Immunization with a DNA vaccine of testis-specific sodium-hydrogen exchanger by oral feeding or nasal instillation reduces fertility in female mice  Tao Liu, Ph.D., Jun-Cheng Huang, Ph.D., Cui-Ling Lu, Ph.D., Jun-Ling Yang, Ph.D., Zhao-Yuan Hu, B.A., Fei Gao, Ph.D., Yi-Xun Liu, Ph.D.  Fertility and Sterility  Volume 93, Issue 5, Pages 1556-1566 (March 2010) DOI: 10.1016/j.fertnstert.2009.03.056 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 tsNHE mRNA expression and its DNA vaccine antibody specialty detection. (1) Representative expression of mtsNHE fragment in mouse uteri immunized with 20 μg pCR-NHE via oral feeding (left) or nasal instillation (right). Antigen expression in the various groups on the days 3, 5, 7, 10, 12, and 14 is shown after the DNA immunization. Lane M shows the DNA marker. The expression of gapdh served as an internal standard for mRNA. (2) Normal mouse sperm was reacted with the antiserum or the vaginal fluid obtained from the DNA vaccine immunized mice and stained with goat anti-mouse IgA, IgG conjugated with FITC, then counterstained with PI for visualizing nuclei. (A) Reacted with serum of pCR3.1 immunized mouse via oral feeding. (B) With vaginal fluid of pCR3.1 immunized mouse via oral feeding. (C) With antiserum of pCR-NHE immunized mouse via oral feeding. (D) With vaginal fluid of the pCR-NHE immunized mouse via oral feeding. (E) With antiserum of the pCR3.1 immunized mice via nasal instillation. (F) With vaginal fluids of the pCR3.1 immunized mice via nasal instillation. (G) With antiserum of pCR-NHE immunized mouse via nasal instillation. (H) With vaginal fluid of pCR-NHE immunized mouse via nasal instillation. (Magnification: × 200.) (3) Mouse testicular sections were reacted with the antiserum or the vaginal fluid obtained from the DNA vaccine immunized mice. The sections were counterstained with hematoxylin for visualization of nucleic. Red shows mtsNHE protein. (A) With the serum of pCR3.1 of immunized mouse via oral feeding. (B) With vaginal fluid of pCR3.1 via oral feeding. (C) With antiserum of pCR-NHE via oral feeding. (D) With vaginal fluid of pCR-NHE via oral feeding. E With antiserum of pCR3.1 via nasal instillation (positive signal can be observed in the whole section). (F) With vaginal fluids of pCR3.1 via nasal instillation. (G) With antiserum of pCR-NHE via nasal instillation. (H) With vaginal fluid of pCR-NHE via nasal instillation. (Magnification: × 200.) (4) Western blot analysis of specification of antibodies via DNA vaccine immunization. The antiserum or vaginal wash from the immunized mice with pCR-NHE or pCR3.1 orally or nasally was used for the total testicular detection, which was separated in 10% SDS-PAGE and probed with the antiserum or vaginal fluid, respectively. Lane 1–4: Probing of the serum or vaginal fluid of orally immunized mice. Lane 5–8: Serum or vaginal fluid of nasally immunized mice. Lane 1: Probed with serum of pCR3.1. Lane 2: Vaginal fluid of pCR3.1. Lane 3: Antiserum of pCR-NHE. Lane 4: Vaginal fluid of pCR-NHE. Lane 5: Serum of pCR3.1. Lane 6: Vaginal fluid of pCR3.1. Lane 7: Antiserum of pCR-NHE. Lane 8: Vaginal fluid of pCR-NHE. Specific brand was detected only in testicular protein by the antisera or the vaginal fluids obtained from the immunized mice with pCR-NHE. Fertility and Sterility 2010 93, 1556-1566DOI: (10.1016/j.fertnstert.2009.03.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Expression of mtsNHE protein in transfected HeLa cells. HeLa cells were transfected with pCR-NHE or pCR3.1 and reacted with the antiserum or vaginal wash obtained from the immunized female mice with tsNHE vaccine. The secondary antibody was conjugated with FITC as described in Materials and Methods. (A) Treated with serum of oral feeding pCR3.1. (B) With vaginal fluid of oral feeding pCR3.1. (C) With antiserum of oral feeding pCR-NHE. (D) With vaginal fluid of oral feeding pCR-NHE. (E) With serum of pCR3.1 nasal instillation. (F) With vaginal fluid of pCR3.1 nasal instillation. (G) With antiserum of pCR-NHE nasal instillation. (H) With vaginal fluid of pCR-NHE nasal instillation. Green fluorescence emitted by activated FITC represents the expression of mtsNHE protein fragment, and blue fluorescence from activated Hoechst represents the cell nuclei. (Magnification: × 400.) Fertility and Sterility 2010 93, 1556-1566DOI: (10.1016/j.fertnstert.2009.03.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Detection of antibody titers specific to mtsNHE by ELISA. Total testicular protein was chosen as the coated antigen. The antiserum and vaginal fluid were diluted from 1:100 to 1:4000, respectively. The antibody titers were pooled and expressed as log10 titers. (A) Serum from pCR3.1 and pCR-NHE immunized mice via oral vaccination. (B) Vaginal fluid from pCR3.1 and pCR-NHE immunized mice via oral feeding. (C) Serum from pCR3.1 and pCR-NHE immunized mice via nasal inoculation. (D) Vaginal fluid from pCR3.1 and pCR-NHE immunized mice via nasal instillation. Fertility and Sterility 2010 93, 1556-1566DOI: (10.1016/j.fertnstert.2009.03.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of antiserum and vaginal fluid on spermatozoa motility and in vitro fertilization. The antisera or vaginal fluids from the immunized mice with pCR-NHE and pCR3.1 orally or nasally were used for sperm motility and in vitro fertilization test as described in Materials and Methods. (A) Spermatozoa motility treated with the antiserum of pCR-NHE orally immunized mouse (63.6% ± 3.7%) was statistically significantly reduced as compared with that of the pCR3.1 group (78.4% ± 8.1%). (B) Sperm motility treated with the vaginal fluid of pCR-NHE orally immunized mouse (60.2% ± 7.9%) as compared with that of the pCR3.1 group (78.7% ± 6.2%). (C) Antiserum of pCR-NHE immunized mice via nasal instillation was reduced from 74.3% ± 7.3% to 60.6% ± 4.9%. (D) Vaginal fluid of pCR-NHE immunized mice via nasal instillation was reduced from 72.3% ± 9.2% to 56.1% ± 8.4%. (E) Antiserum from pCR-NHE orally immunized mouse significantly decreased fertilization rate (from 86.4% ± 6.3% to 62.1% ± 3.4%). (F) Vaginal wash from pCR-NHE orally immunized mouse (from 86.5% ± 2.8% to 56.3% ± 5.5%). (G) Antiserum from pCR-NHE nasally immunized mouse (from 82.3% ± 7.4% to 49.9% ± 3.7%). (H) Vaginal wash from pCR-NHE nasally immunized mouse (from 83.0% ± 5.3% to 54.6% ± 5.1%). ∗Indicates statistically significant difference (P<.05). ∗∗Indicates statistically significant difference (P<.01). Two-cell stage embryos were scored at 24 to 27 hours after insemination during in vitro fertilization test. Fertility and Sterility 2010 93, 1556-1566DOI: (10.1016/j.fertnstert.2009.03.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Effect of pCR-NHE DNA vaccination on number of newborns. Female mice were inoculated with the pCR-NHE or pCR3.1 via oral or nasal vaccination. To evaluate efficacy and safety of the DNA vaccine, the immunized female mice were mated with normal adult males for a 1-week period after immunization. Number of newborns was scored by the method as described in Materials and Methods. (A) Number of newborns (mean ± standard deviation [SD]) of the vaccinated female mice with pCR-NHE via oral feeding was statistically significantly decreased from 11.3 ± 0.7 (pCR3.1) to 5.7 ± 1.1 (pCR-NHE). (B) Number of newborns (mean ± SD) of vaccinated female mice with pCR-NHE via nasal instillation was statistically significantly reduced from 11.0 ± 0.5 (control) to 4.7 ± 0.9 (pCR-NHE). ∗∗Indicates statistically significant difference (P<.01). Fertility and Sterility 2010 93, 1556-1566DOI: (10.1016/j.fertnstert.2009.03.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 6 Sperm agglutination analysis. Agglutination in a tangled pattern was only observed in samples treated with the antisera or the vaginal fluids from the mice immunized with pCR-NHE. (A) Sperm suspensions of normal mouse were mixed with the antiserum of pCR3.1 immunized mouse via oral feeding. (B) With vaginal fluids of pCR3.1 immunized mouse via oral feeding. (C) With antiserum of pCR-NHE immunized mouse via oral feeding. (D) With vaginal fluid of pCR-NHE immunized mouse via oral feeding. (E) With antiserum of pCR3.1 immunized mouse via nasal instillation. (F) With vaginal fluid of pCR3.1 immunized mouse via nasal instillation. (G) With antiserum of pCR-NHE immunized mouse via nasal instillation. (H) With vaginal fluids of pCR-NHE immunized mouse via nasal instillation. (Magnification: × 200.) Fertility and Sterility 2010 93, 1556-1566DOI: (10.1016/j.fertnstert.2009.03.056) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions