Measurements

The assessment of very low levels of ROS needs special caution because the probes themselves and any of the products under study may influence cells or cause interference in the measurement techniques. First of all, most of the probes can affect capacitation, even to the point of inhibition, simply because they react with ROS to produce a signal and, at the same time, they remove too much of these same ROS that are essential to trigger capacitation. An example is that of luminol and lucigenin, two common luminescence probes for the determination of high ROS levels in the semen from infertile men [52-54]. Both probes are then used at concentrations of 200-400 mM, which is very high as compared to the amount of ROS produced by capacitating spermatozoa. As a consequence, the probes themselves block capacita-tion (at least in our experimental conditions) because they scavenge all O2'- present (SOD-like effect) [55] . 2

We chose a modified Cypridina luciferin analog (MCLA) as luminescence amplifier and opted for a suboptimal concentration (20 mM) because it allowed O2'-measurements without affecting capacitation [55] . We first tested MCLA at a lower concentration (10 mM), but the signal was too short due to depletion of the probe; on the other hand, MCLA at higher level (50 mM) blocked capacitation because of extensive O2'- scavenging. Therefore, the assay performed with MCLA at 20 mM allows comparison between sperm samples (control, treated, capacitated, etc.) and a gross, rather than exact, determination of the amounts of O2'- formed [46, 55]. To our approximation, 1 x 106 capacitating human spermatozoa would generate about 1 nmole of O2'- per hour [46].

We confirmed that MCLA-amplified luminescence is specific for O2'- and not modified by H2O2, NO', or ONOO- [46, 55]. Then, as for most probes for O2'-

(cytochrome C, nitroblue tetrazolium, etc.), every sample should be tested in the absence and presence of SOD so that only the SOD-inhibitable signal, actually related to O2'-, is considered [19, 55]. Moreover, because MCLA is highly sensitive, even the incubation media (without or with capacitation inducer or enzymatic modulators) should be tested so that their contribution is subtracted [46, 55].

The levels of O2'- synthesized by spermatozoa is of the same order of magnitude whether capacitation is promoted by FCSu, progesterone, l-Arg, etc. [40, 46, 56], confirming the validity of the data. Then, when comes a situation as for BSA, another known capacitation inducer but that causes a major interference (autoluminescence) in MCLA assay [46, 57], the role of O2'- has to be deduced from the inhibitory effect of SOD on capacitation [5, 46] and the fact that all the other inducers tested promote O2'- formation.

Compounds that appear to modify sperm O2'- production may actually affect the luminescence assay itself rather than cells. For example, yellow compounds, such as 7-nitroindazole (NOS inhibitor), chelerythrine (inhibitor of protein kinase C, PKC), or tyrphostin A47 (inhibitor of protein tyrosine kinase, PTK), often cause interferences in luminescence assays (e.g., striking increase of baseline in the absence of cells) (unpublished observations) and therefore need special care, more controls, and corroboration using other chemicals with similar effects on cells and enzymes. Another example is that of uric acid (end product of xanthine + xanthine oxidase incubation) that inhibits the reaction between O2'- and luminol, and therefore blocks luminescence, even at 10-4 M [35, 36, 58, 59]. These are only examples to stress the importance to assess experimental conditions, always to verify for possible interferences and whenever possible use more than one compound of every class of chemical.

MCLA is cell impermeant and, therefore, useful for evaluation of extracellular O2'- [53], Alternatively, dihydrorhodamine 123 concentrates mainly to the sperm mitochondria allowing intracellular O2'- determination; then, fluorescence increases more in FCSu-treated than in control spermatozoa but addition of SOD, and therefore inhibition of capacitation, does not modify this effect indicating that the raise in O,'- synthesis probably relates simply to a higher metabolic activity [60, 61]. Dihydroethidium, another O2'--specific probe, localizes mostly to the sperm head, but the increased fluorescence appears to correlate with a loss of sperm motility and/ or altered membrane permeability [62] rather than capacitating conditions [46] .

The fluorescent probe dichlorofluorescin diacetate (DCFH-DA) was used to detect H2O2 in spermatozoa from infertile men [63], but this chemical is rather nonspecific and reacts with most ROS. Heparin-induced capacitation in bull spermatozoa is associated with increased formation of H2 O2 as determined by fluorimetry using the p-hydroxyphenylacetic acid-horseradish peroxidase system; the fact that diphenyliodonium, an inhibitor of the sperm oxidase, prevents the rise in H2O2 indicates that H2O2 probably originates from O2'- dismutation [64]. This is an interesting example of how the use of probes and inhibitors can help to determine which of the ROS plays an initiating role.

The classical methods to detect NO, by measuring l-citrulline (end product of NO' formation) or nitrates and nitrites (NO' metabolites) [65, 66] are not sensitive enough for the very low levels of NO' formed during capacitation [45]. Electrodes sensitive to NO' have been used but only with spermatozoa treated with l-Arg (substrate for NOS) and/or A23187 (calcium ionophore) [29]. Electron spin resonance, using the spin trap sodium N-methyl-d-glucamine dithiocarbamate, allows the direct, real-time detection of NO' and evidences that capacitating spermatozoa (BSA as inducer) produce sevenfold higher amounts of NO ' than control cells [43]. However, the need of expensive equipment and not common expertise limits the use of this technique. The fluorescence probes diaminofluorescein-2 (Daf2) and its cell-permeant analog Daf2-diacetate (Daf2-DA) are recognized for their specificity and sensitivity and now extensively used, but not often on capacitating spermatozoa [67, 68],

ROS other than NO' and related to capacitation, O2'-, H2O2 , and ONOO- do not significantly affect Daf2 fluorescence, and Daf2 (up to 25 mM) and Daf2-DA (10 mM) do not alter sperm capacitation nor O2'- production [46]. The extracellular NO' formation of spermatozoa (10 x 106 cells; Daf2 25 mM) incubated with FCSu as inducer appears lower than 1 nmole NO' per hour [46]. It may be that NO' synthesis is really that low, but it may be that the NO' formed aims faster and better at its cellular targets than at Daf2. Sperm intracellular NO' , as detected after loading with Daf2-DA, is localized mostly in mitochondria and related to metabolic activity but is also present in the sperm head (Fig. 4.1a) [46]. The proportion of spermatozoa with a brighter fluorescence in the head (white arrows) increases following a challenge with FCSu as early as 15 min after the beginning of the incubation and peaks at about 1 h (Fig. 4.1a, b). Of interest also is that this proportion of cells with more fluorescent head similarly increases whether capacitation is induced by FCSu, BSA, l-Arg, etc. (Fig. 4.1c); as expected, the NOS inhibitor l-NMMA (1 mM) prevents this effect and capacitation, confirming the role of NO' and its detection with Daf2-DA in spermatozoa [46],

Again, any product added to the sperm incubation medium needs to be tested for possible interferences. For example, the need for PKC in NO' production cannot be assessed with chelerythrine because this chemical strikingly increases (threefold) the fluorescence ofDaf2 incubated with NO'-releasing agent (diethylamine-NONOate, DA-NONOate) in cell-free assay and causes an artificial increase in the fluorescence of sperm heads so that they are all very bright; other PKC inhibitors (calphos-tin and G06976) are, therefore, better choices for studies on spermatozoa [56].

The need for, and production of, both O2'- and NO' in spermatozoa may imply the formation of the peroxynitrite anion (ONOO-), an ROS with longer half-life and higher reactivity but for which there is no specific probe or scavenger for a test in vitro [19] . Indirect evidence for the formation of ONOO- may come from an increase in protein Tyr nitration [19]. Exogenous ONOO- (50 mM) promotes sperm capacitation, the associated Tyr phosphorylation of p80 and p105, as well as Tyr nitration of several sperm proteins [69]. Furthermore, the increase in Tyr nitration of three proteins (105, 116, and 200 kDa) in capacitating spermatozoa (FCSu as inducer) is blocked by SOD and l-NMMA [46], therefore providing an indirect evidence for the endogenous formation of ONOO- from O2'- and NO' during this process (Fig. 4.2). The surprise at this point rather comes from the inhibitory effect of catalase on Tyr nitration (Fig. 4.2); this result is, however, rather coherent with our previous one showing that catalase also blocks capacitation triggered by

Fig. 4.1 Nitric oxide (NO') production in human spermatozoa as evaluated with Daf2-DA. Localization, time course, and regulation. Percoll-washed spermatozoa (400 x 106/mL) are loaded with Daf2-DA (10 mM) for 30 min at 20°C and in the dark; then, they are incubated (20 x 106/mL) in a medium supplemented or not with substances that induce (FCSu 10% v/v; l-Arg, 2.5 mM; BSA, 3 mg/mL) or prevent (SOD 0.1 mg/mL; l-NMMA, 1 mM) capacitation. After incubation at 37°C, spermatozoa are fixed with formaldehyde (2%) and mounted with a mix of glycerol and water containing DABCO (1.5%, w/v) as antifading agent [46]. (a) Fluorescence is concentrated

Fig. 4.1 Nitric oxide (NO') production in human spermatozoa as evaluated with Daf2-DA. Localization, time course, and regulation. Percoll-washed spermatozoa (400 x 106/mL) are loaded with Daf2-DA (10 mM) for 30 min at 20°C and in the dark; then, they are incubated (20 x 106/mL) in a medium supplemented or not with substances that induce (FCSu 10% v/v; l-Arg, 2.5 mM; BSA, 3 mg/mL) or prevent (SOD 0.1 mg/mL; l-NMMA, 1 mM) capacitation. After incubation at 37°C, spermatozoa are fixed with formaldehyde (2%) and mounted with a mix of glycerol and water containing DABCO (1.5%, w/v) as antifading agent [46]. (a) Fluorescence is concentrated

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