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D Brochard
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L Morel
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G Veyssiere
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C Jean
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MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids) is a member of the rat and mouse seminal vesicle secretory protein family. The gene encoding MSVSP99 is under androgenic control and we demonstrate here that this regulation involves a complex interplay of positive and negative regions. First, we show that the promoter region (-387/+16) sufficient to mediate a full androgen induction is a complex enhancer organized in two regulatory regions. These two regions are inactive individually and must act together to confer a 40-fold androgen induction to the MSVSP99 gene and androgen responsiveness is not only dependent on the presence of functional androgen response element (ARE) sequences but results from complex cooperations between ARE and non-ARE sequences forming an androgen response unit. Secondly, we characterized a new regulatory region (-824/-632) that decreases androgen-dependent transcriptional activity of the MSVSP99 promoter. This region, also able to repress the transcriptional activity of the heterologous thymidine kinase promoter, contains a functional promoter on the inverted strand (-826 to -387) and we identified a transcription initiation site located at position -639 with respect to the cap site of the MSVSP99 promoter. Sequence analysis of the flanking DNA also revealed that the MSVSP99 gene is surrounded by long interspersed repeated sequences called LINEs.

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A M Simon
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G Veyssière
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Cl Jean
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ABSTRACT

The gene encoding MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids), an androgen-dependent protein specifically expressed in the mouse seminal vesicle, was isolated and sequenced. A mouse genomic library constructed in the λEMBL12 vector was screened using a full length cDNA probe. One genomic clone was selected, 7·4 kb of which were shown to contain the whole MSVSP99 gene. The complete sequence of the MSVSP99 gene (1·7 kb), plus 0·8 and 0·3 kb of the 5′ and 3′ flanking regions respectively, has been determined. The gene is composed of four exons interrupted by three introns. The size range for the four exons is 47–217 bp, while that of introns is 87–615 bp. The transcription start site was identified as an adenine residue located 21 nucleotides upstream from the ATG start codon. Putative TATA and CAAT boxes were identified, along with a number of regions that shared homologies with known regulatory sequences. These included androgen-responsive elements located in the promoter as well as in the gene sequence. Sequence comparisons with other androgen-responsive genes showed strong homologies between the MSVSP99 gene and the seminal vesicle secretory protein (SVS) family genes (rat SVS II, IV, V and VI). Moreover, some regions were found to be conserved between the MSVSP99 gene and the human semenogelin I and II genes.

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E. Pailhoux
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A. Martinez
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Ch. Jean-Faucher
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G. Veyssière
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Cl. Jean
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ABSTRACT

We have previously characterized an androgen-inducible secretory protein from the mouse vas deferens (MVDP), and a cDNA to its mRNA has been obtained. This report describes altered MVDP gene expression after neonatal exposure to oestrogens. As shown by immunohistochemistry and Western blot analysis, MVDP was missing in the vas deferens from adult mice neonatally exposed to oestrogens. Northern blot analysis showed that the expression of MVDP mRNA was also suppressed. Exogenous testosterone was unable to stimulate MVDP production (either message or protein) in neonatally oestrogenized males. The results suggest that the alterations in gene expression in the oestrogen-exposed vas deferens reflect changes in the programme of differentiation of the organ itself.

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C Guilbaud
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A M Simon
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G Veyssière
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C Jean
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ABSTRACT

We report the cloning and sequencing of a new cDNA sequence encoding a protein from the mouse seminal vesicle. An open reading frame of 297 nucleotides encoded a protein of 99 amino acids with a calculated molecular mass of 11·454 kDa. The first 21 amino acids constituted a signal peptide followed by 78 amino acids encoding the secreted protein. The cDNA sequence comprised a 3′ untranslated region of 226 bp and the polyadenylation signal AATAAA, 19 bp upstream from the poly(A)+ tail. A high degree of homology was found between this protein and members of the family of seminal vesicle secretory (SVS) proteins, especially rat SVS VI. Northern blot analysis indicated the presence of a 0·7 kb mRNA species in the mRNAs of seminal vesicle tissue. Castration resulted in a marked decrease in the level of the 0·7 kb mRNA encoding the protein, whereas administration of testosterone to castrated males restored the 0·7 kb mRNA.

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S Baron
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M Manin
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C Aigueperse
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M Berger
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C Jean
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G Veyssiere
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L Morel
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The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0.25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.

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A Dassouli
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Ch Darne
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S Fabre
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M Manin
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G Veyssière
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Cl Jean
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ABSTRACT

The understanding of androgen-regulated gene expression requires a cell culture system that mimics the functions of cells in vivo. In the present paper we have examined a vas deferens epithelial cell subculture system. Cultured vas deferens epithelial cells have been shown to exhibit polarized properties characteristic of functioning epithelia and to display a high level of androgen receptors. Incubation of cells with androgen caused a decrease in cellular androgen receptor mRNA that was time-dependent. Total suppression was observed after 24 h of exposure to androgen. By contrast, incubation of vas deferens epithelial cells with androgen resulted in a threefold increase in the cellular content of androgen receptor protein, as assayed by ligand binding. In response to androgens, vas deferens epithelial cells expressed mouse vas deferens protein mRNA (MVDP mRNA). Maximum expression of the MVDP gene, at both mRNA and protein levels, was observed after 24 h of androgen induction.

DEAE-dextran transfection conditions were defined using the MMTV-CAT vector. Dihydrotestosterone stimulated the transcription activation of MMTV-CAT gene in vas deferens epithelial cells in a dose- and time-dependent manner. No induction was seen when fragments of the MVDP promoter region were cloned directly in front of the CAT gene and transiently transfected into vas deferens epithelial cells. It was found that cotransfection of cells with MVDP-CAT constructs and with an androgen receptor expression vector resulted in a small but consistent androgen-dependent increase in reporter gene activity. Transiently transfected vas deferens epithelial cells are a suitable model with which to study the effect of androgen on gene regulatory elements.

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