Search PubMed⌕ Search

Biomedical subjects

M Rossato

Publications and source records attributed to M Rossato.

At least 55 records · Page 3Linked to original sources
PubMed1996

Involvement of osmo-sensitive calcium influx in human sperm activation.

Mammalian spermatozoa must undergo capacitation and acrosome reaction before fertilization. To date, the precise mechanisms regulating these complex processes are not well understood but it is generally agreed that they involve an influx of calcium from the extracellular space through, as yet, poorly characterized plasma membrane pathways. Here we present evidence for a novel mechanism to increase intracellular calcium concentration via a calcium influx pathway activated by sperm cell swelling. Activation of this influx pathway by a mild hypo-osmotic shock and the ensuing calcium rise are a potent stimulus for sperm acrosome reaction. Furthermore, hypo-osmolarity-activated spermatozoa are fully competent for oocyte fertilization. During transit along male and, after ejaculation, female genital tracts spermatozoa are known to be exposed to extracellular fluids of widely different osmolarity; thus osmo-sensitive calcium influx could have a crucial regulatory role in the cellular events preceding fertilization.

Acrosome↗
PubMed1996

Congenital hypertrophy of the retinal pigment epithelium (CHRPE) and familial adenomatous polyposis (FAP).

Familial adenomatous polyposis (FAP) is a genetically transmitted disease affecting the colon. It is characterized by the presence of several (at least 100) adenomatous polyps, each able to develop into carcinomas, and by other extra-colonic signs such as skin and bone lesions. Within the framework of research studies to identify phenotyphic markers for early detection of subjects at risk within a family affected by FAP, attention has recently been paid to congenital hypertrophy of the retinal pigment ephitelium (CHRPE). With the aim of evaluating the relationship between FAP and CHRPE, 36 members of 7 FAP families were examined. We found that 43.75% of the subjects presenting CHRPE areas were also affected by FAP, whereas 58.33% of patients affected by FAP had CHRPE. Our findings indicated a lower incidence of CHRPE in FAP patients, compared to other studies reported so far. Moreover, in a control group of 160 healthy individuals we found a CHRPE prevalence of 5.5%.

Adenomatous Polyposis Coli↗
PubMed1996

Mechanism of human sperm activation by extracellular ATP.

We have identified the mechanism whereby extracellular ATP (ATPe) triggers the acrosome reaction in human spermatozoa. This nucleotide opens a ligand-gated ion channel expressed on the sperm plasma membrane. ATPe threshold and 50% effective concentration calculated on the total added ATPe are 0.1 and 2 mM, respectively, corresponding to a free ATP concentration (ATP4-) of 3 and 200 microM, respectively. The ATPe-gated channel is selective for monovalent cations (Na+, choline, and methylglucamine), whereas on the contrary, permeability to Ca2+ is negligible. Isosmolar replacement of extracellular Na+ with sucrose fully blocked ATPe-dependent sperm activation, thus suggesting a mandatory role for Na+ influx. These results show that human sperm express an ATPe-gated Na+ channel that might have an important role in sperm activation before egg fertilization.

Acrosome↗
PubMed1996

Gamma Knife stereotactic radiosurgery for uveal melanoma: clinical results after 2 years.

We report on 36 cases of uveal melanoma treated at our center between March 1993 and September 1995. There were 16 men and 20 women, aged 57 +/- 11 years. The choroid was affected in 35 patients and the ciliary-body in 1. The same preoperative and follow-up protocol was adopted for all cases. The procedure included fixation and positioning of the eye with a retrobulbar injection of long-lasting anesthetic and two extraocular muscle sutures, application of the frame, computed tomography scan localization, dose planning and treatment with the Gamma Knife. The patients were divided into three groups. Group A: 10 patients with a follow-up of 24 +/- 4 months, treated with a high dose (surface dose 58 +/- 9 Gy, maximum dose 81 +/- 15 Gy, mean dose 66 +/- 11 Gy). Group B: 9 patients with a follow-up of 16 +/- 2 months, treated with a lower dose (surface dose 41 +/- 3 Gy, maximum dose 76 +/- 10 Gy, mean dose 53 +/- 11 Gy). Group C: 17 patients with a follow-up of 6 +/- 3 months, treated with a lower dose (surface dose 42 +/- 3 Gy, maximum dose 72 +/- 16 Gy, mean dose: 54 +/- 6 Gy). In group A, we observed marked tumor regression in 9 cases, tumor recurrence in 1 case and severe complications in 5 cases (neovascular glaucoma and/or radiation retinopathy and/or radiation optic neuropathy). In group B, significant local control of the tumor was obtained with minor complications (cotton wool spots hard exudates, intraretinal hemorrhages). In group C, to date we have observed a regression of the tumor in 7 cases and 1 severe complication (neovascular glaucoma). Our data show that uveal melanomas may be adequately controlled by a high radiosurgical dosage (50-70 Gy), though there are significant side effects. Comparable levels of local tumor control may be obtainable using lower doses (40-45 Gy) which would hopefully reduce the rate of complications. However, a longer follow-up is needed for further validation of these results.

Aged↗
PubMed1995

Extracellular ATP activates different signalling pathways in rat Sertoli cells.

1. The present study describes effects of extracellular ATP (ATPe) on plasma membrane potential and cytoplasmic Ca2+ concentrations ([Ca2+]i) in rat Sertoli cells. Sertoli cells in suspension were stimulated with ATPe and other nucleotides and ionic changes were monitored utilizing the fluorescent dyes bis-oxonol and fura-2/AM. ATPe induced a prompt plasma membrane depolarization which was dependent on Na+ influx from the extracellular medium, since it was abolished by omission of extracellular Na+. Depolarization was independent of [Ca2+]i rise as it also occurred in the absence of extracellular Ca2+ and after intracellular Ca2+ stores were discharged with thapsigargin. ATPe also stimulated a rapid and biphasic increase in [Ca2+]i: a prompt spike was followed by a prolonged sustained plateau. The initial spike was dependent on Ca2+ release from intracellular stores since it was also present when cells were incubated in EGTA-supplemented Ca(2+)-free medium and was abolished by pretreatment with ionomycin and thapsigargin, agents that discharge intracellular Ca2+ stores. The sustained phase was dependent on Ca2+ influx from the extracellular medium as it was abolished when cells were incubated in EGTA-supplemented Ca(2+)-free medium. Ca2+ influx was due to activation of voltage-operated calcium channels (VOCCs) since it was abolished by the VOCC inhibitors verapamil and nifedipine or incubation in sucrose medium, an experimental condition which precludes plasma membrane depolarization by ATPe. 2. ATPe-induced rises in intracellular Ca2+ concentration and plasma membrane depolarization were reduced by pretreatment with pertussis toxin, suggesting that ATPe-activated transduction mechanisms are in part under the control of pertussis toxin-sensitive G-proteins. These data show that Sertoli cells possess P2-purinergic receptor subtypes coupled to influx of Na+ and release of Ca2+ from intracellular stores and provide evidence for an activation of different pathways by extracellular ATPe. Activation of these receptors induces Na+ influx that causes a rapid plasma membrane depolarization. Furthermore, ATPe also triggers Ca2+ release from intracellular stores and Ca2+ influx from extracellular space via dihydropyridine-sensitive VOCCs.

Adenosine Triphosphate↗
PubMed1995

Differential modulation by protein kinase C of progesterone-activated responses in human sperm.

Progesterone exerts important effects on human spermatozoa by rapid non genomic mechanisms of action. It has been demonstrated that processes triggered by this steroid are dependent on the activation of calcium influx through the plasma membrane. Beside calcium, progesterone also induces a rapid plasma membrane depolarization that is dependent on an influx of sodium through a putative progesterone-activated channel located on the plasma membrane. In this study we show that protein-kinase C inhibition inhibits calcium influx activated by progesterone, while leaving the depolarizing effect of this steroid unchanged. These results may be explained by the existence of two progesterone receptors on human sperm plasma membrane, one responsible for calcium influx and modulated by protein-kinase C and the other selectively permeable to sodium that is not under protein-kinase C control. Alternatively, protein-kinase C inhibition might change ion selectively of a single progesterone-activated channel, thus decreasing calcium permeability, while leaving sodium permeability unchanged.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Diagnostic and clinical features in azoospermia.

BACKGROUND AND OBJECTIVE: The recent advances in assisted fertilization and gamete micromanipulation techniques have enabled fertilization in some forms of azoospermia; for example, epididymal sperm aspiration in obstructive azoospermia. Therefore knowledge of the specific degree of testicular damage is of primary importance, since other clinical parameters, such as FSH plasma levels and testicular volume, do not discriminate between the different testiculopathies. In order to further characterize the specific testicular conditions present in azoospermia, we have examined a large group of azoospermic subjects on the basis of testicular cytological analysis obtained by fine needle aspiration. DESIGN AND PATIENTS: One hundred and twenty-two infertile, azoospermic men were studied by physical examination, FSH radioimmunoassay, testicular ultrasound examination and fine needle aspiration of the testes. Thirty-five infertile normozoospermic subjects were studied as controls. RESULTS: The cytological analysis identified five different sub-types in azoospermic subjects: I, Sertoli cell-only syndrome; II, hypospermatogenesis; III, spermatogonial and/or spermatocytic arrest; IV, spermatidic arrest; and V, normal germ line. The testicular volume was reduced in groups I and II, while the FSH plasma levels were increased in groups I, II and III, suggesting a primary role of spermatids in the control of FSH secretion. CONCLUSIONS: In azoospermic subjects, testicular cytological analysis permits the identification of different sub-types and this classification may be very important in determining therapy, particularly the choice between surgical treatment and the hypothetical use of assisted fertilization techniques by retrieval of epididymal or intratesticular spermatozoa or spermatids.

Adult↗
PubMed1995

Functional and structural characteristics of human epididymal spermatozoa retrieved by transcutaneous aspiration.

The present paper describes the development of a simple, repeatable technique for transcutaneous aspiration of epididymal spermatozoa in patients with inoperable excretory azoospermia. Some functional and structural parameters of the retrieved spermatozoa were also evaluated. Epididymal spermatozoa from patients and ejaculated spermatozoa from fertile donors showed similar percentages of acrosome-reacted cells after incubation for 6 or 15 h in capacitating medium. Penetration of zona-free hamster oocytes, expressed as the percentage of penetrated ova, was significantly lower (p < 0.01) for spermatozoa from patients than for ejaculated spermatozoa from fertile donors. The percentage of epididymal spermatozoa with stable nuclei in sodium dodecyl sulphate was lower (p < 0.001) than for ejaculated spermatozoa from fertile donors. A positive linear correlation (r = 0.74, p < 0.005) was evident between the oocyte penetration rate and the percentage of spermatozoa with stable nuclei. In conclusion the percutaneous retrieval of epididymal spermatozoa in excretory azoospermia allows collection of motile spermatozoa, which can be evaluated for their structural and functional characteristics before repetition of the technique for retrieval of spermatozoa for assisted fertilization techniques.

Adult↗
PubMed1995

Erythropoietin and testicular steroidogenesis: the role of second messengers.

It has been demonstrated that erythropoietin (EPO) influences rat and human Leydig cell steroidogenesis, stimulating testosterone production through a direct and specific receptor-mediated mechanism. The aim of this study was to investigate the mechanism by which recombinant human erythropoietin (rHuEPO) exerts its stimulatory effect on rat Leydig cells. Recombinant human EPO did not induce, at any dose tested (10(-10) to 10(-13) mol/l), an increase in either cAMP or cGMP, suggesting that in Leydig cells the effect of rHuEPO does not involve the adenylate or guanylate-cyclase systems. The role of transmembrane calcium flux in rHuEPO-stimulated steroidogenesis was studied by evaluating the effect of calcium channel blocker, verapamil, and by the 45Ca2+ uptake method. Verapamil did not influence rHuEPO-induced testosterone secretion and rHuEPO did not modify calcium recycling, indicating that calcium transmembrane flux is not involved in the rHuEPO effect. The protein kinase C inhibitor staurosporine (10, 30, 100 and 300 nmol/l) inhibited rHuEPO-stimulated testicular steroidogenesis in a dose-dependent manner. This indirect evidence suggests that the stimulatory effect of rHuEPO on rat Leydig cells may involve protein kinase C activation.

Alkaloids↗
PubMed1993

Ion fluxes through the progesterone-activated channel of the sperm plasma membrane.

We have characterized ionic changes triggered by progesterone in human spermatozoa. This steroid, which is a fast-acting stimulator of the acrosome reaction, triggered a rapid increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) which was entirely due to influx across the plasma membrane, as it was obliterated by chelation of extracellular Ca2+. Ca2+ fluxes were insensitive to verapamil and pertussis toxin, thus suggesting that they did not occur via voltage-gated channels and did not involve a pertussis toxin-sensitive G protein, and were potentiated in Na(+)-free, choline-containing or methylglucamine-containing medium. Progesterone also caused a depolarization of the plasma membrane in Na(+)-containing as well as in choline- or methyl-glucamine-containing saline; depolarization was larger in the absence of extracellular Ca2+, suggesting that Na+ and Ca2+ fluxes occurred through the same channel. Progesterone was able to trigger the acrosome reaction in the three media investigated (Na+, choline and methylglucamine), provided that extracellular Ca2+ was also present. We conclude that progesterone activates a membrane ion channel that is permeable to monovalent cations as well as to Ca2+.

Biological Transport↗
PubMed1992

Extracellular ATP is a trigger for the acrosome reaction in human spermatozoa.

We tested the effect of extracellular adenosine 5'-triphosphate (ATPo) on the activation of human spermatozoa. ATPo, in a concentration range from 50 microM to 5 mM, induced the acrosome reaction, which, at the optimal concentration of 2.5 mM, was maximal (30-35% of spermatozoa activated) within 60 min of the addition of the nucleotide. At the end of this incubation in the presence of ATPo no decrease in cell motility and viability was observed. Among other purine/pyrimidine nucleotides only the ATP analogue adeny-5'-lyl imidodiphosphate was effective (70% of ATP); a weak (10% of ATP) effect was also observed with CTP and the ATP analogues adenosine 5'-(beta gamma-methylene)triphosphate and adenosine 5'-O-(thiotriphosphate). ATPo did not cause Ca2+ release from intracellular stores, nor it caused Ca2+ influx from the extracellular milieu; on the contrary, it caused a clear, albeit slow, plasma membrane depolarization. ATPo-activated spermatozoa showed a nearly 100% success rate in the standard hamster egg fertilization test. Our results describe a new effect of ATPo in human spermatozoa with relevant potential applications in fertility studies.

Acrosome↗
PubMed1992

Sperm nuclear instability and staining with aniline blue: abnormal persistence of histones in spermatozoa in infertile men.

During mammalian spermiogenesis, replacement of the somatic histones by basic proteins, the protamines, allows normal sperm nuclear condensation. In this study we have evaluated the degree of chromatin compaction in spermatozoa from 191 infertile subjects, affected by different testicular disorders, compare with that in 50 fertile sperm donors (controls). In infertile men, there was a higher percentage of unstable spermatozoa after incubation with sodium dodecyl sulphate (SDS) and of stained spermatozoa after staining with aniline blue (P less than 0.001 vs. controls). Furthermore, a positive linear correlation was found between SDS-unstable spermatozoa and stained spermatozoa (P less than 0.001), suggesting that sperm instability was related to a defect in histone-replacement by sperm-specific nucleoproteins, protamines. When the patients were considered according to pathology, high sperm nuclear instability and a high percentage of stained spermatozoa were detected in groups affected by varicocele, idiopathic infertility and in patients with a history of unilateral cryptorchidism. In the latter group the same alterations were observed even when the cryptorchid testis had been removed during surgery. In the group with a past history of mumps orchitis these parameters did not show any difference when compared with controls.

Adult↗
PubMed1991

Evidence for the involvement of sperm angiotensin converting enzyme in fertilization.

Recently it has been observed that ejaculated human sperm possess high angiotensin converting enzyme (ACE) activity and that this enzyme is released during the process of capacitation. This observation raises the possibility that ACE may be involved in the fertilization process. To verify this hypothesis, we tested the effects of a potent ACE inhibitor, Captopril, on acrosome reaction induced by capacitating medium (3.5% HSA-added BWW) and on ability of human capacitated spermatozoa to penetrate zona-free hamster oocytes. Addition of Captopril (100 nmol l-1) modified neither sperm motility nor viability at any time considered, but significantly reduced the acrosome reaction percentages of sperm incubated in capacitating medium. Furthermore, Captopril significantly reduced the percentage of penetrated oocytes. The mean penetration rates both in the absence and presence of Captopril were 65.5 +/- 4.9% and 26.9 +/- 2.3% (P less than 0.001) respectively. These findings provide evidence that sperm release of ACE during capacitation may have a physiological role in the regulation of the mechanisms that allow sperm acrosome reaction and thus fertilizability.

Acrosome↗
PubMed1990

Quantitative analysis of trace elements in human clear cell carcinoma of the kidney by energy-dispersive X-ray fluorescence.

In a case-control study, 20 cases of renal cell carcinoma (RCC) were analyzed by energy-dispersive X-ray fluorescence in order to establish the concentration of Fe, Cu, Zn and Cd. Patients with RCC were examined and compared with 7 controls from selected autopsies. A significant decrease in Cd and Zn concentration was found in the neoplastic tissue in all cases. In contrast, no significant decrease in Cu concentration was detected in our cases.

Aged↗
PubMedDate not supplied

Identification of functional binding sites for progesterone in rat Leydig cell plasma membrane.

Steroid hormones influence cell functions by binding to intracellular receptors and then acting within the nucleus. There is now evidence that steroids affect cell functions also via interaction with plasma membrane receptors in a number of different cell types. In this regard, progesterone appears to be one of the most active steroids. In this paper, we evaluate the effects of progesterone on rat Leydig cell functions, determining variations of ion homeostasis and testosterone production. This steroid was able to effect a depolarization of the plasma membrane that was due to an influx of sodium (Na+) from the external medium since it was absent when extracellular Na+ was iso-osmotically substituted with choline chloride or sucrose. The determination of intracellular sodium concentration ([Na+]i) with the Na+ -sensitive fluorescent dye sodium-benzofuran-isophtalate (SBFI) confirmed these observations. Progesterone did not modify Leydig cell intracellular calcium concentration ([Ca2+]i) at any dose tested. Furthermore, using a cell impermeant progesterone conjugate, we demonstrated that progesterone was able to stimulate Leydig cell steroidogenesis in a dose-dependent manner. The exclusion of calcium (Ca2+) from the extracellular medium did not modify the depolarizing action of progesterone and its steroidogenetic effect while in Na+ -free medium (sucrose supplemented) progesterone-stimulated effects were completely blunted. Finally, using fluorescence microscopy with a fluorescein isothiocyanate-coupled cell impermeant progesterone conjugate, we identified plasma membrane binding sites for progesterone in rat Leydig cells. These results suggest that rat Leydig cells possess progesterone receptors located on the plasma membrane, which when occupied achieves a plasma membrane depolarization, dependent on an influx of Na+ from the external medium, and the subsequent activation of steroidogenesis.

Animals↗