摘要
为探讨亚慢性染铝对雄性大鼠生殖系统的毒性机理,本试验以60只5周龄Wistar雄性大鼠为研究对象,预饲一周后,随机分成4组:对照组、低剂量组、中剂量组和高剂量组,分别饮用含0、64.18、128.36、256.72 mg/kg B.W.AlCl_3的水溶液,每组15只。染铝120 d处死大鼠取睾丸、附睾组织,观察并记录大鼠临床表现,剖检变化,睾丸、附睾组织显微和超微结构变化,精子数量和精子畸形率的变化;分光光度法和ELISA法检测睾丸中酸性磷酸酶(ACP)、琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)、ATPase和乳酸脱氢酶同工酶(LDH-x)活性;石墨炉原子吸收分光光度法和火焰原子吸收分光光度法测定睾丸中铝(Al)、锌(Zn)、铜(Cu)、铁(Fe)的含量;放射免疫法检测血清中睾酮(T)、卵泡刺激素(FSH)和黄体生成素(LH)水平;免疫组化法检测睾丸雄激素受体(AR)、卵泡刺激素受体(FSHR)和黄体生成素受体(LHR)蛋白表达的变化;实时荧光定量PCR检测睾丸AR mRNA表达的变化;半定量RT-PCR法检测睾丸FSHR和LHR mRNA表达的变化。结果表明:
1光镜观察发现,染铝大鼠附睾细胞间质出现纤维化,柱状上皮细胞溶解、消失;睾丸间质嗜酸性明显增强,生精上皮变薄,曲精小管管腔较窄,各级生精细胞数量减少,管腔内精子较少或缺如。电镜观察发现,染铝大鼠附睾组织细胞表面部分静纤毛融合,线粒体或肿胀或空泡变性,部分粗面内质网膜性结构融合,顶体溶解,精子体部、尾部包膜溶解;睾丸精原细胞、精母细胞间隙增大,核膜皱缩,细胞内可见大小不等的空泡性结构,线粒体肿胀,核周隙增宽。
2大鼠精子数量亚慢性染铝中、高剂量组极显著低于对照组(P<0.01),精子畸形率极显著高于对照组(P<0.01),说明铝蓄积可导致精子损伤。
3亚慢性染铝可使大鼠睾丸ACP、SDH、LDH、LDH-x和ATPase活性不同程度的降低,干扰雄性大鼠生殖系统的能量代谢,抑制生殖细胞的功能。
4大鼠睾丸亚慢性染铝中、高剂量组铝含量极显著高于对照组(P<0.01),Cu含量显著或极显著高于对照组(P<0.05,P<0.01),Zn、Fe含量显著或极显著低于对照组(P<0.05,P<0.01),说明铝可在睾丸中蓄积,干扰睾丸中Zn、Cu、Fe等微量元素的代谢,进而抑制雄性大鼠的生殖能力。
5大鼠亚慢性染铝中、高剂量组血清T水平显著或极显著低于对照组(P<0.05,P<0.01);LH水平显著低于对照组(P<0.05);FSH水平低于对照组,但差异不显著(P>0.05)。睾丸组织AR、FSHR和LHR mRNA及蛋白的表达均呈不同程度的下降,提示亚慢性染铝可降低T、LH水平,干扰睾丸AR、FSHR和LHR mRNA及蛋白的表达,减弱激素与受体的结合,抑制了激素生理作用的发挥,进而阻碍其对睾丸发育和精子生成及成熟的调节。
The toxicity mechanism of sub-chronic aluminum exposure on reproductive system in male rats was explored. In this experiment, 5-weeks rats (n=60) were randomly divided into four groups: the control group, the low dose group, the middle dose group and the high dose group, and orally exposed to 0, 25.67, 128.36 and 256.72 mg/kg aluminum trichloride (AlCl_3) by drinking water for 120 days. Physical condition, cutting inspection changes, microstructure and ultrastructure of the testis and epididymis, sperm count and abnormal sperm rate were observed. The activities of ACP、SDH、LDH、LDHx and ATPase were detected by spectrophotometry. The contests of Al, Zn, Cu and Fe in testis were measured using graphite furnace atomic absorption spectrophotometry and flame atomic absorption spectrophotometry. The levels of T, LH and FSH in serum were detected using 125I radioimmunoassay (RIA) kits. The protein expressions of AR, FSHR and LHR in testes were detected by immunohistochemistry. The mRNA expressions of AR in testes were detected by real-time Q-PCR. The mRNA expressions of FSHR and LHR in testes were detected by semiquantitative RT-PCR. The results were shown as follows:
1 Under optical microscope, cell interstitial fibrosis of the epididymis in Al-treated rats was observed, and columnar epithelial cells dissolved and disappeared; Leydig eosinophils increased significantly, and testicular tubules seminiferous epithelium became thinning. Seminiferous tube was narrow, and sperm quantity within convoluted tubule was little or no. Under electron microscope, stereocilia of cell surface fusion, mitochondria swelling or empty bubble degeneration and rough surface tastes retinal sexual structure fusion were observed in the epididymis of Al-treated rats. Capsule of spermtail and acrosomal dissolved. Intercellular spaces of spermatogoniums and spermatocytes increased and nuclear membrane shrinked.
2 Compared with the control group, the sperm counts in the middle and high groups were decreased (P<0.01), and the sperm abnormal rate was increased (P<0.01), which indicated that Al damaged sperms.
3 The decreases of ACP, SDH, LDH, LDH-x and ATPase activities induced by sub-chronic Al exposure interfered with energy metabolism of reproductive system and then suppressed the function of germ cells.
4 Compared with the control group, the contents of Al and Cu in the middle and high groups were increased (P<0.05, P<0.01), and the contents of Zn and Fe were decreased (P<0.05, P<0.01) in testes, which indicated that Al accumulated in the testes interfered with the metabolism of Zn, Cu, Fe, accordingly inhibited spermatogenesis.
5 Compared with the control group, the serum levels of T and LH in the middle and high groups were decreased (P<0.05, P<0.01), while there was no significant difference in the levels of FSH between the Al-exposre groups and the control group. The results indicated that sub-chronic Al exposure decreased the serum levels of T, LH and the mRNA and protein expressions of AR, FSHR and LHR, which reduced the combination of the hormones and receptors, inhibited the effects of the hormones on testis development and spermatogenesis.
引文
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