Experiment 1 was designed to investigate whether metformin has beneficial effects on boar sperm functionality during preservation at 17 °C. Sperm were preserved in Modena extender with different concentrations of metformin (50, 100, 200, 500 µmol/L) at 17 °C for 13 d. Sperm motility was analyzed every 2 d. Plasma membrane integrity, acrosome integrity, ΔΨm, and cellular ATP levels were detected every 4 d.
Experiment 2 sought to confirm the expression and localization of AMPK protein and p-AMPK in boar sperm and to elucidate whether metformin was involved in the phosphorylation of AMPK. The expression and localization of AMPK and p-AMPK in boar sperm were detected by western blotting and immunofluorescence analysis. The levels of p-AMPK in the metformin and inhibitor (dorsomorphin dihydrochloride, Compound C) groups after 4 h of incubation at 37 °C or after 1, 5, 9, and 13 d at 17 °C were detected by western blot analysis. Samples for localization of AMPK and p-AMPK were from fresh semen.
Experiment 3 was devised to investigate whether metformin also protected boar sperm at 37 °C and whether it exerted its role through regulating AMPK activity. Sperm were preserved in Modena extender at 37 °C for 4 h with 200 µmol/L metformin in the presence or absence of Compound C. Sperm motility, membrane integrity, and ΔΨm were evaluated after 1 and 4 h of incubation at 37 °C. ATP content, glucose uptake capacity, lactate efflux, and lactate dehydrogenase (LDH) activity were detected after 4 h of incubation.
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (USA). Compound C was obtained from MedChemExpress (China). 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) was purchased from the Cayman Chemical Company (USA) and 4′,6-diamidino-2-phenylindole (DAPI) was obtained from the Beyotime Institute of Biotechnology (China).
Modena solution was used as the basic medium, which contained 152.8 mmol/L D-glucose, 26.7 mmol/L trisodium citrate, 11.9 mmol/L sodium hydrogen carbonate, 15.1 mmol/L citric acid, 6.3 mmol/L ethylenediamine tetraacetic acid disodium (EDTA-2Na), 46.6 mmol/L tris (hydroxymethyl)aminomethane (Tris), and 4 g/L bovine serum albumin (BSA) (pH=7.2). The Modena solution was supplemented with penicillin G sodium salt (1 000 IU/mL; Solarbio, China), streptomycin sesquisulfate (1 mg/mL; Solarbio, China), and polymyxin B (400 IU/mL; Amresco, USA), and filtered with a 0.22 μm filter to prevent bacterial contamination.
Seven mature and fertile Duroc boars (aged 15–28 months) were used in this study. The boars were housed individually, maintained under natural daylight, and provided with free access to food and water. The sperm-rich fraction was collected with the gloved hand technique twice a week, with fresh semen placed in a 37 °C bath and delivered to the laboratory within 15 min for the evaluation of sperm motility and concentration. Only semen samples with over 80% total motility were used for this study. The ejaculated semen was diluted by Modena solution containing metformin at a final concentration of 1×108 sperm/mL for the following processes. The liquid storage experiment was performed to determine whether metformin helped maintain sperm functionality during long-term preservation at 17 °C. Moreover, the diluted semen was also incubated at 37 °C. All experimental procedures involving the care and use of animals were approved by the Northwest A&F University Institutional Animal Care and Use Committee.
Sperm motility parameters were evaluated using computer-assisted sperm analysis (CASA; HVIEW, China), as per Zhu et al. (2018). Samples (1 mL) were incubated at 37 °C for 5 min before evaluation. The standard parameter settings were set at 30 frames/s. Total motility was defined as the percentage of sperm with curvilinear velocity (VCL)>10 μm/s, and progressive motility was defined as the percentage of sperm with straight line velocity (VSL)>25 μm/s and straightness of path (STR)≥75%. A minimum of 300 sperm were observed from at least five randomly selected fields with 20 μm CELL-VU® DRM-600 sperm count slides (Millennium Sciences, USA) and a microscopic stage warmer (KITAZATO, Japan).
Based on previous study (Zhu et al., 2019b), sperm membrane integrity and intact acrosomes were determined using a Live/Dead Sperm Viability Kit (Invitrogen™, USA) and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA; Sigma-Aldrich, USA), respectively. Spermatozoa were incubated at 37 °C for 10 min with propidium iodide (PI) and SYBR-14 at a final concentration of 4.8 μmol/L and 0.1 μmol/L, respectively. The spermatozoa were classified into two groups: Group A showing plasma membrane integrity, which only were stained green fluorescence with SYBR-14; Group B showing plasma membrane damage, which only were stained red fluorescence with PI. Furthermore, to evaluate acrosome integrity via FITC-PNA, sperm samples were fixed with absolute methanol and spread onto poly-L-lysine slides and air-dried at room temperature. The stain solution, which included FITC-PNA (100 μg/mL in phosphate-buffered saline (PBS)) and PI (4.8 μmol/L in PBS), was then spread over the slide. Spermatozoa with an intensively bright fluorescence of the acrosomal cap were deemed to have an intact outer acrosomal membrane; spermatozoa with a disrupted fluorescence of the acrosomal cap or no fluorescence of the outer acrosomal membrane were deemed to have a damaged acrosome membrane. The stained sperm were monitored and photographed with an epifluorescent microscope (Nikon 80i, Japan) with a set of filters (400×). A minimum of 1 000 sperm were observed from at least five randomly selected fields for each sample. All samples were identified and evaluated by one observer.
Here, ΔΨm was evaluated using a Mitochondrial Membrane Potential Detection Kit with JC-1 (Beyotime Institute of Biotechnology, China; Zhu et al., 2019b). Briefly, sperm samples (5×106 sperm) were stained with 1×JC-1 (lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) solution at 37 °C for 30 min in the dark. The samples were then centrifuged at 1 000 g at 4 °C for 5 min and washed with JC-1 buffer. Fluorescence intensity of JC-1 (488 nm excitation and 525 nm emission for JC-1-monomer vs 525 nm excitation and 590 nm emission for JC-1-aggregates) was detected by a multi-detection microplate reader (BioTek, Synergy H1, USA). The ΔΨm of the sperm samples was calculated as the fluorescence ratio of JC-1-aggregates (red) to monomer (green). At least three technical replicates were evaluated for each sample.
ATP content of sperm was measured using an ATP Assay Kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions. Briefly, 1 mL aliquots containing 5×107 sperm were centrifuged and re-suspended in ATP assay lysate to release intracellular ATP on ice. Sperm counts were performed for each sample to normalize ATP content to sperm number. Samples were centrifuged at 12 000 g for 10 min at 4 °C. The ATP standard solution (0.5 mmol/L) was diluted to concentrations of 10 nmol/L to 10 µmol/L in succession by ATP assay lysate. Either supernatants or standards (lysate at the same volume as the blank) were added to luciferin/luciferase reagent in opaque 96-wells, and the fluorescence intensity of samples was detected by a multi-detection microplate reader (BioTek, Synergy H1, USA). At least three technical replicates were evaluated for each sample.
Fluorescent 2-deoxy-D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG; Cayman Chemical Company, USA) was used to measure the glucose uptake capacity of sperm, as per Swegen et al. (2016). Briefly, sperm (2×107) were incubated with or without metformin and Compound C in specific Modena extender (contain 30 mmol/L glucose) with 100 µg/mL 2-NBDG at 37 °C for 4 h. The samples were centrifuged at 1 000 g for 3 min at room temperature and re-suspended with PI (1 μL/mL in specific Modena) for 15 min at 37 °C. Subsequently, the samples were analyzed by flow cytometry after a single wash with specific Modena. The geometric mean of fluorescence intensity (GMFI) of 2-NBDG (green fluorescence, FL-1) was used to indicate the cellular glucose up-take capacity measured after dead (red fluorescence positive, FL-2) cells were gated out of the analysis plot. As specified above, flow cytometry gates were set using a boiled sperm sample as a dead (red fluorescence) positive control.
Lactate efflux was evaluated using a Lactate Content Assay Kit (Nanjing Jiancheng, China) following the manufacturer’s instructions. Briefly, the lactate concentration of the Modena medium was measured by indirectly detecting the NADH formed following lactate oxidation by lactate dehydrogenase (LDH) using a multi-detection microplate reader (BioTek, Synergy H1, USA) at 340 nm. A standard curve with increasing concentrations of lactic acid (0, 0.6, 0.9, 1.5, 3 mmol/L) was constructed before the measurement of samples. Sperm counts were performed for each sample to normalize lactate content to sperm number.
LDH activity was evaluated using an LDH activity assay kit (Beyotime Institute of Biotechnology, China) following the manufacturer’s instructions. Briefly, the sperm samples were lysed with LDH release reagent for 1 h at 37 °C. The LDH activity was measured by indirectly detecting the production of NAD+ with a multi-detection microplate reader (BioTek, Synergy H1, USA). The protein concentration of samples was determined using a BCA Protein Assay Kit (TaKaRa, Japan) for the normalization of LDH activity.
Samples under different treatments were first centrifuged at 2 000 g at room temperature for 3 min, then washed with PBS and re-suspended with RIPA buffer containing 1% phenylmethyl sulfonylfluoride (PMSF) and phosphatase inhibitor (HAT, China) and 1% protease inhibitor cocktail (EDTA free, 100×; MedChemExpress, China) for 10 min at 4 °C. Given that the sperm membrane is relatively unbreakable, the samples were lysed by ultrasonication (20 KHz, 750 W, operating at 30% power, six cycles for 5 s on and 5 s off). After 30 min of lysis at 4 °C, the samples were centrifuged at 12 000 g for 10 min at 4 °C. A portion of the supernatant was used to analyze the concentration of total protein, with the rest mixed with 5×SDS loading buffer and boiled at 90 °C for 5 min. According to previous study (Lv et al., 2018), the lysates containing equivalent protein (30 μg) were determined by SDS-PAGE followed by western blotting in compliance with standard procedures using the following primary antibodies: anti-AMPKα rabbit polyclonal antibody detecting the α-1 and α-2 isoforms of the catalytic subunit (Cell Signaling Technology, 1∶1 000) and anti-p-AMPKα1/2 (Thr172) rabbit polyclonal antibody raised against a short amino acid sequence containing Thr172 phosphorylated AMPKα2 of human origin (Santa Cruz Biotechnology, 1∶1 000). The PVDF membranes were stripped and incubated with loading control antibodies overnight at 4 °C. Alpha-tubulin blotted with anti-alpha-tubulin rabbit polyclonal antibody (Proteintech, 1∶5 000) was used as a loading control.
Aliquots of 100 μL (1×107) sperm samples were fixed with 4% paraformaldehyde for 10 min, followed by washing in PBS, permeabilization with 0.25% Triton X-100 in PBS for 10 min, and washing again in PBS. Samples were incubated with 10% BSA and 100 mmol/L glycine in PBS for 1 h at 37 °C and incubated with one of the primary antibodies for AMPK (Cell Signaling Technology, 1∶100) or p-AMPK (Santa Cruz Biotechnology, 1∶100) overnight at 4 °C. Negative control immunostaining was also performed by omitting the primary antibody. The samples were washed and re-suspended with secondary antibody (FITC conjugated goat anti-rabbit IgG from CWBIO, 1∶200) for 2 h at 37 °C. Finally, the samples were washed and re-stained with DAPI (Beyotime Institute of Biotechnology, 1∶1 000) for 10 min. Images were captured using confocal laser scanning microscopy (Leica TCS SP8, Germany). A minimum of 200 sperm were observed from at least five randomly selected fields for each sample.
All values are presented as mean±standard error of the mean (SEM). All data were tested for normality and variance homogeneity prior to statistical analysis. Data were transformed by arc-sin square root transformation when necessary. Data were analyzed by one-way ANOVA (with repeated measures) and the Duncan test was used to perform post hoc analyses. Statistical analysis was determined using the unpaired Student’s t-test for Table 1 and Figures 1, 2. All analyses were performed using SPSS v23.0 for Windows (SPSS Inc., USA). Significant differences among treatments were set at *: P<0.05 and **: P<0.01.
Total motility (%) Control Met - 50 μmol/L Met - 100 μmol/L Met - 200 μmol/L Met - 500 μmol/L 0 d 87.37±1.88 86.65±1.76 86.74±2.75 87.65±0.96 88.90±1.60 1 d 84.20±2.08 83.06±3.32 86.12±2.87 88.17±1.83 85.18±2.57 3 d 82.17±3.11 84.70±3.09 85.63±2.29 85.30±2.09 84.51±2.22 5 d 80.07±2.93* 84.79±2.80 85.03±1.58 86.65±1.44 83.29±2.27 7 d 77.63±3.47b* 83.24±1.67ab 84.26±2.14ab 85.35±1.43a 83.60±1.67ab 9 d 79.93±3.42b* 82.16±2.15ab 84.92±1.94ab 86.31±1.93a 81.35±2.61b 11 d 73.49±2.79c* 77.80±3.17bc 79.68±2.99ab 86.11±2.00a 82.19±2.69ab 13 d 66.82±3.90b* 77.84±2.34a 79.54±1.84a 82.92±2.29a 77.16±2.72a Progressive motility (%) Control Met - 50 μmol/L Met - 100 μmol/L Met - 200 μmol/L Met - 500 μmol/L 0 d 78.46±2.80 78.47±2.58 76.47±2.04 75.34±1.91 76.85±3.56 1 d 72.29±1.97 67.71±4.33 74.03±3.84 73.88±2.76 67.86±3.66 3 d 66.56±3.56* 68.70±2.50 68.11±2.45 71.60±3.05 64.85±1.68 5 d 62.35±2.79b* 68.70±3.90ab 69.53±2.46ab 73.08±3.57a 67.83±2.92ab 7 d 61.48±2.14b* 69.13±2.27ab 67.02±2.53ab 73.25±3.42a 66.34±4.30ab 9 d 55.33±4.81b* 55.56±3.99b 61.59±3.23ab 67.35±3.41a* 60.74±2.48ab 11 d 42.52±4.18c* 49.23±4.13bc 55.83±3.08ab 61.70±3.21a* 55.35±2.40ab 13 d 38.16±2.71c* 43.44±3.10bc 49.00±4.02b 59.26±3.44a* 45.94±2.78b Sperm motility parameters were determined using the CASA system. Values are presented as mean±SEM. Different lower-case letters indicate significant difference (P<0.05) between treatments; asterisks represent significant difference from D0. *: P<0.05, determined by unpaired Student's t-test. n=5. Met: Metformin.
Table 1. Effects of metformin addition on sperm motility parameters during long-term preservation at 17 °C
Figure 1. Effects of metformin addition on sperm plasma membrane integrity, acrosome membrane integrity, mitochondrial membrane potential (ΔΨm), and cellular ATP content during long-term preservation at 17 °C
Metformin improves boar sperm quality via 5′-AMP-activated protein kinase-mediated energy metabolism in vitro
- Received Date: 2020-04-09
- Accepted Date: 2020-07-10
- Available Online: 2020-07-30
Abstract: Sperm are specialized cells that require adenosine triphosphate (ATP) to support their function. Maintaining sperm energy homeostasis in vitro is vitally important to improve the efficacy of boar sperm preservation. Metformin can activate 5′-AMP-activated protein kinase (AMPK) to improve metabolic flexibility and maintain energy homeostasis. Thus, the aim of the present study was to investigate whether metformin can improve boar sperm quality through AMPK mediation of energy metabolism. Sperm motility parameters, membrane integrity, acrosome integrity, mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake, and lactate efflux were analyzed. Localization and expression levels of AMPK and phospho-Thr172-AMPK (p-AMPK) were also detected by western blotting and immunofluorescence. We found that metformin treatment significantly increased sperm motility parameters, ΔΨm, and ATP content during storage at 17 °C. Moreover, results showed that AMPK was localized at the acrosomal region, connecting piece, and midpiece of sperm and p-AMPK was distributed at the post-acrosomal region, connecting piece, and midpiece. When sperm were incubated with metformin for 4 h at 37 °C, sperm motility parameters, ΔΨm, ATP content, p-AMPK, glucose uptake, and lactate efflux all significantly increased, whereas the addition of Compound C treatment, an inhibitor of AMPK, counteracted these positive effects. Together, our results suggest that metformin promotes AMPK activation, which contributes to the maintenance of energy hemostasis and mitochondrial activity, thereby maintaining boar sperm functionality and improving the efficacy of semen preservation.
|Citation:||Rong-Nan Li, Zhen-Dong Zhu, Yi Zheng, Ying-Hua Lv, Xiu-E Tian, De Wu, Yong-Jun Wang, Wen-Xian Zeng. Metformin improves boar sperm quality via 5′-AMP-activated protein kinase-mediated energy metabolism in vitro. Zoological Research. doi: 10.24272/j.issn.2095-8137.2020.074|