Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells

Levallet, Jérôme; Mittre, Hervé; Delarue, B; Carreau, Serge

doi:10.1677/jme.0.0200305

Cited by 52 publications

(23 citation statements)

References 32 publications

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“…7A). Nucleotide sequence analysis of these products revealed that the shorter segment is the same as that reported by Levallet (Levallet et al 1998b) and results from an alternative splicing site on a GT dinucleotide at position 1813-1814 (Fig. 1C).…”

Section: Effects Of T 3 On P450 Arom Mrna Expression: Evidence Of Norsupporting

confidence: 82%

“…1A: upstream Aro-Ex8, 5 -GCTTCTCATCGCAGAGTAT CCGG-3 , located in exon 8; downstream Aro-Ex9, 5 -CAAGGGTAAATTCATTGGGCTTGG-3 , located in exon 9; amplified product 289 bp (Ex9)) (Levallet et al 1998a). However, for the selective detection of altered transcripts with different 3 -end regions (coding for putative inactive protein previously reported for germ cells and rat ovary (Lephart et al 1990, Levallet et al 1998b)), we used the following antisense primers: (1) Aro-Ex10, 5 -GGA ATCGTTTCAAAAGTGTAACCAG-3 , encompassing 23 nucleotides of exon 10 (1949-1973) (Fig. 1A); (2) Aro-Ex9Tr, 5 -GGCTGAAAATACCTGTAGGGA ACTCG-3 , which matches the three nucleotides before the 49 nucleotides lacking region of exon 9 and the first 23 nucleotides at the beginning of exon 10 (Fig.…”

Section: Rt-pcr Assaymentioning

confidence: 99%

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Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells

Pezzi

Panno

Sirianni

et al. 2001

Journal of Endocrinology

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Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coincident with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T 3 ) replacement either in vivo or in vitro. This study, after the immunolocalization of aromatase in cultured rat Sertoli cells, examined the effects elicited by T 3 on this enzyme, by simultaneously investigating three functional levels of aromatase: mRNA expression, protein content, and enzymatic activity. The immunolocalization of cytochrome P450 aromatase (P450 arom) was shown in the cytoplasm of cultured Sertoli cells from 15-and 21-day-old rats. Western blot analysis revealed an enhancement of aromatase protein content upon stimulation with N 6 ,2 -O-dibutyryladenosine-3 :5 -cyclic monophosphate ((Bu) 2 cAMP) that was clearly downregulated by T 3 . The presence of a functional P450 arom protein in purified Sertoli cells was confirmed by the measurement of [ H]androst-4-ene-3,17-dione. With 100 nM T3, a decrease in both P450 arom mRNA levels and aromatase activity was observed. The aromatase enzymatic activity was strongly stimulated by (Bu) 2 cAMP and markedly down-regulated by T 3 . In contrast, the strong increase in aromatase mRNA upon (Bu) 2 cAMP stimulation was apparently unaffected by T 3 administration. This paper shows how the identification of an altered transcript induced by T 3 coding for putative truncated and inactive aromatase protein might explain such a decrease in aromatase activity in T 3 -treated cells. On the basis of these results, it is concluded that at least two mechanisms could be involved in the down-regulatory effect of T 3 on aromatase activity in prepuberal Sertoli cells. The first mechanism is linked to a possible direct modulatory role for T 3 in the regulation of the aromatase promoter, whilst the second one is represented by the induction of altered transcripts coding for truncated and inactive aromatase proteins.

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Section: Effects Of T 3 On P450 Arom Mrna Expression: Evidence Of Norsupporting

confidence: 82%

Section: Rt-pcr Assaymentioning

confidence: 99%

Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells

Pezzi

Panno

Sirianni

et al. 2001

Journal of Endocrinology

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“…Together with oLH, it is clear that testosterone further controls the aromatase both at the transcriptional level and in terms of biological activity. However, with a larger concentration of gonadotropin (50 ng) the Leydig cell estradiol output decreases whereas the amount of transcripts is higher suggesting, therefore, additional regulatory steps and maybe the expression of truncated P450arom mRNAs as shown previously in rat Sertoli and germ cells (Levallet et al 1998b). Consequently, our data related to the positive effects of steroid hormones on the expression of the aromatase gene indicate that besides CRE, androgen responsive elements as well as estrogen responsive elements are necessary for a full expression of the aromatase in Leydig cells.…”

Section: Discussionsupporting

confidence: 52%

Regulation of cytochrome P450 aromatase gene expression in adult rat Leydig cells: comparison with estradiol production

Genissel¹,

Levallet²,

Carreau³

2001

Journal of Endocrinology

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Regulation of aromatase gene expression in purified rat Leydig cells has not yet been investigated. Therefore, using a highly specific quantitative RT-PCR method, we have measured the amount of cytochrome P450 aromatase (P450arom) mRNA and aromatase activity in mature rat Leydig cells submitted to various treatments during 24 h. Estradiol production was enhanced in a dose-related manner in the presence of testosterone, the maximum (28% increase) being obtained with 200 ng/ml. Related to the P450arom mRNA levels, a decrease was observed in the presence of low concentrations (50 and 100 ng/ml) of testosterone, then a 20% increase of the amount of transcripts was recorded for the higher concentrations (200-500 ng/ml). The same result was obtained in the presence of 5 -dihydrotestosterone (an androgen resistant to aromatase activity). The addition of ovine LH (oLH; 0·1-50 ng/ml) to the Leydig cell culture medium induced a dose-related augmentation of estradiol output up to 10 ng/ml oLH, although a decrease was observed with 50 ng/ml when compared with maximal values. mRNA levels slightly decreased in the presence of low concentrations (0·1-1 ng/ml) of oLH, an effect that was abolished by the addition of testosterone; mRNA levels were increased by oLH (5-10 ng/ml) 35 and 75% respectively in the absence and presence of testosterone (when compared with Leydig cells incubated without treatment). With 50 ng/ml oLH, a large augmentation (twofold) of the P450arom mRNA level either without or with testosterone was observed. Dibutyryl cyclic AMP (1 mM) mimicked the effect of oLH. The half-life of the P450arom mRNAs was twofold increased in the presence of testosterone and oLH when compared with the half-life in the absence of treatment (5·8 0·6 h). Taken together, our data have demonstrated that, in freshly isolated Leydig cells from mature rat testes, the regulation of aromatase expression and enzymatic activity is under LH (through cyclic AMP) and steroid control; moreover seminiferous tubule-secreted factor(s) are also involved. Therefore, rat Leydig cell aromatase is controlled at both transcriptional and post-transcriptional steps by endocrine and/or locally produced modulators.

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“…In fact, this study has identified a new 3 0 truncated aromatase mRNA splice variant (DQ004742) expressed in bovine granulosa cells. However, this transcript variant, with orthologues in the sheep, pig and human EST libraries, consists only of exons 1-5 of the aromatase gene, and thus may have no further function following translation (Levallet et al 1998). A new gene with so far unknown functions, OSAP, identified in mouse preovulatory follicles, may be a marker of differentiated follicle development, specifically of increased E synthesis, with gene expression responding to gonadotrophic stimulation (Hennebold et al 2000, Tanaka et al 2003.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes

Mihm¹,

Baker²,

Fleming³

et al. 2008

Reproduction

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This study was designed to identify genes that regulate the transition from FSH-to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P%0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, b; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development.

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Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells

Cited by 52 publications

References 32 publications

Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells

Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells

Regulation of cytochrome P450 aromatase gene expression in adult rat Leydig cells: comparison with estradiol production

Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes

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