[en] In the last decades, molecular methods are becoming increasingly relevant in the assessment of sex, sex differentiation and gametogenesis staging in teleost fish as they can provide more accurate estimates than histological or visual analysis of gonads. This is a requirement of the EU Data Collection Framework as part of the policy to improve fisheries management, where fecundity estimates of commercial fish stocks are of prime interest. In this study, we wanted to analyse the differential transcription distinguishing ovaries and testes in fish by studying the levels of transcription of amino acid-specific transfer RNAs (tRNA). Expression of tRNAs was hypothesised to be a lot higher in ovaries than in testes, as it has already been proved with the other major product of RNA polymerase III activity, 5S rRNA. The teleosts species selected for this study were the zebrafish, Danio rerio, as laboratory model species whose genome has been fully sequenced, and the thicklip grey mullet, Chelon labrosus, as pollution sentinel species that in the Basque coast has displayed cases of intersex with oocytes produced in testis due to exposure to xenoestrogens. Primers for PCR analysis were designed against the multiple genes displayed in the genome of zebrafish targeting specific amino acid tRNAs. Such primers allowed quantification of transcription levels of selenocysteine, suppressor, phenylalanine, tryptophan and alanine tRNAs showing higher transcription levels in testes than in ovaries. These results against expectations could be due to the different cross-amplification of primers across the more than 50 genes per analysed specific tRNA or due to tRNA structural properties that did not allow adequate PCR analysis. No amplification was obtained in the case of C. labrosus with the exception of suppressor tRNA. Small RNA capillary electrophoresis of total RNA, which allows identification of very small RNA fragments up to 200 bp in length, showed higher production of tRNAs, as well as of 5S rRNA, in the ovaries than in testes in both species. The calculated tRNA/5S rRNA index value showed no differences between gonads of both sexes but the tRNA/5.8S rRNA index clearly showed the highest levels in ovaries than in testes. These differences were only significant in the case of C. labrosus (analysed C. labrosus ovaries were all previtellogenic). This proves a high RNA polymerase III activity during the early phases of oogenesis in C. labrosus that results in high levels of tRNA and 5S rRNA. Conversely, RNA polymerase I activity and production of 5,8S, 18S and 28S rRNA is initiated with vitellogenesis which explains low levels in the studied mullet samples. In the meantime, zebrafish ovaries were showing oocytes in all stages of oogenesis and this did not allow significant differences in tRNA/5.8S rRNA index values between gonads. Bulk tRNA production has been proven to be a good marker to identify sex in teleost fish when comparing gonads in the early stages of gametogenesis. Similarly, the tRNA/5.8S rRNA index has shown to be a useful proxy to identify the oogenesis stage in fish ovaries. Further research is necessary to elucidate the usefulness of each specific tRNA in the monitoring and staging of ovarian gametogenesis in teleost fish.

[eu] Azken hamarkadetan, metodo molekularrak gero eta garrantzi handiagoa dute sexu desberdintzapen eta gametogenesiaren faseen identifikazioan arrain teleosteoetan, aurreikuspen zehatzagoak eskaini ahal baitituzte gonaden analisi histologiko edo bisualak baino. Hori EBko "Data Collection Framework" betebeharra da, arrantzaren kudeaketa hobetzeko politika aurrera eramateko, non arrain komertsialen emankortasunaren estimazioa interes handia duen. Ikerketa honetan arrainetan obarioak eta testikuluak bereizten dituen transkripzio diferentziala analizatu nahi genuen amino azido espezifiko transferentziako RNA (tRNA) transkripzio maila aztertuz. tRNAren adierazpena obarioetan testikuluetan baino askoz altuagoa izateko hipotesia zegoen, polimerasa III aktibitatearen beste produktu nagusiarekin, 5S rRNA, frogatu den bezala. Analisi honetarako aukeratutako teleosteo espezieak, hauek izan ziren: zebra arraina, Danio rerio, genoma guztiz sekuentziatuta duen laborategiko eredu espezie gisa, eta hondoetako korrokoia, Chelon labrosus, poluzioaren monitorizatzaile gisa, Euskal kostaldean intersex egoera aurkeztu baitu xenoestrogenoen ondorioz obozitoak ekoiztuz testikuluetan. PCR analisirako primerrak diseinatu ziren D. rerio genomako amino azido espezifiko tRNA geneak kontuan izanda. Primer horiek selenozisteina, supresorea, fenilalanina, triptofanoa eta alanina tRNA transkripzio mailak kuantifikatzea ahalbidetu zuten testikuloetan obarioetan baino transkripzio maila handiagoak erakutsiz. Hondoetako korrokoiaren kasuan ez zen anplifikaziorik lortu supresore tRNAan izan ezik. RNA totalaren RNA txikiko kapilare elektroforesia, 200bp-ko RNA kate txikien identifikazioa ahalbidetzen duena, 5S rRNA eta tRNA produkzio handiagoa erakutsi zuen obarioetan testikuloetan baino bi espezieetan. Kalkulatutako tRNA/5S rRNA indizearen balioak ez zuen ezberdintasunik erakutsi gonaden artean, baina, tRNA/5.8S rRNA indizeak maila altuagoa argi erakutsi zuen obarioetan testikuloetan baino. Ezberdintasun horiek soilik izan ziren esangarriak C. labrosus kasuan (aztertutako C. labrosus obario guztiak prebitelogenikoak ziren). Horrek egiaztatzen du RNA polymerasa IIIren aktibitate handia C. labrosus oogenesiaren fase goiztiarretan tRNA eta 5S rRNA sortzen duena. Alderantziz, RNA polimerasa I-ko aktibitatea eta 5,8S, 18S eta 28S RNAren ekoizpena vitellogenesiarekin batera hasten da, korrokoietan lortutako balio baxuak azaltzen duena. Bitartean, D. rerio obuluak fase oogenesi guztietako obozitoak erakutsi dituzte eta horrek ez du ahalbidetu ezberdintasun esangarririk gonaden arteko tRNA/5.8S rRNA indizean. tRNA produkzioa sexua identifikatzeko markatzaile ona dela frogatu da arrain teleosteoetan gonadak alderatzean gametogenesiaren fase goiztiarretan. Era berean, tRNA/5.8S rRNA indizea arrain obulutegietako oogenesi fasea identifikatzeko baliogarria dela erakutsi da. Ikerketa gehiago egin behar dira zehazteko tRNA espezifiko bakoitzaren erabilgarritasuna monitorizazioan eta arrain teleosteoen obulutegi gametogenesiaren faseen adierazpenean.