摘要
水胺硫磷作为一种新型高效的有机磷杀虫剂,其毒性研究国内外仅见少量急性中毒、迟发性神经病和生物膜毒性作用研究报道[1,2],其对雄性生殖的影响少有报道。本研究采用动物繁殖实验,用水胺硫磷染毒亲代大鼠,观察水胺硫磷对雄性大鼠生殖功能、病理及子代生长发育的影响,为进一步了解水胺硫磷的毒性作用提供科学依据。此外,本次实验结合水胺硫磷对雄性大鼠下丘脑-垂体-性腺轴结构的影响,为探讨水胺硫磷对雄性生殖功能影响的可能机制开拓新的思路。120只健康SD大鼠(雌鼠80只,雄鼠40只)随机分为四组:对照组(0mg/kg)、低剂量组(0.3mg/kg)、中剂量组(3mg/kg)、高剂量组(6mg/kg)。水胺硫磷溶于食油拌入饲料中,大鼠自由摄食。染毒8周后,将雌、雄2∶1合笼交配,每日清晨用阴道涂片法或阴栓法查精子,以查到精子之日作为妊娠0d,孕鼠单笼饲养,自然分娩,产仔为子代,对仔鼠在哺乳的0、4、7、14和21d称量体重并测量身长、尾长和胸围。交配结束后,处死雄性大鼠,取下丘脑、垂体、睾丸、附睾、前列腺和精囊腺,称量并计算脏器系数;采用HE染色和电镜观察组织病理改变;原子吸收光谱法测定睾丸组织中钙、镁、铜、锌含量;采集大鼠附睾尾精子,计数精子总数并计算精子活动率、精子畸形率。从多个方面探讨水胺硫磷对雄性大鼠生殖系统结构及功能的影响。
研究结果显示:1雄性大鼠体重及脏器系数水胺硫磷对雄性大鼠体重增长及脏器系数均无明显影响(P>0.05)。2 HE染色(1)高剂量组下丘脑神经元可见水肿。(2)腺垂体中嗜酸性细胞随染毒剂量增加有逐渐增多的趋势。(3)经水胺硫磷处理后,各剂量组睾丸组织生精细胞的数目及层次随染毒剂量增加逐渐减少;附睾,前列腺,精囊腺未见明显病理改变。3电镜(1)随染毒剂量增加,下丘脑神经元胞浆内线粒体肿胀增多,高剂量组神经元可见水肿;神经胶质细胞未见明显结构改变。(2)腺垂体中生长激素细胞核周隙增宽、内质网扩张及线粒体肿胀剂量依赖性加重;随染毒剂量增加,促性腺激素细胞胞浆内肿胀的线粒体逐渐增多;促肾上腺皮质激素细胞、促甲状腺激素细胞和催乳素细胞均未见明显结构改变。(3)睾丸组织内中、高剂量组支持细胞水肿;精原细胞随染毒剂量增加水肿加重直至精原细胞坏死,膜性结构破坏;各剂量组精母细胞和精子细胞均未见明显结构改变;中、高剂量组精子尾部横切面“9×2+2”微管结构正常,致密纤维缺失。4睾丸组织钙、镁、铜、锌含量雄性大鼠睾丸组织中钙、铜含量有剂量依赖性升高的趋势,中、高剂量组铜含量与对照组相比差异有统计学意义(P<0.05);镁、锌含量随染毒剂量增加而降低,其中高剂量组与对照组相比有统计学差异(P<0.05)。5精子计数、精子活动率和精子畸形率各剂量组精子计数、精子活动率低于对照组,差异有统计学意义(P<0.01和P<0.05);各剂量组精子畸形率均高于与对照组,差异有统计学意义(P<0.01和P<0.05)。6繁殖力指标亲代大鼠高剂量组交配成功率、出生存活率均低于对照组,差异有统计学意义(P<0.05)。7子代生长发育各剂量组仔鼠体重、胸围、身长、尾长均低于对照组,有统计学差异(P<0.05,P<0.01或P<0.001);仔鼠生长发育各指标随染毒剂量增加逐渐降低。
从以上结果可以推测,水胺硫磷可直接导致大鼠睾丸组织损伤,使曲细精管内各级生精细胞数目及层次减少,并能使精原细胞、支持细胞、精子发生超微结构改变,进而精子总数减少,精子活动率降低和精子畸形率增加,从而影响雄性大鼠精子质量。水胺硫磷可通过影响睾丸组织中钙、镁、铜、锌含量干扰精子生成、发育和成熟,最终影响大鼠繁殖能力及子代的质量。此外水胺硫磷可导致下丘脑-垂体-睾丸轴病理结构改变,提示激素分泌异常可能是水胺硫磷影响雄性大鼠生殖功能的机制之一。
Isocarbophos is a new type of efficient organic phosphorus pesticides. Toxicity studies of isocarbophos at home and abroad are only a small number of acute poisoning, delayed neuropathy and toxic effects of biofilm research reports [1,2] and its impact on male reproductive function is reported rarely. In this study, male rats were fed with isocarbophos. The reproductive function and pathology of male rats, the growth and development of offspring were observed in order to provide a scientific basis to further understand the toxicity of isocarbophos. In addition, this experiment also observed pathology of hypothalamus-pituitary-gonadal axis on male rat, in order to explore new ideas of the possible mechanisms that isocarbophos affect male reproductive function.
A total of 120 rats (female: 80, male:40) was randomly divided into four groups: control group(0 mg/kg), low-dose group(0.3 mg/kg), media-dose group(3 mg/kg), high-dose group(6 mg/kg). Rats were exposed to isocarbophos by food mixed with dissolved isocarbophos. After 8-week treatment, male and female rats were mated in rate of 2∶1. Sperm was determined by the method of sperm smear in everyday morning. The day that sperm was found was recorded as the 0 day of pregnancy. The pregnant rat was fed in single cage. The weight, length, tail length and chest circumference of offspring were measured at 0, 4, 7, 14, and 21 days. After mated, male rats were killed. The hypothalamus, pituitary, testis, epididymis, prostate and seminal vesicle were removed, weighed and calculated the organ coefficient; HE staining and electron microscopy were used to observed pathological changes; the content of calcium, magnesium, copper, zinc in testicular tissue were determined by the method of atomic absorption spectrometry; sperm in tail of epididymis were collected and counted; the activity rate and the abnormal rate of sperm were calculated. The experiment discussed effects of isocarbophos on structure and function of reproduction system from a number of aspects.
The results showed that: 1 The body weight and organ coefficient of male rats were not affected by iscarbophos significantly (P>0.05). 2 HE staining (1) Hypothalamic neurons edema can be observed in high dose group. (2)Eosinophil in adenohypophysis increased with the dose increasing. (3) In the groups treated by isocarbophos, the counts and layers of spermatogenic cells obviously decreased with the dose increasing. The pathological changes of epididymis, prostate and seminal vesicle were not observed. 3 Electron microscopy (1) Swelling of mitochondria in hypothalamic neurons increased with the dose increasing, edema of neuron can be observed in high-dose group; glial cells had no obvious structural changes. (2) nuclear-week gap wide, the expansion of the endoplasmic reticulum and mitochondrial swelling in a dose-dependent increase in growth hormone cells; with increasing dose, mitochondrial swelling in gonadotropin cells gradually increased; adrenocorticotropic hormone cells, thyroid-stimulating hormone cells and prolactin cells had no obvious structural changes. (3) Edema of Sertoli cells were observed in middle and high group; edema of spermatogonia aggravated with the dose increaseing, spermatogonium necrosis and destruction of membrane structure can be observed in high-dose-group; loss of dense fiber can be observed in middle-and high-dose group; spermatocytes and sperm cells had no significant structural changes in each dose group. 4 The contents of calcium and copper increased with the dose increasing. The content of copper obviously increased in the middle-and high-dose group, contrast with the control group (P<0.05). On the contrary, the contents of magnesium and zinc decreased with the dose increasing. The contents of magnesium and zinc in high dose group were lower than that in the control group (P<0.05). 5 Sperm count and the rate of sperm activity in treated groups were decreased obviously, contrast with the control group (P<0.01 and P<0.05). The rate of sperm deformity was higher than the control group, the difference was significant statistically (P<0.01 and P<0.05). 6 The rate of success mating and birth rate in high-dose group were lower than the control group (P<0.05). 7 The weight, length, tail length, chest circumference of offspring decreased in treated groups, contrast with the control group (P<0.05, P<0.01 or P<0.001).
The above results clearly indicated that isocarbophos can directly lead to the harm of testicular tissue, decreasion of the counts and layers of spermatoogenic cells, pathological changs of spermatogonia, Sertoli cells and sperm. The damage of testicular tissue resulted in sperm count and the rate of sperm activity decreasing, the rate of sperm abnormality increaseing, thus isocarbophos affected the quality of sperm. Isocarbophos can interfere with sperm production, development and maturity through affecting the contents of calcium, magnesium, copper and zinc in testicular tissue, ultimately affect the breeding capacity of rats and the quality of offspring. In addition, isocarbophos can lead to pathological structural changes of hypothalamus-pituitary-testicular axis, indicated one possible mechanism of isocarbophos affecting reproductive function is that abnormal hormone secretion.
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