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.

Cultures Y-1 mouse adrenal tumor cells exhibited varying degrees of rounding when treated with ACTH (0.5 U/ml) caused rounding, formation of filopodia and numerous thin microvilli, and stimulated steroidgenesis.

No description available.

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%).

Pro-opiomelanocortin (POMC) is the precursor of many neuropeptides which includ adrenocorticotropin (ACTH). ACTH has a biological activity in regulating adrenocortical function. In the present study, we will investigate the effect of POMC gene transfer on adrenal cortex cells in cell cultures and animal models. The study included adrenal cortical H295R cells for adenovirus-mediated gene delivery. The effects of POMC gene on H295R cell steroidogenesis and cell proliferation were investigated. In addition, there were 32 SD rats dividing into three groups. 1) Control, injected with normal saline via tail vein (n = 8); 2) Ad-GFP, injected with adenovirus containing GFP (n=12); 3) Ad-POMC, injected with adenovirus containing recombinant POMC gene (n=12). Body weight (BW) was measured. Adrenals were collected, fixed and a series of sections were cut for stains for PCNA and MC2-R. The plasma cortisol and VEGF levels of rats were measured. The results showed that Ad-POMC delivery significantly increased the ACTH and cortisol levels by 50-100 fold and 20-100% in H295R cells, respectively. In addition, Ad-POMC delivery significantly inhibited the cell proliferation and increased the apoptotic cells. The expression of MC2-R protein of H295R cells was also suppressed after Ad-POMC delivery. In the study of SD rats, the Ad-POMC-treated rats exhibited reduced weight gain compared with other groups in the first 2 weeks; however, there was no significant change in BW between Ad-POMC and Ad-GFP groups during the experimental period. The weight of adrenal in Ad-POMC-treated rats was significantly higher than Ad-GFP group in the 8th week. Comparing the sequential adrenal weights in Ad-POMC group, those in 6th week were significantly higher than in 2nd and 4th weeks. The plasma VEFG levels of Ad-POMC-treated rats were higher than Ad-GFP group in the 8th week. The adrenal sections showed that Ad-POMC treated rats had moreanti-PCNA stained cells than Ad-GFP treated rats in 8th week. However, less anti-MC2R stained cells were found in Ad-POMC treated rats in 8th week. Ad-POMC treated rats had higher plasma cortisol levels than those in Ad-GFP treated rats, however, there were no statistical significances. In conclusion, POMC gene transfer modulates the morphology and function of the adrenal cortex. POMC gene inhibits the H295R cells proliferation by inducing MC2-R down-regulation and cells apoptosis. In SD rat adrenal, however, it stimulates adrenal cortex in biphasic pattern. The rapid growing pattern noted in the later phase may be due to the effect of VEGF. Besides, the physical regulation of cortisol synthesis is much stricter than that of ACTH.

No description available.

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.

>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.

OBJECTIVE: Adrenal steroidogenesis is important for maturation of fetal organ systems and plays a role in triggering parturition in ovine pregnancies. Studies have suggested a differential increase in baseline cortisol between twin gestations near term. Our aim was to further delineate the mechanisms responsible for the differences between the hypothalamic- pituitary-adrenal axes of twin fetuses in vivo. STUDY DESIGN: Surgery was performed on pregnant ewes (n = 6) with twin gestations to implant fetal vascular catheters. After recovery but while the subjects were resting, plasma cortisol concentrations were similar in both fetuses. Fetuses received, intravenously, boluses of adrenocorticotropic hormone at 2 doses, and plasma samples were obtained for analysis of the cortisol response. This stimulation by adrenocorticotropic hormone was then repeated in the same fetuses approximately 4 days later, after the increase of resting daily cortisol values in one but not the other fetus. RESULTS: Cortisol responses to adrenocorticotropic hormone before changes in daily resting cortisol concentrations were indistinguishable between twins. However, after separation of daily resting cortisol values, fetuses in group A (elevated resting cortisol concentration) demonstrated a significantly increased response to stimulation by adrenocorticotropic hormone. CONCLUSION: These results suggest a differential development in response to stimulation by adrenocorticotropic hormone between twin fetuses in vivo as the mechanism responsible for the asynchronous elevation of one twin's resting plasma cortisol concentration.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • The UPLC-MS/MS analyses and quantification of novel 5α-reduced steroids using response factors. • The kinetic characterisation of human steroid 5α-reductase type 1 (SRD5A1), expressed in HEK-293 cells, towards 11OHA4 and 11OHT and their keto derivatives by progress curve analysis. • The subcloning, transformation and functional expression of SRD5A1 in the yeast expression system, P. pastoris. • The conversion of 11OHA4 and 11OHT and their keto derivatives by SRD5A1 expressed in P. pastoris. • The endogenous enzymatic activity in P. pastoris towards the 5α-reduced metabolites in the 11OHA4- and alternate 5α-dione pathways. • The potential application of P. pastoris as a biocatalyst in the production of 5α- reduced C19 steroids. / AFRIKAANSE OPSOMMING: Hierdie ondersoek beskryf: • Die UPLC-MS/MS analise en kwantifisering van nuut-ondekte 5α-gereduseerde steroïede met behulp van responsfaktore. • Die kinetiese karakterisering van menslike steroïed 5α-reduktase tipe 1 (SRD5A1), uitgedruk in HEK-293 selle, vir 11OHA4 en 11OHT en hul ketoderivate deur middel van progressiekurwe-analise. • Die subklonering, transformasie en funksionele uitdrukking van SRD5A1 in die gis P. pastoris. • Die omsetting van 11OHA4 en 11OHT en hul ketoderivate deur SRD5A1 uitgedruk in P. pastoris. • Die omsetting van 5α-gereduseerde steroïede in die 11OHA4 en alternatiewe 5α-dioon paaie deur endogene ensieme in P. pastoris • ‘n Ondersoek na die toepassing van die gisuitdrukkingstelsel as ‘n moontlike OR potensiële biokatalis vir die produksie van 5α-gereduseerde C19 steroïede.