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7. Gene Symbol Direction of. Change. Log2 Expression. Ratio. DDX4. Up. 0.941. DEPDC1. Down. -0.817. DHX29. Down. -1.293. DNAJB4. Up. 0.879. DNMT1.
Supplementary Material

An embryonic atrazine exposure results in reproductive dysfunction in adult zebrafish and morphological alterations in their offspring

Sara E. Wirbisky*, Gregory J. Weber*, Maria S. Sepulveda*†, Tsang-Long Linϒ, Amber S. Jannaschǂ, Jennifer L. Freeman*1 *School of Health Sciences, †Department of Forestry and Natural Resources, ϒDepartment of Comparative Pathobiology and ǂBindley Bioscience Center, Purdue University, West Lafayette, IN, 47907 Table of Contents: I. II. III. IV. V. VI. VII. VIII. IX.

Supplementary Material, Figure S1. Summary of exposure regimen and experimental analysis. Supplementary Material, Figure S2. Sex ratios of adults exposed to atrazine during embryogenesis. Supplementary Material, Figure S3. Average GSI of adult females among treatment groups. Supplementary Material, Figure S4. Follicular staging of adult female ovaries. Supplementary Material, Table S1. Genes altered in adult female gonad tissue in all three atrazine treatments. Supplementary Material, Table S2. Gene enrichment table in adult female gonad tissue at 0.3 ppb atrazine. Supplementary Material, Table S3. Gene enrichment table in adult female gonad tissue at 3 ppb atrazine. Supplementary Material, Table S4. Gene enrichment table in adult female gonad tissue at 30 ppb atrazine. Supplementary Material, Table S5. Primers used in qPCR confirmation of microarray analysis of female gonad tissue.

1

Control

0.3 ppb

3 ppb

30 ppb

Atrazine exposure (1-72 hpf)

Control

0.3 ppb

3 ppb

30 ppb

5-8 months unexposed maturation period

Morphology of offspring at 72 hpf

Histology and hormone analysis

Reproductive potential

64 pairs breed pairs within treatment group†

Transcriptomic analysis

Supplementary Material, Figure S1. Summary of exposure regimen and experimental analysis. Zebrafish were bred and embryos were collected and treated with 0, 0.3, 3, or 30 ppb atrazine from 1-72 hpf. Following the exposure period, embryos were rinsed and allowed to mature under normal conditions until 5-8 mpf. Zebrafish were then bred for three weeks in order to initiate breeding cycles. To assess reproductive potential, single pairs were bred and number of pairs that bred, number of embryos per pair, total number of embryos per treatment, mortality, and hatching rates were assessed. In addition, offspring were collected for morphological assessment at 72 hpf. Adult female zebrafish were then collected for ovarian histology, hormone, and transcriptome analysis. (†16 pairs per replicate; 4 replicates; blue: male fish; pink: female fish)

2

100 90 80

Percent

70 60 50

Female

40

Male

30 20 10 0 0

0.3

3

30

Atrazine Concentration (ppb) Supplementary Material, Figure S2. Sex ratios of adults exposed to atrazine during embryogenesis. Zebrafish were bred and embryos were collected and treated with 0, 0.3, 3, or 30 ppb atrazine from 1-72 hpf. Following the exposure period, embryos were rinsed and allowed to mature under normal conditions until 5-8 mpf. Zebrafish were then separated and counted based upon sex in order to determine sex ratios. No significant differences were observed among the treatment groups for sex ratios (p=0.64; 4 replicates with 60-75 fish per replicate treatment). Error bars are expressed as ± SD.

3

20 18 16

GSI (%)

14 12 10 8 6 4 2 0 0 ppb

0.3 ppb

3 ppb

30 ppb

Atrazine Concentration Supplementary Material, Figure S3. Average GSI of adult females among treatment groups. Adult female zebrafish (4 replicates with 10 fish per replicate treatment) approximately 5-8 mpf were collected from each treatment group (0, 0.3, 3, or 30 ppb atrazine), euthanized, and ovaries dissected. Zebrafish and gonads were weighed and GSI calculated. GSI among the atrazine treatment groups was not statistically different (p=0.11). Error bars are expressed as ± SD.

4

Percent

A

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Atretic Post-Ovulatory Late Vitellogenic Early Vitellogenic Cortical Alveoli Perinuclear

Control 0.3 ppb 3 ppb

30 ppb

Atrazine Concentration

Percent

B

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Atretic Post-Ovulatory Late Vitellogenic Early Vitellogenic Cortical Alveoli Perinuclear

Normal

Swollen

Abdominal Morphology

Supplementary Material, Figure S4. Follicular staging of adult female ovaries. Adult female zebrafish (4 replicates with 10 fish per replicate) approximately 5-8 mpf were collected from each treatment group (0, 0.3, 3, or 30 ppb atrazine), euthanized, and ovaries dissected and prepared for histological analysis. (A) The percent distribution of different oocyte stages across treatments was not significantly different from each other (p>0.05). (B) When comparing oocyte staging between females presenting with swollen abdomens which were unable to release eggs to normal females a significant difference was observed for the number of atretic follicles (p=0.0002).

5 Supplementary Material, Table S1. Genes altered in adult female gonad tissue in all three atrazine treatments. Gene Symbol A2M ABCG4 ABHD12 ACBD6 ACE ACHE ACTL6A ADCY8 ADCYAP1 ADD3 AGA AK1 ALDH2 ALDH3A2 Aldoart1 ALKBH8 AMPD3 ANLN ANTXR2 ANXA13 AP1M2 APC2 APLP1 AQP1 ARFIP2 ARID3B ARNTL2 ASIC2 ATL3 ATP1A1 ATP1B1 ATP6V0A2 ATP6V0C ATP6V1B2 ATPAF2 BBS1 BBS12 BCL11B BET1 BMS1

Direction of Change Down Down Down Up Up Up Down Up Up Up Up Up Down Up Up Up Down Down Up Up Down Down Up Up Down Down Down Up Down Up Down Up Down Down Up Down Up Down Up Down

Log2 Expression Ratio -1.300 -0.621 -0.961 0.943 1.400 1.549 -1.179 0.853 0.597 0.728 0.645 0.857 -0.729 0.947 0.639 1.288 -1.010 -1.154 1.270 0.656 -1.052 -0.825 0.976 0.976 -0.851 -0.985 -1.083 1.542 -2.054 0.822 -0.699 1.537 -1.541 -0.656 0.863 -1.096 1.567 -0.797 0.948 -1.899

6 Gene Symbol C11orf80 C12orf23 C17orf104 C1orf146 C1orf198 C9orf114 C9orf172 CA2 CA8 CADM1 CALB1 CAMK2N2 CAMK4 CAMKV CARTPT CASP8AP2 CBLN1 CCDC69 CCNH CDC27 CDCP1 CDIP1 CENPM CEP70 CHAF1B CIRBP CKM CLDN7 CLIP3 CNN3 CPE CRHBP CSPG5 CTDSP2 CTNND2 CTSF CYP19A1 CYP1B1 CYP27A1 Dclk1 DDC DDX19A DDX20

Direction of Change Down Down Down Down Up Down Up Up Up Down Down Up Up Up Up Down Up Down Down Up Down Down Up Down Down Up Up Down Up Down Up Up Up Up Up Up Up Up Up Down Down Up Down

Log2 Expression Ratio -2.287 -1.068 -1.218 -0.853 0.769 -0.632 1.365 0.959 1.963 -1.366 -1.149 0.813 0.853 0.791 0.869 -1.254 1.170 -0.900 -1.654 0.695 -1.137 -1.018 1.274 -1.146 -0.980 0.626 0.793 -1.504 0.736 -0.727 0.939 0.989 0.973 1.097 0.995 0.887 1.842 0.589 0.638 -0.920 -1.610 0.664 -0.768

7 Gene Symbol DDX4 DEPDC1 DHX29 DNAJB4 DNMT1 DPP7 DPYSL3 DRAM2 DSPP DYNC1LI2 EFCAB1 EIF4G1 ELAVL3 ELFN2 ELOVL6 EN1 ENO4 ERI1 ETNK1 EZH2 FAM13B FAM184B FAM228B FAM69B FAM83D FAM84B FANCF FBXO15 FDXR FKBP1B FLOT2 Folh1 FOXA1 FOXQ1 FOXR1 GABRA6 GAP43 GATA6 GDF6 GGCT GLB1L2 Gm19410 GNB2

Direction of Change Up Down Down Up Down Up Up Up Up Down Down Down Up Up Down Down Down Down Up Up Down Down Up Down Down Down Down Down Down Up Up Down Up Down Up Up Down Up Up Down Up Up Down

Log2 Expression Ratio 0.941 -0.817 -1.293 0.879 -3.820 1.561 0.717 2.180 1.287 -0.806 -0.594 -1.638 0.935 1.477 -1.260 -0.689 -1.873 -1.075 0.819 0.875 -0.970 -0.622 0.642 -1.405 -1.305 -1.280 -1.332 -1.084 -1.134 0.698 0.702 -1.220 0.746 -1.912 1.011 0.907 -1.300 0.588 0.927 -1.836 0.736 0.749 -0.636

8 Gene Symbol GNG13 GNG3 GP1BB GPATCH8 GPR157 GPR85 GPSM2 GREB1L GRIA2 GSAP GSG1L GSR GSS GTSF1 H2AFV H2AFY2 HBE1 HBZ HIF3A HMBS HPGD HTRA1 IFFO1 IGF2BP2 IGFBP1 IGSF9B INSM1 Iqcd IQCJ-SCHIP1 ISLR2 ISY1-RAB43 ITGB5 KCNA1 KCNA4 KCNIP3 KIF1A KIF20B KIF23 KL KLHDC10 KMO KPNA7 KRT17

Direction of Change Up Up Down Up Up Up Up Up Up Down Up Down Down Down Up Up Down Up Down Down Up Up Up Down Up Up Down Down Up Up Down Up Up Up Down Down Down Down Down Up Down Up Up

Log2 Expression Ratio 0.770 0.599 -0.825 0.591 0.960 1.097 1.057 0.595 0.850 -1.324 0.718 -1.217 -0.828 -0.973 1.524 1.375 -0.735 1.065 -1.078 -0.810 0.806 1.031 0.692 -0.784 0.732 0.921 -0.909 -0.754 0.674 1.051 -1.445 0.833 0.940 0.653 -1.433 -0.881 -0.597 -1.118 -1.542 0.705 -1.740 0.739 0.829

9 Gene Symbol L1CAM LDLRAP1 LEPREL4 LNX1 LOC680121 LRRC8D LRRN3 LRRTM2 MAB21L1 MAF MAP2K5 MAPK11 MARVELD2 MBD3 MC2R MCOLN2 MED6 MEF2A METTL20 METTL8 MGAT5 MGST1 MSI1 MTA3 MTMR1 MTX3 MYT1L NAF1 NAT8L NCAPH2 NDRG2 NDRG3 NDRG4 NDUFA4 NDUFS8 NEFL NEGR1 NEK2 NELFCD NEUROD1 NEUROD2 NIM1K NMRK1

Direction of Change Up Down Down Up Up Up Up Up Up Up Up Up Down Up Down Up Up Down Down Up Down Up Up Down Down Up Up Down Up Up Up Up Up Down Down Down Down Down Up Up Down Down Up

Log2 Expression Ratio 0.587 -0.619 -0.652 1.349 1.742 1.209 0.616 0.655 1.220 0.883 0.750 0.759 -0.944 0.680 -1.095 0.666 0.928 -1.186 -0.690 0.647 -1.195 0.699 0.694 -0.820 -1.147 0.791 1.233 -0.870 2.412 1.275 0.959 0.975 1.072 -0.671 -0.629 -0.588 -1.442 -1.622 0.746 0.716 -1.143 -1.139 1.163

10 Gene Symbol NOVA1 NPC2 NPTX1 NRG2 Nrxn3 NSL1 NUF2 ODF3 OGG1 Olfr146 OSBPL11 OSTF1 P2RX4 PANK1 PAQR4 PCDH19 PCDH7 PCDHAC2 PCDHGA12 PCLO PCMT1 PCYT1A PFAS PHLDA2 PICK1 PIK3R1 PIWIL1 PIWIL2 PLP1 PLXNA4 POLE POU3F3 Ppfia4 PPP3R1 PRIMPOL PRTG PTBP1 PTGES PTPRD PTPRS PUSL1 PVALB RAB13

Direction of Change Down Down Down Down Down Up Down Up Up Down Down Down Down Down Down Up Down Down Up Up Up Down Down Down Down Up Down Up Down Up Up Down Up Up Down Up Down Up Down Down Down Down Down

Log2 Expression Ratio -0.862 -0.754 -0.825 -1.586 -1.363 1.280 -1.028 1.975 0.644 -0.674 -1.088 -1.176 -1.348 -0.813 -0.732 0.721 -0.654 -0.995 2.398 0.814 0.953 -1.064 -1.282 -0.742 -0.957 1.050 -0.710 0.745 -1.710 0.789 1.052 -1.568 1.040 0.762 -0.928 0.811 -0.844 1.036 -0.893 -1.002 -0.696 -1.074 -0.977

11 Gene Symbol RAD52 RAD54L RALY RARA RASD1 RASL11B RASSF2 RASSF8 RBM24 RBM26 RBM41 RBMS2 RCAN2 RCAN3 RCC2 RERGL RGL2 RGS20 RGS7BP RIC8A RIMS2 RIPK4 RND1 RNF217 ROBO2 RTEL1 RUNX1T1 SCARB1 SCG2 SCN8A SDK1 SEPT5 SETD6 SEZ6L2 SFRP1 SGCE SH3D19 SHD SLC1A2 SLC1A4 SLC25A19 Slc26a10 SLC27A1

Direction of Change Down Down Up Down Up Up Up Up Down Down Down Up Up Up Down Up Down Down Down Up Down Up Up Down Up Down Up Up Up Up Up Up Up Up Down Up Down Up Down Up Down Down Up

Log2 Expression Ratio -0.854 -1.093 0.861 -0.656 1.254 1.195 2.522 0.626 -1.566 -1.084 -1.696 0.588 0.774 0.935 -0.710 0.855 -1.112 -0.663 -0.646 0.620 -0.776 1.150 2.020 -1.339 0.723 -0.771 0.773 1.240 0.618 1.867 0.798 1.854 0.773 1.090 -0.598 0.627 -1.200 1.646 -0.922 0.961 -1.448 -0.758 0.950

12 Gene Symbol SLC35D1 SLC4A4 SLC9A2 SMARCAL1 SNAI1 SNAP25 SNX12 SNX21 SORBS3 SOX1 SOX10 SPINT1 SPSB4 SRGAP2 ST6GAL1 STAR STARD8 STIL STMN2 STOM STX11 SULT2B1 SUN1 SV2B SVOPL SYCP1 SYNE1 SYNGR3 TAL1 TAL2 TBC1D15 TDP2 TGDS TIAL1 TJP2 TK1 TMBIM1 TMEM160 TMEM178B TMEM35 TMEM51 TNFRSF19 TONSL

Direction of Change Down Up Down Down Down Up Up Down Down Down Down Down Up Up Down Up Down Up Up Down Up Up Up Down Up Down Up Up Up Up Up Down Down Down Down Up Up Down Down Down Down Up Down

Log2 Expression Ratio -0.639 0.699 -0.681 -0.972 -0.889 1.482 0.927 -0.797 -0.771 -1.473 -0.968 -1.223 1.053 0.702 -1.162 1.053 -1.105 1.561 0.844 -0.690 1.813 1.155 1.065 -0.937 1.156 -2.075 1.199 1.023 0.993 1.153 0.675 -0.921 -2.044 -0.657 -0.714 1.367 0.994 -0.873 -0.964 -0.699 -0.889 1.258 -0.715

13 Gene Symbol Tor1aip1 TPPP3 TRABD TRAF2 TRAPPC2L TRIM16 TRIM35 TRPM3 TSPAN15 TSPAN2 TTLL7 TUBA1A TUBA3E TUSC3 UBE2C UBE2J2 UBE4A UBR1 UHRF1 UNC119 UNC119B URB2 USP44 VCAM1 VGF VIM VIP WBSCR27 WDR12 WDR26 WIF1 XRCC2 YAF2 YRDC YWHAG ZC3H18 ZFPM2 ZIC1 ZNF281 ZNF410 ZNF598 ZNF91

Direction of Change Down Up Down Down Up Up Down Down Down Up Down Up Up Down Down Up Down Down Up Down Up Down Down Up Up Up Down Down Down Down Up Up Up Down Down Down Up Down Down Up Down Up

Log2 Expression Ratio -0.885 0.749 -1.068 -0.720 0.838 1.016 -2.052 -1.029 -1.186 2.381 -1.380 0.792 0.855 -1.010 -0.714 0.883 -1.280 -1.190 1.051 -1.181 1.169 -0.923 -0.925 0.713 0.950 0.604 -0.586 -0.784 -1.125 -0.810 0.634 1.054 0.878 -0.863 -1.230 -1.519 0.629 -0.884 -0.791 2.856 -0.817 2.208

14 Supplementary Material, Table S2. Genes enrichment table in adult female gonad tissue at 0.3 ppb atrazine. Physiological System Development and Function – 0.3 ppb NERVOUS SYSTEM DEVELOPMENT AND FUNCTION Morphology of CNS Neurotransmission Growth of neurites Myelination Coordination EMBRYONIC DEVELOPMENT Development of head Development of body axis Growth of embryo BEHAVIOR Behavior Cognition Learning Memory ORGANISMAL SURVIVAL Organismal Death Perinatal Death Neonatal Death

p-valuea

Number of Genesb

2.88E-26 – 3.74E-4

565

5.87E-18 1.96E-22 1.72E-15 4.44E-6 1.94E-9 2.51E-23 – 3.65E-4 2.51E-23 3.33E-21 8.24E-7 5.00E-25 – 1.45E-4 5.00E-25 2.13E-14 3.96E-13 4.10E-6 3.97E-32 – 1.35E-4 3.97E-32 5.99E-11 2.24E-9

135 120 117 38 54 396 203 213 88 260 228 101 94 48 497 491 131 98

a

Derived from the likelihood of observing the degree of enrichment in a gene set of a given size by chance alone. b

Classified as being differentially expressed that relate to the specified function category; a gene may be present in more than one category.

15 Supplementary Material, Table S3. Gene enrichment table of adult female gonad tissue at 3 ppb atrazine. Physiological System Development and Function – 3 ppb NERVOUS SYSTEM DEVELOPMENT AND FUNCTION Growth of neurites Migration of Neurons Morphology of CNS Dendritic Growth and Branching ORGAN MORPHOLOGY Abnormal morphology of gonad Abnormal morphology of internal genitalia Mass of gonad Morphology of brain BEHAVIOR Behavior Cognition Learning Memory ORGANISMAL SURVIVAL Organismal Death Perinatal Death Neonatal Death

p-valuea

Number of Genesb

1.74E-15 – 1.62E-3

260

2.05E-10 4.83E-6 1.74E-15 5.07E-8

57 27 73 27

1.65E-13 – 1.62E-3 6.52E-7 8.84E-6 4.01E-7 1.65E-13 5.42E-18 – 1.56E-3 5.42E-18 1.69E-9 1.78E-8 8.91E-5 1.55E-18 – 3.03E-4 1.55E-18 1.21E-5 1.25E-4

110 41 37 24 65 134 113 49 45 25 233 220 56 41

a

Derived from the likelihood of observing the degree of enrichment in a gene set of a given size by chance alone. b

Classified as being differentially expressed that relate to the specified function category; a gene may be present in more than one category.

16 Supplementary Material, Table S4. Gene enrichment table of adult female gonad tissue at 30 ppb atrazine. p-valuea

Number of Genesb

7.61E-8 – 8.58E-3

184

3.27E-7 1.39E-3 1.67E-6 7.49E-5

48 15 42 32

TISSUE DEVELOPMENT Gonadogenesis Development of genital organ Gametogenesis Development of neurons

8.12E07 – 8.47E-3 3.52E-03 3.40E-3 5.46E-03 8.19E-04

152 32 34 26 16

BEHAVIOR Behavior Cognition Learning Locomotion ORGANISMAL SURVIVAL Organismal Death Perinatal Death Neonatal Death

4.05E-10 – 6.51E-3 4.05E-10 3.30E-5 6.52E-4 4.24E-5 5.31E-10 – 5.71E-3 5.31E-10 2.17E-4 5.85E-5

97 82 34 29 28 171 165 45 37

Physiological System Development and Function – 30 ppb NERVOUS SYSTEM DEVELOPMENT AND FUNCTION Morphology of CNS Synaptogenesis Growth of neurites Differentiation of neurons

a

Derived from the likelihood of observing the degree of enrichment in a gene set of a given size by chance alone. b

Classified as being differentially expressed that relate to the specified function category; a gene may be present in more than one category.

17 Supplementary Material, Table S5. Primers used in qPCR confirmation of microarray analysis of female gonad tissue. Seq. ID* NM_131663.1

Gene Symbol* STAR

Primer Sequence Forward: ATGCGACCAAGTGCAGATGA Reverse: GCCATTCTGTCCCTGAGGTG XM_005169447.1 ACE Forward: AGAGGCATGGGGTCACAAAG Reverse: CTGTGATTGCGAGATTGGCG NM_131154.2 CYP19A1 Forward: CAGGGCATCATATTCAACTCAA Reverse: AGGTGGTGCAGATCTCCATAGT NM_001045256.1 CRHBP Forward: GGATAACGAGATCAGCCCGG Reverse: ACCCTCTACGGCCACCATAT XM_689239.5 HPGD Forward: AGCAAGGAGGGGCAATCATC Reverse: ATGGCTCGTGAAAACCCGAT NM_001045256.1 CYP1B1 Forward: CAACGGTTACCCCATTCCCA Reverse: GCCGAGTGAGAAGATGAGCA NM_001114553.2 VIP Forward: TACCCTTCGCCTCCATGAGA Reverse: TCATCACTGACCCGCTTTCC NM_181601 Β-ACTIN Forward: CTAAAAACTGGAACGGTGAAGG Reverse: AGGCAAATAAGTTTCGGAACAA *Seq IDs are zebrafish and gene symbols are the human homolog.