摘要
目的 检测大鼠睾丸不同发育阶段中锌含量和锌转运体(ZnT)的表达,探讨锌稳态在睾丸发育过程中的作用及机制.方法 将雄性SD大鼠随机分为4周龄组(n=10)、8周龄组(n=10)、12周龄组(n=10)和32周龄组(n=10),分别饲养至相应周龄.取新鲜大鼠附睾尾部精子检测精子总数、活力,睾丸组织HE染色观察病理学变化,化学发光法测定血清睾酮水平,电感耦合等离子体质谱法测定睾丸组织锌含量,Zinquin探针测定大鼠睾丸锌离子水平,酶联免疫吸附法测定含锌酶(ALP、ADH、LDH)活性,实时荧光定量PCR和Western blot检测睾丸组织中锌转运体ZnT1-9 mRNA和蛋白表达.结果 8周龄组大鼠+睾丸内开始出现精子,精子总数随大鼠周龄而增加,8、12周龄组大鼠精子活力和睾酮水平显著高于4、32周龄组大鼠(P＜0.05),8、12、32周龄组大鼠睾丸游离锌离子水平显著高于4周龄组大鼠,12周和32周龄大鼠睾丸ALP活性明显低于4周和8周组(P＜0.05),8、12、32周龄组大鼠睾丸ZnT3 mRNA和蛋白水平显著高于4周组(P＜0.05),8、12、32周龄组大鼠睾丸ZnT4 mRNA水平显著高于4周组(P＜0.05),12、32周龄组大鼠睾丸ZnT8 mRNA和蛋白水平显著高于4周和8周组(P＜0.05).结论 在大鼠睾丸发育过程中,锌+含锌酶和锌转运体(ZnT)发生动态变化,为进一步研究锌稳态对雄性生殖系统发育的影响提供依据.
Abstract
Objective To investigate the role and mechanism of zinc homeostasis in testicular development by detecting zinc content and zinc transporter(ZnT)expression at different developmental stages of rat testis.Methods Male SD rats were randomly divided into four groups including4-week age group(n=10),8-week age group(n=10),12-week age group(n=10)and 32-week age group(n=10).Fresh spermatozoa from the tail of epididymis of rats were collected and their number and motility were measured.The histopathological changes of testis were observed by HE staining and microscope.Serum testosterone was determined by chemiluminescence method.The content of zinc in testicular tissue was determined by inductively coupled plasma mass spectrometry.The level of zinc ion in testis was detected by Zinquin probe.Zinc-containing enzyme activity(ALP,ADH,LDH)was analyzed by enzyme-linked immunosorbent assay.mRNA and protein expression of zinc transporters ZnT1-9 in testicular tissue were detected by real-time fluorescence quantitative PCR and Western Blot.Results Spermatozoa appeared in the testis of the 8-week-old rats,and the total number of spermatozoa was increased with the increase of age.Sperm motility and testosterone level of rats in 8-week and 12-week age groups were all significantly higher than those of rats in 4-week and 32-week age groups(P＜0.05).The level of free zinc ion in testis tissue in 8,12 and 32-week age groups was significantly higher than that of 4-week age group.The activity of ALP in testis of 12 and 32 weeks of age was significantly lower than that of 4 and 8 weeks of age(P＜0.05).The levels of ZnT3 mRNA and protein in testis of rats at 8,12 and 32 weeks of age were significantly higher than those of rats at 4 weeks of age(P＜0.05),and the levels of ZnT4 mRNA in testis of rats at 8,12 and 32 weeks of age were significantly higher than those of rats at 4 weeks of age(P＜0.05).The levels of ZnT8 mRNA and protein in testis in 12 and 32 weeks of age groups were significantly higher than those in 4 and 8 weeks of age groups(P＜0.05).Conclusion Zinc-containing enzymeand zinc transporter(ZnT)change dynamically during testicular development in rats,which provides a basis for further investigation on the effect of zinc homeostasis on male reproductive system development.
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