高表达SEMG1蛋白的弱精子症大鼠模型的建立
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摘要
目的构建一个睾丸中SEMG1蛋白高表达的弱精子症大鼠模型。方法将30只SD大鼠随机分为3组:空白对照组、阴性对照组、及实验组(腹腔注射环磷酰胺),每组10只。实验结束后称其体质量及一侧睾丸质量并计算睾丸指数;睾丸常规石蜡切片,HE染色;取附睾尾制备精子悬液进行精子计数及活力检测; Western blot检测SEMG1及CASPASE-3及BCL-2蛋白表达。结果与对照组相比,实验组大鼠体质量差异无统计学意义(P> 0.05),睾丸质量及睾丸指数较对照组降低(P <0.001),实验组大鼠精子悬液镜下观察见精子数量及精子活力明显降低,HE染色见睾丸生精小管中各级生精细胞排列紊乱,细胞数量减少。Western blot结果显示:与对照组相比,实验组SEMG1及CASPASE-3蛋白表达水平显著升高,BCL-2蛋白表达水平明显降低(P <0.001)。结论成功构建了睾丸中高表达SEMG1蛋白的弱精子症大鼠模型。
Objective To construct a rat model of asthenozoospermia with high expression of SEMG1 genein testis.MethodsThirty SD rats were randomly divided into 3 groups,10 in each group. The blank controlgroup which did not do any treatment,the saline intraperitoneal injection group and the cyclophosphamide intraper-itoneal injection group. At the end of the experiment,the quality and the testicular index was calculated. The testiswas routinely paraffin-embedded and HE staining. The spermatic suspension was prepared for sperm count. Theexpression of SEMG1,CASPE-3 and BCL-2 protein was detected by Western blot.ResultsCompared with thecontrol group,there was no significant difference in the quality of the cyclophosphamide-treated group(P > 0.05).The testicular mass and testicular index were lower than the control group(P < 0.001). Under the microscope,thenumber of spermatozoa was significantly reduced in the cyclophosphamide-treated group. In HE,the spermatogeniccells in the spermatogenic tubules were disordered and the number of cells was reduced. Western blot resultsshowed that compared with the control group,the expression levels of SEMG1 and CASPE-3 in the cyclophospha-mide treatment group were significantly increased,and the expression level of BCL-2 protein was significantlydecreased(P < 0.001).ConclusionA rat model of asthenzoospermia with high expression of SEMG1 gene intestis is successfully constructed.
Objective To construct a rat model of asthenozoospermia with high expression of SEMG1 genein testis.MethodsThirty SD rats were randomly divided into 3 groups,10 in each group. The blank controlgroup which did not do any treatment,the saline intraperitoneal injection group and the cyclophosphamide intraper-itoneal injection group. At the end of the experiment,the quality and the testicular index was calculated. The testiswas routinely paraffin-embedded and HE staining. The spermatic suspension was prepared for sperm count. Theexpression of SEMG1,CASPE-3 and BCL-2 protein was detected by Western blot.ResultsCompared with thecontrol group,there was no significant difference in the quality of the cyclophosphamide-treated group(P > 0.05).The testicular mass and testicular index were lower than the control group(P < 0.001). Under the microscope,thenumber of spermatozoa was significantly reduced in the cyclophosphamide-treated group. In HE,the spermatogeniccells in the spermatogenic tubules were disordered and the number of cells was reduced. Western blot resultsshowed that compared with the control group,the expression levels of SEMG1 and CASPE-3 in the cyclophospha-mide treatment group were significantly increased,and the expression level of BCL-2 protein was significantlydecreased(P < 0.001).ConclusionA rat model of asthenzoospermia with high expression of SEMG1 gene intestis is successfully constructed.
引文
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[10]AGHAIE S,NIKZAD H,MAHABADI J,et al.Protective effect of combined pumpkin seed and ginger extracts on sperm characteristics,biochemical parameters and epididymal histology in adult male rats treated with cyclophosphamide[J].Anat Sci Int,2016,91(4):382-390.
[11]WANG Y N,WANG B,LIANG M,et al.Down-regulation of CatSper1 channel in epididymal spermatozoa contributes to the pathogenesis of asthenozoospermia,whereas up-regulation of the channel by Sheng-Jing-San treatment improves the sperm motility of asthenozoospermia in rats[J].Fertil Steril,2013,99(2):579-587.
[12]SCHUPPE H C,PILATZ A,HOSSAIN H,et al.Urogenital Infection as a Risk Factor for Male Infertility[J].Dtsch Arztebl Int,2017,114(19):339-346.
[13]JANGKHAH M,FARRAHI F,SADIGHI GILANI M A,et al.Effects of varicocelectomy on serum testosterone levels among infertile men with varicocele[J].Int J Fertil Steril,2018,12(2):169-172.
[14]WANG Y,SUN Y,ZHAO X,et al.Downregulation of DJ-1fails to protect mitochondrial complex I subunit NDUFS3 in the testes and contributes to the asthenozoospermia[J].Mediators Inflamm,2018,2018:6136075.
[15]XU Y,FAN Y,FAN W,et al.RNASET2 impairs the sperm motility via PKA/PI3K/calcium signal pathways[J].Reproduction,2018,155(4):383-392.
[16]GILANY K,MOAZENI-POURASIL R S,JAFARZADEH N,et al.Metabolomics fingerprinting of the human seminal plasma of asthenozoospermic patients[J].Mol Reprod Dev,2014,81(1):84-86.
[17]LEGARE C,DROIT A,FOURNIER F,et al.Investigation of male infertility using quantitative comparative proteomics[J].JProteome Res,2014,13(12):5403-14.
[18]陈指龙,张晓春,薛立群,等.环磷酰胺致小鼠生精障碍作用研究[J].动物医学进展,2017,38(5):34-38.
[19]高学勇,王玮,韩咪莎,等.环磷酰胺对大鼠睾丸和附睾的影响[J].解剖学研究,2009,31(1):25-27.
[20]MITRA A,RICHARDSON R T,O′RAND M G.Analysis of recombinant human semenogelin as an inhibitor of human sperm motility[J].Biol Reprod,2010,82(3):489-496.
[21]WANG Z,WIDGREN E E,RICHARDSON R T,et al.Characterization of an eppin protein complex from human semen and spermatozoa[J].Biol Reprod,2007,77(3):476-84.
[22]CARNAHAN S J,JENSEN-SEAMAN M I.Hominoid seminal protein evolution and ancestral mating behavior[J].Am J Primatol,2008,70(10):939-948.
[23]AGUILAR-MAHECHA A,HALES B F,ROBAIRE B.Effects of acute and chronic cyclophosphamide treatment on meiotic progression and the induction of DNA double-strand breaks in rat spermatocytes[J].Biol Reprod,2005,72(6):1297-1304.
[2]BRACKE A,PEETERS K,PUNJABI U,et al.A search for molecular mechanisms underlying male idiopathic infertility[J].Reprod Biomed Online,2018,36(3):327-339.
[3]ROBERT M,GAGNON C.Purification and characterization of the active precursor of a human sperm motility inhibitor secreted by the seminal vesicles:identity with semenogelin[J].Biol Reprod,1996,55(4):813-821.
[4]ROBERT M,GAGNON C.Semenogelin I:A coagulum forming,multifunctional seminal vesicle protein[J].Cell Mol Life Sci,1999,55(6-7):944-960.
[5]YU Q,ZHOU Q,WEI Q,et al.SEMG1 may be the candidate gene for idiopathic asthenozoospermia[J].Andrologia,2014,46(2):158-166.
[6]SILVA E J,HAMIL K G,O′RAND M G.Interacting proteins on human spermatozoa:adaptive evolution of the binding of semenogelin I to EPPIN[J].PLoS One,2013,8(12):e82014.
[7]JONSSON M,FROHM B,MALM J.Binding of semenogelin Ito intact human spermatozoa studied by flow cytometry and surface plasmon resonance[J].J Androl,2010,31(6):560-565.
[8]WANG Z,WIDGREN E E,SIVASHANMUGAM P,et al.Association of eppin with semenogelin on human spermatozoa.[J].Biol Reprod,2005,72(5):1064-1070.
[9]EMADI A,JONES R J,BRODSKY R A.Cyclophosphamide and cancer:golden anniversary[J].Nat Rev Clin Oncol,2009,6(11):638-647.
[10]AGHAIE S,NIKZAD H,MAHABADI J,et al.Protective effect of combined pumpkin seed and ginger extracts on sperm characteristics,biochemical parameters and epididymal histology in adult male rats treated with cyclophosphamide[J].Anat Sci Int,2016,91(4):382-390.
[11]WANG Y N,WANG B,LIANG M,et al.Down-regulation of CatSper1 channel in epididymal spermatozoa contributes to the pathogenesis of asthenozoospermia,whereas up-regulation of the channel by Sheng-Jing-San treatment improves the sperm motility of asthenozoospermia in rats[J].Fertil Steril,2013,99(2):579-587.
[12]SCHUPPE H C,PILATZ A,HOSSAIN H,et al.Urogenital Infection as a Risk Factor for Male Infertility[J].Dtsch Arztebl Int,2017,114(19):339-346.
[13]JANGKHAH M,FARRAHI F,SADIGHI GILANI M A,et al.Effects of varicocelectomy on serum testosterone levels among infertile men with varicocele[J].Int J Fertil Steril,2018,12(2):169-172.
[14]WANG Y,SUN Y,ZHAO X,et al.Downregulation of DJ-1fails to protect mitochondrial complex I subunit NDUFS3 in the testes and contributes to the asthenozoospermia[J].Mediators Inflamm,2018,2018:6136075.
[15]XU Y,FAN Y,FAN W,et al.RNASET2 impairs the sperm motility via PKA/PI3K/calcium signal pathways[J].Reproduction,2018,155(4):383-392.
[16]GILANY K,MOAZENI-POURASIL R S,JAFARZADEH N,et al.Metabolomics fingerprinting of the human seminal plasma of asthenozoospermic patients[J].Mol Reprod Dev,2014,81(1):84-86.
[17]LEGARE C,DROIT A,FOURNIER F,et al.Investigation of male infertility using quantitative comparative proteomics[J].JProteome Res,2014,13(12):5403-14.
[18]陈指龙,张晓春,薛立群,等.环磷酰胺致小鼠生精障碍作用研究[J].动物医学进展,2017,38(5):34-38.
[19]高学勇,王玮,韩咪莎,等.环磷酰胺对大鼠睾丸和附睾的影响[J].解剖学研究,2009,31(1):25-27.
[20]MITRA A,RICHARDSON R T,O′RAND M G.Analysis of recombinant human semenogelin as an inhibitor of human sperm motility[J].Biol Reprod,2010,82(3):489-496.
[21]WANG Z,WIDGREN E E,RICHARDSON R T,et al.Characterization of an eppin protein complex from human semen and spermatozoa[J].Biol Reprod,2007,77(3):476-84.
[22]CARNAHAN S J,JENSEN-SEAMAN M I.Hominoid seminal protein evolution and ancestral mating behavior[J].Am J Primatol,2008,70(10):939-948.
[23]AGUILAR-MAHECHA A,HALES B F,ROBAIRE B.Effects of acute and chronic cyclophosphamide treatment on meiotic progression and the induction of DNA double-strand breaks in rat spermatocytes[J].Biol Reprod,2005,72(6):1297-1304.