Functional studies of acid transporter in cultured rat epididymal cell Wu-Lin Zuo, B.Sc., Jie-Hong Huang, Ph.D., Jia-Jie Shan, M.Sc., Sheng Li, B.Sc., Patrick Y.D. Wong, Ph.D., Wen-Liang Zhou, Ph.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2744-2749 (May 2010) DOI: 10.1016/j.fertnstert.2010.02.042 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Colocalization of H+-ATPase and carbonic anhydrase II (CA II) in primary culture of cauda epididymal epithelial cells. Specialized cells were labeled with (A) the c subunit of H+-ATPase antibody and (B) CA II antibody. (C) Section A and B, and (D) section A, B and phase contrast image were overlaid to demonstrate the coexistence of H+-ATPase and CA II in specialized cells (i.e., clear cells). Magnification, ×150. Fertility and Sterility 2010 93, 2744-2749DOI: (10.1016/j.fertnstert.2010.02.042) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Different intracellular pH (pHi) regulation patterns in clear cells and principal cells. (A) Cells were consecutively challenged three times and allowed to recover normally as control. Two cell types are revealed by adding bafilomycin A1. (B) One is bafilomycin-insensitive, (C) the other is bafilomycin-sensitive. (D) Addition of amiloride showed that pHi recovery of bafilomycin-insensitive cells after acid load could be blocked by the Na+/H+ exchanger inhibitor. (E) Reverse application sequence of bafilomycin A1 and amiloride confirmed these results, and (F) showed that pHi recovery of bafilomycin-sensitive cells was also regulated by Na+/H+ exchanger. These functional assays indicate that the two cell types, bafilomycin-sensitive and bafilomycin-insensitive, were clear and principal cells, respectively. (G) Histogram of pHi recovery rate of presumable principal cells in different conditions. (H) Histogram of pHi recovery rate of presumable clear cells in different conditions. Ctl = control; NH4Cl = 40 mM NH4Cl challenge; bafilo = 100 nM bafilomycin A1; amil = 1 mM amiloride; ∗∗∗P<.001 versus control; +++P<.001 versus bafilomycin A1. Fertility and Sterility 2010 93, 2744-2749DOI: (10.1016/j.fertnstert.2010.02.042) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Dose-dependent inhibition curves of bafilomycin A1 (open circles) and concanamycin (closed circles) on luminal pH acidification. Each point shows the mean ± SEM of five experiments. Fertility and Sterility 2010 93, 2744-2749DOI: (10.1016/j.fertnstert.2010.02.042) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of pH 6.8 (open circles) and 7.4 (closed circles) on rat cauda sperm percent motility. Each point shows the mean ± SEM of 10 experiments. ∗∗P<.01, pH 7.4 versus pH 6.8. Fertility and Sterility 2010 93, 2744-2749DOI: (10.1016/j.fertnstert.2010.02.042) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions