Karlsson O , Svanholm S , Eriksson A , Chidiac J , Eriksson J , Jernerén F , Berg C .

805057 European Research Council

             regulation of gene expression, epigenetic
             regulation of molecular function, epigenetic
             thyroid hormone receptor binding

	Fig. 1. The overall study design and main findings.
	Fig. 2. Histomicrographs showing seminiferous tubules (– – –) from 24-month-old Xenopus tropicalis A-B) F1 control male with all different germ cell stages and clearly structured, organized and distinguished seminiferous tubules with a thin layer of interstitial connective tissue and C) F1 male in the linuron group containing an elevated number of spermatogonia. D) F1 male in the linuron group with disorganized germinal epithelium in the seminiferous tubules, germ cells at different developmental stages scattered across the tubule, and thickened interstitial connective tissue. Arrows indicate interstitial connective tissue. Magnification 40× (A-B) and 20× (C–D). Spg = spermatogonia, Spc = spermatocytes, Spt = spermatids, Spz = spermatozoa.
	Table 1. Biometrics and reproductive system variables including fertility in adult Xenopus tropicalis F1 males after paternal developmental exposure to linuron. a Sexual behavior and fertility assessed at 20 months of age, biometrics, tissue collection and analysis at 24 months of age. b Snout-vent length measured from the tip of the snout to the end of the cloaca. c Gonadosomatic index, calculated as (weight of both testes/body weight) × 100. d The size of the largest spermatocyte nest in each seminiferous tubule was determined using score numbers (1–3) based on the number of spermatocytes: 1 = 1–10, 2 = 10–20, 3 ≥20 spermatocytes/nest. e The number of spermatozoa in the seminiferous tubular lumen was estimated using score numbers (0–3): 0 = no spermatozoa and 1 ≤50%, 2 = 50%, 3 ≥50% of the lumen was occupied by spermatozoa. f Eight testes in linuronF1 and one control testis that lacked distinct germ cell nests, were excluded from further analysis. ⁎p < 0.05 compared to control animals (Mann-Whitney U test or Fisher's exact test). ⁎⁎p < 0.01 compared to control animals (Mann-Whitney U test or Fisher's exact test). ⁎⁎⁎p < 0.001 compared to control animals (Mann-Whitney U test or Fisher's exact test).
	Table 2. Biometrics and reproductive system variables including fertility in adulta Xenopus tropicalis F2 males after grand-paternal developmental exposure to linuron. a All parameters assessed at 12 months of age. b Snout-vent length. Measured from the tip of the snout to the end of the cloaca. c Gonadosomatic index, calculated as (weight of both testis/body weight) ∗ 100. d The size of the largest spermatocyte nest in each seminiferous tubule was determined by using score numbers (1–3) based on the number of spermatocytes: 1 = 1–10, 2 = 10–20, 3 ≥20 spermatocytes/nest. e The number of spermatozoa in the seminiferous tubular lumen was estimated using score numbers (0–3): 0 = no spermatozoa and 1 ≤50%, 2 = 50%, 3 ≥50% of the lumen was occupied by spermatozoa. ⁎p < 0.05 compared to control animals (Mann-Whitney U test). ⁎⁎ p < 0.01 compared to control animals (Mann-Whitney U test).
	Table S1. Fat body fatty acid profiles in 24-month-old Xenopus tropicalis F1 males after paternal developmental exposure to linuron. Fatty acids were compared between control and linuron animals using Mann-Whitney U- test.
	Table S2. Liver fatty acid profiles in 24-month-old Xenopus tropicalis F1 males after paternal developmental exposure to linuron. Fatty acids were compared between control and linuron animals using Mann-Whitney U- test.
	Figure S1. Desaturation index values based on fatty acids 16:1/16:0 (palmitoleic acid/palmitic acid) and 18:1/18:0 (oleic acid/stearic acid) in the fat body (A, B) and liver (C, D) of 24-month-old Xenopus tropicalis F1 males after paternal developmental exposure to linuron. Data are presented as the mean ± SEM. Control n=7; linuronF1 n=28-30.
	Table S3. Fat body fatty acid profiles in 12-month-old Xenopus tropicalis F2 males after grand-paternal developmental exposure to linuron. Fatty acids were compared between control and linuron animals using Mann- Whitney U-test. *p < 0.05 compared to control animals.
	Table S4. Liver fatty acid profiles in 12-month-old Xenopus tropicalis F2 males after grand-paternal developmental exposure to linuron. Fatty acids were compared between control and linuron animals using Mann- Whitney U-test.
	Figure S2. Desaturation index values based on fatty acids 16:1/16:0 (palmitoleic acid/palmitic acid) and 18:1/18:0 (oleic acid/stearic acid) in the fat body (A, B) and liver (C, D) of 12-month-old male Xenopus tropicalis F2 males after grand-paternal developmental exposure to linuron. *p < 0.05 compared to control animals (Mann-Whitney U-test), one outlier removed (Grubbs' test, p <0.05, Z=2.02, considered outlier if >1.96 S.D. from mean). Data are presented as the mean ± SEM. Control n=6-7; linuronF2 n=16.
	Figure S3. Suggested adverse outcome pathway (AOP) for multigenerational anti-androgenic and thyroid toxicity after paternal developmental exposure. Whole lines represent observed key events and dotted lines represent plausible key events. AR = Androgen receptor. TR = Thyroid receptor. Key event is here defined as either a molecular, cellular or tissue response as a consequent of the molecular initiating event leading to the adverse outcome. 1Kojima et al., 2004, 2Orton et al., 2009 ,3Wilson et al., 2009, 4Spirhanzlova et al. 2017, 5Orton et al., 2018

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