Human sperm chromatin undergoes physiological remodeling during in vitro capacitation and acrosome reaction.

Capacitation (CAP) and acrosome reaction (AR) are sequential processes of sperm activation. Beside the known ionic, membrane, and transduction events and final release of proteolytic enzymes that help sperm movement toward the egg, chromatin changes, such as a physiological remodeling, are also possible. Our aims were to ascertain that CAP and AR do not induce DNA damage and to evaluate changes occurring in the human sperm head during these physiological processes using cytochemical stains. Percollpurified spermatozoa from normal donors were incubated in Biggers, Whitten, and Whittingham medium ± fetal cord serum ultrafiltrate (CAP inducer) and then with lysophosphatidylcholine (AR inducer). CAP and AR were associated with increases in aniline blue (AB, for histones; ∼70%) and toluidine blue (TB, for chromatin compaction; ∼40%) staining but had no influence on that of chromomycin A3 (for protamines). The increase (∼40%) in iodoacetamide-fluorescein (IAF, for sulfhydryl groups) staining observed during CAP was absent after AR. CAP and AR did not damage DNA (percentage of DNA fragmentation index remained low) nor affect histone content. CAP, and even more AR, primed sperm heads to decondense (∼80% and ∼140% increases, respectively) when challenged with sodium dodecyl sulfate + dithiothreitol. Interestingly, induced decondensation correlated with all other tests (CAP, AB, TB, and IAF). Therefore, the data strongly support a physiological remodeling of nondamaged human sperm chromatin during CAP and AR, and modifications are probably interlinked and help prepare chromatin for postfertilization events.

Levels of semenogelin in human spermatozoa decrease during capacitation: involvement of reactive oxygen species and zinc.

BACKGROUND: Semenogelin (Sg), the main protein of human semen coagulum, prevents sperm capacitation. The objective of this study was to examine the role of Sg and its mechanism of action. METHODS AND RESULTS: Sg blocked sperm capacitation triggered by various stimuli, via inhibition of superoxide anion (O(2)*-; luminescence assay) and nitric oxide (NO*; tested using diaminofluorescein) generation. Triton-soluble and -insoluble sperm fractions contained Sg and Sg peptides (immunoblotting), the level of which decreased with initiation of capacitation. This drop was prevented by superoxide dismutase and NO* synthase inhibitor and was reproduced by addition of O(2)*- and NO*. Zinc (Zn(2+)) blocked and a zinc chelator (TPEN) promoted the decline in Sg levels. There was a decreased labelling of Sg on the head in capacitating spermatozoa with the two fixation techniques tested (immunocytochemistry). Reactive oxygen species (ROS) (O(2)*- and NO*) caused, these changes, and zinc prevented them. Spermatozoa quickly internalized Sg upon incubation and Sg was then rapidly degraded in a zinc-inhibitable manner. CONCLUSIONS: Sg blocked capacitation mainly via inhibition of ROS generation. Spermatozoa appeared permeable to Sg and processed Sg in a zinc-inhibitable fashion. ROS themselves could promote sperm disposal of Sg which maybe one of the mechanisms that allows initiation of capacitation.

Various protein kinases regulate human sperm acrosome reaction and the associated phosphorylation of Tyr residues and of the Thr-Glu-Tyr motif.

Acrosome reaction (AR) is an exocytotic process of fundamental importance for the spermatozoon to fertilize the oocyte. The mechanisms mediating this process are only partially defined. The aim of the present study was to investigate the role of various kinases and the extracellular signal-regulated kinase (ERK) pathway in the induction of the AR and associated phosphorylation of tyrosine (Tyr) residues and of the threonine-glutamic acid-tyrosine (Thr-Glu-Tyr) motif that occurs in 80 and 105 kDa proteins (p80/p105). Human spermatozoa were capacitated and AR was induced with lysophosphatidylcholine in the presence of inhibitors of various kinases and of the ERK pathway. Phosphorylation of Tyr and of Thr-Glu-Tyr peaked 15 min after the induction of the AR. Both phosphorylations were prevented by inhibitors of protein kinase C, MEK, phosphoinositide 3-kinase and Akt but not by protein kinase A inhibitors. Phosphorylation of Thr-Glu-Tyr, but not Tyr, was decreased by inhibitors of protein tyrosine kinase and Grb2-SH2. All the inhibitors prevented lysophosphatidylcholine-induced AR, indicating the involvement of PKC, PKA, PTK, PI3K, Akt and the ERK pathway. These results show that phosphorylation of Tyr and Thr-Glu-Tyr are associated with the AR and are differently regulated by the various kinases emphasing the complexity of this process.

Phosphorylation of the Arginine-X-X-(Serine/Threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency.

Sperm capacitation is a complex process that involves a protein kinase A (PKA)-dependent tyrosine phosphorylation of proteins. We studied the time-course, the modulation and the cellular localization of the phosphorylation of the Arginine-X-X-(Serine/Threonine) motif, characteristic of PKA substrates, in sperm proteins during capacitation. There was an increased phosphorylation of 80 (p80) and 105 (p105) kDa protein bands in human sperm treated with different capacitation inducers. Phosphorylation of p80 and p105 induced by fetal cord serum ultrafiltrate or the combination of 3-isobutyl-1-methylxanthine and dibutyryl cAMP was prevented by H89 and Rp-adenosine-3',5'-cyclic monophosphorothionate, confirming the involvement of PKA in this effect. Inhibitors of protein kinase C, receptor type tyrosine kinase and mitogen-activated protein kinase kinase did not affect the Arginine-X-X-(Serine/Threonine) motif phosphorylation. Non-receptor type protein tyrosine kinase inhibitors, PP2 and herbimycin A, enzymatic antioxidants and a nitric oxide synthase inhibitor prevented the phosphorylation of p80 and p105 when sperm were incubated with fetal cord serum ultrafiltrate. The phosphorylated Arginine-X-X-Serine/Threonine motif was immunolocalized all along the flagellum and the fluorescent signal was higher in capacitating than in non-capacitating sperm. These results show for the first time the presence of a PKA-dependent phosphorylation of proteins in human sperm capacitation and its upstream modulation by reactive oxygen species and non-receptor type protein tyrosine kinase.

Different signal transduction pathways are involved during human sperm capacitation induced by biological and pharmacological agents.

Human sperm capacitation involves complex signal transduction mechanisms during which double phosphorylation of the threonine-glutamine-tyrosine motif (P-Thr-Glu-Tyr-P) occurs in some sperm proteins. The objective of this study was to investigate the regulation of this process. Fetal cord serum ultrafiltrate (FCSu), follicular fluid ultrafiltrate (FFu), progesterone and a combination of N(6),2'-O-dibutyryl cAMP (dbcAMP; cell permeant analogue of cAMP) and 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase inhibitor) were used as inducers of capacitation alone or in combination with inhibitors of protein kinase A (H89), protein kinase C (chelerythrine), protein tyrosine kinase (tyrphostin A47, PP2) and of dual specificity kinase (MEK-like kinases; PD98059). The level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation induced by FCSu, FFu and progesterone was regulated by a similar signal transduction pathway and involved receptor type protein tyrosine kinase and dual specificity kinase (MEK or MEK-like) but not protein kinase A or C. However, the level of P-Thr-Glu-Tyr-P in these sperm proteins during capacitation induced by dbcAMP+IBMX was mainly mediated through protein kinase A and C and receptor type protein tyrosine kinase, but not by dual specificity kinase. In conclusion, human sperm capacitation induced by some biological and pharmacological agents is regulated through very different signal transduction pathways.

The extracellular signal-regulated kinase (ERK) pathway is involved in human sperm function and modulated by the superoxide anion.

Our aim was to ascertain the role of the extracellular signal-regulated protein kinase (ERK) pathway in human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and its regulation by the superoxide anion (O(2)(-)*). Immunoblotting indicated the presence of Shc, Grb2, Ras(p21), Raf and ERK1 and 2 (ERK1/2) in spermatozoa. Grb2, Ras(p21), Raf and MEK inhibitors dose-dependently prevented sperm capacitation and protein tyrosine phosphorylation, without modifying sperm O(2)(-)* production. Therefore, the whole ERK cascade plays a role in capacitation, downstream of O(2)(-)* but upstream of protein tyrosine phosphorylation. Upon incubation with FCSu, the early (5 min) increase in ERK1/2 activity (as shown by double phosphorylation of the Thr-Glu-Tyr motif) was followed by an important decrease over the next 2 h; superoxide dismutase did not change this pattern. The phosphorylation of the Thr-Glu-Tyr motif present in other sperm proteins (16-33 kDa) also increased (5 min incubation with FCSu) and then progressively decreased, and this effect was regulated by O(2)(-)*, MEK and cAMP. The phospho-Ser/Thr-Pro content (characteristic of ERK1/2 substrates) of Triton-insoluble proteins (75 and 80 kDa) increased during capacitation and also appeared to be regulated by O(2)(-)* and the ERK pathway. Inhibition of ERK1/2 activation reduced lysophosphatidylcholine-induced acrosome reaction and the associated protein tyrosine phosphorylation. These results support a role for the ERK pathway in human sperm function.

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.

Tyrosine nitration in human spermatozoa: a physiological function of peroxynitrite, the reaction product of nitric oxide and superoxide.

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO-) produced from the reaction of nitric oxide (NO.) with superoxide (O2(.-)). Since human spermatozoa are able to produce both NO. and O2(.-) during capacitation in vitro, we investigated whether spontaneous tyrosine nitration of proteins occurs in human spermatozoa and evaluated the physiological effects of peroxynitrite on sperm function. We report here that human spermatozoa, incubated for 8 h under conditions conducive to capacitation, display a reproducible pattern of protein tyrosine nitration. Several proteins with mol. wt of 105-14 kDa become increasingly tyrosine-nitrated after 15 min incubation and then minimal changes are observed. Treatment of capacitated spermatozoa with human follicular fluid or calcium ionophore causes an increase of the nitrotyrosine content of proteins at the mol. wt of 85 kDa. Moreover, exposure of spermatozoa to ONOO- (2.5-50 micromol/l) increases motility and primes spermatozoa to respond earlier to human follicular fluid. ONOO- also increases protein tyrosine nitration and phosphorylation in a concentration-dependent manner. Taken together, these results demonstrate that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of ONOO- modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process.

Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process.

Semenogelin (Sg), the major protein of the human semen coagulum, is present at high concentrations in seminal vesicle secretions. It is degraded by the prostate-specific antigen (PSA) to generate peptides of various biological activities that were found on and inside spermatozoa. Our aim was to determine the effect of Sg on capacitation, which is the series of transformations that spermatozoa must undergo to become fertile. At concentrations of 0.1 to 1.0 mg/mL (600- to 20-fold lower than those of semen), Sg did not affect sperm motility (%) but completely prevented capacitation induced by fetal cord serum ultrafiltrate; a partial inhibition of capacitation was noted with 0.03 mg Sg/mL. There was also a dose-dependent decrease in the tyrosine phosphorylation of fibrous sheath proteins and in the O2-.-related chemiluminescence. Ribonuclease (RNase), which has as high an isoelectric point (pI = 9.7) as Sg (pI = 9.5), also prevented sperm capacitation and O2-.-related chemiluminescence but to a lower extent, suggesting that one mechanism of Sg action on spermatozoa could be related to its positive charge at physiological pH. Sg at 1, but not 0.3 or 0.1 mg/mL, scavenged the O2-. generated by the mix of xanthine + xanthine oxidase and modified the kinetics of the reaction; RNase did not have such effects. Therefore, Sg is a potential scavenger for O2-. but probably also affects the sperm oxidase. Spermatozoa rapidly processed Sg; a high proportion of Sg was degraded after 15 minutes of incubation. The resulting polypeptide patterns were reminiscent of those obtained with PSA as a proteolytic enzyme. These data suggest that Sg, its degradation products, or both may be natural regulators of sperm capacitation and could prevent this process from occurring prematurely. One mechanism by which Sg acts could involve an interference with the O2-. that is normally generated during this process.

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction.

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.

Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa.

This study aimed to demonstrate nitric oxide production by human spermatozoa and to characterize the interaction between nitric oxide and cAMP-related pathway in the control of human sperm capacitation and protein tyrosine phosphorylation. Spermatozoa were incubated in Tyrode's medium with or without bovine serum albumin (BSA), and nitric oxide was measured with the spin trap sodium N-methyl-D-glucamine dithiocarbamate. Under noncapacitating conditions, spermatozoa produced low levels of nitric oxide. However, under capacitating conditions, prominent nitric oxide adduct signals were obtained and a time-dependent increase of nitric oxide production was observed. When spermatozoa were incubated in Tyrode+BSA medium with nitric oxide-releasing compounds, intracellular cAMP concentrations increased to levels higher than those of spermatozoa incubated in Tyrode+BSA alone. In contrast, incubation with nitric oxide synthase inhibitors (N(G)-nitro-L-arginine methyl ester or N(G)-monomethyl L-arginine) decreased intracellular sperm cAMP concentrations. The inhibitory effect observed with N(G)-nitro-L-arginine methyl ester on capacitation and tyrosine phosphorylation of two sperm proteins (105, 81 kDa) was overcome by the presence of cAMP analogs or of a phosphodiesterase inhibitor. These results indicate that nitric oxide is produced by capacitating human spermatozoa and that it may act as a cellular messenger by modulating the cAMP pathway involved in capacitation and protein tyrosine phosphorylation.

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.

Paradoxical effect of reagents for sulfhydryl and disulfide groups on human sperm capacitation and superoxide production.

Spermatozoa must undergo capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human sperm capacitation, superoxide (O2-.) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered sperm capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased sperm production of O2-.. However, SOD did not affect the increase in protein tyrosine phosphorylation of spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced sperm capacitation and O2-. production. TCEP, DTT, and TRX decreased the capacitation-associated tyrosine phosphorylation of sperm proteins. The strong time-dependent increase of sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human sperm capacitation and the mechanisms involved may be complex and multifactorial.

Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates.

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.

Interaction between Ca2+, cyclic 3',5' adenosine monophosphate, the superoxide anion, and tyrosine phosphorylation pathways in the regulation of human sperm capacitation.

In order to fertilize the egg, spermatozoa must go through the capacitation process where they experience Ca2+ uptake, increases in cyclic 3',5' adenosine monophosphate (cAMP) concentrations, superoxide anion production, and protein tyrosine phosphorylation. Although the importance of these processes has been described, the interactions between them, as well as the temporal sequence of these events, remain to be demonstrated. Previous studies from our laboratory have demonstrated that tyrosine phosphorylation of p105 and p81 (p105/81), the two major human sperm phosphotyrosine-containing proteins, was under cAMP and oxygen derivatives regulation. In the present study, we investigated the importance of intra- and extracellular Ca2+, as well as the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the phosphatase inhibitors calyculin A and okadaic acid, in the production of superoxide anion and p105/81 tyrosine phosphorylation. An increase in p105/81 phosphotyrosine content was observed when spermatozoa were incubated in the absence of extracellular Ca2+ or with the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide. However, the human sperm capacitation inducer FCSu (ultrafiltrate of fetal cord serum) requires the presence of the extracellular Ca2+ to induce capacitation, superoxide anion production, and tyrosine phosphorylation of p105/ 81, whereas free intracellular Ca2+ had no effect on these last two processes. The production of superoxide anion by spermatozoa was stimulated by inhibitors of phosphodiesterases and serine/threonine phosphoprotein phosphatases. The tyrosine phosphatase inhibitor vanadate decreased by 40% the FCSu-stimulated superoxide anion production, although it had no effect when used alone. These results suggest that, during sperm capacitation, Ca2+ induces an elevation in cAMP levels; this cAMP, through undefined serine/threonine protein phosphorylation, stimulates the generation of superoxide anion, which, in turn, causes the increase in p105/81 phosphotyrosine contents.

Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion.

Recent evidence indicated that human sperm capacitation is associated with an increased production of superoxide anion (O2.-). To further study the role and importance of O2.- in capacitation, we investigated whether the O2.- generation is a general feature of capacitating spermatozoa, irrespective of the inducer used, and is correlated with capacitation levels and increased tyrosine phosphorylation of two sperm proteins (p105/p81). We also studied the time courses of O2.- production and action. Percoll-washed human spermatozoa were incubated in Ham's F-10 medium, supplemented or not supplemented with various capacitation inducers and in the presence or absence of superoxide dismutase (SOD). Sperm capacitation was measured by induction of the acrosome reaction with lysophosphatidylcholine, O2.- production was measured by chemiluminescence, and tyrosine phosphorylation was measured by immunodetection after electrophoresis and western blotting of sperm proteins. Progesterone and ultrafiltrates of human fetal cord serum, follicular fluid, and seminal plasma individually promoted sperm generation of O2.-, tyrosine phosphorylation of p105/p81, and capacitation. Fetal cord serum ultrafiltrate triggered a fivefold higher O2.- production than the other inducers (1,700 +/- 300 and 300 to 400 mV/10s/8 x 10(6) cells, respectively), a phenomenon possibly associated with the higher potency of this fluid to promote sperm hyperactivation. The production of O2.- by spermatozoa was rapid and transient. SOD prevented sperm capacitation triggered by the inducers mentioned above, but only when SOD was added at the beginning of incubation, and not after 30 minutes, indicating that the O2.- initiates a chain of early events leading to sperm capacitation. NADH and NADPH (5 mM) triggered sperm capacitation and phosphorylation of p105/p81, but these processes were not prevented by SOD or catalase, nor were they associated with an increased O2.- production. Therefore, these cofactors appeared to act by mechanisms different from those used by the other inducers studied. The sperm enzyme responsible for the O2.- generation may be very different from the NADPH oxidase of neutrophils.

Impaired fertility in mice deficient for the testicular germ-cell protease PC4.

PC4 is a member of the proprotein convertase family of serine proteases implicated in the processing of a variety of polypeptides including prohormones, proneuropeptides, and cell surface proteins. In rodents, PC4 transcripts have been detected in spermatocytes and round spermatids exclusively, suggesting a reproductive function for this enzyme. In an effort to elucidate this function, we have disrupted its locus (Pcsk4) by homologous recombination in embryonic stem cells and have produced mice carrying the mutation. In intercrosses of heterozygous mutant mice, there was low transmission of the mutant Pcsk4 allele to the progeny, resulting in lower than expected incidence of heterozygosity and null homozygosity. The in vivo fertility of homozygous mutant males was severely impaired in the absence of any evident spermatogenic abnormality. In vitro, the fertilizing ability of Pcsk4 null spermatozoa was also found to be significantly reduced. Moreover, eggs fertilized by these spermatozoa failed to grow to the blastocyst stage. These results suggest that PC4 in the male may be important for achieving fertilization and for supporting early embryonic development in mice.

Motility of spermatozoa in hydrosalpingeal and follicular fluid of pigs.

Hydrosalpinges were created to collect adequate volumes of fluid during pre-, peri- and postovulatory intervals from the ampulla, ampullary-isthmic junction and the isthmic-utero-tubal junction of the oviducts from Large White gilts that had exhibited at least two natural oestrous cycles. The accumulated fluids, follicular fluid and Butschwiler's medium were compared for their effects on various parameters of boar sperm motility using the CellSoft, computer-assisted, digital image analysis system. Sperm velocity (micron s-1 +/- SEM) was significantly higher (P < 0.05) in follicular fluid (84 +/- 3; n = 5) than in fluids from the ampulla during peri- and early postovulatory intervals, and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals. It was also higher (P < 0.05) than in the fluid from the ampullary-isthmic junction during pre- and early postovulatory intervals; however, sperm velocity in follicular fluid was not significantly different from that in the periovulatory fluid from the ampullary-isthmic junction. The mean lateral head displacement (ALHmean) of spermatozoa was significantly greater in follicular fluid (3.9 +/- 0.3 microns; n = 5) than in fluid from the ampulla during peri- and early postovulatory intervals and from the isthmic-utero-tubal junction during pre- and early postovulatory intervals, and was also higher (P < 0.05) than in fluid from the ampullary-isthmic junction during the preovulatory period, but was not different from the peri- and postovulatory ampullary-isthmic junction fluids. The proportion of spermatozoa exhibiting circular motion was significantly higher (P < 0.05) in the periovulatory fluid from the ampullary-isthmic junction (24 +/- 3%) compared with fluids obtained during preovulatory and early postovulatory periods. Follicular fluid had no effect on the proportion of spermatozoa exhibiting circular motion. The average radius of sperm movement in circular trajectories was higher in follicular fluid than in the periovulatory fluids from the ampulla and ampullary-isthmic junction (P < 0.05). In hydrosalpingeal fluids collected 2-5 days after ovulation, the average radius of movement was greater in the ampulla fluid and ampullary-isthmic junction fluid than in fluid from the isthmic-utero-tubal junction (P < 0.05). These results demonstrate that follicular fluid and oviductal fluids have considerable influences on boar sperm motility. Furthermore, the immediate effect of periovulatory ampullary-isthmic junction fluid in increasing the percentage of spermatozoa swimming in circles (hyperactivated) is relevant, since it is at this time and within this region that fertilization occurs.

Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization.

Capacitation is defined as the series of transformations that spermatozoa normally undergo during their migration through the female genital tract, in order to reach and bind to the zona pellucida, undergo the acrosome reaction, and fertilize the egg. During this process, extensive changes occur in all sperm compartments (head and flagellum; membrane, cytosol, cytoskeleton), factors originating from epididymal fluid and seminal plasma are lost or redistributed and membrane lipids and proteins are reorganized; ion fluxes induce biochemical modifications and controlled amounts of reactive oxygen species are generated; spermatozoa develop hyperactivated motility; and complex signal transduction mechanisms are initiated. The main purpose of capacitation is to ensure that spermatozoa reach the eggs at the appropriate time and in the appropriate state to fertilize these eggs, by finely-controlling the rate of the changes necessary to prime spermatozoa and by activating all the mechanisms needed for the subsequent acrosome reaction. The reversibility of some of the mechanisms leading to sperm capacitation may therefore be a very important aspect of the fine regulation and perfect timing of this process.

Morphological and biochemical changes following acute unilateral testicular torsion in prepubertal rats.

PURPOSE: The role played by reactive oxygen species in the effects of testicular torsion and torsion/detorsion on ipsilateral and contralateral testis was investigated. MATERIALS AND METHODS: Prepubertal rats were submitted to unilateral testicular torsion alone or followed by detorsion for up to 1 week. Morphology and biochemical parameters (thiobarbituric reactive substances, superoxide dismutase-like and catalase-like activities) were evaluated. RESULTS: Torsion (1 or 2 h) alone induced time-dependent morphological damages that worsened progressively after detorsion in ipsilateral testis but had no effect on contralateral testis. The levels of antioxidants against the superoxide anion and hydrogen peroxide decreased in ipsilateral but not in contralateral testis. However, the level of thiobarbituric reactive substances (an indicator of lipid perioxidation) decreased after testicular torsion or torsion/detorsion in both testes. CONCLUSION: These data emphasize that oxidative stress may play a role in testicular damage caused by torsion/detorsion and that biochemical indicators of oxidative stress are more sensitive than histological techniques in detecting modifications in the contralateral testis.