高钼低铜对雄性小鼠生殖功能的影响
摘要
目的:为研究高钼、低铜及两因素协同作用对雄性小鼠生殖功能的影响。
方法:将80只健康雄性昆明种小鼠随机分为4组,每组20只。配制低钼低铜饲料,通过在饮水中添加不同剂量的钼和铜,建立试验模型:对照组(Mo:0mg·L-1,Cu:3mg·L-1)、低铜组(Mo:0mg·L-1,Cu:0mg·L-1)、高钼组(Mo:600mg·L-1,Cu:3mg·L-1)和高钼低铜组(Mo:600 mg·L-1,Cu:0 mg·L-1)。在试验处理的第90天,每组随机处死10只供试小鼠,采集睾丸、附睾等样品,用HE染色、透射电镜、试剂盒、免疫组化、荧光免疫等技术,在显微、超微结构,细胞、蛋白等水平上研究高钼低铜对雄性小鼠生殖功能的影响。
结果:1.睾丸系数的变化:与对照组相比,高钼组睾丸系数降低3.0% (P<0.01) ,高钼低铜组睾丸系数降低6.7%(P<0.01),与高钼组相比,高钼低铜组睾丸系数降低4.0%(P<0.01),与低铜组相比,高钼低铜组睾丸系数降低3.8%(P<0.01)。
2.微核率测定结果:与对照组相比较,高钼组,低铜组,高钼低铜组睾丸微核率分别升高95.1%,78.3%,142%,差异均极显著(P<0.01);高钼低铜组比高钼组睾丸微核率高24.0%(P<0.05) ,高钼低铜组比低铜组微核率高35.8%(P<0.01)。
3.小鼠精液品质测定结果:与对照组相比,高钼组,低铜组,高钼低铜组小鼠附睾内精子密度分别下降25.6%,27.9%,40.9%,差异极显著(P<0.01),高钼低铜组比高钼组降低20.5% (P<0.05);高钼低铜组比低铜组降低18.3%(P<0.05)。高钼组,低铜组,高钼低铜组精子活动率分别比对照组降低12.1%,12.5%,39.5%(P<0.01);高钼低铜组比高钼组精子活动率降低31.1%(P<0.01);高钼低铜组比低铜组精子活动率降低31.2%(P<0.01)。与对照组相比,高钼组,低铜组,高钼低铜组小鼠附睾内精子畸形率分别升高50.0%,45.4%,93.1%差异均极显著(P<0.01);高钼低铜组比高钼组精子畸形率高28.7%(P<0.05);高钼低铜组比低铜组精子畸形率高32.8% (P<0.05);
4.睾丸组织常规切片显示:对照组睾丸组织曲细精管形态规则,结构完整,生精细胞排列紧密,层次分明,管腔内有较多量的精子;低铜组睾丸组织整体结构上没有明显的形态学损伤,仅可观察到少量生精细胞空泡化以及精子凋亡等现象;高钼组睾丸曲细精管形态结构发生明显改变,各级生精细胞和支持细胞数量减少,排列紊乱,空泡化明显,成熟的精子明显减少,甚至消失;高钼低铜组曲精小管结构紊乱、生精细胞和间质细胞变性坏死明显增多,空泡化严重。
5.睾丸组织电镜切片结果:对照组小鼠睾丸组织细胞核膜清晰,内质网均匀分布,精子细胞尾部中段的线粒体排列整齐、结构完整;高钼组部分生精细胞线粒体肿胀、内质网扩张;低铜组的小鼠睾丸组织细胞存在低频率的膜变化,少部分精子细胞顶体膜模糊、不连续;高钼低铜组小鼠睾丸组织细胞核固缩,线粒体肿胀、空泡化,内质网扩张,核膜不清、精细胞顶体脱落等。
6.睾丸抗氧化酶测定结果:高钼组,低铜组小鼠睾丸组织内MDA含量分别比对照组高34.5%,33.3% (P<0.05);高钼低铜组比对照组高111%(P<0.01)。高钼低铜组比高钼组高56.8% (P<0.01),高钼低铜组比低铜组高58.3% (P<0.01)。与对照组相比,高钼组,低铜组,高钼低铜组小鼠睾丸组织内SOD活性分别下降19.8%,19.6%,45.8%,差异均为极显著(P<0.01),高钼低铜组比高钼组降低32.4% (P<0.01),高钼低铜组比低铜组降低31.1% (P<0.01)。高钼组,低铜组睾丸组织内GSH-Px活性分别比对照组降低22.1%,32.8%,差异显著(P<0.05),高钼低铜组比对照组降低45.0%(P<0.01) ,高钼低铜组比高钼组降低29.3%(P<0.05)。
7.睾丸组织免疫组化显示:对照组TNF-α表达平均光密度值为0.582,低铜组TNF-α表达平均光密度值为0.542;高钼组TNF-α表达平均光密度值为0.683,高于对照组17.35%(P<0.01);高钼低铜组TNF-α表达平均光密度值为0.706,高于低铜组30.26%(P<0.01)。
结论:高剂量的钼(600 mg·L-1)对小鼠睾丸组织造成了一定的损伤,影响了小鼠的生殖功能,铜缺乏加剧了钼的生殖毒性。
Objective: To investigate the synergistic action of the molybdenum excessive and copper deficiency on the reproductive function of male mice, the study was carried out.
Methods: Eighty healthy male Kunming mice were randomly divided into 4 groups, n=20. Preparation of diets with low copper and low molybdenum, animal models were established by adding different doses of molybdenum and copper into drinking water: the control group (molybdenum: 0 mg·L-1, Copper: 3 mg·L-1), low copper group ( Mo: 0 mg·L-1, Cu: 0 mg·L-1), high molybdenum group (Mo: 600 mg·L-1, Cu: 3 mg·L-1) ,high molybdenum and low copper group (Mo: 600 mg·L-1, Cu: 0 mg·L-1). On the 90th day, ten donor mice were sacrificed randomly in each group, collected testis and epididymides to study the effects of high molybdenum and low copper on the reproductive function of male mice on the level of microstructure, ultra structure, cell, protein and so on, by the hematoxylin-eosin staining, transmission electron microscope, kit, fluorescent microscope, immunohistochemistry and other techniques.
Results:
1. Changes in mice testis index: the testis index in each high molybdenum group were significantly decreased 3.0%(P<0.01) compared with the control group, in which the index of the high molybdenum and low copper group was very significantly decreased 6.7%(P<0.01) from the control group,and 4.0%(P<0.01) significantly decreased from the high molybdenum group,and significantly decreased 3.8% (P<0.01)from the low copper group.
2. Results of testis micronucleus test: the testis micronucleus rates in each experimental group were very significantly increased compared the control group(P<0.01), the high molybdenum group, the low copper group and the high molybdenum low copper group are respectively increased 95.1%,78.3%,142% compared the control group. The testis micronucleus rate of the high molybdenum low copper group was 24% higher than the high molybdenum group (P<0.05); and that was 35.8% higher than the low copper group (P<0.01).
3. Results of the determination of semen quality: Results of the determination of semen quality: compared with the control group, epididymal sperm density in each experimental group was significantly decreased (P<0.01),the high molybdenum group, the low copper group and the high molybdenum group and the low copper group are respectively decreased 25.6%,27.9%,40.9% compared with the control group; the high molybdenum group was significantly different 20.5% from the high molybdenum low copper group(P<0.05); the low copper group was significantly different 18.3% from the high molybdenum low copper group(P<0.05); Sperm motility rate in each group was significantly lower than the control group(P<0.01),the high molybdenum group, the low copper group and the high molybdenum low copper group are respectively decreased 12.1%,12.5% 39.5% compared the control group; Sperm motility rate in the high molybdenum and low copper group was significantly lower 31.1% than the high molybdenum group(P <0.01) and was significantly lower 31.2% the low copper group(P<0.01); The percentage of abnormal sperm in each group was significantly higher than the control group (P<0.01),the high molybdenum group, the low copper group and the high molybdenum group low copper group are respectively increaed 50.0%,45.4%,93.1% compared the control group (P<0.01); that of the high molybdenum low copper group was 28.7% higher than the high molybdenum group(P<0.05);
4. Normal testicular tissue sections showed that: Testis tissue in the control group has seminiferous tubules, spermatogenic cells and leading cells with normal structure, there are lots of sperms in the lumens. The low copper group can also be observed kinds of structure damage such as vacuole in spermatogenic cell and necrotic with sperm cells. The high molybdenum group made serious damages to seminiferous tubule, spermatogenic cell and interstitial cell, but is not as serious as the high molybdenum and low copper group. There presented more obvious structural damages in the high molybdenum and low copper group such as seminiferous tubules, spermatogenic cells and leading cells.
5. Results of testicular tissue biopsy electron microscopy: In the control group, mouse testis nuclear membrane was clear, and had uniform distribution of the endoplasmic reticulum, the mitochondria of sperm cells in the middle of the tail was neatly arranged and had integrate structure; In the high molybdenum group, some mitochondrial in the germ cell was swelling, endoplasmic reticulum became dilated; In the low copper group, testis cell membrane changed in the presence of low frequency, minority of acrosome membrane of sperm cells was vague and discontinuous; In the high molybdenum and low copper group, there were nuclear pyknosis, mitochondrial swelling, vacuolization, the endoplasmic reticulum expansion, unclear nuclear membrane, sperm cell acrosome loss and so on. 6. Determination results of testis lipid peroxides: compared with the control group,
the content of testis organization MDA in the high molybdenum group low copper group were 34.5%,33.3% higher than the control group respectively(P<0.05); the content of testis organization MDA in the high molybdenum and low copper group was 111% higher than the control group (P<0.01). The content of testis organization MDA in the high molybdenum and low copper group was 56.8% higher than the high molybdenum group (P<0.01) and 58.3% higher than the low copper group(P<0.01). Compared with the control group, the SOD activity of testis organization in the the high molybdenum group, the low copper group and the high molybdenum and low copper group were 19.8%,19.6%,45.8% very significantly lower (P<0.01) than the control group respectively, the SOD activity in the high molybdenum and low copper group was 32.4% lower than the high molybdenum group (P<0.01) and 31.1% lower than the low copper group (P<0.01). The GSH-PX activity of testis organization in the high molybdenum group and the low copper group were 22.1%,32.8% significantly lower than the control group respectively(P<0.05), the GSH-PX activity in the high molybdenum and low copper group was 45.0% lower than the control group(P<0.01) and 29.3% lower than the high molybdenum group (P<0.05).
7.Immunohistochemistry of testicular tissue showed: The expression of TNF-αin the control group was 0.582, that in the low copper group was 0.542; The expression of TNF-αin the high molybdenum group was 0.683, and 17.35% higher than Control group(P<0.01). The expression of TNF-αin the high molybdenum and low copper group was 0.706, and 30.26% higher than the low copper group(P<0.01).
Conclusion:
High molybdenum destroyed the normal structure of testicular and affected the normal function of testis. Deficiency copper intensified the molybdenum toxic action in testis.
方法:将80只健康雄性昆明种小鼠随机分为4组,每组20只。配制低钼低铜饲料,通过在饮水中添加不同剂量的钼和铜,建立试验模型:对照组(Mo:0mg·L-1,Cu:3mg·L-1)、低铜组(Mo:0mg·L-1,Cu:0mg·L-1)、高钼组(Mo:600mg·L-1,Cu:3mg·L-1)和高钼低铜组(Mo:600 mg·L-1,Cu:0 mg·L-1)。在试验处理的第90天,每组随机处死10只供试小鼠,采集睾丸、附睾等样品,用HE染色、透射电镜、试剂盒、免疫组化、荧光免疫等技术,在显微、超微结构,细胞、蛋白等水平上研究高钼低铜对雄性小鼠生殖功能的影响。
结果:1.睾丸系数的变化:与对照组相比,高钼组睾丸系数降低3.0% (P<0.01) ,高钼低铜组睾丸系数降低6.7%(P<0.01),与高钼组相比,高钼低铜组睾丸系数降低4.0%(P<0.01),与低铜组相比,高钼低铜组睾丸系数降低3.8%(P<0.01)。
2.微核率测定结果:与对照组相比较,高钼组,低铜组,高钼低铜组睾丸微核率分别升高95.1%,78.3%,142%,差异均极显著(P<0.01);高钼低铜组比高钼组睾丸微核率高24.0%(P<0.05) ,高钼低铜组比低铜组微核率高35.8%(P<0.01)。
3.小鼠精液品质测定结果:与对照组相比,高钼组,低铜组,高钼低铜组小鼠附睾内精子密度分别下降25.6%,27.9%,40.9%,差异极显著(P<0.01),高钼低铜组比高钼组降低20.5% (P<0.05);高钼低铜组比低铜组降低18.3%(P<0.05)。高钼组,低铜组,高钼低铜组精子活动率分别比对照组降低12.1%,12.5%,39.5%(P<0.01);高钼低铜组比高钼组精子活动率降低31.1%(P<0.01);高钼低铜组比低铜组精子活动率降低31.2%(P<0.01)。与对照组相比,高钼组,低铜组,高钼低铜组小鼠附睾内精子畸形率分别升高50.0%,45.4%,93.1%差异均极显著(P<0.01);高钼低铜组比高钼组精子畸形率高28.7%(P<0.05);高钼低铜组比低铜组精子畸形率高32.8% (P<0.05);
4.睾丸组织常规切片显示:对照组睾丸组织曲细精管形态规则,结构完整,生精细胞排列紧密,层次分明,管腔内有较多量的精子;低铜组睾丸组织整体结构上没有明显的形态学损伤,仅可观察到少量生精细胞空泡化以及精子凋亡等现象;高钼组睾丸曲细精管形态结构发生明显改变,各级生精细胞和支持细胞数量减少,排列紊乱,空泡化明显,成熟的精子明显减少,甚至消失;高钼低铜组曲精小管结构紊乱、生精细胞和间质细胞变性坏死明显增多,空泡化严重。
5.睾丸组织电镜切片结果:对照组小鼠睾丸组织细胞核膜清晰,内质网均匀分布,精子细胞尾部中段的线粒体排列整齐、结构完整;高钼组部分生精细胞线粒体肿胀、内质网扩张;低铜组的小鼠睾丸组织细胞存在低频率的膜变化,少部分精子细胞顶体膜模糊、不连续;高钼低铜组小鼠睾丸组织细胞核固缩,线粒体肿胀、空泡化,内质网扩张,核膜不清、精细胞顶体脱落等。
6.睾丸抗氧化酶测定结果:高钼组,低铜组小鼠睾丸组织内MDA含量分别比对照组高34.5%,33.3% (P<0.05);高钼低铜组比对照组高111%(P<0.01)。高钼低铜组比高钼组高56.8% (P<0.01),高钼低铜组比低铜组高58.3% (P<0.01)。与对照组相比,高钼组,低铜组,高钼低铜组小鼠睾丸组织内SOD活性分别下降19.8%,19.6%,45.8%,差异均为极显著(P<0.01),高钼低铜组比高钼组降低32.4% (P<0.01),高钼低铜组比低铜组降低31.1% (P<0.01)。高钼组,低铜组睾丸组织内GSH-Px活性分别比对照组降低22.1%,32.8%,差异显著(P<0.05),高钼低铜组比对照组降低45.0%(P<0.01) ,高钼低铜组比高钼组降低29.3%(P<0.05)。
7.睾丸组织免疫组化显示:对照组TNF-α表达平均光密度值为0.582,低铜组TNF-α表达平均光密度值为0.542;高钼组TNF-α表达平均光密度值为0.683,高于对照组17.35%(P<0.01);高钼低铜组TNF-α表达平均光密度值为0.706,高于低铜组30.26%(P<0.01)。
结论:高剂量的钼(600 mg·L-1)对小鼠睾丸组织造成了一定的损伤,影响了小鼠的生殖功能,铜缺乏加剧了钼的生殖毒性。
Objective: To investigate the synergistic action of the molybdenum excessive and copper deficiency on the reproductive function of male mice, the study was carried out.
Methods: Eighty healthy male Kunming mice were randomly divided into 4 groups, n=20. Preparation of diets with low copper and low molybdenum, animal models were established by adding different doses of molybdenum and copper into drinking water: the control group (molybdenum: 0 mg·L-1, Copper: 3 mg·L-1), low copper group ( Mo: 0 mg·L-1, Cu: 0 mg·L-1), high molybdenum group (Mo: 600 mg·L-1, Cu: 3 mg·L-1) ,high molybdenum and low copper group (Mo: 600 mg·L-1, Cu: 0 mg·L-1). On the 90th day, ten donor mice were sacrificed randomly in each group, collected testis and epididymides to study the effects of high molybdenum and low copper on the reproductive function of male mice on the level of microstructure, ultra structure, cell, protein and so on, by the hematoxylin-eosin staining, transmission electron microscope, kit, fluorescent microscope, immunohistochemistry and other techniques.
Results:
1. Changes in mice testis index: the testis index in each high molybdenum group were significantly decreased 3.0%(P<0.01) compared with the control group, in which the index of the high molybdenum and low copper group was very significantly decreased 6.7%(P<0.01) from the control group,and 4.0%(P<0.01) significantly decreased from the high molybdenum group,and significantly decreased 3.8% (P<0.01)from the low copper group.
2. Results of testis micronucleus test: the testis micronucleus rates in each experimental group were very significantly increased compared the control group(P<0.01), the high molybdenum group, the low copper group and the high molybdenum low copper group are respectively increased 95.1%,78.3%,142% compared the control group. The testis micronucleus rate of the high molybdenum low copper group was 24% higher than the high molybdenum group (P<0.05); and that was 35.8% higher than the low copper group (P<0.01).
3. Results of the determination of semen quality: Results of the determination of semen quality: compared with the control group, epididymal sperm density in each experimental group was significantly decreased (P<0.01),the high molybdenum group, the low copper group and the high molybdenum group and the low copper group are respectively decreased 25.6%,27.9%,40.9% compared with the control group; the high molybdenum group was significantly different 20.5% from the high molybdenum low copper group(P<0.05); the low copper group was significantly different 18.3% from the high molybdenum low copper group(P<0.05); Sperm motility rate in each group was significantly lower than the control group(P<0.01),the high molybdenum group, the low copper group and the high molybdenum low copper group are respectively decreased 12.1%,12.5% 39.5% compared the control group; Sperm motility rate in the high molybdenum and low copper group was significantly lower 31.1% than the high molybdenum group(P <0.01) and was significantly lower 31.2% the low copper group(P<0.01); The percentage of abnormal sperm in each group was significantly higher than the control group (P<0.01),the high molybdenum group, the low copper group and the high molybdenum group low copper group are respectively increaed 50.0%,45.4%,93.1% compared the control group (P<0.01); that of the high molybdenum low copper group was 28.7% higher than the high molybdenum group(P<0.05);
4. Normal testicular tissue sections showed that: Testis tissue in the control group has seminiferous tubules, spermatogenic cells and leading cells with normal structure, there are lots of sperms in the lumens. The low copper group can also be observed kinds of structure damage such as vacuole in spermatogenic cell and necrotic with sperm cells. The high molybdenum group made serious damages to seminiferous tubule, spermatogenic cell and interstitial cell, but is not as serious as the high molybdenum and low copper group. There presented more obvious structural damages in the high molybdenum and low copper group such as seminiferous tubules, spermatogenic cells and leading cells.
5. Results of testicular tissue biopsy electron microscopy: In the control group, mouse testis nuclear membrane was clear, and had uniform distribution of the endoplasmic reticulum, the mitochondria of sperm cells in the middle of the tail was neatly arranged and had integrate structure; In the high molybdenum group, some mitochondrial in the germ cell was swelling, endoplasmic reticulum became dilated; In the low copper group, testis cell membrane changed in the presence of low frequency, minority of acrosome membrane of sperm cells was vague and discontinuous; In the high molybdenum and low copper group, there were nuclear pyknosis, mitochondrial swelling, vacuolization, the endoplasmic reticulum expansion, unclear nuclear membrane, sperm cell acrosome loss and so on. 6. Determination results of testis lipid peroxides: compared with the control group,
the content of testis organization MDA in the high molybdenum group low copper group were 34.5%,33.3% higher than the control group respectively(P<0.05); the content of testis organization MDA in the high molybdenum and low copper group was 111% higher than the control group (P<0.01). The content of testis organization MDA in the high molybdenum and low copper group was 56.8% higher than the high molybdenum group (P<0.01) and 58.3% higher than the low copper group(P<0.01). Compared with the control group, the SOD activity of testis organization in the the high molybdenum group, the low copper group and the high molybdenum and low copper group were 19.8%,19.6%,45.8% very significantly lower (P<0.01) than the control group respectively, the SOD activity in the high molybdenum and low copper group was 32.4% lower than the high molybdenum group (P<0.01) and 31.1% lower than the low copper group (P<0.01). The GSH-PX activity of testis organization in the high molybdenum group and the low copper group were 22.1%,32.8% significantly lower than the control group respectively(P<0.05), the GSH-PX activity in the high molybdenum and low copper group was 45.0% lower than the control group(P<0.01) and 29.3% lower than the high molybdenum group (P<0.05).
7.Immunohistochemistry of testicular tissue showed: The expression of TNF-αin the control group was 0.582, that in the low copper group was 0.542; The expression of TNF-αin the high molybdenum group was 0.683, and 17.35% higher than Control group(P<0.01). The expression of TNF-αin the high molybdenum and low copper group was 0.706, and 30.26% higher than the low copper group(P<0.01).
Conclusion:
High molybdenum destroyed the normal structure of testicular and affected the normal function of testis. Deficiency copper intensified the molybdenum toxic action in testis.
引文
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[12]杨自军,冉林武,赵树科等.实验性钼中毒绵羊血液酶学指标的变化与锌、铜的保护作用[J].畜牧与兽医,2008,40(4):52-55.
[13]孙素玲,程玲玲.钼对大鼠肝微粒体苯胺羟化酶活力的影响[J].动物医学进展,2007,28(7):29-31.
[14]张元贞.钼对鸡的营养作用[J].饲料与畜牧,1989,(6):22-25.
[15] Pandey, Ratna, Singh, S P, Effects of molybdenum on fertility of male rats [J]. Biom etal, 2002, 15(1):65-72.
[16]杨自军,冉林武,王宏伟等.锌、铜对钼中毒绵羊免疫功能的影响[J].中国兽医学报,2009,29(2):207-209.
[17]肖杰,崔恒敏,杨帆等.高钼对雏鸡肝脏和脾脏抗氧化功能的影响[J].畜牧兽医学报,2010,41(7):883-890.
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[19]樊璞,吴治礼,王继玉等.耕牛钼中毒的研究初报[J].中国兽医杂志,1983,12:5-8.
[20]李三强,龙晶,丁伯良等.天然高钼饮水复制耕牛钼中毒试验研究[J].动物营养学报,1997,(1):64.
[21]吕爱军,穆阿丽,张学胜等.绵羊铜缺乏症的防治措施[J].河北畜牧兽医,2004,20(12):40.
[22]杨帆,崔恒敏,肖杰等.高钼对艾维茵肉鸡法氏囊细胞周期和凋亡影响分析[J].畜牧兽医学报,2010,41(8):1031-1038.
[23] Zamzami N, Marchetti P, Castedo M. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo[J].J Exp Med,1995, 181: 161.
[24]李三强,龙晶.饮用天然高钼水诱发的钼中毒耕牛主要生物酶测定[J].动物营养学报,1998,10(1):29-33.
[25] Merlf, Roger, Michaelas. Modest copper supplementation blocks molybdenosis in cattle[J].Vet Diagn Invest,2006, 18: 566-572.
[26]林曦.家畜病理学[M].第三版.北京:中国农业出版社,1999,206-208.
[27]梁斌,龚新辉,马健等.绵羊铜缺乏症的诊治[J].草食家畜,2009,(4):76-77.
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[29]吴建设,呙于明,杨汉春等.微量元素铜的营养与免疫研究进展[J].国外畜牧科技,1999,26(1):5-9.
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