Transcription of ribogenesis genes in fish gonads: Applications in the identification of stages of oogenesis and in environmental monitoring of intersex condition

Transcription of ribogenesis genes in fish gonads: Applications in the identification of stages of oogenesis and in environmental monitoring of intersex condition Iratxe Rojo-Bartolomé, Ainara Valencia, Ibon Cancio* CBET Research Group, Dept. of Zoology and Animal Cell Biology, Research Centre for Experimental Marine Biology and Biotechnology of Plentzia (PiE-UPV/EHU), University of the Basque Country (UPV/EHU), Areatza, z/g, E-48620 Plentzia, Basque Country, Spain Corresponding author: [email protected] ABSTRACT One of the best described effects of environmental xenoestrogens in fish is the generation of intersex gonads in males. Considering 5S rRNA a marker of the presence of oocytes, a 5S/18S rRNA index was calculated in 296 thicklip grey mullets (Chelon labrosus) from polluted environments. In addition, qPCR analysis of transcription factors gtf3a and ubtf1, related respectively to 5S and 18S rRNA synthesis, was conducted along female-oogenesis. 5S/18S rRNA index identified sex with a threshold value of 0.4521 separating males from females. Histological analysis identified 38 intersex individuals. Intersex severity and 5S/18S rRNA indexes were correlated. 5S/18S rRNA index identified ovarian developmental stage with high 5S rRNA levels during early oogenesis and 18S rRNA relative values increasing towards maturation. gtf3a and ubtf1 transcription levels followed the pattern of 5S rRNA accumulation. Thus, ribogenesis genes provide easy/quantitative methods to molecularly identify the sex, female gametogenic stage and intersex severity in mullets. INTRODUCTION Organisms inhabiting estuarine waters are normally exposed to complex contaminant cocktails with anthropic origin (Boehm and Bischel, 2011). Among other contaminants detected in the aquatic environments, endocrine disrupting chemicals (EDCs) have received special attention after they were first highlighted by the European Environmental Agency in 1997 (EEA, 1997). EDCs were then defined as “chemical pollutants able to interfere with the normal functioning of hormones” (EEA, 2012) and they can cause alterations at different biological organisation levels, from the molecular one to the individual or the (sub)population one (Brander, 2013, WHO/UNEP, 2013). They include a complex array of substances, with different chemical structures and sources (Tyler et al., 1998, López de Alda and Barceló, 2001, Porte et al., 2006, Casals-Casas and Desvergne, 2011, Khetan, 2014) which in industrialized countries mainly arrive to the aquatic environment through the municipal, industrial and hospital wastewater treatment plants effluents (Campbell et al., 2006). Such compounds in complex mixtures, can interact enhancing their potency and biological activity, and in some circumstances, acting in an additive manner (Thorpe et al., 2003). Xenoestrogens are considered EDCs with the ability to mimic estrogens or to cause estrogen-like responses in exposed organisms (Campbell et al., 2006). They alter hormonal homeostasis interfering with normal sexual differentiation and gametogenesis, which in consequence affects the development and reproduction of exposed individuals/populations (Tyler et al., 1998, Goksøyr et al., 2003, Mills and Chichester, 2005). The effects of xenoestrogenic compounds in some aquatic This is the accepted manuscript of the article that appeared in final form in Marine Pollution Bulletin 121(1/2) : 292-301 (2017), which has been published in final form at https://doi.org/10.1016/j.marpolbul.2017.06.027. ©2017 Elsevier under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) organisms are well known, one of the best described responses being the feminization of juvenile and male fish (WHO/UNEP, 2013, Tyler and Jobling, 2008, Goksøyr et al., 2003, Goksøyr, 2006, Bizarro et al., 2014). Sometimes this leads to the generation of intersex gonads, when oocytes differentiate within the normal testicular tissue in gonochoristic fish species (Matthiessen, 2003, Bahamonde et al., 2013, Bizarro et al., 2014, Ortiz-Zarragoitia et al., 2014). Intersex condition has been reported in both freshwater and marine fish related to chemical exposure in highly to moderately contaminated areas (Bahamonde et al., 2013, Ortiz-Zarragoitia et al., 2014). These intersex males display lower reproduction capacity than normal males, with logical consequences in population viability (Jobling et al., 2002b, Harris et al., 2011). Xenoestrogenic effects have been reported also in mugilid fish populations from contaminated estuaries (Ferreira et al., 2004) and intersex gonads have been described in thicklip grey mullets (Chelon labrosus) used as pollution sentinel organisms in estuaries in the Southern Bay of Biscay (Diaz de Cerio et al., 2012, Puy-Azurmendi et al., 2013, Bizarro et al., 2014, Valencia et al., 2017). Oocytes have been found in testes of mullets from Bilbao, Pasaia and Ondarroa harbours, and in estuaries in Deba and in the Biosphere Reserve of Urdaibai in Gernika (Puy-Azurmendi et al., 2013, Bizarro et al., 2014, Valencia et al., 2017). These sites have been associated to waste water treatment plant effluents and/or to industrial activities in harbour areas. Intersex mullets have been described all along the reproductive cycle, with percentages ranging from 3% to 60% of analyzed males depending on the site and month of capture (Ortiz-Zarragoitia et al., 2014). Most intersex mullets present low to moderate intersex severity indexes following the score developed by Jobling et al. (2006) that ranks the severity according to the number and distribution of oocytes presented in the testis. Furthermore, elevated vitellogenin transcript and protein levels have been measured in these males, accompanied by an up-regulation of the aromatase coding gene cyp19a1b in brain (Bizarro et al., 2014). Thus, thicklip grey mullet is considered an important sentinel species for the biomonitoring of exposure to reproduction EDCs in the Southern Bay of Biscay (Ortiz-Zarragoitia et al., 2014). In spite of the increasing number of studies describing the intersex condition in fish in the last decades, the physiological and molecular mechanisms governing the process remain unknown (Abdel-Moneim et al., 2015). In addition, and although it is known that the transcription levels of several genes related to sex differentiation are altered after exposure to xenoestrogens (Bahamonde et al., 2015b), no direct relationship with the intersex condition has been established yet and more research is needed in order to find specific molecular markers of this condition (Bahamonde et al., 2013, Ortiz-Zarragoitia et al., 2014, Abdel-Moneim et al., 2015). In this respect, recently, accumulation of 5S rRNA and of transcripts coding for 5S rRNA accompanying proteins have been studied in mullets from polluted areas (Diaz de Cerio et al., 2012, Valencia et al., 2017). The transcription levels of ribogenesis genes enabled the identification of sex of each individual in a comparative manner, when at least one individual of each sex is present in the study, irrespective of their site of collection and their stage within the reproductive cycle. In addition, such genes were up-regulated in intersex testis in comparison to normal testis. Strong 5S rRNA transcription, specific of ovaries, can be easily identified by a simple electrophoresis of total RNA extracted from the gonads (Diaz de Cerio et al., 2012). The relative amount of 5S to 18S rRNA calculated after electrophoretic analysis (5S/18S rRNA index) identifies the presence of oocytes in gonads and allows distinguishing not only the sex but also the oogenic stage in females, as demonstrated in different commercial fish species of the Bay of Biscay (RojoBartolomé et al., 2016). This is so because 5S rRNA levels relative to 18S rRNA are higher in previtellogenic oocytes than in mature ones (Rojo-Bartolomé et al., 2016). In this context, the 5S/18S rRNA index was applied in the present study for the unambiguous molecular identification of sex, female reproductive stage and intersex severity in mullets collected from polluted sites of the Southern Bay of Biscay during a complete annual reproductive cycle. In addition, transcription levels of the general transcription factor IIIA (gtf3a) and upstream binding transcription factor 1 (ubtf1), genes related to ribogenesis through regulation of 5S and 18S rRNA synthesis, were studied in ovaries of females at different developmental stages. Such analysis could elucidate the possibility of using transcription levels of ribogenesis genes in an environmental monitoring context to assess the impact of xenoestrogenic compounds and the incidence of intersex condition in pollution sentinel fish species. MATERIAL AND METHODS Study area and biological samples From September 2010 to September 2011 twelve to thirty adult (> 20 cm length) thicklip grey mullets (Chelon labrosus) were monthly collected by fishing-rod in the harbour of Pasaia (43°19′35″N, 1°55′9″W), located on the Basque coast (SE Bay of Biscay). Mullets were also sampled in June 2013 and in February 2014 in the estuaries of Gernika (43°19′26″N, 2°40′26″W) and Galindo (43°18′11″N, 2°59′55″W) close the points of discharge of the waste water treatment plants of Gernika and of the Bilbao metropolitan area. The total amount of individuals collected for this study was of 296. All animal manipulations conducted were authorised by competent regional authorities after the evaluation and approval of all protocols by the Ethics and Animal Welfare Commission of the University of the Basque Country (CEBA/152/2010). After capture, individuals were immediately anaesthetized in a saturated ethyl 4-aminobenzoate, sacrificed by decapitation and gonads were removed. A portion of each gonad was embedded in RNA later (Ambion, Life Technologies, Carlsbard, USA), frozen in liquid nitrogen and then stored in the laboratory at − 80 °C until further used. For histological analysis, a portion was taken from the middle part of the gonad from each fish (around 1 cm in length across the whole gonad). This was then fixed in 4% neutral buffered formalin and transported at 4 °C to the laboratory for further processing. Histological analysis of the gonads After 24 h in the fixative, gonads were dehydrated in a graded series of ethanol (70%, 90% and 96%) and embedded in methacrylate resin according to the manufacturer's instructions (Technovit 7100; Heraeus Kulzer GmbH & Co. KG, Werheim, Germany). 6 to 9 resin sections (5 μm) were cut in a 2065 Supercut microtome (Leica Instruments GmbH, Wetzlar, Germany) and stained with hematoxylin/eosin. Sex and the developmental stage of the oocytes in ovaries were determined microscopically, following the gametogenic stage grading described by McDonough et al. (2005) for Mugil cephalus. The stages were as follows. Resting (R), with the presence of atretic oocytes (> 20%) in an otherwise empty ovary with scarce oogonia and lamellae presenting some muscle and connective tissue bundles while ovarian wall look thickened. Perinucleolar (Pn), with inactive ovary containing perinucleolar oocytes and a very thin ovarian wall. Cortical alveoli (Ca), with oocytes at cortical alveoli stage some of them starting vitellogenesis (< 50%). Vitellogenesis (V), with oocytes full of yolk globules and enlarged plasma membrane. Mature/spawning (M), with hydrated oocytes showing coalescence of lipid droplets and very thick oocyte envelope, and presence of some atretic oocytes (< 20%). No full mature or spawning individuals could be identified as spawning occurs in the open sea (Ortiz-Zarragoitia et al., 2014) and samplings were always carried out in estuarine areas. In the case of identified intersex individuals, the intersex severity was established microscopically. For that, up to 9 non-consecutive 5 μm histological sections were completely examined with a 20X objective, dividing each section in several fields of view. To determine the number of oocytes in each histological section, the field of view with the maximum oocyte amount recorded was considered, following the methodology described by Blazer et al. (2007). Then, the intersex severity for each fish was established depending on the mean number of oocytes within all histological sections analyzed per individual and following the index developed by Jobling et al. (2006). Extraction of total RNA, capillary electrophoresis and 5S/18S rRNA index Total RNA was extracted from 50 to 100 mg of tissue using TRIzol® (Invitrogen, Life Technologies) and following the manufacturer's instructions. Obtained RNA was purified using Qiagen RNeasy kit (Qiagen, California, USA) after a DNase digestion step (RNase-Free DNase Set, Qiagen). After purification, the same amount of RNA (250–500 ng), as estimated through absorbance at 260 nm (good quality RNA established at absorbance ratios of 260/280 and 260/230 around 2), was loaded in an Agilent RNA 6000 Nano Kit Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Electropherograms provided by the Bioanalyzer were used to quantify the concentration of the bands corresponding to 5S rRNA and 18S rRNA in each sample using the “Time Corrected Area” of each peak to calculate the 5S/18S rRNA ratio (Rojo-Bartolomé et al., 2016). When the presence of one of the rRNAs was below the levels of detection of the machine a 0.1 value was given to each sample instead of 0 (the lowest recordable value was 0.2). The binary logarithm of the ratio was calculated in order to develop a 5S/18S rRNA index that allowed clear visualization of the differences between testes and ovaries. cDNA synthesis and PCR analysis in ovaries 2 μg of total RNA from each individual gonad were used for cDNA synthesis with the AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies) using random primers, according to manufacturer's instructions. 5S rRNA is a very small transcript and random primers may not adequately retrotranscribe such small nucleic acids. For that reason, and for comparative purposes, an additional retrotranscription assay was performed with RNA from ovaries with perinucleolar (Pn) and vitellogenic (V) oocytes using a reverse primer specific for mullet 5S rRNA (5′-AAGCTTACAGCACCTG-3′). Primers to obtain mullet 45S pre-rRNA sequence fragments were designed through alignment (Clustal Omega, http://www.ebi.ac.uk/Tools/msa/clustalo/) of piscine 45S pre-rRNA sequences available in NCBI (http://www.ncbi.nlm.nih.gov/) and searching for highly conserved nucleotide regions. Properties of designed primers were checked employing the IDT OligoAnalyzer Tool (https://eu.idtdna.com/calc/analyzer). Conventional PCRs were run with 0.8 nM of forward 5′- GAGGCCCTGTAATTGGAATGAG-3′, reverse1 5′-CAAAGTGCGTTCGAAGTGTCGA-3′ or reverse2 5′- AGAGAAGGCGCGAGGACAC-3′ primers. The amplification was run with commercial Taq DNA Polymerase, recombinant Kit and 100 mM dNTP Mix (Invitrogen) for 35 cycles in a 2720 Thermal Cycler (Applied Biosystems, Carlsbard, California, USA). PCR procedure was as follows: 94 °C for 2 min, and 35 cycles with a denaturation step at 94 °C for 30 s, annealing at 61 °C for 30 s and elongation at 72 °C for 30 s. A final step at 72 °C was added for 8 min. Obtained PCR products were sequenced in the SGIker Sequencing Service of the University of Basque Country. Once sequenced, fragments were aligned to obtain a consensus sequence and analyzed using CAP3 (http://pbil.univlyon1.fr/cap3.php) and ClustalW2 tools. Sequences obtained have been published in GenBank (KX358060, KX358061). Specific primers were designed to amplify the fragment ranging from the 18S rRNA 3′-region into the internal transcribed spacer 1 and reaching the 5.8S rRNA 5′-region in cDNA generated from RNA extracted from Pn and V ovaries. This region suffers 45S pre-rRNA internal splicing leading to the generation of the mature 18S rRNA. Thus, the amplification of the sequence in this case is only possible when 18S rRNA and 5.8S rRNA remain together. The fragment was amplified using conventional PCR and employing 0.8 mM of forward 5′-AACCTCAGTGCGTGGCGGA-3′ and reverse 5′-TCCACCGCTAAGAGTAGTCATG-3′ primers. Amplification was performed as described above (annealing temperature 61 °C). Finally, PCR products were analyzed by electrophoresis in ethidium bromide stained agarose gels (1.5%). qPCR analyses in ovaries cDNA obtained by random and specific retrotranscription from ovaries at different developmental stages was quantified in the Synergy HT Multi-Made Microplate Reader (BioTek, Winooski, USA) by Quant-itTM OliGreen® stain (Invitrogen). The quantification was performed in a reaction volume of 100 μl with a theoretical cDNA concentration range of 0.02–0.2 ng/μl, at 485/20 nm excitation and 528/20 nm emission wavelengths. Real PCR input cDNA concentration was calculated using the high-range standard curve according to manufacturer's instructions. 5S and 18S rRNA, gtf3a and ubtf1 transcript levels were quantified (only in ovaries) by SYBR Green® qPCR (Roche, Basel, Switzerland). qPCR was conducted in triplicates using a 7300 Applied Biosystems Thermocycler. The PCR reaction mixtures (20 μl) consisted of 10 μl of 2 × SYBR® Green PCR master mix, appropriate concentration of primers diluted in RNase-free water (see Table 1) and 2 μl cDNA template. qPCR procedure was as follows: 50 °C for 2 min, 95 °C for 10 min, then 40 cycles consisting of a denaturation step at95°C for 15 s and an annealing step for 30 s (temperature for each primer set in Table 1). A dissociation step was added at the end consisting of 15 s at 95 °C, 1 min at annealing temperature and a final step at 95 °C for 15 s. Table 1. Primer sequences used for the qPCR analysis of the transcription levels of 5S rRNA, 18S rRNA, gtf3a and ubtf1 in ovaries of thicklip grey mullets. Gene Forward sequence (5′3′) Reverse sequence (5′3′) Primer (nM) Sample dilution Annealing T (°C) 5S rRNAa Fw-CTTACGGCCATACCACCCTG Rv-GTATTCCCAGGCGGTCTCCC 6.25 1/200 & 1/12800b 60 18S rRNA Fw-GAGGCCCTGTAATTGGAATGAG Rv-TAAGATACGCTATTGGAGCTGGAA 6.25 1/12800 60 gtf3a Fw-CCAGGAGAAGCGATATAAATGTGA Rv-TCGTGATGCTTCAGTTTTCCATG 12.5 1/400 59 ubtf1 Fw-CTCTAAAGCAAAGGTCAGTCCAGA Rv-AATATGAACATGGCTGAGATGGGC 12.5 1/400 60 A Protected under Spanish patent P201130778. B Dilution for cDNA obtained from 5S rRNA specific retrotranscription. All amplification results were normalized with the amount of cDNA charged in the qPCR according to Rojo-Bartolomé et al. (2016) using an adapted ΔCT formula (RQ) with efficiency correction (E): m being the slope of the standard curve of the qPCR reaction. where ∆ CT = CT sample − CT plate internal control. 5S vs. 18S rRNA transcription level indexes were calculated using qPCR results obtained for 5S rRNA from cDNA produced using both random or 5S rRNA specific primers. Statistical analysis To assess whether female and male individuals could be segregated into two statistically different groups according to their 5S/18S rRNA index the R 3.3.1 (2016) software version was used. To generate the clusters a logistic regression was carried out using the balance between specificity and sensitivity. Then, a maximum Area Under the Curve (AUC) was calculated using the “pROC” package version 1.8 available for R (AUC = 0.996). Finally, the 5S/18S index cutpoint value differentiating males and females was obtained. The statistical analyses of the rest of the results were undertaken using SPSS (SPSS Inc., Chicago, Illinois). Data failed in normality and variance equality after applying the Shapiro-Wilk (n < 30) test and Levene's test, both at a 0.05 significance level (p < 0.05). Significant differences between groups were then evaluated using the nonparametric Kruskal-Wallis test. When only two groups were compared (Pn vs. V), data analysis was performed with the non-parametric Mann-Whitney test for two independent samples. In all the cases, significant differences were established at p < 0.05. In addition, Spearman unilateral correlation analysis was performed to compare the histological intersex severity index and the 5S/18S rRNA index. Spearman significant differences were established at p < 0.01. RESULTS 5S/18S rRNA index: identification of sex 5S/18S rRNA index was used to identify the sex of thicklip grey mullet individuals studied during a whole annual cycle (Fig. 1). Of all analyzed individuals (296) histological analysis was only possible in 215 (methacrylate blocks of 81 samples were lost) which were histologically sexed (colored dots in Fig. 1), identifying 91 females, 86 males and 38 intersex individuals. Then two population clusters were distinguished based on their 5S/18S rRNA index values with a cut point value of 0.4251 (Fig. S1). When the 177 histologically sexed males and females were classified using this cluster distribution 95% of females and 100% of males were correctly identified. No male individual showed a 5S/18S rRNA index value higher than the cut point and only two males out of 86 did not show negative values (one in February and another one in June). The majority of the females (86 from 91) showed an index value higher than the cut point, with the exception of only one female in November and four in January. Sample distribution revealed that while all male individuals displayed a narrow normal distribution with an index peak around − 3.0, two populations could be distinguished within females with peaks around 5.0 and 12.0 (Supplementary Fig. S1). Fig. 1. 5S/18S rRNA in gonads of thicklip grey mullets collected during a complete annual cycle. Each red dot corresponds to one histologically identified female individual (n = 91) and each blue dot to a male (n = 86). Each black line corresponds to an individual whose sex was not identified histologically (n = 81). Box in pink groups most of female individuals, and the blue box encompasses 100% of the males. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) This sex distribution was used to classify a total of 81 individuals not sexed histologically (black lines in Fig. 1, Supplementary Fig. S2). 35 individuals displayed a 5S/18S rRNA index value above 0.4251 and thus their probability to be females was 100%. Individuals (46 out of 81) with an index value lower than 0.4251 were males with a 94.5% probability. This applies in the case that none of the 81 individuals were intersex. 5S/18S rRNA index and intersex severity A total of 38 intersex individuals, histologically identified during our samplings in Pasaia, Galindo and Gernika, were analyzed in the present study. They all displayed low to moderate intersex severity (severity indexes 1 to 3) according to the ranking methodology used (Jobling et al., 2006, Blazer et al., 2007). When comparing 5S/18S rRNA index from these intersex individuals and their histologically ranked intersex severity index, a positive correlation (Spearman p < 0.01) with a magnitude of 0.638 was observed between both indexes. As the amount of oocytes in the testes increased, the 5S/18S rRNA index increased (Fig. 2). The lowest 5S/18S rRNA index values were recorded with intersex index values of 1, where testis presented from 1 to 5 oocytes scattered within each testis section, and the highest at intersex index 3, with clusters of 21 to 50 oocytes in each testis section. When comparing intersex individuals with males and females, intersex individuals with lower severity indexes did not show significant differences in 5S/18S rRNA index values with males, while individuals with severity index 3 showed 5S/18S rRNA index values similar to females and different from males (Fig. 2, Supplementary Fig. S2). Fig. 2. Comparison between the 5S/18S rRNA index as calculated from the electropherograms and the histologically calculated intersex severity index in thicklip grey mullets. Box plots represent the data within the 25th and 75th percentiles, with the median indicated by a line, and top and bottom whiskers indicating the minimum and maximum values. Different letters indicate significant differences between groups (Kruskal-Wallis, p < 0.05). M: male, I.I.: intersex severity index, F: female. I.I. ranked from 1 to 3 following the description proposed by Jobling et al. in 2006. The total amount of individuals in each group were; 86 in M, 22 in I.I.1, 7 in I.I.2, 9 in I.I.3 and 91 in F (total n = 215). 5S/18S rRNA index: identification of female reproductive stage 5S and 18S rRNA relative fluctuations allowed distinguishing ovaries at different developmental stages (Fig. 3). At early stages during oogenesis (Pn and Ca) 5S rRNA levels were very high in comparison with 18S rRNA, but as oogenesis advanced (from Pn to V), the relative amount of 5S rRNA decreased and the 18S rRNA relative amount increased in electropherograms. In consequence, the 5S/18S rRNA index that was high at the beginning of the oogenesis (Pn = 6.11 and Ca = 7.69), decreased as oogenesis advanced; the lowest index values being recorded at vitellogenesis (V = 0.90). Values increased again at regressing stage (R = 4.59). Fig. 3. Variations in the relative amount of 5S rRNA, 18S rRNA and 5S/18S index during thicklip grey mullet oogenesis. Representative micrographs of ovaries within each of the developmental stages analyzed are shown (scale bar = 200 nm): R: resting, Pn: perinucleolar, Ca: cortical alveoli, EV: early vitellogenesis and V: vitellogenesis. No individuals with mature hydrated ovaries (M) were available as mullets were never sampled at open sea. The value above each micrograph depicts the mean 5S/18S rRNA index value at each of the analyzed stages. In the total RNA electropherograms (representative of individuals in each stage) the peak at 25 s corresponds to 5S rRNA and the one at 40–45 s corresponds to 18S rRNA. The last peak corresponds to 28S rRNA. [FU]: fluorescence, [s]: time in seconds. To prove that we were really measuring 5S and 18S rRNA levels in the electropherograms and not anything else as it could be argued by somebody, specific rRNA transcription levels were analyzed through qPCR (Fig. 4). Comparison of the 5S/18S rRNA indexes obtained from Bioanalyzer electropherograms (Fig. 4A) and after qPCR analysis (Fig. 4B) showed identical differences between groups. Anyhow, it was noticeable that 5S/18S rRNA index values calculated with qPCR analyses were always negative, this meaning that qPCR always identified higher levels of 18S rRNA than of 5S rRNA, also in Pn ovaries. In order to clarify this inconsistency with the Bioanalyzer results, 5 Pn ovaries and 5 V ovaries were randomly chosen and retrotranscription was carried out using 5S rRNA specific primers followed by a qPCR analysis of 5S rRNA transcription levels. 5S/18S rRNA index after specific 5S rRNA retrotranscription (Fig. 5C) showed the same relative profile obtained with the total cDNA (Pn > V) (Fig. 5B), but displaying positive values, as it happened with the index obtained from the electropherograms (Fig. 5A). CONTRIBUTIONS OF AUTHORS IR conducted the laboratory measurements and drafted the manuscript. AV conducted most of the samplings and participated in histological analysis of the samples. IC designed and supervised the study directing analysis of results and the writing process. ACKNOWLEDGEMENTS Funded through projects of the University of the Basque Country (UFI 11/3), Basque Government (S-PEN13UN101 and IT810-13) and the Spanish MINECO + European ERDF Funds (AGL2012-33477 and AGL2015-63936-R). IR and AV are recipient of a grant for predoctoral studies of the Basque Government. We thank the technical support of the SGiker Service (UPV/EHU) and Prof. Inma Arostegui and Dr. Irantzu Barrio of the Department of Applied Mathematics and Statistics and Operational Research of UPV/EHU for their help with the R software statistical analysis. REFERENCES A. Abdel-Moneim, D.P. Coulter, C.T. Mahapatraa, M.S. Sepúlveda. Intersex in fishes and amphibians: population implications, prevalence, mechanisms and molecular biomarkers. Appl. Toxicol., 35 (2015), pp. 1228-1240 G.T. Ankley, D.C. Bencic, M.S. Breen, T.W. Collette, R.B. Conolly, N.D. Denslow, S.W. Edwards, D.R. Ekman, N. Garcia-Reyero, K.M. Jensen, J.M. Lazorchak, D. Martinovic, D.H. Miller, E.J. Perkins, E.F. 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