Quantitative Changes in Gene Expression in Fetal Rat Testes following Exposure to Di(n-butyl) Phthalate

Norman J. Barlow*,1, Suzanne L. Phillips*, Duncan G. Wallace*, Madhabananda Sar*, Kevin W. Gaido* and Paul M. D. Foster{dagger},2

* CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709; and {dagger} National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received February 9, 2003; accepted March 7, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Di(n-butyl) phthalate (DBP) alters male reproductive development by decreasing testicular testosterone (T) production when fetuses are exposed on gestation days (GD) 12–21. Previous studies have shown altered gene expression for enzymes in the T biosynthetic pathway following exposure to DBP. The objectives of this study were to develop a more detailed understanding of the effect of DBP on steroidogenesis, using a robust study design with increased numbers of dams and fetuses, compared with previous studies, and to explore messenger RNA (mRNA) expression for other critical genes involved in androgen biosynthesis and signaling. Additionally, immunohistochemical localization of protein expression for several key genes was performed to further confirm mRNA changes. Fetal Leydig cell lipid levels were also examined histochemically, using oil red O. Six to seven pregnant Crl:CD(SD)BR rats per group were gavaged with corn oil or DBP at 500 mg/kg/day on GD 12–19. Testicular RNA isolated from three randomly selected GD 19 fetuses per litter was used for real-time RT-PCR for the following genes: scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), P450c17, 17ß-hydroxysteroid dehydrogenase (17ß-HSD), androgen receptor (AR), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), stem cell factor tyrosine kinase receptor (c-kit), stem cell factor (SCF), proliferating cell nuclear antigen (PCNA), and testosterone-repressed prostate message-2 (TRPM-2). mRNA expression was downregulated for SRB1, StAR, P450scc, 3ß-HSD, P450c17, and c-kit following DBP exposure, and TRPM-2 was upregulated. 17ß-HSD, AR, LHR, FSHR, and PCNA were not significantly changed. Immunohistochemical staining for c-kit was seen in fetal Leydig cells, which has not been previously reported. Downregulation of most of the genes in the T biosynthetic pathway confirms and extends previous findings. Diminished Leydig cell lipid content and alteration of cholesterol transport genes also support altered cholesterol metabolism and transport as a potential mechanism for decreased T synthesis following exposure to DBP.

Key Words: di(n-butyl); phthalate; steroidogenesis; male reproductive development; gene expression; fetal testes; litter variability; c-kit.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phthalate esters are used in the plastics, coatings, and cosmetics industries and are widely distributed environmental contaminants (International Programme on Chemical Safety [IPCS], 1992Go, 1997Go). Examining urinary phthalate metabolites, Blount et al.(2000)Go found that the general population appears to be exposed to disproportionately higher amounts of di(n-butyl) phthalate (DBP) compared with other phthalates. In addition, women of childbearing age, 20–40 years old, which is the target population for the teratogenic effects of DBP, had estimated exposures higher than other groups and had six of the eight highest levels found in the study (Blount et al., 2000Go; Kohn et al., 2000Go). A DBP multigenerational study performed by the National Toxicology Program showed DBP to be a male reproductive toxicant that had effects on animals exposed pre- and postnatally, whereas similar reproductive effects were not seen in the parental generation (National Toxicology Program, 1991Go; Wine et al., 1997Go). Furthermore, studies by Mylchreest et al.(1999Go, 2000)Go showed that DBP exposure during gestation had the ability to affect the developing male reproductive tract profoundly in the absence of maternal toxicity. Dams gavaged on gestation days (GD) 12–21 produced male offspring that had multiple malformations of epididymides, vasa deferentia, seminal vesicles, and dorsolateral and ventral prostate lobes that persisted into adulthood. In utero exposure also led to hypospadias, cryptorchidism, decreased anogenital distance (AGD) on postnatal day (PND) 1, and increased areolae retention on PND 13 (Barlow and Foster, 2003Go; Gray et al., 1999Go; Mylchreest et al., 1999Go, 2000Go).

Although DBP and its major metabolite do not bind to the androgen receptor (Foster et al., 2001Go), DBP has been characterized as an antiandrogen because it caused a 66–88% decrease in fetal intratesticular testosterone (T) levels in the rat on GD 18, 19, and 21 (Mylchreest et al., 2002Go; Shultz et al., 2001Go) and had profound effects on the developing male reproductive tract (Barlow and Foster, 2003Go; Gray et al., 1999Go; Mylchreest et al., 1999Go, 2000Go). The mechanism by which DBP caused reduced T levels was through decreased production of androgen by the fetal Leydig cells (LCs) (Lambright et al., 2003Go). Decreased T in the testes may have led to altered differentiation of the Wolffian ducts and induced malformations in those tissues that were then detected in adult offspring (Mylchreest et al., 2002Go). In addition to effects on T-dependent tissues, the epididymides, vasa deferentia, and seminal vesicles, effects were also seen in dihydrotestosterone (DHT)-dependent tissues, the prostate and external genitalia, although those alterations were less prevalent (Barlow and Foster, 2003Go).

Three characteristic histologic lesions diagnosed in fetal testes exposed to DBP were large aggregates of LCs, multinucleated gonocytes, and seminiferous cords that contained increased numbers of gonocytes. Although histologic changes were first observed on GD 17, gross lesions were not detected until GD 19–20, at which time the developing epididymides appeared smaller with decreased coiling of the epididymal duct (Barlow and Foster, 2003Go). GD 19 was chosen for the current study because nearly 100% of the animals exhibited three characteristic DBP-induced lesions at this age, especially increased numbers of LCs (Barlow and Foster, 2003Go). In addition to morphologic lesions on GD 19, this age was chosen because T synthesis is at or near its zenith (Huhtaniemi and Pelliniemi, 1992Go; Tapanainen et al., 1984Go).

Utilizing cDNA microarrays and real-time quantitative RT-PCR, Shultz et al.(2001)Go identified genes in fetal testes whose expression was altered by DBP exposure. They found decreased gene expression on GD 19 for structure-specific recognition protein, prothymosin-{alpha}, heart fatty acid binding protein, P450 side-chain cleavage enzyme (P450scc), scavenger receptor class B-1 (SRB1), and eukaryotic translation initiation factor. The results were based on total RNA from both testes of one fetus per dam and three dams per treatment group. Changes in gene expression seen with microarrays on GD 19 were supported by RT-PCR data for P450scc and SRB1. RT-PCR was also performed on other genes that were not on the microarrays: steroidogenic acute regulatory protein (StAR), P450c17 (17{alpha}-hydroxylase/17,20-lyase), myristoylated alanine-rich C-kinase substrate (MARCKS), testosterone-repressed prostate message-2 (TRPM-2), proliferating cell nuclear antigen (PCNA), and stem cell factor tyrosine kinase receptor (c-kit). Testicular RNA from a single fetus for each of three dams per group was also used for these analyses (Shultz et al., 2001Go).

One objective of the present study was to confirm DBP-induced alterations in fetal testicular messenger RNA (mRNA) expression found by Shultz et al.(2001)Go, utilizing increased numbers of dams per treatment group and more fetuses per dam. Additional genes in the steroidogenic pathway not previously examined, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were added to the analyses to give a more complete picture of the changes in gene expression in the entire steroid biosynthetic pathway. Androgen receptor (AR), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), and stem cell factor (SCF) were also examined for changes in mRNA expression. Immunolocalization of the proteins for StAR, SRB1, TRPM-2, c-kit, and SCF was performed to assess cell localization within the testis and to determine whether changes in mRNA expression were also reflected in altered protein expression. Data from this study indicated that there were a multitude of changes in gene expression for most of the enzymes of the T biosynthetic pathway, for several associated with cholesterol transport, and for other androgen-related genes. These changes in mRNA expression were supported by immunohistochemical localization of selected proteins and by staining for lipids.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Animals were housed in the animal facility of the CIIT Centers for Health Research (CIIT), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. This study followed federal guidelines for the care and use of laboratory animals (National Research Council, 1996Go) and was approved by the Institutional Animal Care and Use Committee at CIIT. Thirteen pregnant CRL:CD(SD)BR rats were time-mated at Charles River Breeding Laboratories, Inc. (Raleigh, NC) and shipped to CIIT on GD 0, the day sperm was detected in the vaginal smear. Dams were assigned to a treatment group by body weight randomization, using Provantis (Instem LSS, Stone, UK), six animals in the control group and seven animals in the DBP-dosed group. Animals were identified by ear tags and cage cards, and housed individually in polycarbonate cages with Alpha-dri cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet NIH-07 (Zeigler Brothers, Gardener, PA) and reverse-osmosis water were provided ad libitum. Animals were kept in a humidity- and temperature-controlled, HEPA-filtered, mass air-displacement room. The room was maintained on a 12-h light-dark cycle at approximately 22±4°C with a relative humidity of approximately 30–70%.

Study design.
Dams were gavaged daily from GD 12 to 19 with corn oil vehicle (1 ml/kg) (Sigma Chemical Co., St. Louis, MO) or DBP (Aldrich Chemical Company, Milwaukee, WI) in corn oil at 500 mg/kg/day. This dose of DBP was selected because a previous morphologic study (Barlow and Foster, 2003Go) had shown that nearly 100% of male fetuses had testicular lesions, especially of LCs, on GD 19 and because this was also the dose of DBP used by Shultz et al.(2001)Go to examine gene expression.

Dam body weights were recorded on GD 9 and daily during the dosing period. The dams from each dose group were euthanized on GD 19 by CO2 asphyxiation and exsanguination via aortic transection. Fetuses were immediately removed from the uterus, weighed, euthanized by decapitation, and sexed by internal examination of the reproductive organs. The right and left testes and epididymides were removed from male fetuses, using a dissecting microscope. The epididymides were separated from the testes using a dissecting microscope with transillumination. Both testes were snap-frozen in liquid nitrogen and stored at -80°C until RNA isolation.

Real-time quantitative RT-PCR.
Total RNA was isolated from both testes using RNA STAT-60 reagent (Tel-Test, Friendswood, TX) according to the manufacturer’s suggested protocol. Total testicular RNA (1 µg) was treated with DNase I (Amersham Pharmacia Biotech, Newark, NJ) at 37°C for 30 min in the presence of RNasin (Applied Biosystems, Foster City, CA). DNase I was heat-inactivated at 75°C for 5 min, and cDNA was synthesized using random hexamers and TaqMan reverse transcription reagents (Applied Biosystems) according to the manufacturer’s suggested protocol. Total RNA from each tissue was separated into four aliquots for reverse transcription (RT), with one aliquot receiving no enzyme and designated to serve as a negative control. Quality of RT reactions was confirmed by comparison of triplicate RT versus no enzyme control for each RNA sample using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer set. Rat-specific primers and probes were designed for the genes of interest (Tables 1Go and 2Go) using Primer Express software (Applied Biosystems) with the following parameters: low Tm = 60°C, high Tm = 64°C, optimum Tm = 62°C, amplicon length = 80–150 base pairs, and primer length = 20–24 base pairs, with an optimum of 22. Production of a single PCR product was confirmed using agarose gel electrophoresis, and primer and probe efficiencies were determined according to manufacturer’s recommended protocol (Applied Biosystems). RT-PCR was performed on an ABI PRISM 7700 Sequence Detection System and on the ABI PRISM 7900HT Sequence Detection System using SYBR Green PCR and TaqMan Universal PCR Master Mix reagent kits according to the manufacturer’s instructions for quantification of gene expression (Applied Biosystems). GAPDH was used as an on-plate internal calibrator for all RT-PCR reactions. Five dams from the control group were used because the sixth dam had only two male fetuses. Five dams were randomly selected from the seven DBP-exposed dams to maintain the same number of animals between control and DBP-exposed groups. RT-PCR was performed in triplicate on three randomly selected fetuses from each dam, for a total of 15 fetuses per group.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Primer Sets for Real-Time Quantitative RT-PCR Analyses
 

View this table:
[in this window]
[in a new window]
 
TABLE 2 Probe Sequences for Real-Time Quantitative RT-PCR Analyses
 
Immunohistochemistry.
GD 19 fetal testes for immunohistochemistry were randomly selected from a prior DBP study that used the same dosing schedule utilized in this study (Barlow and Foster, 2003Go). Testes were immersion-fixed in 10% neutral-buffered formalin for 24 h, transferred to 70% ethanol, processed into paraffin, sectioned at 5 mm, placed on charged slides, and stored at room temperature until processed for immunostaining. The sections were deparaffinized, then treated with 3% H2O2 in methanol for 10 min to suppress endogenous peroxidase activity. Antigen retrieval was performed by heating the sections for 3 min in citrate buffer (1:10 dilution in deionized water, pH 5.5–5.7) (BioGenex, San Ramon, CA). The avidin-biotin peroxidase method was used for immunostaining, as described previously (Sar and Welsch, 1999Go). The sections were incubated with 10% Carnation powdered nonfat milk (Nestlé, Solon, OH) for 10 min followed by 2% normal serum (rabbit or goat) in PBS for 20 min each to reduce nonspecific staining. Tissues were also treated for 15 min each with avidin and biotin to suppress endogenous biotin activities. Incubation occurred overnight at 4°C, with the following primary antibodies: StAR (rabbit polyclonal IgG, 1 mg/ml), c-kit (rabbit polyclonal IgG, 2 mg/ml), SRB1 (goat polyclonal IgG, 1 mg/ml), SCF (goat polyclonal IgG, 2 mg/ml), and TRPM-2 (goat polyclonal IgG, 4 mg/ml).

All primary antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA except StAR, which was purchased from Affinity BioReagents (Golden, CO). The optimal working dilution of each antibody was determined by incubating sections with various concentrations of antibody ranging from 1 to 5 mg/ml. Following incubation with the primary antibody, the slides were washed in PBS for 5 min, followed by incubation with a biotinylated secondary antibody antirabbit IgG or antigoat IgG (1:200), then with avidin-biotin peroxidase (1:200) (Vector Labs, Burlingame, CA) for 30 min at room temperature. The sections were exposed to liquid diaminobenzidine (BioGenex, San Ramon, CA) after a 5-min PBS wash. The slides were then rinsed in distilled water, counterstained with hematoxylin, and mounted with Permount. The specificity of immunostaining was determined by incubating adjacent sections with the preabsorbed antibody, which was prepared by incubating each antibody with four- to fivefold excess of the synthetic peptide used as the immunogen.

Oil red O histochemistry.
Frozen sections from GD 19 testes were cut and placed on slides. Oil red O staining was performed according to Pearse (1996)Go. Skin was used as the positive control with dark red staining seen in subcuticular adipose tissue.

Statistical analyses.
Statistical analyses were conducted using JMP (version 4.0.0, SAS Institute, Cary, NC). Gene expression data were analyzed by a repeated measure ANOVA (nested design), with the dams treated as the experimental unit. Relative expression ratios were calculated using the equation set forth by Pfaffl (2001)Go, in which efficiencies for both the gene of interest and the calibrator, GAPDH, were used. Actual efficiencies were calculated from standard curves of serially diluted cDNA for TaqMan data, whereas efficiencies were assumed to be 100% for both the gene of interest and GAPDH for SYBR Green data. Therefore, 2 was used as the efficiency, as recommended by the manufacturer (Applied Biosystems). An average threshold cycle (CT) for all of the control fetuses was generated for both the gene of interest and GAPDH. Each individual CT was then subtracted from the average CT for both the control and DBP-exposed fetuses, yielding DCTs. These data were entered into the Pfaffl equation


to generate relative expression ratios for each fetus. Because a group control average was used for the gene of interest and GAPDH, the expected expression ratio for each animal in the control group was 1 (or 100% of control). Statistical analyses were performed on the expression ratios of individual fetuses, nested by dam. For data generated using SYBR Green fluorescence, the Pfaffl equation is equal to the equation put forth in Applied Biosystem’s User Bulletin No. 2, 2-{Delta}{Delta}CT. Analyses of relative expression ratios were considered to be statistically significant at p < 0.05.

Because three fetuses per dam and five dams per group were used in this study, we examined the intralitter versus interlitter variability to determine whether there was more variability between fetuses in a litter or whether there was more variability between dams (litter means). The analysis was achieved by comparing the dam and fetus variance components.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oil red O histochemistry.
Oil red O stains lipid deep red within histologic sections. Fetal LCs randomly dispersed throughout the interstitium of control testes contained large numbers of variably sized lipid vacuoles (Fig. 1AGo). The interstitium of DBP-exposed testes contained large aggregates of LCs with red staining lipid vacuoles. However, the size and number of the lipid vacuoles in the LCs of these large clusters were decreased (Fig. 1BGo). Additionally, there were decreased numbers of oil red O positive LCs throughout the rest of the interstitium.



View larger version (142K):
[in this window]
[in a new window]
 
FIG. 1. Histochemical and immunohistochemical staining of gestation day (GD) 19 fetuses from control males (A, C, and E) and males exposed to di(n-butyl) phthalate (DBP) (500 mg/kg/day) (B, D, and F) on GD 12–19. Control testis (A) stained with oil red O for lipids have Leydig cells (LCs) randomly scattered throughout the interstitium that contain large numbers of variably sized, red-staining lipid vacuoles. DBP-exposed testis (B) has several large aggregates of numerous LCs that individually contained fewer lipid vacuoles. Control testis (C) stained for SRB1 with Sertoli cells (arrowheads) lining the seminiferous cords and LCs scattered throughout the interstitium. DBP-exposed testis (D) immunostained for scavenger receptor B-1 shows increased staining of Sertoli cells (arrowheads) lining the cords, compared with control (C), and decreased staining of LCs in a large interstitial aggregate (*). Steroid acute regulatory protein expression is confined to LCs in both the control (E) and DBP-exposed testes (F). There is decreased staining intensity in the DBP-exposed LCs. Original magnification = x500 (A and B) and x250 (C, D, E, and F).

 
Cholesterol transport genes and TRPM-2.
Gene expression for the high-density lipoprotein (HDL) receptor on the cell surface, SRB1, was significantly decreased, ~41% of control (Fig. 2Go). Immunostaining for SRB1 in control testes was localized to the cytoplasm of LCs with low-intensity staining of the cytoplasm of some Sertoli cells (Fig. 1CGo). The staining for this protein was decreased in LCs in DBP-exposed fetuses (Fig. 1DGo). In addition to decreased staining in the LCs, there was marked increase in the cytoplasmic staining of Sertoli cells (Fig. 1DGo). Similar to SRB1, gene expression for StAR, the protein responsible for cholesterol transport to the inner mitochondrial membrane, was also significantly reduced to ~34% of control (Fig. 2Go). Immunostaining for StAR was intense and localized to the cytoplasm of LCs in control and DBP-exposed testes (Figs. 1EGo and 1FGo). However, the staining intensity of LCs in DBP-exposed testes was markedly decreased (Fig. 1FGo). mRNA expression for the androgen-related gene TRPM-2 was significantly increased to approximately 275% of control (Fig. 2Go), which corresponded to immunostaining for this gene. Staining for TRPM-2 protein was observed in the cytoplasm of small numbers of Sertoli cells in control animals (Fig. 3AGo), whereas staining was markedly increased in Sertoli cells of DBP-exposed testes (Fig. 3BGo).



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 2. Real-time quantitative RT-PCR analyses of testicular mRNA on gestation day (GD) 19 for scavenger receptor B-1 (SRB1), steroid acute regulatory protein (StAR), and testosterone-repressed prostate message-2 (TRPM-2) from control and di(n-butyl) phthalate-exposed (500 mg/kg/day) fetuses. Gene expression levels are graphed as relative mRNA expressions (% of control). Gene expressions were analyzed for 15 fetuses per group, nested by dam. Values are nested litter means ± SEM. *Significantly different from control (p < 0.05).

 


View larger version (160K):
[in this window]
[in a new window]
 
FIG. 3. Immunohistochemical staining of gestation day (GD) 19 fetuses from control males (A, C, and E) and males exposed to di(n-butyl) phthalate (DBP) (500 mg/kg/day) (B, D, and F) on GD 12–19. TRPM-2 staining was markedly increased in Sertoli cells (arrowheads) of the DBP-exposed testis (B), whereas minimal staining for TRPM-2 protein was seen in small numbers of Sertoli cells (arrowheads) and Leydig cells (LC) in the control testis (A). Immunostaining for c-kit was prominent in gonocytes (arrows) and LCs of the control testis (C). Although these cells stained for c-kit in the DBP-exposed testis (D), staining intensity was diminished (gonocytes = arrows, LC aggregate = *). Small numbers of LCs, Sertoli cells, and gonocytes were lightly stained for stem cell factor (SCF) in control animals (E), whereas staining intensity was markedly increased in Sertoli cells (arrowheads) in the DBP-exposed testis (F). LCs within a large aggregate (*) have little staining for SCF (F). Original magnification = x500 (A and B) and x250 (C, D, E, and F).

 
Steroidogenic enzymes.
Gene expressions for all the enzymes involved in testosterone biosynthesis from the conversion of cholesterol to pregnenolone (P450scc) to the formation of T from androstenedione (17ß-HSD) were examined. The initial three enzymes that catalyze the first four reactions in T biosynthesis all had significantly decreased expression ratios. P450scc was most severely decreased with a gene expression of ~5% of control (Fig. 4Go). 3ß-HSD and P450c17 were similarly decreased, with gene expressions of ~52% and 59% of control (Fig. 4Go). Whereas the initial three enzymes were significantly decreased, gene expression for 17ß-HSD was increased 42%, relative to control (Fig. 4Go). However, there was a large component of variability in the DBP-exposed fetal testes for 17ß-HSD, and the increased gene expression was not of statistically significant difference from control.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 4. Real-time quantitative RT-PCR analyses of testicular mRNA on gestation day (GD) 19 for P450scc, 3ß-hydroxysteroid dehydrogenase, P450c17, and 17ß- hydroxysteroid dehydrogenase from control and di(n-butyl) phthalate-exposed (500 mg/kg/day) fetuses. Gene expression levels are graphed as relative mRNA expressions (% of control). Gene expressions were analyzed for 15 fetuses per group, nested by dam. Values are nested litter means ± SEM. *Significantly different from control (p < 0.05).

 
Androgen and gonadotropin receptors.
There were no significant differences from control for AR, LHR, or FSHR (Fig. 5Go).



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 5. Real-time quantitative RT-PCR analyses of testicular mRNA on gestation day (GD) 19 for androgen receptor (AR), luteinizing hormone receptor (LHR), and follicle-stimulating hormone receptor (FSHR) from control and di(n-butyl) phthalate-exposed (500 mg/kg/day) fetuses. Gene expression levels are graphed as relative mRNA expressions (% of control). Gene expressions were analyzed for 15 fetuses per group, nested by dam. Values are nested litter means ± SEM. *Significantly different from control (p < 0.05).

 
Gonocyte and Sertoli cell surface proteins and PCNA.
Relative expression ratios for two cell surface proteins were significantly decreased. c-kit is the tyrosine kinase receptor for SCF and is located on the cell surface of gonocytes. Gene expression for this protein was decreased to ~9% of control (Fig. 6Go). Intense c-kit immunostaining was noted in the cytoplasm of gonocytes and LCs in control testes (Fig. 3CGo), whereas the staining was appreciably decreased in both cell types of DBP-exposed testes (Fig. 3DGo). The ligand for c-kit is the Sertoli cell surface protein SCF. Its mRNA expression was also significantly decreased by DBP to ~10% of control (Fig. 6Go). SCF protein was observed in the cytoplasm of Sertoli cells, LCs, and some gonocytes in control testes (Fig. 3EGo). Staining intensity in Sertoli cells was dramatically increased following DBP exposure, whereas the cytoplasm of most of the LCs in the large aggregates did not appear to stain (Fig. 3FGo). There was no statistically significant change in gene expression for the general cell proliferation protein PCNA (Fig. 6Go).



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 6. Real-time quantitative RT-PCR analyses of testicular mRNA on gestation day (GD) 19 for stem cell factor tyrosine kinase receptor (c-kit), stem cell factor (SCF), and proliferating cell nuclear antigen (PCNA) from control and di(n-butyl) phthalate-exposed (500 mg/kg/day) fetuses. Gene expression levels are graphed as relative mRNA expressions (% of control). Gene expressions were analyzed for 15 fetuses per group, nested by dam. Values are nested litter means ± SEM. *Significantly different from control (p < 0.05).

 
Intra- versus interlitter variability.
The variance component for individual fetuses was significantly increased over the dam variance component for 11 of the 13 genes examined, indicating that the variability between fetuses within a litter was greater than the variability between dams. The difference in variability was statistically significant for SRB1 (p < 0.001), P450scc (p < 0.024), P450c17 (p < 0.039), 17ß-HSD (p < 0.001), LHR (p < 0.001), c-kit (p < 0.001), and TRPM-2 (p < 0.001). Although not significant, the dam variance component for 3ß-HSD and FSHR was greater than the fetal variance component.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histologic lesions seen with in utero DBP exposure include large aggregates of abnormal LCs within the interstitium and multinucleated gonocytes and increased numbers of gonocytes in seminiferous cords (Barlow and Foster, 2003Go; Mylchreest et al., 2002Go). A fetal LC and the T biosynthetic pathway within that cell are illustrated in Figure 7Go. Genes examined in this study, the cellular location of their proteins, and their mRNA expression levels in response to DBP are shown (percentage of control). In addition, photomicrographs of control and DBP-exposed fetal testes and a graph of fetal intratesticular T levels found by Shultz et al.(2001)Go are included to highlight all the changes that occur in fetal testes following in utero DBP exposure. Though intratesticular T levels were not measured in this study, the dosing regimen used in the current study has been shown to significantly reduce fetal intratesticular T and decrease production of T by DBP-exposed testes (Fig. 7Go) (Lambright et al., 2003Go; Mylchreest et al., 2002Go; Shultz et al., 2001Go). Decreased androgen synthesis was supported by increased mRNA and protein expression for TRPM-2, an androgen-responsive gene found to be increased during prostatic regression following castration and by the use of antiandrogens (Lakins et al., 1998Go; Wong et al., 1993Go). As the name of the gene states, normal androgen levels repress TRPM-2 expression in the prostate (Leger et al., 1987Go). If androgen status is disrupted and androgen levels drop, there is increased expression of this gene and the corresponding protein (Miyake et al., 2000aGo,bGo).



View larger version (59K):
[in this window]
[in a new window]
 
FIG. 7. Testosterone (T) biosynthetic pathway in a fetal Leydig cell and the effects of DBP on gene expression. Photomicrographs of control and DBP-exposed testes and a graph of the intratesticular T levels on gestation day (GD) 19. Cholesterol is synthesized from extracellular high-density lipoprotein (HDL) cholesteryl esters or from intracellular acetate. Cholesterol is then carried from the outer mitochondrial membrane by steroid acute regulatory protein (StAR), where it is converted to pregnenolone by P450 side-chain cleavage enzyme (P450scc). Pregnenolone is then transported to the smooth endoplasmic reticulum (SER), where it is converted to progesterone by 3ß-hydroxysteroid dehydrogenase (3ß-HSD). Progesterone is converted to 17-hydroxyprogesterone and androstenedione by P450c17. Androstenedione is converted to the end product, T, by 17ß-hydroxysteroid dehydrogenase (17ß-HSD). The percentages in the boxes are relative gene expression ratios (% of control) for mRNA expression of each protein. *Significantly different from control (p < 0.05). Intratesticular T levels were significantly decreased on GD 19 following in utero DBP exposure (Shultz et al., 2001Go). DBP-testes (B) have large areas of abnormal Leydig cells (*), increased numbers of gonocytes within the seminiferous cords (arrows), and multinucleated gonocytes (B, inset), compared with control testes (A). Original magnification = x250 (A and B) and x500 (inset).

 
Proteins directly responsible for T synthesis in fetal rat testes include StAR, P450scc, 3ß-HSD, 17{alpha}-hydroxylase/17,20-lyase, and 17ß-HSD. Additionally, SRB1 is responsible for transport of high-density lipoprotein (HDL) cholesteryl esters into the cell (Cao et al., 1999Go; Trigatti et al., 2000Go). Although cholesterol for steroidogenesis in the fetal testis may be obtained from the conversion of intracellular acetate, the preferred source of cholesterol is uptake of cholesteryl esters from HDL by SRB1 (Andersen and Dietschy, 1978Go; Cao et al., 1999Go). Even so, phthalates have been shown to interfere with cholesterologenesis in the testes and decrease plasma cholesterol levels (Bell, 1982Go). Although conversion of acetate to cholesterol and cholesterol uptake were not measured in the current study, decreased cholesterol uptake into fetal LCs following DBP exposure has been noted (Thompson et al., 2003Go). SRB1 gene expression was significantly decreased in the current study (Fig. 7Go), and DBP likely altered intracellular cholesterol synthesis, both of which may have contributed to decreased intracellular cholesterol levels. There were decreased numbers of LCs staining for lipid, and the LCs of the large aggregates (Fig. 7Go) had decreased lipid staining. Transmission electron microscopy performed in a separate morphologic study on testes exposed to DBP by the same dosing scheme showed decreased lipid vacuoles in LCs within large aggregates (N. J. Barlow, unpublished data). Taken together, these data support alterations in cholesterol synthesis, transport, and storage that likely play a role in decreased testosterone production by fetal LCs.

StAR is necessary for delivery of cholesterol to the inner mitochondrial membrane (Hasegawa et al., 2000Go; Manna et al., 2001Go). Gene expression and protein levels of this cholesterol transport molecule were significantly decreased, relative to control (Fig. 7Go). Studies by Thompson et al.(2003)Go have shown that, in addition to decreased uptake of cholesterol by LCs, there is also decreased uptake of cholesterol into DBP-exposed mitochondria, further supporting altered cholesterol handling in the pathogenesis of decreased T synthesis (Fig. 7Go).

P450scc conversion of cholesterol to pregnenolone is the limiting enzymatic step in T biosynthesis (Miller, 1988Go; Omura and Morohashi, 1995Go). Although alteration of cholesterol transport and metabolism appear to contribute to decreased T synthesis, the significantly decreased level of mRNA expression for P450scc indicates another possible contributor (Fig. 7Go). Decreased expression of P450scc may be partially due to reduced delivery of cholesterol; therefore, gene expression for the protein responsible for conversion to the next intermediate may have been downregulated. However, GD 19 DBP-exposed testes still produce small amounts of T, indicating that T biosynthesis was not completely inhibited. Whether the significantly decreased gene expression of P450scc was due to direct effects of DBP on gene expression for this enzyme or whether there was secondary downregulation following decreased cholesterol delivery is currently unknown. Gene expressions for 3ß-HSD and P450c17 were both significantly decreased (Fig. 7Go). However, Thompson et al.(2003)Go showed that, when DBP-exposed testes were incubated with pregnenolone, progesterone, or 17{alpha}-hydroxyprogesterone, T production increased, though it never attained the same level as control testes. Collectively, these data may indicate that decreased delivery of intermediates leads to decreased gene expression for these two proteins or that proteins levels were still high enough to support steroidogenesis, even though gene expressions were decreased.

Gene expression changes for three receptors responsible for androgen signaling and male reproductive development and function were not significantly different from control. Unlike LHR and FSHR, testicular AR showed a trend for an increase above control. Mylchreest et al. (2002)Go found similar protein expression for AR in DBP-exposed fetal testes. AR protein expression throughout the testis, especially in the large areas of LCs, was increased and double staining with 3ß-HSD found that many of the AR-positive cells were 3ß-HSD-negative, which correlates with the decreased gene expression for 3ß-HSD observed in the current study. Steroidogenesis in fetal LCs is initially independent of luteinizing hormone (LH) (El-Gehani et al., 1998Go; Noumura et al., 1966Go; O’Shaughnessy et al., 1998Go). Although the LHR is first detectable in the fetal rat testis on GD 16.5, significant amounts of LH are not seen until T levels begin to decrease near the end of gestation (El-Gehani et al., 1998Go; Zhang et al., 1994Go). In addition, the male reproductive tract of LHR knockout mice is similar to control animals at birth, further supporting LH-independent production of T (Zhang et al., 2001Go). Given that LH does not play a major role in steroidogenesis before GD 19, we were not surprised that we did not see alterations of gene expression for LHR in the current study.

Lesions in DBP-exposed seminiferous cords include the formation of large, multinucleated gonocytes and an increased number of gonocytes within the developing seminiferous cords (Fig. 7Go). c-kit is the receptor for SCF, both of which are located on gonocytes and Sertoli cells, respectively (Zsebo et al., 1990Go). Mutations in either of these two genes have been shown to disrupt the interaction between the proteins, which leads to a lack of spermatogenesis and infertility in sexually mature animals (Loveland and Schlatt, 1997Go). Gene expressions for both c-kit and SCF were significantly decreased to approximately 10% of control, indicating that altered gene expression for these two proteins may play a part in the formation of multinucleated gonocytes or the increased numbers of gonocytes, although a clear mechanistic link between the reduced expression for these two genes and the histologic lesions has not been definitively made (Fig. 7Go). Immunostaining for c-kit was seen in both gonocytes and fetal LCs in control animals, with decreased protein expression in both cell populations in DBP-exposed testes. Although observed in LCs in early postnatal and adult males, c-kit protein expression has not been previously described in fetal LCs (Fox et al., 2000Go; Loveland and Schlatt, 1997Go; Manova et al., 1990Go; Orth et al., 1996Go). The purpose of c-kit in adult LCs may be related to soluble SCF and regulation of testosterone biosynthesis (Fox et al., 2000Go; Loveland and Schlatt, 1997Go). Whether the same relationship is present in the fetus is not clear at this time. Though it is not known whether c-kit and SCF expression remains low following birth and discontinued exposure to DBP, the early postnatal testicular lesion of decreased germ cells observed in SCF mutant mice is similar to the testicular lesion seen early postnatally following in utero DBP exposure (Barlow and Foster, 2003Go; Brannan et al., 1992Go). Immunostaining for SCF in Sertoli cells of DBP-exposed testes was increased, which was opposite the observed SCF mRNA expression. The reason for the disparity between gene expression data and protein expression is not clear at this time.

DBP at 500 mg/kg/day during gestation is not a likely environmental exposure for humans. The purpose of the high dose of DBP used in this study was to produce increased penetrance of the phenotype, thereby allowing changes in mRNA expression associated with morphologic alterations to be detected. Although gene changes can be readily detected following gestational exposure to 500 mg/kg/day of DBP, the lowest dose of DBP at which these changes occurs remains to be studied. The identification of changes in gene expression is critical to understanding the pathogenesis of phthalate-induced male reproductive tract lesions. Without determining which genes are significantly affected by DBP exposure when a large number of the pups are affected, one would be forced to conduct numerous unnecessary RT-PCR reactions on multiple genes that may not be changed, and at lower dose levels, one would not be able to attribute any gene expression findings to a phenotypic response occurring in far fewer animals. Given the number of pups generated in a typical dose-response study and the large number of genes important for testicular development, one would easily be overwhelmed by the amount of work necessary to collect potentially negative data. A dose-response study is planned, based on the critical genes identified in this experiment.

A generally assumed biologic phenomenon is that fetuses or pups within a litter are more closely related to each other than are offspring from other litters (Elswick et al., 2000Go; Haseman and Hogan, 1975Go; Shirley and Hickling, 1981Go; Williams, 1975Go). Under this principle, it may be assumed that one fetus or pup from a litter is representative of the entire litter. In this study, testes from three male fetuses were used to calculate a litter mean. When statistically analyzed, there was more variability among the three fetuses in the litter for most of the genes than there was between dam litter means. Because we have seen differences in animal sensitivity to DBP following in utero exposure, the intralitter variability is not surprising (Elswick et al., 2000Go; Mylchreest et al., 1999Go, 2000Go). Although the reason for this variability is not known, it may be due to a variety of factors, including uterine blood flow, which may alter the amount of compound being delivered to each fetus (Buelke-Sam et al., 1982Go; Even et al., 1994Go), and the location of each fetus within the uterus, which may lead to increased or decreased exposure to testosterone from neighboring fetuses (Clark et al., 1993Go; Even et al., 1992Go; Nonneman et al., 1992Go). Given the intralitter variability seen in this study, selection of one fetus from a litter may be an inappropriate representation of the entire litter and may lead to erroneous conclusions. Therefore, increased numbers of fetuses or pups from each litter (the entire litter complement, if possible) should be used for statistical analyses.

This study, using a more robust design that included increased numbers of fetuses and dams, confirmed the gene expression changes found by Shultz et al. (2001)Go. Additional genes (including 3ß-HSD, 17ß-HSD, AR, LHR, FSHR, SCF, and PCNA) were examined for changes in gene expression. Taken together, the data correlate with decreased T synthesis by fetal LCs. Whether the changes in gene expression were the primary molecular cause of decreased steroidogenesis or whether the decreased expression of the steroidogenic enzymes was simply a physiologic response to decreased amounts of intermediates is not known. Decreased amounts of lipid within LCs and the decreased gene expression for SRB1 and StAR, in conjunction with data from Thompson et al.(2003)Go and Bell (1982)Go, favor the latter mechanism, although other mechanisms, such as reduced intracellular signaling or lack of appropriate LC differentiation, cannot be completely ruled out. Gene expression for the rate-limiting enzyme in T biosynthesis, P450scc, is decreased to approximately 5% of control; therefore, this decrease may also play a significant part in the mode of action of DBP on the fetal testis. Although gene expressions for many of enzymes in the T biosynthetic pathway were downregulated, gene expression for 17ß-HSD was not statistically different from control, arguing against a wholesale downregulation of all cellular genes in response to exposure to a toxicant. The finding of increased intralitter variability compared with interlitter variability warrants further investigation to determine the number of animals appropriate for gene expression analysis when using a nested study design.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Katie J. Turner and Mrs. Kim P. Lehmann for their assistance with dissections and statistical analyses of the gene expression data. We would also like to thank Drs. Christopher J. Thompson and L. Earl Gray, Jr., for their discussion of data in relationship to the current study. The assistance of Mrs. Renee Thacker and Mr. Otis Lyght of the CIIT histology laboratory was greatly appreciated. Mr. E. Stan Piestrak and Mr. James Mangum were instrumental in the capturing of images and design of figures and illustrations. This study would not have been possible without the assistance of the CIIT animal care staff. Funding was provided in part by the American Chemistry Council and by a National Research Service Award from the National Institute of Environmental Health Sciences (awarded to N.J.B.). This manuscript does not necessarily reflect the views or policies of either of these organizations.


    NOTES
 
1 Present address: Aventis Pharmaceuticals, 1041 Route 202-206, PO Box 6800, Bridgewater, NJ 08807. Back

2 To whom correspondence should be addressed at National Institute of Environmental Health Sciences, PO Box 12233 (MD E1-06), Research Triangle Park, NC 27709. Fax: (919) 541-4634. E-mail: foster2{at}niehs.nih.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Andersen, J. M., and Dietschy, J. M. (1978). Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the rat. J. Biol. Chem. 253, 9024–9032.[ISI][Medline]

Barlow, N. J., and Foster, P. M. D. (2003). Pathogenesis of male reproductive tract lesions from gestation through adulthood following in utero exposure to di(n-butyl) phthalate. Toxicol. Pathol. 31.

Bell, F. P. (1982). Effects of phthalate esters on lipid metabolism in various tissues, cells and organelles in mammals. Environ. Health Perspect. 45, 41–50.[ISI][Medline]

Blount, B. C., Silva, M. J., Caudill, S. P., Needham, L. L., Pirkle, J. L., Sampson, E. J., Lucier, G. W., Jackson, R. J., and Brock, J. W. (2000). Levels of seven urinary phthalate metabolites in a human reference population. Environ. Health Perspect. 108, 979–982.

Brannan, C. I., Bedell, M. A., Resnick, J. L., Eppig, J. J., Handel, M. A., Williams, D. E., Lyman, S. D., Donovan, P. J., Jenkins, N. A., and Copeland, N. G. (1992). Developmental abnormalities in Steel17H mice result from a splicing defect in the steel factor cytoplasmic tail. Genes Dev. 6, 1832–1842.[Abstract]

Buelke-Sam, J., Holson, J. F., and Nelson, C. J. (1982). Blood flow during pregnancy in the rat: II. Dynamics of and litter variability in uterine flow. Teratology 26, 279–288.[ISI][Medline]

Cao, G., Zhao, L., Strangle, H., Hasegawa, T., Richardson, J. A., Parker, K. L., and Hobbs, H. H. (1999). Developmental and hormonal regulation of murine scavenger receptor, class B, type 1. Mol. Endocrinol. 13, 1460–1473.[Abstract/Free Full Text]

Clark, M. M., Bishop, A. M., vom Saal, F. S., and Galef, B. G., Jr. (1993). Responsiveness to testosterone of male gerbils from known intrauterine positions. Physiol. Behav. 53, 1183–1187.[CrossRef][ISI][Medline]

El-Gehani, F., Zhang, F. P., Pakarinen, P., Rannikko, A., and Huhtaniemi, I. (1998). Gonadotropin-independent regulation of steroidogenesis in the fetal rat testis. Biol. Reprod. 58, 116–123.[Abstract]

Elswick, B. A., Welsch, F., and Janszen, D. B. (2000). Effect of different sampling designs on outcome of endocrine disrupter studies. Reprod. Toxicol. 14, 359–367.[CrossRef][ISI][Medline]

Even, M. D., Dhar, M. G., and vom Saal, F. S. (1992). Transport of steroids between fetuses via amniotic fluid in relation to the intrauterine position phenomenon in rats. J. Reprod. Fertil. 96, 709–716.[Abstract]

Even, M. D., Laughlin, M. H., Krause, G. F., and vom Saal, F. S. (1994). Differences in blood flow to uterine segments and placentae in relation to sex, intrauterine location and side in pregnant rats. J. Reprod. Fertil. 102, 245–252.[Abstract]

Foster, P. M. D., Mylchreest, E., Gaido, K. W., and Sar, M. (2001). Effects of phthalate esters on the developing reproductive tract of male rats. Hum. Reprod. Update 7, 231–235.[Abstract/Free Full Text]

Fox, R. A., Sigman, M., and Boekelheide, K. (2000). Transmembrane versus soluble stem cell factor expression in human testis. J. Androl. 21, 579–585.[Abstract/Free Full Text]

Gray, L. E., Jr., Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., and Ostby, J. (1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health 15, 94–118.[ISI][Medline]

Hasegawa, T., Zhao, L., Caron, K. M., Majdic, G., Suzuki, T., Shizawa, S., Sasano, H., and Parker, K. L. (2000). Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol. Endocrinol. 14, 1462–1471.[Abstract/Free Full Text]

Haseman, J. K., and Hogan, M. D. (1975). Selection of the experimental unit in teratology studies. Teratology 12, 165–171.[ISI][Medline]

Huhtaniemi, I., and Pelliniemi, L. J. (1992). Fetal Leydig cells: Cellular origin, morphology, life span, and special functional features. Proc. Soc. Exp. Biol. Med. 201, 125–140.[Abstract]

International Programme on Chemical Safety (IPCS) (1992). Environmental Health Criteria 131. Diethylhexyl Phthalate. World Health Organization, Geneva.

IPCS (1997). Environmental Health Criteria 189. Di-n-butyl Phthalate. World Health Organization, Geneva.

Kohn, M. C., Parham, F., Masten, S. A., Portier, C. J., Shelby, M. D., Brock, J. W., and Needham, L. L. (2000). Human exposure estimates for phthalates. Environ. Health Perspect. 108, A440–A442.

Lakins, J., Bennett, S. A., Chen, J. H., Arnold, J. M., Morrissey, C., Wong, P., O’Sullivan, J., and Tenniswood, M. (1998). Clusterin biogenesis is altered during apoptosis in the regressing rat ventral prostate. J. Biol. Chem. 273, 27887–27895.[Abstract/Free Full Text]

Lambright, C. S., Wilson, V. S., Furr, J. R., Wolf, C. J., Noriega, N., and Gray, L. E., Jr. (2003). Effects of endocrine disrupting chemicals on fetal testes hormone production. The Toxicologist 72, 272.

Leger, J. G., Montpetit, M. L., and Tenniswood, M. P. (1987). Characterization and cloning of androgen-repressed mRNAs from rat ventral prostate. Biochem. Biophys. Res. Commun. 147, 196–203.[ISI][Medline]

Loveland, K. L., and Schlatt, S. (1997). Stem cell factor and c-kit in the mammalian testis: Lessons originating from Mother Nature’s gene knockouts. J. Endocrinol. 153, 337–344.[Abstract/Free Full Text]

Manna, P. R., Roy, P., Clark, B. J., Stocco, D. M., and Huhtaniemi, I. T. (2001). Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis. J. Steroid Biochem. Mol. Biol. 76, 167–177.[CrossRef][ISI][Medline]

Manova, K., Nocka, K., Besmer, P., and Bachvarova, R. F. (1990). Gonadal expression of c-kit encoded at the W locus of the mouse. Development 110, 1057–1069.[Abstract]

Miller, W. L. (1988). Molecular biology of steroid hormone synthesis. Endocr. Rev. 9, 295–318.[ISI][Medline]

Miyake, H., Chi, K. N., and Gleave, M. E. (2000a). Antisense TRPM-2 oligodeoxynucleotides chemosensitize human androgen-independent PC-3 prostate cancer cells both in vitro and in vivo. Clin. Cancer Res. 6, 1655–1663.[Abstract/Free Full Text]

Miyake, H., Nelson, C., Rennie, P. S., and Gleave, M. E. (2000b). Testosterone-repressed prostate message-2 is an antiapoptotic gene involved in progression to androgen independence in prostate cancer. Cancer Res. 60, 170–176.[Abstract/Free Full Text]

Mylchreest, E., Sar, M., Cattley, R. C., and Foster, P. M. D. (1999). Disruption of androgen-regulated male reproductive development by di(n- butyl) phthalate during late gestation in rats is different from flutamide. Toxicol. Appl. Pharmacol. 156, 81–95.[CrossRef][ISI][Medline]

Mylchreest, E., Sar, M., Wallace, D. G., and Foster, P. M. D. (2002). Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate. Reprod. Toxicol. 16, 19–28.[CrossRef][ISI][Medline]

Mylchreest, E., Wallace, D. G., Cattley, R. C., and Foster, P. M. D. (2000). Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to di(n-butyl) phthalate during late gestation. Toxicol. Sci. 55, 143–151.[Abstract/Free Full Text]

National Research Council (1996). Guide for the care and use of laboratory animals. National Academy Press, Washington, D.C.

National Toxicology Program (1991). Di(n-butyl) phthalate reproduction and fertility assessment in CD Sprague-Dawley rats when administered via feed. National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Nonneman, D. J., Ganjam, V. K., Welshons, W. V., and Vom Saal, F. S. (1992). Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice. Biol. Reprod. 47, 723–729.[Abstract]

Noumura, T., Weisz, J., and Lloyd, C. W. (1966). In vitro conversion of 7–3-H-progesterone to androgens by the rat testis during the second half of fetal life. Endocrinology 78, 245–253.[ISI][Medline]

Omura, T., and Morohashi, K. (1995). Gene regulation of steroidogenesis. J. Steroid Biochem. Mol. Biol. 53, 19–25.[CrossRef][ISI][Medline]

Orth, J. M., Jester, W. F., Jr., and Qiu, J. (1996). Gonocytes in testes of neonatal rats express the c-kit gene. Mol. Reprod. Dev. 45, 123–131.[CrossRef][ISI][Medline]

O’Shaughnessy, P. J., Baker, P., Sohnius, U., Haavisto, A. M., Charlton, H. M., and Huhtaniemi, I. (1998). Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology 139, 1141–1146.[Abstract/Free Full Text]

Pearse, A. G. E. (1996). Histotechnology: A self-instructional text. ASCP Press, Chicago, IL.

Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2002–2007.

Sar, M., and Welsch, F. (1999). Differential expression of estrogen receptor-ß and estrogen receptor-{alpha} in the rat ovary. Endocrinology 140, 963–971.[Abstract/Free Full Text]

Shirley, E. A., and Hickling, R. (1981). An evaluation of some statistical methods for analysing numbers of abnormalities found amongst litters in teratology studies. Biometrics 37, 819–829.[ISI][Medline]

Shultz, V. D., Phillips, S., Sar, M., Foster, P. M. D., and Gaido, K. W. (2001). Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate. Toxicol. Sci. 64, 233–242.[Abstract/Free Full Text]

Tapanainen, J., Kuopio, T., Pelliniemi, L. J., and Huhtaniemi, I. (1984). Rat testicular endogenous steroids and number of Leydig cells between the fetal period and sexual maturity. Biol. Reprod. 31, 1027–1035.[Abstract]

Thompson, C. T., Ross, S. M., and Gaido, K. W. (2003). Di(n-butyl) phthalate interferes with fetal testicular steroidogenesis at the level of cholesterol transport and cleavage. The Toxicologist 72, 273.

Trigatti, B., Rigotti, A., and Krieger, M. (2000). The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism. Curr. Opin. Lipidol. 11, 123–131.[CrossRef][ISI][Medline]

Williams, D. A. (1975). The analysis of binary responses from toxicological experiments involving reproduction and teratogenicity. Biometrics 31, 949–952.[ISI][Medline]

Wine, R. N., Li, L. H., Barnes, L. H., Gulati, D. K., and Chapin, R. E. (1997). Reproductive toxicity of di-n-butylphthalate in a continuous breeding protocol in Sprague-Dawley rats. Environ. Health Perspect. 105, 102–107.[ISI][Medline]

Wong, P., Pineault, J., Lakins, J., Taillefer, D., Leger, J., Wang, C., and Tenniswood, M. (1993). Genomic organization and expression of the rat TRPM-2 (clusterin) gene, a gene implicated in apoptosis. J. Biol. Chem. 268, 5021–5031.[Abstract/Free Full Text]

Zhang, F. P., Hamalainen, T., Kaipia, A., Pakarinen, P., and Huhtaniemi, I. (1994). Ontogeny of luteinizing hormone receptor gene expression in the rat testis. Endocrinology 134, 2206–2213.[Abstract]

Zhang, F. P., Poutanen, M., Wilbertz, J., and Huhtaniemi, I. (2001). Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol. Endocrinol. 15, 172–183.[Abstract/Free Full Text]

Zsebo, K. M., Williams, D. A., Geissler, E. N., Broudy, V. C., Martin, F. H., Atkins, H. L., Hsu, R. Y., Birkett, N. C., Okino, K. H., Murdock, D. C., et al. (1990). Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213–224.[ISI][Medline]