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Functional and Proteomic Alterations of F1 Capacitated Spermatozoa of Adult Mice Following Gestational Exposure to Bisphenol A Md Saidur Rahman, Woo-Sung Kwon, Do-Yeal Ryu, Amena Khatun, Polash Chandra Karmakar, Buom-Yong Ryu, and Myung-Geol Pang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00668 • Publication Date (Web): 03 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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Journal of Proteome Research

Functional and Proteomic Alterations of F1 Capacitated Spermatozoa of Adult Mice Following Gestational Exposure to Bisphenol A

Md Saidur Rahman†, Woo-Sung Kwon†, Do-Yeal Ryu†, Amena Khatun†, Polash Chandra Karmakar†, Buom-Yong Ryu†, and Myung-Geol Pang†,*

†

Department of Animal Science and Technology, Chung-Ang University, Anseong, Gyeonggi-do 456-756, Republic of Korea

*

Corresponding Author: E-mail: [email protected]; Tel.: +82.316.70.4841; Fax: +82.31.675.9001

ACS Paragon Plus Environment 1

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ABSTRACT Studies regarding bisphenol A (BPA) exposure and male (in)fertility have conventionally focused on modifications in ejaculated spermatozoa function from exposed individuals. However, mammalian spermatozoa are incapable of fertilization prior to achieving capacitation, the penultimate step in maturation. Therefore, it is necessary to investigate BPA-induced changes in capacitated spermatozoa and assess the consequences on subsequent fertilization. Here, we demonstrate the effect of gestational BPA exposure (50 µg, 5 mg, and 50 mg•kg bw-1•day-1) on the functions, biochemical properties, and proteomic profiles of F1 capacitated spermatozoa from adult mice. The data showed that high concentrations of BPA inhibited motility, motion kinematics, and capacitation of spermatozoa, perhaps because of increased lipid peroxidation and protein tyrosine nitration, and decreased intracellular ATP levels and protein kinase-A activity in spermatozoa. We also found that BPA compromised the rates of fertilization and early embryonic development. Differentially expressed proteins identified between BPA-exposed and control groups play a critical role in energy metabolism, stress responses, and fertility. Protein function abnormalities were responsible for the development of several diseases according to bioinformatics analysis. Based on these results, gestational exposure to BPA may alter capacitated spermatozoa function and the proteomic profile, ultimately affecting their fertility potential.

Keywords: Bisphenol A, spermatozoa, capacitation, sperm function, fertility, proteomics


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INTRODUCTION Endocrine disruptors (EDs) are exogenous chemicals or chemical mixtures in the environment, food, and consumer products that can interfere with regular hormone functions in the body. Because normal growth and development requires a healthy endocrine system, exposure to EDs may predispose individuals to many undesirable consequences.1 Exposure to EDs is associated with reproductive abnormalities, problems in breast development, cancer and neoplasia, neuroendocrine disorders, thyroid and metabolic abnormalities, obesity, and cardiovascular diseases.1 In addition, other studies have shown a direct link between ED exposure and infertility, altered sex ratio at birth, defective embryonic development, low sperm count, altered sperm functions, increased number of morphologically abnormal spermatozoa, sperm DNA damage, and chromatin remodeling.2–7 Bisphenol A (BPA) is an ED with a high worldwide production volume that is used in the manufacture of polycarbonate plastics (i.e. food and water containers) and epoxy resins (i.e. canned food linings). BPA leaches from plastic containers and cans into food or drinks; thus, exposure is ubiquitous. Measurable levels of BPA are detected in the blood, serum, urine, saliva, milk, placental fluid, and maternal/fetal plasma without known environmental exposure to BPA.8 BPA exposure can be initiated during early embryonic life through the maternal placenta.9 Developing fetuses are exposed to high BPA levels stored in maternal lipids, which are metabolized rapidly during pregnancy.9 Maternal exposure to BPA is linked to an increased incidence of hepatic tumors, lung inflammation, Parkinson's disease, coat color variation, and, most relevant here, offspring reproductive abnormalities.10–13 Therefore, whether early exposure to BPA during sensitive developmental stages has adverse consequences on offspring fertility needs to be examined. Recently, we showed that gestational exposure to BPA alters the function and proteome profile of ejaculated spermatozoa in F1 adult mice.5 Other studies also investigated



BPA-induced functional modifications of ejaculated spermatozoa using rat and mouse models.6,7,14 However, mammalian ejaculated spermatozoa cannot fertilize an oocyte before reaching the penultimate step in maturation, known as ‘capacitation’.15–17 During capacitation, the glycoprotein coat and seminal proteins are removed from the acrosomal surface of spermatozoa, thereby permitting the acrosome reaction to occur.15–19 Recently, we showed that capacitated boar spermatozoa represent a more physiologically relevant model than ejaculated spermatozoa for investigating male fertility.18–20 Therefore, studies should examine BPA-induced changes in capacitated spermatozoa and their consequences on subsequent fertilization. Proteomic investigations of spermatozoa are practical tools for distinguishing normal, functional spermatozoa from abnormal spermatozoa.5,6,18,19 Indeed, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) applies high-throughput industrial applications to identify sperm-specific proteins indicative of chemical exposure.5–6 As such, direct comparison of protein expression profiles between control and exposed cells returned a set of protein markers.5,6,18,19 Because mature mammalian spermatozoa are virtually incapable of protein synthesis,5,6,19 the predicted protein biomarkers in spermatozoa offer considerable stability to be used in clinical applications. Simultaneously, to utilize large-scale proteomic data, high-level bioinformatics support is critical for summarizing and interpreting the findings. In the current study, we first investigated the effects of gestational BPA exposure (50 µg, 5 mg, and 50 mg kg bw-1•day-1) on adult sperm function after capacitation and searched for mechanisms of action related to these observed effects. Second, we investigated the modified protein profile in spermatozoa after BPA exposure to determine whether these modifications explained the observed functional alterations in spermatozoa. Third, we used bioinformatics to screen for the clinical significance of proteomic alterations in spermatozoa due to gestational exposure to BPA.


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EXPERIMENTAL SECTION Chemicals, Reagents, and Media. All chemicals and reagents were purchased from SigmaAldrich (St. Louis, MO, USA) unless otherwise mentioned. BPA (≥ 99 % pure) was suspended in corn oil using mild heat in a water bath to obtain the desired molecular concentrations. Modified Tyrode’s medium (97.84 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl2H2O, 0.36 mM NaH2PO4H2O, 5.56 mM

D-glucose,

25 mM NaHCO3, 1.78 mM

CaCl2H2O, 24.9 mM Na-lactate, 50 µg/mL gentamycin, and 0.4% bovine serum albumin) was used as the basic medium (BM) for spermatozoa.5,6,7 The osmolality and pH of BM were maintained at 300 ± 20 mOsm/kg and 7.2 ± 0.2, respectively.5,6

BPA Dose Selection. BPA exposure concentrations were 50 µg, 5 mg, and 50 mg•kg bw1

•day-1. The 50 µg•kg bw-1•day-1 is the tolerable daily intake value of BPA approved by the

European Food Safety Authority.21,22 The tolerable daily intake value was determined based on an overall no-observed-adverse-effect level (NOAEL) of 5 mg•kg bw-1•day-1 and uncertainty factor of 100 (interspecies and interindividual differences).21 The doses of 5 and 50 mg•kg bw-1•day-1 are the NOAEL and lowest-observed-adverse-effect level (LOAEL), respectively, described by the US Environmental Protection Agency.22 The NOAEL value was determined based on body weight changes (particularly the weights of the liver and kidneys) in two- and threegeneration studies using mice23,24 and rats,25 respectively. In contrast, the LOAEL value is based on reproductive and developmental toxicity (delayed puberty in rats and mice) of BPA.21 Control animals were treated with corn oil only. We did not use an estrogenic positive control because BPA functions differently than the estrogenic positive control in CD-1 mice.1, 26



Animal Care, Maintenance, and BPA Exposure. All animal procedures were approved by the Institutional Animal Care and Use Committee of Chung-Ang University, Seoul, Korea (IACUC Number: 2016-00009). Animals were handled humanely with regard for alleviation of suffering. Eight-week-old CD-1 (ICR) male and female mice were purchased (Doo Yeol Biotech, Seoul, Korea) and maintained under constant conditions of temperature (22 ± 2 °C), humidity, ventilation, and light (12-h light/dark period). The mice were housed individually in BPA-free new polysulfone cages with autoclaved bedding materials for 4 weeks before mating. Only paper materials were used in housing facilities; no plastic enrichment was added to limit background BPA exposure. The mice were provided ad libitum access to a soy-free diet (SAM#31; Samtako Co., O-San, Korea) and water from glass bottles throughout the study period. The experimental design is depicted in Figure 1A. The mice were bred by cohabitating at the ratio of two female per male in a single cage. The females were examined daily and confined individually after observation of a vaginal sperm plug [embryonic day 0, (E0)]. The copulated female mice (F0) were randomly assigned into four treatment groups (n = 10 mice/group): group 1: control, group 2: 50 µg, group 3: 5 mg, and group 4: 50 mg•kg bw-1•day-1. The F0 dams were treated daily between E7 and E14 by oral gavage with BPA, depending on the daily body weight changes. Dosing was initiated at E7 to avoid the BPA induced effects on early embryonic development and implantation that occurred prior to E7. Simultaneously, the treatment window was very critical because the primordial germ cell migration, DNA methylation in male and female germ cells, remethylation in male germ cells, sex determination, and epigenetic changes occur during this time.5,27 Following parturition, F1 offspring were weighed and sexed by examining their anogenital distance. Only male offsprings (4–5 pups/litter) were kept with the F0 lactating mothers in each group until postnatal day 21 (PND 21). All mice were weaned on PND 22 and subsequently maintained separately until adulthood (PND 120).


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Animal Sacrifice, Spermatozoa Collection, and Capacitation Induction. On PND 120, the F1 male mice were euthanized using avertin (2,2,2-tribromoethanol). Avertin stock solutions (1.6 g/mL) were prepared by thoroughly mixing 2, 2, 2-tribromoethanol (25 g) and 2-methyl2-butanol (15.5 mL) for 10–12 h with mild heat. The stock solution was supplemented with 0.9% saline (NaCl) to prepare the working solution (20 mg/mL). Intraperitoneal injection of avertin (working solution, ~1.0 mL/mouse) was performed for euthanasia. Spermatozoa were collected from the epididymis as previously described.7,16 Collected spermatozoa were incubated for 90 min in BM supplemented with 0.4 % bovine serum albumin (BSA) at 37°C and 5% CO2 to induce capacitation, as previously described.16,28,29 Additionally, we conducted a preliminary study using control spermatozoa to confirm that incubation of mice spermatozoa for 90 min in BM containing BSA induces capacitation (Supporting Information, Figure S1). All sperm function tests and proteomic analyses were performed using capacitated spermatozoa.

Functional Analyses and Fertility Assessment of F1 Capacitated Spermatozoa. A detailed description of “Detection of viability of F1 capacitated spermatozoa,” “Detection of motility and motion kinematics of F1 capacitated spermatozoa,” “Detection of capacitation status,” “Detection of intracellular ATP, ROS, Ca2+, and cytotoxicity levels,” “Detection of lipid peroxidation of F1 capacitated spermatozoa,” and “Evolution of fertility potential by F1 capacitated spermatozoa,” are provided in the Supporting Information (Supplementary Methods).

Preparation of Spermatozoa for Proteomic Analysis. The detailed procedure for sperm collection and preparation for 2-DE analysis is depicted in Figure 1B. Capacitated



spermatozoa collected from F1 males were washed twice by centrifugation (100 × g for 2.5 min), re-suspended in BM, and allowed to swim-up for 15 min at 37°C. As reported, density gradient centrifugation permits a low level of cell contamination,15,18 but also results in a very low rate of sperm recovery for 2-DE.5,18,30 Therefore, the swim-up technique was used to separate the motile sperm fraction from immature spermatozoa and somatic cells. After collection of the motile sperm fraction, the resulting spermatozoa were carefully checked for immature spermatozoa as well as somatic cells by microscopic examination.

2-DE, Image Analysis, and Identification of Differentially Expressed Proteins in Spermatozoa. The detailed description of “2-DE and image analysis” and “Identification of differentially expressed proteins in spermatozoa” are provided in the Supporting Information (Supplementary Methods). To confirm the 2-DE results, protein tyrosine nitration, and phospho-protein kinase A (PKA) substrate activities were examined by western blotting (see “Western blotting” in the Supporting Information).

Bioinformatics Analysis of Differentially Expressed Proteins. To investigate signaling pathways, related diseases associated to the differentially expressed proteins, and proteinprotein interaction, we utilized the Pathway Studio program (Elsevier, Amsterdam, Netherlands) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, Version 10) (see “Detection of signaling pathways, protein related diseases, and proteinprotein interactions’ in the Supporting Information).

Statistical Analysis. Data from the ≥ 3 independent experiment are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed with SPSS version 23.0 (SPSS, Inc., Chicago, IL, USA) using one-way analysis of variance. Student’s t test was


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used to determine the differences between before and after capacitation. The probabilities of the signaling pathways being associated with the differentially expressed proteins were determined using Fisher’s exact test. Significant differences from the mean are indicated (p < 0.05).



RESULTS Gestational Exposure to BPA Altered the Functions and Fertility of F1 Capacitated Spermatozoa. First, we evaluated whether gestational exposure to BPA affects adult (F1) sperm function after capacitation. Hyperactivated (HYP) motility, straight-line velocity, and wobble were significantly decreased following 5 and 50 mg BPA treatment, whereas the motility and average path velocity of spermatozoa were only affected by 50 mg•kg bw-1•day-1 of BPA exposure (p < 0.05) (Table 1). Interestingly, the number of viable spermatozoa was unaffected by BPA exposure (Table 1). Figure 2(A–C) shows the change in capacitation patterns of F1 spermatozoa following gestational BPA exposure. Both the 5 and 50 mg•kg bw-1•day-1 BPA doses significantly decreased the percentage of capacitated spermatozoa compared to the control (p < 0.05) (Figure 2A). However, the number of the acrosomereacted and non-capacitated spermatozoa were apparently unaffected (Figure 2B and 2C). F1 capacitated spermatozoa collected from different groups were also inspected to evaluate their competence for fertilization (cleavage) and early embryonic development (blastocyst formation) using the in vitro fertilization (IVF) system. Both 5 and 50 mg•kg bw-1•day-1 doses of BPA significantly decreased the rates of fertilization and early embryonic development compared to the control (p < 0.05) (Figure 2D).

Effect of BPA on the Levels of ATP, Reactive Oxygen Species (ROS), [Ca2+]i, Lactate Dehydrogenase (LDH), Lipid Peroxidation, Tyrosine Nitration, and PKA Activities in F1 Capacitated Spermatozoa. Several important biochemical features of spermatozoa such as intracellular levels of ATP, [Ca2+]i, ROS, LDH (as a measure of cytotoxicity), and lipid peroxidation were also measured in F1 capacitated spermatozoa following gestational exposure to BPA. The intracellular ATP levels significantly decreased following exposure to all BPA doses (p < 0.05) (Figure 3A). However, significantly increased levels of lipid


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peroxidation were observed for the 5 and 50 mg•kg bw-1•day-1 BPA doses (p < 0.05) (Figure 3F). Interestingly, other parameters such as the levels of ROS, LDH, and [Ca2+]i were unaffected (Figure 3). To better elucidate oxidative stress mechanism, we also measured nitrotyrosine protein modification in spermatozoa. We found a significant increase in protein tyrosine nitration (~75, 72, and 25 kDa) in F1 capacitated spermatozoa following exposure to 5 and 50 mg•kg bw-1•day-1 BPA doses (Figure 4A). Capacitation events in mammalian spermatozoa are mostly regulated by PKA substrate activity, which is responsible for fertilization.5–7 Therefore, we evaluated the levels of phospho-PKA substrate in F1 capacitated spermatozoa following gestational BPA exposure. Our data showed that treatment with both 5 and 50 mg•kg bw-1•day-1 BPA significantly reduced PKA activity on the substrates of ~28, 23, and 16 kDa in spermatozoa. However, no changes were observed in the low-dose group (Figure 4C).

BPA Induced Differential Protein Expression in F1 Capacitated Spermatozoa. Next, we applied 2-DE coupled with electrospray ionization-MS/MS to evaluate protein profiles in F1 capacitated spermatozoa following gestational exposure to BPA. On average, 285 spots were repeatedly detected in all gels with 181 showing similar expression levels. Among the 104 differentially expressed proteins, 93 showed a dose-dependent expression profile. However, significant (p < 0.05) changes were observed for only 24 spots. Electrospray ionizationMS/MS was used to identify the proteins associated with 15 spots (9 spots were non-detected because of extremely small spot densities). To clarify the dose-dependent expression profiles, we eliminated 9 spots showing non-linear expression between treated and control groups (p < 0.05). A spot summary, identified spots, and normalized spot values are shown in Figure 5A, 5(C–F), and Table 2, respectively [see Supporting Information for additional details (Table S1 and Figure S2)].



In addition, the identified proteins were characterized according to the Gene Ontology annotations in bioinformatics databases (UniProt-GOA and Human Sperm Proteome) shown in Figure 5B. Table 2 shows that the expression levels of cytochrome c oxidase subunit 5B (COX5B), triosephosphate isomerase (TPI-1), ropporin-1 (ROPN1), and fatty acid-binding protein 9 (FABP9) increased, whereas other proteins significantly decreased dosedependently compared to their control levels (p < 0.05). All proteins showed significant differential expression profiles between the control and 50 mg•kg bw-1•day-1 exposure groups (p < 0.05). However, 4 proteins [NADH dehydrogenase 1 alpha subunit 13 (NDUFA13), outer dense fiber protein (ODF), FABP9, and ras-related protein Rab-2A (RAB2A)] were unchanged compared to the control, 5 µg, and 5 mg•kg bw-1•day-1 groups. Simultaneously, 5 proteins [cytochrome c1 heme protein c1 (CYC1), actin-related protein T3 (ACTRT3), ROPN1, peroxiredoxin-5, mitochondrial (PRDX5), and superoxide dismutase (SOD2)] were significantly altered in all treatment groups compared to the control (p < 0.05).

Validation of 2-DE Results by Western Blot Analysis. To confirm the reliability of the proteomics analysis, RAB2A, TPI-1, FABP9, and PRDX5 were selected as representative proteins and analyzed by western blotting (Supporting Information, Figure S3). We detected RAB2A, TPI-1, FABP9, and PRDX5 proteins in F1 capacitated spermatozoa of approximately 24, 27, 15, and 22 kDa, respectively. The protein expression profiles by western blot were consistent with those observed in 2-DE (Table 2).

Bioinformatics Analysis of Differential Protein Profiles. We investigated specific proteinregulated cellular pathways using the Pathway Studio program. Our results demonstrated that differentially expressed proteins in capacitated spermatozoa following BPA exposure were significantly correlated with five canonical pathways such as respiratory chain and oxidative


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phosphorylation, ROS metabolism, necrosis, glutathione metabolism, and notch pathway (p < 0.05) (Supporting Information, Table S2). Simultaneously, to determine the clinical significance of differentially expressed proteins, we detected the diseases regulated by these proteins and their regulatory paradigm using the same program. Our data revealed a functional relationship between the identified proteins and pathogenesis of several diseases including neoplasm, carcinoma, diabetes, Parkinson’s disease, and infertility. These consequences appeared to be caused by increased oxidative stress, lipid peroxidation, apoptosis, and failure of energy metabolism (Figure 6A and Supporting Information, Figure S4). In addition, we also examined the protein-protein interactions between differentially expressed proteins in capacitated spermatozoa following BPA exposure. Our results revealed that 10 proteins [NDUFA13, CYC1, COX5B, ATP synthase subunit O (ATP5O), adenylate kinase isozyme 2 (AK2), SOD2, ACTRT3, TPI-1, prohibitin (PHB), and PRDX5] showed medium (single line) to high (multiple line) interactions with each other in a medium required interaction score (0.4000) search (Figure 6B).



DISCUSSION Capacitation is the functional maturation of the spermatozoon and is required for fertilization. It is associated with several functional adjustments, biochemical modifications, and proteomic changes in spermatozoa.15 Therefore, examining BPA-induced alterations in capacitated spermatozoa is clinically important for understanding BPA toxicity and its corresponding relationship with subsequent fertility. In the current study, we found that BPA may affect sperm functions, related processes, and proteomic profiles of F1 capacitated spermatozoa. Simultaneously, we also evaluated the clinical significance of such modifications in spermatozoa. Exposure to BPA is frequently associated with low sperm count and motility.6,7,31 Prior studies suggested that perinatal exposure to BPA adversely affects adult spermatozoa motility.14 Recently, we showed that gestational exposure to BPA affects spermatogenesis in F1 males by altering the proportion of seminiferous epithelial cells, which subsequently decreases total sperm count and motility.5 Consistent with these finding, we confirmed that BPA also inhibits several motility parameters in capacitated spermatozoa. To investigate the molecular mechanism of BPA-induced motility loss, we measured various biochemical parameters in capacitated spermatozoa, i.e. intracellular ATP, ROS, LDH, Ca2+, lipid peroxidation, protein tyrosine nitration, and PKA activity. Cellular antioxidant defense systems can be overwhelmed by high levels of ROS, subsequently affecting DNA, lipids, and proteins in the cell.32 Sperm motility typically depends on the integrity of its mitochondrial and fibrous sheath. Suleiman et al.33 reported that ROS oxidized phospholipids located in the mitochondrial sheath subsequently impaired sperm motility. Simultaneously, Morielli and Morielli34 reported that treatment of human spermatozoa with extracellular ROS increased tyrosine-nitrated-modified proteins in the fibrous sheath, affecting motility and capacitation ability. In current study, we found no significant changes in ROS levels, which can be


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because a non-specific oxidation-sensitive fluorescent dye (2′,7′-dichlorofluorescein diacetate)35 was used to measure cellular ROS. Therefore, the marginal increase in ROS may have decreased spermatozoa motility via lipid peroxidation and protein tyrosine nitration. Otherwise increased lipid peroxidation and protein tyrosine nitration may directly reduce sperm motility. In addition, decreased ATP levels following BPA treatment may represent mitochondrial dysfunction, which can decrease sperm motility. Mitochondrial dysfunction and decreased ATP levels were also reported in mice spermatozoa following in vitro exposure to BPA (100 µM).6,7 In contrast, it is well-known that HCO3−-dependent PKA activity is involved in regulating sperm motility and capacitation.5,6,36 Increased PKA activity boosts flagellar beat frequency and waveform symmetry of spermatozoa, which is responsible for regulating motility and capacitation.37 Therefore, reduced PKA activity observed in the current study may also predispose sperm to decrease motility and capacitation. Strict regulation of sperm motility is a prerequisite for successful fertilization. Consequently, spermatozoa require HYP motility to provide sufficient strength to penetrate the extracellular matrix of female eggs during fertilization. Quill et al.38 reported that CatSper2 -/- (sperm-specific cation channel) males are completely infertile because of their inability to acquire HYP motility. Therefore, the decreased motility and HYP motility observed in the current study may have directly contributed to the reduced fertilization rates and early embryonic development by F1 spermatozoa following gestational exposure to BPA (5 and 50 mg•kg bw-1•day-1). Alternatively, decreases in the number of capacitated spermatozoa (B pattern), perhaps caused by decreased PKA activity may directly lead to adverse fertility consequences following BPA exposure. Because only capacitated spermatozoa are able to interact with the zona pellucida of the oocyte for fertilization.15,17,19 Decreased litter size from F1 males was also reported in another study following gestational exposure to 50 mg•kg bw-1•day-1 of BPA, while 5 mg•kg bw-1•day-1 treatment resulted in no



adverse effects.5 Although the reasons for these inconsistencies are unclear, the female body may adapt to allow the best sperm for fertilization, which may differ from in the current in vitro examination of fertility potential by IVF. Examination of the protein content in cells, including sperm cells, provides insight into cell function. Mammalian spermatozoa modify their pre-existing protein content during capacitation.15,19 Because capacitation confers sperm with fertilization ability, protein modifications in capacitated spermatozoa following BPA exposure may provide an uninterrupted link with BPA-mediated fertility loss. We demonstrated a significant dosedependent differential expression pattern for 15 proteins in F1 capacitated spermatozoa following gestational exposure to BPA. Most proteins (47 %) were associated with cellular energy metabolism. From this protein pool, six proteins (CYC1, AK2, NDUFA13, ATP5O, TP-1, and COX5B) interact with each other. Previous studies report that ATP5O is present in the flagellum of cauda epididymal spermatozoa.30,39 TPI-1 was detected in both caput and cauda epididymal sperm flagella.39 These proteins play significant roles in oxidative phosphorylation in spermatozoa,39 and subsequently generate ATP to support motility. Therefore, abnormalities in the function of these proteins may disturb energy production machinery in sperm flagella, affecting their motility and fertility. In contrast, CYC1 is an important protein in the mitochondrial electron transport cycle.40 Incomplete processing of the CYC1 gene is linked to an increased production of free radicles (•O2−) in spermatozoa, thereby affecting male fertility.41 Although a literature search did not reveal a reliable correlation between CYC1 function and sperm motility regulation, downregulation of this protein may limit overall cellular energy production capability and thus, alter motility. Steroid dehydrogenase (KE6) and AK2 belong to the energy metabolism group altered following BPA exposure. The KE6 enzyme facilitates ATP production from ADP in the flagella of ejaculated bovine spermatozoa during capacitation.42 Simultaneously, AMP


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generated by the reaction scatters back to the mitochondria to be rephosphorylated to ADP by mitochondrial AK2. Thus, downregulation of both enzymes may contribute to the observed modified function in spermatozoa following BPA exposure. The levels of the structural proteins such as ACTRT3, ODF, and ROPN1 were significantly altered in F1 spermatozoa because of gestational exposure to BPA. The expression levels of ACTRT3 and ROPN1 were changed following exposure to all doses of BPA. Therefore, these candidate proteins may be excellent biomarkers of BPA exposure. ODF expression levels decreased significantly for the highest BPA exposure dose. Cytoskeletal and structural proteins play a critical role in cytoplasmic integrity, cell movement, and structural transduction mechanisms in cells.43 Abnormalities in these protein functions were reported in non-motile, abnormally headed, and infertile spermatozoa.15,43,44 Therefore, alterations in the expression levels of these proteins may directly affect sperm motility and subsequent fertility in exposed individuals. The stress response proteins PRDX5, SOD2, and FABP9 showed altered expression levels in F1 capacitated spermatozoa following BPA exposure. PRDX5 localizes in the acrosome, post-acrosome, and midpiece of human spermatozoa,45 as well as in the head and midpiece of porcine spermatozoa.15 As an antioxidant enzyme, PRDX5 plays a vital role in protecting spermatozoa against harmful endogenous and exogenous peroxide. This protein function is related to the sperm-egg interaction for boar spermatozoa.15 In the present study, the density of PRDX5 was decreased. In contrast, PRDX5 was shown to be upregulated following in vitro exposure to BPA (100 µM) for 6 h.6 Although the exact molecular mechanisms of these discrepancies are unclear, direct in vitro exposure to BPA may responsible for the different results from those noted in F1 capacitated spermatozoa following gestation exposure. In addition, mature spermatozoa are silent in gene transcription and translation.7,19,46 Therefore, upregulation of PRDX5 reported in the previous study6 occurred



via post-translational processes, likely by the phosphorylation of PRDX5, which also differed from the current investigation. Additionally, SOD2 is a prominent antioxidant enzyme highly specialized for ROS scavenging. Clinically, decreased SOD2 activity was found in the spermatozoa and semen of heavy smokers and was associated with low fertility.47 Our results showed that both PRDX5 and SOD2 were significantly decreased in F1 capacitated spermatozoa following gestational exposure to BPA. Therefore, downregulation of these proteins decreased the ability of sperm to maintain stress response mercenaries, subsequently affecting sperm function and fertility. Another protein in this group was FABP9, a germ cell-specific fatty acid-binding protein that protects sperm fatty acids from oxidative damage.44 FABP9-deficient mice are fertile, but exhibit increased spermatozoa morphological abnormalities.44 In the current study, we showed that FABP9 was up-regulated by the 50 mg•kg bw-1•day-1 BPA dose. It is unclear how FABP9 is increased in F1 spermatozoa when other proteins (PRDX5 and SOD2) in this group are decreased. However, gestational exposure to BPA (50 mg•kg bw-1•day-1) may directly influence the genes synthesizing FABP9, resulting in increased expression. Optimal expression and function of a protein are central to normal cell function.6 Thus, substantial increases in FABP9 may represent atypical protein function. Therefore, we hypothesized that gestational BPA exposure affects the levels of stress response proteins, resulting in a loss of spermatozoa normal function and a predisposition to fertilization failure. In addition, the expression of two fertility-related proteins, PHB and RAB2A, were also changed in F1 capacitated spermatozoa by BPA exposure. PHB is a mitochondrial membrane protein of spermatozoa associated with mammalian spermatogenesis.48 Highly conserved PHB ubiquitination has been reported in semen samples from both fertile and infertile men,49 which may play a critical role in fertility. RAB2A is a ras-related protein involved in the acrosome reaction, membrane fusion, and vesicular transport in


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spermatozoa.50 Kwon et al.15 identified fertility-associated expression of this protein in porcine spermatozoa. Because both proteins were downregulated in current study following BPA exposure, BPA may be related to the decreased fertility observed in the current study. Another objective of the current study was to determine the clinical significance of the BPA induced differential protein expression. Prediction of the correlation between protein expression profiles and clinicopathologic conditions may aid clinicians in selecting appropriate theranostic strategies for patients as well as provide future research directions by emphasizing new disease pathways related to toxic exposure. We showed that 7 of 15 identified proteins were significantly correlated with 5 canonical signaling pathways according to Pathway Studio analysis. Proper function and interaction between several signaling pathways are critical for the regulation of cell function.5–7 Therefore, modification of proteins in these pathways may have pathological consequences. To confirm this hypothesis, we searched for protein related diseases using the same program. We found that dysfunction of the identified proteins was related to the pathogenesis of several diseases such as cancer, neoplasm, diabetes, Parkinson’s disease, and infertility. These adverse consequences may be caused by increased oxidative stress in cells, lipid peroxidation, apoptosis, mitochondrial dysfunction, and protein degradation. Notably, even 50 µg•kg bw1

•day-1 BPA was nontoxic to several sperm functions, however, the differential protein

expression at this dose showed a clinical association with several diseases. BPA-induced differential proteins in spermatozoa were also searched in the STRING database to study their interactions. Most proteins displayed strong/medium interactions with each other. Typically, proteins interact with each other or different molecules (e.g. DNA, RNA) in cells to regulate various biological processes related to health and disease.51 There are several mechanisms such as protein co-occurrence, co-expression, co-regulation, and gene-fusions linked with protein-protein interactions.52 For example, the strong interactions between SOD2



and PRDX5 observed in the current study may indicate that the proteins are involved in similar biological processes such as ROS metabolism, oxidative stress response mechanism, and lipid peroxidation, subsequently affecting sperm function following BPA exposure. Based on these observations, we hypothesized that a relationship exists between the occurrence of some diseases in adulthood and developmental exposure to BPA. However, further studies are needed to confirm our preliminary findings.

CONCLUSIONS To the best of our knowledge, this is the first comprehensive study to evaluate the effects of gestational BPA exposure in adulthood (F1) capacitated spermatozoa. BPA at a dose of 50 µg has no or minimal effects, while both 5 and 50 mg•kg bw-1•day-1 have severe toxic effects in capacitated spermatozoa. In addition, there may be a relationship between the occurrence of some diseases in adulthood and developmental exposure to BPA. Thus, capacitated spermatozoa provide an excellent model for investigating the underlying molecular mechanisms of ED exposure and occurrence of some diseases in adulthood.


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ASSOCIATED CONTENT Supporting Information The following files are available free of charge at ACS website http://pubs.acs.org: Supplementary Methods - Complete experimental procedures. Table S1 - List of peptide sequences, precursor charge (m/z), and sequence coverage (%) for each assignment. Table S2 - Signaling pathways significantly correlated with differentially expressed proteins in F1 capacitated spermatozoa from control and BPA-treated groups. Figure S1 - Changes in capacitation status of mice spermatozoa following incubation in basic media (containing 0.4% bovine serum albumen) for 90 min. Figure S2 - Annotated spectra (MS/MS spectrum) for each identified protein. Figure S3 - Western blot of 5 representative proteins. Figure S4 - Disease associations of differentially expressed proteins between control and 50 µg•kg bw-1•day-1 BPA treatment groups as determined using the Pathway Studio program.

Author Contributions MGP, BYR, and MSR designed the study. MSR, WSK, DYR, AK, and PCK performed experiments and analyzed data. MSR generated the figures. MSR and MGP drafted the manuscript. All authors have corrected the manuscript and approved the final version.

Notes The authors declare they have no actual or potential competing financial interests.



Funding This work was supported by Korea Research Fellowship (KRF) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (project no. 2017H1D3A1A02013844) and by the 2014 grant (14162MFDS661) from the Ministry of Food and Drug Safety of the Republic of Korea.


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FIGURE LEGENDS Figure 1. Experimental design for bisphenol A (BPA) exposure, sperm function study, and two-dimensional-gel electrophoresis (2-DE). A, Pregnant mice were administered three different BPA doses (50 µg, 5 mg, and 50 mg•kg bw-1•day-1) by gavage during embryonic days 7–14 (E7–E14). Control mice were treated with corn oil (vehicle). Collected spermatozoa were incubated for 90 min in basic media (BM) supplemented with 0.4 % bovine serum albumin (BSA) to induce capacitation (see text for more detail). Several sperm function tests, fertility assessments, and proteomic analyses were conducted on postnatal day (PND) 120 for F1 spermatozoa following in vitro capacitation. B, Capacitated spermatozoa from F1 males were washed twice and allowed to swim-up. Proteins were extracted from the swim-up sperm fraction using rehydration buffer. Solubilized protein (250 µg) was used for first-dimensional

electrophoresis

using

DryStrips

(pH

3–11).

Second-dimension

electrophoresis was conducted by 12.5% SDS-PAGE. Silver-stained gels were scanned and spots were matched and analyzed using the PDQuest 8.0 program. The target spots were excised and used for in-gel digestion, desalting, and electrospray ionization mass spectrometry (ESI-MS/MS) to identify specific proteins (see text for additional details).

Figure 2. Effect of gestational bisphenol A (BPA) exposure on capacitation status and fertility of F1 capacitated spermatozoa. A, Percentage of live capacitated (B) spermatozoa. B, Percentage of live acrosome-reacted (AR) spermatozoa. C, Percentage of live noncapacitated (F) spermatozoa. D, Percentage of cleavage (fertilization) and blastocyst (early embryonic development) formation in the control and treated groups. Data are the means of four replicate experiments ± SEM (n = 3 mice/replicate). All data were analyzed by one-way analysis of variance. Tukey’s test was used to identify differences between groups. Values


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with different superscript characters (A,B,a,b) indicate significant differences between control and treatment groups (p < 0.05).

Figure 3. Effects of gestational bisphenol A (BPA) exposure on several spermatozoa biochemical parameters. A, ATP bioluminescence intensity (proportional to the levels of intracellular ATP) in control and treated samples. B, Fluorescence intensity [proportional to intracellular reactive oxygen species (ROS) activity] in control and treated samples. C, Absorbance [proportional to the levels of intracellular lactose dehydrogenase (LDH)]. D, Fluorescence intensity [proportional to the levels of intracellular ionic calcium (Ca2+)]. E, Levels of lipid peroxidation (BODIPY C11 mean fluorescence) in BPA-treated and control spermatozoa. Data are the means of four replicate experiments ± SEM (n = 3 mice/replicate). All data were analyzed by one-way analysis of variance. Tukey’s test was used to identify differences between groups. Values with different superscript characters (A,B,C) indicate significant differences (p < 0.05).

Figure 4. Effects of gestational bisphenol A (BPA) exposure on protein tyrosine nitration and PKA activity in F1 capacitated spermatozoa. A, Relative intensity of tyrosine nitrated proteins (approximately 25, 72, and 75kDa) in control and BPA-exposed spermatozoa. B, Representative immunoblotted image of tyrosine nitrated proteins in control and BPAtreated spermatozoa probed with an anti-nitrotyrosine antibody. C, Density of phosphoPKA substrates at approximately 16, 23, and 28 kDa. D, Characteristic western blot image of phospho-PKA substrates. Data are the means of four replicate experiments ± SEM. All data were analyzed by one-way analysis of variance. Tukey’s test was used to identify

differences between groups. Values with different superscript characters (A,B,a,b,α,β) indicate significant differences between control and treatment groups (p < 0.05).



Figure 5. Spot summary, functional characterization of identified proteins, and representative silver nitrate stained image of two-dimensional gel electrophoresis (2-DE). A, Summary of the spots in control and treated samples. B, Pie chart showing the functional characterization of identified proteins. C, Detected protein spots in control spermatozoa (corn oil). D, Detected protein spots in spermatozoa after 50 µg•kg bw-1•day-1 bisphenol A (BPA) treatment. E, Detected protein spots in spermatozoa after 5 mg•kg bw-1•day-1 BPA treatment. F, Detected protein spots in spermatozoa after 50 mg•kg bw-1•day-1 BPA treatment.

Figure 6. Bioinformatics analysis of differentially expressed proteins. A, Regulatory cellular processes and disease associations for differentially expressed proteins in F1 capacitated spermatozoa as determined using the Pathway Studio program. Proteins with significantly increased or decreased expression patterns compared to the control are indicated by different background colors. B, Protein-protein interactions among differentially expressed proteins in F1 capacitated spermatozoa determined by the STRING program. A medium required interaction score (0.4000) was used to illustrate the finding.


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Table 1. Viability and motility parameters for F1 capacitated spermatozoa following gestational exposure to bisphenol A (BPA). BPA (•kg bw-1•day-1) Parameters

Control 50 µg

5 mg

50 mg

Viability (%)

80.11±2.94

80.62±2.15

70.95±1.53

67.51±2.29

MOT (%)

79.50±1.46A

66.07±1.60A,B

68.72±2.00A,B

59.50±5.34B

HYP (%)

30.81±1.20 A

28.02±5.34A,B

17. 00±0.09B

17.71±0.63B

VCL (µm/s)

172.54±6.50

175.28±9.26

156.69±2.53

144.48±9.09

VSL (µm/s)

83.75±3.04A

84.68±2.51A

64.62±3.88B

55.23±4.44B

VAP (µm/s)

82.89±4.29A

75.52±0.48A,B

70.17±3.19A,B

64.53±4.88B

ALH (µm)

7.15±0.38

6.84±0.02

5.39±0.90

6.06±0.33

LIN (%)

50.41±2.53

44.68±2.51

37.96±4.94

38.56±0.83

WOB (%)

60.41±2.53A

51.35±5.43A,B

44.62±2.17B

41.89±2.52B

BPA, bisphenol A; MOT, motility; HYP, hyperactivated motility; VCL, curvilinear velocity; VSL, straight-line velocity; VAP, average path velocity; ALH, mean amplitude of head lateral displacement; LIN, linearity; WOB, wobble. Data are the means of three replicate experiments ± SEM (n = 3 mice/replicate). All data were analyzed by one-way analysis of variance. Tukey’s test was used to identify differences between groups. Mean values with different

superscript

characters

(A,B)

are

significantly


different

(p

30 indicate identity or extensive homology (p

Functional and Proteomic Alterations of F1 ... - ACS Publications

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