Seminars in Thrombosis and Hemostasis, Mar 1, 2007
Semenogelin (Sg), the main component of the human semen coagulum, is an important and versatile p... more Semenogelin (Sg), the main component of the human semen coagulum, is an important and versatile protein acting on several sperm parameters, both as intact or degraded Sg. Sg originates mostly from seminal vesicle and probably is responsible for sperm immobilization in the seminal coagulum. Purified Sg can be cross-linked by transglutaminase or phosphorylated by kinases, but the actual occurrence of these reactions in reproductive physiology is not clear. Experimental evidence demonstrates that prostatespecific antigen (PSA) rapidly cleaves Sg, an event temporally associated with semen liquefaction and initiation of sperm motility. Sg and its degradation peptides participate in various processes including Zn þ 2 shuttling, antibacterial activity, hyaluronidase activation, and so on. Sg inhibits sperm motility at the concentration found in the coagulum, but the rapid processing by PSA allows initiation of movement. The mechanism of Sg action and its targets are not known, but improper Sg degradation decreases fertility. Sg and its degradation peptides block sperm capacitation and associated events at concentrations much lower than those of seminal plasma and could play important role in preventing premature capacitation. The effects of Sg are dependent on time and proteolysis due to PSA, and any imbalance may affect sperm physiology and fertility.
Capacitation of spermatozoa is essential for fertilization, and can be induced by various agents ... more Capacitation of spermatozoa is essential for fertilization, and can be induced by various agents or biological fluids. Previous reports have shown that foetal cord serum (FCS) and the superoxide anion trigger human sperm hyperactivation and capacitation, and that superoxide dismutase (SOD) prevents these processes. We investigated: (1) the capacity of seminal plasma (SP) and follicular fluid (FF) (whole, or fractionated into high and low molecular weight components), in the presence or absence of SOD, to induce the spontaneous acrosome reaction (no stimulant needed, AR) and capacitation (as measured by the lysophosphatidylcholine-induced AR, LPC-AR); (2) a possible relationship between the levels of AR and capacitation obtained with these biological fluids and the superoxide scavenging capacity of the same fluids. The highest levels of LPC-AR were obtained with FF ultrafiltrate (48 f 6%), followed by SP ultrafiltrate (31.9 f 0.8%), FF (30 k 5%), dialysed FF (27 k 4%), and finally, by FCS ultrafiltrate (23 f lo/.), SP (21 f 1%) and dialysed SP (18.9 k 0.8%). A similar order ofpotency for the fluids existed when sperm AR was studied, the levels of AR observed ranging from 26 f 2% to 5.3 f 0.8% after incubation with FF ultrafiltrate and SP respectively. None of these treatments had detrimental effects on sperm motility. In the presence of SOD, there was always an important reduction (52-86%) of the AR and LPC-AR observed. A highly significant inverse linear relationship was observed between the SOD-like activity of the fluids tested and the AR (Y = 0.86, p < 0.001) and LPC-AR (Y= 0.91, p < 0.001) observed in the presence of these fluids. The results suggest that biological fluids contain inducers of the AR and capacitation, and that these probably act by a common mechanism, possibly by direct or indirect induction of an NADPH oxidase in the sperm membrane. The data suggest strongly that the SOD-like activity of a specific fluid is probably one of the most important factors that will determine its capacity to induce the AR and capacitation. Superoxide and human sperm capacitation 259
Capacitation is the series of transformations that spermatozoa undergo in the female genital trac... more 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 calciumcalmodulin-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 IC 50 of 97 Ϯ 3 and 33 Ϯ 3 M when cAMP and cGMP, respectively, were used as substrates. Because the IC 50 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.
Human sperm capacitation involves complex signal transduction mechanisms during which double phos... more 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 ,2Ј-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 dbcAMPϩIBMX 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.
A seminal plasma factor and aprotinin are capable of inhibiting the movement of demembranated rea... more A seminal plasma factor and aprotinin are capable of inhibiting the movement of demembranated reactivated rabbit spermatozoa. These inhibitors can be overcome by the addition of ATP. This reversibility could be explained by a common binding receptor for the inhibitors and MgATP. The respective affinities for the receptor are dependent on whether the microtubules are sliding or resting.
Semenogelin (Sg), the major protein of the human semen coagulum, is present at high concentration... more 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 O 2 Ϫ •-related chemiluminescence. Ribonuclease (RNase), which has as high an isoelectric point (pI ϭ 9.7) as Sg (pI ϭ 9.5), also prevented sperm capacitation and O 2 Ϫ •-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 O 2 Ϫ • generated by the mix of xanthine ϩ xanthine oxidase and modified the kinetics of the reaction; RNase did not have such effects. Therefore, Sg is a potential scavenger for O 2 Ϫ • 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 O 2 Ϫ • that is normally generated during this process.
Spermatozoa undergo a variety of changes during their life that are prerequisites to their matura... more Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian sperm capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating spermatozoa and that i...
Although recent evidence indicated that the production of reactive oxygen species (ROS) by human ... more 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 ca...
Recent evidence indicated that human sperm capacitation is associated with an increased productio... more 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 u...
To characterize the very vigorous type of motility observed in the semen of some infertile men an... more To characterize the very vigorous type of motility observed in the semen of some infertile men and to compare the superoxide anion scavenging capacity of the seminal plasma from these men and that from normal men. Patients consulting for infertility related to sperm motility problems and men presenting as sperm donors. Motility patterns and measurements of sperm motility parameters were evaluated by computer-assisted digital image analysis system. The superoxide anion scavenging capacity of seminal plasma was measured by inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase. Spermatozoa from 9 of 68 semen samples with normal sperm concentration, morphology, and percentage of motility showed the typical motility patterns observed during hyperactivation (HA) and a significant level of HA (16% +/- 3%) as compared with those in semen (2.3% +/- 0.3%) from fertile volunteers. The superoxide anion scavenging ca...
Seminal plasma contains a motility inhibitor of demembranated reactivated spermatozoa. We investi... more Seminal plasma contains a motility inhibitor of demembranated reactivated spermatozoa. We investigated its origin within the reproductive tract. The highest level of inhibitor was detected in seminal vesicle fluids from the three species investigated (bull, rat, rabbit). Significant levels of inhibitor were also observed in prostatic fluids. Testes and epididymal fluids, as well as bulbo-urethral and coagulating gland homogenates were essentially devoid of inhibitor. On a mg protein basis, the inhibitor in seminal vesicle fluid was about four times less active than the inhibitor of seminal plasma. The high level of inhibitor in seminal plasma can not be explained by the synergistic effect of the combination of seminal vesicle, prostatic and epididymal fluids. Dialysis experiments suggested that the high level of inhibitor in seminal plasma was mainly due to the presence of a dialysable activator. This activator is capable of potentiating up to four-fold the inhibitor present in semi...
The presence of motility inhibitors in seminal plasma and within spermatozoa from control and inf... more The presence of motility inhibitors in seminal plasma and within spermatozoa from control and infertile men with poor sperm motility was investigated using demembranated reactivated human spermatozoa. No difference was found in the inhibitory capacities in seminal plasma of patients with poor sperm motility (less than 50%) when compared with that of fertile controls with motility above 50%. No correlation was observed between inhibitory capacity and sperm motility. However, when extracts of spermatozoa from these patients were tested for the presence of inhibitor, it was observed that three of nine patients had an inhibitor in their sperm extract. By contrast, all sperm extracts from fertile control subjects were devoid of inhibitor. It was concluded that the presence of a motility inhibitor in seminal plasma does not explain the poor sperm motility observed in patients. The presence of a motility inhibitor within spermatozoa, however, may represent an important factor in the etiolo...
Levels of protein-carboxyl methylase (PCM) activity were measured in spermatozoa from infertile p... more Levels of protein-carboxyl methylase (PCM) activity were measured in spermatozoa from infertile patients with less than 50% sperm motility and compared with those of normal fertile controls. When spermatozoa were washed by a standard centrifugation procedure, the level of PCM activity in a subgroup of patients with sperm motility ranging from 0% and 20% (24.0 +/- 5.2 pmol/mg protein, mean +/- standard error of the mean) was significantly different from that of controls (35.9 +/- 2.3 pmol/mg). However, when the entire population of patients with sperm motility ranging from 0% to 50% (32.6 +/- 6.2 pmol/mg) was compared with controls, no significant difference was observed in sperm PCM levels. With this standard washing procedure no significant relationship (r = 0.28; P greater than 0.05) between sperm PCM activity and motility was observed. By contrast, when spermatozoa were washed on a Percoll gradient, to eliminate other cellular elements, both groups of patients with 0% to 20% (14....
Proceedings of the National Academy of Sciences, 1997
PC4 is a member of the proprotein convertase family of serine proteases implicated in the process... more 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.
Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermat... more 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 48C 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. Mol. Reprod. Dev. 60: 498±506,
A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure incl... more A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris-HCI containing 0.1 mM D T and chromatographies on SP-Sephadex C-25 and Phenyl-Sepharose CL-4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMl in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMl is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMl effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMl inhibited bull dynein ATPase in a concentrationdependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.
Seminars in Thrombosis and Hemostasis, Mar 1, 2007
Semenogelin (Sg), the main component of the human semen coagulum, is an important and versatile p... more Semenogelin (Sg), the main component of the human semen coagulum, is an important and versatile protein acting on several sperm parameters, both as intact or degraded Sg. Sg originates mostly from seminal vesicle and probably is responsible for sperm immobilization in the seminal coagulum. Purified Sg can be cross-linked by transglutaminase or phosphorylated by kinases, but the actual occurrence of these reactions in reproductive physiology is not clear. Experimental evidence demonstrates that prostatespecific antigen (PSA) rapidly cleaves Sg, an event temporally associated with semen liquefaction and initiation of sperm motility. Sg and its degradation peptides participate in various processes including Zn þ 2 shuttling, antibacterial activity, hyaluronidase activation, and so on. Sg inhibits sperm motility at the concentration found in the coagulum, but the rapid processing by PSA allows initiation of movement. The mechanism of Sg action and its targets are not known, but improper Sg degradation decreases fertility. Sg and its degradation peptides block sperm capacitation and associated events at concentrations much lower than those of seminal plasma and could play important role in preventing premature capacitation. The effects of Sg are dependent on time and proteolysis due to PSA, and any imbalance may affect sperm physiology and fertility.
Capacitation of spermatozoa is essential for fertilization, and can be induced by various agents ... more Capacitation of spermatozoa is essential for fertilization, and can be induced by various agents or biological fluids. Previous reports have shown that foetal cord serum (FCS) and the superoxide anion trigger human sperm hyperactivation and capacitation, and that superoxide dismutase (SOD) prevents these processes. We investigated: (1) the capacity of seminal plasma (SP) and follicular fluid (FF) (whole, or fractionated into high and low molecular weight components), in the presence or absence of SOD, to induce the spontaneous acrosome reaction (no stimulant needed, AR) and capacitation (as measured by the lysophosphatidylcholine-induced AR, LPC-AR); (2) a possible relationship between the levels of AR and capacitation obtained with these biological fluids and the superoxide scavenging capacity of the same fluids. The highest levels of LPC-AR were obtained with FF ultrafiltrate (48 f 6%), followed by SP ultrafiltrate (31.9 f 0.8%), FF (30 k 5%), dialysed FF (27 k 4%), and finally, by FCS ultrafiltrate (23 f lo/.), SP (21 f 1%) and dialysed SP (18.9 k 0.8%). A similar order ofpotency for the fluids existed when sperm AR was studied, the levels of AR observed ranging from 26 f 2% to 5.3 f 0.8% after incubation with FF ultrafiltrate and SP respectively. None of these treatments had detrimental effects on sperm motility. In the presence of SOD, there was always an important reduction (52-86%) of the AR and LPC-AR observed. A highly significant inverse linear relationship was observed between the SOD-like activity of the fluids tested and the AR (Y = 0.86, p < 0.001) and LPC-AR (Y= 0.91, p < 0.001) observed in the presence of these fluids. The results suggest that biological fluids contain inducers of the AR and capacitation, and that these probably act by a common mechanism, possibly by direct or indirect induction of an NADPH oxidase in the sperm membrane. The data suggest strongly that the SOD-like activity of a specific fluid is probably one of the most important factors that will determine its capacity to induce the AR and capacitation. Superoxide and human sperm capacitation 259
Capacitation is the series of transformations that spermatozoa undergo in the female genital trac... more 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 calciumcalmodulin-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 IC 50 of 97 Ϯ 3 and 33 Ϯ 3 M when cAMP and cGMP, respectively, were used as substrates. Because the IC 50 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.
Human sperm capacitation involves complex signal transduction mechanisms during which double phos... more 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 ,2Ј-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 dbcAMPϩIBMX 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.
A seminal plasma factor and aprotinin are capable of inhibiting the movement of demembranated rea... more A seminal plasma factor and aprotinin are capable of inhibiting the movement of demembranated reactivated rabbit spermatozoa. These inhibitors can be overcome by the addition of ATP. This reversibility could be explained by a common binding receptor for the inhibitors and MgATP. The respective affinities for the receptor are dependent on whether the microtubules are sliding or resting.
Semenogelin (Sg), the major protein of the human semen coagulum, is present at high concentration... more 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 O 2 Ϫ •-related chemiluminescence. Ribonuclease (RNase), which has as high an isoelectric point (pI ϭ 9.7) as Sg (pI ϭ 9.5), also prevented sperm capacitation and O 2 Ϫ •-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 O 2 Ϫ • generated by the mix of xanthine ϩ xanthine oxidase and modified the kinetics of the reaction; RNase did not have such effects. Therefore, Sg is a potential scavenger for O 2 Ϫ • 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 O 2 Ϫ • that is normally generated during this process.
Spermatozoa undergo a variety of changes during their life that are prerequisites to their matura... more Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian sperm capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating spermatozoa and that i...
Although recent evidence indicated that the production of reactive oxygen species (ROS) by human ... more 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 ca...
Recent evidence indicated that human sperm capacitation is associated with an increased productio... more 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 u...
To characterize the very vigorous type of motility observed in the semen of some infertile men an... more To characterize the very vigorous type of motility observed in the semen of some infertile men and to compare the superoxide anion scavenging capacity of the seminal plasma from these men and that from normal men. Patients consulting for infertility related to sperm motility problems and men presenting as sperm donors. Motility patterns and measurements of sperm motility parameters were evaluated by computer-assisted digital image analysis system. The superoxide anion scavenging capacity of seminal plasma was measured by inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase. Spermatozoa from 9 of 68 semen samples with normal sperm concentration, morphology, and percentage of motility showed the typical motility patterns observed during hyperactivation (HA) and a significant level of HA (16% +/- 3%) as compared with those in semen (2.3% +/- 0.3%) from fertile volunteers. The superoxide anion scavenging ca...
Seminal plasma contains a motility inhibitor of demembranated reactivated spermatozoa. We investi... more Seminal plasma contains a motility inhibitor of demembranated reactivated spermatozoa. We investigated its origin within the reproductive tract. The highest level of inhibitor was detected in seminal vesicle fluids from the three species investigated (bull, rat, rabbit). Significant levels of inhibitor were also observed in prostatic fluids. Testes and epididymal fluids, as well as bulbo-urethral and coagulating gland homogenates were essentially devoid of inhibitor. On a mg protein basis, the inhibitor in seminal vesicle fluid was about four times less active than the inhibitor of seminal plasma. The high level of inhibitor in seminal plasma can not be explained by the synergistic effect of the combination of seminal vesicle, prostatic and epididymal fluids. Dialysis experiments suggested that the high level of inhibitor in seminal plasma was mainly due to the presence of a dialysable activator. This activator is capable of potentiating up to four-fold the inhibitor present in semi...
The presence of motility inhibitors in seminal plasma and within spermatozoa from control and inf... more The presence of motility inhibitors in seminal plasma and within spermatozoa from control and infertile men with poor sperm motility was investigated using demembranated reactivated human spermatozoa. No difference was found in the inhibitory capacities in seminal plasma of patients with poor sperm motility (less than 50%) when compared with that of fertile controls with motility above 50%. No correlation was observed between inhibitory capacity and sperm motility. However, when extracts of spermatozoa from these patients were tested for the presence of inhibitor, it was observed that three of nine patients had an inhibitor in their sperm extract. By contrast, all sperm extracts from fertile control subjects were devoid of inhibitor. It was concluded that the presence of a motility inhibitor in seminal plasma does not explain the poor sperm motility observed in patients. The presence of a motility inhibitor within spermatozoa, however, may represent an important factor in the etiolo...
Levels of protein-carboxyl methylase (PCM) activity were measured in spermatozoa from infertile p... more Levels of protein-carboxyl methylase (PCM) activity were measured in spermatozoa from infertile patients with less than 50% sperm motility and compared with those of normal fertile controls. When spermatozoa were washed by a standard centrifugation procedure, the level of PCM activity in a subgroup of patients with sperm motility ranging from 0% and 20% (24.0 +/- 5.2 pmol/mg protein, mean +/- standard error of the mean) was significantly different from that of controls (35.9 +/- 2.3 pmol/mg). However, when the entire population of patients with sperm motility ranging from 0% to 50% (32.6 +/- 6.2 pmol/mg) was compared with controls, no significant difference was observed in sperm PCM levels. With this standard washing procedure no significant relationship (r = 0.28; P greater than 0.05) between sperm PCM activity and motility was observed. By contrast, when spermatozoa were washed on a Percoll gradient, to eliminate other cellular elements, both groups of patients with 0% to 20% (14....
Proceedings of the National Academy of Sciences, 1997
PC4 is a member of the proprotein convertase family of serine proteases implicated in the process... more 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.
Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermat... more 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 48C 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. Mol. Reprod. Dev. 60: 498±506,
A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure incl... more A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris-HCI containing 0.1 mM D T and chromatographies on SP-Sephadex C-25 and Phenyl-Sepharose CL-4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMl in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMl is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMl effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMl inhibited bull dynein ATPase in a concentrationdependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.
Uploads
Papers by Eve de Lamirande