Influence of Prenatal Transportation Stress-Induced Differential DNA Methylation on the Physiological Control of Behavior and St

Copyedited by: KD

Journal of Animal Science, 2020, 1–18

doi:10.1093/jas/skz368 Advance Access publication December 6, 2019 Received: 31 August 2019 and Accepted: 5 December 2019 Fetal Programming Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 Fetal Programming Influence of prenatal transportation stress-induced differential DNA methylation on the physiological control of behavior and stress response in suckling Brahman bull calves Brittni P. Littlejohn,†,‡,1 Deborah M. Price,†,‡ Don A. Neuendorff,† Jeffery A. Carroll,|| Rhonda C. Vann,$ Penny K. Riggs,‡ David G. Riley,‡ Charles R. Long,†,‡ Ronald D. Randel,† and Thomas H. Welsh, Jr.‡,2 †Texas A&M AgriLife Research & Extension Center, Overton, TX 75684, ‡Department of Animal Science, Texas A&M University, and Texas A&M AgriLife Research, College Station, TX 77843, ||USDA-ARS, Livestock Issues Research Unit, Lubbock, TX 79403, $Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Raymond, MS 39154

1Current address: Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS 39762

2Corresponding author: [email protected]

Abstract The objective of this experiment was to examine potential differential methylation of DNA as a mechanism for altered behavioral and stress responses in prenatally stressed (PNS) compared with nonprenatally stressed (Control) young bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation (Transported group) or maintained as nontransported Controls (n = 48). From the offspring born to Transported and Control cows, a subset of 28-d-old intact bulls (n = 7 PNS; n = 7 Control) were evaluated for methylation of DNA of behavior and stress response–associated genes. Methylation of DNA from white blood cells was assessed via reduced representation bisulfite sequencing methods. Because increased methylation of DNA within gene promoter regions has been associated with decreased transcriptional activity of the corresponding gene, differentially methylated (P ≤ 0.05) CG sites (cytosine followed by a guanine nucleotide) located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. Among differentially methylated genes (P ≤ 0.05) related to behavior and the stress response were OPRK1, OPRM1, PENK, POMC, NR3C2, TH, DRD1, DRD5, COMT, HTR6, HTR5A, GABRA4, GABRQ, and GAD2. Among altered (P < 0.05) signaling pathways related to behavior and the stress response were Opioid Signaling, Corticotropin-Releasing Hormone Signaling, Dopamine Receptor Signaling, Dopamine-DARPP32 Feedback in cAMP Signaling, Serotonin Receptor Signaling, and GABA Receptor Signaling. Alterations to behavior and stress response–related genes and canonical pathways supported previously observed elevations in temperament score and serum cortisol through weaning in the larger population of PNS calves from which bulls in this study were derived. Differential methylation of DNA and predicted alterations to behavior and stress response–related pathways in PNS compared with Control bull calves suggest epigenetic programming of behavior and the stress response in utero.

Key words: behavior, bovine, epigenetics, prenatal stress, stress response, temperament

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Introduction Animal Procedures Alteration of behavior and stress responsiveness is among the Mature Brahman cows were assigned to 1 of 2 treatment most noted outcomes of prenatal stress (PNS) in mammals groups (Transported: n = 48 and Control: n = 48), which were (Lindsay et al., 2019; Matthews and McGowan, 2019). The balanced by age, parity, and temperament (Littlejohn et al., mechanisms controlling these alterations are still largely 2016). Transported cows were hauled for 2 h at 60 ± 5, 80 ± 5, unclear; however, it is known that stress incurred by a gestating 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation (Price et al., 2015). dam can result in suppression of the placental barrier enzyme Control cows were managed the same as Transported cows

11β-hydroxysteroid dehydrogenase type 2 (11-βHSD2). This with the exception of being transported. Both treatment groups Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 enzyme converts cortisol to an inactive form prior to entering were housed in the same pasture (at the Texas A&M AgriLife fetal circulation; therefore, decreased 11-βHSD2 can result Research and Extension Center at Overton, TX) and fed the in increased fetal exposure to corticosteroids (Benediktsson same diet (Littlejohn et al., 2016). From these groups, 26 male et al., 1997; Stirrat et al., 2018). Such alterations to the fetal and 18 female calves (Control group) were born to Control dams, environment may result in epigenetic modifications, such whereas 20 male and 21 female calves (Prenatally Stressed, as DNA methylation, which appear to be major regulators of PNS, group) were born to Transported dams. Male calves phenotypic changes due to prenatal stress (Szyf, 2012; Lester were maintained as bulls throughout the study. Each calf was and Marist, 2018). Deoxyribonucleic acid methylation is defined manually restrained for less than 5 min for sample collection as a covalent modification in which a methyl group is added at 28 d of age. One 10-mL vacuum tube (BD, Franklin Lakes, NJ) to the carbon 5 position of a cytosine nucleotide (Wyatt, 1950; containing EDTA was used to collect the blood sample from each Razin and Riggs, 1980). Increased methylation of DNA within calf via jugular venipuncture with a sterile 18-gauge needle. the promoter region of a gene has been reported to suppress Leukocytes were isolated and stored at −80 °C until DNA was transcriptional activity and gene expression (Levine et al., 1991; extracted. Samples from bull calves were stratified into groups Tate and Bird, 1993), whereas increased methylation of DNA in based on the following criteria: 1) adequate availability of DNA, the gene body region of a gene has been suggested to result in 2) calf treatment group, and 3) calf sire. From those stratified increased gene expression (Hellman and Chess, 2007). Therefore, groups, DNA samples from 7 PNS and 7 Control bull calves were the presence or absence of methyl groups at specific locations randomly selected for analysis in this study. within a gene can be utilized as a tool to better understand how prenatal environment shaped postnatal phenotype. Behavior Sample Analysis and stress response traits are multifaceted in nature and Processing of blood samples controlled by many components of neural, neuroendocrine, and Blood samples were centrifuged at 2,671 × g for 30 min at 6 °C. endocrine systems (Chan et al., 2018; Bale et al., 2019). Some Leukocytes were isolated and transferred into 2-mL nuclease- of these systems include the hypothalamic–pituitary–adrenal free microcentrifuge tubes. Samples were washed in red blood (HPA) axis and signaling by catecholamines, monoamines, and cell lysis buffer solution repeatedly until a clean cell pellet was opioids. Differential transcript abundance in the adrenal cortex produced. Leukocyte samples were stored at −80 °C until they (Friedrich et al., 2017) and differential methylation of DNA in were thawed for DNA isolation procedures. different brain regions (Cantrell et al., 2019) have been associated with temperament in cattle. Furthermore, emerging literature DNA extraction supports the ability of glucocorticoids to alter methylation of Phenol-chloroform extraction procedures were used to isolate DNA in developing neural cells; specifically, in vitro exposure DNA from each leukocyte pellet for DNA methylation analysis of early neural progenitor cells to dexamethasone resulted in by standard procedures as previously described (Littlejohn et al., genome-wide differences in methylation of DNA (Provençal 2018). Following DNA precipitation, purified DNA was stored at et al., 2019). Because behavior and stress response phenotypes −80 °C and submitted to Zymo Research Corp (Irvine, CA) for are of economic and biologic significance to beef cattle DNA methylation analysis. production, this study sought to understand the mechanisms by which a common stressor for beef cattle, transportation, Library construction for DNA methylation analysis incurred by a pregnant dam could affect behavior and the stress A method of reduced representation bisulfite sequencing, response of offspring. The objective of this study was to identify Methyl-MiniSeq (Zymo Research; Irvine, CA), was utilized to differentially methylated genes from a previously reported evaluate differential methylation of DNA in samples from PNS genome-wide assessment of DNA methylation in prenatally compared with Control bull calves. Libraries were prepared from stressed bull calves that were related to behavior and stress 200 to 500 ng of genomic DNA digested with 60 units of TaqαI responses (Littlejohn et al., 2018). Because behavior and stress and 30 units of MspI (New England Biolabs, Inc., Ipswich, MA) response phenotypes were altered in the larger population of sequentially and then extracted with Zymo Research DNA Clean PNS and Control calves from the same study (Littlejohn et al., & Concentrator-5 kit (Cat#: D4003). Fragments were ligated to 2016), we hypothesized that prenatal transportation stress preannealed adapters containing 5′-methyl-cytosine instead would result in differential methylation of DNA in genes of cytosine according to Illumina’s specified guidelines (www. associated with behavior and stress response phenotypes. illumina.com). Adaptor-ligated fragments of 150–250 bp and 250– 350 bp in size were recovered from a 2.5% NuSieve 1:1 agarose gel (ZymocleanTM Gel DNA Recovery Kit, Zymo Research, Cat#: Materials and Methods D4001). Then, the fragments were bisulfite-treated using the EZ All procedures were in compliance with the Guide for the Care DNA Methylation-Lightning Kit (Zymo Research, Cat#: D5020). and Use of Agricultural Animals in Research and Teaching (FASS, Preparative-scale PCR was performed and the resulting products 2010) and approved by the Texas A&M University Animal Care were purified (DNA Clean & Concentrator—Zymo Research, and Use Committee. Cat#D4005) and sequenced on an Illumina HiSeq. Copyedited by: KD

Littlejohn et al. | 3

Methyl-MiniSeq sequence alignments and data analysis alterations to the physiological systems regulating those Sequence reads from bisulfite-treated EpiQuest libraries were phenotypes. To evaluate potential mechanisms controlling such identified with standard Illumina base-calling software and phenotypic differences, leukocytes were used as a generalized analyzed with a proprietary analysis pipeline (Zymo Research), surrogate cell to evaluate methylation of DNA. The following which is written in Python. Bismark (http://www.bioinformatics. sections evaluate and discuss the potential influence of PNS- babraham.ac.uk/projects/bismark/) was utilized to perform induced differences in methylation of DNA on behavior and the alignment to the UMD 3.1 bovine assembly (Zimin et al., stress response phenotypes in Brahman cattle. 2009). Index files were constructed using the bismark_genome_ preparation command and the entire reference genome. The Influence of Prenatal Stress on Neural, Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 --non_directional parameter was applied while running Bismark. Neuroendocrine, and Endocrine Function All other parameters were set to default. Filled-in nucleotides Many function terms, canonical pathways, and specific genes were trimmed off for methylation calling. The methylation ratio related to behavior, the stress response, and neural function were was defined as the measured number of cytosines (number of altered (P < 0.05) in PNS compared with Control bull calves. A list reads reporting a C) divided by the total number of cytosines of canonical pathways related to behavior, the stress response, (total number of reads reporting a C or T) covered at that site. and neural function that were predicted to be altered in PNS The methylation difference (MD) was calculated by subtracting compared with Control bull calves are presented in Table 1. the average Control methylation ratio at a site from the average A list of IPA-designated genes associated with the Behavior PNS methylation ratio at that same site. Fisher’s exact test or function term that contained CpG sites within the promoter t-test was performed for each CpG site which had at least 5 reads region that were hypomethylated or hypermethylated in PNS coverage. Promoter, gene body and CpG island annotations were compared with Control bulls are presented in Tables 2 and 3, added for each CpG included in the comparison. A summary of respectively. In general, opioid signaling, HPA axis signaling, genome-wide coverage has been previously described (Littlejohn dopamine signaling, serotonin signaling, and γ-aminobutyric et al., 2018). No multiple test correction was performed because acid (GABA) signaling were among the most notable pathways multiple test corrections have been suggested to be too related to behavior, the stress response, and neural function conservative for reduced representation bisulfite sequencing that were predicted to be altered in PNS compared with Control (Kanzleiter et al., 2015). Specifically, in spite of decreasing false bulls and will be discussed in the following paragraphs. positives, multiple test corrections would also unnecessarily increase false negatives (Rothman, 1990; Kanzleiter et al., 2015). Opioid Signaling Instead, CpG site methylation differences and P-value thresholds The Opioid Signaling pathway was the most significant canonical were utilized as criteria for inclusion in the enrichment analysis pathway altered in PNS compared with Control bull calves (Table 1). to assess potential statistical and biological significance. Two genes encoding for opiate agonists (endogenous opioids) contained a hypomethylated CpG site within the promoter Prediction of pathways and functions altered by prenatal stress region, including the proenkephalin (PENK; MD = −0.14) gene An enrichment analysis was performed (analyzed on 3 April encoding for Meta-enkephalin and Leu-enkephalin and the 2018) with Ingenuity Pathway Analysis software (IPA; Redwood pro-opiomelanocortin (POMC; MD = −0.18) gene encoding for City, CA) to assess alterations to signaling pathways in PNS β-endorphin (Figure 1; Table 2). Hypomethylation of these sites compared with Control bull calves. Because methylation of suggests potential increased production of these opiate agonists DNA in mammals primarily occurs within CpG contexts (a in PNS compared with Control bulls. Stress has been reported cytosine nucleotide followed by a guanine nucleotide; Ehrlic to increase production of opiate agonists (Bruchas et al., 2010). et al., 1982), we focused on DNA methylation of cytosines within Because PNS calves from the larger population from which CpG contexts. Only differentially methylated CpG sites (P ≤ 0.05) calves in this study were derived were found to exhibit increased that had a methylation difference that was ≥ 10% and located circulating concentrations of cortisol (Littlejohn et al., 2016), within a promoter region were included in the enrichment an indicator of stress, increased production of opiate agonists analysis. Only CpG sites located within a promoter region were might be expected. Opiate agonists, however, are also known to utilized in the analysis because DNA methylation within the play a role in overriding the physiological response to a stressor, promoter region has been established to regulate gene activity. thereby, acting as a mechanism to aid in recovery from the Specifically, increased methylation of DNA within the promoter stress response (Bali et al., 2015; Valentino and Van Bockstaele, region has been reported to result in suppressed transcriptional 2015). For example, in times of stress POMC production activity and gene expression (Levine et al., 1991; Tate and Bird, increases, giving rise to both adrenocorticotropic hormone 1993). Based on this relationship, hypomethylation within gene (ACTH) and β-endorphin (Mains et al., 1977; Cawley et al., promoter regions was assumed to result in increased gene 2016). Adrenocorticotropic hormone stimulates adrenocortical expression, whereas hypermethylation within gene promoter secretion of cortisol, the product of an activated HPA axis, and regions was assumed to result in decreased gene expression. It β-endorphin binds to mu-opioid receptors in the intercalated is important to note that genes can be inhibitors or activators nucleus of the amygdala to prevent over-activation of the HPA of specific cellular processes. Genes, canonical pathways, and axis (Nakagawasai et al., 1999). Furthermore, enkephalins function groups related to the physiological control of behavior have long been known to play a role in stress adaptation (Van and stress response were identified by IPA software. Loon et al., 1990), and enkephalin knock out models result in increased anxiety-like behavior in rodents (Bérubé et al., 2014). Additionally, the Opiod Receptor Mu 1 (OPRM1) gene contained Results and Discussion a CpG site within the promoter region that was hypomethylated Previously reported alterations to behavior and stress response (MD = −0.20; Figure 1; Table 2) in PNS compared with Control bulls, phenotypes in the larger population from which calves in this suggesting increased gene expression. Mu-opioid receptor (MOR) study were derived (Littlejohn et al., 2016) suggested coinciding pathways have been associated with the stress response and Copyedited by: KD

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Table 1. Canonical pathways related to neural function, behavior, and/or stress response that were significantly altered in PNS compared with Control bull calves1

Ingenuity Canonical -log Pathways (P-value) Ratio Z-score Differentially methylated genes (P ≤ 0.05)

Opioid Signaling 8.48 0.119 0.392 RGS9,PLD2,CAMK1,POMC,AP2A2,SLC12A5,PRKCZ,TH,YES1,LCK,CACNA1E, PRKCE,ADCY8,CACNG6,GNAS,OPRM1,EGR4,CREBBP,CREB3L3,RAC1,ADC Y6,ADCY9,CACNA1B,KCNJ9,ADCY1,PENK,OPRK1,PDE1B,CDKN1B,GNAL Axonal Guidance 5.44 0.078 NaN FZD10,MMP7,NTN4,BMP2,LIMK2,NFATC1,SEMA4C,PRKCZ,NTN1,LIMK1,FG Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 Signaling FR3,PLCD3,ITGA3,RHOD,ADAM19,SMO,PRKCE,ADAM23,ERBB2,ADAMTS 5,ADAMTS4,EPHA7,GNAS,EPHA1,BMP8A,WNT2B,RAC1,TUBA4A,EPHA1 0,WNT10A,PIK3R6,BMP7,SEMA3C,WNT1,GNAL,NTN3 GABA Receptor 5.07 0.139 NaN GABRQ,CACNG6,GNAS,GABRA4,ADCY6,AP2A2,ADCY9,SLC6A11,GAD2,CA Signaling CNA1E,CACNA1B,ADCY1,ADCY8,SLC6A12 Dopamine-DARPP32 4.39 0.101 2.183 PPP1R14C,GNAS,PPP2R5D,CREBBP,CREB3L3,ADCY6,CSNK1D,DRD5,PRKCZ, Feedback in cAMP ADCY9,PLCD3,CACNA1E,PRKG1,DRD1,ADCY1,KCNJ9,PRKCE,ADCY8 Signaling CDK5 Signaling 4.31 0.126 1.732 PPP1R14C,GNAS,EGR1,PPP2R5D,ADCY6,DRD5,MAPK9,ADCY9,ITGA3,DRD1, ADCY1,ADCY8,GNAL CRH Signaling 3.74 0.101 1.291 CACNG6,GNAS,CREB3L3,CREBBP,ADCY6,POMC,PRKCZ,ADCY9,CACNA1E,C ACNA1B,NPR1,ADCY1,SMO,PRKCE,ADCY8 Huntington’s Disease 3.27 0.078 −0.302 NEUROD1,PSMF1,CREBBP,CREB3L3,MAPK9,HDAC10,HIP1,VTI1B,AP2A2,G Signaling PAA1,PRKCZ,ATP5F1E,FGFR3,DNAJC5,HDAC3,CACNA1B,PENK,CASQ1,P IK3R6,PRKCE CREB Signaling in 3.23 0.082 2.138 CACNG6,GNAS,GRIK3,CREBBP,CREB3L3,ADCY6,PRKCZ,FGFR3,ADCY9,PLCD Neurons 3,CACNA1E,CACNA1B,ADCY1,PIK3R6,PRKCE,ADCY8,GNAL,GRIK1 Agrin Interactions 3.22 0.129 0 ITGA3,DVL1,RAC1,MAPK9,ERBB3,DAG1,ERBB2,CTTN,ACTA1 at Neuromuscular Junction Dopamine Receptor 2.97 0.11 1.89 ADCY9,TH,PPP1R14C,DRD1,PPP2R5D,COMT,ADCY1,ADCY6,DRD5,ADCY8 Signaling Adrenomedullin 2.79 0.079 1.5 ADM,TFAP2C,GNAS,ADCY6,MAPK9,SHE,FGFR3,ADCY9,PLCD3,PRKG1,TFA Signaling P2A,NPR1,ADCY1,PIK3R6,RAMP3,ADCY8 Renin-Angiotensin 2.75 0.092 0.707 FGFR3,ADCY9,ADCY1,RAC1,REN,ADCY6,PIK3R6,MAPK9,PRKCE,ADCY8,S Signaling HE,PRKCZ Synaptic Long Term 2.38 0.077 0 CACNG6,GNAS,PPP2R5D,PLA2R1,PRKCZ,PLCD3,CACNA1E,PRKG1,LCAT,CA Depression CNA1B,NPR1,PLA2G4B,PRKCE,GNAL Neuregulin Signaling 2.35 0.097 −1.134 HSP90B1,ITGA3,HBEGF,PRKCE,ERBB3,ERRFI1,ERBB2,CDKN1B,PRKCZ Ephrin A Signaling 2.29 0.113 NaN EPHA7,FGFR3,EPHA10,EPHA1,RAC1,PIK3R6,LIMK1 NRF2-mediated 2.18 0.073 1.134 NQO2,CREBBP,HSPB8,MAPK9,GSTT1,PRKCZ,FGFR3,DNAJC5,ERP29,SCARB1, Oxidative Stress KEAP1,PIK3R6,PRKCE,ACTA1 Response Serotonin Receptor 2.04 0.113 NaN ADCY9,HTR6,HTR5A,ADCY1,ADCY6,ADCY8 Signaling Netrin Signaling 2.01 0.1 NaN CACNG6,PRKG1,CACNA1E,CACNA1B,RAC1,NFATC1,NTN1 P2Y Purigenic Receptor 1.98 0.076 1.508 FGFR3,PLCD3,ADCY9,CREBBP,CREB3L3,ADCY1,ADCY6,PIK3R6,PRKCE,AD Signaling Pathway CY8,PRKCZ Cardiac β-adrenergic 1.87 0.074 1.508 ADCY9,PPP1R14C,CACNA1E,GNAS,PPP2R5D,PDE1B,ADCY1,ADCY6,ADCY8, Signaling PDE11A,PKIG FGF Signaling 1.80 0.084 0.707 FGFR3,FGF4,FGF14,CREBBP,CREB3L3,RAC1,PIK3R6,FGF5 α-Adrenergic Signaling 1.75 0.083 1 ADCY9,GNAS,ADCY1,ADCY6,PRKCE,ADCY8,ADRA1A,PRKCZ RAR Activation 1.74 0.066 NaN ADCY9,BMP2,CREBBP,ADCY1,NR2F2,RAC1,ADCY6,MAPK9,PRKCE,ADCY8,P RKCZ,SMARCD3,CRABP1 NGF Signaling 1.52 0.071 0.333 FGFR3,MAP3K11,CREBBP,CREB3L3,RAC1,PIK3R6,MAPK9,SMPD1,PRKCZ Neuroinflammation 1.50 0.055 1.387 GABRQ,GDNF,GABRA4,CREBBP,CREB3L3,MAPK9,BMPR2,IDE,IRF3,NFATC1,F Signaling GFR3,SLC6A11,GAD2,PLA2G4B,KCNJ9,PIK3R6,WNT1,SLC6A12 Semaphorin Signaling 1.47 0.094 NaN RHOC,RHOD,RAC1,LIMK2,LIMK1 in Neurons Reelin Signaling in 1.42 0.076 NaN FGFR3,YES1,LCK,ITGA3,MAP3K11,PIK3R6,MAPK9 Neurons Ephrin Receptor 1.37 0.062 0 EPHA7,EPHA10,ITGA3,GNAS,EPHA1,CREBBP,CREB3L3,RAC1,LIMK2,GNAL, Signaling LIMK1

1IPA software (analyzed on 3 April 2018) was utilized to determine canonical pathways significantly altered in PNS bull calves. The analysis was conducted using differentially methylated (a ≥10% difference between treatment groups) CpG sites located within promoter regions (P ≤ 0.05). Of the 113 total canonical pathways significantly altered in PNS calves (Littlejohn et al., 2018), 28 were related to neural function, behavior, and/or stress response. Copyedited by: KD

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Table 2. Hypomethylated CpG sites located within promoter regions of genes associated with the Behavior function term (IPA software) in PNS compared with Control bull calves1,2

BTA Gene Location, Mb Methyl Diff P

13 growth hormone secretagogue receptor; GHSR 95.8 −0.22 0.01832 13 glutamate ionotropic receptor kainate type subunit 1; GRIK1 5.4 −0.12 0.01293 2 G protein-coupled receptor 39; GPR39 65.5 −0.27 0.00344 2 phosphodiesterase 11A; PDE11A 18.8 −0.25 0.00409

2 microtubule-associated protein 2; MAP2 97.8 −0.19 0.04946 Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 2 selenoprotein N, 1; SEPN1 127.9 −0.18 0.02758 23 protein tyrosine phosphatase receptor type N; PTPRN 108.0 −0.17 0.04341 23 5-hydroxytryptamine receptor 6; HTR6 133.6 −0.16 0.03218 23 T-box brain transcription factor 1; TBR1 35.1 −0.15 0.00925 23 inhibin subunit beta B; INHBB 72.5 −0.13 0.00929 4 adenylate cyclase 1; ADCY1 77.0 −0.12 0.02256 43 leptin; LEP 93.3 −0.15 0.01131 54 cyclin dependent kinase inhibitor 1B; CDKN1B 97.6 −0.24 0.02051 53 potassium voltage-gated channel subfamily H member 3; KCNH3 30.5 −0.16 0.03118 53,4 phosphodiesterase 1B; PDE1B 25.7 −0.14 0.03023 53,4 vitamin D receptor; VDR 32.5 −0.14 0.04952 63 dopamine receptor D5; DRD5 107.6 −0.15 0.00648 7 PTTG1 regulator of sister chromatid separation, securing; PTTG1 74.1 −0.13 0.00891 73 histone deacetylase 3; HDAC3 54.4 −0.11 0.03307 73,4 POU class 4 homeobox 3; POU4F3 59.5 −0.11 0.03876 84 tenascin C; TNC 106.1 −0.19 0.02080 83 adrenoceptor alpha 1A; ADRA1A 75.4 −0.20 0.04682 9 parkin RBR E3 ubiquitin protein ligase; PARK2 99.6 −0.23 0.01609 9 opioid receptor mu 1; OPRM1 92.2 −0.20 0.00125 104 dopamine receptor D1; DRD1 5.7 −0.19 0.00030 103,4 cysteine dioxygenase type 1; CDO1 4.6 −0.11 0.03595 11 fatty acid binding protein 1; FABP1 47.8 −0.29 0.03130 114 solute carrier family 1 member 4; SLC1A4 63.3 −0.21 0.01018 113 proopiomelanocortin; POMC 74.1 −0.18 0.01736 113 protein kinase C epsilon; PRKCE 27.9 −0.16 0.03505 113,4 calcium voltage-gated channel subunit alpha1 B; CACNA1B 105.4 −0.11 0.01762 123 protocadherin 8; PCDH8 10.9 −0.17 0.04747 134 DnaJ heat shock protein family (Hsp40) member C5; DNAJC5 54.4 −0.12 0.04970 133 glutamic acid decarboxylase 2; GAD2 26.9 −0.12 0.01438 133,4 GNAS complex locus; GNAS 58.0 −0.35 0.01470 133,4 adrenoceptor alpha 1D; ADRA1D 51.4 −0.11 0.03773 14 adenylate cyclase 8; ADCY8 10.8 −0.21 0.03585 144 snail family transcriptional repressor 2; SNAI2 21.6 −0.11 0.03954 143,4 preproenkephalin; PENK 25.2 −0.14 0.04169 153 apelin receptor; APLNR 81.7 −0.23 0.00044 153 adrenomedullin; ADM 42.9 −0.14 0.00546 153 paired box 6; PAX6 63.4 −0.11 0.03895 153,4 potassium voltage-gated channel subfamily C member 1; KCNC1 35.3 −0.18 0.03103 163 hes family bHLH transcription factor 5; HES5 51.6 −0.15 0.00986 163,4 ERBB receptor feedback inhibitor 1; ERRFI1 46.2 −0.13 0.03823 174 neuropeptide Y receptor Y2; NPY2R 2.1 −0.12 0.03787 173 catechol-O-methyltransferase; COMT 74.9 −0.13 0.03446 193 complement C1q like 1; C1QL1 45.3 −0.21 0.00931 193 regulator of G protein signaling 9; RGS9 62.6 −0.21 0.00880 203 T cell leukemia homeobox 3; TLX3 3.1 −0.22 0.00807 203 CART prepropeptide; CARTPT 9.8 −0.14 0.03470 203,4 glial cell derived neurotrophic factor; GDNF 36.6 −0.13 0.02902 21 creatine kinase, mitochondrial 1B; CKMT1B 55.9 −0.14 0.03775 214 cholinergic receptor nicotinic alpha 7 subunit; CHRNA7 30.2 −0.29 0.00054 213 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2; ST8SIA2 14.9 −0.17 0.02089 23 protein phosphatase 2 regulatory subunit B’delta; PPP2R5D 16.6 −0.12 0.00234 25 LIM domain kinase 1; LIMK1 33.8 −0.30 0.01825 254 Rac family small GTPase 1; RAC1 38.8 −0.25 0.02451 26 prolactin releasing hormone receptor; PRLHR 39.2 −0.22 0.00009 26 fucose mutarotase; FUOM 25.9 −0.17 0.01690 263 protein kinase cGMP-dependent 1; PRKG1 8.3 −0.13 0.03617 294 potassium voltage-gated channel subfamily Q member 1; KCNQ1 49.7 −0.15 0.03606 294 tyrosine hydroxylase; TH 50.0 −0.13 0.01405

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Table 3. Hypermethylated CpG sites located within promoter regions of genes associated with the Behavior function term (IPA software) in PNS compared with Control bull calves1,2

BTA Gene Location, Mb Methyl Diff P

1 somatostatin; SST 80.3 0.13 0.03745 2 lin-28 homolog A; LIN28A 127.3 0.12 0.02204 2 neurogenic differentiation 1; NEUROD1 15.0 0.15 0.01520 33 prostaglandin E receptor 3; PTGER3 74.5 0.19 0.04315 4 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, 114.6 0.12 0.02467 Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 subfamily d, member 3SMARCD3 4 5-hydroxytryptamine receptor 5A; HTR5A 117.7 0.20 0.03440 74 mannosidase alpha class 2B member 1; MAN2B1 14.0 0.25 0.00233 73 early growth response 1; EGR1 51.4 0.11 0.02447 93 neuromedin B receptor; NMBR 80.6 0.12 0.03940 93 protein phosphatase 1 regulatory inhibitor subunit 14C; PPP1R14C 88.4 0.12 0.03313 11 receptor accessory protein 1; REEP1 48.4 0.12 0.03986 11 RIC8 guanine nucleotide exchange factor A; RIC8A 107.2 0.14 0.00631 113 leucine rich repeat transmembrane neuronal 1; LRRTM1 55.1 0.14 0.04496 113 RAB11 family interacting protein 5; RAB11FIP5 11.3 0.15 0.03192 123 protocadherin 17; PCDH17 5.2 0.34 0.00044 133,4 solute carrier family 12, member 5; SLC12A5 75.5 0.13 0.04118 133,42 transcription factor 15; TCF15 61.0 0.24 0.00551 143,4 opioid receptor kappa 1; OPRK1 23.4 0.30 0.01213 15 sphingomyelin phosphodiesterase 1; SMPD1 47.3 0.12 0.01855 154 midkine; MDK 77.2 0.20 0.02052 153 cholinergic receptor, muscarinic 4; CHRM4 77.3 0.13 0.00031 153 progesterone receptor; PGR 8.1 0.13 0.02459 16 calcium voltage-gated channel subunit alpha1 E; CACNA1E 64.1 0.11 0.03893 16 renin; REN 1.8 0.11 0.03689 164 DNA fragmentation factor subunit alpha; DFFA 44.0 0.24 0.02651 163 protein kinase C zeta; PRKCZ 51.9 0.11 0.03226 163 disheveled segment polarity protein 1; DVL1 52.4 0.13 0.00297 163 natriuretic peptide B; NPPB 42.7 0.14 0.04007 173 chemerin chemokine-like receptor 1; CMKLR1 66.8 0.10 0.02338 173 nuclear receptor subfamily 3 group C member 2; NR3C2 9.7 0.16 0.02930 18 gigaxonin; GAN 8.0 0.11 0.01103 184 junctophilin 3; JPH3 13.2 0.10 0.03613 19 integrin subunit alpha 3; ITGA3 37.2 0.12 0.00009 193 peptide YY; PYY 44.4 0.17 0.02333 213,4 creatine kinase B; CKB 69.8 0.15 0.00779 223,4 GATA binding protein 2; GATA2 60.0 0.11 0.04886 233 N-ribosyldihydronicotinamide:quinone reductase 2; NQO2 50.5 0.42 0.02622 233,4 transcription factor AP-2 alpha; TFAP2A 45.5 0.17 0.00496 243 G protein subunit alpha L; GNAL 43.1 0.11 0.02352 243 adenylate cyclase activating polypeptide 1; ADCYAP1 36.1 0.14 0.03890 243 neuropilin and tolloid like 1; NETO1 5.4 0.21 0.02225 253,4 CREB binding protein; CREBBP 31.7 0.13 0.03924 294 insulin like growth factor 2; IGF2 50.0 0.17 0.00315 294 cholinergic receptor muscarinic 1; CHRM1 41.9 0.20 0.04241 293 protein tyrosine phosphatase non-receptor type 5; PTPN5 26.2 0.12 0.00280

1Methylation difference (Methyl Diff): defined as the average PNS methylation ratio minus the average Control methylation ratio at a site. 2Average read coverage for the listed hypermethylated sites was 11.4 and 10.1 for Control and PNS bull calves, respectively. 3DNA methylation was located within a CpG island. 4DNA methylation was exclusively located within the promoter region. the HPA axis (Chong et al., 2006). It is also well established that that was hypermethylated (MD = 0.30) in PNS compared with MOR pathways are involved in pain modulation. For example, Control bulls, suggesting decreased gene expression (Table 3). In MOR can be found on GABA interneurons that activate pain- humans, the kappa opioid receptor (KOR) pathway is involved in inhibitory neurons (Al-Hasani and Bruchas, 2011). Also, MOR pro-addictive tendencies in times of stress (Bruchas et al., 2010). signaling activates dopamine signaling pathways, thereby acting Disruption of the KOR pathway with KOR antagonists has been as mood enhancers (Al-Hasani and Bruchas, 2011; Saigusa et al., reported to suppress stress responsiveness and even serve as 2017). This may play a role in the predicted activation of both the treatment for psychiatric disorders (Van’t Veer et al., 2012; Van’t Dopamine Receptor Signaling and Dopamine-DARPP32 Feedback Veer and Carlezon, 2013). Furthermore, activation of KOR by in cAMP Signaling pathways (Table 1). The Opioid Receptor Kappa opiate agonists, such as dynorphin, has been reported to have an 1 (OPRK1) gene contained a CpG site within the promoter region inhibitory influence on dopamine pathways (Spanagel et al., 1994; Copyedited by: KD

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Figure 1. Canonical pathway (Modified from IPA) for Opioid Signaling [−log(P-value) = 8.48; Z-score = 0.392].

Chefer et al., 2005). Therefore, the predicted repression of the Membrane Hyperpolarization and Receptor Internalization, but KOR-mediated opioid pathway coincides with activation of decreased Neurotransmission, Membrane Potential, Synaptic both the Dopamine Receptor Signaling and Dopamine-DARPP32 Transmission, and Analgesic Tolerance in PNS compared with Feedback in cAMP Signaling pathways (Table 1). With regard to Control bulls (Figure 1). Alterations to various components of the the opioid signaling pathway, IPA software predicted increased opioid signaling pathway have been reported to be influenced by Copyedited by: KD

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prenatal stress (Insel et al., 1990; Poltyrev and Weinstock, 1997; and circulating glucose concentrations to be utilized for Vey et al., 2016). For example, rats whose dams were exposed energy expenditure during the stress response (Gerrard and to random instances of noise and light stress during gestation Grant, 2003). Increased cortisol has also been associated with exhibited decreased locomotion after naloxone administration an elevated temperament score in cattle (Curley et al., 2006). compared with Controls (Poltyrev and Weinstock, 1997). Furthermore, single-nucleotide polymorphisms (SNPs) within Furthermore, rats whose dams were exposed to various chronic the POMC gene have been associated with temperament stressors during gestation exhibited increased glucocorticoid in cattle (Garza-Brenner et al., 2017). Predicted increased concentrations, anxiety phenotypes, and preference for glucocorticoid production concurs with increased circulating morphine after withdrawal (Vey et al., 2016). Rats whose dams concentrations of cortisol and more excitable temperaments Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 experienced a combination of heat and restraint stress during observed in the larger population of calves from which bull gestation exhibited differential alterations to binding of opiate calves in this study were derived (Littlejohn et al., 2016). In agonists with MORs in various regions of the brain (Insel et al., addition to POMC, differential methylation of CpG sites within 1990). It is clear that the opioid signaling pathway is sensitive the promoter regions of the following genes (Figure 2B) directly to programming during prenatal development, and that complex or indirectly predicted increased glucocorticoid concentrations: interactions with multiple signaling pathways existed in PNS adrenomedullin (ADM; MD = −0.14), CART prepropeptide bulls regulating behavior and stress response. (CARTPT; MD = −0.14), potassium two pore domain channel subfamily K member 9 (KCNK9; MD = 0.10), lecithin-cholesterol Glucocorticoid Signaling acyltransferase (LCAT; MD = −0.14), protein kinase C epsilon The Corticotropin Releasing Hormone (CRH) Signaling pathway (PRKCE; MD = −0.16), scavenger receptor class B member 1 was predicted to be activated in PNS compared with Control (SCARB1; MD = −0.11), secretogranin V (SCG5; MD = 0.12), bull calves (Table 1; Figure 2). The IPA software predicted somatostatin (SST; MD = 0.13), and tyrosine hydroxylase (TH; increased corticosteroid synthesis, as mediated primarily by MD = −0.13). In addition to these genes, it is important to discuss POMC (MD = −0.18) in the CRH pathway (Figure 2A; Table 2). receptors with which glucocorticoids interact. Perroud et al. As described in the previous section, POMC gives rise to the (2014) reported differential methylation of the glucocorticoid prohormone, ACTH, which acts on the adrenal cortex to cause receptor (GR) gene (NR3C1) and mineralocorticoid receptor (MR) the production of cortisol (Charmandarie et al., 2005). Therefore, gene (NR3C2), along with decreased cortisol concentrations, hypomethylation of a CpG site within the promoter region of decreased GR, and increased MR in children whose mothers POMC is predictive of increased cortisol production (Figure 2A). were affected by the Tutsi genocide during gestation. Although Cortisol is essential in mediating the stress response (Butcher PNS bulls did not exhibit differences in NR3C1 methylation, and Lord, 2004) by increasing lipolysis, amino acid availability, they did exhibit hypermethylation (MD = 0.16) of a CpG site

Figure 2. (A) Canonical pathway (Modified from IPA) for Corticotropin Releasing Hormone Signaling [−log(P-value) = 3.74; Z-score = 1.29]. (B) Function group network (Modified from IPA) for Concentration of Corticosterone (P < 0.0001; Z-score = 2.51). Copyedited by: KD

Littlejohn et al. | 9

within the promoter region of NR3C2 compared with Control cortisol, reduced GR mRNA in the hippocampus, and increased bulls, suggesting decreased gene expression (Table 3). Cortisol POMC mRNA in the pituitary (Kapoor et al., 2008). Pigs whose is known to bind both the GR and the MR but generally has a dams were restrained by snout snare daily for 5 min between higher affinity for the MR (Rupprecht et al., 1993). It is important 84 and 112 d of gestation exhibited elevated circulating to note that GR and MR are differentially expressed in different concentrations of cortisol relative to Control pigs (Collier et al., neuron types (Koning et al., 2019). In general, GR can be found 2011). Blue foxes whose dams were exposed to 1 min of daily throughout the brain, but MR is primarily found in limbic handling stress during the last trimester of pregnancy exhibited system tissues (Seckl et al., 1991; Patel et al., 2000). Emerging heightened signs of distress, such as crawling, fighting, yelping, evidence suggests independent roles of the MR in regulating the and aggression, when handled by a human evaluator (Braastad Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 stress response (Heegde et al., 2015), such as a direct influence et al., 1998). Calves whose dams were transported for 2 h at on neurotransmission in the hippocampus and amygdala 60, 80, 100, 120, and 140 d of gestation exhibited increased in times of stress (Karst et al., 2005). Perhaps increased circulating concentrations of cortisol for a longer duration of cortisol played a role in the potential suppression of NR3C2 time in response to restraint stress and cleared a bolus dose to alter cellular sensitivity to elevations in circulating cortisol of cortisol at a slower rate compared with Control calves (Lay concentrations. It is important to acknowledge that associations et al., 1997b). It is apparent that the CRH signaling pathway is of various prenatal and early life stressors with alterations to sensitive to programming during prenatal development, and it components of the HPA axis are arguably among the most noted is important to note that observed differences in methylation of in the scientific literature and found across species (Lay et al., DNA within genes associated with the HPA axis directly relate 1997a,b; Haussmann et al., 2000; Meaney et al., 2001; Roussel to the most prominent phenotypic differences observed in the et al., 2004; Kertes et al., 2016; Spencer and Deak, 2017). Cord larger population from which bulls in this study were derived, blood, placental tissue, and maternal blood from pregnancies including increased temperament scores and circulating in which mothers experienced chronic stressors or war trauma concentrations of cortisol (Littlejohn et al., 2016). during gestation exhibited differential methylation of various CpG sites within the CRH, corticotropin releasing hormone Dopamine Signaling binding protein (CRHBP), NR3C1, and FKBP prolyl isomerase 5 The Dopamine Receptor Signaling and Dopamine-DARPP32 (FKBP5) genes (Paquette et al., 2014; Kertes et al., 2016). Guinea Feedback in cAMP Signaling pathways were activated in pigs whose dams were exposed to strobe light stressors during PNS bull calves (Table 1). Specifically, a CpG site within the gestation exhibited increased circulating concentrations of promoter region of the tyrosine hydroxylase (TH) gene was

Figure 3. (A) Canonical pathway (Modified from IPA) for Dopamine Receptor Signaling [−log(P-value) = 2.97; Z-score = 1.89]. (B) Function group network (Modified from IPA) for Concentration of Dopamine (P < 0.0001; Z-score = 0.79). Copyedited by: KD

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hypomethylated (MD = −0.13; Table 2; Figure 3A), suggesting had decreased DRD1 gene expression in the nucleus accumbens increased gene expression. Because the TH enzyme is (Zhu et al., 2010). Furthermore, young rats that were separated responsible for the conversion of a precursor hormone in from their mothers for 3-h periods had decreased mid-brain the dopamine production cascade, the predicted increased tyrosine hydroxylase-immunoreactive dopaminergic neurons as expression of TH would result in the prediction of increased juveniles (15 d of age) but increased numbers as adolescents (35 dopamine production and release from the presynaptic neuron d of age) and adults (70 d of age; Chocyk et al., 2011). Adult rats (Figure 3). At the level of the postsynaptic neuron, CpG sites whose dams were exposed to dexamethasone on days 18 and 19 within the promoter region of the dopamine receptor D1 (DRD1; of gestation had decreased dopamine and increased DRD2 gene MD = −0.19) and dopamine receptor D5 (DRD5; MD = −0.15) genes expression in the nucleus accumbens (Rodrigues et al., 2012). Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 were hypomethylated, suggesting increased gene expression Additionally, an SNP within the COMT gene was associated with (Table 2; Figure 3A). The D1-like dopamine receptors, DRD1 emotional and behavioral issues in children whose mothers and DRD5, activate adenylate cyclase; alternatively, the D2-like experienced substantial stress during gestion (Thompson et al., dopamine receptors, dopamine receptor D2 (DRD2), dopamine 2012). The present study reaffirms various reports of altered receptor D3 (DRD3), and dopamine receptor D4 (DRD4), inhibit components of the dopamine signaling pathway in prenatally adenylate cyclase (Neve et al., 2004; Xue et al., 2018). Predicted stressed mammals and suggests activation of multiple increased dopamine release from the presynaptic neuron components of the pre- and post-synaptic dopamine signaling in conjunction with predicted increased DRD1 and DRD5 on pathway. the postsynaptic neuron suggests increased activation of the postsynaptic neuron as mediated by the adenylate cyclase Serotonin Signaling cascade and ultimately phosphorylation of DARPP-32, resulting The Serotonin Receptor Signaling pathway was predicted by in inhibition of protein phosphatase-1 (PP1) and activation of IPA software to be altered in PNS compared with Control bull transcription in the nucleus (Scheggi et al., 2018; Figure 3A). calves (Table 1; Figure 4). Serotonin (5-hydroxytryptamine, 5-HT) Furthermore, SNPs within the DRD1 gene have been associated is a monoamine, specifically an indoleamine, neurotransmitter with behavioral disorders in humans (Andreou et al., 2016), and that is derived from dietary tryptophan and is involved in many SNPs within the DRD2 and DRD3 genes have been associated physiological processes, including neurobehavioral control with temperament in cattle (Garza-Brenner et al., 2017). In (Wilkinson and Brown, 2015; Sahu et al., 2018). Two serotonin addition to the aforementioned genes, differential methylation receptor genes contained differentially methylated CpG sites. of CpG sites within the promoter regions of the following genes The 5-hydroxytryptamine receptor 5A (HTR5A) gene contained a (Figure 3B) directly or indirectly predicted increased dopamine CpG site within the promoter region that was hypermethylated concentrations: adrenoceptor alpha 1D (ADRA1D; MD = −0.11), (MD = 0.20) in PNS compared with Control bulls, suggesting CARTPT (MD = −0.14), cholinergic receptor muscarinic 1 decreased gene expression (Table 3). When serotonin binds to (CHRM1; MD = 0.20), cholinergic receptor muscarinic 4 (CHRM4; HTR5A in the postsynaptic neuron, adenylate cyclase is inhibited MD = 0.13), glial cell line derived neurotrophic factor (GDNF; (Francken et al., 1998). This relationship suggests less inhibition MD = −0.13), leptin (LEP; MD = −0.15), OPRK1 (MD = 0.30), protein of adenylate cyclase by HTR5A (Figure 4). Not only was adenylate tyrosine phosphatase receptor type N (PTPRN; MD = −0.17), and cyclase predicted to be indirectly increased by suppressed TH (MD = −0.13). Additionally, a CpG site within the catechol- HTR5A, but it was also predicted to be directly increased by O-methyltransferase (COMT) gene was hypomethylated differential methylation of the 5-hydroxytryptamine receptor 6 (MD = −0.13; Table 2; Figure 3A) in PNS compared with Control (HTR6) gene. Specifically, the HTR6 gene contained a CpG site bulls, suggesting increased gene expression. The COMT enzyme that was hypomethylated (MD = −0.16) in PNS compared with is responsible for degrading catecholamines (Wilkinson and Control bulls, suggesting increased gene expression (Table 2). In Brown, 2015); therefore, increased expression of COMT would the postsynaptic neuron, HTR6 is known to increase activation potentially result in increased degradation of dopamine, of adenylate cyclase (Unsworth and Molinoff, 1994); therefore, resulting in increased circulating metabolites of dopamine increased activity of HTR6 predicts potential increased and other catecholamines. For example, dopamine metabolites activation of adenylate cyclase (Figure 4). Increased adenylate 3-methyltyramine and homovanillic acid were predicted to be cyclase is known to regulate cell activity by increasing cAMP increased by IPA software (Figure 3A). Such metabolites have needed for activation of the postsynaptic neuron. Serotonin been associated with psychiatric and behavioral disorders and signaling is well known to interact with dopamine and GABA have long been utilized as clinical indicators for disorders such signaling pathways (Fischer and Markus Ullsperger, 2017). Like as schizophrenia and depression (Bowers et al., 1969; Ogawa and dopamine and GABA signaling pathways, components of the Kunugi, 2019). Although various components of the dopamine serotonin signaling pathway have been associated with behavior signaling pathway have been associated with behavior in rodents, in humans, nonhuman primates, and cattle (Raleigh et al., 1980; humans, and cattle (Ray et al., 2006; Keltikangas-Järvinen and Knutson et al., 1998; Garza-Brenner et al., 2017). Dysfunctions in Salo, 2009; Garza-Brenner et al., 2017), dopaminergic regulation serotonin signaling have been associated with disorders such of behavior is complex and multifaceted due to the number and as anxiety (Marcinkiewcz et al., 2016), aggression (Olivier, 2004), diversity of brain regions innervated by dopaminergic neurons autism spectrum disorder (Chugani, 2002), and schizophrenia (Cools, 2008). An increasing amount of literature suggests a (Hashimoto et al., 1991). Single-nucleotide polymorphisms complex sensitivity of the dopamine signaling pathway to within the genes 5-hydroxytryptamine receptor 2A (HTR2A) and programming in utero. Rats whose dams were exposed to noise solute carrier family 18 member A2 (SLC18A2; a monoamine and light stress during gestation spent less time in open areas of transporter) have been associated with temperament in cattle a maze and exhibited altered dopamine activity in left compared (Garza-Brenner et al., 2017). Prenatal stress has been associated with right regions of the prefrontal cortex, caudate nucleus, and with alterations in serotonin receptor binding, serotonin nucleus accumbens (Fride and Weinstock, 1988). Contrary to synthesis, and associated behavioral alterations (Peters, 1986a; predictions in PNS bulls, young rats that were separated from Van den Hove et al., 2006; Miyagawa et al., 2011). Newborns their mothers daily for 6-h periods during the first 2 wk of life whose mothers experienced anxiety or depression during Copyedited by: KD

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Figure 4. Canonical pathway (Modified from IPA) for Serotonin Signaling [−log(P-value) = 2.04; Z-score = NaN].

pregnancy and took serotonin reuptake inhibitor medication (GABA), the most prevalent neurotransmitter in the brain, is an exhibited differential methylation of a CpG site within the inhibitory amino acid neurotransmitter, spanning diverse brain SLC6A4 gene (encodes for a serotonin transporter) in cord regions (Young and Chu, 1990). The glutamic acid decarboxylase blood (Non et al., 2014). Furthermore, mice whose dams were (GAD) enzyme converts the precursor glutamic acid (glutamate) exposed to daily 6-h periods of restraint between 5.5 and 17.5 to GABA (Wilkinson and Brown, 2015; Figure 5). The GAD gene d of gestation exhibited increased anxiety-like behavior and family, specifically the glutamic acid decarboxylase 2 GAD2( ) an increase in cells within the raphe nuclei (Miyagawa et al., gene, contained a CpG site within the promoter region that was 2011), the region of the brain that contains the majority of hypomethylated (MD = −0.12; Table 2; Figure 5) in PNS compared serotonin neuron cell bodies which reach throughout diverse with Control bulls, suggesting increased gene expression. As a regions of the brain (Wilkinson and Brown, 2015). Rats whose result, IPA predicted increased synthesis of GABA within the dams experienced noise and light stress during gestation presynaptic neuron (Figure 5). It is established that GABA acts exhibited decreased binding of the receptor 5-HT1A in the on GABAA receptors on the postsynaptic neuron, which consist ventral hippocampus (Van den Hove et al., 2006). Like predicted of 5 protein molecules to form an ion channel that opens upon alterations to PNS bulls, rats whose dams were exposed to a interaction with GABA to inhibit electrophysiological activity prenatal stressor (daily crowding and saline injections) were (Wilkinson and Brown, 2015). In addition to GABA, tranquilizers, more sensitive to serotonin administration, exhibiting increased anesthetics, barbiturates, and alcohol elicit their effects by behavioral and physiological responses compared with Control acting on GABAA, thereby suppressing anxious behaviors rats (Peters, 1986b). It has been established that the serotonin (Wilkinson and Brown, 2015). The γ-aminobutyric acid receptor signaling pathway is subject to changes induced by the prenatal subunit alpha (GABR-A) gene family encodes for receptors environment; specific results from this study predict increased within the GABAA family. Within the GABR-A family, the neuron sensitivity to serotonin. GABRA4 gene contained a CpG site within the promoter region that was hypermethylated (MD = 0.15), and the GABRQ gene GABA Signaling contained two CpG sites within the promoter region that were The GABA Receptor Signaling pathway was altered in PNS compared hypermethylated (MD = 0.13 and 0.10), suggesting suppressed with Control bull calves (Table 1). Gamma-aminobutyric acid gene expression of GABR-A receptors (Figure 5). Suppression Copyedited by: KD

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Figure 5. Canonical pathway (Modified from IPA) for GABA Receptor Signaling [−log(P-value) = 5.07; Z-score = NaN]. of GABR-A predicts suppression of the inhibitory effects of et al., 2011; Earnheart et al., 2007; Paredes and Aġmo, 1992; GABA on the postsynaptic neuron, which might have played a Stratton et al., 2014). Dysfunctions in GABA signaling have role in the phenotype of increased temperament scores in the been associated with neural and neurobehavioral disorders, larger population of calves in which PNS calves in this study such as depression (Stratton et al., 2014), anxiety (Berger et al., were derived (Littlejohn et al., 2016). Furthermore, due to the 2002; Stratton et al., 2014), and Attention-Deficit/Hyperactivity aforementioned differences in methylation (hypermethylation) Disorder (Bollman et al., 2015). Previous reports suggest an of the MR gene in PNS bulls, it is interesting to note that MR influence of prenatal stress on various components of the GABA and GR have been found to be differentially expressed in GABA- signaling pathway (Ehrlich et al., 2015; Lussier and Stevens, ergic (inhibitory) and glutamatergic (excitatory) neurons in the 2016; Vangeel et al., 2017). Children born to mothers with temporal cortex of humans (Koning et al., 2019). Specifically, high anxiety levels during gestation exhibited genome-wide intermediate to low expression of MR was observed in both differences in DNA methylation, including GABBR1 (GABA-B GABA-ergic and glutamatergic neurons and high expression of receptor subunit 1 gene; Vangeel et al., 2017). Mice whose dams GR was observed in glutamatergic neurons (Koning et al., 2019). underwent restraint and light stress from embryonic day 12 The GABA signaling pathway is involved in the physiological until parturition exhibited increased anxiety-like characteristics control of cognition, motor learning, fear, and behavior (Stagg and developmental delays in GABAergic cell counts (Lussier and Copyedited by: KD

Littlejohn et al. | 13

Stevens, 2016). Furthermore, rats whose dams were exposed to received daily saline injections during gestation; prenatally various gestational stressors between days 9 and 20 of gestation stressed rats exhibited decreased volume, number of neurons, exhibited anxious behaviors and altered gene expression of the and number of glial cells in the basolateral, central, and lateral GABAergic regulators, KCC2 and NKCC1 (chloride transporters; nuclei of the amygdala (Kraszpulski et al., 2006). Rats from the Ehrlich et al., 2015). Although differential methylation of GAD2 same study at 45 d of age exhibited no difference in volume, predicted increased synthesis of GABA in PNS bulls, differential number of neurons, or number of glial cells (Kraszpulski et al., methylation of 3 CpG sites within GABR-A receptors suggests 2006). Prenatal stress has been associated with decreased potential suppression of the inhibitory effects of GABA. neurogenesis in the dentate gyrus portion of the hippocampus (Lemaire et al., 2000; Coe et al., 2003), decreased hippocampal Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 Other neural, neuroendocrine, and endocrine systems volume (Schmitz et al., 2002; Coe et al., 2003), decreased Beyond the genes, signaling pathways, and function terms hippocampal weight (Szuran et al., 1994), and decreased number discussed in the preceding paragraphs, many other systems of granule cells (Lemaire et al., 2000) in the hippocampus. related to neural, neuroendocrine, and endocrine systems Increasing amounts of literature are revealing differential controlling behavior and stress response phenotypes were methylation of DNA as a regulator of neural differences in predicted to be altered in PNS compared with Control bulls due prenatally stressed individuals. For example, Provençal et al. to differential methylation (Tables 1–3). For example, a CpG site (2019) reported altered in vitro neurogenesis in response to within the promoter region of the Secretogranin V (SCG5) gene dexamethasone administration that was associated with altered was hypermethylated (MD = 0.12) in PNS compared with Control DNA methylation and transcript abundance. Furthermore, bulls, suggesting decreased gene expression. Likewise, Cao-Lei DNA methylation enrichment analyses revealed alterations et al. (2014) reported multiple CpG sites within the promoter in Neurogenesis and Neuronal Differentiation function terms region of SCG5 to be hypermethylated in children whose mothers (Provençal et al., 2019). A DNA methylation enrichment analyses experienced the Quebec ice storm of 1998. The SCG5 gene in mice whose dams were exposed to a viral challenge on day encodes for a chaperone protein that is known to transport and 9 or 17 of gestation revealed Neuronal Differentiation to be activate prohormone convertase 2 (PC2), an important enzyme the most enriched gene ontology term, significant subterms of involved in the synthesis of endogenous opioids. Furthermore, which included Gamma-aminobutyric Acidergic Differentiation, PC2 knockout mice are known to exhibit increased MOR in the Central Nervous System Differentiation, Noradrenergic System brain (Lutfy et al., 2016). Also, the Retinoic Acid Receptor (RAR) Differentiation, and Dopamine Differentiation (Richetto et al., activation pathway was predicted to be altered in PNS compared 2017). In the present study, prenatal transportation stress with Control bulls (Table 1). Specifically, the cellular retinoic acid was associated with vast differences in methylation of DNA binding protein 1 (CRABP1) gene contained a CpG site that was within genes related to neural, neuroendocrine, and endocrine hypermethylated (MD = 0.10) in PNS compared with Control pathways controlling behavior and stress response. bulls, suggesting decreased gene expression. Specifically, CRABP1 is known to increase metabolism and degradation of Conclusions retinoic acid; therefore, increased methylation of CRABP1 might suggest decreased retinoic acid (Noy, 2000). Retinoic acid is Prenatal transportation stress was associated with differential involved in nervous system development (Maden and Holder, methylation of DNA within genes related to the physiological 1991) and has been associated with behavioral alterations, such control of behavior and the stress response, which are as hyperactivity in rodents (Cai et al., 2010). Additionally, the multifaceted manifestations of complex interactions between Reelin Signaling in Neurons pathway was predicted to be altered neural, neuroendocrine, and endocrine systems. Altered by differential methylation in PNS compared with Control pathways included but were not limited to opioid signaling, CRH bulls (Table 1). Likewise, rats whose dams underwent daily signaling, dopamine signaling, serotonin signaling, and GABA restraint stress for 2-h periods between embryonic days 11 and signaling, all of which are known to interact to influence aspects 20 exhibited lower numbers of reelin-positive neurons, lower of behavior and the stress response. Association of prenatal reelin gene expression, and altered DNA methylation within transportation stress with genes in behavior and stress response– the promoter region of the reelin gene (Palacios-García et al., related signaling pathways coincided with previously reported 2015). The reelin gene is expressed during cortical development elevations in temperament scores and circulating concentrations in Cajal-Retzius cells and is involved in cortical lamination and of cortisol observed in the larger population of calves from the synaptic maturation (Palacios-García et al., 2015). In general, which bull calves in this study were derived (Littlejohn et al., IPA software predicted activation of the following function 2016). Differential methylation of DNA in different brain regions terms: Quantity of Neurons (P < 0.001; Z-score = 0.861); Quantity has been associated with differences in temperament in cattle of Nervous Tissue (P < 0.001; Z-score = 0.954); Migration of (Cantrell et al., 2019). In our study, leukocytes were used as a Neurons (P < 0.001; Z-score = 1.530); Proliferation of Neuronal generalized surrogate cell type. The use of peripheral tissues as Cells (P < 0.001; Z-score = 1.252); Outgrowth of Neurons surrogate cells for neural tissues in DNA methylation assessment (P < 0.001; Z-score = 0.843); Development of Neurons (P < 0.001; has been widely utilized, especially in human studies in which Z-score = 1.783); and Quantity of Neurotransmitter (P < 0.001; it is not feasible to collect neural tissues. A high concordance of Z-score = 0.922). Prenatal and early life stressors have been DNA methylation between blood cells and brain tissues across associated with diverse differences in neural function and CpG sites has been repeatedly reported (Horvath et al., 2012; physiology. Rats between 80 and 120 d of age whose dams were Tylee et al., 2013; Braun et al., 2019), though the correlation randomly handled in a new environment and received daily of individual CpG site methylation in blood compared with saline injections during gestation exhibited increased volume, brain tissues has been reported to be variable and potentially neuronal density, number of neurons, and number of glial dependent on factors such as gene function, gene region, CpG cells in the lateral nucleus of the amygdala (Salm et al., 2004). abundance within the gene region, and developmental stage in Contrasting results were reported in rats at 25 d of age whose which differential methylation is established (Davies et al., 2012; dams were also randomly handled in a new environment and Bediaga et al., 2017; Braun et al., 2019). More specifically, the Copyedited by: KD

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degree of differential methylation associated with prenatal or Bali, A., P. K. Randhawa, and A. S. Jaggi. 2015. Stress and early life stressors has been observed to be similar in leukocytes opioids: role of opioids in modulating stress-related compared with neural cells (Provençal et al., 2012; Seifuddin behavior and effect of stress on morphine conditioned place et al., 2017). Rhesus monkeys that experienced early life rearing preference. Neurosci. Biobehav. Rev. 51:138–150. doi:10.1016/j. neubiorev.2014.12.018. stress exhibited a significant overlap of differentially methylated Bediaga, N. G., X. Elcoroaristizabal, B. Calvo, I. Inza, A. Perez, CpG sites in T cells compared with prefrontal cortex cells A. Acha-Sagredo, M. Álvare-Álvarez, F. G. Busto, I. A. Artabe, (Provençal et al., 2012). Although the literature supports the use of R. Sevillano, J. A. Lozano, and M. M. de Pancorb. 2017. Blood leukocytes as a surrogate cell to evaluate differential methylation samples as a surrogate for brain samples in methylation Downloaded from https://academic.oup.com/jas/article-abstract/98/1/skz368/5661111 by Texas A&M College Station user on 30 April 2020 of neural tissue, it is important to acknowledge that differential studies. EC Neurology. 5.3:74–90. methylation in leukocytes serves as only a partial representation Benediktsson, R., A. A. Calder, C. R. Edwards, and J. R. Seckl. of differential methylation in neural tissues. These data from 1997. Placental 11 beta-hydroxysteroid dehydrogenase: a key bovine leukocytes warrant future evaluation of the influence of regulator of fetal glucocorticoid exposure. Clin. Endocrinol. prenatal transportation stress on specific neural tissues. Future (Oxf). 46:161–166. doi:10.1046/j.1365-2265.1997.1230939.x. studies will evaluate methylomic and transcriptomic differences Berger, M. A., V. G. Barros, M. I. Sarchi, F. I. Tarazi, and M. C. Antonelli. 2002. Long-term effects of prenatal stress in specific neural tissues of cattle exposed to prenatal stressors. on dopamine and glutamate receptors in adult rat brain. Understanding how the prenatal environment shapes postnatal Neurochem. Res. 27:1525–1533. doi:10.1023/a:1021656607278. behavior and stress response phenotypes may provide novel Bérubé, P., J. F. Poulin, S. Laforest, and G. Drolet. 2014. Enkephalin opportunities for beef cattle improvement. knockdown in the basolateral amygdala reproduces vulnerable anxiety-like responses to chronic unpredictable stress. Neuropsychopharmacology 39:1159–1168. doi:10.1038/ Acknowledgments npp.2013.316. Bollmann, S., C. Ghisleni, S. S. Poil, E. Martin, J. Ball, D. Eich- This work was supported by Texas A&M AgriLife Research, Höchli, R. A. Edden, P. Klaver, L. Michels, D. Brandeis, and Western Regional project TEX03212, Hatch project H-9022, R. L. O’Gorman. 2015. Developmental changes in gamma- Texas A&M University One Health Initiative, and United States aminobutyric acid levels in attention-deficit/hyperactivity Department of Agriculture National Institute of Food and disorder. Transl. Psychiatry. 23(5):e589. doi:10.1038/ Agriculture Award 2018-67015-28131. We also thank Dr. Keith tp.2015.79 Booher from Zymo Research Corporation. Mention of trade Bowers, M. B., Jr, G. R. Heninger, and F. Gerbode. 1969. Cerebrospinal names or commercial products in this article is solely for the fluid 5-hydroxyindoleactiic acid and homovanillic acid purpose of providing specific information and does not imply in psychiatric patients. Int. J. Neuropharmacol. 8:255–262. doi:10.1016/0028-3908(69)90046-x. recommendation or endorsement by the U.S. Department of Braastad, B. O., L. V. Osadchuk, G. Lund, and M. Bakken. 1998. Agriculture. The U.S. Department of Agriculture (USDA) prohibits Effects of prenatal handling stress on adrenal weight and discrimination in all its programs and activities on the basis of function and behaviour in novel situations in blue fox cubs race, color, national origin, age, disability, and where applicable, (Alopex lagopus). Appl. Anim. Behav. Sci. 57:157–169. doi:10.1016/ sex, marital status, familial status, parental status, religion, S0168-1591(97)00114-7 sexual orientation, genetic information, political beliefs, reprisal, Braun, P. R., S. Han, B. Hing, Y. Nagahama, L. N. Gaul, J. T. Heinzman, or because all or part of an individual’s income is derived from A. J. Grossbach, L. Close, B. J. Dlouhy, M. A. Howard, 3rd, et al. any public assistance program. (Not all prohibited bases apply to 2019. Genome-wide DNA methylation comparison between all programs.) Persons with disabilities who require alternative live human brain and peripheral tissues within individuals. Transl. Psychiatry 9:47. doi:10.1038/s41398-019-0376-y. means for communication of program information (Braille, Bruchas, M. R., B. B. Land, and C. Chavkin. 2010. The dynorphin/ large print, audiotape, etc.) should contact USDA’s TARGET kappa opioid system as a modulator of stress-induced and Center at (202) 720-2600 (voice and TDD). To file a complaint of pro-addictive behaviors. Brain Res. 1314:44–55. doi:10.1016/j. discrimination, write to USDA, Director, Office of Civil Rights, brainres.2009.08.062. 1400 Independence Avenue, S.W., Washington, DC. 20250-9410, Butcher, S. K., and J. M. Lord. 2004. Stress responses and innate or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an immunity: aging as a contributory factor. Aging Cell 3:151–160. equal opportunity provider and employer. doi:10.1111/j.1474-9728.2004.00103.x. Cai, L., X. B. Yan, X. N. Chen, Q. Y. Meng, and J. N. Zhou. 2010. 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