**3.4. Assessment of the oxidative stress and apoptosis**

chitecture. Two sperm populations may be found under a fluorescent microscope: sperm with intact membranes devoid of fluorescence and sperm with disordered cell membranes that emit fluorescence [7,35]. This probe further labels sperm round, apoptotic bodies, which are more frequently found in men with decreased sperm quality [14]. Whether these struc‐ tures are indicators of pathological or excessive apoptosis in the male genital tract or simply

Besides the modifications on lipid arrangement in sperm plasma membrane, loss of mem‐ brane integrity also induces disorganization of the membrane proteins. In fact, in defective sperm or after cold-shock, the clustering of the membrane proteins is frequently observed. At fertilization, such modifications can interfere with the exposition of molecular epitopes and compromise receptor-ligand interactions between sperm and the oviductal cells or the oocyte [15,36]. A more conservative approach to test these changes includes the functional *in vitro* gamete interaction tests, such as the oocyte penetration test or the hemi-zona assay (for a quick review see [11]). The *zona pellucida* binding assay tests the ability of spermatozoa to interact with the *zona pellucida* of the oocytes. It is an assay with much variability and it tends to be replaced for the hemi-zona assay, which has the advantage of allowing the com‐ parison between 2 sperm samples (one being used as control) on a single ovum. The oocyte penetration test assesses the fertilizing ability of spermatozoa by evaluating the presence of

cell remnants of similar density to sperm heads is still to prove.

104 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

**Figure 5.** Canine spermatozoa in a HOST test (magnification 100x).

Sperm metabolism in aerobic conditions originates oxidative molecules (reactive oxygen species or ROS - short-lived reactive chemical intermediates), which are highly reactive and oxidize lipids, proteins and glycides. Cells contribute to the maintenance of the oxidative homeostasis by controlling the amount of ROS, converting them into less injuring molecules [40,41]. Excessive ROS production damages the sperm membrane, reduces motility (by de‐ creasing membrane potential), induces irreparable DNA damage and is closely associated with apoptosis [42,43]. Oxidation reaction in the membranes increases ROS, changes mem‐ brane fluidity and compromises its integrity, impairs ion-gradients and lipid-protein inter‐ action and causes changes in proteins [44,45]. The seminal plasma possesses various natural antioxidants that protect spermatozoa against the oxidative stress which are removed when sperm is diluted or submitted to a process for preservation. Spermatozoa are particularly susceptible to lipid peroxidation, and one should be aware that semen manipulation and cryopreservation-thaw procedures accelerate the production of reactive oxygen species. Within the spermatozoa, mitochondria and the plasma membrane are the most sensitive structures to ROS [45].

Lipid peroxidation (LPO) releases membrane polyunsaturated fatty acids that are used as substrates for ROS and hydroxyl radical generation. The most frequent product of LPO is malonaldehyde (MDA) [44]. LPO can be indirectly assessed using a spectrophotometer by measuring thiobarbituric acid reactive (TBAR) substances; the method is based on the meas‐ urement of the complex formed by the reaction of MDA with TBA under a temperature stressor (incubation at 100ºC), which produce a pink-coloured chromogen and is readable at a wavelength of 532 nm. Also, the fluorescent probe BODIPY581/591-C11 (4,4-difluoro-5-(4 phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid) is frequently used in association with flow cytometry to assess LPO in the sperm. BODIPY is a fatty acids sen‐ sitive fluorescent probe that changes fluorescence from red to green in the presence of lipid peroxidation. Its association with a vital probe further allows to evaluate the fluorescence emission ratio in living cells [44,46].

Assessment of additional molecules known to be involved in the apoptosis mechanism, which might work as possible biological markers, can be performed by ICC, Western blotting or even in proteomic studies. Some molecules participating or regulating apoptotic processes in cells have been analysed in sperm and in semen, and its concentration was found to correlate with sperm quality. Among these molecules, TNF localization in pig and canine sperm has been per‐ formed [48,49]. The immunolabelling is limited to the sperm mid-piece in the mitochondrial re‐ gion (Figure 6) and it has been demonstrated that a decrease in TNF immunoreaction is observed in spermatozoa incubated in a capacitating medium. When exposed to TNF, sperma‐ tozoa showed decreased motility, increased PS externalization and chromatin and DNA dam‐

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 107

**Figure 6.** Sperm immunoreactions against TNF. In canine spermatozoa, strong immunolabelling for TNF was found in

An association between infertility and the integrity of DNA content in sperm has been sug‐ gested. The integrity of male DNA is of utmost importance for embryo development and offspring production [13,41]. DNA damage is not usually perceived under classic or ad‐ vanced semen assessment, but has been proposed to be at the origin of infertility in normo‐ spermic individuals. DNA damage (abnormal chromatin structure) may arise from different processes: deficient recombination or packaging during spermatogenesis, apoptosis and oxi‐ dative stress. DNA loss of integrity does not always impair fertilization, but compromises sustainable embryo development, predisposing to embryo losses and abortion [9,15]. DNA fragmentation may be associated with various pathological and environmental conditions [51,52], but also with endogenous mechanisms such as the oxidative stress and apoptosis.

age, changes that are usually associated with apoptosis [50].

sperm mid–piece, in the mitochondrial region.

**3.5. Assessment of DNA integrity**

Additional, currently used methods also include the glutathione peroxidase reaction (where the hydrogen peroxide oxidizes GSH (reduced glutathione) into GSSG (oxidized glutathione) in the presence of glutathione reductase and NADPH results from the con‐ sumption of NADPH in proportion to the peroxide content), by flow cytometry measure‐ ment of the fluorescent intensity of the compounds oxidized by ROS (such as the dichlorofluorescin diacetate- DCFH-DA- or the Hydroethidine- HE), using the gas-liquid chromatography separation of lipid peroxides, followed by its identification by mass spectrometry and by measuring cytotoxic aldehydes through high performance liquid chromatography (HPLC) [44,45].

ROS production can be directly monitored by a luminol or a lucigenin-based chemillumi‐ nescence assay [43,45]. This assay does not distinguish between intracellular and extracel‐ lular ROS, but it differentiates between the production of superoxide and hydrogen peroxide according to the probe used (lucigenin and luminol, respectively for superoxide and hydrogen peroxide). Measurement of chemilluminescence is proportional to ROS ac‐ cumulation [45].

An important side effect of the oxidative stress is apoptosis [42]. The most important changes associated to sperm apoptosis are the externalization of the phosphatidylserine (PS), a molecule usually confined to the inner leaflet of the plasma membrane, the caspase system activation, the DNA fragmentation, the lost of mitochondrial integrity and the in‐ crease of cell membrane permeability [41]. To assess sperm apoptosis it is frequently used the Annexin V, a Ca2+-dependent PS-binding protein that reacts to the PS, which is translo‐ cated to the outer leaflet of the plasma membrane in damaged sperm. Annexin V can be con‐ jugated to fluorochromes such as FITC (Fluorescein isothiocyanate) in flow cytometry analysis. If a vital staining is used, such as the propidium iodide, the combination allows to distinguish between three sperm sub-populations: viable (Annexin-FITC-PI-negative), early apoptotic (Annexin-FITC-positive and PI-negative) and late apoptotic (Annexin-FITC-PIpositive) [7,41].

Caspases are molecules associated with the apoptotic pathway and can be classified as ini‐ tiators or executors; caspase 7 and 9 are initiators, while active caspase 3 is an executor. The determination of the caspase enzymatic activity in sperm extracts, in comparison to the one of neutrophils, can also be used to assess apoptosis in sperm, which may be completed by the semiquantitative determination of active caspase 3 and caspase 7 content, by Western blotting. Caspase activity has been shown to be consistently higher in low motility sperm, in particular, the active caspase 3 [47].

Assessment of additional molecules known to be involved in the apoptosis mechanism, which might work as possible biological markers, can be performed by ICC, Western blotting or even in proteomic studies. Some molecules participating or regulating apoptotic processes in cells have been analysed in sperm and in semen, and its concentration was found to correlate with sperm quality. Among these molecules, TNF localization in pig and canine sperm has been per‐ formed [48,49]. The immunolabelling is limited to the sperm mid-piece in the mitochondrial re‐ gion (Figure 6) and it has been demonstrated that a decrease in TNF immunoreaction is observed in spermatozoa incubated in a capacitating medium. When exposed to TNF, sperma‐ tozoa showed decreased motility, increased PS externalization and chromatin and DNA dam‐ age, changes that are usually associated with apoptosis [50].

**Figure 6.** Sperm immunoreactions against TNF. In canine spermatozoa, strong immunolabelling for TNF was found in sperm mid–piece, in the mitochondrial region.
