Hormonal regulation of testicular steroid and cholesterol homeostasis

The male sex steroid, testosterone (T), is synthesized from cholesterol in the testicular Leydig cell under control of the pituitary gonadotropin LH. Unlike most cells that use cholesterol primarily for membrane synthesis, steroidogenic cells have additional requirements for cholesterol, because it is the essential precursor for all steroid hormones. Little is known about how Leydig cells satisfy their specialized cholesterol requirements for steroid synthesis. We show that in mice with a unique hypomorphic androgen mutation, which disrupts the feedback loop governing T synthesis, that genes involved in cholesterol biosynthesis/uptake and steroid biosynthesis are up-regulated. We identify LH as the central regulatory molecule that controls both steroidogenesis and Leydig cell cholesterol homeostasis in vivo. In addition to the primary defect caused by high levels of LH, absence of T signaling exacerbates the lipid homeostasis defect in Leydig cells by eliminating a short feedback loop. We show that T signaling can affect the synthesis of steroids and modulates the expression of genes involved in de novo cholesterol synthesis. Surprisingly, accumulation of active sterol response element-binding protein 2 is not required for up-regulation of genes involved in cholesterol biosynthesis and uptake in Leydig cells.     

Multiple regulation of testicular steroidogenesis

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.     

Regulation of steroidogenesis in rat Leydig cells in culture: effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin

Rat Leydig cells in primary culture were used as a model system to investigate the effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and the iron-sulfur protein, adrenodoxin. Leydig cells isolated from the testes of mature rats were placed in monolayer culture in the absence of stimulatory factors for 8 days. HCG (10 mIU/ml) or Bt2cAMP (1 mM) were then added to some of the cultures and the incubations were continued for up to 48 h. Testosterone production was increased markedly in cells incubated with hCG or Bt2cAMP. A significant accumulation of pregnenolone in the medium of cells treated with Bt2cAMP was also observed. Both hCG and Bt2cAMP increased the rates of synthesis of cytochrome P-450scc and adrenodoxin. In hCG-treated cells the apparent rate of synthesis of cytochrome P-450scc was increased 13-fold over that of controls after 48 h of incubation; the rate of adrenodoxin synthesis was increased 4-fold by hCG treatment. In Bt2cAMP-treated cells the rate of synthesis of cytochrome P-450scc was 37-fold greater than that of control cells after 48 h of incubation; adrenodoxin synthesis was increased 36-fold over controls. In hCG- and Bt2cAMP-treated cells, the concentration of immunoreactive cytochrome P-450scc and adrenodoxin increased with increasing time of incubation, and were correlated with the stimulatory effects of these agents on cytochrome P-450scc activity and on total steroid production. The results of this study are indicative that the maintenance by LH/hCG of elevated levels of testosterone synthesis by the Leydig cell is mediated, in part, by induction of the synthesis of cytochrome P-450scc and its associated protein, adrenodoxin. Since Bt2cAMP had effects similar to those observed with hCG, it is suggested that the stimulatory effects of hCG on the synthesis of cytochrome P-450scc and adrenodoxin are mediated by increased cyclic AMP formation.     

Interleukin-1 alpha as a potent inhibitor of gonadotropin action in porcine Leydig cells: site(s) of action.

The effects of interleukin on testicular steroidogenesis have been studied in several laboratories, most often by using cultured rat Leydig cells. Several reports have indicated that interleukin-1 beta (IL-1 beta), but not interleukin-1 alpha (IL-1 alpha), exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, we found that IL-1 alpha (and to a lesser extent IL-1 beta), contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action; and we further localized the steroidogenic biochemical step(s) affected by IL-1 alpha. IL-1 alpha inhibited hCG-induced testosterone secretion (about 67%) in a dose- and time-dependent manner. Half maximal and maximal effects were obtained with 4 U/ml (approximately 0.4 ng/ml, 0.3 x 10(-10) M) and 20 U/ml (approximately 2 ng/ml, 1.4 x 10(-10) M) of IL-1 alpha, respectively. The inhibitory effect of IL-1 alpha on gonadotropin action was detected at 6 h and was maximal after 24 h of treatment with the cytokine. The IL-1 alpha inhibitory effect was more potent than that of IL-1 beta: the maximal inhibitory effect of IL-1 beta was obtained with 400 U/ml. Subsequent investigations indicated that IL-1 alpha inhibited different biochemical steps involved in gonadotropin-induced testicular steroidogenesis. In this context, although IL-1 alpha appears to inhibit Leydig cell membrane functions (through a decrease in LH/hCG binding and gonadotropin-induced cAMP production), the antigonadotropin action of the cytokine is probably exerted predominantly at a step(s) located beyond cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats

A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng-1 microgram/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 microgram LH/ml, maximal LH-stimulated testosterone production. After production in the presence of excess 20 alpha-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 microgram LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action.     

Cross-talk between G protein-coupled and epidermal growth factor receptors regulates gonadotropin-mediated steroidogenesis in Leydig cells

Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.     

Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells

Testosterone biosynthesis in Leydig cells is dependent on two cytochrome P450 enzymes, cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha]. The expression of these two enzymes is differentially regulated by LH acting via its second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), and by specific steroid hormones. P450scc is constitutively expressed in normal mouse Leydig cells and in MA-10 tumor Leydig cells. Chronic cAMP stimulation increases the steady state levels of P450scc mRNA and de novo P450scc protein synthesis. In contrast, cAMP is obligatory for de novo synthesis of P450(17 alpha) in normal mouse Leydig cells; P450(17 alpha) synthesis ceases in the absence of luteinizing hormone or cAMP. MA-10 tumor Leydig cells do not express P450(17 alpha) even after treatment with cAMP. The amount of P450(17 alpha) in Leydig cells is negatively regulated by testosterone acting by two distinct mechanisms. At low concentrations, testosterone acts via the androgen receptor to repress cAMP-induced synthesis of P450(17 alpha), whereas at high concentrations this steroid increases the rate of degradation of the enzyme by an oxygen-mediated mechanism. Both constitutive and cAMP-induced synthesis of P450scc protein and steady state levels of mRNA are modulated by glucocorticoids. In normal mouse Leydig cells, glucocorticoids repress P450scc synthesis and steady state levels of P450scc mRNA, whereas glucocorticoids stimulate P450scc synthesis and levels of P450scc mRNA in the tumor Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multistep regulation of Leydig cell function

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.     

Regulation of the cholesterol ester cycle of cultured Leydig tumor cells

The MA-10 Leydig tumor cells take up low-density lipoprotein (LDL) from the medium and store the LDL-derived cholesterol as cholesterol esters that can be subsequently mobilized and used for steroid hormone synthesis. The present studies investigate the mechanisms by which cAMP acutely regulates the cellular content of cholesterol esters. In the absence of cholesterol utilization for steroidogenesis, cAMP stimulates cholesterol ester hydrolysis and ester resynthesis proportionally. The augmentation of ester hydrolysis by cAMP is completely matched by increased activity of the acyl-coenzyme-A:cholesterol acyltransferase and thus does not regulate cellular cholesterol ester concentration per se. The more important action of cAMP is to interrupt the cycle of hydrolysis and ester resynthesis by decreasing cholesterol re-esterification. In cells actively synthesizing steroid hormones, cholesterol reesterification is decreased by 82%. The decrease in cholesterol re-esterification occurs because cAMP directs cholesterol normally destined for re-esterification into steroid synthesis; simply blocking the utilization of cholesterol for steroidogenesis completely prevents net cholesterol ester hydrolysis and increases the cellular rate of cholesterol esterification.     

Stimulation of progesterone production by phorbol-12-myristate-13-acetate in MA-10 Leydig tumor cells

The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain cleavage (CSCC) enzyme was observed in mitochondria isolated from the PMA-treated MA-10 Leydig cells when compared to mitochondria isolated from non-treated cells. However, this stimulation of CSCC activity appears to be of limited importance when compared to the 240-fold increase observed in progesterone production following PMA stimulation. In contrast, the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (alpha-PD) had no effect on either progesterone production or CSCC activity. PMA had no effect on the conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone suggesting that one of the mechanism(s) of PMA action may involve the delivery of cholesterol to the mitochondria and/or the affinity of cholesterol with cytochrome P-450scc. Stimulation of steroidogenesis by PMA was also shown to be inhibited by cycloheximide. When PMA was added together with a submaximal dose of hCG, hCG-stimulated steroidogenesis was inhibited. However, at a maximal dose of human chorionic gonadotropin (hCG), PMA inhibited steroid synthesis at 1 and 2 h but had no significant effect at 3 h. Conversely, PMA had an additive effect on cAMP induced steroidogenesis. It was further demonstrated that PMA resulted in a decrease in the hCG-induced accumulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The possible role of protein kinase C and phospholipids in the regulation of steroid production in rat Leydig cells

We have studied the possible involvement of the activation of calcium-dependent phospholipid-activated protein kinase (PK-C) in the stimulatory action of LHRH on Leydig cells, using 4 beta-phorbol-12-myristate-13-acetate (PMA) and phospholipase C (PL-C). LHRH agonist (LHRH-A) and PL-C had a large synergistic effect on LH-stimulated steroid production, whereas PMA inhibited the effect of LH. However, PMA always caused an increase in steroid production stimulated by various doses of dibutyryl cAMP. LH and PMA stimulated the phosphorylation of 17 and 33 kDa proteins, whereas LHRH-A and PL-C had no effect. Of all effectors used, LH had the most pronounced effect on the synthesis of 14, 27 and 30 kDa proteins. The present results suggest that the mechanisms of action of LHRH-A and PL-C on steroid production in Leydig cells may be similar and different from PMA, and may involve stimulation of a specific type of PK-C or hydrolysis of a specific pool of phospholipids.     

Effects of prolactin on the morphology and function of rat Leydig cells: short-term versus long-term administration

Summary The bolus administration of prolactin (PRL) to adult rats did not cause any apparent change in the basal and luteinizing hormone (LH)-stimulated blood levels of testosterone (as estimated by radioimmune assay). Prolonged PRL infusion did not affect either basal testosterone plasma concentration or the morphology of Leydig cells (as evaluated by electron microscopy and stereology). Conversely, prolonged PRL treatment notably increased the gonadotrophic effects of chronic LH administration; this mainly consisted of a rise in the blood concentration of testosterone and a conspicuous hypertrophy of Leydig cells. The LH-induced increase in the volume of Leydig cells was the result of an increase in the volumes of all the organelles involved in steroid synthesis (i.e., smooth endoplasmic reticulum, peroxisomes and mitochondria). However, the trophic effects of PRL infusion exclusively concerned smooth endoplasmic reticulum and peroxisomes. In the light of these findings, the hypothesis is advanced that the mechanism underlying the gonadotrophic action of PRL involves an enhancement of the endogenous cholesterol synthesis, which could provide an abundance of precursors for testosterone synthesis, the post-cholesterol steps of which, in turn, would be exclusively controlled by LH.     

Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.     

LH action in the Leydig cell: modulation by angiotensin II and corticotropin releasing hormone, and regulation of P450(17) alpha mRNA

Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic function. A number of hormones produced by the Leydig cell (e.g. estrogen, angiotensin, CRF, vasopressin) and the tubular compartment (inhibin, TGF beta), can influence both acute and long-term actions of LH. Conversely, hormones produced in the Leydig cells modulate tubular function (e.g. androgen, beta-endorphin, oxytocin). The LH stimulatory event can be negatively influenced by the action of angiotensin II through the guanyl nucleotide inhibitory unit of adenylate cyclase. We have recently discovered an action of corticotrophin releasing hormone through specific high-affinity low-capacity receptors in the Leydig cells which involves a pertussis toxin insensitive guanyl nucleotide regulatory unit with interaction between signalling pathways and resulting inhibition of LH induced cAMP generation and consequently of steroidogenesis. In contrast to other tissues the CRF receptor in the Leydig cells did not couple to Gs. CRF action is exerted through direct or indirect action of protein kinase C, at the level of the catalytic subunit of adenylate cyclase. Physiological increases in endogenous LH cause positive regulation of membrane receptors and steroidogenesis, while major elevations in circulating gonadotropin can induce down-regulation of LH receptors and desensitization of steroid responses in the adult cell. Gonadotropin-induced desensitization in adult rat tests include an estrogen mediated steroidogenic lesion of the microsomal enzymes 17 alpha-hydroxylase/17,20-desmolase. For further understanding of the regulation of this key enzyme of the androgen pathway the rat P450(17) alpha cDNA was cloned and sequenced. This cDNA expressed in COS-1 cells 17 alpha-hydroxylase/17,20-desmolase activities. From the deduced amino acid sequence, two transmembrane regions were identified, a signal peptide for insertion in the ER, and a 2nd transmembrane region separated from the first by 122 amino acids. The carboxy terminal non-transmembrane region possesses 4 hydrophobic clefts, of which cleft II would contain the putative steroid binding site for both hydroxylase and lyase activities. The rat cDNA was employed to evaluate the hormonal regulation of mRNA levels in adult and fetal Leydig cells. Low dose hCG treatment caused an early increase in mRNA levels followed by a return to control values at later times, while with higher desensitizing doses the initial increase in mRNA was followed by a marked reduction in mRNA at 24 h and a small recovery at 48 h. Fetal rat Leydig cells treated with E2 showed a 70% decrease in P450 mRNA levels, and testosterone production closely followed the changes in mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish

In African catfish, two gonadotropin-releasing hormone (GnRH) peptides have been identified: chicken GnRH (cGnRH)-II and catfish GnRH (cfGnRH). The GnRH receptors on pituitary cells producing gonadotropic hormone signal through inositol phosphate (IP) elevation followed by increases in intracellular calcium concentration (?Ca(2+)(i)). In primary pituitary cell cultures of male African catfish, both cGnRH-II and cfGnRH dose dependently elevated IP accumulation, ?Ca(2+)(i), and the release of the luteinizing hormone (LH)-like gonadotropin. In all cases, cGnRH-II was more potent than cfGnRH. The GnRH-stimulated LH release was not associated with elevated cAMP levels, and forskolin-induced cAMP elevation had no effect on LH release. With the use of pituitary tissue fragments, however, cAMP was elevated by GnRH, and forskolin was able to stimulate LH secretion. Incubating these fragments with antibodies against cfGnRH abolished the forskolin-induced LH release but did not compromise the forskolin-induced cAMP elevation. This suggests that cfGnRH-containing nerve terminals are present in pituitary tissue fragments and release cfGnRH via cAMP signaling on GnRH stimulation, whereas the GnRH receptors on gonadotrophs use IP/?Ca(2+)(i) to stimulate the release of LH.     

Corticotropin-releasing factor receptors and actions in rat Leydig cells

Rat Leydig cells possess functional high affinity receptors for corticotropin-releasing factor (CRF). CRF inhibited human chorionic gonadotropin (hCG)-induced androgen production in cultured fetal and adult Leydig cells in a dose-dependent manner, but it had no effect on basal testosterone secretion. Comparable inhibitory effects of CRF were observed in the presence or absence of 3-isobutyl-1-methylxanthine. CRF treatment caused a marked reduction of steroid precursors of the androgen pathway (from pregnenolone to testosterone) during gonadotropin stimulation, but it did not influence their basal levels. The inhibitory action of CRF on hCG-induced steroidogenesis was fully reversed by 8-bromo-cAMP but was not affected by pertussis toxin. The action of CRF was rapid; and it was blocked by coincubation with anti-CRF antibody. CRF caused no changes in hCG binding to Leydig cells, and in contrast to other target tissues, CRF did not stimulate cAMP production, indicating that CRF receptors are not coupled to Gs in Leydig cells. These studies have demonstrated that CRF-induced inhibition of the acute steroidogenic action of hCG is exerted at sites related to receptor/cyclase coupling or cAMP formation. The inhibitory effects of CRF in the Leydig cell do not occur through the Gi unit of adenylate cyclase, but could involve pertussis toxin-insensitive G protein(s). These observations demonstrate that CRF has a novel and potent antireproductive effect at the testicular level. Since CRF is synthesized in the testis and is present in Leydig cells, it is likely that locally produced CRF could exert negative autocrine modulation on the stimulatory action of luteinizing hormone on Leydig cell function.     

Luteinizing hormone receptor mediates neuronal pregnenolone production via up-regulation of steroidogenic acute regulatory protein expression

The functional consequences of luteinizing hormone/human chorionic gonadotropin signaling via neuronal luteinizing hormone/human chorionic gonadotropin receptors expressed throughout the brain remain unclear. A primary function of luteinizing hormone (LH) in the gonads is the stimulation of sex steroid production. As LH can cross the blood-brain barrier, present in cerebrospinal fluid and is expressed by neuronal cells, we tested whether LH might also modulate steroid synthesis in the brain. Treatment of differentiated rat primary hippocampal neurons and human M17 neuroblastoma cells with LH (100 mIU/mL) resulted in a twofold increase in pregnenolone secretion in both cell types, suggesting an increase in P450scc-mediated cleavage of cholesterol to pregnenolone and its secretion from neurons. To explore how LH might regulate the synthesis of pregnenolone, the precursor for steroid synthesis, we treated rat primary hippocampal neurons with LH (0, 10 and 100 mIU/mL) and measured changes in the expression of LH receptor and steroidogenic acute regulatory protein (StAR). LH induced a rapid (within 30 min) increase in the expression of StAR, but induced a dose-dependent decrease in LH receptor expression. Consistent with these results, the suppression of serum LH in young rats treated with leuprolide acetate for 4 months down-regulated StAR expression, but increased LH receptor expression in the brain. Taken together, these results indicate that LH induces neuronal pregnenolone production by modulating the expression of the LH receptor, increasing mitochondrial cholesterol transport and increasing P450scc-mediated cleavage of cholesterol for pregnenolone synthesis and secretion.