Acetylated α-tubulin is reduced in individuals with poor sperm motility  Shweta Bhagwat, M.Sc., Veena Dalvi, M.Sc., Darshan Chandrasekhar, M.Sc., Tinsu Matthew, M.Sc., Kshitish Acharya, Ph.D., Rahul Gajbhiye, M.B.B.S., Ph.D., Vijay Kulkarni, M.S., Shobha Sonawane, M.Sc., Manish Ghosalkar, B.Sc., Priyanka Parte, Ph.D.  Fertility and Sterility  Volume 101, Issue 1, Pages 95-104.e3 (January 2014) DOI: 10.1016/j.fertnstert.2013.09.016 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Protein expression for α-tubulin and acetylated α-tubulin. The ratio of intensities of the respective protein to that of GAPDH was quantified, and data are expressed as mean ± SEM. (A) Representative Western blot for α-tubulin and acetylated α-tubulin (upper panel). N = normal spermatozoa. A = asthenozoosperm. Densitometric analysis of the bands for α-tubulin and acetylated α- tubulin in normal sperm (n = 9) and asthenozoosperm, n = 10 (lower panel). (B) Flow cytometric analysis for α-tubulin and acetylated α-tubulin in normal men (n = 23) and asthenozoospermic men (n = 20). (C) Flow cytometric analysis for α-tubulin and acetylated α-tubulin from the motile and immotile fraction of normal spermatozoa (n = 6). *P<.05; **P<.005. Values are mean ± SEM. Fertility and Sterility 2014 101, 95-104.e3DOI: (10.1016/j.fertnstert.2013.09.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Direct localization of α-tubulin and acetylated α-tubulin in normal sperm and asthenozoosperm. (A) Double immunostaining was performed using FITC-labeled α-tubulin and Alexa Fluor 568-labeled acetylated α-tubulin antibodies. Positive staining is seen in the flagella for α-tubulin (αT; green signal), acetylated α-tubulin (Ac-αT; red signal) and colocalization of αT, Ac-αT (yellow signal) in normal sperm and asthenozoosperm. (B) Graphical representation for degree of acetylation of α-tubulin (n = 100 sperm per group).**P≤.01. Fertility and Sterility 2014 101, 95-104.e3DOI: (10.1016/j.fertnstert.2013.09.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Expression of the protein and transcript for testis-specific isoforms of α-tubulin: TUBA3C, TUBA4A, and TUBA8 in normal sperm and asthenozoosperm. The ratio of intensities of the respective protein to that of GAPDH was quantified and data expressed as mean ± SEM (n = 6 per group). (A) Representative Western blot for α-tubulin isoforms (upper panel). Densitometric analysis of the bands for the αT isoforms (lower panel). (B) Relative quantity of α-tubulin isoforms (TUBA3C, TUBA4A, TUBA8) as determined by real-time RT-PCR analysis. Values are mean ± SEM. *P<.05. ***P<.0005. N = normal spermatozoa; A = asthenozoosperm. Fertility and Sterility 2014 101, 95-104.e3DOI: (10.1016/j.fertnstert.2013.09.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Representative images for indirect immunofluorescence localization of α-tubulin isoforms in the sperm of normal and asthenozoospermic men. Indirect immunofluorescent localization of TUBA3C, TUBA4A, and TUBA8 (green fluorescence) on the flagella in normal sperm and asthenozoosperm. Blue staining identifies the nuclei. Scale bar: 5 μm. Fertility and Sterility 2014 101, 95-104.e3DOI: (10.1016/j.fertnstert.2013.09.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Protein-protein interactions among the important transcription factors identified with their potential role in the differential regulation of TUBA4A and TUBA3C (http://string-db.org/). Fertility and Sterility 2014 101, 95-104.e3DOI: (10.1016/j.fertnstert.2013.09.016) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions