* CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709; and
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received February 9, 2003; accepted March 7, 2003
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ABSTRACT |
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Key Words: di(n-butyl); phthalate; steroidogenesis; male reproductive development; gene expression; fetal testes; litter variability; c-kit.
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INTRODUCTION |
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Although DBP and its major metabolite do not bind to the androgen receptor (Foster et al., 2001), DBP has been characterized as an antiandrogen because it caused a 6688% decrease in fetal intratesticular testosterone (T) levels in the rat on GD 18, 19, and 21 (Mylchreest et al., 2002
; Shultz et al., 2001
) and had profound effects on the developing male reproductive tract (Barlow and Foster, 2003
; Gray et al., 1999
; Mylchreest et al., 1999
, 2000
). The mechanism by which DBP caused reduced T levels was through decreased production of androgen by the fetal Leydig cells (LCs) (Lambright et al., 2003
). 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., 2002
). 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, 2003
).
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 1920, at which time the developing epididymides appeared smaller with decreased coiling of the epididymal duct (Barlow and Foster, 2003). 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, 2003
). In addition to morphologic lesions on GD 19, this age was chosen because T synthesis is at or near its zenith (Huhtaniemi and Pelliniemi, 1992
; Tapanainen et al., 1984
).
Utilizing cDNA microarrays and real-time quantitative RT-PCR, Shultz et al.(2001) 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-
, 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
-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., 2001
).
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), 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.
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MATERIALS AND METHODS |
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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, 2003) 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)
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 manufacturers 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 manufacturers 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 1 and 2
) using Primer Express software (Applied Biosystems) with the following parameters: low Tm = 60°C, high Tm = 64°C, optimum Tm = 62°C, amplicon length = 80150 base pairs, and primer length = 2024 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 manufacturers 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 manufacturers 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.
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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). 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), 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
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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 Biosystems User Bulletin No. 2, 2-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.
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RESULTS |
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DISCUSSION |
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StAR is necessary for delivery of cholesterol to the inner mitochondrial membrane (Hasegawa et al., 2000; Manna et al., 2001
). Gene expression and protein levels of this cholesterol transport molecule were significantly decreased, relative to control (Fig. 7
). Studies by Thompson et al.(2003)
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. 7
).
P450scc conversion of cholesterol to pregnenolone is the limiting enzymatic step in T biosynthesis (Miller, 1988; Omura and Morohashi, 1995
). 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. 7
). 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. 7
). However, Thompson et al.(2003)
showed that, when DBP-exposed testes were incubated with pregnenolone, progesterone, or 17
-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) 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., 1998
; Noumura et al., 1966
; OShaughnessy et al., 1998
). 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., 1998
; Zhang et al., 1994
). 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., 2001
). 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. 7). c-kit is the receptor for SCF, both of which are located on gonocytes and Sertoli cells, respectively (Zsebo et al., 1990
). 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, 1997
). 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. 7
). 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., 2000
; Loveland and Schlatt, 1997
; Manova et al., 1990
; Orth et al., 1996
). The purpose of c-kit in adult LCs may be related to soluble SCF and regulation of testosterone biosynthesis (Fox et al., 2000
; Loveland and Schlatt, 1997
). 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, 2003
; Brannan et al., 1992
). 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., 2000; Haseman and Hogan, 1975
; Shirley and Hickling, 1981
; Williams, 1975
). 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., 2000
; Mylchreest et al., 1999
, 2000
). 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., 1982
; Even et al., 1994
), 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., 1993
; Even et al., 1992
; Nonneman et al., 1992
). 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). 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)
and Bell (1982)
, 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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