Angiotensin II type 2 receptor is expressed in human sperm cells and is involved in sperm motility  Marta Gianzo, J.D., Iraia Muñoa-Hoyos, J.D., Itziar Urizar-Arenaza, J.D., Zaloa Larreategui, J.D., Fernando Quintana, J.D., Nicolás Garrido, Ph.D., Nerea Subirán, Ph.D., Jon Irazusta, Ph.D.  Fertility and Sterility  Volume 105, Issue 3, Pages 608-616 (March 2016) DOI: 10.1016/j.fertnstert.2015.11.004 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Expression and localization of angiotensin II type 2 receptor in human sperm cells. (A) Midori green–stained 1.5% agarose electrophoresis gels of the RT-PCR products for Agtr2 in human sperm (Spz) and kidney cells (Kd). Amplified fragment using primers specific for the human Agtr2 (177-bp band). Primers without cDNA were used as negative control. (B) Western blotting analysis of AT2R in human sperm (Spz) and kidney (kd). Molecular weights (kDa) of prestained markers proteins are indicated on the left. Western blots representative of those obtained with normozoospermic donors are shown. (C) Sperm population analyzed by flow cytometry. Plots of blank control (red) without both primary and secondary antibodies; negative primary antibody control (green), preabsorbing primary antibody with its specific blocking peptide; negative secondary antibody control (blue), treated with secondary antibody alone and AT2R-positive sperm population (yellow). Nuclei were stained with Hoescht 33258 (n = 4). (D) Immunofluorescence analysis of AT2R in human sperm cells. (D1) Negative primary antibody control (green). (D2) Preabsorbing primary antibody with its specific blocking peptide. (D3) Negative secondary antibody control (blue), treated with secondary antibody alone. (D4) Phase-contrast image of the human sperm cells. Nucleus were stained with Hoescht 33258. Representative photomicrographs are shown; n = 5. Scale bar = 1 μm. Fertility and Sterility 2016 105, 608-616DOI: (10.1016/j.fertnstert.2015.11.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Correlation between the percentage of AT2R-positive cells and sperm concentration. Scatter plots showing the percentage of AT2R-positive spermatozoa related to (A) fresh spermatozoa and (B) prepared spermatozoa concentration. (C) Spearman rank correlation coefficients analyses. ∗∗ P>.01. Fertility and Sterility 2016 105, 608-616DOI: (10.1016/j.fertnstert.2015.11.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Correlation between the percentage of AT2R-positive cells and sperm motility. Scatter plots showing the percentage of AT2R-positive spermatozoa in association with (A) percentage of PR and (B) percentage of IM spermatozoa in fresh semen samples; (C) percentage of PR and (D) percentage of IM spermatozoa in prepared semen samples. (E) Spearman rank correlation coefficients analyses. ∗∗ P>.01. Fertility and Sterility 2016 105, 608-616DOI: (10.1016/j.fertnstert.2015.11.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Percentage of AT2R-positive sperms from men with different etiologies. Graphic representation of the percentage of AT2R-positive sperms from men with varying etiologies, determined by flow cytometry. The graphic shows four different groups: normozoospermic (NZ), asthenozoospermic (AZ), teratozoospermic (TZ), and oligozoospermic (OZ) semen samples. **Highly significant difference between groups (P<.01). Fertility and Sterility 2016 105, 608-616DOI: (10.1016/j.fertnstert.2015.11.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions