Biomedical subjects

A M Ronco

Publications and source records attributed to A M Ronco.

At least 19 recordsLinked to original sources

PubMed2002

Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production.

We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of PLA(2) and G proteins in the release of AA from hCG-stimulated Leydig cells, and under particular conditions, regulation of cAMP production by this fatty acid in these cells.

Acetophenones↗

PubMed2001

The mechanism for lindane-induced inhibition of steroidogenesis in cultured rat Leydig cells.

The in vitro effect of the gamma-isomer of hexachlorocyclohexane, lindane, on rat Leydig cell steroidogenesis was studied. Leydig cells from mature male rats were incubated with human chorionic gonadotropin (hCG, 1 IU) for 3 h at 34 degrees C in the presence of different doses of lindane (2-200 microg/ml; 2-200 ppm). Results demonstrate that lindane produces a dose-dependent inhibition of testosterone production in hCG-stimulated Leydig cells. The decreased testosterone synthesis was accompanied with a half-reduced LH/hCG receptor number without any modification in the K(d) value. In addition, lindane also decreased cAMP production. These effects were not due to a detrimental action of lindane on cell viability. Results of this study demonstrate a direct inhibitory action of lindane on testicular steroidogenesis, at least in part, through a reduction in the classical second messenger production involved in this pathway.

Animals↗

PubMed2001

Hamster sperm glycine receptor: evidence for its presence and involvement in the acrosome reaction.

Recent reports have provided evidence for the presence of amino acid neurotransmitter receptor/chloride channels in human and porcine spermatozoa and their involvement in the acrosome reaction (AR). In this work we investigated whether a glycine receptor (GlyR) was present in golden hamster sperm, and whether it had a role in the hamster AR. The neuronal GlyR agonist glycine, stimulated in a dose-dependent manner, the AR of hamster spermatozoa previously capacitated for at least 3 hr. This stimulation was completely inhibited by 50 microM (+)-bicuculline and by concentrations of strychnine as low as 10-50 nM; both agents are antagonists of neuronal GlyR when used at the concentrations reported in this study. beta-Alanine, another agonist of the neuronal GlyR, also stimulated the AR. The AR-stimulatory effect of this compound was completely abolished by 50 nM strychnine. The inhibitory effect of strychnine on the glycine-induced hamster sperm AR was completely overcome by subsequent treatment with the calcium ionophore ionomycin, demonstrating that the strychnine effect was specific for GlyR. Additional binding studies with (3)[H]-strychnine, the typical radioligand used to detect GlyR in several cells, demonstrated for the first time the presence of specific binding sites for strychnine in the hamster spermatozoa. Interestingly, binding increased during in vitro capacitation, particularly in those sperm suspensions showing high percentages of AR. Taken together these results strongly suggest the presence of a GlyR in the hamster spermatozoa, with a role in the AR when activated.

Acrosome Reaction↗

PubMed2000

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.

Animals↗

PubMed2000

Plasma absorption and ultrastructural changes of rat testicular cells induced by lindane.

This paper describes, for the first time, how topical application in rats of a commercial preparation of lindane widely used in public health, at similar doses and routes of administration as in humans, leads to rapid absorption and accumulation of lindane in the testes. An early peak of absorption was detected in plasma 6 h after topical treatment of male Wistar rats with a commercial preparation of 1% lindane (Plomurol). Higher plasma levels were observed after repetitive doses of 60 mg/kg b.w., the amount recommended for the treatment of scabies and pediculosis in humans in several countries. A residue level of 7.4 +/- 0.67 microg/g was found in testicular tissue 6 h after a single daily topical application for 4 consecutive days. The ultrastructural study of testicular interstitial cells exposed to dermal application of lindane (Plomurol) revealed widespread damage of a great number of Leydig cells, some of which were completely disintegrated.

Administration, Topical↗

PubMed1998

Effect of desialylated human chorionic gonadotropin (hCG) on the bioactivity of rat Leydig cells.

Human chorionic gonadotropin is a glycoprotein hormone that, like LH, stimulates steroidogenesis in gonadal cells. Using a desialylation process. 95 per cent of the sialic acid residues from an intact standard hCG molecule were eliminated and then the electrophoretic properties and the bioactivity of the desialylated hCG were determined. Using rat Leydig cells as a biological model, the binding affinity to LH receptors of Leydig cell membranes, steroidogenic activity and second messenger production were studied. The results indicate that the loss of sialic acid from the hCG molecule slightly increases the binding activity to LH receptors and results in steroidogenic activity with an increased ED50. Cyclic AMP production was significantly reduced however and arachidonic acid release was not observed. Several possible mechanisms that could explain these results are discussed.

Animals↗

PubMed1997

Arachidonic acid release from rat Leydig cells depends on the presence of luteinizing hormone/human chorionic gonadotrophin receptors.

In this work, the involvement of arachidonic acid (AA) in the luteinizing hormone and human chorionic gonadotrophin (LH/hCG) action on Leydig cells was studied. Experiments were first designed to evaluate [14C]AA incorporation into membrane phospholipids. Subsequently, time-course, pulse-chase and dose-response studies of the effect of hCG on [14C]AA release were performed. Results indicated that 4 h was optimal for maximal incorporation of [14C]AA into membrane phospholipids of viable Leydig cells. Pulse-chase experiments and studies performed to evaluate the effect of different doses of hCG on [14C]AA release demonstrated that this hormone stimulates [14C]AA release in a dose-response and time-dependent manner. Furthermore, using a desensitised animal model, a link between the presence of LH/hCG receptors and LH/hCG-stimulated [14C]AA release in Leydig cells could be established. In fact, the amount of [14C]AA released was significantly dependent on, and directly proportional to, the concentration of LH/hCG binding sites. Thus [14C]AA released from intact rat Leydig cells decreased when animals had been previously injected with a high single dose of hCG (desensitised animals), which is known to cause a dramatic decrease in the number of LH/hCG binding sites. These results demonstrate that the mechanism of AA release in Leydig cells depends on LH/hCG-receptor interaction and also suggest that AA could act as an additional intracellular messenger associated with the hormonal action of LH/hCG.

Animals↗

PubMed1994

Sperm phospholipid methyltransferase activity during preparation for exocytosis.

The present report describes experiments to evaluate phospholipid methyltransferase activity in golden hamster spermatozoa incubated under different conditions. Washed cauda epididymal sperm were incubated with taurine, in the presence or absence of epinephrine. At various times, the sperm were separated, and phospholipid methyltransferase activity measured. Also, at each time, aliquots of the sperm suspension were assayed for motility, and acrosome reactions. Some sperm incubated in the presence of taurine and epinephrine were capacitated by 3.5 h, because about 40 per cent of them can undergo the acrosome reaction 10 min after addition of the fusogen lysophosphatidylcholine. In epinephrine-free incubations the fusogen failed to stimulate acrosome reactions. On the other hand, epinephrine stimulated by twofold phospholipid methyltransferase activity from '0 time' incubated sperm, in comparison to that observed in taurine-treated cells. Enzyme activities from both taurine or epinephrine plus taurine-treated cells decreased as the incubation time of the sperm suspension increased. Kinetic properties of the sperm phospholipid methyltransferase activity were modified by the presence of taurine and epinephrine when S-adenosylmethionine was used as the substrate. These results suggest that refined molecular events occur in the sperm cell during the acquisition of fertilizing ability.

Animals↗

PubMed1994

Leydig cell heterogeneity as judged by quantitative cytochemistry of 3 beta-hydroxysteroid dehydrogenase activity in individual rat Leydig cells.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) is one of the key enzymes involved in the steroidogenic pathway of Leydig cells. In this study, quantitative cytochemistry was used to detect the 3 beta-HSD staining intensity in individual rat Leydig cells. The measurement of the intensity of staining was a reliable method reflecting the relative amount of 3 beta-HSD activity. The objective was to determine the presence, basal and hCG-mediated effect of 3 beta-HSD activity in individual Leydig cells. 3 beta-HSD cytochemistry was performed in both, 8 and 12 microns diameter rat Leydig cells. The results showed that both populations of Leydig cells have different basal 3 beta-HSD activity. The 8 microns cells showed a greater basal 3 beta-HSD activity than the 12 microns cells when their optical density values were normalized to their size. A difference in regulation of the enzymatic activity by LH/hCG was observed in the two types of Leydig cells. Incubation of the whole population of Leydig cells with hCG (1IU), decreased the 3 beta-HSD activity in the 8 microns cells, but increased the activity in the 12 microns cells. The results describe for the first time that the 3 beta-HSD activity may be differentially regulated by LH/hCG in Leydig cells.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Human chorionic gonadotropin and free beta subunits stimulate phospholipid methylation in intact rat Leydig cells.

The effect of human chorionic gonadotropin (hCG) on intact Leydig cell phospholipid methylation was studied. Hormonal stimulation of rat Leydig cells increased the incorporation of [methyl-3H]methionine into phospholipids threefold. This effect was observed after 10 minutes of incubation time and was time and dose dependent with a maximal stimulation at 67 ng/ml of hCG. In the presence of hCG, 3H-labeled methyl groups were preferentially incorporated into phosphatidyl-N-monomethylethanolamine. This effect of hCG was not reproduced by dibutyryl cyclic adenosine monophosphate (cAMP), cholera toxin, or forskolin. Purified hCG beta subunit but not hCG alpha subunit had stimulatory activity on Leydig cell phospholipid methylation. We conclude that luteinizing hormone (LH)/hCG stimulates specifically Leydig cell phospholipid methylation, because LH-releasing hormone or [Arg8]-vasopressin did not modify these reactions. We postulate that these reactions are occurring at a cellular level that involves hormone-receptor interaction. It is also suggested that this biological response involves hCG beta subunit receptor interaction and does not require cAMP synthesis.

Animals↗

PubMed1993

Phospholipid methylation decreases in human chorionic gonadotrophin-induced desensitized rat Leydig cells.

Phospholipid methylation in Leydig cells from desensitized rats was studied. The incorporation of L-[methyl-3H]methionine into phospholipids in intact Leydig cells decreased when animals were injected with a single high dose of human chorionic gonadotrophin (hCG). This effect was detected on the first day after hCG injection and remained up to 12 days after treatment. The inhibition was not due to a reduced uptake of L-[methyl-3H]methionine. A decreased phospholipid methylation with unaltered phospholipid methyltransferase activity was observed on days 1, 6 and 12 after the hCG. On day 3 after hCG injection, phospholipid methyltransferase activity and phospholipid methylation in intact Leydig cells were both inhibited by 40%. Also, a minimal amount of LH free receptors and the lowest number of total receptors was observed at this time. Thus, a relationship between the reduced enzymatic activity and the maximal decrease in LH surface receptors is suggested. In addition, the decreased incorporation of L-[methyl-3H]methionine into phospholipids on days 1, 6 and 12 after hCG injection, could be associated with other cellular changes related to the desensitization process.

Animals↗

PubMed1993

Melatonin binding sites in interstitial cells from immature rat testes.

Melatonin binding sites in rat testes interstitial cells were identified using 2-[125I]-iodomelatonin. Saturation studies of cells revealed a single class of high affinity binding sites, with an apparent equilibrium dissociation constant (Kd) of 100 +/- 20 pM, and a total binding capacity (B max) of 3.0 +/- 0.2 x 10(3) melatonin molecules per cell. Binding was reversible and inhibited by non radioactive melatonin. These results suggest that interstitial cells from immature rat testes have specific receptors for melatonin.

Animals↗

PubMed1992

In vitro effect of hCG on steroidogenesis in the testicular tissue from a patient with complete androgen resistance.

The present study was performed to determine the in vitro steroidogenic capacity of a gonadal sample from a patient suffering from a complete androgen resistance syndrome. Testosterone and estradiol production by the testicular tissue from this patient as well as gonadotropin binding to a membrane fraction prepared from this tissue were measured. hCG bound with high affinity but with a very low capacity and the gonadotropin induced a clear dose response for both testosterone and estradiol production. The ED50 of hCG on testosterone and estradiol production were 2.5 and 5.0 nM, respectively. We conclude that estradiol originates from Leydig cell activity, since estradiol synthesis does not depend on testosterone availability and it shows a clear hCG dose response.

Adolescent↗

PubMed1992

"The effect of hCG-induced desensitization on RNA synthesis in rat Leydig cells".

In this study we have examined the effect of hCG-induced desensitization on RNA synthesis in rat Leydig cells. In vitro [3H]-uridine incorporation into RNA decreased after a high, single dose of hCG (100 IU). This effect was maximal after the second and third day of treatment. When Leydig cells were incubated in vitro for 30 min or more, a marked decrease of total and poly(A)+ RNA synthesis was observed. This was not due to reduced cell permeability to the radioactive nucleotide, indicating a truly decreased RNA synthesis during desensitization. The magnitude of this inhibitory effect (73-80%) suggests that desensitization may involve other biological functions of Leydig cell.

Animals↗

PubMed1988

RNA synthesis in the Leydig cells of the mature rat: effect of oestradiol-17 beta.

In view of the evidence that there may be an effect of high concentrations of oestradiol on testicular steroidogenic function, we have investigated the effect of this steroid on [3H]uridine incorporation into RNA by testicular cells. Our results have shown that oestradiol in vitro induced a marked dose-dependent inhibition of RNA synthesis by purified Leydig cells. The concentrations of oestradiol tested varied from 2 to 40 mumol/l; these concentrations also impaired net testosterone synthesis in vitro after human chorionic gonadotrophin (hCG) stimulation. Under the effect of oestradiol, the kinetics of [3H]nucleoside incorporation into RNA were impaired early and the inhibition of RNA synthesis was specific for oestrogenic compounds. It was concluded that, in Leydig cells, oestradiol, in addition to its known inhibitory action on the response of testosterone to hCG, triggers a more extensive response that also includes RNA synthesis in vitro.

Animals↗

PubMed1986

RNA synthesis in Leydig cells during sexual maturation in the rat: effect of LH.

The trophic action of LH on Leydig cells involves the triggering of a number of cellular events including changes in protein synthesis. This latter change has led a number of workers to postulate an effect of LH on RNA synthesis. A direct action of LH on RNA synthesis, however, has been difficult to assess. The aim of the present work was to analyse the effect of LH on RNA synthesis in vitro during sexual development. Studies were performed using purified Leydig cells from rats of 20, 30, 40, 50, 60 and 90 days of age. The results obtained show that basal uridine incorporation into RNA increases in an age-dependent manner in rats from 20 to 60 days of age and then remains unchanged until 90 days of age. A stimulatory effect of LH on RNA synthesis was clearly demonstrated only in the youngest rats (20 and 30 days old). In order to differentiate the effect of LH on different RNA populations, the RNA synthesized by immature and mature rats was analysed using a poly(U)-Sepharose column. In 20-day-old rats, LH stimulated both unbound and poly(A) RNA, although a more marked effect was clearly demonstrated on the latter. On the other hand, LH had an identical effect on both unbound and poly(A) RNA obtained from Leydig cells of 60-day-old rats. This stimulatory effect of LH on RNA synthesis in Leydig cells from immature rats seemed specific, since effectors which act on interstitial cells, such as LH-releasing hormone, [Arg8]-vasopressin and FSH (which may act on macrophages) did not modify RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

The transmethylations inhibitor 3-deazaadenosine, inhibits in vitro testosterone production by rat testis interstitial cells stimulated with HCG.

3-Deazaadenosine (3-DZA), an inhibitor of somatic cell transmethylations, inhibited in vitro HCG-stimulated testosterone synthesis by rat testis interstitial cells. A maximal inhibition of 50% was observed with 100 microM 3-DZA; in addition homocysteine-thiolactone (Hcy) enhanced the inhibitory effect of 3-DZA. On the other hand, when cells were stimulated with dibutyryl cyclic AMP (Bt)2-cAMP, 3-DZA did not exert any effect on the stimulation. The presence of 3-DZA in the incubation medium neither modified HCG Kd values nor the number of its binding sites to Leydig cells. These results demonstrate that inhibitors of transmethylation reactions interfere with hormone-stimulated testosterone synthesis, suggesting that those reactions (presumably phospholipid methylation) at the plasma membrane level are involved in hormone-stimulated testosterone synthesis by rat Leydig cells.

Animals↗

PubMed1984

Effect of luteinizing hormone on RNA synthesis in Leydig cells of immature rats.

Luteotrophic hormone acts on testicular interstitial cells, promoting the activation of several cellular events that culminate in steroids synthesis. Since the interstitial tissue include several cell types, purified Leydig cells were used in this work. Isolated interstitial cells from immature rats were purified through a 0-40% metrizamide gradient. Either LH, HCG or Bt2-cAMP significantly stimulated the incorporation of [3H]uridine into RNA, when compared to control. The effect of HCG on RNA synthesis was developed within 30 min after the addition of the hormone and was dose-dependent. The maximum effect was attained with 10 mIU/ml of HCG. These results indicate that HCG/LH or Bt2-cAMP but not FSH, promote an acute stimulation of RNA synthesis by Leydig cells from immature rats.

Animals↗