**3. Biological markers of sperm function**

**2. Proposed side effects for sperm cryopreservation**

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

cryopreservation [9].

oocyte;

pregnancy.

tion (Figure 2). These include [9,13-15]:

pose to DNA fragmentation.

Sperm cryopreservation is unavoidably linked to a reduction in sperm quality, which has been related to cold shock and freezing damages. The importance of cold shock injuries var‐ ies with the species, the composition of the extender, the cryoprotectant selected and the male, among other factors [10,11]. Seldom more than 50% of the sperm population survives

Deleterious effects of freezing/thawing procedures originate a reduction on the sperm life span due to alterations in the structure and functions of spermatozoa. Side effects in‐ clude altered motility, changes in the plasma membrane and acrosomal integrity and in‐ creased DNA fragmentation. All these alterations induce a reduction of the sperm ability to survive in the female reproductive tract and to interact with the oocyte at fertilization [8,12]. In an attempt to compensate these side effects, seminal doses are usually pre‐ pared with excessive numbers of spermatozoa in order to improve AI fertility [5,8].

Available cryopreservation techniques have a number of potentially detrimental problems, such as physical and chemical injuries that prone the spermatozoa to cell death and dysfunc‐

**•** Capacitation-like changes – after freezing/thawing, sperm behaves as if capacitated, which decreases its ability to survive within the female genital tract and to fuse with the

**•** Motility impairment – a decrease in the motility is observed in post-thawed spermatozoa, which tend to exhibit a variable degree of motility weakening, with subsequent hamper‐

**•** Oxidative damages – which may trigger apoptosis and DNA damage when reaching a given threshold. Apoptosis compromises the mitochondrial function, motility and predis‐

**•** Compromise of the membrane and acrosome integrity - loss of membrane integrity lead to altered ionic transport to the cell, in particular the calcium and water balance, with sub‐ sequent loss of the sperm ability for volume regulation and osmoadaptation. Also, it will compromise protein location and/or exposition on the cell's surface, which negatively af‐ fects sperm survival, sperm binding to oviductal epithelium and interaction between male and female gametes. In addition, restrain of the acrosome integrity may compromise

**•** DNA and chromatin changes, which may not be directly related to fertilization but are often reported to impair sustainable post-syngamy embryonic development and

sperm competence to penetrate the oocyte layers at fertilization;

ing of sperm progression till the oviducts and a decrease on the fertility potential;

The most frequently used methods of sperm analysis have been pleasantly reviewed in a re‐ cent InTech publication [11], driving the main topic of this review into new adjunctive meth‐ ods available to test sperm quality (Figure 3). These tests can be performed as well in freshly ejaculated sperm or in preserved samples. In the former, it would allow to increase the abili‐ ty to predict sperm quality, the selection of donor/sperm for cryopreservation and to assess infertility causes. In the later it could be of utmost interest to study the sperm response to preservation trials, such as the design of a new extender. Further, it could also be of impor‐ tance when studying the sperm response to preservation in new species, where it would al‐ low the identification of the most suitable molecular and functionally-friendly extender or procedure.

### **3.1. Assessment of events associated with sperm capacitation**

For long, it has been accepted that freezing/thawing procedures induce a capacitation-like status that originate losses on the fertilizing potential of spermatozoa. Non-capacitated live sperm cells survive longer in the female genital tract than capacitated sperm [16]. Dysfunc‐ tion of intracellular pathways associated with calcium (Ca2+) predisposes to acrosome insta‐ bility and exocytosis of its content. Regulation of protein function by Ca2+ signalling pathways is central for most sperm functions and infertility is often found when those sig‐ nalling pathways are disturbed [17].

ther in flow cytometry [18] or in cell imaging microscopy, and when combined with other vital staining, such as Hoechst 33258 or 33342 and carboxy-SNARF/PI (carboxy-seminaph‐ thorhodafluor/ propidium iodide), or with the hypoosmotic swelling test they also allow to

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 99

There are other fluorescent tests to evaluate the acrosome, like the chlortetracyclin (CTC) staining, in which fluorescence is activated when there is bounding to free calcium ions. When combined with another fluorescent dye, such as Hoechst 33258, the combined fluores‐ cent staining allows to differentiate from three different sperm populations: the uncapacitat‐ ed and acrosome intact (F-pattern), the capacitated and acrosome intact (B-pattern) and the capacitated and acrosome reacted (AR-pattern) [5]. Today, CTC staining is a routine test to assess the occurrence of the capacitation and the acrosome reaction; it has also been adapted

Additional tests can be performed for assessment of the occurrence of capacitation-like events, using biological markers, molecules known to trigger or participate in the capacita‐ tion reaction. Determining the cholesterol efflux, the protein phosphorylation and changes in intracellular calcium are some of the available methodologies. Nevertheless, for some in‐

Furthermore, the acrosome status may be tested indirectly through calcium assessment or by studying the response of sperm stimulation with calcium ionophores, progesterone or egg vestments [14,15,17,19]. Acrosome defective sperm show poorer responses to calcium

Changes in free Ca2+ concentrations in sperm may be studied by flow cytometry or indirect‐ ly by an ionophore challenge test, the later generating intracellular calcium signals that trig‐ ger the acrosome reaction [14,15,20]. The percentage of reacted spermatozoa is usually determined using a fluorescent dye. Samples with 10 to 30% of reacted spermatozoa have higher fertility potential than samples with less than 10% (this value being considered a

Recently, it was demonstrated that sperm exposition to progesterone induced similar but more rapid Ca2+ signalling pathway, which seems to be independent of a known second messenger system [19]. This behaviour allows the use of this molecule to challenge the sperm acrosome function, as do the ionophore test. For a large number of species, granulosa cells expelled with the oocyte from the ovulatory follicle have the capacity to produce pro‐

Protein phosphorylation can be studied using different approaches. Detection of phospho‐ tyrosine residues in the spermatozoa can be performed by immunocytochemistry (ICC) in a cytology specimen (over silane- or poly-L-lysine-coated slides), using specific antibodies. The reaction is amplified by the use of secondary antibodies and the reaction may be visual‐ ized either with a fluorescent or a non-fluorescent dye. Further, this technique also allows the assessment of sub-cellular changes in the molecule localisation, besides the evaluation of changes in the intensity of immunolabelling [7]. ICC may also extend to other proteins tar‐

gesterone, which can affect the spermatozoa that approaches the egg for fertilization.

distinguish between non-viable and reacted spermatozoa.

dicators, it is still unclear how they correlate with sperm quality.

testing than do the sperm with intact acrosome [6].

to flow cytometry analysis.

threshold) [20].

geting acrosome-related functions.

**Figure 3.** Main objectives for advanced sperm screening are directly related to the assessment of the spermatozoa functions [ICC - immunocytochemistry; HOST - hypoosmotic swelling test; TUNEL - Terminal deoxynucleotidyl transfer‐ ase dUTP nick end labeling].

As it was mentioned, calcium is an important regulator of intracellular activity. Calcium mobilization has been associated with major sperm functions, such as capacitation, acro‐ some reaction and hypermotility. Ca2+ stores in the sperm are located in the acrosome, neck and mitochondria [17]. Release of Ca2+ from its stores triggers the above-mentioned reactions, although it is now suspected that different patterns of calcium release are re‐ sponsible for different functions. For example, hypermotility is associated with an oscilla‐ tory, wave-like pattern of Ca2+ release, while capacitation, acrosome reaction and exocytosis of the content are associated with a burst of intracellular Ca2+ into the cyto‐ plasm [6,17]. Also, the increase in free intracellular Ca2+ is often associated with the stim‐ ulation of different, pH-sensitive ion-channels that have been associated with hypermotility and acrosome reaction. Sperm neck Ca2+ stores seem to be related with the flagella movement, during hyperactivation [17].

Acrosome membrane integrity is commonly assessed with fluorescent conjugated lectins (PNA- Peanut agglutinin- and PSA- Pisum sativum agglutinin). Absence of fluorescence in the living sperm indicates an intact acrosome, whilst fluorescence is indicative of acrosome disrupted or acrosome-reacted sperm [5,11]. Fluorescent conjugated lectins can be used ei‐ ther in flow cytometry [18] or in cell imaging microscopy, and when combined with other vital staining, such as Hoechst 33258 or 33342 and carboxy-SNARF/PI (carboxy-seminaph‐ thorhodafluor/ propidium iodide), or with the hypoosmotic swelling test they also allow to distinguish between non-viable and reacted spermatozoa.

There are other fluorescent tests to evaluate the acrosome, like the chlortetracyclin (CTC) staining, in which fluorescence is activated when there is bounding to free calcium ions. When combined with another fluorescent dye, such as Hoechst 33258, the combined fluores‐ cent staining allows to differentiate from three different sperm populations: the uncapacitat‐ ed and acrosome intact (F-pattern), the capacitated and acrosome intact (B-pattern) and the capacitated and acrosome reacted (AR-pattern) [5]. Today, CTC staining is a routine test to assess the occurrence of the capacitation and the acrosome reaction; it has also been adapted to flow cytometry analysis.

Additional tests can be performed for assessment of the occurrence of capacitation-like events, using biological markers, molecules known to trigger or participate in the capacita‐ tion reaction. Determining the cholesterol efflux, the protein phosphorylation and changes in intracellular calcium are some of the available methodologies. Nevertheless, for some in‐ dicators, it is still unclear how they correlate with sperm quality.

Furthermore, the acrosome status may be tested indirectly through calcium assessment or by studying the response of sperm stimulation with calcium ionophores, progesterone or egg vestments [14,15,17,19]. Acrosome defective sperm show poorer responses to calcium testing than do the sperm with intact acrosome [6].

Changes in free Ca2+ concentrations in sperm may be studied by flow cytometry or indirect‐ ly by an ionophore challenge test, the later generating intracellular calcium signals that trig‐ ger the acrosome reaction [14,15,20]. The percentage of reacted spermatozoa is usually determined using a fluorescent dye. Samples with 10 to 30% of reacted spermatozoa have higher fertility potential than samples with less than 10% (this value being considered a threshold) [20].

**Figure 3.** Main objectives for advanced sperm screening are directly related to the assessment of the spermatozoa functions [ICC - immunocytochemistry; HOST - hypoosmotic swelling test; TUNEL - Terminal deoxynucleotidyl transfer‐

As it was mentioned, calcium is an important regulator of intracellular activity. Calcium mobilization has been associated with major sperm functions, such as capacitation, acro‐ some reaction and hypermotility. Ca2+ stores in the sperm are located in the acrosome, neck and mitochondria [17]. Release of Ca2+ from its stores triggers the above-mentioned reactions, although it is now suspected that different patterns of calcium release are re‐ sponsible for different functions. For example, hypermotility is associated with an oscilla‐ tory, wave-like pattern of Ca2+ release, while capacitation, acrosome reaction and exocytosis of the content are associated with a burst of intracellular Ca2+ into the cyto‐ plasm [6,17]. Also, the increase in free intracellular Ca2+ is often associated with the stim‐ ulation of different, pH-sensitive ion-channels that have been associated with hypermotility and acrosome reaction. Sperm neck Ca2+ stores seem to be related with the

Acrosome membrane integrity is commonly assessed with fluorescent conjugated lectins (PNA- Peanut agglutinin- and PSA- Pisum sativum agglutinin). Absence of fluorescence in the living sperm indicates an intact acrosome, whilst fluorescence is indicative of acrosome disrupted or acrosome-reacted sperm [5,11]. Fluorescent conjugated lectins can be used ei‐

ase dUTP nick end labeling].

flagella movement, during hyperactivation [17].

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

Recently, it was demonstrated that sperm exposition to progesterone induced similar but more rapid Ca2+ signalling pathway, which seems to be independent of a known second messenger system [19]. This behaviour allows the use of this molecule to challenge the sperm acrosome function, as do the ionophore test. For a large number of species, granulosa cells expelled with the oocyte from the ovulatory follicle have the capacity to produce pro‐ gesterone, which can affect the spermatozoa that approaches the egg for fertilization.

Protein phosphorylation can be studied using different approaches. Detection of phospho‐ tyrosine residues in the spermatozoa can be performed by immunocytochemistry (ICC) in a cytology specimen (over silane- or poly-L-lysine-coated slides), using specific antibodies. The reaction is amplified by the use of secondary antibodies and the reaction may be visual‐ ized either with a fluorescent or a non-fluorescent dye. Further, this technique also allows the assessment of sub-cellular changes in the molecule localisation, besides the evaluation of changes in the intensity of immunolabelling [7]. ICC may also extend to other proteins tar‐ geting acrosome-related functions.

While ICC locates the molecule inside the cell, the Western blotting technique (also named immunoblotting) may be used for quantification of the protein. After a gel electrophoresis, the extracted proteins are transposed into a membrane and incubated with a primary anti‐ body for the target molecule (the same as for ICC). The reaction is revealed in an X-ray film or a digital image [21]. The use of a cell or a molecular standard, like for the genomic assays, will allow the relative quantification of the protein content in the sample. However, this technique presents a weakness: the possible degradation of the target protein during sample preparation may cause the visualization of multiple bands of different molecular weight.

drial functionality, which has been correlated with the mitochondrial potential [25]. They are named MitoTracker® and are available in red, green and orange colours. Live sperm cells are suspended to incubate into a solution with the selected probe. Cells may be ana‐ lysed by flow cytometry, by microplate-based analysis or by epifluorescence microscopy, using cytological preparations. The probes diffuse across the plasma membrane and accu‐ mulate in active mitochondria. To determine the percentage of MitoTracker positive sperm, 200 spermatozoa are usually counted per sample, in at least four fields, in a fluorescence mi‐ croscope [7], varying the spectral wavelength with the probe used. The MitoTracker® can be combined with a vital dye, such as the Hoechst 33342, allowing the separation of different sperm sub-populations: the dead spermatozoa, the live mitochondrial non-competent sperm and the live mitochondrial competent spermatozoa. Also belonging to the MitoTracker dyes, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide) is a dualemission, potential-sensitive probe, which emits different fluorescent colours according to the membrane potential (IMM): after incubation JC-1 is captured by functional mitochondria where it stains in green if the IMM is polarized or in orange or red if the IMM is depolar‐ ized. Depolarization of IMM leads to the aggregation of the dye. The ratio of orange-togreen JC-1 fluorescence depends only on the membrane potential, since it is independent of the mitochondrial size, shape or density. Using this staining, a sperm sample can be com‐ posed of different combinations of fluorescent cells according to their mitochondrial inner membrane potential [13]. The different labelling patterns may be correlated with parameters

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 101

A different approach to assess the mitochondrial integrity is to assess the presence of sperm mitochondrial proteins through the use of ICC [7,13]. This approach allows the detection and location of target molecules within the cell and the study of the modifications to the ex‐ pected pattern of immunolabelling. One of the proteins available to study mitochondrial function is the Cytochrome C oxidase or complex IV, which catalyzes the final step in the mitochondrial electron transfer chain. This molecule is regarded as one of the major regula‐ tory molecules for oxidative phosphorylation. As in other ICC, cytological preparations are set to incubate with the specific primary antibody, followed by incubation with the appro‐ priated secondary antibody. The revelation can be obtained with DAB (3,3-Diaminobenzi‐ dine) or using DAPI (4',6-diamidino-2-phenylindole) as fluorochrome, under light or epifluorescence microscopy, respectively. The percentage of stained cells is determined over

Heat Shock Proteins (HSP), which are divided in families, are chaperon proteins involved in the protection of intracellular macromolecules against unfolding and aggregation during thermal and osmotic stress. HSP70 and HSP90, which have been found in the sperm, have important functions in the cellular trafficking of proteins other than the refolding and trans‐ port of client proteins. The role of HSP's on cell signalling in mature sperm is not clearly un‐ derstood. It is known that an active cell metabolism, such as ATP production, is required for the expression of heat sock response [26,27]. HSP 70 and HSP 90 have separated ATPase and client protein-binding sites [27] and also distinct roles in sperm function. It has been shown that HSP70 and 90 are targets for protein phosphorylation, which is activated during capaci‐

such as sperm motility [7].

200 spermatozoa in a minimum of 4 microscope fields.

Mass spectrometry and liquid chromatography, enhancing the separation and the identifica‐ tion of a large number of proteins, are often used for proteomics analysis. However, until now, this method gives a catalogue of hundreds or thousands of proteins that are not easily associated with sperm biological functions [22,23] and consequently there is not a practical interest for the immediate sperm quality assessment. Yet, using specific regions of spermato‐ zoa or particular cell organelles to focus the analysis could turn this approach to be helpful in the assessment of sperm function or specific sperm events.

## **3.2. Assessment of energy metabolism and sperm motility**

Energy metabolism is a key-factor in sperm function. It is supported by ATP pathway, which is found in the background of the most important sperm events, such as hyperacti‐ vation, capacitation and protein phosphorylation of the acrosome reaction. It has been shown that high intracellular ATP values correlate with higher survival and vitality postfreezing/thawing [24], while mitochondrial membrane potential mirrors the sperm quality and a better motility pattern. A primary function associated with mitochondria is the ATP synthesis by oxidative phosphorylation, although, energy might also be obtained by gly‐ cogenolysis in the sperm tail, a necessary complement to sustain energy in the tail and to maintain an effective movement. In humans, a decrease in mitochondrial activity has been found in patients with history of infertility even when normozoospermic [25]. Also, cumu‐ lative evidences suggest that mitochondrial activity is positively correlated with sperm quality and fertility, possibly associated with the fact that healthy mitochondria have a higher membrane potential [25].

The intracellular ATP content may be determined by an enzymatic assay (ATP/NADHlinked enzyme coupling assay) in association with spectrophotometry. On this reaction, the regeneration of hydrolysed ATP is linked to NADH oxidation. The assay measures the dif‐ ferences in NADH, which are proportional to the rate of ATP hydrolysis.

Sperm metabolic function may also be evaluated by assessment of the mitochondrial activi‐ ty. Integrity of the mitochondrial functioning can be assessed using specific dyes for these organelles [7,43]. Earlier, rhodamine 123 (R123) was frequently used to selectively stain functional mitochondria. It is a potentiometric membrane dye that fluoresces only when the proton gradient over the inner mitochondrial membrane (IMM) is built up. When the proton gradient collapses, the aerobic production of ATP fails, and mitochondria remain unstained [13]. More recently, other dyes have been developed, which selectively bind to respirating mitochondria and become fluorescent after oxidation. These can be used to test mitochon‐ drial functionality, which has been correlated with the mitochondrial potential [25]. They are named MitoTracker® and are available in red, green and orange colours. Live sperm cells are suspended to incubate into a solution with the selected probe. Cells may be ana‐ lysed by flow cytometry, by microplate-based analysis or by epifluorescence microscopy, using cytological preparations. The probes diffuse across the plasma membrane and accu‐ mulate in active mitochondria. To determine the percentage of MitoTracker positive sperm, 200 spermatozoa are usually counted per sample, in at least four fields, in a fluorescence mi‐ croscope [7], varying the spectral wavelength with the probe used. The MitoTracker® can be combined with a vital dye, such as the Hoechst 33342, allowing the separation of different sperm sub-populations: the dead spermatozoa, the live mitochondrial non-competent sperm and the live mitochondrial competent spermatozoa. Also belonging to the MitoTracker dyes, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide) is a dualemission, potential-sensitive probe, which emits different fluorescent colours according to the membrane potential (IMM): after incubation JC-1 is captured by functional mitochondria where it stains in green if the IMM is polarized or in orange or red if the IMM is depolar‐ ized. Depolarization of IMM leads to the aggregation of the dye. The ratio of orange-togreen JC-1 fluorescence depends only on the membrane potential, since it is independent of the mitochondrial size, shape or density. Using this staining, a sperm sample can be com‐ posed of different combinations of fluorescent cells according to their mitochondrial inner membrane potential [13]. The different labelling patterns may be correlated with parameters such as sperm motility [7].

While ICC locates the molecule inside the cell, the Western blotting technique (also named immunoblotting) may be used for quantification of the protein. After a gel electrophoresis, the extracted proteins are transposed into a membrane and incubated with a primary anti‐ body for the target molecule (the same as for ICC). The reaction is revealed in an X-ray film or a digital image [21]. The use of a cell or a molecular standard, like for the genomic assays, will allow the relative quantification of the protein content in the sample. However, this technique presents a weakness: the possible degradation of the target protein during sample preparation may cause the visualization of multiple bands of different molecular weight. Mass spectrometry and liquid chromatography, enhancing the separation and the identifica‐ tion of a large number of proteins, are often used for proteomics analysis. However, until now, this method gives a catalogue of hundreds or thousands of proteins that are not easily associated with sperm biological functions [22,23] and consequently there is not a practical interest for the immediate sperm quality assessment. Yet, using specific regions of spermato‐ zoa or particular cell organelles to focus the analysis could turn this approach to be helpful

Energy metabolism is a key-factor in sperm function. It is supported by ATP pathway, which is found in the background of the most important sperm events, such as hyperacti‐ vation, capacitation and protein phosphorylation of the acrosome reaction. It has been shown that high intracellular ATP values correlate with higher survival and vitality postfreezing/thawing [24], while mitochondrial membrane potential mirrors the sperm quality and a better motility pattern. A primary function associated with mitochondria is the ATP synthesis by oxidative phosphorylation, although, energy might also be obtained by gly‐ cogenolysis in the sperm tail, a necessary complement to sustain energy in the tail and to maintain an effective movement. In humans, a decrease in mitochondrial activity has been found in patients with history of infertility even when normozoospermic [25]. Also, cumu‐ lative evidences suggest that mitochondrial activity is positively correlated with sperm quality and fertility, possibly associated with the fact that healthy mitochondria have a

The intracellular ATP content may be determined by an enzymatic assay (ATP/NADHlinked enzyme coupling assay) in association with spectrophotometry. On this reaction, the regeneration of hydrolysed ATP is linked to NADH oxidation. The assay measures the dif‐

Sperm metabolic function may also be evaluated by assessment of the mitochondrial activi‐ ty. Integrity of the mitochondrial functioning can be assessed using specific dyes for these organelles [7,43]. Earlier, rhodamine 123 (R123) was frequently used to selectively stain functional mitochondria. It is a potentiometric membrane dye that fluoresces only when the proton gradient over the inner mitochondrial membrane (IMM) is built up. When the proton gradient collapses, the aerobic production of ATP fails, and mitochondria remain unstained [13]. More recently, other dyes have been developed, which selectively bind to respirating mitochondria and become fluorescent after oxidation. These can be used to test mitochon‐

ferences in NADH, which are proportional to the rate of ATP hydrolysis.

in the assessment of sperm function or specific sperm events.

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

**3.2. Assessment of energy metabolism and sperm motility**

higher membrane potential [25].

A different approach to assess the mitochondrial integrity is to assess the presence of sperm mitochondrial proteins through the use of ICC [7,13]. This approach allows the detection and location of target molecules within the cell and the study of the modifications to the ex‐ pected pattern of immunolabelling. One of the proteins available to study mitochondrial function is the Cytochrome C oxidase or complex IV, which catalyzes the final step in the mitochondrial electron transfer chain. This molecule is regarded as one of the major regula‐ tory molecules for oxidative phosphorylation. As in other ICC, cytological preparations are set to incubate with the specific primary antibody, followed by incubation with the appro‐ priated secondary antibody. The revelation can be obtained with DAB (3,3-Diaminobenzi‐ dine) or using DAPI (4',6-diamidino-2-phenylindole) as fluorochrome, under light or epifluorescence microscopy, respectively. The percentage of stained cells is determined over 200 spermatozoa in a minimum of 4 microscope fields.

Heat Shock Proteins (HSP), which are divided in families, are chaperon proteins involved in the protection of intracellular macromolecules against unfolding and aggregation during thermal and osmotic stress. HSP70 and HSP90, which have been found in the sperm, have important functions in the cellular trafficking of proteins other than the refolding and trans‐ port of client proteins. The role of HSP's on cell signalling in mature sperm is not clearly un‐ derstood. It is known that an active cell metabolism, such as ATP production, is required for the expression of heat sock response [26,27]. HSP 70 and HSP 90 have separated ATPase and client protein-binding sites [27] and also distinct roles in sperm function. It has been shown that HSP70 and 90 are targets for protein phosphorylation, which is activated during capaci‐ tation and capacitation-like response to sperm manipulation, in a reaction that might be as‐ sociated with the nitric oxide synthesis during oxidative stress [28]. Sperm is transcriptionally inactive. Thus, HSP content in the spermatozoa is defined at ejaculation and those proteins must be present in the cytosol to help protecting the sperm from injury [29]. Therefore it is expectable to find a reduction of its amount or intensity of immunoex‐ pression for these molecules (if Western blotting or ICC were used) after the cell attack. In canine ejaculates, a diminished number of sperm cells with low immunoreaction for HSP70 was found in semen of good quality. A reduction of the intensity of immunolabelling for this molecule was found after freezing/thawing (Figure 4), along with dislocation of the im‐ munostaining from the acrosomal area to the sperm tail [30,31]. It has also been found a cor‐ relation between HSP70 immunoreaction in freshly ejaculated sperm and sperm damage after freezing/thawing procedures [30,31].

damages are one of the major side effects of cryopreservation and are irreversible [33]. It is due to changes in the membrane structure and lateral phase separation of the membrane components leading to focal aggregation of proteins, disarrangement of the membrane lip‐

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 103

When introduced in a hypo- or hypertonic environment, cells tend to adjust and reach os‐ motic equilibrium by allowing water and solutes to change across the cell membrane. Sper‐ matozoa, among other cells, have the ability to maintain their volume after osmotic shock [1,34]. It has for long been proved that, for domestic species, cell volume control shows a close positive correlation with fertility [1]. In the ejaculate, there is usually sperm with dif‐ ferent aptitudes and with differences in the ability to respond to osmotic stressors. This is often related with membrane deficiencies in ion channels or signalling pathways that con‐ trol cell volume. The ability to adapt to osmotic changes can be tested by the hypoosmotic test (HOST), an indirect method to assess the membrane integrity, where sperm is incubated in hypoosmotic solutions between 1-60 minutes at 37ºC. Spermatozoa with intact plasma‐ lemma become swollen and present coiled tails when incubated in a sucrose solution (rang‐ ing from 75 to 150 mOsm, according to the species) (Figure 5). After longer exposures, they recover the initial volume [34]. Although currently used for *in vitro* semen assessment, this evaluation is subjective and not quantitatively rigorous. It is also possible that a number of sperm cells may die if prolonged incubation periods are used, biasing the results. However, it becomes more precise if performed with the aid of an electronic cell counter. In this ap‐ proach, known as the volume regulatory test, after the osmotic challenge, sperm passes through a capillary pore and cell volume is determined upon changes in the electric resist‐ ance to passage. The results are expressed as cell frequency distribution for the iso- and the hypoosmotic moments of the test and the amount of displacement of the distribution curve,

Different combinations of fluorescent membrane-impermeable dyes may also be used to as‐ sess the sperm membrane integrity. Most commonly used ones, also show some degree of affinity for DNA, as for Hoechst 33258, propidium iodide (PI) or ethidium homodimer 1 [11]. Alternatively acylated membrane dyes are also used. These dyes can cross the intact cell membrane and be held in the viable spermatozoa. When the plasma membrane is dam‐ aged, the probe leak out of the cell. More recently, fluorescein diacetate (CFDA), carbox‐ yl(methil)-derivates, such as carboxyl-SNARF and SYBR-14 have been used for this purpose (for more detail, see [11]). This sort of probes can be combined and used with flow cytome‐ try. The combination of different patterns allows estimating different degrees of sperm via‐ bility [13]. When combined with PI, green fluorochromes such as CFDA (Carboxyfluorescein diacetate) or SYBER-14 are replaced in the dead spermatozoa by the red fluorescence, which is not found in the membrane intact sperm. Carboxyl-SNARF, a pH-in‐ dicator, stains the live spermatozoa in orange, whilst Hoechst 33258 stains the dead sperma‐

Sperm membrane integrity can also be assessed by the use of merocyanine 540 (MC540), a hydrophylic probe with highly disorganized lipids that shows a high affinity pattern for in‐ stable membranes. This probe allows to monitor the changes in the cell membrane lipid ar‐

ids and increased permeability to solutes [11,33].

which reflects the adaptability of the sampled cells [1].

tozoa in bright-blue [11].

**Figure 4.** Canine sperm immunoreaction against HSP70 (Scale bar = 10 μm). In freshly ejaculated sperm (on the left), labelling for HSP70 is found over the acrosome region while the sperm tail is negative for this molecule. After freezing (on the right), a reduction of the intensity of immunostaining over the acrosome was found, with some negative sperm. In parallel, dislocation of the HSP immunoreactions to the sperm tail was observed.

### **3.3. Assessment of surface membrane integrity**

Integrity of the sperm membrane is essential to sperm survival in the female genital tract and to fertilization [32]. Until placed in the female reproductive tract, spermatozoa are main‐ tained in a hyperosmotic medium. Thereafter, it is passed into an iso-osmotic medium and contacts not only with the genital fluid, but also with the epithelia of the uterus and the ute‐ rine tubes, where it is stored. Further, molecules present on the sperm surface are of utmost significance for the spermatozoa interaction with the female local immune system, binding with the uterine tube epithelium, to cross the oocyte vestments (*cumulus* cells and *zona pellu‐ cida*) and to fertilize the oocyte. The molecular and hormonal local environment possess an important regulatory role on what concerns the sperm functions. However, to fulfil its role the sperm needs to acknowledge those influences and to react accordingly. Cell membrane damages are one of the major side effects of cryopreservation and are irreversible [33]. It is due to changes in the membrane structure and lateral phase separation of the membrane components leading to focal aggregation of proteins, disarrangement of the membrane lip‐ ids and increased permeability to solutes [11,33].

tation and capacitation-like response to sperm manipulation, in a reaction that might be as‐ sociated with the nitric oxide synthesis during oxidative stress [28]. Sperm is transcriptionally inactive. Thus, HSP content in the spermatozoa is defined at ejaculation and those proteins must be present in the cytosol to help protecting the sperm from injury [29]. Therefore it is expectable to find a reduction of its amount or intensity of immunoex‐ pression for these molecules (if Western blotting or ICC were used) after the cell attack. In canine ejaculates, a diminished number of sperm cells with low immunoreaction for HSP70 was found in semen of good quality. A reduction of the intensity of immunolabelling for this molecule was found after freezing/thawing (Figure 4), along with dislocation of the im‐ munostaining from the acrosomal area to the sperm tail [30,31]. It has also been found a cor‐ relation between HSP70 immunoreaction in freshly ejaculated sperm and sperm damage

**Figure 4.** Canine sperm immunoreaction against HSP70 (Scale bar = 10 μm). In freshly ejaculated sperm (on the left), labelling for HSP70 is found over the acrosome region while the sperm tail is negative for this molecule. After freezing (on the right), a reduction of the intensity of immunostaining over the acrosome was found, with some negative

Integrity of the sperm membrane is essential to sperm survival in the female genital tract and to fertilization [32]. Until placed in the female reproductive tract, spermatozoa are main‐ tained in a hyperosmotic medium. Thereafter, it is passed into an iso-osmotic medium and contacts not only with the genital fluid, but also with the epithelia of the uterus and the ute‐ rine tubes, where it is stored. Further, molecules present on the sperm surface are of utmost significance for the spermatozoa interaction with the female local immune system, binding with the uterine tube epithelium, to cross the oocyte vestments (*cumulus* cells and *zona pellu‐ cida*) and to fertilize the oocyte. The molecular and hormonal local environment possess an important regulatory role on what concerns the sperm functions. However, to fulfil its role the sperm needs to acknowledge those influences and to react accordingly. Cell membrane

sperm. In parallel, dislocation of the HSP immunoreactions to the sperm tail was observed.

**3.3. Assessment of surface membrane integrity**

after freezing/thawing procedures [30,31].

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

When introduced in a hypo- or hypertonic environment, cells tend to adjust and reach os‐ motic equilibrium by allowing water and solutes to change across the cell membrane. Sper‐ matozoa, among other cells, have the ability to maintain their volume after osmotic shock [1,34]. It has for long been proved that, for domestic species, cell volume control shows a close positive correlation with fertility [1]. In the ejaculate, there is usually sperm with dif‐ ferent aptitudes and with differences in the ability to respond to osmotic stressors. This is often related with membrane deficiencies in ion channels or signalling pathways that con‐ trol cell volume. The ability to adapt to osmotic changes can be tested by the hypoosmotic test (HOST), an indirect method to assess the membrane integrity, where sperm is incubated in hypoosmotic solutions between 1-60 minutes at 37ºC. Spermatozoa with intact plasma‐ lemma become swollen and present coiled tails when incubated in a sucrose solution (rang‐ ing from 75 to 150 mOsm, according to the species) (Figure 5). After longer exposures, they recover the initial volume [34]. Although currently used for *in vitro* semen assessment, this evaluation is subjective and not quantitatively rigorous. It is also possible that a number of sperm cells may die if prolonged incubation periods are used, biasing the results. However, it becomes more precise if performed with the aid of an electronic cell counter. In this ap‐ proach, known as the volume regulatory test, after the osmotic challenge, sperm passes through a capillary pore and cell volume is determined upon changes in the electric resist‐ ance to passage. The results are expressed as cell frequency distribution for the iso- and the hypoosmotic moments of the test and the amount of displacement of the distribution curve, which reflects the adaptability of the sampled cells [1].

Different combinations of fluorescent membrane-impermeable dyes may also be used to as‐ sess the sperm membrane integrity. Most commonly used ones, also show some degree of affinity for DNA, as for Hoechst 33258, propidium iodide (PI) or ethidium homodimer 1 [11]. Alternatively acylated membrane dyes are also used. These dyes can cross the intact cell membrane and be held in the viable spermatozoa. When the plasma membrane is dam‐ aged, the probe leak out of the cell. More recently, fluorescein diacetate (CFDA), carbox‐ yl(methil)-derivates, such as carboxyl-SNARF and SYBR-14 have been used for this purpose (for more detail, see [11]). This sort of probes can be combined and used with flow cytome‐ try. The combination of different patterns allows estimating different degrees of sperm via‐ bility [13]. When combined with PI, green fluorochromes such as CFDA (Carboxyfluorescein diacetate) or SYBER-14 are replaced in the dead spermatozoa by the red fluorescence, which is not found in the membrane intact sperm. Carboxyl-SNARF, a pH-in‐ dicator, stains the live spermatozoa in orange, whilst Hoechst 33258 stains the dead sperma‐ tozoa in bright-blue [11].

Sperm membrane integrity can also be assessed by the use of merocyanine 540 (MC540), a hydrophylic probe with highly disorganized lipids that shows a high affinity pattern for in‐ stable membranes. This probe allows to monitor the changes in the cell membrane lipid ar‐ 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 cell remnants of similar density to sperm heads is still to prove.

fluorescent spermatozoa heads in the perivitelline space and in the ooplasm after several

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 105

Further, ICC, Western blotting, Chromatography and ELISA (Enzyme-linked immunosorb‐ ent assay) techniques can be used to detect the immunoexpression of particular membrane proteins (like integrins, adhesins or membrane-anchored proteases- ADAM) and to assess possible changes in immunoexpression in defective sperm following a challenging stimulus. Recently, some studies have been presented, concerning the water channels function in sper‐ matozoa and their functions in the cell volume regulation and sperm adaptation to environ‐ mental changes in osmotic pressure. Aquaporins (AQPs) are a family of proteins highly specialized in water permeability and involved in water transport across membranes. It has been demonstrated that AQP3 is an important water channel localized on the principal piece of the sperm tail, which acts like a key-fluid regulator for sperm osmoadaptation, protecting the cell membrane from swelling and mechanical stretching damages [38]. By using a fluo‐ rescence immunocytochemistry approach and flow cytometry, it was found that in AQP3 defective sperm exist an increased proportion of tail bending at cytoplasmic droplet under osmotic stressor conditions, which were associated to membrane rupture and exaggerated cell swelling during HOST, along with decreased sperm motility and reduced fertilization [38]. Additional AQP's have been localized on the sperm of different species. AQP7 and AQP8 may play a role in the glycerol metabolism and water transport respectively, with

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

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

AQP7 showing some association with sperm progressive motility [39].

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

structures to ROS [45].

hours of sperm-oocyte co-incubation [37].

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

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 fluorescent spermatozoa heads in the perivitelline space and in the ooplasm after several hours of sperm-oocyte co-incubation [37].

Further, ICC, Western blotting, Chromatography and ELISA (Enzyme-linked immunosorb‐ ent assay) techniques can be used to detect the immunoexpression of particular membrane proteins (like integrins, adhesins or membrane-anchored proteases- ADAM) and to assess possible changes in immunoexpression in defective sperm following a challenging stimulus.

Recently, some studies have been presented, concerning the water channels function in sper‐ matozoa and their functions in the cell volume regulation and sperm adaptation to environ‐ mental changes in osmotic pressure. Aquaporins (AQPs) are a family of proteins highly specialized in water permeability and involved in water transport across membranes. It has been demonstrated that AQP3 is an important water channel localized on the principal piece of the sperm tail, which acts like a key-fluid regulator for sperm osmoadaptation, protecting the cell membrane from swelling and mechanical stretching damages [38]. By using a fluo‐ rescence immunocytochemistry approach and flow cytometry, it was found that in AQP3 defective sperm exist an increased proportion of tail bending at cytoplasmic droplet under osmotic stressor conditions, which were associated to membrane rupture and exaggerated cell swelling during HOST, along with decreased sperm motility and reduced fertilization [38]. Additional AQP's have been localized on the sperm of different species. AQP7 and AQP8 may play a role in the glycerol metabolism and water transport respectively, with AQP7 showing some association with sperm progressive motility [39].