Steroidogenic acute regulatory protein expression and pregnenolone synthesis in rat astrocyte cultures.

Neurosteroids are steroids synthesised by brain cells. The molecular mechanism of neurosteroidogenesis from cholesterol has not yet been revealed. We studied the potential role of the steroidogenic acute regulatory (StAR) protein in neurosterodogenesis by using rat brain astrocytes. The novelty of the study is that regulation of StAR is described in primary cultures from embryonic mesencephalon and cerebellum regions of the brain. Dibutyryl cyclic AMP (dbcAMP) treatment increased StAR protein expression in astrocyte cultures. This was observed in immunoblots of mitochondrial fractions and by immunocytochemistry. Dual-labelling showed that the cyclic AMP-induced increase in StAR immunofluorescence was localised to mitochondria. In addition, mitochondrial cytochrome P450-side chain cleavage enzyme was demonstrated with a specific antibody, indicating the potential for pregnenolone production in these cells. Radioimmunoassay on ether-extracted conditioned media of control and dbcAMP treated cells demonstrated pregnenolone production by mesencephalic and cerebellar astrocyte cultures. Furthermore, 24-h pregnenolone levels, in the presence of inhibitors of further pregnenolone metabolism, were significantly increased by dbcAMP exposure. A murine StAR promoter-luciferase fusion plasmid was activated by dbcAMP in transiently transfected mesencephalic and cerebellar astrocytes. These novel results indicate that cyclic AMP signalling can regulate StAR expression and pregnenolone production in brain astrocytes, and provide additional insight into the role of StAR in neurosteroidogenesis.

Induction of steroidogenesis in immature rat Leydig cells by interleukin-1alpha is dependent on extracellular signal-regulated kinases.

Interleukin-1alpha (IL-1alpha) plays an important role in the regulation of immune responses as well as in non-inflammatory events in different types of cells. Here we have investigated the involvement of the extracellular signal-regulated kinase (ERK) cascade in IL-1alpha-induced steroidogenesis by primary cultures of immature rat Leydig cells. Our findings indicate that protein kinase C functions as an upstream component of signal transduction from the IL-1 receptor type I (IL-1RI) to the ERK cascade. It was observed that IL-1alpha upregulated both steroidogenic acute regulatory (StAR) protein expression and its phosphorylation when compared with controls. Selective inhibition of these mitogen-activated protein kinases (MAPKs) by UO126 enhanced both the expression and phosphorylation of the StAR protein, but suppressed androgen production by the immature Leydig cells as well as dissipating the mitochondrial electrochemical potential (Psim) in these cells. The evidence that water-soluble cholesterol but not 22R-hydroxycholesterol-stimulated steroidogenesis was inhibited by UO126 suggested that an intact Psim across the inner mitochondrial membrane is required for cholesterol translocation and is positively regulated by the ERK cascade. We propose that activation of ERKs by IL-1alpha plays a dual role in the regulation of steroidogenesis in immature Leydig cells: these MAPKs downregulate StAR expression and phosphorylation, while at the same time they support an intact Psim across the inner mitochondrial membrane, thereby promoting translocation of cholesterol into the mitochondria of the Leydig cell.

Regulation of the steroidogenic acute regulatory protein expression: functional and physiological consequences.

Steroid hormones are synthesized in steroidogenic cells of the adrenal, ovary, testis, placenta and brain and are essential for normal reproductive function and bodily homeostasis. The rate-limiting and regulated step in steroid biosynthesis is the intramitochondrial transport of cholesterol, a process that is mediated by the steroidogenic acute regulatory (StAR) protein. The importance of StAR has been illustrated by analyses of patients with lipoid congenital adrenal hyperplasia (lipoid CAH), an autosomal recessive disorder that markedly disrupts the synthesis of all gonadal and adrenal steroids. Molecular and physio-pathological analyses have demonstrated that alterations in the StAR gene are the only known cause of lipoid CAH. Furthermore, StAR knockout mice have been generated and display a phenotype that is essentially identical to the human condition. Recent advances in tissue-specific and hormone-induced expression of the StAR protein provide insights into a number of human endocrinological health issues including developmental and reproductive abnormalities. Several factors and processes have been demonstrated to influence StAR expression in steroidogenic cells and there is increasing evidence that a transcription factor-binding site-rich region present in the proximal region of the StAR promoter is highly instrumental in StAR gene expression. In this review we focus on the significant findings that have been made with regards to the regulation of StAR expression and also on the clinical and endocrinological consequences of a non-functioning StAR gene.

Oxysterols regulate expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20alphaOHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20alphaOHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.

Specific dose-dependent effects of ethane 1,2-dimethanesulfonate in rat and mouse Leydig cells and non-steroidogenic cells on programmed cell death.

The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively kills Leydig cells is poorly understood. To characterize further the cell-specific actions of EDS, we studied biochemical and morphological changes during apoptosis in different Leydig cell and non-steroidogenic cell models. Rat testicular and H540 tumor Leydig cells were killed by 1-2 mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher concentration of EDS was also necessary for activation of apoptosis in non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney cells were resistant. These variable effects of EDS on apoptosis were independent of new protein synthesis and, interestingly, could be delayed by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also observed chromosomal fragmentation and other hallmarks indicative of apoptosis as evidenced by DNA laddering and fluorescent microscopy. Time-lapse photography with a confocal microscope showed that the time of onset, duration and even the sequence of apoptotic events between individual H540 cells was heterogeneous. When the dose of EDS was gradually increased from 2 to 10 mM, the proportion of cells showing normal apoptotic features gradually decreased. Intriguingly, treatment with 10 mM EDS did not result in death for most cells and was marked by an absence of DNA laddering and ultrastructural features of apoptosis and necrosis. However, incubation with 20 mM EDS resulted in necrosis.These results demonstrated that the effects of EDS on cell survival are not specific to Leydig cells, that different cell types have different sensitivities to EDS and that stimulation of the cAMP pathway may mitigate EDS action. The data obtained with H540 cells further revealed that EDS can induce two types of programmed cell death.

Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1.

Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5'-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMtau (but not CREMalpha and CREMbeta) have qualitatively similar effects on StAR promoter activity in response to (Bu)(2)cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu)(2)cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMtau and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMtau and SF-1 results in enhanced activation of the StAR promoter.

Interleukin-1alpha stimulates steroidogenic acute regulatory protein expression via p38 MAP kinase in immature rat Leydig cells.

We have investigated the involvement of the steroidogenic acute regulatory (StAR) protein in interleukin-1alpha (IL-1alpha)-induced steroidogenesis in immature (40-day-old) and adult Leydig cells in vitro. Further, IL-1alpha-mediated signaling pathway(s) controlling StAR expression in immature Leydig cells were also studied. IL-1alpha stimulated both androgen production and StAR protein expression in a dose- and time-dependent manner in immature but not adult Leydig cells. These effects of IL-1alpha were prevented by pretreatment of the cells with the specific inhibitors of the p38 MAP kinase, SB203580 and PD169316, suggesting that this kinase is an important part of IL-1alpha signaling in the immature Leydig cell. The present results suggest that IL-1alpha, which is constitutively produced by the rat testis from postnatal day 25, is an important paracrine regulator of postnatal Leydig cell maturation. Regulation of StAR protein expression is one of the possible mechanisms by which IL-1alpha contributes to the differentiation of immature Leydig cells into adult cells.

Protein tyrosine phosphatases are involved in LH/chorionic gonadotropin and 8Br-cAMP regulation of steroidogenesis and StAR protein levels in MA-10 Leydig cells.

The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.

Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes.

In the current study we test the hypothesis that liver receptor homologue-1 (LRH; designated NR5A2) is involved in the regulation of steroid hormone production. The potential role of LRH was assessed by first examining expression in human steroidogenic tissues and second by examining effects on transcription of genes encoding enzymes involved in steroidogenesis. LRH is closely related to steroidogenic factor 1 (SF1; designated NR5A1), which is expressed in most steroidogenic tissues and regulates expression of several steroid-metabolizing enzymes. LRH transcripts were expressed at high levels in the human ovary and testis. Adrenal and placenta expressed much lower levels of LRH than either ovary or liver. To examine the effects of LRH on steroidogenic capacity we used reporter constructs prepared with the 5'-flanking region of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A1), 3beta hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha hydroxylase, 17,20 lyase (CYP17), 11beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). Co-transfection of these reporter constructs with LRH expression vector demonstrated that like SF1, LRH enhanced reporter activity driven by flanking DNA from StAR, CYP11A1, CYP17, HSD3B2, and CYP11B1. Reporter constructs driven by CYP11A1 and CYP17 were increased the most by co-transfection with LRH and SF1. Of the promoters examined only HSD3B2 was more sensitive to LRH than SF1. The high level of ovarian and testicular LRH expression make it likely that LRH plays an important role in the regulation of gonadal function.

Dietary soy-phytoestrogens decrease testosterone levels and prostate weight without altering LH, prostate 5alpha-reductase or testicular steroidogenic acute regulatory peptide levels in adult male Sprague-Dawley rats.

Nutritional factors, especially phytoestrogens, have been extensively studied for their potential beneficial effects against hormone-dependent and age-related diseases. The present study describes the short-term effects of dietary phytoestrogens on regulatory behaviors (food/water intake, locomotor activity and body weight), prostate weight, prostate 5alpha-reductase enzyme activity, reproductive hormone levels, and testicular steroidogenic acute regulatory peptide (StAR) levels in adult Sprague-Dawley rats. Animals were fed either a phytoestrogen-rich diet containing approximately 600 microg/g isoflavones (as determined by HPLC) or a phytoestrogen-free diet. After 5 weeks of consuming these diets, plasma phytoestrogen levels were 35 times higher in animals fed the phytoestrogen-rich vs phytoestrogen-free diets. Body and prostate weights were significantly decreased in animals fed the phytoestrogen-rich diet vs the phytoestrogen-free fed animals; however, no significant change in prostate 5alpha-reductase enzyme activity was observed between the treatment groups. Locomotor activity levels were higher in the phytoestrogen-rich vs the phytoestrogen-free animals during the course of the treatment interval. Plasma testosterone and androstenedione levels were significantly lower in the animals fed the phytoestrogen-rich diet compared with animals fed the phytoestrogen-free diet. However, there were no significant differences in plasma LH or estradiol levels between the diet groups. Testicular StAR levels were not significantly different between the phytoestrogen-rich vs the phytoestrogen-free fed animals. These results indicated that consumption of dietary phytoestrogens resulting in very high plasma isoflavone levels over a relatively short period can significantly alter body and prostate weight and plasma androgen hormone levels without affecting gonadotropin or testicular StAR levels. The findings of this study identify the biological actions of phytoestrogens on male reproductive endocrinology and provide insights into the protective effects these estrogen mimics exert in male reproductive disorders such as benign prostatic hyperplasia and prostate cancer.

Tracking the role of a star in the sky of the new millennium.

The steroidogenic acute regulatory protein is indispensable for the biosynthesis of steroid hormones. Steroidogenic acute regulatory protein mediates the rate-limiting step in steroidogenesis, the transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane where it is cleaved to pregnenolone. Its essential role in steroidogenesis was shown when it was discovered that mutations in the steroidogenic acute regulatory protein gene in humans cause the lipoid form of congenital adrenal hyperplasia, a potentially lethal disease resulting from an inability to synthesize steroids. Also, the steroidogenic acute regulatory protein null mouse has a phenotype that is essentially the same as that observed with human mutations. Studies on the regulation of the expression of the steroidogenic acute regulatory protein gene has enjoyed considerable progress, yet the complexity of this regulation indicates that much work remains. The mechanism whereby steroidogenic acute regulatory protein mediates the transfer of cholesterol to the inner mitochondrial membrane remains a mystery, but the recent solving of the structure of the cholesterol transferring domain of a steroidogenic acute regulatory protein homolog coupled with structure-function studies of steroidogenic acute regulatory protein in natural and synthetic membranes has allowed for at least two models to be proposed. This review will briefly attempt to summarize what is currently known about the regulation of the steroidogenic acute regulatory protein gene and its mechanism of action, fully understanding that in both areas considerable gaps in our knowledge remain.

Heat shock interferes with steroidogenesis by reducing transcription of the steroidogenic acute regulatory protein gene.

A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.

Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis.

The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T(3)) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T(3) response requires both on-going mRNA and protein synthesis. In addition, the effects of T(3) were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T(3). Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T(3)-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5'-flanking region of the mouse StAR gene, which identified a region between -254 and -110 bp that was essential for T(3) function. Importantly, it was found that the SF-1 binding site at position -135 bp of the 5'-flanking region was greatly involved in T(3)-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T(3) function. The relevance of T(3)-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.

Elements involved in the regulation of the StAR gene.

The steroidogenic acute regulatory protein (StAR) mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane, the regulated step in steroidogenesis. A most interesting facet of this protein is the manner in which its expression is acutely regulated. In this regard, a number of studies have concentrated on the search for consensus cis regulatory elements within its promoter, and, more importantly, on whether these elements are involved in its expression. This short review will summarize some of the findings that have been reported concerning the nature of how the expression of this gene is regulated.

StAR protein and the regulation of steroid hormone biosynthesis.

Steroid hormone biosynthesis is acutely regulated by pituitary trophic hormones and other steroidogenic stimuli. This regulation requires the synthesis of a protein whose function is to translocate cholesterol from the outer to the inner mitochondrial membrane in steroidogenic cells, the rate-limiting step in steroid hormone formation. The steroidogenic acute regulatory (StAR) protein is an indispensable component in this process and is the best candidate to fill the role of the putative regulator. StAR is expressed in steroidogenic tissues in response to agents that stimulate steroid production, and mutations in the StAR gene result in the disease congenital lipoid adrenal hyperplasia, in which steroid hormone biosynthesis is severely compromised. The StAR null mouse has a phenotype that is essentially identical to the human disease. The positive and negative expression of StAR is sensitive to agents that increase and inhibit steroid biosynthesis respectively. The mechanism by which StAR mediates cholesterol transfer in the mitochondria has not been fully characterized. However, the tertiary structure of the START domain of a StAR homolog has been solved, and identification of a cholesterol-binding hydrophobic tunnel within this domain raises the possibility that StAR acts as a cholesterol-shuttling protein.

StAR protein is increased by muscarinic receptor activation in human luteinized granulosa cells.

Cultured human luteinized granulosa cells (GC) are a unique model to study regulation of the human corpus luteum, e.g. by local factors, including acetylcholine (ACh). GC express functional ACh-receptors of the M1-type (M1R). Priming with the cholinergic agonist carbachol for 24 h, followed by a 24 h stimulation with human chorionic gonadotrophin (hCG), significantly increased progesterone levels (up to 2-fold), compared with hCG stimulation alone. This is likely due to the effect of carbachol, observed after 24 h, to increase the levels of steroid acute regulatory (StAR) protein, as found in Western blots. StAR protein was increased (2-10-fold), while P450 side chain cleavage (SCC) enzyme was unaffected. Thus, one role of muscarinic receptor activation in human GC is regulation of StAR protein and, subsequently, progesterone production.

Assessment of mechanisms of thyroid hormone action in mouse Leydig cells: regulation of the steroidogenic acute regulatory protein, steroidogenesis, and luteinizing hormone receptor function.

Recently, we demonstrated that triiodothyronine (T(3)) stimulated steroid hormone biosynthesis and steroidogenic acute regulatory (StAR) protein expression in mLTC-1 mouse Leydig tumor cells through the mediation of steroidogenic factor 1 (SF-1). We now report a dual response mechanism of T(3) on steroidogenesis and StAR expression, and on LH receptor (LHR) expression and binding in mLTC-1 cells. T(3) acutely (8 h), induced a 260% increase in StAR messenger RNA (mRNA) expression over the basal level which was coincident with an increase in progesterone (P) production. In contrast, chronic stimulation with T(3) (beyond 8 h), resulted in an attenuation of StAR expression and P production. This attenuation was most likely caused by a decrease in cholesterol delivery to the inner mitochondrial membrane as demonstrated by incubations with the hydrophilic steroid precursors, 22R hydroxycholesterol and pregnenolone, which restored P synthesis. In similar studies, chronic treatment with T(3) increased the levels of cytochrome P450scc mRNA by 83%, whereas those of cytochrome P450 17alpha-hydroxylase and 3ss-hydroxysteroid dehydrogenase decreased. The diminished response in steroidogenesis following chronic T(3) exposure was not a result of alterations in StAR mRNA stability, but rather was due to inhibition of transcription of the StAR gene. Similar acute stimulatory and chronic inhibitory responses to T(3) were found when LHR mRNA expression and LHR ligand binding were examined. Transfections with an LHR or StAR promoter/luciferase reporter construct demonstrated that a 173-bp fragment of the LHR promoter containing an SF-1 binding motif was involved in T(3) response, as was the SF-1 recognition site at -135 bp in the StAR promoter. Furthermore, the importance of SF-1 in T(3) function was also verified employing mutation in the bases of SF-1 sequences using electrophoretic mobility shift assays. The potential physiological relevance of these findings was demonstrated when similar responses were obtained in mice rendered hypo and hyperthyroid. Collectively, these observations further characterize the thyroid-gonadal connection and provide insights into the mechanisms for a dual regulatory role of thyroid hormone in Leydig cell functions.

Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells.

We report the generation of stable cell lines obtained by spontaneous immortalization of primary cultures of porcine granulosa cells. Three hundred stable cell lines were obtained from three independent immortalization trials. Two of these cell lines retained the steroidogenic capabilities characteristic of granulosa cells, such as de novo synthesis of progesterone and conversion of androstenedione into estradiol-17beta. All the stable cell lines expressed the P450arom and 3betaHSD genes, confirming their granulosa origin. Moreover, the steroidogenic stable granulosa cells also expressed StAR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells overcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures grown in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal calf serum (FCS). Medium 199 is a formulation richer in nutrients compared to MEM or DMEM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spontaneous immortalization of granulosa cells may be facilitated by using a rich culture formulation supplemented with low concentrations of serum deficient in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the expression of the steroidogenic genes. Therefore, we believe that they are useful models to study the molecular mechanism involved in granulosa cell differentiation and steroidogenesis.

Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene.

Dimethoate is a widely used organophosphate insecticide that has been shown to disrupt reproductive function in animals. Although the pathogenesis of Dimethoate-induced reproductive toxicity remains to be determined, a reduction in serum testosterone levels is thought to play an important role in the development of Dimethoate-induced infertility. Since Leydig cells play a crucial role in male reproductive function by producing testosterone, the mouse MA-10 Leydig tumor cell line was used to determine if Dimethoate can directly block steroid hormone biosynthesis and to identify the site of steroidogenic inhibition. Dimethoate inhibited steroidogenesis in both a dose- and time-dependent manner without affecting total protein synthesis or protein kinase A activity. While it decreased the activity of the P450 side chain cleavage (P450 scc) enzyme, a reduction in the activity of this enzyme alone could not account for the level of Bu(2)cAMP-inhibited progesterone production. Instead, our results suggest that Dimethoate inhibited steroidogenesis primarily by blocking transcription of the steroidogenic acute regulatory (StAR) gene. This finding is significant since StAR protein mediates the rate-limiting and acutely-regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane. This study indicates that StAR may be an important target for environmental pollutants which disrupt steroidogenesis and impair reproductive function.

Effects of lindane on steroidogenesis and steroidogenic acute regulatory protein expression.

Lindane, the gamma isomer of hexachlorocyclohexane (HCH), is one of the oldest synthetic pesticides still in use worldwide. Numerous reports have shown that this pesticide adversely affects reproductive function in animals. Although the pathogenesis of reproductive dysfunction is not yet fully understood, recent reports indicate that lindane can directly inhibit adrenal and gonadal steroidogenesis. Because Leydig cells play a pivotal role in male reproductive function through the production of testosterone, the mouse MA-10 Leydig tumor cell line was used to assess the potential effects of gamma-HCH and its isomers, alpha-HCH and delta-HCH, on steroid production, steroidogenic enzyme expression and activity, and steroidogenic acute regulatory (StAR) protein expression. StAR mediates the rate-limiting and acutely regulated step in hormone-stimulated steroidogenesis, the intramitochondrial transfer of cholesterol to the P450(scc) enzyme. Our studies demonstrate that alpha-, delta-, and gamma-HCH inhibited dibutyryl ([Bu](2)) cAMP-stimulated progesterone production in MA-10 cells in a dosage-dependent manner without affecting general protein synthesis; and protein kinase A or steroidogenic enzyme expression, activity, or both. In contrast, each of these isomers dramatically reduced (Bu)(2)cAMP-stimulated StAR protein levels. Therefore, our results are consistent with the hypothesis that alpha-, delta-, and gamma-HCH inhibited steroidogenesis by reducing StAR protein expression, an action that may contribute to the pathogenesis of lindane-induced reproductive dysfunction.