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Biomedical subjects

D R Aquilano

Publications and source records attributed to D R Aquilano.

13 recordsLinked to original sources
PubMed1998

Altered luteinizing hormone pulsatility in infertile patients with idiopathic oligoasthenozoospermia.

In a previous study, we demonstrated that oligoasthenozoospermic (OAZ) patients had two types of testosterone response to human chorionic gonadotrophin (HCG) administration: group 1 (OAZ-1) had an altered, monophasic (no first peak) response, and group 2 (OAZ-2) had a normal biphasic response. The objective of the present work was to study the luteinizing hormone (LH) pulsatility in OAZ-1 compared with both OAZ-2 and men of proven fertility (PF), in order partly to determine the possible aetiology of the blunted acute testosterone response to HCG in these patients. LH pulsatility was measured in 10 PF, 10 OAZ-1 and 10 OAZ-2 patients, in blood samples taken every 5 min for 6 h in PF, and for 4 h in OAZ patients. LH values were determined by a time-resolved immunofluorometric assay. Frequency and amplitude of the LH pulses were determined by a computer program. LH pulse frequency, expressed as pulses/4 h, was significantly lower in OAZ-1 (1.5+/-0.97) than in PF (2.4+/-0.63) and OAZ-2 (2.4+/-0.84) patients. In six OAZ-1 and two OAZ-2 patients, LH pulsatility was diminished, as they showed less than two pulses/4 h. No statistically significant differences in LH pulse amplitude were found. These results, together with a higher number of OAZ-1 cases found with decreased LH pulsatility, suggest that, at least in a subset of these men, quantitative and/or qualitative alterations of LH secretion might have occurred.

Adult↗
PubMed1985

Gonadotropic regulation of aromatase activity in the adult rat testis.

Aromatase activity during gonadotropin action in vivo and in vitro was examined in purified Leydig cells to further define the early effects of LH and for elucidation of the enzymatic processes involved in the development of late lesions of androgen biosynthetic pathway. Aromatase was measured by the tritiated water release method using [1 beta-3H]testosterone as substrate. The enzyme activity was proportional to the amount of cells (0.1-1.0 X 10(6] incubated, increased with the incubation time (2-60 min), was inhibited by androstatriendione (ED50, 5.0 microM), and showed a Km for testosterone of 1.69 microM. Aromatase activity was stimulated (10-20%; P less than 0.05) 1 h after treatment of rats with a single sc dose of 5 micrograms hCG. This activation preceded the late steroidogenic lesion at the site of 17 alpha-hydroxylase and 17,20-desmolase activity by 2-5 h. A RIA of improved sensitivity (0.5 pg) was developed to detect the very low cellular and secretory levels of estradiol. The testicular contents of testosterone and estradiol showed small increases (P less than 0.05) within 40 and 60 min after hCG treatment, respectively. Testicular testosterone levels reached a peak by 1 h after injection and preceded the peak level of estradiol formation by 2 h. After in vitro treatment of cultured Leydig cells with 100 ng hCG, the aromatase activity was significantly increased within 30 min (P less than 0.05) and then returned to control levels for up to 16 h of culture. A similar temporal pattern for enzyme activation was observed after treatment of cultures with 8-bromo-cAMP or forskolin (23-27% above control; P less than 0.05), while cholera toxin stimulated aromatase activity at 2 h. Net testosterone accumulation in the incubation medium increased 30 min after the hCG treatment and reached a plateau by 4 h. A small but significant increase in estradiol levels (P less than 0.05) was also observed at 30 min, remaining constant until 120 min, which was followed by a sharp rise parallel to that of testosterone. These results suggest that the estradiol-mediated desensitization of the Leydig cell observed after hCG administration is consistent with an early cAMP-dependent activation of aromatase and a further rise in estradiol formation due to increased substrate availability.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Cellular localization of rat testicular aromatase activity during development.

The aromatase activity from purified testicular sources (Leydig and Sertoli cells) of immature (5- and 15-day-old) and adult rats (60-day-old) was investigated by the tritiated water release method in isolated Leydig and Sertoli cells that were morphologically and functionally characterized. Electron micrographs of Sertoli cell preparations from different ages showed no marked changes, except that tight junctions between Sertoli cells normally present in 60-day-old rats were not observed in 5-day-old and rarely found in 15-day-old animals. Leydig cells underwent ultrastructural changes along with development, such as the appearance of thicker nuclear heterochromatin and laminar-like mitochondria. The 15-day-old rat interstitial tissue possessed less than 10% of Leydig cells morphologically similar to those present in the adult, whereas the rest were probably transition cells, since they did not show typical Leydig cell structure but were able to bind [125I]iodo-hCG, as evaluated by autoradiography. The number of LH/hCG-binding sites increased with age in Leydig cells, but was not detectable in Sertoli cells. The highest number of FSH-binding sites in Sertoli cells was observed in the 15-day-old animals. Minor FSH binding was found in Leydig cell preparations, which was consistent with the known LH contamination of the human FSH tracer preparation. cAMP production increased significantly in Leydig cells only after hCG treatment and in Sertoli cells after FSH stimulation. Both types of cells were shown to have the capacity for aromatization. The aromatase activity increased in the Leydig cell but decreased in the Sertoli cell during testicular development. The highest aromatase activity was found in adult rat Leydig cells, and the enzyme activity was significantly higher (2-fold) in purified Leydig cells than in crude interstitial cell preparations. Estradiol production in response to hCG stimulation in vitro was not different from the basal value in 5-day-old rat Leydig cells, but increased significantly in 60-day-old rat Leydig cells. In conclusion, 1) Leydig cells are the major site of estrogen synthesis in adult rat testis; and 2) the low aromatase activity observed in immature rat Leydig cells could partially explain the differential response of the mature and immature rat testis to hCG-induced desensitization.

Aging↗
PubMed1985

Mitochondrial cholesterol availability during gonadotropin-induced Leydig cell desensitization.

In order to define the early lesion (before pregnenolone formation) of the androgen biosynthetic pathway induced by human CG (hCG) or LH in the Leydig cell, we initially have optimized the use of aminoglutethimide to obtain maximal and sustained inhibition of steroidogenesis in vivo and in vitro. Aminoglutethimide inhibited Leydig cell steroidogenesis in vitro at a dose of 100 micrograms/ml. The minimal serum concentration of aminoglutethimide necessary for maximal inhibition of testosterone in vivo was also 100 micrograms/ml (1 h after the ip injection of 20 mg aminoglutethimide). However, testosterone levels were normal 12 h later, coincident with a marked fall in the serum aminoglutethimide levels. The t 1/2 of the circulating aminoglutethimide was 5 +/- 0.7 h on the first day of treatment but was reduced to 3.0 +/- 0.4 and 2.25 +/- 0.35 h at 2 and 3 days of treatment. At the dose eliciting maximal and sustained steroid inhibition (60 mg/day) aminoglutethimide was able to prevent the estradiol-dependent late steroidogenic lesion (after pregnenolone formation) induced by 1 microgram hCG, with no effect on the early lesion (before pregnenolone formation) caused by 10 micrograms hCG. The aminoglutethimide-induced in vivo accumulation of cholesterol in the inner mitochondrial membrane (by 50%) was associated with an increase in the production of testosterone and pregnenolone by the Leydig cell when subsequently incubated in vitro. Similar increases in the steroidogenic capacity were observed after initial exposure of Leydig cells to aminoglutethimide in vitro, even after acid wash to remove the surface-bound endogenous LH. The steroidogenic cholesterol was also increased in desensitized Leydig cells (by 50-70%); however, the conversion of cholesterol to pregnenolone was substantially blocked in animals with the early lesion. Our findings define the requirement of increasing high levels of aminoglutethimide to inhibit cholesterol metabolism and provide a dose schedule suitable for studies on cholesterol availability and inhibition of steroidogenesis in the rat. These results support our proposal that the early lesion observed in desensitized Leydig cells is due to inhibition of the side-chain cleavage activity rather than to a decrease in the amount of metabolically available cholesterol.

Aminoglutethimide↗
PubMed1985

Endocrinological control and cellular localization of rat testicular angiotensin-converting enzyme (EC 3.4.15.1).

Hypophysectomy of prepubescent (3-week-old) rats prevented the pubertal development of testicular, but not pulmonary, angiotensin-converting enzyme (EC 3.4.15.1). Additionally, hypophysectomy resulted in a loss of testicular converting enzyme activity in 10-week-old rats that had achieved puberty and had developed enzyme activity. Hormone regimens consisting of FSH/LH (7.5 U/rat X day), hCG (10 U/rat X day), or testosterone (1 mg/rat X day) were employed to ascertain their ability to maintain activity in hypophysectomized rats. All three of the above hormone regimens, if initiated on the first day after hypophysectomy of 10-week-old rats, were capable of maintaining testicular converting enzyme activity. Centrifugal elutriation of dispersed testicular cells indicated that the majority of enzyme activity in mature rats was associated with the germinal cells, a result consistent with the data accumulated from the hormonal studies. Lastly, [3H]captopril bound specifically to cellular fractions enriched in germinal cells. The above studies suggest that the pituitary gland is required for the development and maintenance of testicular angiotensin-converting enzyme in the rat by stimulating steroidogenesis in the testes. Furthermore, the sensitivity of converting enzyme activity to androgen coupled with the centrifugal elutriation and [3H] captopril binding studies strongly support the notion that testicular converting enzyme is associated with germinal cells.

Animals↗
PubMed1984

An early steroidogenic defect in hormone-induced Leydig cell desensitization.

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [14C]isocaproic acid formation from [26-14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 micrograms hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 microM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The Km value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37 mM after hCG, with no changes in Vmax. Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 microM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID50 of 500 and 280 micrograms protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 +/- 0.23 SD (n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5' ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.

Adrenal Glands↗
PubMed1984

Functional and morphological studies on isolated Leydig cells: purification by centrifugal elutriation and metrizamide fractionation.

Rat interstitial cells were fractionated by centrifugal elutriation to facilitate the purification of Leydig cells for analysis of mechanisms of gonadotropin action in vitro. By this procedure, 10(9) collagenase-dispersed interstitial cells from adult rat testes were separated into 12 fractions in about 1 h. Fractions 1-7 (sedimentation velocities, 1.9-12 mm/h.g) comprised 80-85% of the total cells applied, including erythrocytes, lymphocytes, germinal cells, macrophages, endothelial cells, damaged Leydig cells, and contained less than 4% intact Leydig cells. Fractions 8-12 (sedimentation velocities, greater than 12 mm/h.g) comprised 15-20% of the original cells and contained 90-95% intact Leydig cells. Despite their different sedimentation velocities, the Leydig cell-rich fractions were similar in their LH receptor content (mean +/- SD, 36,115 +/- 4,815 sites/cell) and showed similar 5-fold increases above the original cell preparation in testosterone and cAMP responses to hCG. The pooled Leydig cell-rich fractions (8-12) were further resolved on 16-24% Metrizamide gradients into 5 bands. Bands II-V (density range, 1.075-1.110 g/ml) contained pure Leydig cells, and band I (1.048 g/ml) contained pachytene spermatocytes, the contaminating cell type present in the Leydig cell-rich fractions obtained by elutriation. Each of the 4 Leydig cell-rich bands showed similar morphology and functional activity. Essentially similar results were observed using 14-32% Metrizamide gradients. Leydig cells desensitized in vivo by hCG treatment and isolated by elutriation were also resolved by Metrizamide gradients into 4 bands, but showed a redistribution in the gradient, due to the shift of about 50% of the cells originally present in the heaviest bands to lower density fractions. However, in spite of their changes in density, the Leydig cell bands still showed similar degrees of receptor down-regulation and impairment of the steroid responses to hCG in vitro. This study has demonstrated that centrifugal elutriation is a rapid and effective method for obtaining large quantities of purified (greater than 90%) and active Leydig cells. Further resolution of the Leydig cell-rich fractions in Metrizamide gradients has allowed complete Leydig cell purification, which is not achieved by density gradient centrifugation alone. Since less responsive or inactive Leydig cells displayed various degrees of structural damage, such cells should not be considered as a population of physiological significance.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Changes in ribonucleic acid polymerase activities in gonadotropin-treated Leydig cells: an estradiol-mediated process.

The effects of desensitizing doses of hCG on the activities of Leydig cell DNA-dependent RNA polymerases I, II, and III were investigated after optimization of the enzyme assay. Individual activities were obtained by taking advantage of their different sensitivities to alpha-amanitin. RNA polymerase III was a minor component of the alpha-amanitin-resistant activity at 0.10 micrograms/ml, and was therefore measured together with RNA polymerase I. In adult rats treated with 10 micrograms hCG, sc, RNA polymerases II and I plus III activities of Leydig cells rose within 45 min to 180 +/- 6% and 162 +/- 2% of the control value, and then decreased to control values at 60 min. The initial stimulation of polymerase activities was coincident with maximal increases in testosterone and estradiol levels in plasma. A second and more sustained increase in polymerases II and I plus III activities occurred between 12 and 24 h (212 +/- 12% and 180 +/- 10% of the control value) and was maintained until 48 h after hCG injection. The hCG-induced rise in polymerase activities was due to activation of the enzymes, since chromatin template capacity was unaltered. In animals treated with the antiestrogen tamoxifen, stimulation of RNA polymerase activity by hCG was completely inhibited. Also, hCG did not stimulate polymerase activity in animals treated with aminoglutethimide, which blocks steroid synthesis from cholesterol, or in those treated with adrostatriendione, which inhibits aromatization of testosterone leading to estradiol. Increases in RNA polymerase activities were also achieved by the administration of lower doses of hCG (0.1 and 1 micrograms) and the administration of estradiol (2 micrograms), resembling the pattern seen with 10 micrograms hCG. These studies have indicated that the hCG-induced RNA polymerase activation in the Leydig cell is mediated through nuclear actions of estradiol, since stimulation of the enzymes was prevented by tamoxifen and inhibition of steroid biosynthesis, and was induced by estradiol administration.

Animals↗
PubMedDate not supplied

Radioimmunoassays of human luteinizing and follicle-stimulating hormones using the same radioactive tracer.

The National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) provides the hFSH-I-3 preparation, to be employed as a tracer in the radioimmunoassay (RIA) of human follicle-stimulating hormone (hFSH). The contaminating LH contained in that preparation led us to study whether the iodination of such a material could render it a suitable tracer for RIA of both LH and FSH. hFSH-I-3 was labelled with 125I by the chloramine-T method and was further purified on Sephadex G-75 column. The LH-RIA was performed using this preparation and anti-LH at a final dilution of 1:37,500, with a sensitivity of 3 mIU LH/ml (2nd international reference pattern). The method was validated by comparing the LH values obtained in different serum samples with those obtained using the standard RIA (125I-LH/anti-LH); the correlation coefficient (r) was equal to 0.9988. No LH overestimation due to the putative cross-reaction with FSH was found. This was demonstrated by testing serum samples containing high (greater than 100 mIU/ml) and low (less than 10 mIU/ml) concentrations of FSH before and after the treatment with anti-LH. Under these conditions, serum samples from postmenopausal women, pregnant women, normal men and women in basal conditions and after the LH-RH administration, and from a patient with Klinefelter's syndrome, were evaluated. In conclusion, NIDDK 125I-hFSH-I-3 can be used as a tracer for the radioimmunological quantitation of both hLH and hFSH, which results not only inexpensive, but also allows to reduce the amount of the stored radioactive materials.

Follicle Stimulating Hormone↗
PubMedDate not supplied

Failure of the müllerian regression factor in two patients with complete androgen insensitivity syndrome.

The present paper describes the histological and endocrinologic features of 2 subjects with 46,XY karyotype affected by complete androgen insensitivity syndrome (AIS) with müllerian structures. Both patients had fallopian tubes, but only one had also uterus and presented a seminoma. Serum levels of luteinizing hormone, testosterone and estradiol were high or in the upper part of normal limits, whereas levels of follicle-stimulating hormone were normal. The association between AIS and the presence of müllerian structures observed in these 2 patients might be explained by an impaired synthesis of müllerian regression factor or by a failure in its mechanism of action.

Adolescent↗
PubMedDate not supplied

Altered testicular hormone production in infertile patients with idiopathic oligoasthenospermia.

We studied the kinetics of testicular response to human chorionic gonadotropin (hCG) in oligoasthenospermic and asthenospermic patients (OAZ-AZ). The responses of testosterone (T), androstenedione (A), 17 OH-progesterone (17OHP), and estradiol (E2) were evaluated in 60 OAZ-AZ patients and compared to those of 10 normal men. The responses of T, A, and 17OHP to hCG in the control group displayed a biphasic pattern with an initial peak at 4 hours and a second peak after 24 hours. The E2 response showed a single peak between 24 and 48 hours after hCG administration. OAZ-AZ patients had two types of T responses: group 1 (n = 40) had no first peak and group 2 (n = 20) had a normal response pattern. The response of A was similar to that of T, and the E2 response was normal in both groups. There were three types of 17OHP responses in group 1 (low, high, or normal); however, the 17OHP response was normal in group 2. Treatment of group 1 with aromatase inhibitors (aminoglutethimide or testolactone) induced an improvement of the acute T response only in patients with high or normal 17OHP response to hCG, whereas no effects were observed in patients with low 17OHP response. In group 2, the aromatase inhibitors induced no changes in the T response. These results demonstrate that in some OAZ-AZ patients (group 1, blunted T response) testicular hormone production is altered. They also suggest the presence of two enzyme blocks: one at the 17,20 desmolase level, mediated by E2, and another at early biosynthetic steps, not mediated by E2.

Adult↗