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. 2015 Oct 13;30(12):2725–2736. doi: 10.1093/humrep/dev245

Figure 3.

Figure 3

SCSA analysis of SCF. Percentage DFI of SCF by the SCSA Assay. (a–f) In situ SCF: sperm samples were isolated and diluted with their luminal fluid. Control, untreated epididymal (a) and vas deferens (b) sperm. SCF induced epididymal (c) and vas deferens (d) sperm. SCF reversed with EDTA epididymal (e) and vas deferens (f) sperm; (g–l) reconstituted SCF: sperm were washed and reconstituted in the luminal fluid from the same organ; (g) Control, untreated epididymal sperm; (h) control, untreated vas deferens sperm; (i) SCF induced epididymal sperm; (j) SCF induced vas deferens sperm. (k–n) Mixed SCF: sperm were washed and resuspended in the other organ's luminal fluid: (k) control, epididymal sperm mixed with vas deferens luminal fluid; (l) control, vas deferens sperm mixed with epididymal luminal fluid; (m) epididymal sperm mixed with vas deferens luminal fluid then induced to undergo SCF; (n) vas deferens sperm mixed with epididymal luminal fluid then induced to undergo SCF. (o–r) Sperm induced to undergo SCF in the absence of luminal fluid: sperm were washed and resuspended in TKB; (o) control, epididymal sperm; (p) control, vas deferens sperm; (q) SCF induced epididymal sperm; (r) SCF induced vas deferens sperm. For each bar in the graph, n = 4, and one mouse was used for two experiments. The lines above the bars represent SD. Each experiment was compared with every other experiment in Student's t-test, and these values are shown in Supplementary data, Table SII. The most relevant comparisons are shown as brackets above the bars. These are: (i) SCF in epididymal sperm (c) differs from control (a), and both of these differ from control epididymal sperm incubated with vas deferens fluid (k) and from epididymal sperm induced to undergo SCF with vas deferens fluid (m) (P < 0.05 for all four comparisons); (ii) vas deferens sperm control (b) differs from vas deferens SCF (d) (P < 0.0001); (iii) epididymal sperm induced to undergo SCF (c) has significantly less DNA damage than vas deferens induced to undergo SCF (d) (P > 0.0001); (iv) vas deferens sperm induced to undergo SCF (d) has much more DNA damage than vas deferens sperm induced to undergo SCF in the presence of epididymal fluid (l) (P < 0.0001); (v) vas deferens sperm can undergo SCF without any luminal fluid present (r) but has significantly less DNA damage than vas deferens sperm with luminal fluid (d) (P < 0.0001).