Experimental data-sets on sex reversal and histopathological assessment of potential endocrine disrupting effects of graphene oxide on Japanese medaka (Oryzias latipes) larvae at the onset of maturity

Abstract

The datasets of this article present the experimental parameters resulting from the assessment of sex reversal (SR) as a biomarker of endocrine disrupting effect of graphene oxide (GO), together with the histopathological assessment of ovary, testis, liver and kidneys of medaka larvae. These data sets support the published article “Sex-reversal and histopathological assessment of potential endocrine-disrupting effect of graphene oxide on Japanese medaka (Oryzias larvae) larvae.” The experiments were conducted on one day-post hatch (dph) Japanese medaka fries (orange-red variety) exposed to different concentrations of GO (2.5–20 mg/L) by immersion in embryo-rearing medium (ERM) for 96 h under laboratory conditions (25 ± 1 °C; light cycle 16 h light: 8 h dark). No food was given during the GO-exposure period. Controls (no GO) were identically maintained in ERM. After treatment, the larvae were maintained in balanced salt solution (BSS) with feeding and allowed to grow for 6 more weeks in a GO-free environment. On 47 dph, the larvae were anesthetized in MS-222, and the total length (mm) and body weight (mg) were recorded. For histopathological and phenotypic sex assessments, after sacrifice, the body excluding post-anal tail was preserved in 4% paraformaldehyde containing 0.05% Tween 20; ovary, testis, liver and kidneys were evaluated in 5 µm thick sections stained on haematoxylin eosin (HE) following OECD guidelines. The photomicrographs of sections were made using either an Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. A minimum 3 images of gonads in different regions were further analysed by imagej software and used for counting spermatogonia (SPG) and spermatocytes (SPT) in testis as well as perinucleolar (PNO) and cortical alveolar (CAO) oocytes in ovary. Data were expressed as number of SPG or SPT/mm2 testis and % CAO or PNO in an ovary. Preserved tail in TRI reagent was used for genomic DNA extraction and the genetic sex was assessed by genotyping Y chromosome-specific male sex-determining gene dmy. Two different sets of buffers and primers were used and the reactions were conducted in a thermal cycler. The amplified products were separated in 2% agarose gel containing 0.01% ethidium bromide. The gels were viewed on an UV illuminator and the genotypes were identified by visual inspection. The first primer set amplified a 355 bp product for XY genotypes and no amplification for XX. The second set of primers amplified two products; one at 1249 bp and another at 986 bp for XY, and one product at 1249 bp for XX. Experimental data were expressed as means ± SD or SEM, analysed either by one-way analysis of variance (ANOVA) followed by post-hoc Tukey's multiple comparison test or unpaired parametric ‘t’ test including Welch's correction, if distributed normally (lengths and weights), or by Kruskal-Wallis test followed by Man-Whitney's test as post hoc test, if data (stromal follicles in ovary and SPGs and SPTs in testis) did not meet the criteria of using a parametric test. Statistically significant difference were considered for p ≤ 0.05.