Targets at the Molecular Level

ROS are known to interact with all cellular components [19] and we are used to think of lipid and DNA as first targets for ROS in spermatozoa [143, 144]. However, O2'- and NO' have relatively low reactivity and, especially at the low levels they are generated, do not promote lipid peroxidation or oxidation of DNA bases as the toxic hydroxyl radical [19, 20]. As noted above, ONOO-, which is also very reactive, is endogenously formed during capacitation and causes Tyr nitration of sperm proteins [46]. This modification is associated to capacitation but the cause-effect relationship still needs to be ascertained. Tyr nitration can be toxic when it happens at high levels, but this is not the case during capacitation as sperm motility and viability are not decreased and capacitation proceeds [46] .

The relatively weak reactivity and low amounts of O2'- and NO' formed by spermatozoa point to SH groups (e.g., Cys on proteins) as some of the main targets for ROS during capacitation [56, 101, 145]. We have to realize that a limited number of Cys on proteins are prone to oxidation at low ROS levels. The acidic Cys residues, those with a p^a (acidity constant) lower than 7.0, are present as Cys thiolate anion (Cys-S-) at physiologic pH, which markedly increases their vulnerability to oxidation as compared to most other Cys (Cys-SH, p^a around 8.5) [146-150],

Worth mentioning is that many enzymes related to signal transduction cascades, as those involved in sperm capacitation, have these acidic Cys residues that render them susceptible to ROS modulation. For example, ROS activate adenylyl cyclase, PKA, PKC, and NOS but inhibit protein Tyr and Ser/Thr phosphatases via oxidation of Cys [146-150]. One major advantage for cells to use this oxidation of Cys on proteins is that the resulting products, Cys sulfenic acid, SS bridges, nitrosy-lated Cys (Cys-NO), etc., are readily reduced back to Cys by various cellular reduc-tants [146-150],

The SH/SS couple on sperm proteins is subject to extensive and complex redox modifications during capacitation [101, 145]. First, there is a major time-dependent increase in SH content of Triton-soluble proteins during the first 30-60 min of capacitation [101, 145]. This is quite surprising because capacitation, as an oxidative process, is expected to be rather associated with a decrease in SH content of proteins. However, this could suggest an important rearrangement of SH-carrying proteins on the sperm membrane during the initiation of capacitation [101, 151].

A deeper analysis using two-dimension (2D) gel electrophoresis points out that alterations in SH levels of at least ten proteins during capacitation are both decreases (by 45-95%, five proteins, 20-60 kDa) and increases (by 200-400%, five proteins, 20-60 kDa) (Fig. 4.7) [145]. The time course of these modifications parallels that of O2'- formation by capacitating spermatozoa and, as could be expected, SOD and/or catalase prevent all these changes [145]. The identity of these proteins is still unknown but, as mentioned above, they could be elements of signal transduction pathways.

Sulfhydryl groups such as those of glutathione and Cys of many sperm proteins are relatively labile and expected to be involved in exchange mechanisms, leading to glutathionylation of proteins [152, 153]. This protein modification usually switches off or stabilizes enzyme, and its involvement in sperm capacitation still needs to be ascertained. However, glutathione, reduced (GSH) and even better oxidized (GSSG), prevents the striking increases and redistribution of SH groups on bull sperm surface that occurs during cryopreservation as well as the associated oxidative stress and premature capacitation [151]. Therefore, protein glutathiony-lation could also play a role in the regulation of sperm capacitation.

Enzymes, such as PKC, Ras, adenylyl cyclase, are all susceptible to activation by O2'- and H2O2 [99, 108, 140, 154, 155]. On the other hand, the oxidation of the vicinal Cys residues at the active center of protein phosphatases causes a selective and reversible inactivation of the enzyme 2 142, 148, 156, 157]. Protein Ser/Thr phosphatases, such as PP1, PP2A, and both Ser/Thr- and Tyr-directed classes of ERK protein phosphatases, are frequent targets for ROS [156-161]. This mechanism could, at least in part, explain the increases in phosphorylation levels that occur during capacitation, but this hypothesis still needs to be proven.

Cys residues are also targets for NO' , which results in Cys nitrosylation [162-165]. Exogenous NO' (from S-nitrosocysteine and DA-NONOate) promotes extensive Cys nitrosylation in sperm proteins, but the high number of proteins then modified [166] may be due to exposure of cells to relatively high levels of NO' as compared to what they endogenously produce. In our hands, spermatozoa have only four protein bands indicative of Cys nitrosylation (Fig. 4.8). One of these (21 kDa)

Fig. 4.7 Sulfhydryl groups change during capacitation. Percoll-washed spermatozoa are treated without (BWW medium alone) or with FCSu (10%, v/v) and in the absence or presence of SOD (0.1 mg/mL) or catalase (0.1 mg/mL) for 30 min at 37°C. Then, Triton-soluble sperm proteins are labeled with 3-(ALmaleimidylpropionyl) biocytin, separated by 2D gel electrophoresis, blotted, and probed with streptavidin conjugated to horseradish peroxidase [145]. (a) Digitized image of a blot after scanning. Some spots chosen for their most evident differences in capacitating spermatozoa are identified with a letter, red for those that present an increased intensity with FCSu treatment, green for those with a decrease, and black for those with no change. (b) SOD reverses the effects due to FCSu in all cases; catalase also reverses effects due to FCSu but not those seen on spot H

Fig. 4.7 Sulfhydryl groups change during capacitation. Percoll-washed spermatozoa are treated without (BWW medium alone) or with FCSu (10%, v/v) and in the absence or presence of SOD (0.1 mg/mL) or catalase (0.1 mg/mL) for 30 min at 37°C. Then, Triton-soluble sperm proteins are labeled with 3-(ALmaleimidylpropionyl) biocytin, separated by 2D gel electrophoresis, blotted, and probed with streptavidin conjugated to horseradish peroxidase [145]. (a) Digitized image of a blot after scanning. Some spots chosen for their most evident differences in capacitating spermatozoa are identified with a letter, red for those that present an increased intensity with FCSu treatment, green for those with a decrease, and black for those with no change. (b) SOD reverses the effects due to FCSu in all cases; catalase also reverses effects due to FCSu but not those seen on spot H

Triton- SDS extract soluble of

Triton-in so I lib le

Fig. 4.8 Capacitation induced by FCSu is associated with an early increase in nitrosylated Cys level of sperm proteins. Percoll-washed spermatozoa are incubated for 20 min in the absence or presence of FCSu (10%, v/v). Cells are then extracted with Triton (0.2%) and the residual Triton-insoluble pellet with SDS (sodium dodecylsulfate, 0.1%, w/v). The biotin switch assay is then performed [162] to assess Cys nitrosylation. Both sperm extracts show an increased Cys nitrosyla-tion of a 21 kDa protein in capacitating spermatozoa. Three other proteins (55, 65, and 80 kDa) are also detected in the sperm SDS extract, but there seem to be no differences between control and treated spermatozoa

Triton- SDS extract soluble of

Triton-in so I lib le

Fig. 4.8 Capacitation induced by FCSu is associated with an early increase in nitrosylated Cys level of sperm proteins. Percoll-washed spermatozoa are incubated for 20 min in the absence or presence of FCSu (10%, v/v). Cells are then extracted with Triton (0.2%) and the residual Triton-insoluble pellet with SDS (sodium dodecylsulfate, 0.1%, w/v). The biotin switch assay is then performed [162] to assess Cys nitrosylation. Both sperm extracts show an increased Cys nitrosyla-tion of a 21 kDa protein in capacitating spermatozoa. Three other proteins (55, 65, and 80 kDa) are also detected in the sperm SDS extract, but there seem to be no differences between control and treated spermatozoa is more intense during the beginning of capacitation and we could hypothesize that this is Rasp21, a protein involved in sperm capacitation [5, 17, 74, 166] and known to be subject to Cys nitrosylation [124, 167].

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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