Hydrogen Sulfide and/or Ammonia Reduces Spermatozoa Motility through AMPK/AKT Related Pathways

A number of emerging studies suggest that air pollutants such as hydrogen sulfide (H2S) and ammonia (NH3) may cause a decline in spermatozoa motility. The impact and underlying mechanisms are currently unknown. Boar spermatozoa (in vitro) and peripubertal male mice (in vivo) were exposed to H2S and/or NH3 to evaluate the impact on spermatozoa motility. Na2S and/or NH4Cl reduced the motility of boar spermatozoa in vitro. Na2S and/or NH4Cl disrupted multiple signaling pathways including decreasing Na+/K+ ATPase activity and protein kinase B (AKT) levels, activating Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) and phosphatase and tensin homolog deleted on chromosome ten (PTEN), and increasing reactive oxygen species (ROS) to diminish boar spermatozoa motility. The increase in ROS might have activated PTEN, which in turn diminished AKT activation. The ATP deficiency (indicated by reduction in Na+/K+ ATPase activity), transforming growth factor (TGFβ) activated kinase-1 (TAK1) activation, and AKT deactivation stimulated AMPK, which caused a decline in boar spermatozoa motility. Simultaneously, the deactivation of AKT might play some role in the reduction of boar spermatozoa motility. Furthermore, Na2S and/or NH4Cl declined the motility of mouse spermatozoa without affecting mouse body weight gain in vivo. Findings of the present study suggest that H2S and/or NH3 are adversely associated with spermatozoa motility.

increasing over the last few decades, which raises concerns that it might negatively influence environmental and public health as well as climate change due to the role of NH 3 in PM formation, visibility degradation, and atmospheric deposition of nitrogen within ecosystems 18,20,21 . H 2 S, a colorless, flammable, and foul odored gas, has been considered poisonous for a long time 10,22 . Recently, it has been found to be a signaling molecule regulating insulin secretion/sensitivity and vascular homeostasis to control blood pressure and penile tone 10,22,23 . Furthermore, it is a mediator of dietary restriction 24 . The concentration of H 2 S in blood has been detected to be ~50 μ M in animals and ~600 μ M in asthmatic patients 25 . Although H 2 S acts as a transmitter in biological systems, its poisonous nature is still a problem because it is the predominant sulfur contaminant of natural gas with widespread environmental and occupational exposure from industrial activities 22,26 .
Although NH 3 and H 2 S have not been considered as major gaseous pollutants, they are present in air, carried by PM, and strikingly little is understood about their effects on spermatozoa quality and the underlying mechanisms. Therefore, this current investigation aimed to explore the impact of NH 3 and H 2 S on spermatozoa quality and the underlying mechanisms.

Results
Na 2 S and a combination of NH 4 Cl + Na 2 S decreased boar spermatozoa motility. To examine the effects of NH 4 Cl and/or Na 2 S on boar spermatozoa motility, we treated the spermatozoa with different concentrations of NH 4 Cl, Na 2 S, and a combination of NH 4 Cl + Na 2 S in spermatozoa incubation medium for 24 h. Treatments involving 100, 400, and 1600 μ M NH 4 Cl did not alter spermatozoa motility. However, 25, 50, and 100 μ M Na 2 S treatments and a combination of NH 4 Cl + Na 2 S significantly decreased spermatozoa motility as shown by a dramatic increase in grade D spermatozoa (immotile) and a decrease in grade A + B spermatozoa in a concentration dependent manner [ Fig. 1A; p < 0.05 (grade A + B, grade D): Na 2 S treatment compared to control treatment, Na 2 S + NH 4 Cl treatment compared to control treatment]. Immotile spermatozoa (grade D) increased from 17% in the control to 77% in the Na 2 S-100 μ M treatment and 82% in the NH 4 Cl-1600 μ M + Na 2 S-100 μ M treatment. The straight motile spermatozoa (grade A + B) decreased from 55% in the control to 3% in the Na 2 S-100 μ M treatment and 1% in the NH 4 Cl-1600 μ M + Na 2 S-100 μ M treatment. To assess boar spermatozoa viability after 24 h treatments, we analyzed the living cells with a Live/Dead Sperm Viability Kit using flow cytometry. As presented in Fig. 1B, none of the treatments changed spermatozoa viability. Most treatments increased the protein levels of apoptosis markers Bax and Caspase 8; at the same time, the treatments also elevated the anti-apoptosis markers Bcl-xl and Bcl-2 (Fig. 1C). Boar spermatozoa abnormality rate was very low (2-4% of total cells) and no treatments affected these levels (Fig. 1D). Phosphatidylserine (PS) externalization at the spermatozoon plasma membrane is a process that indicates plasma membrane scrambling. We further investigated the effect of NH 4 Cl and/or Na 2 S on PS externalization at the spermatozoon plasma membrane. The level of PS externalized in spermatozoa plasma membranes was very low (< 10% of total viable spermatozoa) and no treatments disrupted PS externalization (Fig. 1E). To determine the function of mitochondria, we evaluated the spermatozoon mitochondrial membrane potential (△ Ψ m). No treatments affected the population of spermatozoa presenting high △ Ψ m (Fig. 1F), which remained at around 97%. Subsequently, the effects of NH 4 Cl and/or Na 2 S on spermatozoa capacitation were assessed by CTC staining. As reported in Fig. 1G, NH 4 Cl and/or Na 2 S had no evident effects on the percentage of cells showing the F pattern (typical of freshly ejaculated cells), B pattern (typical of capacitated cells), or AR pattern (typical of acrosome-reacted cells). Na 2 S and the combination of NH 4 Cl + Na 2 S increased ROS formation in boar spermatozoa. To investigate the effects of NH 4 Cl and/or Na 2 S on ROS production, we analyzed the ROS levels (H 2 O 2 ) in spermatozoa using flow cytometry. Na 2 S and the combination of NH 4 Cl + Na 2 S increased the level of H 2 O 2 after 24 h of treatment. Na 2 S-50 μ M, NH 4 Cl-400 μ M + Na 2 S-50 μ M, and NH 4 Cl-1600 μ M + Na 2 S-100 μ M significantly increased H 2 O 2 levels by 20-40% ( Fig. 2A, p < 0.05). At the same time, the anti-oxidant enzymes catalase, total-SOD (super oxide dismutase), and GPX1 (glutathione peroxidase) were determined (Fig. 2B). All treatments increased SOD protein levels in spermatozoa. The combination of NH 4 Cl and Na 2 S elevated the protein levels of GPX1; however, NH 4 Cl or Na 2 S alone did not affect GPX1. NH 4 Cl treatments elevated the protein level of catalase; however, Na 2 S or combinations of NH 4 Cl and Na 2 S treatments did not affect catalase protein levels.

Na 2 S and/or NH 4 Cl decreased ATPase, increased TAK1, and activated AMPK in boar spermatozoa.
The AMP-activated protein kinase (AMPK) pathway plays a crucial role in boar spermatozoa motility. The combination of NH 4 Cl and Na 2 S treatments dramatically increased AMPK protein levels by ~5-fold (Fig. 3A). The phosphorylated form (Thr 172 ) of AMPK was elevated by NH 4 Cl alone, Na 2 S alone, and the combination of NH 4 Cl + Na 2 S treatments (Fig. 3A). Moreover, the protein levels of the phosphorylated form (Thr 172 ) of AMPK in the NH 4 Cl + Na 2 S combination treatments were higher than those in the treatments of NH 4 Cl or Na 2 S alone. The NH 4 Cl + Na 2 S treatments synergistically increased the phosphorylated form (Thr 172 ) of AMPK (Fig. 3A). AMPK can be activated through the TAK1, CaMKK, or LKB1 pathways, or by changing the ratio of AMP/ATP. NH 4 Cl, Na 2 S, and combinations of NH 4 Cl + Na 2 S increased the protein levels of TAK1 (Fig. 3B); however, none of the treatments altered CaMKKα /β , and Na 2 S, furthermore, the combination of NH 4 Cl + Na 2 S treatment decreased LKB1 level (data not shown). The activity of Na + /K + -ATP synthesis enzymes (Na + /K + -ATPase) was decreased by Na 2 S and the combination NH 4 CL + Na 2 S treatments. The activity of Na + /K + -ATPase in the combination treatments was even lower than that in Na 2 S treatments (Fig. 3C). Although NH 4 Cl treatments decreased Na + /K + -ATPase activity compared to the control treatment, the difference was not significant. The data were confirmed by the protein level of ATPase5β , which was decreased by Na 2 S and the combination NH 4 Cl + Na 2 S treatments in Western blotting analysis (Fig. 3D).
ATP addition partially rescued boar spermatozoa motility. To test whether ATP deficiency was the major reason for the decrease in boar spermatozoa motility caused by Na 2 S and/or NH 4 Cl, ATP rescue experiments were performed with addition of ATP. The addition of ATP only slightly elevated spermatozoa motility. NH 4 Cl-1600 + ATP-2 mM elevated the percentage of grade A + B motility compared to the NH 4 Cl-1600 treatment alone (p < 0.05). NH 4 Cl-400 + Na 2 S-50 μ M + ATP-1 mM and NH 4 Cl-400 + Na 2 S-50 μ M + ATP-2 mM increased the percentage of grade A + B motility compared to the NH 4 Cl-400 + Na 2 S-50 μ M treatment alone (p < 0.05; Fig. 3E).

Na 2 S and/or NH 4 Cl activated the PI3K, ERK, and PTEN pathways and inhibited the AKT pathway in boar spermatozoa. PI3K/AKT/ERK pathways play vital roles in cell biology and the activation of AKT
inhibits AMPK activation. The levels of these proteins were determined by Western blotting and immunofluorescent staining. Na 2 S-50 (100 μ M) and the combination of NH 4 Cl + Na 2 S treatments increased PI3K protein levels (Figs 4 and 5A). However, Na 2 S and the combination of NH 4 Cl + Na 2 S treatments decreased protein levels of both AKT and the phosphorylated form (Thr 308 ) of AKT (p-AKT; Figs 4 and 5B). ERK 1+2 protein level remained unchanged by all treatments (Figs 4 and 5C). However, NH 4 Cl-400, 1600 μ M, Na 2 S-25, 50, 100 μ M, and combinations of NH 4 Cl + Na 2 S treatments increased the phosphorylated form (Thr 197 + Thr 202 ) of ERK 1 (Fig. 4). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an inhibitor for activation of AKT from PI3K. All treatments increased PTEN and the phosphorylated form (Ser 380 + Thr 382 + Thr 383 ) of PTEN (p-PTEN; Fig. 4).
Combination of Na 2 S and NH 4 Cl decreased mouse spermatozoa motility in vivo. In order to confirm the data obtained from boar spermatozoa in vitro, mice were treated with different concentrations of NH 4 Cl and/or Na 2 S (in vivo studies). The combination NH 4 Cl-50 mg/kg + Na 2 S-50 mg/kg treatment significantly decreased mouse spermatozoa motility by decreasing the population of grade A + B spermatozoa (Fig. 6A, p < 0.05). Furthermore, Na 2 S-50 mg/kg and NH 4 Cl-10 + Na 2 S-10 treatments significantly increased the population of grade D (immotile) spermatozoa (Fig. 6A, p < 0.05). Moreover, the Na 2 S-50 mg/kg treatment promoted the number of abnormal mouse spermatozoa (Fig. 6B, p < 0.05). Na 2 S-50 + NH 4 Cl-50 mg/kg also elevated spermatozoa abnormalities; however, the difference was not significant at p < 0.05. Finally, no treatments disrupted mouse body weight gain during the experimental period (Fig. 6C).

Discussion
Numerous emerging studies suggest that air pollutants may cause a decline in the motility of human spermatozoa 1-5 . NH 3 and H 2 S are air pollutants that can be free or bound to air PM 8,9,16,17 . In the current study, H 2 S donor Na 2 S and/or NH 3 donor NH 4 Cl decreased boar spermatozoa motility in vitro and reduced mouse spermatozoa motility in vivo, which confirmed our hypothesis that air pollutants might reduce spermatozoa motility. However, Na 2 S and/or NH 4 Cl did not change boar spermatozoa viability, abnormality rate, plasma membrane integrity, mitochondrial membrane potential, or capacitation status. This suggests that the toxicity of NH 4 CL and/or Na 2 S treatments (used in current investigation) might be not very high just that spermatozoa motility was declined while the spermatozoa survival was not impaired. This phenomenon was also found by the epidemiology studies 1-5 .  Although epidemiological studies have observed that air pollutants decrease spermatozoa motility, the underlying mechanisms are currently unknown. In this investigation, multiple signaling pathways were involved in the reduction of boar spermatozoa motility through the impact of Na 2 S and/or NH 4 Cl. These pathways are all connected to the AMPK pathway. AMPK, an energy status sensor, regulates cellular energy homeostasis, mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. AMPK is expressed in the ovaries, testes, and spermatozoa; it regulates gonadal steroidogenesis and is thus involved in fertility 27 . Most interestingly, it modulates spermatozoa motility and other functions 28,29 . The increase in AMP: ATP or ADP: ATP ratio can activate AMPK by phosphorylation at Thr 172 (p-AMPK). It has also been found that Ca 2+ / calmodulin-dependent protein kinase (CaMKK β ), transforming growth factor (TGF β ) activated kinase-1 (TAK1), and LKB1 could activate AMPK 28,30 . TAK1 can be activated by cytokines. In the current study NH 4 Cl and/or Na 2 S increased TAK1. In line with previous studies where exposure to low levels of airborne irritants stimulated the markers of airway inflammation, where high level of H 2 S were detected in asthma patients, and exposure to different size fractions of PM activated inflammation and oxidative stress signals, Na 2 S and/or NH 4 Cl might elevate inflammatory factors to activate TAK1, which in turn enhances AMPK activation 25,31,32 . Combinations of Na 2 S + NH 4 Cl significantly elevated the protein levels of AMPK and p-AMPK (Thr 172 ) in boar spermatozoa. Na 2 S alone or NH 4 Cl alone stimulated p-AMPK (Thr 172 ), but not AMPK protein in boar spermatozoa. Na 2 S and combinations of NH 4 Cl + Na 2 S diminished the activity of Na + /K + -ATPase in boar spermatozoa. Consistent with the notion that AMPK is very sensitive to the AMP: ATP ratio, our data suggested that NH 4 Cl and/or Na 2 S might decrease ATP production in spermatozoa by inhibiting ATPase, which consequently activated AMPK to reduce spermatozoa motility 28 .
Because boar sperm motility is sensitive to oxidative stress and H 2 O 2 is the major free radical mediating direct ROS effects in boar spermatozoa, H 2 O 2 was measured in boar spermatozoa after Na 2 S and/or NH 4 Cl treatments 33 . Interestingly, Nguyen et al. 34 observed that AMPK stimulated intracellular anti-oxidative defense enzymes in chicken spermatozoa. In the current study, Na 2 S and the combination of Na 2 S + NH 4 Cl elevated H 2 O 2 in boar spermatozoa in vitro. However, at the same time the antioxidant enzymes SOD and GPX1 were also increased by N 2 S and the combinations of Na 2 S + NH 4 Cl. These results indicated that N 2 S and the combinations of Na 2 S + NH 4 Cl might promote oxidative stress; on the other hand, in order to defend against the stress, the boar spermatozoa might activate antioxidant systems to suppress ROS. The PI3K/AKT pathways play critical roles in controlling cell survival and spermatozoa motility. Sagare-Patil et al. 35 found that the PI3K-AKT pathway is required for motility and hyper-activation in human spermatozoa and Gallardo Bolaños et al. 36 observed that p-AKT preserved stallion spermatozoa motility. Moreover, activation of AKT inhibits AMPK phosphorylation (Thr 172 ) 30 ; the PTEN signaling pathway regulates cell proliferation, cell-cycle progression, and cell survival; and ROS, ATP deficiency, and AMPK activation promote PTEN expression and nuclear accumulation [37][38][39][40] . However, PTEN is a negative regulator of AKT. In the current study, Na 2 S and the combinations of Na 2 S + NH 4 Cl increased the protein level of PI3K; however, these treatments decreased AKT and p-AKT levels. Na 2 S and/or NH 4 Cl treatments also elevated the protein levels of PTEN and p-PTEN in boar spermatozoa. This data suggested that the increase in H 2 O 2 , ATP deficiency, and AMPK activation stimulated the PTEN pathway, which consequently inhibited AKT activation. On the other hand, the decrease in AKT stimulates AMPK activation 30 . Furthermore, AKT inhibition might reduce spermatozoa motility due to its importance in controlling spermatozoa motility 35,36 . PI3K/ERK pathway is very important for cell survival and growth too 35,41 . PI3K might activate ERK pathway to increase p-ERK in the spermatozoa. It also was found that spermatozoa viability was not altered by NH 4 CL and/ or Na 2 S treatment. Therefore p-ERK might play crucial role in the sperm survival.
It is known that mammalian spermatozoa are transcriptionally and translationally inactive. However, it has been found that post-translational modifications and protein acquisition/degradation play very import role in spermatozoa in order to response to the changes in the epididymis and female tract 42 . In current study, NH 4 CL and/or Na 2 S treatments altered many proteins or phosphorylated proteins in spermatozoa which might be due to the post-translational modifications and protein acquisition/degradation. The combination of Na 2 S-50 mg/kg + NH 4 Cl-50 mg/kg treatment significantly decreased mouse spermatozoa motility by reducing the percentage of grade A + B spermatozoa and elevating grade D (immotile) spermatozoa in vivo, which agreed with the data from boar spermatozoa in vitro. The data further indicated that exposure to one component of air pollution may not be excessively problematic; however, a combination of two or more components of air pollution might synergistically pose problems to human health.
In conclusion, the data from boar spermatozoa in vitro demonstrated that H 2 S and/or NH 3 disrupted multiple signaling pathways to diminish spermatozoa motility. The main points include a decrease in ATP production and AKT levels, activation of AMPK and PTEN, and an increase in ROS. The increase in ROS might activate PTEN, which in turn diminished AKT activation. The ATP deficiency (indicated by reduction in Na + /K + ATPase activity), TAK1 activation, and AKT deactivation stimulate AMPK, which results in a decline in spermatozoa motility (Fig. 7). And the in vivo data with mouse spermatozoa confirmed the in vitro results. Findings of the present study suggest that H 2 S and/or NH 3 may be adversely associated with spermatozoa quality, particularly spermatozoa motility.

Collection of boar semen and preparation of spermatozoa samples for different treatments.
Porcine spermatozoa incubation medium powder was purchased from Hangzhouyuefeng Bio-engineering CO., Ltd. (Hangzhou, China). The medium can maintain pH, osmolarity, ion balance, and buffering to sustain spermatozoa activity for 7-14 d. Semen samples from Duroc boars (2-3 y old) were commercially obtained from a Regional Porcine Company (Hengshengyuan CO., Ltd., Qingdao, China). All boars were housed in individual pens in an environmentally controlled building (15-25 °C) according to Regional Government and national regulations. Artificial insemination took place using preserved liquid semen from boars of demonstrated fertility. Fresh ejaculates were collected with the gloved hand technique and stored at 17 °C before use 28 . Semen samples from 3-5 animals were pooled each time to minimize individual boar variation and the samples had > 80% morphologically normal spermatozoa. Subsequently, the semen was diluted with incubation medium to a concentration ~40 × 10 6 cell/ml and then the cells were treated with NH 3 donor NH 4 Cl (Cat #:09718; Sigma-Aldrich Co. LLC. St. Louis, MO, USA) and/or H 2 S donor Na 2 S (Cat #:431648; Sigma-Aldrich Co. LLC.) 43,44 . There were ten treatment groups: (1)  Mouse exposure to NH 4 CL and/or Na 2 S. This investigation was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Qingdao Agricultural University IACUC (Institutional Animal Care and Use Committee). Animals were housed under a light: dark cycle of 12:12 h and at a temperature of 23 °C and humidity of 50-70%. Mice were handled humanely during the experiments and in order to minimize fighting, two mice were housed per shoebox-type cage with a solid floor and woodchip bedding. Mice had constant access to food (chow diet) and water, and bedding was changed every other day 45 . Mice were exposed to NH 4 Cl and/or Na 2 S via oral gavage. The NH 4 Cl and/or Na 2 S dosing solution was freshly prepared on a daily basis in phosphate buffered saline (PBS) solution and given to animals as described previously 24,46 . There were 7 treatments (8 mice/treatment): (1) vehicle control (PBS); (2) NH 4 Cl-10 mg/kg BW; (3) NH 4 Cl-50 mg/kg BW; (4) Na 2 S-10 mg/kg BW; (5) Na 2 S-50 mg/kg BW; (6) NH 4 Cl-10 mg/kg + Na 2 S-10 mg/kg BW; (7) NH 4 Cl-50 mg/kg BW + Na 2 S-50 mg/kg BW. The volume of gavage for each mouse was 0.1 ml/d. The gavage took place every morning for 30 d starting at 25 d of age. pH measurement. The pH of the samples was determined by a pH-meter (PB-10; Satorium; Germany). The electrode was thoroughly washed every time with distilled water before and after sample detection. 3-5 replicates were assessed for every sample 47 . Evaluation of spermatozoa motility by computer-assisted sperm analysis system. Spermatozoa motility was assessed by the computer-assisted sperm assay (CASA) method according to World Health Organization guidelines 48 . After 24 h of treatment, boar spermatozoa were incubated at 37.5 °C for 30 min then samples were placed in a pre-warmed counting chamber (MICROPTIC S.L., Barcelona, Spain). After euthanized, spermatozoa was collected from cauda epididymis of mice and suspended in DMEM/F12 medium with 10% FBS and incubated at 37.5 °C for 30 min then samples were placed in a pre-warmed counting chamber 49 . The Micropic Sperm class analyzer (CASA system) was used in this investigation. It was equipped with a 20-fold objective, a camera adaptor (Eclipse E200, Nicon, Japan), and a camera (acA780-75gc, Basler, Germany), and it was operated by an SCA sperm class analyzer (MICROPTIC S.L.). The classification of sperm motility was as follows: grade A linear velocity > 22 μ m s −1 ; grade B < 22 μ m s −1 and curvilinear velocity > 5 μ m s −1 ; grade C curvilinear velocity < 5 μ m s −1 ; and grade D immotile spermatozoa 48 .
Morphological observations of spermatozoa. After 24 h of treatment and subsequent incubation at 37.5 °C for 30 min, the boar spermatozoa were stained with Eosin Y (1%) as described by Shin et al. 50 . Briefly, the extracted caudal epididymis from mice were placed in RPMI and finely chopped and then Eosin Y (1%) was added for staining. Spermatozoa abnormalities were classified into head or tail morphological abnormalities: two heads, two tails, blunt hooks, and short tails (for each treatment group sample, 3-6 repeats). Analysis of boar spermatozoa viability by flow cytometry. As described by Hurtado de Llera et al. 28 , fluorescent staining using the Live/Dead spermatozoa viability kit (Cat #: L7011; Thermo Fisher scientific Inc., Waltham, MA, USA) was performed to measure boar spermatozoa viability following the manufacturer's instructions. Briefly, 5 μ l of SYBR-14 (20 μ mol/l) was added to 1 ml of spermatozoa sample in PBS with 40 × 10 6 cells/ml and incubated for 10 min at room temperature (RT) in darkness. And then 10 μ l of propidium iodide (PI; 2.4 mM) was added into the sample and incubated for another 10 min at RT in darkness. After incubation, spermatozoa were analyzed by the flow cytometer. The fluorescence values of SYBR-14 were collected in the FL1 sensor using a 525 nm bad pass filter, whereas PI fluorescence was collected in the FL3 sensor using a 620 nm bad pass filter. The viable spermatozoa were expressed as the average of the percentage of SYBR14 + /PI − spermatozoa ± SEM.

Flow cytometry analysis. A FACSCalibur
Evaluation of phosphatidylserine externalization at the outer leaflet plasma membrane of boar spermatozoa by flow cytometry. The spermatozoa plasma membrane phosphatidylserine (PS) externalization was detected by Annexin-V-FITC (Cat #: FA101; Beijing TransGen Biotech Co., Ltd.; Beijing, China) to specifically detect PS translocation from the inner to the outer leaflet of the boar spermatozoa plasma membrane as described by Hurtado de Llera et al. 28 . Briefly, after 24 hr treatment, boar spermatozoa cells were collected and resuspended in 1× Annexin V binding buffer (100 μ l). Then 5 μ l Annexin V and 5 μ l PI were added into the Scientific RepoRts | 6:37884 | DOI: 10.1038/srep37884 samples following by incubation for 15 min in the darkness at RT. After incubation, 400 μ l of binding buffer were added to each sample and mixed before flow cytometry analysis. The fluorescence values of probes Annexin V-FITC and PI were collected in the FL1 and FL3 sensors using a 520 and 620 nm bad pass filter, respectively. The results are expressed as the average of the percentage of Annexin V + /PI − spermatozoa ± SEM.

Analysis of boar spermatozoa mitochondrial membrane potential (△Ψm) by flow cytometry.
The mitochondrial membrane potential (△ Ψ m) was measured by the specific probe JC-1 (5,5′ ,6,6′ -tetrachloro-1,1′ ,3,3′ tetraethylbenzymidazolyl carbocyanine iodine; JC assay kit, Cat #: M34152; Thermo Fisher scientific Inc., Waltham, MA, USA) 28 . Briefly, after 24 hr treatment, the sperm cells was collected and resuspended in 1 ml PBS, and 10 μ l of 200 μ M JC-1 solution was added into the samples following incubation at 37 °C for 30 min. The samples were analyzed by flow cytometry analysis. The fluorescence value was collected using a 525 nm bad pass filter and the percentage of orange stained cells was recorded to be the population of spermatozoa with a high mitochondrial membrane potential. The data were present as the average percentage of high △ Ψ m spermatozoa ± SEM.
Determination of capacitation status. Sperm cells were tested with chlortetracycline (CTC; Cat #: 16663-5; Cayman Chemical, Ann Arbor, Michigan, USA) assay to assess the capacitation status as well as acrosome reaction as described by Bucci et al. 51 (Table S1). Secondary donkey anti-goat Ab (Cat no.: A0181) was purchased from Beyotime Institute of Biotechnology (Shanghai, P.R. China), and goat anti-rabbit (Cat no.: A24531) Abs were bought from Novex ® by life technologies (USA). Fifty micrograms of total protein per sample were loaded onto 10% SDS polyacrylamide electrophoresis gels. The gels were transferred to Polyvinylidene Fluoride (PVDF) membrane at 300 mA for 2.5 hr at 4 °C. Then, the membranes were blocked with 5% BSA for 1 hr at RT, followed by three washes with 0.1% Tween-20 in TBS (TBST). The membranes were incubated with primary Abs diluted with 1:500 in TBST with 1% BSA overnight at 4 °C. After three washes with TBST, the blots were incubated with the HRP-labeled secondary goat anti-rabbit or donkey anti-goat Ab respectively for 1 hr at RT. Then, the blots were imaged after three washes.

Detection of protein levels and location in spermatozoa using immunofluorescent staining.
Hurtado de Llera et al. 53 have reported the methodology for immunofluorescent staining of spermatozoa. After 24 hr treatment boar spermatozoa were fixed in 4% paraformaldehyde for 1 hr, then the cells were spread onto poly-L-lysine coated microscope slides and air-dry. After three washings with PBS (5 min each) spermatozoa were incubated with 2% (vol/vol) Triton X-100 in PBS for 1 hr at RT. Then, after three washes with PBS, the cells were blocked with 1% (wt/vol) BSA and 1% goat serum in PBS for 30 min at RT, then incubation with primary antibodies PI3K, p-AKT and ERK (1:100) diluted in blocking solution overnight at 4 °C. The following morning, after three washes with PBS Tween 20 (0.5%) the slides were incubated with Alexa Fluor 546 goat anti-rabbit IgG (1:200) for 30 min in darkness at RT. The negative controls samples were incubated with secondary antibody and without primary antibody. Slides were washed with PBS Tween-20 three times and then incubated with DAPI (4.6-diamidino-2-phenylindole hydrochloride, 100 ng/ml) as nuclear stain for 5 min. After brief wash with ddH 2 O, the slides were covered with an anti-fading mounting medium (Vector, Burlingame, USA). Fluorescent images were obtained with Leica Laser Scanning Confocal Microscope (LEICA TCS SP5 II, Germany).
Measurement of Na + /K + -ATPase activity. The activity of Na + /K + -ATPase was determined by the kit from Nanjing Jiancheng Biochemistry Co. (Nanjing, China) following the manufacturer's instruction 54 . Briefly, after 24 hr treatment, the spermatozoa were collected and lysed in 0.9% NaCl. Then the enzyme activity in the lysate was determined spectrophotometrically with the kit. The protein concentration was measured by the BCA method.
Scientific RepoRts | 6:37884 | DOI: 10.1038/srep37884 ATP rescue experiment. To test whether ATP can rescue the inhibitory effect of Na 2 S and/or NH 4 Cl on boar spermatozoa motility, 1 or 2 mM ATP (Cat #:10519979001; Sigma-Aldrich Co. LLC) was added to the incubation solution when the spermatozoa were treated with Na 2 S/NH 4 Cl 55 . Then after a 24 h treatment, spermatozoa motility was determined using the CASA method.
Statistical analysis. The data were statistically analyzed by SPSS statistical software (IBM Co., NY) using ANOVA. Comparisons between groups were tested by One-Way ANOVA analysis and the LSD test. All groups were compared with each other for every parameter (mean ± SEM). Differences were considered significant at p < 0.05.