Effects of Bisphenol A on Gene Expression and Apoptosis of Leydig Cells in the Mouse Testis

생쥐 정소에서 비스페놀 에이 (Bisphenol A)가 Leydig Cell의 유전자 발현과 세포자멸사에 주는 영향

  • Eo, Jin-Won (Department of Biomedical Science & Technology, Konkuk University) ;
  • Lim, Hyun-Jung (Department of Biomedical Science & Technology, Konkuk University)
  • 어진원 (건국대학교 의생명과학과) ;
  • 임현정 (건국대학교 의생명과학과)
  • Published : 2008.09.30

Abstract

Objective: Environmental chemicals alter reproduction, growth, and survival by changing the normal function of the endocrine system. Bisphenol A (BPA), one of the endocrine disruptors, is known to be an estrogen receptor agonist. Therefore, we hypothesized that BPA may affect male reproduction including spermatogenesis in the mouse testis. Methods: We used 7-week-old ICR mice. The first experiment group received BPA in sesame oil (vehicle, 1 mg/kg, 10 mg/kg, and 100 mg/kg) by i.p. injection and mice were sacrificed 24 hr later. The second experiment group received BPA (vehicle, 10 ${\mu}g/kg$, 1 mg/kg, and 100 mg/kg) daily for 14 days by subcutaneous injection. Expression of cell type-specific marker genes in the testis was evaluated by RT-PCR. Histological analysis, immunofluorescence staining, and TUNEL staining were also performed. Results: RT-PCR analyses showed that expression of luteinizing hormone receptor (LHR), a marker gene for the Leydig cell, was notably decreased in the testes of high dose-exposed mice. No obvious difference in the histology of testes was noted among treatment groups. Immunostaining of LHR in the first experiment group did not show noticeable difference in LHR protein expression in Leydig cells. Immunohistochemistry also revealed heightened expression of the immunoreactive Bax in the treatment group, and this was accompanied by positive TUNEL staining in the interstitial area within testis where Leydig cells reside. Conclusions: Our result suggests that BPA affects Leydig cell functions by altering gene expression and by increasing apoptosis in the mouse testis.

목 적: 본 연구에서는 에스트로겐 수용체의 agonist로 작용할 수 있는 내분비계 교란물질인 bisphenol A (BPA)가 정소 내 세포들의 유전자 발현 및 세포자멸사에 미치는 영향을 관찰하였다. 연구방법: 그룹 I의 7주령 수컷 생쥐들에는 sesame oil에 녹인 BPA를 1 mg/kg, 10 mg/kg, 100 mg/kg를 1회 복강주사하여 급성 영향을 조사하였고, 그룹 II의 생쥐들에는 BPA 10 ${\mu}g/kg$, 1 mg/kg, 100 mg/kg를 하루에 1회씩 14일간 피하주사하여 장기적 효과를 보았다. 생쥐의 정소를 채취하여 정소의 다양한 표지유전자들의 발현을 RT-PCR로 조사하였고, 조직학적 관찰, 형광면역염색법과 TUNEL 염색을 수행하였다. 결 과: RT-PCR 결과, 정소의 표지유전자 중 특이적으로 Leydig cell의 표지유전자인 LHR가 고농도의 BPA 처리군에서 감소되어 있었다. 정원세포 표지유전자인 ERM은 장기간 BPA를 주사 받은 생쥐들의 정소에서 감소되었다. 1회의 BPA를 주사 받은 그룹에서는 조직학적 영향은 없는 것으로 보였다. 또한 세포자멸사의 표지인 Bax가 Leydig cell이 존재하는 정소의 interstitial area에서 BPA에 의해 증가된 것을 관찰하였으며 이는 TUNEL 염색이 positive하게 나타난 부분과 일치하였다. 결 론: 본 연구는 BPA가 정소에서 주로 Leydig cell에 영향을 주며 이는 Leydig cell이 에스트로겐 수용체와 에스트로겐 합성효소를 발현하는 내분비 세포라는 사실에 부합되는 것으로, 차후 그 분자적 기작을 밝히는 연구로 이어져야 할 것이다.

Keywords

References

  1. Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 2005; 308: 1466-9 https://doi.org/10.1126/science.1108190
  2. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. Bisphenol-A: and estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 1993; 132: 2279-86 https://doi.org/10.1210/en.132.6.2279
  3. Steinmetz R, Brown NG, Allen DL, Bigsby RM, Ben-Jonathan N. The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo. Endocrinology 1997; 138: 1780-6 https://doi.org/10.1210/en.138.5.1780
  4. Gould JC, Leonard LS, Maness SC, Wagner BL, Conner K, Zachalewski T, Safe S, McDonnell DP, Gaido KW. Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiol. Mol Cell Endocrinol 1998; 142: 203-14 https://doi.org/10.1016/S0303-7207(98)00084-7
  5. Ashby J, Tinwell H. Uterotrophic activity of bisphenol A in the immature rat. Environ Health Perspect 1998; 106: 719-20 https://doi.org/10.2307/3434259
  6. Bergeron RM, Thompson TB, Leonard LS, Pluta L, Gaido KW. Estrogenicity of bisphenol A in a human endometrial carcinoma cell line. Mol Cell Endocrinol 1999; 150: 179-87 https://doi.org/10.1016/S0303-7207(98)00202-0
  7. Schafer TE, Lapp CA, Hanes CM, Lewis JB, Wataha JC, Schuster GS. Estrogenicity of bisphenol A and bisphenol A dimethacrylate in vitro. J Biomed Mater Res 1999; 45: 192-7 https://doi.org/10.1002/(SICI)1097-4636(19990605)45:3<192::AID-JBM5>3.0.CO;2-A
  8. Papaconstantinou AD, Umbreit TH, Fisher BR, Goering PL, Lappas NT, Brown KM. Bisphenol-A induced increase in uterine weight and alterations in uterine morphology in ovariectomized B6C3F1 mice: role of the estrogen receptor. Toxicol Sci 2000; 56: 332-9 https://doi.org/10.1093/toxsci/56.2.332
  9. Palmer BD, Palmer SK. Vitellogenin induction by xenobiotic estrogens in the red-eared turtle and African clawed frog. Environ. Health Perspect 1995; 103(Suppl.4): 19-25
  10. Anderson MJ, Olsen H, Matsumura F, Hinton DE. In vitro modulation of $17{\beta}$-estradiol-induced vitellogenin synthesis and estrogen receptor in rainbow trout (Oncorhynchus mykiss) liver cells. Aquatic Toxicol 1996; 137(2): 210-8
  11. Crews D, Bergeron JM, McLachlan JA. The role of estrogen in turtle sex determination and the effect of PCBs. Environ Health Perspect 1995; 103(Suppl.7): 73-7 https://doi.org/10.1289/ehp.95103s773
  12. Egeland GM, Sweeney MH, Fingerhut MA, Halperin WE, Wille KK, Schnorr TM. Serum dioxin 2,3,7,8 Tetrachlorodibenzop-dioxin 2,3,7,8-Tcdd and total serum testosterone and gonadotropins in occupationally exposed men. Am J Epidem 1992; 136: 1014
  13. Egeland GM, Sweeney MH, Fingerhut MA, Wille KK, Schnorr TM, Halperin WE. Total serum testosterone and gonadotropins in workers exposed to dioxins. Am J Epidem 1994; 139(3): 272-81 https://doi.org/10.1093/oxfordjournals.aje.a116994
  14. Haishima Y, Hayashi Y, Yagami T, Nakamura A. Elution of bisphenol-A from hemodialyzers consisting of polycarbonate and polysulfone resins. J Biomed Mater Res 2001; 58: 209-15 https://doi.org/10.1002/1097-4636(2001)58:2<209::AID-JBM1009>3.0.CO;2-7
  15. Hiroi H, Tsutsumi O, Momoeda M, Takai Y, Osuga Y, Taketani Y. Differential interactions of bisphenol A and 17 beta-estradiol with estrogen receptor alpha (ER alpha) and ER beta. Endocr J 1999; 46(6): 773-8 https://doi.org/10.1507/endocrj.46.773
  16. Funabashi T, Sano A, Mitsushima D, Kimura F. Bisphenol A increases progesterone receptor immunoreactivity in the hypothalamus in a dose-dependent manner and affects sexual behaviour in adult ovariectomized rats. J Neuroendocrinol 2003; 15: 134-40 https://doi.org/10.1046/j.1365-2826.2003.00872.x
  17. Ashby J, Tinwell H, Haseman J. Lack of effects for low lose levels of bisphenol A and diethylstilbestrol on the prostate gland of CF1 mice exposed in utero. Regul Toxicol Pharmacol 1999; 30: 156-66 https://doi.org/10.1006/rtph.1999.1317
  18. Cargen SZ, Waechter JM Jr, Dimond SS, Breslin WJ, Butala JH, Jekat FW, Joiner RL, Shiotsuka RN, Veenstra GE, Harris LR. Normal reproductive organ development in Wistar rats exposed to bisphenol A in the drinking water. Regul Toxicol Pharmacol 1999; 50: 36-44
  19. Nagao T, Saito Y, Usumi K, Kuwagata M, Imai K. Reproductive function in rats exposed neonatally to bisphenol A and estradiol benzoate. Reprod Toxicol 1999; 13(4): 303-11 https://doi.org/10.1016/S0890-6238(99)00017-9
  20. Nagao T, Saito Y, Usumi K, Yoshimura S, Ono H. Low dose bisphenol A does not affect reproductive organs in estrogen-sensitive C57BL/6N mice exposed at the sexually mature, juvenile, or embryonic stage. Reprod Toxicol 2002; 16: 123-30 https://doi.org/10.1016/S0890-6238(02)00003-5
  21. Atanassova N, McKinnell C, Turner KJ, Walker M, Fisher JS, Morley M, Millar MR, Groome NP, Sharpe RM. Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels. Endocrinology 2000; 141: 3898-907 https://doi.org/10.1210/en.141.10.3898
  22. Kloas W, Lutz I, Einspanier R. Amphibians as a model to study endocrine disruptors: II Estrogenic activity of environmental chemicals in vitro and in vivo. The Sci of the Total Env 1999; 225(1-2): 59-68 https://doi.org/10.1016/S0048-9697(99)80017-5
  23. Welshons WV, Nagel SC, Thayer KA, Judy BM, Vom Saal FS. Low-dose bioactivity of xenoestrogens in animals: fetal exposure to low doses of methoxychlor and other xenoestrogens increases adult prostate size in mice. Toxicol Ind Health 1999; 15(1-2): 12-25 https://doi.org/10.1177/074823379901500103
  24. vom Saal FS, Cooke PS, Buchannan DL, Palanza P, Thayer KA, Nagel SC, Parmigiani S, Welshons WV. A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behaviour. Toxicol Ind Health 1998; 14: 239-60 https://doi.org/10.1177/074823379801400115
  25. Steinmetz R, Brown NG, Allen DL, Bigsby RM, Ben-Jonathan N. The environmental estrogen bisphenol A stimulates prolactin release in vitro and vivo. Endocrinology 1997; 138: 1780-6 https://doi.org/10.1210/en.138.5.1780
  26. Delbes G, Levacher C, Duguemec C, Racine C, Pakarinen P, Habert R. Endogenous Estrogens Inhibit Mouse Fetal Leydig Cell Development via Estrogen Receptor ${\alpha}$. Endocrinology 2005; 146(5): 2454-61 https://doi.org/10.1210/en.2004-1540
  27. Oulad-Abdelqhani M, Bouillet P, Decimo D, Gansmuller A, Heyberger S, Dolle P, Bronner S, Lutz Y, Chambon P. Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-reponsive gene. J Biol Chem 1996; 135(2): 469-77
  28. Schrans-Stassen BH, Saunders PT, Cooke HJ, de Rooij DG. Nature of the spermatogenic arrest in Dazl-/- Mice. Biol Reprod 2001; 65(3): 771-6 https://doi.org/10.1095/biolreprod65.3.771
  29. Venables JP, Ruggiu M, Cooke HJ. The RNA-binding specificity of the mouse Dazl protein. Nucleic Acids Research 2001; 29(12): 2479-83 https://doi.org/10.1093/nar/29.12.2479
  30. Lee J, Hong J, Kim E, Kim K, Kim SW, Krishnamurthy H, et al. Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis. Mol Hum Reprod 2004; 10(4): 259-64 https://doi.org/10.1093/molehr/gah037
  31. Chen C, Ouyang W, Griqura V, Zhou Q, Carnes K, Lim H, et al. ERM is required for transcriptional control of the spermatogonial stem cell niche. Nature 2005; 436: 1030-4 https://doi.org/10.1038/nature03894
  32. Balvers M, Spiess AN, Domagalski R, Hunt N, Kinic E, Mukhopadhyay AK, Hanks E, Charlton HM, Ivell R. Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary. Endocrinology 2006; 139: 2960-70 https://doi.org/10.1210/en.139.6.2960
  33. vom Saal FS, Hughes C. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environ Health Perspect 2005; 113(8): 926-33 https://doi.org/10.1289/ehp.7713
  34. O'Donnell L, Robertson KM, Jones ME, Simpson ER. Estrogen and spermatogenesis. Endocr Rev 2001; 22(3): 289-318 https://doi.org/10.1210/er.22.3.289
  35. Song KH, Lee K, Choi HS. Endocrine disrupter bisphenol a induces orphan nuclear receptor Nur77 gene expression and steroidogenesis in mouse testicular Leydig cells. Endocrinology 2002; 143(6): 2208-15 https://doi.org/10.1210/en.143.6.2208
  36. Nair R, Shaha C. Diethylstilbestrol induces rat spematogenic cell apoptosis in vivo through increased expression of spermatogenic cell Fas/FasL system. J Biol Chem 2003; 278(8); 6470-81 https://doi.org/10.1074/jbc.M209319200
  37. Fritz WA, Cotroneo MS, Wang J, Eltoum IE, Lamartiniere CA. Dietary diethylstilbestrol but not genistein adversely affects rat testicular development. J Nutr 2003; 133(7): 2287-93
  38. Zha H, Reed JC. Heterodimerization-independent Functions of Cell Death Regulatory Proteins Bax and Bcl-2 in Yeast and Mammalian Cells. J Biol Chem 1997; 272(50): 31482-8 https://doi.org/10.1074/jbc.272.50.31482
  39. Xu J, Osuga Y, Yano T, Morita Y, Tang X, Fujiwara T, Takai Y, Matsumi H, Koga K, Taketani Y, Tsutsumi O. Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells. Biochem Biophys Res Commun 2002; 292(2): 456-62 https://doi.org/10.1006/bbrc.2002.6644
  40. Iida H, Maehara K, Doiguchi M, Mori T, Tamada F. Bisphenol A-induced apoptosis of cultured rat Sertoli cells. Reprod Toxicol 2003; 17(4): 457-64 https://doi.org/10.1016/S0890-6238(03)00034-0