短期热量限制对雄性大鼠睾丸形态和抗氧化能力的影响
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摘要
本文旨在研究短期热量限制对雄性大鼠睾丸形态和抗氧化能力的影响。将20只6周龄Wistar雄性大鼠随机分为对照组(C)和短期热量限制组(CR)(n=10),对照组自由采食,热量限制组按对照组食物消耗量的60%进行饲喂。连续饲养7周后称重,观察精神状态,处死,采集睾丸组织。对大鼠睾丸组织进行HE染色、紧密连接蛋白(ZO-1)免疫荧光染色;通过生化技术测定超氧化物歧化酶、谷胱甘肽过氧化物酶活力及过氧化产物丙二醛含量;荧光定量PCR检测ZO-1、超氧化酶歧化酶、过氧化氢酶、谷胱甘肽巯基转移酶和转录因子Nrf2 mRNAs的表达变化。与对照组相比,短期热量限制组大鼠体重上升趋势减缓,精神状态好于对照组,曲细精管生长良好,基膜完整,管内各级生精细胞排列整齐,生精上皮普遍可见有6-8层细胞。短期热量限制组与对照组相比ZO-1 mRNAs表达显著升高,同时在免疫荧光结果中观察到ZO-1蛋白有较强的阳性反应。此外,短期热量限制组睾丸组织相较于对照组丙二醛含量显著降低(P<0.05),超氧化酶歧化酶和谷胱甘肽过氧化物酶活力显著升高(P<0.05);短期热量限制组相较于对照组抗氧化编码基因SOD、CAT、GST和Nrf2 mRNAs水平显著升高(P<0.05)。短期热量限制可以促进大鼠睾丸支持细胞间的紧密连接,改善精子发育微环境,提高大鼠睾丸组织的抗氧化能力,减少自由基对细胞的毒性作用,改善大鼠生殖机能。
The present study was conducted to examine the short-term calorie restriction effects( CR) on reproduciton in male rats.Twenty adult male Wistar rats were separated to two groups randomly,including calorie restriction group and control group. Controls were raised ad libitum and treatments were raised with 60% of the food expended by control rats. After seven weeks,the rats were weighed and observed the mental state. Then rats were executed and the testicular tissues were embedded into paraffin stained with hematoxylin and eosin( HE). Immunostaining was carried out with mice polyclonal antibodies to ZO-1. Then,oxidative stress parameters,including MDA,T-SOD and GSH-Px,were measured. Total RNA was extracted from testicular tissues and the expression of mRNA of ZO-1,SOD,CAT,GST and Nrf2 were measured by real-time quantitative PCR. The results showed that the body weight of short-term restricion group was lighter than control group. However,rats had better mental state in short-term calorie restricion group. No significant differences were observed between the short-term calorie restricion group and the control group about the diameter of seminiferous tubule. Rats had good seminiferous tubule and intact basilemma in short-term calorie restricion group. Various kinds of the germ cells were neatly arranged in the seminiferous tubules.There were six to eight layer cells in seminiferous epithelium. The expression of ZO-1 mRNAs was significantly increased in the short-term calorie restriction group compared with the control group,and the positive reaction of ZO-1 protein was observed. MDA was reduced in calorie restricion groups( P<0. 05). However,T-SOD and GSH-Px were increased because of calorie restricion( P<0. 05). The expression of mRNAs of SOD,CAT,GST,Nrf2 were increased by calorie restricion treatment( P<0. 05). The results suggest that short-term calorie restriction can promote tight junctions between testicular cells and improve reproduction through enhancing the ability of antioxidation and decreasing the poisonousness of free radical to cells.
The present study was conducted to examine the short-term calorie restriction effects( CR) on reproduciton in male rats.Twenty adult male Wistar rats were separated to two groups randomly,including calorie restriction group and control group. Controls were raised ad libitum and treatments were raised with 60% of the food expended by control rats. After seven weeks,the rats were weighed and observed the mental state. Then rats were executed and the testicular tissues were embedded into paraffin stained with hematoxylin and eosin( HE). Immunostaining was carried out with mice polyclonal antibodies to ZO-1. Then,oxidative stress parameters,including MDA,T-SOD and GSH-Px,were measured. Total RNA was extracted from testicular tissues and the expression of mRNA of ZO-1,SOD,CAT,GST and Nrf2 were measured by real-time quantitative PCR. The results showed that the body weight of short-term restricion group was lighter than control group. However,rats had better mental state in short-term calorie restricion group. No significant differences were observed between the short-term calorie restricion group and the control group about the diameter of seminiferous tubule. Rats had good seminiferous tubule and intact basilemma in short-term calorie restricion group. Various kinds of the germ cells were neatly arranged in the seminiferous tubules.There were six to eight layer cells in seminiferous epithelium. The expression of ZO-1 mRNAs was significantly increased in the short-term calorie restriction group compared with the control group,and the positive reaction of ZO-1 protein was observed. MDA was reduced in calorie restricion groups( P<0. 05). However,T-SOD and GSH-Px were increased because of calorie restricion( P<0. 05). The expression of mRNAs of SOD,CAT,GST,Nrf2 were increased by calorie restricion treatment( P<0. 05). The results suggest that short-term calorie restriction can promote tight junctions between testicular cells and improve reproduction through enhancing the ability of antioxidation and decreasing the poisonousness of free radical to cells.
引文
[1]Lopez-Lluch G,Hunt N,Jones B,et al.Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency[J].Proc Natl Acad Sci USA,2006,103(6):1768-1773.
[2]Koubova J,Guarente L.How does calorie restriction work?[J].Genes Dev,2003,17(3):313-321.
[3]Mccay C M,Crowell M F,Maynard L A.The effect of retarded growth upon the length of life span and upon the ultimate body size.1935[J].Nutrition,1989,5(3):155-171,172.
[4]Mattison J A,Colman R J,Beasley T M,et al.Caloric restriction improves health and survival of rhesus monkeys[J].Nat Commun,2017(8):14063.
[5]Rocha J S,Bonkowski M S,Masternak M M,et al.Effects of adult onset mild calorie restriction on weight of reproductive organs,plasma parameters and gene expression in male mice[J].Anim Reprod.2012,9(1):40-51.
[6]Sreekumar R,Unnikrishnan J,Fu A,et al.Effects of caloric restriction on mitochondrial function and gene transcripts in rat muscle[J].Am J Physiol Endocrinol Metab,2002,283(1):E38-E43.
[7]Sitzmann B D,Brown D I,Garyfallou V T,et al.Impact of moderate calorie restriction on testicular morphology and endocrine function in adult rhesus macaques(Macaca mulatta)[J].Age(Dordr),2014,36(1):183-197.
[8]陈晓纯,罗丽莉,许锦阶,等.短期能量限制对成年雌性大鼠生殖寿命的影响[J].汕头大学医学院学报,2011,24(03):141-144.
[9]石良艳,田勇,赖志文,等.不同程度热量限制对卵巢储备功能的影响[J].中国妇幼保健,2014,29(17):2786-2790.
[10]Merry B J.Molecular mechanisms linking calorie restriction and longevity[J].Int J Biochem Cell Biol,2002,34(11):1340-1354.
[11]Gredilla R,Barja G.Minireview:the role of oxidative stress in relation to caloric restriction and longevity[J].Endocrinology,2005,146(9):3713-3717.
[12]Lane M A,Tilmont E M,De Angelis H,et al.Short-term calorie restriction improves disease-related markers in older male rhesus monkeys(Macaca mulatta)[J].Mech Ageing Dev,2000,112(3):185-196.
[13]Ingram D K,Cutler R G,Weindruch R,et al.Dietary restriction and aging:the initiation of a primate study[J].J Gerontol,1990,45(5):B148-B163.
[14]Ottinger M A,Mobarak M,Abdelnabi M,et al.Effects of calorie restriction on reproductive and adrenal systems in Japanese quail:are responses similar to mammals,particularly primates?[J].Mech Ageing Dev,2005,126(9):967-975.
[15]Roth G S,Ingram D K,Lane M A.Calorie restriction in primates:will it work and how will we know?[J].J Am Geriatr Soc,1999,47(7):896-903.
[16]Kealy R D,Lawler D F,Ballam J M,et al.Effects of diet restriction on life span and age-related changes in dogs[J].J Am Vet Med Assoc,2002,220(9):1315-1320.
[17]Sohal R S,Weindruch R.Oxidative stress,caloric restriction,and aging[J].Science,1996,273(5271):59-63.
[18]Kristal B S,Yu B P.An emerging hypothesis:synergistic induction of aging by free radicals and Maillard reactions[J].J Gerontol,1992,47(4):B107-B114.
[19]Walford R L,Spindler S R.The response to calorie restriction in mammals shows features also common to hibernation:a cross-adaptation hypothesis[J].J Gerontol A Biol Sci Med Sci,1997,52(4):B179-B183.
[20]Lee C K,Klopp R G,Weindruch R,et al.Gene expression profile of aging and its retardation by caloric restriction[J].Science,1999,285(5432):1390-1393.
[21]Nelson J F,Karelus K,Bergman M D,et al.Neuroendocrine involvement in aging:evidence from studies of reproductive aging and caloric restriction[J].Neurobiol Aging,1995,16(5):837-843,855-856.
[22]Smith B E,Braun R E.Germ cell migration across Sertoli cell tight junctions[J].Science,2012,338(6108):798-802.
[23]Guan Y,Watson A J,Marchiando A M,et al.Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells[J].Am J Physiol Cell Physiol,2011,300(6):C1404-C1414.
[2]Koubova J,Guarente L.How does calorie restriction work?[J].Genes Dev,2003,17(3):313-321.
[3]Mccay C M,Crowell M F,Maynard L A.The effect of retarded growth upon the length of life span and upon the ultimate body size.1935[J].Nutrition,1989,5(3):155-171,172.
[4]Mattison J A,Colman R J,Beasley T M,et al.Caloric restriction improves health and survival of rhesus monkeys[J].Nat Commun,2017(8):14063.
[5]Rocha J S,Bonkowski M S,Masternak M M,et al.Effects of adult onset mild calorie restriction on weight of reproductive organs,plasma parameters and gene expression in male mice[J].Anim Reprod.2012,9(1):40-51.
[6]Sreekumar R,Unnikrishnan J,Fu A,et al.Effects of caloric restriction on mitochondrial function and gene transcripts in rat muscle[J].Am J Physiol Endocrinol Metab,2002,283(1):E38-E43.
[7]Sitzmann B D,Brown D I,Garyfallou V T,et al.Impact of moderate calorie restriction on testicular morphology and endocrine function in adult rhesus macaques(Macaca mulatta)[J].Age(Dordr),2014,36(1):183-197.
[8]陈晓纯,罗丽莉,许锦阶,等.短期能量限制对成年雌性大鼠生殖寿命的影响[J].汕头大学医学院学报,2011,24(03):141-144.
[9]石良艳,田勇,赖志文,等.不同程度热量限制对卵巢储备功能的影响[J].中国妇幼保健,2014,29(17):2786-2790.
[10]Merry B J.Molecular mechanisms linking calorie restriction and longevity[J].Int J Biochem Cell Biol,2002,34(11):1340-1354.
[11]Gredilla R,Barja G.Minireview:the role of oxidative stress in relation to caloric restriction and longevity[J].Endocrinology,2005,146(9):3713-3717.
[12]Lane M A,Tilmont E M,De Angelis H,et al.Short-term calorie restriction improves disease-related markers in older male rhesus monkeys(Macaca mulatta)[J].Mech Ageing Dev,2000,112(3):185-196.
[13]Ingram D K,Cutler R G,Weindruch R,et al.Dietary restriction and aging:the initiation of a primate study[J].J Gerontol,1990,45(5):B148-B163.
[14]Ottinger M A,Mobarak M,Abdelnabi M,et al.Effects of calorie restriction on reproductive and adrenal systems in Japanese quail:are responses similar to mammals,particularly primates?[J].Mech Ageing Dev,2005,126(9):967-975.
[15]Roth G S,Ingram D K,Lane M A.Calorie restriction in primates:will it work and how will we know?[J].J Am Geriatr Soc,1999,47(7):896-903.
[16]Kealy R D,Lawler D F,Ballam J M,et al.Effects of diet restriction on life span and age-related changes in dogs[J].J Am Vet Med Assoc,2002,220(9):1315-1320.
[17]Sohal R S,Weindruch R.Oxidative stress,caloric restriction,and aging[J].Science,1996,273(5271):59-63.
[18]Kristal B S,Yu B P.An emerging hypothesis:synergistic induction of aging by free radicals and Maillard reactions[J].J Gerontol,1992,47(4):B107-B114.
[19]Walford R L,Spindler S R.The response to calorie restriction in mammals shows features also common to hibernation:a cross-adaptation hypothesis[J].J Gerontol A Biol Sci Med Sci,1997,52(4):B179-B183.
[20]Lee C K,Klopp R G,Weindruch R,et al.Gene expression profile of aging and its retardation by caloric restriction[J].Science,1999,285(5432):1390-1393.
[21]Nelson J F,Karelus K,Bergman M D,et al.Neuroendocrine involvement in aging:evidence from studies of reproductive aging and caloric restriction[J].Neurobiol Aging,1995,16(5):837-843,855-856.
[22]Smith B E,Braun R E.Germ cell migration across Sertoli cell tight junctions[J].Science,2012,338(6108):798-802.
[23]Guan Y,Watson A J,Marchiando A M,et al.Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells[J].Am J Physiol Cell Physiol,2011,300(6):C1404-C1414.