Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.     

The regulatory mechanism of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as tonic food to improve body condition in Chinese society for a long time. We have previously demonstrated that TM reduced in vitro hCG-treated steroidogenesis in MA-10 mouse Leydig tumor cells without any toxicity effect. In the present study, the mechanism how TM suppressed hCG-treated steroidogenesis in MA-10 cells was investigated. MA-10 cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents with or without TM to clarify the effects. TM significantly suppressed progesterone production with the presences of forskolin (10 and 100 microM) or dbcAMP (0.5 and 1mM), respectively, in MA-10 cells (p<0.05), which indicated that TM suppressed steroidogenesis after PKA activation along the signal pathway. Beyond our expectation, TM induced the expression of steroidogenic acute regulatory (StAR) protein with or without hCG treatments. However, TM profoundly decreased P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activities without any influences on the expression of both enzymes. These inhibitions on steroidogenic enzyme activities might counteract the stimulation of StAR protein expression. In conclusion, results suggest that TM suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.     

Time-related effects of arginine vasopressin on steroidogenesis in cultured mouse Leydig cells

The dose and time treatment effects of arginine vasopressin (AVP) on basal and hCG-stimulated testosterone accumulation by purified mouse Leydig cells in primary culture were examined. Pretreatment for 24 h of Leydig cells with AVP caused a stimulation of the acute (3 h) basal testosterone accumulation. In these conditions, progesterone accumulation was also increased. The stimulatory effect of AVP (10(-11)-10(-5) M) on testosterone accumulation was dose-dependent and as little as 10(-11) M-AVP caused significant stimulation whilst maximal effect was achieved with 10(-7) M. Oxytocin (10(-6) M) also showed a stimulation of testosterone accumulation in basal conditions, but the other peptides tested at the same concentration (neurotensin, somatostatin and substance P) did not have any effect. When Leydig cells were exposed to AVP for a longer period (48 or 72 h), the increase in basal testosterone accumulation disappeared. AVP treatment of Leydig cells for 72 h led to a significant and dose-dependent reduction in the hCG-responsiveness without altering the slope of the hCG dose-response curve. This inhibitory effect, which was also observed when AVP-pretreated Leydig cells were acutely challenged for 3 h with 8-bromo-cAMP, was accompanied by a concomitant increase in progesterone accumulation. These results indicate that AVP can exert a dual effect on mouse Leydig cells: stimulatory on basal testosterone accumulation during short-term exposure (24 h) and inhibitory on the response to hCG stimulation after long-term treatment (72 h). They provide additional evidence that neurohypophysial peptides directly affect Leydig cell steroidogenesis.     

Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.     

Specific low density lipoprotein receptors in pig Leydig cells. Role of this lipoprotein in cultured Leydig cells steroidogenesis

Freshly prepared and cultured pig Leydig cells were shown to possess specific binding sites for iodinated human low density lipoprotein (LDL). Binding of LDL was followed by internalisation. Both processes were inhibited by unlabelled LDL but not high density lipoprotein (HDL). The number of LDL binding sites was enhanced by prior treatment of cultured cells with hCG. Addition of LDL to the culture medium caused a large enhancement of the rate of steroid secretion (testosterone and dehydroepiandrosterone sulfate) both in the presence or absence of hCG. On the contrary, HDL decreased the steroid output, both in the presence or absence of hCG. We concluded that LDL cholesterol rather than cholesterol synthesized $\text{de novo}$ by the cells, is the major substrate for androgen production by pig Leydig cells in culture.     

Control of steroidogenesis in Leydig cells.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to stimulate steroidogenesis not only via cyclic AMP but also other pathways which include arachidonic acid and leukotrienes and regulation of chloride and calcium channels. The same stimulatory pathways may lead to desensitization and down-regulation of the LH receptor and steroidogenesis. The LH receptor exists in a dynamic state, being truncated, or internalized, degraded or recycled. Desensitization is controlled by protein kinase C (PKC) in the rat and by cyclic AMP dependent protein kinase and PKC in the mouse Leydig cells. Using an adapted anti-sense oligonucleotide strategy we have shown that the cytoplasmic C-terminal sequence of the LH receptor is essential for desensitization to occur. In contrast, these sequences of the LH receptor are not required for the stimulation of cyclic AMP and steroid production. We have also shown that the extracellular domain of the LH receptor is secreted from the Leydig cell and may act as a LH-binding protein.     

Leydig cells, thyroid hormones and steroidogenesis

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

The role of chloride ions in the regulation of steroidogenesis in rat Leydig cells and adrenal cells

The role of chloride ions in the regulation of steroidogenesis in rat Leydig cells and adrenal cells has been investigated. It was found that the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) inhibited LH but not dibutyryl cAMP (dbcAMP)-stimulated steroidogenesis in the Leydig cells. This was found to be via an inhibition of cAMP production, because both LH- and forskolin-stimulated cAMP productions were inhibited by DIDS. The exclusion of chloride ions enhanced steroidogenesis during incubation of Leydig cells and adrenal cells with dbcAMP. The adrenal cells were found to be more sensitive to dbcAMP than Leydig cells and the enhancing effects of chloride removal were higher. In the presence of chloride ions, near maximum steroidogenesis was achieved with approximately 60 μM and 1 mM dbcAMP in the adrenal and Leydig cells, respectively. In the absence of chloride ions the concentrations required decreased approximately 50-fold and 10-fold, respectively. It is concluded that although LH may regulate DIDS sensitive chloride channels, the enhanced stimulation of cAMP-mediated steroidogenesis by chloride exclusion is not mediated via these channels. We propose a model based on the present and previous studies [1] with Leydig tumour (MA10) cells i.e. that intracellular chloride ion depletion enhances the action of cAMP on protein synthesis which results in increased synthesis of the Steroidogenic Acute Regulator (StAR) protein and consequently increased steroidogenesis.     

Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells

Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to inositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA), a tumor promoting agent, could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA (100 mU and 12.5 microM, respectively) augmented hCG induced T secretion, while at higher concentrations (PLC: 500 mU and AA: 200 microM) they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5, 8, 11, 14 Eicosatetraynoic acid (ETYA), a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. We conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclooxygenase products.     

Nicotine and cotinine inhibit steroidogenesis in mouse Leydig cells

Cigarette smoking alters plasma testosterone concentrations in men. The objectives of this study were to determine if nicotine and cotinine, two alkaloid products of cigarettes, affect luteinizing hormone(LH)-stimulated steroidogenesis in isolated adult mouse Leydig cells. Leydig cells from adult Swiss-Webster mice were isolated by linear density gradient and incubated (95% O2, 5% CO2) in minimum essential medium at 37 C for 3 hours with LH (10 ng) and with or without nicotine or cotinine (10(-5)-10(-7) M). Both nicotine and cotinine produced dose response inhibition (P less than 0.05) of LH-stimulated testosterone production (50-70%). The addition of 8-bromo-3',5'-cyclic monophosphate (cAMP, 500 uM) stimulated steroidogenesis comparable to LH in the absence of the alkaloids, but both nicotine and cotinine significantly (P less than 0.05) reduced testosterone production in response to cAMP, suggesting that the alkaloids inhibit testosterone production in response to LH distal to the formation of cAMP. In MEM without calcium, LH-stimulated testosterone synthesis was decreased, and neither nicotine nor cotinine significantly affected steroidogenesis. The addition of a calcium ionophore in MEM with normal calcium content enhanced (P less than 0.05) the inhibitory effects of nicotine and cotinine on LH-responsive steroidogenesis. A calcium channel blocking agent, verapamil, at 10uM significantly (P less than 0.05) reversed the inhibition of LH-stimulated testosterone production produced by both alkaloids when incubated in the medium with a normal calcium concentration. These results suggest that nicotine and cotinine either affect intracellular calcium content or block the effects of calcium on steroidogenesis in mouse Leydig cells.     

Effect of phorbol ester and phospholipase C on LH-stimulated steroidogenesis in purified rat Leydig cells

When the phorbol ester, 4 beta-phorbol-12-myristate-13-acetate (PMA) or bacterial phospholipase C (PL-C) is added to a preparation of purified adult rat Leydig cells, containing 2 mM CaCl2, a time- and dose-dependent decreases of LH-stimulated testosterone production is observed. After a 3 h stimulation with oLH (100 ng/ml), PMA (100 ng/ml) and PL-C (1.6 U/ml) do not affect the cell viability or the hCG specific binding, while cAMP accumulation is significantly reduced; cAMP-stimulated steroidogenesis is diminished only in the presence of PL-C. These observations suggest that in vitro: (i) activated Ca2+- and phospholipid-dependent protein kinase is implicated in the regulation of rat Leydig cell steroidogenesis by LH at a step before the adenylate cyclase; (ii) phospholipids play an important role in cAMP-stimulated testosterone synthesis.     

Effect of prostaglandins on hormonal function of cultured rat Leydig cells

The effect of PGF2 alpha and its analogues on androgen production and activity of delta 5,3 beta-hydroxysteroid dehydrogenase in rat Leydig cells in vitro was investigated. Prostaglandin of the F type inhibit the enzyme activity and hormone secretion by cultured Leydig cells. This effect was considerably stronger in Leydig cells isolated from mature rats, than by Leydig cells from immature animals.     

The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.     

Effect of growth hormone and corticotropin on steroidogenesis in cultured rat adrenocortical cells

Primary cultures of rat adrenocortical cells responded to corticotropin (ACTH; 10 microU/ml) with peak steroid production within 24 h which declined thereafter. In the presence of ACTH and growth hormone (GH; 10 micrograms/ml), steroid production was significantly greater than with ACTH alone and was better maintained over several days. This latter response was not due to changes in cell number or multiplication and required several days to develop. GH also interacted with 10(-6) and 10(-5) M dibutyryl cyclic AMP (dbcAMP) to augment synthesis of corticosterone. At maximal doses of both ACTH and dbcAMP, GH did not have an additional effect on steroid production. In conclusion, GH has a stimulatory effect on steroid production when added in vitro but it is unlike the response seen in vivo in that it is less sensitive, additive rather than synergistic, and without effect on cell growth and multiplication.     

Regulatory mechanism of Cordyceps sinensis mycelium on mouse Leydig cell steroidogenesis

We demonstrate the mechanism by which Cordyceps sinensis (CS) mycelium regulates Leydig cell steroidogenesis. Mouse Leydig cells were treated with forskolin, H89, phorbol 12-myristate 13-acetate, staurosporine, or steroidogenic enzyme precursors with or without 3 mg/ml CS; then testosterone production was determined. H89, but not phorbol 12-myristate 13-acetate or staurosporine, decreased CS-treated Leydig cell steroidogenesis. CS inhibited Leydig cell steroidogenesis by suppressing the activity of P450scc enzyme, but not 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, 20alpha-hydroxylase, or 17beta-hydroxysteroid dehydrogenase enzymes. Thus, CS activated the cAMP-protein kinase A signal pathway, but not protein kinase C, and attenuated P45scc enzyme activity to reduce human chorionic gonadotropin-stimulated steroidogenesis in purified mouse Leydig cells.     

Fate determination of fetal Leydig cells

Leydig cell (LC) is one of the most important somatic cell types in testis, which localized in the interstitium between seminiferous tubules. The major function of Leydig cells is to produce steroid hormone, androgens. LC differentiation exhibits a biphasic pattern in rodent testes, which are divided into two different temporal mature populations, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). FLCs are transiently present in fetal testes and undergo involution or degeneration after birth. FLCs are completely devoid and replaced by ALCs in adult testes. Comparing to ALCs, FLCs display unique morphology, ultrastructure and functions. The origin of FLCs has been debated for many years, but it is still a mystery. Many factors have been reported regulating the specification, proliferation and differentiation of FLCs. FLCs degenerate in a few weeks postnatally, however, the underlying mechanism is still unknown. In this review, we will focus on the fate determination of FLCs, and summarize the resent progress on the morphology, ultrastructure, function, origin and involution of FLCs.