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BBOP (CAS registry number 117-83-9) was purchased from Fusheng Industrial Co. (Shanghai, China). An Immulite 2000 total testosterone kit was obtained from Sinopharm (Hangzhou, China). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA). A reverse transcription kit and SYBR green real-time polymerase chain reaction (qPCR) kit were purchased from Takara (Otsu, Japan). A BCA protein assay kit was purchased from Beyotime (Shanghai, China). Primer information for qPCR detection is shown in Supplementary Table S1 (available in www.besjournal.com) and antibody information for immunohistochemistry and western blotting is shown in Supplementary Table S2 (available in www.besjournal.com).
Symbol Gene name Primer Sequences (5’to 3’) bp Accession No. Lhcgr Luteinizing hormone receptor Forward
ReverseCTGCGCTGTCCTGGCC
CGACCTCATTAAGTCCCCTGAA102 NM_012978 Star Steroidogenic acute regulatory protein Forward
ReverseCCCAAATGTCAAGGAAATCA
AGGCATCTCCCCAAAGTG187 NM_031558 Scarb1 Scavenger receptor class B member 1 Forward
ReverseATGGTACTGCCGGGCAGAT
CGAACACCCTTGATTCCTGGTA117 NM_031541 Cyp11a1 Cholesterol side chain cleavage Forward
ReverseAAGTATCCGTGATGTGGG
TCATACAGTGTCGCCTTTTCT127 NM_017286 Cyp17a1 17α-Hydroxylase/17,20-lyase Forward
ReverseTGGCTTTCCTGGTGCACAATC
TGAAAGTTGGTGTTCGGCTGAAG90 NM_012753 Hsd3b1 3β-Hydroxysteroid dehydrogenase 1 Forward
ReverseCCCTGCTCTACTGGCTTGC
TCTGCTTGGCTTCCTCCC189 NM_001007719 Hsd17b3 17β-Hydroxysteroid dehydrogenase 3 Forward
ReverseTGAAAGTTGGTGTTCGGCTGAAG
TGAAAGTTGGTGTTCGGCTGAAG202 NM_054007 Hsd11b1 11β-Hydroxysteroid dehydrogenase 1 Forward
ReverseGAAGAAGCATGGAGGTCAAC
GCAATCAGAGGTTGGGTCAT133 NM_017080 Sox9
FshrSRY-box transcription factor 9
Follicle-stimulating hormone receptorForward
Reverse
Forward
ReverseGCAGCGTGGGGTTGTG
TGGATGATTGGGATGGTCA
CAAAAGTCCAGCCCAATACC
AACCCCGACATAATCTTCA172
327NM_138547
NM_199237Dhh Desert hedgehog Forward
ReverseAACCCCGACATAATCTTCA
CTCGTCCCAACCTTCAGT150 NM_053367 Nr5a1 Nuclear receptor subfamily 5 group A1 Forward
ReverseCAGAGCTGCAAAATCGACAA
CCCGAATCTGTGCTTTCTTC187 NM_001191099 Insl3 Insulin-like 3 Forward
ReverseGTGGCTGGAGCAACGACA
AGAAGCCTGGTGAGGAAGC102 NM_053680 Sod1
Sod2
Cat
Gpx1
BCL2
BaxSuperoxide dismutase 1
Superoxide dismutase 2
Catalase
Glutathione peroxidase 1
BCL2 apoptosis regulator
BCL2 associated X apoptosis regulatorForward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
ReverseGCCGTGTGCGTGCTGAAGG
TGTAATCTGTCCTGACACCACAACTG
TCCCTGACCTGCCTTACGACTATG
TCGTGGTACTTCTCCTCGGTGAC
AGCGGATTCCTGAGAGAGTGGTAC
CTGTGGAGAATCGGACGGCAATAG
TGCAATCAGTTCGGACATCAGGAG
CTCACCATTCACCTCGCACTTCTC
AGCGTCAACAGGGAGATGTC
TATGCACCCAGAGTGATGCA
GACGCATCCACCAAGAAGCTGAG
GCTGCCACACGGAAGAAGACC99
130
147
129
204
134NM_017050.1
NM_017051.2
NM_012520.2
NM_030826.4
NM_016993
NM_017059Lhb Luteinizing hormone subunit beta Forward
ReverseCTGCTGCTGAGCCCAAGTGT TGCTGGTGGTGAAGGTGATG 400 NM_012858 Fshb Follicle stimulating hormone subunit beta Forward
ReverseCATTCACCCACCCTTGTCTT
GCTCCTCCTCACTACCTGTC326 NM_001007597.2 Gnrhr Gonadotropin releasing hormone receptor Forward
ReverseCTTGAAGCCCGTCCTTGG
GCGATCCAGGCTAATCAC440 NM_031038 Rps16
GapdhRibosomal protein S16
Glyceraldehyde-3-phosphate dehydrogenaseForward
Reverse
Forward
ReverseAAGTCTTCGGACGCAAGAAA
TTGCCCAGAAGCAGAACAG
GTCCATGCCATCACTGCCACTC
GATGACCTTGCCCACAGCCTTG148
132NM_001169146
NM_017008.4Table S1. qPCR primer information
Antibody Species Vendor (City, State) Dilution WB HS GAPDH Mouse Cell Signaling Technology (Danvers, MA) 1:1,000 ND CYP11A1 Rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 1:200 HSD11B1 Rabbit Abcam (San Francisco, CA) 1:1,000 1:200 INSL3 Rabbit Abcam (San Francisco, CA) 1:1,000 ND DHH Mouse Santa Cruz Biotechnology (Dallas, TX) 1:100 ND pmTOR rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND mTOR rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND ERK1/2 mouse Cell Signaling Technology (Danvers, MA) 1:1,000 ND pERK1/2 mouse Cell Signaling Technology (Danvers, MA) 1:5,000 ND AKT1 Rabbit Cell Signaling Technology (Danvers, MA) 1:2,000 ND pAKT1 Rabbit Cell Signaling Technology (Danvers, MA) 1:5,000 ND AKT2 Rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND pAKT2 Rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND BAX Rabbit Abcam (San Francisco, CA) 1:1,000 ND BCL2 Rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND SOD1 Rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND SOD2 Rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND PGC1α Rabbit Abcam (San Francisco, CA) 1:1,000 ND SIRT1 Rabbit Cell Signaling Technology (Danvers, MA) 1:1,000 ND Note. ND = Not detected; WB = Western blot; HS = Histochemical staining. Table S2. Antibody information
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Male Sprague-Dawley rats [28 days of age (post weaning)] were from the Shanghai Laboratory Animal Center (Shanghai, China). All animal procedures were approved by the Ethics Committee of Animal Care and Use of Wenzhou Medical University (protocol number: wydw2014-0057). Rats were raised in clear polycarbonate cages under the following conditions: a 12-h dark/12-h light cycle, temperature range of 21–25 °C, and a humidity range of 45%–55%. After 7 days of acclimation, the rats were 35 days of age. Thirty rats were randomly assigned to five groups (with 6 animals per group) receiving BBOP at 0 (corn oil as control vehicle), 10, 100, 250, or 500 mg/kg bw per day. The dosage range was chosen on the basis of a previous study on the developmental/reproductive toxicity of BBOP, which induced abnormal changes in the morphology of fetal Leydig cells after in utero exposure through gavage for 8 days at doses as high as 500 mg/kg bw per day[8]. Different doses of BBOP were prepared by dissolving BBOP in corn oil and were administered at a rate of 2 mL/(kg·day). The administration duration (from PND 35 to 56) spanned the pubertal development of Leydig cells in rats[10]. Rats were weighed daily. At the end of administration, the rats were euthanized by CO2 asphyxiation. The testis and epididymis were harvested and weighed. Pituitary glands were collected. One testis, one epididymis, and the pituitary gland were stored at –80 °C. The other testis was immersed in Bouin’s fixative. Trunk blood was harvested for the collection of serum.
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An Immulite 2000 total testosterone kit was used to measure serum testosterone concentrations at the Department of Medical Chemistry of our hospital, as previously documented[18].
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Enzyme linked immunosorbent assay kits for rat LH or FSH were used to detect rat serum LH and FSH concentrations, as previously documented[19]. We prepared samples and standards in pre-coated anti-LH or anti-FSH antibody plates and added biotinylated anti-LH or anti-FSH solution, and then peroxidase-conjugated IgG solutions to the plates. After the detection substrate was added, the plates were read at 450 nm within 30 min. The lower detection limit of LH and FSH was 0.1 mIU/mL; the detection was within the linear range, and the co-variation in detection of LH and FSH was within 10%.
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For equivalent detection of the pixel density of each antigen, the testis was prepared as a tissue array according to a previously described protocol[19]. The testis was cut into pieces and dehydrated in ethanol and xylene. The dehydrated pieces were embedded in paraffin, and 1.5 mm2 of testis tissue was randomly selected from each sample and assembled in a tissue array mold holding 30 samples (6 per group). The cross section (6 μm) was cut. Ten slides were randomly collected for immunohistochemistry analysis with an immunohistochemical kit (Vector Laboratories, Inc., Burlingame, CA) as previously described[19]. Each slide was boiled at 60 °C for 2 h, de-waxed, and rehydrated with xylene and alcohol. Peroxidase activity was blocked with 3% hydrogen peroxide. Antigens were repaired in citric acid, and the slides were blocked with 10% goat serum for 30 min. CYP11A1, which is expressed in all cells of the Leydig cell lineage[9]; HSD11B1, which is expressed in mature Leydig cells[20]; and SOX9, which is expressed in Sertoli cells[21], were selected as biomarkers for immunohistochemical staining. Anti-CYP11A1, anti-HSD11B1, and anti-SOX9 primary antibodies and subsequently conjugated secondary antibody were incubated with the slides at 4 °C. Diaminobenzidine was added for color development. The sections were counterstained with Mayer hematoxylin. The slides were scanned with a NanoZoomer-XR (Hamamatsu, Japan) at 0.23 μm per pixel to generate digital images.
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The digital images were displayed in Image-Pro Plus software version 7 (Media Cybernetics, Silver Spring, MD). Cells with positive staining were picked, and Leydig cells and Sertoli cells were identified. The number of CYP11A1-positive Leydig cells, HSD11B1-positive Leydig cells, and SOX9-positive Sertoli cells was calculated through stereological techniques as previously described[22].
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Leydig cell size increases with maturity[23]. The Leydig cell morphological metrics (cell size, nuclear size, and cytoplasmic area) were calculated as previously described [24]. Immunohistochemical staining for Leydig cells was performed. The digital images were displayed in Image-Pro Plus software. The peripheral contours of a Leydig cell and its nucleus were drawn, and the surface areas of the Leydig cell and its nucleus were exported into a Microsoft Excel spreadsheet by the software. The cytoplasmic size was calculated by subtraction of the nuclear area from the Leydig cell size. Fifty Leydig cells were randomly selected, and the mean value of each sample was calculated.
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Total RNAs in testis and pituitary samples were prepared with a TRIzol extraction kit, as previously described[25]. The concentration of total RNA was determined by the optical density (OD) value at 260 nm with a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, CA). The purity of total RNA was calculated according to the OD260/OD280 ratio. Total RNA was used to prepare cDNA templates through reverse transcription[25]. The expression of each gene was measured by qPCR as described previously[19]. The following genes were analyzed. Leydig cell genes: 1) a growth factor (Insl3), 2) receptors (Lhcgr and Scrab1), 3) a cholesterol transport regulator (Star), 4) androgen synthases (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3), 5) a glucocorticoid steroidogenic enzyme (Hsd11b1), and 6) a transcription factor (Nr5a1); Sertoli cell genes: 1) a growth factor (Dhh), 2) a receptor (Fshr), and 3) a transcription factor (Sox9); and antioxidant enzyme genes: Sod1, Sod2, Gpx1, and Cat; anti-apoptotic and pro-apoptotic genes: 1) an anti-apoptosis gene (Bcl2) and 2) a pro-apoptosis gene (Bax); pituitary genes: 1) hormones (Lhb and Fshb), and 2) a receptor (Gnrhr); and internal house-keeping genes: Rps16 and Gapdh. After qPCR, Ct values were exported into an Excel spreadsheet for standards and samples, and the expression levels of the target genes were determined according to the standard curve as previously described[25]. No statistical difference in Rps16 or Gapdh was observed between groups. The gene Rps16 was finally selected for normalization [19].
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Western blotting was used for the detection of testicular proteins as previously described[19]. Briefly, tissue lysate was prepared, and protein was quantified with a BCA kit. An aliquot of protein (30 μg) from each sample was added to a 10%–12% PAGE gel, and electrophoresis was performed. The proteins were electrically transferred onto a nitrocellulose membrane. The membranes were subjected to western blotting to detect the following proteins: CYP11A1, HSD11B1, INSL3, DHH; mTOR, AKT1, AKT2, ERK1/2, GSK3β, phosphorylated mTOR (pmTOR), phosphorylated AKT1 (pAKT1), phosphorylated AKT2 (pAKT2), phosphorylated GSK3β (pGSK3β), phosphorylated ERK1/2 (pERK), SIRT1, and GAPDH (an internal control). Enhanced chemiluminescence was detected with an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). The protein density was quantified in Image-Lab (Bio-Rad), and GAPDH was used for normalization.
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Semi-quantitative immunohistochemical measurement of protein density was performed as previously described[26]. In brief, the digital images were displayed in Image-Pro plus software, and the protein was selected, and a nearby negative background area was selected for subtraction. The pixel density of CYP11A1 and SOX9 was exported by the software to determine the protein levels in individual cells. Fifty pairs of densities per sample were measured and calculated for mean value as a sample size. Optical density (in arbitrary units) represents the ratio of IOD/area for proteins.
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MDA is a decomposition product of tri-unsaturated fatty acid hydroperoxides and is a biomarker of ROS[27]. The amount of MDA in testicular homogenates was measured with a kit (Solarbio Science and Technology Co, Beijing, China) as previously described[27]. In brief, 100 mg of testicular tissue was prepared for homogenization in the extraction solution. The solution was centrifuged at 8,000 ×g in a 4 °C centrifuge for 10 min. The supernatant was used for measurement of MDA with the kit. The plates were read at 450, 532, and 600 nm with a Bio-Tek microplate reader (Shanghai, China). The amount of MDA (mmol/g testis) was calculated with the following Formula: 5 × (12.9 × (ΔA532 – ΔA600) – 2.58 × Δ450)/0.1.
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To study the direct effects of BBOP, we used 35-day-old male Sprague Dawley rats to purify Leydig cells, as previously described[9]. The testes were removed and perfused with a collagenase D solution (0.1 mg/mL) through the testicular artery. The testis was further digested with collagenase D (0.25 mg/mL) and DNase (0.25 mg/mL). The cell suspension was filtered with a 100 μm nylon mesh and centrifuged through a Percoll gradient, and cells in the density range of 1.070–1.088 g/mL were harvested. HSD3B1 staining was performed as previously described to determine the purity of Leydig cells[28]. Leydig cells were transferred to a 12-well culture plate at a cell density of 1.0 × 106 cells/well, and 1 mL of DMEM/F12 medium was added to the well. After 24 h, the cells attached to the bottom of the well, and the medium was switched to DMEM/F12 medium containing BBOP (0, 50, 100, and 500 μmol/L) for another 24 h. The highest concentration, 500 µmol/L, was water soluble.
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ROS levels were measured with a DCFH-DA test kit (Solarbio, Beijing, China), as previously described[29]. In brief, after Leydig cells were harvested, they were incubated with DCFH-DA in a dark room at 37 °C for 20 min, and then the fluorescence intensity was measured in a flow cytometer for ROS. The apoptosis rate was measured with an Annexin V-FITC/PI Apoptosis Detection Kit (MultiSciences Biotech Co., Hangzhou, China) as previously described[29]. Leydig cells were incubated with FITC-labeled Annexin V and PI. The apoptotic rate was measured with a flow cytometer.
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Data are expressed as mean ± standard error (SEM). GraphPad 8 (San Diego, CA) was used for statistical analysis and graphing. Statistical evaluation was performed with one-way analysis of variance followed by Dunnett’s post hoc multiple comparison test. The significance level was set as P < 0.05.
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Rats were exposed to BBOP (0, 10, 100, 250, or 500 mg/kg bw per day) through daily gavage during puberty (PND 35 to PND 56, Figure 1A). Compared with the control, exposure to BBOP for 21 days did not affect body weight or testis weight in rats at PND 56. BBOP significantly decreased the weight of the epididymis at doses of 250 (P < 0.05) and 500 (P < 0.01) mg/kg (Table 1). No mortality was observed in any rat group, and no abnormal behavior was observed.
Figure 1. Regimen and serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels after BBOP exposure in puberty. Panel A, BBOP (0, 10, 100, 250, or 500 mg/kg bw per day) was administered via gavage; Panels B–D, serum T, LH, and FSH levels, respectively. Mean ± SEM, n = 6; *P < 0.05 and **P < 0.01 in the BBOP group versus control (0 mg/kg).
Parameters BBOP [mg/(kg·day)] 0 10 100 250 500 Number 6 6 6 6 6 mortality (%) 0 0 0 0 0 BW (g) before 213.3 ± 9.3 218.3 ± 8.2 211.8 ± 11.8 204.3 ± 8.0 218.0 ± 4.8 BW (g) after 316.8 ± 20.2 321.7 ± 24.4 335.0 ± 14.4 317.5 ± 14.2 304.3 ± 18.3 Testis weight (g) 1.55 ± 0.08 1.45 ± 0.11 1.53 ± 0.10 1.53 ± 0.10 1.47 ± 0.09 Relative testis weight (g) 1.54 ± 0.07 1.42 ± 0.14 1.543 ± 0.10 1.543 ± 0.10 1.479 ± 0.07 Both epididymis weight (g) 0.81 ± 0.07 0.73 ± 0.04 0.76 ± 0.05 0.72 ± 0.05* 0.70 ± 0.03** Note. BW = body weight. Values are mean ± SEM, n = 6. *, **Indicate significant difference when compared to the control [0 mg/(kg·day) BBOP] at P < 0.05 and 0.01, respectively. Table 1. General parameters of toxicity after treatment of BBOP in puberty
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BBOP affected serum testosterone levels, which were significantly lower than those in the control groups after treatment with BBOP at 250 and 500 mg/kg bw per day (P < 0.05, Figure 1B). Therefore, BBOP delays puberty onset by inhibiting testosterone biosynthesis. To check whether BBOP affects the development of Leydig cells by acting on the pituitary in the hypothalamus-pituitary testis axis, we measured serum LH and FSH levels. BBOP at 500 mg/kg bw per day significantly decreased the levels of LH (P < 0.01, Figure 1C) and FSH (P < 0.05, Figure 1D), mg/kg bw per day thus indicating that BBOP at the highest dose inhibited gonadotropin secretion.
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Sertoli cells were stained with anti-SOX9 antibody, which detected SOX9, a biomarker of Sertoli cells[30]. Compared with the control, BBOP did not alter the number of SOX9+ Sertoli cells (Supplementary Figure S1, available in www.besjournal.com). Because Sertoli cells do not proliferate after PND 21[31], the unaltered Sertoli cell number after BBOP treatment indicated that BBOP did not induce Sertoli cell apoptosis. CYP11A1, a steroidogenic enzyme in all cells in the Leydig cell lineage[9], is used as a universal biomarker to detect all Leydig cells. HSD11B1 is a steroidogenic enzyme in mature Leydig cells [32] that is used as a specific biomarker to detect Leydig cell maturity. BBOP exposure at 500 mg/kg bw per day markedly decreased the number of CYP11A1+ Leydig cells mg/kg bw per day (P < 0.01, Figure 2). However, no significant difference was observed in HSD11B1+ Leydig cells between the BBOP groups and the control (Supplementary Figure S2, available in www.besjournal.com). Therefore, BBOP affects the number of CYP11A+/HSD11B1- progenitor Leydig cells.
Figure 2. Immunohistochemical staining of CYP11A1 and the number of Leydig cells (LC). The LC number was normalized to that of Sertoli cells (SC). Panels A–E, CYP11A1 staining of the 0, 10, 100, 250, and 500 mg/kg bw per day BBOP groups, respectively. Black arrow indicates CYP11A1. Scale bar = 20 μm. Panel F, quantitative number. Mean ± SEM, n = 6. **P < 0.01 in the BBOP group versus the control (0 mg/kg).
Figure S1. Immunohistochemical staining of SOX9 and Sertoli cell (SC) number in the testis after BBOP exposure from postnatal day (PND) 35 to 56 Panels A–E, SOX9 staining for 0, 10, 100, 250, and 500 mg/kg BBOP group, respectively; Black arrow points to SOX9 staining; Scale bar = 20 μm; Panel F, quantitation of SC number; Mean ± SEM, n = 6. There was no significant difference in the BBOP group versus the control (0 mg/kg).
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The expression of Leydig cell genes (Insl3, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Nr5a1) and Sertoli cell genes (Dhh, Fshr, and Sox9) was determined by qPCR. BBOP significantly decreased the transcript levels of Cyp11a1 at doses of 10 mg/kg or higher (P < 0.01), and the mRNA levels of Insl3, Hsd11b1, and Dhh at a dose of 500 mg/kg bw per day (P < 0.05, Figure 3), thus indicating that BBOP interferes with the expression of some Leydig cell and Sertoli cell genes. Because BBOP decreased the levels of serum LH and FSH, we asked whether BBOP might affect the expression of the pituitary genes Lhb, Fshb, and Gnrhr in the pituitary gland. BBOP markedly down-regulated the expression of Lhb and Fshb at doses of 10 mg/kg and above (P < 0.01), whereas it up-regulated the expression of Gnrhr at doses of 250 and 500 mg/kg bw per day (P < 0.001, Figure 3), thereby indicating that BBOP interferes with pituitary gene expression.
Figure 3. Gene expression in the testis and pituitary gland after BBOP exposure. The expression of the testis genes Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Nr5a1, Sox9, Dhh, and Fshr and of the pituitary genes Gnrhr, Lhb, and Fshb (in the dotted box) was determined by qPCR. The mRNA levels were normalized to those of Rsp16 (internal control). Mean ± SEM, n = 6. *P < 0.05, **P < 0.01 and ***P < 0.001 in the BBOP group versus the control (0 mg/kg).
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Protein levels of INSL3, CYP11A1, HSD11B1, and DHH in the testis were detected by western blotting. BBOP significantly decreased CYP11A1 levels at doses of 100 mg/kg (P < 0.05) and above (P < 0.01, Figure 4A–B), and significantly decreased the levels of INSL3, HSD11B1, and DHH at a dose of 500 mg/kg bw per day (P < 0.05, Figure 4A–B). The density of CYP11A1 and SOX9 represented the protein levels in individual cells. Semi-quantitative immunohistochemical staining used to calculate CYP11A1 and SOX9 pixel density indicated that BBOP significantly decreased CYP11A1 density at doses of 100 mg/kg or above (P < 0.001, Figure 5) without affecting SOX9 density (Supplementary Figure S3, available in www.besjournal.com), in agreement with the western blot data.
Figure 4. Protein levels in the testis after BBOP exposure. Panel A, western blotting image. Panel B, quantitative results for CYP11A1, HSD11B1, INSL3, and DHH, normalized to GAPDH (the internal control). Mean ± SEM, n = 3–4. *P < 0.05 and **P < 0.01, in the BBOP group versus the control (0 mg/kg).
Figure 5. Semi-quantitative immunohistochemical measurement of CYP11A1 density and Leydig cell (LC) morphological metrics. Panels A–E, CYP11A1 staining for 0, 10, 100, 250, and 500 mg/kg bw per day BBOP, groups, respectively. Black arrow indicates CYP11A1 staining; scale bar = 20 μm. Panel F, CYP11A1 density. Panels G–I, LC size, nuclear size, and cytoplasm size. Mean ± SEM, n = 6. *P < 0.05 and ***P < 0.001 in the BBOP group versus the control (0 mg/kg).
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More mature Leydig cells have larger cell sizes [23]. Both the 250 and 500 mg/kg bw per day doses of BBOP significantly decreased the Leydig cell size and cytoplasmic size (P < 0.05, Figure 5) without affecting nuclear size. Thus, the maturation of Leydig cells is delayed after exposure to BBOP in puberty.
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We measured the expression of antioxidant genes (Sod1, Sod2, Gpx1, and Cat), an anti-apoptotic gene (Bcl2), and a pro-apoptotic gene (Bax). BBOP significantly down-regulated the expression of Sod1 and Bcl2 at a dose of 250 (P < 0.05) or 500 (P < 0.01) mg/kg, and the expression of Sod2 at a dose of 500 mg/kg bw per day (P < 0.01), but up-regulated Bax expression at a dose of 500 mg/kg bw per day (P < 0.01, Figure 6). MDA is the end product of lipid peroxidation caused by excessive ROS. MDA was significantly greater in the 250 (P < 0.01) and 500 (P < 0.001) mg/kg bw per day groups than the control group (Figure 6B). We further detected the protein levels of SOD1, SOD2, BCL2, and BAX. BBOP treatment significantly decreased SOD1, SOD2, and BCL2 levels at a dose of 250 (P < 0.05) and 500 (P < 0.01) mg/kg, whereas it markedly elevated BAX levels at a dose of 100 mg/kg (P < 0.05) or higher (P < 0.001, Figure 6C).
Figure 6. Expression of antioxidant enzymes, apoptosis-associated genes, and malondialdehyde (MDA) amounts after BBOP exposure. Panel A, expression of Sod1, Sod2, Gpx1, Cat, Bax, and Bcl2 in the testis, normalized to Rsp16. Panel B, testicular level of MDA. Panel C, western blotting image. Panel D, quantitative results of antioxidant enzymes and apoptosis-associated proteins. Mean ± SEM, n = 6 for mRNAs and MDA and n = 3 for western blotting. *P < 0.05, **P < 0.01, and ***P < 0.001 in the BBOP group versus the control (0 mg/kg).
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We measured the SIRT1 levels and total protein levels of AKT1, AKT2, ERK1/2, mTOR, GSK3β, and their phosphorylated proteins by western blotting. BBOP at 250 (P < 0.05) and 500 mg/kg bw per day significantly decreased SIRT1 levels mg/kg bw per day (P < 0.01, Figure 7). BBOP did not affect total protein levels of AKT1, AKT2, ERK1/2, mTOR, and GSK3β (Figure 7). However, BBOP at 10 mg/kg or higher doses (P < 0.01) significantly decreased pAKT1 levels; consequently, the pAKT1/AKT1 ratio decreased at these doses (P < 0.01). The pAKT2, pERK1/2, and pmTOR levels decreased at a BBOP dose of 500 mg/kg bw per day (P < 0.05), thereby decreasing the pAKT2/AKT2, pERK/ERK1/2, and pmTOR/mTOR ratios (P < 0.05). The pGSK3β levels decreased at BBOP doses of 100 mg/kg or higher (P < 0.01), thus significantly decreasing the pGSK3β/GSK3β ratios (P < 0.01, Figure 7).
Figure 7. SIRT1, AKT1, AKT2, ERK1/2, mTOR, and GSK3β and their phosphorylated protein levels in the rat testis. Panel A, western blotting image. Panels B–G: quantification of AKT1, AKT2, ERK1/2, mTOR, and GSK3β, and their phosphorylated proteins, as well as SIRT1. The protein levels were normalized to those of GAPDH (internal control). Mean ± SEM, n = 3. *P < 0.05 and **P < 0.01 in the BBOP group versus the control (0 mg/kg).
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We cultured Leydig cells from 35-old rats in the presence of various concentrations (50–500 μmol/L) of BBOP. We measured ROS production by cytology with a DCFH-DA kit and apoptosis of Leydig cells with an Annexin V/PI kit at the end of treatment. BBOP significantly induced ROS production at doses of 100 μmol/L (P < 0.01) and 500 μmol/L (P < 0.001, Figure 8). BBOP also markedly increased the apoptotic rate at doses of 100 μmol/L (P < 0.05) and 500 μmol/L (P < 0.001, Figure 8). These results indicated that BBOP directly induces ROS and apoptosis in primary Leydig cells.
Figure 8. Effects of BBOP on reactive oxygen species (ROS) and apoptosis in Leydig cells from 35-day-old rats. Leydig cells were cultured for 24 h. Panel A: ROS fluorescence spectrum. Panel B: quantification of ROS levels. Panel C: apoptosis spectrum. Panel D: quantification of the apoptosis rate. Mean ± SEM, n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 in the BBOP group versus the control (0 μmol/L).
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Reagents
Animals and Experimental Design
Measurement of Serum Testosterone Concentrations
Measurement of Serum LH and FSH Levels
Immunohistochemistry
Counting Leydig Cell and Sertoli Cell Numbers
Measurement of Leydig Cell Morphological Metrics
Total RNA Purification and qPCR
Western Blotting
Semi-quantitative Immunohistochemical Measurement of CYP11A1 and SOX9 Density
Detection of MDA Content in Testicular Homogenates
Isolation and Culture of Leydig Cells
Measurement of ROS by DCFH-DA and Apoptosis by Annexin V/PI
Statistical Analysis
Weights of the Body, Testis, and Epididymis after BBOP Exposure
BBOP Decreases Serum Testosterone Levels in Vivo
BBOP Does not affect Sertoli Cell Number but affects the Number of CYP11A1+Leydig Cells in Vivo
BBOP affects Gene Expression in Leydig and Sertoli Cells and the Pituitary Gland
BBOP Interferes with Protein Levels in Leydig and Sertoli Cells
BBOP Decreases Leydig Cell Size and Cytoplasmic Size
BBOP Induces ROS and Interferes with the Expression of Antioxidant- and Apoptosis-Associated Genes in Vivo
BBOP Alters Several Pathways in Vivo
BBOP induces ROS and Apoptosis in Primary Leydig Cells
22068Supplementary Materials.pdf |