Experimental immunological infertility effect of anti-GAPDH-2 antibodies on the fertility of female mice Yong Wang, Ph.D., Ning Zhang, Ph.D., Xiaodong.

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Experimental immunological infertility effect of anti-GAPDH-2 antibodies on the fertility of female mice Yong Wang, Ph.D., Ning Zhang, Ph.D., Xiaodong Zhang, Ph.D., Shiying Miao, Shudong Zong, S.S. Koide, Ph.D., Linfang Wang, Ph.D. Fertility and Sterility Volume 92, Issue 6, Pages 2020-2027 (December 2009) DOI: 10.1016/j.fertnstert.2008.09.001 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Assessment of the specificity of anti-GAPDH-2 antibodies. (A) Recombinant His-GAPDH-2 was expressed in E. coli BL21(DE3) and purified by affinity purification method. Lysates of bacteria before (uninduced) and after (induced) IPTG induction, and the purified His-GAPDH-2 (MW 56 kD)(purified), were separated by 12% SDS-PAGE and stained with coomassie blue. (B, C) Assessment of the specificity of anti-GAPDH-2 antibodies for endogenous proteins in human or mouse sperm samples by Western blot. Anti-GAPDH-2 antibodies reacted specifically with GAPDH-2 (human sperm) (MW 56 kD) or GAPDS (mouse sperm) (MW 69 kD), showing no cross-reactivity with anti-GAPDH. (D, E) Immunostaining of human sperm, testis sections, and mouse sperm using anti-GAPDH-2 antibodies. The panels labeled I, III, and V show the results of staining of GAPDH-2 or GAPDS in sperm tails, visualized by FITC fluorescence. The panels labeled II, IV, and VI show the results of staining with preimmune serum. MW is an abbreviation of molecular weight. Fertility and Sterility 2009 92, 2020-2027DOI: (10.1016/j.fertnstert.2008.09.001) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Uterine horns containing embryos excised from female mice treated with anti-GAPDH-2 or preimmune sera (10 animals per group). Fertility and Sterility 2009 92, 2020-2027DOI: (10.1016/j.fertnstert.2008.09.001) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Sperm agglutination analysis. Agglutination in a tangled pattern was observed exclusively in the sample incubated with antiserum from the mice immunized with His-GAPDH-2. (A) Sperm suspensions mixed with antiserum from the mice immunized with His-GAPDH-2. (B) Sperm suspensions mixed with the preimmunized serum. (C) Sperm suspensions mixed with PBS. Fertility and Sterility 2009 92, 2020-2027DOI: (10.1016/j.fertnstert.2008.09.001) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Detection of the antibody titers specific to GAPDH-2 in serum or uterine fluid by ELISA. (A) Serum samples (1:20,000 to 1:320,000) from the mice immunized with GAPDH-2 or control mice. (B) Uterine fluid samples (1:500 to 1:16,000) from the mice immunized with GAPDH-2 or control mice. The antibody titers were expressed as an OD (490 nm) value. The data represented the average value from three experiments. Fertility and Sterility 2009 92, 2020-2027DOI: (10.1016/j.fertnstert.2008.09.001) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions