Rat Testicular Tissue Research Articles

Aromatase activity in a rat Leydig cell tumor.

Male Wistar-Furth rats bearing the transplantable LTW(m) Leydig cell tumor have elevated serum estradiol (E2) concentrations. We measured the ability of these tumors to aromatize testosterone (T) to E2 by two methods. First, tumor minces were incubated with [7-3H]T, and the resultant [3H]E2 and [3H]estrone were purified and measured. In addition, tumor cell cultures were incubated with [1 beta-3H]T, and the resultant [3H]H2O was determined as a measure of aromatization. Tumor minces aromatized more actively than normal rat testicular tissue (3.30 +/- 0.15% of the T added was converted to E2 by the tumor vs. 0.30 +/- 0.25% by normal testis). Most of the aromatizing actitivity was localized to the microsomes. Using cell cultures the maximum velocity was 6.1 pmol/h X 5 X 10(5) cells, and the Km was 98 nM. In neither minces nor cell cultures were we able to show stimulation of aromatization with hCG, (Bu)2cAMP, or phorbol esters, although we could show stimulation by these agents in normal testicular cells. We were unable to inhibit the aromatase activity with human beta-endorphin or stimulate it with naloxone. However, we were able to inhibit the aromatase activity with 4-hydroxy-4-androstene-3,17-dione. We conclude that the LTW(m) rat Leydig cell tumor has an active autonomous aromatase system that is not responsive to compounds affecting the adenylate cyclase-cAMP system. It can be inhibited by 4-hydroxy-4-androstene-3,17-dione, a competitive-suicide inhibitor of the aromatase enzyme(s).

Purine phosphoribosyltransferase (EC 2.4.2.7 and 2.4.2.8) and purine de novo synthesis activity in rat testicular tissue at different stages of development, and their correlation with the circulating levels of gonadotrophins and testosterone, and with structural changes

The overall activity of the purine de novo synthesis pathway and the activities of purine phosphoribosyltransferase in the rat testis were measured at different ages and were correlated with histological observations. Similar studies of the concentration of circulating gonadotrophins and testosterone were performed. The purine phosphoribosyltransferase activities were between two and three orders of magnitude greater than purine de novo synthesis. The peak activity of the purine de novo synthesis pathway coincided with the first appearance of meiosis in the spermato-cytes immediately before the luteinising hormone (LH) level rose to its peak. The highest activity of the hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) — catalysed purine salvage pathway coincided with the first appearance of mature spermatozoa in the tubules just after the occurrence of peak levels of follicle-stimulating hormone (FSH). These findings are linked to the development of testicular atrophy in cases of severe HPRT deficiency in man.

Changes in germ cell adenylate cyclase and protein carboxyl methylase activities in rat testicular tissue during bilateral cryptorchidism and after orchidopexy.

Soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase are two enzymes that are primarily localized in haploid germ cels of rat testicular tissue, and both enzymes exhibit an increase in activity in association with sexual maturation. Experimental cryptorchidism (surgery at 17 days of age) in immature rats prevented the age-dependent increase in the activity of these two testicular enzymes. After orchidopexy at 34 days of age the activities of these two enzymes increased to normal control values in association with testicular growth. These observations show that biochemical markers such as soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase can be used to follow germ cell differentiation.

The steroid C-17,20-lyase complex in isolated Graafian follicles: effects of human chorionic gonadotropin.

Androstenedione synthesis was studied in isolated rat preovulatory follicles and compared with that of rat testicular tissue using [14C]progesterone together with 17 alpha-hydroxy-[3H]progesterone as substrates in the presence of NADH or NADPH as cofactors. The amount of androstenedione formed was measured by addition of carrier, reisolation, and crystallization to constant specific activity. The labeling patterns of androstenedione and 17 alpha-hydroxyprogesterone (17-OHP) confirmed that both tissues preferentially catalyzed the synthesis of androstenedione from progesterone rather than from 17-OHP. It appears, therefore, that free 17-OHP was not an obligatory intermediate in this reaction. When hCG (5 IU) was administered sc and the follicles were isolated 3 h later, androstenedione synthesis was inhibited whether NADH or NADPH was added as cofactors. By contrast, 17-hydroxylase activity was inhibited only with NADH as cofactor. Hence, the gonadotropin, with NADH as cofactor, specifically reduced progesterone incorporation into androstenedione without affecting incorporation of 17-OHP. Thus, hCG appears to affect androstenedione production from progesterone at two different sites of the lyase complex.

Analysis of profiles of unconjugated steroids in rat testicular tissue by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric (GC-MS) method for analysis of unconjugated steroids in a rat testis is described. A combined solvent-solid extraction procedure, utilizing Lipidex 1000 and Sep-Pak C 18, gives a 25-fold purified extract. Steroids in this extract are fractionated by straight phase high-performance liquid chromatography (HPLC) on a LiChrosorb DIOL column in n-hexane-2-propanol, 92:8 (v/v). Four fractions are collected and the steroids are converted to tert-butyldimethylsilyl (TBDMS), 3-enol-TBDMS, and mixed TBDMS-trimethylsilyl (TMS) derivatives using TBDMS- and TMS-imidazole with sodium formate as catalyst under conditions suitable for the steroids present in the respective fractions. The derivatives are purified by reversed phase HPLC in 100% methanol and are analyzed by GC-MS, using selected ion monitoring of the major ions of high mass. For quantification, a mixture of known amounts of ten 14C-labelled steroids, [ 3H]estradiol and [ 2H 3]estradiol are added to the testis homogenate. The mean concentrations (ng/g wet wt) of the twelve steroids determined were: 4-androstene-3,17-dione, 4.0; testosterone, 127; 17β-hydroxy-5α-androstan-3-one, 4.5; 5α-andro-stane-3α,17β-diol, 5.7; 5α-androstane-3β,17β-diol, 1.5; progesterone, 5.5; 17α-hydroxyprogesterone, 14.4; 3β-hydroxy-5-androsten-17-one, 0.07; 5-androstene-3β,17β-diol, 0.25; 3β-hydroxy-5-pregnen-20-one, 10.3; 3β,17β-dihydroxy-5-pregnen-20-one, 0.95; and estradiol, 0.025. Variations between animals were large whereas testes from the same animal in most cases had similar steroid concentrations.

Inhibition of 3β-hydroxy-δ 5-steroid dehydrogenase in rat testicular tissue by mercuric chloride

Histochemical investigations of 3β-hydroxy-Δ 5-steroid dehydrogenase (Δ 5-3βOHD) activity in the testicular tissue of rats administered mercuric chloride (HgCla) at dosages of 0.05 mg kg and 0.1 mg kg (i.p.) daily for 90 days reveal a graded inhibition of Δ 5-3βOHD activity which was positively related to dosage and to the duration of treatment with this compound. This phenomenon may be mainly responsible for the inhibition of spermatogenesis, observed as a toxic manifestation of mercury compounds.

Changes in Gonadotropin and Isoproterenol Stimulated Adenylate Cyclase Activities in Rat Testicular Tissue during Cryptorchidism

Changes in Gonadotropin and Isoproterenol Stimulated Adenylate Cyclase Activities in Rat Testicular Tissue during Cryptorchidism

The effects of hypophysectomy and testosterone treatment on the composition and metabolism of testicular lipids

The effects of hypophysectomy and of testosterone administration on lipid composition and metabolism of rat testicular tissue have been investigated. Increased concentrations of triacylglycerols and cholesterol were observed in testes of hypophysectomized compared to control (non-hypophysectomized) rats on the eighth day posthypophysectomy. Administration of testosterone maintained the concentrations of these lipids at about normal levels. The concentration of phospholipids was not affected by the hypophysectomy. Incorporation of 14C from 1-[14C] linoleate into testicular lipids was determined 24 hours after intratesticular injection. In hypophysectomized compared to control rats there was more 14C in C 16:0, C 20:2 and C 20:3 and less 14C in C 20:4 and C 22:4 of both phospholipids and triacylglycerols. After intratesticular injection of 1-[14C] eicosatrienoate there was more 14C in C 16:0 and C 20:3 and less 14C in C 20:4 and C 22:4 of both phospholipids and triacylglycerols of hypophysectomized compared to control rats. Intratesticular injection of 1-[14C]-arachidonate resulted in less 14C incorporation in C 22:4 in testes of hypophysectomized than in those of control rats. Treatment with testosterone did not affect the metabolism of any of the 14C-substrates. These results indicate that the testicular desaturation of C 20:3 to arachidonate, requiring a delta 5 desaturase, is inhibited by hypophysectomy and that testosterone by itself may control the concentrations of some testicular lipid classes but not the metabolism of the polyenoic acids.

The effect of zinc ion on adenylate cyclase in rat testicular tissue.

The effect of zinc ion on adenylate cyclase in rat testicular tissue.

Changes in isoproterenol-stimulated adenylate cyclase activity in rat testicular tissue during cryptorchidism and after orchidopexy.

Cryptorchidism was associated with increased responsiveness of the isoproterenol-sensitive adenylate cyclase in membrane particles from rat testis. Abdominal testes from uni- and bilaterally cryptorchid rats showed the same activities. The change in isoproterenol-responsive adenylate cyclase was independent of the age at which the animals were made cryptorchid. The isoproterenol response was maximal 3-4 weeks after the rats were made cryptorchid. By 2-3 months after orchidopexy the isoproterenol response in the rat testis had decreased to normal control values.

Effect of zinc ion on human chorionic gonadotropin-stimulated in vitro production of cyclic AMP and testosterone by rat testis.

This study explores the effects of several divalent metal ions on the in vitro production of cyclic AMP, cyclic GMP, and testosterone by rat testicular tissue and on the amount of binding of [125I]human chorionic gonadotropin (hCG) in the testis. Zn2+ at concentrations of 10(-6) to 10(-4) M enhanced the hCG-stimulated production of cyclic AMP and testosterone, but only in the presence of Ca2+ X [125I]hCG binding to rat testicular tissue was not affected by Zn2+ X Cu2+, Ni2+, Co2+, and Mn2+ did not increase cyclic AMP or testosterone production in concentrations of 10(-7) to 10(-3) M and even inhibited them at a high concentration (10(-2) M). Cyclic GMP production was not affected by these divalent ions. These results suggest that Zn2+ may play an important role in rat testicular steroidogenesis.

Direct effect of androgens on progesterone binding and metabolism in rat testis microsomes.

A possible site of action at which androgens may control their own biosynthesis in rat testicular tissue in terms of an intratesticular feedback mechanism is investigated. It is shown that both progesterone (Ks = 0.45 microM) and testosterone (Ks = 14.7 microM) induce spectral changes at microsomal cytochrome P-450; these spectral effects are not additive and therefore both steroids may act on the same species of cytochrome P-450. This hypothesis is supported by the observation of competitive inhibition by testosterone of progesterone binding to solubilized microsomal proteins (Ki = 10.0 microM) and of progesterone conversion to androgens (Ki = 14.3 microM). It is concluded that rat testicular androgen biosynthesis is subject to feedback regulation not only via the pituitary-testicular axis but also by direct action of androgens on microsomal reactions dependent on progesterone-binding cytochrome P-450.

Dissociation of receptor-bound human chorionic gonadotropin from rat testicular membranes in vitro as a high molecular weight complex and its inhibition by heavy metals and alkylating agents

Intratesticular administration of the heavy metals, Zn 2+, Cu 2+, Cd 2+, and the alkylating agents, N-ethylmaleimide, iodocacetamine, increased the in vivo uptake of human 125I-labelled chorionic gonadotropin (CG) into rat testicular tissue. The results of gel filtrations and Scatchard plot analyses suggested that this effect is not due to an increase in the binding affinity of the hormone, but rather to an increased stability of receptor-human [ 125I]CG complex in the plasma membranes of the target cells. In order to elucidate this possibility in vitro, the release of radioactivity from testicular particles pre-labelled in vivo with human [ 125I]CG was studied under defined incubation conditions. The release was found to be dependent upon temperature and pH, with optimum at pH 8.0–8.5, being about 40% of the total radioactivity subjected to incubation at 37°C. No such release was observed from liver particles (referred to as nonspecific binding). About 80% of the radioactivity released from testicular particles was eluted from a Sephacryl S-300 column as a high molecular weight human CG comples between the intact human [ 125I]CG and the solubilized receptor- human [ 125I]CG complex. The release of this human CG complex was selectively inhibited by the addition of heavy metals and alkylating agents without affecting the release of the other particle proteins, but not by the other enzyme activity modulating agents in the incubation medium. The results show that incubation of testicular particles pre-labelled in vivo with human [ 125I]CG results in dissociation of the receptor-hormone complex and a release of a high molecular weight human CG complex. The process, which seems to proceed via fragmentation of the receptor molecule, is significantly inhibited by heavy metals and alkylating agents. These findings are compatible with our in vivo results presented here, and support the view that fragmentation of the receptor molecule is an important aspect in the removal of the receptor-bound human CG from the target cells.

The acylation of lysophosphatidylcholine in the rat testicular tissue : The combined activity of acyl-CoA synthetase and lysolecithin acyltransferase

The activity of lysophosphatidylcholine acyltransferase (EC 2.3.1.23) in combination with acyl-CoA synthetase (EC 6.2.1.3) has been determined in the homogenates and subcellular fractions of rat testis. The enzyme activity was found to be maximal at pH 7.4 ATP and CoASH were required for optimal incorporation of [1-14C] oleic acid into phosphatidylcholine. The sulfhydryl-binding reagents showed inhibitory effect on the acyltransferase activity. Dibutyryl cyclic AMP and beta-mercaptoethanol did not affect the enzyme activity. Subcellular distribution patterns of markers, marker enzymes and lysolecithin acyltransferase have shown that the acyltransferase activity was found to be predominantly localized in the microsomal fraction, though significant activity was also present in the mitochondrial fraction. These findings, together with our previous studies on testicular phospholipases A, suggest that the deacylation-reacylation cycle is operative in rat testicular tissue.

In Vitro Effect of Inhibin on Cyclic AMP‐Phosphodiesterase Activity in Rat Testes

We have previously reported that incubation of testicular slices with inhibin in the presence of 6 × 10−8 M oF SH resulted in a dose‐related reduction in accumulation of cyclic AMP due to a decrease in adenyl cyclase activity. The present study demonstrates that inhibin enhances the cyclic AMP‐phosphodiesterase activity in rat testicular tissue. These results suggest an additional mechanism by which cyclic AMP levels in gonadal tissue could be regulated by inhibin.

Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops).

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for 125 I-hCG, (0.9-2.5 × 109 M-1 , and 0.7-1.64 × 109 M-1 respectively, for green and rhesus monkeys) and low binding capacity (0.343-0.682 fmol/mg and 0.198-0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 109 M-1 ) and low binding capacity (1.04 fmol/mg tissue homogenate) for 125 I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.

Inhibition of 123I-labeled human chorionic gonadotropin binding to gonadal receptors by a factor obtained from rat testicular tissue.

Inhibition of 123I-labeled human chorionic gonadotropin binding to gonadal receptors by a factor obtained from rat testicular tissue.

Effect of vitamin A deficiency on luteinizing hormone receptors and adenosine 3',5'-monophosphate-mediated steroidogenesis in rat testicular tissue.

AbstractWeanling male Sprague-Dawley rats were maintained on a vitamin A (retinol)-deficient retinoic acid-supplemented diet for a period of 8 to 14 weeks. After 10 weeks, Leydig cell-enriched preparations from vitamin A-deficient animals had 45% fewer gonadotropin receptors for human luteinizing hormone (hLH). Testicular slices from vitamin A-deficient animals demonstrated decreased formation of adenosine 3′5′-monophosphate (cyclic AMP) and decreased testosterone content in response to hLH stimulation compared to vitamin A-supplemented rats (P < 0.01). Our findings suggest that changes which cause hyporesponsiveness in testosterone production from vitamin A-deficient rats can be attributed to a reduction in gonadotropin testicular receptors for hLH and a decreased cyclic AMP production. Testicular morphology was not altered during the first 10 weeks of the experiment, although, after 14 weeks on the experimental diet there was marked degeneration of tubules, cessation of spermatogenesis, and testicular a...

Inhibitory Effects of &amp;karsi028;&lt;sup&gt;9&lt;/sup&gt;-Tetrahydrocannabinol on Glycolytic Substrates in the Rat Testis

Experiments conducted with a rat testicular tissue indicated that delta 9-tetrahydrocannabinol (THC) produced a dose-dependent inhibition of glucose metabolism. Incubation of testicular tissue with different U-14C-fructose concentrations and 0.3 mM THC also significantly inhibited 14CO2 production; however, testicular utilization of 214C-pyruvate remained unaffected by THC. In other experiments, 0.3 mM THC significantly decreased tissue concentrations of dihydroxyacetone phosphate, fructose-1,6-diphosphate, and glucose-6-phosphate by 23, 31 and 29%, respectively. Uptake studies with 14C-2-deoxy-D-glucose showed a 43% reduction in uptake of this substrate by testicular tissue in the presence of 0.3 mM THC. These studies demonstrate that THC has an inhibitory effect on the utilization of energy substrates by the testis. Sites of THC action may be the membrane uptake step, the hexokinase step, and/or possible the phosphofructokinase step. It is proposed that this inhibition of testicular glycolysis could deprive the cells of their energy reserves and thereby may disrupt many gonadal functions.

Composition of gangliosides in ovine testis

1. 1. Gangliosides were identified as constituents of ovine testis and compared with ganglioside content of bovine, porcine and rat testicular tissue. 2. 2. Eight chromatographically distinguishable gangliosides were found and the structures of seven of these gangliosides were determined. 3. 3. Gangliosides GM 3, GD 3, GM 1 and GD 1a were most abundant in ovine testicular tissue. 4. 4. All even carbon fatty acids from 14:0 to 26:0 were contained in ganglioside fractions while the only odd carbon acids found in any quantity were 23:0 and 25:0. The only unsaturated fatty acids observed were 16:1 and 18:1.