CYP19 mRNA Research Articles

The onset of cortisol synthesis and the stress response is independent of changes in CYP11B or CYP21 mRNA levels in larval red drum ( Sciaenops ocellatus)

Although cortisol plays an important role in teleost development, the onset of cortisol production and the cortisol stress response in teleosts remain poorly understood. Here we have reported basal cortisol levels and the development of the cortisol stress response in larval red drum ( Sciaenops ocellatus). We isolated partial nucleic acid sequences encoding two key corticosteroidogenic enzymes, CYP11B and CYP21 and assessed ontogenetic patterns of their mRNA levels relative to basal and stress-induced cortisol production. Basal cortisol was first detected 3 days post-hatch (DPH) and reached a maximum at 9 DPH. Cortisol did not increase in response to an acute stressor prior to 6 DPH. From 6 DPH forward, stress caused significant increases in larval cortisol content. Stress-induced cortisol levels in 6–9 DPH larvae were highest 1 h post-stress. In larvae 11 DPH and older, the highest cortisol measurements occurred 0.5 h post-stress. Elevated cortisol was still evident after 3 h in 6 DPH larvae. From 11 DPH onward, basal cortisol levels were reestablished in larvae by 1 h post-stress. CYP11B and CYP21 transcripts were detected in red drum 12 h prior to hatching and in all post-hatch larvae examined. Changes in CYP11B and CYP21 mRNA levels did not occur in association with the ontogenetic appearance of cortisol, or the onset of the stress response. As larvae developed, the dynamics of the cortisol stress response matured from a low magnitude, slow recovery response, to a response similar to that observed in juvenile and adult fish.

Expression of Estrogenicity Genes in a Lineage Cell Culture Model of Human Breast Cancer Progression

The mRNA levels of estrogen receptors (ERα and ERβ), cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1 and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β‐hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1 and catechol‐O‐methyltransferase (COMT) were examined in MCF7 and in MCF10A‐derived cell lines representing a lineage cell culture model for breast cancer progression. Relative to MCF7, MCF10A‐derived cells expressed low levels of ERα but comparable levels of ERβ. In benign parental MCF10A breast epithelial cells, SULT1E1 mRNA was induced in confluent relative to subconfluent cell cultures. Proestrogenic 17βHSD1 mRNA was expressed in all of the MCF10A lineage cell lines while 17βHSD2 mRNA was most abundant in MCF10A cells. CYP1B1, SULT1A1 and SULT2B1 were expressed in MCF7 cells, while STS and COMT were widely detected across cell lines. Treatment with the potent histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1, SULT2B1 and CYP19 mRNA, but suppressed CYP1B1, STS, COMT, 17βHSD1 and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to ER‐positive MCF7 cells, key determinants of breast estrogen metabolism are differentially regulated during neoplastic progression. Supported by ES05823 and ES06636.

Dehydroepiandrosterone formation is independent of cytochrome P450 17α-hydroxylase/17, 20 lyase activity in the mouse brain

Cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) is a microsomal enzyme reported to have two distinct catalytic activities, 17α-hydroxylase and 17, 20 lyase, that are essential for the biosynthesis of peripheral androgens such as dehydroepiandrosterone (DHEA). Paradoxically, DHEA is present and plays a role in learning and memory in the adult rodent brain, while CYP17 activity and protein are undetectable. To determine if CYP17 is required for DHEA formation and function in the adult rodent brain, we generated CYP17 chimeric mice that had reduced circulating testosterone levels. There were no detectable differences in cognitive spatial learning between CYP17 chimeric and wild-type mice. In addition, while CYP17 mRNA levels were reduced in CYP17 chimeric compared to wild-type mouse brain, the levels of brain DHEA levels were comparable. To determine if adult brain DHEA is formed by an alternative Fe 2+-dependent pathway, brain microsomes were isolated from wild-type and CYP17 chimeric mice and treated with FeSO 4. Fe 2+ caused comparable levels of DHEA production by both wild-type and CYP17 chimeric mouse brain microsomes; DHEA production was not reduced by a CYP17 inhibitor. Taken together these in vivo studies suggest that in the adult mouse brain DHEA is formed via a Fe 2+-sensitive CYP17-independent pathway.

Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERalpha and ERbeta) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenases (17betaHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERalpha-positive luminal MCF7 breast cancer cells. Low levels of ERalpha and ERbeta mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17betaHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17betaHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17betaHSD1, and 17betaHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERalpha, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERbeta, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

Inhibition of Adrenocortical Carcinoma Cell Proliferation by Steroidogenic Factor-1 Inverse Agonists

Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and reverse transcription quantitative PCR were used. SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.

Inhibition of Adrenocortical Carcinoma Cell Proliferation by Steroidogenic Factor-1 Inverse Agonists

ABSTRACT Context: Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). Objective: Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). Main Outcome Measures: Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and real-time quantitative PCR were used. Results: SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. Conclusion: SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.

Expression profile of CYP19 in human breast cancer cell lines treated with indole-3-carbinol, 3,3'-diindolylmethane, and sulforaphane.

Abstract Abstract #1113 Breast cancer is the most frequently diagnosed cancer in Western women. Most cases (up to 75%) are estrogen-dependent. The estrogen-independent cases are more invasive, more difficult to treat, and they are connected with worse prognosis. The biologically active estrogens important in the proliferation of breast cancer are produced from inactive precursors by the action of aromatase encoded by CYP19 gene. Conventional treatment (e.i. hormonal therapy) is expensive and for advanced cancer ultimately unsuccessful. That is why early diagnosis and prevention of this disease is urgently needed. A new hope is brought by chemoprevention – the concept of early intervention by usage of different agents inhibiting the development of invasive cancer. Recent epidemiological migrant studies showed that consumption of raw or short cooked cabbage and sauerkraut is connected with significant reduction of breast cancer incidences. Indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM) – a major in vivo acid-catalyzed condensation product of I3C, and sulforaphane (SUL) are examples of numerous active chemicals in cabbage.
 The aim of the present study was to investigate the effect of I3C, DIM and SUL on the profile of CYP19 expression in human breast cancer estrogen-dependent MCF-7 cell line. Cells were treated with these compounds at the concentrations relevant to those observed in human plasma. The total RNA extraction from cell cultures after 144 hours of incubation with different substances concentrations and reverse transcriptase reaction preceded the cDNA synthesis. The screening of cDNA from aromatase mRNA was performed using real-time PCR assay. The results showed that I3C in the dose of 30 μM reduced the expression of CYP19 in MCF-7 cell line. In contrast, higher dose of I3C (50 μM) induced its expression. The latter results confirmed the observations of the other authors indicating reciprocal I3C effect on carcinogenesis. SUL in the doses of 5μM and 20μM diminished the level of CYP19 mRNA. Similar trend was observed after treatment with 5 μM and 10 μM of DIM. Since the down-regulation of CYP19 gene causes the reduction in estrogens synthesis the usage of I3C, DIM, and/or SUL could be well-founded as preventive agents against breast carcinoma development. Further investigation on estrogen-independent MDA-MB-231 cell line which is under way are necessary to compare the possible application of I3C, DIM, and SUL as a chemopreventive agents depending on the hormonal character of the tumour. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1113.

생후 발달과정 동안 숫 흰쥐의 Efferent Ductules과 부정소에서 Cytochrome P450 Aromatase(Cyp19) 발현 양상 분석

본 연구는 생후 발달과정에서 숫 흰쥐의 생식기인 Efferent ductules(EDs)과 부정소에서 cytochrome P450 aromatase(Cyp19) 유전자의 발현과 연령별 발현 양상을 알아보는데 목적이 있다. 조직으로부터 추출된 total RNA는 역 전사 반응을 통해 cDNA로 바뀌어진 후 real-time PCR 기법을 사용하여 부위별 그리고 연령별로 Cyp19 유전자 발현을 알아보았다. Cyp19 유전자의 발현은 EDs에서 90일령을 제외한 7일, 14일, 그리고 30일령 등 성숙기 이전의 모든 연령에서 나타났으며, 부정소에서는 7일령을 제외한 모든 연령에서 보여졌다. 특히 부정소에서 Cyp19의 발현은 부정소의 각 부위별로 특이한 양상을 보였다. 본 연구 결과를 통해 남성 생식기의 EDs와 부정소의 각 부분에서 Cyp19 유전자 발현은 연령과 부위에 따라 차별적으로 조절되는 것을 알 수 있었다. The present study was performed to determine expression of cytochrome P450 aromatase(Cyp19) in the efferent ductules(EDs) and the epididymis of male rat reproductive tract at different postnatal ages. Total RNAs isolated were reverse-transcribed, and cDNAs were utilized for real-time PCR analysis. In the EDs, the Cyp19 transcript was expressed at all prepubertal ages with the highest level at 14 days of age, but not at 90 days of age. Expression of Cyp19 mRNA in the epididymis was found at all age groups, except 7 days of age. Distinct expression patterns of Cyp19 transcript were shown in each segment of the epididymis. These results indicate that expression of Cyp19 gene in the excurrent duct of male reproductive tract is differentially regulated in age-dependent and segment-specific manners.

Steroidogenic enzyme gene expression in the brain of the parthenogenetic whiptail lizard, Cnemidophorus uniparens

The steroidogenic enzyme CYP17 is responsible for catalyzing the production of androgenic precursors, while CYP19 converts testosterone to estradiol. De novo neurosteroidogenesis in specific brain regions influences steroid hormone dependent behaviors. In the all-female lizard species Cnemidophorus uniparens, individuals alternately display both male-like mounting and female-like receptivity. Mounting is associated with high circulating concentrations of progesterone following ovulation (PostOv), while receptivity is correlated with estrogen preceding it (PreOv). At a neuroanatomical level, the preoptic area (POA) and ventromedial nucleus of the hypothalamus (VMN) are the foci of the male-typical mounting and female-typical receptivity, respectively. In this study, we indirectly test the hypothesis that the whiptail lizard brain is capable of de novo neurosteroidogenesis by cloning fragments of the genes encoding two steroidogenic enzymes, CYP17 and CYP19, and examining their expression patterns in the C. uniparens brain. Our data indicate that these genes are expressed in the C. uniparens brain, and more importantly in the POA and VMN. Using radioactive in situ hybridization, we measured higher CYP17 mRNA levels in the POA of PostOv lizards compared to receptive PreOv animals; CYP19 mRNA levels in the VMN did not change across the ovarian cycle. To our knowledge, these are the first data suggesting that the reptilian brain is capable of de novo steroidogenesis. This study also supports the idea that non-gonadal sources of steroid hormones locally produced in behaviorally relevant brain loci are central to the mediation of behavioral output.

Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

BackgroundP450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis.MethodsChanges in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E) POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells.ResultsPOR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E), such as those found in ABS patients, had much less effect on aromatase activity than expression of wild-type POR proteins. Knockdown of endogenous POR protein in KGN human granulosa cells led to reduced estrone production, indicating that endogenous POR affected aromatase activity.ConclusionWe demonstrated that the expression of POR, together with that of aromatase, was regulated by gonadotropins, and that its induction could up-regulate aromatase activity in the ovary, resulting in a coordinated increase in estrogen production.

Estrogen biosynthesis in human H295 adrenocortical carcinoma cells

Adrenocortical carcinoma is an uncommon malignancy and feminizing symptoms secondary to adrenal estrogen-secretion are extremely rare. The direct secretion of estradiol by adrenocortical tumors requires, in addition to the expression of aromatase (CYP19), the expression of one or more of the reductive 17beta-hydroxysteroid dehydrogenases. The expression of CYP19 transcripts and protein were markedly induced in the H295 adrenocortical carcinoma cell line after treatment with either forskolin or vasoactive intestinal peptide (VIP). Western immunoblotting demonstrated a marked induction of the CYP19 protein of characteristic size after only a short (6h) treatment period with VIP or forskolin. The CYP19 mRNA transcripts were derived from both promoters PII (Ic) and I.3 (Id) after treatment with both agents. The reductive type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) was also constitutively expressed in the H295 cells but neither its mRNA transcript nor protein levels were altered after forskolin or VIP treatment. Western immunoblotting of an estrogen-secreting adrenal carcinoma revealed notable levels of both aromatase and AKR1C3 expression while an aldosterone-producing adrenal adenoma lacked aromatase expression and showed a reduced level of AKR1C3 expression. Immunohistochemistry of the carcinoma-bearing adrenal revealed localization of AKR1C3 not only in the tumor but also principally in the zona reticularis of the normal adrenal tissue. Adrenal aromatase and AKR1C3 expression therefore appear to be features of adrenocortical malignancies that are associated with biosynthesis of active estrogen.

Differential Effects of High and Low Steroidogenic Factor-1 Expression on CYP11B2 Expression and Aldosterone Production in Adrenocortical Cells

Steroidogenic factor-1 (SF-1/Ad4BP/NR5A1) plays a major role in regulating steroidogenic enzymes. We have previously shown that SF-1 inhibits aldosterone synthase (CYP11B2) reporter gene activity. Herein, we used the H295R/TR/SF-1 adrenal cells that increase SF-1 in a doxycycline-dependent fashion. Cells were incubated with or without doxycycline to induce SF-1 and then treated with angiotensin II (Ang II). Aldosterone was measured by immunoassay. SF-1 mRNA was silenced by small interfering RNA (siRNA) by Nucleofector technology. mRNA levels were measured by real-time RT-PCR. Ang II treatment without doxycycline increased aldosterone production by 11.3-fold and CYP11B2 mRNA by 116-fold. Doxycycline treatment increased SF-1 mRNA levels by 3.7-fold and inhibited Ang II-induced aldosterone by 84%. Doxycycline treatment inhibited Ang II-stimulated CYP11B2 mRNA levels by 86%. Doxycycline decreased basal CYP11B2 promoter activity by 68%. Doxycycline inhibited Ang II stimulation by 85%. Ang II increased CYP21 mRNA expression by 4.6-fold, whereas doxycycline inhibited induction by 69%. In contrast, doxycycline treatment increased CYP11B1 mRNA by 1.7-fold in basal cells and increased Ang II induction by 3.6-fold. SF-1-specific siRNA significantly reduced SF-1 mRNA expression as compared with cells treated with control siRNA. SF-1 siRNA reversed doxycycline stimulation of CYP B1 and its inhibition of CYP11B2. However, in H295R/TR/SF-1 cells without doxycycline treatment, both CYP11B1 and CYP11B2 mRNAs were significantly decreased, suggesting that both enzymes require a minimal level of SF-1 for basal expression. In summary, SF-1 overexpression dramatically inhibited CYP11B2 expression and decreased aldosterone production. The opposing effects of SF-1 on CYP11B1 and CYP11B2 suggest that the regulation of SF-1 activity may play a role that determines the relative ability to produce mineralocorticoid and glucocorticoid.

Complex assembly on the human CYP17 promoter

Optimal steroid hormone biosynthesis occurs via the integration of multiple regulatory processes, one of which entails a coordinate increase in the transcription of all genes required for steroidogenesis. In the human adrenal cortex adrenocorticotropin (ACTH) activates a signaling cascade that promotes the dynamic assembly of protein complexes on the promoters of steroidogenic genes. For CYP17, multiple transcription factors, including steroidogenic factor-1 (SF-1), GATA-6, and sterol regulatory binding protein 1 (SREBP1), are recruited to the promoter during activated transcription. The ability of these factors to increase CYP17 mRNA expression requires the formation of higher order coregulatory complexes, many of which contain enzymatic activities that post-translationally modify both the transcription factors and histones. We discuss the mechanisms by which transcription factors and coregulatory proteins regulate CYP17 transcription and summarize the role of kinases, phosphatases, acetyltransferases, and histone deacetylases in controlling CYP17 mRNA expression.

Time-Response Effects of Testicular Gene Expression Profiles in Sprague-Dawley Male Rats Treated with Di(n-Butyl) Phthalate

Phthalate esters were reported to damage fetal and postnatal testes of experimental animals, but the molecular mechanisms underlying these effects remain unknown. The time-response effects of di(n-butyl) phthalate (DBP) on the expression patterns of the testicular genes in male Sprague-Dawley rats were examined for different periods of exposure (1, 7, 14, or 28 d). The steroidogenic- or spermatogenic-related gene expression patterns were measured using reverse-transcription polymerase chain reaction (RT-PCR). After 28 d of exposure, the serum concentrations of DBP and monobutyl phthalate (MBP) increased in a dose-dependent manner, and were significantly higher in the DBP-treated rats than in the control rats. Liver weight was increased markedly at 28 d after DBP exposure at 750 mg/kg/d. Testicular weight was reduced significantly after 14 and 28 d of exposure. DBP (750 mg/kg/d) produced a significant increase in scavenger receptor class B1 (SR-B1) and steroidogenic acute regulatory (StAR) mRNA after 14 and 28 d of exposure. The level of cytochrome P-450 (P450) side-chain cleavage (P450scc) mRNA decreased in the group treated with DBP at 750 mg/kg/d at 7 d. After 14 and 28 d of exposure, there was an apparent increase in P450scc mRNA. High doses of DBP significantly increased the Cyp17 mRNA level after 28 d of exposure. At 7 d, a significant decrease in Cyp19 mRNA was observed only in the group exposed to 750 mg/kg/d DBP. In addition, DBP significantly decreased the levels of a spermatid-specific gene (Spag4) and lactate dehydrogenase A (LDHA) mRNA after 7 d of exposure. The levels of androgen receptor (AR), estrogen receptor-alpha (ER-alpha), and retinoid X receptor-gamma (RXR-r) expression decreased significantly in a time- or dose-dependent manner. DBP significantly increased the peroxisome proliferator-activated receptor-gamma (PPAR-r) and phosphorylated extracellular-signal-regulated kinase (p-ERK1/2) levels in the testis. These results suggest that the acute and chronic effects of DBP on the steroidogenic pathways in the testes show mechanistically distinct patterns. Data thus provide some insights into the molecular mechanisms underlying DBP-induced testicular dysgenesis.

The Src tyrosine kinase pathway regulates thecal CYP17 expression and androstenedione secretion

In order to evaluate the role of Src tyrosine kinase in thecal cell steroidogenesis, a pharmacological approach was utilized by treating enriched populations of mouse ovarian theca-interstitial cells in vitro with a direct Src kinase inhibitor, PP2. Inhibition of Src with PP2 increased both basal and forskolin-stimulated androstenedione secretion, and increased cytochrome P450 17-alpha hydroxylase-lyase (CYP17) promoter activity and steady state mRNA. PP2 did not change thecal levels of StAR mRNA. Inhibition of mitogen-activated protein kinase kinase, a downstream regulator of Src activity, using PD98059 also increased forskolin-stimulated secretion of androstenedione above forskolin alone, but had no effect on basal secretion of androstenedione. Src inhibition increased mitogen-activated protein kinase phosphatase-1 protein and decreased phosphorylation of SF-1, which correlated with increased CYP17 promoter activity and mRNA levels. These results implicate Src tyrosine kinase in the regulation of CYP17 and thecal androgen secretion.

Benzo( a)pyrene decreases brain and ovarian aromatase mRNA expression in Fundulus heteroclitus

The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo( a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100 μg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3-month-old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100 μg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development.

Steroidogenic gene expression during sex determination in the frog Rana rugosa

Rana rugosa is unique among frog species in that it has two distinct types of sex chromosomes in two separate forms (XX/XY and ZZ/ZW). Treatment with sex steroids can reverse its gender from female to male or male to female. This phenomenon makes it a novel model for studying gonadal differentiation. The physiological role of sex steroids in sex differentiation in amphibians is yet unclear, however. To address this issue, we cloned the cDNAs of 17βHSD types 8 ( 17βHSD8) and 12 ( 17βHSD12), 5α-reductase type 1 ( 5αRed1), and the steroidogenic acute regulatory protein known as StAR in the steroidogenic pathway. Then, we measured the mRNA levels of these genes during sex differentiation by real-time RT-PCR. The levels of CYP11A1, 3βHSD, CYP17 and CYP19 mRNA were also measured by real-time RT-PCR. As a result, we detected transcripts of all such genes except for that of 17βHSD8 in the indifferent gonad before the onset of sex determination. The expression of CYP17 occurred in indifferent gonads in both sexes; and its transcript levels were much higher in the male gonads. By contrast, the levels for CYP19 were much higher in the female gonads. CYP11A1, 3βHSD, 17βHSD12, 5αRed1 and StAR showed no sexually dimorphic expression during gonadal sex differentiation. Taken together, the results suggest that CYP17 has a major influence on testis development and that CYP19 plays a similar role in ovary development. However, the factors that up-regulate their expression remain to be identified.

Excess Estrogen Sulfoconjugation Causes a Weak Sign of Delivery in Somatic Cell Clone Recipient Cows.

This study was conducted to elucidate a factor causing a weak sign of delivery and a prolonged gestation, which are frequently observed in cows carrying somatic clone fetus, in spite of the maturity of fetal size or the occurrence of large offspring syndrome. Till date, several investigators have suggested morphological and functional abnormalities of the placenta in clone fetus in the various gestational stages. However, the roles for the placenta in the final process of clone pregnancy, namely placental estrogen synthesis and regulation of estrogen activity with sulfoconjugation, have not been fully understood. In IETS (2007 in Kyoto, Japan), we reported that prepartum rise of estrone and estradiol-17b concentrations in clone pregnancy was subtle, but estrone-sulfate concentration in clone pregnancy was higher than that in cows carrying fetus derived from in vivo embryo. In this study, we analyzed mRNA expression levels of estrogen-related genes, estrogen sulfotransferase (SULT1E1), estrogen sulfatase (STS) and aromatase (CYP19), in placental tissues, cotyledon (COT) and caruncle (CAR). Placentomes were obtained immediately after purtrition from cows, which delivered clone and in vivo calves after induction of parturition (gestation length was 284 to 289 days). Correlation analyses between mRNA abundances in placental tissues and maternal estrogen concentrations at parturition were performed using the data obtained from clones. Localization of gene expression in placentome was examined with in situ hybridization (SULT1E1 and STS) and immunohistochemistry (CYP19). Number of binucleate cells (BNC), existing in placentome, was counted from sections stained with hematoxylin-eosin. mRNA abundance of SULT1E1 was tended to be high in COT compared with CAR, clone pregnancy showed significantly higher (p<0.05) expression level than in vivo pregnancy. In situ hybridization signal of SULT1E1 was detected in binucleate cells. SULT1E1 mRNA abundance was negatively and positively correlated with maternal estrone (p<0.05) and estrone-sulfate (p<0.05) at parturition, respectively. mRNA abundances of STS and CYP19 were significantly higher in CAR and COT, respectively. In situ hybridization and immunohistochemistry showed that BNC and carunclar epithelial cells expressed both genes. Expression levels of STS and CYP19 mRNA were similar between clone and in vivo pregnancy, and were positively correlated (p<0.05) with maternal estradiol-17b at parturition of clones. Population of BNC in placentome did not differ between clone (vaginally delivered clone; 18.8}1.4/0.1mm2, cesarean section delivered clone; 18.6}1.7/0.1mm2) and in vivo (18.9}1.8/0.1mm2) pregnancy. Therefore, it can be surmised that increased SULT1E1 mRNA abundance in clone placental tissue was due to an increased transcription of the gene, rather than to an increased proportion of SULT1E1 producing BNC. The results obtained here suggested that excess estrogen sulfoconjugation in placenta was reasonable for the confusion of the ratio of unconjugated (active) and sulfoconjugated (inactive) estrogens. The hormonal abnormality would cause the weak sign of delivery and the prolonged gestation in cows carrying clone.

A positive feedback pathway of estrogen biosynthesis in breast cancer cells is contained by resveratrol

Cytochrome P450 (CYP) 19 enzyme or aromatase catalyses the rate-determining step of estrogen synthesis. The transcriptional control of CYP19 gene is highly specific in different cell types, for instance, Promoter I.3/II is commonly used for regulation in breast cancer cells. Recently, a positive feedback pathway for estrogen synthesis has been identified in ERα expressing SK-BR-3 cells. CYP19 mRNA abundance and activity are increased in this pathway and the promoter usage is switched from Promoter I.3/II to I.1 through a non-genomic process. In the present study, effect of the phytocompound resveratrol on this Promoter I.1-controlled expression of aromatase was investigated. Results indicated that resveratrol reduced the estradiol-induced mRNA abundance in SK-BR-3 cells expressing ERα. Luciferase reporter gene assays revealed that resveratrol could also repress the transcriptional control dictated by Promoter I.1. Since the ERE-driven luciferase activity was not repressed by resveratrol, the nuclear events of estrogen were unlikely to be suppressed by resveratrol. Instead the phytochemical reduced the amount of ERK activated by estradiol, which could be the pathway responsible for Promoter I.1 transactivation and the induced CYP19 expression. The present study illustrated that resveratrol impeded the non-genomic induction of estrogen on CYP19.

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1–100 ppb atrazine or 0.27 ppb (1 nM) 17β-estradiol (E 2) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E 2, but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1–100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.