Hydroxylation of 19-norandrogens by porcine Leydig cells.

The metabolism of 19-norandrogens by porcine Leydig cells was investigated. Non-radioactive 19-norandrostenedione (19-Nor A) and [3H]19-nortestosterone (19-Nor T) were used as substrates in incubations with cell preparations from mature male pigs. Steroid products were separated by reversed-phase HPLC and material in selected peaks was rechromatographed before attempts to identify them by GC-MS. Both 11 beta- and 6 beta-hydroxylated derivatives of 19-Nor A were found and a third product (11-oxo-19-Nor A) was tentatively identified. The profile of radioactive metabolites from [3H]19-Nor T also favours the view of a capacity for hydroxylation of 19-norandrogens by porcine Leydig cells. The significance of these findings together with our earlier report of direct 11 beta-hydroxylation of C19 steroids by such cells remains to be examined.     

Aromatization of 19-norandrogens by equine testicular microsomes

In the stallion testis, aromatase activity was localized in the microsomal fraction. Androgen aromatization occurred through the loss of 1 beta,2 beta hydrogen atoms and appeared to involve free sulfhydryl groups. A single enzyme system seemed to aromatize androgen and norandrogen at the same rate while having a much lower affinity for norandrogens.     

Formation of C19 11-hydroxysteroids by porcine Leydig cells.

In a previous study, we reported the presence of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone in testicular vein blood from mature male pigs. Since C19 steroids with an oxygen function at C11 have not been recorded as products of steroid biosynthesis in normal mammalian testes, we have examined their possible production in purified preparations of porcine Leydig cells. Both androstenedione and cortisol were added as substrates in studies using cell incubations of Leydig cells from mature boars (greater than 8 months old). Steroids were recovered from media by solid-phase extraction and separated by reversed-phase high performance liquid chromatography. Peaks corresponding to retention times of authentic standard steroids were seen for both 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone from each substrate. Generally, lesser amounts of C19 11-oxosteroids were noted also. Definitive confirmation was made by gas chromatography - mass spectrometry for 11 beta-hydroxyandrostenedione in the media.     

Synthesis and aromatization of 19-norandrogens in the stallion testis

The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost entirely conjugated after biosynthesis in vitro. The sequence of appearance of steroids in the peripheral blood after a single injection of 10,000 IU hCG suggests that, in the equine testis, 19-norandrogens are produced by a specific C10-19 desmolase (estrene synthetase), stimulable by hCG. 19-Nortestosterone was aromatized into estradiol-17 beta by stallion testicular microsomes. The affinity of the aromatase for 19-nortestosterone was very low compared to that for testosterone. At low and presumably physiological levels, and at a high testosterone/19-nortestosterone ratio, testosterone did not inhibit 19-nortestosterone aromatization by more than 53%. Thus, 19-nortestosterone may be aromatized in vivo in the testis in spite of the endogenous concentrations of androgens. However, the low velocity of 19-nortestosterone aromatization by testicular microsomes at roughly physiological concentrations suggests that 19-norandrogen aromatization may only participate slightly in the testicular estrogen production. These results suggest that in the equine testis, two aromatizing enzyme systems may exist: one which aromatizes both androgens and 19-norandrogens, and a minority system more specific for 19-norandrogens.     

Internalization and recycling of lutropin receptors upon stimulation by porcine lutropin and human choriogonadotropin in porcine Leydig cells

Porcine lutropin (pLH) and human choriogonadotropin (hCG) recognize the same hormonal receptor and elicit the same steroidogenic response in porcine Leydig cells in primary culture. We compared the variation in the number of occupied and free receptors present on the cell surface under short-term stimulation by the two hormones at 35 degrees C. Both hormones produced a rapid dose-dependent decrease in the total number of the gonadotropin receptors present on the cell surface with a half-life of 8-10 min. This decrease was reversible upon hormone removal and receptors were recovered on the cell surface with the same half-life of 8-10 min. With pLH, the receptors were recycled in a free state, but in the presence of hCG the receptors were recycled in an occupied state. This difference could be related to the higher affinity of hCG for the receptor in 150 mM NaCl buffer (Ka = 1.6 X 10(9) for hCG and 1.5 X 10(7) for pLH) and higher stability to acid pH of the hCG-receptor complex (dissociation pK = 3.7 for hCG and 4.5 for pLH).     

Studies with purified immature porcine Leydig cells in primary culture

The steroidogenic capacity of purified immature porcine Leydig cells in culture was studied over several days. The cells were obtained by fractionating crude testicular interstitial cell suspensions on a discontinuous Percoll gradient (d = 1.037, 1.042, 1.052, 1.098 g/ml), and characterized by specific binding of 125I-human chorionic gonadotropin (hCG), testosterone (T) and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to hCG, and the enzymatic determination of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The Leydig cells were recovered in a density band between 1.052-1.068 g/ml and grown in a chemically defined medium (Mather et al., 1981). In the absence of hCG, T production was low throughout the 6 days of culture. However, in response to hCG (10 mIU/ml), the cultured Leydig cells showed a progressive increase in T synthesis, which reached a maximum at Days 3-4. 8-Br-cAMP (1 mM) induced a comparable rise in T production to that obtained with hCG throughout the culture period. In contrast, 8-Br-cAMP induced a near maximal increase in dehydroepiandrosterone (DHEA) production from Day 1. This paper demonstrates that purified immature porcine Leydig cells in primary culture are a valuable model to study the ontogeny of Leydig cell function.     

Interactions between immature porcine Leydig and Sertoli cells in vitro

Summary Interactions between Leydig and Sertoli cells, as well as a stimulatory effect of FSH on Leydig cell activity, have been reported in many studies. In order to investigate these interactions, the ultrastructure of immature pig Leydig cells under different culture conditions has been studied. When cultured alone in a chemically defined medium, there is a marked regression of the Leydig cell smooth endoplasmic reticulum and a swelling of the mitochondria. Addition of FSH or hCG does not prevent these phenomena. Co-culturing of Leydig cells with Sertoli cells from the same animal maintains the smooth endoplasmic reticulum at the level seen in vivo and in freshly isolated Leydig cells. The addition of FSH to the co-culture stimulates its development and increases Leydig cell activity, as assessed by an increase in hCG binding sites and an increased steroidogenic response to hCG. These results suggest that Sertoli cells exert a trophic effect on Leydig cells, and that the stimulatory effect of FSH on Leydig cell function is mediated via the Sertoli cells. These results reinforce the concept of a local regulatory control of Leydig cell steroidogenesis.Post-Doctoral fellow supported by CIRIT, Generalitat de Catalunya, Spain     

Different patterns of metabolism determine the relative anabolic activity of 19-norandrogens

Testosterone, the principal androgen secreted by Leydig cells, exerts a wide range of actions including growth of the male reproductive tract (androgenic effects) and growth of non-reproductive tissues such as muscle, kidney, liver, and salivary gland (anabolic effects). As androgenic steroids were discovered some were found to have relatively more anabolic than androgenic activity. The results reviewed in this report suggest that these differences result, in part, from the differential metabolism of the steroids in individual tissues and the varied activities of the individual metabolites. In the accessory sex organs (e.g. the prostate) testosterone is 5-reduced to dihydrotestosterone (DHT) which, due to its higher affinity for androgen receptors (AR), amplifies the action of testosterone. In contrast, when 19-nortestosterone (NT) is 5-reduced, its affinity for AR decreases, resulting in a decrease in its androgenic potency. However, their anabolic potency remains unchanged since significant 5-reduction of the steroids does not occur in the muscle. 7-methyl-19-nortestosterone (MENT) does not get 5-reduced due to steric hindrance from the 7-methyl group. Therefore, the androgenic potency of MENT is not amplified as happens with testosterone. These metabolic differences are responsible for the increased anabolic activity of NT and MENT compared to testosterone. Part of the biological effects of testosterone are mediated by its aromatization to estrogens. The fact that MENT is also aromatized to 7-methyl estradiol, a potent estrogen, in vitro by human placental and rat ovarian aromatase suggests that some of the anabolic actions of MENT may be mediated by this estrogen.     

Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF)

The effects of fibroblast growth factor (FGF) on testicular aromatase activity has been studied using primary cultures of porcine Leydig cells. After culture for 3 days in the absence or presence of FGF, the ability of the cells to produce estrogen was examined in a 4h-test period in which either (a) hCG (10(-9) M) or (b) androstenedione (3 x 10(-6) M) was added to the medium. FGF produced a 3- to 20-fold increase in estrogen formation from endogenous or exogenous substrate during the test period, in spite of a marked decrease (approximately equal to 60%) in [125I]-hCG binding and no significant change in testosterone concentration. Stimulation of estrogen secretion by FGF was dose-(ED50 approximately equal to 2 ng/ml) and time-dependent, the first and maximal effects were observed after 12h and 48h, respectively. Preliminary tests with several other factors (insulin, EGF, TGF-beta, FSH and hCG) showed that hCG alone directly stimulated aromatase activity. From these findings a role is suggested for FGF as a paracrine/autocrine agent in the control of estrogen secretion by Leydig cells.     

Effect of catecholamines on porcine Sertoli and Leydig cells in primary culture

The accumulation by purified immature porcine Leydig and Sertoli cells of cyclic adenosine 3',5'-monophosphate in the presence of 1-methyl-3-isobuthylxathine was studied and their respective testosterone and 17 beta-estradiol production in response to catecholamines was assessed in vitro. These substances increased both basal and FSH-stimulated cyclic adenosine 3',5'-monophosphate accumulation in Sertoli cells. In contrast, catecholamines slightly enhanced basal cyclic adenosine 3',5'-monophosphate production but inhibited its human chorionic gonadotropin-stimulated accumulation by Leydig cells. Catecholamines had no effect on basal and stimulated testosterone release by these cells, while dopamine inhibited 17 beta-estradiol synthesis by Sertoli cells. Using various alpha- and beta-adrenergic agonists and antagonists, beta-receptors, likely of the beta 1-subtype, were shown to be present in both cell lines. Taken together these data suggest the presence of a cyclic adenosine 3',5'-monophosphate-linked adrenergic receptor in porcine Leydig and Sertoli cells, the role of which remains to be determined.     

Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells.

In the present study, we evaluated the effect of the homodimer activin A on immature porcine Leydig cell functions in primary culture. Activin A (0.5-100 ng/ml) reduced hCG-stimulated dehydroepiandrosterone (DHEA) accumulation in a dose- and time-dependent manner, with a maximal inhibitory effect (58% decrease) at 20 ng/ml (8 x 10(-10) M). Activin A was found not to control steroidogenesis, either through a modulation of the gonadotropin LH/hCG binding or low-density lipoprotein cholesterol binding and internalization. However, activin A significantly decreased pregnenolone (p less than 0.002) and DHEA (p less than 0.001) formation (evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase [3 beta-HSDI]activity) in Leydig cells maximally stimulated with hCG (3 ng/ml, 3 h) or incubated in the presence of 22R-hydroxycholesterol (5 micrograms/ml, 2 h). These findings indicate that activin A probably exerts a partial inhibitory effect on cholesterol side-chain cleavage cytochrome P450 (P450scc) activity. On the other hand, activin A significantly (p less than 0.001) enhanced the conversion of exogenous pregnenolone and DHEA (500 ng/ml) but not of progesterone and androstenedione (500 ng/ml) into testosterone, suggesting that activin A potentially enhances 3 beta-HSDI activity in Leydig cells. Activin A action on 3 beta-HSDI activity was found to be closely related to that of transforming growth factor-beta 1 (TGF beta 1), since both activin A (20 ng/ml) and TGF beta 1 (2 ng/ml) induced a comparable and non-additive increase in 3 beta-HSDI activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lutropin is processed much more rapidly than human choriogonadotropin by porcine Leydig cells in primary culture

Lutropin (LH) and human choriogonadotropin (hCG) share the same receptor and stimulate testosterone production in porcine Leydig cells in primary culture. Cells were pulsed with [125I]LH or [125I]hCG. During the chase, more than 80% of cell-bound LH consisted in internalized material which was degraded and excreted (half-time: 25 min) NH4Cl largely inhibited this degradation. On the contrary, hCG remained essentially bound to the cell surface and was not degraded by the cells with or without NH4Cl up to 160 min.     

Lutropin is processed much more rapidly than human choriogonadotropin by porcine Leydig cells in primary culture

Lutropin (LH) and human choriogonadotropin (hCG) share the same receptor and stimulate testosterone production in porcine Leydig cells in primary culture. Cells were pulsed with [125I]LH or [125I]hCG. During the chase, more than 80% of cell-bound LH consisted in internalized material which was degraded and excreted (half-time : 25 min) NH4Cl largely inhibited this degradation. On the contrary, hCG remained essentially bound to the cell surface and was not degraded by the cells with or without NH4Cl up to 160 min.     

Human leukocyte interferon inhibits human chorionic gonadotropin stimulated testosterone production by porcine Leydig cells in culture

Pretreatment of primary porcine Leydig cell cultures with human leukocyte interferon suppressed the subsequent hCG-stimulated testosterone production in a dose-dependent manner, with an ED50 at 13 IU/ml. The treatment had no effect on hCG-binding to its receptor, and the inhibition of testosterone production was not abolished by 8Br-cAMP addition. The results indicate that the site of interferon action on hCG-stimulated testosterone production in primary cultures of porcine Leydig cells is located distal to cAMP formation.     

Mouse Leydig tumor cells produce C-19 steroids,including testosterone

The mouse Leydig tumor cells (MLTC-1) were derived from a transplantable Leydig cell tumor carried in C57BL/6 mice. The original cell line (M5480) produced testosterone and little progesterone. However, it was later shown that there were two subtypes of the cell line, one producing mainly progesterone and termed M5480P and the other which produced androgens and termed M5480A. MLTC-1 cells are reportedly derived from the former. We studied the production of testosterone by MLTC-1 cells using a specific and sensitive testosterone RIA, tandem mass spectrometry (TMS) and examined the expression of mRNA of some key enzymes involved in steroidogenesis. Although the molar yields were 1:20:60 for testosterone, androstenedione and progesterone, respectively, in response to human chorionic gonadotropin (hCG), testosterone measured by our RIA accounted for 94% of the testosterone immunoreactivity. Both MLTC-1 and Balb/c Leydig cells expressed Steroidogenic Acute Response (StAR) protein mRNA in response to hCG. Cytochrome P450 17alpha-hydroxylase/17,20-lyase mRNA was expressed constitutively in MLTC-1 and Balb/c Leydig cells. Whereas the latter expressed 17beta-hydroxydehydrogenase/17-ketoreductase isoform Type 3mRNA in response to hCG, MLTC-1 cells expressed isoform Type 7 constitutively. The absence of isoform Type 3 in MLTC-1 cells thus may account for the low conversion of androstenedione to testosterone in this cell line. However, with a very specific and sensitive RIA even the low production of testosterone becomes meaningful. In conclusion MLTC-1 cells produce testosterone.     

Aromatization and 19-hydroxylation of androgens by rat brain cytochrome P-450

The oxidative metabolism of androgens in the rat brain includes aromatization preceded by the requisite 19-hydroxylation. We have examined the transformation of [19-C3H3]androstenedione and [4-14C]testosterone by the semipurified cytochrome P-450 fraction of the rat brain. [19-C3H3]Androstenedione generated tritiated water and formic acid in a ratio of 8 to 1 indicating that 19-hydroxylation in the brain far exceeds that necessary for aromatization. This was confirmed by the results of the 14C-testosterone incubation in which the 14C labeled 19-hydroxy and 19-oxo derivatives which were isolated exceeded the yield of 14C-estrogens by several fold. Thus the rat brain has the capacity to form in situ 19-hydroxylated androgens which are not available to it from the circulation.     

Partial characterization of steroid sulfohydrolase and steroid sulfotransferase activities in purified porcine Leydig cells

Subcellular fractions of purified pig Leydig cells from 7 different animals have been investigated with respect to their abilities to catalyze the sulfation of several steroids and the hydrolysis of the sulfated forms of these same steroids. Considerable estrone sulfate sulfohydrolase of pH optimum 7.5 and high apparent Km was found to be concentrated in the 105,000 g pellet but no evidence was obtained, in any subcellular fraction, for the presence of any activity toward the 3-sulfate of pregnenolone, dehydroepiandrosterone (DHA) or delta 5-androstene-3 beta,17 beta-diol (androstenediol). Cytosolic sulfotransferase activity toward estrone, pregnenolone, DHA and androstenediol was present in each animal. The activity toward these 4 substrates was eluted from a gel filtration column as a single peak of apparent molecular weight 43 KDa. Upon chromatofocusing, a sharp estrogen sulfotransferase peak of apparent pI 6.1 and pH optimum 9.5, was clearly separated from the neutral steroid sulfotransferase which eluted over a more acidic pH range in a manner suggestive of the presence of several isozymes. This latter, which exhibited a wide pH optimum range between 6 and 8.5, was most active toward androstenediol, and least active toward pregnenolone. The estrogen sulfotransferase exhibited Michaelis-Menten kinetics (apparent Km = 4 microM). The neutral steroid sulfotransferase activity increased in velocity with increasing androstenediol or DHA concentration up to 1 microM beyond which considerable substrate inhibition occurred. It appears from these data that neutral steroid sulfates synthesized in the pig Leydig cell are not subject to enzymic desulfation in the same cells.     

Rapid in vitro desensitization of the testosterone response in rat Leydig cells by sub-active concentrations of porcine luteinizing hormone

We have studied in rat Leydig cells, the effect of sub-active concentrations of porcine LH on the subsequent stimulation of the cAMP and testosterone production by a sub-maximal concentration of pLH or hCG. We found that extremely low concentrations of pLH (0.01-2.0 ng/ml) were able to induce rapidly a partial but highly significative desensitization of the testosterone response without affecting the cyclic AMP response. These data indicate that desensitization of the steroidogenic response might be due to some lesion beyond cAMP formation or at the level of one discrete compartment of cyclic AMP, directly involved in the control of steroidogenesis. Moreover, our data strongly suggest that the basal circulating concentrations of LH can exert an inhibitory control on the testosterone response to LH pulses in vivo.     

Dual internalization pathway for lutropin and choriogonadotropin in porcine Leydig cells in primary culture

The role of the high affinity receptor in the internalization of porcine lutropin (pLH) and human choriogonadotropin (hCG) by porcine Leydig cells in primary culture during short-term stimulation by the two hormones was investigated. The fate of the hormones was followed either by electron microscopy (with colloidal gold-labeled hormones) or by measurement of the cellular distribution of [125I]pLH and [125I]hCG. With both techniques, the internalization of pLH was found to be one order of magnitude greater than hCG, though the recycling rate of the high affinity receptors was the same with both hormones. However, when the cell surface was progressively depleted of its high affinity receptors by preincubation with increasing doses of hCG or pLH, the internalization of [125I]pLH remained high and largely independent of the number of high affinity receptors still available on the cell surface, while that of [125I]hCG was found to be proportional to this number. The endocytosis of [125I]pLH could only be inhibited by the simultaneous presence of micromolar concentrations of unlabeled pLH, hCG or alpha or beta subunits of ovine LH (oLH). The intact alpha-hCG subunit and the deglycosylated alpha-oLH subunit were less potent, while beta-hCG and deglycosylated beta-oLH had no significant effect. These results could be explained by the existence of a "carrier" or "scavenger" receptor for LH, but with a low affinity (congruent to 3.10(6) M-1) and present in excess on the cell surface as compared to the high affinity receptor. The possible physiological significance of this receptor is discussed.