PNPLA5基因敲除对大鼠睾丸形态学及精子运动能力的影响
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摘要
旨在研究PNPLA5基因对雄性大鼠繁殖性能的影响。本研究以PNPLA5敲除型雄性大鼠(KO)为试验组,野生型大鼠(WT)为对照组,分别饲喂普通饲料,并采集大鼠附睾精子及睾丸组织。利用繁殖试验检测配种后雌鼠的妊娠率及平均产仔数,精子运动性能分析检测精子运动能力,Western blot检测精子中线粒体功能相关细胞色素C(cytochrome C,Cytc)与细胞色素C氧化酶亚基IV(cytochrome c oxidase subunit IV,COX IV)的表达水平,HE染色检测睾丸组织形态。结果表明:与野生型雄性大鼠相比,PNPLA5敲除型雄鼠与野生型雌鼠配种后,雌鼠的妊娠率极显著下降(P<0.01);精子的曲线运动速度显著降低(P<0.05)及渐进运动精子的百分率极显著降低(P<0.01);PNPLA5敲除型大鼠精子中细胞色素C表达量下调,细胞色素C氧化酶亚基IV表达量上调,但差异均不显著(P>0.05);PNPLA5基因缺失引起大鼠睾丸组织结构疏松,曲细精管中各阶段生精细胞排列紊乱,上皮细胞脱落至管腔。综上表明,本研究发现PNPLA5基因敲除能够引起雄性大鼠繁殖能力降低,为家畜繁殖研究提供重要的参考依据。
The aim of this study was to investigate the effect of PNPLA5 gene on male rats' fertility.We used male PNPLA5 knockout rats(KO)as the experimental group and wild type rats(WT)as the control group,feeding them with regular diet.The epididymis spermatozoa and testis tissue were collected.We detected the pregnancy rate and average litter size by breeding assay,the sperm motility by Computer Aided Sperm Analysis(CASA),the expression level of mitochondria function related proteins(cytochrome C,Cytc and cytochrome C Oxidase subunit IV)in sperms were detected by Western blot,and histochemical morphology of testis tissue by HE staining.The results showed that PNPLA5 knockout significantly reduced the pregnancy rate of the mated female rats(P<0.01),significantly decreased the curvilinear velocity of sperm(P<0.05)and percentage of sperm with progressive movement(P<0.01)compared to the wild type male rats.We also found that the PNPLA5 knockout downregulated the expression level of Cytc and upregulated expression level of COX IV in sperms compared with those in wild type rats(WT),but no significant difference were observed between 2groups(P>0.05).In addition,the histological structure of testis of PNPLA5 knockout rats(KO)appeared loose,unarranged,and epithelial cells detached to the lumen.In conclusion,this study shows that PNPLA5 knockout reduces the fertility of male rats and it may provide an important evidence for understanding the animal reproductive performance.
The aim of this study was to investigate the effect of PNPLA5 gene on male rats' fertility.We used male PNPLA5 knockout rats(KO)as the experimental group and wild type rats(WT)as the control group,feeding them with regular diet.The epididymis spermatozoa and testis tissue were collected.We detected the pregnancy rate and average litter size by breeding assay,the sperm motility by Computer Aided Sperm Analysis(CASA),the expression level of mitochondria function related proteins(cytochrome C,Cytc and cytochrome C Oxidase subunit IV)in sperms were detected by Western blot,and histochemical morphology of testis tissue by HE staining.The results showed that PNPLA5 knockout significantly reduced the pregnancy rate of the mated female rats(P<0.01),significantly decreased the curvilinear velocity of sperm(P<0.05)and percentage of sperm with progressive movement(P<0.01)compared to the wild type male rats.We also found that the PNPLA5 knockout downregulated the expression level of Cytc and upregulated expression level of COX IV in sperms compared with those in wild type rats(WT),but no significant difference were observed between 2groups(P>0.05).In addition,the histological structure of testis of PNPLA5 knockout rats(KO)appeared loose,unarranged,and epithelial cells detached to the lumen.In conclusion,this study shows that PNPLA5 knockout reduces the fertility of male rats and it may provide an important evidence for understanding the animal reproductive performance.
引文
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[20]WANG H R,ZHAO R,GUO C,et al.Knockout of BRD7results in impaired spermatogenesis and male infertility[J].Sci Rep,2016,6:21776.
[21]YAN J T,ZHANG H,LIU Y,et al.Germline deletion of huntingtin causes male infertility and arrested spermiogenesis in mice[J].J Cell Sci,2016,129(3):492-501.
[2]LAKE A C,SUN Y,LI J L,et al.Expression,regulation,and triglyceride hydrolase activity of Adiponutrin family members[J].J Lipid Res,2005,46(11):2477-2487.
[3]WILSON P A,GARDNER S D,LAMBIE N M,et al.Characterization of the human patatin-like phospholipase family[J].J Lipid Res,2006,47(9):1940-1949.
[4]LIU Y,GAO Q,ZHANG X,et al.PNPLA5-knockout rats induced by CRISPR/Cas9exhibit abnormal bleeding and lipid level[J].J Integr Agric,2017,16(1):169-180.
[5]SAARELA J,JUNG G,HERMANN M,et al.The patatin-like lipase family in Gallus gallus[J].BMCGenomics,2008,9:281.
[6]BRASAEMLE D L.Thematic review series:adipocyte Biology.The perilipin family of structural lipid droplet proteins:stabilization of lipid droplets and control of lipolysis[J].J Lipid Res,2007,48(12):2547-2559.
[7]NOTARI L,BALADRON V,AROCA-AGUILAR JD,et al.Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor[J].JBiol Chem,2006,281(49):38022-38037.
[8]CHEN Q,PENG H Y,LEI L,et al.Aquaporin 3is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration[J].Cell Res,2011,21(6):922-933.
[9]LANGE L A,HU Y N,ZHANG H,et al.Whole-exome sequencing identifies rare and low-frequency coding variants associated with LDL cholesterol[J].Am J Hum Genet,2014,94(2):233-245.
[10]DUPONT N,CHAUHAN S,ARKO-MENSAH J,et al.Neutral lipid stores and lipase PNPLA5contribute to autophagosome biogenesis[J].Curr Biol,2014,24(6):609-620.
[11]PADRN R S,MS J,ZAMORA R,et al.Lipids and testicular function[J].Int Urol Nephrol,1989,21(5):515-519.
[12]ERGN A,KSE S K,AYDOS K,et al.Correlation of seminal parameters with serum lipid profile and sex hormones[J].Arch Androl,2007,53(1):21-23.
[13]FAN Y,LIU Y,XUE K,et al.Diet-induced obesity in male C57BL/6 mice decreases fertility as a consequence of disrupted blood-testis barrier[J].PLoSOne,2015,10(4):e0120775.
[14]王巧刚,陈黎亚,盛礼建.精子线粒体与男性不育的相关性研究进展[J].中国现代医生,2017,55(4):160-163.WANG Q G,CHEN L Y,SHENG L J.Advances in studies on the correlation between sperm mitochondria and male infertility[J].China Modern Doctor,2017,55(4):160-163.(in Chinese)
[15]ALMEHMADI Y,YASSIN A A,NETTLESHIP JE,et al.Testosterone replacement therapy improves the health-related quality of life of men diagnosed with late-onset hypogonadism[J].Arab J Urol,2016,14(1):31-36.
[16]VENDRAMINI V,SASSO-CERRI E,MIRAGLIA SM.Amifostine reduces the seminiferous epithelium damage in doxorubicin-treated prepubertal rats without improving the fertility status[J].Reprod Biol Endocrinol,2010,8:3.
[17]VENDRAMINI V,CEDENHO A P,MIRAGLIA SM,et al.Reproductive function of the male obese Zucker rats:alteration in sperm production and sperm DNA damage[J].Reprod Sci,2014,21(2):221-229.
[18]LI N C,WEI X X,HU Y L,et al.Aerobic exercise blocks interleukin-6levels and germ cell apoptosis in obese rats[J].Andrologia,2018,50(2):e12880.
[19]CAMPOS-SILVA P,FURRIEL A,COSTA W S,et al.Metabolic and testicular effects of the long-term administration of different high-fat diets in adult rats[J].Int Braz J Urol,2015,41(3):569-575.
[20]WANG H R,ZHAO R,GUO C,et al.Knockout of BRD7results in impaired spermatogenesis and male infertility[J].Sci Rep,2016,6:21776.
[21]YAN J T,ZHANG H,LIU Y,et al.Germline deletion of huntingtin causes male infertility and arrested spermiogenesis in mice[J].J Cell Sci,2016,129(3):492-501.