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CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709-2137
Address all correspondence and requests for reprints to: Dr. Kevin Gaido, CIIT Centers for Health Research, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709-2137. E-mail: Gaido{at}ciit.org.
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Introduction |
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Phthalate esters are commonly used as plasticizers (8, 9) and can be found in such disparate products as cosmetics (10) and infant formula (11). As such, the potential for human exposure to phthalates is quite high, particularly among those using polyvinyl chloride-based medical devices (12, 13). Phthalate esters are metabolized in the gut to the corresponding monoester and alcohol (14, 15, 16), with toxicity ascribed to the monoester metabolite (17, 18). A study of urinary levels of phthalate ester metabolites in the general population showed that women of childbearing age (20–40 yr) displayed significantly higher levels of monobutyl phthalate (MBP) than all other groups examined (19). The high levels of MBP in women of reproductive age are a matter of concern given the clear adverse effects of the diester precursor DBP on male reproductive development in animal models (3, 11, 20).
Endocrine-disrupting chemicals are defined as exogenous agents that interfere with normal endocrine signaling (21). Typically, endocrine-disrupting chemicals are thought to work at the level of the estrogen or androgen receptor. However, phthalate esters do not interact with the androgen receptor (4, 22). Rather, these chemicals disrupt testosterone synthesis by the fetal testis (6, 20, 22), probably through diminished expression of several genes in the cholesterol transport and testosterone biosynthesis pathways (20, 23). Cholesterol, synthesized de novo in the testis or acquired from serum lipoproteins via scavenger receptor B1 (SR-B1), is transported from the outer to the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Transport across the mitochondrial membrane is the rate-limiting step of testosterone biosynthesis (24). A six-carbon moiety is cleaved from cholesterol by the side-chain cleavage enzyme (P450scc) to generate the steroid pregnenolone. The remaining enzymatic reactions required for production of testosterone take place in the smooth endoplasmic reticulum (Fig. 1
) (25). Expression of SR-B1, StAR, P450SCC, 3ß-hydroxysteroid dehydrogenase (HSD), and cytochrome p450c17 (CYP17) have all been shown to be diminished in the fetal testis after treatment with DBP (20, 23).
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. Animals were euthanized by CO2 anesthesia and exsanguination by abdominal aorta transection. Fetuses were removed by Cesarean section and euthanized by decapitation. The sex of fetuses was determined by internal inspection of the gonads. Female fetuses were discarded. Testes for explant culture were removed and placed in ice-cold PBS supplemented with penicillin and streptomycin. All other testes were snap-frozen in liquid nitrogen and stored at -70 C.
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). The supplement concentrations used in this study have been shown to be maximally stimulating in Leydig cell cultures (29). 3H-cholesterol was purchased from PerkinElmer Life Sciences, Inc. (Boston, MA). SR-B1 has been shown to efficiently transport nonlipoprotein cholesterol (30). All other supplements were purchased from Sigma.
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counter (Packard Instrument Co., Downers Grove, IL).
RNA isolation and cDNA synthesis
Total RNA was isolated from frozen tissues using STAT-60 reagent (Tel-Test, Inc., Friendswood, TX) according to manufacturer’s instructions. Each total RNA sample was checked for integrity and DNA contamination by measurement of OD and size-fractionation of 18S and 28S rRNA on an agarose gel. All reagents for reverse transcription were purchased from Applied Biosystems (Foster City, CA) unless otherwise noted. After isolation, total RNA was incubated for 1 h at 37 C, in a reaction mixture containing ribonuclease (RNase) inhibitor, dithiothreitol, 5x transcription buffer, and RQ1 RNase-free deoxyribonuclease (Promega Corp., Madison, WI). Deoxyribonuclease was inactivated by incubating for 5 min at 75 C. One microgram of total RNA was reverse-transcribed for 65 min at 42 C in a 20-µl reaction containing 5 mM MgCl2, 1x GeneAmp PCR buffer II [50 mM KCl, 10 mM Tris-HCl (pH 8.3)], 1 mM each deoxynucleotide transferase, random hexamers, 20 U RNase inhibitor, and 50 U murine leukemia virus reverse transcriptase. The reverse transcriptase reaction was terminated by heating to 95 C for 5 min; 0.4 µl cDNA was used for subsequent PCRs.
Real-time quantitative RT-PCR
Real-time quantitative RT-PCR was performed on an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). cDNA prepared as described above was amplified in a 25 µl reaction mix containing 1x SYBR Green PCR Master Mix (Applied Biosystems) and 64 nM each primer. After a 10-min Taq activation step at 95 C, reactions were subjected to 50 cycles of 15 sec denaturation at 94 C, and 1 min annealing/extension at 60 C. Primers were purchased from Operon, Inc. (Alameda, CA). After PCR, reaction products were melted for 3 min at 95 C, and then the temperature was lowered to 50 C in 0.5 C increments, 10 sec per increment. Optical data were collected over the duration of the temperature drop, with a dramatic increase in fluorescence occurring when the strands reannealed. This was done to ensure that only one PCR product was amplified per reaction. Relative expression of the RT-PCR products was determined using the method described by Pfaffl (31). One of the control samples was chosen as the calibrator sample and used in each PCR. Each sample was run in triplicate and the mean Ct used for determination of relative expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization as described previously (20). Primer pairs used are as follows: GAPDH sense 5'-GAAGGTGAAGGTTCGGAGTC-3' and antisense 5'-GAAGATGGTGATGGGATTTC-3'; P450SCC sense 5'-TTCCCATGCTCAACATGCCTC-3' and antisense 5'-ACTGAAAATCACATCCCAGGCAG-3'; CYP17 sense 5'-TGGCTTTCCTGGTGCACAATC-3' and antisense 5'-TGAAAGTTGGTGTTCGGCTGAAG-3'; StAR sense 5'-ACCACATCTACCTGCACGCCAT-3' and antisense 5'-CCTCTCGTTGTCCTTGGCTGAA-3'; SR-B1 sense 5'-CCATTCATGACACCCGAATCCT-3' and antisense 5'-TCGAACACCCTTGATTCCTGGT-3'. The following primers were used to amplify the full-length rat SF-1 cDNA: sense 5'-CGAATTCACCATGGACTATTCGTACGACG-3', and antisense 5'-GCGGCCGCAGTCTGCTTGGCCTGCAGCATC-3'. The sense primer was designed to include an EcoR1 restriction site and a Kozak initiation sequence. The antisense primer contained a Not1 restriction site downstream from the coding region. Thermal cycling parameters were 2 min at 95 C; 1 cycle of 30 sec denaturation at 94 C, 60 sec annealing at 42 C, and 90 sec extension at 72 C; 40 cycles of 30 sec denaturation at 94 C, 30 sec annealing at 60 C, and 90 sec extension at 72 C; and 1 cycle of 10 min at 72 C.
Immunoblotting
For immunoblot analysis, total protein was extracted from paired testis by homogenizing in 50 µl lysis buffer [0.1 M Tris-HCl (pH 8.0), 0.05 M EDTA, 0.1 M NaCl, 1% wt/vol sodium dodecyl sulfate, 1% wt/vol sarcosyl] supplemented with Complete Protease Inhibitor Cocktail (Roche Molecular Biochemicals, Mannheim, Germany). Total protein was quantitated using the bicinchoninic acid protein assay reagent (Sigma). Thirty micrograms of total protein were run on SDS-PAGE and transferred to polyvinylidene difluoride membranes. The following antibodies were used to probe the membrane: rabbit antimouse SR-B1 (Novus Biologicals, Inc., Littleton, CO), rabbit antirat cytochrome P450SCC (U.S. Biological, Swampscott, MA), rabbit antirat StAR (Affinity Bioreagents, Inc., Golden, CO), and rabbit antiporcine CYP17 (a kind gift of Dr. D. B. Hales, University of Illinois at Chicago, Chicago, IL) (32). Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies to rabbit IgG (Amersham Biosciences, Piscataway, NJ) and ECL Plus Western Blotting Detection Reagents (Amersham) and visualized with a MultiImage Light Cabinet (Alpha Innotech Corp., San Leandro, CA). Quantitation of expressed protein levels was done using FluorChem 8000 software (Alpha Innotech).
Cholesterol transport
After overnight culture, explants were washed twice in ice-cold PBS. Samples for whole-cell cholesterol uptake assessment were homogenized in lysis buffer [0.1 M Tris-HCl (pH 8.0), 0.05 M EDTA, 0.1 M NaCl, 1% wt/vol sodium dodecyl sulfate, 1% wt/vol sarcosyl] and incubated for 30 min at 65 C. Samples for mitochondrial uptake assessment were prepared as described (33). Testes were incubated for 10 min on ice in a hypotonic buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1.5 mM MgCl2], followed by homogenization. The homogenate was then spiked with 0.4 vol 2.5x buffer [525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2]. This was centrifuged twice at 1000 x g for 10 min at 4 C. The supernate was then centrifuged at 10,000 x g for 20 min at 4 C. The mitochondrial pellet was resuspended in lysis buffer and incubated at 65 C for 30 min. Total DPM were counted on a Tri-Carb 1900CA Liquid Scintillation Analyzer (Packard Instrument Co.).
Cloning
For expression of the full-length rat SF-1 cDNA, purified PCR products were cloned into pGEM-T Easy Vector (Promega Corp.) for sequence verification. Clones that matched the predicted sequence for SF-1 were digested with EcoR1 and Not1 (both purchased from New England Biolabs, Beverly, MA) and ligated into expression vector pEF1/V5 HisA (Invitrogen, Corp., Carlsbad, CA). This vector adds the V5 epitope tag onto the expressed protein. Expression of SF-1 was confirmed by immunoblotting.
Cell culture
HepG2 cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in phenol red-free MEM (Mediatech, Inc., Herndon, VA) with 4 mM L-glutamine, 1 mM sodium pyruvate (all Life Technologies), and 10% charcoal-stripped fetal bovine serum (HyClone, Logan, UT). MA-10 cells were provided by Dr. Mario Ascoli, University of Iowa (Iowa City, IA). Cells were maintained in Waymouth MB 752/1 media supplemented with 4 mM L-glutamine, 62.5 µg/ml gentamicin, 20 mM HEPES, pH 7.4 (all Life Technologies), and 15% horse serum (Atlanta Biologicals, Inc., Norcross, GA). Both cell lines were maintained at 37 C in an atmosphere of 5% CO2/95% air.
Transfection and luciferase assay
HepG2 or MA-10 cells were grown to 80% confluence in 24-well plates. Plasmid DNA was mixed with TransIT-LT1 Transfection reagent (Mirus Corp., Madison, WI) in 0.65 ml phenol red-free MEM (Mediatech, Inc.) and added to cells. Our laboratory has obtained the following luciferase reporter constructs: StAR (from Dr. Doug Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center); SR-B1 (from Dr. Kaoru Miyamoto, Department of Biochemistry, Fukui Medical University, Fukui, Japan); and CYP17 (from Dr. Synthia Mellon, Department of Obstetrics, Gynecology, and Reproductive Sciences and the Metabolic Research Unit, University of California). A ß-galactosidase plasmid was used as a transfection control. Cells were transfected with reporter constructs with or without the SF-1 expression vector in the presence of 10-5 M MBP (Aldrich) or dimethylsulfoxide control. After 48 h, cells were lysed in 65 µl lysis buffer [25 mM Tris/EDTA (pH 7.8), 10% glycerol, 0.5% Triton X-100, 3 mM dithiothreitol]. One hundred microliters of Luciferase Assay Reagent (Promega Corp.) were added to 20 µl of lysate and luminescence read on an LMax microplate luminometer (Molecular Devices Corp., Sunnyvale, CA). Thirty microliters of lysate were used for ß-galactosidase activity determination. Eighty micrograms of chlorophenol red-ß-D-galactopyranoside (CPRG, Roche Molecular Biochemicals, Indianapolis, IN) in 170 µl of CPRG buffer [60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM ß-mercaptoethanol (pH 7.8)] were added to lysate and absorbance read at 575 nm at 1-min intervals for 30 min in a SpectraMax340 microplate reader (Molecular Devices Corp.) to obtain the Vmax for the reaction. Luciferase activity was normalized to ß-galactosidase activity.
Statistical analyses
The Student’s t test or one-way ANOVA with Tukey post hoc analysis were performed using JMP statistical software (SAS Institute, Inc., Cary, NC), version 5.0.1. P < 0.05 was considered to be statistically significant.
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). The mean testosterone concentration per DBP-treated testes was actually slightly lower on gd 18 than on gd 17, although this difference was not statistically significant. The percentage difference in testicular testosterone between control and treated testes was much higher on gd 18 than on gd 17. Testosterone concentration in the gd 17 testis was 46.6% of that seen in the untreated control, whereas the level at gd 18 was 17.8% of that seen in the control samples. The previously observed differences in expression of mRNA for SR-B1, StAR, P450SCC, and CYP17 after DBP exposure (20, 23) were detected at gd 17, although the diminished expression of P450SCC mRNA was not significant at this gestational day (Fig. 2B). As was the case with testosterone, the percentage difference between DBP-treated and control gene expression was considerably less at gd 17 than at gd 18. Each of the genes demonstrated a similar relative expression level at both gd 17 and 18, but the mean percentage of expression of the four genes relative to age-matched control was 46.4% in the gd 17 DBP-exposed fetuses vs. 15.4% in the gd 18 fetuses exposed to DBP.
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). However, testosterone concentration was 3.6-fold higher 48 h after cessation of DBP exposure relative to the 24-h recovery group, indicating that the steroidogenic capability of the testis was not irreversably affected by DBP. The suppression of transcription by DBP was also a transient effect because mRNA levels for SR-B1, StAR, P450SCC, and CYP17 all returned to control levels 48 h after DBP withdrawal (Fig. 3B).
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). CYP17 and StAR were not detected in the 24-h recovery group. SR-B1 protein showed the most rapid recovery from phthalate exposure, with protein expression 67% that of the control animals 48 h after withdrawal of DBP (Fig. 3D). StAR, P450SCC, and CYP17 protein were all expressed at a similar level (approximately 40–45% of control expression) at this time point. The level of protein expression for StAR and CYP17, both 40% of the control level, correlated well with observed testosterone production by testes in the 48-h recovery group (36% of control steroidogenesis).
To determine whether DBP exposure leads to diminished testicular testosterone through interference with a critical event in male reproductive development, pregnant dams were dosed with corn oil from gd 12–19 or with 500 mg/kg DBP. The start of DBP treatment was shifted from gd 12 1 d later in gestation for each dose group, so that the final group was dosed only on gd 19 approximately 3 h before the animals were killed. Testosterone was repressed in all DBP dose groups (Fig. 4A). The mean testosterone concentration in the fetuses dosed only on gd 19 was 44% of that seen in the control group. The mean testosterone concentration of all the other dose groups was 13% of control levels. mRNA levels were similarly repressed at all time points (Fig. 4B). The only genes that did not show significantly diminished expression relative to control were CYP17 in the gd 19 dose group and P450SCC in the gd 12–19 dose group, although the lack of significance in the latter group is attributable to one sample displaying 3-fold higher expression than all of the other samples in this group. Expression of these four genes was significantly higher in the group dosed only on gd 19 when compared with all the other groups exposed to DBP, except P450SCC in the gd 12–19 and gd 17–19 dose groups. Each of these groups had one sample that showed elevated expression. The pattern of protein expression followed that of mRNA expression, with minimal protein expressed in the animals dosed gd 12–19 through gd 18–19 and moderately decreased expression in the animals dosed exclusively on gd 19 (Fig. 4, C and D).
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). This suggests that there was little functional diminishment of SR-B1 activity after DBP exposure. There is a significant decrease in the amount of cholesterol transported across the mitochondrial membrane in the testes from DBP-treated fetuses, indicating that the observed decrease in StAR mRNA correlated with diminished protein function.
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). However, the magnitude of the response was significantly less in the treated explants, with testosterone production approximately 40% of the control value for each molecule (nontransformed values). Bt2-cAMP was a more efficient stimulant than was LH, with the fold increase over control double that seen with LH for both control and the DBP-exposed explants. The magnitude of the increase in steroidogenesis was actually greater in the DBP group, with the fold increase over unstimulated explants 2.5x that of the corn oil controls in response to both LH and Bt2-cAMP.
Determination of enzymatic steps in steroidogenesis affected by DBP
Explants were supplemented with steroidal precursors to testosterone to pinpoint the steps in the testosterone synthesis pathway altered by DBP. Both control and DBP-exposed explants showed a significant increase in testosterone production after exposure to all supplements. However, the level of testosterone production corresponding to each supplement in the DBP-exposed explants was significantly lower than the accompanying control, except in the case of androstenedione (Fig. 5C). These data suggest that 17ß-HSD is the only enzyme in the testosterone biosynthesis pathway unaffected by exposure to DBP.
Effects of MBP on SF-1-mediated transcription
The genes SR-B1, StAR, P450SCC, and CYP17 have all been shown previously to be regulated by the transcription factor SF-1 (34). However, our laboratory has shown SF-1 gene expression not to be affected after exposure to DBP. To ascertain whether the mechanism of DBP toxicity might be through interference with SF-1-mediated transcription, we cloned the rat SF-1 cDNA into a mammalian expression vector and coexpressed this construct in HepG2 cells along with reporter plasmids for StAR, SR-B1, or CYP17. In each case, SF-1 increased transcription of the reporter construct. However, monobutyl phthalate, the metabolite of DBP to which reproductive toxicity has been ascribed (17, 18), had no effect on SF-1-mediated transcription (Fig. 6). This was also the case when reporter assays were performed in the mouse Leydig tumor cell line MA-10 (not shown).
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Discussion |
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Our data indicated that the effects of DBP on testicular mRNA expression and steroidogenesis were evident as early as gd 17. The lack of a statistically significant down-regulation of testosterone production by DBP at gd 16 reported by Shultz et al. (20) is probably due to the low level of steroidogenesis in control animals at this developmental time point. The Shultz study demonstrated significant decreases in testicular progesterone concentration, StAR mRNA expression, and SR-B1 mRNA expression. Steroidogenesis commences with the differentiation of fetal Leydig cells at gd 15 (36) and increases gradually until gd 18, at which time there is a rapid increase to the peak of testosterone production at gd 19 (37). Our findings correlate well with previous reports of maldevelopment of the reproductive tract in male fetuses exposed in utero on gd 15–17 to DBP or its monoester metabolite MBP (38, 39) and also with data indicating that DEHP, another phthatlate ester that causes testicular toxicity, interferes with testosterone production at gd 17 (22).
The rapid functional recovery of the testis after phthalate exposure could provide insight into the mechanism of phthalate transcriptional regulation. The elimination half-life of phthalate monoesters is approximately 12 h (40). Assuming an equivalent elimination half-life for animals exposed in utero, the level of toxic phthalate to which the fetus is exposed should be below the previously reported NOAEL for DBP of 50 mg/kg·d (27) 48 h after cessation of treatment. Our results demonstrate that transcription returns to normal as toxic phthalate is cleared from the system. This indicates that the mechanism of transcriptional repression by DBP involves direct interaction of DBP or its metabolites with the factors responsible for regulation of the genes responsible for testosterone synthesis. It is not clear at this time whether DBP acts directly on Leydig cells or on the production of molecules by other testicular cell populations that stimulate steroidogenesis in the Leydig cells. Future studies will aim to determine this point of interaction.
Although mRNA expression had returned to control levels 48 h post DBP treatment, testosterone levels as well as levels of SR-B1, StAR, P450SCC, and CYP17 protein were still significantly lower than those seen in control animals. The observed differences between mRNA and protein expression may simply have been a result of translation lagging transcription. Previous studies have shown that protein expression of CYP17, P450SCC (41), and StAR (42) corresponds to mRNA expression in stimulated Leydig cells. However, peak expression of StAR protein in MA-10 Leydig tumor cells occurs approximately 2 h after peak expression of the mRNA (42), and expression of StAR protein has been shown to increase up to 20 h after mRNA expression has reached its peak in granulosa cells (43) and 16 h after mRNA peaks in Y-1 adrenal cells (44). A more detailed time course study of DBP recovery will determine whether the exposed testes are capable of complete recovery of steroidogenesis.
The effects of DBP treatment become evident rapidly, with full repression of steroidogenesis apparent 24 h after first exposure and some repression apparent as early as 3 h after exposure. These data agree with previously reported in vitro and in vivo studies on testicular toxicity of phthalate esters. MA-10 Leydig tumor cells are less responsive to human chorionic gonadotropin-stimulated progesterone production after 24-h exposure to mono(2-ethylhetyl)phthalate (MEHP) (45). Primary cultures of Leydig cells display diminished testosterone production 2 h after dosing with MEHP (46), and expression of mRNA for TRPM2, a gene negatively regulated by androgens, is elevated within 3 h of MEHP gavage in 28-d-old rats (47). It has been suggested that the toxic effects of phthalates on Sertoli cells (48) and Leydig cells (22) are a result of arrested development of these cells. Our results indicate that phthalate esters exert toxic effects on fetal Leydig cells via transcriptional repression well after development of full testosterone biosynthetic capability by these cells.
Despite diminished mRNA and protein expression for SR-B1, there was not a significant reduction in whole-cell cholesterol transport observed in testis explants after in utero exposure to DBP. This finding is in contrast to the findings of Barlow et al. (23), who showed diminished Leydig cell lipid content after in utero exposure to DBP. A possible explanation for this discrepancy is that SR-B1 activity is not required for basal steroidogenesis in the fetal testis. Most of the cholesterol used in steroidogenesis is produced de novo in the adult testis, with selective uptake of cholesteryl esters from high-density lipoprotein particles increasing only after prolonged stimulation with gonadotropins, a treatment regimen that also results in increased expression of SR-B1 (49). Although the source of cholesterol for the fetal rat Leydig cell has not been clearly defined, studies indicate that there is high de novo synthesis of cholesterol in several tissues of the rat embryo (50, 51, 52) and that the human fetal testis uses cholesterol produced internally as a testosterone precursor (53). In this study, cholesterol uptake by the explants was assessed under basal culture conditions. Supplementing the cultures with LH or Bt2-cAMP led to dramatic increases in testosterone production. Culture of the explants under stimulating conditions should lead to greater cholesterol uptake on the whole-cell level and, consequently, a greater disparity in selective uptake between control and DBP-exposed testis. Alternatively, the lack of difference in whole-cell cholesterol transport between the control and DBP-exposed explants might indicate that the amount of SR-B1 protein in the DBP-exposed explants, although decreased, was still sufficient for movement of cholesterol across the cell membrane.
There was a significant difference in mitochondrial transport of 3H-cholesterol between control and DBP-exposed testes, indicating that the observed repression of StAR mRNA and protein by DBP was of functional importance. Transport of cholesterol from the outer to the inner mitochondrial membrane by the StAR protein is the rate-limiting step of acute steroidogenesis (24) and as such represents a key point in the regulation of testosterone synthesis. StAR protein is rapidly expressed in response to stimulating signals such as gonadotropins (54). However, in regard to basal steroidogenesis in the fetal testis cultures, the observed 28% decrease in mitochondrial transport alone is unlikely to account for the 83% reduction in testosterone production after DBP exposure.
Both control and DBP-exposed cultures demonstrated significant increases in testosterone production in response to testosterone precursors. The response of the DBP-exposed explants was lower than that of the controls for all supplements, with the exception of explants supplemented with androstenedione. This finding correlates well with the reported lack of change in expression of 17ß-HSD mRNA after in utero exposure to DBP (23). These results suggest that the combined effects of diminished expression of StAR, P450SCC, 3ß-HSD, and CYP17 led to the observed alterations in steroidogenesis after DBP exposure.
DBP-exposed explants responded to stimulation with both LH and Bt2-cAMP. However, as was the case with steroid precursors, the response of explants was dampened in DBP-exposed testes. These data suggest that the reduction in steroidogenesis was not due to altered responsiveness to LH or cAMP signaling but rather due to reduced expression of the downstream factors necessary for steroidogenesis. The factors involved in regulating steroidogenesis in the fetal testis are unknown. Testosterone production by the fetal testis begins on gd 15 (36) and peaks on gd 19 (37). However, LH levels in the fetus do not reach appreciable levels until gd 19.5 (37), indicating that fetal testicular steroidogenesis, unlike that of mature Leydig cells, is initiated by factors other than LH. Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide have both been shown to stimulate testosterone production by fetal Leydig cells (37). However, mRNA expression of these genes and their receptors are not altered in the fetal testis after DBP exposure (our unpublished observations). Future studies will employ this explant culture system to identify potential regulators of fetal steroidogenesis.
All the genes shown to have diminished expression after DBP exposure are regulated by the orphan nuclear receptor SF-1 (34). SF-1 mRNA expression is unchanged by treatment with phthalate esters (20). Numerous studies have shown interactions between SF-1 and other nuclear proteins leading to potentiation or attenuation of SF-1-mediated transcription (55, 56, 57, 58, 59). However, MBP, the metabolite of DBP to which reproductive toxicity has been ascribed (17, 18), did not affect the SF-1-regulated transcription of SR-B1, StAR, or CYP reporter constructs in either HepG2 or MA-10 cells. Because MA-10 cells are steroidogenic (45), any cofactors required for expression of these genes are most likely present in this cell line. This finding indicates that MBP does not interfere with transcriptional regulation by preventing SF-1 binding to response elements or recruitment of cofactors. Because there are differences in the regulation of steroidogenesis between fetal and adult Leydig cell populations (36, 37) and in the susceptibility of these cell types to phthalate ester toxicity (5, 11, 60), exposure to DBP might lead to altered gene expression through interaction with a fetal Leydig cell-specific transcriptional regulator. Alternatively, DBP treatment could lead to expression of a novel transcriptional repressor in the fetal testis. The future aim of our laboratory is to elucidate the mechanism of transcriptional repression by DBP.
In summary, these studies demonstrate that suppression of testosterone production in the fetal testis by DBP is coincident with diminished transcription of several genes in the cholesterol transport and steroidogenesis pathways as early as gd 17. The effect of DBP on gene expression is rapid and independent of the stage of development of the fetal Leydig cell. Also, diminished expression of the genes and proteins necessary for testicular steroidogenesis is reversed as DBP and its metabolite are cleared. The rapid and reversible effect of DBP on steroidogenesis indicates that DBP directly interferes with the signaling processes necessary for maintenance of steroidogenesis or with the transcriptional regulators required to maintain coordinate expression of the genes involved in cholesterol transport and testosterone biosynthesis. Future work will focus on determining the extent of testicular recovery after cessation of DBP treatment and on determining the minimal timing of exposure to DBP required for altered expression of the relevant genes.
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Acknowledgments |
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Footnotes |
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Abbreviations: Bt2-cAMP, Dibutyryl cAMP (Bt2-cAMP)DBP, di(n-butyl) phthalate; CYP17, cytochrome p450c17; gd, gestational day; HSD, 17ß-hydroxysteroid dehydrogenase; MBP, monobutyl phthalate; MEHP, mono (2-ethylhetyl)phthalate; P450scc, side-chain cleavage enzyme; RNase, ribonuclease; SF-1, steroidogenic factor-1; SR-B1, scavenger receptor B1; StAR, steroidogenic acute regulatory protein.
Received October 31, 2003.
Accepted for publication November 5, 2003.
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) by an androgen receptor-mediated mechanism. J Biol Chem 262:11200–11206 in the regulation of mouse Leydig cell steroidogenesis. Endocrinology 132:2438–2444This article has been cited by other articles:
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  C. McKinnell, R. T. Mitchell, M. Walker, K. Morris, C. J.H. Kelnar, W. H. Wallace, and R. M. Sharpe Effect of fetal or neonatal exposure to monobutyl phthalate (MBP) on testicular development and function in the marmoset Hum. Reprod., September 1, 2009; 24(9): 2244 - 2254. [Abstract] [Full Text] [PDF]
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A. J. Kuhl, S. M. Ross, and K. W. Gaido CCAAT/Enhancer Binding Protein {beta}, But Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis Endocrinology, December 1, 2007; 148(12): 5851 - 5864. [Abstract] [Full Text] [PDF]
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K. W. Gaido, J. B. Hensley, D. Liu, D. G. Wallace, S. Borghoff, K. J. Johnson, S. J. Hall, and K. Boekelheide Fetal Mouse Phthalate Exposure Shows that Gonocyte Multinucleation is Not Associated with Decreased Testicular Testosterone Toxicol. Sci., June 1, 2007; 97(2): 491 - 503. [Abstract] [Full Text] [PDF]
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S. Plummer, R. M. Sharpe, N. Hallmark, I. K. Mahood, and C. Elcombe Time-Dependent and Compartment-Specific Effects of In Utero Exposure to Di(n-butyl) Phthalate on Gene/Protein Expression in the Fetal Rat Testis as Revealed by Transcription Profiling and Laser Capture Microdissection Toxicol. Sci., June 1, 2007; 97(2): 520 - 532. [Abstract] [Full Text] [PDF]
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 C. J. Thompson, S. M. Ross, J. Hensley, K. Liu, S. C. Heinze, S. S. Young, and K. W. Gaido Differential Steroidogenic Gene Expression in the Fetal Adrenal Gland Versus the Testis and Rapid and Dynamic Response of the Fetal Testis to Di(n-butyl) Phthalate Biol Reprod, November 1, 2005; 73(5): 908 - 917. [Abstract] [Full Text] [PDF]
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C. McKinnell, R. M. Sharpe, K. Mahood, N. Hallmark, H. Scott, R. Ivell, C. Staub, B. Jegou, F. Haag, F. Koch-Nolte, et al. Expression of Insulin-Like Factor 3 Protein in the Rat Testis during Fetal and Postnatal Development and in Relation to Cryptorchidism Induced by in Utero Exposure to Di (n-Butyl) Phthalate Endocrinology, October 1, 2005; 146(10): 4536 - 4544. [Abstract] [Full Text] [PDF]
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E. Kleymenova, C. Swanson, K. Boekelheide, and K. W. Gaido 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, September 1, 2005; 73(3): 482 - 490. [Abstract] [Full Text] [PDF]
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J. M. Naciff, K. A. Hess, G. J. Overmann, S. M. Torontali, G. J. Carr, J. P. Tiesman, L. M. Foertsch, B. D. Richardson, J. E. Martinez, and G. P. Daston Gene Expression Changes Induced in the Testis by Transplacental Exposure to High and Low Doses of 17{alpha}-Ethynyl Estradiol, Genistein, or Bisphenol A Toxicol. Sci., August 1, 2005; 86(2): 396 - 416. [Abstract] [Full Text] [PDF]
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C. J. Bowman, K. J. Turner, M. Sar, N. J. Barlow, K. W. Gaido, and P. M. D. Foster Altered Gene Expression During Rat Wolffian Duct Development following Di(n-Butyl) Phthalate Exposure Toxicol. Sci., July 1, 2005; 86(1): 161 - 174. [Abstract] [Full Text] [PDF]
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I. K. Mahood, N. Hallmark, C. McKinnell, M. Walker, J. S. Fisher, and R. M. Sharpe Abnormal Leydig Cell Aggregation in the Fetal Testis of Rats Exposed to Di (n-Butyl) Phthalate and Its Possible Role in Testicular Dysgenesis Endocrinology, February 1, 2005; 146(2): 613 - 623. [Abstract] [Full Text] [PDF]
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J. C. Corton and P. J. Lapinskas Peroxisome Proliferator-Activated Receptors: Mediators of Phthalate Ester-Induced Effects in the Male Reproductive Tract? Toxicol. Sci., January 1, 2005; 83(1): 4 - 17. [Abstract] [Full Text] [PDF]
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K. Boekelheide Cracking the Nut Toxicol. Sci., September 1, 2004; 81(1): 1 - 2. [Full Text] [PDF]
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, P < 0.01 when compared 24-h recovery samples by one-way ANOVA with Tukey post hoc analysis. B, Real-time RT-PCR was performed as described in Fig. 2
