F. F. G. Rommerts

Rapid Androgen Actions on Calcium Signaling in Rat Sertoli Cells and Two Human Prostatic Cell Lines: Similar Biphasic Responses Between 1 Picomolar and 100 Nanomolar Concentrations

Abstract Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2–3 min duration) increase in intracellular calcium levels was observed within 20–30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1–1000 pM induced transient calcium increases with ED50 values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED50 values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17β were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.

Occurrence and localization of 5α-steroid reductase, 3α- and 17β-hydroxysteroid dehydrogenases in hypothalamus and other brain tissues of the male rat☆

Abstract 1. 1. The presence of steroid-converting enzymes in different brain areas as well as the subcellular distribution of these enzymes have been studied. 2. 2. Identification of metabolites following incubations of various steroids with brain tissue indicated that 5α-steroid reductase (EC 1.3.1.99) and 3α- and 17β-hydroxysteroid dehydrogenases (E.C. 1.1.1.50 and EC 1.1.1.51) are present. 3. 3. The subcellular localizations of these steroid-converting enzymes were studied with ultracentrifugation techniques. From the comparison of the specific activities of steroid-converting enzymes with marker enzymes (NADH-cytochrome c reductase and lactate dehydrogenase) and other characteristic parameters (RNA, DNA and protein content) it was concluded that hydroxysteroid dehydrogenases are present in the soluble fraction and the 5α-steroid reductase in the microsomes. 4. 4. Ratios of the specific activities of 5α-steroid reductase in different brain tissues relative to the specific activity in total brain were: hypophysis, 0.3; hypothalamus, 1.0; cerebellum, 1.6; cortex, 0.3. No significant differences were found between the specific activities of 17β-hydroxysteroid dehydrogenase in the different brain tissues.

Absence of a nuclear androgen receptor in isolated germinal cells of rat testis

Abstract Androgen receptors are known to be present in the seminiferous tubules of rat testis and in the present study it has been attempted to compare the binding of [ 3 H] testosterone to androgen receptors in male germinal cells and Sertoli cells. Cell preparations enriched in germinal cells and Sertoli cells were isolated from testicular tissue of 30–35-day-old rats. The cell preparations were either obtained from intact rats and labelled in vitro with [ 3 H] testosterone or were obtained from the testes of hypophysectomized rats which were labelled in vivo with [ 3 H]testosterone prior to the isolation of the cells. The nuclear fractions of the labelled cell preparations were extracted with 0.4 M KCl and the extracts were fractionated by sucrose density gradient centrifugation to estimate specific binding of radioactive steroid. Specific binding of radioactive steroid to nuclear androgen receptors was observed in Sertoli cell preparations but not in preparations of germinal cells (spermatocytes and round spermatids).

THE SECRETION, MEASUREMENT, AND FUNCTION OF A TESTICULAR LHRH‐LIKE FACTOR

In this paper we have demonstrated the presence and secretion within the testis of a factor that is immunologically distinct from LHRH, but that has receptor-binding and bioactive features similar to LHRH. The major source of this LHRH-like factor is probably the Sertoli cell and, as its site of action appears to be restricted to the Leydig cells, it is possible that it is one of the means by which the Sertoli cell has been postulated to regulate the Leydig cell. All of the available evidence suggests that the action of the LHRH-like factor on the Leydig cell is inhibitory, and it may mediate some or all of the negative effects on the Leydig cell of LH and hCG. The precise role of the LHRH-like factor, particularly under physiological conditions (i.e., low level stimulation with LH), remains to be determined, but the availability of methods for its extraction, measurement, and inactivation should enable this problem to be resolved within the near future. Moreover, the recognition that Leydig cell function may be regulated by local factors within the testis should open up new avenues of research that may lead to improved methods for the regulation of fertility in the male.

Immunohistochemical detection of transforming growth factor-α 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)

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.

[22] Preparation of isolated leydig cells

Publisher Summary This chapter discusses relevant observations on the preparation, purification, and characterization of isolated Leydig cells and explains that different in vitro preparations of Leydig cells have been used for studies on the biochemical mechanisms involved in regulation of steroidogenesis. The chapter also explains that several alternative methods for cell isolation and characterization have been developed and the techniques have been used to isolate Leydig cells from porcine and mouse testes and Leydig cell tumors. A careful investigation and standardization of the methods appears necessary, because for Leydig cells isolated from adult rats, a large variation in the capacity for steroid production has been observed. The recovery of Leydig cells after the isolation procedure is low, and there are indications that extensive cell damage can occur during tissue dispersion. When isolated cells are used for investigations on the biochemical mechanisms involved in the regulation of steroidogenesis, it is essential to work with homogeneous preparations of viable cells and prevent various degrees of functional heterogeneity and other artifacts, which may be caused by the isolation procedure.

Secretion of proteins by Sertoli cell enriched cultures: effects of follicle stimulating hormone, dibutyryl cAMP and testosterone and correlation with secretion of oestradiol and androgen binding protein.

Abstract Sertoli cells from 21–23-day-old rats were maintained in a chemically defined medium with or without hormones for 7 days with daily renewal of medium. It was found that in addition to secretion of oestradiol-17β and androgen binding protein (ABP), radioactive proteins were present in the culture medium after incubation of the cells with [ 3 H]leucine. These labelled proteins are secreted into the medium by the Sertoli cells. Lactate dehydrogenase was also present in culture medium as a secretion product from Sertoli cells. Stimulation of the secretion of the labelled proteins, ABP and oestradiol-17β at the 6th or 7th day could be observed when cells had been cultured in the presence of either testosterone, follicle stimulating hormone (FSH) or dibutyryl cyclic adenosine monophosphate (DcAMP) for 5 or 6 days. Addition of luteinizing hormone (LH) had no effect. One peak in the electrophoretic profile of labelled proteins decreased when cells had been cultured in the presence of FSH or DcAMP, but testosterone had no effect. No other effects of hormones on the profiles were observed. Germinal cells, interstitial cells or testicular fibroblasts did not contribute significantly to the quantity of the radioactive proteins. During the culture period and even in the presence of FSH or testosterone, decreasing daily production of ABP but a constant or increasing daily production of oestradiol-17β from testosterone was observed.

Modulation of steroidogenic activities in testis leydig cells

Recent developments in the regulation of testicular Leydig-cell function support the following conclusions. (1) Enzymic activities involved in steroid production in the testis are mainly localized in Leydig cells. The aromatase enzyme complex for oestrogen production appears to be localized in Leydig cells as well as in Sertoli cells. (2) LH- (or hCG-) induced alterations of Leydig cells depend on dose and duration of exposure of the cell to the hormone. Locally produced oestradiol is probably involved in the inhibition of steroidogenesis. (3) The stimulatory action of LH on Leydig cells involves different proteins in concert with the activation of the cleavage activity of mitochondrial cholesterol side-chain. However, most of the functional properties of these proteins are yet unknown. (4) Different populations of Leydig cells are present in the testis. These different cell populations can be characterized by quantitatively and qualitatively different responses to hormones.

The development of rat Leydig cell progenitors in vitro: how essential is luteinising hormone?

UNLABELLED Luteinising hormone (LH) appears to be important for the establishment of the adult-type Leydig cell population. The role of LH in the initial steps of stem Leydig cell/precursor cell differentiation is less clear. The aim of the present study was to elucidate the role of LH in the differentiation of spindle-shaped mesenchymal-like cells into Leydig cell progenitors. Interstitial cells were isolated from rat testes at three different ages reflecting different phases in development, and cultured in the presence of increasing concentrations of LH (ranging from 0.01 to 10 ng/ml). Cells were isolated from 10-day-old rats when stem Leydig cells/precursor cells are abundant; 13-day-old rats when the first 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-positive Leydig cell progenitors have developed in the strain of rats used in this study; and 18-day-old rats just prior to the wave of progenitor proliferation. Immunohistochemistry revealed that before stem Leydig cells differentiate into progenitor cells, they acquire functional LH receptors and become precursor cells. This was confirmed by an in vivo immunohistochemical double-labelling experiment. Addition of LH to the cultures increased the percentage of LH/3beta-HSD-labelled Leydig cell progenitors, while the percentage of cells solely expressing the LH receptor decreased. Cell proliferation was negligible, suggesting that the increase in 3beta-HSD-positive cells is the result of precursor cell differentiation. When interstitial cells were isolated from 13-day-old rats and to a lesser extent from 10-day-old rats, a small proportion of the precursors could develop into progenitor cells independent of the presence of LH. IN CONCLUSION before Leydig stem cells differentiate into 3beta-HSD-positive progenitor cells, they acquire LH receptors and become precursor cells. LH appears to be essential, even at very low doses for the differentiation of these precursor cells into 3beta-HSD-positive progenitors, although a subpopulation of precursor cells can develop into progenitors independently of LH.