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L L Ewing

Publications and source records attributed to L L Ewing.

At least 37 records · Page 2Linked to original sources
PubMed1989

Maintenance of testosterone production by purified adult rat Leydig cells for 3 days in vitro.

Using a preparation of highly purified, adult rat Leydig cells and conditions of culture which we found to optimize testosterone production during 24 h, we sought to maintain optimal testosterone production for 3 d. Leydig cells cultured on Cytodex 3 beads at 19% O2 in Dulbecco's modified Eagle's medium-Ham's nutrient mixture F12 (1:1; vol/vol) containing 0.5 mg/ml total bovine lipoproteins (less than 1.222 g/ml) with maximal luteinizing hormone (LH) stimulation failed to maintain a constant amount of testosterone for 3 d. These cells did however secrete a similar amount of total delta 4-3-ketosteroids on each of the 3 culture d, indicating that their viability was preserved. The predominance of progesterone and 170H-progesterone relative to the amount of androstenedione found on Days 2 and 3 suggested that the activity of the cytochrome P450 C17-hydroxylase-C17,20-lyase enzyme in the smooth endoplasmic reticulum was diminished when Leydig cells were maintained in our primary culture for longer than 24 h. Decreasing the oxygen tension of the cultures from 19 to 5%, and decreasing the concentration of LH used to stimulate the Leydig cells from 100 to 0.1 ng/ml, were necessary to achieve maintenance of testosterone secretion without accumulation of other delta 4-3-ketosteroids during a 3-d period. Cells cultured in this fashion were still able to respond to maximal LH stimulation during Day 3, producing as much testosterone as if cultured for 24 h on Day 1 at 19% O2 with 100 ng/ml LH stimulation.

17-alpha-Hydroxyprogesterone↗
PubMed1989

Kinetic studies on the development of the adult population of Leydig cells in testes of the pubertal rat.

The objective of this study was to determine whether postnatal increases in rat Leydig cell number result from differentiation of precursor cells, division of existing Leydig cells, or both. Our approach was 1) to examine changes in the absolute number of Leydig cells and potential precursor cells (macrophages, pericytes, and mesenchymal, endothelial, and myoid cells) per testis on day 19 of gestation (day -2) and days 7, 14, 21, 28, and 56 postpartum; 2) to examine the frequency with which mesenchymal and Leydig cells divide during prenatal and postnatal development; and 3) to identify and examine the fate of the progeny of Leydig and mesenchymal cell divisions during prenatal and postnatal development. Stereological methods were used to show that mesenchymal cells comprised 44% of the total interstitial cell population and Leydig cells 16% on day -2, whereas by day 56 postpartum the relationship had reversed; mesenchymal cells comprised 3% and Leydig cells 49%. These results suggested a precursor-product relationship between mesenchymal and Leydig cells because no such reciprocal relationship was observed between Leydig cells and macrophages, pericytes, endothelial, or myoid cells. Autoradiographic analysis of [3H]thymidine incorporation into mesenchymal and Leydig cells was consistent with this interpretation. In a series of pulse-chase experiments, the percentage of labeled mesenchymal and Leydig cells was measured after a single injection of [3H]thymidine on days 2, 14, 28, and 56 postpartum, each followed by sampling at timed intervals (between 1 h and 14 days) thereafter. Starting on day 14, the percentage of labeled Leydig cells was approximately 1% immediately after injection of [3H]thymidine and increased significantly to approximately 6% by 6 days after injection. No such increase was observed when rats were similarly injected starting on days 2, 28, and 56 postpartum. The rise in Leydig cell labeling between days 14 and 28 postpartum did not result in a decline in the number of silver grains over labeled Leydig cell nuclei, indicating that the increase in the percentage of labeled cells was not caused by Leydig cell division. These observations led us to conclude that the increase in Leydig cell labeling from days 14 to 28 was the result of recruitment from a compartment of labeled mesenchymal cells. In contrast, our analysis indicated that from day 28 postpartum and thereafter until the mature number of Leydig cells is attained, Leydig cells are generated by division of morphologically recognizable Leydig cells.

Animals↗
PubMed1989

Restoration of advanced spermatogenic cells in the experimentally regressed rat testis: quantitative relationship to testosterone concentration within the testis.

We examined the effect of exogenously administered testosterone (T) on the quantitative restoration of advanced spermatogenic cells in adult rat testes rendered azoospermic by treating rats with polydimethylsiloxane (PDS) implants of T and estradiol (E). Experimental rats received PDS-TE implants for an initial 8-week period; control rats received empty implants. By 8 weeks of PDS-TE treatment, rats became severely oligospermic, and the T concentration within the seminiferous tubule fluid (STF) was reduced approximately 80% (from 57.8 ng/ml in controls to 9.6 ng/ml). After the initial 8-week PDS-TE treatment, PDS-TE implants were removed from one group of rats; a second group of PDS-TE-implanted rats received an additional PDS-T implant of 24 cm. Eight weeks after the removal of PDS-TE implants or the implantation of additional T, testis weight and numbers of advanced spermatogenic cells were restored to those of control rats. The STF T concentration 8 weeks after the removal of PDS-TE implants also was restored to that in control rats. In contrast, the STF T concentration increased to only 40% of control values in the rats that received an additional T implant. Despite this 60% reduction in T concentration compared to the control value, advanced spermatogenic cell number was restored to a value indistinguishable from that of intact controls. These observations indicate that spermatogenesis can be quantitatively restored in PDS-TE-implanted rats with exogenously administered T, and moreover, that this restoration does not require the high T concentration found in the STF of intact control rats.

Animals↗
PubMed1989

Maintenance of advanced spermatogenic cells in the adult rat testis: quantitative relationship to testosterone concentration within the testis.

The studies described herein were designed to examine whether there is a threshold concentration of testosterone (T) within the seminiferous tubules that is required to maintain spermatogenesis in the rat, or alternatively, whether there is a dose-response relationship between the intratesticular T concentration and the maintenance of spermatogenesis. T was administered to intact adult male rats via sustained release polydimethylsiloxane capsules in order to experimentally clamp T at well defined concentrations within the seminiferous tubules. Implantation of T-filled capsules of increasing sizes resulted in linear increases in T concentrations in serum, interstitial fluid, and seminiferous tubule fluid (STF). We examined the effect of step decreases in intratesticular T concentration on the numbers of advanced spermatogenic cells maintained by the testis over a 2-month period. Quantitatively complete spermatogenesis was maintained despite an 80% reduction in the STF T concentration (to approximately 13 ng/ml) from control values. The ability of the testis to maintain complete spermatogenesis was extremely sensitive to further decreases in STF T concentration. Thus, reduction of the STF T concentration from approximately 13 to 9 ng/ml resulted in a reduction in the number of advanced spermatids that were maintained in the testis by approximately 100 x 10(6). Reduction of the STF T concentration to approximately 4 ng/ml resulted in a further reduction in the number of advanced spermatids per testis by 100 x 10(6). Taken together, these data support the contention that there is far more T present within the seminiferous tubules of intact rat testes than is required to maintain quantitatively normal spermatogenesis and reveal for the first time that there is a dose-response relationship between the STF T concentration and the quantitative maintenance of advanced spermatogenic cells in the rat testis.

Animals↗
PubMed1988

Comparison of components of the testis interstitium with testosterone secretion in hamster, rat, and guinea pig testes perfused in vitro.

Components of the testis and cytoplasmic organelles in Leydig cells were quantified with morphometric techniques in hamster, rat, and guinea pig. Testosterone secretory capacity per gram of testis and per Leydig cell in response to luteinizing hormone (LH) (100 ng/ml) stimulation was determined in these three species from testes perfused in vitro. Numerous correlations were measured among structures, and between structures and testosterone secretion, to provide structural evidence of intratesticular control of Leydig cell function. Testosterone secretion per gm testis and per Leydig cell was significantly different in the three species: highest in the guinea pig, intermediate in the rat, and lowest in the hamster. The volume of seminiferous tubules per gm testis was negatively correlated, and the volumes of interstitium, Leydig cells, and lymphatic space per gm testis were positively correlated with testosterone secretion. No correlations were observed between volumes of blood vessels, elongated spindleshaped cells, or macrophages per gm testes and testosterone secretion. The average volume of a Leydig cell and the volume and surface area of smooth endoplasmic reticulum (SER) and peroxisomes per Leydig cell were positively correlated, and the volume of lysosomes and surface area of inner mitochondrial membrane per Leydig cell were negatively correlated with testosterone secretion. No correlations were observed between volume and surface area of rough endoplasmic reticulum (RER), Golgi apparatus, and lipid, and volume of ribosomes, cytoplasmic matrix, and the nucleus with testosterone secretion per Leydig cell. These results suggest that Leydig cell size is more important than number of Leydig cells in explaining the difference in testosterone-secreting capacity among the three species, and that this increase in average volume of a Leydig cell is associated specifically with increased volume and surface area of SER and peroxisomes. An important unresolved question is what is the role of peroxisomes in Leydig cell steroidogenesis.

Analysis of Variance↗
PubMed1988

Optimizing testosterone production by purified adult rat Leydig cells in vitro.

We sought to establish conditions that increased the duration of testosterone production by fully differentiated adult rat Leydig cells in primary culture. A freshly isolated suspension of highly purified adult rat Leydig cells produced 83 ng testosterone/10(6) Leydig cells.h-1 when incubated in Medium 199 in a 1.5 ml microfuge tube with shaking for 3 h with a maximally stimulating concentration of ovine luteinizing hormone (LH). Unfortunately, adult rat Leydig cells that were allowed to attach only to a plastic culture dish flattened out, and testosterone production diminished rapidly. Leydig cells in Dulbecco's modified Eagles' medium-Ham's F12 (1:1; vol/vol) containing Cytodex 3 beads pre-equilibrated in culture medium containing fetal bovine serum attached to the beads and remained viable, but produced only 30 ng testosterone/10(6) Leydig cells.h-1 when incubated for 24 h with similar stimulation. Leydig cells similarly cultured and maximally stimulated with LH, responded to bovine lipoproteins (less than 1.222 g/ml) producing 105 ng of testosterone/10(6) Leydig cells.h-1 when incubated with 1 mg/ml bovine lipoprotein. Therefore, lipoproteins maintain the steroidogenic capacity of purified adult rat Leydig cells in primary culture for 24 h.

Animals↗
PubMed1988

Identification of phytoestrogens in the urine of male dogs.

It is becoming increasingly apparent that dietary factors may play a role in the etiology of hormone dependent neoplasias. It has been hypothesized that estrogens play some role in the etiology of benign prostatic hyperplasia (BPH) in the canine. The presence of estrogen receptor binding activity in a fraction of canine urine purified by high performance liquid chromatography (HPLC) that did not correspond to estriol, estradiol, estrone or any of their primary metabolites was observed in the present study. We used thermospray-mass spectrometry and GC-MS to identify the phytoestrogens daidzein, equol, formononetin and genistein in HPLC purified fractions of urine obtained from male beagles. Using the same techniques we also confirmed the presence of daidzein and genistein in the commercial diet fed to these same dogs. Using the immature rat uterine cytosol estrogen receptor assay, relative binding affinities of 0.08, 1.1, less than 0.01 and 3.9% were obtained for daidzein, equol, formononetin and genistein, respectively when compared to estradiol (100%). In conclusion, phytoestrogens are present in urine of male beagles. Moreover, the commercial diet fed to these dogs contains isoflavones which can be converted to equol by intestinal microflora. These results suggest the need for investigations of phytoestrogens (e.g. equol) excreted into the urine daily and its relationship to the incidence and severity of BPH in the dog.

Animal Feed↗
PubMed1988

Aromatase inhibition in the dog. I. Effect on serum LH, serum testosterone concentrations, testicular secretions and spermatogenesis.

Chronic treatment of intact male beagles with an orally active nonsteroidal aromatase inhibitor (4-(5,6,7,8-tetrahydroimidazo [1,5a] pyridin-5-yl) benzonitrile hydrochloride; CGS 16949; CIBA-GEIGY) at a dosage of 2.5 mg./kg. per day for six months resulted in increased (p less than 0.01) serum LH and testosterone concentrations compared to placebo-fed controls. The increases in serum LH and testosterone concentrations occurred by one week of treatment and were maintained over the six month period. Testes of CGS 16949A fed dogs obtained at termination of the experiment when perfused in vitro in the presence of a maximally stimulating concentration of LH secreted nondetectable amounts of estradiol and estrone and higher (p less than 0.01) amounts of testosterone, androstenedione and dihydrotestosterone than testes of control dogs. Despite these changes in androgen secretion there was no evidence on any effect of aromatase inhibition upon spermatogenesis. These data support the hypothesis that in the dog, estrogens play a major role in negative feedback of the hypothalamic-pituitary-testicular axis.

Animals↗
PubMed1988

Aromatase inhibition in the dog. II. Effect on growth, function, and pathology of the prostate.

To evaluate the role of aromatase inhibitors in the treatment of benign prostatic hyperplasia (BPH), the effects of a potent, oral, nonsteroidal aromatase inhibitor [4-(5,6,7,8-tetrahydroimidazo[1,5a]pyridin-5yl)benzonitrile; CGS-16949A, CIBA-GEIGY] on canine BPH have been studied. Twelve mature beagles with enlarged prostates were divided into two groups of similar age, prostatic size, and prostatic histology. Dogs were given either CGS-16949A at a dosage of 2.5 mg./kg./day p.o. (n = 6) or an oral placebo (n = 6) for 25 weeks. Treatment with aromatase inhibitor had no significant effect on prostatic weight nor on total DNA content when compared to controls (p greater than 0.1). Semen volume did not change with treatment, and prostatic tissue concentrations of testosterone, dihydrotestosterone (DHT), and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) were similar for the treated and control beagles (p greater than 0.1). Protein concentration in the ejaculate, however, was significantly greater for the dogs receiving CGS-16949A (p less than 0.01). Histologically, there was no difference in the incidence or type of BPH between the two treatment groups. Beagles treated with aromatase inhibitor, however, did have a more severe inflammatory infiltrate of the prostatic parenchyma than the control dogs (p less than 0.01). The mean serum testosterone concentration for the beagles treated with aromatase inhibitor was 10 times greater than that for the control animals (p less than 0.01). The endocrine effect of this aromatase inhibitor in the male canine is presented in the preceding paper. Taken together, these results suggest that inhibition of endogenous aromatase activity is not an effective treatment for spontaneous BPH in the intact canine.

Animals↗
PubMed1988

Progesterone secretion by corpora lutea of the isolated perfused rabbit ovary during pseudopregnancy.

An ovarian in vitro perfusion method was adapted to examine rabbit corpus luteum (CL) function during pseudopregnancy. Ovaries were perfused in vitro with tissue culture Medium 199 with or without 3% bovine serum albumin (BSA). Samples were obtained from both arterial and venous cannulae, allowing rates of progesterone secretion to be determined. Two perfusion methods were compared: a closed system in which perfusion medium continuously recirculated through the tissue, and an open system in which the ovarian vein cannula was left outside the perfusion chamber. The addition of 3% BSA was found to prevent edema and distortion of the interstitial space, and to result in increased progesterone secretion. With the closed perfusion system, the progesterone secretion rate measured over the 6-h perfusion was significantly higher on Day 11 than on Day 1 of pseudopregnancy and had declined significantly on Day 18. There was no difference in secretion rates whether the perfusion system was open or closed, and the results obtained with both systems closely approximated in vivo progesterone secretion rates. Measurements of CL tissue progesterone content before and after in vitro perfusion indicated that the changes in progesterone secretion seen during pseudopregnancy resulted from differences in the synthesis and secretion of progesterone and not from leakage of progesterone already present in the CL prior to perfusion. Taken together, these results indicate that the modified in vitro perfused rabbit ovary preparation described herein is an appropriate model to examine progesterone secretion by ovaries bearing CL.

Animals↗
PubMed1988

Effect of adrenocorticotropin and other proopiomelanocortin-derived peptides on testosterone secretion by the in vitro perfused testis.

Several lines of evidence have demonstrated the presence of POMC-derived peptides in testicular tissue. Consequently, this study was designed to determine the effects of ACTH and other POMC-derived peptides on testosterone secretion by the in vitro perfused testis. Infusion of synthetic human ACTH-(1-24) at a concentration of 500 ng/ml increased (P less than 0.01) testosterone secretion over that in nontreated controls by in vitro perfused rabbit and guinea pig testes, but not by rat, hamster, or dog testes. This increase in testosterone secretion after ACTH treatment, however, was less (P less than 0.01) than that after the infusion of a maximally stimulating concentration of ovine LH (100 ng/ml). Using rabbit testes, the testosterone response to ACTH was demonstrated to be dose dependent and specific, since similar responses were observed after the infusion of synthetic human ACTH-(1-39) and purified porcine ACTH-(1-39) and since no response was observed after the infusion of a variety of ACTH fragments. Infusion of alpha MSH, beta-endorphin, or naloxone had no effect (P greater than 0.10) on testosterone secretion by either rabbit or rat testes at the doses used. These data show that rabbit and guinea pig, but not rat, hamster, and dog, testes secrete testosterone in response to ACTH.

Adrenocorticotropic Hormone↗
PubMed1988

The effect of inhibition of aromatase enzyme activity on Leydig cell number and ultrastructure in beagles.

We have shown previously that administration of an orally active competitive aromatase inhibitor 4-(5,6,7,8-tetrahydroimidazo [1,5a] pyridin-5-yl) benzonitrile monohydrochloride to adult male beagles increases peripheral blood LH and testosterone concentrations, and that testes from treated dogs produce more testosterone when perfused in vitro than age-matched controls. In the present study we posed the question of whether the increased testosterone secretion by testes from these same aromatase-treated dogs was due to Leydig cell hypertrophy or hyperplasia, and if the latter, whether cytoplasmic organelles are increased. Beagles were treated with the inhibitor at a dosage of 2.5 mg/kg.day for 25 weeks and were euthanized by an overdose of iv sodium pentobarbital; testes were perfusion-fixed, embedded, and sectioned for stereological analysis. There were no significant differences in testis volume and absolute volumes of seminiferous tubules, blood vessels, lymphatic space, macrophage cells, and mesenchymal cells between the control and treated dogs. In contrast absolute interstitium volume and the absolute volume of Leydig cells per testis were significantly increased (P less than 0.05) in treated dogs. This increased Leydig cell volume per testis was due to increased volume of individual Leydig cells rather than to increases in Leydig cell number per testis. Additional studies showed that the surface area per Leydig cell of smooth endoplasmic reticulum, outer and inner mitochondrial membranes, and membranes of lipid droplets per testis were significantly higher (P less than 0.05) in the treated dogs as compared to the controls. In summary, the results of this study lead us to conclude that aromatase inhibition in the mature dog causes Leydig cell hypertrophy rather than hyperplasia and increased surface area per Leydig cell of subcellular organelles that contain enzymes involved in steroid biosynthesis.

Animals↗
PubMed1988

Effect of long term deprivation of luteinizing hormone on Leydig cell volume, Leydig cell number, and steroidogenic capacity of the rat testis.

Leydig cells atrophy, losing cytoplasmic volume and the capacity for testosterone secretion, within 1-2 weeks of LH deprivation. We investigated the effects of long term (0-16 weeks) LH deprivation on the volume of an average Leydig cell, the volume of Leydig cells per testis, the number of Leydig cells per testis, and testosterone secretion by in vitro perfused testes. Endogenous LH was suppressed in adult rats by testosterone/estradiol-filled (TE) Silastic implants. The presence of Leydig cells in testes was verified by 1) morphological examination using light and electron microscopy, 2) histochemical localization of 3 beta-hydroxysteroid dehydrogenase activity (3 beta HSD), and 3) conversion of pregnenolone to progesterone by in vitro perfused testes. Marked quantitative differences existed in Leydig cell morphology among control and treated rats. The volume of an average Leydig cell and the total volume of Leydig cells per testis decreased (P less than 0.01) rapidly and progressively after TE implantation. At 16 weeks, the average Leydig cell lost 90% of its cytoplasmic volume and 65% of its nuclear volume. Analysis of variance failed to detect a significant decline in Leydig cell number per testis, despite a 16% reduction from the value in control rats (22.2 +/- 1.5 x 10(6)) in rats treated for 16 weeks (18.7 +/- 1.5 x 10(6)). After TE implantation, LH-stimulated testosterone secretion by in vitro perfused testes diminished (P less than 0.01) rapidly to 5% of the control values at 1 week and less than 0.3% of the control value from 4-16 weeks. In contrast, 25% of 3 beta HSD activity was retained (P less than 0.01 vs. controls) at 16 weeks, based on the rate of pregnenolone conversion to progesterone. Moreover, testes of treated rats secreted progesterone at a rate twice that of controls, when the steroid secretion rates were expressed per volume of Leydig cell cytoplasm. Loss of the testosterone-secreting capacity of testes after LH withdrawal was associated with a loss in the volume, but not a significant loss in the number, of Leydig cells. Thus, LH was required to maintain the differentiated structure and function of Leydig cells, but was not required to maintain the overwhelming majority of Leydig cells in the adult rat testis through 16 weeks. Moreover, at least one steroidogenic enzyme, 3 beta HSD, was retained by Leydig cells after long term LH deprivation.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Initial trial of slow-release testosterone microspheres in hypogonadal men.

The duration of hormone release from testosterone-loaded poly(DL-lactide-co-glycolide) microspheres was assessed in eight hypogonadal men. Eight weeks before receiving testosterone (T) microspheres, patients were withdrawn from their prior androgen therapy in order to minimize the contribution of that therapy to hormone levels measured following microsphere injection. Blood was obtained for hormone measurements immediately before and weekly for 14 weeks after the administration of the T microspheres. A biphasic release of hormone was observed; a significant increase in total T and free T concentration (compared to preinjection values) was observed during week 1 (P less than 0.01) and between weeks 6 and 11 after injection (P less than 0.05). A similar pattern was detected in estradiol and dihydrotestosterone concentrations, whereas T-binding globulin levels showed a small, reciprocal fall after the injection of microspheres (P = not significant [NS]). After receiving the T microspheres, seven of eight hypogonadal men reported the return of normal sexual function; the eight patient withdrew from the study after 5 weeks. It was concluded that T microspheres are a promising new method of androgen replacement therapy for hypogonadal men.

Adult↗
PubMed1987

Leydig cell differentiation during maturation of the rat testis: a stereological study of cell number and ultrastructure.

In an effort to further understand the basis for the changes in steroidogenesis known to occur during sexual maturation in the rat, we examined by quantitative morphologic methods the number and ultrastructure of Leydig cells in fetal rats (days 18-20 of gestation) and in rats from days 2 to 3 of age through adult. Quantitative light microscopic analyses indicated that Leydig cell number, when expressed per unit volume of testis, was very high in fetal rat testes, fell significantly in testes of days 2 to 3 rats, and subsequently rose significantly. When Leydig cell number was expressed per testis rather than per unit volume of testis, the results indicated that testes of fetal rats and rats of days 2 to 3 contained the same number of Leydig cells; after the neonatal period, significant increases in Leydig cell number per testis occurred in concert with increases in testis weight. Quantitative electron microscopic studies revealed significant differences in the ultrastructure of fetal and adult populations of Leydig cells. For example, Leydig cells of fetal and neonatal rats contained abundant lipid, whereas Leydig cells of weeks 7 to 8 and adult rats contained little. Stereological analyses also revealed dramatic changes in smooth endoplasmic reticulum and inner mitochondrial membrane surface areas during sexual maturation, both per cell and per testis. These findings are discussed with respect to the steroidogenic capacity of the testis during sexual maturation.

Animals↗
PubMed1987

Photoperiodic variation of Leydig cell numbers in the testis of the golden hamster: a possible mechanism for their renewal during recrudescence.

Golden hamster testes regress after short day exposure. The present study asks: 1) are Leydig cell numbers depleted during short days, and 2) if so, how are they replenished during recrudescence. Control hamsters were shown 14 h of light and 10 h of dark (LD 14:10) for 10 weeks (n = 12). Testicular regression was induced by LD 6:18 for 10 weeks (n = 4), and recrudescence by switching regressed hamsters to LD 14:10 for 3 and 5 weeks (n = 8 for each group). All hamsters were injected with [3H]thymidine [3 microCi/gm body wt., intraperitoneally (i.p.)] 1 h or 2 weeks before sacrifice. Leydig cell number per testis was determined by stereological analysis of sections of perfusion-fixed testes, and labeling indices were determined by autoradiography. Leydig cell numbers were reduced significantly from 18.2 X 10(6) in control to 9.0 X 10(6) in regressed testes (p less than 0.05); then increased to 14.0 X 10(6) and 17.9 X 10(6) in 3- and 5-week recrudesced hamsters. The labeling index was nondetectable (n.d.) for regressed hamsters. In control and recrudescing hamsters the labeling index was measured at two times (t1 = 1 h vs. t2 = 2 weeks post-injection): in controls, t1 = 0.22 +/- 0.15% (mean +/- SEM) vs. t2 = 0.28 +/- 0.22%; in 1 week recrudesced, n.d. vs. 1.92 +/- 0.77% (p less than 0.05); at 3 wk, n.d. vs. 4.58 +/- 1.74% (p less than 0.05); at 5 weeks, 1.92 +/- 0.61% vs. 2.25 +/- 0.59%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure.

Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.

Aldehyde-Lyases↗