Biomedical subjects

M Luconi

Publications and source records attributed to M Luconi.

At least 19 recordsLinked to original sources

PubMed2005

Peripheral regulatory mechanisms in erection.

The most important pathway underlying the penile erection is the nonadrenergic/noncholinergic signalling, which through the release of nitric oxide (NO), leads to an intracellular increase of cyclic GMP (cGMP), the main secondary messenger mediating tumescence in the penis. Interestingly, both cGMP formation and degradation are affected by testosterone (T). In fact, beyond the well-known role of T in regulating sexual desire and NO release, recent experimental evidences from our group showed that T also regulates the expression of phosphodiesterase type 5 (PDE5), the hydrolytic enzyme involved in cGMP breakdown. This antithetic role of T seems to be the main way through which the peripheral hormonal regulation of penile erections occurs, allowing an important synchronization between erectile processes and sexual desire. Hence, erections are still possible in hypogonadal conditions where a decreased cGMP formation, because of impaired NO production, is counterbalanced by a reduced cGMP hydrolysis. The purpose of this review is to describe evidences about the peripheral role of T in regulating penile erection and to justify the importance to test T plasma levels in those patients with erectile dysfunction who do not respond to PDE5 inhibitors.

Animals↗

PubMed2005

Identification, localization and functional in vitro and in vivo activity of oxytocin receptor in the rat penis.

We recently found that the oxytocin receptor (OTR) is expressed in the human and rabbit corpus cavernosum and mediates contractility in vitro. The present study extended our investigations to the rat, and explored whether OTR regulates penile detumescence in vivo. Real-time RT-PCR quantitatively characterized the distribution of OTR mRNA in the male genital tract. Specific transcripts for OTR were expressed in all the tissues investigated. Penile expression of OTR was comparable to that observed in testis and prostate. Western blot analysis detected a single band of the expected molecular mass for OTR in all tissues examined, including rat penis. Expression of OTR protein in rat penile extracts was further confirmed by binding studies, using the OTR selective radiolabeled ligand 125I-OTA (K(d) = 17 +/- 6.5 pM, B(max)=15.7 +/- 5 fmoles/mg protein). OTR was immunolocalized to the endothelial and smooth muscle compartments of cavernous spaces and blood vessels. In rat corpus cavernosum strips, oxytocin (OT) and an OTR selective agonist ([Thr4,Gly7]OT) induced identical increases in tension, while different vasopressin agonists were less active. In vivo, OT intra-cavernous injection (ICI) dose-dependently inhibited intracavernous pressure (ICP) increase elicited by either electrical stimulation of the cavernous nerve or ICI of papaverine with similar IC(50)s (117.7 +/- 37 mU). The OTR antagonist, atosiban, counteracted the contractile effect of OT both in vitro and in vivo. Atosiban alone significantly increased ICP at lower stimulation frequencies (2 Hz = P<0.001 and 4 Hz = P<0.05 vs control), but not at the maximal frequency (16 Hz). Our data showed that OTR is present in the rat penis and mediates contractility both in vitro and in vivo, therefore suggesting a role for OT in maintaining penile detumescence.

Animals↗

PubMed2004

Tyrosine phosphorylation of the a kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate.

Mammalian testicular spermatozoa are immotile, thus, to reach the oocyte, they need to acquire swimming ability under the control of different factors acting during the sperm transit through the epididymis and the female genital tract. Although bicarbonate is known to physiologically increase motility by stimulating soluble adenylate cyclase (sAC) activity of mammalian spermatozoa, no extensive studies in human sperm have been performed yet to elucidate the additional molecular mechanisms involved. In this light, we investigated the effect of in vitro addition of bicarbonate to human spermatozoa on the main intracellular signaling pathways involved in regulation of motility, namely, intracellular cAMP production and protein tyrosine phosphorylation. Bicarbonate effects were compared with those of the phosphatidyl-inositol-3 kinase inhibitor, LY294002, previously demonstrated to be a pharmacological stimulus for sperm motility. Bicarbonate addition to spermatozoa results in a significant increase in sperm motility as well as in several hyperactivation parameters. This stimulatory effect of bicarbonate and LY294002 is mediated by an increase in cAMP production and tyrosine phosphorylation of the A kinase anchoring protein, AKAP3. The specificity of bicarbonate effects was confirmed by inhibition with 4,4'-di-isothiocyanostilbene-2,2'-disulfonic acid. We remark that, in human spermatozoa, bicarbonate acts primarily through activation of sAC to stimulate tyrosine phosphorylation of AKAP3 and sperm motility because both effects are blunted by the sAC inhibitor 2OH-estradiol. In conclusion, our data provide the first evidence that bicarbonate stimulates human sperm motility and hyperactivation through activation of sAC and tyrosine phosphorylation of AKAP3, finally leading to an increased recruitment of PKA to AKAP3.

A Kinase Anchor Proteins↗

PubMed2004

Human spermatozoa as a model for studying membrane receptors mediating rapid nongenomic effects of progesterone and estrogens.

In the past few years, besides the classical genomic effects of steroid hormones, a plethora of so called rapid non genomic effects have been described in different cell types, which are too rapid to be due to activation of gene expression. Although some of these effects might involve the same nuclear steroid receptors acting on different cellular signalling, others have been ascribed to poorly characterized membrane receptors. Several rapid nongenomic effects of progesterone (P) and estrogens (E) have been recently demonstrated in human spermatozoa. They seem to be mediated by the steroid binding to specific receptors on plasma membrane different from the classical ones. In particular, P has been demonstrated to stimulate calcium influx, tyrosine phosphorylation of sperm proteins, including extracellular signaling regulated kinases, chloride efflux and cAMP increase, finally resulting in activation of spermatozoa through induction of capacitation, hyperactivated motility and acrosome reaction. Conversely, E, by acting rapidly on calcium influx and on protein tyrosine phosphorylation, seem to modulate sperm responsiveness to P. Several attempts have been used to characterize the putative membrane receptors for P (mPR) and E (mER) in spermatozoa, however their isolation still remains elusive. However, in the past few years our laboratory has obtained several evidences supporting the existence and functional activity of mPR and mER in human spermatozoa. To characterize these membrane receptors, we used two antibodies directed against the ligand binding domains of the classical receptors, namely c262 and H222 antibodies for PR and ER respectively, hypothesizing that these regions should be conserved between nongenomic and genomic receptors. In western blot analysis of sperm lysates the antibodies detected a band of about 57 kDa for PR and of 29 kDa for ER, excluding the presence of the classical receptors. On live human spermatozoa, both antibodies were able to block the calcium and AR response to P and E respectively, whereas, antibodies directed against different domains of the classical PR and ER were ineffective. Moreover, c262 antibody also blocks in vitro human sperm penetration of hamster oocytes. Taken together all these data strongly support the existence of mPR and mER different from the classical ones, mediating rapid effects of these steroid hormones in human spermatozoa.

Animals↗

PubMed2004

Phosphatidylinositol 3-kinase inhibition enhances human sperm motility and sperm-zona pellucida binding.

Various signalling pathways are involved in the regulation of sperm motility, capacitation, acrosome reaction and sperm-zona binding. Recent data pointed out an important role for phosphatidylinositol 3-kinase (PI3K) in human sperm motility. However, no study as of yet has been carried out to determine the effect of sperm treatment with the PI3K inhibitor LY294002 on other sperm parameters. In the present study, we investigated the role of PI3K on human sperm motility, acrosome reaction and sperm-oocyte binding by using this inhibitor. We demonstrate that in vitro incubation of washed unselected spermatozoa with LY294002 increased the percentage motility and progressive motility in asthenozoospermia patients as evaluated by computer-aided sperm analysis. The compound furthermore did not influence the acrosome reaction, whilst it (further) slightly enhanced sperm-oocyte binding. Our results therefore imply that PI3K negatively affects sperm motility and oocyte binding and might suggest a possible therapeutic role for PI3K inhibitors in the treatment regime for asthenozoospermia.

Acrosome Reaction↗

PubMed2003

Effects of hypoxia on endothelin-1 sensitivity in the corpus cavernosum.

The penis remains in a hypo-oxygenated, flaccid state for a large majority of the time. In this study, we investigated the effect of changing oxygen tension on the expression and functional activity of endothelin-1 (ET-1) receptors in the penis. Experiments were performed in rabbit and human corpora cavernosa (CC) as well as in human fetal penile tissue and cell cultures [human fetal penile endothelial cells (hfPECs) and human fetal smooth muscle cells (hfPSMCs)]. Endothelin A (ETA) receptors are expressed by both endothelial and muscular cells in all tissues investigated. Only penile endothelial cells express endothelin B (ETB) receptors, which are further turned on during experimental hypoxia. In addition, hypoxia also allows ETB expression in the muscular compartment without affecting ETA expression. This hypoxia-induced over-expression of ETB decreased the contractile activity of ET-1 and increased ETB-mediated relaxation. The latter was essentially related to increased ETB-mediated nitric oxide formation in hfPEC and even in hfPSMC. Hypoxia also induced a time-dependent down-regulation of RhoA and Rho kinase (ROK) expression which, in turn, participated in the decreased contractile activity of ET-1 in the hypoxic penile tissue. Accordingly, during hypoxia, an ROK inhibitor, Y27632, was less effective in relaxing ET-1-precontracted strips. In conclusion, prolonged (24 h) hypoxia stimulated several counter-regulatory mechanisms in penile tissue, including up-regulation of ETB and down-regulation of RhoA/ROK pathways, which may help to preserve CC hypo-oxygenation, allowing smooth muscle relaxation and, most probably, penile erection.

Animals↗

PubMed2003

Expression of functional estrogen receptors in human fetal male external genitalia.

It is generally assumed that male genital development is determined by androgens on a default program leading to female genitalia. Female genitalia virilization is due to high levels of androgens, whereas feminization is linked to reduction or lack of fetal androgen. Excess androgen determines sex reversion in female, whereas excess estrogen does not cause male feminization. In the present study, we investigate the presence of androgen receptors (AR) and estrogen receptors (ER) in human fetal penile tissue and in a cellular model of human fetal penile smooth muscle cells (hfPSMC). By immunohistochemistry, we showed the presence of ER and AR in the developing penile tissue of male fetuses. Besides the presence of AR, hfPSMC showed ERalpha/beta as demonstrated by RT-PCR, Western blot, and binding techniques. These receptors are functionally active because cell stimulation with 17beta-estradiol increased progesterone receptor B expression and inhibited hfPSMC growth, both effects being reversed by tamoxifen. Conversely, cell proliferation was stimulated by R1881 and testosterone, an effect enhanced by letrozole. These findings are the first demonstration of the presence of functional ER in differentiating male external genitalia and indicate a possible novel inhibitory role of estrogens in the regulation of the development of these sex structures.

Aromatase Inhibitors↗

PubMed2003

How do sperm swim? Molecular mechanisms underlying sperm motility.

Motility is a characteristic function of the male gamete, which allows spermatozoa to actively reach and penetrate the female gamete in organisms with internal and external fertilization. Sperm motility is acquired under the control of many extrinsic and intrinsic factors and is based on the specialized structure of the sperm flagellum. After a brief overview of how the sperm flagellum is organized and works to support cell motility, the present review focuses on the molecular mechanisms and factors involved in the development and maintenance of sperm motility. Data obtained both in organisms with external fertilization, such as fishes and sea urchin, and with internal fertilization, such as Mammals, are critically analyzed. In particular, a great attention has been put on the ionic mechanisms and on the involvement of protein kinases and phosphatases in regulation of sperm motility. A brief overview of the pharmacological and physiological molecules which have been studied for their possible application as therapeutic molecules for in vitro treatment of defects of sperm motility in asthenozoospermic human subjects, is presented. Moreover, we show some preliminary data obtained in our laboratory on the involvement of the phosphatydilinositol 3-kinase and the A kinase anchoring protein (AKAP3) in regulation of motility in human spermatozoa. The last section is dedicated to hyperactivation, a peculiar pattern of motility which is developed in association with capacitation occurring during sperm transit through the female genital tract and which can also be obtained in vitro by incubation in defined media.

Animals↗

PubMed2002

Identification, localization and functional activity of oxytocin receptors in epididymis.

Oxytocin (OT) is a neurohypophysial hormone with unclear physiological functions in the male. Several previous studies indicated that OT might have a role in the ejaculatory process, stimulating sperm release from the epididymal storage. In this study we investigated on the presence and function of OT receptor (OTR) in rabbit and human epididymis. By using RT-PCR, Western and binding studies, we found that OTR gene and protein is expressed in the human epididymis and stimulates in vitro contractility. The immunolocalization of OTR suggests that the receptor is not only present in the smooth muscle cells of the human epididymis but also in the epithelial compartment. Experiments performed in rabbit epididymal epithelial (rEE) cells in culture indicate that OT induces the release of an other potent stimulator of epididymal contractility, endothelin-1 (ET-1), Blocking the ET(A) subtype of the ET-1 receptors, by using a specific antagonist (BQ-123), partially counteracts the contractile effect of OT, suggesting positive interactions between the two peptides in regulating epididymal contractility. Finally, to investigate whether an acute OT administration increases sperm release also in humans, we treated oligozoospermic patients with an intravenous bolus of OT (2.5 IU), just before sperm collection. In a small, single blind study, we found that OT almost doubled sperm retrieval when compared with vehicle administration. Our results indicate that OT might have physiological functions also in the male, controlling epididymal motility and sperm progression through the male genital tract.

Adult↗

PubMed2002

Endothelium-dependency of yohimbine-induced corpus cavernosum relaxation.

Development and maintenance of penile erection requires the relaxation of the smooth muscle cells in the cavernous bodies and is essentially mediated by nitric oxide (NO). The penile flaccid state is conversely maintained by the alpha adrenergic neuroeffector system and by other vasoconstrictors, such as endothelin-1 (ET-1). In this study we examined the mechanisms involved in yohimbine-induced relaxation in human and rabbit corpora cavernosa (CC). We essentially found that yohimbine not only blocks contractions induced by adrenergic agonists, but also by non-adrenergic substances, such as ET-1. This effect was unrelated to antagonism at the level of ET receptors, because yohimbine did not affect ET-1-induced increase in intracellular calcium in isolated CC cells. Conversely, our data suggest that yohimbine counteracts ET-1-induced contractions by interfering with NO release from the endothelium. In fact, yohimbine-induced CC relaxation was inhibited by the mechanical removing of the endothelium and by blocking NO formation or signalling via guanylate cyclase and cGMP formation. Conversely, yohimbine activity was strongly increased by inhibiting cGMP degradation. In an experimental model of hypogonadism, performed on rabbits by chronic treatment with a long-lasting GnRH agonist, the relaxant yohimbine activity was also decreased, but completely restored by androgen supplementation. This effect was evident only in preparations in which the main source of NO was present (endothelium) or in which NO formation was not impaired by L-NAME. Our data indicate that the relaxant effect of yohimbine is both endothelium and androgen-dependent. This might justify the lack of efficacy of this drug in treatment of some form of organic erectile dysfunction.

Adrenergic alpha-Agonists↗

PubMed2002

Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells.

We report for the first time that penile smooth muscle cells (SMC) not only respond to, but also synthesize, endothelin-1 (ET-1), one of the main regulators of SMC activity. Immunohistochemical studies indicated that, beside endothelial cells (EC), SMC of the human adult and fetal penis also express ET-1 and its converting enzyme, ECE-1. Accordingly, cultures of adult penile stromal cells express these genes. We also prepared and characterized penile SMC from human fetuses. These cells express SMC specific markers such as alpha smooth muscle actin and phosphodiesterase type 5A3 along with hallmarks of androgen-dependent cells (androgen receptor and 5alpha reductase type 2). Human fetal penile SMC (hfPSMC) are immunopositive for ET-1 and release ET-1. ET-1 expression in hfPSMC was strongly increased by several factors such as transforming growth factor-beta1 (TGF-beta1), interleukin-1alpha (IL-1alpha), ET-1 itself and prolonged (24 h) hypoxia. This latter condition not only affected ET-1 expression but also responsiveness. While at normal oxygen tension, hfPSMC responded to ET-1 with a decreased proliferation mediated by the endothelin-A receptors and TGF-beta1; however, during hypoxia, ET-1 stimulated cell growth. Accordingly, prolonged hypoxia up-regulated endothelin-B receptor mRNA expression. In conclusion, our results indicate that in penile tissues SMC produce ET-1 and that such production is modulated by factors involved in penile physiology and tissue remodelling. In addition, the hfPSMC we have characterized might be a useful model for studying biochemical aspects of the human erectile process in vitro.

Aspartic Acid Endopeptidases↗

PubMed2001

Effects of estrogenic compounds on human spermatozoa: evidence for interaction with a nongenomic receptor for estrogen on human sperm membrane.

Estrogens play an important role in the development and regulation of the male reproductive system. We have earlier shown that a nongenomic receptor for estradiol present on sperm plasma membrane mediates the effects exerted by this hormone on sperm intracellular calcium concentrations ([Ca(2+)](i)), as well as on the biological response to progesterone (P). In particular, 17 beta-estradiol (17betaE(2)) shows an inhibitory effect on P-mediated calcium influx and acrosome reaction (AR). In the present study, the effects of different anti-estrogens and xenoestrogens on [Ca(2+)](i) and AR stimulated by P have been investigated in human spermatozoa in order to better define the pharmacological characteristics of the sperm membrane estrogen receptor. The anti-estrogens tamoxifen (Tx) and ICI 164384 (ICI) induce only a slight increase of [Ca(2+)](i), which, however, as in the case of 17betaE(2), results in a reduction of P-stimulated calcium influx. Moreover, both the compounds reduce the calcium response to 17betaE(2) without affecting 17betaE(2)-inhibition of calcium response to P. Concerning AR, Tx alone does not alter either spontaneous or P-stimulated AR but partially revert the inhibitory effect of 17betaE(2). These results indicate that the two estrogens act as pharmacological agonists of the membrane estrogen receptors of human spermatozoa. On the other hand, the xenoestrogens bisphenol A (BPA) and octyiphenol polyethoxilate (OP) do not exert any direct effect on calcium fluxes and AR in human spermatozoa either in basal conditions or in response to P challenge. Moreover, although these environmental estrogens have been suggested to mimic estrogen effects in the other cell types, probably acting through genomic receptors, in human spermatozoa they do not interfere with 17betaE(2) binding to its membrane receptor and with the short-term effects exerted by this steroid. In conclusion, our data indicate that the membrane receptor for estradiol in human spermatozoa shows both biochemical and pharmacological differences respect to the genomic receptor.

Acrosome Reaction↗

PubMed2001

Spectrophotometric determination of trace aluminium content in parenteral solutions by combined cloud point preconcentration-flow injection analysis.

A cloud point preconcentration and flow injection (FI) analysis methodology for aluminium(III) determination has been developed. The analyte in the initial aqueous solution was complexed with Chrome Azurol S (CAS) in the presence of the cationic surfactant benzyldimethyltetradecylammonium chloride (BDTAC). The absorption spectroscopic characteristics of the ternary complex [Al(III)-CAS-BDTAC] were examined in detail. The preconcentration step was carried out by means of the non-ionic surfactant polyethylene glycol p-nonylphenyl ether (PONPE 7.5). The enriched analyte solution was injected into an FI system using an HPLC pump. The chemical variables affecting the analytical performance of the combined methodology were studied and optimised. The developed approach was successfully applied to the determination of trace amounts of aluminium in parenteral solutions without previous treatment. Under the optimum experimental conditions, 99.9% extraction was achieved for a preconcentration factor of 50. The limit of detection was 1.12 x 10(-7) mol(-1). The calibration plot was linear over at least two orders of magnitude of aluminium concentration. The developed coupled methodology, which thoroughly satisfies the typical requirements for pharmaceutical control processes, is appropriate for monitoring the aluminium concentration in parenteral nutrition.

Aluminum↗

PubMed2001

Phosphatidylinositol 3-kinase inhibition enhances human sperm motility.

BACKGROUND: The number of spermatozoa with forward motility after capacitation procedures represents the limiting factor for application of IVF versus intracytoplasmatic sperm injection (ICSI) procedure in cases of oligoasthenozoospermia. The possibility of increasing this number may thus be of help to the patient. A complex array of signalling pathways is involved in the regulation of sperm motility and recent data pointed out an important role for kinase/phosphatase-regulated phosphorylation of proteins. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K), a lipid and protein kinase involved in the regulation of several biological aspects of somatic cells, on human sperm motility by using the specific PI3K inhibitor LY294002. METHODS AND RESULTS: We demonstrated that in-vitro incubation of swim-up selected or unselected human spermatozoa with LY294002 determined an increase of percentage forward motility in all the treated samples. The effect was dose-dependent with an EC(50) of 1.09 +/- 0.54 micromol/l. LY294002 also increased sperm movement characteristics and hyperactivation as evaluated by computer-assisted motion analyser. The compound was also able to overcome the detrimental effect of hydrogen peroxide and lithium chloride on sperm motility. CONCLUSIONS: Our results suggest a negative role for PI3K in the development and maintenance of sperm motility and suggest a possible use of PI3K inhibitors to enhance motility in cases of asthenozoospermia.

Chromones↗

PubMed2000

Uteroglobin reverts the transformed phenotype in the endometrial adenocarcinoma cell line HEC-1A by disrupting the metabolic pathways generating platelet-activating factor.

Uteroglobin, originally named blastokinin, is a protein synthesized and secreted by most epithelia, including the endometrium. Uteroglobin has strong anti-inflammatory properties that appear to be due, at least in part, to its inhibitory effect on the activity of the enzyme phospholipase A(2). In addition, recent experimental evidence indicates that uteroglobin exerts antiproliferative and antimetastatic effects in different cancer cells via a membrane receptor. The human endometrial adenocarcinoma cell line HEC-1A does not express uteroglobin. Thus, we transfected HEC-1A cells with human uteroglobin cDNA. The transfectants showed a markedly reduced proliferative potential as assessed by impaired plating efficiency as well as by reduced growth in soft agar. Cytofluorimetric analysis clearly indicated that in uteroglobin-transfected cells the time for completion of the cell cycle was increased. We previously demonstrated that HEC-1A cells actively synthesize platelet-activating factor, one of the products of phospholipase A(2) activity. In addition, we demonstrated that platelet-activating factor stimulates the proliferation of these cells through an autocrine loop. In uteroglobin transfectants, the activity of phospholipase A(2) and platelet-activating factor acetyl-transferase, which are involved in the synthesis of platelet-activating factor, was significantly reduced compared with wild-type and vector-transfected cells (p < 0.05). Our results indicate that enforced expression of uteroglobin in HEC-1A cells markedly reduced their growth potential and significantly impaired the synthesis of platelet-activating factor, an autocrine growth factor for these cells. These data suggest that one possible mechanism for the recently observed antineoplastic properties of uteroglobin may be the inhibition of the synthesis of platelet-activating factor.

Acetyltransferases↗

PubMed2000

Intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and acrosome reaction.

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 properties of spermatozoa render these cells able to penetrate oocyte investments and 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 expose 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 have been partially defined, and appear to involve modifications of intracellular calcium and other ions, lipid transfer and phospholipid remodeling in sperm plasma membrane as well as changes in protein phosphorylation. Some of the kinases and phosphorylated proteins that are involved in the processes of capacitation and acrosome reaction have been now characterized. Characterization of sperm receptors to physiological inducers of acrosome reaction is in progress. This review summarizes the main signal transduction pathways involved in capacitation and acrosome reaction.Furthermore, the mechanisms underlying sperm DNA fragmentation are also briefly reviewed.

Acrosome Reaction↗

PubMed2000

A novel functional estrogen receptor on human sperm membrane interferes with progesterone effects.

We have identified an estrogen receptor of approximately 29 kDa apparent molecular weight in human sperm membranes by ligand and Western blot analysis, respectively, using peroxidase-conjugated E(2) and an antibody directed against the ligand binding region of the genomic receptor (alphaH222). Such receptor is functional since 17betaE(2) induces a rapid and sustained increase of intracellular calcium concentrations ([Ca(2+)](i)) which is completely eliminated by preincubation with alphaH222. 17betaE(2) effects on calcium are clearly mediated by a membrane receptor, as they are reproduced by the membrane-impermeable conjugate of the hormone BSA-E(2). Dose-response curve for this effect is biphasic with EC(50)s in the nanomolar and micromolar range. In addition to calcium increase, 17betaE(2) stimulates tyrosine phosphorylation of several sperm proteins including the 29-kDa protein band. Preincubation of human sperm with 17betaE(2) inhibits calcium and acrosome reaction increases in response to progesterone. We conclude that estrogens may play a role in the modulation of non-genomic action of progesterone (P) in human sperm during the process of fertilization.

Acrosome Reaction↗

PubMed2000

Progesterone action in a murine Leydig tumor cell line (mLTC-1), possibly through a nonclassical receptor type.

In a recent report we demonstrated that a high (micromolar) concentration of progesterone (P) specifically down-regulates LH receptor (R) expression and function in murine Leydig tumor cells. The aim of the present study was to characterize further the putative novel R, mediating these P effects in the murine Leydig tumor cell line, mLTC-1. The binding of [3H]P to these cells revealed a high (Kd, approximately 9.3 nmol/liter) and a low affinity (Kd, approximately 284 nmol/liter) component, and the binding displayed with specificity (P > dehydroepiandrosterone > 17-OHP). The binding was apparently different from that of the classical nuclear PR in the following ways. 1) The P/glucocorticoid antagonist RU 486 did not compete with [3H]P binding to the mLTC-1 cells. 2) No expression of the classical PR messenger RNA was detected, despite clear P binding to these cells, by Northern hybridization or RT-PCR. 3) An antibody against the C-terminal end of the classical PR (alpha c-262) revealed in mLTC-1 cells several molecular size protein bands between 45-57 kDa on Western hybridization, whereas these immunoreactive proteins were faintly recognized by another antibody (alpha-PR) directed toward the NH2-terminal region of the classical PR. The sizes of the immunoreactive molecules were relatively similar to those detected using the same antibodies in human sperm lysates, but were at variance with the classical PR (120, 94, and 60 kDa), detected with these antibodies in human uterus. The immunoreactive proteins bound peroxidase-labeled-P, which could be displaced in the presence of a 10-fold excess of free P. 4) An immediate increase in the intracellular free calcium level was observed after P treatment in cultured mLTC-1 cells, whereas it also increased the 45Ca2+ entry within 15 min in these cells. 5) Increasing doses of P (0.1-10 micromol/liter) demonstrated significant inhibition of LH receptor messenger RNA levels in a dose-dependent manner in mLTC-1 cells. In conclusion, a nonclassical PR is expressed and functional in these cells, and it is clearly distinct from the classical nuclear PR. It is apparent that recently reported inhibitory effects of P on LH receptor gene expression and function are mediated through this novel type PR in mouse Leydig cells.

Animals↗