| Environmental endocrine disruptors are of persistent toxicity, latent hazard and bioaccumulation. They have become a common concern as their effect can be amplified through biological concentration and harm human health directly or indirectly. As a common environmental endocrine disruptor, DEHP is widely used in food packages, containers, medical supplies and children’s toys. It can cause diseases such as infertility, sexual precocity and uterine bleeding and thus arouse concerns from the society and scholars. Purpose:To explore the effect of DEHP on the hypothalamus-pituitary-ovarian axis in pubertal female rats, reveal the reproductive toxicity of DEHP on pubertal female rats and its mechanism, and provide scientific evidence for the evaluation of toxicity and toxic mechanism of DEHP on reproductive system. Methods:Female Wistar rats(21 days old, 60 ± 10 g) were purchased from the Experimental Animal Center of Jilin University. They were allowed at least 7-day acclimatization-period and observed for signs of illness before starting the experimental procedures, and their body weight was measured every day. The 48 rats were randomly apportioned into 4 groups(n = 12, each). The experiment lasted for approximately 4 weeks including the period of adaptation to the feeding regime. Rats were administered via oral gavage 0, 250, 500, or 1000 mg/kg/d DEHP(Sinopharm Chemical Reagent, purity>99%) in 0.1 m L corn oil/20 g body weight for up to 4 weeks.The rats’ body weights, food and water consumption and vaginal opening time were recorded every day. After 4 weeks, the rats were weighed and killed by decapitation on the anestrus. Blood was collected from the eyeballs and the serum was separated. The hypothalamus, pituitary, ovary and uterus of each rat were also dissected, weighed, and collected immediately after decapitation. The organ coefficients were counted. The histopathological changes of the hypothalamus, pituitary, ovary and uterus of the pubertal female rats were observed by HE staining. The level of Gn RH in the hypothalamus was quantified by using enzyme-linked immunoabsorbent assay method. The serum hormone levels were quantified by radioimmunoassay. Real-time reverse transcription-PCR was used to verify the expression of m RNA of Gn RH, Kiss-1 and GPR54 in the hypothalamus-pituitaryovarian axis. The location of Gn RH, Kiss-1 and GPR54 in the hypothalamuspituitary-ovarian axis was observed by immunohistochemical staining. The quantitative expression of proteins related to the hypothalamus- pituitary- ovarian axis was detected by Western blot. Results:1. The general toxicity and reproductive toxicity of DEHP in pubertal female rats(1) The rats treated with DEHP appeared less active and less spirited, moved more slowly, had untidy and lusterless fur and lost hair. These changes were most obvious in the group of 1000 mg/kg/d DEHP.(2) Compared with the control group, the food consumption and body weight of rats treated with DEHP were significantly higher(P<0.05), while their water consumption significantly decreased(P<0.05).(3) Compared with the control group, the vaginal opening time of rats treated with DEHP was significantly shorter(P<0.05), while the time of anestrus and estrous cycle was significantly longer(P<0.05).(4) The ovaries of the rats treated with DEHP were congestive and swelling. And the volume of the ovaries and uterus became bigger.(5) Compared with the control group, the coefficient of the ovary of the rats treated with DEHP was significantly higher(P<0.05).2. Effects of DEHP on histopathological changes of the hypothalamus-pituitary-ovarian axis in pubertal female rats(1) For both the exposure groups and the control group, the glial cells, neurons and nerve fibers were normal. The bodies of neuronal cells were polygonal, the nuclei were big, round and at the center of the cell, and the nucleoli were big, round and hyperchromatic. The cytoplasm was full of small dark purple particles or granular Nissl bodies. The bodies and nuclei of glial cells were big. The bodies of oligodendrocyte were small and round or oval-shaped.(2) For both the exposure groups and the control group, there were osinophils, basophils, chromophobe cells and capillaries in pars distalis of the pituitary.(3) For the exposure groups, the ovarian granulosa cells were of loose structure, decreased number and unclear outline and the nuclei were of different sizes. Some of the ovarian granulosa cells presented changes such as karyopyknosis and karyorrhexis.(4) For the exposure groups, the endometrium cells presented phenomena such as pseudostratified nuclei, epithelial hyperplasia and endothelial fibrosis. The luminal epithelial cells were loose and irregular and the nuclei were of different sizes. The glands in the lamina propria atrophied, and the number was reduced.3. Effects of DEHP on hormone levels of the hypothalamus-pituitary-ovarian axis in pubertal female rats(1) Compared with the control group, the Gn RH level in the hypothalamus was significantly higher in the exposure groups(P<0.05). And the Gn RH level rose with the increase of dose of DEHP, which indicated a dose-response relationship.(2) Compared with the control group, the levels of FSH, LH and T were significantly lower in rats treated with DEHP(P < 0.05), the P level was significantly higher in rats treated with DEHP(P < 0.05), and there was no significant difference in the level of E2(P>0.05).4. Effects of DEHP on the gene expression levels of the hypothalamus-pituitary-ovarian axis in pubertal female rats(1) Compared with the control group, there were no significant difference in the m RNA levels of Gn RH and GPR54 in the hypothalamus of the rats treated with DEHP(P>0.05), and the level of Kiss-1 was significantly lower in the rats treated with DEHP(P<0.05).(2) Compared with the control group, the m RNA level of Gn RHR in the pituitary was significantly lower in rats treated with 250 mg/kg/d DEHP(P<0.05). The level in the pituitary was significantly higher in the rats treated with 1000 mg/kg/d DEHP compared with the rats in all other groups(P<0.05). Compared with the control group, the m RNA levels of FSH and LH in the pituitary were significantly lower in the rats treated with DEHP(P<0.05).(3) Compared with the control group, the m RNA levels of FSHR and LHR in the ovary were significantly lower in the rats treated with DEHP(P<0.05), while the level of ERα was significantly higher in those treated with DEHP(P<0.05).(4) Compared with the control group, the m RNA levels of Gn RHR and ERα in the uterus were significantly higher in the rats treated with DEHP(P < 0.05).5. Effects of DEHP on the protein expression levels in the hypothalamus-pituitary-ovarian axis in pubertal female rats(1) Compared with the control group, the protein levels of Gn RH, Kiss-1 and GPR54 in the hypothalamus were significantly higher in the rats treated with DEHP(P<0.05).(2) Compared with the control group, the protein level of Gn RHR in the pituitary was significantly higher in rats treated with 250 mg/kg/d DEHP(P<0.05), while that was significantly lower after treatment with 1000 mg/kg/d DEHP. The level of Gn RHR was significantly lower in the rats treated with 500 mg/kg/d DEHP compared with the control group(P<0.05). The levels of FSH and LH were in the pituitary were significantly lower in the rats treated with DEHP compared with the control group(P<0.05).(3) Compared with the control group, the protein levels of FSHR and ERα in the ovary were significantly lower in the rats treated with DEHP(P<0.05), and there was no significant difference in the level of LHR(P>0.05).(4) Compared with the control rats, the protein level of Gn RHR in the uterus was significantly lower in the rats treated with 250 and 500 mg/kg/d DEHP(P<0.05). The level of Gn RHR was significantly higher in the rats treated with 1000 mg/kg/d DEHP compared with the rats in all other groups(P<0.05). Compared with the control rats and the rats treated with 250 mg/kg/d DEHP, the protein level of ERα in the uterus was significantly lower in the rats treated with 500 mg/kg/d DEHP(P<0.05). The level of ERα was significantly higher in the rats treated with 1000 mg/kg/d DEHP compared with the rats in all other groups(P<0.05). Conclusion:1. DEHP exposure could increase the food consumption and body weight, shorten the time of vaginal opening, and prolong the estrous cycle of pubertal female rats.2. DEHP exposure could increase the organ coefficient of the ovary organ pubertal female rats, cause the ovary to become congestive and swelling and bigger in volume, and result in the damage of ovarian granulosa cells to different extent. DEHP could cause the uterus of pubertal female rats to become swelling and bigger in volume, and result in the damage of endometrium to different extent. These suggest that ovary and uterus might be the main target organs of DEHP.3. DEHP exposure could increase the level of Gn RH in the hypothalamus of pubertal female rats and the level rises with the increase of the dose of DEHP exposure, which indicates a dose-response relationship. DEHP exposure could also cause the decrease of the levels of FSH, LH and T and the increase of the level of P. These suggest that DEHP could exert the effect of endocrine disruption on the hypothalamus-pituitary-ovarian axis in pubertal female rats.4. DEHP exposure could affect the m RNA and protein expression levels of Gn RH, Kiss-1, GPR54, Gn RHR, FSH, LH, FSHR, LHR and ERα in the hypothalamus-pituitary-ovarian axis in pubertal female rats, interfering with the endocrine regulation of the hypothalamus-pituitary-ovarian axis and affecting the development and reproductive function of the gonad.5. DEHP exposure may increase the m RNA and protein expression levels of Gn RHR in the uterus of pubertal female rats, suggesting that DEHP may interfere with the reproductive function by affecting the expression level of Gn RHR in the uterus.In summary, DEHP may have reproductive toxicity on pubertal female rats. It may affect the synthesis and secretion of sex hormones, damage the gonad and reproductive endocrine of female rats and produce female reproductive toxicity through disrupting the endocrine regulation of the hypothalamus-pituitary-ovarian axis. |
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