Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print September 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.062950
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow [Supplemental Data]
Right arrow All Versions of this Article:
77/6/978    most recent
biolreprod.107.062950v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Johnson, K. J
Right arrow Articles by Gaido, K. W
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, K. J
Right arrow Articles by Gaido, K. W
Agricola
Right arrow Articles by Johnson, K. J
Right arrow Articles by Gaido, K. W
BIOLOGY OF REPRODUCTION 77, 978–989 (2007)
DOI: 10.1095/biolreprod.107.062950
© 2007 by the Society for the Study of Reproduction, Inc.

Mapping Gene Expression Changes in the Fetal Rat Testis Following Acute Dibutyl Phthalate Exposure Defines a Complex Temporal Cascade of Responding Cell Types1

Kamin J Johnson  2, Janan B Hensley , Michael D Kelso , Duncan G Wallace , and Kevin W Gaido 

The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709

ABSTRACT

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1–3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1–3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell.

fetal, gene expression, Leydig, Leydig cells, peritubular myoid, phthalate, rat, Sertoli, Sertoli cells, testis, toxicology

INTRODUCTION

Phthalates are a class of industrial chemicals used in a variety of consumer products, and human exposure via the skin, inhalation, or ingestion is ubiquitous [1]. In the fetal rat, high-dose-level exposure to some phthalate congeners lowers testosterone levels and produces testicular dysgenesis [2, 3]. In the neonatal primate, a decrease in blood testosterone levels is observed [4]. Along with decreased expression of the Leydig cell product insulin-like factor 3 (INSL3) [5, 6], lowered testosterone levels lead to a variety of downstream effects, including cryptorchidism and maldevelopment of male reproductive tissues [7, 8]. Within the phthalate-induced dysgenic fetal testis, focal accumulations of Leydig cells are observed interspersed among malformed seminiferous cords containing multinucleated gonocytes [3, 8]. Prepubertal phthalate exposure leads to a different testicular phenotype that is characterized by early Sertoli cell histopathology followed by germ cell apoptosis and sloughing [9]. Human phthalate exposure also may cause reproductive abnormalities; in human epidemiology studies, phthalate body burden has been correlated with alterations in sperm quality as well as testosterone-dependent endpoints [10–13], suggesting phthalate exposure may adversely affect human testicular development.

Despite approximately 50 years of mechanistic research on phthalate-induced testicular toxicity, the initial phthalate molecular and cellular targets remain unknown. In the prepubertal exposure model, the Sertoli cell was the acknowledged cellular target due to in vivo histopathology within 3 h of high-dose-level exposure and in vitro alterations in Sertoli cell function [9]. However, the rapid effects of fetal phthalate exposure on Leydig cell function have reopened the phthalate target cell debate [14]. Testicular gene profiling studies following acute fetal or prepubertal phthalate exposure have identified a conserved pattern of expression changes, with the first gene expression change detected 1 h after exposure [15, 16]. Using these genomics data as a starting point, the goal of the research reported here was to discover the fetal rat testis phthalate target cell(s) by localizing the expression of a panel of phthalate biomarker genes using in situ hybridization (ISH).

MATERIALS AND METHODS

Experimental Animals and Phthalate Exposure

This body of research incorporated the results of two phthalate exposure studies. Data shown in Figure 2 were derived from one phthalate exposure study, whereas all other data were collected from a second phthalate exposure study.


Figure 02
View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 Quantitative PCR expression analysis of potential early phthalate exposure biomarker genes. Testis mRNA levels were determined using SYBR Green-based qPCR 2 h after a single DBP exposure at dose levels ranging from 0.1 to 100 mg/kg body weight. Data are represented as the mean ± SEM. *P < 0.05.

Animal husbandry and usage were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Hamner Institutes for Health Science. Timed-pregnant Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were purchased at gestational day 12 (GD12); GD0 was defined as the day females were found with a vaginal plug. Animals were acclimatized from GD12 until study commencement, allowed unlimited access to rodent chow (NIH-07; Zeigler Brothers, Gardners, PA) and reverse-osmosis water, and housed in polycarbonate cages in the Hamner Institutes animal care facility. Within this facility, room light (12L:12D), temperature (18°C–25°C), and humidity (30%–70%) were controlled.

Allocation of animals to each study group was based upon body weight randomization. On GD19, dams were dosed via oral gavage with corn oil vehicle (Sigma-Aldrich, St. Louis, MO) or dibutyl phthalate (DBP; Aldrich Chemical Co., Milwaukee, WI) in a total volume of 1 ml/kg. Purity and concentration of all dosing solutions were verified by using a Hewlett-Packard 5890 gas chromatograph. The highest DBP dose level (500 mg/kg body weight) was chosen based upon previously defined rapid changes in gene expression [16]. At the appropriate times thereafter, dams were killed with carbon dioxide, fetuses killed by decapitation, and testes frozen in plastic tubes or Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and stored at –80°C until endpoint analysis.

Testicular Testosterone Measurement

Fetal testis testosterone concentrations were determined using a method modified from vom Saal [17]. This protocol estimated the recovery of extracted testosterone for each assay using 3H-testosterone (PerkinElmer, Wellesley, MA); for all samples, recovery estimates were 96%. Two testes per dam from different fetuses were combined for analysis. Vehicle control and 1, 10, and 100 mg/kg DBP groups contained five dams, whereas the 500 mg/kg DBP groups contained three or four dams. After testis homogenization in 100 µl PBS containing 0.1% gelatin, a 10-µl aliquot was taken to determine protein concentration using the BCA protein assay (Pierce, Rockford, IL). The remaining homogenate was extracted three times with ethylacetate and chloroform (4:1) in a total volume of 1 ml. Extracts were dried under nitrogen and resuspended in 100 µl methanol. Testosterone was measured in duplicate 25-µl aliquots using a Double Antibody-125I RIA Kit (catalog no. 07–189105; MP Biomedicals, Costa Mesa, CA). The pellet was counted in a Cobra D5005 gamma counter (Packard Instrument Co., Downers Grove, IL), and testosterone values were obtained by comparison to a standard curve. Final testosterone values for each dam were obtained by averaging the duplicate aliquot values. According to the manufacturer, cross-reactivity of the kit to other steroids is less than 5%. The lower detection limit of our assay was 0.025 ng testosterone per milliliter sera (data not shown).

Quantitative PCR

Quantitative PCR (qPCR) was performed using SYBR Green (Tel-Test Inc., Friendswood, TX; Fig. 2) and Taqman chemistries (Applied Biosystems, Foster City, CA; Fig. 3). With SYBR Green, total RNA was isolated from fetal testes using STAT-60 reagent following the manufacturer's protocols. With Taqman chemistries, total RNA was isolated from fetal testes using an RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocols. Subsequent reverse transcription reactions, quality control for reverse transcriptase reactions, and SYBR Green or Taqman qPCR reactions were performed as described [15, 18]. SYBR Green and Taqman primers are shown in Tables 1 and 2. Analysis of the qPCR data was conducted using the equation set forth by Pfaffl [19], in which efficiencies were used for both the gene of interest and the calibrators (glyceraldehyde-3-phosphate dehydrogenase or 18S ribosomal RNA). The average Ct of samples run in duplicate or triplicate was used to establish expression relative to the calibrator. For data shown in Figure 2, testes from the fetuses of six control dams and four DBP-treated dams per dose level were analyzed. For Figure 3 data, the number of dams within each group was: controls (n = 11); 1, 10, and 100 mg/kg DBP (n = 5); and 500 mg/kg DBP (n = 3 or 4).


Figure 03
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 Quantitative PCR expression analysis of potential early phthalate exposure biomarker genes. Messenger RNA levels of these genes were determined using Taqman-based qPCR at 1, 3, or 6 h after a single DBP exposure at dose levels of 1, 10, 100, or 500 mg/kg. Data are expressed relative to control (set to 1) and depicted as the mean ± SEM. *P < 0.05.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1 SYBR Green qPCR primer sequences.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 2 qPCR Taqman Assays.

The names of genes analyzed were: Btg2 (B-cell translocation gene 2); Cdh1 (cadherin 1); Cebpd (CCAAT/enhancer-binding protein [C/EBP], delta); Ctgf (connective tissue growth factor); Cx3cl1 (chemokine [C-X3-C motif] ligand 1); Cxcl1 (chemokine [C-X-C motif] ligand 1); Cxcl10 (chemokine [C-X-C motif] ligand 10); Cyp17a1 (cytochrome P450, family 17, subfamily a, polypeptide 1); Dhcr7 (7-dehydrocholesterol reductase); Dusp6 (dual-specificity phosphatase 6); Edg3 (endothelial differentiation sphingolipid G-protein-coupled receptor 3); Edn1 (endothelin 1); Egr1 (early growth response 1); Egr2 (early growth response 2); Fabp3 (fatty acid-binding protein 3); Fos (FBJ murine osteosarcoma viral oncogene homolog); Fzd2 (frizzled homolog 2); Grb14 (growth factor receptor-bound protein 14); Ier3 (immediate early response 3); Junb (Jun-B oncogene); Kit (kit oncogene); Ldlr (low-density lipoprotein receptor); Map3k8 (mitogen-activated protein kinase kinase kinase 8); Nfkb1 (nuclear factor of kappa light chain gene enhancer in B cells 1); Nfkb2 (nuclear factor of kappa light polypeptide gene enhancer in B cells 2); Nfkbia (nuclear factor of kappa light chain gene enhancer in B cells inhibitor, alpha); Nr0b1 (nuclear receptor subfamily 0, group B, member 1); Nr4a1 (nuclear receptor subfamily 4, group A, member 1); Nr4a3 (nuclear receptor subfamily 4, group A, member 3); Star (steroidogenic acute regulatory protein); Stat3 (signal transducer and activator of transcription 3); Stc1 (stanniocalcin 1); Stc2 (stanniocalcin 2); Thbs1 (thrombospondin 1); Tnfrsf1a (tumor necrosis factor receptor superfamily, member 1a); Tnfrsf11b (tumor necrosis factor receptor superfamily, member 11b); Tnfrsf12a (tumor necrosis factor receptor superfamily, member 12a); Wnt4 (wingless-related MMTV integration site 4); and Zfp36 (zinc finger protein 36).

Statistical Analysis

Statistical significance (P < 0.05) of the qPCR and testosterone data was determined in Prism4 software (GraphPad Software Inc., San Diego, CA) by one-way ANOVA and a Dunnett post-hoc test.

In Situ Hybridization

Unless stated otherwise, all ISH reagents and labware were obtained from Roche Applied Science (Indianapolis, IN) or Sigma Chemical Co. (St. Louis, MO). ISH was performed on 10-µm fresh-frozen testis cryosections using digoxigenin-labeled riboprobes of approximately 700-base pair lengths. Cryosections from control and 1-, 3-, and 6-h DBP-treated animals were placed on a single glass slide; within each control or treatment group, testes from two to four litters were examined. All treated animals examined by ISH were exposed to 500 mg/kg DBP. In vehicle control animals, no ISH differences were observed among animals exposed to corn oil for 1, 3, or 6 h (data not shown). To produce the riboprobe templates, a PCR fragment was generated using fetal rat testis cDNA and Expand Hi Fidelity polymerase. PCR primer sequences for each riboprobe are shown in Table 3. After cloning the PCR fragment into pCRII-TOPO (Invitrogen, Carlsbad, CA), the identities and orientations of the cloned fragments were determined by restriction analysis and DNA sequencing (data not shown). Using BamHI- or EcoRV-linearized and agarose gel-purified plasmid DNA as a template, sense and antisense digoxigenin-labeled riboprobes were generated using SP6 or T7 RNA polymerase and a DIG RNA Labeling Kit. After DNaseI digestion, riboprobes were purified using a mini Quick Spin RNA Column and quantified.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 3 ISH template generation primer sequences.

A detailed description of the ISH protocol is available online (see Supplementary Data at www.biolreprod.org). Unless stated otherwise, all ISH steps were performed at room temperature. Cryosections were fixed in PBS containing 4% paraformaldehyde for 10 min, washed in PBS, and acetylated for 10 min in 10 mM triethanolamine (pH 8.0) containing 0.25% acetic anhydride. After washing in PBS, sections were dehydrated in a series of 70%–100% ethanol solutions. The remaining ISH steps were performed using a liquid handling robot with a slide holder designed for ISH (Tecan Genepaint System, Zurich, Switzerland) [20]. Endogenous peroxidases were quenched with 0.7% hydrogen peroxide in methanol for 25 min, and sections were exposed to 0.2 N hydrochloric acid for 10 min, followed by prehybridization buffer (50% formamide, 5x sodium chloride/sodium citrate [SSC], 0.01 mg/ml transfer RNA, and 0.1 mg/ml heparin). Next, sections were incubated in Ambion (Austin, TX) In Situ Hybridization Buffer (catalog number B8807G) for 15 min, followed by another 15 min at 64°C. After dilution to 300 ng/ml in hybridization buffer containing 0.1 mg/ml dithiothreitol and 0.01 mg/ml transfer RNA, riboprobes were denatured by incubation at 80°C for 30 sec, followed by 64°C for 10 min. Riboprobes were hybridized at 64°C for a total of 5.5 h, with a second riboprobe aliquot added after 2.5 h. All stringency washes were performed at 62°C and consisted of 5x SSC (25 min), 2x SSC with 50% formamide (50 min), 1x SSC with 50% formamide (60 min), and 0.1x SSC (32 min). Next, sections were incubated in the following solutions: 1) NTE (5 mM EDTA, 10 mM Tris, 500 mM NaCl, pH 8.0) containing 20 mM iodoacetamide for 20 min; 2) 4% normal sheep serum (Invitrogen) in TNT (100 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween-20) for 60 min; 3) TNB (TNT containing 0.5% Blocking Reagent; catalog number FP1020; Perkin-Elmer Life Sciences, Boston, MA) for 20 min; 4) maleate wash buffer (100 mM maleate, pH 7.5, 150 mM NaCl) for 10 min; 5) maleate wash buffer containing 1% Blocking Reagent (Roche catalog number 1096176) for 20 min; and 6) TNB for 40 min. Digoxigenin on the sections was detected with sheep anti-DIG-POD antibody (Roche catalog number 1207733) diluted 1:500 in TNB and incubated for 60 min.

The anti-DIG-POD antibody was detected with tyramide signal amplification coupled to either a fluorescent or colorimetric output. All data shown in this manuscript are from fluorescence-based signal detection, but colorimetric detection produced similar results (data not shown). For fluorescent detection, the TSA-Plus Cyanine 3 System (Perkin-Elmer Life Sciences) was used according to the manufacturer's protocol. After postfixing in PBS containing 4% paraformaldehyde, sections were mounted with Vectashield containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Fluorescent sections were viewed with an Axiovert200 epifluorescent microscope and digital images captured at 10x, 20x, or 40x magnification via an Axiocam HRm camera (Carl Zeiss Inc., Thornwood, NY). All of the ISH images for a given gene shown in Figures 5–7 were captured with the same exposure time, with the exception of Egr1 and Fos in 1-h DBP samples. Because of the intense signal in these two samples, the exposure times were reduced relative to vehicle control and 3- and 6-h DBP exposure. ISH data for Junb and Ctgf are not shown because of technical difficulties in generating this information.


Figure 05
View larger version (52K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 In situ hybridization of genes induced predominantly in Sertoli cells. All treated animals were exposed to 500 mg/kg DBP. Analysis of Edn1 (A–D), Tnfrsf12a (E, F), and Ier3 (G–I) is shown. Edn1 sense riboprobe (A) produced a general background signal with somewhat increased interstitial cell signal (arrowhead) and was similar to vehicle control testes probed with an Edn1 antisense riboprobes (B). DBP exposure for 3 h induced Edn1 expression in seminiferous cords (C; arrowhead). In D, higher magnification of a single seminiferous cord at this exposure time point stained with DAPI (blue) and Edn1 antisense riboprobe (red) confirmed expression in Sertoli cells (asterisks denote Sertoli cell nuclei and arrowhead denotes peritubular myoid cell nuclei). Tnfrsf12a expression in vehicle control samples was observed in Sertoli cells (E; arrowheads), and DBP exposure for 3 h dramatically increased expression in Sertoli cells (F; arrowhead). Weak Ier3 Sertoli cell expression in vehicle control testes was observed (G; arrowhead), which was increased 3 and 6 h after DBP exposure (arrowheads in H and I); DBP exposure also induced Ier3 expression in a subset of interstitial cells (arrows in H and I). Scale bar in A equals 150 µm and is the same for all other panels except D, in which the scale bar equals 10 µm.


Figure 07
View larger version (80K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 7 In situ hybridization of genes induced in a subset of interstitial cells as well as peritubular myoid cells. All treated animals were exposed to 500 mg/kg DBP. Analysis of Nr4a1 (A–F), Cxcl10 (labeled cxcl10 in I), (G–J), and Stc1 (K–N) is shown. In vehicle control testes, no specific signal was seen for Nr4a1 (A) or Cxcl10 (G); however, specific interstitial Stc1 expression using the antisense riboprobe was observed (arrow in L) compared with vehicle control testes probed with the Stc1 sense riboprobes (K). At 1 h of DBP exposure, Nr4a1 expression was increased in the interstitial compartment (B; arrow). An adjacent cryosection immunostained with CYP11A1 (Leydig cells) and laminin (seminiferous cords) antibodies is shown (C). At 3 h of DBP exposure, interstitial cell expression of Nr4a1 (D; arrow), Cxcl10 (H; arrow), and Stc1 (M; arrow) was seen. In I, an overlay is shown of the 3 h DBP Cxcl10 signal (red) with Leydig cell-specific CYP11A1 immunostaining (green) from an adjacent cryosection. For Nr4a1 and Cxcl10, peritubular myoid cell expression also was observed at 3 h of DBP exposure (D and H; arrowheads). In E, peritubular cell expression of Nr4a1 (red) was confirmed by colocalization with DAPI (blue); arrowheads denote the location of two peritubular myoid cell nuclei. Six hours after DBP exposure, Nr4a1 expression remained in interstitial cells (F; arrow) but not in peritubular myoid cells; Cxcl10 expression was still seen in interstitial cells (J; arrow) and peritubular myoid cells (J; arrowhead); and maximal interstitial cell Stc1 expression was observed (N; arrow). Scale bar in A equals 150 µm and is the same for all other panels except E, in which the scale bar equals 50 µm.

Immunostaining

Fluorescent immunostaining was performed at room temperature (except where noted) as previously described [21]. In brief, 7-µm cryosections were fixed for 10 min in PBS containing 4% paraformaldehyde, blocked for 30 min in PBS containing 5% normal goat serum and 0.1% bovine serum albumin (blocking buffer), incubated overnight at 4°C in primary antibody diluted in blocking buffer, incubated in secondary antibody for 45 min diluted in blocking buffer, mounted, viewed, and images captured as for fluorescent ISH. Primary antibodies and dilutions were as follows: mouse anti-laminin (catalog number MAB1920; Chemicon International Inc., Temecula, CA) diluted 1:5000; rabbit anti-CYP11A1 (catalog number AB1294; Chemicon International) diluted 1:500; mouse anti-alpha smooth muscle actin (clone 1A4; Sigma Chemical Co.) diluted 1:200; mouse anti-rat macrophage (clone F-6-J; Abcam, Cambridge, MA) diluted 1:100; mouse anti-rat macrophage (clone Ki-M2R; BMA Biomedicals, Augst, Switzerland) diluted 1:200; and mouse anti-CD163 (clone ED2; BMA Biomedicals) diluted 1:200. Secondary antibodies and dilutions were goat anti-mouse Alexa594 and goat anti-rabbit Alexa488 (Invitrogen) diluted 1:500. The laminin antibody binds to the tunica albuginea and the basement membranes of both blood vessels and seminiferous cords. To remove unwanted laminin signal and show only the vasculature or seminiferous cords, the clone stamp tool in Photoshop 6.0 (Adobe Systems Inc., San Jose, CA) was employed using the signal from the DAPI channel as a guide. In a similar manner, signals from the tunica albuginea and vasculature were removed from the alpha-smooth muscle actin signal to show only peritubular myoid cells.

RESULTS

Testicular Testosterone Levels

Because phthalates can decrease fetal rat testis steroidogenesis, testis testosterone levels were examined at various time points up to 6 h after a single DBP gavage exposure (Fig. 1). Although 500 mg/kg DBP exposure produced a trend toward reduced testosterone levels at 6 h, no consistent, time-dependent decrease in testosterone was observed. This equivocal result was corroborated by an overall lack of reduced expression for Star, the rate-limiting steroidogenic gene (see below). Dose levels below 500 mg/kg either had no significant effects or suggested a transient increase in testicular testosterone.


Figure 01
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 Dose and time response of GD19 testis testosterone levels after DBP exposure. Testosterone level in the control group (mean, 0.2384; SEM, 0.05382) is represented by the dashed line. At 1, 3, or 6 h after a single DBP dose of either 1, 10, 100, or 500 mg/kg body weight, total testosterone/testis was determined. Data are represented as the mean ± SEM. *P < 0.05.

Identification of Phthalate Biomarker Genes

Data from two phthalate genomics studies were used as a starting point to identify biomarker genes [15, 16]. Genes for qPCR-based dose and time response experiments were chosen based upon robust testis expression following acute fetal and/or prepubertal phthalate exposure, their broad spectrum of cellular functions, and their potential importance in the phthalate toxicity mechanism. From these results, 34 candidate genes with increased expression within 12 h after phthalate exposure were chosen for further analyses. In addition, five genes with reduced expression (Cyp17a1, Dhcr7, Nr0b1, Star, and Stc2) also were examined to determine their temporal and dose response in relationship to candidate biomarker genes.

Quantitative PCR analysis of a subset of these candidate genes 2 h after gavage exposure to DBP dose levels ranging from 0.1 to 100 mg/kg identified eight genes with significantly increased and dose-dependent expression: Ctgf, Egr1, Fos, Nr4a1, Nr4a3, Stc1, Thbs1, and Tnfrsf11b (Fig. 2). At the highest dose level, mRNAs for these genes were increased approximately 2- to 8-fold. While most of these eight genes were increased within 2 h only after 100 mg/kg DBP exposure, significantly higher levels of Nr4a1 and Thbs1 were noted at 30 mg/kg and 1 mg/kg, respectively. Tnfrsf12a and Cxcl1 were induced 2- to 4-fold at DBP dose levels at or above 10 mg/kg, but the large experimental variation precluded statistical significance. Expression changes of all other genes were either insignificant or were not dose dependent.

In conjunction with the 2-h dose-response analysis, expression of 19 candidate biomarker genes was analyzed at 1, 3, and 6 h after exposure to 1, 10, 100, and 500 mg/kg DBP (Fig. 3). As a whole, the gene expression pattern at these dose levels and time points corroborated data shown in Figure 2. Regarding the interplay of dose level and time, significantly increased gene expression generally was delayed at lower dose levels. At 1 mg/kg DBP, none of the genes displayed significantly increased expression at any time point; the biologic relevance of significantly reduced expression for some genes 1 h after 1 mg/kg DBP exposure remains to be determined.

Leydig cell-specific genes known to be reduced hours after fetal rat phthalate exposure (Cyp17a1, Dhcr7, and Star) [16] also showed reduced expression here (Fig. 3). For Cyp17a1 and Dhcr7, significant mRNA level reductions were observed 3 h after 100 and 500 mg/kg DBP. DBP effects on Star expression were variable, being reduced significantly at 10 and 100 mg/kg but not at 500 mg/kg.

With the exception of Fos, the criteria used to identify biomarker genes were as follows: 1) greater than 2-fold significantly increased expression at any time point following 10 mg/kg DBP exposure or significantly increased expression by 3 h after 100 mg/kg DBP; and 2) similar dose and time response after both fetal and prepubertal phthalate exposure [15, 16]. Using these standards, 11 phthalate biomarker genes were culled from the original list of 34 candidate genes. These were Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a. Although Fos was not examined in the data reported in Figure 3 for technical reasons, it was added to the list of biomarker genes because of its robust and early expression after both high-dose-level fetal and prepubertal phthalate exposure and its induction 2 h after fetal 100 mg/kg DBP exposure [15, 16].

Fetal Rat Testis ISH of Phthalate Biomarker Genes

To help identify the cellular origins of ISH patterns, the patterns of fetal rat testis landmarks at GD19 were examined (Fig. 4). Using an antibody against alpha-smooth muscle actin, peritubular myoid cells were observed surrounding seminiferous cords (Fig. 4A). This pattern was similar to that of the seminiferous cord basement membrane visualized by laminin immunostaining (Fig. 4B). Generally, CYP11A1 immunostaining (which detects Leydig cells) was found within the central portion of the testis as well as surrounding seminiferous cords (Fig. 4, A–C). Testis vasculature was interspersed throughout the tissue, including areas between seminiferous cords, but large vessels were concentrated just under the tunica albuginea (Fig. 4B). Using two antibodies against rat macrophages, no antibody-positive macrophages were observed in GD19 rat testis (Fig. 4C), although numerous macrophages were detected in the interstitium of prepubertal rat testis (Fig. 4D).


Figure 04
View larger version (136K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 Morphology of cellular landmarks in the fetal testis. Testes were immunostained with cell-specific or compartmental marker antibodies to show the distribution of cellular features. A–C) GD19 testes stained with antibodies for alpha-smooth muscle actin (detecting peritubular myoid cells), CYP11A1 (labeling Leydig cells), laminin (to localize the seminiferous cord basement membrane and vasculature), and macrophage markers (using Ki-M2R and ED2 antibodies). D) PND28 testis stained with antibodies for CYP11A1 (labeling Leydig cells) and macrophage markers (using Ki-M2R and ED2 antibodies). Bar = 75 µm.

Following phthalate exposure, gene induction was observed in five compartmental patterns, including Sertoli cell, peritubular myoid cell, a sparse interstitial pattern, a relatively ubiquitous interstitial pattern, and an interstitial Leydig cell pattern. Within the time frame of the experiments, no changes in testis histology after DBP exposure were apparent by examining DAPI-stained cryosections. Sense negative controls were performed for all genes at all time points and produced a diffuse background appearance with the occasional brighter interstitial area; representative negative control ISH signals using the Edn1 and Stc1 sense probes are shown in Figures 5A and 7K. In testis cryosections from vehicle control animals, expression of Tnfrsf12a and Ier3 was detected in Sertoli cells (Fig. 5, E and G), but no specific Edn1 signal was seen (Fig. 5B). For Edn1 and Tnfrsf12a, DPB-induced gene expression was confined to Sertoli cells beginning 3 h after exposure (Fig. 5, C and F). Ier3 showed similar Sertoli cell expression kinetics to Edn1 and Tnfrsf12a, but signal was observed in some interstitial cells (Fig. 5, H and I). Sertoli cell expression in controls and DBP-treated testes was confirmed by colocalization with DAPI-stained nuclei. A ring of Sertoli cell nuclei around the periphery of seminiferous cords was obvious, and Edn1, Ier3, and Tnfrsf12a signals were juxtaposed to Sertoli cell nuclei. The Sertoli cell Edn1 pattern colocalized with DAPI is shown in Figure 5D. Six hours after DBP exposure, Sertoli cells still expressed increased levels of Edn1, Ier3, and Tnfrsf12a, but expression was much weaker compared with 3 h DBP (Fig. 5I and data not shown).

Peritubular myoid cells responded immediately following DBP exposure by increasing expression of Thbs1, Dusp6, Egr1, and Fos. In testes from vehicle control animals, peritubular myoid cells produced Thbs1, Egr1, and Fos (Fig. 6, A, H, and L). One hour after DPB dosing, Thbs1 and Dusp6 were increased only in peritubular myoid cells (Fig. 6, B and F), whereas Fos and Egr1 were induced in these cells as well as interstitial cells (Fig. 6, I and M). The Fos and Egr1 interstitial cell pattern was spread throughout the compartment but did not extend to the tunica albuginea. At 3 h, the Thbs1 and Dusp6 peritubular myoid cell signals were maximal (Fig. 6, C and G). Colocalization of the Thbs1 pattern with DAPI confirmed expression in peritubular myoid cells (Fig. 6D). Similarly, expression of Thbs1, Dusp6, Egr1, and Fos within peritubular myoid cells was verified for all vehicle and DBP-exposed groups by colocalization with DAPI fluorescence (data not shown). Six hours after DBP exposure, peritubular myoid cell expression of all four genes returned to levels seen in vehicle control testes (data not shown); however, Egr1 expression in the interstitium remained elevated (Fig. 6K).


Figure 06
View larger version (76K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 In situ hybridization of genes induced widely in interstitial cells and peritubular myoid cells. All treated animals were exposed to 500 mg/kg DBP. Analysis of Thbs1 (A–D), Dusp6 (E–G), Egr1 (H–K), and Fos (L–N) is shown. In vehicle control testes, Thbs1 (A), Egr1 (H), and Fos (L) displayed peritubular myoid cell expression (arrowheads). No specific signal was seen with Dusp6 antisense in vehicle control testis (E). One hour following DBP exposure, increased expression of all four genes was observed in peritubular myoid cells (arrowheads in B, F, I, and M). For Egr1 and Fos, widespread expression in the interstitial compartment was seen at 1 h of DBP exposure (arrows in I and M). Three hours following DBP exposure, peritubular myoid cell expression of Thbs1 and Dusp6 was maximal (arrowheads in C and G). Peritubular myoid cell expression of Thbs1 at 3 h was confirmed by colocalization with DAPI; in D, a high-magnification image of a seminiferous cord shows Thbs1 ISH signal (red) surrounding DAPI-stained (blue) peritubular myoid cell nuclei (arrowhead) but not Sertoli cell nuclei (asterisks). Induction of Egr1 (J) and Fos (N) expression was still observed 3 h after DBP exposure in the interstitium (arrows) and peritubular myoid cells (arrowheads). Six hours after exposure, Egr1 expression was still elevated in interstitial cells (arrow in K). Scale bar in A equals 150 µm and is the same for all other panels except D, in which the scale bar equals 10 µm.

Unlike other genes examined, Nr4a1 and Cxcl10 displayed expression predominantly within a relatively sparse interstitial cell population. At some time points following DBP exposure, peritubular myoid cell induction also was seen. In vehicle controls, Nr4a1 and Cxcl10 ISH patterns were indistinguishable from sense controls (Fig. 7, A and G). One hour after DBP exposure, no change in Cxcl10 was seen (data not shown), but Nr4a1 was induced in a subset of interstitial cells (Fig. 7B). This pattern was similar to that of Leydig cells (observed by CYP11A1 immunostaining of an adjacent section; Fig. 7C). By 3 h of DBP exposure, Cxcl10 was induced in interstitial cells (Fig. 7H) and, similarly to Nr4a1, this Cxcl10 pattern was confined to areas containing CYP11A1-positive Leydig cells (Fig. 7I). For both Cxcl10 and Nr4a1, peritubular myoid cells showed expression beginning at 3 h (Fig. 7, D, E, and H). At 6 h of DBP exposure, peritubular myoid cell expression still was observed for Cxcl10 but not Nr4a1, and interstitial cell expression of both genes remained (Fig. 7, F and J).

A pattern typical of Leydig cells was observed for the DBP-induced expression of Stc1. In vehicle control animals, Stc1 localized to interstitial cells, with a portion of these cells apparently expressing increased amounts of Stc1 (Fig. 7L). No change from control was seen at 1 h DBP (data not shown), but an obvious Stc1 induction in interstitial cells was seen at 3 and 6 h following DBP gavage (Fig. 7, M and N). The distribution pattern of these Stc1-positive interstitial cells mirrored that of CYP11A1-positive Leydig cells.

For all genes except Stc1, high-magnification images showing colocalization of the ISH signal with DAPI are available; in addition, representative low-magnification ISH images of all genes at all time points are available (see Supplementary Figs. 1 and 2, available at www.biolreprod.org).

DISCUSSION

The first consistently observed molecular event after in vivo rodent phthalate exposure is an alteration of testicular gene expression [16, 22]. Following oral gavage of a pregnant rat, the fetal testis contains detectable phthalate monoester metabolite within a few minutes [23], and microarray studies following high-dose-level exposure have identified a subset of genes responding with increased expression by 1–6 h [15, 16]. Here, we have used these previous genome-wide microarray gene expression data as a starting point to identify a cohort of early phthalate exposure biomarker genes showing increased expression in whole-testis extracts within 6 h of DBP administration at dose levels as low as 10 mg/kg. For comparison, combined median human exposure estimates to toxic phthalate congeners are approximately 5 µg/kg/day, with a subset of humans exposed to levels one or two orders of magnitude higher [24, 25]. To identify the fetal rat testicular cell types responding immediately upon 500 mg/kg DBP exposure, expression of the early biomarker genes was determined using ISH.

The identified early phthalate exposure biomarker genes have a number of characteristics. They encode a variety of protein functional classes, including transcription factors (Egr1, Fos, Junb, and Nr4a1), regulators of intracellular signaling (Ier3 and Dusp6), a receptor (Tnfrsf12a), and several secreted proteins (Ctgf, Cxcl10, Edn1, Stc1, and Thbs1). All of the genes analyzed when identifying biomarker genes displayed an expression change by 6 h of 500 mg/kg DBP exposure; however, many were not localized via ISH because either the expression change was relatively modest or it was observed only at 100 or 500 mg/kg DBP. All biomarker genes showed a similar acute increase in expression following either fetal or prepubertal rat phthalate exposure [15]. Their increased expression levels preceded or were coincident with decreased expression of Leydig cell genes involved in cholesterol metabolism and steroidogenesis (Cyp17a1, Dhcr7, and Star). Within the time frame of these experiments, Star mRNA levels were variable and did not show a dose-dependent expression change. Along with the inherent variability of testosterone measurements, the observed maintenance of Star mRNA levels likely accounted for the lack of statistically significant time- and dose-dependent decreases in testicular testosterone levels.

Phthalate exposure produced an interesting testis gene expression response related to time and dose. At decreasing dose levels, mRNA levels of all genes studied showed an increasing lag time prior to being affected. Fetal rat plasma phthalate levels of phthalate monoester (the toxic metabolite) increase at similar rates within the first hour of gavage exposure regardless of the administered dose level, but lower dose levels achieve a correspondingly lower maximal concentration [23]. Consequently, the switch to altered gene expression was not related to the maximal fetal plasma phthalate monoester level, but rather the cumulative dose (area under the toxicokinetic curve). This suggests that DBP dose levels lower than the lowest observed effect level reported here (10 mg/kg) may produce significant gene expression changes in the some of the identified biomarker genes at time points greater than 6 h or after repeated dosing.

Based upon previous mechanistic studies of prepubertal and fetal rat phthalate-induced testis injury, we hypothesized that either Sertoli cells or Leydig cells would be the testicular cell type initially targeted by DBP. Numerous studies employing postnatal rat phthalate exposures as well as isolated cells from postnatal testes suggested that Sertoli cells were the primary phthalate target cell [9]. For example, vacuolation of Sertoli cell endoplasmic reticulum and alterations in Sertoli cell protein localization were observed by 2–3 h after high-dose-level prepubertal phthalate exposure [26–28]. Additionally, in vitro studies using primary Sertoli cell cultures or Sertoli/gonocyte cocultures showed that acute phthalate monoester exposure could inhibit signaling downstream of the FSH receptor, reduce Sertoli cell proliferation, and induce detachment of gonocytes [29–31]. In contrast, direct adverse effects of phthalates on postnatal primary Leydig cell function generally are not reported or occur in vitro only at phthalate concentrations (≥1 mM) that may be cytotoxic [32, 33], although a recent report showed increased steroidogenesis following in vitro phthalate exposure of postnatal Leydig cells [33]. In the past several years, the possibility that fetal Leydig cells could be directly targeted by phthalates has emerged [14]. Studies have documented effects on fetal rat testis testosterone levels and steroidogenic gene expression within 1–3 h of high-dose-level DBP exposure, raising the possibility that the fetal Leydig cell is a direct target [16].

Our data show that the acute testicular genetic response to phthalate is complex and proceeds through a cascade involving multiple cell types. As supported by the analysis of altered gene expression presented here, the initial testicular phthalate target cell may reside in the interstitial compartment or be the peritubular myoid cell. These two cellular compartments were the first to respond (at 1 h of 500 mg/kg DBP exposure) with significantly elevated expression of Dusp6, Egr1, Fos, Nr4a1, and Thbs1 (Table 4). Identification of peritubular myoid cell expression was unequivocal after colocalization of the ISH signal with DAPI-stained nuclei. The first readily apparent change in Leydig cell gene expression (induction of Stc1) occurred at 3 h, and elevated Stc1 expression coincided precisely in dose level and time with repression of Cyp17a1 in Leydig cells. For Nr4a1 and Cxcl10, gene induction was concentrated in punctate interstitial areas containing Leydig cells, but their expression pattern was less extensive compared with the Leydig cell marker CYP11A1. One potential cell type exhibiting Nr4a1 and Cxcl10 expression is the stem Leydig cell [34], although the presence of this cell population in the fetal testis is undocumented. Unlike Nr4a1 and Cxcl10, interstitial cell expression of Egr1 and Fos was diffuse, raising the possibility that a cell type other than Leydig cells produced these two transcription factors. Our data suggest that Sertoli cells are not a proximal target, since the first Sertoli cell genetic response was not seen until 3 h after exposure. Gonocytes and endothelial cells did not respond at any time point examined, and macrophages were not detected in the GD19 testis. Although we did not observe macrophages in the GD19 testis, two other groups have detected a small number of testis macrophages at this fetal age [35, 36].


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 4 Summary of regional gene expression following acute 500 mg/kg DBP exposure.*

If phthalate exposure modulates the biology of fetal peritubular myoid cells, as our data suggest, what role might this have regarding the testicular phenotypic outcomes of phthalate exposure? Two potential outcomes supported by the scientific literature are abnormal morphogenesis of seminiferous cords and decreased Sertoli cell proliferation. After fetal phthalate exposure, malformed seminiferous cords develop, which are characterized by anastomotic outlines containing intracordal Leydig cells [3, 37]. Because peritubular myoid cells appear to be important for seminiferous cord formation [38], disruption of peritubular myoid cell function by phthalates may produce improper cord morphogenesis, as suggested previously [37]. Fetal phthalate exposure reduces Sertoli cell numbers, and paracrine signaling from fetal peritubular myoid cells downstream of the androgen receptor appears to modulate Sertoli cell numbers [39]. Indeed, deletion of the androgen receptor in peritubular myoid cells results in smaller testes and oligospermia, a phenotype typical of lowered Sertoli cell numbers [40, 41].

Previously, we speculated on the functional consequences of phthalate-induced expression of some the biomarker genes identified here [15, 22], but we will briefly discuss their potential mechanistic role in phthalate testicular toxicity. Repression of Leydig cell-differentiated functions (steroidogenesis and Insl3 expression) is a major phenotypic outcome of fetal phthalate exposure. The transcription factors FOS and NR4A1 and the secreted proteins STC1 and CXCL10 have been implicated in hormone biosynthesis processes in at least one steroidogenic cell type, although the fetal Leydig cell has not been examined. Depending upon the study, Fos expression is associated with increased or decreased expression of Star [42, 43]. NR4A1, EDN1, and STC1 can positively regulate steroid production [44–46], whereas postnatal Leydig cell Cxcl10 overexpression has been associated with decreased steroidogenesis [47]. Finally, NR4A1 can induce Leydig cell Insl3 expression. Defining the crucial factor(s) of phthalate exposure leading to lowered fetal Leydig cell expression of steroidogenic and Insl3 genes requires additional mechanistic information.

Peritubular myoid and Sertoli cells likely cooperate to generate seminiferous cords via a process of cell migration, proliferation, intercellular interactions, and cell-matrix interactions. Thbs1 encodes a matricellular protein implicated in a variety of tissue remodeling processes, growth factor activation, and cell-matrix interactions [48], and its overexpression in peritubular myoid cells following phthalate exposure may lead to altered seminiferous cord formation. During postnatal life, peritubular myoid cell contractility is regulated by Sertoli cell EDN1 secretion [49]; despite this known function, the developmental consequence of Edn1 overexpression following fetal phthalate exposure in relation to peritubular myoid cell function or seminiferous cord morphogenesis remains speculative. Phthalate-induced reduction of Sertoli cell proliferation may contribute to altered seminiferous cord formation, and because Ier3 and Tnfrsf12a are induced in Sertoli cells and known regulators of cell proliferation [50, 51], overexpression of these two genes may contribute to decreased Sertoli cell numbers in the phthalate-exposed testis.

In conclusion, this report adds two important contributions toward determining the mechanism of phthalate-induced fetal testis injury. These are: 1) the identification of a suite of fetal testis genes exhibiting altered expression early after phthalate exposure and at relatively low dose levels; and 2) revealing a complex cascade of the earliest responding testicular cell types in vivo, which suggests the importance of interstitial mesenchymal and peritubular myoid cells (rather than Sertoli cells and perhaps Leydig cells) in the initial testicular molecular events following phthalate exposure. Based upon these results, we propose that phthalates functionally alter peritubular myoid cell biology, leading to perturbation of Leydig cell-differentiated processes (testosterone and INSL3 synthesis) and Sertoli cell-differentiated processes (physiological support of germ cell development).

FOOTNOTES

1Supported by the Long-Range Research Initiative of the American Chemistry Council. Back

Correspondence: 2Kamin J. Johnson, Nemours Biomedical Research, Alfred I. duPont Hospital for Children, 1600 Rockland Rd., Wilmington, DE 19803. FAX: 302 651 6782; e-mail: johnson{at}medsci.udel.edu

Received: 18 May 2007.

First decision: 24 June 2007.

Accepted: 15 August 2007.

REFERENCES

  1. Silva MJ, Barr DB, Reidy JA, Malek NA, Hodge CC, Caudill SP, Brock JW, Needham LL, Calafat AM. Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999–2000 Environ Health Perspect 2004 112331–338[Medline]
  2. Foster PM. Disruption of reproductive development in male rat offspring following in utero exposure to phthalate esters Int J Androl 2006 29140–147 discussion 181–145[CrossRef][Medline]
  3. Mahood IK, Hallmark N, McKinnell C, Walker M, Fisher JS, Sharpe RM. Abnormal Leydig cell aggregation in the fetal testis of rats exposed to di (n-butyl) phthalate and its possible role in testicular dysgenesis Endocrinology 2005 146613–623[Abstract/Free Full Text]
  4. Hallmark N, Walker M, McKinnell C, Mahood IK, Scott H, Bayne R, Coutts S, Anderson RA, Greig I, Morris K, Sharpe RM. Effects of monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants from the rat: comparison with effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human Environ Health Perspect 2007 115390–396[Medline]
  5. Wilson VS, Lambright C, Furr J, Ostby J, Wood C, Held G, Gray LE Jr. Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis Toxicol Lett 2004 146207–215[CrossRef][Medline]
  6. McKinnell C, Sharpe RM, Mahood K, Hallmark N, Scott H, Ivell R, Staub C, Jegou B, Haag F, Koch-Nolte F, Hartung S. Expression of insulin-like factor 3 protein in the rat testis during fetal and postnatal development and in relation to cryptorchidism induced by in utero exposure to di (n-Butyl) phthalate Endocrinology 2005 1464536–4544[Abstract/Free Full Text]
  7. Mylchreest E, Cattley RC, Foster PM. Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl) phthalate: an antiandrogenic mechanism Toxicol Sci 1998 4347–60[Abstract/Free Full Text]
  8. Parks LG, Ostby JS, Lambright CR, Abbott BD, Klinefelter GR, Barlow NJ, Gray LE Jr. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat Toxicol Sci 2000 58339–349[Abstract/Free Full Text]
  9. Boekelheide K, Johnson KJ, Richburg JH. Sertoli Cell Toxicants Academic Press 2005
  10. Hauser R, Meeker JD, Singh NP, Silva MJ, Ryan L, Duty S, Calafat AM. DNA damage in human sperm is related to urinary levels of phthalate monoester and oxidative metabolites Hum Reprod 2007 22688–695[Abstract/Free Full Text]
  11. Pan G, Hanaoka T, Yoshimura M, Zhang S, Wang P, Tsukino H, Inoue K, Nakazawa H, Tsugane S, Takahashi K. Decreased serum free testosterone in workers exposed to high levels of di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP): a cross-sectional study in China Environ Health Perspect 2006 1141643–1648[Medline]
  12. Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, Mao CS, Redmon JB, Ternand CL, Sullivan S, Teague JL. Decrease in anogenital distance among male infants with prenatal phthalate exposure Environ Health Perspect 2005 1131056–1061[Medline]
  13. Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, Schmidt IM, Suomi AM, Virtanen HE, Petersen DV, Andersson AM, Toppari J, et al. Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age Environ Health Perspect 2006 114270–276[Medline]
  14. Boekelheide K. Cracking the nut Toxicol Sci 2004 811–2[Free Full Text]
  15. Lahousse SA, Wallace DG, Liu D, Gaido KW, Johnson KJ. Testicular gene expression profiling following prepubertal rat mono-(2-ethylhexyl) phthalate exposure suggests a common initial genetic response at fetal and prepubertal ages Toxicol Sci 2006 93369–381[Abstract/Free Full Text]
  16. Thompson CJ, Ross SM, Hensley J, Liu K, Heinze SC, Young SS, Gaido KW. Differential steroidogenic gene expression in the fetal adrenal gland versus the testis and rapid and dynamic response of the fetal testis to di(n-butyl) phthalate Biol Reprod 2005 73908–917[Abstract/Free Full Text]
  17. vom Saal FS, Quadagno DM, Even MD, Keisler LW, Keisler DH, Khan S. Paradoxical effects of maternal stress on fetal steroids and postnatal reproductive traits in female mice from different intrauterine positions Biol Reprod 1990 43751–761[Abstract]
  18. Lehmann KP, Phillips S, Sar M, Foster PM, Gaido KW. Dose-dependent alterations in gene expression and testosterone synthesis in the fetal testes of male rats exposed to di (n-butyl) phthalate Toxicol Sci 2004 8160–68[Abstract/Free Full Text]
  19. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR Nucleic Acids Res 2001 29e45[Abstract/Free Full Text]
  20. Yaylaoglu MB, Titmus A, Visel A, Alvarez-Bolado G, Thaller C, Eichele G. Comprehensive expression atlas of fibroblast growth factors and their receptors generated by a novel robotic in situ hybridization platform Dev Dyn 2005 234371–386[CrossRef][Medline]
  21. Johnson KJ, Boekelheide K. Dynamic testicular adhesion junctions are immunologically unique. I. Localization of p120 catenin in rat testis Biol Reprod 2002 66983–991[Abstract/Free Full Text]
  22. Gaido KW, Hensley JB, Liu D, Wallace DG, Borghoff S, Johnson KJ, Hall SJ, Boekelheide K. Fetal mouse phthalate exposure shows that gonocyte multinucleation is not associated with decreased testicular testosterone Toxicol Sci 2007 97491–503[Abstract/Free Full Text]
  23. Fennell TR, Krol WL, Sumner SC, Snyder RW. Pharmacokinetics of dibutylphthalate in pregnant rats Toxicol Sci 2004 82407–418[Abstract/Free Full Text]
  24. Weuve J, Sanchez BN, Calafat AM, Schettler T, Green RA, Hu H, Hauser R. Exposure to phthalates in neonatal intensive care unit infants: urinary concentrations of monoesters and oxidative metabolites Environ Health Perspect 2006 1141424–1431[Medline]
  25. Kohn MC, Parham F, Masten SA, Portier CJ, Shelby MD, Brock JW, Needham LL. Human exposure estimates for phthalates Environ Health Perspect 2000 108A440–A442[Medline]
  26. Creasy DM, Beech LM, Gray TJ, Butler WH. The ultrastructural effects of di-n-pentyl phthalate on the testis of the mature rat Exp Mol Pathol 1987 46357–371[CrossRef][Medline]
  27. Richburg JH, Boekelheide K. Mono-(2-ethylhexyl) phthalate rapidly alters both Sertoli cell vimentin filaments and germ cell apoptosis in young rat testes Toxicol Appl Pharmacol 1996 13742–50[CrossRef][Medline]
  28. Lahousse SA, Beall SA, Johnson KJ. Mono-(2-ethylhexyl) phthalate rapidly increases celsr2 protein phosphorylation in HeLa cells via protein kinase C and casein kinase 1 Toxicol Sci 2006 91255–264[Abstract/Free Full Text]
  29. Gray TJ, Gangolli SD. Aspects of the testicular toxicity of phthalate esters Environ Health Perspect 1986 65229–235[Medline]
  30. Lloyd SC, Foster PM. Effect of mono-(2-ethylhexyl)phthalate on follicle-stimulating hormone responsiveness of cultured rat Sertoli cells Toxicol Appl Pharmacol 1988 95484–489[CrossRef][Medline]
  31. Li LH, Jester WF Jr, Orth JM. Effects of relatively low levels of mono-(2-ethylhexyl) phthalate on cocultured Sertoli cells and gonocytes from neonatal rats Toxicol Appl Pharmacol 1998 153258–265[CrossRef][Medline]
  32. Jones HB, Garside DA, Liu R, Roberts JC. The influence of phthalate esters on Leydig cell structure and function in vitro and in vivo Exp Mol Pathol 1993 58179–193[CrossRef][Medline]
  33. Ge RS, Chen GR, Dong Q, Akingbemi B, Sottas CM, Santos M, Sealfon SC, Bernard DJ, Hardy MP. Biphasic effects of postnatal exposure to diethylhexylphthalate on the timing of puberty in male rats J Androl 2007 28513–520[Abstract/Free Full Text]
  34. Ge RS, Dong Q, Sottas CM, Papadopoulos V, Zirkin BR, Hardy MP. In search of rat stem Leydig cells: identification, isolation, and lineage-specific development Proc Natl Acad Sci U S A 2006 1032719–2724[Abstract/Free Full Text]
  35. Hutson JC. Changes in the concentration and size of testicular macrophages during development Biol Reprod 1990 43885–890[Abstract]
  36. Livera G, Delbes G, Pairault C, Rouiller-Fabre V, Habert R. Organotypic culture, a powerful model for studying rat and mouse fetal testis development Cell Tissue Res 2006 324507–521[CrossRef][Medline]
  37. Fisher JS, Macpherson S, Marchetti N, Sharpe RM. Human ‘testicular dysgenesis syndrome’: a possible model using in-utero exposure of the rat to dibutyl phthalate Hum Reprod 2003 181383–1394[Abstract/Free Full Text]
  38. Martineau J, Nordqvist K, Tilmann C, Lovell-Badge R, Capel B. Male-specific cell migration into the developing gonad Curr Biol 1997 7958–968[CrossRef][Medline]
  39. Scott HM, Hutchison GR, Mahood IK, Hallmark N, Welsh M, De Gendt K, Verhoeven G, O'Shaughnessy P, Sharpe RM. Role of androgens in fetal testis development and dysgenesis Endocrinology 2007 1482027–2036[Abstract/Free Full Text]
  40. Zhang C, Yeh S, Chen YT, Wu CC, Chuang KH, Lin HY, Wang RS, Chang YJ, Mendis-Handagama C, Hu L, Lardy H, Chang C. Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells Proc Natl Acad Sci U S A 2006 10317718–17723[Abstract/Free Full Text]
  41. Orth JM, Gunsalus GL, Lamperti AA. Evidence from Sertoli cell-depleted rats indicates that spermatid number in adults depends on numbers of Sertoli cells produced during perinatal development Endocrinology 1988 122787–794[Abstract/Free Full Text]
  42. Shea-Eaton W, Sandhoff TW, Lopez D, Hales DB, McLean MP. Transcriptional repression of the rat steroidogenic acute regulatory (StAR) protein gene by the AP-1 family member c-Fos Mol Cell Endocrinol 2002 188161–170[CrossRef][Medline]
  43. Manna PR, Eubank DW, Stocco DM. Assessment of the role of activator protein-1 on transcription of the mouse steroidogenic acute regulatory protein gene Mol Endocrinol 2004 18558–573[Abstract/Free Full Text]
  44. Luo CW, Kawamura K, Klein C, Hsueh AJ. Paracrine regulation of ovarian granulosa cell differentiation by stanniocalcin (STC) 1: mediation through specific STC1 receptors Mol Endocrinol 2004 182085–2096[Abstract/Free Full Text]
  45. Robert NM, Martin LJ, Tremblay JJ. The orphan nuclear receptor NR4A1 regulates insulin-like 3 gene transcription in Leydig cells Biol Reprod 2006 74322–330[Abstract/Free Full Text]
  46. Ergul A, Glassberg MK, Majercik MH, Puett D. Endothelin-1 promotes steroidogenesis and stimulates protooncogene expression in transformed murine Leydig cells Endocrinology 1993 132598–603[Abstract/Free Full Text]
  47. Nagpal ML, Chen Y, Lin T. Effects of overexpression of CXCL10 (cytokine-responsive gene-2) on MA-10 mouse Leydig tumor cell steroidogenesis and proliferation J Endocrinol 2004 183585–594[Abstract/Free Full Text]
  48. Hiscott P, Paraoan L, Choudhary A, Ordonez JL, Al-Khaier A, Armstrong DJ. Thrombospondin 1, thrombospondin 2 and the eye Prog Retin Eye Res 2006 251–18[CrossRef][Medline]
  49. Romano F, Tripiciano A, Muciaccia B, De Cesaris P, Ziparo E, Palombi F, Filippini A. The contractile phenotype of peritubular smooth muscle cells is locally controlled: possible implications in male fertility Contraception 2005 72294–297[CrossRef][Medline]
  50. Wu MX. Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis Apoptosis 2003 811–18[CrossRef][Medline]
  51. Blanco-Colio LM, Martin-Ventura JL, Munoz-Garcia B, Moreno JA, Meilhac O, Ortiz A, Egido J. TWEAK and Fn14. New players in the pathogenesis of atherosclerosis Front Biosci 2007 123648–3655[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Toxicol SciHome page
K. J. Johnson, S. M. McCahan, X. Si, L. Campion, R. Herrmann, and J. S. Barthold
The orl Rat with Inherited Cryptorchidism Has Increased Susceptibility to the Testicular Effects of In Utero Dibutyl Phthalate Exposure
Toxicol. Sci., October 1, 2008; 105(2): 360 - 367.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow [Supplemental Data]
Right arrow All Versions of this Article:
77/6/978    most recent
biolreprod.107.062950v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Johnson, K. J
Right arrow Articles by Gaido, K. W
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, K. J
Right arrow Articles by Gaido, K. W
Agricola
Right arrow Articles by Johnson, K. J
Right arrow Articles by Gaido, K. W

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS