Morphological Changes of Mouse Ovary by X-Ray Irradiation

방사선 조사선량에 따른 생쥐 난소의 형태학적 변화

  • Yoon, Chul-Ho (Department of Radiologic Technology, Dongnam Health College) ;
  • Choi, Jong-Woon (Department of Radiologic Technology, Daejeon Health Science College) ;
  • Yoon, Surk-Hwan (Department of Radiologic Technology, Dongnam Health College)
  • 윤철호 (동남보건대학 방사선과) ;
  • 최종운 (대전보건대학 방사선과) ;
  • 윤석환 (동남보건대학 방사선과)
  • Published : 2007.12.30

Abstract

This research was performed to investigate the morphological changes of folliculus ovary according to the radiation dose. The whole body radiation of 200 cGy, 400 cGy, and 600 cGy was given to the each groups of 5 months-aged female mouse. Various staining methods used in this research are: Hematosylin-Eosin method, and immunohistochemistrical methods using BrdU, TUNEL, p53, p21, PCNA and inhibin. The minute structural changes of folliculus ovary were observed through an electron microscope with high magnification. The morphological changes of growing folliculus ovary became distinct as the dose of X-rays increased. Especially, the nuclei of granular cells showed manifest condensation and the changes of the transparent zone were distinct. As a result of histochemical reaction according to Masson's trichrome method and reticular fiber method, the changed granular cells, the deformed basilar membrane of folliculus ovary and the abnormal arrangement of the reticular fiber were observed. In the reaction of BrdU, the granular cells of normal folliculus ovary with positive reaction rapidly decreased according to the increase of the dose of X-rays. In TUNEL study, granular cells showing positive reaction in retarded folliculus ovary were expanded to growing folliculus ovary and primordial folliculus ovary according to the increase of the dose of X-rays. In case of 600 cGy of X-rays, oocyte underwent apoptosis. In p53 immunohistochemistry, p53 manifested to be stronger as the dose of X-rays increased. p53 reactivity was manifested distinctively in all cells comprising folliculus ovary following irradiation of 600 cGy. p21 was manifested in granular cells of folliculus ovary and showed very positive reaction around follicular antrum according to the increase of the dose of X-rays. In PCNA, positive reaction was manifested in growing folliculus ovary, mature folliculus ovary and primordial folliculus ovary, but the extent of the reaction decreased as the dose of the X-rays decreased. The finding that the reaction of granular cells around folliculus ovary was stronger than that near follicular membrane indicates that what was damaged first by X-ray was the cells near folliculus ovary and follicular antrum. The reactivity of $inhibin-{\alpha}$ showed difference according to the growing stage of folliculus ovary: $inhibin-{\alpha}$ showed the most strong reaction in mature folliculus ovary with follicular antrum. There was strong reaction in granular cells around follicular membrane but $inhibin-{\alpha}$ did not occur at all in theca cells comprising follicular membrane. $Inhibin-{\alpha}$ in ovary tissue exposed to 400 cGy of X-rays was manifested more strongly than in ovary tissue exposed to 600 cGy of X-rays, which was related to the phenomenon that granular cells of mature folliculus ovary underwent necrosis or apoptosis increasingly due to X-rays. In an electron microscope with high magnification, nuclei and protoplasm of granular cells in growing folliculus ovary abruptly underwent minute structural changes according to the increase of dose of X-rays. Cell residue, by-product of cell decease, neutrophil and macrophage around follicular antrum were observed. The minute structural changes in granular cells showed typical characteristics of apoptosis: the increase of electronic density due to nuclear condensation, fragmentation of nuclei and atrophy of protoplasm. Necrosis of cells was identified but it was not so remarkable. Macrophage with apoptotic bodies was scattered. Proportional to the radiation dose, we found that the generation of heterogeneous substance of normal ovary texture's follicular fluid, the emergence of dyeing characteristic in the basilar membrane of folicle, the generation of apoptosis, and the transformation of macrophages, etc. From this results, we can infer the possible radiation hazard on the ovary of cervix cancer patient with radiation therapy.

References

  1. Fridovich I, Superoxide dismutase. In: Bergmyer, ed. Methods of Enzymatic Analysis. Berlin, Academic Verlag, 1986;61-97
  2. Kitazawa T, Streilein JW, Studies on delayed systemic effects of ultraviolet B radiation on the induction of contact hypersensitivity, 3. dendritic cells from secondery lymphoid organs are deficient in interleukin-12 production and capacity to promote activation and differentiation of Thelper type 1 cells. Immunology, 2000;296-304
  3. Emerit J, Chaudiere J, CRC Handbook of free radical and antioxidants in biomedicine. Florida; CRC Press, 1989;177
  4. Bowman PD, CRC Hand Book of Cell Biology of Aging. Florida; CRC Press, 1986:117
  5. Aardal N, Circamnnual variation of circadian periodicity in murine colony-forming cells. Exp Hematol, 1984;12:61-67
  6. Bernabei PA, Balzi MM, Saccardi R, Becciolini A, Ferrini PR, Time dependent sensitivity of rat CFU-GM to totalbody irradiation. Haematologica, 1992;77:21-24
  7. Mosse I, Kostrova L, Subbot S, Maksimenya I, Melanin decreases clastogenic effect of ionizing radiation in human and mouse somatic cells and modifies the radioadaptive response. Radiat Environ Biophys, 2000;39:47-52 https://doi.org/10.1007/PL00007685
  8. Hassell JR, Dratt RM, Elevated levels of CAMP alters the effect of epidermal growth factor in vitro on programmed cell death in the secondary palatial epithelium. Exp Cell Res, 1977;106:55-62 https://doi.org/10.1016/0014-4827(77)90240-3
  9. Kerr JFR, Searle J, Harmon BV, Bishop CJ, Apoptosis. In: Perspectives on Mammalian Cell Death Potten. England, Oxford University Press, 1987;35
  10. Cohen JJ, Duke RC, Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death. J Immunol, 1984;132:38-42
  11. Fortune JE, Ovarian follicular growth and development in mammal. Biol Reprod, 1994;50:225-232 https://doi.org/10.1095/biolreprod50.2.225
  12. Falck B, Site of production of estrogen in rat ovary as studied in micro-transplant. Acta Physiol Scand, 1959;163:1-100 https://doi.org/10.1046/j.1365-201x.1998.00367.x
  13. Ingram DL, Atresia. In: Zuckerman S, ed. The Ovary. New York, Academic Press, 1962;247-273
  14. Peters H, McNatty KP, The ovary. London; Granada Publishing, 1980;113-128
  15. Tsafriri A, Braw RH, Experimental approaches to atresia in mammals. In: Oxford Review of Reproductive Biology, Vol 6, Oxford Univ Press, 1984;226-265
  16. Braw RH, Bar-Ami S, Tsafriri A, Effect of hypophysectomy on atresia of rat preovulatory follicles. Biol Reprod, 1981;25:989-996 https://doi.org/10.1095/biolreprod25.5.989
  17. Sadrkhanloo R, Hofeditz C, Erickson GF, Evidence for widespread atresia in the hypophysectomized estrogentreated rat. Endocrinology, 1987;120:146-155 https://doi.org/10.1210/endo-120-1-146
  18. Bagnell CA, Mills TM, Costtoff A, Mahesh VB, A model for the study of androgen effects on follicular atresia and ovulation. Biol Reprod, 1982;27:1727-1737
  19. Gore-Langton RE, Amstrong DT, Follicular steroidogenesis and its control. In: Knobil E, Neil J, eds. The physiology of reproduction. New York, Raven Press, 1988;331-385
  20. Uilenbroek JT, Woutersen PJ, Vanderschoot P, Atresia of preovulatory follicles: Gonadotropin binding and steroidogenic activity. Biol Reprod, 1980;23:219-229 https://doi.org/10.1095/biolreprod23.1.219
  21. CJ, Greewald GS, In vitro effects of luteinizing hormone on induced atretic graffian follicles in the hamster. Biol Reprod, 1983;28:849-859 https://doi.org/10.1095/biolreprod28.4.849
  22. Hirshfield AN, Effect of a low dase of pregnent mare's serum gonadotropin on follicular recruitment and atesia in cycling rats. Biol Reprod, 1986;35:113-118 https://doi.org/10.1095/biolreprod35.1.113
  23. Naib ZM, The effects of irradiation and other therapies. In: Exfoliative Cytopathology, Little Brown Co. 1985;575-586
  24. Ramzy I, Effects of radiation and chemotherapy. In: Clinical Cytopathology and Aspiration Biopsy, Fundamental Principles and ractice, Appleton, Connecticut. 1990;107-116
  25. Dobson RL, Felton JS, Female germ cell loss from radiation and chemical exposures. Am J Indust Med, 1983;4:175-190 https://doi.org/10.1002/ajim.4700040116
  26. Erickson BH, Effects of ionizing radiation and chemical on mammalian reproduction. Vet Hum Toxicol, 1985;27:409-416
  27. Mandl AM, The radiosensitivity of germ cell. Biol Rev, 1964;39:288-371 https://doi.org/10.1111/j.1469-185X.1964.tb01161.x
  28. Lindop PJ, The effects of radiation on rodent and human ovaries. Proc Soc Med, 1969;62:144-148
  29. Ronnback C, Effects on fetal ovaries after protracted, external gamma irradiation as compared with those internal deposition. Acta Radiol Oncolo, 1983;22:465-471 https://doi.org/10.3109/02841868309135972
  30. Gregg RL, Humphrey HD, Thames JR, Meyn RE, The response of chinese hamster ovary cells to fast neutron radiotherapy beams III. Variation in relative biological effectiveness with position in the cell cycle. Radiat Res, 1978;76: 283-291 https://doi.org/10.2307/3574779
  31. Dobson RL, Cooper MF, Tritium toxicity: Effect of lowlevel $3H_{2}O$ exposure on developing female germ cells in the mouse. Radiat Res, 1974;58:91-100 https://doi.org/10.2307/3573952
  32. Lavu A, Reddy PP, Reddi OS, $^{125}I$ induced micronuclei and sperm head abnormalities in mice. Int J Radiat Biol, 1985;47:249-253 https://doi.org/10.1080/09553008514550371
  33. Kerr JFR, Wyllie AH, Currie AR, Apoptosis: A basic biological phenomenon with wide-ranging implication in tissue kinetics. Br J Cancer, 1972;26:239-257 https://doi.org/10.1038/bjc.1972.33
  34. Satow YH, Hori JY, Lee M, Ohtaki S, Nakamura SN, Okada S, Effect of tritiated water on female germ cells: mouse oocyte killing and RBE. Int J Radiat Biol, 1989;56:293-299 https://doi.org/10.1080/09553008914551461
  35. Savill J, Dransfield I, Hogg N, Haslett C, Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature, 1990;343:170-173 https://doi.org/10.1038/343170a0
  36. Kerr JFR, Searle J, Harmon BV, Bishop CJ, Apoptosis. In: Perspectives on Mammalian Cell Death Potten. England, Oxford University Press, 1987;35
  37. Ryan R, Follicular atresia. Some speculations of biochemical markers and mechanisms. In: Schwartz NB, Hunzicker-Dunn M, eds. Dynamics of Ovarian Funtio. Raven Press, New York, 1987;1-11
  38. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccadi C, A vrapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Meth, 1991;139:271-279 https://doi.org/10.1016/0022-1759(91)90198-O
  39. Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie AH, Analysis and discrimination of necrosis and apoptosis(programmed cell death) by mutiparameter flow cytometry. Bichim Biophys Acta, 1992;1133:275-285 https://doi.org/10.1016/0167-4889(92)90048-G
  40. Billig H, Furuta I, Hsueh AJW, Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology, 1993;133:2204-2212 https://doi.org/10.1210/en.133.5.2204
  41. Kutluk O, Robert S, Nelson F, Proliferation cell nuclear antigen make the initiation of follicular growth in the rat. Biol Reprod, 1995;53:295-301 https://doi.org/10.1095/biolreprod53.2.295
  42. Xiong Y, Connolly T, Futcher B, Beach D, Human D-type cylin. Cell, 1991;65:691-699 https://doi.org/10.1016/0092-8674(91)90100-D
  43. Xiong Y, Zhang H, Beach D, D-type cyclin associated with multiple protein kinase and the DNA replication and repair factor PCNA. Cell, 1992;71:505-514 https://doi.org/10.1016/0092-8674(92)90518-H
  44. Cantley LC, Auger KR, Carpenter C, Duokworth B, Graziani A, Kapeller R, Soltof S, Oncogenes and signal transduction. Cell, 1991;64:281-302 https://doi.org/10.1016/0092-8674(91)90639-G
  45. Gunji H, Kharbanda S, Kufe D, Inducation of internucleosomal DNA fragmentation in human myeloid leukemia cells by 1-6-D-arabinofuranosylcytosine. Cancer Res, 1991;51:741-743
  46. Estman-Reks SB, Vedeckis WV, Glucocorticoid inhibition of c-myc, c-myb and c-ki-ras expression in a mouse lymphoma cell line. Cancer Res, 1986;46:2457-2462
  47. Buttyan R, Zakeri Z, Lockshin R, Wolgemuth D, Cascade induction of c-fos, c-myc and heat shock 70 KD transcripts during regression of the rat ventral prostate gland. Mol Endocrinol, 1988;2:650-657 https://doi.org/10.1210/mend-2-7-650
  48. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ, Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature, 1990;348:334-336 https://doi.org/10.1038/348334a0
  49. Owen-Schaub LB, Yonehara S, Crump WL, Grimm EA, DNA fragmentation and cell death is selectively triggered in activated human lymphocytes by Fas antigen engagement. Cell Immunol, 1992;140:197-205 https://doi.org/10.1016/0008-8749(92)90187-T
  50. Watanabe-Fukunaga R, Brannan CI, Itoch N, Yonehara S, Copeland NG, Jenkins NA, Nagata S, The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J Immunol, 1992;148:1274-1279
  51. Yeh HJ, Silos-Santiago I, Wang YX, George RJ, Snider WD, Deuel TF, Developmental expression of the plateletderived growth factor ${\alpha}$-receptor gene in mammalian central nervous system. Proc Natl Acad Sci USA, 1993;90:1952-1956
  52. Miyashita T, Krajewski S, Krajewski M, Wang HG, Lin HK, Liebermann DA, Hoffman B, Reed JC, Tumor supperessor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene, 1994;9:1799-1805
  53. EI-Deiry WS, Tokino T, Velculesco VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B, A potential mediator of p53 tumor suppression. Cell, 1993;75:817-825 https://doi.org/10.1016/0092-8674(93)90500-P
  54. Xiong Y, Hannom GJ, Zhang H, Casso D, Kobayaxhi R, Beach D, p21 is a universal inhibitor of cyclin kinases. Nature, 1993a;66:701-704
  55. Xiong Y, Zhang H, Beach D, Subunit rearrangment of the cyclin-dependent kinase is associated with cellular transformation. Genes Dev, 1993b;7:1572-1583 https://doi.org/10.1101/gad.7.8.1572
  56. Tilly KI, Banerjee S, Banerjee PP, Tilly JL, Expression of the p53 and the Wilm's tumor suppressor genes in the rat ovary: Gonadotropin repression in vivo and immunohistochemical localization of nuclear p53 protein to apoptotic granulosa cells to atrtic follicles. Endocrinology, 1995;136:1394-1402 https://doi.org/10.1210/en.136.4.1394
  57. Chung WK, Radiation-induced apoptosis in the crypt cells of mouse jejunum. Med Sci, 1997;10:119-125
  58. Umadevi P, Hossain M, Induction of solid tumours in the Swiss albino mouse by low-dose foetal irradiation. Int J Radiat Biol, 2000;76:95-99 https://doi.org/10.1080/095530000139050
  59. Kapoor G, Sharan RN, Srivastava PN, Histopathologic changes in the ovary following acute and chronic lowlevel tritium exposure to mice in vivo. Int Radiat J Biol, 1985;47:197-203 https://doi.org/10.1080/09553008514550271