|Home | About | Journals | Submit | Contact Us | Français|
Diethylhexylphthalate (DEHP) has been classified as an antiandrogen. However, whether in utero and lactational exposures of DEHP affect Leydig cells has not been well established. In the present study, the effects of DEHP exposures on fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) were assessed. Pregnant dams of Long-Evans rats were treated with 0, 10, and 750 mg/kg body weight DEHP from Gestational Day 12.5 to Postnatal Day (PND) 21.5. Fetal Leydig cell clustering and FLC-specific gene expression were examined. Anogenital distances (AGDs) of male pups were assessed at PND 2. Serum testosterone levels of male pups and mRNA levels of ALC-specific genes were measured at PNDs 21 and 49. The AGDs of male pups were significantly shorter in the group treated with 750 mg/kg DEHP (mean ± SEM, 3.68 ± 0.16 mm) compared with control (4.62 ± 0.13 mm). The FLCs were aggregated after 10 and 750 mg/kg DEHP exposures. Several FLC-specific genes, including luteinizing hormone receptor (Lhcgr) and steroidogenic enzyme genes, were downregulated at both doses. Serum testosterone levels were significantly lower compared with control at PND 21 after treatment of 10 or 750 mg/kg DEHP, and continued to be lower even up to 49 days postpartum at the higher dose. The mRNA levels for Lhcgr and steroidogenic enzyme genes were significantly lower at both doses of DEHP at PND 21, whereas there were no significant differences for these genes at PND 49. In conclusion, in utero and continued lactational exposures to DEHP exert long-term disruption of steroidogenesis of ALCs.
Phthalates are plasticizers, which are present in many consumer products, such as cosmetics, children's toys, and medical tubing. Increasing public concern over lack of regulation on their use in the United States, in contrast to the European Union and 14 other countries, has arisen in response to reports that exposures to phthalates may be linked to abnormal reproductive development in the human male [1–3]. Epidemiological studies show statistical correlations between urinary concentrations of phthalate monoesters, the primary metabolites of phthalates, and incidence of anomalies such as cryptorchidism and shortened anogenital distance (AGD) of newborn males [3, 4], suggesting that the Leydig cell function is disrupted.
There are two populations of Leydig cells during the rodent life span: fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). Fetal Leydig cells differentiate from stem cells outside of the gonadal cords. Leydig cell marker cholesterol side-chain cleavage enzyme (P450scc) is detectable in rodents beginning on Gestational Day (GD) 12.5 in rodents. Fetal Leydig cells have maximal steroidogenic capacity on GD 19  and secrete testosterone that is critical for inducing sexual development . Fetal Leydig cells also play a critical role in the descent of the testis by synthesizing androgen and insulin-like growth factor 3 (INSL3) . Fetal Leydig cell numbers are at peak abundance around birth, and then FLCs involute gradually after Postnatal Day (PND) 7 . Because the majority of FLCs dedifferentiate or undergo apoptosis after birth [9, 10], they are unlikely to contribute significantly to postnatal testosterone production.
The ALC population originates from progenitor Leydig cells between PNDs 11 and 14, and their numbers peak around PND 21. Progenitor Leydig cells are spindle-shaped cells and produce markers of Leydig cell-differentiated function, including P450scc, 3β-hydroxysteroid dehydrogenase (3βHSD1)  and P450-dependent 17α-hydroxylase/C17–20-lyase (P450c17), and a truncated form of the luteinizing hormone receptor (LHCGR) [12, 13]. Progenitor Leydig cells gradually differentiate into ALCs around PND 49 . Adult Leydig cells produce primarily testosterone that is required for spermatogenesis and maintaining male secondary sexual characteristics in the adult. Testosterone is metabolized to dihydrotestosterone in testis and other peripheral tissues by 5α-reductase (SRD5A) . Adult Leydig cells also produce INSL3 , which is possibly associated with spermatogenesis because its receptor is located on the sperm , but its function has not been fully defined.
Diethylhexyl phthalate (DEHP), the most abundant phthalate in the environment, has known adverse effects on androgen synthesis and FLC function in rodent models . Another phthalate, di(n-butyl) phthalate (DBP), has been shown to have a similar effect on FLC function [19, 20]. The effects of DEHP on the reproductive tract depend on the duration of exposure. The effects of in utero and lactational exposures to DEHP on the adult population of Leydig cells is yet unknown. The object of the present study was to measure and compare effects of in utero and lactational DEHP exposures on both fetal and adult populations of Leydig cells.
25-[26,27-3H]Hydroxycholesterol, [7-N-3H]pregnenolone, and [1β,2β-N-3H]androst-4-ene-3,17-dione were purchased from DuPont-New England Nuclear (Boston, MA). [1,2,6,7-N-3H]Progesterone and [1,2,6,7-3H]testosterone were purchased from GE Biosciences Corp. (Piscataway, NJ). Nonradioactive steroids were purchased from Sigma Chemical Co. (St. Louis, MO).
Long-Evans rats (Charles River, Wilmington, MA) were used in these studies because an extensive toxicological database is available for this strain in studies of endocrine disrupters and testicular function . All animal procedures were performed in accordance with the policies of The Rockefeller University's Animal Care and Use Committee. Adult pregnant dams were treated from GD 12.5 to PND 21 with either 0 (control), 10, or 750 mg/kg DEHP (Sigma-Aldrich Co. Ltd.) in 1 ml/kg corn oil administered daily by oral gavage. This dose range was selected because of effects measured in Leydig cells in adult animals in previous studies [22–24]. A total of 36 pregnant dams were used: 25 were treated with DEHP and 11 with corn oil. The body weights and AGDs of male pups were measured at PND 2. Male pups were killed by inhalation of carbon dioxide at birth for FLC analysis or at PNDs 21 and 49 for ALC population analysis. Testes were removed, weighed, and placed in liquid nitrogen. Representative testes were subsequently used for immunohistochemical analysis of FLC distribution and real-time PCR analysis of Leydig cell mRNA levels.
Frozen testes from three groups were placed in the same blocks and sectioned (8 μm) using a cryostat. Fetal Leydig cells were measured by immunohistochemical detection of 3βHSD using rabbit polyclonal antibodies against 3βHSD by the avidin-biotin method (Vectastain Elite ABC Kit; PK-6101; Vector Laboratories Inc., Burlingame, CA) according to the manufacturer's instructions. Endogenous peroxidase was blocked with 0.5% H2O2 in methanol for 30 min. The sections were then incubated with anti-3βHSD antibody (diluted 1:200) for 1 h at room temperature. The antibody-antigen complexes were visualized with diaminobenzidine (Peroxidase Substrate Kit, SK-4100; Vector Laboratories), resulting in brown cytoplasmic staining in positively labeled Leydig cells. The sections were counterstained with Mayer hematoxylin, dehydrated in graded concentrations of alcohol, and coverslipped with resin (Permount; SP15–100; Fisher Scientific Co.). In control slides, sections were incubated with nonimmune rabbit immunoglobulin G (IgG) using the same working dilution as the primary antibody. The Leydig cell numbers in each cluster were counted according to the staining of 3βHSD.
Serum testosterone was measured by a previously described tritium-based radioimmunoassay validated for use with rat antiserum [22, 23]. The covariance of intraassay and interassay for testosterone was less than 15%.
Total RNA was extracted from rat testes in Trizol according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). First-strand synthesis and real-time PCR were performed as described previously . Ribosomal protein S16 (Rps16) mRNA levels were assayed in all samples as internal controls. The 13 Leydig cell and 4 Sertoli cell genes analyzed and the primers for the following genes were described in our previous study , including receptor genes luteinizing hormone receptor (Lhcgr) and KIT (Kit); steroidogenic transporters and enzymes scavenger receptor class B member 1 (Scarb1), steroidogenic acute regulatory protein (Star), P450scc (Cyp11a1), 3βHSD isoform 1 (Hsd3b1), P450c17 (Cyp17a1), and 17β-hydroxysteroid dehydrogenase isoforms 3 (17βHSD3) and 12 (Hsd17b3 and Hsd17b12), and SRD5A isoform 1 (Srd5a1); Leydig cell function marker insulin-like growth factor 3 (Insl3); cell proliferation marker proliferation cell nuclear antigen (Pcna); cell communication connexin 43 (Gja1); and Sertoli cell markers clusterin (Clu), androgen receptor (Ar), follicle-stimulating hormone receptor (Fshr), and stem cell factor (Kitl). The functions of these gene products were reported in our previous study .
The testes were homogenized in 1 ml of ice-cold 0.1 M PBS (pH 7.2) containing 0.25 M sucrose. Supernatants were collected by centrifugation at 700 × g for 30 min. Supernatants were used to measure enzyme activities of steroidogenic enzymes P450c17, 3βHSD, 17βHSD3, and SRD5A1. For STAR protein and P450scc enzyme activity analysis, the resultant supernatant was centrifuged at 9000 × g for 30 min to isolate mitochondria. The protein concentrations were determined using a kit (no. 500-0006; Bio-Rad Laboratories Inc., Hercules, CA) with bovine serum albumin as a standard.
Mitochondrial protein (25 μg) was boiled in equal volumes of sample loading buffer, a Tris-Cl buffer (pH 6.8) containing 20% glycerol, 5% SDS, 3.1% dithiothreitol, and 0.001% bromophenol blue. Protein samples were electrophoresed on 10% polyacrylamide gels containing SDS. Proteins were electrophoretically transferred onto nitrocellulose membranes, and after 30-min exposure to 10% nonfat milk to block nonspecific binding, the membranes were incubated with a STAR antibody (1:1000; kindly donated by Dr. Stocco, Texas Tech Health Sciences Center, Lubbock, TX). The membranes then were washed and incubated with a 1:5000 dilution of second antibody that was conjugated to horseradish peroxidase (HRP; anti-rabbit Ig, HRP-linked whole antibody produced in donkey; Amersham Biosciences Corp., Piscataway, NJ). The washing step was repeated, and immunoreactive bands were visualized by chemiluminescence using a kit (ECL; Amersham, Arlington Heights, IL). Then, the antibody in the membrane was stripped away and probed again by an antibody against β-actin (no. A2228; 1:1000; Sigma). The second anti-mouse Ig, HRP-linked whole antibody produced in sheep (Amersham Biosciences), was used, and the actin band was visualized by chemiluminescence. Protein levels were measured by densitometry of the films and normalized to β-actin.
Activity of P450scc was determined by measuring the conversion of side-chain-labeled 25-[26,27-3H]hydroxycholesterol to radioactive 4-hydroxyl-4-methyl-pentanoic acid, as previously described . Mitochondrial proteins were incubated in a total volume of 0.5 ml of medium containing 1 mCi 25-[26,27-3H]-hydroxycholesterol (1 μM 25-hydroxy-cholesterol). Incubations were performed for 30 min at 34°C, and at the end of the incubation, 0.5 ml of NaOH was added. The mixture was extracted twice with 2 ml of chloroform and mixed with neutral alumina to remove nonmetabolized substrate, and an aliquot was removed for measurement by liquid scintillation counting.
Testosterone biosynthetic enzymes activities P450c17, 3βHSD1, and 17βHSD3, and testosterone-metabolizing enzyme SRD5A were determined by thin-layer chromatography as described previously . The reaction mixtures (total volume of 250 μl) containing 25–160 μg of protein, 0.2 mM cofactors (NAD+ for 3βHSD1, and NADPH for P450c17, 17βHSD3, and SRD5A), and 10–1000 nM steroid substrates (radiolabeled and cold substrates) were incubated in shaking water bath at 37°C for 1 to 3 h. The substrates were: pregnenolone (for 3βHSD1), progesterone (for P450c17), androstenedione (for 17βHSD3), and testosterone (for SRD5A). The preliminary experiment was conducted to determine the linear reaction curve using different concentrations of proteins at different time periods. The steroids were extracted from reaction mixture with 1 ml of ice-cold ether, and the organic layer was evaporated under nitrogen gas. The extract of steroids was suspended in 70 μl of ether and then spotted on thin-layer plates (Baker-Flex, Phillipsburg, NJ). The steroids were separated chromatographically in chloroform:methanol (97:3, v/v) for 3βHSD1, 17βHSD3, and SRD5A, as well as chloroform:ether (7:1, vol/vol) for P450c17. The radioactivity was measured with a scanning radiometer (System 200/AC3000; Bioscan Inc., Washington, DC). The conversion of steroid to product was calculated as a percentage of the total radioactivity found in the product. All assays were repeated in triplicate.
Values are expressed as mean ± SEM, and data were analyzed using one-way ANOVA with Dunnett comparison of all columns versus control column using GraphPad Prism (version 4; GraphPad Software Inc., San Diego, CA). Data for Leydig cell cluster number per testis were log transformed before statistical analysis using the ANOVA test due to skewed distribution of the data.
Pregnant dams were exposed from GD 12.5 to PND 21 to a low dose of DEHP (10 mg/kg, normal environmental exposure level) and to a higher dose (750 mg/kg) that was shown previously to induce lower testosterone in the fetal and adult rats. None of the following parameters were affected at GD 21.5 in the treated animals exposed to DEHP for 10 days: body weights of dams, birth rates, the numbers of pups per dam, and the male:female sex ratio of pups (Table 1).
The AGDs and body weights of male pups at PND 2 were significantly reduced (P < 0.001) at a dose of 750 mg/kg DEHP (Table 2). Increased FLC clustering was obvious in both the 10 and 750 mg/kg DEHP treatment groups (Fig. 1) compared with controls. The aggregation was most pronounced in the 750 mg/kg DEHP group, with the average FLC numbers per cluster higher than 15, compared with three in the control group (Fig. 1D). Fetal Leydig cell function was further evaluated by analysis of Leydig cell-specific genes by real-time PCR (Fig. 2). A panel of genetic markers was selected to assess cell type-specific function in the testis after in utero exposure to DEHP. Transcript levels of 16 testicular mRNAs were measured by real-time PCR. As shown in Figure 2, cholesterol transporters and steroidogenic enzymes, including Scarb1, Star, and Hsd17b12, were significantly reduced, even at 10 mg/kg DEHP exposure. At higher concentrations, luteinizing hormone receptor gene (Lhcgr), testosterone biosynthetic enzymes Cyp17a1 and Hsd17b3, testis descent gene Insl3, and cell junction gene Gja1 also were reduced. Sertoli cell genes, including Kitl, Clu, and Fshr were evaluated for comparison, and Clu and Fshr were found to be reduced only at 750 mg/kg DEHP group, suggesting that Sertoli cells are less sensitive to DEHP exposure than FLCs.
At 11 days postpartum, the second generation of Leydig cells consists of differentiated progenitor Leydig cells. Progenitor Leydig cells differentiate into ALCs at around PND 49. We measured serum testosterone levels at 21 and 49 days postpartum. The serum testosterone levels were significantly reduced after exposures to 10 and 750 mg/kg DEHP at PND 21 (Fig. 3). The exposures to DEHP were discontinued at PND 21. However, serum testosterone level was lower at 750 mg/kg DEHP exposure at PND 49. This suggests that the disruption to ALC function resulting from in utero and lactational exposures to DEHP persisted as long as 4 wk after cessation of DEHP exposures.
A panel of genetic markers was selected to assess cell type-specific function in the testis after continued lactational exposure to phthalate. As shown in Figure 4, mRNA levels of Lhcgr and Kit signaling proteins; cholesterol transporters and steroidogenic enzymes, including Scarb1, Star, Cyp17a1, Hsd17b3, and Srd5a1; and Leydig cell gene Insl3 were significantly reduced at 10 or 750 mg/kg DEHP exposure at PND 21. Levels of mRNA for the cell junction gene Gja1 and Sertoli cell genes Clu, Kitl, and Fshr were also compared with levels after DEHP exposure. Kitl and Fshr were reduced at both doses of DEHP treatment at PND 21, suggesting that at this stage, Sertoli cells are also altered by DEHP exposures. The proliferating marker Pcna was significantly reduced at PND 21 after both doses of DEHP exposures. However, after cessation of DEHP, no genes were downregulated at PND 49. The activities of four testosterone biosynthetic enzymes, P450scc, P450c17, 3βHSD1, and 17βHSD3, and one testosterone metabolic enzyme, SRD5A, were also examined (Fig. 5). P450scc, P450c17, 3βHSD1, and 17βHSD3 enzyme activity levels reflected the lower mRNA levels. Messenger RNAs of Srd5a1, the gene highly expressed in prepubertal testis at PND 21, and of SRD5A were significantly reduced, suggesting that the function of PLCs is perturbed. Although there was a significant decrease in serum testosterone levels at PND 49 for the 750 mg/kg DEHP group, no lesions were found in gene expression related to testosterone biosynthetic pathways. However, SRD5A enzyme activity was significant higher in 750 mg/kg DEHP-treated testis. STAR (30 kDa) protein levels were also examined and found to follow the trend of mRNA levels.
Abnormal FLC aggregation was observed when DEHP exposure started at GD 12.5, which was similar to what was observed when DEHP exposure started at GD 2 . It has been reported that high-dose exposures to DEHP in utero result in focal disruptions in the structure of the seminiferous epithelium, as well as abnormal aggregations of FLCs. Because of their large size, the FLC aggregations were at first thought to represent Leydig cell hyperplasia or neoplasia [28, 29]. The aggregations are also evident when rats are exposed to 500 mg/kg DBP [30, 31]. Stereological analysis, however, does not support the concept that Leydig cell numbers are increased in the aggregations [26, 32].
Reduced AGDs are considered to be a reliable marker of decreased testosterone levels . In human newborn boys, higher phthalate levels also have been reported to be associated with decreased AGDs . This means that phthalates, such as DEHP, may act as antiandrogens and might affect FLC steroidogenesis in humans. Significant downregulation of steroidogenic pathway components, including cholesterol-transporting proteins Scarb1 and Star and steroidogenic enzyme genes, such as Hsd17b12, was observed in low doses of DEHP (10 mg/kg) in the present study. Disruption of FLC function was consistent with the significant reduction in testicular testosterone levels that were seen after high-dose exposures . The reduction of Lhcgr, Scarb1, Star, Cyp17a1, Hsd17b3, Hsd17b2, and Insl3 in fetal testis was associated with reduced FLC numbers after in utero exposure to 750 mg/kg DEHP in our previous report . Similar changes in mRNA levels also were reported in a recent publication in which Sprague-Dawley rats were exposed in utero to 234 mg/kg or more DEHP . The high-dose effects of DEHP also were clearly shown in experiments using 500 mg/kg DBP, another phthalate ester that is structurally related to DEHP . Decreased AGD in male fetuses has been observed following exposure of Wister and Sprague-Dawley pregnant dams to either DBP or DEHP at doses of 375–750 mg/kg and above [35, 36]. Nipple retention and the increased frequencies of underdeveloped epididymides, testicular atrophy, hypospadias, and ectopic or absent testes were also seen in animals exposed to phthalates at 250 mg/kg per day or higher [36–38]. However, steroidogenic activity in FLCs peaks 1 to 2 days prior to birth on Day 19 of gestation . The testosterone produced at this time is critical for male secondary sexual differentiation (i.e., development of the penis and sex accessory glands) . Thus, disruption of FLC function during this period will disrupt male secondary sexual development.
Fetal Leydig cells also produce INSL3, which binds to RXFP2, relaxin/insulin-like family peptide receptor 2 (previous symbol LGR8). INSL3 specifically binds RSFP2 in the gubernaculum  and, together with androgen, induces scrotal descent of the testis . It now appears that INSL3 is the critical hormone responsible for early-stage descent of the testis from the abdominal to inguinal position, as shown by the loss-of-function mutation in mice that prevents this process. For this reason, interference with the development of FLCs may be a precipitating cause of cryptorchidism.
In utero and lactational exposures to DEHP caused significantly reduced serum testosterone levels in male rats at PND 21 at a dose of 10 mg/kg (Fig. 3). This dosage regimen also reduced Lhcgr, cholesterol-transporting protein genes Star and Scarb1, as well as steroidogenic enzyme genes Cyp17a1, Hsd17b3, and Srd5a1 (Fig. 4) at PND 21. The lowest observed adverse effect level for the inhibition of testosterone production following a continued lactational DEHP exposure was 10 mg/kg per day at PND 21. This level is within the suggested environmental exposure levels of the human population, such as in the neonatal intensive care unit. Serum testosterone was not recovered after the higher-dose exposure of DEHP (750 mg/kg) at PND 49 when exposure to DEHP was stopped at PND 21 (Fig. 3). Previously, we reported that in utero exposures to DEHP (100 mg/kg) alone caused significant decreases of serum testosterone levels postnatally . The present in utero and lactational exposure regimen continues to support the notion that in utero and lactational exposure to DEHP could lead to dysfunction of ALC populations in the adult, as corroborated in a recently published study . The decreased Leydig cell capacity was correlated with downregulation of most genes related to Leydig cell regulation (such as Lhcgr) and steroidogenic pathways (such as Scarb1, Star, Cyp17a1, and Hsd17b3) at PND 21. Genes that are directly related to testosterone biosynthesis in Leydig cells were not reduced at PND 49, although there was a significant decrease in serum testosterone after higher DEHP (750 mg/kg) exposures. The absence of correlation after in utero DEHP exposures between reduced serum testosterone levels in the adult testis and genes related to Leydig cell steroidogenesis has been reported elsewhere . Interestingly, there were controversies about reproductive outcomes in adult male rats after in utero and lactational exposure to phthalates. Moore et al.  observed that in utero and lactational exposure to DEHP at 750 mg/kg significantly reduced the weights of prostate, an androgen-dependent gland, and sperm counts in the epididymis. The weight of the seminal vesicle plus coagulating glands (androgen-dependent tissues) in adult rats was also significantly reduced after exposure to 475 mg/kg DEHP, although only this dose caused significant serum testosterone levels . Doses below 400 mg/kg DEHP had no effects on weights of these androgen-dependent organs and testosterone levels [38, 41]. However, the gene profiles of in utero and lactational exposures to DEHP in our study were a little bit different from those reported by Culty et al. . We observed the downregulation of these Leydig cell genes, including Lhchr, Kit, Scarb1, Star, Cyp17a1, Hsd17b3, Srd5a1, Insl3, Gja1, and Ar at PND 21 and no changes at PND 49 (Fig. 4). In contrast, Culty et al.  observed the upregulation of these Leydig cell genes, including Cyp11a1, Cyp17a1, Hsd17b3, and Insl3, at both PND 21 and PND 60 . These differences have resulted from different dosage regimens and timing, in utero exposure in the Culty et al. study  but in utero and lactational exposures in our study. We speculate that the different dosage regimen might affect different Leydig cell populations, possibly only FLCs and stem Leydig cells in the Culty et al. study  but FLCs and stem as well as progenitor Leydig cells in our study.
The cause of lower serum testosterone after the cessation of DEHP exposures is not yet known. One possible cause may be increased testosterone metabolism; for example, SRD5A activity was significantly higher in 750 mg/kg DEHP-treated testis, and it led to lower testosterone levels (Fig. 5).
In conclusion, the present study shows that DEHP exposure in utero caused FLC dysfunction. Furthermore, ALC dysfunction was evident in mature animals that had been exposed to DEHP as pups during the lactational period.
The authors thank the services of the Population Council's Cell Biology and Flow Cytometry Facility.
1Supported in part by National Institute of Environmental Health Sciences grant R01 ES10233 to M.P.H. and R.-S.G.