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Biomedical subjects

M N Ritta

Publications and source records attributed to M N Ritta.

At least 19 recordsLinked to original sources
PubMed2004

Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen.

Cryopreservation imposes irreversible damage to sperm membranes, such as swelling and disruption of plasma and acrosome membranes, changes in membrane fluidity, altered influx of calcium, and changes in enzyme activity. Morphological integrity of the sperm plasma membrane has been widely studied using different techniques, including exposure of spermatozoa to hypoosmotic solutions (provides information concerning the biochemical activity of the sperm tail membrane), supravital test using eosin stain (yields information regarding sperm head membrane integrity), and Trypan-blue Giemsa stain (TBG; reveals both sperm plasma membrane and acrosome integrity). The objective of this study was to combine these tests in order to provide information about the integrity of the whole sperm surface, as well as acrosome status, and determine if the results of these tests were associated with sperm in vitro fertilizing ability. Stepwise regression analyses yielded a model in which fertility (maintain variable) was expressed as a combination of the results of different spermatological parameters (independent variables). The results of a test combining supravital eosin staining of samples previously submitted to hypoosmotic swelling test (STHOS) accounted for the greatest proportion of variation in fertilization rates (78%). Inclusion of the results of dual staining with TBG increased the proportion of variation in fertility rate that could be accounted for to 82%. Therefore, sperm plasma membrane integrity and function, and acrosome integrity can be considered important variables for normal sperm function and STHOST and TBG could be used for the prognosis of the potential fertility of bovine semen samples used for IVF or AI.

Acrosome↗
PubMed2004

In vitro effect of gamma-aminobutyric acid on bovine spermatozoa capacitation.

Sperm capacitation is defined as the maturational changes that render a sperm competent for fertilization and occurs in the female reproductive tract. Identification of the factor/s that regulate sperm capacitation would allow the understanding of these phenomena. Among these factors, gamma-aminobutyric acid (GABA) has recently become as a putative modulator of sperm function. The aim of this study was to explore the presence of a GABAergic regulation of bovine sperm capacitation as well as the possible intracellular mechanisms involved. GABA was detected in fresh semen by a sensitive radioreceptor assay (spermatozoa, 0.064 +/- 0.003 nmoles/10(6) cells; seminal plasma, 23.21 +/- 1.16 nmoles/ml). Scatchard analysis of [(3)H]-muscimol binding to sperm membranes yielded a linear plot consistent with a single population of binding sites (K(d) = 3.87 nM, B(max) = 417 fmol/mg prot.). [(3)H]-muscimol specific binding to sperm membranes was significantly inhibited by the GABA A receptor (GABA A-R) antagonist bicuculline and by the agonists muscimol and isoguvacine. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of capacitated spermatozoa (chlortetracycline assay). We observed a significant increment on intracellular calcium and cyclic 3',5' adenosine monophosphate (cAMP) concentrations induced by GABA, being the cation influx abolished when the cell suspensions were coincubated with the antagonists bicuculline or picrotoxin. It is concluded that GABA induces sperm capacitation through an intracellular mechanism dependent on calcium influx and cAMP accumulation mediated by a specific GABA A-R.

Animals↗
PubMed1998

Occurrence of GABA and GABA receptors in human spermatozoa.

Gamma-aminobutyric acid (GABA) concentrations in seminal plasma and washed spermatozoa from normal donors were assessed by a sensitive radioreceptor assay, and were detectable in both fractions. Specific binding of [3H]-muscimol was shown to be dependent on protein concentration, temperature and incubation time. [3H]-muscimol specific binding to human sperm membranes was significantly inhibited by the GABA type A receptor (GABA(A)) antagonist, bicuculline, and by the GABA(A) agonists, muscimol and isoguvacine, but not by the GABA type B receptor (GABA(B)) agonist baclofen. Scatchard analysis of [3H]-muscimol binding yielded a linear plot consistent with a single population of binding sites with a dissociation constant in the low nanomolar range. Incubation with GABA at a high micromolar concentration for 3 h under capacitating conditions resulted in an increase in the percentage of spermatozoa showing hyperactivated motility as assessed by computerized motility analyser. However, low micromolar concentrations of the GABA(A) agonist, muscimol, were sufficient to significantly increase sperm hyperactivity. These results suggest that the effect of GABA on human sperm motility might be mediated through a specific GABA(A) receptor.

Bicuculline↗
PubMed1993

Effect of bilateral denervation of the immature rat testis on testicular gonadotropin receptors and in vitro androgen production.

We have studied the effect of superior spermatic nerve (SSN) section on testicular gonadotropin receptors and in vitro androgen production by immature rat testis. Bilateral testicular denervation had no effect on testicular weight, serum androgens, LH, FSH and PRL levels. Denervation resulted in a significant inhibition of hCG stimulated in vitro androgen production. A reduction in the number of testicular LH receptors was observed after SSN section, while FSH binding sites remained unchanged. These results indicate that the number of LH receptors and testicular steroidogenic response to hCG are influenced by nerves reaching the testis.

Androgens↗
PubMed1991

Effect of long term diazepam administration on testicular benzodiazepine receptors and steroidogenesis.

We evaluated the effect of acute and chronic diazepam administration on testicular peripheral type benzodiazepine receptors (PBZD-R), serum testosterone and LH levels and the "in vitro" androgen production in response to Ro 5-4864, a PBZD-R agonist. The chronic diazepam treatment induced a significant fall in plasma testosterone concentration while LH levels remained unchanged. The number of PBZD-R was reduced by 37% and low concentrations (10(-8)-10(-6) M) of Ro 5-4864 failed to stimulate "in vitro" androgen production. The acute diazepam administration caused a significant increase in plasma testosterone levels while no changes were observed in LH concentrations and testicular PBZD-R. These results further suggest a modulatory role of PBZD-R on testicular steroidogenic activity.

Androgens↗
PubMed1991

Coexistence of gamma-aminobutyric acid type A and type B receptors in testicular interstitial cells.

The existence of specific gamma-aminobutyric acid (GABA)ergic receptors in testicular interstitial cells was investigated in the present study. Specific binding of [3H]GABA to interstitial cell membranes was found to be time- and temperature-dependent and varied according to Ca2+ concentration present in the incubation medium. We analyzed the ability of different GABAergic agonists and antagonists to displace the bound radioactivity. In the absence of Ca2+ (1 mM EDTA), GABA and the GABAergic agonist isoguvacine displaced the bound radioactivity. When the radioligand assay was performed in the presence of 2.5 mM CaCl2, the [3H]GABA specifically bound increased twofold. Under such conditions, the specific GABAergic agonist baclofen, as well as GABA and isoguvacine, displaced the [3H]GABA bound. Saturation analysis revealed the presence of a population of GABAA binding sites with a KD value of 45.2 nM and a maximal number of binding sites of 57.4 fmol/mg of protein. The maximal binding increased on addition of 2.5 mM CaCl2 to 102 fmol/mg of protein, indicating the existence of a second population of GABAergic receptors, i.e., type B, with essentially the same affinity. In addition, the incubation of testicular interstitial cells with GABA and baclofen resulted in an increase in androgen production. These results support a functional role of GABA in the neuroendocrine control of the male gonad.

Ammonium Chloride↗
PubMed1990

Peripheral-type benzodiazepine receptors are highly concentrated in mitochondrial membranes of rat testicular interstitial cells.

The binding of 3H-RO 5-4864 to the peripheral-type benzodiazepine receptors (PBZDR) in rat testicular interstitial cells (TIC) was characterized. The binding was saturable, reversible and showed a single high-affinity (Kd = 5.02 +/- 0.86 nM) class of binding sites. The maximal binding capacity (Bmax) in crude mitochondrial fractions (77.6 +/- 9.1 pmol/mg protein) represents the highest density of PBZDR in tissues thus far studied. In comparison with the crude mitochondrial fraction the subcellular fractionation of TIC revealed a 2-fold enrichment of 3H-RO 5-4864 binding sites to the purified mitochondria (Bmax = 140 +/- 23 pmol/mg protein). The ability of various drugs to displace 3H-RO 5-4864 from TIC binding sites was examined and the inhibition constants (Ki) for RO 5-4864, PK 11195, diazepam and flunitrazepam were 3.5, 4.4, 159, and 353 nM, respectively, whereas clonazepam and RO 15-1788 were inefficient in displacing 3H-RO 5-4864 (Ki greater than 10 microM). This pharmacological profile is characteristic of PBZDR described in other tissues. In conclusion, rat TIC possess a very high concentration of PBZDR primarily associated with mitochondrial membranes.

Animals↗
PubMed1989

Testicular interstitial cells as targets for peripheral benzodiazepines.

We evaluated the 'in vitro' effect of a selective peripheral benzodiazepine (BZD) receptor agonist, Ro 5-4864, on basal and hCG-stimulated androgen production by testicular interstitial cell suspensions. Ro 5-4864 (10(-9)-10(-5) M) induced a significant increment of basal testosterone release into the medium. In addition, under conditions of hCG stimulation, Ro 5-4864 (10(-7) M) induced a potentiated response to the gonadotropin in a dose-dependent manner. The selective peripheral BZD antagonist PK 11195 fully prevented the stimulatory effect of Ro 5-4864. On the other hand, clonazepam, a central BZD agonist, failed to affect androgen production significantly, whereas diazepam (10(-5)-10(-4) M), which binds to both central and peripheral BZD receptors, was able to induce a significant increment of basal and hCG-stimulated testosterone production. These results suggest that under our experimental conditions Ro 5-4864 exerts an effect on testicular steroidogenesis, presumably through binding to the previously described peripheral-type BZD receptor.

Animals↗
PubMed1988

Effects of thyroxine on oestrogen receptor concentrations in anterior pituitary and hypothalamus of hypothyroid rats.

The effects of thyroxine (T4) were studied on the concentration of oestrogen receptors in the anterior pituitary gland and hypothalamus of ovariectomized euthyroid and hypothyroid rats. A group of rats was made hypothyroid by the administration of 131I. Seven days after ovariectomy, animals were separated into five groups: I, euthyroid controls; II, hypothyroid controls; III, hypothyroid and injected with oestradiol benzoate (10 micrograms/day for 10 days); IV, hypothyroid and injected with T4 (4 micrograms/day for 10 days) and V, hypothyroid and injected with both oestradiol and T4 as described above. In group I, oestrogen receptor levels in pituitary cytosol were 44.4 +/- 3.4 (S.D.) fmol/mg protein and in the nucleus 47.7 +/- 4.0 fmol/mg DNA. In group II the respective values were 12.8 +/- 1.7 fmol/mg protein (P less than 0.01) and 12.7 +/- 1.7 fmol/mg DNA (P less than 0.01 compared with group I). In group III, cytosolic receptor concentrations decreased when compared with those in group II (P less than 0.05), whereas nuclear receptor concentrations rose significantly (P less than 0.01). Group IV had both pituitary cytosolic and nuclear receptors increased (P less than 0.01 compared with group II). In group V there were no changes in cytosolic receptor concentrations but a significant (P less than 0.01) rise in nuclear receptors as compared with group II. Hypothalamic oestrogen receptors in untreated hypothyroid rats (group II) were unchanged in the cytosol and diminished (P less than 0.01) in the nucleus in relation to euthyroid controls (group I). Thyroxine, but not oestrogen, was effective in increasing the concentration of cytosolic receptors (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effect of GABA and benzodiazepines on testicular androgen production.

We have evaluated the effect of Ro5-4864, a selective probe to label peripheral type benzodiazepine receptor, on "in vitro" testicular androgen production. Decapsulated testes from adult rats showed a significant increase in the basal and hCG-stimulated testosterone secretion into the medium in response to 10(-5) M, 10(-6) M, and 10(-7) M Ro5-4864. In addition, we have studied the changes in testicular GABA content at three different ages and we found its highest concentration at 31 days of age. When we evaluated the effect of GABA on "in vitro" androgen production at different stages of gonadal maturation we observed that the highest concentration of GABA (10(-6) M) was able to modify the basal and hCG-stimulated androgen production from adult (60 days) and pubertal (45 days) testes. In addition, when prepubertal testes (31 days) were incubated under basal conditions, 10(-6) M GABA induced a significant increment of androstanediol production, while the stimulatory effect of hCG was reduced in the presence of the same GABA concentration. The present results suggest that GABA plays a physiological role in the regulation of rat testicular androgen production depending on the stage of sexual maturation.

Age Factors↗
PubMed1983

The role of prostaglandins in neuroendocrine junctions: studies in the pineal gland and the hypothalamus.

This article discusses current experimental evidence indicating a role for prostaglandins (PGs) in pineal and median eminence neuroendocrine junctions. Both tissues release PGs, particularly of the E series, upon exposure to norepinephrine (NE) and through alpha-adrenoceptors. Exposure of pineal and median eminence explants to nanomolar concentrations of PGE2 augments melatonin and GnRH release, respectively. In the pineal gland, this effect appears to be linked to the stimulation of adenylate cyclase. Both in vivo and in vitro PG synthesis inhibitors impair the hormone release elicited by NE. In the pineal gland, PGE2 also constitutes a trans-synaptic negative signal for NE release. PGs receptors are present in pineal and hypothalamic membranes.

Animals↗
PubMed1982

Involvement of alpha-adrenoceptors in norepinephrine-induced prostaglandin E2 release by rat pineal gland in vitro.

Rat pineal glands incubated in vitro with 10 or 100 microM norepinephrine (NE) released 51% and 415% more prostaglandin E2 (PGE2) to the medium than in the absence of NE. Phentolamine (10 microM) prevented fully the effect of NE at both concentrations, whereas propranolol failed to affect it significantly. After superior cervical ganglionectomy (SCGx), NE-induced PGE2 release was significantly higher than in sham-operated controls--an effect also blocked by phentolamine but not by propranolol. These results suggest that NE releases PGE2 in rat pineals via alpha-adrenoceptors, acting at a post-synaptic site, and that SCGx induces alpha-adrenergic supersensitivity in the pinealocytes.

Adrenergic Fibers↗
PubMed1982

Norepinephrine stimulates testosterone aromatization and inhibits 5 alpha reduction via beta-adrenoceptors in rat pineal gland.

The possible modulatory role of the sympathetic nervous system on testosterone aromatization and 5 alpha reduction by rat pineal gland was examined in vitro. NE (10 microM) added to pineal organ cultures increased by 72% the conversion of [14C]testosterone into estradiol and depressed by 39 and 53% that into the 5 alpha-reduced metabolites 5 alpha-dihydrotestosterone (5-DHT) and 5 alpha-androstanediol (5 alpha-diol). Both effects of NE were negated by the addition of the beta-adrenoceptor antagonist propranolol but not by the alpha-adrenoceptor antagonist phentolamine. Dibutyryl cAMP (0.1 mM) mimicked the effect of NE on pineal [14C]testosterone metabolism; it also mimicked the NE-induced inhibition of [14C]progesterone reduction to 5 alpha-pregnanedione and 3 alpha-hydroxy-5 alpha-pregnan-20-one by rat pineal gland explants. At the end of the dark phase of the daily photoperiod, pineal aromatization of testosterone was significantly higher, and 5 alpha reduction lower, than in rats killed at noon. Pineal glands obtained from rats subjected to superior cervical ganglionectomy 12 h earlier exhibited increased conversion of [14C]testosterone into estradiol, and depressed synthesis of 5 alpha-reduced metabolites, as compared with their respective sham-operated controls. 3 days after ganglionectomy a diminished testosterone aromatization was found. These results suggest that the increased release of NE from pineal sympathetic nerve endings stimulates testosterone aromatization and inhibits 5 alpha reduction via a beta-adrenoceptor.

5-alpha Reductase Inhibitors↗
PubMed1981

Prostaglandin E2 increases adenosine 3',5'-monophosphate concentration and binding-site occupancy, and stimulates serotonin-N-acetyltransferase activity in rat pineal glands in vitro.

The effects of prostaglandins (PGs) on rat pineal metabolism were examined in vitro. PGE2 (0.01-1 microM) increased the activity of serotonin-N-acetyltransferase (SNAT), the stimulation curve exhibiting a maximum at 0.1 microM. PGE1 increased SNAT activity only at the highest dose (1 microM) whereas PGF2 alpha, 15-keto-PGF2 alpha or PGI2 did not affect the enzymic activity. The stimulation of SNAT activity brought about by PGE2 in pineals from ganglionectomized rats was greater than in sham-operated controls at all the doses studied, suggesting that the observed effect is predominantly post-synaptic. Only PGE2 significantly increased pineal cAMP accumulation in vitro at doses between 0.01 and 1 microM, and depressed the unoccupied cAMP-binding sites in pineal 900 g supernatants. The total number of cAMP-binding sites remained unaltered after incubation of PGE2. The present observations together with the previously reported NE-induced release of PGs in incubated pineal glands, the occurrence of pineal PG-binding sites and the indomethacin blockade of the nocturnal rise of pineal SNAT and melatonin content, support a role for PGs in the control of melatonin synthesis.

Acetyltransferases↗
PubMed1980

Prostaglandin E release by rat medial basal hypothalamus in vitro. Inhibition by melatonin at submicromolar concentrations.

Melatonin depressed PGE release by rat medial basal hypothalamus (MBH) in vitro at concentrations 10(-8) M or greater. The two-fold increase in PGE release induced by adding 10(-4) M norepinephrine to MBH explants was also impaired by melatonin at micromolar or greater concentrations. These data support the view that melatonin is a physiological inhibitor of PG synthesis in MBH.

Animals↗