Mitochondrial damage are involved in Aflatoxin B1-induced testicular damage and spermatogenesis disorder in mice

Journal Pre-proofs Mitochondrial damage are involved in Aflatoxin B1-induced testicular damage and spermatogenesis disorder in mice Wanyue Huang, Zheng Cao, Qiucheng Yao, Qiang Ji, Jian Zhang, Yanfei Li PII: DOI: Reference:

S0048-9697(19)35069-7 https://doi.org/10.1016/j.scitotenv.2019.135077 STOTEN 135077

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Science of the Total Environment

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12 September 2019 14 October 2019 18 October 2019

Please cite this article as: W. Huang, Z. Cao, Q. Yao, Q. Ji, J. Zhang, Y. Li, Mitochondrial damage are involved in Aflatoxin B1-induced testicular damage and spermatogenesis disorder in mice, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.135077

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Mitochondrial damage are involved in Aflatoxin B1-induced

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testicular damage and spermatogenesis disorder in mice

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Wanyue Huanga, Zheng Caoa, Qiucheng Yaob, Qiang Jia, Jian Zhanga, Yanfei Lia1

4

a

5

Disease Prevention and Treatment, College of Veterinary Medicine, Northeast Agricultural

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University, Harbin 150030, China.

7

b

Key Laboratory of the Provincial Education, Department of Heilongjiang for Common Animal

College

of

Agriculture,

Guangdong

Ocean

University,

Zhanjiang

524000,

China

Abbreviations: AFB1, Aflatoxin B1; ATP, adenosine triphosphate; T, testosterone; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; TEM, transmission electron microscope; H2O2, hydrogen peroxide; SOD, superoxide dismutase; GSH, glutathione; cDNA, complementary DNA; ETC, electron transfer chain; nDNA, nuclear genes; mtDNA, mitochondrial genes; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator 1α; Nrf1, Nuclear respiratory factor 1; Tfam, transcription of mitochondrial transcription factor A; Mfn1, mitofusins 1; Opa1, optic atrophy 1; Drp1, Dynamin related protein 1; Fis1, fission 1; OXPHOS, oxidative phosphorylation; complex I, NADH dehydrogenase; complex II, succinate-ubiquinone oxidoreductase; complex III, ubiquinone-cytochrome c reductase; complex IV, cytochrome c oxidase. 1 Corresponding author at: College of Veterinary Medicine, Northeast Agricultural University, NO. 600, Changjiang Road, Harbin 150030, China. E-mail address: [email protected] (Y.F. Li); Tel.: +86 18846180665; fax: +86 451 55191672. 1

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ABSTRACT

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Aflatoxin B1 (AFB1) is an unavoidable environmental pollutants, which seriously endangers

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human and animal health. AFB1 has male reproductive toxicity, yet the underlying mechanisms

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remain inconclusive. Mitochondra are a kind of crucial organelle for maintaining spermatogenesis

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in testis. Thus, we hypothesized that AFB1 can impair mitochondria to aggravate testicular

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damage and spermatogenesis disorder. To verify this hypothesis, 48 male mice were

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intragastrically administered with 0, 0.375, 0.75 or 1.5 mg/kg body weight AFB1 for 30 days,

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respectively. In this study, we found AFB1 caused testicular histopathological lesions and

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spermatogenesis abnormalities, with the elevation of oxidative stress (increased H2O2, whereas

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dcreased SOD and GSH). Significant mitochondria structure damage of germ cells and Leydig

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cells, MMP loss, ATP contents reduction, and inhibited activities of mitochondrial complexes I-IV

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in mice testis were found in AFB1 treatment groups. Besides, AFB1 inhibited mitochondrial

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biogenesis and mitochondrial dynamics, presenting as the decreased mRNA and protein

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expressions of PGC-1α, Nrf1, Tfam, Drp1, Fis1 Mfn1 and Opa1. The results revealed that the

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mitochondrial damage were involved in AFB1-induced testicular damage and spermatogenesis

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disorder, providing a considerable direction to clarify potential mechanisms of AFB1 reproductive

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toxicity.

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Key words: Aflatoxin B1 (AFB1); Mitochondria; Testicular damage; Spermatogenesis disorder;

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Mice

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1. Introduction

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Male reproduction is the foundation for maintaining the human race multiplying and social

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stability. However, estimated 48.5 million (15%) couples globally face the threat of infertility in

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recent years, and about half of infertility cases are attributed to men (Nakada et al., 2006). Male

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reproductive abnormality is to a certain extent due to mycotoxin exposure (Eze et al., 2018;

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Supriya et al., 2014). Mycotoxins has been documented as unavoidable environmental

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contaminants (Kolpin et al., 2014). Aflatoxins, a class of mycotoxins produced by Aspergillus

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flavus and Aspergillus parasiticus, frequently contaminate cereal crops such as corn, beans,

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peanuts, and dried fruits (Williams et al., 2004). Approximately 5 billion people globally were at a

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risk of chronic exposure to aflatoxins in developing countries (Heather et al., 2006). Aflatoxin B1

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(AFB1) is deemed as the most toxic among the various aflatoxins and their metabolites, owning

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carcinogenic, mutagenic, and teratogenic properties (Williams et al., 2004). Recently, reproductive

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toxicity of AFB1 has received much attention. High level of AFB1 residue was found in the

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seminal plasma of boars and caused fertility disorders (Picha et al., 1986). Several studies reported

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that AFB1 can cause testicular damage, preclude testosterone synthesis and decrease sperm

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quality, eventually impaire fertility (Agnes and Akbarsha, 2003; Supriya et al., 2014).

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Nevertheless, the underlying mechanisms of AFB1-induced reproductive toxicity are not fully

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understood.

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Male fertility is dependent upon the successful perpetuation of spermatogenesis, which

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occurs within the seminiferous tubules of the testis (Yang et al., 2019). Therefore, healthy male

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reproduction is highly dependent on the normal structure and function of testis. Testis can produce

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male steroids and male gametes in male reproductive system, while testicular damage block 3

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spermatogenesis and sex steroids secretion to cause infertility (Jiang et al., 2018; Yang et al.,

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2019). Oxidative stress is a fundamental pathogenesis of testicular dysfunction due to their high

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content of polyunsaturated lipids in cellular membranes and the presence of potential reactive

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oxygen species (ROS) generating systems (Turner and Lysiak, 2008). Increased oxidative stress

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resulted in testicular morphology destruction and spermatogenesis disorder in mice (Yang et al.,

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2019). Meanwhile, oxidative stress is also being confirmed a major pathogenesis of AFB1-induced

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reproductive toxicity (Abu El-Saad and Mahmoud, 2009; Aitken and Roman, 2008).

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Mitochondra, the primary targets of oxidative stress, are susceptible to attack by

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internal/exogenous ROS, and then lead to mitochondrial structural and functional damage (Zorov

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et al., 2014). Meanwhile, mitochondria comprise approximately 15-22% of the total cellular

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volume delivering 90% of the energy required. Testicular mitochondria are responsible for

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adenosine triphosphate (ATP) synthesis and sperm quality to maintain male reproductive

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functions (Ramalho-Santos et al., 2009; Rajender et al., 2010). Furthermore, the mitochondria of

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Leydig cells in testis are main sites to form testosterone (T) for maintain spermatogenesis,

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including maintenance of the blood-testis-barrier, meiosis, sertoli-spermatid adhesion and sperm

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release (Smith and Walker, 2014; Walker, 2009). When testicular mitochondria damaged, energy

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metabolism obstruction and cellular dysfunction were occurred, resulting in cell death and sperm

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function impairment (Zhang et al., 2019; Zhang et al., 2018). Thus it can be seen that the normal

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mitochondria in testicular tissue are the basis of spermatogenesis. Limited evidence indicated that

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AFB1 impaired mitochondrial membrane potential (MMP) in bovine sperm (Komsky-Elbaz et al.,

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2017), and decreased mitochondrial content of cells in mice testis by mitochondrial specific

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staining (Yasin and Mazdak, 2018). There are still needed systematic researches to clarify the 4

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effects of AFB1 on mitochondria and its role in male reproductive system.

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Mitochondria are controlled by mitochondrial biogenesis and dynamics (Scarpulla, 2011;

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Serasinghe and Chipuk, 2017). Mitochondrial biogenesis contributes to renew or adapt

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mitochondrial population during periods of damage or intensified energy demands, and impaired

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mitochondrial biogenesis can exacerbate mitochondrial dysfunction (Zhu et al., 2013).

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Mitochondrial dynamics involved fusion and fission two opposite forces (Meyer et al., 2017). The

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disproportion between fusion and fission in the mitochondria led to lipid peroxidation, excessive

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ROS production, the drop of MMP, inhibited respiration function, and lowered ATP production

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(Calkins and Reddy, 2011).

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AFB1 had reproductive toxicity. However, there were few reports about the effects of AFB1

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on mitochondria in mice testis. Therefore, the aim of our study was to investigate whether

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impaired structure and function of mitochondria were involved in AFB1-induced testicular damage

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and spermatogenesis disorder.

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2. Materials and methods

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2.1 Animals and Treatment

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Forty-eight male kunming mice (6 weeks old) weighing 20-22 g were purchased from the

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Experimental Animal Centre of Harbin Medical University and housed in the Biomedical

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Research Center, Northeast Agricultural University, China. The animals were maintained under a

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well-ventilated room with the temperature, humidity-controlled (24±2°C, 60%-80%) atmosphere

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and 12 h light/dark cycles. Distilled water and sterilized food for mice were available ad libitum.

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After one week of acclimatization period, all the mice were weighed and randomly divided into

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four groups (12 per group): Group 1 (Control group, CG) was orally administered with the vehicle 5

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(0.2 mL/animal/day corn oil). Group 2 (Low-dose group, LG) was orally administrated with AFB1

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(≥99.8%, Qingdao Pribolab Pte. Ltd, China) at 0.375 mg/kg body weight. Group 3 (Mid-dose

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group, MG) were treated with AFB1 at 0.75 mg/kg body weight. Group 4 (High-dose group, HG)

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were treated with AFB1 at 1.5 mg/kg body weight. The doses of AFB1 in this study were

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determined according to the LD50 of AFB1 (9 mg/kg body weight) for male mice and can cause

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testis damage (Cao et al., 2017).

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After 30 days treatment, mice were weighed and euthanatized. All the testis and

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epididymides were weighed immediately after harvesting. Next, the left testis and cauda

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epididymides were fixed for histopathological examination and spermatogenesis scoring, and the

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right cauda epididymides were removed for sperm analysis and the right testis were stored at

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-80°C to detect for other parameters in this study. All the use of the animals and experimental

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procedures were approved by the Animal Ethics Committee of the Northeast Agricultural

106

University (Harbin, China).

107

2.2. Testicular and epididymal volume detection

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Testicular and epididymal volume (n=12 per group) was calculated by formula as followed:

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Volume (cm3) = weight / density (g/cm3). The density of testis and epididymides is 0.93 g/cm3

110

reported as (Yang et al., 2006).

111

2.3 Histopathology observation and spermatogenesis scoring

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The fixed testis and cauda epididymides were embedded in paraffin, and sectioned to slides

113

with a thickness of 5 μm. Then the tissues were stained with hematoxylin and eosin (HE) for

114

histological analysis. The final images were captured by a digital section scanner (Pannoramic

115

MIDI, Hungary). The spermatogenesis was graded using Johnsen Score, which gives a score of 6

116

1-10 according to the presence or absence of the spermatogenic cell types (Johnsen, 1970). In each

117

cross-section, 20 randomly selected seminiferous tubules were scored at 400×, the mean score was

118

determined for each group. Three animals were randomly selected from each group for the

119

examination.

120

2.4 Ultrastructure observation of mice testis

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Testicular tissues were processed for ultrastructural evaluation. Briefly, the testis were cut

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into proper size (approximately 1 mm3) with razor blades, and then fixed with 2.5%

123

glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 for 2 h, then rinsed three times in the buffer.

124

Later, the tissues were post-fixed for 80 min in 1% OsO4, dehydrated through ascending graded

125

series of acetone, and embedded in epoxy resin. The ultra-thin sections were cut and stained with

126

uranyl acetate and lead citrate. Transmission electron microscope (TEM) images were obtained

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using a transmission electron microscope (HT7650, Hitachi, Japan). Three animals were randomly

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chosen from each group for ultrastructural examination.

129

2.5 Sperm morphology observation

130

The right cauda epididymides (n=6 per group) were cut into small pieces in physiological

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saline (37°C, 5% CO2) to obtain the sperm as reported by Supriya et al. (Supriya et al., 2014). The

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sperm suspension was smeared on a slide and then stained by Quick sperm stain kit (D029,

133

Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions.

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Sperm presenting abnormalities in head, neck/mid-piece, and tail were assessed and examined at

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400× for morphological abnormalites in each sample.

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2.6 Oxidative stress markers measurement

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Testicular tissues (n=6 per group) were homogenized in pre-cooled physiological saline. The 7

138

10% tissue homogenized solution was centrifuged at 3500×g (4°C) for 15 min to obtain tissue

139

supernatant and then detected hydrogen peroxide (H2O2), superoxide dismutase (SOD) and

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glutathione (GSH) following the instructions of corresponding commercial kits (A064-1, A001-2

141

and A006-2 Nanjing Jiancheng Bioengineering Institute, China), respectively.

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2.7 Mitochondria isolation of mice testicular tissue

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The Tissue Mitochondria Isolation Kit (C3606, Beyotime institute of biotechnology, Jiangsu,

144

China) was used to isolate testis mitochondria. Briefly, testis tissue was removed and washed once

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in chilled PBS. Tissue was cut into pieces and placed into mitochondrial isolation reagent A for

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homogenates. The homogenate composition was centrifuged at 600 g for 5 min and the

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supernatant was subjected to centrifugation at 11 000 g for 10 min (4°C). The remaining sediment

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consisted of mitochondria and was suspended in storage buffer.

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2.8 Measurement of membrane potential in mitochondria

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The mitochondria membrane potential of testicular tissue was detected by JC-1 fluorescent

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probe staining method (C2006, Beyotime institute of biotechnology, Jiangsu, China) according to

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the manufacturer's instructions. 0.1 mg of purified mitochondria was diluted to 0.1 mL with a

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stock solution, and added it to 0.9 mL of JC-1 staining working solution diluted 5 times by JC-1

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staining buffer (1×), pipetted and mixed. The fluorescence value was measured by a fluorescence

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microplate reader (M200 Pro, Tecan, Switzerland) at an excitation wavelength of 485 nm and an

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emission wavelength of 590 nm.

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2.9 Electron Transport Chain Complex activities analysis

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The activities of Electron Transport Chain Complex I-IV were measured using the

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corresponding Electron Transport Chain Complex Assay Kit (BC0510, BC3230, BC3240, 8

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BC0940, Beijing Solarbio Science & Technology Co., Ltd, China) following the manufacturer's

161

instructions. The absorbance was measured by a visible spectrophotometer (D-7 Manjing Philes

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instrument Co., Ltd., China) at 340 nm, 605 nm, 550 nm or 550 nm for complexes I, II, III and IV,

163

respectively.

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2.10 Assessment of ATP content

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ATP content was measured with the ATP assay kit according to the manufacturer instructions

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(S0026, Beyotime institute of biotechnology, Jiangsu, China). Briefly, 20 mg testis tissues were

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homogenized in ATP detection lysis buffer and then subjected to centrifugation at 12, 000 r/min at

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4°C for 5 min and the supernatant was used to measure ATP concentration by Centro XS3 LB 960

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luminometer (Berthold Technologies, Germany), and the ATP concentration was calculated using

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the ATP standard curve and expressed as nmol/mg protein.

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2.11 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis

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Total RNA was extracted from mice testis using Trizol Reagent (No. 9109 Takara, Japan)

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according to the manufacturer’s instructions. RNA amount and quality were determined using the

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spectrophotometer (GeneQuant 1300 GE, USA). Samples with ratios of absorbance at 260/280 nm

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between 1.8 and 2.0 were judged to be of acceptable quality and integrity. Total RNA was reverse

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transcribed in a total volume of 80 μL using TransScript FirstStrand complementary DNA

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(cDNA) Synthesis SuperMix (Roche, CH, Switzerland) according to the manufacturer’s

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instructions. The primer sequences are shown in Table 1. PCR was performed in LineGene 9620

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real-time PCR detection system (Bioer, China) using SYBR Green PCR core reagents (Roche,

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CH, Switzerland). The housekeeping gene GAPDH was used as a reference gene for

181

normalization. Data were analyzed according to the 2-ΔΔCt method (Huang et al., 2017). The results 9

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were expressed as the relative mRNA levels.

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Table 1 Primer sequences and amplification lengths of destination fragments.

Gene

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Number

PGC-1α

NM_008904.2

Nrf1

NM_001164226.1

Tfam

NM_009360.4

Mfn1

NM_024200.4

Opa1

NM_001199177.1

Drp1

NM_001025947.2

Fis1

NM_001163243.1

GAPDH

NM_001289726.1

Upstream and downstream primer sequence

Primer

Product

lengh

lengh

(bp)

(bp)

UP: 5′- TCATGTGACTGGGGACTGTAG -3′

21

LOW: 5′- ACCAGAGCAGCACACTCTATG -3′

21

UP: 5′- ACGTTGGATGAGTACACGACG -3′

21

LOW: 5′- GTACTTTCGCACCACATTCTCC -3′

22

UP: 5’ CCCCTCGTCTATCAGTCTTGTC 3’

22

LOW: 5’ TTCTGGTAGCTCCCTCCACA 3’

20

UP: 5′- GGAGTACATGGAGGATGTGCG -3′

21

LOW: 5′- CTGGCATCCCCTGAGCTTTA -3′

20

UP: 5’ GCTGGCAGAAGATCTCAAGAAAG 3’

23

LOW: 5’ GCACGGAAAAGGGTAGAACG 3’

20

UP: 5’ CAGCGGATATGCTGAAGGCATTA 3’

23

LOW: 5’ GCAGGTTCAAGTCAGCAAAGT 3’

21

UP: 5’ AAGTATGTGCGAGGGCTGTT 3’

20

LOW: 5’ TGCCTACCAGTCCATCTTTCT 3’

21

UP: 5′- GGTTGTCTCCTGCGACTTCA -3′

20

LOW: 5′- TGGTCCAGGGTTTCTTACTCC -3′

21

200

126

190

164

192

116

103

183

2.12 Preparation of protein extracts and western blot analysis

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Western blot analysis was carried out according to previous study with modification (Huang

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et al., 2017). Whole protein of testis was extracted with superactive RIPA lysis buffer (P0013B

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Beyotime, China). The protein concentration was determined by the BCA analysis kit (P0010S

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Beyotime, China). The protein (30 μg) was separated by 8-15% sodium dodecyl sulfate

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polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, Bedford,

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USA). These membranes were blocked with 5% fat-free milk in PBST at 37°C for 3 h, and

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incubated overnight at 4°C with primary antibodies recognizing PGC-1α (A12348 Abclonal), Nrf1 10

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(A5547 Abclonal), Tfam (A13552 Abclonal), Mfn1 (PB0263 BOSTER), Opa1 (PB0773

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BOSTER), Drp1 (WL03028 Wanleibio), Fis1 (DF12005 Affinity) and GAPDH (AC002

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Abclonal). Subsequently, the primary antibodies were incubated with corresponding secondary

195

antibodies (1: 5000 sc-2357 and sc-516102 Sigma-Aldrich, USA) for 2 h at 37°C. The bands were

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quantified using the Image J software.

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2.13 Statistical analysis

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All analyses were performed with the SPSS 22.0 software (SPSS Incorporated, USA). Data

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were expressed as mean ± standard deviation (mean ± SD). Statistical significance was analyzed

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using one-way ANOVA followed by LSD test as the post-hoc test. In all statistical comparisons,

201

values of P < 0.05 were considered significant, and values of P < 0.01 were considered markedly

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significant. *P < 0.05 and **P < 0.01 symbol for the significance of differences versus the CG.

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3. Results

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3.1. AFB1 caused testicular damage and spermatogenesis disorder in mice

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As shown in Fig. 1B and 1D, the final body weight of the mice and epididymides volume

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were significantly decreased after AFB1 treatment in the MG and HG compared with the CG (P <

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0.05, P < 0.01), and there were no significant differences between the LG and the CG. The mice

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testis volume was significantly reduced in all AFB1-exposed groups compared with the CG (P <

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0.05, P < 0.01) (Fig. 1A and C). HE staining and sperm staining were used for the morphological

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analysis of testis and sperm to reveal the effect of AFB1 on the testicular structure and

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spermatogenesis. The HE staining of testis displayed testicular structure from the CG showed

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well-organised seminiferous tubules lined by normal spermatogenic epithelium and its lumen 11

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filled with sperm with normal interstitial tissue and Leydig cells. On the contrary, the atrophic

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seminiferous tubules, vacuolar degenerative changes of spermatogenic epithelium, as well as

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reduced numbers of sperm and Leydig cells were found in all AFB1-exposed groups (Fig. 1E). As

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expected, Johnsen scores of the MG and HG were significantly lower than that of the CG, and

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there were no significant differences between the LG and the CG (Fig. 1G). From the HE staining

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of cauda epididymidis (Fig. 1F), the sperm density was obvious reduced in all AFB1 treatment

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groups. Even worse, abnormal structure of sperm was observed after AFB1 exposure as shown in

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Fig. 1G, including head deformities (a-d), folded tails (e-i) and flagellar kinked (j) (Fig. 1H).

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3.2. AFB1 induced oxidative stress in mice testis

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As shown in Fig. 2, H2O2 levels were significantly increased, whereas GSH levels were

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significantly reduced in all AFB1-exposed groups compared with the CG (P < 0.01). AFB1

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significantly decreased SOD activities in the MG and HG compared with the CG (P < 0.01), and

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there were no significant differences between the LG and the CG.

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3.3. Ultrastructural evaluation

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TEM examination indicated normal Leydig cells and germ cells ultrastructure of mice testis

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in the CG with intact nuclear membrane, normal structure of mitochondria and endoplasmic

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reticulum (Fig. 3). In contrast, severe impairment ultrastructure of testicular cells were observed in

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AFB1-exposed groups, including nuclear fragmentation, chromatin condensation and the obvious

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swelling and degeneration of mitochondria. The significant impairment of the mitochondria were the

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mitochondrial cristae and matrix. The mitochondrial cristae membranes were breaked and became

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vague and even disappeared. The mitochondrial matrix was also become invisible.

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3.4. AFB1 reduced MMP, mitochondrial complexes I-IV activities and ATP contents 12

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As shown in Fig. 4A, it was observed that AFB1 exposure significantly decreased MMP

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compared to the CG (P < 0.05, P < 0.01). As shown in Fig. 4B-F, the complex I and IV activities,

237

and ATP contents in the MG and HG were significantly decreased compared to the CG (P < 0.01),

238

and there were no significant differences between the LG and the CG. The activities of complexes

239

II and III were significantly decreased in all AFB1-exposed groups as compared to the CG (P <

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0.05, P < 0.01).

241

3.5. AFB1 inhibited mitochondrial biogenesis in mice testis

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As shown in Fig. 5, compared with the CG, the protein expressions of PGC-1α and Tfam,

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and Tfam mRNA expression were significantly decreased in the MG and HG (P < 0.05, P < 0.01),

244

and there were no significant differences between the LG and the CG. Nrf1 protein expression and

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the mRNA expressions of PGC-1α and Nrf1 were significantly decreased in all AFB1-exposed

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groups compared with the CG (P < 0.01).

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3.6. AFB1 inhibited mitochondrial dynamics in mice testis

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As shown in Fig. 6, the protein expressions of Fis1, Mfn1 and Opa1, and the mRNA

249

expressions of Drp1, Mfn1 and Opa1 were significantly decreased in all AFB1-exposed groups

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compared with the CG (P < 0.05, P < 0.01). Drp1 protein expression and Fis1 mRNA expression

251

were significantly decreased in the MG and HG compared with the CG (P < 0.05, P < 0.01), and

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there were no significant differences between the LG and the CG.

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4. Discussion

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In this study, AFB1 exposure caused obvious testicular microstructure and ultrastructure

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lesions and spermatogenesis abnormalities, as well as triggered oxidative stress in mice testis. The 13

256

damaged mitochondrial ultrastructure structure, MMP loss and inhibited activities of testicular

257

mitochondrial complexes I-IV were found in AFB1-exposed mice, accompanied with a drop in

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ATP generation. Besides, AFB1 induced mitochondrial biogenesis inhibition and mitochondrial

259

dynamics disruption, presenting as the decreased mRNA and protein expressions of PGC-1α,

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Nrf1, Tfam, Drp1, Fis1 Mfn1 and Opa1. These results revealed that impaired the structure and

261

function of testicular mitochondria were involved in AFB1-induced testicular damage and

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spermatogenesis disorder.

263

Compelling evidence has accumulated that oral feeding or intraperitoneal administration of

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AFB1 to experimental animals produced adverse effects on male reproduction, and testicular tissue

265

was the main target organ (Agnes and Akbarsha, 2003; Verma and Nair, 2002). Studies have

266

shown that testicular atrophy and structural disorder are hallmarks of the spermatogenic damage in

267

mice (Li et al., 2015). In the present study, we found AFB1 significant reduced mice weight and

268

the volume of testis and epididymides. The microscopic examination of testicular tissue obtained

269

from AFB1-treatment groups revealed that shriveled seminiferous tubule and the diminution of

270

spermatogenic cells, which were in conformity with the result of Johnsen Score. Cell gap grew

271

bigger than the CG with significantly reduced Leydig cells. Moreover, testicular cells

272

ultrastructure displayed serious injury, particularly obvious mitochondrial damage. Meanwhile,

273

the sperms in cauda epididymides were significantly reduced and abnormal sperms significantly

274

increased. These results indicated AFB1 induced testicular damage and spermatogenesis disorder

275

in mice.

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Oxidative stress is the key mechanism of AFB1-induced testicular damage (Abu El-Saad and

277

Mahmoud, 2009). AFB1 can trigger excessive oxidative stress and further cause germ cells 14

278

apoptosis, ultimately impair testicular structure and spermatogenesis in mice (Yasin and Mazdak,

279

2018). Among a great variety of ROS, H2O2 has been recognized as the most toxic oxidizing

280

species for human spermatozoa (Aitken et al., 1993). H2O2 can decrease SOD activities and

281

cellular GSH contents, and this two antioxidant enzymes in semen are considered as the part of

282

first line defence against ROS (Parviz and R John, 2011). Our current results showed that AFB1

283

significantly elevated H2O2 levels, whereas reduced SOD activities and GSH levels in mice testis,

284

indicating AFB1 triggered oxidative stress. And we speculated that the AFB1-caused testicular

285

damage and spermatogenesis disorder may be resulted from oxidative stress.

286

Mitochondria are double-membrane organelles that play a fundamental role in regulation of

287

energy metabolism in testis, and mitochondrial damage are associated with infertility

288

(Ramalho-Santos et al., 2009; Wallace, 1999). ATP are main synthesized through the electron

289

transfer chain (ETC) in mitochondrial inner membrane to maintain cellular function and sperm

290

maturation (Rajender et al., 2010). In addition, intact mitochondria in testicular Leydig cells with

291

active respiration are key for T production, T is one of the most important androgens and it is

292

essential for the initiation of spermatogenesis as well as testis development (Allen et al., 2006;

293

Roberts and Chauvin, 2019). These evidences confirm the important role of mitochondria in

294

male reproductive function. Mitochondria are susceptible to be impaired by oxidative stress,

295

resulting in membrane structure damage and then decreased MMP (Zorov et al., 2014). Stable

296

MMP is the basis for maintaining oxidative phosphorylation of mitochondria (Zorova et al., 2018),

297

while MMP loss promotes mitochondrial dysfunction and is regarded as an early requirement in

298

cell apoptosis (Zamzami et al., 1995). Studies found AFB1 evoked mitochondrial ROS generation,

299

decreased MMP and induced apoptosis in broiler hepatocytes and cardiomyocytes (Liu and Wang, 15

300

2016; Wang et al., 2017). Consistent with the above studies, we found significant mitochondria

301

swelling with cristae disappearance in germ cells and Leydig cells via TEM observation and

302

decreased MMP in AFB1-exposed mice testis, indicating AFB1 damaged mitochondria structure.

303

Mitochondrial number and structure can be regulated by mitochondrial biogenesis and

304

dynamics. Mitochondrial biogenesis is accomplished by dual regulation of nuclear genes (nDNA)

305

and mitochondrial genes (mtDNA) (Duchen, 2004). The majority of mitochondrial proteins (more

306

than 1500) are encoded by nuclear genes, and mtDNA only encode 13 proteins, all of them are

307

essential subunits of respiratory complexes I, III, IV and V (Bohr, 2002; Scarpulla, 2011). Thus it

308

can be seen, mitochondrial biogenesis can expand mitochondrial mass, promote mitochondrial

309

oxidative phosphorylation and ATP synthesis, which are closely related to the regulation of

310

mitochondrial function. This process is dependent on the interplay between transcription factors

311

and coactivators (Scarpulla, 2011). Peroxisome proliferator-activated receptor gamma coactivator

312

1α (PGC-1α) is a master regulator of mitochondrial biogenesis and function (Liang and Ward,

313

2006). Several studies found PGC-1α overexpression can promote mitochondrial biogenesis and

314

increase mitochondrial respiratory chain activities in vivo and vitro (Srivastava et al., 2009;

315

Viscomi et al., 2011). Nuclear respiratory factor 1 (Nrf1), a PGC1-downstream-target, is

316

associated with the expression of nuclear genes encoding subunits of the mitochondrial respiratory

317

complexes (Scarpulla, 2008). Furthermore, Nrf1 can regulate the transcription of mitochondrial

318

transcription factor A (Tfam). Tfam binds to mtDNA and initiates the expression of mtDNA gene

319

products encoding subunits of oxidative phosphorylation (OXPHOS) enzyme complexes

320

(Vernochet et al., 2012). From our results, AFB1 exposure decreased the mRNA and protein

321

expressions of PGC-1α, Nrf1 and Tfam, suggesting AFB1 disturbed testicular mitochondrial 16

322

biogenesis and then impaired mitochondrial respiratory function in mice. In addition, several lines

323

of evidence proved PGC-1α are powerful suppressors of ROS. PGC-1α null mice have an

324

increased sensitivity to oxidative stress, while PGC-1α overproduction dramatically protects

325

neural cells from oxidative-stressor-mediated death (St-Pierre et al., 2006). Suppression of

326

PGC-1α also significantly decreased the T secretion in goat Leydig cells, as well as reduced the

327

expressions of key steroidogenesis related genes, while overexpression of PGC-1α showed the

328

opposite effects (An et al., 2019). These evidences indicated that decreased PGC-1α expression

329

may aggravated AFB1-induced reproductive toxicity via inhibiting T production and promoting

330

oxidative stress-mediated mitochondrial damage and cell death.

331

Mitochondria are dynamic organelles which continuously undergo fission/fusion processes

332

for remodeling the mitochondrial morphology, number and subcellular distribution to satisfy

333

cellular energy requirements (Meyer et al., 2017). Mitochondrial fusion of individual

334

mitochondria into dynamic network can improve mitochondrial function, whereas mitochondrial

335

fission has abilities to eliminate damaged mitochondria via mitochondrial autophagy (mitophagy)

336

(Meyer et al., 2017). Mitochondrial fusion allows efficient mixing of mitochondrial content and

337

form a mesh of highly interconnected filaments in an membrane-anchored mitofusins 1 (Mfn1)

338

and optic atrophy 1 (Opa1)-dependent manner. Mfn1 mediates fusion of the outer mitochondrial

339

membrane, while Opa1 mediates fusion of the inner mitochondrial membrane (Luz et al., 2017).

340

Disruption of mitochondrial fusion causes mitochondrial dysfunction and loss of respiratory

341

capacity both in yeast and in mammalian cells, thereby will be unable to participate in ATP

342

synthesis (Hsiuchen et al., 2005; Westermann, 2012). Opa1 can also help the maintenance of

343

mitochondrial cristae structure, whereas loss of Opa1 commit cells to apoptosis without any 17

344

stimulus (Olichon et al., 2003). Shao et al. found CuSO4 exposure disturbed the mitochondrial

345

dynamics balance with decreased gene and protein levels of Mfn1, Mfn2 and Opa1, and

346

eventually caused testicular damage in chickens (Shao et al., 2019). Besides, mitochondrial fusion

347

(Opa1, Mfn2)-deficient nematodes were hypersensitive to AFB1 and significantly inhibited its

348

growth, supporting the importance of mitochondrial fusion in limiting AFB1 toxicity (Luz et al.,

349

2017). Coincident with the above studies, we found that AFB1 exposure significantly lowered the

350

mRNA and protein expressions of Mfn1 and Opa1, demonstrating AFB1 inhibited mitochondrial

351

fusion. The inhibition of mitochondrial fusion induced by AFB1 was also in parallel with the

352

phenomenon of mitochondrial structural damage, decreased respiratory capacity and ATP levels

353

loss in mice testis. Furthermore, MMP loss also leads to Opa1 degradation and inhibit

354

mitochondrial fusion (Head et al., 2009), therby speculated that AFB1 interfered mitochondrial

355

fusion was partly involved with decreasing MMP in mice testis. Dynamin related protein 1 (Drp1)

356

is the major executor in fission, which is recruited to the mitochondrial surface, forms helical

357

oligomers and undergoes self-interaction mediated GTP hydrolysis to drive the scission of both

358

inner and outer mitochondrial membranes (Serasinghe and Chipuk, 2017). Owing Drp1 is mostly

359

cytosolic, there is a requirement for a mitochondrial membrane anchored receptor to recruit Drp1

360

to the mitochondria for fission, such as mitochondrial fission 1 (Fis1) (Serasinghe and Yoon,

361

2008). It has been shown that impaired mitochondria theoretically undergo asymmetric

362

mitochondrial fission, and then the fragmented mitochondria are removed by selective mitophagy,

363

the process has benefits to maintain a healthy mitochondrial population and bioenergetic capacity

364

(Twig et al., 2008). Ikeda et al. found Drp1 loss induced accumulation of damaged mitochondria

365

in vitro and vivo, which induced mitochondrial dysfunction, cell apoptosis, even mice death (Ikeda 18

366

et al., 2015). The current data concur with the previous studies, significantly lowered mRNA and

367

protein expressions of Drp1 and Fis1 were found in AFB1-exposed mice testis, demonstrating

368

AFB1 may inhibit the clearance of abnormal mitochondria, and resulted in mitochondrial

369

dysfunction.

370

Damage to the mitochondrial structure is bound to disturb its normal function. ATP level

371

directly reflects the mitochondrial functional state. ATP is mainly produced by OXPHOS through

372

the mitochondrial ETC (Chinopoulos and Adam-Vizi, 2010). The ETC is mainly includs NADH

373

dehydrogenase

374

ubiquinone-cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) (Zorov

375

et al., 2014). These enzymes transfer electrons, establishing a proton gradient to drive the

376

synthesis of ATP (Duchen, 2004). Damage to any enzymes of ETC can inhibit ATP generation

377

and result in mitochondria energy metabolism disorder (Cuperus et al., 2010). Shi et al. found that

378

the activities of hepatic mitochondrial complexes I-IV were significantly decreased after ducklings

379

were treated with AFB1, eventually lead to hepatic mitochondrial dysfunction (Shi et al., 2012).

380

AFB1 exposure inhibited mitochondrial complex II and IV activities in rat liver, thereby disrupting

381

mitochondrial respiratory functions (Sajan et al., 1995). Baldissera et al. found AFB1 decreased

382

cerebral ATP levels in silver catfish (Baldissera et al., 2018). Similarly, our results presented that

383

AFB1 exposure inhibited activities of complexes I-IV and ATP generation, indicating AFB1

384

impaired mitochondrial function and cut off energy supply. Furthermore, the MMP is generated

385

by complexes I, III and IV during OXPHOS (Zorova et al., 2018), indicating that the drop of

386

MMP induced by AFB1 may be because of the damaged the ETC of mitochondria. Besides ATP,

387

ROS are generated as by-products during the process of ATP production in mitochondria, whereas

(complex

I),

succinate-ubiquinone

19

oxidoreductase

(complex

II),

388

excessive ROS levels cause irreversible mitochondrial impairment, insuffcient ATP generation

389

and eventually induce cell death (Zhao et al., 2019). Xu et al. found decreased ATP production

390

and induced liver injury were related with ROS accumulation in rat liver mitochondrial (Xu et al.,

391

2017). Thus, we speculated that AFB1-induced mitochondrial dysfunction and ATP loss may be

392

due to excessive ROS production.

393

In conclusion, the mitochondrial damage were involved in AFB1-induced testicular damage

394

and spermatogenesis disorder in mice, which shed new light on the potential mechanisms of

395

reproductive toxicity caused by AFB1.

396

Conflicts of interest

397

398

399 400

The authors declare no competing financial interest.

Acknowledgments This study was supported by the “Young Talent” Project of Northeast Agricultural University (18QC44);

and

the

National

Youth

20

Foundation

of

China

(31902332).

401

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28

572 573 574 575 576 577 578 579

Fig. 1 AFB1 caused testicular damage and spermatogenesis disorder in mice. Male mice were treated with 0, 0.375, 0.75 or 1.5 mg AFB1/kg body weight for 30 d. (A) Representative photographs of testis, (B) The final body weight of the mice, (C-D) The mice testis and epididymides volume, (E) Representative micrograph of seminiferous tubule in mice testis (200×). (F) Representative micrograph of cauda epididymides (200×), (G) Johnsen score in each group, (H) Representative micrograph of sperm morphology (400×). CG control group, LG low-dose group, MG mid-dose group, HG high-dose group. The data were expressed as mean ± SD. *P < 0.05 versus

CG,

**P

<

29

0.01

versus

CG.

580 581

Fig. 2 AFB1 induced oxidative stress in mice testis. Male mice were treated with 0, 0.375, 0.75 or 1.5 mg AFB1/kg

582

body weight for 30 d. (A) H2O2 levels, (B) SOD activities, (C) GSH levels. CG control group, LG low-dose group,

583

MG mid-dose group, HG high-dose group. The data were expressed as mean ± SD. *P < 0.05 versus CG, **P <

584

0.01

versus

30

CG.

585 586

Fig. 3 AFB1 caused testicular ultrastructural damage in mice. Male mice were treated with 0, 0.375, 0.75 or 1.5 mg

587

AFB1/kg body weight for 30 d. (A) The ultrastructure of Leydig cells in mice testis, (B) The ultrastructure of germ

588

cells in mice testis. CG control group, LG low-dose group, MG mid-dose group, HG high-dose group. Red arrows

589

indicate

abnormal

mitochondria.

(Bar

31

=

2

μm)

590 591

Fig. 4 AFB1 reduced MMP, mitochondrial complexes I-IV activities and ATP contents. Male mice were treated

592

with 0, 0.375, 0.75 or 1.5 mg AFB1/kg body weight for 30 d. CG control group, LG low-dose group, MG mid-dose

593

group, HG high-dose group. The data were expressed as mean ± SD. *P < 0.05 versus CG, **P < 0.01 versus CG.

32

594 595

Fig. 5 AFB1 inhibited mitochondrial biogenesis in mice testis. Male mice were treated with 0, 0.375, 0.75 or 1.5

596

mg AFB1/kg body weight for 30 d. (A) Representative image of PGC-1α, Nrf1 and Tfam. (B) Relative protein

597

expression levels to GAPDH. (C) PGC-1α, Nrf1 and Tfam mRNA expressions. CG control group, LG low-dose

598

group, MG mid-dose group, HG high-dose group. The data were expressed as mean ± SD. *P < 0.05 versus CG,

599

**P

<

0.01

33

versus

CG.

600 601

Fig. 6 AFB1 inhibited mitochondrial dynamics in mice testis. Male mice were treated with 0, 0.375, 0.75 or 1.5 mg

602

AFB1/kg body weight for 30 d. (A) Representative image of Drp1, Fis1, Mfn1 and Opa1. (B) Relative protein

603

expression levels to GAPDH. (C) Drp1, Fis1, Mfn1 and Opa1 mRNA expressions. CG control group, LG low-dose

604

group, MG mid-dose group, HG high-dose group. The data were expressed as mean ± SD. *P < 0.05 versus CG,

605

**P

<

0.01

34

versus

CG.

606 607

Graphical Abstract

35