-PAGE. The images detected using αNUP62 and Ts4 (within dotted lines) were overlaid using Adobe Photoshop Elements 7 with Ts4 (green) and αNUP62 pAb (red).