**3.2.6 Apoptotic changes**

Apoptosis is a physiological mechanism required for any organism function. In contrast to necrosis, apoptosis is a process, where cells play an active role in their own death. Apoptosis comprising of a complex phenomenon that includes three stages: induction, execution and degradation. The most significant changes related to apoptosis are the externalization of the phosphatidylserine (PS), DNA fragmentation, caspase activation, loss of mitochondrial membrane potential, and increase in sperm membrane permeability (Bratton et al., 1997; Glander & Schaller, 1999; Martin et al., 2004;). Several pathways are reported for mammalian cell apoptosis. These include the intrinsic, extrinsic, and apoptosis-inducing factors. During the early phases of disturbed membrane function, asymmetry of the membrane phospholipids occurs, before the integrity of the plasma membrane is

Methods of Assessment of Cryopreserved Semen 561

affected. Although, spermatozoa with DNA damage may be able to fertilize an oocyte, that could potentially disturb (epi)genetic regulation of the early embryo and block its further

DNA damage can be evaluated at different levels. One of the usually used methods, developed for detecting changes in the chromatin structure of DNA integrity, is the sperm chromatin structure assay (SCSA) (Chohan et al., 2006). The SCSA is a flow cytometric method for identification of changes in the DNA status. It is based on the assumption that a structurally abnormal sperm chromatin shows a higher susceptibility to acid denaturation (Evenson et al., 2002). The SCSA method utilizes the metachromatic properties of acridine orange (AO). This stain fluoresces in the green band when intercalates into the intact double-stranded DNA helix, and in the red band when associated with single strand denaturated DNA and RNA. After denaturation of chromatin by decreased pH, the spermatozoa with structurally abnormal chromatin fluorescence is detected in the red band (Fig. 6f) (Bochenek et al., 2001). The fertility data have been shown to correlate with the results obtained from the SCSA of human (Evenson et al., 1980), bull (Ballachey et al., 1988; Karabinus et al., 1990), stallion (Love & Kenney, 1998) and boar semen (Evenson et al., 1994). SCSA was also used for dog semen assessment (Garcia-Macias et al, 2006) and for evaluation of freezing-thawing effect on chicken and goose DNA status (Partyka et al. 2010; Partyka et

Another method to detect DNA defragmentation is TUNEL assay, which allows to incorporate of fluorescent nucleotide analogs by a terminal nucleotide transferase into single stranded DNA areas at the 3-OH termini (Chohan et al., 2006). Ramos & Wetzels (2001) using this method have shown that DNA damage is limited in functional human

The alternative method for detecting the DNA damage at the level of individual cells is the single-cell DNA gel electrophoresis assay (COMET). Although this method does not use such equipment as flow cytometry, application of fluorescent DNA specific stain is required. In COMET assay spermatozoa are spread on a surface covered with an agarose gel, and treated with a solution that lyses the cell components leaving the DNA immobilized in the agarose. They are then subjected to a DNA denaturation process, followed by electrophoresis, causing DNA fragments to migrate away from the main bulk of nuclear DNA. After staining with propidium iodide or ethidium bromide, cells with DNA strand breaks, display a comet-like shape, with the undamaged DNA located in the head of the comet and the fragmented DNA dispersed through the tail. Image analyses provide information on the extent of strand breaks in the DNA molecule. Several studies, conducted with different techniques, including comet assay, showed a negative relationship between the fertilization potential of spermatozoa and alterations at the level of genetic material. In particular in humans, infertility has been associated with higher levels of DNA damage in sperm compared to fertile subjects (Irvine et al., 2000). Fraser & Strzeżek (2007) have shown that the freezing–thawing process provoked sperm chromatin destabilization rendering the boar spermatozoa more vulnerable to DNA fragmentation. COMET assay has also been recently used for the evaluation of cryopreserved avian semen (Madeddu et al., 2010;

development (Lewis & Aitken, 2005).

spermatozoa resulting from a swim-up procedure.

al., 2011b).

Gliozzi et al., 2011).

progressively damaged (Martin et al., 1995). When the cell membrane is disturbed, the phospholipid PS is translocated from the inner to the outer leaflet of the plasma membrane (Desagher & Martinou, 2000).

It is widely known that the cryopreservation usually causes sublethal cryodamage to spermatozoa, decreasing post-thaw cell viability. The freezing-thawing of human (Glander & Schaller, 1999), bull (Martin et al., 2004), and boar (Pena et al., 2003), stallion (Ortega Ferrusola et al., 2008), and dog (Kim et al., 2010) spermatozoa induces membrane PS translocation, what demonstrates that cryopreservation leads to apoptosis. Therefore, detecting early phases of membrane dysfunction, or initial phases of apoptosis of viable spermatozoa, would be important when evaluating stressed spermatozoa, such as those subjected to freezing and thawing, and would be useful for controlling freezing procedures in semen.

Annexin V is calcium-dependent phosphatidylserine (PS) binding protein conjugated with fluorochrome – FITC or Alexa Fluor®. The properties of Annexin V allow for detection of externally exposed PS. In ejaculated spermatozoa PS is confined to the cytoplasmatic side of the plasma membrane (Gadella et al., 1999). Different categories of apoptotic, necrotic and viable cells can then be sorted out using AnnexinV with PI, through flow cytometer (Fig. 6e), or visually evaluated using fluorescent microscope.

After induction of apoptosis, mitochondrial pores are being opened, leading to a decrease in mitochondrial membrane potential. Therefore, described above JC-1 dye is used for monitoring of apoptotic changes in spermatozoa, too (Ortega Ferrusola et al., 2009). Mentioned above, the opening of mitochondrial pores causes the release of proapoptotic factors into the cytoplasm, where they are activated. These factors – caspases, are central components in the apoptosis signaling cascade. The detection of activated caspases in living spermatozoa can be performed using fluorescence labeled inhibitors of caspases (FLICATM). It allows investigating caspase activation in semen samples with regard to a single cell. The FLICATM reagent is comprised of 3 segments—it includes a green (FAM 5 carboxyfluorescein) fluorescent label; an amino acid peptide inhibitor sequence targeted by the active caspase; and a fluoromethylketone group (FMK), which acts as a leaving group and forms a covalent bond with the active enzyme. Fluorescence labeled inhibitors of caspases are cell permeable and noncytotoxic (cited by Grunewald et al., 2009). Martin et al. (2004) showed that cryopreservation of bovine spermatozoa induced the significant increase in the proportion of cells with active caspases, which were mainly detected in the intermediate piece of spermatozoa.
