Unknown,Transcriptomics,Genomics,Proteomics

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Fsh controls gene expression in fish both independently of and through steroid mediation

ABSTRACT: We recently demonstrated that Fsh modifies in vitro the testicular transcriptome of rainbow trout at early stages of spermatogenesis. Some of the regulated genes were found related to pathways highly relevant for the testicular functions i.e. spermatogenesis and steroidogenesis. Unlike in mammals, in fish both gonadotropins are able to efficiently stimulate the steroidogenesis, likely through a direct interaction with their cognate receptors present on the Leydig cells. In this context, we wondered whether the effects of Fsh on the tubular compartment were mediated through the production of steroids. To address this issue we performed in vitro incubations of testis explants in the presence of Fsh alone or in combination with an inhibitor of the steroidogenesis, the trilostane. Trilostane significantly reduced or suppressed the response to Fsh of many genes showing that important aspects of the Fsh action on fish testis is indirect and requires the production of steroids. Interestingly, most of the genes regulated in response to Fsh through the steroid mediation were similarly regulated by Lh and many were also found regulated by androgen treatment. On the other hand, for many other genes the response to Fsh was not affected in the presence of trilostane. A majority of those genes were also preferentially regulated by Fsh, as compared to Lh, suggesting that specific regulatory effects of Fsh were independent of steroid production. Finally antagonistic effects between Fsh and steroids were found, in particular for genes encoding clue factors of steroidogenesis or for genes of the Igf system that tended to be inhibited by androgens. Testes were collected from all-male population rainbow trout at early stages of spermatogenesis.Testes were chopped in small pieces, in sterile conditions and then pooled and mixed. Testis fragments were randomly distributed (60-80 mg per well) on Nunc polycarbonate membrane inserts in 24-well plates filled with 300 ÂµL of modified L15 culture medium supplemented with 2% Ultroser SF. Incubation were performed in six replicates for 96 hours, at 12Â°C, in the absence or presence of purified trout Fsh alone (500 ng/mL) or in combination with 10 Âµg/mL trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase. A pre-incubation with trilostane was done for 1 hour before adding Fsh.

ORGANISM(S): Oncorhynchus mykiss

SUBMITTER: Elisabeth Sambroni

PROVIDER: E-GEOD-46458 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear, and a FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3- mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development. Experiment Overall Design: Immature rat granulosa cells were affected with either control (GFP) or FOXO1 mutant adenovirus (FOXOA3 and FOXOA3-mDBD) for 4 hours. Then the cells were treated with either FSH or vehicle for 24 hours. The gene expression profiles for the four treatment samples (control, control +FSH, FOXOA3 + FSH, FOXOA3-mDBD + FSH) were compared using Rat Genome 230.2 Arrays with one array per sample.

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Fsh controls gene expression in fish both independently of and through steroid mediation

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