Huei-Wen Chen, Ph.D., Wen-Sheng Jiang, M.S., Chii-Ruey Tzeng, M.D.

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Nitric oxide as a regulator in preimplantation embryo development and apoptosis Huei-Wen Chen, Ph.D., Wen-Sheng Jiang, M.S., Chii-Ruey Tzeng, M.D. Fertility and Sterility Volume 75, Issue 6, Pages 1163-1171 (June 2001) DOI: 10.1016/S0015-0282(01)01780-0

Figure 1 Immunocytochemical staining of eNOS and iNOS on embryos by day 3 of incubation. The fixed zona-free embryos were stained with the following FITC-conjugated polyclonal antibody: (A) Negative control and (B) eNOS and (C) iNOS. ×400. Chen. Nitric oxide regulates embryo viability. Fertil Steril 2001. Fertility and Sterility 2001 75, 1163-1171DOI: (10.1016/S0015-0282(01)01780-0)

Figure 2 In vitro preimplantation development of mouse embryos on day 4 after culture in medium without treatment (□, control) or in media supplemented with different concentrations of l-NAME (NOS inhibitor; = 0.1 μM; = 1.0 μM; ■ = 10.0 μM). Stage of development for each embryo was assessed daily, and the percentages of embryos at different developmental stages within each group are presented. Total number of embryos per treatment group was 80–100. Values are mean ± SE. ∗Significantly different from control value, P<.05 by paired Student’s t-test. Chen. Nitric oxide regulates embryo viability. Fertil Steril 2001. Fertility and Sterility 2001 75, 1163-1171DOI: (10.1016/S0015-0282(01)01780-0)

Figure 3 In vitro preimplantation development of mouse embryos in medium without treatment (□, control) and in media supplemented with different concentrations of SNP ( = 0.1 μM; = 1.0 μM; ■ = 10.0 μM). The degeneration (fragment, arrest, and lysis) for each embryo was assessed daily, and the percentage of degenerated embryos within each group are presented. Total number of embryos per treatment group was 80–100. Values are mean ± SE. ∗Significantly different from control value, P<.05 by paired Student’s t test. Chen. Nitric oxide regulates embryo viability. Fertil Steril 2001. Fertility and Sterility 2001 75, 1163-1171DOI: (10.1016/S0015-0282(01)01780-0)

Figure 4 In vitro preimplantation development of mouse embryos in medium without treatment (control, panels A and C) or in media supplemented with 10-μM SNP (NO donor, panels B and D) were collected for the study of apoptosis. These representative embryos were examined by annexin-V (green fluorescence; panels A and B) or TUNEL reaction (green fluorescence), and propidium iodide (red fluorescence) counterstained (panels C and D), ×200. The normal embryo showed no staining with annexin-V (A) or TUNEL (C), but the fragmented embryo had strong green-fluorescence (B; annexin V positive) or yellow fluorescence (D, TUNEL positive, double stained with TUNEL and propidium iodide). In the annexin V–positive cells, the stain localized in the cell membrane, and the staining localization of TUNEL-positive cells was in the cell nuclei rather than in their cytoplasm. Chen. Nitric oxide regulates embryo viability. Fertil Steril 2001. Fertility and Sterility 2001 75, 1163-1171DOI: (10.1016/S0015-0282(01)01780-0)

Figure 5 Immunocytochemical localization of p53 (A and B) and Bax (C and D) on embryos after SNP treatment is shown as follows: control embryo (A and C) and fragmented embryo (10-μM SNP treatment for 4 days, panels B and D); ×200. The brown-colored cells (or fragments) indicated the p53- or Bax-positive staining, and the negative cells were visualized by counterstaining with methyl green (green colored). Chen. Nitric oxide regulates embryo viability. Fertil Steril 2001. Fertility and Sterility 2001 75, 1163-1171DOI: (10.1016/S0015-0282(01)01780-0)

Figure 6 Hypothetical model for the role of NO in embryo implantation, in which NO/cGMP pathway may increase blood supply and muscle relaxation in the endometrium and also be involved in embryo development (A). In the patients with endometriosis, the inflammatory cells may produce excess NO to induce embryo apoptosis through the p53/Bax pathway (B). Chen. Nitric oxide regulates embryo viability. Fertil Steril 2001. Fertility and Sterility 2001 75, 1163-1171DOI: (10.1016/S0015-0282(01)01780-0)