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Indrajit Chowdhury, Kelwyn Thomas, Anthony Zeleznik and Winston E Thompson

450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase ( cyp19a1 ) ( Miller & Bose 2011 ). All steroidogenic pathways begin in the mitochondria, where regulation of the cellular steroidogenic capacity occurs through key enzymatic rate

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G Schuler, Y Dezhkam, L Tenbusch, MC Klymiuk, B Zimmer and B Hoffmann

aromatase and estrogen receptors, point to significant differences between mammalian species concerning the roles of estrogens for male reproductive functions (for review see Cooke et al . 2017 ). The expression of estrogen receptors in porcine germ cells

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Khampoune Sayasith, Kristy A Brown, Jacques G Lussier, Monique Doré and Jean Sirois

62 283 –291. Bao B , Garverick HA, Smith GW, Smith MF, Salfen BE & Youngquist RS 1997 Changes in messenger ribonucleic acid encoding luteinizing hormone receptor, cytochrome P450-side chain cleavage, and aromatase are

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S C Sharma, P Purohit and A J Rao

ABSTRACT

Inhibition of aromatase, a key enzyme in the biosynthesis of oestradiol-17β, by the addition of 1,4,6-androstatrien-3,17-dione resulted in a significant increase in the levels of immunoreactive human chorionic gonadotrophin (hCG) in the medium and tissue. This increase was partially reversed by the simultaneous addition of oestradiol-17β. These effects on the levels of immunoreactive hCG were also reflected by the increased levels of mRNA specific for the α and β subunits of hCG following the addition of the aromatase inhibitor. However, addition of tamoxifen resulted in a drastic decrease in the levels of both the messages. Based on these results, it is suggested that the synthesis of hCG is negatively modulated by oestradiol-17β in the human placenta.

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G. Verhoeven, J. Cailleau and I. D. Morris

ABSTRACT

Ethane 1,2-dimethane sulphonate (EDS) selectively destroys Leydig cells in the interstitium of the testis of adult rats. The toxic activity of this compound is much less obvious in the immature rat testis. We examined the effects of EDS, its monomethyl derivative and busulphan on cultured interstitial cells, percoll-purified Leydig cells, Sertoli cells and peritubular cells derived from immature rats. The studies with interstitial cells and Leydig cells showed that EDS (40–160 μg/ml) blocked the conversion of C21 and androgen precursors into testosterone and androstenedione. Higher concentrations of this compound also inhibited the production of C21 steroids and the LH-induced production of cyclic AMP (cAMP). The observed effects required a latent period of at least 8 h and were slowly reversible. Isolated cells were more sensitive to EDS than monolayer cultures. Reaggregation cultures were even less sensitive. EDS was markedly more effective on immature Leydig cells than its monomethyl derivative and busulphan. In cultured Sertoli cells FSH-inducible aromatase activity, cAMP production, androgen-binding protein (ABP) production and the secretion of a paracrine factor with Leydig cell-stimulatory activity were markedly reduced by busulphan. In these cells, busulphan was clearly more active than EDS and its monomethyl derivative. The production of paracrine factors which increase ABP production and decrease FSH-inducible aromatase activity in Sertoli cells was studied as a parameter of the effects of alkane sulphonates on peritubular cells. Only busulphan markedly decreased the production of these paracrine factors. It is concluded that EDS displays a selective toxicity to Leydig cells derived from immature animals and that, apart from its effects on germ cells, busulphan may also directly impair the function of Sertoli cells and peritubular cells.

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K. J. Doody, E. D. Lephart, D. Stirling, M. C. Lorence, R. R. Magness, M. J. McPhaul and E. R. Simpson

ABSTRACT

We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17α-hydroxylase cytochrome P-450 (P-45017α ), aromatase cytochrome P-450 (P-450AROM) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)+-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of ∼1·9 kb was detected at low levels, while 3β-HSD mRNA of 1·7 kb was in relatively high abundance throughout the oestrous cycle. While P-45017α mRNA of 1·9 kb and P-450AROM of 2·7, 2·2 and 1·7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14–21 of gestation). Levels of P-45017α mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3β-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-45017α mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG. Further RNA blot analysis utilizing a 3′ portion of the P-450AROM cDNA as probe, which contains an intronic segment instead of the steroid-binding region, revealed that the two mRNA species of lower molecular weight hybridized to this probe. In contrast, only the largest aromatase mRNA band hybridized to a probe specific for the steroid-binding region. These findings indicate that a majority of the P-450AROM mRNA species in rat ovarian tissue during the oestrous cycle and throughout pregnancy represents alternatively spliced products, which presumably lack the ability to encode for aromatase activity. These results indicate that the pattern of steroid secretion in the rat can be explained, in part, by the differential expression of mRNA species encoding the various key steroidogenic enzymes throughout the ovarian cycle, during pregnancy and following administration of gonadotrophins.

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C Kalbe, R Furbass, M Schwerin and J Vanselow

Cyp19 encodes aromatase cytochrome P450, the key enzyme of oestrogen biosynthesis. In the bovine placenta, the majority of Cyp19 transcripts include a 5' untranslated region which is encoded by exon 1.1; this suggests that its 5'-flanking region is the predominant placental promoter. The aim of the present investigation was to examine the promoter activity of this region and to map cis-acting regulatory elements in order to improve our understanding of the complex regulation of this gene within the placenta. As an initial approach, human JEG-3 choriocarcinoma cells were transiently transfected with reporter-gene constructs consisting of different 5'-flanking sequences of exon 1.1 fused to the luciferase gene as a reporter. To localise and further characterise functional cis-acting elements, targeted point mutations and electrophoretic mobility-shift experiments were used. The data demonstrate, for the first time, (1) that the bovine exon 1.1 5'-flanking sequence is an active promoter, (2) that 404 bp of this region are sufficient for constitutive reporter-gene expression in JEG-3 cells and (3) that the region includes at least two different enhancer elements; the data also suggest (4) that one of these elements consists of the E-box motif CATGTG and that the second enhancer element includes the half-site hexameric sequence AGGTCA and additional nucleotides flanking this element upstream.

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R G Sutcliffe, A J Russell, C R W Edwards and A M Wallace

Understanding of the principal pathways of steroid hormone biosynthesis was established over two decades ago through advances in steroid radioisotopic and chromatographic techniques. When the enzymes of individual pathways could be examined in more detail, the dissection of the complex pattern of enzyme activities began. At many points, separate pathways employ precisely the same enzyme for equivalent catalytic steps, e.g. for 21-hydroxylase, 11 β-hydroxylase, aromatase and several dehydrogenases (Orth et al. 1992). A further economy was found for 17α-hydroxylase and 17,20-lyase activities, which co-purify with the same P450c17 polypeptide. This enzyme was later cloned and expressed in tissue culture cells, revealing that, contrary to the enzyme in rat, human and cattle, 17α-hydroxylase cannot convert 17α-hydroxyprogesterone to androstenedione (Bradshaw et al. 1987, Fevold et al. 1989). Further complexity emerged with the existence of multiple tissue-specific forms of 5α-reductase (Wilson et al. 1993), and 3β-, 11β- and 17β-hydroxysteroid dehydrogenases, most of which

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PJ Chedrese, MR Rodway, CL Swan and C Gillio-Meina

We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 microM androstenedione elevated the levels of estradiol-17beta to 0.028, 0.3 and 1.21 pg/microg protein, respectively. The level of expression of 3betaHSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.

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M. Simoni, G. F. Weinbauer, R. K. Chandolia and E. Nieschlag

ABSTRACT

Testicular androgens are known to influence not only the secretion but also the bioactivity and molecular composition of pituitary FSH. In the present study, we investigated the effects of chronic androgen blockade and castration on the molecular heterogeneity of the gonadotrophin. Groups of male adult rats (five animals per group) received one of the following treatments: vehicle, the non-steroidal anti-androgens casodex (20 mg/kg per day) or flutamide (20 mg/kg per day), or castration. After 8 weeks, the animals were killed and individual pituitary homogenates fractionated by isoelectric focusing (IEF) on sucrose density gradients in the pH range 2·5–8. FSH was measured by radioimmunoassay (RIA) in the individual fractions and by in-vitro bioassay (Sertoli cell aromatase bioassay) in pools of fractions which were combined according to pH intervals of 0·5 units. Bioactive and immunoreactive FSH were also measured in sera and unfractionated pituitary extracts. Testosterone and inhibin were assayed in sera by RIA.

A significant increase in serum immunoreactive and bioactive FSH was demonstrated in flutamide-treated and castrated animals, whereas the pituitary content of bioactive FSH remained unchanged in the four groups. Serum testosterone and inhibin were undetectable in castrated animals and significantly increased in those treated with flutamide. By RIA, the IEF profiles of the flutamide-treated and castrated rats showed a significant reduction of the FSH isoforms with 3·5<pI<4, with a significant increase in the isoforms with pI>4 only in the castrated group. By bioassay, there was a significant decrease in the isoforms with 3·5<pI<4 in both casodex- and flutamide-treated animals, with no significant differences between the two groups. Castration caused a further significant shift in the relative distribution of FSH isoforms towards the less acidic components, with a significant increase in the isoforms with 5<pI<5·5 not attained by androgen blockade alone.

These results suggest that the effects of long-lasting castration on pituitary FSH heterogeneity cannot be entirely reproduced by the androgen blockade. Since inhibin, eliminated by castration but not by androgen blockade, is a major regulator of FSH in the male rat, we speculate that it might not only influence FSH secretion but also modulate its qualitative properties.