Warning note on male infertility treatment.
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Cytoplasm↗
Biomedical subjects
C Foresta
Publications and source records attributed to C Foresta.
Capacitative calcium entry in rat Sertoli cells.
Depletion of intracellular Ca2+ stores activates an influx of Ca2+ from the extracellular medium. This capacitative Ca2+ entry as originally proposed by Putney in 1986 can be studied with drugs that inhibit sarco/endoplasmic reticulum ATPase. In the present study we examined the effects of depletion of internal Ca2+ stores on Ca2+ influx in rat Sertoli cells utilizing thapsigargin and cyclopiazonic acid. Both inhibitors induced a rapid and dose-dependent rise in [Ca2+]i that was dependent on an influx of Ca2+ from the extracellular medium since it was rapidly blocked by the addition of the Ca2+ chelating agent EGTA. In the absence of external Ca2+ thapsigargin and cyclopiazonic acid still produced an increase in [Ca2+]i that was lower than that observed in Ca2+ medium and was transient since [Ca2+]i returned to basal levels by few minutes. In these experimental conditions readdition of Ca2+ induced a rapid rise in [Ca2+]i supporting a role for Ca2+ influx. Increase of plasma membrane permeability to Ca2+ induced by thapsigargin and cyclopiazonic acid were confirmed by the ability of Mn2+ to permeate through Ca2+ channels and to quench intracellular fura-2 fluorescence after challenge with these inhibitors. Mn2+ induced influx was blocked by La3+, a well known blocker of Ca2+ channels. These results demonstrate that internal Ca2+ stores depletion induce Ca2+ influx from the extracellular medium in rat Sertoli cells providing evidence for the existence of capacitative Ca2+ entry in these cells.
Animals↗
Functional and cytologic features of the contralateral testis in cryptorchidism.
OBJECTIVE: To characterize the testicular cytologic pictures in cryptorchild and contralateral testis related to seminal pattern. DESIGN: Controlled clinical study. SETTING: Andrological and urologic academic setting. PATIENTS: One hundred nine patients orchidopexied because of unilateral cryptorchidism compared with 35 normospermic subjects. INTERVENTIONS: Bilateral testicular fine-needle aspiration cytology. MAIN OUTCOME MEASURES: Seminal parameters; testicular cytologic features; plasma levels of FSH, LH, and T; and ultrasound testicular examination. RESULTS: The cytologic analysis revealed an important quantitative and qualitative impairment of spermatogenic line in all the excryptorchid testes. These alterations are present in the contralateral testes only when azoospermia or severe oligozoospermia were observed, whereas in moderate oligozoospermic subjects a normal tubular status was evident. A compensatory activity in normally descended testis of normozoospermic patients was present. CONCLUSIONS: In unilateral cryptorchidism, a frequent testicular damage in the orchidopexied and in the contralateral descended testis suggests that this condition is the end point of different pathological conditions, including testicular intrinsic (congenital) and extrinsic (anatomical) causes. Fine-needle aspiration cytology of both testes represents a tool in the assessment of the tubular status in excryptorchid subjects.
Adolescent↗
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↗
Male infertility and ICSI: are there limits?
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Chromosome Aberrations↗
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↗
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↗
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↗
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↗
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↗
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↗
Caution in use of interferon for male infertility.
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Humans↗
Fine needle testicular biopsy--one or more punctures?
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Biopsy, Needle↗
Immunocytochemical localization of epidermal growth factor receptors in human testis from infertile subjects.
OBJECTIVE: To investigate the immunolocalization of the epidermal growth factor receptor (EGFR) in normal and pathological human testis by immunocytochemical technique. DESIGN: Cytologic specimens were obtained by bilateral fine needle aspiration (FNA) of the testis and stained in May Grünwald-Giemsa for the cytologic analysis; immunolocalization of EGFR was analyzed on duplicate slides from each testis using two anti-EGFR monoclonal antibodies and peroxidase-antiperoxidase technique. SETTING: Infertility center of an academic unit. PATIENTS: A total of 42 infertile patients, affected by various testicular diseases. The control group was made up of 10 normal sperm patients with autoimmune infertility and cytologic picture of normal spermatogenesis. INTERVENTIONS: Exogenous FSH was administered 75 IU IM on alternate days for 3 months on 16 of the infertile patients who showed oligospermia and normal FSH plasma levels. Semen analysis and testicular FNA (and after cytologic and immunocytochemical studies) were repeated at 3 months of treatment. MAIN OUTCOME MEASURES: Luteinizing hormone and FSH plasma levels were determined by RIA methods; qualitative and quantitative parameters for the cytologic evaluation are reported in our previous works. RESULTS: The cytologic analysis permitted identification of seven classes of infertile subjects, characterized by different cytologic pictures. Epidermal growth factor receptor immunostaining evidenced weak positivity on Sertoli and germ cells (with the exception of spermatozoa) in the presence of normal germ line and normal FSH plasma levels and strongly intense positivity in the presence of serious hypospermatogenesis, spermatogonial or spermatocytic arrest, and Sertoli cell-only syndrome. These conditions were characterized by higher FSH plasma levels than normal controls. All of the subjects who received exogenous FSH, with moderate hypospermatogenesis or spermatidic arrest, showed on Sertoli and germ cells a weak EGFR immunostaining before the treatment and intense immunostaining after the treatment. CONCLUSIONS: These results confirm recent demonstrations of EGFRs in human testis and evidence different EGFR immunostaining in the presence of various degrees of testicular damage, suggesting a role of this growth factor in growth and differentiation of the germ cells throughout spermatogenesis. The observation that intense EGFR immunostaining was found in subjects showing high FSH plasma levels and in all of the patients who received exogenous FSH, supports a possible role of this gonadotropin in the modulation of the EGFR expression.
Adult↗
Erythropoietin stimulates testosterone production in man.
Human recombinant erythropoietin (rHuEPO) treatment improves sexual function in end-stage renal failure patients with a still-debated mechanism. Experimental data suggested that rHuEPO was able to stimulate rat Leydig steroidogenesis; therefore, it has been suggested that rHuEPO may induce its effects in humans by acting on gonadal steroid production. Thirteen young adult males (age range, 16-28 yr) catheterized at peripheral and left internal spermatic venous levels during a contrast study for varicocele, were studied. In five subjects, rHuEPO (60 IU/kg, up to a maximum of 4000 IU total) was injected over 1 min in the cubital vein. Similarly, in other five patients, 50 micrograms GnRH were infused. In three subjects, 2 mL saline were injected, as controls. Plasma LH, FSH, and testosterone (T) levels were then determined at -15, 0, 15, 30, 45, 60, 90, and 120 min simultaneously in peripheral and spermatic venous blood. rHuEPO infusion did not have any effect on plasma LH and FSH levels in peripheral or spermatic veins. Similarly, rHuEPO infusion did not affect peripheral T concentration, but increased (approximately 400% vs. controls; P < 0.05) spermatic T levels. GnRH infusion induced an increase in plasma LH and FSH levels in both peripheral and spermatic veins. After GnRH infusion, an increase of approximately 12-fold (P = 0.05-0.001) in T was observed only at the spermatic venous level, without any peripheral T variation. These findings show that rHuEPO was able to influence testicular steroidogenesis by stimulating T production in man, whereas the absence of any effect on gonadotropin secretion suggests that rHuEPO might act directly on human Leydig cell function.
Adolescent↗
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↗
Simvastatin influences testicular steroidogenesis in human.
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Adult↗
The role of calcium ions in rat Leydig cell steroidogenesis induced by atrial natriuretic peptide.
The aim of this study was to clarify whether calcium ions play a role in the mechanisms by which rat atriopeptin II exerts its stimulatory effects on rat Leydig cell steroidogenesis. The absence of calcium in the medium and the calcium channel-blocker drug, verapamil, both significantly inhibited testosterone production in rat atriopeptin II-treated rat Leydig cells. Similar effects were observed on human chorionic gonadotropin-stimulated Leydig cells. Furthermore, 8-Bromoguanosine 3':5' cyclic monophosphate, the putative second messenger of atrial natriuretic peptide, significantly increased rat Leydig cell steroidogenesis, determining a stimulatory effect comparable to that obtained with rat atriopeptin II. Calcium-free medium and verapamil inhibited this stimulatory effect. The addition of a calcium ionophore. A-23187, significantly counteracted the inhibitory effect of verapamil on rat atriopeptin II-induced and 8-Bromoguanosine 3':5' monophosphate-induced testosterone production. Furthermore, rat atriopeptin II significantly stimulated the calcium flux through the plasma membrane, as evaluated by the 45Ca2+ uptake method. This effect was strongly inhibited by the addition of verapamil. These data underline that calcium ions may play a role in the mechanisms by which atriopeptin II stimulates rat Leydig steroidogenesis, although other post-receptor systems cannot be excluded.
Analysis of Variance↗