Biomedical subjects

L Bonaccorsi

Publications and source records attributed to L Bonaccorsi.

14 recordsLinked to original sources

PubMed1998

Nongenomic effects of progesterone on spermatozoa: mechanisms of signal transduction and clinical implications.

Progesterone (P) is one of the physiological stimuli of human sperm acrosome reaction. It is present in high levels at the site of fertilization (cumulus oophorus) and has been describe to affect several sperm functions including motility, capacitation and acrosome reaction. The effects of the steroid, which is present in high levels in the cumulus matrix that surrounds the oocyte, are mediated by an increase of intracellular calcium concentrations, efflux of chloride, stimulation of activity of phospholipases and phosphorylation of proteins. These effects are due to activation of a rapid/nongenomic pathway. Two different types of receptors for P, distinct from the genomic ones, have been recently identified on the surface of human spermatozoa. The affinities of P for these receptors are respectively in the nano- and in the micromolar range. Sperm responsiveness to progesterone is impaired in subfertile patients and is strictly correlated to the ability of fertilize the oocyte. In addition, the determination of sperm responsiveness is predictive of fertilizing ability with a positive predictive value of 90% and can be clinically useful for the preliminary assessment of the male partner to select the appropriate assisted reproductive technique.

Calcium↗

PubMed1998

Progesterone-stimulated intracellular calcium increase in human spermatozoa is protein kinase C-independent.

Indirect studies suggested that protein kinase C (PKC) has a role in sperm motility and the acrosome reaction. Physiological inducers of the sperm acrosome reaction include progesterone, which can increase intracellular calcium ([Ca2+]i), tyrosine phosphorylation of proteins and chloride efflux in human spermatozoa. PKC may be involved in progesterone-stimulated acrosome reaction, although controversial results have been obtained concerning the effect of PKC inhibition on progesterone-stimulated [Ca2+]i increase. In the present study, we investigated the direct effect of progesterone on the activity of PKC, as well as the effect of a panel of PKC inhibitors on progesterone-stimulated [Ca2+]i increase and tyrosine phosphorylation of proteins. We found that progesterone stimulates sperm PKC activity and that PKC inhibition with staurosporine and bisindolylmaleimide partially reversed the effect of progesterone on acrosome reaction, indicating an involvement of the enzyme in the effect of the steroid. We next evaluated the effect of three different PKC inhibitors (sangivamycin, staurosporine and bisindolylmaleimide) on progesterone-stimulated [Ca2+]i increase. Neither short-term (15 min) nor long-term (90 min) preincubation with any of the three compounds had a substantial effect on the stimulatory effect of progesterone on sperm [Ca2+]i. Nor was responsiveness to progesterone affected by either short-term (determining activation of PKC) or long-term (determining down-regulation of PKC) incubation with the tumour promoter phorbol myristate acetate (PMA), a known non-physiological stimulator of PKC. These results indicate that progesterone-stimulated calcium influx is independent of PKC activation. In addition, we found that preincubation with PKC inhibitors had a stimulatory effect per se on tyrosine phosphorylation of sperm proteins. When compared with the appropriate control, the effect of progesterone on tyrosine phosphorylation was slightly (but not significantly) reduced by the inhibitors, sangivamycin, staurosporine and bisindolylmaleimide, but was significantly inhibited by calphostin C. These results do not permit a final conclusion on the involvement of PKC in progesterone-stimulated tyrosine phosphorylation of sperm proteins. However, the lack of effect of PMA on tyrosine phosphorylation indicates that PKC stimulation is not sufficient to induce this effect. In conclusion, our results indicate that PKC plays a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is downstream to stimulation of calcium influx by the steroid.

Calcium↗

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↗

PubMed1997

Protein tyrosine kinase, mitogen-activated protein kinase and protein kinase C are involved in the mitogenic signaling of platelet-activating factor (PAF) in HEC-1A cells.

We have recently demonstrated that the phospholipid platelet-activating factor (PAF) mediates an autocrine proliferative loop in the endometrial adenocarcinoma cell line HEC-1A. In the present study we investigated the signaling pathways involved in PAF-mediated increase of proliferation in these cells. In particular, we studied the effect of PAF on protein tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity, as well as the effect of protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibition on PAF-induced increase of c-fos gene expression and thymidine incorporation in HEC-1A cells. We found that PAF induced a rapid, time- and dose-dependent increase of tyrosine phosphorylation of a subset of proteins of 42, 44, 78, 99, and 150 kDa molecular weight. We also found that PAF increased tyrosine phosphorylation and activity of p42 MAPK, suggesting the involvement of this important intermediary enzyme in the proliferative effect of PAF. The effect of PAF on c-fos gene expression was not prevented by pre incubation with the PTK inhibitors genistein or methyl-2,5-dihydroxycinnamate, whereas was strongly affected by PKC down regulation after long term incubation with PMA or by PKC inhibition with sangivamycin. We also found that genistein and methyl 2,5-dihydroxycinnamate decreased both basal and PAF-stimulated [3H]thymidine uptake in these cells. Similar results were obtained with PD 098059, a specific inhibitor of MAP kinase cascade. PAF-stimulated [3H]thymidine uptake was also prevented by PKC down regulation after long term exposure to PMA and PKC inhibition with the two inhibitors sangivamycin and bis-indolylmaleimide. In conclusion, our results indicate that PAF-induced mitogenesis in HEC-1A cells is mediated by the activation of multiple signaling pathways, involving PTK, MAPK, and PKC activation.

Adenocarcinoma↗

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

Platelet-activating factor enhances production of insulin-like growth factor binding proteins in a human adenocarcinoma cell line (HEC-1A)

We have recently demonstrated the existence of an autocrine growth loop driven by platelet-activating factor (PAF) in the human endometrial adenocarcinoma. cell line HEC-1A. To investigate a possible cooperation between PAF and the insulin-like growth factor (IGF) system in this cell line, the effect of PAF on insulin-like growth factor binding protein (IGFBP) production as well as binding and biological activities of IGF-I, IGFv-II, and the analog Des(1-3)IGF-I have been evaluated. Analysis of self- and cross-displacement curves of [125I]IGF-I binding to HEC-1A cells indicates the presence of a single class of binding sites, with affinity constants of 1-4 nM for IGF-I and IGF-II and 70 nM for Des(1- 3)IGF-I, which binds to IGFBPs with lower affinity. Insulin does not apparently bind to this binding site. Moreover, the addition of increasing concentrations of IGF-I leads to a paradoxical increase of binding. These results indicate a similarity of this binding site to IGFBPs. The presence of IGFBPs has been demonstrated by Western ligand blot analysis of HEC-1A conditioned medium which shows the presence of two bands of 32-34 and 40-45 kDa. By Western immunoblotting analysis, the two bands were respectively identified as IGFBP-2 and IGFBP-3. Incubation with PAF (1 microM) highly increases the release of the two IGFBPs from the cells. Such an effect is inhibited by the PAF receptor antagonist L659,989, by the PKC inhibitor sangivamycin, and by the tyrosine kinase inhibitor genistein. PAF also induces a time-dependent increase of mRNA expression for IGFBP-3, suggesting an effect on synthesis of this protein. IGF-I and IGF-II (0.1-100 nM) are almost ineffective in inducing [3H]thymidine incorporation, whereas a slight proliferative effect is observed with Des(1-3)IGF-I which also increases PAF synthesis. These data demonstrate a modulatory action of PAF on IGFBP secretion in HEC-1A cells and indicate that the IGF system plays a minor, if any, modulatory role on proliferation of this cell line.

Adenocarcinoma↗

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↗

PubMed1995

Tyrosine kinase inhibition reduces the plateau phase of the calcium increase in response to progesterone in human sperm.

Progesterone (P) has previously been shown to induce a rapid increase in [Ca2+]i as well as tyrosine phosphorylation of proteins in human spermatozoa. Both these effects are essential for induction of the acrosome reaction by P. We investigated a possible relationship between the P-induced calcium increase and tyrosine kinase activation, by evaluating the effect of the tyrosine kinase inhibitor genistein on these two effects. We found that preincubation with genistein abolished P-induced tyrosine phosphorylation of two sperm proteins of 97 and 75 kDa molecular weight and significantly inhibited the plateau phase of P-induced [Ca2+]i increase without affecting the peak phase. Conversely, the plateau phase was enhanced by the tyrosine phosphatase inhibitor Na3VO4. The effect of genistein was specific for P, since no inhibition was observed on the [Ca2+]i increase induced by thapsigargin, an inhibitor of endoplasmic Ca(2+)-ATPase previously shown to mobilize Ca2+ in spermatozoa. These results indicate that tyrosine kinase activation is involved in the generation of the plateau phase of Ca2+ influx induced by P, and suggest the possibility that two different pathways are involved in the induction of Ca2+ entry by P in human sperm.

Biological Transport↗

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

Platelet-activating factor mediates an autocrine proliferative loop in the endometrial adenocarcinoma cell line HEC-1A.

We investigated the synthesis and biological effects of platelet-activating factor (PAF) in the human endometrial cancer cell line HEC-1A. We found that HEC-1A cells actively synthesize and release PAF, as demonstrated by both [3H]acetate incorporation into PAF and gas chromatography-mass spectrometry studies. HEC-1A cells not only synthesize but also respond to PAF. Indeed, in fura-2-loaded cells, PAF stimulates [Ca2+]i increase with a median effective concentration of 5.6 nM. Furthermore, PAF induces a time-dependent expression increase of the nuclear protooncogene c-fos with a median effective concentration of 130 nM and stimulates DNA synthesis (median effective concentration, 700 nM). All of these effects are inhibited by the PAF receptor antagonist L659,989. Radioligand binding studies indicated the presence of two populations of PAF receptors with affinity constants in the nanomolar and micromolar range. Since the PAF antagonist per se inhibits DNA synthesis and cell proliferation, we suggest that PAF supports an autocrine growth circuit in HEC-1A cells. On the contrary, in the uterine leiomyosarcoma cell line SK-UT-1, which does not express specific binding sites for PAF, neither this phospholipid nor its receptor antagonist affect DNA synthesis. Our results provide evidence for the existence of an autocrine proliferative loop involving PAF in the endometrial cancer cell line HEC-1A.

Acetyl-CoA C-Acetyltransferase↗

PubMed1994

Platelet-activating factor in human endometrium.

Platelet-activating factor (PAF) is a phospholipid actively produced by human endometrium and deeply involved in the processes of ovoimplantation and labor. We recently found that PAF represents a new autocrine growth factor for a human adenocarcinoma cell line, HEC-1A. Indeed, biologically active PAF is synthesized by HEC-1A cells, under progesterone control. In HEC-1A cells, PAF regulates intracellular calcium concentration ([Ca2+]), DNA synthesis and expression of early oncogenes. All these effects are blocked by the receptor antagonist L659,989. However, while nanomolar concentrations of PAF mobilize [Ca2+], only micromolar concentrations affect cell growth, suggesting heterogeneity of PAF receptors or signaling. Two distinct populations of PAF receptors are present in HEC-1A cells, which bind PAF in nanomolar and micromolar concentrations, respectively. Since HEC-1A cells are producing elevated concentrations of PAF and micromolar concentrations of the PAF antagonist L659,989 inhibit cell proliferation, an autocrine role for PAF is suggested in HEC-1A cells.

Adenocarcinoma↗

PubMedDate not supplied

Endothelin-1 and its receptors in human testis.

We have previously found the presence of endothelin (ET) receptor and ET-like immunoreactivity in rat testis. We now extend our studies from rat to human testis. We found expression of a specific transcript for ET-1 and ET-1-like immunoreactivity in human testis. Positive staining was confined to the Sertoli cells of the tubular compartment, although few peritubular and interstitial cells were also stained. We also identified specific ETA and ETB receptor transcripts in human testis; ETA expression was more abundant than the ETB expression. Mathematical analysis of multiple self- and cross-competition studies among [125I]ET-1, [125I]ET-3, and analogues confirmed the presence of the ETA and ETB isoreceptors. In testicular homogenates, the ETA receptor was sevenfold more concentrated than the ETB receptor. In order to localize the receptors, we performed [125I]ET-1 autoradiography. Binding sites were mostly concentrated into the seminiferous tubules, although interstitial and peritubular myoid cells were also positive. Within the seminiferous tubules, [125I]ET-1 binding sites were confined to primary and secondary spermatocytes and early spermatids, whereas Sertoli cells were negative. We were unable to demonstrate the presence of functional ET receptors in ejaculated spermatozoa. Because ET-like immunoreactivity was present in Sertoli cells, we next asked whether authentic ET-1 is present in human seminal fluid and represents a good index for Sertoli cell function. Reverse-phase high-performance liquid chromatography analysis of ET-like immunoreactivity in seminal fluid indicated that most of the detected peptides correspond to the ET-1 precursor, big-ET-1. The seminal concentration of ET-like immunoreactivity was similar in normospermic, oligospermic, azoospermic, and vasectomized men, indicating that ETs are produced in different parts of the male genital tract and that they do not represent an useful tool for the diagnosis of male reproductive diseases. In conclusion, this study demonstrated, for the first time, the presence of ET-1 and its receptors in human testis.

Adult↗