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Results are presented concerning apoptosis markers, which shed light on the effects …


Biology Articles » Cryobiology » Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm » Figures

Figures
- Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm

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FIG. 1. Optical microscopy study of U937 without (A) and with (B) induction of apoptosis by 25 µM VP-16. Arrows show cells with a typical apoptotic pattern. Scale bar = 50 µM

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FIG. 2. Flow cytometry study of {Delta}{Psi}m. A, B) Typical cytograms of DiOC6(3) staining in living cells (PI). The population with a normal {Delta}{Psi}m (a) is shown. A) U937 cells without treatment (3), treated with FCCP (50 mM, 30 min) (1) or with VP-16 (25 µM, 6 h) (2). B) Cryopreserved (2) and freshly ejaculated spermatozoa without treatment (3) or treated with FCCP (50 mM, 30 min) (1). C) Means of the various populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P D) Location of DiOC6(3) fluorescence in fresh spermatozoon using fluorescence microscopy. Scale bar = 5 µM

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FIG. 3. Flow cytometry study of the activation of proteins from the caspase family. Typical cytograms of FITC-VAD-FMK/PI in U937 incubated with 25 µM VP-16 or not (A) and in fresh or cryopreserved ejaculated spermatozoa (B). a) Dead cells are PI+; (b) living nonapoptotic cells are FITC-VAD-FMK/PI; and (c) living apoptotic cells are FITC-VAD-FMK+/PI. C) Means of the different populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P D) Location of FITC-VAD-FMK fluorescence in a cryopreserved spermatozoon using fluorescence microscopy. Scale bar = 5 µM

figure 3

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FIG. 4. Flow cytometry study of the phosphatidylserine externalization. Typical cytograms of annexin V-FITC/PI in U937 incubated with 25 µM VP-16 or not (A) and in human spermatozoa incubated with 10 µM A23187 or not (B) and in fresh or cryopreserved bull ejaculated spermatozoa (C). a) Dead cells are PI+; (b) living nonapoptotic cells are annexin V-FITC/PI; and (c) living apoptotic cells are annexin V-FITC+/PI. All these cytograms are representative of three independent assays

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FIG. 5. Flow cytometry study of the permeability to Yo-Pro-1 and to PI. Typical cytograms of Yo-Pro-1/PI in U937 incubated with 25 µM VP-16 or not (A) and in fresh or cryopreserved ejaculated spermatozoa (B). a) Dead cells are PI+, (b) living nonapoptotic cells are Yo-Pro-1/PI, and (c) living apoptotic cells are Yo-Pro-1+/PI. C) Means of the different populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P

figure 5

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FIG. 6. Flow cytometry study of DNA fragmentation and nucleus condensation of bovine spermatozoa. A) Typical cytograms of TUNEL assay in fresh or cryopreserved ejaculated spermatozoa. Negative control was obtained by omitting the TdT enzyme and DNA fragmentation in positive control was obtained by DNase digestion (10 IU, 1 h, 37°C). a) Populations with a DNA fragmentation, (b) with a normally condensed nucleus, and (c) with a decondensed nucleus. B) Means of the different populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Dead (PI+) and viable populations were determined by a Sybr14/PI assay before fixation. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P C) Correlation between the DNA fragmentation in normally condensed and in decondensed nuclei. Thirty-two samples were studied (16 fresh and 16 cryopreserved)

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FIG. 7. Flow cytometry study of DNA fragmentation and nucleus condensation of bovine spermatozoa. Ten pairs of ejaculated sperm were analyzed immediately after thawing or after 30 h of incubation in PBS at 37°C. DNA fragmentation and nucleus condensation was determined using TUNEL assay, and dead (PI+) and viable populations were determined by a Sybr14/PI assay before fixation. Error bars indicate standard deviation. *, Difference between 0 h and 30 h of incubation was significant (P

figure 7

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Source: Biology of Reproduction 71, 28–37 (2004).


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