Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Electron Microscopic analysis of step 19 spermatids in Stage VII of spermatogenesis: 1 Represents control spermatid showing single and double membranes bound vesicular structure (E) (Magnification-18,500X). 2 And 3 represents spermatid with well-defined chromatin (S) showing distinct tubulobulbar complex formation (T) along with membrane bound vesicular structure (E) (Magnification-23,000X for 2 and 13,000X for 3). 4 Represents treated spermatid with normal chromatin (S) showing normal Ectoplasmic specialization (ES) (Magnification 49,000).5 represents spermatid with a normal ectoplasmic specialization (ES) on the upper surface and a pronounced absence of ES on the lower surface indicated by arrow (Magnification 30,000). 6 Represents phagocytosed spermatids (PS) with the absence of Ectoplasmic specialization around it (Magnification 6800). 7 Represents sperm tails (arrow) in the vicinity of round spermatids (RS) (Magnification- 6800).
• Supplemental Figure 2a & 2b - Supplementary Figure 2a: Magnified image of Figure 2 showing F-actin in treated stage VII and IX: ‘A, C’ represent the counterstained image (red-Propidium iodide) for ‘B, D’ showing positive F-actin (green) staining respectively. Arrows in ‘B’ indicate delayed step 19 spermatids at the base of the epithelium lacking positive stain. Arrows in ‘D’ represents failed step 19 spermatids lacking positive stain.

  Supplementary Figure 2b: Stage specific localization of F-actin: ‘A, C, E, G, I, K, M, O, Q’ represent the counterstained image (red-propidium iodide) for ‘B, D, F, H, J, L, N, P, R’ showing positive F-actin (green) staining respectively. Left panel represent control where as right represent treated as indicated. Stages of spermatogenesis are indicated in extreme left of the image. ‘Lu’ represents luminal edge. Bar = 10 μm.

• Supplemental Figure 3 - Stage specific localization of Arp 3: ‘A, C, E, G, I, K, M, O, Q, S, U, W,’ represent the counterstained image (red- propidium iodide) for ‘B, D, F, H, J, L, N, P, R, T, V, X’ showing positive Arp 3 (green) staining respectively. Left panel represent control where as right represent treated as indicated. Stages of spermatogenesis are indicated in extreme left of the image. ‘Lu’ represents luminal edge, ‘Ds’ represent delayed spermatid and ‘Fl’ represent failed spermatid. Bar = 10 μm.
• Supplemental Figure 4 - Distribution of G-actin in Stage VII of spermatogenesis: ‘A-B’ is low magnification images, ‘C-D’ are high magnification images, ‘E-F’ negative control. ‘A, C, E’ are counterstained image (red-propidium iodide) for ‘B, D, F’ showing positive G-actin (green) staining respectively. Staining was seen in the ventral concavity of the step-19 spermatid in stage VII. ‘Lu’ represents luminal edge. Bar = 10 μm. Staining for G-actin was done as follows. Excised testis was placed in Tissues Tek OCT compound and frozen in liquid nitrogen. 8 μm cryosections were taken and post fixed with chilled acetone. Sections were blocked with iT FX compound and then incubated it with Dnase labelled Alexa 488.
  Colocalisation studies for Arp3 and F-actin: Images i–iv are represent fragmented step 19 spermatids obtained from treated samples, where one spermatid (upper) is stained partially positive for Arp 3 and F actin while the other spermatid (lower) shows absence of both Arp 3 and F actin. ‘i’ represent DAPI ( blue) counterstain. ‘ii’ represent Arp staining ( green) ‘iii’ represents F-actin staining (red) and ‘iv’ represent merged image. Bar = 10 μm.
• Supplemental Figure 5 - Stage specific localization of β-tubulin: ‘A, C, E, G, I, K, M, O, Q’ represent the counterstained image (red-propidium iodide) for ‘B, D, F, H, J, L, N, P, R’ showing positive β-tubulin (green) staining respectively. Left panel represent control where as right represent treated as indicated. Stages of spermatogenesis are indicated in extreme left of the image. ‘Lu’ represents luminal edge, ‘Ds’ represents delayed spermatid and ‘Fl’ represents failed spermatid. Bar = 10 μm.
• Supplemental Figure 6 - Haematoxylin-Eosin staining and F-actin Distribution in Stage IX in rats treated with 100 μg/kg/day 17 β Estradiol for 20 days:
  A- Haematoxylin-Eosin staining of testicular section showing stage IX. ‘Bs’ represents basement membrane, ‘Lu’ represents the lumen and ‘El’ represents elongating spermatids. Bar = 40 μm.
  B- Phalloidin staining for F-actin. ‘a’ represents counterstained images (red-propidium iodide) for ‘b’ showing positive F-actin (green) staining. ‘Lu’ represents lumen and ‘Fl’ represents failed spermatids. Bar = 10 μm.
  Note: The disorganized F-actin staining as compared to the organised staining seen in Supplementary Fig 2 (b) J.