Zearalenone Delays Rat Leydig Cell Regeneration

Zearalenone Delays Rat Leydig Cell Regeneration Abstract Zearalenone (ZEA), a fungal mycotoxin, is present in a wide range of human foods. By virtual screening, we have identified that ZEA is a potential endocrine disruptor of Leydig cells. The effect of ZEA on Leydig cell development is still unclear. The objective of the present study was to explore whether ZEA affected Leydig cell developmental process and to clarify the underlying mechanism. Adult male Sprague-Dawley rats (60 days old) were randomly divided into three groups and these rats received a single intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to eliminate all Leydig cells. Seven days after EDS treatment, rats intratesticularly received normal saline (control) or 150 or 300 ng/testis/day ZEA for 21 days. Immature Leydig cells isolated from 35-day-old rats were treated with ZEA (0.05–50 μM) for 24 h in vitro. In vivo ZEA exposure lowered serum testosterone levels, reduced Leydig cell number, and decreased Leydig cell–specific gene or protein expression levels possibly via downregulating the steroidogenic factor 1 (Nr5a1) expression. ZEA in vitro inhibited androgen production and steroidogenic enzyme activities in immature Leydig cells by downregulating expression levels of cholesterol side cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), and steroid 5α-reductase 1 (Srd5a1) at a concentration as low as 50 nM. In conclusion, ZEA exposure disrupts Leydig cell development and steroidogenesis possibly via downregulating Nr5a1. zearalenone, Leydig cells, steroidogenic factor 1, ethane dimethane sulfonate, regeneration Zearalenone (ZEA), also known as F-2 toxin, is one of the most widely distributed estrogenic mycotoxins produced by several Fusarium (Kuiper-Goodman et al., 1987; Muller et al., 1997; Tashiro et al., 1980). ZEA is found in a number of cereal crops worldwide, such as wheat, maize, barley, oats, rice, and sorghum (Kuiper-Goodman et al., 1987). Humans expose to ZEA directly via contaminated foods or indirectly through products derived from animals exposed to mycotoxins. ZEA is found to have toxicity to liver, immune system, and development (Kuiper-Goodman et al., 1987). Moreover, many studies demonstrated that ZEA bound to estrogen receptors and activated estrogen responsive genes (Lemke et al., 1999; Tashiro et al., 1980), leading to abnormal development of female reproductive tracts (Tiemann and Danicke, 2007). ZEA might also be an endocrine disruptor for male reproductive system. Previous studies showed that mice exposed to 150 and 0.15 μg/l ZEA had lower sperm concentration and increased morphologically abnormal spermatozoa (Zatecka et al., 2014) and that rats exposed to 5 mg/kg ZEA had disrupted spermatogenesis (Kim et al., 2003). ZEA also inhibited testosterone biosynthesis of mouse Leydig cells through cross-talk between estrogen receptors and Nur77 (also called NR4A1) signaling (Liu et al., 2014) and inducing apoptosis of Leydig cells via an endoplasmic reticulum stress dependent signaling pathway (Lin et al., 2015). However, the effect of ZEA on Leydig cell development is still unclear. Leydig cells are the primary cells that contribute to about 95% circulatory androgen level. The androgen production depends not only on the function of Leydig cells but also on their number. In the rat, pubertal development of Leydig cells is conceptually defined into four stages: stem (during the whole lifespan), progenitor (at postnatal day 21), immature (between postnatal days 28 and 35), and adult Leydig cells (after postnatal day 56) (Ye et al., 2017). The pubertal development of Leydig cells can be mimicked by eliminating Leydig cells in the adult rat testis using a drug called ethane dimethane sulfonate (EDS) (Rommerts et al., 1988). EDS is a drug that specifically kills rat Leydig cells (Rommerts et al., 1988). Seven days after intraperitoneal injection of 75 mg/kg EDS, Leydig cells in the interstitium of rat testis are completely eliminated (Rommerts et al., 1988). Leydig cell regeneration starts on the 14th day post-EDS and newly regenerated spindle-shaped progenitor Leydig cells are recognized on the 21st day post-EDS (Rommerts et al., 1988). The regenerated progenitor Leydig cells begin to express luteinizing hormone (LH) receptor (LHCGR) for LH-stimulated response, scavenger receptor class B member 1 (SCARB1) for binding to high-density lipoprotein to transport cholesterol into the cells and steroidogenic acute regulatory protein (STAR) for transporting cholesterol from cytosol into the inner membrane of mitochondria to provide steroidogenic substrate, and cholesterol side chain cleavage enzyme (CYP11A1) in the inner membrane of the mitochondria to catalyze cholesterol into 22R-hydroxycholesterol (22R) and further into pregnenolone (P5). P5 is diffused into the smooth endoplasmic reticulum, where it is converted into progesterone (P4) by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), and the latter is converted into androstenedione (D4) by 17α-hydroxylase/17, 20-lyase (CYP17A1) (Rommerts et al., 1988). The regenerated progenitor Leydig cells are differentiated into immature Leydig cells on the 28th day post-EDS, which are identical to those isolated from pubertal testis and which begin to express 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) for the last-step biosynthesis of testosterone from androstenedione (Guo et al., 2013). Immature Leydig cells also highly express androgen metabolizing enzymes, including steroid 5α-reductase 1 (SRD5A1), which converts testosterone into dihydrotestosterone (DHT), and the latter is further metabolized into 5α-androstanediol (DIOL) by 3α-hydroxysteroid dehydrogenase (AKR1C14) (Guo et al., 2013). Interestingly, a glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) begins to express in the immature Leydig cells and continues to maintain the high level later on (Guo et al., 2013) and this enzyme can be a good biomarker for Leydig cells at the advanced stage (immature and adult Leydig cells). EDS-treated rat is a good model to study the effects of many endocrine disruptors on Leydig cell development. In the present study, we exposed rats to ZEA from post-EDS day 7 to day 28 and investigated its effects on early-stage Leydig cell regeneration and treated immature Leydig cells isolated from 35-day-old rats with ZEA to investigate its direct effects. MATERIALS AND METHODS Chemicals and animals ZEA was purchased from J&K Scientific (Beijing, China). SYBR Green qPCR Kit and BCA Protein Assay Kit was obtained from Takara (Otsu, Japan). Trizol Kit was purchased from Invitrogen (Carlsbad, CA). EDS was purchased from Pterosaur Biotech (Hangzhou, China). Immulite2000 Total Testosterone Kit was purchased from Sinopharm Group Medical Supply Chain Services Co (Hangzhou, Zhejiang, China). Radio immunoprecipitation assay buffer was obtained from Bocai Biotechnology (Shanghai, China). The manufacturers of antibodies were listed in Supplementary Table 1. Male Sprague-Dawley rats (60 days of age) were purchased from Shanghai Animal Center (Shanghai, China). All animal studies were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University and were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Animal administration Eighteen 54-day-old male Sprague-Dawley rats were raised in a 12 h dark/light cycle temperature at 23 ± 2°C and relative humidity of 45%–55%. Water and food were provided ad libitum. Animals were adjusted for 6 days before they were randomly divided into 3 groups: control (0 ng/testis/day, n = 6), low dose (150 ng/testis/day, n = 6), and high dose (300 ng/testis/day, n = 6). EDS was dissolved in a mixture of dimethyl sulfoxide and deionized sterile water (1:3, v/v). All rats received a single intraperitoneal injection of 75 mg/kg EDS to eliminate all Leydig cells as previously described (Guo et al., 2013). ZEA was dissolved in normal saline for intratesticular injection. Seven days post-EDS, each testis of a rat received an intratesticular injection of 0, 150, or 300 ng ZEA/testis/day for 21 days. Body weight of each rat was recorded every 3 days. Rats were sacrificed on the 28th day post-EDS by asphyxiation with CO2. Trunk blood was collected, placed in a gel glass tube, and centrifuged at 1500 × g for 10 min to collect serum samples. Serum samples were labeled and stored at –80°C until hormone [testosterone, LH, and follicle-stimulating hormone (FSH)] analysis. Furthermore, each pair of testes was separated and weighted. One testis each animal was frozen in the liquid nitrogen and stored at –80°C for subsequent gene and protein expression analysis. The contralateral testis was punched three holes using a G27 needle and then fixed in Bouin’s solution for immunohistochemical analysis. Immature Leydig cell isolation Eighteen 35-day-old male Sprague-Dawley rats were sacrificed by asphyxiation with CO2. Testes were removed and Leydig cells were purified as described previously (Ge and Hardy, 1998). In brief, animals were sacrificed in CO2 tank, testes were removed, perfused with a M199 solution containing 0.1 mg/ml collagenase via the testicular artery, digested with a mixture of 0.25 mg/ml collagenase and 0.25 mg/ml DNase for 15 min, filtered with 100-μm nylon mesh, and the cells were separated under Percoll gradient as previously described (Ge and Hardy, 1998). The cells with density of 1.070–1.088 g/ml were collected and washed. Purities of Leydig cell fractions were evaluated by histochemical staining for HSD3B1 activity, with 0.4 mM etiocholanolone as the steroid substrate and NAD+ as a cofactor (Payne et al., 1980). The purities of immature Leydig cells were >95%. Leydig cell culture Immature Leydig cells were seeded into the 6-well culture plates after isolation with a density of 105 cells per well. Leydig cells were treated with 0, 0.05, 0.5, 5, and 50 μM ZEA in 2.0 ml DMEM: F12 medium (Gibco, Grand Island, NY) for 24 h. Cells were harvested for the analysis of Leydig cell mRNA and protein levels. Because immature Leydig cells produced about 80% DIOL and 10% testosterone (Ge and Hardy, 1998), media were harvested for the measurement of testosterone and DIOL concentrations. To further dissect the action site(s) of ZEA on androgen biosynthesis and metabolism, we isolated immature Leydig cells and added hormone (LH, 10 ng/ml), signaling compound (8Br-cAMP, 8BR, 10 mM) and steroidogenic enzyme substrates, including those of CYP11A1 (22R, 5 µM), HSD3B1 (pregnenolone, P5, 5 µM), CYP17A1 (progesterone, P4, 5 µM), HSD17B3 (androstenedione, D4, 5 µM), SRD5A1 (testosterone, T, 5 µM), and AKR1C14 (DHT, 5 µM), and measured the medium DIOL and T concentrations and then compared them with the control (no treatment, basal). The substrate information was listed in Supplementary Table 2. After isolation, the purified immature Leydig cells were seeded into 24-well culture plate with a density of 0.05 × 106 cells per well. About 50 μM ZEA was coincubated with these compounds. LH (10 ng/ml) and 8BR (10 mM) were used to act as a maximum stimulation to induce Leydig cell steroidogenesis as previously described (Liu et al., 2015, 2014). Because 8BR can penetrate the cell membrane, therefore it is used to replace the intracellular cAMP, which is impermeable. 22R, pregnenolone, progesterone, androstenedione, testosterone, and DHT were used as the respective substrate of the enzyme: CYP11A1, HSD3B1, CYP17A1, HSD17B3, SRD5A1, and AKR1C14. Because 22R can readily penetrate cell and mitochondrial membrane, it is used to replace cholesterol as the substrate for CYP11A1. Media were collected for DIOL and testosterone assay after 3 h incubation. Measurement of DIOL and testosterone levels by RIA DIOL and testosterone concentrations in the medium were measured with the tritium-based RIA as described previously (Ge and Hardy, 1998) using the commercial RIA kits (IBL). The minimum detectable concentration of the assay for DIOL and testosterone was 5 pg/ml. The internal control contains 100 pg/ml DIOL or testosterone dissolved in the same culture media. Interassay and intraassay variations of DIOL and testosterone were within 15%. ELISA for serum LH and FSH levels Serum LH and FSH levels were measured with their ELISA kits according to the manufacturer’s instructions (Chemicon, CA). In brief, 200 μl samples and 50 μl assay diluent were added to precoated 96-well plates. The plates were incubated for 2 h at room temperature, and washed 5 times with washing buffer. About 100 μl peroxidase-conjugated IgG anti-LH or -FSH of solution was added into each well for 2 h at room temperature. Then, plates were washed 5 times. Then, 100 μl substrate buffers were added into each well, and incubated in the dark place for 30 min at room temperature. The enzyme reaction was stopped by 50 μl stop solution. The quantification of LH and FSH levels was obtained by a microplate reader at 550 nm with correction wavelength at 450 nm. Western blot analysis Testes were homogenized and boiled in equal volumes of sample loading buffer, a Tris–Cl buffer (pH 6.8), containing 20% glycerol, 5% sodium dodecyl sulfate, 3.1% dithiothreitol, and 0.001% bromophenol blue. Homogenized samples (50 μg protein) were electrophoresed on 10% polyacrylamide gels containing sodium dodecyl sulfate. Proteins were electrophoretically transferred onto nitrocellulose membranes, and the membranes incubated with 5% nonfat milk for 1 h to block nonspecific binding. Then, the membranes were incubated with primary antibodies against the following antigens: HSD3B1, HSD11B1, HSD17B3, NR5A1, FSHR, and ACTB (listed in Supplementary Table 1). The membranes were washed and incubated with a 1:5000 dilution of goat antirabbit antiserum that was conjugated to horseradish peroxidase. The washing step was repeated, and immunoreactive bands were visualized by chemiluminescence using an ECL kit (Amersham, Arlington Heights, IL). The intensity of proteins was quantified using ImageJ software. The Leydig and Sertoli cell proteins were adjusted to ACTB, a house-keeping protein. RNA isolation and real-time PCR Total RNAs were purified from the testes and immature Leydig cells using the Trizol Kit according to the manufacturer’s instructions, and the concentration of RNA was measured by reading OD value at 260 nm. The first strand (cDNA) was reversely transcribed and used as the template for qPCR analysis as previously described (Li et al., 2014). The expression levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, Srd5a1, Nrd5a1, Hsd11b1, and Akr1c14) and Sertoli cells (Dhh and Fshr) were measured using a SYBR Green qPCR Kit. The gene name and primer sequences were listed in Supplementary Table 3. The qPCR reaction mixture had 10 μl of SYBR Green mix, 1.6 μl forward and reverse primer mix, 400 ng diluted cDNA sample, and 5–8 μl RNase-free water. The reaction was processed by the following program: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. The Ct value was read and the expression level of a target gene was calculated using a standard curve method as previously described (Li et al., 2014). The mRNA levels were adjusted to Rps16, a house-keeping gene, for internal control. These primers have been tested to detect the respective mRNA levels in our previous study (Wu et al., 2017). Immunohistochemistry and enumeration of Leydig and Sertoli cells One testis per rat was used for immunohistochemical staining (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. Six testes per group were randomly selected and cut into 8 discs, and each disc was cut to 2 pieces. One piece each testis was randomly selected and dehydrated in ethanol and xylene and then embedded in paraffin in a tissue array as previously described (Wu et al., 2017). Six micrometer-thick transverse sections were cut and mounted on glass slides. Approximately 10 sections were used. Avidin-biotin immunohistochemical stainings for CYP11A1 (all Leydig cells) or HSD11B1 (Leydig cells at the advanced stage) or SOX9 (Sertoli cells) were conducted following manufacturer’s instructions. Antigen retrieval was conducted by a microwave irradiation in 10 mM (pH 6.0) citrate buffer for 10 min. Then, endogenous peroxidase was blocked with 0.5% of H2O2 in methanol for 30 min. Sections were incubated with either CYP11A1 or HSD11B1 or SOX9 polyclonal antibody (diluted 1:200) for 1 h at room temperature. Diaminobenzidine was used for visualizing the antibody-antigen complexes, which positively label Leydig cells by brown cytoplasmic staining. Mayer hematoxylin was adopted as the counterstaining. The sections were dehydrated in graded concentrations of alcohol and cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). Nonimmune rabbit IgG was used in the incubation of negative control sections with working dilution the same as the primary antibody. The cells with CYP11A1 staining in the interstitial area represent Leydig cells, whereas cells with HSD11B1 staining in the interstitial area represent the Leydig cells at the advanced stage (Phillips et al., 1989). The cells with SOX9 staining in the seminiferous tubules represent Sertoli cells. The enumeration of Leydig cells and Sertoli cells was performed as previously described (Wu et al., 2017). The total number of Leydig and Sertoli cells was calculated by multiplying the number of Leydig cells counted in a known fraction of the testis by the inverse of the sampling probability. Computer-assisted image analysis of cell size and nuclear size Leydig cells were identified by staining HSD11B1 as above. The Leydig cell size, nuclear size, and cytoplasmic size were calculated as previously described (Liu et al., 2016a). Six randomly selected fields in each of 3 nonadjacent sections per testis were captured using a BX53 Olympus microscope (Tokyo, Japan) equipped with a digital camera interfaced to a computer. The images that were displayed on the monitor represented partial area of a testis. Cell size and nuclear size were estimated using the image analysis software (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). More than 50 Leydig cells were evaluated in each testis. The cell size and nuclear size were recorded as μm3 and cytoplasmic size was calculated by cell size minus nuclear size. Semi-quantitative measurement of CYP11A1 and HSD11B1 CYP11A1 and HSD11B1 are the proteins of Leydig cells. Immunohistochemical stainings of CYP11A1 and HSD11B1 were performed as above. Target protein density and background area density were measured using the image analysis software as previously described (Liu et al., 2016b). More than 50 Leydig cells were evaluated in each testis and the protein density of each sample was averaged. Calculation of Leydig cell proliferation The Leydig cell proliferation was judged by immunofluorescent staining of PCNA in Leydig cells after dual stainings of PCNA (for proliferating cell) and CYP11A1 (the Leydig cells) in testis collected on post-EDS day 28, when the regenerated Leydig cells were at the stage of immature Leydig cells that have proliferating capacity. The sections in the tissue array assembled above were used. Sections were sequentially incubated with the primary antibodies of CYP11A1 and PCNA for 60 min. Then, the fluorescent secondary antibody (Alexa-conjugated antirabbit or antimouse IgG, 1:500) was used to label Leydig cells (CYP11A1, cytoplasmic staining in green color) and proliferating cells (PCNA, nuclear staining in red color). Images were taken with a fluorescent microscopy. The percentage of the cells with CYP11A1/PCNA double staining was calculated. Statistical analysis All data are presented as mean and standard errors (SEM). Statistical significance was analyzed using one-way ANOVA followed by ad hoc Turkey’s multiple comparisons between groups. Statistical analysis was performed using GraphPad Prism (version 6, GraphPad Software Inc, San Diego, CA). A p < .05 was considered statistically significant. RESULTS General Toxicological Parameters of ZEA To analyze the general parameters of ZEA toxicity, body and testis weights were recorded at the end of in vivo ZEA treatment (Table 1). ZEA did not significantly affect body weights at both doses. ZEA did not affect testis weights at 150 ng ZEA/testis group. However, the testis weight at 300 ng ZEA/testis group was significantly lower than that of the control (Table 1). No mortalities and abnormal activities were observed in rats of any groups. Table 1. General Toxicological Parameters After Treatment of Zearalenone Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Mean ± SEM, n = 6. ** Significant difference of zearalenone group when compared with control (0 ng/testis) on post-EDS day 28 at p < .01. Table 1. General Toxicological Parameters After Treatment of Zearalenone Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Mean ± SEM, n = 6. ** Significant difference of zearalenone group when compared with control (0 ng/testis) on post-EDS day 28 at p < .01. ZEA Lowers Testosterone Levels In Vivo Rats were treated with ZEA for 21 days starting on the 7th day post-EDS, when only stem Leydig cells were present (Figure 1A). Sera were collected on post-EDS day 28 for hormone (testosterone, LH, and FSH) analysis (Figs. 1B–D). ZEA significantly decreased testosterone levels at 150 and 300 ng/testis doses (Figure 1B). This result indicates that ZEA delays Leydig cell regeneration. Further analysis showed that ZEA had no effect on LH (Figure 1C) and FSH (Figure 1D) levels, suggesting that pituitary hormone secretion is not affected by ZEA. Figure 1. View largeDownload slide Regimen of zearalenone (ZEA) and serum hormone levels. A, ZEA regimen; B, serum testosterone (T); C, serum luteinizing hormone (LH); D, serum follicle-stimulating hormone (FSH). Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 1. View largeDownload slide Regimen of zearalenone (ZEA) and serum hormone levels. A, ZEA regimen; B, serum testosterone (T); C, serum luteinizing hormone (LH); D, serum follicle-stimulating hormone (FSH). Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). ZEA Reduces Leydig Cell Number In Vivo All Leydig cells were identified by staining CYP11A1, a biomarker for all Leydig cells in the Leydig cell lineage (Ge and Hardy, 1998). Leydig cells at immature and adult stages were identified by HSD11B1 because it began to express in Leydig cells after postnatal day 28 (Phillips et al., 1989) and in regenerated Leydig cells after post-EDS day 28 (Guo et al., 2013). Brown cytosolic staining in the interstitium showed the CYP11A1 positive or HSD11B1 positive cell. We counted the cell number of CYP11A1 positive cells (all Leydig cells) and HSD11B1 positive cells (Leydig cells at the advanced stage) (Figs. 2I and 2J). We found that CYP11A1 positive Leydig cell numbers were not significantly reduced after ZEA treatment (Figs. 2A, 2C, 2E, and 2G). However, HSD11B1 positive Leydig cell numbers were significantly reduced after ZEA treatment (Figs. 2B, 2D, 2F, and 2H). This indicates that the differentiation of Leydig cells from the progenitor stage into the immature stage is blocked by ZEA. Meanwhile, ZEA at 300 ng/testis reduced Leydig cell and cytoplasmic volumes (Figs. 2K and 2L), further confirming that Leydig cells are at more immature stage. However, ZEA did not alter the SOX9-positive Sertoli cell number (data not shown). Figure 2. View largeDownload slide Immunohistochemical staining of CYP11A1 and HSD11B1 in rat testis sections on post-EDS day 28 after ZEA treatment. CYP11A1: A, C, E, G; HSD11B1: B, D, F, H. (A and B): control; (C and D): 150 ng/testis ZEA; (E and F): 300 ng/testis ZEA. (G and H): the negative control. (I and J): quantification of CYP11A1 and HSD11B1 positive cell numbers. (K) Cell volume. (L) Cytoplasmic volume. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. Figure 2. View largeDownload slide Immunohistochemical staining of CYP11A1 and HSD11B1 in rat testis sections on post-EDS day 28 after ZEA treatment. CYP11A1: A, C, E, G; HSD11B1: B, D, F, H. (A and B): control; (C and D): 150 ng/testis ZEA; (E and F): 300 ng/testis ZEA. (G and H): the negative control. (I and J): quantification of CYP11A1 and HSD11B1 positive cell numbers. (K) Cell volume. (L) Cytoplasmic volume. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. ZEA Downregulates Leydig and Sertoli Cell mRNA Levels In Vivo We measured the mRNA levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, Srd5a1, Nr5a1, and Hsd11b1) and Sertoli (Dhh and Fshr) cell genes on post-EDS day 28. ZEA at 300 ng/testis reduced mRNA levels of Leydig cell genes, including Hsd3b1, Hsd17b3, Srd5a1, Nr5a1, and Hsd11b1 without affecting the levels of Lhcgr, Scarb1, Star, Cyp11a1, and Cyp17a1 (Figure 3). ZEA at 300 ng/testis dose also decreased Fshr level without affecting Dhh level (Figure 3). These results suggest that both Leydig and Sertoli cell gene expression is affected by ZEA at the higher dose. Figure 3. View largeDownload slide Gene expression levels of the testes in rats with or without ZEA treatment. Genes in the Leydig cell: (A) Lhcgr, (B) Scarb1, (C) Star, (D) CYP11a1, (E) Cyp17a1, (F) Hsd3b1, (G) Hsd17b3, (H) Srd5a1, (I) Nr5a1, and (J) Hsd11b1. Genes in the Sertoli cell: (K) Dhh and (L) Fshr. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 3. View largeDownload slide Gene expression levels of the testes in rats with or without ZEA treatment. Genes in the Leydig cell: (A) Lhcgr, (B) Scarb1, (C) Star, (D) CYP11a1, (E) Cyp17a1, (F) Hsd3b1, (G) Hsd17b3, (H) Srd5a1, (I) Nr5a1, and (J) Hsd11b1. Genes in the Sertoli cell: (K) Dhh and (L) Fshr. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). ZEA Reduces Protein Levels in Leydig and Sertoli Cells In Vivo We measured the levels of Leydig (HSD3B1, HSD11B1, HSD17B3, and NR5A1) and Sertoli (FSHR) cell proteins in the testes collected on post-EDS day 28. ZEA lowered these protein levels in parallel with their mRNA levels (Figure 4). Furthermore, we used a semiquantitative analysis of HSD11B1 density in the individual cell and found that ZEA lowered HSD11B1 level significantly at 300 ng/testis (Figs. 5D–F and 5H). These results suggest that ZEA delays Leydig cell regeneration. Figure 4. View largeDownload slide Protein levels of the testis in rats with or without ZEA treatment. Protein expressions in the testis: (A) western blot band. (B) Quantification of protein levels. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 4. View largeDownload slide Protein levels of the testis in rats with or without ZEA treatment. Protein expressions in the testis: (A) western blot band. (B) Quantification of protein levels. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 5. View largeDownload slide Semiquantitative analysis of CYP11A1 and HSD11B1 in single Leydig cell after ZEA treatment. Immunohistochemical images (1) CYP11A1: A, B, C and (2) HSD11B1: D, E, F. (G) and (H): quantification of CYP11A1 and HSD11B1 density. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. Figure 5. View largeDownload slide Semiquantitative analysis of CYP11A1 and HSD11B1 in single Leydig cell after ZEA treatment. Immunohistochemical images (1) CYP11A1: A, B, C and (2) HSD11B1: D, E, F. (G) and (H): quantification of CYP11A1 and HSD11B1 density. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. ZEA Has No Effect on Immature Leydig Cell Proliferation In Vivo Immature Leydig cells on post-EDS day 28 have the capacity of proliferation (Guo et al., 2013). PCNA is a nuclear matrix protein for cell proliferation. We stained Leydig cells using CYP11A1 antibody and the proliferating cell using PCNA antibody. There was no difference in PCNA-positive Leydig cells between control and ZEA-treated groups (Figs. 6A–D), indicating that the reduced HSD11B1-positive Leydig cell number is not contributed by the proliferation of Leydig cells. Figure 6. View largeDownload slide Immunohistochemical staining for PCNA and Leydig cells after ZEA treatment. Immunohistochemical images: A, B, C. Quantification: PCNA-positive Leydig cells (D). Mean ± SEM, n = 6. No significant difference was identified. Scale bars = 50 μm. Figure 6. View largeDownload slide Immunohistochemical staining for PCNA and Leydig cells after ZEA treatment. Immunohistochemical images: A, B, C. Quantification: PCNA-positive Leydig cells (D). Mean ± SEM, n = 6. No significant difference was identified. Scale bars = 50 μm. ZEA Inhibits Androgen Production in Immature Leydig Cell In Vitro To further dissect the direct effect of ZEA on immature Leydig cells, we treated immature Leydig cells with 0.05, 0.5, 5, and 50 μM ZEA for 24 h. ZEA concentration-dependently lowered androgen (DIOL + testosterone) levels (Figure 7). We further measured the mRNA levels in Leydig cells after ZEA treatment (Figure 8). ZEA significantly reduced Cyp11a1, Hsd3b1, Srd5a1 mRNA levels at ≥0.5 μM and Hsd17b3 mRNA level at 50 μM. We further examined the effects of ZEA in basal, LH-stimulated, and 8BR-stimulated conditions. ZEA at 50 μM inhibited androgen (DIOL) production in all cases (Figs. 9A–C). To explore the specific sites by which ZEA may affect androgen production, we tested all enzymatic steps by providing the cells with different enzyme substrates. The enzyme name, substrate name, and concentrations were listed in Supplementary Table 2. ZEA (50 µM) inhibited androgen production in 22R-, P5-, and D4-supplemented Leydig cells, indicating that ZEA inhibits CYP11A1, HSD3B1, and HSD17B3 enzyme activities. ZEA (50 µM) also inhibited androgen production in testosterone-supplemented Leydig cells, suggesting that SRD5A1 is inhibited by ZEA. Figure 7. View largeDownload slide Concentration-dependent effects of ZEA on basal androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. The levels of testosterone and DIOL were measured. Mean ± SE, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). Figure 7. View largeDownload slide Concentration-dependent effects of ZEA on basal androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. The levels of testosterone and DIOL were measured. Mean ± SE, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). Figure 8. View largeDownload slide Effects of ZEA on expression levels of steroidogenesis related genes in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. Leydig cell genes: (A) Lhcgr, (B) Scarb1, (C) Star, (D) Cyp11a1, (E) Hsd3b1, (F) Cyp17a1, (G) Hsd17b3, (H) Srd5a1, and (I) Akr1c14. Mean ± SEM, n = 6. *p < .05, **p < .01, ***p < .001 indicates significant differences when compared with the control (0 μM). Figure 8. View largeDownload slide Effects of ZEA on expression levels of steroidogenesis related genes in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. Leydig cell genes: (A) Lhcgr, (B) Scarb1, (C) Star, (D) Cyp11a1, (E) Hsd3b1, (F) Cyp17a1, (G) Hsd17b3, (H) Srd5a1, and (I) Akr1c14. Mean ± SEM, n = 6. *p < .05, **p < .01, ***p < .001 indicates significant differences when compared with the control (0 μM). Figure 9. View largeDownload slide Effects of ZEA on the androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 50 μM ZEA in the presence of 10 ng/ml LH, 10 mM 8bromo-cAMP (8BR), 5 μM substrate: 22 R-hydroxycholesterol (22 R), pregnenolone (P5), progesterone (P4), androstenedione (D4), testosterone (T), dihydrotestosterone (DHT) 24 h. The levels of DIOL were measured. Mean ± SEM, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). Figure 9. View largeDownload slide Effects of ZEA on the androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 50 μM ZEA in the presence of 10 ng/ml LH, 10 mM 8bromo-cAMP (8BR), 5 μM substrate: 22 R-hydroxycholesterol (22 R), pregnenolone (P5), progesterone (P4), androstenedione (D4), testosterone (T), dihydrotestosterone (DHT) 24 h. The levels of DIOL were measured. Mean ± SEM, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). DISCUSSION ZEA is an estrogenic mycotoxin produced by several Fusarium (Kuiper-Goodman et al., 1987; Muller et al., 1997; Tashiro et al., 1980). Humans can expose to ZEA via direct ingestion or indirect ingestion of animal products containing ZEA or its metabolites. Therefore, there are possible health concerns about its adverse effects on general population. The potentially undesired effects of ZEA on Leydig cell regeneration were examined in the present study. The effects of ZEA on Leydig cell regeneration were examined in vivo using an EDS-treated Leydig cell-depleted rat model. In this model, Leydig cells can completely regenerate from stem Leydig cells after a single intraperitoneal injection of 75 mg/kg EDS to the rat (Rommerts et al., 1987; Teerds et al., 1989). The regenerated Leydig cells on the 28th day post-EDS were identical to those isolated from pubertal rats (Guo et al., 2013). We observed that ZEA lowered HSD11B1-positive Leydig cell numbers without affecting CYP11A1-positive Leydig cells on post-EDS day 28, indicating that ZEA mainly delays the differentiation of progenitor Leydig cells into immature Leydig cells, because CYP11A1 is a biomarker for all Leydig cells, including progenitor, immature, and adult Leydig cells (Ge and Hardy, 1998), whereas HSD11B1 is a biomarker for Leydig cells at the advanced stage (immature and adult Leydig cells) in either normal rats (Phillips et al., 1989) or EDS-treated rats (Guo et al., 2013). The regenerated Leydig cells on the 28th day post-EDS are the immature Leydig cells (Guo et al., 2013). At this time, the reduced number of HSD11B1 positive Leydig cells without affecting the number of CYP11A1 positive Leydig cells indicated that the differentiation of progenitor Leydig cells into immature Leydig cells was delayed by ZEA. ZEA suppressed steroidogenic enzymes and reduced androgen production without affecting pituitary LH secretion as shown in the unchanged LH level. Furthermore, in vitro study also proved that ZEA potently inhibited Leydig cell specific gene expression and steroidogenesis. In the testis, Cyp11a1 was unchanged whereas Hsd11b1 and Srd5a1 were lower in the 300 ng/testis ZEA group than those in the control. This could represent the differentiated status of Leydig cells during the regeneration after ZEA treatment. Hsd11b1 was only expressed in immature or adult Leydig cells (Phillips et al., 1989) and Srd5a1 was expressed in the highest level in immature Leydig cells during Leydig cell development (Ge and Hardy, 1998). The lower expression of Hsd11b1 and Srd5a1 indicates that ZEA blocks the differentiation of progenitor Leydig cells into immature Leydig cells. In vitro ZEA treatment also significantly downregulated Srd5a1 expression. Interestingly, in vitro ZEA treatment downregulated Cyp11a1 expression although it did not affect its expression in vivo. This discrepancy for Cyp11a1 between rat testes and immature Leydig cells in response to ZEA is still unclear. ZEA has been classified as an estrogenic compound, because it is structurally similar to estradiol and it can bind to estrogen receptors. A previous study demonstrated that it bound to estrogen receptor α being an agonist receptor (Nikov et al., 2000). Although ZEA has an affinity for estrogen receptors with 100–1000 times less than estradiol, ZEA can act through estrogen receptor (Nikov et al., 2000) to transactivate estrogen receptor responsive genes in vivo (Mehmood et al., 2000) and in vitro (Mayr, 1988). Estrogens have been proven to be the negative regulator of Leydig cell regeneration as shown by the evidence that estradiol (Abney and Myers, 1991; Chen et al., 2014) and another estrogen-like compound methoxychlor (Chen et al., 2014) can significantly delay the regeneration of rat Leydig cells in the EDS-treated model. Estrogen receptor α is highly expressed in the progenitor and immature Leydig cells during the pubertal development. The effects of ZEA on Leydig cell regeneration could be resulted from the direct action on Leydig cells because the intratesticular injection of ZEA was adopted in the current study. Abney and Myers also reported the blockade of Leydig cell regeneration after the estradiol treatment happened from day 5 through 30 post-EDS but did not take place before day 5 or after day 30, indicating that this early-stage Leydig cell regeneration is more sensitive to estradiol (Abney and Myers, 1991). Because estradiol inhibited the hCG-stimulation of Leydig cell regeneration after coadministration with hCG, this blockade of estradiol via the pituitary effects was excluded, further indicating that estrogen can inhibit Leydig cell regeneration within the testis (Abney and Myers, 1991). The blockade of steroidogenesis of estradiol or estrogen-like compound methoxychlor metabolite may be acted via estrogen receptor α because the estrogen receptor blockade reversed estrogen-induced suppression of steroidogenesis (Akingbemi et al., 2004). The nuclear mechanism of estrogen receptor α involves estrogen binding to the receptors in the cytosol and receptor-ligand complex translocation into the nucleus and binding to specific response elements known as estrogen response elements in the promoters of target genes, such as many steroidogenic enzymes in Leydig cells (Bjornstrom and Sjoberg, 2005). Apparently, ZEA can downregulate Star and steroidogenic enzyme (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) gene expression in mice (Liu et al., 2014; Yang et al., 2007). Beside estrogen receptor-mediated action, other nuclear receptors may also involve in ZEA-mediated effects. The present study indicated that the effect of ZEA might partially act via downregulating the expression of NR5A1, an important transcription factor for Leydig cell development. In vivo ZEA exposure significantly downregulated NR5A1 expression at 300 ng/testis dose (Figure 4). NR5A1 is very critical for Leydig cell development. Null mutation of NR5A1 caused gonadal agenesis (Sadovsky et al., 1995). Indeed, NR5A1 can convert stem cells or fibroblasts into the Leydig cell lineage by promoting the expression of LHCGR and other steroidogenic enzymes (CYP11A1, HSD11B1, CYP17A1, and HSD17B3) (Yang et al., 2017). Many studies demonstrated that Cyp11a1, Cyp17a1, Hsd3b1, and Hsd17b3 promoters had NR5A1 binding sites (Chen et al., 1999; Hu et al., 2001; Sandhoff et al., 1998; Schimmer et al., 2011). NR4A1 (also called Nur77) is another important transcriptional factor that is similar to NR5A1 to regulate Leydig cell steroidogenesis (Martin et al., 2008; Martin and Tremblay, 2005). Star and Hsd3b promoters contained NR4A1 binding elements (Martin et al., 2008; Martin and Tremblay, 2005). It was reported that ZEA also downregulated NR4A1 expression (Liu et al., 2014), thus possibly decreasing the expression levels of Star and steroidogenic enzymes. In the present study, we could not rule out the indirect action of ZEA although FSH level was not altered. However, ZEA (300 ng/testis) indeed suppressed FSHR level. This indicates that ZEA can disturb Leydig cell regeneration indirectly via lowering the FSHR level. FSHR is located in the Sertoli cell (Ottenweller et al., 2000) and FSHR knockout in mice could reduce Leydig cell numbers and delay the puberty (O'Shaughnessy et al., 2012). The present study clearly demonstrated that in vivo exposure to ZEA delayed Leydig cell regeneration and lowered androgen production. ZEA (as low as 50 nM) also in vitro significantly lowered Cyp11a1, Hsd3b1, Hsd17b3, and Srd5a1 mRNA levels as well as androgen production. In conclusion, ZEA exerts impairments on rat Leydig cell regeneration at lower concentrations (as low as 50 nM) possibly via significantly lowering NR5A1 expression and thus lowering expression levels of some important steroidogenic enzymes and decreasing androgen production. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING National Natural Science Foundation of China (NSFC) (81730042 to R.G.); Health and Family Planning Commission of Zhejiang Province (11-CX29 to R.G.); Zhejiang Provincial NSF (LQ16H040005); Wenzhou Science & Technology Bureau (Y20140661). REFERENCES Abney T. O. , Myers R. B. ( 1991 ). 17β-Estradiol inhibition of Leydig cell regeneration in the ethane dimethylsulfonate-treated mature rat . J. Androl. 12 , 295 – 304 . Google Scholar PubMed Akingbemi B. T. , Sottas C. M. , Koulova A. I. , Klinefelter G. R. , Hardy M. P. ( 2004 ). Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells . Endocrinology 145 , 592 – 603 . 10.1210/en.2003-1174 en.2003-1174 [pii]. Google Scholar CrossRef Search ADS PubMed Bjornstrom L. , Sjoberg M. ( 2005 ). Mechanisms of estrogen receptor signaling: Convergence of genomic and nongenomic actions on target genes . Mol. Endocrinol. 19 , 833 – 842 . me.2004-0486 [pii] 10.1210/me.2004-0486. Google Scholar CrossRef Search ADS PubMed Chen B. , Chen D. , Jiang Z. , Li J. , Liu S. , Dong Y. , Yao W. , Akingbemi B. , Ge R. , Li X. ( 2014 ). Effects of estradiol and methoxychlor on Leydig cell regeneration in the adult rat testis . Int. J. Mol. Sci. 15 , 7812 – 7826 . ijms15057812 [pii] 10.3390/ijms15057812. Google Scholar CrossRef Search ADS PubMed Chen S. , Shi H. , Liu X. , Segaloff D. L. ( 1999 ). Multiple elements and protein factors coordinate the basal and cyclic adenosine 3′, 5′-monophosphate-induced transcription of the lutropin receptor gene in rat granulosa cells . Endocrinology 140 , 2100 – 2109 . 10.1210/endo.140.5.6722. Google Scholar CrossRef Search ADS PubMed Ge R. S. , Hardy M. P. ( 1998 ). Variation in the end products of androgen biosynthesis and metabolism during postnatal differentiation of rat Leydig cells . Endocrinology 139 , 3787 – 3795 . 10.1210/endo.139.9.6183. Google Scholar CrossRef Search ADS PubMed Guo J. , Zhou H. , Su Z. , Chen B. , Wang G. , Wang C. Q. , Xu Y. , Ge R. S. ( 2013 ). Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment . Reproduction 145 , 371 – 380 . Google Scholar CrossRef Search ADS PubMed Hu M. C. , Hsu N. C. , Pai C. I. , Wang C. K. , Chung B. ( 2001 ). Functions of the upstream and proximal steroidogenic factor 1 (SF-1)-binding sites in the CYP11A1 promoter in basal transcription and hormonal response . Mol. Endocrinol. 15 , 812 – 818 . 10.1210/mend.15.5.0636. Google Scholar CrossRef Search ADS PubMed Kim I. H. , Son H. Y. , Cho S. W. , Ha C. S. , Kang B. H. ( 2003 ). Zearalenone induces male germ cell apoptosis in rats . Toxicol. Lett. 138 , 185 – 192 . Google Scholar CrossRef Search ADS PubMed Kuiper-Goodman T. , Scott P. M. , Watanabe H. ( 1987 ). Risk assessment of the mycotoxin zearalenone . Regul. Toxicol. Pharmacol. 7 , 253 – 306 . Google Scholar CrossRef Search ADS PubMed Lemke S. L. , Mayura K. , Ottinger S. E. , McKenzie K. S. , Wang N. , Fickey C. , Kubena L. F. , Phillips T. D. ( 1999 ). Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay . J. Toxicol. Environ. Health A 56 , 283 – 295 . Google Scholar CrossRef Search ADS PubMed Li L. , Bu T. , Su H. , Chen Z. , Liang Y. , Zhang G. , Zhu D. , Shan Y. , Xu R. , Hu Y. et al. , . ( 2014 ). Inutero exposure to diisononyl phthalate caused testicular dysgenesis of rat fetal testis . Toxicol. Lett. 232 , 466 – 474 . S0378-4274(14)01476-3 [pii] 10.1016/j.toxlet.2014.11.024. Google Scholar CrossRef Search ADS PubMed Lin P. , Chen F. , Sun J. , Zhou J. , Wang X. , Wang N. , Li X. , Zhang Z. , Wang A. , Jin Y. ( 2015 ). Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticulum stress-dependent signalling pathway . Reprod. Toxicol. 52 , 71 – 77 . 10.1016/j.reprotox.2015.02.007. Google Scholar CrossRef Search ADS PubMed Liu H. C. , Zhu D. , Wang C. , Guan H. , Li S. , Hu C. , Chen Z. , Hu Y. , Lin H. , Lian Q. Q. et al. , . ( 2015 ). Effects of etomidate on the steroidogenesis of rat immature Leydig cells . PLoS One 10 , e0139311. 10.1371/journal.pone.0139311 PONE-D-15-06330 [pii]. Google Scholar CrossRef Search ADS PubMed Liu Q. , Wang Y. , Gu J. , Yuan Y. , Liu X. , Zheng W. , Huang Q. , Liu Z. , Bian J. ( 2014 ). Zearalenone inhibits testosterone biosynthesis in mouse Leydig cells via the crosstalk of estrogen receptor signaling and orphan nuclear receptor Nur77 expression . Toxicol. In Vitro 28 , 647 – 656 . S0887-2333(14)00025-3 [pii] 10.1016/j.tiv.2014.01.013. Google Scholar CrossRef Search ADS PubMed Liu S. , Li C. , Wang Y. , Hong T. , Song T. , Li L. , Ye L. , Lian Q. , Ge R. S. ( 2016a ). In utero methoxychlor exposure increases rat fetal Leydig cell number but inhibits its function . Toxicology 370 , 31 – 40 . S0300-483X(16)30215-3 [pii] 10.1016/j.tox.2016.09.009. Google Scholar CrossRef Search ADS Liu S. , Mao B. , Bai Y. , Liu J. , Li H. , Li X. , Lian Q. , Ge R. S. ( 2016b ). Effects of methoxychlor and its metabolite hydroxychlor on human placental 3beta-hydroxysteroid dehydrogenase 1 and aromatase in JEG-3 cells . Pharmacology 97 , 126 – 133 . 000442711 [pii] 10.1159/000442711. Google Scholar CrossRef Search ADS Martin L. J. , Boucher N. , Brousseau C. , Tremblay J. J. ( 2008 ). The orphan nuclear receptor NUR77 regulates hormone-induced StAR transcription in Leydig cells through cooperation with Ca2+/calmodulin-dependent protein kinase I . Mol. Endocrinol. 22 , 2021 – 2037 . me.2007-0370 [pii] 10.1210/me.2007-0370. Google Scholar CrossRef Search ADS PubMed Martin L. J. , Tremblay J. J. ( 2005 ). The human 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase type 2 promoter is a novel target for the immediate early orphan nuclear receptor Nur77 in steroidogenic cells . Endocrinology 146 , 861 – 869 . en.2004-0859 [pii] 10.1210/en.2004-0859. Google Scholar CrossRef Search ADS PubMed Mayr U. E. ( 1988 ). Estrogen-controlled gene expression in tissue culture cells by zearalenone . FEBS Lett. 239 , 223 – 226 . 0014-5793(88)80921-9 [pii]. Google Scholar CrossRef Search ADS PubMed Mehmood Z. , Smith A. G. , Tucker M. J. , Chuzel F. , Carmichael N. G. ( 2000 ). The development of methods for assessing the in vivo oestrogen-like effects of xenobiotics in CD-1 mice . Food Chem. Toxicol. 38 , 493 – 501 . S0278-6915(00)00022-3 [pii]. Google Scholar CrossRef Search ADS PubMed Muller H. M. , Reimann J. , Schumacher U. , Schwadorf K. ( 1997 ). Fusarium toxins in wheat harvested during six years in an area of southwest Germany . Nat. Toxins 5 , 24 – 30 . 10.1002/(SICI)(1997)5: 1< 24:: AID-NT4> 3.0.CO; 2-#. Google Scholar CrossRef Search ADS PubMed Nikov G. N. , Hopkins N. E. , Boue S. , Alworth W. L. ( 2000 ). Interactions of dietary estrogens with human estrogen receptors and the effect on estrogen receptor-estrogen response element complex formation . Environ. Health Perspect. 108 , 867 – 872 . sc271_5_1835 [pii]. Google Scholar CrossRef Search ADS PubMed O'Shaughnessy P. J. , Monteiro A. , Abel M. ( 2012 ). Testicular development in mice lacking receptors for follicle stimulating hormone and androgen . PLoS One 7 , e35136. 10.1371/journal.pone.0035136. Google Scholar CrossRef Search ADS PubMed Ottenweller J. E. , Li M.-T. , Giglio W. , Anesetti R. , Pogach L. M. , Huang H. F. S. ( 2000 ). Alteration of follicle-stimulating hormone and testosterone regulation of messenger ribonucleic acid for sertoli cell proteins in the rat during the acute phase of spinal cord injury . Biol. Reprod. 63 , 730 – 735 . 10.1095/biolreprod63.3.730. Google Scholar CrossRef Search ADS PubMed Payne A. H. , Wong K. L. , Vega M. M. ( 1980 ). Differential effects of single and repeated administrations of gonadotropins on luteinizing hormone receptors and testosterone synthesis in two populations of Leydig cells . J. Biol. Chem. 255 , 7118 – 7122 . Google Scholar PubMed Phillips D. M. , Lakshmi V. , Monder C. ( 1989 ). Corticosteroid 11β-dehydrogenase in rat testis . Endocrinology 125 , 209 – 216 . Google Scholar CrossRef Search ADS PubMed Rommerts F. F. , Teerds K. , Themmen A. P. , van Noort M. ( 1987 ). Multiple regulation of testicular steroidogenesis . J. Steroid Biochem. 27 , 309 – 316 . Google Scholar CrossRef Search ADS PubMed Rommerts F. F. , Teerds K. J. , Hoogerbrugge J. W. ( 1988 ). In vitro effects of ethylene-dimethane sulfonate (EDS) on Leydig cells: Inhibition of steroid production and cytotoxic effects are dependent on species and age of rat . Mol. Cell. Endocrinol. 55 , 87 – 94 . Google Scholar CrossRef Search ADS PubMed Sadovsky Y. , Crawford P. A. , Woodson K. G. , Polish J. A. , Clements M. A. , Tourtellotte L. M. , Simburger K. , Milbrandt J. ( 1995 ). Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids . Proc. Natl. Acad. Sci. U.S.A. 92 , 10939 – 10943 . Google Scholar CrossRef Search ADS PubMed Sandhoff T. W. , Hales D. B. , Hales K. H. , McLean M. P. ( 1998 ). Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1 . Endocrinology 139 , 4820 – 4831 . Google Scholar CrossRef Search ADS PubMed Schimmer B. P. , Tsao J. , Cordova M. , Mostafavi S. , Morris Q. , Scheys J. O. ( 2011 ). Contributions of steroidogenic factor 1 to the transcription landscape of Y1 mouse adrenocortical tumor cells . Mol. Cell. Endocrinol. 336 , 85 – 91 . 10.1016/j.mce.2010.11.024. Google Scholar CrossRef Search ADS PubMed Tashiro F. , Kawabata Y. , Naoi M. , Ueno Y. ( 1980 ). Zearalenone-Estrogen Receptor Interaction and RNA Synthesis in Rat Uterus ( Preuser H. J. , Ed), Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, IA, pp. 311 – 320 . Stuttgart : Fischer . Teerds K. J. , de Rooij D. G. , Rommerts F. F. , van den Hurk R. , Wensing C. J. ( 1989 ). Proliferation and differentiation of possible Leydig cell precursors after destruction of the existing Leydig cells with ethane dimethyl sulphonate: The role of LH/human chorionic gonadotrophin . J. Endocrinol. 122 , 689 – 696 . Google Scholar CrossRef Search ADS PubMed Tiemann U. , Danicke S. ( 2007 ). In vivo and in vitro effects of the mycotoxins zearalenone and deoxynivalenol on different non-reproductive and reproductive organs in female pigs: A review . Food Addit. Contam. 24 , 306 – 314 . 10.1080/02652030601053626. Google Scholar CrossRef Search ADS PubMed Wu X. , Guo X. , Wang H. , Zhou S. , Li L. , Chen X. , Wang G. , Liu J. , Ge H. S. , Ge R. S. ( 2017 ). A brief exposure to cadmium impairs Leydig cell regeneration in the adult rat testis . Sci. Rep. 7 , 6337. 10.1038/s41598-017-06870-0 10.1038/s41598-017-06870-0 [pii]. Google Scholar CrossRef Search ADS PubMed Yang J. , Zhang Y. , Wang Y. , Cui S. ( 2007 ). Toxic effects of zearalenone and alpha-zearalenol on the regulation of steroidogenesis and testosterone production in mouse Leydig cells . Toxicol. In Vitro 21 , 558 – 565 . S0887-2333(06)00238-4 [pii] 10.1016/j.tiv.2006.10.013. Google Scholar CrossRef Search ADS PubMed Yang Y. , Li Z. , Wu X. , Chen H. , Xu W. , Xiang Q. , Zhang Q. , Chen J. , Ge R. S. , Su Z. et al. , . ( 2017 ). Direct reprogramming of mouse fibroblasts toward Leydig-like cells by defined factors . Stem Cell Rep. 8 , 39 – 53 . S2213-6711(16)30272-7 [pii] 10.1016/j.stemcr.2016.11.010. Google Scholar CrossRef Search ADS Ye L. , Li X. , Li L. , Chen H. , Ge R. S. ( 2017 ). Insights into the development of the adult Leydig cell lineage from stem Leydig cells . Front. Physiol. 8 , 430. 10.3389/fphys.2017.00430. Google Scholar CrossRef Search ADS PubMed Zatecka E. , Ded L. , Elzeinova F. , Kubatova A. , Dorosh A. , Margaryan H. , Dostalova P. , Korenkova V. , Hoskova K. , Peknicova J. ( 2014 ). Effect of zearalenone on reproductive parameters and expression of selected testicular genes in mice . Reprod. Toxicol. 45 , 20 – 30 . 10.1016/j.reprotox.2014.01.003. Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. 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Abstract

Abstract Zearalenone (ZEA), a fungal mycotoxin, is present in a wide range of human foods. By virtual screening, we have identified that ZEA is a potential endocrine disruptor of Leydig cells. The effect of ZEA on Leydig cell development is still unclear. The objective of the present study was to explore whether ZEA affected Leydig cell developmental process and to clarify the underlying mechanism. Adult male Sprague-Dawley rats (60 days old) were randomly divided into three groups and these rats received a single intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to eliminate all Leydig cells. Seven days after EDS treatment, rats intratesticularly received normal saline (control) or 150 or 300 ng/testis/day ZEA for 21 days. Immature Leydig cells isolated from 35-day-old rats were treated with ZEA (0.05–50 μM) for 24 h in vitro. In vivo ZEA exposure lowered serum testosterone levels, reduced Leydig cell number, and decreased Leydig cell–specific gene or protein expression levels possibly via downregulating the steroidogenic factor 1 (Nr5a1) expression. ZEA in vitro inhibited androgen production and steroidogenic enzyme activities in immature Leydig cells by downregulating expression levels of cholesterol side cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), and steroid 5α-reductase 1 (Srd5a1) at a concentration as low as 50 nM. In conclusion, ZEA exposure disrupts Leydig cell development and steroidogenesis possibly via downregulating Nr5a1. zearalenone, Leydig cells, steroidogenic factor 1, ethane dimethane sulfonate, regeneration Zearalenone (ZEA), also known as F-2 toxin, is one of the most widely distributed estrogenic mycotoxins produced by several Fusarium (Kuiper-Goodman et al., 1987; Muller et al., 1997; Tashiro et al., 1980). ZEA is found in a number of cereal crops worldwide, such as wheat, maize, barley, oats, rice, and sorghum (Kuiper-Goodman et al., 1987). Humans expose to ZEA directly via contaminated foods or indirectly through products derived from animals exposed to mycotoxins. ZEA is found to have toxicity to liver, immune system, and development (Kuiper-Goodman et al., 1987). Moreover, many studies demonstrated that ZEA bound to estrogen receptors and activated estrogen responsive genes (Lemke et al., 1999; Tashiro et al., 1980), leading to abnormal development of female reproductive tracts (Tiemann and Danicke, 2007). ZEA might also be an endocrine disruptor for male reproductive system. Previous studies showed that mice exposed to 150 and 0.15 μg/l ZEA had lower sperm concentration and increased morphologically abnormal spermatozoa (Zatecka et al., 2014) and that rats exposed to 5 mg/kg ZEA had disrupted spermatogenesis (Kim et al., 2003). ZEA also inhibited testosterone biosynthesis of mouse Leydig cells through cross-talk between estrogen receptors and Nur77 (also called NR4A1) signaling (Liu et al., 2014) and inducing apoptosis of Leydig cells via an endoplasmic reticulum stress dependent signaling pathway (Lin et al., 2015). However, the effect of ZEA on Leydig cell development is still unclear. Leydig cells are the primary cells that contribute to about 95% circulatory androgen level. The androgen production depends not only on the function of Leydig cells but also on their number. In the rat, pubertal development of Leydig cells is conceptually defined into four stages: stem (during the whole lifespan), progenitor (at postnatal day 21), immature (between postnatal days 28 and 35), and adult Leydig cells (after postnatal day 56) (Ye et al., 2017). The pubertal development of Leydig cells can be mimicked by eliminating Leydig cells in the adult rat testis using a drug called ethane dimethane sulfonate (EDS) (Rommerts et al., 1988). EDS is a drug that specifically kills rat Leydig cells (Rommerts et al., 1988). Seven days after intraperitoneal injection of 75 mg/kg EDS, Leydig cells in the interstitium of rat testis are completely eliminated (Rommerts et al., 1988). Leydig cell regeneration starts on the 14th day post-EDS and newly regenerated spindle-shaped progenitor Leydig cells are recognized on the 21st day post-EDS (Rommerts et al., 1988). The regenerated progenitor Leydig cells begin to express luteinizing hormone (LH) receptor (LHCGR) for LH-stimulated response, scavenger receptor class B member 1 (SCARB1) for binding to high-density lipoprotein to transport cholesterol into the cells and steroidogenic acute regulatory protein (STAR) for transporting cholesterol from cytosol into the inner membrane of mitochondria to provide steroidogenic substrate, and cholesterol side chain cleavage enzyme (CYP11A1) in the inner membrane of the mitochondria to catalyze cholesterol into 22R-hydroxycholesterol (22R) and further into pregnenolone (P5). P5 is diffused into the smooth endoplasmic reticulum, where it is converted into progesterone (P4) by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), and the latter is converted into androstenedione (D4) by 17α-hydroxylase/17, 20-lyase (CYP17A1) (Rommerts et al., 1988). The regenerated progenitor Leydig cells are differentiated into immature Leydig cells on the 28th day post-EDS, which are identical to those isolated from pubertal testis and which begin to express 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) for the last-step biosynthesis of testosterone from androstenedione (Guo et al., 2013). Immature Leydig cells also highly express androgen metabolizing enzymes, including steroid 5α-reductase 1 (SRD5A1), which converts testosterone into dihydrotestosterone (DHT), and the latter is further metabolized into 5α-androstanediol (DIOL) by 3α-hydroxysteroid dehydrogenase (AKR1C14) (Guo et al., 2013). Interestingly, a glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) begins to express in the immature Leydig cells and continues to maintain the high level later on (Guo et al., 2013) and this enzyme can be a good biomarker for Leydig cells at the advanced stage (immature and adult Leydig cells). EDS-treated rat is a good model to study the effects of many endocrine disruptors on Leydig cell development. In the present study, we exposed rats to ZEA from post-EDS day 7 to day 28 and investigated its effects on early-stage Leydig cell regeneration and treated immature Leydig cells isolated from 35-day-old rats with ZEA to investigate its direct effects. MATERIALS AND METHODS Chemicals and animals ZEA was purchased from J&K Scientific (Beijing, China). SYBR Green qPCR Kit and BCA Protein Assay Kit was obtained from Takara (Otsu, Japan). Trizol Kit was purchased from Invitrogen (Carlsbad, CA). EDS was purchased from Pterosaur Biotech (Hangzhou, China). Immulite2000 Total Testosterone Kit was purchased from Sinopharm Group Medical Supply Chain Services Co (Hangzhou, Zhejiang, China). Radio immunoprecipitation assay buffer was obtained from Bocai Biotechnology (Shanghai, China). The manufacturers of antibodies were listed in Supplementary Table 1. Male Sprague-Dawley rats (60 days of age) were purchased from Shanghai Animal Center (Shanghai, China). All animal studies were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University and were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Animal administration Eighteen 54-day-old male Sprague-Dawley rats were raised in a 12 h dark/light cycle temperature at 23 ± 2°C and relative humidity of 45%–55%. Water and food were provided ad libitum. Animals were adjusted for 6 days before they were randomly divided into 3 groups: control (0 ng/testis/day, n = 6), low dose (150 ng/testis/day, n = 6), and high dose (300 ng/testis/day, n = 6). EDS was dissolved in a mixture of dimethyl sulfoxide and deionized sterile water (1:3, v/v). All rats received a single intraperitoneal injection of 75 mg/kg EDS to eliminate all Leydig cells as previously described (Guo et al., 2013). ZEA was dissolved in normal saline for intratesticular injection. Seven days post-EDS, each testis of a rat received an intratesticular injection of 0, 150, or 300 ng ZEA/testis/day for 21 days. Body weight of each rat was recorded every 3 days. Rats were sacrificed on the 28th day post-EDS by asphyxiation with CO2. Trunk blood was collected, placed in a gel glass tube, and centrifuged at 1500 × g for 10 min to collect serum samples. Serum samples were labeled and stored at –80°C until hormone [testosterone, LH, and follicle-stimulating hormone (FSH)] analysis. Furthermore, each pair of testes was separated and weighted. One testis each animal was frozen in the liquid nitrogen and stored at –80°C for subsequent gene and protein expression analysis. The contralateral testis was punched three holes using a G27 needle and then fixed in Bouin’s solution for immunohistochemical analysis. Immature Leydig cell isolation Eighteen 35-day-old male Sprague-Dawley rats were sacrificed by asphyxiation with CO2. Testes were removed and Leydig cells were purified as described previously (Ge and Hardy, 1998). In brief, animals were sacrificed in CO2 tank, testes were removed, perfused with a M199 solution containing 0.1 mg/ml collagenase via the testicular artery, digested with a mixture of 0.25 mg/ml collagenase and 0.25 mg/ml DNase for 15 min, filtered with 100-μm nylon mesh, and the cells were separated under Percoll gradient as previously described (Ge and Hardy, 1998). The cells with density of 1.070–1.088 g/ml were collected and washed. Purities of Leydig cell fractions were evaluated by histochemical staining for HSD3B1 activity, with 0.4 mM etiocholanolone as the steroid substrate and NAD+ as a cofactor (Payne et al., 1980). The purities of immature Leydig cells were >95%. Leydig cell culture Immature Leydig cells were seeded into the 6-well culture plates after isolation with a density of 105 cells per well. Leydig cells were treated with 0, 0.05, 0.5, 5, and 50 μM ZEA in 2.0 ml DMEM: F12 medium (Gibco, Grand Island, NY) for 24 h. Cells were harvested for the analysis of Leydig cell mRNA and protein levels. Because immature Leydig cells produced about 80% DIOL and 10% testosterone (Ge and Hardy, 1998), media were harvested for the measurement of testosterone and DIOL concentrations. To further dissect the action site(s) of ZEA on androgen biosynthesis and metabolism, we isolated immature Leydig cells and added hormone (LH, 10 ng/ml), signaling compound (8Br-cAMP, 8BR, 10 mM) and steroidogenic enzyme substrates, including those of CYP11A1 (22R, 5 µM), HSD3B1 (pregnenolone, P5, 5 µM), CYP17A1 (progesterone, P4, 5 µM), HSD17B3 (androstenedione, D4, 5 µM), SRD5A1 (testosterone, T, 5 µM), and AKR1C14 (DHT, 5 µM), and measured the medium DIOL and T concentrations and then compared them with the control (no treatment, basal). The substrate information was listed in Supplementary Table 2. After isolation, the purified immature Leydig cells were seeded into 24-well culture plate with a density of 0.05 × 106 cells per well. About 50 μM ZEA was coincubated with these compounds. LH (10 ng/ml) and 8BR (10 mM) were used to act as a maximum stimulation to induce Leydig cell steroidogenesis as previously described (Liu et al., 2015, 2014). Because 8BR can penetrate the cell membrane, therefore it is used to replace the intracellular cAMP, which is impermeable. 22R, pregnenolone, progesterone, androstenedione, testosterone, and DHT were used as the respective substrate of the enzyme: CYP11A1, HSD3B1, CYP17A1, HSD17B3, SRD5A1, and AKR1C14. Because 22R can readily penetrate cell and mitochondrial membrane, it is used to replace cholesterol as the substrate for CYP11A1. Media were collected for DIOL and testosterone assay after 3 h incubation. Measurement of DIOL and testosterone levels by RIA DIOL and testosterone concentrations in the medium were measured with the tritium-based RIA as described previously (Ge and Hardy, 1998) using the commercial RIA kits (IBL). The minimum detectable concentration of the assay for DIOL and testosterone was 5 pg/ml. The internal control contains 100 pg/ml DIOL or testosterone dissolved in the same culture media. Interassay and intraassay variations of DIOL and testosterone were within 15%. ELISA for serum LH and FSH levels Serum LH and FSH levels were measured with their ELISA kits according to the manufacturer’s instructions (Chemicon, CA). In brief, 200 μl samples and 50 μl assay diluent were added to precoated 96-well plates. The plates were incubated for 2 h at room temperature, and washed 5 times with washing buffer. About 100 μl peroxidase-conjugated IgG anti-LH or -FSH of solution was added into each well for 2 h at room temperature. Then, plates were washed 5 times. Then, 100 μl substrate buffers were added into each well, and incubated in the dark place for 30 min at room temperature. The enzyme reaction was stopped by 50 μl stop solution. The quantification of LH and FSH levels was obtained by a microplate reader at 550 nm with correction wavelength at 450 nm. Western blot analysis Testes were homogenized and boiled in equal volumes of sample loading buffer, a Tris–Cl buffer (pH 6.8), containing 20% glycerol, 5% sodium dodecyl sulfate, 3.1% dithiothreitol, and 0.001% bromophenol blue. Homogenized samples (50 μg protein) were electrophoresed on 10% polyacrylamide gels containing sodium dodecyl sulfate. Proteins were electrophoretically transferred onto nitrocellulose membranes, and the membranes incubated with 5% nonfat milk for 1 h to block nonspecific binding. Then, the membranes were incubated with primary antibodies against the following antigens: HSD3B1, HSD11B1, HSD17B3, NR5A1, FSHR, and ACTB (listed in Supplementary Table 1). The membranes were washed and incubated with a 1:5000 dilution of goat antirabbit antiserum that was conjugated to horseradish peroxidase. The washing step was repeated, and immunoreactive bands were visualized by chemiluminescence using an ECL kit (Amersham, Arlington Heights, IL). The intensity of proteins was quantified using ImageJ software. The Leydig and Sertoli cell proteins were adjusted to ACTB, a house-keeping protein. RNA isolation and real-time PCR Total RNAs were purified from the testes and immature Leydig cells using the Trizol Kit according to the manufacturer’s instructions, and the concentration of RNA was measured by reading OD value at 260 nm. The first strand (cDNA) was reversely transcribed and used as the template for qPCR analysis as previously described (Li et al., 2014). The expression levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, Srd5a1, Nrd5a1, Hsd11b1, and Akr1c14) and Sertoli cells (Dhh and Fshr) were measured using a SYBR Green qPCR Kit. The gene name and primer sequences were listed in Supplementary Table 3. The qPCR reaction mixture had 10 μl of SYBR Green mix, 1.6 μl forward and reverse primer mix, 400 ng diluted cDNA sample, and 5–8 μl RNase-free water. The reaction was processed by the following program: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. The Ct value was read and the expression level of a target gene was calculated using a standard curve method as previously described (Li et al., 2014). The mRNA levels were adjusted to Rps16, a house-keeping gene, for internal control. These primers have been tested to detect the respective mRNA levels in our previous study (Wu et al., 2017). Immunohistochemistry and enumeration of Leydig and Sertoli cells One testis per rat was used for immunohistochemical staining (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. Six testes per group were randomly selected and cut into 8 discs, and each disc was cut to 2 pieces. One piece each testis was randomly selected and dehydrated in ethanol and xylene and then embedded in paraffin in a tissue array as previously described (Wu et al., 2017). Six micrometer-thick transverse sections were cut and mounted on glass slides. Approximately 10 sections were used. Avidin-biotin immunohistochemical stainings for CYP11A1 (all Leydig cells) or HSD11B1 (Leydig cells at the advanced stage) or SOX9 (Sertoli cells) were conducted following manufacturer’s instructions. Antigen retrieval was conducted by a microwave irradiation in 10 mM (pH 6.0) citrate buffer for 10 min. Then, endogenous peroxidase was blocked with 0.5% of H2O2 in methanol for 30 min. Sections were incubated with either CYP11A1 or HSD11B1 or SOX9 polyclonal antibody (diluted 1:200) for 1 h at room temperature. Diaminobenzidine was used for visualizing the antibody-antigen complexes, which positively label Leydig cells by brown cytoplasmic staining. Mayer hematoxylin was adopted as the counterstaining. The sections were dehydrated in graded concentrations of alcohol and cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). Nonimmune rabbit IgG was used in the incubation of negative control sections with working dilution the same as the primary antibody. The cells with CYP11A1 staining in the interstitial area represent Leydig cells, whereas cells with HSD11B1 staining in the interstitial area represent the Leydig cells at the advanced stage (Phillips et al., 1989). The cells with SOX9 staining in the seminiferous tubules represent Sertoli cells. The enumeration of Leydig cells and Sertoli cells was performed as previously described (Wu et al., 2017). The total number of Leydig and Sertoli cells was calculated by multiplying the number of Leydig cells counted in a known fraction of the testis by the inverse of the sampling probability. Computer-assisted image analysis of cell size and nuclear size Leydig cells were identified by staining HSD11B1 as above. The Leydig cell size, nuclear size, and cytoplasmic size were calculated as previously described (Liu et al., 2016a). Six randomly selected fields in each of 3 nonadjacent sections per testis were captured using a BX53 Olympus microscope (Tokyo, Japan) equipped with a digital camera interfaced to a computer. The images that were displayed on the monitor represented partial area of a testis. Cell size and nuclear size were estimated using the image analysis software (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). More than 50 Leydig cells were evaluated in each testis. The cell size and nuclear size were recorded as μm3 and cytoplasmic size was calculated by cell size minus nuclear size. Semi-quantitative measurement of CYP11A1 and HSD11B1 CYP11A1 and HSD11B1 are the proteins of Leydig cells. Immunohistochemical stainings of CYP11A1 and HSD11B1 were performed as above. Target protein density and background area density were measured using the image analysis software as previously described (Liu et al., 2016b). More than 50 Leydig cells were evaluated in each testis and the protein density of each sample was averaged. Calculation of Leydig cell proliferation The Leydig cell proliferation was judged by immunofluorescent staining of PCNA in Leydig cells after dual stainings of PCNA (for proliferating cell) and CYP11A1 (the Leydig cells) in testis collected on post-EDS day 28, when the regenerated Leydig cells were at the stage of immature Leydig cells that have proliferating capacity. The sections in the tissue array assembled above were used. Sections were sequentially incubated with the primary antibodies of CYP11A1 and PCNA for 60 min. Then, the fluorescent secondary antibody (Alexa-conjugated antirabbit or antimouse IgG, 1:500) was used to label Leydig cells (CYP11A1, cytoplasmic staining in green color) and proliferating cells (PCNA, nuclear staining in red color). Images were taken with a fluorescent microscopy. The percentage of the cells with CYP11A1/PCNA double staining was calculated. Statistical analysis All data are presented as mean and standard errors (SEM). Statistical significance was analyzed using one-way ANOVA followed by ad hoc Turkey’s multiple comparisons between groups. Statistical analysis was performed using GraphPad Prism (version 6, GraphPad Software Inc, San Diego, CA). A p < .05 was considered statistically significant. RESULTS General Toxicological Parameters of ZEA To analyze the general parameters of ZEA toxicity, body and testis weights were recorded at the end of in vivo ZEA treatment (Table 1). ZEA did not significantly affect body weights at both doses. ZEA did not affect testis weights at 150 ng ZEA/testis group. However, the testis weight at 300 ng ZEA/testis group was significantly lower than that of the control (Table 1). No mortalities and abnormal activities were observed in rats of any groups. Table 1. General Toxicological Parameters After Treatment of Zearalenone Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Mean ± SEM, n = 6. ** Significant difference of zearalenone group when compared with control (0 ng/testis) on post-EDS day 28 at p < .01. Table 1. General Toxicological Parameters After Treatment of Zearalenone Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Parameters Dosage (ng/testis) 0 150 300 Body weight 382.03 ± 34.86 381.93 ± 34.87 372.74 ± 38.63 Testis weight 3.04 ± 0.25 2.78 ± 0.48 2.10 ± 0.59** Mean ± SEM, n = 6. ** Significant difference of zearalenone group when compared with control (0 ng/testis) on post-EDS day 28 at p < .01. ZEA Lowers Testosterone Levels In Vivo Rats were treated with ZEA for 21 days starting on the 7th day post-EDS, when only stem Leydig cells were present (Figure 1A). Sera were collected on post-EDS day 28 for hormone (testosterone, LH, and FSH) analysis (Figs. 1B–D). ZEA significantly decreased testosterone levels at 150 and 300 ng/testis doses (Figure 1B). This result indicates that ZEA delays Leydig cell regeneration. Further analysis showed that ZEA had no effect on LH (Figure 1C) and FSH (Figure 1D) levels, suggesting that pituitary hormone secretion is not affected by ZEA. Figure 1. View largeDownload slide Regimen of zearalenone (ZEA) and serum hormone levels. A, ZEA regimen; B, serum testosterone (T); C, serum luteinizing hormone (LH); D, serum follicle-stimulating hormone (FSH). Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 1. View largeDownload slide Regimen of zearalenone (ZEA) and serum hormone levels. A, ZEA regimen; B, serum testosterone (T); C, serum luteinizing hormone (LH); D, serum follicle-stimulating hormone (FSH). Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). ZEA Reduces Leydig Cell Number In Vivo All Leydig cells were identified by staining CYP11A1, a biomarker for all Leydig cells in the Leydig cell lineage (Ge and Hardy, 1998). Leydig cells at immature and adult stages were identified by HSD11B1 because it began to express in Leydig cells after postnatal day 28 (Phillips et al., 1989) and in regenerated Leydig cells after post-EDS day 28 (Guo et al., 2013). Brown cytosolic staining in the interstitium showed the CYP11A1 positive or HSD11B1 positive cell. We counted the cell number of CYP11A1 positive cells (all Leydig cells) and HSD11B1 positive cells (Leydig cells at the advanced stage) (Figs. 2I and 2J). We found that CYP11A1 positive Leydig cell numbers were not significantly reduced after ZEA treatment (Figs. 2A, 2C, 2E, and 2G). However, HSD11B1 positive Leydig cell numbers were significantly reduced after ZEA treatment (Figs. 2B, 2D, 2F, and 2H). This indicates that the differentiation of Leydig cells from the progenitor stage into the immature stage is blocked by ZEA. Meanwhile, ZEA at 300 ng/testis reduced Leydig cell and cytoplasmic volumes (Figs. 2K and 2L), further confirming that Leydig cells are at more immature stage. However, ZEA did not alter the SOX9-positive Sertoli cell number (data not shown). Figure 2. View largeDownload slide Immunohistochemical staining of CYP11A1 and HSD11B1 in rat testis sections on post-EDS day 28 after ZEA treatment. CYP11A1: A, C, E, G; HSD11B1: B, D, F, H. (A and B): control; (C and D): 150 ng/testis ZEA; (E and F): 300 ng/testis ZEA. (G and H): the negative control. (I and J): quantification of CYP11A1 and HSD11B1 positive cell numbers. (K) Cell volume. (L) Cytoplasmic volume. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. Figure 2. View largeDownload slide Immunohistochemical staining of CYP11A1 and HSD11B1 in rat testis sections on post-EDS day 28 after ZEA treatment. CYP11A1: A, C, E, G; HSD11B1: B, D, F, H. (A and B): control; (C and D): 150 ng/testis ZEA; (E and F): 300 ng/testis ZEA. (G and H): the negative control. (I and J): quantification of CYP11A1 and HSD11B1 positive cell numbers. (K) Cell volume. (L) Cytoplasmic volume. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. ZEA Downregulates Leydig and Sertoli Cell mRNA Levels In Vivo We measured the mRNA levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, Srd5a1, Nr5a1, and Hsd11b1) and Sertoli (Dhh and Fshr) cell genes on post-EDS day 28. ZEA at 300 ng/testis reduced mRNA levels of Leydig cell genes, including Hsd3b1, Hsd17b3, Srd5a1, Nr5a1, and Hsd11b1 without affecting the levels of Lhcgr, Scarb1, Star, Cyp11a1, and Cyp17a1 (Figure 3). ZEA at 300 ng/testis dose also decreased Fshr level without affecting Dhh level (Figure 3). These results suggest that both Leydig and Sertoli cell gene expression is affected by ZEA at the higher dose. Figure 3. View largeDownload slide Gene expression levels of the testes in rats with or without ZEA treatment. Genes in the Leydig cell: (A) Lhcgr, (B) Scarb1, (C) Star, (D) CYP11a1, (E) Cyp17a1, (F) Hsd3b1, (G) Hsd17b3, (H) Srd5a1, (I) Nr5a1, and (J) Hsd11b1. Genes in the Sertoli cell: (K) Dhh and (L) Fshr. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 3. View largeDownload slide Gene expression levels of the testes in rats with or without ZEA treatment. Genes in the Leydig cell: (A) Lhcgr, (B) Scarb1, (C) Star, (D) CYP11a1, (E) Cyp17a1, (F) Hsd3b1, (G) Hsd17b3, (H) Srd5a1, (I) Nr5a1, and (J) Hsd11b1. Genes in the Sertoli cell: (K) Dhh and (L) Fshr. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). ZEA Reduces Protein Levels in Leydig and Sertoli Cells In Vivo We measured the levels of Leydig (HSD3B1, HSD11B1, HSD17B3, and NR5A1) and Sertoli (FSHR) cell proteins in the testes collected on post-EDS day 28. ZEA lowered these protein levels in parallel with their mRNA levels (Figure 4). Furthermore, we used a semiquantitative analysis of HSD11B1 density in the individual cell and found that ZEA lowered HSD11B1 level significantly at 300 ng/testis (Figs. 5D–F and 5H). These results suggest that ZEA delays Leydig cell regeneration. Figure 4. View largeDownload slide Protein levels of the testis in rats with or without ZEA treatment. Protein expressions in the testis: (A) western blot band. (B) Quantification of protein levels. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 4. View largeDownload slide Protein levels of the testis in rats with or without ZEA treatment. Protein expressions in the testis: (A) western blot band. (B) Quantification of protein levels. Mean ± SEM, n = 6. *p < .05, **p < .01 indicate significant differences when compared with the control (0 mg/kg). Figure 5. View largeDownload slide Semiquantitative analysis of CYP11A1 and HSD11B1 in single Leydig cell after ZEA treatment. Immunohistochemical images (1) CYP11A1: A, B, C and (2) HSD11B1: D, E, F. (G) and (H): quantification of CYP11A1 and HSD11B1 density. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. Figure 5. View largeDownload slide Semiquantitative analysis of CYP11A1 and HSD11B1 in single Leydig cell after ZEA treatment. Immunohistochemical images (1) CYP11A1: A, B, C and (2) HSD11B1: D, E, F. (G) and (H): quantification of CYP11A1 and HSD11B1 density. Mean ± SEM, n = 6. *p < .05 indicates a significant difference when compared with the control (0 mg/kg). Scale bars 50 μm. ZEA Has No Effect on Immature Leydig Cell Proliferation In Vivo Immature Leydig cells on post-EDS day 28 have the capacity of proliferation (Guo et al., 2013). PCNA is a nuclear matrix protein for cell proliferation. We stained Leydig cells using CYP11A1 antibody and the proliferating cell using PCNA antibody. There was no difference in PCNA-positive Leydig cells between control and ZEA-treated groups (Figs. 6A–D), indicating that the reduced HSD11B1-positive Leydig cell number is not contributed by the proliferation of Leydig cells. Figure 6. View largeDownload slide Immunohistochemical staining for PCNA and Leydig cells after ZEA treatment. Immunohistochemical images: A, B, C. Quantification: PCNA-positive Leydig cells (D). Mean ± SEM, n = 6. No significant difference was identified. Scale bars = 50 μm. Figure 6. View largeDownload slide Immunohistochemical staining for PCNA and Leydig cells after ZEA treatment. Immunohistochemical images: A, B, C. Quantification: PCNA-positive Leydig cells (D). Mean ± SEM, n = 6. No significant difference was identified. Scale bars = 50 μm. ZEA Inhibits Androgen Production in Immature Leydig Cell In Vitro To further dissect the direct effect of ZEA on immature Leydig cells, we treated immature Leydig cells with 0.05, 0.5, 5, and 50 μM ZEA for 24 h. ZEA concentration-dependently lowered androgen (DIOL + testosterone) levels (Figure 7). We further measured the mRNA levels in Leydig cells after ZEA treatment (Figure 8). ZEA significantly reduced Cyp11a1, Hsd3b1, Srd5a1 mRNA levels at ≥0.5 μM and Hsd17b3 mRNA level at 50 μM. We further examined the effects of ZEA in basal, LH-stimulated, and 8BR-stimulated conditions. ZEA at 50 μM inhibited androgen (DIOL) production in all cases (Figs. 9A–C). To explore the specific sites by which ZEA may affect androgen production, we tested all enzymatic steps by providing the cells with different enzyme substrates. The enzyme name, substrate name, and concentrations were listed in Supplementary Table 2. ZEA (50 µM) inhibited androgen production in 22R-, P5-, and D4-supplemented Leydig cells, indicating that ZEA inhibits CYP11A1, HSD3B1, and HSD17B3 enzyme activities. ZEA (50 µM) also inhibited androgen production in testosterone-supplemented Leydig cells, suggesting that SRD5A1 is inhibited by ZEA. Figure 7. View largeDownload slide Concentration-dependent effects of ZEA on basal androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. The levels of testosterone and DIOL were measured. Mean ± SE, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). Figure 7. View largeDownload slide Concentration-dependent effects of ZEA on basal androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. The levels of testosterone and DIOL were measured. Mean ± SE, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). Figure 8. View largeDownload slide Effects of ZEA on expression levels of steroidogenesis related genes in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. Leydig cell genes: (A) Lhcgr, (B) Scarb1, (C) Star, (D) Cyp11a1, (E) Hsd3b1, (F) Cyp17a1, (G) Hsd17b3, (H) Srd5a1, and (I) Akr1c14. Mean ± SEM, n = 6. *p < .05, **p < .01, ***p < .001 indicates significant differences when compared with the control (0 μM). Figure 8. View largeDownload slide Effects of ZEA on expression levels of steroidogenesis related genes in rat immature Leydig cells. Rat immature Leydig cells were cultured with 0.5–50 μM ZEA for 24 h. Leydig cell genes: (A) Lhcgr, (B) Scarb1, (C) Star, (D) Cyp11a1, (E) Hsd3b1, (F) Cyp17a1, (G) Hsd17b3, (H) Srd5a1, and (I) Akr1c14. Mean ± SEM, n = 6. *p < .05, **p < .01, ***p < .001 indicates significant differences when compared with the control (0 μM). Figure 9. View largeDownload slide Effects of ZEA on the androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 50 μM ZEA in the presence of 10 ng/ml LH, 10 mM 8bromo-cAMP (8BR), 5 μM substrate: 22 R-hydroxycholesterol (22 R), pregnenolone (P5), progesterone (P4), androstenedione (D4), testosterone (T), dihydrotestosterone (DHT) 24 h. The levels of DIOL were measured. Mean ± SEM, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). Figure 9. View largeDownload slide Effects of ZEA on the androgen production in rat immature Leydig cells. Rat immature Leydig cells were cultured with 50 μM ZEA in the presence of 10 ng/ml LH, 10 mM 8bromo-cAMP (8BR), 5 μM substrate: 22 R-hydroxycholesterol (22 R), pregnenolone (P5), progesterone (P4), androstenedione (D4), testosterone (T), dihydrotestosterone (DHT) 24 h. The levels of DIOL were measured. Mean ± SEM, n = 6. ***p < .001 indicates significant difference when compared with the control (0 μM). DISCUSSION ZEA is an estrogenic mycotoxin produced by several Fusarium (Kuiper-Goodman et al., 1987; Muller et al., 1997; Tashiro et al., 1980). Humans can expose to ZEA via direct ingestion or indirect ingestion of animal products containing ZEA or its metabolites. Therefore, there are possible health concerns about its adverse effects on general population. The potentially undesired effects of ZEA on Leydig cell regeneration were examined in the present study. The effects of ZEA on Leydig cell regeneration were examined in vivo using an EDS-treated Leydig cell-depleted rat model. In this model, Leydig cells can completely regenerate from stem Leydig cells after a single intraperitoneal injection of 75 mg/kg EDS to the rat (Rommerts et al., 1987; Teerds et al., 1989). The regenerated Leydig cells on the 28th day post-EDS were identical to those isolated from pubertal rats (Guo et al., 2013). We observed that ZEA lowered HSD11B1-positive Leydig cell numbers without affecting CYP11A1-positive Leydig cells on post-EDS day 28, indicating that ZEA mainly delays the differentiation of progenitor Leydig cells into immature Leydig cells, because CYP11A1 is a biomarker for all Leydig cells, including progenitor, immature, and adult Leydig cells (Ge and Hardy, 1998), whereas HSD11B1 is a biomarker for Leydig cells at the advanced stage (immature and adult Leydig cells) in either normal rats (Phillips et al., 1989) or EDS-treated rats (Guo et al., 2013). The regenerated Leydig cells on the 28th day post-EDS are the immature Leydig cells (Guo et al., 2013). At this time, the reduced number of HSD11B1 positive Leydig cells without affecting the number of CYP11A1 positive Leydig cells indicated that the differentiation of progenitor Leydig cells into immature Leydig cells was delayed by ZEA. ZEA suppressed steroidogenic enzymes and reduced androgen production without affecting pituitary LH secretion as shown in the unchanged LH level. Furthermore, in vitro study also proved that ZEA potently inhibited Leydig cell specific gene expression and steroidogenesis. In the testis, Cyp11a1 was unchanged whereas Hsd11b1 and Srd5a1 were lower in the 300 ng/testis ZEA group than those in the control. This could represent the differentiated status of Leydig cells during the regeneration after ZEA treatment. Hsd11b1 was only expressed in immature or adult Leydig cells (Phillips et al., 1989) and Srd5a1 was expressed in the highest level in immature Leydig cells during Leydig cell development (Ge and Hardy, 1998). The lower expression of Hsd11b1 and Srd5a1 indicates that ZEA blocks the differentiation of progenitor Leydig cells into immature Leydig cells. In vitro ZEA treatment also significantly downregulated Srd5a1 expression. Interestingly, in vitro ZEA treatment downregulated Cyp11a1 expression although it did not affect its expression in vivo. This discrepancy for Cyp11a1 between rat testes and immature Leydig cells in response to ZEA is still unclear. ZEA has been classified as an estrogenic compound, because it is structurally similar to estradiol and it can bind to estrogen receptors. A previous study demonstrated that it bound to estrogen receptor α being an agonist receptor (Nikov et al., 2000). Although ZEA has an affinity for estrogen receptors with 100–1000 times less than estradiol, ZEA can act through estrogen receptor (Nikov et al., 2000) to transactivate estrogen receptor responsive genes in vivo (Mehmood et al., 2000) and in vitro (Mayr, 1988). Estrogens have been proven to be the negative regulator of Leydig cell regeneration as shown by the evidence that estradiol (Abney and Myers, 1991; Chen et al., 2014) and another estrogen-like compound methoxychlor (Chen et al., 2014) can significantly delay the regeneration of rat Leydig cells in the EDS-treated model. Estrogen receptor α is highly expressed in the progenitor and immature Leydig cells during the pubertal development. The effects of ZEA on Leydig cell regeneration could be resulted from the direct action on Leydig cells because the intratesticular injection of ZEA was adopted in the current study. Abney and Myers also reported the blockade of Leydig cell regeneration after the estradiol treatment happened from day 5 through 30 post-EDS but did not take place before day 5 or after day 30, indicating that this early-stage Leydig cell regeneration is more sensitive to estradiol (Abney and Myers, 1991). Because estradiol inhibited the hCG-stimulation of Leydig cell regeneration after coadministration with hCG, this blockade of estradiol via the pituitary effects was excluded, further indicating that estrogen can inhibit Leydig cell regeneration within the testis (Abney and Myers, 1991). The blockade of steroidogenesis of estradiol or estrogen-like compound methoxychlor metabolite may be acted via estrogen receptor α because the estrogen receptor blockade reversed estrogen-induced suppression of steroidogenesis (Akingbemi et al., 2004). The nuclear mechanism of estrogen receptor α involves estrogen binding to the receptors in the cytosol and receptor-ligand complex translocation into the nucleus and binding to specific response elements known as estrogen response elements in the promoters of target genes, such as many steroidogenic enzymes in Leydig cells (Bjornstrom and Sjoberg, 2005). Apparently, ZEA can downregulate Star and steroidogenic enzyme (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) gene expression in mice (Liu et al., 2014; Yang et al., 2007). Beside estrogen receptor-mediated action, other nuclear receptors may also involve in ZEA-mediated effects. The present study indicated that the effect of ZEA might partially act via downregulating the expression of NR5A1, an important transcription factor for Leydig cell development. In vivo ZEA exposure significantly downregulated NR5A1 expression at 300 ng/testis dose (Figure 4). NR5A1 is very critical for Leydig cell development. Null mutation of NR5A1 caused gonadal agenesis (Sadovsky et al., 1995). Indeed, NR5A1 can convert stem cells or fibroblasts into the Leydig cell lineage by promoting the expression of LHCGR and other steroidogenic enzymes (CYP11A1, HSD11B1, CYP17A1, and HSD17B3) (Yang et al., 2017). Many studies demonstrated that Cyp11a1, Cyp17a1, Hsd3b1, and Hsd17b3 promoters had NR5A1 binding sites (Chen et al., 1999; Hu et al., 2001; Sandhoff et al., 1998; Schimmer et al., 2011). NR4A1 (also called Nur77) is another important transcriptional factor that is similar to NR5A1 to regulate Leydig cell steroidogenesis (Martin et al., 2008; Martin and Tremblay, 2005). Star and Hsd3b promoters contained NR4A1 binding elements (Martin et al., 2008; Martin and Tremblay, 2005). It was reported that ZEA also downregulated NR4A1 expression (Liu et al., 2014), thus possibly decreasing the expression levels of Star and steroidogenic enzymes. In the present study, we could not rule out the indirect action of ZEA although FSH level was not altered. However, ZEA (300 ng/testis) indeed suppressed FSHR level. This indicates that ZEA can disturb Leydig cell regeneration indirectly via lowering the FSHR level. FSHR is located in the Sertoli cell (Ottenweller et al., 2000) and FSHR knockout in mice could reduce Leydig cell numbers and delay the puberty (O'Shaughnessy et al., 2012). The present study clearly demonstrated that in vivo exposure to ZEA delayed Leydig cell regeneration and lowered androgen production. ZEA (as low as 50 nM) also in vitro significantly lowered Cyp11a1, Hsd3b1, Hsd17b3, and Srd5a1 mRNA levels as well as androgen production. In conclusion, ZEA exerts impairments on rat Leydig cell regeneration at lower concentrations (as low as 50 nM) possibly via significantly lowering NR5A1 expression and thus lowering expression levels of some important steroidogenic enzymes and decreasing androgen production. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING National Natural Science Foundation of China (NSFC) (81730042 to R.G.); Health and Family Planning Commission of Zhejiang Province (11-CX29 to R.G.); Zhejiang Provincial NSF (LQ16H040005); Wenzhou Science & Technology Bureau (Y20140661). REFERENCES Abney T. O. , Myers R. B. ( 1991 ). 17β-Estradiol inhibition of Leydig cell regeneration in the ethane dimethylsulfonate-treated mature rat . J. Androl. 12 , 295 – 304 . Google Scholar PubMed Akingbemi B. T. , Sottas C. M. , Koulova A. I. , Klinefelter G. R. , Hardy M. P. ( 2004 ). Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells . Endocrinology 145 , 592 – 603 . 10.1210/en.2003-1174 en.2003-1174 [pii]. Google Scholar CrossRef Search ADS PubMed Bjornstrom L. , Sjoberg M. ( 2005 ). Mechanisms of estrogen receptor signaling: Convergence of genomic and nongenomic actions on target genes . Mol. Endocrinol. 19 , 833 – 842 . me.2004-0486 [pii] 10.1210/me.2004-0486. Google Scholar CrossRef Search ADS PubMed Chen B. , Chen D. , Jiang Z. , Li J. , Liu S. , Dong Y. , Yao W. , Akingbemi B. , Ge R. , Li X. ( 2014 ). Effects of estradiol and methoxychlor on Leydig cell regeneration in the adult rat testis . Int. J. Mol. Sci. 15 , 7812 – 7826 . ijms15057812 [pii] 10.3390/ijms15057812. Google Scholar CrossRef Search ADS PubMed Chen S. , Shi H. , Liu X. , Segaloff D. L. ( 1999 ). Multiple elements and protein factors coordinate the basal and cyclic adenosine 3′, 5′-monophosphate-induced transcription of the lutropin receptor gene in rat granulosa cells . Endocrinology 140 , 2100 – 2109 . 10.1210/endo.140.5.6722. Google Scholar CrossRef Search ADS PubMed Ge R. S. , Hardy M. P. ( 1998 ). Variation in the end products of androgen biosynthesis and metabolism during postnatal differentiation of rat Leydig cells . Endocrinology 139 , 3787 – 3795 . 10.1210/endo.139.9.6183. Google Scholar CrossRef Search ADS PubMed Guo J. , Zhou H. , Su Z. , Chen B. , Wang G. , Wang C. Q. , Xu Y. , Ge R. S. ( 2013 ). Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment . Reproduction 145 , 371 – 380 . Google Scholar CrossRef Search ADS PubMed Hu M. C. , Hsu N. C. , Pai C. I. , Wang C. K. , Chung B. ( 2001 ). Functions of the upstream and proximal steroidogenic factor 1 (SF-1)-binding sites in the CYP11A1 promoter in basal transcription and hormonal response . Mol. Endocrinol. 15 , 812 – 818 . 10.1210/mend.15.5.0636. Google Scholar CrossRef Search ADS PubMed Kim I. H. , Son H. Y. , Cho S. W. , Ha C. S. , Kang B. H. ( 2003 ). Zearalenone induces male germ cell apoptosis in rats . Toxicol. Lett. 138 , 185 – 192 . Google Scholar CrossRef Search ADS PubMed Kuiper-Goodman T. , Scott P. M. , Watanabe H. ( 1987 ). Risk assessment of the mycotoxin zearalenone . Regul. Toxicol. Pharmacol. 7 , 253 – 306 . Google Scholar CrossRef Search ADS PubMed Lemke S. L. , Mayura K. , Ottinger S. E. , McKenzie K. S. , Wang N. , Fickey C. , Kubena L. F. , Phillips T. D. ( 1999 ). Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay . J. Toxicol. Environ. Health A 56 , 283 – 295 . Google Scholar CrossRef Search ADS PubMed Li L. , Bu T. , Su H. , Chen Z. , Liang Y. , Zhang G. , Zhu D. , Shan Y. , Xu R. , Hu Y. et al. , . ( 2014 ). Inutero exposure to diisononyl phthalate caused testicular dysgenesis of rat fetal testis . Toxicol. Lett. 232 , 466 – 474 . S0378-4274(14)01476-3 [pii] 10.1016/j.toxlet.2014.11.024. Google Scholar CrossRef Search ADS PubMed Lin P. , Chen F. , Sun J. , Zhou J. , Wang X. , Wang N. , Li X. , Zhang Z. , Wang A. , Jin Y. ( 2015 ). Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticulum stress-dependent signalling pathway . Reprod. Toxicol. 52 , 71 – 77 . 10.1016/j.reprotox.2015.02.007. Google Scholar CrossRef Search ADS PubMed Liu H. C. , Zhu D. , Wang C. , Guan H. , Li S. , Hu C. , Chen Z. , Hu Y. , Lin H. , Lian Q. Q. et al. , . ( 2015 ). Effects of etomidate on the steroidogenesis of rat immature Leydig cells . PLoS One 10 , e0139311. 10.1371/journal.pone.0139311 PONE-D-15-06330 [pii]. Google Scholar CrossRef Search ADS PubMed Liu Q. , Wang Y. , Gu J. , Yuan Y. , Liu X. , Zheng W. , Huang Q. , Liu Z. , Bian J. ( 2014 ). Zearalenone inhibits testosterone biosynthesis in mouse Leydig cells via the crosstalk of estrogen receptor signaling and orphan nuclear receptor Nur77 expression . Toxicol. In Vitro 28 , 647 – 656 . S0887-2333(14)00025-3 [pii] 10.1016/j.tiv.2014.01.013. Google Scholar CrossRef Search ADS PubMed Liu S. , Li C. , Wang Y. , Hong T. , Song T. , Li L. , Ye L. , Lian Q. , Ge R. S. ( 2016a ). In utero methoxychlor exposure increases rat fetal Leydig cell number but inhibits its function . Toxicology 370 , 31 – 40 . S0300-483X(16)30215-3 [pii] 10.1016/j.tox.2016.09.009. Google Scholar CrossRef Search ADS Liu S. , Mao B. , Bai Y. , Liu J. , Li H. , Li X. , Lian Q. , Ge R. S. ( 2016b ). Effects of methoxychlor and its metabolite hydroxychlor on human placental 3beta-hydroxysteroid dehydrogenase 1 and aromatase in JEG-3 cells . Pharmacology 97 , 126 – 133 . 000442711 [pii] 10.1159/000442711. Google Scholar CrossRef Search ADS Martin L. J. , Boucher N. , Brousseau C. , Tremblay J. J. ( 2008 ). The orphan nuclear receptor NUR77 regulates hormone-induced StAR transcription in Leydig cells through cooperation with Ca2+/calmodulin-dependent protein kinase I . Mol. Endocrinol. 22 , 2021 – 2037 . me.2007-0370 [pii] 10.1210/me.2007-0370. Google Scholar CrossRef Search ADS PubMed Martin L. J. , Tremblay J. J. ( 2005 ). The human 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase type 2 promoter is a novel target for the immediate early orphan nuclear receptor Nur77 in steroidogenic cells . Endocrinology 146 , 861 – 869 . en.2004-0859 [pii] 10.1210/en.2004-0859. Google Scholar CrossRef Search ADS PubMed Mayr U. E. ( 1988 ). Estrogen-controlled gene expression in tissue culture cells by zearalenone . FEBS Lett. 239 , 223 – 226 . 0014-5793(88)80921-9 [pii]. Google Scholar CrossRef Search ADS PubMed Mehmood Z. , Smith A. G. , Tucker M. J. , Chuzel F. , Carmichael N. G. ( 2000 ). The development of methods for assessing the in vivo oestrogen-like effects of xenobiotics in CD-1 mice . Food Chem. Toxicol. 38 , 493 – 501 . S0278-6915(00)00022-3 [pii]. Google Scholar CrossRef Search ADS PubMed Muller H. M. , Reimann J. , Schumacher U. , Schwadorf K. ( 1997 ). Fusarium toxins in wheat harvested during six years in an area of southwest Germany . Nat. Toxins 5 , 24 – 30 . 10.1002/(SICI)(1997)5: 1< 24:: AID-NT4> 3.0.CO; 2-#. Google Scholar CrossRef Search ADS PubMed Nikov G. N. , Hopkins N. E. , Boue S. , Alworth W. L. ( 2000 ). Interactions of dietary estrogens with human estrogen receptors and the effect on estrogen receptor-estrogen response element complex formation . Environ. Health Perspect. 108 , 867 – 872 . sc271_5_1835 [pii]. Google Scholar CrossRef Search ADS PubMed O'Shaughnessy P. J. , Monteiro A. , Abel M. ( 2012 ). Testicular development in mice lacking receptors for follicle stimulating hormone and androgen . PLoS One 7 , e35136. 10.1371/journal.pone.0035136. Google Scholar CrossRef Search ADS PubMed Ottenweller J. E. , Li M.-T. , Giglio W. , Anesetti R. , Pogach L. M. , Huang H. F. S. ( 2000 ). Alteration of follicle-stimulating hormone and testosterone regulation of messenger ribonucleic acid for sertoli cell proteins in the rat during the acute phase of spinal cord injury . Biol. Reprod. 63 , 730 – 735 . 10.1095/biolreprod63.3.730. Google Scholar CrossRef Search ADS PubMed Payne A. H. , Wong K. L. , Vega M. M. ( 1980 ). Differential effects of single and repeated administrations of gonadotropins on luteinizing hormone receptors and testosterone synthesis in two populations of Leydig cells . J. Biol. Chem. 255 , 7118 – 7122 . Google Scholar PubMed Phillips D. M. , Lakshmi V. , Monder C. ( 1989 ). Corticosteroid 11β-dehydrogenase in rat testis . Endocrinology 125 , 209 – 216 . Google Scholar CrossRef Search ADS PubMed Rommerts F. F. , Teerds K. , Themmen A. P. , van Noort M. ( 1987 ). Multiple regulation of testicular steroidogenesis . J. Steroid Biochem. 27 , 309 – 316 . Google Scholar CrossRef Search ADS PubMed Rommerts F. F. , Teerds K. J. , Hoogerbrugge J. W. ( 1988 ). In vitro effects of ethylene-dimethane sulfonate (EDS) on Leydig cells: Inhibition of steroid production and cytotoxic effects are dependent on species and age of rat . Mol. Cell. Endocrinol. 55 , 87 – 94 . Google Scholar CrossRef Search ADS PubMed Sadovsky Y. , Crawford P. A. , Woodson K. G. , Polish J. A. , Clements M. A. , Tourtellotte L. M. , Simburger K. , Milbrandt J. ( 1995 ). Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids . Proc. Natl. Acad. Sci. U.S.A. 92 , 10939 – 10943 . Google Scholar CrossRef Search ADS PubMed Sandhoff T. W. , Hales D. B. , Hales K. H. , McLean M. P. ( 1998 ). Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1 . Endocrinology 139 , 4820 – 4831 . Google Scholar CrossRef Search ADS PubMed Schimmer B. P. , Tsao J. , Cordova M. , Mostafavi S. , Morris Q. , Scheys J. O. ( 2011 ). Contributions of steroidogenic factor 1 to the transcription landscape of Y1 mouse adrenocortical tumor cells . Mol. Cell. Endocrinol. 336 , 85 – 91 . 10.1016/j.mce.2010.11.024. Google Scholar CrossRef Search ADS PubMed Tashiro F. , Kawabata Y. , Naoi M. , Ueno Y. ( 1980 ). Zearalenone-Estrogen Receptor Interaction and RNA Synthesis in Rat Uterus ( Preuser H. J. , Ed), Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, IA, pp. 311 – 320 . Stuttgart : Fischer . Teerds K. J. , de Rooij D. G. , Rommerts F. F. , van den Hurk R. , Wensing C. J. ( 1989 ). Proliferation and differentiation of possible Leydig cell precursors after destruction of the existing Leydig cells with ethane dimethyl sulphonate: The role of LH/human chorionic gonadotrophin . J. Endocrinol. 122 , 689 – 696 . Google Scholar CrossRef Search ADS PubMed Tiemann U. , Danicke S. ( 2007 ). In vivo and in vitro effects of the mycotoxins zearalenone and deoxynivalenol on different non-reproductive and reproductive organs in female pigs: A review . Food Addit. Contam. 24 , 306 – 314 . 10.1080/02652030601053626. Google Scholar CrossRef Search ADS PubMed Wu X. , Guo X. , Wang H. , Zhou S. , Li L. , Chen X. , Wang G. , Liu J. , Ge H. S. , Ge R. S. ( 2017 ). A brief exposure to cadmium impairs Leydig cell regeneration in the adult rat testis . Sci. Rep. 7 , 6337. 10.1038/s41598-017-06870-0 10.1038/s41598-017-06870-0 [pii]. Google Scholar CrossRef Search ADS PubMed Yang J. , Zhang Y. , Wang Y. , Cui S. ( 2007 ). Toxic effects of zearalenone and alpha-zearalenol on the regulation of steroidogenesis and testosterone production in mouse Leydig cells . Toxicol. In Vitro 21 , 558 – 565 . S0887-2333(06)00238-4 [pii] 10.1016/j.tiv.2006.10.013. Google Scholar CrossRef Search ADS PubMed Yang Y. , Li Z. , Wu X. , Chen H. , Xu W. , Xiang Q. , Zhang Q. , Chen J. , Ge R. S. , Su Z. et al. , . ( 2017 ). Direct reprogramming of mouse fibroblasts toward Leydig-like cells by defined factors . Stem Cell Rep. 8 , 39 – 53 . S2213-6711(16)30272-7 [pii] 10.1016/j.stemcr.2016.11.010. Google Scholar CrossRef Search ADS Ye L. , Li X. , Li L. , Chen H. , Ge R. S. ( 2017 ). Insights into the development of the adult Leydig cell lineage from stem Leydig cells . Front. Physiol. 8 , 430. 10.3389/fphys.2017.00430. Google Scholar CrossRef Search ADS PubMed Zatecka E. , Ded L. , Elzeinova F. , Kubatova A. , Dorosh A. , Margaryan H. , Dostalova P. , Korenkova V. , Hoskova K. , Peknicova J. ( 2014 ). Effect of zearalenone on reproductive parameters and expression of selected testicular genes in mice . Reprod. Toxicol. 45 , 20 – 30 . 10.1016/j.reprotox.2014.01.003. Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. 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Toxicological SciencesOxford University Press

Published: Apr 16, 2018

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