5S rRNA PROFILING AND APPLICATION IN IDENTIFICATION OF SEX AND OVARIAN DEVELOPMENT STAGE IN TELEOSTS  

In the process of gametogenesis, oocytes and spermatocyte undergo significantly diverse molecular, cellular, and structural changes. One of these molecules involved considerably in oogenesis is 5S ribosome RNA (5S rRNA). The dynamic feature of oocyte 5S rRNA during oogenesis had been studied in some fish species in 1970s, but has been overlooked until recently. In the oocytes of some amphibian and teleost species, rDNA is amplified ≈1000-fold and 5S rRNA and transfer RNA (tRNA) constituted more than 90% of the RNA content approximately at the end of primary growth or early cortical alveolus stage, and decrease to normal level as the development of ovary. The dynamic feature of 5S rRNA in early stages of ovarian development could be served as a potential approach to determine sex and oogenesis stage for large amount of samples with comparably less input. Meanwhile, overwhelming accumulation of 5S rRNA also cause problems in RNA-seq analysis. Incomplete or incorrect depletion of these unwanted and problematic RNA for in-depth transcriptomics analyses, can lead to insufficient coverage of transcripts of interest, and result in misassembly, incorrect quantification of gene expression and inaccurate conclusion. As far as we know, only a small proportion of scientists have realized the massive unwanted 5s rRNA in developing/resting ovaries of fish and anurans, and subsequent problem in transcriptomics analysis. There is no commercial kit targeting at this issue yet. The objectives of the present study are to: 1) evaluate the application of 5s RNA profiling in accurate sex identification and oogenesis stage determination in fish with asynchronous or synchronous ovaries; 2) alert researchers who are working on ovary transcriptomics in fish and anurans that incorrect depletion of 5s rRNA may lead to misassembly and incorrect quantification of gene expression.

Asynchronous ovaries (bluegill and largemouth bass) and synchronous ovaries (yellow perch and channel catfish) as well as testicular tissues were collected. One part of gonad was embedded in RNAlater® for RNA isolation and the other part was fixed in Prefer for histological analysis. Livers and brains were also collected in some samples as a somatic tissue control. Isolated RNA samples were evaluated by Nanodrop 1000 and the same amount of RNA was loaded in an Agilent RNA 6000 Nano Kit Bioanalyzer for capillary electrophoresis. Generated data were analyzed using Agilent 2100 Expert. Time corrected area of each peak in electrophoresis was used to calculate the 5S/18S rRNA ratio and establish a correlation with ovary development. Our data demonstrate that 5S/18S rRNA ratio could be served as an index to differentiate females from males in early development of gonads and determine ovary development stages, and a large sample size can be completed in a short time, with small amount of samples (e.g. 20-50 mg), and without laborious histology work. It is also worthy noting that the dynamic feature of 5S/18S rRNA ratio is species-specific.