B.A. Cooke

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].

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

A Leydig cell tumour: a model for the study of lutropin action.

The properties of cells isolated from a Leydig cell tumour have been compared with normal rat testis Leydig cells. These cells were found to be similar in the following respects: 1. Lutropin-stimulated cyclic AMP and testosterone production. 2. Lutropin-activated protein kinase activity followed by phosphorylation of endogenous proteins of mol. wts. 57,000and 14,000. 3. Parallel lutropin dose vs. response curves for phosphorylation of the endogenous proteins and for testosterone production. 4. Two forms of isoenzyme, cyclic AMP dependent protein kinase, present. They differed mainly with respect to the lutropin-stimulated testosterone production, which was much lower in the tumour cells compared with the normal adult testis Leydig cells (4.6 +/- 1.1 and 114 +/- 16 ng testosterone/10(6) cells per 2 h, respectively). However, the lutropin-stimulated steroid production in the tumour cells was quantitatively comparable with the normal rat Leydig cell when the metabolism of pregnenolone in intact cells and mitochondria was inhibited by addition of SU-10603 and/or cyanoketone. It is concluded that the Leydig cell tumour used in this study can be used to investigate certain aspects of lutropin action where large quantities of cells are required.

Effects of luteinizing hormone, follicle stimulating hormone, prostaglandin e1 and other hormones on adenosine-3′:5′-cyclic monophosphate and testosterone production in rat testis tissues

Abstract The site and specificity of LH action on cAMP and testosterone production in rat testis have been investigated. In tissue from hypophysectomized rats LH added in vitro specifically stimulated cAMP production in interstitial tissue, whereas FSH added in vitro specifically stimulated cAMP production in seminiferous tubules. No synergistic effect of FSH and LH was found in either tissue. LH markedly stimulated testosterone production in the interstitial tissue whereas only a very small increase was obtained in the seminiferous tubules using tissue from hypophysectomized rats. These results suggest that seminiferous tubules do not significantly contribute to testis testosterone biosynthesis. No stimulatory effect of testosterone production attributable to FSH could be demonstrated in either tissue or in whole testis tissue when this hormone was added alone or with LH in vitro. LH-stimulated testosterone production in total testis tissue from intact rats, when expressed in terms of mg of interstitial tissue present, was two times higher compared with interstitial tissue obtained by dissection. Addition of seminiferous tubules to the interstitial tissue did not increase the testosterone production. In contrast cAMP production per mg interstitial tissue present was the same in all three preparations. Prostaglandin E1 caused a small but significant increase in cAMP production in interstitial tissue but had no effect on testosterone biosynthesis. None of the other hormones tested (oestradiol-17β, ACTH, GH, prolactin, testosterone) had any effect on cAMP or testosterone production by rat testes preparations in vitro.

Evidence for the involvement of lutropin-independent RNA synthesis in Leydig cell steroidogenesis.

The effect of incubating purified Leydig cells in Eagles medium and the subsequent effect of the RNA synthesis inhibitors, actinomycin D and cordycepin, on lutropin-stimulated testosterone synthesis have been investigated. The inhibiting effect was found to be inversely related to the time of preincubation; with cells preincubated for 0, 1, 2 and 3 h with Eagles medium only, followed by 2-h incubation with lutropin with and without actinomycin D, testosterone synthesis was inhibited by 37 +/- 4, 31 +/- 3, 18 +/- 4 and 14 +/- 3% respectively (means +/- s.e.m., n = 5). In cells that had been preincubated for 3 h there was no significant effect of actinomycin D on testosterone synthesis during the first hour of incubation with lutropin. Thereafter the inhibition increased with time reaching a maximum of 30% after 5 h. The effects of preincubation were not due to endogenous lutropin in the Leydig cells because cells isolated from hypophysectomized rats gave similar results. The inhibition of [3H]uridine incorporation into the Leydig cell RNA was 80 +/- 1% with 8 microgram/ml actinomycin D. Increasing the concentration of this inhibitor to 80 microgram/ml did not significantly increase the inhibition of [3H]uridine incorporation or lutropin-stimulated steroidogenesis in preincubated and non-preincubated cells. With cordycepin the inhibition of both RNA synthesis and lutropin-stimulated testosterone synthesis in non-preincubated cells were the same; with 25.1--251 microgram/ml approx. 30--70% resp. With preincubated cells (3 h), 0--50% inhibition of testosterone synthesis was obtained respectively. The inhibitory effect of actinomycin D oimilar to that obtained with lutropin. These observations suggest that during preincubation and independently of lutropin, synthesis of intermediates, including RNAs required for stimulation of steroidogenesis, takes place and that subsequent stimulation of steroidogenesis by lutropin occurs without further de novo RNA synthesis. These results provide evidence for a permissive role of specific RNA and protein synthesis in the action of lutropin on testosterone synthesis in the Leydig cell.

LH induction of a specific protein (LH-IP) in rat testis leydig cells

The available evidence suggests that LH stimulation of testosterone production in rat testis Leydig cells involves protein(s) with a short half life. This evidence is based on the effects of inhibitors of protein and RNA synthesis on LH stimulated testosterone production [l-3] , particularly the rapid effect of cycloheximide, which causes a decrease in testosterone synthesis following first order kinetics with a half life of 13 min [4]. Recent work in our laboratory has shown that two proteins which are synthesized in rat testis Leydig cells and which can be detected using polyacrylamide gel electrophoresis, may be impottant in the regulation of testosterone production by LH; one of these proteins has a short half life (about 11 min) and is present in the particulate fraction of the Leydig cell, but is not under the influence of LH; the other protein (referred to as LH-IP, LH-induced protein) can be detected 2 h after the addition of LH to Leydig cells and has a half life longer than 30 min [5]. We now wish to report that the second protein (LH-IP) can be induced by LH or dibutyryl-CAMP but not by testosterone or follicle stimulating hormone (FSH). Dose response studies have also shown that the induction of LH-IP and the stimulation of testosterone production require approximately the same concentrations of LH. Incubation of the Leydig cells with actinomycin D prevented the induction of LH-IP by LH.

The testis: Biochemical actions of trophic hormones and steroids on steroid production and spermatogenesis

Abstract The present paper summarizes recent new information on steroid production and spermatogenesis in the rat testis, with respect to: 1. The cellular and subcellular localization of testicular steroid production. 2. Biochemical factors which appear to be related to the effect of trophic homones on testicular steroid production. 3. Some biochemical observations on Sertoli cells and germinal cells in the seminiferous tubules and relationships with steroid hormones.

Role of cyclic amp in steroidogenesis in leydig cells: ‘Discrepancies’ between effects of luteinizing hormone and cholera toxin

The mechanisms by which luteinizing hormone (LH) controls steroidogenesis in testis Leydig cells are not well defined. The available evidence suggests that in similarity with the adrenal gland, activation of adenylate cyclase [l-3], protein kinase [3,4] and protein synthesis [5-71 are involved. However, in contrast to the adrenal gland, steroidogenesis in trophic hormone (LH, hCG)-stimulated Leydig cells has been shown: (1) To proceed more slowly (20-30 min is required before increases in steroidogenesis can be detected [2,8,9] (compared with 24 s 3 min in the adrenal gland [ 1 O] ). (2) To be maximal without increases in cyclic AMP being detectable [ I] . These data suggest that additional or different mechanisms not involving cyclic AMP may be operating in the Leydig cell. In the present study the kinetics of cyclic AMP and testosterone production in LH-stimulated Leydig cells have been reinvestigated and compared with the effects of cholera toxin. The latter has previously been shown to mimic the action of hormones at their target cells through stimulation of cyclic AMP production [l l-l 51 and thus is a useful tool in elucidating the role of this nucleotide in hormone action. It has been found that by preincubating Leydig cells before addition of LH a rapid increase in testos-

Mechanism of LH and FSH Action on the Testis

The testes fulfil a dual role analogous to that of the ovaries i.e. an endocrine function in the formation of steroid hormones and a germinal one in the production of spermatozoa. The seminiferous tubules are the site of spermatogenesis and they occupy approximately 85% of the gland volume. They are highly coiled (in the rat there are about 6 separate tubules) and are embedded in connective tissue containing the Leydig (interstitial) cells, the site of steroidogenesis. In contrast to the seminiferous tubules, the Leydig cells have an excellent blood supply. The whole testis is surrounded by a dense white inelastic capsule, the tunica albuginea.

Actions of Lutropin (LH) and cAMP in the Regulation of Testicular Steroid Production

The two main physiological functions of the mammalian testis are localized in different tissue compartments; spermatogenesis occurs within the seminiferous tubules and steroid hormone production within the interstitial tissue. Within the interstitial tissue the Leydig cells are the main, if not the only source of de novo steroid production. Trophic hormones from the pituitary are intimately involved in the maintenance and regulation of these testis functions. Follitropin (FSH) is known to have specific effects on the Sertoli cells of the seminiferous tubules, but has no known effect on cells in the interstitial tissue. Lutropin (LH) specifically interacts with Leydig cells in the interstitial compartment, but has no direct effects on the seminiferous tubules. The testis Leydig cells have become increasingly interesting as a model for studying the biochemical mechanisms involved in the actions of trophic hormones on steroid production, because recent technical developments have made it possible to study specific hormone-biochemical parameters in specific isolated testis cells.