褪黑素对热应激猪精子质量的影响
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摘要
为研究褪黑素(Melatonin,MLT)对热应激引起的猪精子质量下降的缓解作用及其机制。首先用免疫荧光方法检测MLT受体在猪精子上的存在和分布;然后将稀释后的精子分为对照组(ck)、热应激组(H)、MLT组(M)、热应激+MLT组(H+M)、热应激+MLT受体拮抗剂Luzindole组(H+L)和热应激+MLT受体拮抗剂Luzindole+MLT组(H+L+M)6个组,处理结束后检测各组精子活率、活力、线粒体活性、钙离子水平以及顶体完整率。结果显示:猪精子中存在MLT受体MT1和MT2,且均分布于精子头部;与对照组相比热应激组各项指标显著下降,MLT组精子活率没有显著改变(P>0.05),但精子活力、线粒体活性、钙离子水平和顶体完整率显著升高(P<0.05)。添加MLT受体拮抗剂Luzindole后精子活力、线粒体活性和钙离子水平显著下降(P<0.05),顶体完整率呈一定程度下降,但差异不显著(P>0.05)。以上结果表明:热应激会导致猪精子质量显著下降,并且MLT能通过受体途径改善热应激对猪精子质量的影响,但同时也存在非受体介导的作用。
The aim of this study is to investigate the alleviation effect of the melatonin( MLT) on influence of heat stress on sperm quality and its mechanism. We firstly detected the existence and distribution of MLT receptors MT1 and MT2 in boar sperm via immunofluorescence analysis. Then we divided the diluted fresh boar sperm into six groups: control group( C),MLT treated group( M),heat stressed group( H),co-treated with heat stress and MLT group( H+M),co-treated with heat stress and luzindole group( H+L),and co-treated with heat stress,luzindole and MLT group( H+L+M). Then we detected sperm viability,motility,mitochondrial activity,calcium concentration and acrosome integrity. The results showed that heat stress significantly reduced the quality of sperm,and MLT did not significantly( P>0. 05) improve the sperm viability of the heat stressed boar,but significantly enhanced motility,mitochondrial activity,calcium level and acrosomal integrity. Moreover,luzindole treatment could attenuate these effects of MLT to some degree via blocking its receptor. These results suggested that heat stress impaired boar sperm quality and administration of MLT to heat stressed sperm alleviate the adverse effect of heat stress via receptor mediated and non-receptor mediated action.
The aim of this study is to investigate the alleviation effect of the melatonin( MLT) on influence of heat stress on sperm quality and its mechanism. We firstly detected the existence and distribution of MLT receptors MT1 and MT2 in boar sperm via immunofluorescence analysis. Then we divided the diluted fresh boar sperm into six groups: control group( C),MLT treated group( M),heat stressed group( H),co-treated with heat stress and MLT group( H+M),co-treated with heat stress and luzindole group( H+L),and co-treated with heat stress,luzindole and MLT group( H+L+M). Then we detected sperm viability,motility,mitochondrial activity,calcium concentration and acrosome integrity. The results showed that heat stress significantly reduced the quality of sperm,and MLT did not significantly( P>0. 05) improve the sperm viability of the heat stressed boar,but significantly enhanced motility,mitochondrial activity,calcium level and acrosomal integrity. Moreover,luzindole treatment could attenuate these effects of MLT to some degree via blocking its receptor. These results suggested that heat stress impaired boar sperm quality and administration of MLT to heat stressed sperm alleviate the adverse effect of heat stress via receptor mediated and non-receptor mediated action.
引文
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[26]马红,王文涛,刘娣.离心方法对冷冻后民猪精子质量的影响[J].中国兽医学报,2016,36(10):1779-1802.
[27]韩明铭,金一,方南洙,等.双糖对冻融后猪精子质量的影响[J].吉林农业大学学报,2010,32(1):86-88,94.
[28]Reiter R J.Oxidative damage in the central nervous system:protection by melatonin[J].Prog Neurobiol,1998,56:359-384.
[2]Padilla L,Matsui T,Kamiya Y,et al.Heat stress decreases plasma vitamin C concentration in lactating cows[J].Livest Sci,2006,101(1):300-304.
[3]Mstl E,Palme R.Hormones as indicators of stress[J].Domest Anim Endocrinol,2002,23:67-74.
[4]Rockett J C,Mapp F L,Garges J B,et al.Effects of hyperthermia on spermatogenesis,apoptosis,gene expression,and fertility in adult male mice[J].Biol Reprod,2001,65(1):229-239.
[5]Paul C,Teng S,Saunders P T.A single,mild,transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes,which Induces germ cell death[J].Biol Reprod,2009,80(5):913-919.
[6]Shiraishi K,Takihara H,Matsuyama H.Elevated scrotal temperature,but not varicocele grade,reflects testicular oxidative stress-mediated apoptosis[J].World J Urol,2010,28(3):359-364.
[7]Mete F,Kilic E,Somay A,et al.Effects of heat stress on endocrine functions&behaviour in the pre-pubertal rat[J].Indian J Med Res,2012,135:233-239.
[8]Rodriguez C,Mayo J C,Sainz R M,et al.Regulation of antioxidant enzymes:a significant role for melatonin[J].J Pineal Res,2004,36(1):1-9.
[9]Woo M M,Tai C J,Kang S K,et al.Direct action of melatonin in human granulosa-luteal cells[J].J Clin Endocrinol Metab,2001,86(10):4789-4797.
[10]La Vignera S,Condorelli R,Vicari E,et al.Effects of varicocelectomy on sperm DNA fragmentation,mitochondrial function,chromatin condensation,and apoptosis[J].J Androl,2012,33(3):389-396.
[11]Tamura H,Nakamura Y,Korkmaz A,et al.Melatonin and the ovary:physiological and pathophysiological implications[J].Fertil Steril,2009,92(1):328-343.
[12]Nakai A,Ishikawa T.Cell cycle transition under stress conditions controlled by vertebrate heat shock factors[J].EMBO J,2001,20(11):2885-2895.
[13]Sonna L A,Fujita J,Gaffin S L,et al.Invited review:Effects of heat and cold stress on mammalian gene expression[J].J Appl Physiol,2002,92(4):1725-1742.
[14]Banks S,King S A,Irvine D S,et al.Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa[J].Reproduction,2005,129(4):505-514.
[15]Pashkov A N,Popov S S,Semenikhina A V,et al.Glutathione system and activity of NADPH-generating enzymes in the liver of intact rats and animals with toxic hepatitis receiving melatonin[J].Bull Exp Biol Med,2005,139(5):565-568.
[16]Apel K,Hirt H.Reactive oxygen species:metabolism,oxidative stress,and signal transduction[J].Annu Rev Plant Biol,2004,55:373-399.
[17]Ikeda M,Kodama H,Fukuda J,et al.Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress[J].Biol Reprod,1999,61(2):393-399.
[18]Reiter R J,Paredes S D,Manchester L C,et al.Reducing oxidative/nitrosative stress:a newly-discovered genre for melatonin[J].Crit Rev Biochem Mol Biol,2009,44(4):175-200.
[19]Milczarek R,Hallmann A,Sokoowska E,et al.Melatonin enhances antioxidant action of alpha-tocopherol and ascorbate against NADPH-and iron-dependent lipid peroxidation in human placental mitochondria[J].J Pineal Res,2010,49(2):149-155.
[20]Makker K,Agarwal A,Sharma R.Oxidative stress&male infertility[J].Indian J Med Res,2009,129(4):357-367.
[21]Kim B,Park K,Rhee K.Heat stress response of male germ cells[J].Cell Mol Life Sci,2013,70(15):2623-2636.
[22]Rodriguez-Osorio N,Kim I J,Wang H,et al.Melatonin increases cleavage rate of porcine preimplantation embryos in vitro[J].J Pineal Res,2007,43(3):283-288.
[23]Zhang Y,Cook A,Kim J,et al.Melatonin inhibits the caspase-1/cytochrome c/caspase-3 cell death pathway,inhibits MT1 receptor loss and delays disease progression in a mouse model of amyotrophic lateralsclerosis[J].Neurobiol Dis,2013,55:26-35.
[24]Tan D X,Reiter R J,Manchester L C,et al.Chemical and physical properties and potential mechanisms:melatonin as a broad spectrum antioxidant and free radical scavenger[J].Curr Top Med Chem,2002,2(2):181-197.
[25]Lee Y M,Chen H R,Hsiao G,et al.Protective effects of melatonin on myocardial ischemia/reperfusion injury in vivo[J].J Pineal Res,2002,33:72-80.
[26]马红,王文涛,刘娣.离心方法对冷冻后民猪精子质量的影响[J].中国兽医学报,2016,36(10):1779-1802.
[27]韩明铭,金一,方南洙,等.双糖对冻融后猪精子质量的影响[J].吉林农业大学学报,2010,32(1):86-88,94.
[28]Reiter R J.Oxidative damage in the central nervous system:protection by melatonin[J].Prog Neurobiol,1998,56:359-384.