Our data show that phthalate-induced fetal testis endocrine disruption is closely associated with reductions in Leydig cell SREBP2-dependent lipid metabolism gene expression. In the rat, phthalate exposure lowers fetal testis expression of genes in several metabolic pathways. In contrast, mouse phthalate exposure reduces gene expression in only a subset of metabolic pathways, and lipid metabolism gene expression is induced. A recent publication analyzing fetal testis expression microarray data after rat phthalate exposure arrived at similar conclusions to ours concerning the inhibition of several metabolic pathways (Ovacik et al., 2010
), but our pathway data extend these observations by comparing the rat data to a high dose-level DBP exposure in the mouse. The resistance to phthalate-induced endocrine disruption observed in the mouse is not because of a lack of molecular effects in the fetal testis. Significant expression changes in several hundred genes were observed in mouse testis after both acute and multiple day DBP exposure. Pathway analysis showed that mouse phthalate exposure reduces gene expression within many metabolic pathways including amino acid, energy, carbohydrate, and certain lipid pathways, but, significantly, inhibition does not extend to the steroidogenic or cholesterologenic metabolic branches. In fact, multiple day DBP250 exposure in the mouse increased fetal testis expression of steroidogenic and cholesterologenic genes. Thus, DBP exposure appears to reduce the activity of several metabolic pathways (carbohydrate, amino acid, and energy) in both mouse and rat fetal testis, but lipid metabolism pathways are increased in the mouse and decreased in the rat.
The mechanism behind the differential rat and mouse phenotypes is unknown, but we hypothesize a species-specific affect of DBP exposure on fetal Leydig cell SREBP2 activity. Endocrine disruption is correlated with both reduced Leydig cell SREBP2 immunoexpression and reduced expression of lipid metabolism genes regulated by SREBP2 transcriptional activity. Furthermore, decreased fetal testis testosterone levels and SREBP2-dependent gene expression have similar time dependence, dose-response, and species dependence. SREBP2 is widely recognized as being the major transcription factor regulating genes controlling cholesterol biosynthesis, and this pathway displayed reduced expression in rat fetal testis as early as 3 h after DBP500 exposure. This timing approximates that of steroidogenic gene expression inhibition, which is likely a major driver of reduced fetal testis testosterone production. Likewise, cholesterologenic and steroidogenic gene expression displayed similar dose-responses after GD12–20 dosing, although expression of some cholesterologenic genes (Hmgcr and Insig1) was inhibited modestly at DBP100, whereas no significant changes in steroidogenic mRNA amounts were observed at this dose level. Finally, the congruency between effects of DBP on steroidogenesis and cholesterologenesis was supported by similar dose-dependent reductions in rat fetal testis testosterone and total cholesterol levels.
Compared with steroidogenic gene expression and testosterone levels, rat DBP exposure did not reduce expression of cholesterologenic genes, SREBP2 protein, or total cholesterol to the same extent in total testis preparations. For example, testosterone levels after DBP500 exposure were reduced by 90%, but total cholesterol was reduced only 20%. We speculate that this result stems from DBP profoundly targeting lipid metabolism in Leydig cells but having modest, if any, effects on lipid metabolism in other testicular cell types. This conclusion is supported by data showing that DBP primarily reduces rat fetal testis lipid metabolism gene expression in the interstitial compartment while having little effect on these genes in seminiferous cords (Plummer et al., 2007
). Unlike testosterone production and steroidogenic gene expression, which are Leydig cell-specific functions, nearly all mammalian cells have the capacity to generate cholesterol de novo
, and we observed SREBP2 expression in all testis cell types. Therefore, using whole testis as starting material to examine DBP-induced inhibition of lipid metabolism occurring mainly in Leydig cells would dilute the response. This may be the reason for failing to detect significant reductions in total testis SREBP2 via Western blotting but observing a significant decrease in Leydig cell SREBP2 immunoexpression using a technique that targeted quantification to Leydig cells.
In previous publications, others have suggested a potential role of SREBP inhibition in phthalate-induced endocrine disruption (Lehmann et al., 2004
; Shultz et al., 2001
). Lehmann et al. (2004)
examined fetal rat testis Srebf1
mRNA levels after DBP500 exposure, and, like our results, Srebf1
mRNA was not reduced. To our knowledge, the studies presented here are the first to examine the effects of phthalate exposure on SREBP2 expression. Androgen receptor antagonist exposure of prostate cell lines can reduce SREBP activation (Heemers et al., 2006
), and an adult mouse model with elevated testosterone levels shows increased testicular expression of cholesterologenic genes (Eacker et al., 2008
). These data suggest that phthalates may reduce fetal testis SREBP activity secondary to reduced testosterone production (Shultz et al., 2001
). Arguing against this hypothesis are data indicating that the androgen receptor may not be expressed in fetal Leydig cells (Majdic et al.
, 1995; but for a dissenting conclusion, see Mylch reest et al., 2002
) and data showing very few changes in expression of fetal rat testis cholesterol synthesis genes occur following in utero
exposure to the androgen receptor antagonist flutamide (Mu et al., 2006
). Using a fetal rat testis culture model, we have observed no effect of androgen or flutamide exposure on expression of cholesterologenic genes (Kamin Johnson, unpublished observations). Furthermore, cholesterologenic and steroidogenic gene changes after phthalate exposure share similar kinetics, suggesting changes in one pathway do not lead to changes in the other. We conclude from these data that reduced androgen receptor activity after phthalate exposure does not play a major role in inhibition of the fetal Leydig cell cholesterologenic pathway.
In the rat model, inhibition of testicular cholesterologenic gene expression occurred despite a reduction in cholesterol levels. At first glance, these observations imply a dysregulation in the testis of the negative feedback loop modulating SREBP activity and cholesterol levels. However, the SREBP-controlling negative feedback loop is sensitive to sterol concentration within the endoplasmic reticulum membrane (Goldstein et al., 2006
; Radhakrishnan et al., 2008
), whereas cholesterol can be stored in lipid droplets separated from the endoplasmic reticulum membrane. Leydig cells contain abundant lipid droplets for cholesterol storage, and the diminution of Leydig cell lipid droplets after phthalate exposure (Barlow et al., 2003
; Lehmann et al., 2004
) likely accounts for the lowered testis cholesterol content. To account for the simultaneous reduction in both testicular cholesterol content and SREBP-dependent cholesterologenic gene expression, we hypothesize that phthalate exposure decreases storage of cholesterol in lipid droplets while concomitantly increasing the endoplasmic reticulum cholesterol content. Because oxysterol derivatives of cholesterol also negatively regulate SREBP processing and gene expression activity (Gale et al., 2009
; Radhakrishnan et al., 2007
), another potential mechanism operating is a phthalate-induced increase in oxysterol production.
Because SREBP2 is a transcription factor and reduced steroidogenic gene expression appears to contribute to phthalate-induced testosterone reductions, the question arises: does SREBP2 modulate steroidogenic gene expression in fetal Leydig cells? Although no data are available in this cell type, SREBP can increase steroidogenic gene expression in other cell types. Mammalian Star
promoters contain consensus SREBP binding sites. In nonsteroidogenic human cell lines, both SREBP1 and SREBP2 can bind to the Star
promoter and increase expression of transfected Star
promoter/reporter constructs (Christenson et al., 2001
; Shea-Eaton et al., 2001
). In a human adrenocortical cell line, mutation of the putative SREBP binding site in the Cyp17a1
promoter and knockdown of SREBP1 both decrease transcriptional activity of a transfected Cyp17a1
promoter (Ozbay et al., 2006
). Thus, the potential exists for SREBP transcriptional activity to increase steroidogenic gene expression in fetal Leydig cells. Defining how SREBP2 is regulated and its functional activity in fetal Leydig cells may provide clues about the phthalate molecular target as well as the signaling pathways governing fetal Leydig cell steroidogenesis.
Although some epidemiological studies suggest that phthalates may perturb human Leydig cell hormone production (Main et al., 2006
; Swan, 2008
), no direct evidence of human fetal testis endocrine disruption by phthalates is available. Because the experiments presented here did not test human tissue, these data do not speak directly to the endocrine disruption susceptibility of human fetal testes. However, our results could be used to help define the sensitivity of human fetal testes to phthalate endocrine. The question of human sensitivity is highlighted by the differential endocrine disruption observed between mice and rats (Gaido et al., 2007
). Unfortunately, the signaling processes controlling rodent fetal Leydig cell hormone production during the critical window of reproductive tract programming are not known (Scott et al., 2009
), which impedes understanding of the phthalate endocrine disruption mechanism. We show that endocrine disruption is associated with reduced gene expression of SREBP2-dependent pathways and hypothesize that this may contribute to reduced steroidogenesis. Should an experimental system recapitulating rat sensitivity and amenable to using human tissue become available, SREBP2-dependent gene expression analysis could help determine susceptibility to endocrine disruption.