Dissertations / Theses on the topic 'Steroidogenesis'

A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control. In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes. Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*. A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P. The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times. The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ã-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP. Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).

Mitochondrial steroidogenic cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin for catalytic activity. Previous in vitro data suggests these co-factors are key regulators of CYP enzyme activity. However, this has never been studied in vivo. Zebrafish have emerged as model to study human steroidogenesis as they have conserved steroidogenic genes, molecular mechanisms and endocrine tissues. This project aimed to establish zebrafish as an in vivo model for endocrine development and its disorders, and to investigate the influence of mitochondrial redox regulation on steroid hormone production. This study involved the identification and characterisation of zebrafish mitochondrial steroidogenic CYP enzymes and their ferredoxin co-factors. Through implementation of recent genomic editing methods including Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regulatory Interspaced Short Palindromic Repeat Cas9 nuclease (CRISPR/Cas9) system, and steroid hormone analysis from whole zebrafish extracts by liquid chromatography/tandem mass spectrometry, essential mitochondrial redox components required for zebrafish glucocorticoid production were identified. Overall, this work has helped established zebrafish as a model to study the pathophysiological consequences of steroid hormone disease and provided insights into the mechanism of mitochondrial redox regulation of steroid hormone production.

Cortisol and dehydroepiandrosterone (DHEA) are steroids produced by the zona fasciculata (ZF) and reticularis (ZR), respectively, of the adrenal cortex. Both steroids are upregulated in response to adrenocorticotropic hormone (ACTH). Cortisol is a glucorticoid that is important in the regulation of inflammation and metabolism. DHEA is an adrenal androgen important in fetal growth and puberty but tends to decrease gradually after puberty in both men and women. DHEA has various effects on metabolism and immune function including inhibiting the effects of cortisol on some tissues. During the acute phase of stress, cortisol and DHEA rise due to an increase in ACTH released from the anterior pituitary. In contrast, during chronic stress, cortisol remains elevated but DHEA and ACTH levels decrease. Likewise, stress causes serum levels of IL-6 to increase. IL-6 increases cortisol release from the human and bovine adrenal cortex. IL-6 also decreases DHEA release from zona reticularis of the bovine adrenal gland. In humans the effect of IL-6 on DHEA production is still uncertain. To determine a possible mechanism of IL-6 on the zona fasciculata and reticularis, human H294R cells and bovine adrenal tissue were incubated in serum free medium containing IL-6, at various concentrations and incubation intervals. At the end of the incubation interval, mRNA or protein was extracted from the cells or tissue. Standard PCR, real time PCR, and western blot assays were used to determine the effects of IL-6 on the enzymes involved in cortisol and DHEA synthesis, steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), and dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1). In human H295R cells and bovine zona fasciculata cells IL-6 caused an increase in SF-1, StAR, P450scc, 17α hydroxylase, 3β-hydroxysteroid dehydrogenase type 2 (3β HSD2), 21 hydroxylase, and 11β hydroxylase mRNA and protein. IL-6 caused DAX-1 mRNA and protein to decrease. These effects were manifest in a time dependent manner. Dose response treatments incubated for 60 min increased SF-1, StAR, P450scc, 17α hydroxylase, 3β HSD2, 21 hydroxylase, and 11β hydroxylase but there was not significant change between the different treatments of IL-6. The bovine zona reticularis stimulated with IL-6 showed a decrease in SF-1, StAR, P450scc, 17α hydroxylase, and 3β HSD2 with an increase in DAX-1 mRNA and protein. This response was manifest in a time dependent manner for both mRNA and protein, and the effect was dose-dependent for mRNA but not protein levels within the 60 min time period. These data provide a mechanism by which increased stress, physical or emotional, which increases IL-6 serum level, could increase cortisol and decrease DHEA. This would account for decreased immune function, increased blood pressure, and changes in metabolism.

Die vorliegende Arbeit untersucht die Steroidogenese und die Expression Leydig-Zellspezifischer Gene in Kulturen postnataler fetaler Leydig-Zellen (PFLC). Die Stimulation von PFLC mit hCG und (Bu)2cAMP bewirkt eine Steigerung der Testosteronproduktion in vitro. Es wurde eine zeitabhängige Abschwächung der Testosteronproduktion durch (Bu)2cAMPstimulierte PFLC beobachtet. Diese war begleitet von einer Akkumulation von Progesteron im Kulturmedium und einer Suppression der Expression von P450c17 auf dem translatorischen Level. Während der Kultivierung verloren PFLC ihre Fähigkeit der Expression Leydig-Zell-spezifischer Gene (z.B. 3βHSD, P450c17, Insl3). Dieses Phänomen konnte durch Stimulation mit (Bu)2cAMP rückgängig gemacht werden. Außerdem zeigte sich, dass PDGFα allein und in Kombination mit (Bu)2cAMP signifikant die Proliferation der PFLC in vitro stimulierte. Die vorliegende Arbeit deutet darauf hin, dass cAMP-aktivierte Signalkaskaden eine wichtige Rolle in der Regulation von Differenzierung und Funktion von PFLC spielen.

Production of hormones in the ovary is controlled by endocrine, paracrine, autocrine and intracrine influences. Similar controls may exist in the placenta. I wished to investigate the involvement of second messengers in the action of hormones in control of hormonogenesis in rat ovary and human placenta. The second messengers involved in the action of gonadotropin-releasing hormone (GnRH) and prostaglandin (PG) F₂[formula omitted] were investigated in rat granulosa and luteal cells. As well, the endocrine role of GnRH in the placenta and the possible second messengers involved were investigated. Monolayer cultures of rat granulosa and luteal cells and human placental cells were prepared. Rat granulosa cells were mechanically dispersed; rat luteal cells were enzymatically dispersed with collagenase and DNase. Rat granulosa cells were treated during the first 24 hours in culture; rat luteal cells were treated up to 3 days after dispersion. Radioimmunoassay of medium was used to determine the effect of treatments on hormone production. Studies which examined the effect of hormones on the intracellular free calcium concentration ([Ca²⁺]i) in single cells using the calcium sensitive fluorescent dye, Fura-2, were done in monolayer rat granulosa and luteal cell cultures. Human placental cells, from first trimester and term placentae, were dispersed using trypsin-DNase or collagenase-DNase. Cells were cultured for 2 days prior to treatment. The effects of treatments on production of steroid (progesterone and estrogen), glycoprotein (human chorionic gonadotropin; hCG) and protein (human placental lactogen; hPL) hormones were determined by radioimmunoassay of the medium. In rat granulosa and luteal cell cultures, I examined the effect of a number of hormones and second messengers. Effects of follicle-stimulating hormone (FSH), luteinizing hormone (LH), cyclic adenosine monophosphate (cAMP), GnRH and PGF₂[formula omitted] on ovarian hormonogenesis have been previously reported. Changes in cytosolic free calcium concentrations ([Ca²⁺]i) in response to PGF₂[formula omitted] were measured in single rat granulosa and luteal cells. I found that in 34% of granulosa cells, and 53% of luteal cells, there was a 3 to 4 fold increase in resting [Ca²⁺]i within 30 seconds of administration of PGF₂[formula omitted]. Many cells which responded to PGF₂[formula omitted] also responded to GnRH (39% of granulosa cells; 67% of luteal cells). The immediate source of the increased [Ca²⁺]i appeared to be common intracellular stores. No change in hormone production in response to GnRH in placental cell cultures was seen. Trypsin dispersion may have damaged cell surface receptors, therefore the effect of second messengers on hormone production in these cultures was examined. In term and first trimester trophoblast cultures, I observed the following effects with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP): inhibited estrogen production from the supplied androgen precursors; stimulated hCG production; stimulated hPL production in first trimester placental cell cultures (hPL was not measured in enough term cultures to determine the effect of 8-br-cAMP), and stimulated progesterone production. I also investigated the effects of activators and inhibitors of the phosphoinositide (PtdIns(4,5)P₂) breakdown second messenger pathway (TPA, A23187, arachidonic acid); no effects of these agents were seen. Other hormones suspected of having endocrine, paracrine or autocrine effects in the placenta were tested without effect. I conclude that GnRH and PGF₂[formula omitted] cause increases in [Ca²]i in rat ovarian cells, from common intracellular stores of calcium, and that the production of hormones by the human placenta may be under regulation of an agent or agents which induce production of cAMP.
Medicine, Faculty of
Obstetrics and Gynaecology, Department of
Graduate

Both acute and chronic steroidogenesis is regulated by adrenocorticotropin (ACTH), a principal regulator of the adrenal cortex. A number of studies have demonstrated that Steroidogenic Acute Regulatory (StAR) protein plays a crucial role in facilitating cholesterol transfer from the outer to the inner mitochondrial membrane where the first step of cholesterol conversion to steroid hormones occurs. This work has evaluated the relationships between the expression of StAR protein and signalling pathways of ACTH-induced steroidogenesis in primary cultures of bovine adrenal zona fasciculata (ZF) cells. A novel sheep anti-bovine peptide StAR polyclonal antibody has been characterised and optimised for Western immunoblotting. A newly formulated protocol based on enhanced chemiluminescence methodology provided a linear, reproducible and sensitive approach to detect and quantify StAR protein by molecular imaging analysis. The expression level of StAR protein after ACTH treatment of adrenal ZF cells showed that levels of StAR were insensitive to ACTH in freshly isolated cells due to the high initial levels of the protein. The cell responsiveness to ACTH was remarkable, however, after the basal levels of StAR protein diminished to relatively lower levels after 2 days of culture. Concentration-response curves demonstrated that, in general, increasing concentrations of ACTH resulted in increasing in cortisol output in parallel with increases of cyclic adenosine 3', 5'-monophsopathe (cAMP) production; the maximal effects of ACTH on cortisol and cAMP levels exhibited with 10^-8 M ACTH treatment after both 1 and 6 hr. Marked changes in StAR protein occurred at 6 hr. attaining a maximal level with 10^-8 M ACTH. Interestedly, at 1 and 6 hr the elevation of cortisol levels were significantly altered compared to the basal levels at 10^-12 M ACTH without any notable increase in cAMP. The time courses for ACTH treatment showed that at10^-8 M ACTH (a supraphysiological concentration) there was a strong correlation between cortisol and StAR protein induction, suggesting that newly synthesised StAR protein may be a major mediator for steroidogenesis.

La produzione locale ed il metabolismo degli steroidi nel Sistema Nervoso Centrale, la Neurosteroidigenesi, si pensa possa giocare un ruolo chiave nello sviluppo e nel corretto funzionamento del cervello. Un numero in crescita di studi sembra confermare l’importanza di questo processo svelando diverse condizioni fisio-patologiche dove i neurosteroidi sembrano un punto cardine. Sebbene in tempi recenti ci siano stati degli sforzi isolati di confermare diversi enzimi chiave della steroidogenesi in specifiche strutture cerebrali, ad oggi nessuno studio sistematico è stato effettuato per comprendere le complesse pathways della steroidogenesi cerebrale. La difficoltà di questi studi è stata resa maggiore sino ad ora dalla bassa espressione genica degli enzimi e degli ormoni coinvolti, e dalla difficoltà nel reperire tessuto umano encefalico. Lo scopo della nostra ricerca è stato di quantificare tramite la Real-Time quantitative PCR (qPCR) un set di 63 geni di specifico interesse nella steroidogenesi, dalla biosintesi de-novo a partire dal colesterolo, ai passaggi metabolici chiave per formare i neurosteroidi bioattivi (Cytochrome P450 family, Aldo-Keto reductase family, hydroxysteroid dehydrogenase family, hormone receptors, GABA receptors...). Le PCR quantitative sono state eseguite su RNA estratto dal materiale umano congelato (116 campioni, 9 tessuti da 24 autopsie in 7 gruppi d’età ed accoppiati per i diversi sessi) da controlli “non dementi” ottenuti dalla Netherlands Brain Bank, per un totale oltre le 20000 reazioni chimiche. Il tessuto umano raccolto per lo studio includeva: cervelletto, nucleo caudato, giro frontale mediale, giro frontale superiore, giro superiore occipitale, giro superiore parietale, giro cingolato, talamo (pulvinar) e materia bianca. Globalmente parlando, abbiamo esaminato la domanda controversa di quali steroidi sono prodotti direttamente nel cervello umano e quali siano prodotti negli organi periferici endocrini, come il surrene, e poi successivamente modificati nel tessuto cerebrale. Per questo fine abbiamo analizzato l’espressione di enzimi chiave coinvolti nella formazione di corticosteroidi e degli ormoni sessuali. In maniera opposta al controllo positivo surrenalico, le espressioni degli mRNA CYP17A1 (converte gli steroidi C21 in C19), SULT2A1 (DHEA sulfotransferasi), CYP11β1 (11β-idrossilasi) e CYP11β2 (aldosterone sintetasi) non sono stati trovati nel cervello umano (con l’eccezione del cervelletto). In aggiunta grossi livelli di espressione sono stati riscontrati per STS (sulfatasi steroidea) enzima chiave per attivare i solfati steroidei non biologicamente attivi nelle aree esaminate nello studio. Possiamo quindi ipotizzare che la trasformazione periferica e non la sintesi de-novo siano la fonte primaria di aldosterone, cortisolo e DHEA nel cervello umano. Riportiamo inoltre la prima cartografia su larga scala della steroidogenesi steroidea cerebrale che sembra suggerire un dimorfismo sessuale tessuto-specifico negli enzimi neurosteroidogenetici come l’aromatasi, il P450scc (Cholesterol side-chain cleavage enzyme), STS (steroid sulfatase), 3β-HSD [trasformazione del pregnenolone e DHEA in progesterone ed androstenedione rispettivamente], AR (androgen receptor), ESR (estrogen receptor), CYP21A2 (trasformazione del progesterone e 17-α idrossiprogesterone in 11-deossicorticosterone and 11-deossicortisolo), e molti recettori GABA. Concludendo, questa è la prima volta che una ricerca quantitativa e comprensiva sulla trascrizione genetica della steroidogenesi nel cervello umano sia stata portata a termine, usando un approccio metodologico di largo uso, negli stessi “set” di campioni individuali, rendendo così possibile un confronto diretto tra tessuto cerebrale nei due sessi. I risultati della nostra espressione genica hanno mostrato per la prima volta un dimorfismo sessuale nella sintesi steroidea nel cervello umano. I neurosteroidi posso quindi avere effetti immediati e sesso-specifici su alcune pathways neuronali. Il nostro lavoro sembra indicare che la neurosteroidogenesi sia un processo ubiquitario nel sistema nervoso centrale e non limitato a strutture specifiche. La differenza riscontrata nei set enzimatici nelle diverse regioni cerebrali indica un’interazione molto complessa tra di esse. Questo processo generalizzato è differente in strutture specifiche con ruoli importanti come il cervelletto.
Local production and metabolism of steroids in the Central Nervous System: Neurosteroidogenesis, is believed to be a crucial process in normal brain development and function. Increasing number of studies tend to confirm the importance of this process, through the number of physiopathological conditions in which neurosteroids seem to play a key role. Although in recent years isolated efforts have sought to establish the expression of several key steroidogenic enzymes in specific brain structures, to date, no systematic study has been undertaken to understand the intricate pathways of neurosteroidogenesis in the brain. Such efforts have so far been hindered technically by low enzymatic gene expression and hormones production. The difficult access to human encephalic tissue is also a major drawback for this kind of studies. The aim of our work was to quantify by the means of Real-Time quantitative PCR (qPCR) a set of 63 genes of interest corresponding to a broad selection of steroidogenic enzymes, implicated in de novo biosynthesis from cholesterol, so well as key transformation steps for bioactive neurosteroids (Cytochrome P450 family, Aldo-Keto reductase family, hydroxysteroid dehydrogenase family, hormone receptors, GABA receptors...). qPCRs have been performed on RNA extracted from fresh frozen material (116 samples, 9 tissues from 24 autopsies in 7 age groups paired by sex), from non-dement controls obtained from the Netherlands Brain Bank, for a total of more than 20,000 reactions. Human brain tissues harvested for this study included cerebellum, caudate nucleus, medial frontal gyrus, superior frontal gyrus, superior occipital gyrus, superior parietal gyrus, cingulate gyrus, thalamus (pulvinar) and white matter. Overall, we investigated the controversial question of which steroids are directly produced in the human brain and which are produced in peripheral endocrine organs like the adrenal gland and subsequently modified in the brain tissue, we analyzed the expression of key enzymes involved in corticosteroid and sex steroids formation. In contrast to the adrenal gland, that served as positive control, CYP17A1 [conversion of C21 steroids into C19 steroids], SULT2A1 (“DHEA sulfotransferase”), CYP11β1 (11β-hydroxylase) and CYP11β2 (aldosterone synthase) mRNAs expressions were not detected in the human brain (with the exception of the cerebellum). Furthermore, strong mRNA expression of STS (steroid sulfatase) has been confirmed in the areas examined in the study. We therefore conclude that local peripheral transformation, and not de novo synthesis, could be the main source of aldosterone, cortisol and DHEA in the human brain. We reported the first large scale undertaking of human brain cartography which suggested a tissue-specific sexual dimorphism in gene expression of some neurosteroidogenic enzymes, such as P450 aromatase, P450scc (Cholesterol side-chain cleavage enzyme), STS (steroid sulfatase), 3β-HSD [transformation of pregnenolone and DHEA into progesterone and androstenedione respectively], AR (androgen receptor), ESR (estrogen receptor), CYP21A2 [transformation of progesterone and 17-α hydroxyprogesterone to 11-deoxycorticosterone and 11-deoxycortisol], and many GABA receptors. Neuroactive steroids are endogenous neuromodulators. They have potent effects on neurotransmission mediated by γ-aminobutyric acid type A (GABAA) receptors. In this work, statistical analysis revealed significant sex differences of mRNA expression in human thalamus. The expression of STS, 3β-HSD2, CYP19A1, HSD11β1, AR, GR, and GABRA4 was higher in women, while CYP21A2, HSD17β3, HSD11β2, PGR and GABRδ were more expressed in men. We therefore hypothesize that two sex-dependant pathways inhibit the neurotransmission via interaction with the GABAA receptor to modulate the flow of visceral information to the thalamus. Women appear to preferentially modulate GABRA4 through synthesis of DHEA and estrogen, while the formation of TH-PROG, TH-DOC and their precursors was the men tendency with GABRδ modulation. THPROG and THDOC are potent modulators of the GABAA receptor. GABA mediates most of the inhibitory neurotransmission in the mammalian brain. Both THDOC and THPROG have significant sedative effects in vivo. THDOC is a metabolite of the mineralocorticoid DOC and is responsible for the sedative and anti-seizure activity of DOC in animal models. DOC can be metabolised from progesterone, and CYP21A2 mediates this conversion. CYP21A2 mRNA was detected in all samples studied. In the human brain, we found a higher gene expression of CYP21A2 in men than women (with the exception of cerebellum and caudate nucleus). In absence of CYP11B2 which converts DOC in corticosterone, we can conclude that THDOC is the principal DOC metabolite. In men brain, the tendency seems to be the formation of progesterone metabolites which act as potent modulators of GABAR. Recently the Purkinje cell, an important cerebellar neuron, has been identified as a major site for neurosteroid formation in vertebrates. The cerebellum contains more than half the neurons in the brain. Interestingly, gene expression profile of the cerebellum seems to be unusual compared to the other brain specimens analyzed. Indeed, this is the only tissue that expresses genes of de novo synthesis like CYP17A1, SULT2A1 or CYP11B2. Furthermore, we observed the strongest mRNA expression of key genes: 3β-HSD2 and GABRδ (δ-containing GABAR are the most sensitive to modulation by steroids). These data suggest that the cerebellum could have a crucial role in the steroidogenesis in human brain. To conclude, this is the first time, to our knowledge, that a comprehensive quantitative survey of steroidogenic gene transcription in the human CNS has been performed, using a common methodological approach in the same set of individual samples, permitting direct comparison of brain tissue and sex. Our gene expression results demonstrated for the first time a sexual dimorphism on steroid synthesis in the human brain. Neurosteroids can have immediate and sex specific effects on selected neuronal pathways. Our work tends to show that neurosteroidogenesis is an ubiquitous process in the CNS, and is not limited to specific structures. The difference in the enzymatic set in the different regions studied suggests a complex interplay among them. This generalized process is not incompatible with the existence of specialized structures with more predominant roles like the cerebellum.

Sertraline is a selective serotonin reuptake inhibitor (SSRI) that is widely used for the treatment of depression and anxiety. Due to the abundant therapeutic use of sertraline, low levels have been detected in municipal wastewater effluents suggesting that aquatic organisms may be exposed. The purpose of this study was to evaluate the steroidogenic effects of sertraline on larval (FHM) and adult female fathead minnows (FFHM), Pimephales promelas. Larval FHM were exposed to 0.1, 1, and 10 µg/L sertraline for 28 days and analyzed via RT-qPCR for differential expression of 11β-Hydroxysteroid dehydrogenase (11β-HSD), 20β-Hydroxysteroid dehydrogenase (20β-HSD), aromatase (CYP19), and nuclear thyroid receptor alpha (TRα). FFHM were exposed to 3 or 10 µg/L sertraline for 7 days with the brain and ovary excised at exposure termination. Juvenile FHM exposed to 0.1 μg/L sertraline had a significant upregulation of both 20β-HSD and TRα. FFHM exposed to 10 µg/L sertraline had a significant upregulation of 11β-HSD expression in brain tissue, while no steroidogenic changes were observed in the FFHM ovary. Similarly, in FFHM brain tissue, CYP19 and 20β-HSD expression levels were significantly higher in fish exposed to 10µg/L sertraline compared to control. The significance of these findings with respect to survival, growth and reproduction are currently unknown, but represent future research needs.

Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid reductases (17KSRs) modulate the biological activity of certain estrogens and androgens by catalyzing dehydrogenase and reductase reactions between 17β-hydroxy and 17-ketosteroids. In the present study, cDNAs encoding mouse and rat 17HSD/KSR1 were cloned in order to study the role of rodent type 1 enzyme in ovarian estradiol (E2) biosynthesis and its enzymatic characteristics. Both rat and mouse 17HSD/KSR1 were expressed in granulosa cells of developing follicles, where diethylstilbestrol and follicle-stimulating hormone stimulated follicular maturation and up-regulated the expression of 17HSD/KSR1, whereas human chorionic gonadotropin caused luteinization of follicles and down-regulation of the enzyme. In line with this, the rodent type 1 enzymes are not expressed in the corpus luteum (CL). Mouse 17HSD/KSR1 showed substrate specificity different from that of the human counterpart. The mouse type 1 enzyme catalyzed the reaction from androstenedione to testosterone at least as efficiently as estrone (E1) to E2, while human 17HSD/KSR1 clearly preferred the E1 to E2 reaction. A mouse mammary epithelial cell line was found to possess strong estrogenic 17KSR activity. A novel type of 17HSD/KSR responsible for this activity was expression-cloned on the basis of its ability to convert E1 to E2 and it was chronologically named 17HSD/KSR7. Interestingly, it showed 89 % identity with a rat protein called prolactin receptor-associated protein (PRAP), which is expressed in the CL. Enzymatic characterization showed that both mouse 17HSD/KSR7 and PRAP efficiently catalyzed the reaction from E1 to E2. The mouse type 7 enzyme was most abundantly expressed in the ovary and placenta. Similar primary structure, enzymatic characteristics, and tissue distribution of mouse 17HSD/KSR7 and PRAP suggest that PRAP is rat 17HSD/KSR7. Further studies showed that in rat ovaries 17HSD/KSR7 is primarily expressed in the middle and second half of pregnancy, in parallel with E2 secretion from the CL. Using in situ hybridization, cell-specific expression of 17HSD/KSR7 was studied in the mouse ovary, uterus and placenta. In the mouse ovary, the enzyme was expressed exclusively in the CL. In the uterus on day 5 post coitum (p.c.), the type 7 enzyme was expressed in the decidua, mostly in the inner zone of antimesometrial decidua. Between day 8 and 9 p.c. the enzyme was abundant in decidua capsularis of the developing placenta, after which expression moved to the basal zone. On days 12 and 14 p.c., mouse type 7 enzyme was abundantly expressed in the spongiotrophoblasts, where expression decreased towards parturition. Altogether, rodent 17HSD/KSR7 is a new 17HSD/KSR which is involved in the biosynthesis of E2 in the ovaries. In addition, E2 produced locally in the decidua and placenta by the type 7 enzyme may have a role in decidualization and/or implantation and placentation.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Jun. 4, 2010). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Toxicology." Discipline: Toxicology; Department/School: Medicine.

Inhibition of steroidogenic enzymes, which are involved in the biosynthesis of active estrogens, represents an attractive approach to treating hormone-dependent breast cancer. Synthetic routes to novel steroid-based inhibitors of two key enzymes, steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD type 1), are described. Biological evaluation of the derivatives in vitro indicated that most of the compounds synthesised were active against the selected targets to varying degrees. Particular attention was given to assessing the estrogenicity of potential inhibitors, since agonist activity at the estrogen receptor is undesired in the treatment of estrogen-dependent pathologies. Modifications to the D-ring of estrone-3-O-sulfamate EMATE, a potent STS inhibitor, successfully afforded a 3-sulfamoyloxy-16,17-seco-estra-l,3,5(10)-triene16,17-imide template, from which a series of N-alkylated analogues were prepared. The N-propyl and N-pyridin-3-ylmethyl derivatives were 18 times more potent than EMATE in vitro, with IC50S of 1 nM in placental microsomes. In vivo, these compounds inhibited 99% of rat liver sulfatase activity at an oral dose of 10 mg/kg. Importantly, they were also devoid of estrogenic activity. The SAR for the derivatives synthesised is discussed and a QSAR was established. Substrate-based design of 17β-HSD type 1 inhibitors resulted in the identification of several lead compounds, mostly D-ring modified estrogens. The potential of 16-alkylidenes derivatives of estradiol as enzyme-generated irreversible inhibitors was examined. Small modifications to the estra-l,3,5(10)-triene nucleus were investigated. Both studies were assisted by molecular modelling, using the crystal structure of the enzyme. D-Ring fused heterocyclic derivatives of estrone were prepared and an SAR was built around N-alkylated 16,17-fused pyrazoles. The Nethoxymethyl derivative emerged as a potent inhibitor of 17β-HSD type 1 in vitro without being estrogenic. A preliminary study focused on the design of dual inhibitors of STS and 17P-HSD type 1 is also reported here. The synthetic work was supported by single crystal X-ray analyses.

Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome.

The consequences of soy isoflavone consumption on steroidogenesis were examined in F1 male rats from a multi-generation reproduction study investigating the effects of diets varying in isoflavone content. F1 male rats were obtained from a multi-generation study where the parental generation was fed diets containing alcohol-washed soy protein supplemented with increasing amounts of Novasoy, a commercially available isoflavone supplement. A control group was maintained on a soy-free casein-based diet (AIN93G). Testicular and serum androgen levels were assayed with commercial kits and were approximately doubled at postnatal day (PND) 120 for rats fed a diet of elevated levels of isoflavones. Steroidogenic enzyme activities were significantly increased at PND 28 and immunohistochemistry revealed approximately 25% greater numbers of Leydig cells stained for steroidogenic factor 1 at both PND 28 and 120 amongst rats fed elevated levels of isoflavones, resembling high human consumption rates. These findings show that F1 male rats continuously exposed to a mixture of dietary soy isoflavones from conception onwards exhibit altered gene expression at PND 28, which may lead to increased microsomal steroidogenic enzyme activity at this age and serum and testicular androgen profiles in adulthood. (Abstract shortened by UMI.)

Thesis (PhD)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: This study describes: - the characterization and comparison of the enzymatic activity of both Angora and ovine 3βHSD expressed in non-steroidogenic COS-1 cells. The apparent Km and Vmax values for the metabolism of PREG, 17-OHPREG and DHEA were determined; - the characterization of steroid metabolites produced by COS-1 cells coexpressing either Angora or ovine 3βHSD together with Angora CYP17, in the presence and absence of overexpressed Cyt-b5, following the metabolism of PREG and 17-OHPREG. 3βHSD was identified as an additional factor in causing hypocortisolism in the South African Angora goat; - the influence of Cyt-b5 on the enzymatic activity of both Angora and ovine 3βHSD coexpressed in non-steroidogenic COS-1 cells; - the influence of purified ovine live Cyt-b5 and anti-Cyt-b5 IgG on adrenal microsomal 3βHSD activity. Cyt-b5 was shown to specifically augment 3βHSD activity which represents the first documentation of such augmentation in any species; - the overexpression and purification of Angora 3βHSD using a baculovirus expression system coupled with a detergent based enzyme purification method; - the characterization of both substrate and co-factor kinetics for the individual dehydrogenase and isomerase activities of purified 3βHSD, in the presence and absence of purified ovine liver Cyt-b5. Cyt-b5 was shown to increase the affinity of 3βHSD towards NAD+ during the dehydrogenase reaction whilst having no significant influence on the isomerase reaction. This represents the first documentation of Cyt-b5 influencing co-factor binding in any member of the -ydroxysteroid dehydrogenases; - the FRET analysis of COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b5-eYFP fusion proteins, suggesting an allosteric interaction between 3βHSD and Cyt-b5.
AFRIKAANSE OPSOMMING: Hierdie studie beskryf: - die karakterisering en vergelyking van die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD, wat uitgedruk was in nie-steroïed genererende COS-1 selle. Die Km en Vmax waardes tydens die metabolisme van PREG, 17-OHPREG en DHEA was bepaal; - die karakterisering van steroïed metaboliete gegenereer deur COS-1 selle wat Angora of skaap 3βHSD uitdruk saam met Angora CYP17, in die aanwesigheid of afwesigheid van sitochroom b5, na die metaboliseering van PREG en 17-OHPREG. 3βHSD was geïdentifiseer as ‘n bydraende faktor in die oorsaak van hipokortisolisme in die Suid-Afrikaanse Angorabok; - die invloed van sitochroom b5 op die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD wat saam uitgedruk was in nie-steroïed genererende COS-1 selle; - die invloed van gesuiwerde skaap lewer sitochroom b5 en sitochroom b5 teenstof op mikrosomale 3βHSD aktiwiteit. Dit is getoon dat sitochroom b5 die aktiwiteit van 3βHSD spesifiek verhoog. Hierdie studie verteenwoordig die eerste dokumentasie van so ‘n verhoging in enige spesie; - die uitdrukking en suiwering van Angora 3βHSD deur middel van ‘n bakulo-virus sisteem gekoppel aan ‘n detergent gebaseerde ensiem suiwerings metode; - die karakterisering van beide substraat en ko-faktor kinetika vir die afsonderlike dehidrogenase en isomerase aktiwiteite van gesuiwerde 3βHSD, in die aanwesigheid of afwesigheid van gesuiwerde sitochroom b5. Dit is getoon dat sitochroom b5 die affiniteit van 3βHSD teenoor NAD+ tydens die dehidrogenase reaksie verhoog sonder om ‘n beduidende invloed op die isomerase reaksie te hê. Hierdie studie verteenwoordig die eerste dokumentasie van sitochroom b5 wat ko-faktor binding beïnvloed in enige lid van die hidroksisteroïed dehidrogenase familie van ensieme; - die analise van FRET sein in COS-1 selle wat beide 3βHSD-eCFP en Cyt-b5- eYFP fusie proteïene uitdruk. Die resultate stel voor dat sitochroom b5 3βHSD aktiwiteit beïnvloed deur middel van ‘n allosteriese meganisme.

Autosomal recessive triple A syndrome is caused by mutations in the AAAS gene encoding the protein ALADIN. The disorder manifests with the triad of adrenocorticotropin-resistant adrenal insufficiency, achalasia of the stomach cardia and impaired tear production (alacrima) in combination with progressive neurological impairment of the central, peripheral and autonomic nervous systems. ALADIN is part of the nuclear pore complex acting as a scaffold nucleoporin. In this work the role of ALADIN in the human adrenocortical tumour cell line NCI-H295R1 was investigated. These cells were engineered to either over-express or down-regulate AAAS by inducible stable transfection. Alterations in steroidogenic gene expression and functional consequences were determined. In addition, the role of ALADIN on cell viability and oxidative stress response was analysed. Using both the human adrenal NCI-H295R1-TR AAAS knock-down and over-expression models the potential impairment of the nuclear import of aprataxin, DNA ligase 1 and ferritin heavy chain 1 was investigated. For this YFP-specific vectors transiently transfected into the cell lines were employed. The findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore I demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, I show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. I conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting the knock-down cell model as an important in vitro tool for studying the adrenal phenotype in triple A syndrome. In an approach to identify new interaction partners of ALADIN, co-immunoprecipitation followed by proteome analyses using mass spectrometry was conducted in a GFP-ALADIN over-expression model using the human adrenocortical tumor cell line NCI-H295R. These results were verified in co-immunoprecipitation assays of endogenous ALADIN using NCI-H295R wild-type cells. The results suggest a possible interaction between ALADIN and microsomal flavoprotein cytochrome P450 oxidoreductase and progesterone receptor membrane compartment 2. Co-localisation analyses of these findings were done using immunofluorescence. The data are suggestive for an involvement of ALADIN in the export of nuclear-encoded mitochondrial proteins. Regulation of adrenocortical steroidogenesis is complex and there is increasing evidence that oxidative stress due to ROS accumulation and mitochondria are significantly involved. Furthermore, there may be an important cross-talk between functional organelles comprising nucleus, ER and mitochondria which presumably involves lipid metabolism. The goal of this work was to elucidate the function of ALADIN for the cellular oxidative stress response and its possible consequences for adrenocortical steroidogenesis in triple A syndrome patients
Mutationen im AAAS Gen verursachen die autosomal rezessive Krankheit Triple-A-Syndrom. AAAS kodiert das Nukleoporin ALADIN, welches Bestandteil des nukleären Porenkomplexes ist. Phänotypische Charakteristika des Triple-A-Syndroms sind Nebennierenrinden-Insuffizienz, Achalasie des unteren Speiseröhrenschließmuskels und eine fehlende Tränenproduktion (Alakrimie). Diese Symptome sind kombiniert mit progredienten neurologischen Störungen des zentralen, peripheren und autonomen Nervensystems. In dieser Arbeit wurde die Rolle von ALADIN in der humanen Karzinom-Zelllinie NCI-H295R1 untersucht. Diese Nebennierenrinden-Zellen wurden stabil transfiziert und mit einem induzierbaren Expressionssystem modifiziert, so dass sie AAAS entweder überexprimierten oder herunterregulierten. In NCI-H295R1-Zellen wurden Veränderungen der Genexpression von Enzymen der Steroidogenese und funktionelle Konsequenzen der Überexpression oder Herunterregulation von ALADIN gemessen. Des Weiteren wurde die Rolle von ALADIN auf die Zellviabilität und die Redox-Homöostase analysiert. ALADIN überexprimierende und herunterregulierte Zellen wurden verwendet, um die potentielle Behinderung des nukleären Imports von Proteinen zu untersuchen, welche den Zellkern gegen oxidativen Stress schützen (z.B. Aprataxin, DNA-Ligase 1 und Ferritin Heavy Chain 1). Dazu wurden YFP-spezifische Vektoren transient in diese Zellen gebracht. Mit den Ergebnissen dieser Arbeit wurde gezeigt, dass die Herunterregulation von AAAS eine Verminderung der Genexpression von CYP17A1 und CYP21A2 und deren Elektronendonor Cytochrom P450 Oxidoreduktase bewirken. Die Biosynthese der Vorläufermetabolite von Kortisol und Aldosteron ist in diesen Zellen ebenfalls vermindert. Des Weiteren zeigen die ALADIN-defizienten NCIH295R1-Zellen eine erhöhte Sensitivität gegenüber oxidativem Stress und eine veränderte Redox-Homöostase nach der Behandlung mit Paraquat. Darüber hinaus konnte in dieser Studie auch gezeigt werden, dass herunterregulierte ALADIN NCI-H295R1-Zellen einen verminderten Zellkernimport von Aprataxin, DNA-Ligase 1 und Ferritin heavy chain 1 besitzen. Aus diesen Ergebnissen kann geschlussfolgert werden, dass ALADIN-defiziente Nebennierenzellen eine verminderte Stressantwort auf oxidativen Stress besitzen; dies führt schlussendlich zu einer veränderten Steroidogenese. Das beschriebene ALADIN knock-down Modell in NCI-H295R1-Zellen ist ein wichtiges in vitro Werkzeug, um die Pathogenese der Nebennierenveränderungen im Triple-A-Syndrom zu erforschen. Neue Interaktionspartner von ALADIN wurden mit Hilfe von Co-Immunpräzipitation gefolgt von Proteom-Analysen durch Massenspektrometrie in einem GFP-ALADIN Überexpressionsmodell in NCI-H295R charakterisiert. Die Ergebnisse wurden durch Experimente auf endogenem Niveau in NCI-H295R-Wildtypzellen verifiziert. Mit diesen Daten wird in dieser Arbeit erstmals eine Interaktion zwischen ALADIN und dem Flavoprotein Cytochrom P450 Oxidoreduktase und Progesterone Receptor Membrane Compartment 2 nachgewiesen. Diese Ergebnisse wurden mit Co-Lokalisierungsanalysen durch Immunfluoreszenzfärbung von ALADIN und Cytochrome P450 Oxidoreduktase ergänzt. Außerdem gibt die Arbeit Hinweise darauf, dass ALADIN als Nukleoporin an dem nuklearen Export mitochondrialer Vorläuferproteine beteiligt ist. Die Regulation der Steroidogenese in der Nebennierenrinde ist komplex und es existieren zahlreiche Hinweise darauf, dass oxidativer Stress aufgrund der Ansammlung reaktiver Sauerstoffradikale und. dass die Mitochondrien involviert sind. Außerdem ist ein funktionelles Zusammenspiel verschiedener Organellen, darunter Nukleus, ER und Mitochondrien, von großer Bedeutung. Das Ziel dieser Arbeit war die Identifizierung der Funktion von ALADIN in der zellulären oxidativen Stressantwort und die möglichen Konsequenzen für die Steroidogenese in der Nebennierenrinden in Triple-A-Syndrom-Patienten

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Sep. 3, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Environmental Science and Engineering." Discipline: Environmental Sciences and Engineering; Department/School: Public Health.

Abstract The prevalence of the isolated ultrasonographic finding of polycystic ovaries (PCO) in the Finnish population and among women with a history of gestational diabetes (GDM) and changes in the present carbohydrate metabolism were investigated in the present study. One aim of this study was to investigate the prevalence of the recently discovered variant type LH (v-LH) in PCOS and to compare patient cohorts from Finland, the Netherlands, the United Kingdom and the United States of America. In addition, this study attempted to evaluate the nature of the ovarian streoidogenic response of women with PCOS to exogenously administered human chorionic gonadotrophin (hCG), human menotrophin (hMG) and follicle stimulating hormone (FSH). The effect of metformin on ovarian steroidogenesis was also studied. The prevalence of PCO was significantly higher in younger (≤ 35 years, 21.6%) than among older women (in ≥ 36 years, 7.8%). The overall prevalence of PCO in Finnish women was 14.2%. Women with previous GDM revealed a high prevalence of PCO (39.4%). The carrier frequency of the v-LHb allele in the entire study population was 18.5%. The frequency of the v-LH carrier was significantly lower in obese PCOS subjects in the Netherlands (2.0%) and Finland (4.5%). Women with previous GDM had impaired insulin sensitivity and β-cell function. They also had higher adrenal androgen secretion than the control women. Women with PCO and previous GDM had marked hyperinsulinemia which was not explained by obesity. Obese PCOS women achieved peak peripheral serum T concentrations at 48 hours after a hCG injection, preceded by peak levels of 17-OHP and E2 at 24 hours. In contrast, all steroids measured in the control women reached their maximum serum concentrations at 96 hours. HMG stimulated the production of ovarian androgens more efficiently than a urinary FSH after pituitary suppression with a gonadotrophin releasing hormone agonist (GnRHa). In conclusion, the prevalence of PCO is common in healthy Finnish women and even more common in women with a history of GDM. The ultrasonographic appearance of PCO may be a predictive factor with regards abnormal glucose tolerance during and after pregnancy and, these women should therefore be advised as to possible consequences. The high overall frequency of the v-LH allele in women in general and its low frequency in obese PCOS patients suggests that v-LH plays a role in reproductive functions and may counteract the pathogenesis of PCOS in obese individuals. The differences observed in steroid responses to hCG between normal and PCOS women might be explained by higher theca cell activity or mass in polycystic ovaries. Women with PCOS did not show a distinctly exaggerated steroidogenic response to hMG or FSH administration compared with control women. FSH administration also resulted in increased A and T production.

The present studies were undertaken to determine the involvement of protein kinase C (PKC) in the regulation of rat granulosa cell steroidogenesis in vitro. The effects of PKC activators (1-oleoyl-2-acetylglycerol (OAG); 1,2-dioctanoylglycerol (DiC$\sb8$) and phorbol 12-myristate 13-acetate (TPA)) and inhibitors (DL-Sphingosine (ESP) and 1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine free base (H$\sb7)\rbrack$ on basal and FSH-, (Bu)$\sb2$cAMP-, forskolin- and calcium ionophore A23187-stimulated pregnenolone (P$\sb5$), progesterone (P) and 20$\alpha$-hydroxy-pregn-4-en-3-one (20$\alpha$-OH-P) secretion by granulosa cells were studied. OAG, when continually present in the culture medium (MEM), significantly stimulated P$\sb5$, P and 20$\alpha$-OH-P secretion during 6 to 24 h culture periods. It also markedly increased the conversion of exogenous P$\sb5$ to P and 20$\alpha$-OH-P and exogenous P to 20$\alpha$-OH-P during 24 h cultures. Pretreatment of granulosa cells with TPA for 1 h or treatment for up to 6 h resulted in a significant increase in P$\sb5$, P and 20$\alpha$-OH-P secretion. Except for 20$\alpha$-OH-P production, which was stimulated by the phorbol ester during all culture periods studied, secretion of P$\sb5$ and P (in the presence or absence of exogenous hormones and the inhibitors of steroidogenic enzymes) were substantially inhibited by TPA during a 24 h incubation. However, when granulosa cells were incubated with both OAG (20 $\mu$g/ml) and TPA (40 ng/ml), progestin secretion was increased irrespective of the duration of incubation. PKC inhibitors dose-dependently suppressed the stimulatory effect of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) with complete inhibition noted at 100 $\mu$M of H$\sb7$ and 10 $\mu$M of ESP. Diacylglycerols and TPA exerted divergent effects on FSH-, (Bu)$\sb2$cAMP- and forskolin-stimulated progestin secretion. FSH-stimulated accumulation of P$\sb5$ throughout the culture periods (1-24 h) was markedly increased by OAG (20 $\mu$g/ml) but inhibited by TPA (40 ng/ml). OAG (5-80 $\mu$g/ml) and DiC$\sb8$ (20 $\mu$g/ml) significantly enhanced FSH-induced progestin secretion during 6 h and 24 h culture periods and increased steroid synthesis in 24 h cultures in the presence of (Bu)$\sb2$cAMP or forskolin. In contrast, TPA significantly inhibited FSH- and (Bu)$\sb2$cAMP-stimulated progestin secretion during both 6 h and 24 h of incubation. Pretreatment of granulosa cells with TPA (40 ng/ml) for 20 h to down-regulate PKC, decreased progestin secretion during subsequent incubation with FSH (150 ng/ml) and prevented any stimulation by OAG (20 $\mu$g/ml). The effects of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) on FSH-induced steroid secretion appeared to be additive when both PKC activators were present together and differed significantly from those when OAG and TPA were present with FSH separately. Diolein (a nonpermeable diacylglycerol), 4$\alpha$-phorbol 12,13-didecanoate and phorbol 13-monoacetate (two phorbol esters with no tumor promoting activity) did not influence basal or FSH-stimulated steroid secretion by granulosa cells. (Abstract shortened by UMI.)

Enzyme-catalyzed reactions are important to regulate steroidogenesis and nuclear receptor activation. The present investigation examines the role of steroid metabolism catalyzed by CYP7B1 for regulation of hormone receptor activation and the effects of vitamin D on enzymatic regulation of steroidogenesis. The study reports data indicating that CYP7B1 can regulate estrogenic signaling by converting estrogens into inactive or less active metabolites. Similar results were obtained for CYP7B1-mediated metabolism of some androgen receptor ligands, indicating that CYP7B1 can be involved also in the regulation of androgenic signaling. CYP7B1 substrates and metabolites were found to exert androgenic effects in a cell line-specific manner. Furthermore, cell line differences were observed in the expression pattern for androgen receptor comodulators. This thesis reports that 1α,25-dihydroxyvitamin D3 alters the gene expression and enzyme activity of CYP21A2 and CYP17A1 leading to suppressed production of aldosterone, dehydroepiandrosterone and androstenedione in adrenocortical cells. These are novel findings on vitamin D action. A mechanism is reported for the vitamin D-mediated regulation of the CYP21A2 gene. Data indicate that vitamin D receptor interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF) are key comodulators in this novel vitamin D receptor (VDR)-mediated mechanism. Furthermore, the results indicate that altered expression levels of VDIR and WSTF can shift the suppressing effect of vitamin D to a stimulatory effect. Also, epigenetic components were found to be involved in the effects of vitamin D on CYP21A2 transcriptional rate. In addition, a functional vitamin D response element was identified in the CYP21A2 promoter. This study also reports that 1α,25-dihydroxyvitamin D3 affects sex hormone production in a tissue-specific way. Gene expression and enzyme activity of aromatase were found to be downregulated in cells derived from breast, but not in cells derived from prostate and adrenal cortex. The production of estradiol and dihydrotestosterone was altered in a tissue-selective manner following vitamin D treatment. These findings are of importance for the discussion on vitamin D as a potential anti-breast cancer agent.

Follicular growth and maturation are tightly regulated processes, which involve the participation of endocrine, autocrineparacrine factors and intracellular molecules. Due to the numerous research efforts, a large number of regulators and their mechanisms of regulation of follicular growth and differentiation have been established. Although the abnormal expression and activities of some of these regulators are believed to be associated with ovarian dysfunction diseases, such as polycystic ovarian syndrome (PCOS), the etiology and pathogenesis of this syndrome are not completely understood. In this thesis, we have identified two novel regulators of follicular growth and differentiation and examined the cellular and molecular mechanisms that contribute to the folliculogenesis. We present here that chemerin reduces FSH-induced steroidogenic enzyme expression and steroid hormone production in follicles and granulosa cells. Prohibitin expression is upregulated by chemerin and knockdown of prohibitin attenuates the suppressive role of chemerin on steroidogenesis, an action regulated by Akt. Using an androgenized rodent model, we also present the dysregulation of chemerin and prohibitin and their association with dysregulated follicular steroidogenesis. Our data and preliminary clinical studies demonstrate the potential involvement of chemerin and prohibitin in the etiology of PCOS. These studies significantly improve the knowledge of ovarian functions and the pathophysiology of PCOS, and provide important clues for the development of novel diagnosis biomarkers and new treatment strategies for this complex syndrome.

Plasticizers aid in processing, and impart flexibility to plastics. Their broad use and tendency to leach out of polymers have rendered them ubiquitous in the environment. Phthalate plasticizers, in particular di-(2-ethylhexyl) phthalate (DEHP), are known to cause male reproductive tract defects in animal models. It has been assumed that DEHP metabolite, mono-(2-ethylhexyl) phthalate (MEHP), is the active compound, however the bioactivity of metabolites such as 2-ethylhexanol, 2-ethylhexanal and 2-ethylhexanoic acid, has not been thoroughly investigated. The aim of this study was to test the effects of these compounds in a mouse Leydig tumour cell line, MA-10 cells. DEHP, MEHP and 2-ethylhexanal decreased cell viability, as well as steroidogenic potential as quantified by an enzyme-linked immunosorbent assay and gene expression analysis. Interestingly, 2-ethylhexanal was the most potent steroidogenic disruptor, which offered an intriguing contribution to the search for the mechanism(s) of phthalate toxicity and raised doubts that MEHP is the single active metabolite.
Il est connu que les phtalates utilisés comme plastifiants et plus particulièrement les phtalates di-(2-éthylhexyle) (DEHP) sont à l'origine des malformations de l'appareil reproducteur masculin dans les modèles d'animaux. Ces plastifiants sont essentiels lors de la fabrication des plastiques pour leur donner de la flexibilité. Dus à une utilisation excessive de ces plastifiants et à leur capacité à s'échapper des plastiques existant, ces plastifiants sont maintenant omniprésents dans l'environnement. Dans cette étude, on fait l'hypothèse que le métabolite de DEHP, le phtalate mono-(2-éthylhexyle) (MEHP), est la molécule active, bien qu'il n'existe aucune autre étude concernant les métabolites comme 2-éthylhexanol, 2-éthylhexanal et l'acide 2-éthylhexanoïque. Le but de cette étude est d'évaluer les effets de ces produits chimiques sur une lignée cellulaire tumorale interstitielle du testicule de souris, les cellules MA-10. On a montré que le DEHP, le MEHP et le 2-éthylhexanal ont diminué la viabilité des cellules, aussi bien que la stéroïdogénèse qui a été quantifiée par la méthode ELISA et par l'analyse de l'expression génétique. Contre toute attente, le 2-éthylhexanal s'est avéré le plus puissant acteur dans la dégradation de la stéroïdogénèse, ce qui a ouvert une nouvelle avenue de recherche concernant le ou les mécanisme(s) impliqués dans la toxicité des plastifiants phtalates et a mis en doute le fait que le MEHP est le seul métabolite actif.

>Magister Scientiae - MSc
Endocrine disrupting compounds (EDCs) are exogenous compounds/chemicals in the environment that interfere with the synthesis, secretion, distribution and function or elimination of natural hormones in the body. Environmental estrogens are a subclass of EDCs that may mimic or inhibit the effect of endogenous estrogen and can therefore influence developmental and reproductive health in humans and animals. EDCs have been reported to adversely affect the reproductive, immune, endocrine and nervous systems of wildlife and humans. The effects of EDCs include gonadal abnormalities, altered male/female sex ratios, reduced fertility and cancers of the male and female reproductive tract to mention a few. These effects are difficult to detect. Although it is essential to screen for EDCs in aqueous environmental samples, most countries have failed to implement this as part of their routine water quality monitoring programs due to various constraints such as the high cost of assays and the lack of infrastructure and skills required to do the assays. Therefore, there is a clear need for more user-friendly, more economically viable and time saving assays that can be used for routine monitoring of environmental EDCs. The aim of this study was to investigate the comparison between chemical and tissue culture methods to monitor environmental estrogens. 28 environmental water samples were collected from various sites around South Africa and analyzed for EDCs using a battery of rapid in vitro tests. Samples collected for the current study were selected based on various human impacts and also to give approximately 50% high and 50% low estrogen values. The 28 environmental water samples were separated into two groups based on the estradiol ELISA. The estradiol ELISA was chosen because estradiol is the principal estrogen found in all mammalian species during their reproductive years. For this separation, an estradiol level of 5 pg/ml was used as cut-off. Of the 28 samples investigated, 15 had estradiol levels higher than 5 pg/ml and were designated as high estradiol. The remaining 13 samples contained estradiol at 5 pg/ml or less and they were designated as low estradiol. The first objective of this study was to compare different rapid ELISAs for EDC monitoring to determine if the data obtained with these assays are similar/identical. The data obtained from the estrogenic ELISAs was related/similar and showed good correlation with each other. This is because the different estrogens are very similar and also due to the fact that the same sub-group in the population (the reproductively active females) is secreting these hormones. Therefore, an estradiol rapid assay was proposed as a first screening system for estrogens in samples. Even though there was a positive correlation between the estradiol rapid assay and testosterone rapid assay, separation of samples based on estradiol levels wasn’t a good predictor of testosterone levels in the samples. A testosterone rapid assay was therefore recommended as necessary to screen for androgens in samples. The positive correlation between the estradiol rapid assay and progesterone rapid assay was expected because both estradiol and progesterone are secreted and excreted by the same population sub-group (reproductively active females). This study also demonstrated a good predictability of separating samples containing progesterone using the estradiol ELISA. Progesterone is secreted by pregnant women, a sub-group of the reproductively active females. It is advised that a progesterone rapid assay be included to screen samples for progestogens. The second objective of this study was to compare estradiol rapid ELISAs with a bioassay for anti-androgenicity using mouse testicular cell cultures. The mouse testicular cell testosterone synthesis bioassay to monitor anti-androgenicity of the samples showed no correlation between the ELISA data for estrogens. This study shows that anti-androgenic effects need to be monitored independently because the data for estrogenic compounds cannot be used as a predictor for anti-androgenic effects. This demonstrated the need for the inclusion of a mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity of water samples. In summary, due to the different mechanisms of action of EDCs, this study recommended a battery of assays to monitor for EDCs. The battery of assays suggested is: ●Estradiol ELISA as a rapid assay to screen for estrogens. ●Testosterone ELISA as a rapid assay to screen for androgens. ●Progesterone ELISA as a rapid assay to screen for progestogens. ●Mouse testicular cell testosterone synthesis bioassay to screen for androgenicity and anti-androgenicity.