Dynamic alterations in the expression and localization of ACTL7a during capacitation in mouse spermatozoa  Jun Fu, Ph.D., Wei Song, Ph.D., Shudong Zong, Samuel S. Koide, Ph.D., M.D., Shiying Miao, Linfang Wang  Fertility and Sterility  Volume 99, Issue 3, Pages 882-888 (March 2013) DOI: 10.1016/j.fertnstert.2012.11.005 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Increase in the level of ACTL7a after treatment of mouse spermatozoa with activators that induce capacitation. (A) Western blots showing the ACTL7a protein bands before and after capacitation in mouse spermatozoa. The spermatozoa were incubated for 30 minutes in capacitation buffer containing NaCl, KCl, CaCl2, MgCl2, NaH2PO4, Na pyruvate, Na lactate, HEPES, BSA, and NaHCO3. (C) Western blots showing the ACTL7a protein bands after the incubation of the mouse spermatozoa in various capacitation buffers for 30 minutes. (E, G) Western blots showing the ACTL7a protein bands in the mouse spermatozoa treated with 1 mM dbcAMP or 50 μM H89 in HCO3−-deficient buffer (E) or HCO3− buffer (G) for 10 minutes. (I) Western blots showing the ACTL7a protein bands in the mouse spermatozoa treated with 5 μM PMA, 50 μM H89, or 0.1 nM GF in HCO3−-deficient buffer for 30 minutes. (K) Western blots showing the ACTL7a protein bands in the mouse spermatozoa treated with 1 mM dbcAMP or 0.1 nM GF in HCO3− buffer for 10 minutes. The buffers used in panels E, G, I, and K contained the following: NaCl, KCl, MgCl2, NaH2PO4, Na pyruvate, Na lactate, HEPES, and BSA. (B, D, F, H, J, L) The relative expression of ACTL7a is expressed as the ratio of the amount of ACTL7a to the amount of β-actin. *P<.05, **P<.01, and ***P<.001. Fertility and Sterility 2013 99, 882-888DOI: (10.1016/j.fertnstert.2012.11.005) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Alteration in the localization of ACTL7a in mouse spermatozoa after induction of capacitation. (A) Red represents staining with the anti-ACTL7a antibodies, green represents acrosome staining using lectin, and blue represents DAPI staining. The localization of ACTL7a in the mouse spermatozoa was altered after treatment with 25 mM NaHCO3 or 1 mM dbcAMP for 15 minutes. (C) The localization of F-actin did not change. Red represents positive staining with anti-ACTL7a antibodies, green represents phalloidin staining for F-actin, and blue represents DAPI staining. (B and D) Enlarged images of the stained spermatozoa indicated by the arrows in panels (A) and (C). Bars measure 15 μm. Fertility and Sterility 2013 99, 882-888DOI: (10.1016/j.fertnstert.2012.11.005) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Schematic diagram depicting the alteration in the localization of ACTL7a in mouse spermatozoa after induction of capacitation. (A) Schematic diagram depicting the two morphological patterns of localization of ACTL7a. In the type I pattern, the ACTL7a is restricted to the acrosome of the mouse spermatozoa. In the type II pattern, the ACTL7a is dispersed throughout the entire head of the spermatozoa. (B) Bar graphs depicting the percentages of the type II pattern in noncapacitated and capacitated spermatozoa. **P<.01 and ***P<.001. Fertility and Sterility 2013 99, 882-888DOI: (10.1016/j.fertnstert.2012.11.005) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Hypothetical schematic diagram showing the biochemical events involved in the dynamic localization of ACTL7a in mouse spermatozoa undergoing capacitation. Fertility and Sterility 2013 99, 882-888DOI: (10.1016/j.fertnstert.2012.11.005) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions