孕期DEHP暴露對F1代雄性小鼠睪丸早期發(fā)育的影響及機(jī)制研究
發(fā)布時間：2019-06-05 08:25
【摘要】：鄰苯二甲酸二(2-乙基-己基)酯(DEHP)作為環(huán)境中豐富存在的內(nèi)分泌干擾物,易于遷入環(huán)境,不易被降解,已受到人們的普遍重視�，F(xiàn)有實驗證明DEHP會造成神經(jīng)、甲狀腺、腎臟、心臟、生殖發(fā)育等多方面毒性,與雌性相比雄性生殖系統(tǒng)對DEHP更為敏感。目前為止,已證明DEHP暴露可以造成雄性個體附睪、睪丸、前列腺的濕重顯著降低,肛殖距顯著減小,并能引發(fā)隱睪等多種生殖損傷。其具體損傷機(jī)制包括影響雄鼠睪丸DNA甲基化狀態(tài)、誘導(dǎo)生精細(xì)胞發(fā)生凋亡、抑制生精細(xì)胞增殖、影響雄激素的合成、誘導(dǎo)生精細(xì)胞產(chǎn)生DNA損傷等。本實驗在ICR小鼠懷孕的第9.5天到第15.5天通過灌胃進(jìn)行不同濃度的DEHP暴露(75mg/kg/d,225mg/kg/d,675mg/kg/d),分別在F1代雄性小鼠胚胎期第18.5天以及出生后14、28、35天取材,應(yīng)用HE染色和透射電鏡技術(shù),在組織學(xué)水平和超微結(jié)構(gòu)水平研究孕期DEHP暴露對F1代雄性胎鼠和青春期小鼠造成的睪丸病理損傷;并進(jìn)一步應(yīng)用Western blot、TUNEL檢測、免疫組織化學(xué)檢測等實驗方法,從內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)失衡與內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡、細(xì)胞增殖和睪酮合成及代謝等方面探究DEHP造成雄性生殖損傷的具體作用機(jī)制。結(jié)果發(fā)現(xiàn)孕期DEHP暴露后造成:1、胚胎期生精小管數(shù)目顯著減少,出生后生精小管管間和生精細(xì)胞間間隙增大、空泡化、斷層、染色異常和脫落的細(xì)胞數(shù)目增加、生精細(xì)胞排列紊亂。2、睪丸生精細(xì)胞內(nèi)質(zhì)網(wǎng)明顯腫脹;內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白GRP78相對表達(dá)量在出生后14d和28d顯著下調(diào),35d顯著上調(diào)。3、內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)細(xì)胞凋亡的標(biāo)志分子Cleaved Caspase 12的蛋白相對表達(dá)量,在出生后28d和35d顯著上調(diào);出生后35d睪丸細(xì)胞凋亡數(shù)目明顯增加;睪丸中PCNA表達(dá)陽性的生精小管比例顯著下調(diào)。4、間質(zhì)細(xì)胞內(nèi)脂滴數(shù)明顯減少;部分線粒體形態(tài)異常;STARD10的蛋白相對表達(dá)量,在胚胎18.5d 675mg/kg/d劑量組顯著下調(diào);雄激素結(jié)合蛋白ABP1的表達(dá)量明顯下降。綜上所述,孕期DEHP暴露對發(fā)育早期的F1代雄性小鼠睪丸會造成比較明顯的生殖損傷,可能是通過以下途徑造成的:(1)改變內(nèi)質(zhì)網(wǎng)的形態(tài)結(jié)構(gòu),擾亂生精細(xì)胞內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài),引發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激,并通過內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)細(xì)胞凋亡;(2)抑制生精細(xì)胞增殖;(3)影響間質(zhì)細(xì)胞內(nèi)睪酮合成,降低雄激素在體內(nèi)的有效濃度等。本研究初步探究了孕期DEHP暴露對F1代雄性小鼠睪丸早期發(fā)育的損傷及損傷機(jī)制,并首次以小鼠為動物模型將內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)失衡、內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的生精細(xì)胞凋亡作為DEHP誘導(dǎo)雄性生殖損傷的具體機(jī)制之一,為進(jìn)一步了解人類孕期DEHP暴露與男性后代生殖系統(tǒng)損傷及生殖障礙的關(guān)系提供理論基礎(chǔ)。
[Abstract]:Bis (2-ethyl-hexyl) phthalate (DEHP), as a rich endocrine disruptor in the environment, is easy to move into the environment and is not easy to be degraded. Existing experiments have shown that DEHP can cause nerve, thyroid, kidney, heart, reproductive development and other toxicity, and the male reproductive system is more sensitive to DEHP than female. So far, it has been proved that DEHP exposure can significantly reduce the wet weight of epididymis, testes and prostate, reduce the anal distance, and cause a variety of reproductive injury such as cryptorchidymis. The specific injury mechanism includes affecting the DNA methylation status of male rat testes, inducing apoptosis of spermatogenic cells, inhibiting the proliferation of spermatogenic cells, affecting the synthesis of androgen, inducing DNA damage of spermatogenic cells, and so on. Different concentrations of DEHP (75mg kg / d, 225mg kg / d, 675mg kg / d) were exposed to different concentrations of DEHP (75mg kg / d, 225mg kg / d, 675mg kg / kg / The samples of F1 generation male mice were collected on the 18.5 day of embryo stage and 14, 28 and 35 days after birth, respectively. HE staining and transmission electron microscope were used. The pathological damage of testes induced by DEHP exposure during pregnancy to F1 generation male fetal mice and adolescent mice was studied at the level of histology and ultrastructure. Furthermore, the steady state imbalance of endoplasmic reticulum and the apoptosis mediated by endoplasmic reticulum stress were further studied by Western blot,TUNEL detection and immunohistochemical detection, and other experimental methods were used to detect the steady state of endoplasmic reticulum and apoptosis mediated by endoplasmic reticulum stress. Cell proliferation, testosterone synthesis and metabolism were used to explore the specific mechanism of male reproductive damage induced by DEHP. The results showed that after DEHP exposure during pregnancy, the number of spermatogenic tubules decreased significantly, the space between seminal tubules and spermatogenic cells increased, vacuolation, tomography, abnormal staining and the number of exfoliated cells increased. The arrangement of spermatogenic cells was disordered. 2, the endoplasmic reticulum of spermatogenic cells in testes was obviously swollen. The relative expression of endoplasmic reticulum stress-related protein GRP78 was significantly down-regulated on the 14th and 28th day after birth, and up-regulated on the 35th day after birth. 3. The relative expression of Cleaved Caspase-12, a marker of apoptosis mediated by endoplasmic reticulum stress, was significantly up-regulated on the 28th and 35th day after birth. On the 35th day after birth, the number of apoptosis of testicular cells was significantly increased, the proportion of spermatogenic tubules with positive expression of PCNA in testes was significantly down-regulated. 4, the number of lipid droplets in Leydig cells was significantly decreased, and some of the mitochondria were abnormal. The relative expression of STARD10 protein was significantly down-regulated and the expression of androgen-binding protein ABP1 was significantly decreased in 18.5 days of embryo 675mg/kg/d dose group. To sum up, DEHP exposure during pregnancy can cause obvious reproductive damage to the testes of F1 male mice in the early stage of development, which may be caused by the following ways: (1) changing the morphology and structure of endoplasmic reticulum and disturbing the homeostasis of endoplasmic reticulum in spermatogenic cells. Endoplasmic reticulum stress was induced and apoptosis was mediated by endoplasmic reticulum stress. (2) inhibit the proliferation of spermatogenic cells, (3) affect testosterone synthesis in Leydig cells, reduce the effective concentration of androgen in vivo and so on. In this study, the damage and mechanism of early testicular development in F1 male mice induced by DEHP exposure during pregnancy were investigated, and the steady state imbalance of endoplasmic reticulum in mice was used as an animal model for the first time. Endoplasmic reticulum stress-mediated spermatogenic cell apoptosis is one of the specific mechanisms of male reproductive injury induced by DEHP, which provides a theoretical basis for further understanding the relationship between DEHP exposure during pregnancy and reproductive system injury and reproductive disorder in male offspring.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114
本文編號：2493384
[Abstract]:Bis (2-ethyl-hexyl) phthalate (DEHP), as a rich endocrine disruptor in the environment, is easy to move into the environment and is not easy to be degraded. Existing experiments have shown that DEHP can cause nerve, thyroid, kidney, heart, reproductive development and other toxicity, and the male reproductive system is more sensitive to DEHP than female. So far, it has been proved that DEHP exposure can significantly reduce the wet weight of epididymis, testes and prostate, reduce the anal distance, and cause a variety of reproductive injury such as cryptorchidymis. The specific injury mechanism includes affecting the DNA methylation status of male rat testes, inducing apoptosis of spermatogenic cells, inhibiting the proliferation of spermatogenic cells, affecting the synthesis of androgen, inducing DNA damage of spermatogenic cells, and so on. Different concentrations of DEHP (75mg kg / d, 225mg kg / d, 675mg kg / d) were exposed to different concentrations of DEHP (75mg kg / d, 225mg kg / d, 675mg kg / kg / The samples of F1 generation male mice were collected on the 18.5 day of embryo stage and 14, 28 and 35 days after birth, respectively. HE staining and transmission electron microscope were used. The pathological damage of testes induced by DEHP exposure during pregnancy to F1 generation male fetal mice and adolescent mice was studied at the level of histology and ultrastructure. Furthermore, the steady state imbalance of endoplasmic reticulum and the apoptosis mediated by endoplasmic reticulum stress were further studied by Western blot,TUNEL detection and immunohistochemical detection, and other experimental methods were used to detect the steady state of endoplasmic reticulum and apoptosis mediated by endoplasmic reticulum stress. Cell proliferation, testosterone synthesis and metabolism were used to explore the specific mechanism of male reproductive damage induced by DEHP. The results showed that after DEHP exposure during pregnancy, the number of spermatogenic tubules decreased significantly, the space between seminal tubules and spermatogenic cells increased, vacuolation, tomography, abnormal staining and the number of exfoliated cells increased. The arrangement of spermatogenic cells was disordered. 2, the endoplasmic reticulum of spermatogenic cells in testes was obviously swollen. The relative expression of endoplasmic reticulum stress-related protein GRP78 was significantly down-regulated on the 14th and 28th day after birth, and up-regulated on the 35th day after birth. 3. The relative expression of Cleaved Caspase-12, a marker of apoptosis mediated by endoplasmic reticulum stress, was significantly up-regulated on the 28th and 35th day after birth. On the 35th day after birth, the number of apoptosis of testicular cells was significantly increased, the proportion of spermatogenic tubules with positive expression of PCNA in testes was significantly down-regulated. 4, the number of lipid droplets in Leydig cells was significantly decreased, and some of the mitochondria were abnormal. The relative expression of STARD10 protein was significantly down-regulated and the expression of androgen-binding protein ABP1 was significantly decreased in 18.5 days of embryo 675mg/kg/d dose group. To sum up, DEHP exposure during pregnancy can cause obvious reproductive damage to the testes of F1 male mice in the early stage of development, which may be caused by the following ways: (1) changing the morphology and structure of endoplasmic reticulum and disturbing the homeostasis of endoplasmic reticulum in spermatogenic cells. Endoplasmic reticulum stress was induced and apoptosis was mediated by endoplasmic reticulum stress. (2) inhibit the proliferation of spermatogenic cells, (3) affect testosterone synthesis in Leydig cells, reduce the effective concentration of androgen in vivo and so on. In this study, the damage and mechanism of early testicular development in F1 male mice induced by DEHP exposure during pregnancy were investigated, and the steady state imbalance of endoplasmic reticulum in mice was used as an animal model for the first time. Endoplasmic reticulum stress-mediated spermatogenic cell apoptosis is one of the specific mechanisms of male reproductive injury induced by DEHP, which provides a theoretical basis for further understanding the relationship between DEHP exposure during pregnancy and reproductive system injury and reproductive disorder in male offspring.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114
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