Aflatoxin B1 impairs sperm quality and fertilization competence

Accepted Manuscript Title: Aflatoxin B1 impairs sperm quality and fertilization competence Authors: A. Komsky-Elbaz, M. Saktsier, Z. Roth PII: DOI: Reference:

S0300-483X(17)30330-X https://doi.org/10.1016/j.tox.2017.11.007 TOX 51975

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Toxicology

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Please cite this article as: Komsky-Elbaz, A., Saktsier, M., Roth, Z., Aflatoxin B1 impairs sperm quality and fertilization competence.Toxicology https://doi.org/10.1016/j.tox.2017.11.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Aflatoxin B1 impairs sperm quality and fertilization competence Running title: AFB1 effects on sperm fertilization competence

A. Komsky-Elbaz1, M. Saktsier1, and Z. Roth1,*

1

Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and

Environment, The Hebrew University, Rehovot 76100, Israel

*Correspondence: Zvi Roth, E-mail: [email protected] Prof. Zvi Roth, Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, the Hebrew University, Rehovot 76100, Israel. Phone: 972-8-9489103; Fax: 972-8-9465763

Abstract Aflatoxins are poisonous byproducts of the soilborne fungus Aspergillus, involved in the decomposition of plant materials. Aflatoxins can be found in various food products, such as maize, sorghum, millet, rice and wheat. AFB1 is the most toxic of these, classified as a carcinogen and mutagen for both humans and animals. AFB1 has been detected in human cord blood and placenta; however, its toxic effect on sperm is less known. The current study examines sperm responses associated with AFB1 exposure. These included acrosome integrity and function, mitochondrial polarity, DNA fragmentation,

fertilization

competence

and

early

embryonic

development.

Spermatozoa were obtained from bull ejaculate and epididymis and capacitated in vitro

for 4 h with 0, 0.1, 1, 10 and 100 µM AFB1. Following capacitation, acrosome reaction (AR) was induced by Ca2+ ionophore. The integrity and functionality of sperm were examined simultaneously by florescent staining. A Halosperm DNA fragmentation kit was used to evaluate DNA integrity. An in-vitro culture system was used to evaluate fertilization competence and blastocyst formation rate, using bovine oocytes. Findings indicate dose-responsive variation among compartments to AFB1 exposure. Sperm viability, expressed by integrity of the plasma membrane, was lower in sperm isolated from ejaculate or epididymis after culturing with AFB1. Exposure to AFB1 reduced the proportion of sperm from the epididymis tail undergoing acrosome reaction induced by Ca2+ ionophore. AFB1 impaired mitochondrial membrane potential (ΔΨm) in sperm isolated from ejaculate and the epididymis tail. Exposing ejaculated sperm to AFB1 increased the proportion of sperm with fragmented DNA and reduced the proportion of embryos that cleaved to the 2- to 4-cell stage, 42 h postfertilization, however, the proportion of embryos that developed to blastocysts, 7 days postfertilization, did not differ among groups. The findings explore the harmful effects of AFB1 on sperm viability, ΔΨm and DNA integrity associated with fertility competence. We postulate that AFB1-induced fragmentation in paternal DNA might have a carryover effect on the quality of developing embryos. Further evaluation for the quality of blastocysts derived from sperm exposed to AFB1 is warranted.

Key words: Aflatoxin B1 / sperm / acrosome reaction / mitochondrial membrane potential / embryonic development / blastocyst.

1. Introduction

Aflatoxins are low molecular weight compounds produced by the fungi Aspergillus flavus and Aspergillus parasiticus (Dai et al., 2017; Kew, 2013). Under humid conditions, these fungi grow on food grains, fruits, nuts and other crops (Schenzel et al., 2012; Strosnider et al., 2006). Aflatoxin contamination can occur at any stage of food production, from preharvest to storage (Iimura et al., 2017; Shuaib et al., 2010) i.e., present in the food chain (Verma, 2004). According to the US Food and Drug Administration, the permissible amount of aflatoxins in human foods is 4–30 ppb (0.01–0.1 µM), while grains for animal feeding can have up to 300 ppb (1 µM) (Williams et al., 2004). Aflatoxins are of great concern to public health because they accumulate in the body and can be found in edible tissues, such as liver and muscles, as well as in animal food products such as milk and eggs (Fan et al., 2015; Giovati et al., 2015; Monson et al., 2016; Rajkovic et al., 2007; Verma and Nair, 2001; Yuan et al., 2016). Moreover, aflatoxins has been found in human maternal breast milk, and maternal and cord blood (Shuaib et al., 2010), and can apparently enter the developing fetus in humans and animals (Verma, 2004). These unavoidable food contaminants are highly stable chemicals (Williams et al., 2004) and are usually found as a mixture of aflatoxin B1 (AFB1), AFB2, AFG1 and AFG2 (Wu et al., 2016). Of these, AFB1 is considered the most toxic for mammals owing to its hepatotoxic, teratogenic, mutagenic and immunosuppressive properties (Raisuddin et al., 1993; Shen et al., 1994; Macé et al., 1997; Meissonnier et al., 2008). AFB1 is known as a carcinogenic compound (CarvajalMoreno, 2015; Groopman et al., 1996) and might lead to aflatoxicosis following chronic exposure (Agnes and Akbarsha, 2003). AFB1 is also defined as an endocrine disruptor as it affects the cytochrome P450 enzymes, involved in steroid synthesis (Storvik et al., 2011).

Aflatoxins were found in 40% of semen samples collected from infertile men, relative to only 8% of those collected from fertile men (Ibeh et al., 1994). The former were associated with abnormal sperm count and morphology, as well as reduced motility (Ibeh et al., 1994; Nduka et al., 2001). Oral administration of AFB1 (50 µg/kg BW per day) for 35 days resulted in spermatotoxic effects on mouse epididymal sperm, expressed as reduced sperm concentration and motility and increased sperm abnormalities (Agnes and Akbarsha, 2003). Intramuscular injection of AFB1 (0.2–0.25 ml/day for 55 days) into male rats resulted in extrusion of the outer dense fibers and axonemal microtubule doublets of the cauda epididymal sperm flagellum (Faisal et al., 2008), suggesting negative effects on spermatozoa throughout the early stages of spermatogenesis. Spermatogenesis is an intricate and coordinated process by which thousands of spermatozoa are produced daily within the testis seminiferous tubules. Spermatozoa gain fertilization ability during their transit through the epididymal compartments. They are then stored in the tail of the epididymis until ejaculation (Cornwall, 2014). As spermatogenesis is a lengthy process, it is a potential target for environmental toxins at various developmental stages. Certain toxins, including AFB1, may pass through the blood–testis barrier and negatively affect spermatogenesis (Ataman et al., 2014a). Nevertheless, the direct effect of AFB1 on spermatozoa has been less studied and the existing data are limited to basic parameters of sperm characterization. In the current study, we examined the direct effects of AFB1 on acrosome reaction (AR), mitochondrial membrane potential and DNA integrity. In addition, we performed an invitro examination of the effect of AFB1 exposure on sperm fertilization capacity and early embryonic development.

2. Materials and methods 2.1. Reagents and materials All reagents were purchased from Sigma (Rehovot, Israel), unless otherwise specified. AFB1 (lot #1162-65-8, purity: ≥98.9%) was purchased from Cayman Chemical (Ann Arbor, MI, USA). A 10 mM stock solution of AFB1 was prepared in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions (Sigma). Sperm membrane integrity was evaluated using fluorimetric probes as previously described (Komsky-Elbaz and Roth, 2016): (1) double-stranded DNA by 4',6-diamidino-2-phenylindole (DAPI); (2) plasma membrane integrity by propidium iodide (PI); (3) AR by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC–PSA); (4) mitochondrial membrane potential (ΔΨm) by 5,5',6,6'-tetra-chloro1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide fluorescent probe (JC-1; ENZOBiochem, New York, NY, USA). The culture media Hepes–Tyrode's lactate (TL) and in-vitro fertilization–TL (IVF–TL) were prepared in our laboratory as previously described (Kalo and Roth, 2017). Standard oocyte maturation medium (OMM) was made up of TCM-199 and Earle's salts supplemented with 10% (v/v) heat-inactivated fetal calf serum (Promega, Madison, WI, USA), 0.2 mM sodium pyruvate, 50 μg/μl gentamicin, 2.2 g/l sodium bicarbonate, 2 μg/ml 17-ß estradiol and 1.32 μg/ml FSH (Folltropin-V; Bioniche Animal Health, Belleville, Ontario, Canada). Potassium simplex optimized medium (KSOM) contained 95 mM NaCl, 2.5 mM KCl, 0.35 mM KH2PO4, 0.2 mM MgSO4·7H2O, 0.8% (v/v) sodium lactate, 0.2 mM sodium pyruvate, 0.2 mM D(+)glucose, 25 mM NaHCO3, 0.01 mM phenol red, 1 mM L-glutamine, and 0.01 mM EDTA supplemented with 1.7 mM CaCl2·2H2O, 0.1 mg/ml polyvinyl alcohol, 100

U/ml penicillin-G, 0.1 mg/ml streptomycin, 10 μl/ml essential amino acids and 5 μl/ml nonessential amino acids (Life Technologies, Carlsbad, CA, USA).

2.2. Sperm preparation Bovine sperm was supplied by "Sion" Artificial Insemination Center (Hafetz-Haim, Israel). All of the experiments were performed in accordance with the 1994 Israeli guidelines for animal welfare. Ejaculated bull sperm was obtained with an artificial vagina, and the "swim up" technique was applied to obtain motile sperm. Sperm cells were washed three times by centrifugation (600g for 10 min at 25ºC) in NKM buffer (110 mM NaCl, 5 mM KCl, 20 mM MOPS [3-N-morphilino propanesulfonic acid, pH 7.4]) and allowed to swim up after the last wash. The washed cells were counted and maintained at 39ºC until use. Only semen that contained at least 80% motile sperm cells was used in the experiments.

2.3. Extraction of epididymal spermatozoa Bovine testes were brought from the slaughterhouse in 4ºC saline solution. The epididymis was recovered from the testes immediately upon arrival to the laboratory. The epididymis head, body and tail were dissected to facilitate sperm release from each individual compartment. The epididymis content was transferred into tubes and washed twice by centrifugation (600g for 10 min at 25ºC) in NKM buffer. The washed cells were counted and maintained at 39ºC until use.

2.4. Sperm capacitation In-vitro capacitation of bovine sperm was induced as described previously (Parrish et al., 1999; Winer, 1988). Briefly, sperm pellets were resuspended to a final concentration

of 108 cell/ml in mTALP (modified Tyrode's solution containing 100 mM NaCl, 3.1 mM KCl, 1.5 mM MgCl2, 0.92 mM KH2PO4, 25 mM NaHCO3, 20 mM HEPES pH 7.4, 0.1 mM sodium pyruvate, 21.6 mM sodium lactate, 10 IU/ml penicillin, 1 mg/ml bovine serum albumin, 20 µg/ml heparin, 2 mM CaCl2). Cells were incubated in mTALP for 4 h at 39ºC with 5% CO2. Sperm capacitation state was confirmed by examining the sperm's ability to undergo AR, induced by addition of 20 µM Ca++ ionophore A23187 for an additional 20 min of incubation. Note that sperm from epididymis head and body does not have the ability to capacitate (Aitken et al., 2007). Thus, only sperm retrieved from the epididymis tail or ejaculate was subjected to in-vitro capacitation.

2.5. Treatments Sperm (108 cell/ml) was exposed to 0.1, 1, 10 or 100 µM final concentration of AFB1. These concentrations were guided by data from well-known experimental model studies (Adedara et al., 2014; Faisal et al., 2008; Feng et al., 2016; Liu et al., 2014; Zimmermann et al., 2014). Following the manufacturer's instructions, AFB1 was dissolved in DMSO (0.01% v/v maximal concentration). This vehicle was not found to have a deleterious effect on sperm viability at the final concentrations used in the current study. Sperm incubation was performed in mTALP with or without AFB1 at 39ºC under an atmosphere of 5% CO2 in air. Sperm were analyzed after 0, 2 and 4 h of incubation, and an additional 20-min incubation with 20 µM Ca++ ionophore as a positive control for AR.

2.6. Simultaneous fluorimetric assessment of sperm membranes Simultaneous fluorimetric assessment of sperm membranes (plasma, acrosomal and mitochondrial) was performed as described previously (Celeghini et al., 2007), with

some modifications. Briefly, sperm pellets were resuspended to a final concentration of 25 x 106 cell/ml in mTALP. A 150-µl aliquot of semen diluted in mTALP medium was put into a warmed microcentrifuge tube. DAPI (17 µl of a 0.1 mg/ml solution) was added and the sample was incubated for 10 min at 37ºC. The sample was then centrifuged and 100 µl mTALP medium was added to the pellet. In addition, 3 µl of PI (0.5 mg/ml), 2 µl of JC-1 (153 µM) and 50 µl of FITC–PSA (1 mg/ml) were added. The sample was incubated for 10 min at 37ºC, then centrifuged and the pellet was resuspended in 40 µl mTALP medium. A 10-µl sample was put on a glass slide, coverslipped and immediately evaluated by epifluorescent microscopy (Nikon Eclipse, TE2000-u, Tokyo, Japan) using Nis Elements software (Nikon, Tokyo, Japan) and equipped with a digital camera (Nikon DXM1200F), with excitation at 450–490 nm and emission at 515–565 nm using a triple filter. At least 200 sperm cells were examined per slide and classified based on the fluorescence emitted from each probe. The cells were scored by a single skilled individual using ImageJ software (version 1.47v, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) and manual counting (Fig. 1).

2.7. Assessment of DNA fragmentation Sperm DNA damage was assessed with the sperm chromatin dispersion assay, known as the Halosperm G2® kit (Halotech DNA, S.L., Madrid, Spain), according to the manufacturer's instructions. Briefly, the sperm sample was diluted in NKM medium to a maximum concentration of 20 million/ml. Eppendorf tubes were placed in a water bath at 90–100°C for 5 min to fully melt the agarose (100 μl per tube). After 5 min of incubation, 50 μl of the diluted semen sample was transferred to the tube with melted agarose and mixed gently with a pipette. An 8-μl drop of the cell suspension was

immediately placed in the sample well and covered with a coverslip. Slides were then placed on a cold plate in the refrigerator at 4°C for 5 min to solidify the agarose with the embedded sperm cells. After taking the slides out of the refrigerator, the coverslips were gently removed and the slides were placed horizontally in an elevated position. Then denaturing agent was applied, fully covering the well, and incubated for 7 min. The slides were then immersed in a lysis solution and incubated for 20 min. After washing with abundant distilled water for 5 min, the slides were dehydrated in increasing concentrations of ethanol (70 and 100% for 2 min each) and then air-dried. For the bright-field microscopy in this sperm chromatin dispersion assay, the slides were covered with two staining solutions for 7 min each. Slides were then washed and allowed to dry. Spermatozoa with a big or medium-sized haloes were considered normal, without fragmented DNA, whereas spermatozoa with a small or no halo and exhibiting degeneration were considered to have fragmented DNA.

2.8. In-vitro embryo production Oocytes were matured in OMM as previously established in our laboratory (Kalo et al., 2015). Briefly, ovaries were obtained from a local abattoir and transported to the laboratory within 60 to 90 min in physiological saline solution (0.9% w/v NaCl at 38.5°C with 50 μg/ml penicillin–streptomycin). Cumulus oocyte complexes (COCs) were aspirated from 3- to 8-mm follicles with an 18-gauge needle attached to a 10-ml syringe. COCs were collected into Hepes–TL supplemented with 0.3% (w/v) bovine serum albumin, 0.2 mM sodium pyruvate and 0.75 mg/ml gentamicin (Hepes–TALP) at 38.5°C. At the end of the collection, COCs (n = 30–60 per group; 6 replicates) with at least three layers of cumulus surrounding a homogeneous cytoplasm were selected for in-vitro maturation.

At the end of maturation, COCs were washed three times in Hepes–TALP and transferred in groups of 30 oocytes to 4-well plates containing 600 μl IVF–TALP and 25 μl PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 μM epinephrine in 0.9% NaCl) per well. For IVF, COCs were co-incubated with spermatozoa capacitated in the absence or presence of 10 µM AFB1 (~1 × 106) for 18 h at 38.5°C in a humidified atmosphere with 5% CO2. After fertilization, putative zygotes were denuded of cumulus cells by gentle vortexing in Hepes–TALP containing 1000 U/ml hyaluronidase, and placed in groups of 10 in 25-μl droplets of KSOM. All embryo droplets were overlaid with mineral oil and cultured for 7 days at 38.5°C in an atmosphere of humidified air with 5% CO2, 5% O2. Cleavage rates were evaluated 42–44 h postfertilization according to the proportion of 2- to 4-cell-stage embryos, and blastocyst-formation rates were evaluated according the proportion of embryos developed to blastocysts 7 days postfertilization.

2.9. Statistical analysis All values for incidence of the various classes of spermatozoa in the fluorimetric assessment were converted to percentages and normalized to their percentage at T0 (time 0, preincubation time point) before analysis. Data were analyzed by JMP-7 software (SAS Institute Inc., 2004, Cary, NC, USA) and an ANOVA model using concentration, time and their interaction as fixed effects, and bull as a random effect. Posthoc comparisons were performed by Contrast t-test (LS-MEANS Student's t-test). Data are expressed as mean ± SD of the percentages. Data of the proportion of cleavage to 2- to 4-cell-stage embryos and blastocyst-formation rate were arcsine-transformed before being subjected to one-way ANOVA followed by Tukey–Kramer test. Data are presented as mean ± SEM. Overall comparison of 7-day blastocyst distribution between

groups for incidence data was performed by chi-square followed by Pearson test. For all analyses, P < 0.05 was considered significant; P-values between 0.05 and 0.1 were also reported as trends that might be real and worthy of note. For each set of experiments, sperm samples from at least three bulls were tested.

3. Results A total of 57,891 sperm cells were evaluated using simultaneous fluorimetric assessment of their membranes (plasma, acrosomal and mitochondrial): 19,402 cells were obtained from ejaculates and the rest of the cells were obtained from the epididymis tail, body and head (16,143, 11,257 and 11,089, respectively; Table I). DNA integrity was evaluated in 1,957 ejaculated spermatozoa. The maximal concentration of the solvent used as vehicle (0.01% DMSO) did not have any significant effect on any of the examined parameters when tested as a solvent-effect control.

3.1. Effect of AFB1 on ejaculated sperm Exposure to 100 µM AFB1 for 2 h increased the proportion of dead sperm relative to the control group (25.4 ± 9.3 vs. 17.2 ± 12.5; P ≤ 0.05; Fig. 2A). Exposure to AFB1 for 4 h (1, 10 or 100 µM) increased the proportion of dead sperm relative to the control group (27.0 ± 12.0, 28.6 ± 10.1, 28.8 ± 3.4%, respectively, vs. 15.2 ± 12.6%; P ≤ 0.006; Fig. 2A). Exposure to 10 µM AFB1 for 2 h significantly increased the ΔΨm of sperm isolated from ejaculate relative to the control group (2.19 ± 2.24% vs. 0.25 ± 0.22%, P ≤ 0.02; Fig. 2B). AFB1 had no significant effect on pseudo- or Ca++ ionophore-induced AR of sperm isolated from ejaculate (Fig. 2C).

3.2. Effect of AFB1 on epididymal sperm

AFB1 increased the proportion of sperm with damaged plasma membranes in a doseresponsive manner (Fig. 3). Exposing sperm isolated from the head of the epididymis to AFB1 (1, 10 or 100 µM) significantly elevated the proportion of dead sperm after 2 h, relative to the control group (21.0 ± 1.7, 20.6 ± 2.4 and 22.1 ± 6.5, respectively, vs. 11.4 ± 3.1%, P ≤ 0.003; Fig. 3A'). The same pattern was recorded after 4 h of incubation with 1 or 10 µM AFB1, relative to the control group (22.2 ± 12.4 and 25.9 ± 9.1, respectively, vs. 14.3 ± 6.7%, P ≤ 0.02; Fig. 3A'). Sperm isolated from the body of the epididymis were affected only by 10 µM AFB1, expressed as a higher proportion of dead cells relative to the control group (23.1 ± 13.6 vs. 7.6 ± 0.3%, P ≤ 0.05). The rate of dead sperm at 2 h did not differ from that recorded at 4 h of incubation (19.6 ± 7.0 vs. 7.4 ± 1.9%, P ≤ 0.05; Fig. 3A''). Exposing sperm isolated from the tail of the epididymis to AFB1 (10 or 100 µM) significantly elevated the proportion of dead sperm after 2 h, relative to the control group (14.4 ± 1.4 and 17.7 ± 3.6%, respectively, vs. 6.8 ± 4.7%, P ≤ 0.02; Fig. 3A''') and after 4 h of exposure to 1, 10 or 100 µM (16.3 ± 0.2, 24.1 ± 10.0 and 19.2 ± 5.0%, respectively, vs. 8.2 ± 2.2%, P ≤ 0.0001; Fig. 3A'''). Exposure to 1 µM AFB1 for 4 h significantly increased the ΔΨm of sperm isolated from the epididymis tail relative to the control group (0.12 ± 0.09% vs. 0.02 ± 0.02%, P ≤ 0.05; Fig. 3B). AFB1 had no significant effect on pseudo-AR of sperm isolated from the epididymis tail. In contrast, AFB1 significantly decreased the proportion of sperm that reacted to Ca++ ionophore and underwent induced AR after incubation with the highest AFB1 concentration (100 µM), compared to the control group (33.0 ± 9.2 vs. 50.1 ± 11.0%, P ≤ 0.05; Fig. 3C).

3.3. Effect of AFB1 on DNA fragmentation in ejaculated sperm

Exposing ejaculated sperm to 10 µM AFB1 for 4 h significantly increased the proportion of sperm with fragmented DNA compared to the control group (68.0 ± 16.1 vs. 15.5 ± 1.3%, P ≤ 0.001; Fig. 4).

3.4. Effect of sperm exposure to AFB1 on fertilization and early embryonic development Cleavage and blastocyst-formation rates were evaluated after fertilization of 493 oocytes. The proportion of oocytes that were fertilized and cleaved to 2- to 4-cell-stage embryos was lower after fertilization with sperm exposed to 10 µM AFB1 as compared to the control group (70.9 ± 3.9 vs. 85.1 ± 2.8%, P < 0.005; Fig. 5). Blastocyst-formation rate did not differ from controls after fertilization with sperm exposed to AFB1 (19.4 ± 7.6 vs. 18.1 ± 9.3%, not significant; Fig. 6), nor did the distribution into different embryonic stages (early blastocyst and blastocyst).

4. Discussion The gradual decline in fertility of human and farm animals over the past few decades coincides with intensive industrial and agricultural development (Bousquet et al., 2004; Swan et al., 2000). Multiple environmental factors affect sperm function and fertility (Agnes and Akbarsha, 2003; Mathuria and Verma, 2008). The findings of the current study demonstrate direct effects of foodborne toxins on spermatozoa while exploring the potential hazards associated with AFB1 exposure. In particular, exposure of sperm to low concentrations of AFB1 for a few hours resulted in decreased sperm viability and hyperpolarization of the mitochondrial membrane, most markedly with ejaculated sperm, suggesting that not only the earlier stages of spermatogenesis are affected.

Exposure of fresh semen to 10 µM AFB1 prefertilization resulted in sperm DNA damage and impaired fertilization competence, expressed by reduced proportion of oocytes that cleaved to 2- and 4-cell stage embryos. Although the mechanism underlying cellular damage caused by AFB1 has not been fully elucidated, the findings shed light on some potential alterations caused by AFB1 exposure. These include impairment of (1) membrane integrity, (2) mitochondrial function, (3) DNA integrity and (4) fertilization competence.

4.1. Effect of AFB1 on cell viability Cell viability depends mainly on the integrity and functionality of cell membranes. Therefore, AFB1-induced damage in the plasma membrane might reduce cell viability and function, which in turn can lead to cell death. Here we report that exposure to low concentrations of AFB1 (1 or 10 µM) reduced the viability of sperm isolated from the epididymis compartments (i.e., head, body and tail) and of those isolated from ejaculate. This was determined by PI fluorescent dye, which penetrates cells with damaged membranes and binds to the DNA. Direct lysis of the sperm cell membrane and loss of lysozyme, an enzyme that facilitates spermatozoon penetration of the oocyte, have been suggested (Ibeh et al., 1994; Shuaib et al., 2010). Other studies have documented AFB1reduced viability of ram spermatozoa (Tajik et al., 2007) and broiler lymphocytes (Zimmermann et al., 2014). Exposure to high doses of AFB1 (4000 µg/l) for 48 h reduced the viability of human corneal epithelial cells (Bossou et al., 2017). On the other hand, feeding with lower concentrations of AFB1 (300–500 µg/l) for 3 weeks did not affect the viability of broiler breeder sperm (Manafi et al., 2012). While not clear, the discrepancy between these studies might be due to differences in cell types and aflatoxin digestibility and metabolism between animal species (Ataman et al., 2014a).

4.2. Effect of AFB1 on mitochondria The mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species production, calcium signaling and apoptosis. Alterations in mitochondrial function are associated with physiological dysfunction, including male and female infertility (Ramalho-Santos et al., 2009). Here we report a transient increase in ΔΨm (hyperpolarization) due to AFB1 exposure. Mitochondrial hyperpolarization was noted when sperm isolated from the ejaculate was exposed to 10 µM AFB1. A similar effect was noted for sperm isolated from the epididymis tail and exposed to 1 µM AFB1, suggesting that sperm from the latter origin is more sensitive to the toxin. Sperm undergoes maturation as it passes through the epididymis, involving mitochondrial activation (Aitken et al., 2007). In the current study, the effect of AFB1 was only evaluated in sperm isolated from the tail because sperm from the tail of the epididymis, but not the head or body compartments, contains active mitochondria. Nevertheless, a toxic effect on the mitochondria at early stages of sperm maturation should not be ruled out. Toxin-induced alteration in mitochondrial polarity has been reported for various cells exposed to other compounds. For instance, exposing human liver carcinoma HepG2 cells to 0.1 µM bisphenol A for 72 h resulted in mitochondrial hyperpolarization (Huc et al., 2012). Bovine spermatozoa exposed to 1 µM of the pesticide atrazine or its metabolite diaminochlorotriazine (1 µM) expressed higher membrane potential (Komsky-Elbaz and Roth, 2016). AFB1 administration (0.75 mg/kg BW per day) for 30 days disrupted the mitochondrial membrane and induced cellular apoptosis in rat cardiomyocytes (Ge et al., 2017). A study in chickens reported that dietary AFB1 (0.6 mg/kg BW per day) for 21 days induces excessive apoptosis in bursa of Fabricius cells via mitochondrial apoptotic pathways (Yuan et al., 2016).

Taken together, toxin-induced cellular stress can lead to transient mitochondrial hyperpolarization and increased production of free radicals, which in turn might lead to apoptosis.

4.3. Effect of AFB1 on sperm capacitation and acrosome reaction During their ascent in the female reproductive tract, spermatozoa undergo capacitation, a maturation process which provides the sperm with the ability to bind to the zona pellucida and to then undergo AR (Aitken et al., 2007; Cornwall, 2014). Both capacitation and AR are essential processes for fertilization (Breitbart, 2003; Patrat et al., 2000). We determined the spermatozoon's ability to undergo spontaneous or pseudo-AR (i.e., AR induced without a known, controlled stimulation) (Abou-haila and Tulsiani, 2009) vs. induced AR (Maravilla-Galván et al., 2009) to examine the stimulatory or inhibitory effects of environmental compounds. Acrosomal damage was previously reported to be significantly higher in ejaculated sperm of rams given feed containing 250 µg/day AFB1 for 3 weeks (Ataman et al., 2014b). In the current study, AFB1 significantly decreased the proportion of sperm that reacted to Ca++ ionophore and underwent induced AR, but this was noted only for sperm obtained from the epididymis tail that exposed to 100 µM AFB1. Acrosome reaction in ejaculated sperm was not affected, most likely due to protective components present in the seminal fluid (Poiani, 2006). Taken together, it seems that the acrosome is not the main target of AFB1.

4.4. Effect of AFB1 on DNA fragmentation DNA is a target molecule for various drugs, pesticides, and toxic compounds (Ma et al., 2017). Here we report, for the first time, on DNA damage in mammalian sperm,

induced by AFB1. Exposing ejaculated sperm to 10 µM AFB1 increased the proportion of sperm with damaged DNA by 4-fold, determined by Halosperm kit. Our findings are in line with previous studies conducted in somatic cells. Human hepatocytes exposed to 10 µM AFB1 for 24 h expressed a high proportion of cells with DNA damage (Yang et al., 2016). A similar pattern was reported for human epithelioid lung cells exposed to 0.82 µM AFB1 (Wang and Cerutti, 1979). Exposure of Caenorhabditis elegans to 10 µM AFB1 resulted in DNA damage in the nematode cells and induced apoptosis in the germ line (Feng et al., 2016). In-vitro studies have shown that AFB1 reduces DNA copy number variants in human leukocytes (Harutyunyan et al., 2015) and increases DNA fragmentation level in broiler lymphocytes (Zimmermann et al., 2014). One potential mechanism for DNA damage that fits our experimental model is direct interaction of AFB1 with the cell DNA, generating reversible non-covalent interactions (Ma et al., 2017). Alternatively, an indirect effect of the AFB1 metabolite is suggested for in-vivo models. In the liver, AFB1 is metabolized by cytochrome P450 enzymes into the genotoxic metabolite 8,9-epoxide-AFB1 (AFBO). Binding of AFBO to DNA forms AFB1–DNA adducts (Feng et al., 2016). A high amount of adducts was found in human placenta and cord blood samples from term, uncomplicated pregnancies, due to dietary exposure (Hsieh and Hsieh, 1993). Binding of AFB1 to DNA in mammalian spermatogenic cells (Sotomayor and Sega, 2000) and epididymal sperm (Faisal et al., 2008) has been suggested to interfere with normal spermatogenesis, which might further result in abnormal sperm and reduced fertilization competence. Taken together, it seems that AFB1 can act directly or indirectly on the sperm, in a developmental stagedependent manner. The proportion of sperm with DNA fragmentation correlates with male fertility and is considered a practical parameter for characterizing semen quality (Sergerie et al.,

2005). During fertilization, the spermatozoon delivers paternal components to the oocyte which are believed to be crucial for oocyte activation and zygote formation, and to further affect embryonic development (Dogan et al., 2015). In-vitro fertilization of rat oocytes with epididymal sperm, capacitated with AFB1 (2–16 ppb), resulted in significantly lower fertilization rates relative to fertilization with untreated sperm (Ibeh et al., 2000). However, in the latter study, embryonic development was not evaluated. Moreover, fertilization was only performed with epididymal sperm, and the effect on the final spermatogenesis product, the spermatozoa, was not evaluated. In the current study, bovine oocytes were fertilized with sperm isolated from fresh ejaculate and capacitated with 10 µM AFB1. The proportion of oocytes that fertilized and cleaved into 2- and 4-cell stage embryos (i.e., first two cleavages) was lower than for the control, but the proportion of cleaved oocytes that further developed to the blastocyst stage did not differ between groups. It is possible that the reduced cleavage rate reported here expresses a delay in embryonic development rather than fertilization failure. In support of this assumption, it has been recently reported that sperm with DNA damage can fertilize the oocyte. Moreover, soon after fertilization, a repair process starts in the oocyte (Uppangala et al., 2016), suggesting that the embryo can repair DNA damage of sperm origin. On the other hand, it has been recently reported that a high proportion of sperm with DNA damage is associated with altered metabolism in the developing blastocyst (Uppangala et al., 2016). These findings suggest that damaged DNA not only reduces sperm fertilization competence, but also negatively affects embryonic development (Ioannou et al., 2016). Given that in the current study the proportion of blastocysts developed from AFB1-treated sperm (i.e., with damaged DNA) did not differ from that in the control group, genomic instability in these embryos cannot be excluded (Adiga et al., 2010).

5. Summary The findings explore the potential hazards associated with exposure of sperm to AFB1. The current study provides evidence that exposure to low AFB1 concentrations for a few hours results in decreased sperm viability, hyperpolarization of the mitochondrial membrane and increased DNA fragmentation. In light of sperm's sensitivity to AFB1, good-quality semen may be damaged by AFB1 through passage in female reproductive tract. Given the importance of sperm DNA integrity to the developing embryo, the quality of these embryos remains an open question. Further evaluation, at the RNA and protein levels, should be performed to evaluate a possible carryover effect to the blastocyst stage.

Authors’ roles A.K.-E. and Z.R. developed the concept, designed the experiments and prepared the manuscript. A.K.-E. and M.S. carried out the experiments, data organization and statistical analyses. All authors read and approved the final manuscript.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgements The authors would like to thank ‘Sion’ Artificial Insemination Center (Hafetz-Haim, Israel) for their help and cooperation, and Prof. Hillary Voet for the statistical consultation.

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Figure captions

Figure 1 Epifluorescence photomicrography of sperm cells stained with fluorescent probes. (A) Live sperm cell with DAPI staining of nucleus and low mitochondrial membrane potential (ΔΨm), stained by fluorescent probe JC-1. (B) Dead sperm cell with damaged plasma membrane stained by PI fluorescent probe and damaged acrosome stained by FITC–PSA fluorescent probe. (C) Live sperm cell with damaged acrosome and low ΔΨm. (D) Acrosome-reacted sperm cell with residual upper staining and high ΔΨm. (E) Acrosome-reacted sperm cell with residual equatorial staining. Scale bars = 10 µM.

Figure 2 Effect of AFB1 on ejaculate sperm. Spermatozoa were isolated from fresh ejaculate and incubated for 4 h with 0.1, 1, 10 or 100 µM AFB1 dissolved in DMSO. (A) Sperm viability was determined with PI fluorescent probe. (B) Mitochondrial membrane potential (ΔΨm) was determined with JC-1 fluorescent probe and presented as the mean proportion of red-stained (high potential) to green-stained (low potential) sperm. (C) Acrosome reaction was determined with FITC–PSA fluorescent probe. Data are presented as mean proportion ± SD, calculated for 3 replicates. At least 200 sperm were analyzed per group.

Figure 3 Effect of AFB1 on sperm isolated from the epididymis. Spermatozoa were isolated from epididymis compartments: head [A'], body [A''] and tail [A''', B, C] and incubated for 4 h with 0.1, 1, 10 or 100 µM AFB1 dissolved in DMSO. (A', A'', A''') Sperm viability was determined with PI fluorescent probe. (B) Mitochondrial membrane potential (ΔΨm) was determined with JC-1 fluorescent probe and presented

as the mean proportion of red-stained (high potential) to green-stained (low potential) sperm. (C) Acrosome reaction was determined with FITC–PSA fluorescent probe. Data are presented as mean proportion ± SD, calculated for 3 replicates. At least 200 sperm were analyzed per group.

Figure 4 Effect of AFB1 on sperm DNA fragmentation. (A) Spermatozoa were incubated for 4 h with 10 µM AFB1 dissolved in DMSO. (B) Sperm DNA fragmentation was determined with the Halosperm kit. (B') Degenerating spermatozoa with small or no halo were considered to have fragmented DNA, while (B'') spermatozoa with large or medium-sized haloes were considered normal, with no fragmented DNA. Data are presented as means ± SD, calculated for 3 replicates, with 200 sperm per group. *P < 0.0001.

Figure 5 Effect of sperm exposure to AFB1 on fertilization. Sperm isolated from fresh semen was exposed to 100 µM AFB1 for 4 h. Bovine oocytes were matured for 22 h and fertilized for 18 h with AFB1-treated or control sperm (~1 × 106). Presented is the percentage of oocytes that cleaved into 2- to 4-cell stage embryos, 42–44  h postfertilization. The experiment included 6 replicates with 30–60 oocytes per replicate per experimental group. Data are presented as means ± SEM. *P < 0.005.

Figure 6 Effect of sperm exposure to AFB1 on early embryonic development. Bovine oocytes were fertilized for 18 h with AFB1-treated or control sperm and incubated for 7 days. Presented is the percentage of embryos that developed to the blastocyst stage on day 7 postfertilization and the distribution to various embryonic stages, out of total

(A, C) or cleaved (B, D) oocytes. The experiment included 6 replicates with 30–60 oocytes per replicate per experimental group. Data are presented as means ± SEM.

Table I. Summary of AFB1 effects on sperm features. Spermatozoa isolated from fresh ejaculate and epididymis compartments (head, body and tail) were incubated for 4 h with or without 1, 10 or 100 µM AFB1. Spermatozoa were collected after 0, 2 and 4 h of incubation (T0, T2, T4, respectively); viability, pseudo- and Ca++-induced acrosome reaction (AR) and mitochondrial membrane potential (ΔΨm) were examined by simultaneous fluorimetric assessment. The table summarizes the exposure times and the AFB1 doses that had significant effects and warranted further investigation. Shaded squares are parameters that were not examined. Epididymis

Ejaculate

Head

Body

Tail

1, 10, 100 µM (T2)

10 µM (T2)

10, 100 µM (T2)

100 (T2)

10 µM (T4)

1, 10, 100 µM (T4)

1, 10, 100 µM (T4)

AR

100 µM (Ca++)

-

ΔΨm

1 µM (T4)

10 µM (T2)

Viability

1, 10 µM (T4)