Title: Identification and Transcriptional Modulation of the Largemouth Bass, Micropterus salmoides, Vitellogenin Receptor during Oocyte Development by Insulin and Sex Steroids 1 Short Title: Transcriptional Modulation of LMB Vtgr Summary sentence: The sex steroids 17β-estradiol and 11-ketotestosterone repress insulin- induced vitellogenin receptor mRNA expression in previtellogenic oocytes of largemouth bass.

Fish vitellogenin synthesized and released from the liver of oviparous animals is taken up into oocytes by the vitellogenin receptor. This is an essential process in providing nutrient yolk to developing embryos to ensure successful reproduction. Here we disclose the full length vtgr cDNA sequence for largemouth bass (LMB) that reveals greater than 90% sequence homology with other fish vtgr sequences. We classify LMB Vtgr as a member of the low density lipoprotein receptor superfamily based on conserved domains and categorize as the short variant which is devoid of the O-glycan segment. Phylogenetic analysis places LMB Vtgr sequence into a wellsupported monophyletic group of fish Vtgr. Real-time PCR showed greatest levels of LMB vtgr mRNA expression occurred in previtellogenic ovarian tissues. In addition, we reveal the effects of insulin, 17beta-estradiol (E2) and 11-ketotestosterone (11-KT) in modulation of vtgr, esr, and ar mRNAs in previtellogenic oocytes. Insulin increased vtgr expression levels in follicles ex vivo while exposure to E2 or 11-KT did not result in modulation of expression. However, both steroids were able to repress insulin-induced vtgr transcript levels. Co-exposures of insulin and E2 or insulin and 11-KT increased ovarian esr2b and ar mRNA levels, respectively, which suggest a role for these nuclear receptors in insulin-mediated signaling pathways. These data provide the first evidence for the ordered stage-specific expression of LMB vtgr during the normal reproductive process and the hormonal influence of insulin and sex steroids on controlling vtgr transcript levels in ovarian tissues.