Gene Expression Profiling Following In Utero Exposure to Phthalate ...

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Feb 23, 2005 - Diazepam binding inhibitor. 1371519at. 0.39. Etfdh. Electron-transferring-flavoprotein dehydrogenase. 1368336at. 1.65. Fdx1. Ferredoxin 1.

BIOLOGY OF REPRODUCTION 73, 180–192 (2005) Published online before print 23 February 2005. DOI 10.1095/biolreprod.104.039404

Gene Expression Profiling Following In Utero Exposure to Phthalate Esters Reveals New Gene Targets in the Etiology of Testicular Dysgenesis1 Kejun Liu,3,4 Kim P. Lehmann,3 Madhabananda Sar,3 S. Stanley Young,4 and Kevin W. Gaido2,3 CIIT Centers for Health Research3 and National Institute for Statistical Sciences,4 Research Triangle Park, North Carolina 27709 ABSTRACT

(undescended testis), hypospadias (malformed penis), reduced fertility, and testicular cancer [1, 4]. Cryptorchidism and hypospadias are the two most common birth defects in boys, and there is growing concern that testicular cancer and male-related fertility problems are on the rise [5–7]. Testicular dysgenesis syndrome was coined to link abnormal fetal testicular development, as a result of genetic mutation or environmental disturbance, with adverse male reproductive function [8, 9]. Many of the same male reproductive tract abnormalities associated with testicular dysgenesis in humans also occur in male rats exposed gestationally to some phthalate esters [10–16]. Male rat fetuses exposed to di-(n-butyl) phthalate (DBP) or diethylhexyl phthalate (DEHP) in utero develop a number of reproductive tract abnormalities, including underdeveloped or absent reproductive organs, hypospadias, cryptorchidism, decreased anogenital distance (AGD), retained nipples, and decreased sperm production [10, 12– 15]. The fetal testes of DBP- and DEHP-exposed males are characterized by abnormal, poorly formed seminiferous cords containing multinucleated gonocytes. Mature male rats exposed to DBP in utero had smaller testes, degeneration of seminiferous epithelium, and decreased spermatogenesis [14, 17]. The effects of DBP and DEHP on male reproductive tract development are due, in part, to decreased testosterone synthesis as a result of a reduction in expression of genes involved in cholesterol transport and testosterone synthesis [12, 18–20]. However, it is unlikely that reduced androgen signaling alone can explain the myriad of effects that phthalate esters have on the developing testis, because competitive receptor antagonists do not have identical effects on the developing testis [21]. Thus, phthalates may serve as a useful class of compounds for the study of the underlying mechanisms in the development of testicular dysgenesis [13, 15]. To identify the genes and gene networks targeted in the development of testicular dysgenesis, we examined global gene expression in the fetal testis following in utero exposure to a panel of phthalate esters, which included several phthalates that have been shown to similarly disrupt male rat reproductive development and several phthalates that have been shown not to affect male rat reproductive development following in utero exposure [16, 21]. We show that developmentally toxic phthalates targeted gene pathways associated with steroidogenesis, lipid and cholesterol homeostasis, insulin signaling, transcriptional regulation, and oxidative stress. These findings confirm and extend our previously published results with DBP and show that other developmentally toxic phthalates target the same pathways, thus indicating the importance of these pathways in normal male reproductive tract development and the etiology of testicular dysgenesis.

Male reproductive tract abnormalities associated with testicular dysgenesis in humans also occur in male rats exposed gestationally to some phthalate esters. We examined global gene expression in the fetal testis of the rat following in utero exposure to a panel of phthalate esters. Pregnant Sprague-Dawley rats were treated by gavage daily from Gestational Days 12 through 19 with corn oil vehicle (1 ml/kg) or diethyl phthalate (DEP), dimethyl phthalate (DMP), dioctyl tere-phthalate (DOTP), dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), or benzyl butyl phthalate (BBP) at 500 mg/kg per day. Testes were isolated on Gestational Day 19, and global changes in gene expression were determined. Of the approximately 30 000 genes queried, expression of 391 genes was significantly altered following exposure to the developmentally toxic phthalates (DBP, BBP, DPP, and DEHP) relative to the control. The developmentally toxic phthalates were indistinguishable in their effects on global gene expression. No significant changes in gene expression were detected in the nondevelopmentally toxic phthalate group (DMP, DEP, and DOTP). Gene pathways disrupted include those previously identified as targets for DBP, including cholesterol transport and steroidogenesis, as well as newly identified pathways involved in intracellular lipid and cholesterol homeostasis, insulin signaling, transcriptional regulation, and oxidative stress. Additional gene targets include alpha inhibin, which is essential for normal Sertoli cell development, and genes involved with communication between Sertoli cells and gonocytes. The common targeting of these genes by a select group of phthalates indicates a role for their associated molecular pathways in testicular development and offers new insight into the molecular mechanisms of testicular dysgenesis.

developmental biology, male sexual function, testis, testosterone, toxicology

INTRODUCTION

Male reproductive tract development is a complex process driven primarily by fetal testicular production of multiple hormones and signaling factors [1–3]. Disturbance in the timing or concentration of these factors, either through genetic mutation or by pharmaceutical or environmental interference, can have a dramatic effect on the developing male reproductive tract and can result in cryptorchidism Supported by National Institutes of Health grant R21 ES011754-01. Correspondence: Kevin W. Gaido, CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, NC 27709. FAX: 919 558 1300; e-mail: [email protected]

1 2

Received: 22 December 2004. First decision: 20 January 2005. Accepted: 18 February 2005. Q 2005 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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GENE TARGETS IN TESTICULAR DYSGENESIS

MATERIALS AND METHODS

Animals Sprague-Dawley outbred CD rats were time-mated at Charles River Laboratories, Inc. (Raleigh, NC) and shipped to CIIT on Gestation Day 0 (GD 0), the day sperm was detected in the vaginal smear. Dams were assigned to a treatment group by body weight randomization using Provantis (Instem LSS, Stone, U.K.) to ensure equal weight distribution among groups with 10 animals in the control group and 5 animals in each of the treated groups. Animals were housed in the animal facility of the CIIT Centers for Health Research, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, in a room with controlled humidity and temperature, HEPA-filtered, mass airdisplacement. The room was maintained on a 12L:12D cycle at approximately 22 6 48C with a relative humidity of approximately 30%–70%. Animals were identified by ear tags and cage cards, and housed individually in polycarbonate cages with Alpha-dri cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet NIH-07 (Zeigler Brothers, Gardener, PA) and reverse-osmosis water were provided ad libitum. This study followed federal guidelines for the care and use of laboratory animals and was approved by the Institutional Animal Care and Use Committee at CIIT.

Study Design Dams were treated by gavage daily from GD 12 to GD 19 with corn oil vehicle (1 ml/kg; Sigma Chemical Company, St. Louis, MO) or diethyl phthalate (DEP), dimethyl phthalate (DMP), dioctyl tere-phthalate (DOTP), dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), or benzyl butyl phthalate (BBP) (Aldrich Chemical Company, Milwaukee, WI) in corn oil at 500 mg/kg per day. Purity and concentration of all doses were verified using a Hewlett-Packard 5890 gas chromatograph. The dose level was chosen based on our previous studies showing that DBP at 500 mg/kg per day produced significant changes in gene expression in the male offspring without maternal toxicity or fetal death [18–20]. In addition, a study by Gray et al. [16] indicated that doses as high as 750 mg/kg per day for DEP, DMP, DOTP, DEHP, and BBP did not induce maternal toxicity or reduced litter sizes. Dam body weights were recorded at GD 7 and daily during the dosing period. All dams were killed on GD 19 by carbon dioxide asphyxiation. In all subsequent analyses, the litter was considered the experimental unit. Fetuses were removed by cesarean delivery, weighed, and AGD was measured using a dissecting microscope and micrometer lens (accuracy 0.05 mm). All fetuses were killed by decapitation and sexed by internal examination of the reproductive organs. The right and left testes were removed from male fetuses, snap-frozen in liquid nitrogen, and stored at 2808C.

AGD Analysis A linear mixed model was used to analyze the AGD data using the R package nlme [22]. Body weight was correlated with AGD and included in the model as a quantitative covariate. A mixed model-based t-test was then constructed to test whether the mean of a treatment group was significantly different from that of the control.

Microarray Hybridization Testes from individual fetuses were homogenized in RNA Stat-60 reagent (Tel-Test, Friendswood, TX) and RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Complementary DNA was synthesized from 2 mg of total RNA and purified using the RiboAmp OA 1 Round RNA Amplification kit (Arcturus, Mountain View, CA) according to the manufacturer’s protocol. Equal amounts of purified cDNA per sample were used as the template for subsequent in vitro transcription reactions for cRNA amplification and biotin labeling using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Inc., Farmingdale, NY). Complementary RNA was purified and fragmented according to the protocol provided with the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). All GeneChip arrays (RAE230A and RAE230B) were hybridized, washed, stained, and scanned using the Complete GeneChip Instrument System according to the Affymetrix Technical Manual. A pilot study was performed to assess two sources of technical variability: chip-to-chip and labeling-to-labeling variability. Total RNA was

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isolated from the testes of a single pup and divided equally into three labeling reactions. One of these labeling reactions was then divided equally onto two chips for a total of four chips. Second and third pilot studies were performed to assess intralitter and interlitter (i.e., biological) variability in the control and DBP-exposed groups. For these studies, total RNA was isolated from eight pups representing four litters. Each RNA sample was used to generate a single labeling reaction, which was hybridized to a single chip for a total of eight chips per pilot study. For the phthalate profile study, total RNA was isolated from three individual pups, each from a different litter, from each of the eight treatment groups. RNA samples were not pooled in any way. Each individual RNA sample was then hybridized to a single chip giving a total of 24 A chips. After hybridization, the hybridization mix was removed from the A chips and immediately applied to 24 B chips. Final analysis also included chips from the pilot studies, giving a final total of 32 A chips and 28 B chips.

Microarray Analyses A robust singular value decomposition algorithm was used to summarize the expression levels based on the probe level data (unpublished results). After normalization, the first robust principal component of the perfect match was extracted and the mean of the fitted value was used as the signal intensity. A log2 transformation was performed to stabilize the variance. A separate procedure was conducted to summarize the presence or absence of expression of each probe sets within each chip. MAS 5 (Affymetrix) was used to calculate a detection P value for each probe set within each chip. A log transformation was performed on the P values to stabilize the variance. For the pilot data, a one-way analysis of variance (ANOVA) was used to estimate two variance components for each probe set: the variance due to between-litter variability, and the confounded error variance due to within-litter variability and technical variability. The estimation was performed separately on eight chips from the control and DBP-exposed groups. Three ANOVA-based models were used for the profile data to select genes with significant differences in expression under the different treatment conditions. A one-way ANOVA was used to compare overall expression across all treatment groups, a Dunnett test was used to compare each phthalate-treated group to control, and a two-way nested ANOVA was also performed to compare gene expression between the developmentally toxic group (DBP, DEHP, DPP, and BBP) and the nontoxic group (control, DEP, DMP, and DOTP). A Bonferroni adjustment was used to control for family-wise error rate due to multiple testing comparisons [23].

Real-Time, Quantitative Reverse Transcription-Polymerase Chain Reaction Total RNA was isolated from the testes of six individual fetuses from six dams from the control group, and from three individual fetuses from three dams from each of the treated groups. Subsequent reverse transcription (RT) reactions, quality control for RT reactions, and quantitative realtime polymerase chain reactions (PCRs) were performed as described previously [18, 19]. Rat-specific primers were designed (supplemental material, Table 2) using Primer Express software (Applied Biosystems, Foster City, CA) with the following parameters: low Tm 5 598C, high Tm 5 628C, optimum Tm 5 608C, amplicon length 5 75 to 150 base pairs (bp), and primer length 5 12 to 25 bp, optimum length 5 20 bp. Statistical analysis of the RT-PCR data were conducted using JMP version 5.0.1 (SAS Institute, Cary, NC). Log2-transformed relative expression ratios were calculated as described using the equation set forth by Pfaffl [24] in which efficiencies for both the gene of interest and the calibrator, glyceraldehyde-3-phosphate dehydrogenase (Gapd), were used. RT-PCR data were analyzed by the Dunnett test comparing the relative expression ratios from each treatment group to the control. The error term for the Dunnett test was generated by a one-way ANOVA. Data were also analyzed by a two-way nested ANOVA comparing the relative expression ratios from the nontoxic to the toxic group. Analyses of relative expression ratios were considered to be statistically significant if P , 0.05.

Immunohistochemistry GD19 fetal testis from control and DBP-treated rats were immersionfixed in 10% neutral-buffered formalin for 24 h and then transferred to 70% ethanol. The tissues were embedded in paraffin, sectioned at 5 mm, placed on charged slides, and stored at room temperature until processed. The sections were heated in a microwave for 3 min or 9 min in citrate

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TABLE 1. Anogenital distances (AGD) in male fetuses following phthalate exposure. Treatment Control DEP DMP DOTP DBP DEHP BBP DPP

AGD (mm)

SEM

P value

1.61 1.64 1.57 1.63 1.40 1.44 1.44 1.11

0.02 0.03 0.02 0.03 0.03 0.03 0.03 0.05

0.779 0.638 0.725 ,0.001 0.001 ,0.001 ,0.001

buffer (1:10 dilution in deionized water, pH 5.5–5.7; BioGenex, San Ramon, CA) for antigen retrieval and processed for immunostaining by the avidin-biotin peroxidase method as described previously [25]. The sections were incubated with the primary antibodies: DAX-1 (rabbit polyclonal immunoglobulin G [IgG], 1:2000), testin (rabbit polyclonal IgG, 1:400), GRB14 (goat polyclonal IgG, 1:1000), dopa decarboxylase (DDC; rabbit polyclonal IgG, 1:100), and CEBPB (goat polyclonal IgG, 1:2000) overnight at 48C. All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) except DDC, which was obtained from Novus Biologicals, and testin, which was a gift from Dr. C.Y. Cheng (Population Council, New York, NY). After incubation with the primary antibodies, the slides were washed in PBS followed by incubation with a biotinylated secondary antibody, anti-rabbit IgG or anti-goat IgG (1:200), then with avidin-biotin peroxidase (1:200) (Vector Labs, Burlingame, CA) for 30

FIG. 1. Heat map of 391 significant genes based on two-way nested ANOVA model using Bonferroni correction. Data were normalized to z-scores for each gene. Red/green indicate an increase/decrease in gene expression relative to the universal mean for each gene. Phthalates: diethyl (DEP), dimethyl (DMP), dioctyl tere- (DOTP), dibutyl (DBP), diethylhexyl (DEHP), dipentyl (DPP), and benzyl butyl (BBP).

min at room temperature [25]. The sections were then treated with liquid diaminobenzidine (BioGenex), washed in water, counterstained with hematoxylin, and mounted with Paramount. Antibody specificity was confirmed by either excluding incubation with the secondary antibody or incubating with antibody preabsorbed with the appropriate antigen. The antiCEBPB immunostaining was performed in frozen sections as described previously [25] because the antibody did not show specific immunoreaction in paraffin sections.

RESULTS

AGD Analysis

Anogenital distances were significantly reduced in male fetuses exposed to DBP, BBP, DPP, and DEHP relative to control (Table 1). DMP, DEP, and DOTP had no significant effect on AGD. These results are in agreement with previously published studies [16, 17]. Microarray Analyses

Three different statistical tests were compared for identifying significantly changed genes. A one-way ANOVA was used to detect all the probe sets with a significant difference in at least one treatment group. A post hoc Dunnett test was then performed to test the difference of the mean for each phthalate treatment group compared with that of

GENE TARGETS IN TESTICULAR DYSGENESIS TABLE 2. Classification of known genes that are significantly altered in the fetal testis after phthalate exposure.a Affymetrix ID no.

Fold change (log2)

Lipd and cholesterol homeostatis 1372973pat 20.48 1367777pat 20.21 1375852pat 20.47 1367932pat 21.03 1368189pat 20.73 1387783papat 20.37

Symbol

Lss Decr1 Hmgcr Hmgcs1 Dhcr7 Acaa1

Gene name

Aacs Cpt1b Echs1 Fdps Fdft1 Acsl4 Fads1 Fads2 Elovl5 Elovl6 ldh1 Idi1 Mvd Ebp Pnliprp2 Pcyt2 Sqle Scd1 Sc4mol Sc5d Tpi1 Ephx1 Me1 Slc25a1 Slc25a20

2,3-Oxidosqualene: lanosterol cyclase 2, 4-Dienoyl CoA reductase 1, mitochondrial 3-Hydroxy-3-methylglutaryl-Coenzyme A reductase 3-Hydroxy-3-methyiglutaryl-Coenzyme A synthase 1 7-Dehydrocholesterol reductase Acetyl-Coenzyme A acyltransferase 1 (peroxisomal 3-oxoacyl-Coenzyme A thiolase) Acetoacetyl-CoA synthetase Carnitine palmitoyltransferase 1b Enoyl coenzyme A hydratase, short chain 1 Farensyl diphosphate synthase Farnesyl diphosphate farnesyl transferase 1 Fatty acid Coenzyme A ligase, long chain 4 Fatty acid desaturase 1 Fatty acid desaturase 2 ELOVL family member 5, elongation of long-chain fatty acids ELOVL family member 6, elongation of long-chain fatty acids Isocitrate dehydrogenase 1 Isopentenyl-diphosphate delta isomerase Mevalonate pyrophosphate decarboxylase Phenylalkylamine CA21 antagonist (emoparnil) binding protein Pancreatic lipase-related protein 2 Phosphate cytidylyltransferase 2, ethanolamine Squalene epoxidase Stearoyl-Coenzyme A desaturase 1 Sterol-C4-methyl oxidase-like Sterol-C5-desaturase (fungal ERG3, delta-5-desaturase)-like Triosephosphate isomerase 1 Epoxide hydrolase 1 Malic enzyme 1 Solute carrier family 25, member 1 Solute carrier family 25 (carnitine/acylcarnitine translocase), member 20

Lipid, sterol and cholesterol transport 1368103pat 0.38 1370391pat 20.31 1398892pat 20.26 1367660pat 20.49 1368697pat 20.23 1376089pat 20.79 1386956pat 21.91 1368406 pat 22.45 1370296pat 20.17 1368840pat 20.45

Abcg1 Crabp2 re1 Fabp3 Fabp6 Ldlr Scarb1 Star Scp2 Torid

ATP-binding cassete, sub-family G (WHITE), member 1 Cellular retinoic acid-binding protein 2 Epididymal secretory protein 1 Fatty acid-binding protein 3 Fatty acid-binding protein 6 Low-density lipoprotein receptor Scavenger receptor class B, member 1 Steroidogenic acute regulatory protein Sterol carrier protein 2 TORID

Steroidogenesis 1368011pat 1386904papat 1368468pat 1387123pat 1370235pat 1371519pat 1368336pat 1368578pat 1369553pat 1387233pat 1387423pat 1387109pat

20.37 20.30 21.07 21.76 20.38 20.39 21.65 20.50 0.28 20.32 21.39 20.64

Fdxr Cyb5 Cyp11a1 Cyp17a1 Dbi Etfdh Fdx1 Hsd3b1 Hsd17b3 Hsd17b7 Lhcgr Por

Adrenodoxin reductase Cytochrome b5 Cytochrome P450, family 11, subfamily A, polypeptide 1 Cytochrome P450, family 17, subfamily A, polypeptide 1 Diazepam binding inhibitor Electron-transferring-flavoprotein dehydrogenase Ferredoxin 1 Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 Hydroxysteroid 17-beta dehydrogenase 3 Hydroxysteroid 17-beta dehydrogenase 7 Luteinizing hormone/choriogonadotropin receptor P450 (cytochrome) oxidoreductase

Transcription factors 1387087pat 1368321pat 1398333pat 1386935pat 1368488pat 1369478pat 1369148pat 1388782pat 1377013pat

20.60 0.77 20.21 0.30 0.31 20.37 20.14 0.17 20.31

Cebpb Egr1 Epas1 Nr4a1 Nfil3 Dax-1 Tcf1 Tcf21 Wbscr14

CCAAT/enhancer-binding protein (C/EBP), beta Early growth response 1 Endothelial PAS domain protein 1 Nuclear receptor subfamily 4, group A, member 1 (NGFI-B, Nurr77) Nuclear factor, interleukin 3, regulated Nuclear receptor subfamily 0, group B, member 1 (Nr0b1) Transcription factor 1 Transcription factor 21 Williams-Beuren syndrome chromosome region 14 homolog (human)

Signal transduction 1368574pat 1392664pat 1369100pat

20.30 20.90 0.45

Adra1b Admr Nalp6

Adrenergic receptor, alpha 1b Adrenomedullin receptor Angiotensin/vasopressin receptor

1368126pat 1367742pat 1367829pat 1367667pat 1367839pat 1387101pat 1367857pat 1368453pat 1388348pat 1372318pat 1369954pat 1368878pat 1368020pat 1386990pat 1387516pat 1368100pat 1387017pat 1370355pat 1368275pat 1387926pat 1367603pat 1387669papat 1370067pat 1367773pat 1398249pat

20.37 0.23 20.18 20.73 20.58 20.60 20.80 20.42 20.17 20.40 20.52 20.85 20.41 20.64 20.28 20.20 20.59 20.58 21.02 20.32 20.24 20.57 20.67 20.27 20.23

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TABLE 2. Continued Affymetrix ID no.

Fold change (log2)

1387908pat 1370203pat 1389801pat 1387889pat 1370449pat 1368618pat 1367894pat 1387124pat 1388241pat 1367682pat 1387744pat 1383294pat 1370384papat 1368495pat 1367690pat 1396101pat 1369579pat 1388750pat

20.52 20.48 20.19 20.48 20.37 0.68 20.77 21.00 21.56 0.20 20.56 21.06 21.02 20.36 20.23 0.98 21.18 20.23

Rasd1 Frag1 Fgfr4 Folr1 P2ry14 Grb14 Insig-1 Inha Insl3 Mdk Nppc Pdyn Prlr Rln1 Ssr4 Stc1 Stc2 Tfrc

Symbol

RAS, dexamethasone-induced 1 (Dexras1) FGF receptor-activating protein 1 Fibroblast growth factor receptor 4 Folate receptor 1 (adult) Purinergic receptor P2Y, G-protein coupled, 14 (Gpr105) Growth factor receptor-bound protein 14 Insulin-induced gene 1 (CL-6) Inhibin alpha Insulin-like 3 Midkine Natriuretic peptide precursor C Prodynorphin Prolactin receptor Relaxin 1 Signal sequence receptor 4 Stanniocalcin 1 Stanniocalcin-2 Transferrin receptor

Gene name

Oxidative stress 1386908pat 1389832pat 1367774pat 1370952pat 1370080pat 1368025pat 1367612pat 1367591pat 1368806pat 1370172pat 1368322pat

20.20 20.42 20.96 20.40 20.27 21.02 20.36 20.53 20.45 20.51 20.33

Glrx1 Gsto1 Gsta1 Gstm2 Hmox1 Ddit4 Mgst1 Prdx3 Sepp1 Sod2 Sod3

Glutaredoxin 1 (thioltransferase) Glutathione S-transferase omega 1 Glutathione S-transferase, alpha 1 Glutathione S-transferase, mu 2 Heme oxygenase 1 DNA-damage-inducible transcript 4 (RTP801) Microsomal glutathione S-transferase 1 Peroxiredoxin 3 Selenoprotein P, plasma, 1 Superoxide dismutase 2, mitochondrial Superoxide dismutase 3, extracellular

Cytoskeleton 1373081pat 1371530pat 1373130pat 1368093pat 1384183pat 1370697papat 1367918pat 1370165pat 1379936pat 1386931pat 1386893pat 1367592pat 1370875pat

20.22 0.28 0.64 20.72 20.27 0.26 0.32 0.21 0.36 0.26 0.33 0.77 0.20

Baiap2 Krt2-8 Myom2 Myh6 Myrip Nexn Prkcbp1 Smpx Tpm1 Tnni3 Tnni1 Tnnt2 Vil2

Brain-specific angiogenesis inhibitor 1-associated protein 2 Keratin complex 2, basic, gene 8 (keratin 8) Myomesin 2 Myosin heavy-chain, polypeptide 6 Myosin VIIA- and Rab-interacting protein Nexilin Protein kinase C-binding protein (Fez-1) Small muscle protein, X-linked Tropomyosin 1, alpha Troponin 1, type 3 Troponin I, slow isoform Troponin T2 Villin 2

Unclassified 1368378pat 1367999pat 1383469pat 1387376pat 1386998pat 1367982pat 1368512papat 1370964pat 1387925pat 1367814pat 1387331pat 1387665pat 1368523pat 1387133pat 1386921pat 1368293pat 1369852pat 1370026pat 1372299pat 1370406papat 1372031pat 1368064papat 1377064pat 1382434pat 1391272pat 1368622pat 1368307pat 1372002pat

21.03 20.82 20.43 20.50 20.44 21.01 0.48 20.82 20.24 20.24 20.60 20.24 0.31 20.77 0.59 0.21 20.51 0.22 20.81 0.19 0.27 21.14 0.39 20.52 20.25 0.28 20.32 20.36

Fthfd Aldh2 Aldh1a3 Aox1 Aldoc Alas1 Enpep Ass Asns Atp1b1 Atp4b Bhmt Cadps Calb2 Cpe Cpz F10 Cryab Cdkn1c Daf Dab2 Ddc Dusp6 Entpd5 Wfdc2 Fbp2 Ggtl3 Gja1

10-Formyltetrahydrofolate dehydrogenase Aldehyde dehydrogenae 2 Aldehyde dehydrogenase family 1, subfamily A3 Aldehyde oxidase 1 Aldolase C, fructose-biphosphate Aminolevulinic acid synthase 1 Glutamyl aminopeptidase Arginosuccinate synthetase Asparagine synthetase ATPase Na1/K1 transporting beta 1 polypeptide ATPase H1/K1 transporting, beta polypeptide Betaine-homocysteine methyltransferase Ca21-dependent activator protein for secretion Calbindin 2 Carboxypeptidase E Carboxypeptidase Z Coagulation factor 10 Crystallin, alpha B Cyclin-dependent kinase inhibitor 1C, p57 Decay-accelarating factor Disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) Dopa decarboxylase Dual-specificity phosphatase 6 Ectonucleoside triphosphate diphosphohydrolase 5 WAP four-disulfide core domain 2 Fructose bisphosphatase 2 Gamma-glutamyltransferase-like 3 Gap junction membrane channel protein alpha 1

GENE TARGETS IN TESTICULAR DYSGENESIS

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TABLE 2. Continued Affymetrix ID no.

Fold change (log2)

1371799pat 1372649pat 1370202pat 1387139pat 1367648pat 1387223pat 1387922pat 1398296pat 1367796pat 1387631pat 1369008papat 1386891pat 1379374pat 1370336pat 1371133papat 1387915pat 1367615pat 1367802pat 1390036pat 1398771pat

20.30 0.41 20.45 20.58 20.39 20.38 20.27 20.56 0.28 20.46 20.14 20.36 20.97 20.33 20.33 20.13 23.75 20.45 20.38 20.48

Gaa Hspb7 Hrasls3 Hao3 Igfbp2 Aadat Lgl1 Mir16 Mgat1 Hpgd Olfm1 Pbp Prg1 Okl38 Prkar2b Ratsg2 Svs5 Sgk Slc16a6 Slc3a2

1387057pat 1387778pat 1371358pat 1383686pat 1370672papat 1370457pat 1370881pat 1376036pat 1389253pat 1368474pat 1368854pat

20.68 20.27 20.42 20.16 20.27 0.59 20.33 20.35 20.32 20.63 20.62

Slc7a8 Sdf4 SC2 Syngr1 Dnm2 Tes Tst LOC314323 Vnn1 Vcam1 Vsnl1

a

Symbol

Gene name Glucosidase, alpha; acid (Pompe disease, glycogen storage disease type II) Heat shock 27kD protein family, member 7 (cardiovascular) HRAS-like suppressor 3 Hydroxyacid oxidae (glycolate oxidase) 3 Insulin-like growth factor-binding protein 2 Amminoadipate aminotransferase Late gestation lung protein 1 Membrane-interacting protein of RGS16 Mannoside acetylglucosaminyltransferase I NAD-dependent 15-hydroxprostaglandin dehydrogenase Olfactomedin 1-related ER localized protein Phosphatidylethanolimine-binding protein Plasticity-related gene 1 Pregnancy-induced growth inhibitor Protein kinase, cAMP-dependent regulatory, type II beta Ratsg2 Seminal vesicle secretion 5 Serum/glucocorticoid-regulated kinase Solute carrier family 16 (monocarboxylic acid transporters), member 6 Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 Solute carrier family 7 (cationic amino acid transporter, y1 system), member 8 Stromal cell-derived factor 4 Synaptic glycoprotein SC2 Synaptogyrin 1 Dynamin 2 Testis derived transcript (testin) Thiosulfate sulfurtransferase Transporter Vanin 1 Vascular cell adhesion molecular 1 Visinin-like 1

Bold indicates genes investigated further by RT-PCR.

the control. A Tukey test was then applied for all pairwise comparisons. No significant pairwise comparisons within the developmentally toxic group (DBP, BBP, DPP, and DEHP) or nontoxic group (DMP, DEP, and DOTP) were detected. Because of the high degree of similarity between the controls and the nondevelopmentally toxic phthalates and between each of the developmentally toxic phthalates, we performed a two-way nested ANOVA between the pooled control and nontoxic group and the pooled developmentally toxic group. The gene lists generated by oneway ANOVA, which identified genes with significantly different expression in at least one group relative to all other groups, and the Dunnett test, which identified genes with significantly different expression in any of the phthalatetreated groups relative to control, were determined to be subsets of the list generated by two-way nested ANOVA. Two additional filters were performed on the list: 1) duplicate probe sets representing the same gene were removed from the list, and 2) probe sets designated by Affymetrix as being capable of nonspecific hybridization to multiple transcripts were also removed from the list. The final gene list contained 391 unique probe sets (supplemental material, Table 1). A heat map was generated to compare the expression levels of the 391 genes between the different treatment groups relative to a universal mean (Fig. 1). Gene Ontology

The 391 significant unique probe sets were classified initially using the Database for Annotation, Visualization and Integration Discovery, available from the National Institute of Allergy and Infectious Diseases (available at http:// david.niaid.nih.gov/david) [26]. The probe sets were further

classified based on extensive literature searches. Gene ontology classifications with overlapping gene lists such as genes involved in lipid, sterol, and cholesterol synthesis and metabolism were combined. Genes listed in gene ontology classifications that contained three or fewer genes were grouped under the unclassified category. The list of 391 significant genes contained 225 unknown and uncharacterized transcribed sequences, which were not considered in the classification. Of the remaining 167, the largest gene ontology classification (31 genes) was of genes related to lipid, sterol, and cholesterol homeostasis (Table 2). Additional gene ontology classification groups include genes involved in lipid, sterol, and cholesterol transport (10 genes); steroidogenesis (12 genes); transcription factors (9 genes); signal transduction (22 genes); oxidative stress (11 genes); and cytoskeleton-related (13 genes). Quantitation of Gene Expression by Real-Time RT-PCR

Gene expression levels of genes from Table 2 were further examined by real-time RT-PCR. Data were analyzed by a two-way nested ANOVA, which compared the control and nontoxic phthalate groups to the toxic phthalate group and by the Dunnett test, which compared all phthalate-treated groups to the control. Of the 18 genes from Table 2 that were analyzed by RT-PCR, 16 showed a statistically significant change in expression between the toxic and nontoxic groups by two-way nested ANOVA, and 13 showed statistically significant changes in at least one treatment group by the Dunnett test (Figs. 2–4). Two genes from Table 2, decay accelerating factor (Daf) and hydroxysteroid (17-beta) dehydrogenase 3 (Hsd17b3), were not significantly different by RT-PCR in any of the treatment groups

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FIG. 2. Quantitative RT-PCR analyses of steroidogenic-related genes from control and phthalate-exposed fetuses. Gene expression values from phthalate-exposed testes are expressed relative to control values and represent the average 6 SEM from three or six separate rat fetuses from different dams per treatment group or control group, respectively. A) Hydroxysteroid (17-beta) dehydrogenase 7 (Hsd17b7), (B) luteinizing hormone/choriogonadotropin receptor (Lhcgr), (C) low-density lipoprotein receptor (Ldlr), (D) epididymal secretory protein 1 (re1). *P , 0.05 by Dunnett test comparing each treatment group to control. P , 0.05 by one-way ANOVA comparing the toxic and nontoxic groups.

by either method of statistical analysis (data not shown). Gene expression of Hsd17b7 was significantly reduced by the developmentally toxic phthalate group (Fig. 2A). Additional genes involved in regulating steroidogenesis and maintaining cholesterol homeostasis were also reduced following exposure to the developmentally toxic phthalates, including luteinizing/choriogonadotropic hormone receptor (Lhcgr; Fig. 2B), low-density lipoprotein receptor (Ldlr; Fig. 2C), and epididymal secretory protein 1 (re1; Fig. 2D (also known as Niemann-Pick disease, type C2 [npc2]). Seminal vesicle secretion 5 (Svs5) an androgen receptorregulated gene had the most significant change in expression by both microarray analysis and by RT-PCR (Fig. 3A). Several genes involved in insulin signaling were identified as significantly changed by microarray and were further investigated by RT-PCR. Insulin-induced gene 1 (Insig1; Fig. 3B) was significantly reduced by each of the developmentally toxic phthalates, whereas growth factor receptor bound protein 14 (Grb14), a negative regulator of insulin signaling, was significantly increased in all four toxic treatments by the Dunnett test. Protein kinase C binding protein 1 (Prkcbp1), a kinesin-associated protein involved in vesicular transport, and testin (Tes), a marker of germ cell-Sertoli cell junction disruption, were both induced in the developmentally toxic phthalate group as determined by two-way nested ANOVA but not significantly by any single treatment group as determined by the Dunnett test (Fig. 3, D and E). In contrast, expression of dopa decarboxylase (Ddc), which is involved in neurotransmitter synthesis and a potential androgen receptor coactivator, was significantly reduced in the developmentally toxic phthalate group as

determined by two-way nested ANOVA but not significantly in any single treatment group as determined by the Dunnett test (Fig. 3F). Several transcription factors, including nuclear receptor subfamily 0, group B, member 1 (Nr0b1, also known as Dax-1); CCAAT/enhancer binding protein beta (Cebpb); nuclear factor interleukin 3 regulated (Nfil3); nuclear receptor subfamily 4, group A, member 1 (Nr4a1, also known as NGFI-B); and transcription factor 1 (Tcf1, also known as Hnf-1a) were significantly changed in gene expression in the developmentally toxic group as determined by two-way nested ANOVA and by at least one of the toxic groups by the Dunnett test (Fig. 4). Immunohistochemistry

Immunohistochemistry was performed on fetal testicular tissue from control and DBP-exposed fetal testis. Nuclear DAX-1 expression was detected in the interstitial cell and peritubular cell populations as well as in the gonocytes in control fetal testis (Fig. 5A). DAX-1 expression was strongly reduced in the gonocyte population following DBP exposure but apparently not in the peritubular or interstitial cell compartments (Fig. 5B). In contrast, we observed a dramatic increase of testin protein levels in Sertoli cells in DBP-exposed testis relative to control (Fig. 5, C and D). Nuclear CEBPB protein was detected in the interstitial cells in control testis and its expression was reduced following DBP exposure (Fig. 5, E and F). GRB14 (Fig. 5, G and H) and dopa decarboxylase protein (Fig. 5, I and J) expression were reduced in interstitial cells in DBP-treated testis com-

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FIG. 3. Quantitative RT-PCR analyses of selected genes from control and phthalate-exposed fetuses. Gene expression values from phthalate-exposed testes are expressed relative to control values and represent the average 6 SEM from three or six separate rat fetuses from different dams per treatment group or control group, respectively. A) Seminal vesicle secreted protein 5 (Svs5), (B) insulin induced gene 1 (Insig1), (C) growth factor receptor bound protein 14 (Grb14), (D) protein kinase C-binding protein 1 (Prkcbp1), (E) testin, (F) dopa decarboxylase (Ddc). *P , 0.05 by Dunnett test comparing each treatment group to control. P , 0.05 by one-way ANOVA comparing the toxic and non-toxic groups.

pared with control testis. However, GrRB14 expression was increased in Sertoli cells of DBP-treated testis. DISCUSSION

In this study we show that four phthalates that have similar effects on the developing male rat reproductive tract (DBP, DEHP, BBP, and DPP) are indistinguishable in their effects on gene expression in the developing fetal testis. These phthalates targeted pathways directly and indirectly related to Leydig cell production of testosterone as well as additional pathways that are important for normal interaction and development between Sertoli cells and gonocytes. The nondevelopmentally toxic phthalates (DMP, DEP, and

DOTP) had no significant effects on gene expression. These results confirm and extend our previous findings on the effects of DBP on expression of genes involved in cholesterol transport and steroidogenesis, and also identify new gene targets involved in lipid and cholesterol homeostasis, oxidative stress, Sertoli cell development, and interaction between Sertoli cells and gonocytes. The common targeting of these molecular networks by reproductively toxic phthalates and not by the nontoxic phthalates suggests that these pathways should be examined in cases of human testicular dysgenesis. Normal male reproductive tract development and function requires the coordinate integration of multiple and

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FIG. 4. Quantitative RT-PCR analyses of transcriptional regulators from control and phthalate-exposed fetuses. Gene expression values from phthalateexposed testes are expressed relative to control values and represent the average 6 SEM from three or six separate rat fetuses from different dams per treatment group or control group, respectively. A) Nuclear receptor subfamily 4, group A, member 1 (Nr4a1); (B) nuclear receptor subfamily 0, group B, member 1 (Dax-1); (C) early growth response 1 (Egr1); (D) transcription factor 1 (Tcf1); (E) nuclear factor 3, interleukin 3 regulated (Nfil3); (F) CCAAT/ enhancer binding protein beta (Cebpb). *P , 0.05 by Dunnett test comparing each treatment group to control. P , 0.05 by one-way ANOVA comparing the toxic and nontoxic groups.

sometimes overlapping signaling pathways [1–3] and the investigation of single genes or gene pathways by mutational analysis, gene knockouts, or transgenics, may not necessarily provide a complete understanding of the role of any gene or gene pathway [27, 28]. Our results show that testicular dysgenesis following phthalate exposure is associated with altered expression of almost 400 gene targets in at least three different cell types (Leydig, Sertoli, gonocyte). Some of the key genes and gene pathways implicated in phthalate-induced testicular dysgenesis are presented in Figure 6. Testicular Steroidogenesis Fetal testicular testosterone production is essential for normal male reproductive tract development, and impair-

ment of fetal testicular testosterone production, or blockade of the androgen receptor, leads to cryptorchidism, hypospadias, and reduced fertility [10, 29–33]. To maintain a high level of testosterone production the fetal Leydig cell must have an abundant supply of cholesterol, which can either be imported from the extracellular space or synthesized intracellularly from lipids [34]. Previously, we showed that DBP targeted two genes (Scarb1 and Star) involved in transporting cholesterol and several genes (Cyp11a1, also known as P450scc, Hsd3b1, and Cyp17a1) involved in converting cholesterol to testosterone. In the current study we confirm our previous results with DBP and extend our findings to numerous additional genes involved in cellular processes for uptake, transport, and synthesis of sterols, lipids, and cholesterol and the conversion of cholesterol to testos-

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189 FIG. 5. Immunohistochemical analyses of DAX-1, testin, CEBPB, GRB14, and dopa decarboxylase expression in control and phthalate-exposed fetal testis. Immunohistochemical staining of GD 19 testis from control (A, C, E, G, and I) and DBP-exposed males (500 mg/kg per day) (B, D, F, H, and J). A) In control testis, gonocytes (Go) showed strong nuclear immunostaining for DAX-1 while DBP-exposed testis (B) had little or no immunostaining of gonocytes. C) Sertoli cells (S) and gonocytes (Go) showed reduced staining for testin in control testis when compared with the DBP-exposed (D) testis. Interstitial cells (IC) showed strong nuclear immunostaining for CEBPB (E), while DBP-exposed testis (F) had little or no immunostaining of interstitial cells. GRB14 immunostaining was increased in Sertoli cells following DBP exposure (H) relative to control (G). Dopa decarboxylase immunostaining was reduced in the interstitial cell compartment following DBP exposure (J) relative to control (I). Magnification 3280.

terone. We also show that other phthalates that have a similar effect on male reproductive development target the same lipid, cholesterol, and steroidogenic pathways. While the precise factor or factors that initiate and drive fetal Leydig cell testosterone production are not known, a number of factors have been identified that can either induce or inhibit Leydig cell testicular testosterone production, including several that are altered in expression following exposure to the developmentally toxic phthalates. Both angiotensin and vasopressin have been shown to inhibit testosterone production by Leydig cells [35–37] and the induction of the dual angiotensin and vasopressin receptor (Nalp6), together with aminopeptidase A, an enzyme re-

sponsible for converting angiotensin II to angiotensin III, following phthalate exposure, suggests a role for either angiotensin or vasopressin in the suppression of testosterone synthesis following phthalate exposure. Natriuretic peptide precursor type C (Npc, also known as Cnp) is a positive regulator of testosterone synthesis [38] and its expression is reduced following phthalate exposure. Luteinizing hormone receptor (Lhcgr) gene expression is also reduced following phthalate exposure, which may indicate a reduction in LH signaling. Luteinizing hormone is the primary driver of testicular testosterone production in the pubertal and adult rat [39–41]. Although LH is not believed to play a role in the initiation of fetal testicular testosterone produc-

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FIG. 6. Developmentally toxic phthalate esters target multiple pathways in the developing fetal testis. In fetal Leydig cells, molecular pathways associated with lipid and cholesterol synthesis and transport and steroidogenesis are reduced, resulting in a dramatic reduction in testosterone synthesis. Insulin-like 3 (Insl3) production by fetal Leydig cells is also reduced and this reduction is likely involved in phthalate-induced cryptorchidism [68]. A reduction in alpha inhibin production likely plays a role in altered Sertoli cell maturation and function; this altered maturation together with phthalate-induced disruption in Sertoli-gonocyte interaction likely plays a role in the development of multinucleated gonocytes. Scarb1, scavenger receptor class B, member 1; Ldlr, low-density lipoprotein receptor; Vldlr, very-low-density lipoprotein receptor; Cd36, Cd36 antigen (also known as fatty acid translocase); Lhcgr, luteinizing/choriogonadotropic hormone receptor; Cnp, Natriuretic peptide precursor type C; Nalp6, angiotensin/vasopressin receptor; Insl3,, insulin-like 3; Gja1, gap junction alpha-1; Kit, stem cell factor receptor; Fgf, fibroblast growth factor.

tion, it may be involved in regulating testosterone production in the later stages of fetal testicular development [40], although the presence of an LH-like molecule acting through this receptor in earlier stages of fetal testicular development cannot be ruled out. Additional factors peripherally associated with active steroidogenesis are also reduced. The reduction in expression of transferrin receptor, which is required for iron uptake [42], together with aminolevulinic acid, the rate-limiting enzyme in porphyrin and heme synthesis [43], and thiosulfate sulfur transferase, which is involved in the synthesis and modification of iron-sulfur containing proteins such as ferredoxin [44], are likely due to the reduced requirement for iron-containing enzymes such as the cytochrome P450s, which are involved in testosterone synthesis. Free radical formation is a normal occurrence during steroidogenesis [45] and it is likely that the reduction in expression of genes associated with protecting the cell from oxidative stress such as glutathione transferase and superoxide dismutase are due to a reduction in oxidative stress following reduction of testosterone synthesis. Cebpb, a member of a family of transcription factors that bind to a common DNA recognition site [46], is reduced in interstitial cells following exposure to developmentally toxic phthalates. Cebpb may play a role in the regulation of fetal testicular lipogenesis and steroidogenesis [46]. However, Cebpb null male mice are fertile, indicating that there may be transcriptional overlap with other Cebp family members. Cebpb also mediates cellular responses induced by environmental stressors such as ozone [47], hypoxia [48], and asbestos [49], and Cebpb may be reduced in fetal Leydig cells as a consequence of lower oxidative stress levels due to reduced steroidogenesis following phthalate exposure. Disruption of the Sertoli Cell-Germ Cell Interaction

Fetal exposure to DBP or DEHP disrupts normal seminiferous cord development [10, 12, 15–17]. The seminiferous cords are increased in diameter and malformed, nor-

mal contact between Sertoli cells and germ cells (gonocytes) is disrupted (unpublished results), and many cords contain large multinucleated gonocytes. The large multinucleated gonocytes eventually disappear postnatally, but malformed seminiferous tubules appear to be a permanent effect and the Sertoli cells in these areas appear immature [14]. We have previously shown that stem cell factor signaling is reduced following DBP exposure [18, 20]. Stem cell factor secreted by Sertoli cells is essential for normal germ cell migration, proliferation, and survival. In this study, we show that phthalates target key genes associated with normal Sertoli cell-gonocyte interaction, including testin, Gja1, Grb14, and Dax-1. Testin is a Sertoli cell secretory glycoprotein that upon secretion tightly binds to receptors on the Sertoli cell membrane at Sertoli-germ cell gap junctions [50, 51]. Gap junctions form intercellular plasma membrane channels and are required for normal interaction between Sertoli cells and germ cells [52, 53]. In gap junction protein GJA1 (also known as connexin 43) knockout mice, the fetal testes are small and have reduced number of primordial germ cells [54]. While the function of testin is not known, disruption in the Sertoli-germ cell gap junction results in a surge of testin production [55]. The reduction in Gja1 and surge in testin indicate a disruption in normal communication between Sertoli cells and gonocytes following phthalate exposure. GRB14 is an adapter protein that interferes with insulin and Fgf signaling [56–59]. Insulin signaling is required for normal testicular development [28] and both insulin-like growth factor and FGF act as germ cell growth and survival factors [1, 60, 61]. While the precise role of Grb14 in fetal testis development remains to be determined, enhanced expression of Grb14 within the seminiferous cords together with a reduction in fibroblast receptor activating protein 1 (Frag1) and fibroblast growth factor receptor 4 (Fgfr4) suggest an interference in insulin or FGF signaling between Sertoli cells and gonocytes. Transcription factor Dax-1 is dramatically reduced in

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gonocytes exposed to developmentally toxic phthalates. Dax-1 plays a central role in testicular development [62– 64]. Testis cords are disorganized and incompletely formed in Dax-1 deficient mice [65]. Sertoli cell differentiation is impaired, the number of peritubular myoid cells is reduced, and the basal lamina is disrupted, leading to incompletely formed testis cords in Dax-1 deficient mice [65]. Regions of Leydig cell hyperplasia are also apparent [65]. The similarities between Dax-1 deficiency and phthalate exposure implicate Dax-1 in phthalate-induced testicular dysgenesis. Exposure to developmentally toxic phthalates also results in a reduction in inhibin alpha gene expression. Inhibin is a heterodimeric hormone composed of alpha and beta subunits produced in the testis by fetal Leydig cells and later by Sertoli cells [66]. Sertoli cells fail to differentiate, continue to proliferate, and ultimately develop Sertoli cell tumors in inhibin alpha knockout mice [67]. The reduction in inhibin production likely plays a role in the failure of Sertoli cells to differentiate properly following phthalate exposure [14, 15]. Together, these results indicate that in addition to reducing Leydig cell testosterone synthesis, developmentally toxic phthalates also target Sertoli cells and gonocytes, and disrupt normal interactions between Sertoli cells and gonocytes, leading to malformed tubules, multinucleated gonocytes, and ultimately, reduced fertility. Expression of numerous other genes and transcribed sequences were also altered following exposure to DBP, BBP, DEHP, and DPP. The role of many of these genes in the developing fetal testis remains to be determined. However, our results demonstrate that phthalate esters may serve as useful compounds for elucidating the role of these genes in normal testicular development and testicular dysgenesis. The mechanism by which these phthalates alter gene expression also remains to be determined. Given the long-term treatment used in this study, many of the gene expression changes may be downstream events rather than direct targets of the phthalates. Studies to determine the timing and the doseresponse for gene expression changes are currently underway.

9. 10.

11. 12.

13. 14. 15. 16.

17. 18.

19. 20. 21.

22.

ACKNOWLEDGMENTS

23.

We thank Drs. Kamin Johnson, Elena Kleymenova, and Mel Andersen for their critical review of the manuscript.

24. 25.

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