ROSGenerating Enzymes in Human Sperm Capacitation NOS and Oxidase One or Two Enzymes

The three isoforms of NOS (epithelial, neuronal, and inducible) are found in spermatozoa both in the head and flagellum (immunoblotting and immunocytochemis-try data) [30, 31, 66] . The oxidase responsible for sperm O.'- generation during capacitation is, however, still elusive. It should be at the membrane level because O2'- is released in the extracellular milieu and SOD (cell impermeant) prevents capacitation [55] . The first consideration given to the possibility that the sperm enzyme is of the same type as the NADPH oxidase of neutrophils (NOX1) is now

Fig. 4.4 ROS-induced ROS production in human spermatozoa. Percoll-washed spermatozoa are incubated without or with FCSu (10%, v/v), the combination of xanthine (0.1 mM) and xanthine oxidase (0.1 U/mL) (X + XO), DA-NONOate, SOD (0.1 mg/mL), l-NMMA (1 mM), H2O2 (25 mM), or ONOO- (25 mM). Capacitation is evaluated after 3.5 h of incubation by induction of the acrosome reaction [2]. NO' production is estimated by the percentage of spermatozoa with a bright head (see Fig. 4.1) [46]. Net SOD-inhibitable luminescence (MCLA as a probe) [55] is the measure of O2'- synthesis. (a) Capacitation due to X+XO is blocked by the NOS inhibitor l-NMMA and that induced by DA-NONOate (0.1 mM) is prevented by SOD. (b) Spermatozoa treated with FCSu or X + XO produce higher levels of NO" (*p < 0.05). SOD blocks this effect confirming the involvement of O,'- in NO' synthesis. H2O2 , but not ONOO-, also promotes NO' synthesis but to a lower extent (#p < 0.05) than FCSu or X + XO. (c) l-NMMA blocks sperm O2'-generation due to FCSu and DA-NONOate (25 mM) promotes O2'- formation, substantiating the role of NO' in O2'- synthesis. Spermatozoa treated with DA-NONOate or FCSu produce similar amounts of O2'-. H2O2 and ONOO- do not have such an effect

Fig. 4.4 ROS-induced ROS production in human spermatozoa. Percoll-washed spermatozoa are incubated without or with FCSu (10%, v/v), the combination of xanthine (0.1 mM) and xanthine oxidase (0.1 U/mL) (X + XO), DA-NONOate, SOD (0.1 mg/mL), l-NMMA (1 mM), H2O2 (25 mM), or ONOO- (25 mM). Capacitation is evaluated after 3.5 h of incubation by induction of the acrosome reaction [2]. NO' production is estimated by the percentage of spermatozoa with a bright head (see Fig. 4.1) [46]. Net SOD-inhibitable luminescence (MCLA as a probe) [55] is the measure of O2'- synthesis. (a) Capacitation due to X+XO is blocked by the NOS inhibitor l-NMMA and that induced by DA-NONOate (0.1 mM) is prevented by SOD. (b) Spermatozoa treated with FCSu or X + XO produce higher levels of NO" (*p < 0.05). SOD blocks this effect confirming the involvement of O,'- in NO' synthesis. H2O2 , but not ONOO-, also promotes NO' synthesis but to a lower extent (#p < 0.05) than FCSu or X + XO. (c) l-NMMA blocks sperm O2'-generation due to FCSu and DA-NONOate (25 mM) promotes O2'- formation, substantiating the role of NO' in O2'- synthesis. Spermatozoa treated with DA-NONOate or FCSu produce similar amounts of O2'-. H2O2 and ONOO- do not have such an effect

Table 4.2 Oxidases from spermatozoa and neutrophils (NOX1) are very different

Sperm oxidase

Neutrophil NADPH oxidase, NOX1

Very low levels of O2'- produced

Synthesis of O2'- over hours

Aims at cell signaling and transduction

Diphenyliodonium inhibits at 100 mM

p67phox, p47phox, p21rac not found

PMA does not activate O2'- formation

NADPH does not activate

FCSu, FFu, progesterone stimulate

Zn2+, 50 mM, totally inhibits O2'- production

High levels of O2'- produced Burst of O2'- synthesis for 30-60 min Aims at defense against pathogens Diphenyliodonium inhibits at 10 mM PKC, ERK, PTK, etc. needed for activation p67phox, p47phox, p21rac are parts of the oxidase PMA stimulates the oxidative burst NADPH is essential

FCSu, FFu, progesterone have very little effect Zn2+, 150 mM, causes 15% drop in O2'- formed Sg, 1 mg/mL, blocks 44% of O2'- production invalidated because of several important differences (Table 4.2). Components of the neutrophil oxidase are not found in human spermatozoa neither by PCR analysis [73] nor by immunoblotting (antibodies raised against p67phox, p47phox. and p21'ac; unpublished data). Mouse spermatozoa possess p67phox, p47phox, and p40phox but not p22phox and the oxidase activity decreases as epididymal maturation proceeds [27] , suggesting that this oxidase may not be that related to capacitation. Also, the time course of the two oxidases is very different [55] . Moreover, contrarily to what is seen with the neutrophil oxidase [19, 25], activation of the sperm enzyme is independent of signal transduction cascades involving PTK, PKC, extracellular signalregulated kinase (ERK) pathway, etc. [55, 56, 74] . Several NOX (NADH/NADPH oxidase family of enzymes) isoforms are found in reproductive (male and female) tissues, but NOX activation processes are so highly variable between cell types and tissues [25,26] that they cannot be considered as plausible candidates as the oxidase involved in sperm capacitation. As a supplementary example, PKC activation drives NOX5 of pachytene spermatocytes and mature equine spermatozoa [75] but not the sperm oxidase involved in capacitation [56] .

Over the years, we evidenced several other differences between sperm and neu-trophil oxidases (Table 4.2). When evaluated with MCLA-amplified luminescence, neutrophils produce during the first 5 min after stimulation with PMA (phorbol 12-myristate 13-acetate, 100 nM) about 250-fold higher amounts of O2'- than spermatozoa during their first 30 min of capacitation [76] . Also, progesterone, FCSu, and FFu (follicular fluid ultrafiltrate) stimulate sperm O2'- synthesis [40] , but have almost no effect on neutrophils [76]. Furthermore, natural inhibitors of sperm capacitation and O.'- generation, such as zinc ions (Zn'+] and semenogelin (Sg) (section below), inhibit the sperm oxidase much more efficiently (higher extent at lower concentration) than that of neutrophils [76].

Standing at this point, we tried to put together all our observations on the oxidase and NOS involved in sperm capacitation, on the close interaction between these enzymes and/or the ROS produced (see above), as well as on the confirmed association among ROS (O2'- and NO'), capacitation, and its related events (e.g., Tyr phosphorylation of p80/p105). We realized that this could imply that a single enzyme is responsible for the production of the two ROS. NOS are versatile enzymes and, even if they are best known to generate NO' from the conversion of l-Arg to l- citrulline, they can also reduce nitrite to NO' under anoxia or even become oxidases. NOS possess two functionally distinct domains, one being an oxygenase (N-terminal) and responsible for NO' formation and the other being a reductase (C-terminal) that synthesizes O2'- [32-34, 77, 78]. NOS produce NO', O2'-, or both ROS simultaneously depending on the isoform present as well as various conditions, such as availability of substrate (l-Arg, oxygen) and cofactor (NADPH, tetra-hydrobiopterin) and uncoupling due to oxidation of Cys residues at the active center and consequent release of Zn2+ [32-34, 77, 78].

Whether this double activity of NOS prevails during capacitation is not known. On one side, discrepancies between the formation and action of O2'- and NO' would not favor a model in which NOS generates both ROS. As stated above, the time courses for the formation and need of these ROS diverge significantly [46]. Also, O2'- acts upstream, and NO' downstream and/or upstream, of the cAMP/PKA couple [46]' Finally, the double phosphorylation of the Thr-glutamic acid-Tyr (Thr-Glu-Tyr) motif that increases in sperm proteins from 1 h of capacitation is under the control of NO' but not of O2'- and H2O2 (see section below) [79, 80]. However, even with these differences, we cannot completely rule out the possibility that sperm NOS may generate both ROS. Capacitation may involve more than one NOS each having specific regulators, locations, and targets; it is also possible that O2'- and NO' synthesis is switched on/off sequentially and/or that NOS produces simultaneously O2'- and NO' at some time points of capacitation.

Our hypothesis model is rather that the sperm oxidase at the cell membrane is initiator of ROS production and that the ROS synthesized activate an intracellular NOS to provide NO' for long-term effect. The localization of NOS in all sperm compartments [30] would support this idea. Whether O2'- and/or NO' are synthesized sequentially or simultaneously at the initiation of capacitation may not be of paramount importance considering the obvious cross talk between ROS and their generators. This would rather represent another situation in which more than one mechanism exists to give flexibility to the process and to guarantee that capacitation and the subsequent fertilization occur.

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