Etude comparative des éléments régulateurs du promoteur II du gène de l'aromatase dans les cellules de granulosa et les cellules lutéales de lapine

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

Differential Regulation of Two 3′ End Variants of P450 Aromatase Transcripts and of a New Truncated Aromatase Protein in Rabbit Preovulatory Granulosa Cells

International audienceIn rabbit granulosa cells, two cytochrome P450 aromatase (P450 arom) mRNAs issued from promoter II were described: a full-length and a truncated transcript. Western blot analysis showed two P450 arom proteins with apparent molecular masses of 53 and 46 kDa, which are consistent with the predicted theoretical sizes of proteins encoded by these two transcripts. To examine the involvement of the truncated transcript in the regulation of P450 arom gene expression, the level of each transcript was specifically quantified in cultured granulosa cells by competitive quantitative RT-PCR. FSH induced a dose-dependent increase in both estradiol production and P450 arom mRNAs levels with a much more enhancement in the full-length mRNA. The half-life of the transcripts could not explain this differential regulation. Upon dibutyryl cAMP stimulation, the full-length mRNA was less abundant than the truncated one. In contrast, Western blot analysis revealed a stimulation of the 53-kDa protein content, whereas the 46-kDa protein amount was apparently unaffected. TGF beta in FSH-stimulated conditions decreased both estradiol production and P450 arom transcripts levels. TGF beta did not modify estradiol production and aromatase protein amounts induced by dibutyryl cAMP, whereas the two P450 arom mRNAs levels were increased. In conclusion, we report for the first time that a protein encoded by a truncated P450 arom mRNA could be involved in the regulation of estrogen production. Moreover, we show that the two P450 arom mRNAs are regulated in a differential manner, probably through hormonal control of the alternative splicing

The absence of a functional nuclear receptor element A (NREA) in the promoter II of the aromatase P450 gene in rabbit granulosa cells

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Expression of the rabbit cytochrome P450 aromatase encoding gene uses alternative tissue-specific promoters

International audienceThe aim of the present study was to analyse the tissue-specific expression of various promoter-derived transcripts from the gene encoding rabbit aromatase cytochrome P450. A new promoter, named I.r, was identified, and promoters II and I.r were sequenced. Promoter I.r-derived transcripts were found in preovulatory granulosa cells, corpus luteum, placenta and adipose tissue. An alternative splice variant of this transcript was found with tissue-specific preference. Tissue-specific expression of promoter-derived variants was studied in the ovary before and after ovulation. While the level of promoter II-derived transcript decreased dramatically after ovulation, that of promoter I.r-derived transcript remained unchanged, indicating that promoter II and promoter I.r were not controlled by a single regulation system. The existence of this dual system of regulation suggests that the rabbit ovary could be a useful model to study the promoter-specific regulation of aromatase

Quantification of cytochrome P450 aromatase transcripts before and during luteal phase in rabbit

International audienceThe aim of the present study was to quantify the promoter II- and I.r-derived transcripts of P450 aromatase gene during follicular stages and during corpus luteum formation in the rabbit. An ovulatory dose of hCG induced, first the disappearance of 90% of aromatase transcripts since 6 h before ovulation, and second a gradual decrease during pseudopregnancy. Individual quantification of both the promoter-derived transcripts showed that promoter II-derived transcript was the main transcript expressed both during follicular phase and pseudopregnancy, but kinetics of disappearance were not similar between both the promoter-derived transcripts. Moreover, hCG up-regulates aromatase expression in vitro in luteal tissue but estradiol, which was without effect on aromatase expression in preovulatory granulosa cells, down-regulates this expression in luteal tissue. In conclusion, the regulation of P450 aromatase in rabbit is mainly under control of promoter II regardless of which cyclic stage is studied. Moreover, we reported an opposite effect of estradiol on aromatase expression in vitro between follicular and luteal cells

Unusual clinical description of adult with Timothy syndrome, carrier of a new heterozygote mutation of CACNA1C

International audienceCANAC1C encodes for the main cardiac L-type calcium channel and mutations on it lead to a prolonged QT interval in Timothy Syndrome (TS). We provide a new de novo constitutional heterozygote missense variation in CACNA1C in a living adult woman, also carrier of the known c.2146-1G>C heterozygous variation of PKP2 inherited from her father. To our knowledge, this patient is the first to have the two variations in these genes. Theses clinical and molecular findings expand the clinical and molecular spectrum of TS and show the interest of next generation sequencing or whole exome sequencing in rare disorders, atypical or novel phenotype

Aromatase expression in the normal human adult adrenal and in adrenocortical tumors: biochemical, immunohistochemical, and molecular studies

International audienceObjective: The aromatase enzyme catalyzes the final stage of estrogen biosynthesis pathway from androgens. Its expression in the adrenal is poorly Studied except for the rare estrogen-producing adrenocortical tumors. In order to further characterize aromatase expression in the adrenal, we evaluated the aromatase enzyme activity, Cyp19a1 gene expression level, and promoter utilization in normal adrenal tissues and in adrenocortical secreting tumors. Design and Methods: Six normal adult adrenals(NA), 2 feminizing adrenal tumors(FT), I0cortisol-producing adenomas with overt (CS, n = 4) or sub-clinical Cushing syndrome (SCS, n = 6) and 3 aldosterone-producing adenomas (APA) were studied. Tissue aromatase activity was determined by the tritiated ((3)H)-water method. Total aromatase mRNA were measured by a competitive RT-PCR. Promoter regions PIT and PIA-derived transcripts were also studied in NA, FT, and other steroid-producing tumors by a semi-quantitative comparative RT-PCR. Immunofluorescence analysis was performed in normal human adrenal tissues. Results: Aromatase activity was detected in NA tissues and in all tumor subtypes, at high levels in both FT In NA, aromatase immunofluorescence was detected in the cytoplasm of steroidogenic cells, mainly from zona reticularis. Compared with NA, aromatase transcript levels were similar in CS kind APA, lower in SCS and similar or higher in FIR Promoter analysis suggested predominant PII utilization in NA, APA, and SCS, but similar PII and P1.4 utilization in CS tumors. Conclusion: Aromatase is expressed at, similar levels in normal adrenal and in adrenocortical tumors, but at variably high levels in FT. Different promoter utilization patterns are found among tumor subtypes

BMP system expression in GCs from polycystic ovary syndrome women and the in vitro effects of BMP4, BMP6, and BMP7 on GC steroidogenesis.

International audienceBACKGROUND:The bone morphogenetic proteins (BMPs) are growth factors involved in the folliculogenesis. Alteration in their expression may compromise the reproductive process in disease such as the polycystic ovary syndrome (PCOS). This study investigated the expression and role of granulosa cell (GC) BMP from normal cycling and PCOS women.METHODS AND RESULTS:This prospective study was performed in GCs obtained from 14 patients undergoing IVF: i) six women with normal ovulatory cycles and tubal or male infertility and ii) eight women with PCOS. BMP2, BMP4, BMP5, BMP6, BMP7, and BMP8A and their receptors BMPR1A, BMPR1B, and BMPR2 were identified by RT-PCR in GCs from normally cycling and PCOS women. BMP4, BMP6, and BMP7 expressions were confirmed by immunohistochemistry. Quantitative transcript analysis showed the predominant expression of BMP6. In GCs from PCOS women, an overexpression of BMP6 (P<0.01) and BMPR1A mRNA (P<0.05) was observed. GC culture experiments demonstrated that basal estradiol (E₂) production was threefold higher but FSH-induced E₂ increment was twofold lower in PCOS compared with controls. In PCOS, BMP6 and BMP7 exerted a stimulatory effect on basal E₂ production while BMP4 and BMP6 inhibited FSH-induced E₂ production. FSH receptor and aromatase expression were not different between both groups.CONCLUSION:The BMP system is expressed in human GCs from normal cycling and PCOS women. The BMP may be involved in reproductive abnormalities found in PCOS