H.J. van der Molen

Evaluation of a radioimmunoassay for testosterone estimation

Abstract A radioimmunoassay technique, which is essentially a modification of the method described by Furuyama et al.[1], has been evaluated for the determination of testosterone in human peripheral plasma and rat testis tissue. The antiserum used was raised against testosterone-3-(0-carboxymethyl)-oxime-bovine serum albumin in female rabbits. It had an association constant of 5.5 × 109 1/mol, 4°C, at a dilution of 1 in 20,000. The procedure involved addition of [3H]-testosterone internal standard, extraction and chromatography of the plasma extracts on alumina micro-columns prior to assay. Testis tissue extracts were not chromatographed. Known amounts of standard testosterone were subjected to the same procedures. After incubation with antiserum for 16 h at 4°C total recovery from the extraction, chromatography (when used) and incubation procedures were measured in order to correct for losses. Either toluene scintillation fluid, dextran-coated charcoal or polyethylene glycol were used to separate free and bound testosterone. For human plasma as well as for testis tissue a good correlation was observed between results obtained with radioimmunoassay and a gas chromatographic method using electron capture detection of testosterone chloroacetate[12].

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 use of affinity matrices in the purification of inhibin from bovine follicular fluid

The protein associated with inhibin-like activity in bovine follicular fluid was purified 80- to 120-fold after successive adsorptions on different affinity matrices, i.e. Matrex gel red A, phenyl sepharose, omega-aminohexyl agarose and concanavalin-A sepharose. Partial characterization of the active protein resulted in the conclusion that inhibin from bovine follicular fluid is a hydrophobic glycoprotein with an apparent molecular weight between 60 000 and 70 000 daltons. An antiserum, raised against an 80-fold purified preparation, prevented the inhibin-like action of bovine follicular fluid on pituitary cells in vitro.

Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.

Binding of oestradiol by the nuclear fraction of rat testis interstitial tissue

In most steroid target tissues the steroids are bound specifically by proteins from the cytosol and nuclear fraction. Available information indicates that the steroids are generally taken up by the target cell in a process which includes at least two steps: first binding to a receptor protein in the cytosol, and secondly the transfer of the steroid-cytoplasmic receptor complex to the nucleus [l] . In a previous publication [2] we have reported the presence of an oestradiol receptor in the cytoplasmic fraction of rat testis interstitial tissue. It was of interest to investigate if the observed binding of oestradiol in this tissue is limited to only cytoplasmic sites. Furthermore the possibility was considered that in testicular interstitial cells a comparable situation exists as in uterine tissue where in addition to cytoplasmic binding a large part of the oestradiol is associated with the nuclear fraction [3,4]. In the present paper we report that after in vivo or in vitro administration of oestradiol, this steroid can be bound by the nuclear fraction of rat testis interstitial cells and that the cytoplasmic fraction is required for specific retention of oestradiol by the nuclear fraction from a cell-free system.

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.

Cyproterone acetate prevents translocation of the androgen receptor in the rat prostate

The translocation of the androgen receptor in prostatic tissue has been studied under the influence of different ligands (testosterone, methyltrienolone and cyproterone acetate) in vivo and in vitro. Nuclear and cytoplasmic androgen receptors were estimated using an exchange assay with [3H]methyltrienolone ( [3H]R1881) 1 h and 16 h after injection in castrated rats of either 100 micrograms testosterone (T), 10 mg cyproterone acetate (CA) or the combination of T and CA. Within 1 h after T administration, nuclear receptor levels increased with a concomitant depletion of cytosol receptors. In the CA-treated rats nuclear receptor levels were not different from those of control castrated animals and there was no depletion of cytosol receptors. The combined treatment of T and CA resulted in a partial depletion of cytosol receptors and a simultaneous increase of nuclear receptors. The absence of an increase in nuclear androgen receptors in CA-treated animals cannot be explained by a delay in translocation, because even 16 h after CA injection, only a very small number of nuclear receptors were detectable. Incubation of minced prostatic tissue with [3H]CA or [3H]R 1881 resulted in receptor translocation only in the R1881 incubations and confirmed the in vivo results. Competition studies with different steroids and cytosol receptor (non-activated, 8S form in low salt gradient) or nuclear receptor (activated 3.6S form in high salt gradient) of prostatic tissue show that CA can compete with R1881 for specific androgen-binding sites with a similar relative binding affinity for both receptor preparations. The present results provide evidence that CA prevents translocation of the androgen receptor to the nucleus, although CA can be bound with similar affinities to the nuclear receptor and the cytoplasmic receptor. We propose that the anti-androgenic action of CA involves an inhibition of receptor translocation.

Endogenous production of steroids by subcellular fractions from total rat testis and from isolated interstitial tissue and seminiferous tubules

Abstract The endogenous production of pregnenolone and testosterone in subcellular fractions of rat testis tissue was estimated. A medium containing cofactors was used and the incubations were carried out for 120 min at 33 °C. Only mitochondrial fractions produced the estimated steroids. Seminiferous tubules and interstitial tissue were isolated from rat testis tissue using a wet dissection technique. Mitochondrial fractions were isolated from the homogenized isolated tissue compartments and production of pregnenolone and testosterone after incubation of these fractions was estimated. The interstitial mitochondrial fraction produced 1200–2600 pmoles per mg mitochondrial protein per 2 h which was at least 60 times higher than the production in similar fractions obtained from seminiferous tubules. Production in the mitochondrial fraction of whole testis tissue was 300–600 pmoles per mg protein per 2 h.

Effect of luteinizing hormone on 3′,5′-cyclic AMP and testosterone production in isolated interstitial tissue of rat testis

It has been shown that human chorionic gonadotrophin (HCG) and luteinizing hormone (LH) will stimulate steroidogenesis in testes in viva [ 11. The intracellular mediator of this trophic hormone action is thought to be 3’S’-cyclic AMP (CAMP) because in vitro experiments have shown that i) HCG or LH stimulates CAMP and testosterone production in testes [2-71, ii) the increase in CAMP production precedes the increase in testosterone production [3] and iii) dibutyryl-CAMP stimulates testicular testosterone synthesis in vitro [2, 3, 51 and in vivo [l]. However, because of the different cell types present in testes only tentative conclusions can be drawn. It is possible, for example, that CAMP production is stimulated in cell types that are not involved in steroidogenesis. In vitro studies with separated testis tissues have shown that LH specifically stimulates CAMP production in the interstitial tissue [7] and that this tissue is the main site of testosterone biosynthesis [8]. It was therefore decided to investigate the effect of LH on the relationship between CAMP and testosterone synthesis in isolated interstitial tissue in vitro. The results obtained are in accordance with CAMP being an intracellular mediator of LH action. Both CAMP and testosterone production in interstitial tissue were stimulated by LH and the increase in CAMP preceded the increase in testosterone production. The addition of glucose was found to increase the production of testosterone in LH-stimulated interstitial tissue. The magnitude of the observed increased testosterone