Method for the Molecular and Quantitative Identification of Oocytes and Their Developmental Stage in Teleost Fish Gonads

Abstract

Fish display diverse reproductive strategies and their gametogenesis is influenced by numerous genetic, physiological and environmental factors. The analysis of 5S rRNA expression levels in gonads has been proposed as useful method for the molecular identification of the presence of oocytes in fish tissues. The present method provides an easy and unbiased approach to analyse the expression of tRNAs and 5S RNA in teleost gonads and stablish the presence and developmental stage of oocytes. Total RNA extracted from gonads is analysed through capillary electrophoresis in a Bioanalyzer 2100 (Agilent Technologies) using Small RNA Assays. Electropherograms allow quantifying the concentrations of tRNAs, 5S rRNA and 5.8S rRNA per sample and calculate their tRNA/5.8S rRNA and 5S/5.8S rRNA indices. Both indices clearly differentiate ovaries from testes and can be used to identify testes that present oocytes due to exposure to environmental xenoestrogens. The tRNA/5.8S and 5S/5.8S indices show the highest values in ovaries in previtellogenic stage, values decreasing as they advance towards maturity.Detailed molecular method to sex fish and quantitatively identify the maturity stage of femalestRNA levels in gonads can help in the study of teleost reproduction (female fecundity assessment, molecular gonad sexing) and environmental health assessment

Keywords: tRNA, 5S rRNA, tRNA/5.8S rRNA index, fish gonads, intersex, oocyte, fish sexing, ovary staging