Stimulated Testosterone Production Research Articles

A search for immunoglobulins inhibiting gonadal cell steroidogenesis in premature ovarian failure.

Premature ovarian failure (POF) may be caused by the action of circulating gonadotrophin receptor-blocking antibodies. Luteinizing hormone (LH)-stimulated testosterone production from mouse Leydig cells and follicle stimulating hormone (FSH)-stimulated oestradiol production from immature rat Sertoli cells were therefore studied in the presence of protein-G purified immunoglobulin G (IgG) samples from control subjects (n = 9), infertile women with elevated early follicular phase FSH levels but otherwise normal menstrual cycles (n = 10), and patients with POF (n = 10) or Graves' disease (n = 10). A saturating and subsaturating (78% for LH; 60% for FSH) dose of each hormone was chosen for study. A commercial preparation of human IgG (Sigma IgG, 0.75 mg/ml) employed as negative control had no effect on basal or gonadotrophin-stimulated steroidogenesis. In its presence, saturating doses of LH (2 IU/l) and FSH (20 IU/l) gave rise to 11.2 +/- 0.8 (n = 7) and 25.1 +/- 5.8 (n = 8) fold increases in steroid secretion. IgG (0.75 mg/ml) had no effect in the four groups on LH-stimulated testosterone outputs using a saturating (2 IU/l) or subsaturating (1 IU/l) dose of hormone. For example, LH (2 IU/l)-stimulated testosterone production was 94% (83-96 median; interquartile range) and 88% (81-99) of the Sigma IgG control for control and POF groups respectively. However, four out of nine IgG samples from the normal subjects (mean +/- SEM = 86 +/- 6%), two out of 10 of the high FSH group (77 +/- 4%), five out of 10 with Graves' disease (86 +/- 3%) and six out of 10 with POF (76 +/- 6%) gave rise to LH (2 IU/l)-stimulated testosterone outputs which were lower (P < 0.05) than that of Sigma IgG control. Using the identical set of patients and an IgG concentration of 0.25 mg/ml, the FSH-stimulated oestradiol outputs of the four groups were similar when using either the saturating (20 IU/l) or subsaturating (5 IU/l) dose of the hormone. Thus, the percentage of FSH (20 IU/l)-stimulated oestradiol production of the Sigma IgG control was 81 (66-89 median, interquartile range) and 50 (38-84) for control and POF groups respectively. However, once again individual patients had inhibitory IgGs such that four out of nine (controls), three out of 10 (high FSH group), four out of 10 (Graves' disease) and six out of 10 (POF patients) inhibited (P < 0.05) FSH (20 IU/l)-stimulated oestradiol secretion by 52 +/- 9 (mean +/- SEM), 44 +/- 7, 52 +/- 6 and 41 +/- 6% respectively. Of the patients with inhibitory IgGs the extent of inhibition of gonadotrophin-stimulated steroid secretion was similar between the groups. In conclusion, there is little evidence to suggest that immunoglobulins blocking gonadotrophin receptors are a mechanism for POF in a large proportion of women suffering from this condition.

Mechanisms of Action of Free Arachidonic Acid on Ovarian Steroid Production in the Goldfish

This study explores the mechanisms by which free arachidonic acid (AA) affects ovarian steroidogenesis by full-grown prematurational follicles of the goldfishin vitro.AA (6–400 μM) stimulated testosterone production and this action was mediated by prostaglandin E2(PGE2). The steroidogenic actions of AA and the corresponding increase in the production of PGE2were blocked by inhibitors of the cyclooxygenase pathway (indomethacin, ETYA). Exogenous PGE2(20–2000 ng/ml) also stimulated steroid production. In the presence of human chorionic gonadotropin (hCG), AA had differential effects. AA potentiated the steroidogenic actions of low dosages of hCG (0.1 IU/ml), while with maximal gonadotropin (1–10 IU/ml) stimulation a high concentration of AA (400 μM) attenuated steroid production in spite of elevated PGE2concentrations. HCG did not induce PGE2synthesis, nor did it affect the PGE2production obtained with AA-treated follicles. The steroidogenic induction by AA via PGE2was mediated in part by Ca2+since the calcium channel blocker nifedipine (25 μM) inhibited stimulated steroid production by both AA and PGE2. The conversion of AA to PGE2does not require Ca2+since PGE2production by AA-treated follicles was not affected by nifedipine. However, treatment with the calcium ionophore A23187 (1 μM) potentiated the stimulatory actions of AA on steroid and prostaglandin production. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1 mM) potentiated the stimulatory actions of AA on testosterone production but had no effect on the conversion of AA to PGE2. The steroidogenic actions of AA and PGE2involve both transcription and translation since stimulated steroidogenesis was inhibited by actinomycin D and cycloheximide (1–10 μg/ml). The conversion of AA to PGE2was also blocked by these inhibitors. These results underscore the importance of AA and PGE2in the regulation of ovarian steroidogenesis in the goldfish.

Evidence that heparin binding autocrine factors modulate testosterone production by the adult rat Leydig cell

Androgen production by adult rat Leydig cells is stimulated by pituitary LH but can also be modulated in vitro by paracrine stimulatory and inhibitory factors, many of which belong to growth factor families. Their actions are mediated through cell surface or extracellular matrix proteoglycans and the aim of this study was to determine the role of cell surface heparan sulfate proteoglycans in the regulation of testosterone secretion by adult rat Leydig cells. The presence of sodium chlorate (25 mM) and protamine sulfate (10 μg/ml) inhibited testosterone production by LH stimulated cells by over 50%, but had no effect on unstimulated cells. The LH responsiveness and testosterone production returned to normal after these agents were removed from the culture media. No significant difference in LH receptor numbers at the end of the culture period was seen between sodium chlorate treated and untreated cells. Testosterone production by dibutryl-cAMP stimulated Leydig cells was also inhibited by sodium chlorate. The addition of heparin inhibited testosterone production by LH stimulated cells in a dose-dependent manner, however, in unstimulated Leydig cells heparin stimulated testosterone production to up to 50% of that seen in LH stimulated cells. These data suggest that cell surface heparan sulfate proteoglycans modulate testosterone production by adult Leydig cells in vitro, and that this may involve the autocrine actions of heparin binding growth factors on the Leydig cells.

Sex hormone-binding protein, hyperinsulinemia, insulin resistance and noninsulin-dependent diabetes.

Possible data complicating sex hormones, especially testosterone, in the etiology of cardiovascular disease and noninsulin-dependent diabetes mellitus (NIDDM) comes from the much higher rates of cardiovascular disease in men than in women. Pharmacological administration of anabolic steroids to both men and women increases glucose and insulin concentrations and also insulin resistance. In vivo assessment of sex hormones and binding proteins in both premenopausal and postmenopausal women has suggested that increased free testosterone and decreased sex hormone-binding globulin (SHBG) is associated with higher glucose and insulin concentrations. In a few studies, increased insulin resistance has been associated with decreased SHBG levels. Some data suggests that visceral fat mediated the associates of sex hormones with insulin in women. Little prospective data is available on the association of sex hormones to the development of NIDDM in women but in two studies, low SHBG concentrations predicted NIDDM in Gothenburg and San Antonio. Recently, attention has focused on the role of sex hormones in relation to insulin in men. Surprisingly, higher levels of testosterone have been associated with improved cardiovascular risk factors (such as high-density lipoprotein cholesterol) and lower glucose and insulin levels. Total testosterone and SHBG have been associated with defects in nonoxidative glucose disposal and upper body adiposity in normoglycemic Finnish men. The latter observation is of interest since specific defects in nonoxidative glucose disposal are observed in normoglycemic relatives of subjects with NIDDM. The temporal relationship between sex hormones and insulin has been controversial. The traditional view of sex hormones increasing insulin resistance has been challenged in women by studies showing that insulin stimulates androgen production in the ovary. Recent data [JCEM 1995;80:654-658] suggests that insulin stimulates testosterone production and suppresses SHBG production in normal and obese men. On the other hand, administration of testosterone to centrally obese hypogonadal middle-aged men has improved insulin sensitivity.

Inhibition of gonadotropin‐stimulated ovarian steroid production by polyunsaturated fatty acids in teleost fish

The effects of the polyunsaturated fatty acids (PUFAs)--eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA)--on in vitro steroid production by full-grown prematurational ovarian follicles from goldfish and rainbow trout were investigated. EPA and DHA inhibited gonadotropin-stimulated testosterone production in a dose-related manner, but AA was inhibitory only at the highest dose tested (400 microM). AA alone stimulated testosterone production by increasing cAMP production, but the effects of other PUFAs alone were marginal. The inhibitory actions by PUFAs were not restricted to long-chain PUFAs, as linoleic and linolenic acids had similar actions in the goldfish. The inhibitory action of EPA on testosterone production was reversible upon removal of the PUFA from medium. Testosterone production stimulated by the addition of the cAMP analogues, dibutyryl cAMP, and 8-bromo cAMP, was attenuated by PUFAs, suggesting that they act at a site distal to cAMP formation. A post-cAMP site regulating cholesterol availability may be involved as testosterone production induced by addition of 25OH-cholesterol was not affected by the PUFAs in either fish species. Together, these findings underscore the importance of lipids in ovarian physiology and suggest that PUFAs may participate in the regulation of ovarian steroidogenesis in teleost fish.

Insulin regulates testosterone and sex hormone-binding globulin concentrations in adult normal weight and obese men.

There are no studies in vivo on the effects of insulin on androgens and sex hormone-binding globulin (SHBG) in men. We, therefore, investigated the effects of insulin suppression on testosterone and SHBG in two groups of eight nondiabetic adult obese men and six healthy normal weight men who underwent diazoxide treatment (100 mg, three times daily) for 7 days. Blood samples for hormone determination were obtained before the subjects had been selected for the study, immediately before diazoxide administration, and on the last day of treatment. A 24-h oral glucose tolerance test was also performed for glucose, insulin, and C-peptide determinations before and on the last day of treatment. Only one subject experienced significant side-effects, and no significant changes in mean body weight were found during the treatment. Diazoxide administration worsened glucose tolerance in several subjects and reduced fasting and glucose-stimulated insulin levels by approximately 50% in both control and obese subjects. No significant difference was present between historical and pretreatment hormone values in either group. Moreover, there were no differences in pretreatment gonadotropin and SHBG concentrations between the two groups, whereas testosterone (free and total) levels were lower in the obese than in the control subjects. After diazoxide administration, testosterone (free and total) decreased slightly, but significantly, whereas LH and SHBG significantly increased in both groups. Diazoxide treatment increased estradiol levels in controls, but not in obese men. In conclusion, these results indicate that in vivo, insulin is capable of stimulating testosterone production and, simultaneously, of inhibiting SHBG concentrations in both normal weight and obese men.

Enzymic remodelling of the N- and O-linked carbohydrate chains of human chorionic gonadotropin. Effects on biological activity and receptor binding.

The effects of altered terminal sequences in human chorionic gonadotropin (hCG) N- and O-linked glycans on receptor binding and signal transduction were analyzed using forms of hCG with remodelled carbohydrate chains. hCG derivatives were obtained by enzymic removal of the alpha 3-linked sialic acid residues followed by alpha 6-sialylation, alpha 3-galactosylation or alpha 3-fucosylation of uncovered Gal beta 1-->4GlcNAc (LacNAc) termini, or alpha 3-sialylation of Gal beta 1-->3GalNAc sequences. Also a form that carried GalNAc beta 1-->4-GlcNAc units, which are typical for pituitary hormone oligosaccharides, was derived by enzymic desialylation and degalactosylation followed by beta 4-N-acetylgalactosaminylation. The potency to stimulate testosterone production and the binding to the lutotropin/choriogonadotropin receptor of the preparations were compared with those of native and desialylated hCG (as-hCG). The decrease in bioactivity caused by desialylation of hCG was only restored upon alpha 6-sialylation of the Gal beta 1-->4GlcNAc beta 1-->-2Man alpha 1-->3Man branch of the N-linked glycans. This was without a major effect on receptor binding. Further alpha 6-sialylation, occurring at the Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6Man branch, resulted in a bioactivity below a level found with as-hCG, concomitant with a decreased receptor binding affinity. Similarly alpha 3-galactosylation of the Gal beta 1-->4GlcNAc beta 1-->2-Man alpha 1-->6Man branch yielded a hCG derivative that showed decreased bioactivity and receptor binding. alpha 3-Fucosylation of native as well as as-hCG also led to a decreased activity. Re-alpha 3-sialylation of the O-linked chains on as-hCG had little effect on the bioactivity and receptor binding. Hormone preparations with GalNAc beta 1-->4GlcNAc termini showed lower bioactivity and receptor affinity than as-hCG. It is concluded that the Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3Man- rather than the Gal beta 1-->4GlcNAc beta 1-->2-Man alpha 1-->6Man branch of the N-linked glycans on hCG plays an essential role in signal transduction, whereas the latter branch can potentially interfere with receptor binding. Furthermore attachment of sialic acid, but not of other sugars, to the first branch fulfils the requirement for the full expression of bioactivity, while sialylation of the O-linked chains is of minor importance.

Human chorionic gonadotropin protects Leydig cell function against 2,3,7,8-tetrachlorodibenzo- p-dioxin in adult rats: role of leydig cell cytoplasmic volume

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) alters testicular steroidogenesis and reduces Leydig cell volume and number; however, human chorionic gonadotropin (hCG) stimulates testosterone production, increases the number and volume of Leydig cells and prevents TCDD's inhibition of testosterone production. The objective of this study was to determine if hCG protects Leydig cell function by maintaining sufficient Leydig cell cytoplasmic volume in TCDD-treated rats. Adult, male Sprague Dawley rats were divided into six treatment groups. Half of the animals received TCDD in corn oil (50 μg/kg body wt) and half received corn oil alone on Day 7 only. Additionally the rats received daily treatment of saline for 14 days, saline for 7 days and then hCG for 7 days, or hCG for 14 days. Rats were sacrificed on Day 14 and tissues collected. The decapsulated left testes were incubated in Eagles MEM for 2 h to determine basal production of testosterone and for 2 additional hours after the addition of hCG (100 IU) to the culture media. The right testis was evaluated by stereology to determine the volume of Leydig cells. Body weight was reduced ( P < 0.01) in each TCDD-treated group; whereas, testicular weight was not affected by TCDD or hCG treatment. hCG prevented the TCDD-induced reduction in prostate and seminal vesicles weights. TCDD reduced the total volume of Leydig cell cytoplasm in saline-treated rats, but hCG eliminated the TCDD-induced reduction in Leydig cell cytoplasmic volume. hCG prevented the TCDD-induced reduction in Leydig cell function for both the 7- and 14-day treatments. Variation in the total volume of Leydig cell cytoplasm induced by the various treatment combinations was positively correlated with stimulated testosterone production in vitro ( r = 0.486; P < 0.01) and the weight of the androgen sensitive organs (seminal vesicles, r = 0.562, P < 0.01; prostate, r = 0.380, P < 0.05). These data support the hypothesis that hCG prevents the TCDD-reduced Leydig cell function by maintaining sufficient Leydig cell cytoplasm and organelle content.

Erythropoietin and testicular steroidogenesis: the role of second messengers.

It has been demonstrated that erythropoietin (EPO) influences rat and human Leydig cell steroidogenesis, stimulating testosterone production through a direct and specific receptor-mediated mechanism. The aim of this study was to investigate the mechanism by which recombinant human erythropoietin (rHuEPO) exerts its stimulatory effect on rat Leydig cells. Recombinant human EPO did not induce, at any dose tested (10(-10) to 10(-13) mol/l), an increase in either cAMP or cGMP, suggesting that in Leydig cells the effect of rHuEPO does not involve the adenylate or guanylate-cyclase systems. The role of transmembrane calcium flux in rHuEPO-stimulated steroidogenesis was studied by evaluating the effect of calcium channel blocker, verapamil, and by the 45Ca2+ uptake method. Verapamil did not influence rHuEPO-induced testosterone secretion and rHuEPO did not modify calcium recycling, indicating that calcium transmembrane flux is not involved in the rHuEPO effect. The protein kinase C inhibitor staurosporine (10, 30, 100 and 300 nmol/l) inhibited rHuEPO-stimulated testicular steroidogenesis in a dose-dependent manner. This indirect evidence suggests that the stimulatory effect of rHuEPO on rat Leydig cells may involve protein kinase C activation.

Antibody against a Peptide Corresponding to the Extracellular Domain of the Luteinizing Hormone/Chorionic Gonadotropin Receptor Stimulates Testosterone Production in Rat Leydig Cells

In order to study the function of LH/CG receptor, we prepared four synthetic peptides (RP1, 2, 3, and 4 for residues 242-255, 49-66, 493-504, and 573-584) corresponding to extracellular domains of rat ovary LH/CG receptor and raised antibodies against RP1 and RP2 (anti-receptor peptide IgGs). These peptides and IgGs were assayed for inhibition of 125I-labeled hCG binding to Leydig cell membrane receptors. However, neither the peptides nor the IgGs inhibited hCG binding to the receptor at doses up to 10(-3) and 10(-5) M, respectively. On the other hand, although anti-receptor peptide IgGs did not show inhibition of hCG binding to the receptor, it was found that one of the IgGs (anti-RP1IgG) was bound to the cell membrane and stimulated testosterone production in rat Leydig cells. F(ab')2 fragment lacking the N-linked sugar chain in the IgG molecule, whose sugar chains are similar to those of hCG, was prepared from anti-RP1IgG. Anti-RP1F(ab')2 was still bound to the cell membrane but no longer stimulated testosterone production. These results suggested that when LH/CG receptor binds to a glycoprotein, even if it is different from the native ligand hCG, it may interact with sugar chains of the glycoprotein to cause signal transduction. Thus, a lectin-like domain in or near the receptor may play a key role in the receptor function.

Erythropoietin stimulates testosterone production in man.

Human recombinant erythropoietin (rHuEPO) treatment improves sexual function in end-stage renal failure patients with a still-debated mechanism. Experimental data suggested that rHuEPO was able to stimulate rat Leydig steroidogenesis; therefore, it has been suggested that rHuEPO may induce its effects in humans by acting on gonadal steroid production. Thirteen young adult males (age range, 16-28 yr) catheterized at peripheral and left internal spermatic venous levels during a contrast study for varicocele, were studied. In five subjects, rHuEPO (60 IU/kg, up to a maximum of 4000 IU total) was injected over 1 min in the cubital vein. Similarly, in other five patients, 50 micrograms GnRH were infused. In three subjects, 2 mL saline were injected, as controls. Plasma LH, FSH, and testosterone (T) levels were then determined at -15, 0, 15, 30, 45, 60, 90, and 120 min simultaneously in peripheral and spermatic venous blood. rHuEPO infusion did not have any effect on plasma LH and FSH levels in peripheral or spermatic veins. Similarly, rHuEPO infusion did not affect peripheral T concentration, but increased (approximately 400% vs. controls; P < 0.05) spermatic T levels. GnRH infusion induced an increase in plasma LH and FSH levels in both peripheral and spermatic veins. After GnRH infusion, an increase of approximately 12-fold (P = 0.05-0.001) in T was observed only at the spermatic venous level, without any peripheral T variation. These findings show that rHuEPO was able to influence testicular steroidogenesis by stimulating T production in man, whereas the absence of any effect on gonadotropin secretion suggests that rHuEPO might act directly on human Leydig cell function.

Inhibition of steroidogenesis in neonatal Leydig cells by unknown factor(s) present in spent media of androgen-treated cultured testicular cells from adult rats.

Treatment of cultured testicular cells from adult rats with 5 alpha-dihydrotestosterone (DHT; 10(-6) M) or the synthetic androgen methyltrienolone (R1881; 10(-6) M) inhibited Leydig cell 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas no effect of both androgens on cultured cells derived from neonatal animals could be observed. The inhibitory effect of DHT or R1881 on Leydig cell 3 beta-HSD enzyme activity, however, was abolished when adult cells were cultured in the presence of the anti-androgen cyproterone acetate (CPA; 10(-6) M) or the protein synthesis inhibitor cycloheximide (CX; 1 microgram/ml). Testicular cells from adult animals were also cultured in the presence of the different treatments described above, and the spent media was collected and thereafter used as conditioned culture medium (CCM) in subsequent experiments performed with neonatal cells. Dispersed testicular cells from neonatal rats were cultured for 12 days in McCoy's 5a medium of in CCM derived from R1881-treated adult cells, and fresh culture medium or CCM was replaced every 2 days. The human chorionic gonadotropin (hCG)-stimulated testosterone production of neonatal cells was abolished in the presence of CCM derived from R1881-treated adult cells. Nevertheless, the steroidogenic response to hCG recovered when neonatal cells were cultured for two additional days in McCoy's 5a medium. Treatment of neonatal cells with increasing concentrations of hCG (0.1-10 ng/ml) resulted in a dose-dependent augmentation in Leydig cell 3 beta-HSD enzyme activity and testosterone production.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor-mediated stimulatory effect of atrial natriuretic factor, brain natriuretic peptide, and C-type natriuretic peptide on testosterone production in purified mouse Leydig cells: activation of cholesterol side-chain cleavage enzyme.

We have investigated the mechanism by which different natriuretic peptides stimulate steroidogenesis in purified mouse Leydig cells. In addition to atrial natriuretic factor (ANF), we show that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) also stimulate testosterone production in these cells. Testosterone production was increased dramatically to 14-fold with ANF (EC50 = 0.3 nM) and 15-fold with BNP (EC50 = 0.2 nM); however, the CNP-stimulated level of testosterone production was only 2.5-fold compared with controls. ANF and BNP enhanced the stimulatory effect of LH on testosterone production. The C-ANF(4-23) (a truncated form of ANF) had no effect on testosterone production in these cells. ANF, BNP, and CNP stimulated the production of intermediate precursors of testosterone biosynthesis, which included progesterone, 17 alpha-hydroxy progesterone, androstenedione, pregnenolone, 17 alpha-hydroxy pregnenolone, and dehydroepiandrosterone sulfate. The conversion of pregnenolone and progesterone to testosterone was also significantly enhanced after treatment of Leydig cells with these peptides. All three natriuretic peptides (ANF, BNP, and CNP) stimulated the activity of particulate guanylate cyclase by 8.4-, 8.5-, and 4.8-fold and the accumulation of intracellular cGMP by 52-, 58-, and 19-fold, respectively. The cGMP inhibitor LY83583 attenuated both the generation of cGMP as well as testosterone in response to these natriuretic peptides, suggesting the involvement of cGMP as a second messenger. Leydig cells were found to contain high affinity and low capacity binding sites for ANF [dissociation constant (Kd), 2.0 x 10(-10) M; maximum binding capacity (Bmax). 20 fmol/1 x 10(5) cells], BNP (Kd, 2.2 x 10(-10) M; Bmax, 19 fmol/1 x 10(5) cells), and CNP (Kd, 3.1 x 10(-10) M; Bmax, 8.6 fmol/1 x 10(5) cells). The results presented here document that a family of different natriuretic peptides stimulates Leydig cell steroidogenesis in receptor-mediated fashion, beginning at the cholesterol side-chain cleavage enzyme. The data also show that these peptide hormones induce testosterone production in mouse Leydig cells by involving both delta 4- and delta 5-pathways of steroidogenesis.

Receptor-mediated stimulatory effect of atrial natriuretic factor, brain natriuretic peptide, and C-type natriuretic peptide on testosterone production in purified mouse Leydig cells: activation of cholesterol side-chain cleavage enzyme

We have investigated the mechanism by which different natriuretic peptides stimulate steroidogenesis in purified mouse Leydig cells. In addition to atrial natriuretic factor (ANF), we show that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) also stimulate testosterone production in these cells. Testosterone production was increased dramatically to 14-fold with ANF (EC50 = 0.3 nM) and 15-fold with BNP (EC50 = 0.2 nM); however, the CNP-stimulated level of testosterone production was only 2.5-fold compared with controls. ANF and BNP enhanced the stimulatory effect of LH on testosterone production. The C-ANF(4-23) (a truncated form of ANF) had no effect on testosterone production in these cells. ANF, BNP, and CNP stimulated the production of intermediate precursors of testosterone biosynthesis, which included progesterone, 17 alpha-hydroxy progesterone, androstenedione, pregnenolone, 17 alpha-hydroxy pregnenolone, and dehydroepiandrosterone sulfate. The conversion of pregnenolone and progesterone to testosterone was also significantly enhanced after treatment of Leydig cells with these peptides. All three natriuretic peptides (ANF, BNP, and CNP) stimulated the activity of particulate guanylate cyclase by 8.4-, 8.5-, and 4.8-fold and the accumulation of intracellular cGMP by 52-, 58-, and 19-fold, respectively. The cGMP inhibitor LY83583 attenuated both the generation of cGMP as well as testosterone in response to these natriuretic peptides, suggesting the involvement of cGMP as a second messenger. Leydig cells were found to contain high affinity and low capacity binding sites for ANF [dissociation constant (Kd), 2.0 x 10(-10) M; maximum binding capacity (Bmax). 20 fmol/1 x 10(5) cells], BNP (Kd, 2.2 x 10(-10) M; Bmax, 19 fmol/1 x 10(5) cells), and CNP (Kd, 3.1 x 10(-10) M; Bmax, 8.6 fmol/1 x 10(5) cells). The results presented here document that a family of different natriuretic peptides stimulates Leydig cell steroidogenesis in receptor-mediated fashion, beginning at the cholesterol side-chain cleavage enzyme. The data also show that these peptide hormones induce testosterone production in mouse Leydig cells by involving both delta 4- and delta 5-pathways of steroidogenesis.

Regulation of prostaglandin E and F production in the goldfish testis

AbstractThe present studies have investigated the putative role of activators of different signal transduction pathways in the regulation of prostaglandin (PG) production by goldfish testis pieces in vitro. The protein kinase C activator (PKC) phorbol 12‐myristate 13‐acetate (PMA: 1.56‐400 nM) and calcium ionophore A23187 (62.5‐4000 nM) stimulated PGE and F production in a dose and time‐dependent fashion. There was no synergism between A23187 and PMA in stimulating PG production. PGE levels were at least 4.5 times higher than PGF levels. The phorbol ester 4α phorbol didecanoate (400 nM), which does not activate PKC, had no effect on PGE production. The synthetic diacylglycerol 1‐oleoyl‐2‐acetyl‐sn‐glycerol (100 μM), but not 1,2‐dioctanoyl‐sn‐glycerol (100 μ), stimulated PGE production. Although the gonadotropin analog human chorionic gonadotropin (1 IU/ml), adenylate cyclase activator forskolin (2 μM) and testosterone (5 ng/ml) had no effect, the cyclic nucleotide phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (1 mM) and cyclic adenosine‐3′,5′‐monophosphate analog dibutyryladenosine‐3′,5′‐monophosphate (2 mM) inhibited PMA (400 nM) and A23187 (1 μM)‐stimulated PGE production. PMA and A23187 (400 nM and 1 μM, respectively)‐stimulated PGE production was inhibited by phospholipase A2 inhibitors 4‐bromophenacyl bromide, quinacrine and chloroquine (all 6.25‐400 μM) and by diacylglycerol lipase inhibitor RHC 80267 (10–100 μM), suggesting that both lipases are involved in the release of precursor fatty acids for PG synthesis by goldfish testis tissue. These data, in conjunction with earlier studies showing that PGE stimulates testosterone production by goldfish testis pieces, lend support to the contention that locally produced PGs of the E series modulate testicular steroidogenesis in teleosts. © 1993 Wiley‐Liss, Inc.

Catecholamines Stimulate Testicular Steroidogenesis in Vitro in the Siberian Hamster, Phodopus Sungorus1

We have examined direct effects of catecholamines on testicular testosterone production in a seasonally breeding species, the Siberian hamster, Phodopus sungorus. Testicular parenchyma from gonadally active long photoperiod (LD)-exposed and gonadally regressed short photoperiod (SD)-exposed animals was incubated for 6 h with norepinephrine, epinephrine, beta-adrenoceptor agonist isoproterenol, or alpha-adrenoreceptor agonist phenylephrine (all at 10 microM), as well as with various concentrations of norepinephrine (10 nM-10 microM), and 10 microM norepinephrine with or without hCG (0.7, 3.1, and 12.5 mIU/ml). In addition, effects of alpha-adrenoreceptor antagonist prazosin and beta-adrenoreceptor antagonist propranolol (50 microM) were tested in the incubations containing 10 microM norepinephrine. In the incubations of testes from both LD and SD Siberian hamsters, norepinephrine was most effective in stimulating testosterone production, followed by epinephrine and phenylephrine, while isoproterenol failed to increase testosterone accumulation. The stimulatory effects of norepinephrine were dose-dependent and were prevented by coincubation with prazosin, but not affected by coincubation with propranolol. In combination with various doses of hCG, norepinephrine failed to stimulate testosterone production above the levels obtained with hCG alone. These data indicate that the testicular receptors mediating the action of catecholamines on testicular steroidogenesis in Phodopus sungorus are of the alpha 1-subtype, a result in accordance with a previous study in the golden hamster. However, the results of the present study are strikingly different from the findings obtained in the golden hamster in terms of the effects of photoperiod on the responsiveness of testicular steroidogenesis to catecholamines.(ABSTRACT TRUNCATED AT 250 WORDS)

Tylosin inhibits the steroidogenesis in mouse Leydig cells “in vitro”

Former investigations in rats revealed effects of tylosin on the pituitary gonadal axis. After 15 days treatment the animals showed reduced weights of the seminal vesicles, increased pituitary weights, diminished LH/FSH stores in the pituitary and reduced peripheral levels of LH. To investigate if, in addition, the antibiotic exerts a direct effect on the steroidogenesis, the reactivity of Leydig cells was determined: a) after 8 days pretreatment of the donor mice in vitro; b) after addition of the antibiotic in vitro; c) after addition of the antibiotic in vitro in presence of dibutyryl cyclic AMP or hydrocholesterol. Tylosin caused an inhibition of basal and stimulated testosterone production, all the same if it was applied in vivo or in vitro. Moreover the increase in testosterone production caused by dibutyryl cyclic AMP was inhibited as well as that caused by addition of hydrocholesterol. These data give rise to the suspicion that tylosin inhibits directly intracellular steps of testosterone biosynthesis of Leydig cells.

Stimulation of testosterone production by atrial natriuretic peptide in isolated mouse Leydig cells results from a promiscuous activation of cyclic AMP-dependent protein kinase by cyclic GMP

The aim of this study was to examine the possibility that atrial natriuretic peptide-stimulated testosterone production by mouse Leydig cells results from an activation of cAMP-dependent protein kinase (kinase A) by cGMP. In these cells, both 8Br-cGMP and 8Br-cAMP could stimulate testosterone production, though the latter was approximately 50-fold more potent. Following the stimulation of the cells with the atrial peptide, a dose-related decrease in the cellular protein-bound cAMP accompanied by a concomitant increase in the protein-bound cGMP was observed. The steroidogenesis stimulated by both human chorionic gonadotrophin (hCG) and atrial peptide was inhibited in a dose-dependent manner by a cAMP antagonist, adenosine 3′,5′-cyclic monophosphothioate, Rp-isomer (RpcAMPS). In a cell-free [ 3H]cAMP binding assay, we have shown that unlabelled cGMP and RpcAMPS could competitively inhibit the [ 3H]cAMP binding, confirming that cAMP, RpcAMPS and cGMP could bind to the same binding protein. Finally, in a cell-free kinase A assay system, we have demonstrated that in lysates prepared from either atrial peptide or hCG-stimulated cells, the cellular kinase A was activated to an equal extent. We conclude from the data obtained that cGMP can bind to the cAMP-binding sites of kinase A and thereby brings about a promiscuous activation of this kinase. This appears to be an underlying mechanism by which atrial peptide hormone is able to stimulate the steroidogenesis in mouse Leydig cells.

Catecholamines stimulate testicular testosterone release of the immature golden hamster via interaction with alpha- and beta-adrenergic receptors.

Several lines of evidence suggest that catecholamines are involved in the regulation of the development of the testis. We have therefore investigated the ability of testicular parenchyma (decapsulated pieces of testes) from 18 to 20-day-old golden hamsters to respond to catecholaminergic stimuli in vitro. Norepinephrine and epinephrine, as well as the beta-receptor agonist isoproterenol and the alpha-adrenoreceptor agonist phenylephrine were able to significantly stimulate testicular testosterone production. Dopamine and serotonin were not effective. The stimulatory action of norepinephrine on testosterone production was dependent on the concentration. In incubations of testes with human chorionic gonadotropin (hCG) and norepinephrine, no synergistic effects on testosterone release were observed. The stimulatory effect of norepinephrine could be partially blocked by incubation with beta-receptor antagonist propranolol, or with alpha-receptor antagonist prazosin, while a combination of propranolol and prazosin completely inhibited the norepinephrine-induced testosterone production. Moreover, isoproterenol and phenylephrine in combination stimulated testosterone more than either drug did alone. Measurements of concentrations of norepinephrine and epinephrine in testicular homogenates revealed higher values for these catecholamines than in the plasma, implying that catecholamine levels in the interstitial spaces of the testis might be in the range of concentrations effectively stimulating testosterone production in vitro. This suggests that in the immature testis of the golden hamster, catecholamines acting through both alpha- and beta-adrenergic receptors may be potent physiological stimulators of testosterone production.

Direct and indirect effects of murine interleukin-2, gamma interferon, and tumor necrosis factor on testosterone synthesis in mouse Leydig cells.

It was recently observed that treatment of patients with a high dosage of human interleukin (IL-2) resulted in suppression of plasma concentrations of testosterone. A murine model was developed to assess the direct and indirect effects of murine IL-2 and the secondarily released cytokines, gamma interferon (INF gamma), and tumor necrosis factor (TNF alpha), on testosterone production in isolated Leydig cells. Pretreatment for 24 hours with IL-2 (100 to 500 IU/ml) or INF gamma (100 to 1000 IU/ml) significantly decreased testosterone production in response to luteinizing hormone (LH; P < 0.02 and 0.005, respectively). The combinations of INF gamma with either TNF alpha or IL-2 produced enhanced suppressive effects on Leydig cell testosterone production. Steroidogenic precursors (22-hydroxycholesterol, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone) restored testosterone secretion to control levels after preincubation with INF gamma or TNF alpha. In contrast, the inhibition of testosterone synthesis produced by either IL-2 or INF gamma plus TNF alpha could be reversed by 17 alpha-hydroxypregnenolone and dehydroepiandrosterone, but not by 22-hydroxycholesterol (P < 0.01). Dibutyryl cyclic adenosine monophosphate was also ineffective in reversing the inhibitory effects of these cytokines on synthesis. Although IL-2 directly inhibited synthesis in isolated Leydig cells, it stimulated testosterone production (P < 0.005) in minced murine testes. This suggests that IL-2 releases regulatory factors from other cells that were able to overcome the direct inhibitory effect of IL-2. This stimulatory effect was not caused by INF gamma and TNF alpha because INF gamma alone or with TNF alpha inhibited (P < 0.005) testosterone production in minced testes.(ABSTRACT TRUNCATED AT 250 WORDS)