Fig 1: (a) Western blot analysis of PRDX6 abundance in GFE and PFE sperms. The samples derived from the same experiment and all blots were processed in parallel. The bars represented the mean values ± SEM of the intensity of all the bands per lane expressed in arbitrary units (A.U.) using sperm in three independent experiments. (b) The effect of PRDX6 (0.0, 0.01, 0.1 and 1 mg/L) on the frozen-thawed sperm motility of Mediterranean buffaloes. (c) The effect of PRDX6 on the ROS levels of frozen-thawed sperm. (d) The effect of PRDX6 on the DNA oxidation of frozen-thawed sperm. (e) The effect of PRDX6 on the lipid oxidation of frozen-thawed sperm. (f) The effect of PRDX6 on the total antioxidant capacity of frozen-thawed sperm. The t test was used to compare data between two groups and variance (ANOVA) was used to compare data between multiple groups. *P < 0.05, **P < 0.01
Fig 2: (a) Acrosome reacted sperm arising from the CTC fluorescence assay. AR pattern, with dull fluorescence over the whole head except for the thin, bright punctate band of fluorescence in the equatorial segment (arrow). (b) Uncapacitated sperm arising from the chlortetracycline fluorescence assay. F pattern, with fluorescence over the whole head (arrow). (c) Capacitated sperm arising from the chlortetracycline fluorescence assay. B pattern, with a fluorescence-free band in the post acrosomal region (arrow). (d) The effect of PRDX6 on fertility capacity of frozen-thawed sperm. (e) Mitochondrial activity of sperm by JC-1 staining. The data were analyzed by t test *P < 0.05, **P < 0.01, *** P < 0.001
Fig 3: Graphs with corresponding representative Western blot analyses of PRDX5 under various electrophoretic conditions: (A) Native PAGE, showing a significant increase in the normalized intensity of the native band after cryopreservation (p < 0.01); (B) Non-denaturing PAGE, showing an increase in the normalized intensity of the native band after cryopreservation (p < 0.001). Lines f1-f4 correspond to fresh sperm samples, while c1-c4 correspond to cryopreserved sperm samples
Fig 4: EV content, size, and PRDX5/PRDX6 expression in SEC fractions from bovine seminal plasma. In bovine seminal plasma SEC fractions 4–7, 72% of the detected particles were membranous (Cell Mask Green positive, CMG +). Of these, 61% expressed at least one EV-related surface marker (CD9 or CD63). The population marked in purple represents CMG + and CD63 + particles, the green population represents CMG + and CD9 + particles, and the yellow population represents CMG + , CD63 + , and CD9 + particles (A). EVs from SEC fractions 4–7 were sized between 100 and 200 nm (B). Western blot analysis of proteins separated by non-reducing SDS-PAGE, using anti-PRDX5 (C) and anti-PRDX6 (D) antibodies, demonstrated the presence of PRDX5 in the exosomal vesicle (EV) fraction, with no detectable signal in the exosome-depleted seminal plasma (SP). Arrows indicate 8 reactive bands for PRDX5 with wide MW range for PRDX5 in the EV fraction. In contrast, PRDX6 was detected in both the EV fraction (EVs) and the exosome-depleted seminal plasma (SP), with arrows indicating 3 reactive bands for PRDX6 with high MW for PRDX6, although the signal was weaker in both
Fig 5: Presence of PRDX5 and PRDX6 on the sperm surface and within the entire sperm cell. Bar charts depict fluorescence intensity of fluorescently labeled anti-PRDX5 antibodies in non-permeabilized (A) and permeabilized (B) sperm samples, as well as anti-PRDX6 antibodies in non-permeabilized (C) and permeabilized (D) sperm samples. Data are presented as the mean ± SD (n = 8 sperm samples). ***p < 0.001
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