The differential expression of two paralog factor IIIA genes in teleosts: gtf3ab as a marker of ovarian development

11th International Symposium on Reproductive Physiology of Fish (ISRPF-2018), New frontiers in reproductive diversity in a changing environment, 3-8 June 2018, Manaus, Brazil.-- 1 pageIntroduction. Stockpiling of molecules such as rRNAs in fish oocytes is essential to assist ribosomal assembly and protein synthesis in the newly formed embryo. In this way, the massive 5S rRNA expression during fish oogenesis serves as oocyte differentiation marker in the intersex testis formed after exposure to xenoestrogens. This 5S rRNA production is allowed by the general transcription factor IIIA (gtf3a) and as in Xenopusone single gtf3agene but two transcripts exist, one of them oocyte specific, we wanted to study the possible existence of paralog gtf3agenes in teleosts. Methods. Teleost genomes in ENSEMBL were analyzed to identify gtf3a orthologs. Two paralogs were identified so a synteny analysis was performed to understand the origin of the gene duplication event in teleosts. Then, transcription levels of gtf3aa and gtf3ab were analyzed by qPCR in tissues of adult zebrafish (D. rerio, ZF) and during the first 30 hours of embryo development. Transcription levels were also quantified in whole larvae (26 & 61 dpf), either masculinized or feminized after methyltestosterone (MT) and 17β-estradiol (E2) exposures, and compared to cyp19a1a, dmrt1 and amh levels. Finally, the promoter methylation level of both genes was studied in testis and ovary by bisulphite sequencing. Results and Discussion. Teleost genomes present two gtf3aparalog genes. Gtf3ab aroused from the teleost specific genome duplication event, with specific expression in ZF oocytes. Instead, gtf3aa is ubiquitously expressed in all tissues tested. No gonads were observable at 26 dpf in the ZF studied, exposed or not to E2 or MT, with no gtf3abtranscription but with detectable gtf3aalevels. 61 dpf E2 feminized ZF showed transcription of gtf3aband gtf3aain whole body analyses, while MT masculinized juveniles only transcribed gtf3aa. Female gtf3abtranscription coincided with that of ovarian cyp19a1aand opposite to that of amh and dmrt1. Maternal gtf3abtranscripts were present in zygote but disappeared after embryo genome activation. Opposite, the transcription of gtf3aabegan with the activation of the zygotic genome (~8 hpf). Bisulfite sequencing of the promoters of both gtf3agenes is currently ongoing. Conclusion. As 26 days exposure to E2 induced no gtf3abtranscription in whole juveniles, but transcripts were detected upon ovarian development at 61 dpf, we can consider that gtf3abtranscription is a consequence of oocyte production in fish and not a direct result of E2 exposure. Thus, gtf3abexpression constitutes a plausible marker of feminization in ZFFunded: Basque Gov. (IT810-13), UPV/EHU (UFI 11/37), Spanish MINECO & EU-FEDER/ERDF (AGL2015-63936-R & AGL2015-73864)Peer Reviewe