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Anti-ACTL7a antibodies: a cause of infertility

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Presentation on theme: "Anti-ACTL7a antibodies: a cause of infertility"— Presentation transcript:

1 Anti-ACTL7a antibodies: a cause of infertility
Jun Fu, Ph.D., Yong Wang, Ph.D., Kin Lam Fok, Ph.D., Dantong Yang, Ph.D., Yi Qiu, Hsiao Chang Chan, Ph.D., Samuel S. Koide, Ph.D., M.D., Shiying Miao, Linfang Wang  Fertility and Sterility  Volume 97, Issue 5, Pages e8 (May 2012) DOI: /j.fertnstert Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

2 Figure 1 Immunolocalization of ACTL7a in various mouse tissues. Expression of ACTL7a was determined at transcriptional (A) and protein (B) levels. The tissues studied were muscle (M), liver (Li), spleen (S), lung (Lu), kidney (K), brain (B), heart (H), testes (T), epididymidis (Ep), ovary (O), and uterus (U). ACTL7a was present in the testis of a 3–8-week-old mouse at the transcriptional level (C) and in the testis of a 4–8-week-old mouse at the protein level (D). ACTL7a was not detected at the transcriptional (E) or protein (F) levels in GC1, GC2, TM3, and TM4 cell lines. (G) Positive immunostaining for ACTL7a was observed in spermatids but not in spermatocytes. ACTL7a was located in the cytoplasm of early round spermatids and later elongated spermatids, and in the acrosome of late round spermatids, early elongated spermatids, and later elongated spermatids. (H) Red represents interactions with the anti-ACTL7a antibodies, green represents lectin staining of acrosomes, and blue represents DAPI staining. Note that the acrosomes are stained in the human spermatozoa, whereas the acrosomes and tails are stained in mouse spermatozoa. (I) Red represents positive staining with anti-ACTL7a antibodies, green represents phalloidin staining of F-actin, and blue represents DAPI staining. Fertility and Sterility  , e8DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

3 Figure 2 The effect on the fertility of female or male mice after active immunization with ACTL7a. (A) Spermatozoa agglutination assay showing the clumping of mouse and human spermatozoa treated in vitro with rabbit antiserum against ACTL7a or purified anti-ACTL7a antibodies. The clumping of spermatozoa was not observed in spermatozoa treated with PBS, preimmunized serum, or untreated rabbit IgG. (B) Electrophoresis pattern of recombinant His-ACTL7a (molecular weight 55.1 kDa). M = marker; 7a-fl = full-length ACTL7a. (C) Results of the fertility assay of female mice. Uterine horns excised from intact untreated female mice (blank), PBS-treated female mice (control), and purified ACTL7a-treated female mice (ACTL7a). (D) Average number of developed embryos in the blank, control, and ACTL7a groups. The P value for the difference between the control and the ACTL7a groups was (E) Effect of active immunization with ACTL7a on the fertility of male mice. Immunized male mice were mated with normal female mice. Uterine horns containing the embryos were excised from the female mice that were mated with the control and ACTL7a-treated males. (F) Average number of embryos counted in the female mice mated with the control and ACTL7a-treated males. The P value for the difference between the control and the treated groups was (G) Hematoxylin and eosin staining of sections of mouse testis. (H) Average diameters of the seminiferous tubules of the testes in control and treated males. Note the reduction in the size of the tubules in the treated males. (I) Abnormalities in the development of the acrosome in spermatids in the immunized animals. (J) Decrease in the expression of the zpbp gene in the immunized males determined at the transcriptional level. ∗P<.05. Fertility and Sterility  , e8DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

4 Figure 3 Identification of anti-ACTL7a antibodies in the sera of vasectomized men using ELISA. Wells of plates were coated with ACTL7a full-length (A, B) and N-terminal (C, D) proteins (100 ng per well). The sera were diluted to 1:500 (A), 1:1,000 (B), 1:50 (C), and 1:500 (D) and were used as the primary antibody to react with the antigen. At all dilutions, the P values of the differences between the control and the vasectomized sera were <.05. Fertility and Sterility  , e8DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

5 Supplemental Figure 1 Identification of ACTL7a by 2D analysis and mass spectroscopy. (A) Two-dimensional profile gel of human spermatozoa proteins. (B, C) Western blot assays using a composite sera from 10 normal adult fertile men as the control antibody. (D, E) Western blot assays using serum 1426 from a vasectomized male as the primary antibody. (F, G) Enlarged copies of the rectangular sections in Western blots (D and E). Spot 6 was identified as the antigen that immunoreacted with serum (H) Enlarged copy of the rectangular section from the gel in (A). (I) The peptide sequences of ACTL7a identified and compared with the deduced amino acid sequence of the human spermatozoa ACTL7a. The matched peptides are shown in bold red. Fertility and Sterility  , e8DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

6 Supplemental Figure 2 Preparation and detection of anti-ACTL7a antibodies and validation of antibody specificity. (A) Electrophoresis pattern of ACTL7a-N (N-terminal portion of ACTL7a). The purified N-terminal portion of ACTL7a, composed of 70 amino acids, was used to prepare the antibodies. (B) Assessment of the specificity of the interaction of anti-ACTL7a antibodies with mouse testis proteins extracted, mouse spermatozoa, and human spermatozoa. The proteins were analyzed by Western blot. Anti-ACTL7a antibodies reacted specifically with ACTL7a in mouse spermatozoa (molecular weight 49.5 kDa) and human spermatozoa (molecular weight 49 kDa). Fertility and Sterility  , e8DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

7 Supplemental Figure 3 Anti-ACTL7a antibodies block the development of elongated spermatids. Samples of anti-ACTL7a antibodies and normal rabbit IgG were instilled into different sides of the seminiferous tubules of the testes of each mouse. After 2 days the mice were killed and sections of the testes were subjected to hemotoxylin and eosin staining. In the normal IgG-treated side, elongated spermatids were observed in two of the three testes (mouse 2 and 3), whereas elongated spermatids were lacking in the antibody-treated sides in all three mice. Fertility and Sterility  , e8DOI: ( /j.fertnstert ) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions


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