Perfluorododecanoic acid blocks rat Leydig cell development during

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Perfluorododecanoic acid blocks rat Leydig cell development during prepuberty Yong Chen, Huitao Li, Jiaying Mo, Xiuxiu Chen, Keyang Wu, Fei Ge, Leikai Ma, Xiaoheng Li, Xiaoling Guo, Junzhao Zhao, and Ren-Shan Ge Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00241 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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Chemical Research in Toxicology

Perfluorododecanoic acid blocks rat Leydig cell development during prepuberty Yong Chen 1,a, Huitao Li 1,a, Jiaying Mo 1,a, Xiuxiu Chen 1, Keyang Wu 1, Fei Ge 1, Leikai Ma 2, Xiaoheng Li 1, Xiaoling Guo 2,*, Junzhao Zhao 2,*, Ren-Shan Ge 1,2,* 1

Department of Anesthesiology, 2 Department of Fertility Center, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xueyuan West Road, Wenzhou, Zhejiang 325027, China a

These authors contributed to this work equally.

*Correspondence: Ren-shan Ge, Department of Anesthesiology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xueyuan West Road, Wenzhou, Zhejiang 325027, China, Email [email protected] *Correspondence: Department of Anesthesiology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xueyuan West Road, Wenzhou, Zhejiang 325027, China, Xiaoling Guo, [email protected] *Correspondence: Junzhao Zhao, Department of Obstetrics and Gynecology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xueyuan West Road, Wenzhou, Zhejiang 325027, China, Email [email protected]

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Abstract Perfluorododecanoic acid (PFDoA) has been used as a surfactant and may have reproductive toxicity. However, whether PFDoA influences Leydig cell development during prepuberty remains unknown. In the present study, 21-day-old male Sprague Dawley rats were gavaged 0, 5 or 10 mg/kg PFDoA from postnatal day 21 to 35. PFDoA decreased the serum concentrations of testosterone, luteinizing hormone, and follicle-stimulating hormone at doses of 5 and 10 mg/kg without influencing Leydig cell number and proliferation. However, PFDoA down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, and Hsd11b1) or their proteins. PFDoA dose-dependently reduced SIRT1 and PGC-1 levels. PFDoA did not affect AMPK and AKT2 levels, but decreased their phosphorylation. We also treated primary progenitor Leydig cells purified from prepubertal rat testes with PFDoA for 24 h. It in vitro lowered viability and decreased mitochondrial membrane potential of progenitor Leydig cells, but it stimulated the generation of the intracellular reactive oxygen species (ROS) and induced Leydig cell apoptosis at 10 M. In conclusion, PFDoA blocks rat Leydig cell development during the prepubertal period possibly via targeting AMPK/SIRT1/PGC-1 and AKT2 signaling pathways.

Keywords: Leydig cells; perfluorododecanoic acid; development; steroidogenesis; ROS; Apoptosis; Mitochondrial membrane potential

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Introduction Perfluorododecanoic acid (PFDoA) is one compound of the perfluorinated chemicals. It is used in commercial and industrial products as a surfactant and lubricant in paper wrapping, textile manufacturing, food packaging, and fire retarding because it possesses unique characteristics of surface activity 1. Heavy use of perfluorinated chemicals causes extensive distribution in the food chain and wildlife, and human can expose to these chemicals via food package and foods and they are detectable in human blood samples

2, 3.

PFDoA was also detectable in the milk of

humans and serum samples with the highest concentrations of 9.7 and 22 pg/ml, respectively 4, 5. PFDoA exposure might cause nephrotoxicity and liver toxicity and alter gene expression in the metabolic pathways 6, 7, 8. Furthermore, several studies also claimed that PFDoA had reproductive toxicity via blocking androgen secretion in the testis and down-regulating the expression of steroidogenic genes

9, 10.

However, the exact

mechanisms for PFDoA-mediated reproductive toxicity require further investigation. Leydig cells are the specific testicular cells that exist in testicular interstitial compartment and secrete 95–99% of circulatory testosterone 11. Male sexual maturity depends upon the developmental progress of the Leydig cells at the pubertal phase. In rats, progenitor Leydig cells emerge on postnatal day 21, and their progeny continues to differentiate such that by 35 days postpartum, progenitor Leydig cells differentiate to round cells and form a population of Leydig cells referred as immature Leydig cells. The prepubescent period, between postnatal day 21 and 35, is a very critical

4


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period for amplification of Leydig cell numbers and initial differentiation of Leydig cells

12, 13.

In the current study, we interrogated whether PFDoA influences Leydig

cell development and dissected its mechanism(s). Materials and Methods Chemicals PFDoA was obtained from J&K Scientific Ltd (Beijing, China). Collagenase D, DNase, and Percoll were obtained from Sigma-Aldrich (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kits for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were purchased from Chemicon (Emecula, CA). DMEM/F12

media

were

obtained

from

Gibco

(Grand

Island,

NY).

2’7’-Dichlorofluorescin diacetate (DCFH-DA), a fluorescence dye, was purchased from Qcbio Science & Technologies (Shanghai, China). Immunohistochemical staining kit came from Vector Laboratories (Burlingame, CA). Animals Twenty-four male Sprague Dawley rats (ages of 14 days) were from Shanghai Laboratory Animal Center (Shanghai, China). The Wenzhou Medical University's Animal Care and Use Committee approved this study and we performed the animal study by complying with the Guide for the Care and Use of Laboratory Animals. Animal experiment Rats were fed in a 12-h dark-light cycle and temperature of 23 ± 2 oC and relative humidity of 45-55%. After acclimating for one week, these young rats were randomly divided into three groups: control (0 mg/kg/day), low dose of PFDoA (5 mg/kg/day),

5



and high dose of PFDoA (10 mg/kg/day) (eight animals a group). PFDoA was suspended in the vehicle (3% Tween 20) 14. Rats were daily administered orally 0, 5 or 10 mg/kg PFDoA. We selected this dosage range of PFDoA based on a previous study

15.

After 14 days, rats were killed by CO2 on postnatal day 35. We

weighed each testis and collected serum samples for testosterone, LH, and FSH assays. We collected one testis for immunohistochemical analysis after Bouin's solution fixation and the other testis for gene and protein expression analysis after freezing. Testosterone measurement We detected serum testosterone concentrations using a commercial RIA kit (IBL, USA) as described previously

16.

This kit has the lowest testosterone concentration

detection limit of 5 pg/ml. Inter-assay variation of testosterone was within 15%. ELISA for serum LH and FSH levels Serum levels of LH and FSH hormones were measured by commercial ELISA kits as previously described 17. Briefly, serum sample and an assay diluent were put to a 96-well plate and the mixture was incubated for 2 h at room temperature. After the washing step, peroxidase-conjugated IgG anti-LH or anti-FSH antibody was transferred to the mixture. After 2-h reaction and washing, each LH or FSH substrate solution was added and the reaction was processed for 30 min and then stopped. The plate was subjective to the detection of LH or FSH at a wavelength of 450 nm. Progenitor Leydig cell isolation Eighteen male Sprague-Dawley rats (21 days of age) were killed by asphyxiation

6


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with CO2. Progenitor Leydig cell isolation procedure was adopted according to a previous study

16.

Briefly, we digested the testis with the collagenase and DNase,

removed the tissue impurity through 100 μm nylon mesh and separated the cells after a centrifugation of Percoll gradient at a higher speed. Cell fraction between 1.07-1.088 g/ml was aspirated into a new tube. Leydig cell purity was performed using a histochemical staining of 3-hydroxysteroid dehydrogenase 1 (HSD3B1) as previously described 18. Progenitor Leydig cells (95% purity) was obtained. Progenitor Leydig cell culture Cells were seeded at a density of a million cells per well into a 6-well culture plate. Leydig cells were incubated with 0, 0.1, 1, and 10 μM PFDoA. PFDoA was dissolved in ethanol with its final concentration of 0.1%. The PFDoA concentration range was selected based on the in vivo study, in which PFDoA was found to easily get into the blood with serum level of 24 g/ml (39 M) after a single oral exposure to 50 mg/kg PFDoA

19.

The collected cells were used to detect cell viability,

apoptosis, ROS, and m levels. CCK-8 assay Cell kit-8 (CCK-8) was purchased to detect cell viability. Briefly, 100 μl of 104 cells/ml progenitor Leydig cells were added into a 96-well plate and incubated for 24 h. Then, cells were treated with a range of (0.1, 1 and 10 μM) of PFDoA for 24 h at 37 °C. Subsequently, 10 μl CCK-8 was added and the plate was incubated for 4 h at 37 °C. The absorbance at 450 nm was measured with a microplate reader (Thermo, MA).

7



Annexin V and PI assay Cells at a density of 106 cells per well were added into a 6-well culture plate and cultured for 24h and then treated with a range (0, 0.1, 1 and 10 µM) of PFDoA for 24 h. We applied the Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences) to judge early and lately apoptosis. Briefly, after washing step, cells were resuspended in the annexin V-binding buffer and stained with FITC-labeled Annexin V and PI for 15 min and were subjective to the Flow Cytometer (BD FACSAria, Rockville, MD) for analysis. Measurement of cellular ROS ROS levels were detected using indirect measurement with DCFH-DA assay kit according to the manufacturer's instructions. Briefly, cells (106 cells) were pippetted into a 6-well plate and cultured for 24h and then treated with a range of (0, 0.1, 1, and 10 µM) PFDoA for 24 h. Cells were incubated with DCFH-DA in dark room for 20 min at 37°C and then subjective to the detection of fluorescence intensity in the Flow Cytometer. Measurement of mitochondrial membrane potential (m) A JC-1 m assay kit was used according to manufacturer’s instruction. Briefly, cells (106 cells) were pippetted and cultured for 24h, and then treated with PFDoA (0, 0.1, 1 and 10 µM) for 24 h. After a washing step, cells were incubated with JC-1 (5 mM) for 30 min and subjective to the detection of fluorescence emissions in the flow cytometry. m was calculated by red/green fluorescence ratio.

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Immunohistochemical staining of testis Testis fixation, tissue-array preparation, and immunohistochemical staining were performed as previous study

17.

In brief, after embedding, fixation of paraffin and

dehydration, testis was subjective to sectioning at 6 m. The sections were subjective to the antigen display in the microwave heating and then endogenous peroxidase blockade at 0.5% H2O2. Two Leydig cell biomarkers, cholesterol side chain cleavage enzyme (CYP11A1) and 11-hydroxysteroid dehydrogenase 1 (HSD11B1) were selected to identify Leydig cells. One Sertoli cell biomarker SOX9 was selected to identify Sertoli cells 20. CYP11A1, HSD11B1 or SOX9 polyclonal antibody diluted at 1:200 was used. Diaminobenzidine was selected to identify antigen-antibody complex (brown staining). Immunofluorescent staining of PCNA in the cells was performed for investigating cell proliferation. Sections were sequentially treated with CYP11A1 and PCNA antibody for each 90 min at room temperature. After washing, fluorescent secondary antibody (Alexa-conjugated anti-rabbit IgG, 1:500; Alexa-conjugated anti-rat IgG, 1:500) to added, and then the sections were co-stained with DAPI. Sections were subjective to the detection of fluorescence in a microscope (Olympus, Japan). Counting Leydig and Sertoli cell number by a stereological method We performed a fractionator method to enumerate each cell types according to a previous study

21.

Each testis of a rat was sampled about ten testis sections. The

histochemical staining was executed as above. We multiplied the number of cells

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counted in a known fraction of the testis by the reciprocal of the sampling probability to calculate total number of cells. Real-time PCR (RT-qPCR) Total RNA extraction was done using a TRIzol kit (Invitrogen, CA). The concentration of total RNA was read at 260 nm. RNAs were reversely transcribed into the cDNAs by a reverse transcription reagent kit (Invitrogen, CA). A SYBR Green qPCR Kit (Takara, Otsu, Japan) was used to detect RNA levels. Genes represent Leydig cell specific genes are: membrane receptor gene (LH receptor, Lhcgr), cholesterol-transporting

genes

(high-density

lipoprotein

receptor,

Scarb1;

steroidogenic acute regulatory protein, Star; translocator protein, Tspo), steroidogenic enzyme

genes

(CYP11A1,

Cyp11a1;

HSD3B1,

Hsd3b1;

17-hydroxylase/C17,C20-lyase, Cyp17a1; HSD11B1, Hsd11b1; 17-hydroxysteroid dehydrogenase, Hsd17b3; nuclear receptor 5a1, Nr5a1). Genes represent Sertoli cell-related genes are: SOX9 (Sox9) and dessert hedgehog (DHH, Dhh). The qPCR reaction mixture includes10 μl SYBR Green Mix, 1.6 μl forward and reverse primer mix, 1 g diluted cDNAs, and 5-8 μl RNase-free water. The qPCR was set as follows: 95 oC for 2 min, followed by 40 cycles of 95 oC for 10 s, and 59 oC for 30 s. The Ct value was collected, and the expression level of the target mRNA was calculated using a standard method as previously described 22. The relative expression level was standardized by Rps16, the house-keeping gene. Our previous studies have shown that Rps16 is stable during the development and under the stimulation of chemicals

14, 23.

Melting curve was examined for the quality of qPCR amplification for each sample.

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The primers were listed in Supplementary Table S1. Western blotting Testis was homogenized at the ice-cold PBS to obtain a total protein samples. A BCA Assay Kit (Takara, Japan) was used to measure the protein concentration. Protein samples were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane. After being blocked with 5% fat-free milk, the membrane was incubated with the primary antibodies against the following antigens: LHCGR, STAR， SCARB1, CYP11A1, CYP17A1, SIRT1, PGC-1, pAMPK, AMPK, pAKT2, AKT2, and ACTB (Antibody information was listed in Supplementary Table S2). After washing, HRP-conjugated anti-rabbit or anti-goat IgG secondary antibody (1:2000, Bioword, USA) was added and incubated and the immunoreactive bands were identified by an ECL chemiluminescence kit (Amersham, Arlington Heights, IL). The intensity of protein was quantified by Image Lab software and adjusted to the ACTB, the internal control protein. Our previous studies have shown that ACTB is stable during the Leydig cell development and under the stimulation of chemicals 14, 23. Quantitative immunohistochemical measurement of CYP11A1, HSD11B1, and SOX9 The level of a protein in the tissue not only relies on its expression level but also on the cell number. Quantitative immunohistochemical staining was used to detect the intensity of CYP11A1 and HSD11B1 per Leydig cell and SOX9 per Sertoli cell. Image-Pro 6 Plus software was adopted to measure the intensity of the target protein and background area according to a previous study 24.

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Statistical analysis The mean ± standard errors (SE) were used for data presentation. Statistical analysis was performed using GraphPad Prism (version 6, GraphPad Software, San Diego, CA) with one-way ANOVA followed by ad-hoc Dunnett's multiple comparison. The p < 0.05 was considered a significant difference. Results General reproductive toxicity We gavaged rats PFDoA for 14 days. PFDoA significantly decreased the body weights and testis weights of rats at a dose of 10 mg/kg (Table 1). These results are in accordance with those observed in a previous study 10. PFDoA decreases serum testosterone, LH, and FSH concentrations in vivo. After 14-day exposure, PFDoA significantly decreased the levels of testosterone (Fig.1A), LH (Fig.1B), and FSH (Fig.1C) in the serum samples at doses of 5 and 10 mg/kg with testosterone concentrations only 0.304  0.0309 and 0.238  0.027 ng/ml versus the control (1.453  0.231 ng/ml). This indicates that PFDoA interferes with pituitary-testis axis. PFDoA does not change Leydig and Sertoli cell number in vivo. CYP11A1 is the first androgen-synthetic enzyme in the Leydig cell lineage

16.

HSD11B1 begins exclusively located in immature Leydig cells after 28 days postpartum

25.

Therefore, we enumerated both CYP11A1 and HSD11B1-positive

Leydig cells (Supplementary Fig. S1). PFDoA did not alter Leydig cell number. SOX9 is a transcription factor of Sertoli cells 26. Rat Sertoli cell growth starts in the

12


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testis of the male embryo and stops 21 days postpartum and Sertoli cells maintains the steady levels after 21 days of age. SOX9-positive number was not changed by PFDoA (Supplementary Fig.S1). PFDoA down-regulates gene expression in Leydig cells in vivo. We interrogated the mRNA levels of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Nr5a1) and Sertoli cell genes (Sox9 and Dhh). As shown in Fig.2, PFDoA down-regulated the expression of Lhcgr and Cyp11a1 at doses of 5 and 10 mg/mg, and Scarb1, Star, Cyp17a1, and Hsd11b1 at 10 mg/kg without affecting Hsd3b1, Hsd17b3, and Nr5a1. PFDoA did not affect the expression of Sertoli cell genes (Sox9 and Dhh). PFDoA decreases protein expression levels in Leydig cells in vivo. We measured the protein levels (LHCGR, SCARB1, STAR, CYP11A1, CYP17A1, HSD11B1, and SOX9) using both Western blotting and semi-quantitative immunohistochemical staining. PFDoA decreased LHCGR, SCARB1, STAR, CYP11A1, CYP17A1, and HSD11B1 levels, which were in parallel with their mRNA levels (Fig.3A, B). After a semi-quantitative analysis of CYP11A1 and HSD11B1 and SOX9 intensities in the individual cell, we found that PFDoA decreased CYP11A1 at doses of 5 and 10 mg/kg and HSD11B1 level at 10 mg/kg and had no effect on SOX9 intensity at both doses (Fig.4). PFDoA has no effect on proliferative capacity of Leydig cells in vivo Leydig cells during the prepubertal period have highest mitotic capacity

12.

PCNA is a nuclear matrix protein for cell mitosis. The Leydig cells were identified

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Page 14 of 35

using anti-CYP11A1 antibody, and the proliferating cells were identified using anti-PCNA antibody. There were a few PCNA-positive Leydig cells in all three groups, indicating that these Leydig cells are at the mitotic stage. However, there was no significant difference between the three groups (Supplementary Fig.S2). Effect of PFDoA on metabolic-related and phosphorylation proteins Steroidogenic cells have abundant mitochondria, which not only provide the site for steroidogenic enzyme CYP11A1 but also generate enough energy for androgen synthesis. Although many signaling pathways are associated with Leydig cell androgen synthesis, the signaling pathways for the regulation of mitochondrial function are also critical. Metabolic sensors AMPK and SIRT1 are two important gatekeepers

of

the

master

regulator

of

the

mitochondria,

PGC-1

27.

AMPK/SIRT1/PGC-1 controls the metabolic homeostasis of Leydig cells and androgen synthesis

28.

We measured the protein levels of SIRT1, PGC-1, AMPK,

and pAMPK levels. PFDoA significantly decreased SIRT1 levels at doses of 5 and 10 mg/kg and PGC-1 at 10 g/kg (Fig.3C, D). Although PFDoA did not change AMPK levels, but it inhibited its phosphorylation as shown by the reduction of pAMPK amount and pAMPK/AMPK ratio at 10 mg/kg. ATK is also thought to mediate fibroblast growth factor 9-stimulated steroidogenesis in Leydig cells 29. Therefore, we measured pAKT2 and AKT2 levels in the PFDoA-treated testis (Fig.3C, D). Indeed, PFDoA decreased pAKT2 levels and pAKT2/AKT2 ratio at 10 mg/kg, although the AKT2 levels were not changed (Fig.3C, D).

14


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PFDoA reduces Leydig cell viabilities in vitro. Progenitor Leydig cells were treated with 0-10 μM PFDoA in vitro and their viability was detected by CCK-8. As shown in Fig.5A, the viability of progenitor Leydig cells was significantly blocked by 10 μM PFDoA. DCFH-DA was used to identify the intracellular ROS levels in progenitor Leydig cells after PFDoA treatment (Fig.5B). PFDoA significantly stimulated ROS levels at 10 M (Fig.5C). Annexin V/PI assay was carried out to assess the effect of PFDoA on apoptosis rate of progenitor Leydig cells (Fig.5D). PFDoA significantly increased apoptosis rate of progenitor Leydig cells at 10 M (Fig.5F). The results suggest that PFDoA stimulates ROS generation, induces apoptosis and lowers viability of progenitor Leydig cells at the higher concentration (10 M). PFDoA reduces Leydig cell m in vitro. As m loss was associated with early apoptosis and mitochondria function

30,

JC-1 probe was used to calculate the effects of PFDoA on m loss in progenitor Leydig cells. JC-1 can specially enter the mitochondria as a fluorescent probe. While the m is low, it exists as the monomer, which has green fluorescence. When the m is high, JC-1 forms polymers, which has red fluorescence. PFDoA significantly caused m loss in progenitor Leydig cells at 10 μM (Fig.5E). Discussion There is an wide application of PFDoA as a surfactant in industrial and consumer products organs

32.

31,

leading to its accumulation in the environment, wildlife, and human

PFDoA has been detected in milk and serum samples of humans with the

15



highest concentrations of 9.7 and 22 pg/ml 4, 5. Although previous studies showed that PFDoA damaged rat Leydig cell function at adulthood

15, 33, 34,

this study clearly demonstrated that PFDoA also blocked the

differentiation of Leydig cells during the prepubertal period. PFDoA apparently decreased the body weight for about 20% at the high dose (10 mg/kg) and this body-weight loss might lead to a systemic toxicity such as LH and FSH levels. However, PFDoA did not affect the body weight at low dose (5 mg/kg) and caused the serum testosterone level as low as that at high dose, indicating that the testosterone reduction is not caused by the body weight loss. There are Leydig cells, Sertoli cells, peritubular myoid cells and germ cells in the testis. However, PFDoA did not influence Leydig and Sertoli cell number. Thus, in the present study, we measured the expression levels of the genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Hsd11b1) that were exclusively expressed in Leydig cells, gene Nr5a1 that was exclusively expressed in Leydig and Sertoli cells, as well as genes (Sox9 and Dhh) that were exclusively expressed in Sertoli cells using the whole testis. We found that PFDoA impaired Leydig cell differentiation via down-regulating Lhcgr, Scarb1, Star, Cyp11a1, and Cyp17a1 and their proteins, thus resulting in the lower testosterone secretion. PFDoA acts via AMPK/SIRT1/PGC-1 and phosphorylation of AKT2 to disturb the mitochondrion, thus causing loss of m, stimulating ROS regeneration and apoptosis rate of progenitor Leydig cells. PFDoA had no effects on Leydig cell proliferation in vivo as judged by

16


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unchanged PCNA-labeling index of Leydig cells. However, in vitro study showed that PFDoA did not reduce Leydig cell viability by CCK-8 assay until 10 M, indicating that high concentration is required to inhibit its viability and in vivo PFDoA treatment using the highest dose (10 mg/kg) may not reach such level. Serum level of PFDoA can reach 24 g/ml (39 M) after a single oral exposure to 50 mg/kg, indicating that 10 mg/kg PFDoA could lead to about 8 M in the serum19. Previous study in adult male rats that were orally exposed to 5 or 10 mg/kg PFDoA for 14 days showed significantly reduced testosterone and LH levels as well as the down-regulated expression of Star, Cyp11a1, and Cyp17a1

15.

In the present

study, we also demonstrated that PFDoA exposure to prepubertal rats suppressed testosterone levels and down-regulated the expression of Leydig cell genes such as Lhcgr and Scarb1 15. The effects of in vitro and in vivo PFDoA treatment may be different, depending on dose and duration. However, the negative effects of both in vitro and in vivo PFDoA treatment may be comparable. Shi et al. showed that PFDoA down-regulated Leydig cell steroidogenesis-related gene expression and inhibited testosterone production in Leydig cells

35.

Shi et al. also demonstrated that PFDoA disrupted

mitochondrial function of the testis in vivo 15, 34. Indeed, the current study showed that PFDoA caused m loss in rat progenitor Leydig cells at 10 M (Fig.5E). Interestingly, 24-h PFDoA treatment did not cause m loss in rat adult Leydig cells even at 100 M

34.

This again indicates that progenitor Leydig cells are more

vulnerable to the insult of PFDoA. Although the exact mechanism is not clear for the

17



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sensitivity of progenitor Leydig cells to PFDoA, one possible explanation is that progenitor Leydig cells have much lower expression of antioxidative proteins than adult Leydig cells

36.

Indeed, PFDoA induced significant amount of ROS in

progenitor Leydig cells at 10 M (Fig.5C). Mitochondrion is a critical organelle that is responsible for the energy generation. In the Leydig cells, the mitochondrion has another important function that is the catalysis of the first-step steroidogenesis from cholesterol by CYP11A1, a mitochondrial inner membrane enzyme 16. AMPK and SIRT1 are two important metabolic sensors of the master regulator of the mitochondrion, PGC-1

27.

PGC-1

is an important transcriptional

co-activator for energy regulation. PGC-1 interacts with many transcription factors that promote -oxidation of fatty acids for the generation of energy

37.

AMPK is a

heterotrimeric protein complex that consists of α, β, and γ subunits to regulate ATP levels after activation by PGC-1 38 39. SIRT1 is an NAD-dependent class III histone deacetylase and takes roles on many biological activities

40

and interacts with

acetylated PGC-1 and deacetylates it to increase phosphorylated PGC-1 activity, thus promoting mitochondrial biogenesis and maintain its function 41. AMPK, SIRT1, and PGC-1 are present in Leydig cells of rodents, indicating that

they

regulate

the

mitochondrial

biogenesis

and

metabolism

37

42.

AMPK/SIRT1/PGC-1 controls metabolic homeostasis of Leydig cells and steroidogenesis 28. Indeed, phosphorylated AMPK up-regulated STAR and CYP11A1 expression and stimulated testosterone production in Leydig cells 42.

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The current study demonstrated that PFDoA-treated rats exhibited altered expression levels of SIRT1 and PCG-1 as well as phosphorylated AMPK, which were significantly lower than those in the control at a dose of 10 mg/kg, suggesting that mitochondrial function in the testicular cells is disrupted. Some diseases such as the metabolic syndrome suppress testicular mitochondrial function by decreasing SIRT1/ PGC-1, thus increasing oxidative stress. Likewise, streptozotocin-induced type I diabetes in mice decreased SIRT1/PGC-1 levels, leading to increased ROS levels

43.

Indeed, in vitro PFDoA treatment significantly increased ROS levels in

progenitor Leydig cells (Fig.5C) and induced apoptosis rates (Fig.5F) and caused loss of m (Fig. 5G). Besides AMPK/SIRT1/PGC-1 signaling, AKT signaling may also be involved in Leydig cell development

44, 45.

The AKT signaling kinase is evolutionarily

conserved and plays a critical role in the phosphoinositide 3-kinase (PI3K) signaling pathway 46. AKT regulates numerous cellular processes, including cell differentiation, proliferation, and apoptosis

47.

Many hormones and growth factors, such as insulin

and insulin-like growth factor 1), stimulate AKT. AKT2 is highly expressed in insulin-responsive tissues and mainly regulates glucose metabolism 48. In the present study, PFDoA decreased the phosphorylated AKT2 (pAKT2) without affecting total AKT2 levels at 10 mg/kg. Long-term (110 days) low doses of PFDoA (0.5 mg/kg) also caused significant decrease in pAKT2 levels and PFDoA also lowered pAKT2 levels in adult Leydig cells in vitro at 10 and 100 M observation

34

34.

Our data and previous

indicate that decreased phosphorylation of AKT2 may be associated

19



with the delay of Leydig cell development. Although PFDoA can significantly inhibit testosterone levels when rats were exposed to 5 and 10 mg/kg doses, the highest levels in human blood is only around 22 pg/ml

4, 5

and such concentration range may not reach the inhibitory level. Further

human epidemiological investigation is required. In conclusion, PFDoA blocks the Leydig cell development during prepuberty. Both AMPK/SIRT1/PGC-1 and AKT2 signaling may mediate the PFDoA action. Competing interests The authors declared that no competing interests exist. Acknowledgements Supported by NSFC (81730042 to R.S.G., 81701426 to X.L.G., 81601264 to X.H.L.) and Health and Family Planning Commission of Zhejiang Province (2017KY483 to X.H.L., 2018KY523 and 2017KY473 to X.L.G., 11-CX29 to R.S.G.) as well as Zhejiang Provincial NSF (LY15H310008 to R.S.G.).

Supporting information Antibody information was listed in Supplementary Table S1. The primer information was listed in Supplementary Table S2. Effects of PFDoA on Leydig cell number and Sertoli cell number were listed in Supplementary Figure S1. PCNA and CYP11A1 co-staining in rat testis sections were listed in Supplementary Figure S2. Abbreviations list PLCs

Progenitor Leydig cells

20


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LH

Luteinizing hormone

LHCGR

Luteinizing hormone receptor

SCARB1

High-density lipoprotein receptor

STAR

Steroidogenic acute regulatory protein

CYP11A1

Cytochrome P450 cholesterol side-chain cleavage enzyme

HSD3B1

Hydroxy--5-steroid dehydrogenase-3-steroid -isomerase 1

CYP17A1

Cytochrome P450 family 17 subfamily A polypeptide 1

HSD17B3

Hydroxysteroid (17) dehydrogenase 3

HSD11B1

Hydroxysteroid (11) dehydrogenase 1

SIRT1

NAD+-dependent type III deacetylase sirtuin 1

PGC-1α

Peroxisome proliferator-activated receptor coactivator 1α

AKT

Protein kinase B

AMPK

Adenosine 5’-monophosphate (AMP)-activated protein kinase

FSH

Follicle-stimulating hormone

SOX9

SRY box 9

PCNA

Proliferating cell nuclear antigen

DHH

Desert hedgehog

NR5A1

Nuclear receptor steroidogenic factor 1

RPS16

Ribosomal protein S16

21



ACTB

β-Actin

ΔΨm

Mitochondrial membrane potential

ROS

Reactive oxidative species

MTT

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide

PFDoA

Perfluorododecanoic acid

22


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