| 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. |
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