RHO/ROCK信号通路在精子抗冷冻损伤中的作用
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摘要
目的:探讨RHO/ROCK信号通路在人精子抗冷冻损伤中的作用,为高效精液冷冻保护剂的研制提供理论依据。方法:选取健康精液25份,每份精液分为新鲜组、对照组与RHO通路抑制剂组(抑制剂组)。检测冷冻前后各组精液精子活力、精子存活率、精子膜完整率、正常形态精子百分率、精子DNA碎片指数(DFI)、精子顶体酶活性及精子线粒体膜电位变化;免疫荧光染色检测RHOa/ROCK蛋白在精子中的表达。结果:抑制剂组精液冷冻复苏后精子活力[(57.50±6.83)%vs(51.20±7.70)%,P=0.002]、精子存活率[(60.24±5.53)%vs(52.87±5.07)%,P=0.001]、精子膜完整率[(67.10±4.43)%vs(59.78±5.56)%,P=0.001]、正常形态精子百分率[(7.46±1.28)%vs(4.83±1.11)%,P=0.001]、精子DFI[(18.87±4.07)%vs(27.64±6.64)%,P=0.001]、精子线粒体膜电位(63.11±2.97 vs 56.30±4.28,P=0.001)指标均明显优于对照组;抑制剂组冷冻后精子顶体酶活性与对照组差异无统计学意义(98.30±11.33 vs 97.65±9.31,P>0.05)。免疫荧光染色显示RHOa/ROCK蛋白在精子头、颈部广泛表达。结论:RHO/ROCK信号通路在精子冷冻损伤中具有一定作用,抑制其通路活性可明显提高精子抗冷冻损伤的能力。
Objective: To investigate the influence of the Rho/ROCK signaling pathway on the anti-cryodamage ability of human sperm and provide some theoretical evidence for the development of high-efficiency semen cryoprotectants. Methods: We collected semen samples from 25 healthy males, each divided into a fresh, a normal cryopreservation control and an Rho-inhibition group. Before and after freezing, we detected sperm motility, viability, membrane integrity, morphology, DNA fragmentation index(DFI), acrosomal enzyme activity(AEA) and mitochondrial membrane potential(MMP) and determined the expressions of RhoA and ROCK proteins in the sperm by immunofluorescence staining. Results: Compared with the normal cryopreservation control, the frozen-thawed sperm of the Rho-inhibition group showed significantly increased sperm motility( [51.20 ± 7.70]% vs [57.50 ± 6.83]%, P = 0.002), survival rate( [52.87 ± 5.07]% vs [60.24 ± 5.53]%, P = 0.001), membrane integrity([59.78±5.56]% vs [67.10 ± 4.43]%, P = 0.001), percentage of morphologically normal sperm([4.83 ± 1.11]% vs [7.46 ± 1.28], P = 0.001) and MMP(56.30 ± 4.28 vs 63.11 ± 2.97, P = 0.001), but decreased DFI([27.64 ± 6.64]% vs [18.87 ± 4.07]%, P = 0.001). There was no statistically significant difference in the AEA of the frozen-thawed sperm between the control and Rho-inhibition groups(97.65 ± 9.31 vs 98.30 ± 11.33, P > 0.05). Immunofluorescence staining revealed extensive expressions of RhoA and ROCK proteins in the head and neck of the sperm. Conclusion: The Rho/ROCK signaling pathway plays a role in the cryodamage to human sperm, and inhibiting the activity of Rho/ROCK can significantly improve the ability of sperm to resist cryodamage. Natl J Androl, 2019, 25(4): 322-328
Objective: To investigate the influence of the Rho/ROCK signaling pathway on the anti-cryodamage ability of human sperm and provide some theoretical evidence for the development of high-efficiency semen cryoprotectants. Methods: We collected semen samples from 25 healthy males, each divided into a fresh, a normal cryopreservation control and an Rho-inhibition group. Before and after freezing, we detected sperm motility, viability, membrane integrity, morphology, DNA fragmentation index(DFI), acrosomal enzyme activity(AEA) and mitochondrial membrane potential(MMP) and determined the expressions of RhoA and ROCK proteins in the sperm by immunofluorescence staining. Results: Compared with the normal cryopreservation control, the frozen-thawed sperm of the Rho-inhibition group showed significantly increased sperm motility( [51.20 ± 7.70]% vs [57.50 ± 6.83]%, P = 0.002), survival rate( [52.87 ± 5.07]% vs [60.24 ± 5.53]%, P = 0.001), membrane integrity([59.78±5.56]% vs [67.10 ± 4.43]%, P = 0.001), percentage of morphologically normal sperm([4.83 ± 1.11]% vs [7.46 ± 1.28], P = 0.001) and MMP(56.30 ± 4.28 vs 63.11 ± 2.97, P = 0.001), but decreased DFI([27.64 ± 6.64]% vs [18.87 ± 4.07]%, P = 0.001). There was no statistically significant difference in the AEA of the frozen-thawed sperm between the control and Rho-inhibition groups(97.65 ± 9.31 vs 98.30 ± 11.33, P > 0.05). Immunofluorescence staining revealed extensive expressions of RhoA and ROCK proteins in the head and neck of the sperm. Conclusion: The Rho/ROCK signaling pathway plays a role in the cryodamage to human sperm, and inhibiting the activity of Rho/ROCK can significantly improve the ability of sperm to resist cryodamage. Natl J Androl, 2019, 25(4): 322-328
引文
[1] 高庆强,陈海,陈赟,等.Rock蛋白抑制剂法舒地尔对前列腺癌PC3、DU145细胞侵袭、迁移和凋亡的影响.中华男科学杂志,2016,22(6):483-490.
[2] Qu CQ,Li DW,Shen N,et al.Effect of glutathione and Y27632 on the viability of cryopreserved porcine adipose-derived stem cells.Cryo Letters,2014,35(4):308-311.
[3] Shen W,Wang L,Pi R,et al.L-F001,a multifunctional ROCK inhibitor prevents paraquat-induced cell death through attenuating ER stress and mitochondrial dysfunction in PC12 cells.Biochem Biophys Res Commun,2015,464(3):794-799.
[4] Finney EL,Johnson EK.Challenges in fertility preservation among male adolescent and young adult cancer survivors.Pediatr Blood Cancer,2018,65(7):e27042.
[5] Williams DHT.Fertility preservation in the male with cancer.Curr Urol Rep,2013,14(4):315-326.
[6] Arav A,Saragusty J.Directional freezing of sperm and associated derived technologies.Anim Reprod Sci,2016,169:6-13.
[7] 平述煌,杨世华.精子冷冻保存技术及研究进展.中国比较医学杂志,2011,21(3):67-71.
[8] Usuga A,Rojano BA,Restrepo G.Association of the cysteine-rich secretory protein-3 (CRISP-3) and some of its polymorphisms with the quality of cryopreserved stallion semen.Reprod Fertil Dev,2018,30(3):563-569.
[9] Reddy VS,Yadav B,Yadav CL,et al.Effect of sericin supplementation on heat shock protein 70 (HSP70) expression,redox status and post thaw semen quality in goat.Cryobiology,2018,84:33-39.
[10] He Y,Li H,Wang K,et al.Loss of protein kinase 2 subunit alpha 2 (CK2alpha') effect ram sperm function after freezing and thawing process.Anim Reprod Sci,2017,181:9-15.
[11] Ferreira LM,Garcia-Herreros M,Domingos A,et al.Prion protein 2 (dublet) gene (PRND):Role in ovine semen capacitation,cryopreservation and fertility.Reprod Fertil Dev,2017,29(5):985-997.
[12] Fukuda M,Sakase M,Fukushima M,et al.Changes of IZUMO1 in bull spermatozoa during the maturation,acrosome reaction,and cryopreservation.Theriogenology,2016,86(9):2179-2188e3.
[13] Wilson H K,Faubion MG,Hjortness MK,et al.Cryopreservation of brain endothelial cells derived from human induced pluripotent stem cells is enhanced by Rho-associated coiled coil-containing kinase inhibition.Tissue Eng Part C Methods,2016,22(12):1085-1094.
[14] Bit A,Kumar A,Singh AK,et al.Crosstalk between substrates and Rho-associated kinase inhibitors in cryopreservation of tissue-engineered constructs.Stem Cells Int,2017,2017:1380304.
[15] Hinsch KD,Habermann B,Just I,et al.ADP-ribosylation of Rho proteins inhibits sperm motility .FEBS Lett,1993,334(1):32-36.
[16] Osaki JH,Espinha G,Magalhaes YT,et al.Modulation of RhoA GTPase activity sensitizes human cervix carcinoma cells to gamma-radiation by attenuating DNA repair pathways.Oxid Med Cell Longev,2016,2016:6012642.
[17] Srougi MC,Burridge K.The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.PLoS One,2011,6(2):e17108.
[18] Dubash AD,Guilluy C,Srougi MC,et al.The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals.PLoS One,2011,6(2):e17380.
[19] Wang Y,Zhang H,Yang Z,et al.Rho kinase inhibitor,fasudil,attenuates contrast-induced acute kidney injury.Basic Clin Pharmacol Toxicol,2018,122(2):278-287.
[20] Ziegler V,Henninger C,Simiantonakis I,et al.Rho inhibition by lovastatin affects apoptosis and DSB repair of primary human lung cells in vitro and lung tissue in vivo following fractionated irradiation.Cell Death Dis,2017,8(8):e2978.
[21] Ikeda H,Kume Y,Tejima K,et al.Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats.Am J Physiol Gastrointest Liver Physiol,2007,293(4):G911-7.
[22] Sun XZ,Li SY,Tian XY,et al.Effect of Rho kinase inhibitor fasudil on the expression ET-1 and NO in rats with hypoxic pulmonary hypertension.Clin Hemorheol Microcirc,2019,71(1):3-8.
[23] Shen YL,Shi YZ,Chen GG,et al.TNF-alpha induces Drp1-mediated mitochondrial fragmentation during inflammatory cardiomyocyte injury.Int J Mol Med,2018,41(4):2317-2327.
[24] Kumar M,Bansal N.Fasudil hydrochloride ameliorates memory deficits in rat model of streptozotocin-induced Alzheimer's disease:Involvement of PI3-kinase,eNOS and NFkB.Behav Brain Res,2018,351:4-16.
[25] He Y,Xia P,Jin H,et al.Lycopene ameliorates transplant arteriosclerosis in vascular allograft transplantation by regulating the NO/cGMP pathways and Rho-associated kinases expression.Oxid Med Cell Longev,2016,2016:3128280.
[26] Ding R,Han J,Zhao D,et al.Pretreatment with Rho-kinase inhibitor ameliorates lethal endotoxemia-induced liver injury by improving mitochondrial function.Int Immunopharmacol,2016,40:125-130.
[27] Martorell-Riera A,Segarra-Mondejar M,Reina M,et al.Mitochondrial fragmentation in excitotoxicity requires ROCK activation.Cell Cycle,2015,14(9):1365-1369.
[28] Romarowski A,Battistone MA,La Spina FA,et al.PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis.Dev Biol,2015,405(2):237-249.
[29] De La Sancha CU,Martinez-Cadena G,Lopez-Godinez J,et al.Rho-kinase (ROCK) in sea urchin sperm:Its role in regulating the intracellular pH during the acrosome reaction.Biochem Biophys Res Commun,2007,364(3):470-475.
[30] Ren F,Feng T,Dai G,et al.Lycopene and alpha-lipoic acid improve semen antioxidant enzymes activity and cashmere goat sperm function after cryopreservation.Cryobiology,2018,84:27-32.
[31] Losano JD A,Angrimani DSR,Rui BR,et al.The addition of docosahexaenoic acid (DHA) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls.Zygote,2018,26(3):199-206.
[32] 李世佳,苏卫东,邱丽君,等.白藜芦醇对人精子冷冻损伤的保护作用.中华男科学杂志,2018,24(6):499-503.
[33] Jang W G,Kim HS,Park KG,et al.Analysis of proteome and transcriptome of tumor necrosis factor alpha stimulated vascular smooth muscle cells with or without alpha lipoic acid.Proteomics,2004,4(11):3383-3393.
[34] Cao X,Luo T,Luo X,et al.Resveratrol prevents AngII-induced hypertension via AMPK activation and RhoA/ROCK suppression in mice.Hypertens Res,2014,37(9):803-810.
[35] 易龙,张乾勇,糜漫天.RHO GTP酶在Ω-3多不饱和脂肪酸抑制人前列腺癌PC-3细胞转移中的作用.癌症,2007,26(12):1281-1286.
[2] Qu CQ,Li DW,Shen N,et al.Effect of glutathione and Y27632 on the viability of cryopreserved porcine adipose-derived stem cells.Cryo Letters,2014,35(4):308-311.
[3] Shen W,Wang L,Pi R,et al.L-F001,a multifunctional ROCK inhibitor prevents paraquat-induced cell death through attenuating ER stress and mitochondrial dysfunction in PC12 cells.Biochem Biophys Res Commun,2015,464(3):794-799.
[4] Finney EL,Johnson EK.Challenges in fertility preservation among male adolescent and young adult cancer survivors.Pediatr Blood Cancer,2018,65(7):e27042.
[5] Williams DHT.Fertility preservation in the male with cancer.Curr Urol Rep,2013,14(4):315-326.
[6] Arav A,Saragusty J.Directional freezing of sperm and associated derived technologies.Anim Reprod Sci,2016,169:6-13.
[7] 平述煌,杨世华.精子冷冻保存技术及研究进展.中国比较医学杂志,2011,21(3):67-71.
[8] Usuga A,Rojano BA,Restrepo G.Association of the cysteine-rich secretory protein-3 (CRISP-3) and some of its polymorphisms with the quality of cryopreserved stallion semen.Reprod Fertil Dev,2018,30(3):563-569.
[9] Reddy VS,Yadav B,Yadav CL,et al.Effect of sericin supplementation on heat shock protein 70 (HSP70) expression,redox status and post thaw semen quality in goat.Cryobiology,2018,84:33-39.
[10] He Y,Li H,Wang K,et al.Loss of protein kinase 2 subunit alpha 2 (CK2alpha') effect ram sperm function after freezing and thawing process.Anim Reprod Sci,2017,181:9-15.
[11] Ferreira LM,Garcia-Herreros M,Domingos A,et al.Prion protein 2 (dublet) gene (PRND):Role in ovine semen capacitation,cryopreservation and fertility.Reprod Fertil Dev,2017,29(5):985-997.
[12] Fukuda M,Sakase M,Fukushima M,et al.Changes of IZUMO1 in bull spermatozoa during the maturation,acrosome reaction,and cryopreservation.Theriogenology,2016,86(9):2179-2188e3.
[13] Wilson H K,Faubion MG,Hjortness MK,et al.Cryopreservation of brain endothelial cells derived from human induced pluripotent stem cells is enhanced by Rho-associated coiled coil-containing kinase inhibition.Tissue Eng Part C Methods,2016,22(12):1085-1094.
[14] Bit A,Kumar A,Singh AK,et al.Crosstalk between substrates and Rho-associated kinase inhibitors in cryopreservation of tissue-engineered constructs.Stem Cells Int,2017,2017:1380304.
[15] Hinsch KD,Habermann B,Just I,et al.ADP-ribosylation of Rho proteins inhibits sperm motility .FEBS Lett,1993,334(1):32-36.
[16] Osaki JH,Espinha G,Magalhaes YT,et al.Modulation of RhoA GTPase activity sensitizes human cervix carcinoma cells to gamma-radiation by attenuating DNA repair pathways.Oxid Med Cell Longev,2016,2016:6012642.
[17] Srougi MC,Burridge K.The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.PLoS One,2011,6(2):e17108.
[18] Dubash AD,Guilluy C,Srougi MC,et al.The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals.PLoS One,2011,6(2):e17380.
[19] Wang Y,Zhang H,Yang Z,et al.Rho kinase inhibitor,fasudil,attenuates contrast-induced acute kidney injury.Basic Clin Pharmacol Toxicol,2018,122(2):278-287.
[20] Ziegler V,Henninger C,Simiantonakis I,et al.Rho inhibition by lovastatin affects apoptosis and DSB repair of primary human lung cells in vitro and lung tissue in vivo following fractionated irradiation.Cell Death Dis,2017,8(8):e2978.
[21] Ikeda H,Kume Y,Tejima K,et al.Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats.Am J Physiol Gastrointest Liver Physiol,2007,293(4):G911-7.
[22] Sun XZ,Li SY,Tian XY,et al.Effect of Rho kinase inhibitor fasudil on the expression ET-1 and NO in rats with hypoxic pulmonary hypertension.Clin Hemorheol Microcirc,2019,71(1):3-8.
[23] Shen YL,Shi YZ,Chen GG,et al.TNF-alpha induces Drp1-mediated mitochondrial fragmentation during inflammatory cardiomyocyte injury.Int J Mol Med,2018,41(4):2317-2327.
[24] Kumar M,Bansal N.Fasudil hydrochloride ameliorates memory deficits in rat model of streptozotocin-induced Alzheimer's disease:Involvement of PI3-kinase,eNOS and NFkB.Behav Brain Res,2018,351:4-16.
[25] He Y,Xia P,Jin H,et al.Lycopene ameliorates transplant arteriosclerosis in vascular allograft transplantation by regulating the NO/cGMP pathways and Rho-associated kinases expression.Oxid Med Cell Longev,2016,2016:3128280.
[26] Ding R,Han J,Zhao D,et al.Pretreatment with Rho-kinase inhibitor ameliorates lethal endotoxemia-induced liver injury by improving mitochondrial function.Int Immunopharmacol,2016,40:125-130.
[27] Martorell-Riera A,Segarra-Mondejar M,Reina M,et al.Mitochondrial fragmentation in excitotoxicity requires ROCK activation.Cell Cycle,2015,14(9):1365-1369.
[28] Romarowski A,Battistone MA,La Spina FA,et al.PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis.Dev Biol,2015,405(2):237-249.
[29] De La Sancha CU,Martinez-Cadena G,Lopez-Godinez J,et al.Rho-kinase (ROCK) in sea urchin sperm:Its role in regulating the intracellular pH during the acrosome reaction.Biochem Biophys Res Commun,2007,364(3):470-475.
[30] Ren F,Feng T,Dai G,et al.Lycopene and alpha-lipoic acid improve semen antioxidant enzymes activity and cashmere goat sperm function after cryopreservation.Cryobiology,2018,84:27-32.
[31] Losano JD A,Angrimani DSR,Rui BR,et al.The addition of docosahexaenoic acid (DHA) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls.Zygote,2018,26(3):199-206.
[32] 李世佳,苏卫东,邱丽君,等.白藜芦醇对人精子冷冻损伤的保护作用.中华男科学杂志,2018,24(6):499-503.
[33] Jang W G,Kim HS,Park KG,et al.Analysis of proteome and transcriptome of tumor necrosis factor alpha stimulated vascular smooth muscle cells with or without alpha lipoic acid.Proteomics,2004,4(11):3383-3393.
[34] Cao X,Luo T,Luo X,et al.Resveratrol prevents AngII-induced hypertension via AMPK activation and RhoA/ROCK suppression in mice.Hypertens Res,2014,37(9):803-810.
[35] 易龙,张乾勇,糜漫天.RHO GTP酶在Ω-3多不饱和脂肪酸抑制人前列腺癌PC-3细胞转移中的作用.癌症,2007,26(12):1281-1286.
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