Biomedical subjects

C Krausz

Publications and source records attributed to C Krausz.

52 records · Page 3Linked to original sources

PubMed1998

Identification and characterization of functional nongenomic progesterone receptors on human sperm membrane.

The presence of functional nongenomic progesterone (P) receptors in human spermatozoa has been investigated by equilibrium binding studies in intact spermatozoa, ligand blot and Western blot analysis of sperm lysates, as well as determination of the effects of the steroid on sperm intracellular Ca2+ concentrations. Binding experiments were performed using progesterone-11alpha-glucuronide-[125I]iodotyramine as tracer. Computer analysis of competition curves using different steroids as competitors indicated the presence of two distinct binding sites for P. The high affinity site (Kd in the nanomolar range) appears to be specific for P, whereas the low affinity one (Kd in the micromolar range) binds with equal affinity 11beta-hydroxyprogesterone (11betaOHP) and 17alpha-hydroxyprogesterone (17alphaOHP). A significant correlation exists among affinity constants (as determined by binding studies) and EC50 values for the effects of P, 11betaOHP, and 17alphaOHP on intracellular Ca2+ in fura-2-loaded spermatozoa, strongly indicating the involvement of P-binding sites in the biological effect of the steroid. In particular, dose-response curves for P were biphasic, with an EC50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11betaOHP and 17alphaOHP were monophasic, with an EC50 just in the micromolar range. Ligand blot analysis of sperm total lysates performed with peroxidase-conjugated P revealed the presence of two binding proteins of 54 and 57 kDa that were specific for P. Indeed, peroxidase-conjugated P binding was blocked by the simultaneous presence of the unconjugated steroid. Using alpha c262 antibody, which is directed against the P-binding domain of genomic receptor, we detected two proteins of similar molecular mass (54 and 57 kDa), whereas using antibodies directed against the DNA-binding and N-terminal domains of the genomic P receptors, the two proteins were not detected. In addition, p54 and p57 appear to be mostly localized in sperm membranes and virtually absent in the cytoplasm. The involvement of these proteins in the biological effects of P is indicated by the strong inhibitory effect of alpha c262 on P-induced acrosome reaction of capacitated human spermatozoa.

Acrosome↗

PubMed1996

Human sperm activation during capacitation and acrosome reaction: role of calcium, protein phosphorylation and lipid remodelling pathways.

Two processes, namely capacitation and acrosome reaction, are of fundamental importance in the fertilization of oocyte by spermatozoon. Physiologically occurring in the female genital tract, capacitation is a complex process, which renders the sperm cell capable for specific interaction with the oocyte. During capacitation, modification of membrane characteristics, enzyme activity and motility property of spermatozoa render these cells responsive to stimuli that induce acrosome reaction prior to fertilization. Physiological acrosome reaction occurs upon interaction of the spermatozoon with the zona pellucida protein ZP3. This is followed by liberation of several acrosomal enzymes and other constituents that facilitate penetration of the zona and exposes molecules on the sperm equatorial segment that allows fusion of sperm membrane with the oolemma. The molecular mechanisms and the signal transduction pathways mediating the processes of capacitation and acrosome reaction are only partially defined, and appear to involve modifications of intracellular calcium and other ions, lipid transfer and phospholipid remodelling in sperm plasma membrane as well as changes in protein phosphorylation. The human and mouse sperm receptor for ZP3 has been recently sequenced and cloned. This receptor exhibits sequence homology with proto-oncogenes that mediate proliferation and differentiation in somatic cells. This review summarizes the main signal transduction pathways involved in capacitation and acrosome reaction.

Acrosome Reaction↗

PubMed1996

Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization.

We have recently reported, in a small cohort of subjects, that acrosome reaction (AR) and intracellular free calcium ([Ca2+]i) increase in response to progesterone were significantly correlated with in-vitro fertilization (IVF) rate. In the present study we extended these results to 90 subjects undergoing IVF. We confirm that both parameters were highly significantly correlated with the fertilization rate (P < 0.001). In particular, significantly lower responses to progesterone were detected in subjects with a fertilization rate < 50%, further enlightening the functional significance of sperm responsiveness to progesterone with respect to the process of fertilization. Moreover, we report here that both tests are highly discriminant of fertilization success, with positive predictive values > 90% for [Ca2+]i values which increase by > 1.2-fold and AR inducibility > 7% (cut-off values). Conversely, AR following challenge with the calcium ionophore A23187 was less significantly correlated with the percentage fertilization rate (P < 0.05), and showed lower predictive values than response to progesterone. All these tests ([Ca2+]i increase in response to progesterone, AR in response to progesterone and to A23187) appear highly sensitive and moderately specific. The positive predictive value may rise to > 95% when the combination of two tests ([Ca2+]i and inducibility of AR in response to progesterone) is considered. No correlation with fertilization rate has been found for spontaneous AR or basal [Ca2+]i. In conclusion, we propose that assessment of human sperm responsiveness to progesterone may be clinically useful in predicting fertilizing ability in vitro.

Acrosome↗

PubMed1996

Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to obtain fertilization of the oocyte in vivo and in vitro. Although most of the biochemical/ biophysical events that occur during capacitation in vitro have been characterized, the molecular mechanisms underlying these complex events are still obscure. Increases of intracellular free Ca2+ concentrations ([Ca2+]i) and protein tyrosine phosphorylation have previously been demonstrated during in vitro capacitation of human spermatozoa. In the present study we investigated the relationship between extracellular/intracellular Ca2+, protein tyrosine phosphorylation, and tyrosine kinase and phosphatase activities during sperm capacitation. We report that the increase in tyrosine phosphorylation of several protein bands that occurs during sperm capacitation is independent of the presence of Ca2+ in the external medium and, at least partially, of the increase in [Ca2+]i occurring during the process. Indeed, the spontaneous increase in phosphorylation was still present in Ca(2+)-free/EGTA-containing-medium and in the presence of the intracellular Ca2+ chelator BAPTA/AM. Moreover, phosphorylation of proteins and protein tyrosine kinase (PTK) activity was enhanced if spermatozoa were incubated in Ca(2+)-free medium, suggesting the presence of Ca(2+)-inhibited tyrosine kinase(s) in human sperm. This hypothesis is further substantiated by the lower tyrosine phosphorylation observed after incubation with the ionophore A23187 and the endoplasmic Ca(2+)-ATPase inhibitor thapsigargin, which promote Ca2+ influx in human sperm. The ability of the cells to undergo acrosome reaction in response to progesterone, which can be considered a functional endpoint of capacitation, was highly compromised when spermatozoa were incubated in Ca(2+)-free medium or in the presence of EGTA, confirming that Ca2+ is required for sperm capacitation. Conversely, in the presence of erbstatin, a inhibitor of tyrosine kinase activity, which blunts tyrosine phosphorylation during capacitation, response to progesterone was maintained, suggesting that tyrosine phosphorylation must be kept at a low level (physiologically by the presence of Ca2+ in the external medium, or pharmacologically by the presence of erbstatin) in order to obtain response to progesterone. This mechanism may be important in vivo during sperm transit in the female genital tract to ensure appropriate timing of full capacitation in the proximity of the oocyte.

Acrosome↗

PubMed1995

Stimulation of protein tyrosine phosphorylation by platelet-activating factor and progesterone in human spermatozoa.

Tyrosine phosphorylation of proteins is involved in several sperm functions, including capacitation, motility, and acrosome reaction of spermatozoa. This study was undertaken to determine changes of tyrosine phosphorylation during 'in vitro' capacitation as well as the ability of platelet-activating factor (PAF) and progesterone (P), two known activators of sperm functions, to stimulate tyrosine phosphorylation of human sperm proteins. Spermatozoa were capacitated in BSA-containing medium and incubated with PAF (10-1000 nM) and progesterone (0.1-1 microgram/ml). After SDS-PAGE, sperm proteins were transferred to nitrocellulose and tyrosine phosphorylated proteins immunodetected by reacting with anti-phosphotyrosine antibody. The antibody mainly reacted with two proteins of approximately 97 and 75 kDa. The level of phosphorylation increased in these two proteins as a function of capacitation time, with a maximum between 120 and 180 min. In addition, phosphorylation in these two proteins was increased in capacitated spermatozoa by treatment with progesterone and PAF and was greatly reduced by pre-incubation with the tryosine kinase inhibitor erbstatin. Furthermore, pre-incubation with the two tyrosine kinase inhibitors erbstatin and genistein inhibited the induction of acrosome reaction by progesterone and, partially, by PAF. Our results suggest a role for tyrosine kinase(s) in the mechanism of capacitation and activation of human spermatozoa by PAF and progesterone.

Acrosome↗

PubMed1995

Actions of progesterone on human sperm: a model of non-genomic effects of steroids.

Non-genomic actions of steroids have been extensively studied in the last few years. Among these actions, the non-genomic effect of progesterone (P) on human spermatozoa appears to be very promising, in view of the dramatic effect of this steroid on intracellular calcium, activation of tyrosine kinase, and induction of acrosome reaction. We have shown that the ability of spermatozoa to respond to P increases during the process of capacitation and is not counteracted by the P-receptor antagonist RU486 nor by the GABAA antagonists bicuculline and picrotoxin. We have also shown that P increases tyrosine phosphorylation of a sperm protein of about 97 kDa, suggesting activation of tyrosine kinase(s). In addition, we found that P induces a perturbation of sperm membrane phospholipid metabolism resulting in an increase of synthesis of platelet-activating factor and liberation of arachidonic acid. Results of these biochemical studies indicate that P is able to stimulate several signal transduction pathways in human sperm. We have also investigated responsiveness to P in sperm of oligozoospermic subjects as well as of men undergoing an in vitro fertilization (IVF) program. Our results show that the percentage increases of intracellular calcium and acrosome reaction in response to P is significantly reduced in oligozoospermic men as well as in subjects with reduced fertilization rate. Moreover, in the latter subjects response to P is highly significant correlated to fertilization rate of oocytes. These studies indicate that a biochemical alteration of sperm in their capacity to respond to P might be responsible for reduced fertilizing ability.

Acrosome↗

PubMed1995

Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa are correlated with in-vitro fertilization.

In this study we have investigated responsiveness to progesterone in spermatozoa from a group of unselected male partners of couples undergoing in-vitro fertilization (IVF). We evaluated progesterone-stimulated intracellular Ca2+ ([Ca2+]i) and percentage increase in acrosome reaction in the same sperm sample used for oocyte inseminations. [Ca2+]i was measured with a fluorimetric method, while the acrosome reaction was assessed using a fluorescent probe (fluorescein isothiocyanate-labelled peanut lectin). The average percentage [Ca2+]i as well as the rate of increase in the frequency of acrosome reaction following progesterone challenge were significantly lower (P < 0.005) in the group of patients with a fertilization rate < 50%. In addition, significant correlations between the fertilization rate and the progesterone-stimulated [Ca2+]i and acrosome reaction increases (r = 0.78 and r = 0.79 respectively) were observed. Furthermore, in cases of fertilization failure, no increase of [Ca2+]i or acrosome reaction was observed in response to progesterone with the exception of one case. Our results indicate that [Ca2+]i and acrosome reaction increases in response to progesterone can be of value in the prediction of sperm fertilizing ability. As the two parameters were significantly correlated to each other (r = 0.86), the two assays have similar IVF predictive value and might be used interchangeably as a diagnostic tool in the assignment of male patients to the different kinds of assisted fertilization techniques.

Acrosome↗

PubMed1994

Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions.

In this study, we have examined the relationship between creatine phosphokinase (CPK), a biochemical measure of human sperm quality (Huszar et al., 1988a,b, 1990; Huszar and Vigue, 1994), and a marker for the presence of residual cytoplasm in human spermatozoa, glucose-6-phosphate dehydrogenase (G6PDH). We then determined whether the diagnostic potential of these enzymes was related to the capacity of the sperm suspensions to generate reactive oxygen species (ROS) and/or the presence of leukocytes and precursor germ cells. Across the data set as a whole, G6PDH and CPK were highly correlated with each other and, to a lesser extent, with the generation of ROS. Contamination of the sperm suspensions with leukocytes might have contributed to these associations, since the presence of such cells was also significantly correlated with CPK, G6PDH, and ROS. However, even after the leukocytes had been carefully removed, G6PDH was still highly correlated with CPK (r = 0.794), indicating that both criteria were providing similar information of the cytosolic component of human sperm suspensions. In the absence of leukocyte contamination, CPK and G6PDH activities were also correlated with the presence of precursor germ cells, and this association may, in part, explain the diagnostic value of these criteria. An additional component of their prognostic value may be reflected in the statistically significant association observed between G6PDH activity and ROS generation. A possible mechanism for such an association is suggested, which should be amenable to experimental verification.

Biomarkers↗

PubMed1994

Stimulation of oxidant generation by human sperm suspensions using phorbol esters and formyl peptides: relationships with motility and fertilization in vitro.

OBJECTIVES: To investigate the influence of reactive oxygen species generated by human spermatozoa and contaminating leukocytes on sperm movement and fertilization in vitro. DESIGN: A chemiluminescence technique, using luminol and peroxidase, was used to monitor the generation of reactive oxygen species by human sperm suspensions and the results were correlated with sperm movement and the fertilization of human ova in vitro. SETTING: Diagnostic Andrology Laboratory and IVF Clinic. PATIENTS: Infertile couples undergoing IVF therapy. RESULTS: An N-formyl-methionyl-leucyl-phenylalanine (FMLP) provocation test was used to demonstrate that the presence of leukocytes in 28.5% of the sperm preparations was associated with elevated levels of spontaneous reactive oxygen species production, impaired movement, and a reduced capacity for fertilization in vitro. In the absence of leukocytes, exposure to phorbol ester stimulated a burst of reactive oxygen species generation by human spermatozoa, the magnitude of which was correlated highly with a loss of sperm motility but not with fertilization rates observed in the concurrent IVF cycle. CONCLUSION: Leukocyte contamination of human sperm preparations can be detected readily by FMLP-induced, luminol-dependent chemiluminescence and the results have an important bearing on the fertilizing capacity of the spermatozoa in vitro.

Fertilization↗

PubMed1994

Effect of platelet-activating factor on motility and acrosome reaction of human spermatozoa.

The effect of platelet-activating factor (PAF) on motility parameters and induction of the acrosome reaction in human spermatozoa was investigated in 36 unselected men with different degrees of initial sperm motility. The characteristics of sperm movement were assessed by computer-assisted sperm analysis (Hamilton-Thorn Motility Analyser) and the percentage of acrosome-reacted spermatozoa was evaluated after 1 h incubation with PAF (10 nM) and staining with fluorescent peanut lectin. We found that short-term (4 h max) incubation with PAF significantly enhanced total and progressive sperm motility as well as acrosome reaction. An increase of sperm motility in response to PAF was present in 16 out of the 25 subjects studied (defined as responders) and was inversely correlated with basal motility. In the 11 samples (six responders and five non-responders) where the incubation with PAF was prolonged overnight, an increase of sperm motility was present in all the subjects studied. Similarly, an increase in numbers of acrosome reactions in response to 10 nM PAF was present in 20 out of the 26 subjects examined, and was inhibited by the PAF receptor antagonist L659 989. Our results indicate a possible physiological role for PAF in fertilization and suggest a potential use of PAF in in-vitro fertilization techniques in cases of reduced sperm motility.

Acrosome↗

PubMed1994

Simultaneous measurement of sperm LDH, LDH-X, CPK activities and ATP content in normospermic and oligozoospermic men.

Total lactate dehydrogenase (LDH), LDH-X, creatine phosphokinase (CPK) activities and ATP content were measured in washed spermatozoa from 188 normospermic and 94 oligozoospermic men. These four biochemical parameters were significantly higher (p < 0.001) in spermatozoa from oligozoospermic men than in those from normospermic men and were related inversely to sperm concentration. In oligozoospermic men the activity of sperm LDH and LDH-X was related significantly to sperm CPK activity (r = 0.5486 and r = 0.4442, respectively), whereas this relationship was less evident in normospermic men (r = 0.4124 and r = 0.3844, respectively). Sperm ATP content was related weakly to sperm LDH, LDH-X and CPK activities both in oligozoospermic and normospermic men. After separation on a discontinuous (40% and 80%) Percoll gradient LDH, LDH-X, CPK activities and ATP content in 80% separated sperm fractions from 48 normospermic and 30 oligozoospermic men were found to be significantly lower than in the initial semen sample in both groups of subjects. The 80% separated sperm fractions from oligozoospermic and normospermic men did not differ in their LDH or LDH-X activities or ATP content, whereas sperm CPK activity was still significantly higher in oligozoospermic men. These data suggest that, of the four parameters studied, high levels of intracellular CPK activity are the most relevant biochemical feature of spermatozoa from oligozoospermic men.

Adenosine Triphosphate↗

PubMed1993

Stimulation of platelet-activating factor synthesis by progesterone and A23187 in human spermatozoa.

The presence of platelet-activating factor (PAF) has been demonstrated recently in mammalian spermatozoa, together with evidence for a role of this phospholipid in enhancing sperm motility and fertilizing ability. To investigate whether PAF synthesis and release occurs in human spermatozoa following incubation with stimuli that induce acrosome reaction, spermatozoa were incubated with progesterone and A23187, two known inducers of the exocytotic event. PAF synthesis (remodelling pathway) was assessed by [3H]acetate incorporation into PAF. Treatment of spermatozoa with progesterone and A23187 resulted in an increase of [3H]acetate incorporation into PAF. Most of the newly synthesized [3H]PAF formed in response to acrosome reaction was found in the supernatant, suggesting a release of the phospholipid from spermatozoa. PAF-like material extracted from human spermatozoa was able to induce aggregation of rabbit platelets and showed identical retention time and the same ion m/e values as authentic PAF when analysed with g.c.-m.s. Lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) activity in human spermatozoa was also studied and showed similar kinetic parameters to those described for other cell systems. Stimulation of spermatozoa with progesterone and A23187 induced an increase of [3H]arachidonic acid release, suggesting an activation of phospholipase A. In conclusion, our results demonstrated increased production and release of PAF in human sperm following stimulation with progesterone and A23187 and suggest a role for this phospholipid in the activation of spermatozoa.

Acetyltransferases↗

PubMed1992

Development of a technique for monitoring the contamination of human semen samples with leukocytes.

OBJECTIVE: To determine whether receptors for the N-formyl chemotactic peptide, FMLP, exist on the surface of human spermatozoa and regulate reactive oxygen species generation by these cells. DESIGN: Chemiluminescent analysis of reactive oxygen species generation by suspensions of human spermatozoa before and after removal of leukocytes using a magnetic cell separation technique. SETTING: Academic Research Institute. PATIENTS: Unselected male volunteers. RESULTS: Human sperm suspensions responded to FMLP and phorbol ester (PMA) with a burst of reactive oxygen species production. Autoradiographic analyses employing 3H FMLP and chemiluminescence studies involving the selective removal of leucocytes with anti-CD 45-coated magnetic beads demonstrated that the FMLP responses were because of leukocyte contamination. In contrast, reactive oxygen species production in response to PMA appeared to reflect the oxidant-generating capacity of both leukocytes and spermatozoa. CONCLUSION: The only cells present in the human ejaculate possessing detectable receptors for FMLP and capable of generating reactive oxygen species in response to this reagent come from the leukocyte population. Luminol-dependent, FMLP-induced, chemiluminescence provides a rational basis for monitoring the presence of leukocytes in suspensions of human spermatozoa.

Cell Fractionation↗

PubMedDate not supplied

Difficulties in achieving vs maintaining erection: organic, psychogenic and relational determinants.

Achieving and maintaining a penile erection are two essential components of the male sexual response. It has recently been suggested that distinct molecular mechanism could underlie the two disturbances. The aim of the present study is to verify possible clinical differences on pathogenetic factors underlying difficulties of achieving and maintaining an erection. We studied a consecutive series of 560 patients (aged 51.9+/-12.8 y old) reporting erectile dysfunction (ED), using SIEDY structured interview. Patients were classified into two distinct categories: those with difficulties in maintaining, rather than achieving, an erection (sample A) and those with main problems in achieving an erection (sample B). A complete physical examination and a series of metabolic, biochemical, hormonal, psychometric, penile vascular tests and nocturnal penile tumescence and rigidity evaluations (NPT) were also performed. Sample B patients showed a higher prevalence of organic conditions related to ED, when compared with sample A as confirmed by higher SIEDY scale 1 scores (3[1-5] vs 1[0.1-3] for sample B vs sample A, respectively; P < 0.0001) which explores organic component of ED and higher prevalence of pathological instrumental parameters. No difference among groups was observed for SIEDY scale 2 (relational component) and SIEDY scale 3 (intrapsychic component) of ED. In conclusion, this study shows for the first time that patients with difficulties in maintaining erection are less likely to be affected by organic disturbances interfering with sexual function, when compared with those unable to achieve a valid erection.

Adult↗

PubMedDate not supplied

Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa.

Progesterone induced a rapid, long-lasting, dose-dependent increase of intracellular free calcium concentration ([Ca2+]i) in human sperm capacitated overnight. This effect was not counteracted by the cytosolic progesterone receptor antagonist RU486 (1 mumol/L) nor by the GABA-A receptor antagonists bicuculline (10 mumol/L) and picrotoxin (50 mumol/L). Also, the rank order of potency of several progestative steroids on [Ca2+]i differed from that previously reported for uterine intracellular progesterone receptor or for P-GABA interaction in the central nervous system, indicating a different pathway for progesterone stimulation of human sperm. Modifications of basal and progesterone-stimulated [Ca2+]i during sperm capacitation were also studied. A progressive, parallel increase of basal and progesterone-stimulated [Ca2+]i in capacitating spermatozoa was found. In particular, progesterone-stimulated [Ca2+]i increased from a basal concentration of 147% +/- 17% at 10 minutes to 327% +/- 65% after 120 minutes of incubation in capacitating medium. This increase was well correlated with basal [Ca2+]i (r = 0.93). In contrast, basal and progesterone-stimulated [Ca2+]i concentrations were constantly low in spermatozoa incubated in noncapacitating medium. In capacitated spermatozoa, initial responsiveness to progesterone and basal [Ca2+]i was higher than in capacitating and noncapacitated samples, and remained constant throughout the duration of the experiment. The progressive, parallel increase of [Ca2+]i and response to progesterone observed during in vitro capacitation of human spermatozoa might be physiologically relevant in vivo during capacitation of sperm in the female genital tract.

Bicuculline↗

PubMedDate not supplied

Decreased responsiveness to progesterone of spermatozoa in oligozoospermic patients.

Spermatozoa from oligozoospermic subjects are characterized by a reduced in vitro ability to penetrate hamster oocytes and by a decreased responsiveness to physiological stimuli that trigger the acrosome reaction. One of the first steps in the induction of the acrosome reaction is an increase of intracellular free calcium concentrations ([Ca2+]i). It has been recently shown that progesterone (P) is able to increase [Ca2+]i in capacitated human sperm at concentrations similar to those found in follicular fluid. We evaluated sperm [Ca2+]i increase in response to P (0.1 micrograms/ml) in 19 normo- and 17 oligozoospermic subjects. The average percentage of [Ca2+]i increase over the basal level was significantly lower in spermatozoa from oligozoospermic subjects when compared to normozoospermic subjects (138.7 +/- 8.22% increase in oligo- versus 263.3 +/- 39.7% increase in normozoospermic subjects; P < 0.001). Progesterone-stimulated [Ca2+]i increase was significantly correlated with sperm motility (r = 0.54), sperm concentration (r = 0.96), and sperm morphology (% of normal forms) (r = 0.49). In addition P induced a significant increase of acrosome-reacted spermatozoa in normospermic patients (n = 10), whereas no significant effect was observed in spermatozoa from oligozoospermic men (n = 7). Taken together, these results indicate that spermatozoa from oligozoospermic men have a reduced ability to initiate the cascade of events that lead to the acrosome reaction in response to a physiological stimulus, such as P, and might contribute to explaining the reduced fertilizing capacity of these patients.

Acrosome↗