Metformin improves boar sperm quality via 5′-AMP-activated protein kinase-mediated energy metabolism in vitro
摘要: 精子的运动、获能及顶体反应等过程需要大量的能量。维持精子能量稳态对于改善猪精子体外保存的效果至关重要。二甲双胍通过激活AMP依赖的蛋白激酶（AMPK）维持细胞内能量代谢的稳态。因此，本研究旨在探究二甲双胍能否通过AMPK调控能量代谢而改善猪精子的品质。本研究对猪精子活力参数、质膜完整性、线粒体膜电位、ATP含量、葡萄糖摄取量和乳酸外排量等指标进行检测，并运用蛋白质免疫印迹和免疫荧光技术检测AMPK与磷酸化AMPK（172位点；p-AMPK）在精子中的定位和表达水平。结果显示，在17 °C保存过程中，添加二甲双胍显著提高精子活力、线粒体膜电位和ATP含量。同时，发现AMPK定位于精子的顶体、头尾连接段和中段，且p-AMPK分布于顶体后区、头尾连接段和中段。此外，添加二甲双胍显著提高37 °C孵育4h后的精子活力、线粒体膜电位、ATP含量、p-AMPK水平、葡萄糖摄取量和乳酸外排量。然而，添加AMPK抑制剂Compound C会抑制二甲双胍对精子的作用。综上所述，二甲双胍能够激活AMPK，进而维持猪精子能量代谢和线粒体的活性，从而改善精子功能与精液保存的效果。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 immunofluorescence and western blotting. 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.
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
A, C: Sperm were treated with 0, 50, 100, 200, and 500 µmol/L metformin, respectively, in Modena medium. Sperm membrane integrity was evaluated using SYBR-14/PI kit at D1, D5, D9, and D13 (n=5) (A). Sperm acrosome integrity was evaluated using a FITC-PNA kit at D1, D5, D9, and D13 (n=3) (C). B, D: Sperm were treated with or without 200 µmol/L metformin in Modena medium. Sperm ΔΨm was evaluated using a JC-1 kit at D1, D5, D9, and D13 (n=5) (B). Cellular ATP content was evaluated using an ATP bioluminescence assay kit at D1, D5, D9, and D13 (D). Before ATP extraction, sperm counts were executed to normalize ATP content (n=5). Graph bars represent mean±SEM. Different lower-case letters indicate significant difference (P<0.05). Asterisks represent significant difference from control. *: P<0.05, **: P<0.01, determined by unpaired Student's t-test; Met: Metformin.
Figure 2. Subcellular localization of AMPK protein and phospho-Thr172 AMPK in boar sperm and AMPK phosphorylation analyzed by western blotting
A, B: Fresh sperm were stained green with anti-AMPK antibody or anti-phospho-Thr172-AMPKα antibody, and sperm nuclei were stained with DAPI (blue). AMPK and p-AMPK proteins were detected with secondary antibody FITC conjugated goat anti-rabbit immunoglobulin G. Negative control: Primary antibody was not added. Images were visualized using confocal laser scanning microscopy. Serial images of right panels were obtained from a unique sperm. C, E: Sperm were treated with or without 200 µmol/L metformin at D1, D5, D9, and D13 of preservation at 17 °C. D, F: Sperm were incubated for 4 h at 37 °C with or without 200 µmol/L metformin in Modena medium, Modena+100 µmol/L AMPK inhibitor Compound C. C, D: Western blotting using anti-AMPK antibody and anti-phosphor (Thr172)-AMPKα antibody. Loading control was performed for each experiment in same membrane using anti-α tubulin antibody (n=5). E, F: Densitometric quantitation of phosphor (Thr172)-AMPKα bands obtained in C, D. Values obtained for phosphor (Thr172)-AMPKα bands were normalized with values of α-tubulin (n=3). Graph bars represent mean±SEM. Asterisks represent significant difference from control. *: P<0.05, **: P<0.01, determined by unpaired Student’s t-test; Different lower-case letters indicate significant difference (P<0.05).
Figure 3. Effects of metformin and Compound C on sperm motility, membrane integrity, and ΔΨm
Sperm were incubated for 1 h and 4 h at 37 °C with or without metformin in Modena medium, Modena+100 µmol/L Compound C. A, B: Total motility and progressive motility were recorded using CASA system (n=3). C: Membrane integrity was evaluated using SYBR-14/PI kit (n=3). D: Mitochondrial membrane potential was evaluated using a JC-1 kit (n=5). Graph bars represent mean±SEM. Different lower-case letters indicate significant difference (P<0.05). Met: Metformin; CC: Compound C; Met+CC: Metformin+Compound C.
Figure 4. Effects of metformin and Compound C on sperm ATP content, glucose uptake, extracellular lactate content, and lactate dehydrogenase activity
Sperm were incubated for 4 h at 37 °C with or without metformin in Modena medium, Modena+100 µmol/L Compound C. A: Cellular ATP content was evaluated using an ATP bioluminescence assay kit. Before ATP extraction, sperm counts were executed to normalize ATP content (n=3). B: Glucose uptake capability was measured using green fluorescence D-glucose analogue, 2-NBDG, in glucose-free medium. Results are presented as geometric mean of fluorescence intensity of live cells as measured by flow cytometry (n=3). C: Extracellular lactate content was evaluated using a lactate content assay kit, and sperm counts were performed to normalize lactate content (n=3). D: Relative lactate dehydrogenase activity was evaluated using an LDH activity assay kit and results are shown as percentage of untreated control sample (n=3). Graph bars represent mean±SEM. Different lower-case letters indicate significant difference (P<0.05). Met: Metformin; CC: Compound C; Met+CC: Metformin+Compound C.
Figure 5. Proposed model of regulation of metformin on boar sperm function via activation of AMPK
Metformin enters the intracellular space by the transporter, resulting in AMPK activation, which facilitates glucose uptake and lactate efflux and maintains mitochondrial activity, thereby maintaining sperm functionality and improving the efficacy of boar sperm preservation in vitro.
Table 1. Effects of metformin addition on sperm motility parameters during long-term preservation at 17 °C
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.
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