Androgen Biosynthetic Pathway Research Articles

Abstract 3444: Role of AKR1C3 in converting reservoirs of DHEA-S into potent androgens that drive prostate cancer cell growth

Abstract Castration resistant prostate cancer (CRPC) treatment aims to stop a resurgence of androgen receptor (AR) signaling with a class of therapeutics known as androgen receptor signaling inhibitors (ARSI). Through inhibition of CYP17A1, Abiraterone (Abi) prevents an upstream step in androgen biosynthesis, while Enzalutamide (ENZ) acts as an AR antagonist. Despite the early success of ARSI, a subset of patients develop resistance and succumb to the disease. One major mode of resistance is thought to occur when steroidogenic enzymes that are upregulated in CRPC convert weaker circulating precursors to testosterone (T) and dihydrotestosterone (DHT) to restore AR signaling. In this study, we identify a mechanism driving intratumoral steroidogenesis that involves AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase type 5). AKR1C3 is a major steroidogenic enzyme in the prostate that is involved in all androgen biosynthetic pathways to T and DHT, upregulated in CRPC patients, and is a component of Abi and ENZ drug resistance. Previously with Taplin et al. we observed in patients on Leuprolide that T was reduced to 5 ng/dL, and the addition of Abi lowered DHEA-S by 90% (from 230,000 ng/dL to 22,000 ng/dL). However, the DHEA-S that remained was 4,000x greater than circulating T, making it a reservoir for intratumoral steroidogenesis post Abi treatment. Here, we demonstrate that post-Abi concentrations of DHEA-S, and castrate levels of DHEA-S (post-leuprolide but pre-Abi treatment), which also corresponds to DHEA-S levels patients on ENZ are 1) converted to T in an AKR1C3-dependent manner in prostate cancer (PCa) cells, and 2) sufficient to stimulate AKR1C3-dependent cell growth. Our observations are consistent across both primary and metastatic PCa cell lines, CWR22PC and DuCaP, respectively. Androgen measurements were made using our highly sensitive and specific stable isotope dilution liquid chromatography tandem mass spectrometry (SID-LC-MS/MS) method. Additionally, we demonstrate AKR1C3-dependence using genetic and pharmacological approaches with an AKR1C3 stable knockdown for each cell line and two AKR1C3 competitive inhibitors. Taken together, we have identified a mechanism of resistance to ARSIs common to both primary and metastatic cell lines that operates through AKR1C3’s intratumoral conversion of DHEA-S to potent androgens that drive prostate cancer growth. Citation Format: Andrea J. Detlefsen, Ling Duan, Clementina Mesaros, Trevor M. Penning. Role of AKR1C3 in converting reservoirs of DHEA-S into potent androgens that drive prostate cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3444.

A novel prognostic model for prostate cancer based on androgen biosynthetic and catabolic pathways.

Prostate cancer (PCa) is one of the most common malignancies in males globally, and its pathogenesis is significantly related to androgen. As one of the important treatments for prostate cancer, androgen deprivation therapy (ADT) inhibits tumor proliferation by controlling androgen levels, either surgically or pharmacologically. However, patients treated with ADT inevitably develop biochemical recurrence and advance to castration-resistant prostate cancer which has been reported to be associated with androgen biosynthetic and catabolic pathways. Thus, gene expression profiles and clinical information of PCa patients were collected from TCGA, MSKCC, and GEO databases for consensus clustering based on androgen biosynthetic and catabolic pathways. Subsequently, a novel prognostic model containing 13 genes (AFF3, B4GALNT4, CD38, CHRNA2, CST2, ADGRF5, KLK14, LRRC31, MT1F, MT1G, SFTPA2, SLC7A4, TDRD1) was constructed by univariate cox regression, lasso regression, and multivariate cox regression. Patients were divided into two groups based on their risk scores: high risk (HS) and low risk (LS), and survival analysis was used to determine the difference in biochemical recurrence-free time between the two. The results were validated on the MSKCC dataset and the GEO dataset. Functional enrichment analysis revealed some pivotal pathways that may have an impact on the prognosis of patients including the CDK-RB-E2F axis, G2M checkpoint, and KRAS signaling. In addition, somatic mutation, immune infiltration, and drug sensitivity analyses were performed to further explore the characteristics of HS and LS groups. Besides, two potential therapeutic targets, BIRC5 and RHOC, were identified by us in prostate cancer. These results indicate that the prognostic model may serve as a predictive tool to guide clinical treatment and provide new insight into the basic research in prostate cancer.

Urinary androgens excretion patterns and prostate cancer in Mexican men.

Epidemiological studies related to androgens and prostate cancer (PC) have focused on serum determination of testosterone, androstenedione (A4), and DHEA, with inconsistent results. Herein, we hypothesized that differences in androgen biosynthetic and metabolic pathways, rather than differences in specific androgen concentrations, are associated with prostatic carcinogenesis. Therefore, spot urine samples from 111 incident PC cases with Gleason score at diagnosis and 227 healthy population controls, were analyzed. Urinary androgen concentrations (nanograms/milligrams of creatinine) were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). Using a factor analysis, we identified three androgen urinary excretion patterns. In a subsample, we evaluated a modification effect of the androgen receptor (AR) CAG polymorphism. Pattern I, characterized by A4 and testosterone hydroxylated metabolites (11β-OHT; 2β-OHT; 15β-OHT; 2α-OHT; 6β-OHT), was associated with high PC odds among carriers of AR gene (CAG)>19 repeats (OR: 3.67 95% CI: 1.23-11.0; P for interaction= 0.009). Conversely, higher testosterone excretion (pattern III), was marginally associated with lower (OR: 0.35 95% CI: 0.12-1.00, P for trend= 0.08) poorly differentiated PC (Gleason ≥8). No clear association was observed with pattern II (DHEA; 16α and 16β-OHT). Our results were consistent with the previous evidence which suggests that the C11-oxy backdoor pathway is important for prostatic carcinogenesis. Androgen urine excretion analysis could be useful for PC diagnosis, treatment, and prognosis; however, further studies with a larger number of samples and the urinary determination of 11-ketoandrogens are necessary.

GATA4 is a key regulator of steroidogenesis and glycolysis in mouse Leydig cells.

Transcription factor GATA4 is expressed in somatic cells of the mammalian testis. Gene targeting studies in mice have shown that GATA4 is essential for proper differentiation and function of Sertoli cells. The role of GATA4 in Leydig cell development, however, remains controversial, because targeted mutagenesis experiments in mice have not shown a consistent phenotype, possibly due to context-dependent effects or compensatory responses. We therefore undertook a reductionist approach to study the function of GATA4 in Leydig cells. Using microarray analysis and quantitative RT-PCR, we identified a set of genes that are down-regulated or up-regulated after small interfering RNA (siRNA)-mediated silencing of Gata4 in the murine Leydig tumor cell line mLTC-1. These same genes were dysregulated when primary cultures of Gata4(flox/flox) adult Leydig cells were subjected to adenovirus-mediated cre-lox recombination in vitro. Among the down-regulated genes were enzymes of the androgen biosynthetic pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a). Silencing of Gata4 expression in mLTC-1 cells was accompanied by reduced production of sex steroid precursors, as documented by mass spectrometric analysis. Comprehensive metabolomic analysis of GATA4-deficient mLTC-1 cells showed alteration of other metabolic pathways, notably glycolysis. GATA4-depleted mLTC-1 cells had reduced expression of glycolytic genes (Hk1, Gpi1, Pfkp, and Pgam1), lower intracellular levels of ATP, and increased extracellular levels of glucose. Our findings suggest that GATA4 plays a pivotal role in Leydig cell function and provide novel insights into metabolic regulation in this cell type.

Steroidogenesis of the testis – new genes and pathways

Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5α-3α reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3αHSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated.

Development of adrenal cortical zonation and expression of key elements of adrenal androgen production in the chimpanzee (Pan troglodytes) from birth to adulthood

The basis for the pattern of adrenal androgen production in the chimpanzee, which resembles that of humans, is poorly defined. We characterized the developmental zonation and expression of elements of the androgen biosynthetic pathway in the chimpanzee adrenal. The newborn adrenal contained a broad fetal zone (FZ) expressing CYP17, SULT2A1, and Cytochrome B5 (CB5) but not HSD3B; the outer cortex expressed HSD3B but not SULT2A1 or CB5. During infancy, the FZ involuted and the HSD3B-expressing outer cortex broadened. By 3years of age, a thin layer of cells that expressed CB5, SULT2A1, and CYP17 adjoined the medulla and likely represented the zona reticularis; the outer cortex consisted of distinct zonae fasiculata and glomerulosa. Thereafter, the zona reticularis broadened as also occurs in the human. The adult chimpanzee adrenal displayed other human-like characteristics: intramedullary clusters of reticularis-like cells and also a cortical cuff of zona fasiculata-like cells adjoining the central vein.

Aflatoxin B1 disrupts the androgen biosynthetic pathway in rat Leydig cells

The present study investigated if Aflatoxin B1 (AFB1), a potent and naturally occurring mycotoxin, interferes with the steroidogenic pathway in rat Leydig cells. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone (T) that maintains the male phenotype and support fertility. Leydig cells, isolated from 35-day-old male Long-Evans rats (Rattus norvegicus), were incubated with AFB1 at 0, 0.01, 0.1, 1, 10μM followed by measurement of T secretion by radioimmunoassay and analysis of protein expression in western blots. Results demonstrated that AFB1 suppressed testosterone secretion in a dose-dependent manner and inhibited expression of cholesterol transporter steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes [(3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase enzyme (HSD17B3)]. Protein expression analysis showed that AFB1 treatment increased ERK phosphorylation but suppressed p38 MAPK and JNK activation in Leydig cells. AFB1-induced inhibition of Leydig cells was alleviated by co-treatment with the ERK inhibitor UO 126, implying that ERK mediates, at least in part, the inhibitory effects of AFB1 in Leydig cells. The findings highlight potential extra-hepatic effects of aflatoxin exposure and indicate that exposure to AFB1 has significant reproductive health implications for consumers of contaminated products even under conditions of low dietary toxin levels.

Increased protein expression of LHCG receptor and 17 -hydroxylase/17-20-lyase in human polycystic ovaries

Does the expression of LHCG receptor (LHCGR) protein and key enzymes in the androgen biosynthetic pathway differ in normal human versus polycystic ovarian tissue? LHCGR and 17α-hydroxylase/17-20-lyase (CYP17A1) protein levels are increased in polycystic ovaries (PCOs). The predominant source of excess androgen secretion in women with polycystic ovary syndrome (PCOS) is ovarian theca cells but few studies have directly assessed the presence and abundance of protein for key molecules involved in androgen production by theca, including LHCGR and the rate-limiting enzyme in androgen production, CYP17A1. This is a laboratory-based, cross-sectional study comparing protein expression of key molecules in the androgen biosynthetic pathway in archived ovarian tissue from women with normal ovaries (n = 10) with those with PCOs (n = 16). A quantitative morphometric study was performed using sections of archived human ovaries (n = 26) previously characterized as normal or polycystic. The distribution and abundance of LHCGR, CYP17A1, 3β-hydroxysteroid dehydrogenase type 2 (3βHSDII) and 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5) proteins were evaluated by immunohistochemistry and quantified. A higher proportion of theca cells from anovulatory PCO expressed LHCGR protein when compared with control ovaries (P = 0.01). A significant increase in the intensity of immunostaining for CYP17A1 was identified in antral follicles in sections of PCO compared with ovaries from normal women (P = 0.04). As the study used formalin-fixed ovarian tissue sections, it was not possible to carry out studies 'in vitro' using the same ovarian tissues in order to also demonstrate increased functional activity of LHCGR and CYP17A1. The data are in keeping with the results of previous studies in isolated theca cells and support the notion of an intrinsic abnormality of theca cell androgen production in women with PCOS. The research was supported by a Programme Grant, G0802782, from the Medical Research Council (MRC) UK and by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. F.V.C was supported by Capes Foundation (Brazilian Ministry of Education). The authors have no conflicts of interest to disclose.

Intratumoral conversion of adrenal androgen precursors drives androgen receptor-activated cell growth in prostate cancer more potently than de novo steroidogenesis

Despite an initial response to hormonal therapy, patients with advanced prostate cancer (PC) almost always progress to castration-resistant disease (CRPC). Although serum testosterone (T) is reduced by androgen deprivation therapy, intratumoral T levels in CRPC are comparable to those in prostate tissue of eugonadal men. These levels could originate from intratumoral conversion of adrenal androgens and/or from de novo steroid synthesis. However, the relative contribution of de novo steroidogenesis to AR-driven cell growth is unknown. The relative contribution of androgen biosynthetic pathways to activate androgen receptor (AR)-regulated cell growth and expression of PSA, FKBP5, and TMPRSS2 was studied at physiologically relevant levels of adrenal androgen precursors and intermediates of de novo androgen biosynthesis in human prostate cancer cell lines, PC346C, VCaP, and LNCaP. In PC346C and VCaP, responses to pregnenolone and progesterone were absent or minimal, while large effects of adrenal androgen precursors were found. VCaP CRPC clones overexpressing CYP17A1 did not acquire an increased ability to use pregnenolone or progesterone to activate AR. In contrast, all precursors stimulated growth and gene expression in LNCaP cells, presumably resulting from the mutated AR in these cells. Our data indicate that at physiological levels of T precursors PC cells can generally convert adrenal androgens, while de novo steroidogenesis is not generally possible in PC cells and is not able to support AR transactivation and PC growth.

Abstract 1632: Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease.

Abstract Androgen deprivation therapy (ADT) is frontline treatment for metastatic prostate cancer (PCa). ADT initially blocks disease progression but eventually allows the outgrowth of castration resistant disease (CRPC) that is refractory to the diminished systemic androgen levels in ADT-treated patients. CRPC is associated with acquired intratumoral steroidogenesis (IS), a process that increases the androgen levels in the local microenvironment of prostate tumors and enables further tumor growth and progression. IS has mainly been attributed to a steroidogenic process linked to CRPC cells and here we present evidence that benign tumor-associated prostate stromal cells (PrSCs) can also contribute to IS through a paracrine Hedgehog (HH) tumor microenvironment associated with increased production and release ofHedgehog ligands (Hhs) from prostate cancer cells in response to ADT (Chen, et al., Mol Cancer, 9: 89, 2010). Primary human PrSCs upregulate expression of a large repertoire of steroidogenic genes upon treatment with HH chemical agonists, SAG (100 nM) or Hg 1.5 (1 nM). These effects are Gli2-dependent (Levina, et al., Prostate, 72: 817, 2012) and they can be inhibited by the HH antagonist, KAAD-cyclopamine. Previously we showed that this upregulation significantly increased output of testosterone (T) in PrSCs fed DHEA precursor. More comprehensive analysis (via LC/MS/MS) of androgen outputs from PrSCs fed 22 OH-cholesterol showed that Hg1.5 significantly increased the outputs of Progesterone, DHEA, androsterone, T and dihydrotestosterone (DHT) showing that HH signaling activity effects outputs throughout the androgen biosynthetic pathway. Upregulation of the RDH5 gene by Hg1.5 suggests that the effects on production of DHT from PrSCs might be generated by increased synthesis through both classical and back-door pathways. On a weight-to-weight basis, Hg1.5-stimulated PrSCs produce higher T and DHT outputs than LNCaP cells or an androgen growth-independent variant, LNCaP-AI. Finally, we found that treatment of Sonic Hh-producing LNCaP cells with simvastatin reduced the ability of conditioned medium from these cells to induce steroidogenic gene expression from PrSCs. In summary, this work provides evidence for a stromal cell contribution to IS in CRPC mediated by a paracrine-active HH signaling tumor microenvironment induced by ADT-treatment and identifies potential treatment strategies to interfere with this process. Citation Format: Amy A. Lubik, Hans Adomat, Emma S. Guns, Ralph Buttyan. Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1632. doi:10.1158/1538-7445.AM2013-1632

Androgen biosynthetic pathways in the human prostate

It is well recognized that there are two androgens, namely testosterone (T) and dihydrotestosterone (DHT); T plays an important role in the testis and muscle, and DHT is crucial for the development, function and pathology of the prostate. It is generally thought that DHT is produced from the 5alpha-reduction of circulating T before being inactivated by 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) that converts DHT into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol). However, the presence of various steroidogenic enzymes in the prostate as well as the availability at high levels of various steroid precursors such as dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) strongly suggest the existence of additional pathways involved in the biosynthesis and metabolism of DHT. Because steroidogenesis could be different in different species, data from the literature obtained from various human, dog, rat and mouse prostate tissues, as well as primary cells and prostatic cancer cell lines, provide a somewhat confusing picture. In the present chapter, we review the data in order to provide a clearer picture of the pathways involved in DHT biosynthesis and metabolism in the human prostate.

The backdoor pathway to dihydrotestosterone

Dihydrotestosterone (DHT) is the androgen responsible for formation of the male external genitalia during embryogenesis and for most androgen-mediated events at male puberty. In most circumstances, testosterone (T) derived from the testis is converted to DHT by 5α-reductase type 2 in genital skin and prostate. By contrast, the testes of pouch young of the tammar wallaby and immature postnatal testes of several species synthesize 5α-androstane-3α,17β-diol, which is the proximal precursor of DHT in androgen-target tissues. Human steroidogenic enzymes efficiently catalyze all the required steps in a route to DHT that does not involve the T intermediate, called the ‘backdoor pathway’. This alternative pathway of DHT production appears to explain how potent androgens are produced in some normal and pathological conditions when the conventional androgen-biosynthetic pathways fail to account completely for the of patterns androgen synthesis that are observed. Dihydrotestosterone (DHT) is the androgen responsible for formation of the male external genitalia during embryogenesis and for most androgen-mediated events at male puberty. In most circumstances, testosterone (T) derived from the testis is converted to DHT by 5α-reductase type 2 in genital skin and prostate. By contrast, the testes of pouch young of the tammar wallaby and immature postnatal testes of several species synthesize 5α-androstane-3α,17β-diol, which is the proximal precursor of DHT in androgen-target tissues. Human steroidogenic enzymes efficiently catalyze all the required steps in a route to DHT that does not involve the T intermediate, called the ‘backdoor pathway’. This alternative pathway of DHT production appears to explain how potent androgens are produced in some normal and pathological conditions when the conventional androgen-biosynthetic pathways fail to account completely for the of patterns androgen synthesis that are observed.

Lack of defects in androgen production in children with hypospadias.

Formation of the male urethra requires the synthesis of testosterone, its activation to dihydrotestosterone (DHT) in genital skin, and binding of DHT to the androgen receptor. Defects in any of those steps can cause hypospadias. To determine whether defects exist in the production of androgens in individuals with hypospadias, we examined enzymatic function of 3beta-hydroxysteroid dehydrogenase (3betaHSD), P450c17 (17alpha-hydroxylase and 17,20 lyase activity), and type 3 17betaHSD. Sixty-eight subjects participated in the study: 48 patients had hypospadias, and 20 had normal male genitalia. Subjects were stratified into groups based on age and severity of hypospadias, as defined by location of the urethral meatus after correction of penile curvature. Hormonal values in boys with hypospadias were compared by nonparametric analysis with those in age-matched controls. Controls excluded individuals with cryptorchidism, micropenis, known endocrine defects, or receiving steroid supplementation. Morning fasting serum levels of pregnenolone, progesterone, 11-deoxycorticosterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione, androstenediol, testosterone, and DHT were determined. To focus on the proximal steps in androgen biosynthesis, 12 individuals with hypospadias underwent standard ACTH stimulation. No significant differences in the androgen precursors and metabolites were found between controls and individuals with hypospadias. The response to ACTH was variable without a significant difference between the patients with different degrees of hypospadias and/or published controls. These data indicate that enzymatic defects in the steroidogenic steps from cholesterol to DHT are not a common etiology of hypospadias. Routine abnormalities in the androgen biosynthetic pathway are an unlikely cause of any degree of hypospadias in boys without accompanying cryptorchidism or genital malformations.

Colocalization of P450c17 and cytochrome b5 in androgen-synthesizing tissues of the human.

Androgens are an integral part of human physiology. The de novo production of androgens is generally limited to the adrenal cortex and the gonads. Androgen synthesis by these steroidogenic tissues requires the bifunctional enzyme cytochrome P450c17, which catalyzes both 17 hydroxylase and 17,20 lyase activities. 17,20-lyase activity is relevant to the regulation of androgen production, and is allosterically modulated through the action of an accessory protein, cytochrome b5 (CytB5). Our objective was to determine the cellular localization of P450c17 and CytB5 in androgen-synthesizing tissues of the human. Immunohistochemical analyses of P450c17 and CytB5 were performed on fetal and adult human adrenals, ovaries, and testes. In the fetal adrenal, CytB5 and P450c17 were both found in the cells of the fetal zone, but not in the neocortex. In the adult adrenal, the zona fasciculata was immunoreactive for P450c17 only, whereas the zona reticularis was immunopositive for both P450c17 and CytB5. In the adult gonads, P450c17 and CytB5 were colocalized in the Leydig cells of the testis, theca interna cells of the follicle, theca lutein cells, and isolated cell clusters in the ovarian stroma. Whereas P450c17 and CytB5 were colocalized in the Leydig cells of the fetal testes, there was no immunostaining for either in the midgestational fetal ovary. Our findings of colocalization of CytB5 and P450c17 are strongly supportive of the view that CytB5 plays an important role in the regulation of the androgen biosynthetic pathway in the fetal and adult human.

Decreased steroidogenesis and cAMP production in vitro by Leydig cells isolated from rats made hypothyroid during adulthood

The Leydig cell function of adult male rats made hypothyroid with 6-propyl-2-thiouracil (6-PT, 0.1% w/v in drinking water for 1 month) was studied and compared with that of age-matched controls. After 6-PT treatment, a slight, non-significant decrease in serum testosterone was observed, but no changes in testis weight or number of Leydig cells were noted. The in vitro function of Leydig cells was therefore investigated during incubation for 3 h in the presence or absence of several stimuli: LH (30 mIU/mL), forskolin (FK 1 microM), isobutylmethylxanthine (IBMX, 100 microM), GnRH (100 nM) or FK 1 microM + IBMX 100 microM. Irrespective of the stimulus, cells from hypothyroid rats secreted less cyclic AMP, 17-hydroxyprogesterone, androstenedione and testosterone. No differences in LH receptors were noted between the groups. Prolonged incubation with triiodothyronine (5-250 ng/mL) or thyroxine (5-250 ng/mL) for 3, 16, 24 or 48 h did not affect testosterone secretion in either group; however, administration of IGF-I (8 ng/mL for 24 h) resulted in increased spontaneous and stimulated testosterone production in both groups. However, when hypothyroid animals were supplemented in vivo with thyroxine a full recovery of Leydig cell function in vitro was noted. (1) Leydig cells from rats made hypothyroid during adulthood produce less testosterone in vitro, both spontaneously and in response to cAMP and non-cAMP-mediated stimuli; (2) this is due to a reduction in cAMP production and in the activity of the enzymes in the androgen biosynthetic pathway, and not to changes in LH receptors; (3) direct administration of thyroid hormones did not improve testosterone secretion in either group, while incubation with IGF-I did.

Sertoli cell conditioned media modulate the androgen biosynthetic pathways in rat Leydig cell primary cultures.

Increasing evidence suggests the presence of local modulators that contribute to testicular function regulation in rats. The seminiferous tubules and Sertoli cell factors influence the steroidogenic activity of Leydig cells. Addition of Sertoli cell conditioned media to primary cultures of adult rat Leydig cells produces in a dose-dependent manner an inhibition of testosterone production with a concomitant decrease in the C21 delta 4-intermediates of the steroidogenic pathway, modifying the C21/C19 ratios in normal cells. The nature and mechanisms of action of this putative modulating activity are currently under study.

Genetic basis of endocrine disease 2: congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Most cases of congenital adrenal hyperplasia, the inherited inability to synthesize cortisol, are caused by a deficiency of the 21-hydroxylase activity required to convert 17-hydroxyprogesterone to 11-deoxycortisol. Poor synthesis of cortisol results in chronic stimulation of the adrenal cortex by corticotropin with consequent overproduction of cortisol precursors. Some of these precursors are shunted into the androgen biosynthetic pathway causing the signs and symptoms of androgen excess seen in this disorder. The disease occurs in a wide spectrum of clinical variants, including a severely affected form with a concurrent defect in aldosterone biosynthesis (“salt wasting” type), a form with apparently normal aldosterone biosynthesis (“simple virilizing” type) and a mild “nonclassic” form that may be asymptomatic or may be associated with symptoms of androgen excess developing during childhood or at puberty (1). The disorder is inherited as a monogenic autosomal recessive trait located within the HLA major histocompatibility complex on chromosome 6~21.3. There are a number of associations between the different forms of 21-hydroxylase deficiency and specific combinations of HLA antigens, or haplotypes. In particular, the unusual haplotype A3;Bw47;DR7 is associated with about 10% of classic (i.e. salt wasting or simple virilizing) 21-hydroxylase deficiency alleles, whereas HLA-B14;DRl is associated with 70% of alleles for nonclassic disease. Based on these associations and on studies of pedigrees containing patients with the different forms of the disease, it appears that these forms are inherited as allelic variants. Classic disease occurs in approximately l/10,000 births, whereas nonclassic disease occurs in approximately 1% of the general population (2). The molecular genetic basis of 21-hydroxylase deficiency has been thoroughly studied. The 21-hydroxylase enzyme is a microsomal cytochrome P450 termed P45OXXI or P45Oc21. The structural gene encoding

Tetrahydroisoquinoline alkaloids mimic direct but not receptor-mediated inhibitory effects of estrogens and phytoestrogens on testicular endocrine function. Possible significance for Leydig cell insufficiency in alcohol addiction

Possible effects of various tetrahydroisoquinolines (TIQs) on rat testicular endocrine function were tested in vitro in order to prove whether these compounds, some of which have been claimed to accumulate in alcoholics, may be mediators of the development of Leydig cell insufficiency, a well-known side-effect of ethanol ingestion. TIQ effects on different levels of regulation of testis function were compared in vitro with estrogen effects, since both classes of compounds have structural similarities. Gonadotropin-stimulated testosterone production by testicular Leydig cells was inhibited by tetrahydropapaveroline and isosalsoline, the IC 50 values (30 μM) being comparable to those of estradiol (3 μM), 2-hydroxyestradiol (10 μM), and the phytoestrogens, coumestrol (15 μM) and genistein (7 μM); salsolinol (85 μM) and salsoline (240 μM) were less effective, and salsolidine was ineffective. None of these TIQs interacted significantly with testicular estrogen receptor as analyzed by estradiol displacement. However, tetrahydropapaveroline, isosalsoline and salsolinol competitively inhibited (ki 130–150 μM) substrate binding to cytochrome P450XVII, one key enzyme of androgen biosynthesis, with similar efficiency as the estrogens did (Ki 50–110 μM); salsoline and salsolidine were again much less effective. Since the efficient TIQ concentrations in this system are identical with those reported to generate central-nervous effects, it is concluded that certain TIQs may amplify peripheral inhibitory effects of ethanol on testicular endocrine function by their interaction with at least one enzyme of the androgen biosynthetic pathway.

143 IN VITRO METABOLIC STUDIES OF TESTICULAR TISSUE IN FAMILIAL MALE PSEUDOHERMAPHRODITISM DUE TO 17-KETOREDUCTASE DEFICIENCY

Kinetic analyses of progesterone and androsienedione metabolism were performed in testicular tissue of 5 male pseudohermaphrodites (MPH) with familial 17-ketoreductase deficiency previously described (J. Steroid Blochem.. 19:663-674. 1983). and compared to 2 normal controls. The age of the patients ranged between 3-3/4 and 35 years. Testicular tissue was obtained during surgery tor male genitoplasty in 4 cases, and during castration in one individual, who had been reared unequivocally as a female. While the testicular tissue of the 2 prepubertal MPH metabolized progesterone to androstenedione only, and that in a limited extent, the 3 postpuberlal MPH metabolized progesterone to 16α- and 16β-hydroxyprogesterone (16-OHP). 17-hydroxyprogesterone. androsienedione and to testosterone. These MPH metabolized androsienedione to testosterone as well, to a comparable extent at the controls. The Michaelis constant of these reactions was similar for the MPH and the controls. An 7.8 to 12.8-fold increase in the production of 16-OHP, and a 4.6 to 11.5-fold inhibition of the 17-hydroxylase was found in the testes of the MPH. The preference of androsienedione production by the testes of 2 MPH was examined using a equimolar concentration of [14C]-progesterone and [H3]-pregnenolone as substrates. While the flow of substrates in the normal testes was equal or slightly preferable through the Δ-4 pathway, a more than 8-times preference of the Δ-5 pathway was noted in the testes of the MPH. A large accumulation of DHEA was found upon omission of NAD, the 3β-styeroid dehydrogenase-isomerase cofactor, supporting the contention that in MPH androsienedione is mainly produced through the Δ-5 pathway. Further support for this was the finding that the 3H/14C ratio of androsienedione and testosterone produced from both substrates was 8-times higher in MPH than in controls. MPH due to 17-ketoreductase deficiency appears to be a mixture of complex metabolic aberrations In the androgen biosynthetic pathway.

Mitochondrial cholesterol availability during gonadotropin-induced Leydig cell desensitization.

In order to define the early lesion (before pregnenolone formation) of the androgen biosynthetic pathway induced by human CG (hCG) or LH in the Leydig cell, we initially have optimized the use of aminoglutethimide to obtain maximal and sustained inhibition of steroidogenesis in vivo and in vitro. Aminoglutethimide inhibited Leydig cell steroidogenesis in vitro at a dose of 100 micrograms/ml. The minimal serum concentration of aminoglutethimide necessary for maximal inhibition of testosterone in vivo was also 100 micrograms/ml (1 h after the ip injection of 20 mg aminoglutethimide). However, testosterone levels were normal 12 h later, coincident with a marked fall in the serum aminoglutethimide levels. The t 1/2 of the circulating aminoglutethimide was 5 +/- 0.7 h on the first day of treatment but was reduced to 3.0 +/- 0.4 and 2.25 +/- 0.35 h at 2 and 3 days of treatment. At the dose eliciting maximal and sustained steroid inhibition (60 mg/day) aminoglutethimide was able to prevent the estradiol-dependent late steroidogenic lesion (after pregnenolone formation) induced by 1 microgram hCG, with no effect on the early lesion (before pregnenolone formation) caused by 10 micrograms hCG. The aminoglutethimide-induced in vivo accumulation of cholesterol in the inner mitochondrial membrane (by 50%) was associated with an increase in the production of testosterone and pregnenolone by the Leydig cell when subsequently incubated in vitro. Similar increases in the steroidogenic capacity were observed after initial exposure of Leydig cells to aminoglutethimide in vitro, even after acid wash to remove the surface-bound endogenous LH. The steroidogenic cholesterol was also increased in desensitized Leydig cells (by 50-70%); however, the conversion of cholesterol to pregnenolone was substantially blocked in animals with the early lesion. Our findings define the requirement of increasing high levels of aminoglutethimide to inhibit cholesterol metabolism and provide a dose schedule suitable for studies on cholesterol availability and inhibition of steroidogenesis in the rat. These results support our proposal that the early lesion observed in desensitized Leydig cells is due to inhibition of the side-chain cleavage activity rather than to a decrease in the amount of metabolically available cholesterol.