Time Course of ROS Formation

Spermatozoa start to produce O2'- and NO' as soon as they are incubated with a capacitation inducer [43, 46, 552 . ROS production reaches a maximum at about 30 min for O.'- and 1 h for NO' and then decreases slowly over the next hours

Fig. 4.1 (continued) in sperm mitochondria and head. The heads have a low (short yellow arrows) or a high (2ong white arrows) fluorescence. (b' The percentage of spermatozoa with a bright fluorescent head, indicative of a higher NO' synthesis, increases in FCSu-treated spermatozoa (red bars) as early as 15 min after the beginning of incubation and is higher (*) than that of untreated spermatozoa (white bars) at all times (p< 0.05, n = 5). (c) The percentage of spermatozoa with a bright fluorescent head increases with the three capacitation inducers (FCSu, l-Arg, BSA) tested here but not when conditions (SOD or l-NMMA) prevent this process. These tests are done with 1 h of incubation since it allows the best difference between control and capacitating spermatozoa

Fig. 4.3 Time course of ROS production in relation to in vitro capacitation and hyperactivation. Tendency curves are proposed rather than numbers in order to give a global idea on how each of the processes varies over time. Percoll-washed spermatozoa are treated with FCSu as capacitation inducer. ROS (O-'-, NO', and ONOO-) are determined as described in the text. Capacitation is evaluated by the lysophosphatidylcholine-induced acrosome reaction followed by labeling with Pisum sativum agglutinin conjugated with fluorescein isothiocyanate [2, 55]. Sperm hyperactivation is measured with the CellSoft™ (Cryo Resources, Montgomery, NY) computer-assisted digital image analysis system [2, 55]. Production of O2'- (black line) and NO' (red line) starts immediately at the beginning of the incubation and reaches a maximum at 30 min [2, 55] and 1 h [46], respectively, and then slowly decreases over the next hours. Protein Tyr nitration (green line), as a measure of ONOO- formation, is maximal at 3 h and then decreases [46]. Sperm hyperactivation (gray line) in vitro is observed between 1 and 3 h and the level of capacitation (blue line) is increasing over time [2, 55]

Fig. 4.3 Time course of ROS production in relation to in vitro capacitation and hyperactivation. Tendency curves are proposed rather than numbers in order to give a global idea on how each of the processes varies over time. Percoll-washed spermatozoa are treated with FCSu as capacitation inducer. ROS (O-'-, NO', and ONOO-) are determined as described in the text. Capacitation is evaluated by the lysophosphatidylcholine-induced acrosome reaction followed by labeling with Pisum sativum agglutinin conjugated with fluorescein isothiocyanate [2, 55]. Sperm hyperactivation is measured with the CellSoft™ (Cryo Resources, Montgomery, NY) computer-assisted digital image analysis system [2, 55]. Production of O2'- (black line) and NO' (red line) starts immediately at the beginning of the incubation and reaches a maximum at 30 min [2, 55] and 1 h [46], respectively, and then slowly decreases over the next hours. Protein Tyr nitration (green line), as a measure of ONOO- formation, is maximal at 3 h and then decreases [46]. Sperm hyperactivation (gray line) in vitro is observed between 1 and 3 h and the level of capacitation (blue line) is increasing over time [2, 55]

(Fig. 4.3). This time course for O2'- and NO' appears independent of the triggering agent. Differences between O2'- and NO' arise when we look at the period of time for which these ROS are essential. Addition of SOD to spermatozoa 30 min after the beginning of incubation does not anymore prevent capacitation [40, 46] , indicating that extracellular O2'- plays a role only at early steps in this process. On the other hand, NOS inhibitors (l-NAME or l-NMMA) block capacitation even if added after 1-4 h of incubation [42, 46], attesting the need for NO- for the whole process. Data obtained with dibutyryl-cAMP (dbcAMP, a cell-permeant analog of cAMP), a model compound that promotes capacitation but bypasses the initial steps of this event, confirm these assumptions. Then, NOS inhibitors (l-NAME or l-NMMA), but not SOD, prevent capacitation due to dbcAMP [43, 46] .

At this point, it is important to realize that ROS generation involved in capacitation, at least for what we can detect, is extracellular for O2'-, but in the sperm head for NO' [46]. Therefore, we expect O2'- to aim at a target on the cell membrane since this ROS, being ionized, should not easily diffuse through plasma membrane. On the other hand, NO' is not charged and a part of what spermatozoa synthesize could pass across cell membranes and act both at intra- and extracellular levels. We can also hypothesize that spermatozoa possess more than one NOS, that one could be on the membrane and the other intracellular, and that each of the NOS acts at different times and on specific targets.

Protein Tyr nitration reaches a maximum 3 h after the beginning of capacitation (Fig. 4.3) [46], a time at which both O2'- and NO' synthesis are slowly decreasing. A plausible hypothesis is that this reflects an accumulation of protein modifications that are reversible but on a slow rate.

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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