Fish Multigeneration Test Development at EPA

Transcription

1 Fish Multigeneration Test Development at EPA Rodney Johnson ORD National Health and Environmental Effects Research Laboratory Mid-Continent Ecology Division-Duluth Photo image area measures 2 H x 6.93 W and can be masked by a collage strip of one, two or three images. The photo image area is located 3.19 from left and 3.81 from top of page. Each image used in collage should be reduced or cropped to a maximum of 2 high, stroked with a 1.5 pt white frame and positioned edge-to-edge with accompanying images. Office of Research and Development National Health and Environmental Effects Research Lab, Mid-Continent Ecology Division, Duluth MN

2 Purpose of Multigeneration Fish Tests Test Development Develop datasets for developing efficient Tier II and short-term test protocols. Risk Assessment Evaluate transgenerational effects of EDCs determine if present if so, magnitude of effect Evaluate relationships between diagnostic endpoints of EDCs and adverse reproductive outcomes 1

3 Transgenerational Effects Definition: a between-generation increase or decrease in sensitivity of the test organism to the test agent Requirements for evaluation Same test conditions for each generation - chemical concentration - life-stage - endpoint 2

4 Positive Attributes of Medaka for Multigeneration Tests Simple ID of genetic sex of individuals Small aquaria and pair spawning optimize replication improve statistical power Short life-cycle 3

5 Medaka Female (XX) Male (XY) Complete life cycle ~ 10 weeks Small (adults ~0.3 to 0.5 grams) Sexually dimorphic (fins and body shape) Male sex-determination gene (DMY) identified Spawns daily (~25 to 35 eggs per spawn) Genome sequenced Gene arrays available Large historical literature database 4

6 Test protocol and data analysis based on genotypic sex (DMY) Select breeding pairs: 1 male (XY), 1 female (XX) Endpoints analyzed by sex genotype (XX) or (XY) Gonad histology phenotype (ovary or testis) Liver vitellogenin mrna phenotype (high female or low male) Secondary Sex: anal fin papillae phenotype (present in males, absent in females) 5

7 Medaka Multigeneration Test Timeline Exp Design 6 treatments 6 replicates/treatment Generation F0 F1 F2 Exp. Weeks Gamete exp Development Reproduction Development Reproduction Stage Objective Description Endpoints F0 Gen adult Evaluate F0 reproduction Gamete loading for F1 Start 5 wk exposure with 12 wk old breeding pairs Collect embryos 3 rd wk Incubate two spawns/pair (total n=40) Adults terminated after 5 wks Fecundity/fertility Growth Histopathology (optional) Embryos F1/F2 Assess embryo viability Start F1 and F2 generations Evaluate incubators for mortality Transfer hatchlings from incubator to aquarium Mortality Hatch Juvenile F1/F2 Evaluate ELS survival Setup development reps Randomly select and distribute 10 fish/tank Terminate extra fish Early life-stage survival Sub-adult F1/F2 Set up reproductive pairs Assess reproductive devel. Determine genotypic sex of all fish Randomly select 1 breeding pair/ replicate Evaluate remainder for effects on reproductive development (8/rep) Weight/length Gonadal sex (histology and molecular) 2 0 Sex char (anal fin papillae) Liver vitellogenin Adult F1 & F2 Assess reproduction Histopathology Pair breeding (4d/wk for 5 wks; n=20) Histopathology (n=12/treatment) gonad, liver, kidney Fecundity/fertility Growth Histopathology

8 Chemistry Requirements Consistent stock solutions without carrier solvents Saturator columns (solids) Liquid-liquid saturator Stir-plate methods Weekly measurement of chemical concentrations in exposure tanks in all treatments Maintain chemical concentration in exposure tanks across generations 7

9 Medaka Exposure System Safety Air flow control Spill containment Easy physical access spawning feeding cleaning embryo incubation 8

10 Medaka Exposure System Photoperiod: 16L- 8D Temperature: 24 C 48 aquaria (2 liter) 12 incubator aquaria (2-liter) 72 incubators Aeration for each incubator 9

11 Incubator Incubators in exposure tank 10 Procedure: 20 eggs/incubator (one spawn) 2 incubators/pair Eggs separated with screen Gentle aeration Dead eggs removed daily Aeration stopped day-7 Normal hatch ~ 95% Unhatched eggs discarded day-13

12 Fecundity Start 12 th week post-fertilization Collect Eggs Daily 4d/wk;5wks Remove from female and siphon tank Count and evaluate (dissecting scope) fecundity = eggs/pair/day fertility = fertile eggs/pair/day 11

13 DNA and RNA Isolation Methods DNA is isolated from fin clip (any tissue is suitable) Does not need to be destructive ; i.e. breeding pairs RNA is isolated from liver for Vtg analysis DNA and RNA isolation using commercially available kits Based upon selective binding of a silica-gel based membrane Provide high quality DNA and RNA without hazardous reagents Other methods available 12

14 Method Summary DMY Determination DMY present on Y chromosome of every male somatic cell Taqman assay, a variant of real-time PCR to detect DMY Provides higher throughput and better QC Requires two specific primers, a Taqman probe, and real-time thermocycler Included in reaction are primers/probe to detect 18S DNA Verifies the presence of DNA in the sample (positive control for every well) PCR followed by gel electrophoresis can also be used 13

15 Vitellogenin Quantification Method Summary Real-time quantification of Vtg mrna Uses two primers and a Taqman probe Forward primer: 5 AGG CAG TTT CTA AGG GCG AAC 3 Reverse primer: 5 TGA ATG GGC ATA ATC TTT GTG ATT 3 Probe: 5 FAM-CCA GTG CTT CAA ATG CGA GCA-BHQ 3 Obtain copies of Vtg mrna per ng total RNA Commercial ELISA Vtg Kits are available Real-time PCR has much larger dynamic range In the end, real-time PCR is cheaper and more efficient 14

16 Endpoint: Anal fin papillae Control male male phenotype Estrogen-exposed male female phenotype 15

17 Histopathology 16 Fix Davidson s Embed paraffin Step-section dorsal to ventral (transverse-oblique) Kidney: 12 µm intervals Gonad: males 600 µm, 9 50 µm intervals females 300 µm, 9 40 µm intervals Pathology evaluation Kidney and Liver Gonad Male: Testicular oocytes Interstitial cell hyperplasia/hypertrophy Hypoplasia Increased spermatogonia Mineralization Degeneration Female: Oocyte hyperplasia/hypoplasia Perifollicular cell hyperplasia-hypertrophy Oocyte atresia Decreased yolk formation

18 Endpoint: Sex reversal Phenotype: female (ovary) Genotype: female (XX) Phenotype: male (testis) Genotype: male (XY) Control Octylphenol 50 ppb Phenotype: female (ovary) Genotype: male (XY) Phenotype: female (ovary) Genotype: male (XY) 17 Breeding Adult F1 Octylphenol 100 ppb Juvenile F2 Octylphenol 50 ppb

19 Octylphenol Effects on Male Vitellogenin Production Vtg (mrna copies/ng total RNA) 1E+06 1E+06 8E+05 6E+05 4E+05 2E+05 0E-01 F0 Male Vitellogenin Mean±SD Mean±0.95 Conf. Interval Significant Male = Female = -2E Concentration ( g/l) 5E+05 F1 Male Vitellogenin 1E+06 F2 Male Vitellogenin Vtg (mrna copies/ng total RNA) 4E+05 3E+05 2E+05 1E+05 0E-01-1E+05-2E+05 Mean±SD Mean±0.95 Conf. Interval Vtg (mrna copies/ng total RNA) 1E+06 8E+05 6E+05 4E+05 2E+05 0E-01 Mean±SD Mean±0.95 Conf. Interval Significant no samples available Concentration ( g/l) Concentration ( g/l)

20 F0 Male Anal Fin Papillae 140 Octylphenol Effects on Secondary Sex Characters Anal Fin Papillae (number/fin) Mean±SD Mean±0.95 Conf. Interval Male = Female = Concentration ( g/l) 60 F1 Male Anal Fin Papillae 70 F2 Male Anal Fin Papillae Anal Fin Papillae (number/fin) Mean±SD Mean±0.95 Conf. Interval Significant Anal Fin Papillae (number/fin) Mean±SD Mean±0.95 Conf. Interval Significant ( = 0.05) Significant ( = 0.10) no samples available Concentration ( g/l) Concentration ( g/l)

21 n = 6 F0 Gonad Phenotype n = 6 n = 6 Octylphenol Effects on Male Gonads n = 6 0 g/l 6 g/l 13 g/l n = 6 n = 6 Testis Ovary Testicular oocytes 26 g/l 52 g/l 102 g/l F1 Gonad Phenotype F2 Gonad Phenotype n = 27 n = 26 n = 11 n = 27 n = 21 n = 12 0 g/l 6 g/l 13 g/l 0 g/l 6 g/l 13 g/l n = 31 n = 34 n = 10 n = 24 n = g/l 52 g/l 102 g/l 26 g/l 52 g/l

22 30 F0 Fecundity Octylphenol Effects on Fecundity Mean Eggs per Pair/Day Mean±SD Mean±0.95 Conf. Interval Significant ( < 0.05) Significant ( 0.10) Concentration ( g/l) Mean Eggs per Pair/Day F1 Fecundity Mean±SD Mean±0.95 Conf. Interval Significant Mean Eggs per Pair/Day F2 Fecundity Mean±SD Mean±0.95 Conf. Interval Significant ( = 0.10) no samples available Concentration ( g/l) Concentration ( g/l)

23 Summary of Octylphenol Effects on Genetic Males (XY) and Pair Fecundity Vitellogenin (copies x 10 6 mrna/total RNA) Cont 6 µg/l 13 µg/l 26 µg/l 52 µg/l 102 µg/l F0 gen F1 gen F2 gen Anal Fin Papillae (number) Cont 6 µg/l 13 µg/l 26 µg/l 52 µg/l 102 µg/l F0 gen F1 gen F2 gen increased no change decreased Summary F0 least sensitive (2x or more) F2 more sensitive than F1 (0 to 4x) Development most sensitive Gonadal Sex Reversal (XY males with ovaries) Cont 6 µg/l 13 µg/l 26 µg/l 52 µg/l 102 µg/l F0 gen 0/6 0/6 0/6 0/6 0/6 0/6 F1 gen 0/27 0/26 0/11 1/31 12/34 10/11 F2 gen 0/27 0/21 0/12 3/24 16/27 Fecundity (fertile eggs/pair/day) Cont 6 µg/l 13 µg/l 26 µg/l 52 µg/l 102 µg/l F0 gen F1 gen F2 gen

24 Conclusions Development (organization) more sensitive than activation Embryo and early life-stage exposure important to fecundity outcome 8 wk endpoints in F1 and F2 as sensitive as 16 wk endpoints Transgenerational effects ambiguous (sensitivity F2 >F1) Octylphenol: secondary sex and fecundity Trenbolone: vitellogenin, but not fecundity 23