ToxSci Advance Access originally published online on March 14, 2007
Toxicological Sciences 2007 97(2):491-503; doi:10.1093/toxsci/kfm049
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Fetal Mouse Phthalate Exposure Shows that Gonocyte Multinucleation is Not Associated with Decreased Testicular Testosterone
* The Hamner Institutes for Health Sciences Centers for Health Research, PO Box 12137, Research Triangle Park, North Carolina 27709
Integrated Laboratory Systems, Research Triangle Park, North Carolina 27709
Brown University, Providence, Rhode Island 02912
1 To whom correspondence should be addressed. Fax: (919) 558-1300. E-mail: gaido{at}thechamner.org.
Received November 22, 2006; accepted February 22, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary DataREFERENCES |
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The rat has been explored in detail for its in utero susceptibility to male reproductive tract malformation following phthalate exposure. Few other species have been studied in detail, and it is important for both mechanistic and risk assessment purposes to understand the species specificity of this response. We investigated the response of the fetal mouse testis to phthalate exposure and compared these results with those previously obtained from the rat. Initial experiments using a variety of phthalate congeners (monobutyl phthalate, di-(n-butyl) phthalate, or mono (2-ethylhexyl) phthalate) and exposure paradigms did not reduce fetal mouse testis testosterone levels. Pharmacokinetic data after a single 500 mg/kg di-(n-butyl)-phthalate (DBP) exposure on mouse gestation day (gd) 18 demonstrated that the concentrations and kinetics of the active metabolite monobutyl phthalate (MBP) in fetal and maternal plasma were similar to the rat. After a single 500 mg/kg or multiple day 250 mg/kg fetal mouse DBP exposure, rapid and dynamic changes in testis gene expression were observed, including induction of immediate early genes. Unlike the rat, expression of genes involved in cholesterol homeostasis and steroidogenesis were not decreased and were increased in a few cases. Similar to the rat, however, a 250- or 500-mg DBP/kg/day mouse exposure from gd 16 through 18 significantly increased seminiferous cord diameter, the number of multinucleated gonocytes per cord, and the number of nuclei per multinucleated gonocyte. Together, these results demonstrate that fetal mouse and rat phthalate exposure both induce immediate early gene expression and disrupt seminiferous cord and gonocyte development. This response in the mouse occurs without a measurable decrease in testicular testosterone, suggesting that altered seminiferous cord formation and gonocyte multinucleation may not be mechanistically linked to lowered testosterone.
Key Words: phthalate; di(n-butyl); in utero exposure; male reproductive development; antiandrogen; molecular mechanisms; steroidogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary DataREFERENCES |
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Phthalate esters are a class of industrial chemicals used as plasticizers to impart flexibility in vinyl plastics and as fixatives and stabilizers in such disparate products as cosmetics, adhesives, and lubricants. Phthalate esters can leach out over time from these products leading to widespread, low level human exposure. The Center for Disease Control now routinely monitors the general population for phthalate exposure as part of their National Health and Nutrition Examination Survey (Silva et al., 2004
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Administration of some phthalate esters to rats during pregnancy causes male reproductive tract malformations, including underdeveloped or absent reproductive organs, malformation of the external genitalia, cryptorchidism, decreased anogenital distance, and diminished sperm count (Barlow et al., 2004; Gray et al., 2000; Mylchreest et al., 1999). The fetal testes of these phthalate-exposed males are characterized by abnormal, poorly formed seminiferous cords containing multinucleated gonocytes. We have shown that developmentally toxic phthalate congeners target a common set of genes and molecular pathways in the developing rat testes and that the effects of these phthalates on extratesticular 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 (Barlow et al., 2003; Liu et al., 2005; Thompson et al., 2004). The conspicuous phthalate-induced inhibition of fetal testosterone synthesis coupled to the critical role of testosterone in male reproductive development and postnatal spermatogenesis raises the possibility that phthalate effects on fetal testicular development are downstream of reduced testosterone synthesis. However, reduced androgen signaling alone cannot account for the effects that phthalate esters have on the developing testis since competitive receptor antagonists have little effect on gene expression and development of the rat fetal testis (Mu et al., 2006).
The time frame and signaling pathways associated with fetal testicular and male reproductive tract development are similar between the mouse and the rat with major developmental events occurring in the mid to late gestational period (Barsoum et al., 2006; Livera et al., 2006; O'Shaughnessy et al., 2006; Staack et al., 2003). Migrating mesonephros–derived myoid cells and resident pre-Sertoli cells organize seminiferous cords in the developing fetal testis between gestation day (gd) 11.5 and 12.5 in the mouse, and gd 12.5 and 13.5 in the rat, to produce two compartments: an interstitial area containing vascular cells and steroidogenic precursors and seminiferous cords containing peripheral Sertoli cells and centralized germ cells. Coincident with cord formation at gd 12.5 in the mouse and 13.5 in the rat, the first sign of steroidogenic gene expression (Cyp11a1) is observed, marking the differentiation of interstitial Leydig cells (Livera et al., 2006; O'Shaughnessy et al., 2006). Although the major testicular compartments have formed by gd 13 in the mouse and gd 14 in the rat, the testis is not a static tissue and numerous important events occur following this time point that drive both testis and urogenital organ development. Testosterone production begins at approximately gd 14, peaks at gd 15–17 in the mouse and gd 17–19 in the rat, and declines rapidly thereafter. Testosterone drives extratesticular male reproductive tract development and together with insulin-like growth factor 3, also produced by fetal Leydig cells, drives testicular descent through a two-step process. In both the mouse and rat, cord formation continues throughout late gestation.
The majority of studies investigating the in utero effects of phthalates have focused on the rat. Few other species have been examined, and it has not been determined how specific this response is to the rat. The adult mouse appears relatively insensitive to the testicular effects of phthalates (Curto and Thomas, 1982; Gray et al., 1982), and gaining a better understanding of the differences in response to di-(n-butyl)-phthalate (DBP) between the mouse and the rat will aid in determining the mechanism by which DBP produces its effects and help in the cross-species extrapolation for risk assessment purposes. To obtain a better understanding of the in utero response to phthalates in the mouse, we investigated the cellular and molecular responses of the fetal mouse testis to DBP and compared these results with results previously obtained from the rat. We show that the early genomic events that occur in the mouse after in utero DBP exposure are similar to those we previously described in the rat. However, the long-term genomic events in the mouse differed from the rat in that pathways involved in cholesterol homeostasis and steroidogenesis were not widely targeted in the mouse. Despite an overall lack of an effect on testicular testosterone, mouse in utero DBP exposure impaired seminiferous cord formation and induced gonocyte multinucleation. Together, these data suggest that (1) the phthalate-induced decrease in fetal testicular testosterone is species-dependent and (2) altered fetal mouse seminiferous cord development and gonocyte multinucleation following phthalate exposure are not driven by reductions in testicular testosterone.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary DataREFERENCES |
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Study Design
Fetal testicular testosterone analysis.
Pregnant time-mated CD-1 mice were purchased from Charles River Laboratories, Inc. (Raleigh, NC). C57Bl/6J and C3H/HeJ mice were purchased from Jackson Laboratories (Bar Harbor, ME) and mated at The Hamner Institutes for Health Sciences (CIIT) Centers for Health Research. Gd 0 was the day sperm were detected in the vaginal smear. Animals were housed in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at CIIT Centers for Health Research in a humidity- and temperature-controlled, high-efficiency particulate air-filtered (HEPA) filtered, mass air-displacement room. The room was maintained on a 12-h light-dark cycle at approximately 18°–26°C with a relative humidity of approximately 30–70%. Rodent diet NIH-07 (Ziegler Brothers, Gardners, PA) and reverse osmosis water were provided ad libitum. Animals were acclimatized for the time period prior to dosing. This study was approved by the Institutional Animal Care and Use Committee of the CIIT Centers for Health Research and followed federal guidelines for the care and use of laboratory animals.
Allocation of animals to control or phthalate treatment groups was done by weight randomization. For DBP and monoethylhexyl phthalate (MEHP) exposures, dams were dosed by oral gavage with 1 ml/kg corn oil vehicle (Sigma Chemical Co., St Louis, MO) or phthalate congener (DBP: Aldrich Chemical Co., Milwaukee, WI; MEHP: TCI America, Portland, OR). For MBP oral gavage exposure, MBP was dissolved in a basic water solution that was brought to neutral pH prior to use (Kremer et al., 2005). Dosages and dosing periods are listed in Table 1. Purity and concentration of all dosing solutions were verified using a Hewlett-Packard 5890 gas chromatograph.
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Pharmacokinetics and gene expression analysis.
CD-1 mice were dosed on gd 18 with corn oil vehicle (n = 6) or 500 mg/kg DBP 2, 4, and 8 h (n = 3 per dose group) prior to sacrifice. Animals were euthanized by CO2 anesthesia and exsanguinated by abdominal aorta transaction. Fetuses were removed by cesarean section and euthanized by decapitation. The sex of the fetuses was determined by internal inspection of the gonads. Testes were snap frozen in liquid nitrogen and stored at – 70°C for later RNA isolation. Fetal blood was collected from both male and female fetuses using heparinized capillary tubes. Blood was pooled from each litter, and plasma was prepared by centrifugation. In a multiple dose study, CD-1 mice were dosed daily from gd 14 to gd 17 with corn oil vehicle (n = 6) and 250 mg/kg DBP (n = 6). On gd 17, 2 h after dosing, animals were euthanized, exsanguinated, and fetuses removed, and testes were collected and snap frozen.
Multiple dose exposure for histopathology.
A multiple dose study for histopathology used pregnant time-mated C57Bl6 mice purchased from Charles River Laboratories, (Boston, MA). Animals were housed in the animal care facility at Brown University in a humidity- and temperature-controlled, HEPA filtered, mass air-displacement room maintained on a 12-h light-dark cycle at approximately 68°F–73°F and 35–70% humidity. Animals were given water ad libitum and fed Purina Rodent Chow 5001. All procedures involving animals were performed with approval of Brown University's Institutional Animal Care and Use Committee in compliance with National Institute of Health guidelines. Allocation of animals to control and DBP dose groups was done by weight randomization. Dams were dosed by oral gavage with 1 ml/kg corn oil (Supervalu Inc., MN) or DBP (Aldrich Chemical Co.). DBP dosing solutions were prepared at concentrations of 500 and 250 mg/ml corn oil. Animals were dosed with corn oil or DBP on gd 16, 17, and 18. Pups born on gd 19 were euthanized by CO2 anesthesia and decapitation. Pups that were not born by gd 19 were removed by cesarean section following CO2 euthanization of the mother, and the fetuses were euthanized by decapitation. The sex of each pup was determined by internal inspection of the gonads using a Nikon SMZ-U dissection microscope, and the testes were removed for analysis. The right testes of each male pup were collected in Davidson's fixative and processed for plastic embedding using Technovit 7100 Glycolmethacrylate (Heraeus Kulzer, Friedrichsdorf, Germany). Sections (6 µm) were cut and stained with hematoxylin and eosin (H&E). Photographs were taken using a Zeiss Axiovert 35 microscope equipped with a Spot Diagnostic digital camera and RT software (Sterling Heights, MI). Images were analyzed with METAMorph Image series 6.1 software (Molecular Devices Corp., Sunnyvale, CA); each seminiferous cord was assessed for diameter, the number of multinucleated gonocytes, and the number of nuclei per multinucleated gonocyte. For statistical analysis, the pup means for each dam were averaged, and the dams were compared by a one-way ANOVA (number of dams: control, n = 4; 250 mg/kg, n = 5; 500 mg/kg, n = 6).
Testosterone Radioimmunoassay
Fetal testis testosterone concentrations were determined using a method modified from vom Saal (vom Saal et al., 1990). This protocol estimated the recovery of extracted testosterone for each assay using 3H-testosterone (PerkinElmer, Wellesley, MA); for all samples, recovery estimates were 
95%. When possible, two testes per dam from different fetuses were combined for analysis. After testis homogenization in 100 µl of phosphate-buffered saline containing 0.1% gelatin, the 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 of methanol. Testosterone was measured in duplicate 25 µl aliquots using a Double Antibody-125I RIA Kit (Catalog #07-189105; MP Biomedicals, Costa Mesa, CA). Cross-reactivity of this kit to other steroids is less than 5%. 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.
Analysis of MBP in Plasma Samples
MBP was quantified in plasma by liquid chromatography/mass spectrometry (Applied Biosystems, Foster City, CA) using the method described previously (Kremer et al., 2005). Sample preparation and analysis are described in detail in this publication in which quantitation was performed using selected reaction monitoring of precursor-product ion transitions at m/z 221.1–77.1 for MBP and 225.1–79.1 for 13C4-MBP (internal standard added to plasma samples).
Microarray Hybridization
Testes from individual fetuses were homogenized in RNA Stat 60 reagent (Tel-Test, Inc., Friendswood, TX), and RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) following manufacturer's protocol. RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and optical density measured on a NanoDrop ND 1000 (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized from 2.5 or 3µg total RNA and purified using the Affymetrix One-Cycle Target Labeling and control reagents kit (Affymetrix, Santa Clara, CA) according to manufacturer's protocol. Equal amounts of purified cDNA per sample were used as the template for subsequent in vitro transcription reactions for complementary RNA (cRNA) amplification and biotin labeling using the Affymetrix GeneChip IVT labeling kit (Affymetrix) included in the One-Cycle Target Labeling kit (Affymetrix). cRNA was purified and fragmented according to the protocol provided with the GeneChip Sample Cleanup module (Affymetrix). All GeneChip arrays were hybridized, washed, stained, and scanned using the Complete GeneChip Instrument System according to the Affymetrix Technical Manual. Microarray data were analyzed by a linear mixed model with SAS Microarray Solution software as previously described (Lahousse et al., 2006). Perfect-match–only data were normalized to a common mean on a log2 scale, and a linear mixed model was then applied for each probe-set (Chu et al., 2002). Restricted maximum likelihood was used for estimating the parameters for both the fixed and random effects. Significance was determined using mixed model based F-tests (p < 0.05).
Gene Ontology
Probe sets were classified initially using Database for Annotation, Visualization, and Integration Discovery available from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (Dennis et al., 2003). The probes sets were further classified using Ingenuity Pathways Analysis software (Ingenuity Systems, Redwood City, CA) and by additional 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary DataREFERENCES |
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In a preliminary series of experiments to determine the sensitivity of mice to inhibition of fetal testicular steroidogenesis by phthalates, pregnant mice were exposed to various dose levels of DBP, MBP, or MEHP for various lengths of time and evaluated for effects on fetal testicular testosterone (Table 1). No reduction in testicular testosterone was observed at phthalate exposure paradigms that did not induce systemic toxicity (dam mortality and/or fetal resorption). Even when dosing with 1500 mg DBP/kg/day from gd 14 through 16, fetal testicular testosterone concentration was not significantly different from control. Preliminary qRT-PCR analysis (data not shown) from this series of experiments also detected no decrease in expression of two genes associated with steroidogenesis (Cyp11a1 and Scarb1) that are targeted in the fetal rat testis following DBP exposure.
To determine if this lack of response was due to reduced delivery of the active phthalate monoester metabolite to the fetus, mouse maternal and fetal plasma were examined at 2, 4, and 8 h following a single administration of 500 mg DBP/kg/day to the dam on gd 18. These results were compared to results previously obtained from pregnant rats administered a single dose of DBP (500 mg/kg/day) on gd 19 (Fig. 1) (Kremer et al., 2005). Although the concentration of MBP in maternal and fetal plasma appears to be higher in mice compared to rats, there was greater variability in the plasma levels measured in mice. Thus, the plasma concentrations of MBP in mice were equal to or greater than the concentration in maternal and fetal rats.
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In addition to reducing rat fetal testicular testosterone production, phthalates have dramatic effects on the developing testis, such as reduced Sertoli cell maturation, abnormal cord formation, and multinucleated gonocyte formation. In our initial series of experiments, some testicular histopathology was observed but not quantitated (data not shown). To determine whether DBP produced similar histopathology in the mouse, we examined in greater detail the fetal testes following H&E staining. Dosing pregnant mouse dams with 250 or 500 mg/kg DBP from gd 16 to 18, with analysis on gd 19, resulted in obvious alterations in the seminiferous cords, including increased cord diameter, number of multinucleated gonocytes, and the number of nuclei per multinucleated gonocyte (Fig. 2). Quantitation of these changes showed significant increases in cord diameter, percent of cord cross-sections with multinucleated gonocytes, and number of nuclei per multinucleated gonocyte at both the 250 and 500 mg/kg dose levels (Fig. 3). These histopathology results were similar to previous results obtained from rats (Barlow and Foster, 2003
; Kleymenova et al., 2005).
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Previous studies from our lab established that administration of a single dose of DBP to the pregnant rat dam resulted in the altered expression of numerous genes in the fetal testis within hours of exposure (Thompson et al., 2005). To determine whether the mouse fetal testis was similarly targeted, we examined the fetal testes from the pharmacokinetic study for global changes in gene expression. As with the rat, the mouse fetal testes showed rapid and dynamic changes in gene expression following DBP exposure. The genes with altered expression include many of the same immediate early genes that are targeted in the rat fetal testes following phthalate exposure (Table 2). Species differences in gene expression were noted as well (Tables 3 and 4, Supplemental Table 1). Specifically, genes essential for testosterone synthesis including Scarb1 (scavenger receptor class B, member 1; also known as Sr-b1), Star (steroidogenic acute regulatory protein), Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1; also known as P450scc), Cyp17a1 (cytochrome P450 family 17, subfamily a, polypeptide 1; also known as Cyp17), and Dhcr7 (7-dehydrocholesterol reductase) are reduced in expression in the rat within 3–6 h after dosing with DBP. These same genes were not altered in expression up to 8 h after DBP dosing in the mouse (Table 3). In contrast, numerous additional genes involved in cell growth and proliferation as well as genes involved in cell survival/apoptosis and DNA replication and repair were reduced within 4 h after DBP administration to mice (Table 4, Supplemental Table 1). Additional studies are needed in the mouse to determine whether a change in gene expression following DBP exposure correlates with protein expression as it does in the rat.
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Significantly more genes had altered expression in the fetal mouse testes as compared to our previous results in the fetal rat (Table 5). This may be due, in part, to the increased number of probes on the mouse Affy chip (45,102 probe sets) as compared to the rat A and B chip (31,257 total combined probe sets) used in the previous study. The increased number of gene expression alterations may also be an indication of toxicity in the mouse. A similar dose of MEHP (500 mg/kg/day) given daily from gd 14 to 16 or 11 to 16 in our preliminary studies caused between 6 and 86% fetal resorption, respectively (Table 1).
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In a previous study, we established that dosing pregnant rats from gd 12 to 19 with dose levels of DBP of 50 mg/kg/day and above were sufficient to significantly alter fetal testicular steroidogenic gene expression and suppress testosterone synthesis. To determine whether these cellular and molecular events are similarly targeted in the mouse after multiple daily dosing, we examined global gene expression and histopathology in the fetal mouse testis following treatment of the pregnant dam with 250 mg/kg/day from gd 14 to 17 (Table 5). This dose level did not induce fetal resorption relative to controls (data not shown). For gene expression analysis, the fetal testes were collected 2 h after the final dose. We did not detect a decrease in genes involved in cholesterol and lipid homeostasis and steroidogenesis even after the several days dosing period. In fact, several genes associated with these pathways were induced, including Dhcr7, acetoacetyl-CoA synthetase, and StAR-related lipid transfer domain containing 4. While immediate early genes were still induced, remarkably few genes were reduced in expression in the mouse fetal testes compared with rat fetal testes following multiple dose DBP exposure (Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary DataREFERENCES |
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In the rat model, it has not been possible to separate the effects of phthalate esters on fetal Leydig cell androgen production from effects on Sertoli cell function or germ cell multinucleation. This, together with the known importance of androgens for so many other aspects of male reproductive tract development, has led some to hypothesize that the effect of phthalates on Sertoli cell function and multinucleated germ cell formation is secondary to reduced androgen production by the Leydig cell. We now show that, at least in the mouse, the effects of phthalates on seminiferous cord formation and multinucleated germ cell induction can occur in the absence of a measurable decrease in testosterone production. Combined with our previously published study showing a lack of an effect of competitive androgen receptor antagonists on fetal rat testis morphological development and gene expression, these results provide evidence that the effect of phthalates on Sertoli cell function and germ cell multinucleation may be mechanistically distinct from phthalate-induced suppression of fetal Leydig cell steroidogenesis.
It is unlikely this difference in response to DBP between the mouse and the rat is due to pharmacokinetic differences. We show that in both the mouse and rat, DBP is rapidly metabolized to MBP, and MBP distributes quickly to the fetus reaching a peak concentration approximately 2 h after dosing. MBP clearance from maternal and fetal plasma is also similar in the mouse and rat.
Both the fetal mouse and rat quickly respond to DBP with increased testicular expression of a number of immediate early genes including early growth response 1, early growth response 2, Ier3 (immediate early response 3), Nr4a1 (nuclear subfamily 4, group A, member 1; also known as Nur77), as well as many others. Nonreproductively toxic phthalates do not induce immediate early gene expression in the rat (Liu et al., 2005). Interestingly, immediate early gene induction also occurs in the prepubertal rat testis following DBP exposure (Lahousse et al., 2006). Sertoli cell-germ cell interaction is also disrupted in the pubertal testis, although the ultimate response, germ cell sloughing into the lumen accompanied by extensive apoptosis, is different from the fetal testicular response. Together, these results suggest that immediate early gene induction is associated with testicular response to reproductively toxic phthalates, although the ultimate end point varies with species and age.
Six hours following DBP exposure in the fetal rat testis, there is a significant reduction in many of the cellular processes required for transporting and synthesizing sterols, lipids, and cholesterol (Thompson et al., 2004). This is accompanied by reduced expression of steroidogenic enzymes necessary for converting cholesterol to testosterone (Thompson et al., 2004). Fetal testicular testosterone production is essential for normal male reproductive tract development, and impairment of fetal testicular testosterone production or function in both the rat and mouse during mid to late gestation leads to male reproductive tract alterations such as malformed or absent epididymis, hypospadias, reduced anogenital distance, and nipple retention (Gray et al., 1999; McIntyre et al., 2002; Sharpe, 2001). The steroidogenic pathway is apparently not targeted in the mouse following DBP exposure, and similar effects on extratesticular reproductive tract development are not observed.
While the exact factor or factors that initiate and drive fetal Leydig cell testosterone production are not known, we previously hypothesized that several factors altered in expression following exposure to the developmentally toxic phthalates could play a role in steroidogenesis including angiotensin which has been shown to inhibit testosterone production by Leydig cells (Dufau et al., 1989; Tahri-Joutei et al., 1991). The induction in the rat fetal testis following phthalate exposure of the dual angiotensin/vasopressin receptor, together with aminopeptidase A, an enzyme responsible for converting angiotensin II to angiotensin III, suggested a possible role for angiotensin in the suppression of testosterone synthesis (Liu et al., 2005). These genes were not similarly altered in expression in the mouse, and the role of angiotensin in the rodent fetal testes remains to be determined. Stanniocalcin 1 (Stc1) is a polypeptide hormone produced by fetal testicular interstitial cells (Stasko and Wagner, 2001) and has been shown to be a negative regulator of granulosa cell differentiation and progesterone synthesis in the ovary (Luo et al., 2004). Expression of Stc1 is upregulated in the rat fetal testis following phthalate exposure (Liu et al., 2005). Stc1 was also transiently induced in the mouse, which could indicate that Stc1 is not involved in regulation of testicular testosterone concentration, at least in the mouse.
Two positive regulators of steroidogenesis, naturietic peptide precursor type C (El-Gehani et al., 2001) and Lhcgr (luteinizing/choriogonadotropin receptor), are also reduced in the rat following phthalate exposure (Liu et al., 2005). Lhcgr is the primary driver of testicular testosterone production in the pubertal and adult rat (Habert et al., 2001; Migrenne et al., 2001). While Lhcgr is not thought to play a role in the initiation of fetal testicular testosterone production, it is involved in regulating testosterone production in the later stages of fetal testicular development (Migrenne et al., 2001). These genes were not targeted by DBP in the mouse, which adds additional support for their role in the steroidogenic response in the fetal rat testis.
In addition to reducing fetal Leydig cell testosterone production in the rat, DBP also targets the Sertoli cell resulting in abnormal cytoskeletal features and a disruption of Sertoli-germ cell contacts (Kleymenova et al., 2005). Perhaps as a result, cord formation in the fetal rat testis is disrupted and large multinucleated germ cells are formed. Here, we show that in the mouse, like in the rat, fetal exposure to DBP alters normal seminiferous cord development. The seminiferous cords are increased in diameter, and many cords contain large multinucleated gonocytes.
One indicator of altered contact between Sertoli cells and germ cells in both the mouse and the rat following DBP exposure may be the increase in Tes (testis derived transcript; also known as testin) gene expression (Table 2). Tes is a Sertoli cell secretory glycoprotein that upon secretion tightly binds to receptors on the Sertoli cell membrane at Sertoli-germ cell junctions (Cheng et al., 1989). While the function of Tes is not known, disruption in the Sertoli-germ cell junction results in a surge of testin production and as a result, Tes serves as a sensitive marker of Sertoli-germ cell interaction (Grima et al., 1997).
Previously, we proposed that the reduction in inhibin production may play a role in the failure of Sertoli cells to differentiate properly following DBP exposure (Liu et al., 2005). Inhibin is a heterodimeric hormone composed of alpha and beta subunits that is produced in the testis mainly by Sertoli cells (Bardin et al., 1989). Sertoli cells fail to differentiate, continue to proliferate, and ultimately develop Sertoli cell tumors in inhibin alpha knockout mice (Matzuk et al., 1992). In the rat, fetal exposure to DBP resulted in a reduction in Inhibin alpha gene expression. However, inhibin expression was not altered in the mouse, and the role of inhibin in the rodent testicular response to DBP remains to be determined.
In both mice and rats, fetal exposure to DBP resulted in an increase in Grb14 (growth factor receptor-bound protein 14) gene expression. Grb14 is an adapter protein that interferes with insulin and fibroblast growth factor (FGF) signaling (Cariou et al., 2004; Kasus-Jacobi et al., 1998). Insulin signaling is required for normal testicular development (Nef et al., 2003), and both insulin-like growth factor and FGF act as germ cell growth and survival factors (Hirai et al., 2004; Ozkurkcugil et al., 2004). Sprouty proteins also antagonize FGF receptor signaling, and Spry1 (sprouty homolog 1 [Drosophila]) is induced in both the rat and mouse, while Spry2 (sprouty homolog 2 [Drosophila]) and Spry4 (sprouty homolog 4 [Drosophila]) are also induced in the mouse. Additionally, DBP exposure resulted in a reduction in FGF receptor–activating protein 1 in both mouse and rat. Together, these results suggest that interference in FGF signaling may play a role in testicular dysgenesis.
In the rat, Nr0b1 (nuclear receptor subfamily 0, group B, member 1; also known as Dax1) gene expression is reduced following DBP exposure. Dax1 plays a central role in testicular development (Ludbrook and Harley, 2004; Meeks et al., 2003). Testis cords are disorganized and incompletely formed in Dax1-deficient mice (Meeks et al., 2003). 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 Dax1-deficient mice (Meeks et al., 2003). Regions of Leydig cell hyperplasia are also apparent (Meeks et al., 2003). We proposed that Dax1 may play a central role in the etiology of phthalate-induced testicular dysgenesis (Liu et al., 2005). However, Dax1 expression was not altered in the fetal mouse testis suggesting that Dax1 may not be not involved in the effects of DBP on mouse Sertoli cells and gonocytes.
A recent study presented evidence that in utero exposure to DBP delayed germ cell development in rat fetal testis and that this delayed germ cell development was associated with reduced germ cell number postnatally, whereas induction of germ cell multinucleation appeared to be a separate event that was not directly associated with reduced germ cell number postnatally (Ferrara et al., 2006). Whether this DBP-induced delay in germ cell development occurs in the mouse fetal testes remains to be determined.
In summary, we show that fetal mouse DBP exposure induces immediate early gene expression, disrupts seminiferous cord development, and causes an increase in both the number of multinucleated germ cells and the number of nuclei per germ cell. Unlike the rat, this response occurs in the absence of measurable disruption of testicular testosterone concentrations suggesting that these events may be mechanistically distinct. By being able to separate the effects of DBP on testicular development from its effects on testicular testosterone, the mouse may serve as a useful model for studying normal testicular development and the mechanism by which DBP disrupts this process.
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Supplementary Data |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary DataREFERENCES |
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Supplementary table contains genes altered only in the mouse but not in the rat following acute DBP exposure. Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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Supported by National Institutes of Health grant R21 ES13020 and the American Chemistry Council.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary DataREFERENCES |
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Bardin CW, Morris PL, Shaha C, Feng ZM, Rossi V, Vaughan J, Vale WW, Voglmayr J, Chen CL. Inhibin structure and function in the testis. Ann. N. Y. Acad. Sci. (1989) 564:10–23.[Web of Science][Medline]
Barlow NJ, Foster PM. Pathogenesis of male reproductive tract lesions from gestation through adulthood following in utero exposure to di (n-butyl) phthalate. Toxicol. Pathol. (2003) 31(4):397–410.[CrossRef][Web of Science][Medline]
Barlow NJ, McIntyre BS, Foster PM. Male reproductive tract lesions at 6, 12, and 18 months of age following in utero exposure to di(n-butyl) phthalate. Toxicol. Pathol. (2004) 32(1):79–90.
Barlow NJ, Phillips SL, Wallace DG, Sar M, Gaido KW, Foster PM. Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate. Toxicol. Sci. (2003) 73(2):431–441.
Barsoum I, Yao HH. The road to maleness: From testis to Wolffian duct. Trends Endocrinol. Metab. (2006) 17:223–228.[CrossRef][Web of Science][Medline]
Cariou B, Bereziat V, Moncoq K, Kasus-Jacobi A, Perdereau D, Le Marcis V, Burnol AF. Regulation and functional roles of Grb14. Front. Biosci. (2004) 9:1626–1636.[Web of Science][Medline]
Cheng CY, Grima J, Stahler MS, Lockshin RA, Bardin CW. Testins are structurally related Sertoli cell proteins whose secretion is tightly coupled to the presence of germ cells. J. Biol. Chem. (1989) 264(35):21386–21393.
Chu TM, Weir B, Wolfinger R. A systematic statistical linear modeling approach to oligonucleotide array experiments. Math. Biosci. (2002) 176(1):35–51.[CrossRef][Web of Science][Medline]
Curto KA, Thomas JA. Comparative effects of diethylhexyl phthalate or monoethylhexyl phthalate on male mouse and rat reproductive organs. Toxicol. Appl. Pharmacol. (1982) 62(1):121–125.[CrossRef][Web of Science][Medline]
Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database for annotation, visualization, and integrated discovery. Genome Biol. (2003) 4(5):P3.[CrossRef][Medline]
Dufau ML, Ulisse S, Khanum A, Buczko E, Kitamura M, Fabbiri A, Namiki M. LH action in the Leydig cell: Modulation by angiotensin II and corticotropin releasing hormone, and regulation of P450(17) alpha mRNA. J. Steroid Biochem. (1989) 34(1–6):205–217.[CrossRef][Web of Science][Medline]
El-Gehani F, Tena-Sempere M, Ruskoaho H, Huhtaniemi I. Natriuretic peptides stimulate steroidogenesis in the fetal rat testis. Biol. Reprod. (2001) 65(2):595–600.
Ferrara D, Hallmark N, Scott H, Brown R, McKinnell C, Mahood KI, Sharpe RM. Acute and long-term effects of in utero exposure of rats to di(n-butyl) phthalate on testicular germ cell development and proliferation. Endocrinology (2006) 147(11):5352–5362.
Gray LE, Ostby J, Furr J, Price M, Veeramachaneni DNR, Parks L. Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol. Sci. (2000) 58(2):350–365.
Gray LE Jr, Ostby J, Monosson E, Kelce WR. Environmental antiandrogens: Low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol. Ind. Health. (1999) 15(1–2):48–64.
Gray TJ, Rowland IR, Foster PM, Gangolli SD. Species differences in the testicular toxicity of phthalate esters. Toxicol. Lett. (1982) 11(1–2):141–147.[CrossRef][Web of Science][Medline]
Grima J, Zhu L, Cheng CY. Testin is tightly associated with testicular cell membrane upon its secretion by sertoli cells whose steady-state mRNA level in the testis correlates with the turnover and integrity of inter-testicular cell junctions. J. Biol. Chem. (1997) 272(10):6499–6509.
Habert R, Lejeune H, Saez JM. Origin, differentiation and regulation of fetal and adult Leydig cells. Mol. Cell. Endocrinol. (2001) 179(1–2):47–74.[CrossRef][Web of Science][Medline]
Hirai K, Sasaki H, Yamamoto H, Sakamoto H, Kubota Y, Kakizoe T, Terada M, Ochiya T. HST-1/FGF-4 protects male germ cells from apoptosis under heat-stress condition. Exp. Cell Res. (2004) 294(1):77–85.[CrossRef][Web of Science][Medline]
Kasus-Jacobi A, Perdereau D, Auzan C, Clauser E, Van Obberghen E, Mauvais-Jarvis F, Girard J, Burnol AF. Identification of the rat adapter Grb14 as an inhibitor of insulin actions. J. Biol. Chem. (1998) 273(40):26026–26035.
Kleymenova E, Swanson C, Boekelheide K, Gaido KW. Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact. Biol. Reprod. (2005) 73(3):482–490.
Kremer JJ, Williams CC, Parkinson HD, Borghoff SJ. Pharmacokinetics of monobutylphthalate, the active metabolite of di-n-butylphthalate, in pregnant rats. Toxicol. Lett. (2005) 159(2):144–153.[CrossRef][Web of Science][Medline]
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) 93(2):369–381.
Liu K, Lehmann KP, Sar M, Young SS, Gaido KW. Gene expression profiling following in utero exposure to phthalate esters reveals new gene targets in the etiology of testicular dysgenesis. Biol. Reprod. (2005) 73(1):180–192.
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) 324(3):507–521.[CrossRef][Web of Science][Medline]
Ludbrook LM, Harley VR. Sex determination: A ‘window’ of DAX1 activity. Trends Endocrinol. Metab. (2004) 15(3):116–121.[CrossRef][Web of Science][Medline]
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) 18(8):2085–2096.
Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alpha-inhibin is a tumor-suppressor gene with gonadal specificity in mice. Nature (1992) 360(6402):313–319.[CrossRef][Medline]
McIntyre BS, Barlow NJ, Foster PMD. Male rats exposed to linuron in utero exhibit permanent changes in anogenital distance, nipple retention, and epididymal malformations that result in subsequent testicular atrophy. Toxicol. Sci. (2002) 65(1):62–70.
Meeks JJ, Crawford SE, Russell TA, Morohashi K, Weiss J, Jameson JL. Dax1 regulates testis cord organization during gonadal differentiation. Development (2003) 130(5):1029–1036.
Migrenne S, Pairault C, Racine C, Livera G, Geloso A, Habert R. Luteinizing hormone-dependent activity and luteinizing hormone-independent differentiation of rat fetal Leydig cells. Mol. Cell. Endocrinol. (2001) 172(1–2):193–202.[CrossRef][Web of Science][Medline]
Mu X, Liu K, Kleymenova E, Sar M, Young SS, Gaido KW. Gene expression profiling of androgen receptor antagonists in the rat fetal testis reveals few common gene targets. J. Biochem. Mol. Toxicol. (2006) 20(1):7–17.[CrossRef][Web of Science][Medline]
Mylchreest E, Sar M, Cattley RC, Foster PMD. Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol. Appl. Pharmacol. (1999) 156(2):81–95.[CrossRef][Web of Science][Medline]
Nef S, Verma-Kurvari S, Merenmies J, Vassalli JD, Efstratiadis A, Accili D, Parada LF. Testis determination requires insulin receptor family function in mice. Nature (2003) 426(6964):291–295.[CrossRef][Medline]
O'Shaughnessy PJ, Baker PJ, Johnston H. The fetal Leydig cell—differentiation, function, and regulation. Int. J. Androl. (2006) 29:90–95. discussion 105–108.[CrossRef][Web of Science][Medline]
Ozkurkcugil C, Yardimoglu M, Dalcik H, Erdogan S, Gokalp A. Effect of insulin-like growth factor-1 on apoptosis of rat testicular germ cells induced by testicular torsion. BJU Int. (2004) 93(7):1094–1097.[CrossRef][Web of Science][Medline]
Sharpe RM. Hormones and testis development and the possible adverse effects of environmental chemicals. Toxicol. Lett. (2001) 120(1–3):221–232.[CrossRef][Web of Science][Medline]
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) 112(3):331–338.[Web of Science][Medline]
Staack A, Donjacour AA, Brody J, Cunha GR, Carroll P. Mouse urogenital development: A practical approach. Differentiation (2003) 71:402–413.[CrossRef][Web of Science][Medline]
Stasko SE, Wagner GF. Stanniocalcin gene expression during mouse urogenital development: A possible role in mesenchymal-epithelial signaling. Dev. Dyn. (2001) 220(1):49–59.[CrossRef][Web of Science][Medline]
Tahri-Joutei A, Fillion C, Bedin M, Hugues JN, Pointis G. Local control of Leydig cell arginine vasopressin receptor by naloxone. Mol. Cell. Endocrinol. (1991) 79(1–3):R21–R24.[CrossRef][Web of Science][Medline]
Thompson CJ, Ross SM, Gaido KW. Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanism. Endocrinology (2004) 145(3):1227–1237.
vom Saal M, 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) 43:751–761.[Abstract]
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