Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa

Accepted Manuscript Title: Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa Author: Linqing Zhen Lirui Wang Jieli Fu Yuhua Li Na Zhao Xinhong Li PII: DOI: Reference:

S0890-6238(15)30046-0 http://dx.doi.org/doi:10.1016/j.reprotox.2015.11.001 RTX 7205

To appear in:

Reproductive Toxicology

Received date: Revised date: Accepted date:

18-8-2015 14-10-2015 2-11-2015

Please cite this article as: Zhen Linqing, Wang Lirui, Fu Jieli, Li Yuhua, Zhao Na, Li Xinhong.Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa.Reproductive Toxicology http://dx.doi.org/10.1016/j.reprotox.2015.11.001 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.

Hexavalent chromium affects sperm motility by influencing protein tyrosine phosphorylation in the midpiece of boar spermatozoa

Linqing Zhen#，Lirui Wang#， Jieli Fu，Yuhua Li，Na Zhao，Xinhong LI*

(Shanghai Key Lab of Veterinary Biotechnology，School of Agriculture and Biology， Shanghai Jiaotong University，Shanghai 200240，China)

Highlights 

High Cr concentrations decreased boar sperm motility in vitro



Cr affected mitochondrial activity, decreased the generation of ATP, and subsequently down-regulated protein tyrosine phosphorylation in the middle piece of boar sperm



Cr may affected boar sperm motility by impairing protein tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro.



This study is expected to provide a theoretical basis for the development of livestock industry and interesting clues for further study on reproductive toxicity.

Abstract Hexavalent chromium reportedly induces reproductive toxicity and further inhibits male fertility in mammals. In this study, we investigated the molecular mechanism by which hexavalent chromium affects motility signaling in boar spermatozoa in vitro. The results indicated that Cr(VI) decreased sperm motility, protein phosphorylation, mitochondrial membrane potential (ΔΨm) and metabolic enzyme activity starting at 4 μmol/mL following incubation for 1.5 h. Notably, all parameters were potently inhibited by 10 μmol/mL Cr, while supplementation with the dibutyryl-cAMP (dbcAMP) and the 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibition of protein phosphorylation. Interestingly, high concentrations of

Cr

(>10

μmol/mL)

increased

the

tyrosine

phosphorylation

of

some

high-molecular-weight proteins in the principle piece but decreased that in the middle piece associated with an extreme reduction of sperm motility. These results suggest that chromium affects boar sperm motility by impairing tyrosine phosphorylation in the midpiece of sperm by blocking the cAMP/PKA pathway in boar sperm in vitro

Keywords:

chromium;

phosphorylation

boar;

sperm

capacitation;

signal

pathway;

protein

1. Introduction Chromium is well recognized as an essential trace element in the diets of domestic animals because it decreases fat and increases lean deposition in the carcass in fodder

[1]

. However, being a necessary trace element for animals, chromium has a

role in maintaining proper carbohydrate, lipid, protein and nucleic acid metabolism by increasing the action of insulin and even has a positive role in enhancing the ability of anti-stress and immunocompetence

[2-4]

. Therefore, chromium is commonly added to

animal feed to enhance the carcass quality, growth performance, and meat quality and to decrease the fat content

[5, 6]

. Research has revealed that dietary supplementation

with chromium picolinate throughout gestation can increase the body mass gain, the number of piglets born alive and the concentration of colostrum [7]. Elevated levels of chromium in bone, kidneys, liver and ovaries have been found in pigs fed supplemental chromium testis

[9]

[8]

. The enrichment of chromium has also been observed in

. Kidneys have been observed to contain the greatest Cr concentration, while

the largest increase in Cr concentration is in the ovaries [8-9]. Importantly, the half-life of chromium, 40 months in serum and 129 months in urine, is long

[10]

. Therefore,

long-term dietary chromium supplementation may easily cause the accumulation of chromium in domestic animal organs. In toxicological research, however, hexavalent chromium has not only been reported to produce acute and chronic toxicity, allergic dermatitis, carcinogenicity, genotoxicity, cytotoxicity, and immunotoxicity, but to also lead to general environmental toxicity

[11]

. Still, it is unclear whether the long-term

feeding of chromium in the diet has adverse effects on livestock. However, there was a lack of negative responses in the growth performance in pigs fed with 5000 μg/kg of supplemental Cr for 75 days

[8]

, but it was proven that tissue development and organ

function were seriously affected in rats

[12]

, aquatic animals

[13]

and humans

[14]

exposed to chromium. Therefore, the safe use of chromium in animal fodder needs further research. Chromium could induce reproductive toxicity in the male reproductive system of humans and experimental animals, such as a reduction in sperm count and motility or an increased level of abnormal sperm [15, 16]. Moreover, testosterone and gonadotropin

levels were significantly altered

[9]

. The development, morphology and function of

testis were severely affected, which significantly decreased the male reproductive capacity

[17]

. The altered secretion of sex hormones and oxidative stress are the two

main factors underlying the reproductive toxicity induced by chromium. It is well documented that testosterone and gonadotropin hormones are responsible for the [18]

normal growth and function of accessory sex organs

. The consequent

accumulation of chromium in the testis could disrupt the blood-testis and then affect the normal function of Sertoli cells, causing an increase in FSH levels and a decrease in testosterone levels, which directly regulate sperm number

[19, 20]

. Oxidative stress

caused by chromium is well established as a major factor responsible for male infertility

[21, 22]

. Once inside the cell, Cr(VI) ultimately reduces to the Cr(III) form

through the formation of reactive intermediates such as the pentavalent and tetravalent forms. High levels of ROS may lead to the generation of oxidative stress, including DNA damage, lipid peroxidation and protein modification

[23]

. Increased Cr in testis

induces tissue damage that impacts spermatozoa formation, and it can directly cause DNA damage in spermatogenic cells and mature sperm

[24]

. All of these effects

decrease sperm numbers, increase an abnormal sperm rate and finally inhibit male fertility

[22]

. Though many reports have proven that chromium is toxic to the male

reproductive system, the effects on domestic animal reproductive systems and the underlying molecular mechanism are still unclear. Sperm motility, including hyperactivation, is one of the macro-indexes of spermatozoa associated with sperm protein phosphorylation, which is key to successful fertilization and which plays a pivotal role in regulating sperm physiological function, such as sperm capacitation, hyperactivation, acrosome reaction, etc.

[25, 26]

. The strengths of hyperactivation and protein phosphorylation are

regarded as the two main landmarks of reproductive potential in mammalian sperm in vitro. To the best of our knowledge, there have been few reports on the effects on boar sperm caused by chromium in vitro. Especially, whether the accumulation of chromium in blood and tissues adversely impacts boar male fertility. Therefore, in the present study, sperm motility and parameters, protein phosphorylation and related

enzyme activity were analyzed to explore the effects of chromium on sperm motility and protein phosphorylation in boar sperm in vitro. Our study will provide some guidelines for the proper use of chromium in livestock and the exploration of the mechanism underlying reproductive toxicity caused by chromium.

2. Materials and Methods 2.1 Materials Hexavalent chromium (Cr6+, K2CrO4) was purchased from national medicines (China).

Molecular

weight

markers,

N-[2-(p-bromocinnamylamino)

ethyl]-5-isoquinoline sulfonamide (H-89), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl-cAMP (dbcAMP) were acquired from Bio-Rad (USA). PVDF membranes was purchased from Millipore (Billerica, MA). Phospho-PKA substrate (RRXS*/T*) (100G7E) rabbit mAb, anti-phosphotyrosine antibody, anti-α-tubulin antibody, anti-rabbit

IgG

HRP-conjugated

secondary

antibody

and

anti-mouse

IgG

HRP-conjugated secondary antibody were purchased from Cell Signaling Technology. Alexa 555-conjugated anti-rabbit antibody, Alexa 488-conjugated anti-mouse antibody, peanut agglutinin (PNA) and propidium iodide (PI) were purchased from Molecular Probes (Invitrogen). The chemiluminescence detection kit (ECL) was from GE healthcare. Other chemical products were purchased from Sigma-Aldrich (St. Louis, MO).

2.2 Media The basal medium was modified Whitten’s (MW), and the non-capacitating (N-Cap) medium consisted of 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM NaH2PO4, 137 mM NaCl, 5.55 mM glucose and 2.0 mM sodium pyruvate. Medium containing 25 mM NaHCO3, 2 mM CaCl2 and 0.4% BSA was designated the capacitating (Cap) medium, as described by Tardif et al. (2002) [27]. The pH was adjusted to ~7.4 using 1 mM NaOH. The medium was maintained at 37°C until the beginning of the experiment, and the pH was checked again and adjusted if necessary.

2.3 Sperm collection and incubation Semen was routinely obtained from eight mature (2-3 year-old) and sexually mature Duroc boars of the same rearing conditions using the manual method; these boars were selected solely for high sperm quality and proven fertility (over 80% pregnancy rates). Then, the semen were counted and collected by 800×g for 5 min at room temperature, and the sperm pellets were re-suspended. Non-capacitating media was used in the experiment at 37°C inside a sterile collection recipient and transported to the lab for the experiments. An equal sperm suspension was diluted in different media to a final concentration of 2-5×107 cells/ml. To study the effects of chromium on boar sperm in vitro, we designed three experimental groups. Group I: Cr6+ was added to a final concentration of 0.1, 0.5, 1, 2, 4, 6, 8, and 10 μmol/mL in the N-Cap medium. In this group we detected sperm motility, GAPDH activity, ATP level, MMP and protein phosphorylation. Group II: Cr6+ was added to a final concentration of 0.1, 1, 10, 20, 50, and 100 μmol/mL in the N-Cap medium.

Group III: Cr6+ was added to

a final concentration of 0.1, 0.5, 1, 2, 4, 6, 8, and 10 μmol/mL in the Cap medium. In group II and III, we detected sperm protein phosphorylation. Group Ⅳ: Different concentrations of dbcAMP (0.1, 0.25, 0.5, 1.0 mM) and 1.0 mM IBMX added to the N-Cap medium with the addition of 10 μmol/mL Cr6+, to detect the effects of dbcAMP on boar sperm protein phosphorylation Another treatment group: N-cap, Cap, 0.5, 10, 100 μmol/mL Cr in the N-Cap medium, sperm incubated in this group were used to detect the location and effects of Cr on sperm protein phosphorylation. Then, the sperm were incubated in different media at 37°C in a humidified atmosphere for up to 90 min [27, 28], and gently shake every quarter to prevent precipitation.

2.4 Measurement of sperm kinematics At the end of incubation or treatment, the sperm kinematics were assessed using light microscopy and CASA (TOX IVOS, Hamilton Thorne Research, Inc., Beverly, MA, USA). Aliquots (5 μL) of sperm samples were pipetted into disposable counting chambers (standard count, 4-chamber, 20 micron slides, Leja, NieuwVennep, the Netherlands). The following parameters were measured: sperm motility (MOT),

curvilinear velocity (VCL), average path velocity (VAP), and straight-line velocity (VSL), progressive (PRO), hyperactivation, amplitudeof lateral head displacement (ALH), beat cross of frequency (BCF), percentage of linearity (LIN), percentage of straightness (STR). Sperm with hyperactivated motility was sorted by the criteria, VCL> 97μm/s, ALH> 3.5μm, LIN < 32%, WOB < 71%. (WOB=VAP×100/VCL, LIN=VSL×100/VCL, STR=VSL×100/VAP), established by Harald Schmidt and Günter Kamp

[29]

. A minimum of 9 fields per sample was evaluated, and an

independent observer scored at least 200 cells for each measurement.

2.5 Measurement of GAPDH activity, ATP assay and cAMP assay 2.5.1 GAPDH activity Boar sperm were collected after 90 min of incubation and washed with cold PBS after treatment with or without SACH, and the supernatant was discarded by aspiration. A total of 600 mL of sonication buffer consisting of 0.3% HCAPS, 150 mM NaCl, 1 mM DTT, 10 mg/mL aprotinin and 10 mg/mL leupeptin was added, and the suspension was sonicated 3 times on ice. The GAPDH enzyme reaction occurred in the mixture described by Welch et al. [30] containing 0.25 mM NAD, 3.3 mM DTT, 0.3 mM glyceraldehyde-3-phosphate, 5 mM potassium fluoride, 0.5 mM oxalate, 15 mM sodium pyrophosphate, 30 mM sodium arsenate, and 0.1 mM oxamate, and the change in absorbance at 340 nm was read immediately.

2.5.2 ATP assay ATP concentrations were extracted from spermatozoa analyzed by a bioluminescent ATP assay kit (FLASC, Sigma). In brief, 1×106 boar sperm were collected and washed with PBS after 90 mim of incubation, and ATP was released by a lysis reagent. Sample ATP extractions and serial dilutions of an ATP standard were mixed with luciferase reagent, and the luminescence was measured immediately. The generated signal was compared to standard ATP dilutions [31].

2.5.3 cAMP assay The DetectXcAMP Chemiluminescent Immunoassay kits (Arbor Assays, Ann Arbor, MI, USA) were used to measure the boar sperm’s cAMP level after 90 mim of incubation. According to the instructions of the kit, 1 ×106 boar sperm were collected after incubation and washed with PBS by centrifuging at 6000×g at 4°C for 15 min, followed by lysis and acetylation. Luminescence was measured by a microplate reader (Molecular Devices SpectraMax M5, USA).

2.6 Flow cytometry evaluation of mitochondrial membrane potential (ΔΨm) The fluorescent lipophilic cationic dye JC-1 (Bio-rad, CAT: No55130, USA) was used to measure the state of MMP as previously described [32, 33]. In mitochondria with low ΔΨm (inactivity or death), JC-1 forms monomers that emit green light (525–530 nm) when excited at 488 nm. In mitochondria with high ΔΨm (high activity), JC-1 forms multimeric aggregates that emit orange light (wavelength of 590 nm) when excited at 488 nm. A JC-1 stock solution was prepared in DMSO. After 90 mim of incubation, the sperm suspension (1 mL) was diluted to 1 to 2~3×106 sperm/mL and stained with 0.5 mL of a JC-1 working solution. The cells were incubated in the JC-1 working solution for 15 min at 37°C in a CO2 incubator. The cells were washed twice with 1× assay buffer following incubation and then analyzed using a FACScan FCM. A total of 10,000 spermatozoa were collected and analyzed at a flow rate of 100 to 200 cells/s. Green fluorescence (480–530 nm) was measured in the FL-1 channel and orange–red fluorescence (580–630 nm) was measured in the FL-2 channel. The value of FL2/FL1 was recorded.

2.7 SDS-PAGE and Western blot After incubation, sperm were pelleted by centrifugation at 12500×g for 5 minutes. The supernatant was discarded, and the pelleted sperm were re-suspended in 800 μL of PBS (8 g NaCl, 0.2 g KH2PO4, 1.15 g Na2HPO4, 0.2 g KCl, and DDH2O to a total of 1 L) and centrifuged more than three times. The sperm pellets were suspended in 100 μL of 5×sample buffer (1.67 mL DDH2O, 0.5 mM, pH 6.8, 5.83 mL

Tris-base, 2.5 mL glycerol, 833 mg SDS, 1 mg Br-phenol blue, total 10 mL) and placed in boiling water for 4 minutes. Following extraction, the sperm suspension was centrifuged at 13500×g for 15 minutes. The supernatant was transferred to a new tube, and β-mercaptoethanol was added to a final concentration of 10%, which was then placed in boiling water for 3 minutes. At last, the protein samples were either used immediately or stored frozen at -80°C. Sperm protein extracted from 1×106/ml sperm were separated by SDS-PAGE electrophoresis using 10% polyacrylamide gels. The separated polypeptides were electrophoretically transferred to PVDF membranes (Millipore) at 90 volts for 2.5 h. Following the transfer, the membranes were blocked with 1.0% BSA in (1×) T-TBS (30 mM Tris-base, 0.8% (w/v) NaCl, pH 7.5, 0.1% (v/v) Tween 20) for 1 hour at room temperature. After washing 3 times for 10 min each with (1×) T-TBS, the membranes were incubated first with antibodies overnight at 4°C following dilutions in TBST: anti-phospho-tyrosine,

1:10000,

anti-phospho-PKA

substrate,

1:4000,

and

anti-α-tubulin, 1:5000. Washing the membranes with (1×) TBST 3 times was followed by blotting with their corresponding secondary antibodies for 1 h at 4°C. Following the secondary antibody incubation, the membranes were washed 3 times for 10 min each. Then, proteins were visualized using an enhanced chemiluminescence detection kit (ECL plus, CAT：RPN2232, GE) following the manufacturer's instructions and exposure to an ECL film. Western blot intensity was quantitated with ImageJ software.

2.8 Immunofluorescence Following incubation, sperm were centrifuged for 5 min at 800×g and washed with 500 μL of PBS. After centrifugation, the pellets were re-suspended in 500 μL of PBS, and the cell concentrations were adjusted to 5×107/ml spermatozoa per milliliter. A total of 20 μL of the samples were placed onto normal slides, smeared, and air-dried for approximately 30 min on ice. After air-drying, the sperm were fixed with 3.7% formaldehyde in PBS for 20 min at room temperature, washed with PBS (3 washes, each for 5 min) and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After

washing with PBS five times, the sperm were blocked with 1% BSA in PBS for 2 h at room temperature, and then incubated with the respective primary antibody (1:200) in PBS containing 1% BSA at 4°C overnight. After incubation, the sperm were washed 5 times each for 5 min with PBS and incubated with Alexa 555-conjugated anti-rabbit antibody or Alexa 488-conjugated anti-mouse antibody in 1% BSA in PBS for 2 h at room temperature; these solutions also contained Alexa 488-conjugated PNA (1:100) to stain acrosomes and PI to detect the cell nuclei of sperm. After 4 washings with PBS, the samples were mounted onto slides with an antifade solution from Molecular Probes. In the end, samples were evaluated using a confocal fluorescence microscope (Leica, SP5) equipped with a 20× objective [34].

2.9 Statistical analysis Mean values and standard error of the mean (SEM) were calculated for the sperm characteristics under different incubation treatments. All experiments were carried out with at least three independent repeats. The results are presented as the mean ± SEM. The means of control and experimental groups were compared by analysis of variance (ANOVA) using SPSS 17.0 software to determine statistically significant differences (P < 0.05).

3. Results 3.1 Effects on boar sperm motility Sperm motility is an important aspect of ejaculate quality that determines fertilization success. Fig 1 shows that the capacitated sperm motility and parameters were higher than the non-capacitated control. Compared with the control, a low concentration of Cr (0.1-1 μmol/ml) increased the sperm motility and parameters to some extent, but there was no significant increase (P > 0.05). As the Cr concentration increased, the boar sperm motility decreased. The MOT of sperm treated with 4 μmol/ml Cr (51.8%) was significantly lower than that of the control (71.8%) (P < 0.05). In particular, the hyperactivated motility of sperm incubated with 10 μmol/ml Cr was extremely inhibited (Fig S1).

3.2 Effects on metabolism Generally, sperm motility is directly regulated by energy resources, including glycolysis and oxidative phosphorylation

[18]

. Thus, we investigated the ATP levels

and GAPDH activity in boar sperm treated with Cr. Fig 2 shows that a low Cr concentration (1.0 μmol/mL) increased the ATP content and GAPDH activity (P < 0.05), which were still lower than that in the capacitated sperm. However, the ATP level in sperm started to decrease at 8 μmol/mL, and GAPDH started to decrease at 6 μmol/ml. Significantly, the ATP level (1.725 nmol/108 sperm) and GAPDH activity (2.098 unit/109 sperm) in sperm treated with 10 μmol/ml Cr were decreased by 45.8% and 46.9%, respectively. To prove the involvement of mitochondria in the ATP content reduction, we determined the mitochondrial membrane potential (ΔΨm), which is a marker of mitochondrial integrity and function

[35]

. In the present study, the value of FL1/FL2

stands for ΔΨm (Fig 3). The value of FL1/FL2 (1.3) in capacitated boar sperm was significantly higher than that (0.86) in control cells (P < 0.05). However, the mitochondria in sperm incubated with different Cr concentrations showed a tendency of depolarized ΔΨm. Markedly, 4 μmol/mL Cr resulted in decreased ΔΨm compared with the control (P < 0.05). Notably, the reduction of ΔΨm in sperm treated with 10 μmol/mL reached 64.7%. These results indicated that Cr could destroy the mitochondrial integrity and then depolarize the ΔΨm of boar sperm. Given these data, we reasoned that Cr affects the intracellular ATP content by impairing glycolysis and mitochondrial integrity and function in boar sperm.

3.3 Effects on protein phosphorylation in boar spermatozoa In addition, the protein phosphorylation of sperm is a critical event in capacitation that can regulate sperm motility, while PTP is recognized as a hallmark of capacitation [25]. Therefore, we analyzed the protein phosphorylation in boar sperm treated with Cr and found that protein phosphorylation was affected by Cr in a concentration-related pattern in boar sperm. The level of PTP in capacitated sperm was noticeably higher than that in the

control (Fig 4A). Compared with the control, a low Cr concentration (<1 μmol/mL) increased PTP, but a high dose of Cr (10 μmol/mL) substantially decreased it. Considering the 27 kDa brand for example, the PTP states of these proteins increased treated with 0.1-1.0 μmol/ml Cr but decreased at high Cr concentrations (>1 μmol/mL), particularly in the sperm incubated with 10 μmol/mL Cr (P < 0.05) (Fig 4B). At the same time, we investigated the effects of chromium on P-PKAs, the upstream event of PTP. Fig. 5A shows that the P-PKAs level in capacitated sperm was higher than in the control. A low concentration of Cr (<1 μmol/mL) increased the P-PKAs; however, 10 μmol/ml Cr noticeably decreased the P-PKAs in boar sperm. To define the effects of Cr on P-PKAs, western blots were quantified using Image J (Fig 5B). Moreover, our results showing that the level of P-PKAs change tendency did not exist significant differences between boar sperm incubated in the presence or absence of CO2 (Fig S2). To further investigate the effects of chromium on protein phosphorylation, we assessed PTP and P-PKAs in boar sperm exposed to 20, 50, and 100 μmol/mL Cr. As is shown in Fig 6A and 6B, in no-capacitated medium, a high Cr concentration (> 10 μmol/mL) increased the phosphorylation of some high-molecular-weight proteins but decreased the phosphorylation of some small-molecular-weight proteins (<30 kDa). Typically, 100 μmol/mL Cr dramatically increased the PTP. In addition, the P-PKAs and PTP started to decrease at 0.5 μmol/mL Cr in the no-capacitated medium. Similar to the boar sperm incubated in N-Cap medium, this decrease was associated with an increase in Cr concentration, and 10 μmol/mL Cr significantly decreased the P-PKAs and PTP in boar sperm incubated in Cap medium (Fig 7A, 7B). Cr at 100 μmol/mL increased the phosphorylation, especially PTP of some high-molecular-weight proteins. However, sperm motility was potently decreased when the Cr concentration was higher than 10 μmol/mL. Moreover, 100 μmol/mL may be the lethal concentration of Cr to boar sperm according to the analysis of sperm motility (data not shown). dbcAMP and IBMX increased the protein phosphorylation; in contrast, H-89 decreased the P-PKAs and PTP in boar sperm. Such as the inhibition of 20kDa,

45kDa and 100kDa proteins P-PKAs induced by H89 (Fig S3).

3.5 The role of the cAMP/PKA pathway in the inhibition of protein phosphorylation It is well established that PTP in sperm is principally mediated by the cAMP/PKA pathway [36]. To investigate whether this pathway was affected by Cr, we assayed the cAMP level in sperm exposed to Cr and the effects of dbcAMP and IBMX on protein phosphorylation in sperm treated with 10 μmol/mL Cr. As shown in Fig 8, 1.0 μmol/ml Cr increased the cAMP level; in contrast, a high dose of Cr (>4 μmol/mL) reduced the cAMP level in boar sperm. Notably, the cAMP level (2.267 pmol/106 sperm) in sperm treated with 10 μmol/mL Cr decreased by 39.8%，while increased to 5.35±0.34 pmol/106 sperm in 100μmol/mL Cr treated group (data not given). The effects of adding the regulatory factors in the cAMP/PKAs signal pathway, dbcAMP, IBMX and H-89, on the protein phosphorylation associated with sperm capacitation are shown in Fig 9. P-PKAs and PTP increased extremely with the addition of dbcAMP and IBMX in the presence of 10 μmol/mL Cr. Protein phosphorylation increased, which was correlated with the increase of the dbcAMP concentration, but H-89 obviously inhibited the P-PKAs and PTP. Our results indicate that Cr affects protein phosphorylation, at least in part, through the cAMP/PKA pathway in boar sperm.

3.6 Immunofluorescence localization of P-PKAs and PTP in boar spermatozoa Furthermore, we detected the immunofluorescence localization of P-PKAs and PTP to explore the target location of Cr in boar sperm. Fig 10 shows that P-PKAs and PTP were mainly located in the equatorial segment of the head, the middle piece and the principle piece of the flagellum in the boar spermatozoa. In addition, the appearance of PTP was observed in the postacrosomal region. Moreover, the P-PKAs and PTP in the capacitated sperm were higher than in the control. Compared with the control, 0.5 μmol/mL Cr increased the P-PKAs and PTP in the middle piece of boar

sperm. However, 10 μmol/mL Cr reduced the P-PKAs and PTP in the middle piece of the sperm flagellum. Surprisingly, almost no PTP was detected in the middle piece of sperm exposed to 100 μmol/mL Cr. However, the PTP in the principal piece dramatically increased with the presence of 100 μmol/mL Cr. These data suggest that Cr mainly affected protein phosphorylation in the middle piece and principal piece of boar sperm

4. Discussion In the present study, we have shown that hexavalent chromium can lead to reproductive toxicity in boar spermatozoa in vitro. In animal husbandry, dietary Cr supplementation is applied to accelerate body growth, enhance carcass quality and increase lean body mass and productive performance in domestic animals such as pigs [5-7]

. Previously, it had been described that Cr addition resulted in the elevation of Cr

concentration in bone, kidney, liver and ovaries in pigs, without the evaluation of the Cr content in testis

[7]

. However, concentrated Cr was observed in the testis of male

rats treated with Cr, which subsequently decreased the testis’ relative weight and sperm motility, correlated with the increase in Cr dose [9]. In addition, various studies have demonstrated that the development and morphological structure of testis were affected by Cr, accompanied by decreased sperm quality

[15, 16]

. Thus, we reasoned

that chromium could accumulate in testis and then affect the reproductive function of boars supplied with dairy Cr for long periods of time. However, there were few reports on the effects of Cr on the male fertility of pigs. Therefore, the present study was undertaken to evaluate the effects of hexavalent chromium on boar sperm in vitro for the first time. The results indicate that sperm motility and protein phosphorylation were actually affected by chromium, which hopefully offers some interesting clues for further study on reproductive toxicity to facilitate domestic animal breeding. The present work shows that low concentrations of Cr (0.1-1.0 μmol/mL) increased protein phosphorylation and enzyme activity, which play a key role in the male reproductive system of boars. Parallel results have been shown in previous research, including an increased number of live-born piglets, an increased litter

weaned mass and an increased concentration of Cr in the colostrum throughout gestation regarding the productive performance in sow supplemented with 400 ppb dietary chromium through gestation for 110 days [7]. In addition, the increased content of GH mRNA and secretion of GH promoted the growth performance in finishing pigs treated with 200 μg/kg dietary chromium for 35 days [37]. This was followed by a demonstration of increased protein deposition and growth performance in sows with Cr supplementation

[6]

. It is well documented that Cr improves glycometabolism and

ATP levels by enhancing the sensitivity of insulin in cells

[3]

. The present results are

generally consistent with the idea that a certain concentration of Cr can improve the metabolism and reproductive function in boars. Furthermore, we have extended the study to assess the effects of high-dose Cr on boar sperm in vitro. Chromium has been reported to easily accumulate in tissues including kidney, ovary and testis

[8]

. In this way, we assumed that Cr enrichment in

testis, caused by dairy chromium supplementation for long periods of time, could ultimately affect the reproductive function of pigs. In the present study, when the concentration of Cr was higher than 4 μmol/mL, it inhibited the sperm motility, metabolism and protein phosphorylation of boar sperm, correlated with the increase in Cr concentration. In particular, the 10 μmol/mL Cr treatment decreased the most in comparison to the control. It is not difficult to conclude that a high Cr concentration can affect male boar fertility. There are two main established factors, sex hormone levels and oxidative stress, responsible for male infertility caused by Cr

[9]

. Notably,

the change in sex hormone levels affects sperm motility. For instance, testosterone is necessary for the development of accessory sex organs [38]. Additionally, FSH and LH can impair the formation of sperm by regulating testis activity

[39]

. A previous study

reported that increased FSH and decreased LH and testosterone serum levels were detected in Cr-treated rats, correlated with a decrease in sperm number and sperm motility

[9]

. However, accessory sex organ weight and sperm count were restored by

curcumin by ameliorating the decrease of testosterone in rats

[40]

. Oxidative stress is

regarded as the main factor responsible for male infertility. Once inside the cells, hexavalent chromium is usually associated with the over-production of ROS, which

leads to oxidative stress including DNA damage, lipid peroxidation and mitochondrial oxidative damage [1]. These deleterious effects in testis result in the decrease of sperm count and motility, which is crucial for male fertility. Additionally, many studies have claimed that the elevation of ROS decreases sperm motility [41-43]. The reduction of intracellular ATP may be induced by the suppression of mitochondrial activity and glycolysis, as the ΔΨm and GAPDH activity were decreased in sperm treated with high doses of Cr. A previous study has shown that hexavalent chromium is a powerful inducer of cytochrome c (cytC) release to the cytosol

[40]

, which indicates that a high Cr content in boar sperm may impair

mitochondrial activity, subsequently reducing the synthesis of ATP in the midpiece of sperm [45]

[44]

. GAPDH is a key enzyme in glycolysis, which occurs along the sperm tail

. Many studies have suggested that glycolysis is an important pathway to generate

ATP in sperm cells and provides energy for the movement of sperm [46]. Moreover, it is widely known that intracellular ATP not only directly influences sperm motility but also takes part in regulating the activation of protein kinase, including PKA [47]. In addition, Chio et al. reported that sperm exposed to Cr-induced cytochrome c up-regulation were capable of activating tyrosine phosphorylation in sperm proteins for capacitation in non-capacitated media

[40]

. Zhang et al. suggested

that (S)-a-chlorohydrin inhibited sperm protein tyrosine phosphorylation, which may play a role in male rat infertility associated with SACH

[48]

. Given these reports, we

reasoned that the mechanism underlying the reproductive toxicity caused by chromium might be connected with sperm protein phosphorylation. In mammals, sperm motility mainly depends on the structure and physiological properties of sperm and the activity of hyperactivation during capacitation

[49, 50]

. In

particular, the tyrosine phosphorylation of sperm has a close connection with capacitation, and PTP is recognized as a hallmark of capacitation [36, 51]. Therefore, we detected the protein phosphorylation in boar sperm to explore the chromium-induced reproductive toxicity mechanism and found a decrease in sperm motility correlated with the inhibition of protein phosphorylation. It has been well documented that the cAMP/PKA signal pathway regulates P-PKAs, which subsequently regulate PTP and

activate sperm motility [25, 47]. In this pathway, ‘soluble’ adenylyl cyclase (sAC) could catalyze ATP transformed into cAMP, which would then increase the phosphorylation of PKA substrates by activating protein kinase A (PKA). Subsequently, the downstream cascades are up-regulated, resulting in the elevation of PTP, which is necessary for sperm hyperactivity and capacitation

[25]

. In the present study, 10

μmol/mL Cr significantly decreased the cAMP level, P-PKAs and PTP in boar sperm. However, adding dbcAMP and 0.1 mM IBMX to the medium with the presence of 10 μmol/mL Cr restored the P-PKAs and PTP. Even a dose-dependent increase in P-PKAs and PTP was observed in the dbcAMP-treated boar sperm. Therefore, adding dbcAMP and IBMX could alleviate the inhibition of P-PKAs and PTP in boar sperm caused by 10 μmol/mL Cr. Furthermore, we detected the immunofluorescence localization of P-PKAs and PTP in boar spermatozoa and found that 10 μmol/mL Cr significantly decreased protein phosphorylation in the midpiece and principle piece, which are primarily responsible for sperm motility

[44, 49]

. It is therefore most likely that chromium

impaired the sperm motility correlated with the change in protein tyrosine phosphorylation regulated by the cAMP/PKA signal pathway in the tail of boar sperm. Just as shown in Fig. 11, the decrease (or increase) in the ATP level and GAPDH activity in boar sperm treated with a high (or low) concentration of Cr was associated with a reduction (or elevation) in the cAMP content, which subsequently inhibited (or promoted) the cAMP/PKA pathway and down-regulated (or up-regulated) the downstream PTP. In addition, the PTP started to decrease at 0.5 μmol/mL Cr in the Cap medium, whereas it began to decrease at 4.0 μmol/mL Cr in the N-Cap medium. Taking capacitation regulatory factors such as Ca2+, BSA and HCO3- into consideration, we reasoned that the difference in PTP reduction induced by Cr in boar sperm specifically incubated in Cap medium and N-Cap medium may be caused by Ca2+ in the Cap medium. It has been well studied that Ca2+ has an effect on protein tyrosine phosphorylation, which is crucial for sperm capacitation

[36, 47, 52]

. Because the

structure of Cr is similar to that of Ca2+, Cr may be transformed into cells through

Ca2+ ion channels, significantly increasing the intracellular Cr content. Interestingly, as shown in our results, 100 μmol/mL Cr dramatically decreased PTP in the midpiece, but it significantly increased PTP in the principle piece of boar sperm. The decrease in PTP in the midpiece may be associated with the mitochondrial damage induced by high doses of Cr. Moreover, the increase in PTP in the principle piece was similar to the research of Harayama et al., in which adding cBiMPs significantly enhanced the PTP in the principle piece of sperm

[53]

. Harayama et al.

took ADCY 10 (sAC) mainly located in the connection of the sperm head and tail and in the principle piece, and the increase in PTP was induced by cBiMPs though the [54-56]

cAMP/PKA pathway

. Similar to cBiMPs, hexavalent chromium can readily

cross cellular membranes with the help of nonspecific anion carriers concentration

of

Cr

induced

high-molecular-weight

proteins

tyrosine-phosphorylated

upon

the such

the

elevation as

p93,

increased

of

cAMP

p175, cAMP

p220 in

[9]

, then high

level. are

sperm

Some strongly

[57]

.

The

dephosphorylating of PDK1, detected in the principal piece, also can potently induce protein tyrosine phosphorylation

[56]

. Moreover, translocation of tyrosine kinase or

modification of the protein substrates that make their TK phosphorylation sites available, can also increase PTP in boar sperm

[57]

. Directly, a high concentration of

Cr may activate protein tyrosine kinase (PTK) such as TK-32 and auto phosphorylated on tyrosine resulted protein tyrosine-phosphorylation in the principle piece of boar sperm [58].

5. Conclusions In summary, our data demonstrate that Cr affects boar sperm motility in vitro. High Cr concentrations decreased sperm motility. The reason for this may be that Cr affected mitochondrial activity, decreased the generation of ATP, and subsequently down-regulated PTP in the middle piece of boar sperm by impairing the cAMP/PKA pathway. In conclusion, the current study evaluated the effects of Cr on sperm motility and the underlying mechanisms in domestic animals in vitro through the detection of sperm protein phosphorylation. This study is expected to provide a theoretical basis

for the development of livestock industry. Moreover, our research is expected to provide interesting clues for further study on reproductive toxicity, thus facilitating domestic animal breeding.

Conflicts of interest The authors have no conflicts of interest to declare. #, these authors contributed equally to this work.

Acknowledgements This work was supported by special fund for agro-scientific research in the public interest of china (200903056) and key project of Shanghai Municipal Agricultural Commission (2014-7-2-5).

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Fig. 1 Concentration-effects of Cr on boar sperm motility and parameters. Sperm were incubated in no-capacitated, capacitated and a series of Cr concentrations for 90 min. Sperm motility was detected by CASA. (A) sperm motility (%), (B) VSL(μm/s), (C) VAP (μm/s), (D) VCL (μm/s). (N) no-capacitated control; (Cap) capacitated control. (n=9, mean ±SEM, *p<0.05 compare with no-capacitated control).

Fig. 2 Effects of chromium on ATP level and GAPDH activity of boar sperm. Boar sperm were incubated in no-capacitated media with different Cr concentrations as indicated at the bottom of the figure for 90 min before ATP level and GAPDH activity were determined. (A) The ATP level in sperm. (B) The activity of GAPDH in sperm. Each experimental was performed at least three times and subjected to statistical analysis. (n=6, mean ±SEM, *p < 0.05 compare with no-capacitated control).

Fig. 3 Effects of different Cr concentrations on boar sperm mitochondria membrane potential. Sperm were incubated in no-capacitated, capacitated and a series of Cr concentrations for 90 min. (A) Typical double fluorescence dot plot form flow cytometric analysis of ΔΨm. The left one represents high ΔΨm and the right one stands for low ΔΨm. (B) The value of FL2/FL1 in sperm treated with different Cr concentrations. (n=6, mean ±SEM, *p < 0.05 compare with no-capacitated control)

Fig.4 Western blot of protein tyrosine phosphorylation affected by different Cr concentrations. Sperm were incubated in no-capacitated media with different Cr concentrations as indicated at the bottom of the figure for 90 min. (A) Western blot analysis was performed using anti-phosphotyrosine antibody. Equal loading was monitored using a monoclonal anti-α-tubulin antibody. The experiment was performed at least three times and the picture is the repeatable and typical result. (B) Optical density of PTP after incubated with different Cr concentrations. Levels of phosphorylation were quantitated by densitometry analysis of representative bands (27kDa). (n=3, mean ±SEM, *p < 0.05 compare with no-capacitated control)

#

Fig.5 Western blot of P-PKA substrates affected by different Cr concentrations. Sperm were incubated in no-capacitated media with different Cr concentrations as indicated at the bottom of the figure for 90 min. (A) Western blot analysis was performed using anti-phospho-PKA substrate antibody. Equal loading was monitored using a monoclonal anti-α-tubulin antibody. The experiment was performed at least three times and the picture is the repeatable and typical result. (B) Optical density of P-PKA substrates after incubated with different Cr concentrations. Levels of phosphorylation were quantitated by densitometry analysis of each line contained in the region marked by #. (n=3, mean ±SEM, *p < 0.05 compare with no-capacitated control)

Fig. 6 Effects of different Cr concentrations on boar sperm incubated in no-capacitated medium. Sperm were incubated in no-capacitated media with different Cr concentrations as indicated at the bottom of the figure for 90 min. Western blot analysis was performed using anti-phospho-PKA substrate antibody (A) and anti-phosphotyrosine antibody (B). Equal loading was monitored using a monoclonal anti-α-tubulin antibody. The experiment was performed at least three times and the picture is the repeatable and typical result.

Fig. 7 Effects of different Cr concentrations on boar sperm incubated in capacitated medium. Sperm were incubated in capacitated media with different Cr concentrations as indicated at the bottom of the figure for 90 min. Western blot analysis was performed using anti-phospho-PKA substrate antibody (A) and anti-phosphotyrosine antibody (B). Equal loading was monitored using a monoclonal anti-α-tubulin antibody. The experiment was performed at least three times and the picture is the repeatable and typical result.

Fig. 8 Effects of chromium on cAMP level in boar sperm. Boar sperm were incubated in no-capacitated media with different Cr concentrations as indicated at the bottom of the figure for 90 min before cAMP level were determined. Each experimental was performed at least three times and subjected to statistical analysis. (n=6, mean ±SEM, *p < 0.05 compare with no-capacitated control).

Fig.9 dbcAMP and IBMX restored the inhibition of protein phosphorylation by Cr. Boar sperm were incubated with 10μmol/ml Cr, and in the presence or absence of dbcAMP and IBMX for 90 min, Western blot analysis was performed using anti-phospho-PKA substrate antibody (A) and anti-phosphotyrosine antibody (B). Equal loading was monitored using a monoclonal anti-α-tubulin antibody. The experiment was performed at least three times and the picture is the repeatable and typical result.

Fig.10 Immunofluorescence localization of protein phosphorylation affected by Cr. (A)Immunofluorescence localization of P-PKAs substrates (red) in boar sperm cultivated in no-capacitated, capacitated, and 0.5, 10, 100μmol/ml Cr in the no-capacitated medium, acrosome (green) and the merged picture. (B) Immunfluoresce localization of PTP (green) in boar sperm cultivated in no-capacitated, capacitated, and 0.5, 10, 100μmol/ml Cr in the no-capacitated medium, cell nucleus (red) and the merged picture. Localization of acrosome was determined using PNA and nucleic was determined using (Propidium iodide) PI. Sperm cells were visualized using confocal laser scanning microscope (×400).

Fig.11 The model by which Cr affects cAMP/PKA mediated protein phosphorylation through blocking glycolysis in boar spermatozoa during capacitation. Low concentration of Cr (L) increased the mitochondrial and GAPDH activity and high concentration of Cr (H) inhibited the mitochondrial and GAPDH activity. The effects on mitochondrial and GAPDH activity induced by Cr results in the change of intracellular ATP content and subsequently regulate P-PKAs and PTP. Partly, IBMX and dbcAMP reverse the inhibitions of P-PKAs and PTP.