Materials and Methods
- Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm



The Live/Dead Sperm Viability Kit, the Vybrant Apoptosis Assay Kit, and 3,3'-dihexylocarbocyanine iodide (DiOC6(3)) were purchased from Molecular Probes (Montluçon, France); propidium iodide (PI), etoposide (VP-16), and carbonyl cyanide m-chlorophenylhydrazone (FCCP) were from Sigma (Saint Quentin Fallavier, France); the CaspACE FITC-VAD-FMK In Situ Marker was from Promega (Charbonnières-les-Bains, France); the Annexin V-FLUOS Staining Kit was from Roche (Meylan, France); the Apo BrdU Kit was from Phoenix Flow Systems (San Diego, CA); DNase was from Qbiogene (Illkirch, France); RPMI-1640 was from Eurobio (Les Ulis, France); and Biociphos Medium was from IMV Technologies (L'Aigle, France).

Semen Collection and Cryopreservation

Procedures relating to the care and use of animals were approved by the French Ministry of Agriculture and Fishing according to the French regulations for animal experimentation (guideline 19/04/1988). Twenty-six healthy bulls (Charolais) were used in this study. Semen was collected with an artificial vagina. The volume of each ejaculate was measured and the sperm cell concentration assessed under light microscopy. After collection, ejaculate was divided into two parts. Less than 2 h after collection, the first part was analyzed with SYBR-14/PI, DiOC6(3)/PI, Annexin V-FITC/PI, Yo-Pro-1/PI, and caspase inhibitor/PI or fixed for terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL) assay. Immediately after collection, the second part was diluted to 100 x 106 sperm/ml in Biociphos Medium prewarmed at 37°C. The semen was cooled to 4°C over 3 h and frozen in liquid nitrogen vapor for 10 min before being plunged into liquid nitrogen. The cryopreservation method was adapted from previously described protocols [16, 22, 23]. Prior to the various analyses, cryopreserved samples were thawed at 37°C for 1 min. Ten thawed ejaculates were diluted to 10 x 106 cells/ml in PBS and either incubated at 37°C for 4 h for caspase inhibitor/PI analysis or for 30 h for viability analysis and TUNEL assay.

Culture and Apoptosis Induction in U937

U937 cells were grown in RPM1-1640 supplemented with 10% heat-inactivated neonatal calf serum. The medium included penicillin and streptomycin. Cells were maintained at 37°C, in a water-saturated atmosphere of 95% air and 5% CO2. Apoptosis was induced after 6 h of incubation in 25 µM VP-16. Before analysis, the U937 samples were centrifuged for 5 min at 6000 rpm and washed twice with prewarmed 37°C PBS 1x to eliminate culture medium.

Viability Assay

Sperm sample viability was assessed using the Live/Dead Sperm Viability Kit. Briefly, ejaculates were diluted to 1 x 106 sperms/ml in prewarmed 37°C PBS. Then samples were incubated at 37°C for 20 min with 100 nM SYBR-14 and 12 µM PI before flow cytometric analysis [24].

DiOC6(3)/PI Assay

DiOC6(3) was used to detect mitochondrial membrane potential. In 1 ml of PBS, 1 x 106 cells were diluted. DiOC6(3) was added up to a final concentration of 90 nM [25]. The test was validated using FCCP, an uncoupler of mitochondrial oxidative phosphorylation, which makes the inner mitochondrial membrane permeable for protons and induces dissipation of {Delta}{Psi}m. In this control, before adding DiOC6(3), 4 µl of 50 mM FCCP was added and the cells were incubated for 30 min at room temperature (RT). The tubes were gently mixed and incubated for 15 min at RT and 6 µM PI was added to each tube. Flow cytometry or fluorescence microscopy analysis was conducted within 10 min.

Caspase Inhibitor/PI Assay

The CaspACE FITC-VAD-FMK In Situ Marker was used to detect active caspases. The structure of the cell-permeable caspase inhibitor peptide VAD-FMK (Val-Ala-Asp-Phe-Met-Lys) conjugated to FITC allows delivery of the inhibitor into the cell, where it binds to activated caspases, serving as an in situ marker for apoptosis [26]. In 0.5 ml of PBS, 0.5 x 106 cells were diluted. Then 1 µl of FITC-VAD-FMK (5 mM) was added. The tubes were gently mixed and incubated for 20 min at RT in the dark. Then the cells were washed twice with PBS and the pellets were resuspended in 500 µl of PBS. To each tube was added 6 µM PI. Flow cytometry or fluorescence microscopy analysis was conducted within 10 min.

Annexin V-FITC/PI Assay

Annexin V-FITC/PI assay was conducted on U937 and human and bovine sperm cells. Human sperm was obtained from normospermic men by masturbation after obtaining informed written consent. The Annexin-V-FLUOS Staining Kit was used to detect the PS translocation from the inner to the outer leaflet of the plasma membrane. Following manufacturer instructions, for each assay, 1 x 106 cells were washed and then diluted in 100 µl of annexin V buffer. Then 5 µl of annexin V-FITC was added to the sample. The tubes were incubated for 15 min at RT in the dark. Then 400 µl of additional binding buffer and PI was added to each tube. Flow cytometry analysis was conducted within 10 min.

Yo-Pro-1/PI Assay

The Vybrant Apoptosis Assay Kit was used to detect changes in plasma membrane permeability to Yo-Pro-1 [27]. The 1 x 106 cells were diluted in 1 ml of PBS. Then 1 µl of Yo-Pro-1 (100 µM) was added. The tubes were gently mixed and incubated for 20 min at RT and 6 µM PI was added to each tube. Flow cytometry analysis was conducted within 10 min.


Nucleus status was studied using the TUNEL assay. The polymerization of labeled nucleotides in DNA breaks gave information on DNA fragmentation [28] and the varying amounts of PI molecules incorporated into each permeabilized cell gave information on the nucleus condensation level [29]. The DNA fragmentation was assessed using the Apo BrdU Kit. Spermatozoa were diluted to a 5 x 106 cells/ml concentration, centrifuged at 1000 x g for 5 min, fixed, and permeabilized in 70% ethanol at –20°C for more than 12 h. After two washes with 1 ml of PBS, the elongation reaction was performed by incubating the sperm in 50 µl of labeling solution containing the TdT enzyme and dUTP, for 1 h at 37°C. For each experimental set, a negative control was prepared by omitting TdT from the reaction mixture. Two subsequent washes were performed to stop the reaction. To perform the labeling reaction, the highly dUTP-specific fluorescein-PRB1 antibody was incubated with sperm for 30 min at RT in the dark. Before flow cytometry analysis, the sperm was washed twice with PBS, labeled with 150 µM PI, and filtered. Positive controls were prepared as described above but with an additional treatment with 10 IU DNase I for 1 h at 37°C before the elongation reaction.

Flow Cytometry Analysis

Analysis was performed using the FACS Vantage SE cell-sorter (BD Biosciences, San Jose, CA). Fluorochromes were excited with the 488-nm line of the Enterprise laser (Coherent, San Jose, CA). Green and red fluorescence were detected using FL1 and FL3 detectors, respectively, through a bandpass (BP) 530/30 nm and a BP 695/40 nm filter. All data were analyzed with Cell Quest Pro 3.1 software (BD Biosciences).

For viability, DiOC6(3)/PI, caspase inhibitor/PI, Yo-Pro-1/PI, and annexin V-FITC/PI, 10 000 events were analyzed. FL1 and FL3 fluorescence signals were recorded after logarithmic amplification. For TUNEL assay, 15 000 events were recorded at a flow rate stabilized at 200–300 cells/sec. Cell doublets and debris were excluded using an FL3-A vs. FL3-w gate. Analysis of DNA fragmentation was performed using an FL3-A vs. FL1-H cytogram. FL1 and FL3 fluorescence signals were, respectively, recorded before logarithmic and linear amplification. Percentage DNA fragmentation of normally condensed and decondensed nuclei were quantified through gates drawn on an FL3-A histogram.


Before examination under a DMRB microscope (Leica Microsystems, Wetzlar, Germany), all samples were washed twice with PBS. Green and red fluorescence were, respectively, detected using L5 (BP 440–520 nm) and N 2–1 (BP 515–560 nm) filters. Images were captured by a CollSnapfx camera (Roper Scientific, Evry, France) using Meta Imaging 4.6.6. software (Universal Imaging, Downingtown, PA).

Statistical Methods

Statistical analyses were performed using the Statistica 6.0 program (StatSoft, Tulsa, OK). Population means for fresh and cryopreserved spermatozoa were compared by t-test for dependent samples. Values are presented as mean ± SD and were considered statistically significant when P

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