Actions of the endocrine disrupter methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth
Actions of the endocrine disrupter methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth
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DOI：
10.1016/s0890-6238(01)00124-1
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发表时间：
2001-05-01
影响因子：
3.3
通讯作者：
Skinner, MK
中科院分区：
文献类型：
--
作者：
Cupp, AS;Skinner, MK
本研究探讨了甲氧氯胺及其雌激素代谢产物2，2-二(对羟基苯基)-1，1，1-三氯乙烷(HPTE)对发育中的大鼠生精索形成和生长的影响。胚胎和出生后早期的发育中的睾丸可能对激素活性物质比发育后期更敏感。胚胎第13天(E13)的睾丸器官培养用0.2、2或20um甲氧氯胺或1.3、6、15、30或60um I HPTE处理，以检测对脐带形成的影响。NO浓度可完全抑制脐带的形成。然而，在甲氧氯胺浓度为7和20um时，脐带形成异常，脐带数量减少，出现“肿胀”的脐带。脊髓肿胀是由于脊髓横截面内细胞数量的增加和脊髓间质细胞数量的减少所致。用HPTE处理胚胎第13天(E13)的睾丸，在3um和6um的浓度下引起异常的脐带形成，在15、30和60um的浓度下完全抑制脐带的形成。除了雌激素代谢物HTPE外，甲氧氯胺还可以被代谢成抗雄激素化合物。因此。目的：测定甲氧氯胺对睾丸发育的潜在作用范围。不同浓度的雌二醇、睾酮和一种抗雄激素(氟他胺)被用来确定它们对E13睾丸器官培养形态的影响。雌二醇(1um)和氟他胺(0.1um)均能抑制E13睾丸器官培养中生精索的形成。因此。甲氧氯胺可能通过雄激素和/或雌激素受体作用于生精索的形成。逆转录-聚合酶链式反应(RT-PCR)证实，从胚胎第14天(E14)到出生后第5天(P5)，雌激素受体α(ERα)mRNA的存在，而雌激素受体β(ERβ)的mRNA直到胚胎发育到16天才出现。雄激素受体(AR)的表达在睾丸发育的E14至P5期间存在，但在E14和E16的表达水平明显降低。免疫组织化学分析显示Era定位于E14~P5的生精索细胞，而E16和P0的间质细胞中有ERP的表达。雄激素受体定位于生殖细胞和间质细胞。甲氧氯胺的影响。在出生后第0天(PO)的睾丸细胞培养中，用胸腺嘧啶核苷掺入法检测HPTE、雌二醇和睾酮对围产期睾丸细胞生长的影响。甲氧氯胺(0.002，0.02.和0.2um)和HPTE(2和20um)刺激PO睾丸细胞胸腺嘧啶核苷掺入的方式与雌二醇(0.01、0.1和1um)相似。此外，睾酮(0.1um)也能刺激PO睾丸细胞的胸腺嘧啶核苷掺入。观察表明，甲氧氯胺及其代谢物HPTE可以改变正常胚胎睾丸的发育和生长。甲氧氯胺和HPTE的作用可能部分是通过已证实存在于发育中的睾丸的类固醇受体来调节的。(C)2001 Elsevier Science Inc.保留所有权利。
The current study examines the actions of methoxychlor and its estrogenic metabolite, 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on seminiferous cord formation and growth of the developing rat testis. The developing testis in the embryonic and early postnatal period is likely more sensitive to hormonally active agents than at later stages of development. Embryonic day 13 (E13) testis organ cultures were treated with either 0.2, 2, or 20 muM methoxychlor or 1. 3, 6, 15, 30, or 60 muM I HPTE to examine effects on cord formation. No concentration of methoxychlor completely inhibited cord formation. However, cord formation was abnormal with the presence of a reduced number of cords and appearance of "swollen" cords at the 7 and 20 muM concentrations of methoxychlor. The swollen cords were due to an increase in the number of cells in a cord cross section and reduction of interstitial cell numbers between cords. Treatment of embryonic day 13 (E13) testes with HPTE caused abnormal cord formation at the 3 muM and 6 muM concentrations, and completely inhibited cord formation at the 15, 30, and 60 muM concentrations. In addition to the estrogenic metabolite HTPE, methoxychlor can also be metabolized into anti-androgenic compounds. Therefore. to determine the spectrum of potential actions of methoxychlor on testis development. different concentrations of estradiol, testosterone, and an anti-androgen (flutamide) were utilized to determine their effects on E13 testis organ culture morphology. Estradiol (1 muM) and flutamide (0.1 muM) both inhibited seminiferous cord formation in E13 testis organ cultures. Therefore. methoxychlor may be acting through the androgen and/or estrogen receptors to elicit its actions on seminiferous cord formation. Reverse transcription polymerase chain reaction (PCR) (RT-PCR) confirmed the presence of estrogen receptor alpha (ER alpha) mRNA from embryonic day 14 (E14) through postnatal day 5 (P5) while estrogen receptor beta (ER beta) mRNA did not appear until approximately E16 of trstis development. Androgen receptor (AR) expression was present from E14 through P5 of testis development, but at apparently reduced levels at E14 and E16. Immunohistochemical analysis localized ERa to the cells of the seminiferous cords at E14 though P5 while ERP was present in cells of the interstitium at E16 and PO. Androgen receptor was localized to germ and interstitial cells. The effects of methoxychlor. HPTE, estradiol, and testosterone on cell growth of perinatal testes was determined with a thymidine incorporation assay in postnatal day zero (PO) testis cell cultures. Methoxychlor (0.002, 0.02. and 0.2 muM) and HPTE (2 and 20 muM) stimulated thymidine incorporation in PO testis cell cultures in a similar manner to estradiol (0.01, 0.1, and 1 muM). In addition, testosterone (0.1 muM) also stimulated thymidine incorporation in PO testis cultures. Observations suggest that methoxychlor and its metabolite HPTE can alter normal embryonic testis development and growth. The actions of methoxychlor and HPTE are likely mediated in part through the steroid receptors confirmed to be present in the developing testis. (C) 2001 Elsevier Science Inc. All rights reserved.