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  Indian J Med Microbiol
 

Figure 1: (a–e) Comparison of PLCζ visualization in frozen-thawed spermatozoa from fertile donors between in-house and AUM methods. (a) Mean PLCζ levels in spermatozoa from 14 fertile individual donors. RF intensity of PLCζ was compared between AUM and in-house methods, and between methods with and without AUM reagents. (b) Overall mean PLCζ levels in spermatozoa from a total of 14 fertile donors. Data represent mean ± s.e.m., *P < 0.05, **P < 0.01, and ***P < 0.001, with one-way ANOVA and Bonferroni correction. (ci) Comparison of PLCζ visualization in spermatozoa from a fertile donor (donor 3) between “in-house” and AUM methods. (c) Confocal microscopy visualization of PLCζ. The in-house method exhibited better RF intensity of PLCζ in the equatorial segment of the sperm head than AUM methods. Extensive RF intensity of PLCζ was observed in the midpiece of AUM-treated (AT or HCl) sperm (red arrows). Yellow arrows indicate PLCζ. Representative images for BF microscopy, DAPI, FITC-PLCζ staining, and overlay showing sperm expressing PLCζ. Scale bars = 5 μm. (d) Overall mean RF intensity of PLCζ in spermatozoa from donor 3. Data represent mean ± s.e.m. (e) Proportions of spermatozoa exhibiting PLCζ. Data represent mean values. **P < 0.01 and ***P < 0.001, with one-way ANOVA and Bonferroni correction. (f–i) Comparison of PLCζ localization in spermatozoa. PLCζ localization was classified into eight patterns and compared between spermatozoa stained with (f) non-AUM, (g) AT-AUM, (h) HCl-AUM, and (i) in-house methods. Data represent mean percentages. ***P < 0.001 with one-way ANOVA and Bonferroni correction. PLCζ: phospholipase C zeta; AUM: antigen unmasking method; RF: relative fluorescence; a.u.: arbitrary units; s.e.m.: standard error of mean; ANOVA: analysis of variance; DAPI: 4'-6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; BF: bright field; AT: acidic Tyrode's solution.

Figure 1: (<b>a–e</b>) Comparison of PLCζ visualization in frozen-thawed spermatozoa from fertile donors between in-house and AUM methods. (<b>a</b>) Mean PLCζ levels in spermatozoa from 14 fertile individual donors. RF intensity of PLCζ was compared between AUM and in-house methods, and between methods with and without AUM reagents. (<b>b</b>) Overall mean PLCζ levels in spermatozoa from a total of 14 fertile donors. Data represent mean ± s.e.m., <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01, and <sup>***</sup><i>P</i> < 0.001, with one-way ANOVA and Bonferroni correction. (<b>c</b>–<b>i</b>) Comparison of PLCζ visualization in spermatozoa from a fertile donor (donor 3) between “in-house” and AUM methods. (<b>c</b>) Confocal microscopy visualization of PLCζ. The in-house method exhibited better RF intensity of PLCζ in the equatorial segment of the sperm head than AUM methods. Extensive RF intensity of PLCζ was observed in the midpiece of AUM-treated (AT or HCl) sperm (red arrows). Yellow arrows indicate PLCζ. Representative images for BF microscopy, DAPI, FITC-PLCζ staining, and overlay showing sperm expressing PLCζ. Scale bars = 5 μm. (<b>d</b>) Overall mean RF intensity of PLCζ in spermatozoa from donor 3. Data represent mean ± s.e.m. (<b>e</b>) Proportions of spermatozoa exhibiting PLCζ. Data represent mean values. <sup>**</sup><i>P</i> < 0.01 and <sup>***</sup><i>P</i> < 0.001, with one-way ANOVA and Bonferroni correction. (<b>f–i</b>) Comparison of PLCζ localization in spermatozoa. PLCζ localization was classified into eight patterns and compared between spermatozoa stained with (<b>f</b>) non-AUM, (<b>g</b>) AT-AUM, (<b>h</b>) HCl-AUM, and (<b>i</b>) in-house methods. Data represent mean percentages. <sup>***</sup><i>P</i> < 0.001 with one-way ANOVA and Bonferroni correction. PLCζ: phospholipase C zeta; AUM: antigen unmasking method; RF: relative fluorescence; a.u.: arbitrary units; s.e.m.: standard error of mean; ANOVA: analysis of variance; DAPI: 4'-6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; BF: bright field; AT: acidic Tyrode's solution.