Temporal expression and transcriptional regulation of the vitellogenin receptor gene in largemouth bass (Micropterus salmoides)

Fish vitellogenin (VTG) synthesized and released from the liver of oviparous animals is taken up into oocytes, in part, by the vitellogenin receptor (VtgR). Some studies have focused on the functional role of VtgR in fish; however the transcriptional control of this gene has not been investigated. Depending on the signaling pathways involved, the VtgR gene may be a downstream target of endocrine disrupting compounds (EDCs). The objectives of this study are to identify and characterize the mRNA expression and regulation of the VtgR gene in largemouth bass (LMB) ovarian tissue. To begin this work, VtgR cDNA was amplified from female gonadal tissue using rapid amplification of cDNA ends (RACE). With this technique the VtgR cDNA was cloned and verified by BLAST which revealed greater than 90% similarity with other fish VtgR cDNA sequences. We then designed a quantitative realtime PCR (qRT-PCR) assay to measure the temporal expression of VtgR mRNA in gonadal tissue during the annual reproductive cycle. VtgR mRNA levels were most highly expressed (5x10 6 copies/μg RNA) in tissues containing predominantly pre-vitellogenic oocytes, whereas the levels considerably decrease when the majority of oocytes become vitellogenic. We compared this expression profile to those obtained in ovary tissue for the three estrogen receptors, ERα, ERβ2 and ERβ1. Similar to VtgR, all three ERs were highly expressed when pre-vitellogenic oocytes were most abundant. The levels of ERβ1 (5.5x106 copies/μg RNA) and ERβ2 (4.3x106 copies/μg RNA) were much more highly expressed compared to ERα (0.7x10 6 copies/μg RNA). Based on this data, we hypothesize the VtgR gene is regulated partially by LMB ERs in ovarian tissue, specifically ERα1 and ERβ2. To further investigate this notion we cloned a portion of the VtgR promoter using genome walking approaches. We tested the regulation of this promoter by ERs using a luciferase reporter gene assay.;Results from these experiments suggest ERs do not control the synthesis of VtgR, but other factors are predominantly involved. In addition to enhancing our basic understanding of the endocrine system in fish, this work will also uncover the potential of the VtgR as a target of EDCs.