**3.2.3 Mitochondrial function**

556 Current Frontiers in Cryobiology

33258 (Christensen et al., 2004; Donoghue et al., 1995; Hewitt & England, 1998; Partyka et al., 2010; Peña et al., 1998; Rijsselaere et al, 2005; Sirivaidyapong et al., 2000). SYBR-14 and CFDA, usually used detectors of live cells, are membrane-permeant and non-fluorescent compounds, which are immediately deacylated and thus rapidly converted into high fluorescent compounds by intracellular esterases. These green fluorochromes are maintained intracellular by intact membranes (Peña et al., 1998; Silva & Gadella, 2006). As plasma membrane deteriorate at cell death, cells lose their ability to resist the influx of red fluorescent PI. PI replaces or quenches green fluorochromes (Garner & Johnson, 1995). Live, viable, intact spermatozoa show a green fluorescence (CFDA, SYBR-14, calcein-AM) while dead stain red (PI) (Fig. 6a). Carboxy-SNARF is pH indicator which stains live spermatozoa orange, while bisbenzimide stain Hoechst 33258 labels dead spermatozoa bright blue (Hewitt & England, 1998). The last one requires flow cytometric analysis with a laser that operates in the ultraviolet light range and is less commonly used in andrology laboratory,

SYBR-14/PI fluorochromes have been found to be more sensitive in comparison with conventional method of live-dead cell assessment. The advantage of the use of fluorochromes is the possibility to assess the semen without the interference of fat particles and others material present in the extended semen (Rijsselaere et al., 2005). The detection of the third subpopulation i.e. moribund spermatozoa is the next advantage of this method. Additionally the simultaneous assessment of several functions of spermatozoa may be done in the same specimen by simultaneous staining of sperm cells with fluorescent lectins PNA

Acrosome is the acidic secretory organelle filled with hydrolytic enzymes. Assessment of the acrosomal status is a very important part of semen evaluation, in the view of the role of this structure in the maintenance of spermatozoal ability to penetrate the egg's zona pellucida (in mammals), or the egg envelope (in birds) and the ability to fuse with the egg plasma membrane. Cells must retain a normal acrosome to ensure that the acrosome reaction may occur at the suitable time to facilitate fertilization (Esteves et al., 2007). Also the determination of the acrosome status in cryopreserved sperm is of the fundamental importance as cryopreservation directly damages sperm membrane, which could be

Acrosomal status may be assessed using lectins, such as peanut agglutinin from *Arachis hypogaea* (PNA) or *Pisum Sativum* agglutinin (PSA), conjugated with different fluorescent probes like fluorescein isothiocyanate (FITC), phycoerythryn (PE) or Alexa Fluor®, (Graham et al., 1990; Kawakami et al., 2002; Nagy et al., 2004; Partyka et al., 2010; Peña et al., 2001; Rijsselaere et al., 2005). For human sperm concanavalin A lectins (ConA) is used as well (Holden et al., 1990). The PNA labelling is specific for the outer acrosomal membrane and it binds to β-galactose moieties. Whereas the PSA is labelling α-mannose and α-galactose moieties of the acrosomal matrix. The absence of the fluorescence on the living sperm is indicative for an intact acrosome, and fluorescence is indicative for acrosome disruption or acrosome reaction (Silva & Gadella, 2006). Since PNA agglutinin displays less non-specific binding to other areas of the spermatozoa, it leads some researchers to favour this over PSA (Graham, 2001). Lectins may be also combined with Hoechst 33258, carboxy-SNARF/PI,

however alternatively it may be applied within fluorescent microscope.

or PSA for acrosome evaluation with PI for dead cell assessment.

**3.2.2 Acrosomal membrane integrity** 

followed by a loss of the acrosomal matrix contents.

The motility of spermatozoa subjected to cryopreservation is reduced by reason of some changes in the active transport and the permeability of the plasma membrane in the tail region (Blesbois et al., 2008; Watson, 1995). A reduction of spermatozoa motility may also be triggered by a change in the availability of energy or an injury of the axonemal elements. Moreover, it has been noted that the alterations in the ultrastructure of mitochondria occurring during cryopreservation are followed by a loss of the internal mitochondrial structure of frozen-thawed spermatozoa (Watson, 1995).

Rhodamine 123 (R123) is the potentiometric membrane dye which is used to selectively stain functional mitochondria. It fluoresces only when the proton gradient over the inner mitochondrial membrane (IMM) is built up and unstained sperm do not contain functional mitochondria (Garner et al., 1997; Gravance et al., 2001). Also the group of Mitotrackers: Mitotracker Deep Red, Red, Orange and Green selectively label the respirating mitochondria. Thus, these probes are suitable to discriminate sperm with deteriorated mitochondria from sperm in which oxidative respiration occurs (Gadella & Harrison, 2002; Garner et al., 1997).

Some of mitotrackers such as 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) change their fluorescent properties due to changes in the potential of IMM. JC-1 is a lipophilic cationic fluorescent carbocyanine dye that is internalized by all functioning mitochondria, where it fluoresces green. However, as the concentration of JC-1 inside the mitochondria increases (highly functional mitochondria), the stain forms aggregates which fluoresce orange. Hereby, population of spermatozoa can be divided into high (orange staining), moderate (orange and green) and low (green) mitochondrial potential groups after IMM depolarisation (Fig. 6c) (Garner et al. 1999; Gravance et al., 2000).
