Biomedical subjects

F F Rommerts

Publications and source records attributed to F F Rommerts.

At least 37 records · Page 2Linked to original sources

PubMed1990

Short-term stimulatory effect of Sertoli cell conditioned medium on Leydig cell steroidogenesis is not mediated by inhibin.

Addition of concentrated rat Sertoli cell conditioned medium (rSCCM) to isolated Leydig cells from immature rats stimulated steroid production more than 13-fold within 4 h. LH-stimulated steroidogenesis was not enhanced by addition of rSCCM. The biological activity of the concentrated rSCCM was higher after incubation of Sertoli cells with FSH, whereas FSH alone did not stimulate steroid production. This effect of rSCCM was not due to inhibin, since highly purified 32 kDa rat inhibin, in doses equivalent to those present in rSCCM, had no effect on steroidogenesis during the 4 h incubation period. Furthermore, inhibin could be separated from the Leydig cell stimulating factor by anion-exchange chromatography. These results indicate a short-term paracrine control of Leydig cell steroidogenesis by Sertoli cell derived factors, which differ from inhibin.

Animals↗

PubMed1990

Regulation of steroidogenesis in subclones of the MA-10 mouse Leydig tumor cell line.

We have previously reported the isolation of a subclone of the MA-10 mouse Leydig tumor cell line (MA-10 LP) which secretes less than 10% of the steroid synthesized by the parent, accumulates comparable amounts of cAMP and has equivalent cholesterol side-chain cleavage activity as the parent population (Kilgore and Stocco (1989) Endocrinology 124, 1210-1216). In the present study we show that addition of exogenous sterol carrier protein 2 (SCP2) to isolated mitochondria was not able to overcome the deficient steroid response of MA-10 LP. We have also demonstrated that human chorionic gonadotropin (hCG)-stimulated cellular events which activate steroid production by subsequently isolated mitochondria require ongoing protein synthesis, release of intracellular calcium and are mediated through the calcium-calmodulin complex. Additionally, mitochondrial sonicates from hCG-stimulated parent cells were able to stimulate steroid production by intact mitochondria isolated from unstimulated parent cells, whereas sonicates from similarly treated MA-10 LP had no effect on steroid production in these mitochondria. Together these data suggest that hCG induces changes in the mitochondria of the parent stock which are not induced to the same extent in the mitochondria of MA-10 LP.

Animals↗

PubMed1990

Immunohistochemical detection of transforming growth factor-alpha in Leydig cells during the development of the rat testis.

In this paper the localization of transforming growth factor alpha (TGF-alpha) is described in the rat testis at various stages throughout development, e.g. neonatal, prepubertal, and adult, in order to examine somatic cells and germinal cells at different stages of differentiation. This was done by immunoperoxidase staining using a monoclonal antibody that does not cross-react with epidermal growth factor (EGF). In sections of testes from neonatal rats, intense staining was present in Leydig cells. In the cells of the seminiferous tubules the staining was faint or undetectable. At the time when many mesenchymal cells differentiate into Leydig cells in the 21-day-old rat, TGF-alpha was visualized in most but not all of the identifiable Leydig cells. In interstitial cell cultures derived from 21-day-old rats, the majority of the Leydig cells contained TGF-alpha, but in a proportion of the Leydig cells TGF-alpha was undetectable. No staining was apparent in Sertoli cells and germ cells in seminiferous tubules or in Sertoli cell cultures derived from 21-day-old rats. Under these in vitro conditions it was found that peritubular-myoid cells also possessed TGF-alpha immunoreactivity. In the adult testis all Leydig cells stained positively for TFG-alpha, whereas no staining was found in the cells of the seminiferous tubules. Treatment of adult rats with ethylene-1,2-dimethane-sulfonate (EDS) resulted in the destruction of Leydig cells and the loss of all positively stained for TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Development of a new Leydig cell population after the destruction of existing Leydig cells by ethane dimethane sulphonate in rats: an autoradiographic study.

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [3H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [3H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [3H]thymidine at days 2, 3 or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered. In a second series of experiments, [3H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Effects of hypophysectomy and human chorionic gonadotrophin on Leydig cell function in mature rats.

The biochemical activities involved in the maintenance of Leydig cell functions, and the effects of hypophysectomy and human chorionic gonadotrophin (hCG) on these functions are largely unknown. In the present study, adult hypophysectomized rats were used as a model to determine the effects of these treatments on a number of biochemical and morphological parameters. After 33 days of hypophysectomy, the morphology of the Leydig cells had been drastically altered. In addition, alpha-naphthol and beta-naphthol esterase activity as well as the steroidogenic capacity of the Leydig cells were greatly reduced at this time. In contrast, the level of sterol carrier protein 2 (SCP2), a Leydig cell-specific protein, was affected by hypophysectomy much less than the other parameters measured. Two daily injections of hCG to rats hypophysectomized for 31 days resulted in no change in the morphology of the Leydig cells, or in their proliferative activity. Non-specific esterase activities were also unaffected by 2 days of treatment with hCG. However, two injections of hCG to rats hypophysectomized for 31 days resulted in nearly complete restoration of steroidogenic capacity, and a 3.5-fold increase in the level of SCP2. These findings indicate that hypophysectomy results in significant morphological and biochemical changes in Leydig cells, and that hCG is capable of restoring some of these capacities within a short time.

3-Hydroxysteroid Dehydrogenases↗

PubMed1989

The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland.

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membrane-free cytosol as compared to 19% in the adrenal gland. In the latter tissue, the transfer protein is mainly found in the lysosomal/peroxisomal and the microsomal fraction at a level of 1093 and 582 ng per mg total protein, respectively (i.e., 17% and 35% of the total), and to a lesser extent in the mitochondrial fraction (11% of the total). Of all the membrane fractions isolated, the microsomal fraction from the liver and the mitochondrial fraction from the adrenal gland have the lowest levels of the transfer protein (i.e., 168 ng and 126 ng per mg total protein, respectively). These low levels correlate poorly with the active role proposed for this transfer protein in the conversion of cholesterol into bile acids and steroid hormones in these fractions. Using immunoblotting, it was demonstrated that in addition to the transfer protein (14 kDa) a cross-reactive 58 kD protein was present in the supernatant and the membrane fractions of both tissues. Cytochemical visualization in adrenal tissue with specific antibodies against the nonspecific lipid transfer protein showed that immunoreactive protein(s) were present mainly in the peroxisome-like structures.

Adrenal Glands↗

PubMed1989

Albumin, a biologically active protein acting on Leydig cells.

The objective of the present study was to characterize further the albumin fraction of rat testicular fluid (rTF), which can enhance luteinizing hormone (LH)-stimulated pregnenolone production by immature Leydig cells in vitro. Testicular fluid, obtained from 300 rat testes was fractionated by sequential ammonium sulfate precipitation, dye-affinity, anion-exchange chromatography, chromatofocussing and an additional heat treatment. The final fraction showed a single band when analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel and isoelectric focussing gel. The protein had a molecular weight of 67 kDa, an isoelectric point of 5.0 and was identified as albumin after Western blotting using an antibody against rat serum albumin. Albumin in this fraction gave a dose-dependent (0.1-2% protein, w/v) increase in LH-induced pregnenolone production, up to 4-fold, and the increase in specific bioactivity when compared to rTF was 1.4-fold. Selective depletion of albumin from testicular fluid was used as another approach to confirm that albumin itself is the main biologically active component in rTF. rTF from mutant analbuminemic rats (albumin content 0.02 mg/ml) did not stimulate LH-induced steroid production in our assay, in contrast to rTF from normal rats (albumin content 40 mg/ml). Albumin fractions obtained from rat, bovine and human sera were also effective in stimulation of steroid production in the presence of LH, in contrast to chicken serum albumin which gave no stimulation. The stimulatory effect of albumin is not caused by bound fatty acids, nor by the presence of modified forms of albumin such as testibumin or the albumin-bilirubin complex. Our results indicate that Leydig cells are more active in steroid production when surrounded by high but physiological concentrations of albumin.

Albumins↗

PubMed1989

Measurement of steroidogenesis in rodent Leydig cells: a comparison between pregnenolone and testosterone production.

The efficiency and specificity of inhibition of pregnenolone metabolism in mature, immature rat Leydig cells, mouse and tumour Leydig cells by SU-10603, a 17 alpha-hydroxylase inhibitor and epostane (WIN-32729), a 3 beta-hydroxysteroid dehydrogenase inhibitor, were studied. Metabolism of [14C]pregnenolone by mature rat Leydig cells was inhibited for more than 95% in the presence of 20 microM SU-10603 and 5 microM epostane. The sum of the different steroids produced by Leydig cells from immature rats incubated in the presence of a 5 alpha-reductase inhibitor was only 80% of pregnenolone production in the presence of SU-10603 and epostane. Pregnenolone metabolism could also be inhibited in tumour Leydig cells but not in mouse Leydig cells. Pregnenolone and testosterone production by Leydig cells from mature rats were similar when steroidogenesis is maximally stimulated by luteinizing hormone (LH). However, in the presence of LH and bovine serum albumin (bSA), or 22 R-hydroxycholesterol and bSA, pregnenolone production was 1.7- and 6-fold higher respectively, than testosterone production. The data show that for measuring the steroidogenic activity of Leydig cells estimation of pregnenolone production is more reliable than measuring testosterone production. At high activities of the cholesterol side-chain cleavage (CSCC) the conversion of pregnenolone into testosterone may become the rate-limiting step for testosterone production. Under all conditions the conversion of cholesterol into pregnenolone is the (hormonal regulated) rate-determining step for steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗

PubMed1989

Leydig cell number and function in the adult cynomolgus monkey (Macaca fascicularis) is increased by daily hCG treatment but not by daily FSH treatment.

Daily treatment of adult cynomolgus monkeys with 450 i.u. hCG for 16 days resulted in a significant 163% increase in the number of Leydig cells, and a 9-fold rise in plasma testosterone concentrations. The number of proliferating Leydig cells was very low, even after 16 days of treatment with hCG. Daily FSH administration (2 injections of 15 i.u. per day) did not have any effect on the number of Leydig cells or plasma testosterone values. It can be concluded, therefore, that in adult cynomolgus monkeys daily hCG treatment results in an increase in the number of Leydig cells, which is mainly caused by the differentiation of precursor cells. Since plasma testosterone concentrations were increased to an even higher extent, the steroid production per Leydig cell was also stimulated.

Animals↗

PubMed1989

Effects of pure FSH and LH preparations on the number and function of Leydig cells in immature hypophysectomized rats.

The effects of pure FSH and/or LH preparations on the number of Leydig cells and their function in immature hypophysectomized rats have been investigated. As a result of hypophysectomy at the age of 17-18 days, the number of recognizable Leydig cells per testis decreased, as did the steroidogenic capacity in vivo and in vitro. Treatment with 64 micrograms FSH on both 22 and 23 days of age, did not affect the number of recognizable Leydig cells. In contrast, two injections of LH (10 micrograms) caused a sixfold increase in the number of Leydig cells, but had a negative effect on spermatogenesis. These stimulatory and inhibitory effects of LH diminished when FSH was added. Treatment with FSH for 7 days caused a twofold increase in the number of Leydig cells when compared with hypophysectomized controls. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and esterase activity in Leydig cells also increased under the influence of FSH. The pregnenolone production per Leydig cell in the presence of 5-cholesten-3 beta,22(R)-diol (22R-hydroxycholesterol) as substrate showed a sevenfold increase. Plasma testosterone levels 2 h after injection of human chorionic gonadotrophin in intact rats and hypophysectomized FSH-treated rats were the same. Following LH treatment for 7 days, the number of Leydig cells proved to be 11 times higher, and 3 beta-HSD and esterase activity were not different from intact controls. The testicular pregnenolone production was four- to fivefold higher when compared with untreated hypophysectomized rats. However, pregnenolone production per Leydig cell in LH-treated rats was only slightly different from the hypophysectomized controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Proliferation and differentiation of possible Leydig cell precursors after destruction of the existing Leydig cells with ethane dimethyl sulphonate: the role of LH/human chorionic gonadotrophin.

The influence of LH levels on the proliferation and differentiation of possible Leydig cell precursors was investigated in adult rats, after the destruction of the existing Leydig cells with the cytotoxic drug ethane dimethyl sulphonate (EDS). In rats bearing a testosterone implant which prevented the rise in plasma LH levels and kept them within the normal range after the destruction of the Leydig cells, the proliferative activity of possible Leydig cell precursors still increased seven- to eightfold 2 days after EDS administration. Apparently, in this situation, locally produced factors, and not LH, may play a role in the stimulation of proliferation. The proliferative activity of the possible precursor cells could be further stimulated by treating rats with daily injections of human chorionic gonadotrophin (hCG) following EDS administration. It was concluded that the proliferative activity of possible Leydig cell precursors is probably regulated by both paracrine and endocrine factors. Almost no Leydig cells were formed in the rats bearing a testosterone implant during the first 4 weeks after EDS administration. When these rats were treated with hCG, starting 28 days after administration of EDS, a substantial number of Leydig cells was found after 2 days, and these cells also showed 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and alpha-naphtyl esterase (alpha-NE) activity. When hCG treatment was started at 14 or 21 days after EDS administration, some cells with the nuclear characteristics of Leydig cells were present after 2 days, but no 3 beta-HSD or alpha-NE activity could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1989

Transplantation and subsequent recovery of small amounts of isolated Leydig cells.

A technique is described for transplantation of a few million Leydig cells in a gelatin sponge. Within 7 days after subcutaneous transplantation in the neck, a spongeous tissue develops within the gelatin matrix. This tissue contains Leydig cells, fibroblasts, and blood vessels. Isolated Leydig cells were recovered after collagenase dispersion of this tissue. Steroid production by these isolated cells can be stimulated by LH, but the response is less than with fresh Leydig cells isolated directly from testicular tissue. The biochemical properties of Leydig cells devoid of surrounding Sertoli cells can be applied to long-term studies.

Animals↗

PubMed1989

Turnover time of Leydig cells and other interstitial cells in testes of adult rats.

The aim of this study was to investigate the turnover of Leydig cells and other interstitial cells in the adult rat testis. Normal adult rats received injections of [3H]thymidine at 9:00 and 21:00 for 2, 5, or 8 days. The percentage of labeled Leydig cells, which was initially low (0.8% +/- 0.2%), gradually increased during treatment to 1.4% +/- 0.3%. The percentage of labeled peritubular cells was considerably higher and increased from 1.4% +/- 0.1% to 3.5% +/- 0.4% during [3H]thymidine treatment. The remaining interstitial cells were the most actively proliferating cells: the percentage of labeled cells increased from 2.4% +/- 0.2% to 7.5% +/- 0.8% during [3H]thymidine treatment. Leydig cells, peritubular cells, and the remaining interstitial cells in the adult rat testis undergo proliferation. By means of a linear regression analysis and an arcsin transformation, an estimation of the time interval needed to replace various types of interstitial cells was obtained. Taking into account the 95% confidence interval, the turnover time of Leydig cells ranged from 142 to 2823 days. The calculated turnover time for the peritubular cells was 85 to 257 days.

Animals↗

PubMed1988

Intracellular redistribution of SCP2 in Leydig cells after hormonal stimulation may contribute to increased pregnenolone production.

Sterol carrier protein2 (SCP2) also designated non specific lipid transfer protein (nsL-TP), added to tumour Leydig cell mitochondria as a pure compound or in cytosolic preparations, stimulates pregnenolone production two- to three-fold. This stimulation can be abolished by addition of anti rat SCP2 but not by preimmune IgG-antibodies. SCP2- levels in the cytosol are increased in less than two minutes after addition of lutropin (LH). This increased SCP2 level may contribute to stimulation of steroid production in intact cells. After hormonal stimulation the subcellular distribution of SCP2 changes. A two-fold increase of SCP2- levels in the supernatant fraction and four-fold decrease in extracts of the particulate fraction was observed 30 min after stimulation of tumour Leydig cells with LH and subsequent fractionation. This apparent shift of SCP2 can be explained by an altered association with membranes or a true relocation of the protein from the particulate to the supernatant fractions under the influence of the hormone.

Animals↗

PubMed1988

Regulation of sterol carrier protein2 (SCP2) levels in the soluble fraction of rat Leydig cells. Kinetics and the possible role of calcium influx.

The rate-determining step in steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme. The transport of substrate for this reaction may be facilitated by sterol carrier protein2 (SCP2). In rat testis tissue SCP2 is specifically localized in the Leydig cells and tissue levels of SCP2 are regulated by luteinizing hormone (LH). The present study concerns short-term regulation of SCP2 in isolated rat Leydig cells. Levels of SCP2 in the membrane-free supernatant are increased 2-fold already after 2 min incubation with LH and remain elevated for 24 h. The same response occurs with cells preincubated in the presence of cycloheximide for 4 h. SCP2 levels are also 2-fold increased after incubation with dibutyryl cAMP or 4 beta-phorbol 12-myristate 13-acetate (PMA) whereas these compounds stimulate steroid production 5.5- and 2-fold respectively. Luteinizing hormone releasing hormone (LHRH), which can stimulate steroid production more than 3-fold, does not influence SCP2 levels, neither are SCP2 levels altered when LH is added in the presence of the Ca2+-channel blocker diltiazem or in the absence of extracellular Ca2+. A restoration of the LH effect on SCP2 levels was already obtained in the presence of 1 microM extracellular Ca2+. These results suggest that Ca2+ influx through the plasma membrane may play an important role in the control of SCP2 levels. In most of the experiments no correlation between steroid production and SCP2 levels could be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

In vitro effects of ethylene-dimethane sulfonate (EDS) on Leydig cells: inhibition of steroid production and cytotoxic effects are dependent on species and age of rat.

A number of proteins in Leydig cells isolated from immature rats, mature rats and tumour tissue as well as protein in Sertoli cells, hepatocytes and blood plasma are alkylated after incubation of cells with 14C-labelled ethylene-dimethane sulfonate (EDS) for 5 h. LH-stimulated and 22R-hydroxycholesterol-supported steroid production by Leydig cells from immature and mature rats and from rat tumour tissue but not from testes of mature mice was strongly or completely inhibited after incubation with EDS. EDS had no effects on ATP levels in Leydig cells from mature rats after an incubation period of 24 h but ATP levels were almost zero after 72 h. In Leydig cells from tumour tissue the ATP level was decreased to 10% of the original value after 24 h EDS and decreased further during the following 48 h period. In Leydig cells from immature rats EDS had no effect on ATP levels after 72 h incubation. Ultrastructural evidence of cell damage by EDS was observed in cells from mature rats and tumour tissue but not in cells from immature rats. Discrepancies between biochemical and morphological indications for cell damage were noticed after 24 h incubation with EDS but not after 72 h. The results show that EDS exerts a direct inhibitory effect on both mature and immature rat Leydig cells but does not affect LH-stimulated steroid production by mouse Leydig cells. A cytotoxic response to EDS develops in rat Leydig cells during maturation. However, the molecular basis for these very specific effects of EDS on Leydig cells is at present not understood.

Adenosine Triphosphate↗

PubMed1988

The albumin fraction of rat testicular fluid stimulates steroid production by isolated Leydig cells.

Rat testicular fluid (rTF), but not rat serum (rS) or plasma (rP) can further increase within 4 h maximally luteinizing hormone (LH)-stimulated or 22 R-hydroxycholesterol-supported pregnenolone production by immature rat Leydig cells in vitro. This effect of rTF is dose dependent (0.05-1.2% protein, w/v) with an increase up to 4-fold. The objective of the present study was to isolate and characterize the bioactive factor(s) in rTF. After sequential ammonium sulfate fractionation, gel filtration chromatography on Superose 12 and affinity chromatography on concanavalin A-Sepharose it was shown that the albumin fraction was a major biologically active fraction in rTF. The relative specific activity in these fractions was never greater than 1.3-1.4, which is in agreement with the purification factor required to obtain pure albumin from rTF. Commercially obtained albumin fractions from human, bovine and rat sera, up to 99% purity, also increased Leydig cell steroid production more than 3-fold when added in concentrations between 0.1 and 1% w/v in combination with LH or 22R-hydroxycholesterol. Other proteins such as hemoglobin and ovalbumin were not effective in stimulation of steroid production. Bovine serum albumin (bSA, fraction V) at concentrations of 0.25 and 1.0% (w/v), had no or minor effects on LH-stimulated steroid production by rat granulosa cells or adrenocorticotrophic hormone (ACTH)-stimulated steroid production by rat adrenal cells. These findings indicate that albumin itself or minor compounds copurified with albumin represent the main biologically active component in rTF for short-term stimulation of Leydig cell steroid production. Since bioactivity could not be demonstrated in serum which contains similar amounts of albumin as rTF, inhibitory compounds may be present in rat serum.

Albumins↗

PubMed1988

Functional properties of developing rat Leydig cells after treatment with ethylene dimethanesulphonate (EDS).

The development of a new population of Leydig cells after specific elimination of existing Leydig cells in mature rats by ethylene dimethanesulphonate (EDS) was characterized by investigating the testicular activities of 5 alpha-reductase and non-specific esterase, the serum concentrations of 3 alpha-androstanediol and testosterone and the Leydig cell morphology. Plasma concentrations of both androgens were strongly reduced up to 15 days after administration of EDS. Thereafter, in contrast to the gradual and continuous increase of serum testosterone values, the changes in serum 3 alpha-androstanediol were transient, with the highest level on Day 35. The temporal pattern of testicular 5 alpha-reductase activity was almost similar to that of serum 3 alpha-androstanediol. The testicular esterase activity increased gradually from Day 25 until Day 76. The temporal changes in steroid concentrations and enzyme activities after EDS administration indicate that the development of the Leydig cells in EDS-treated rats occurs in a fashion similar to that in pubertal rats. However, the numerous lipid droplets and large nuclei in these Leydig cells indicate that these cells may also be classified as fetal cells. It is concluded that, after treatment with EDS, fetal and pubertal characteristics are present in Leydig cells. It is, however, unknown whether both characteristics are present in one or in two distinct cell populations.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗