体外X-射线照射对小鼠生精功能影响的研究
摘要
课题背景
近年来，由于环境污染和电离辐射等原因，男性生殖健康问题如不育、流产、死胎、先天性畸形等而就诊的患者有不断增多趋势。电离辐射包括X射线，中子，质子，α或β粒子，γ射线。人们接触较多的是X射线。据国外流行病学调查，长期接触放射线的男性，生殖健康问题的发生率较普通人群高。如何降低此发生率一直是人们关注的问题。抑制素B是精子发生的血清标记物，和精子的生成状态密切相关。正常男性的抑制素B值为135.6pg／ml。血清抑制素B的测定简便。那么，它对放射线工作者睾丸生精功能状态的检测是否敏感有效呢?线粒体作为细胞三羧酸循环和氧化磷酸化进行的场所，是提供能量物质ATP的主要细胞器。线粒体损伤，细胞代谢无法正常进行，线粒体是细胞内最易受损伤的一个敏感细胞器。电镜下观察线粒体功能情况可以作为评判细胞早期损伤的一个指标。恒和猴在一定剂量的放射线照射后，睾丸活检可见曲细精管的扩张，生精细胞数量的明显减少，支持细胞变形，排列紧密。但未见以抑制素B来评估放射性睾丸生精功能损伤的文献报道。
浙江大学硕士学位论文
研究目的
分析血清抑制素B和辜丸生精功能之间的关系，了解放射线对皋丸生精细胞、
支持细胞和间质细胞的线粒体的损害。为从事放射线职业的男性工作者，特别是
有生育要求的，提供检测肇丸生精功能状态的有效简便方法。探索修复辜丸生精‘
功能的途径。
研究方法
10一12周的ICR雄性小鼠40只，随机分为对照组和实验组各20只。实验组小
鼠2%戊巴比妥钠0.1 sml腹腔内注射麻醉后固定于放射床，以x一射线3.SGy单剂
量快速照射辜丸，分别于X射线照射后2天，7天，21天，42天分批处死小鼠，
眼球取血，高速离心取血清，以ELISA双抗体夹心法测定血清抑制素B值及放射
免疫法测定血清FSH，LH，T等性激素值;同时解剖出辜丸，计辜丸的重量。光
镜标本以 10%福尔马林固定后切片，染色:电镜标本以2.5%的戊二醛固定后切片，
染色。光镜观察肇丸结构并对翠丸生精细胞包括精原细胞、精母细胞、精子细胞、
精子，支持细胞，间质细胞进行计数，电镜观察生精细胞及支持细胞的超微结构
形态及细胞各超微结构的改变，细胞内线粒体功能情况。
结果
翠丸X一射线照射后2天，小鼠辜丸重量无明显减轻，光镜下辜丸结构未发生
明显变化，电镜下可发现生精细胞及支持细胞的线粒体功能受到了损害，同时血
清抑制素B值下降(P<0.05)。肇丸X一射线照射后7天，小鼠辜丸重量显著减轻
(P<0 .05)，光镜下肇丸精子细胞及精子数量显著减少(P 细胞及支持细胞的线粒体功能损害进一步加重。肇丸X一射线照射后21天，小鼠
翠丸重量继续显著减轻(P<0.05)，光镜下精原细胞数量回升，其他生精细胞仍维持
于低水平。电镜下各细胞边缘空泡状改变，细胞膜破裂，细胞器外流。精子数量
继续减少，出现较多的畸形精子，血清FSH水平升高。辜丸X一射线照射后42天，
小鼠翠丸重量与照射后21天比较无显著差异(P>0 .05)。光镜下精子细胞及精子仍
显著下降(P<0 .05).电镜下见线粒体功能开始恢复，间质细胞增生，精子数量极
少。
浙江大学硕士学位论文
结论
x--射线照射小鼠攀丸可引起生精细胞及支持细胞的损伤，间质细胞对X射线
不敏感，在照射后一段时间可出现增生;线粒体是细胞内最易受损伤的一个敏感
细胞器。辜丸重量及血清FsH可在一定程度上反映辜丸的生精功能，但准确性与
及时性受限制。抑制素B是一个能早期反映生精功能受损的血消指标。
BACKGROUND
Problem of reproduction health of masculinity such as infertility, abortion, fetal deatru
congenital malformation has a tendency to be increased because of environmental pollution and ionization radiation in recent years. lonization radiation involves X-ray , neutro, obligation, a or (3 particle, y-ray. The most frequently that people contact is X-ray. An epidemiological study from abroad said that incidence of problem of reproduction health in man who are contact X-ray for a long-term is more higher than common people. So people pay close attention to how to reduce the incidence. Inhibin B is a serum markers of spermatogenesis. The level of inhibin B in normal man is 135. 6pg/ml . Assay of Inhibin B is not difficult. So we want to know whether inhibin B is a sensitive marker for reflecting the functional status of spermatogenesis in man who has a working in X-ray enviroment. As a locus of tricarboxylic acid cycle and oxidative phosphorylation, Mitochondrion is a main organell that provid ATP. Metabolism of cells can not procee in normal when mitochondrion are injuries. Mitochondrion is athe most
sensitive organell for injuries. Observing the functional condition of mitochondrion through electron microscope can be used as a maker of earlier period injures. After exposure to a dosage of X-ray i n monkeys, testicle biopsy showed dilataltion of seminiferous tubule, significantly decreasing of quantity of spermatogenic cells, deformation of Sertoli's cells. Up to now, there is no study reported that inhibin B act as a maker to evaluate radioactivity injuries of spermatogenesis.
OBJECTIVE
To learn alteration of the function of mitochondria after exposure to X-ray and to explore the status and role of inhibinB on screening injuries of spermatogenesis in mice. MATERIAL AND METHODS
10-12weeks age ,40 male mice were randomly divided into X-ray group and control group. Mice were given 2% soluble pentobarbitone 0.15ml through intraperitoneal injection .The dose of X-ray was 3.5Gy and was given mono-dosage. The mice were killed in batch in days of 2, 7, 21, 42 after X-ray exposure. Taking blood from eyeball and high speed centrifugation to obtain blood serum. Enzyme-linked immunoadsordent assay inhibin B and radio- Immunization assay the levels of FSH, LH, T. with dissection of testis and calculation the weight of testis. The sample for light microscope was fixed with 10% formaldehyde solution by turns with filleting and staining ; the sample for electron microscope was fixed with 2.5% glutaral by turns with filleting and staining. Observing structure of testis with light microscope and counting Sertoli's cell - interstitial cell and spermatogenic cell which include spermatogenous cell, spermatocyte, spermatid, Sperm. Observing the ultrastructure of spermatogenic cell and Sertoli's cell with electron microscope and pay attention to the function of mitochondrion at the same time. RESULTS
Two days after X-ray exposure, the weight of testis was not descent and no significant change was found by light microscope. But lesion of mitochondrion in spermatogenic cell and Sertoli's cell was found by electron microscope. The tite of inhibin B was descent at the same time(P<0.05). Seven days after X-ray exposure, the weight of testis was significantly descent(P<0.05). The quantity of spermatid and sperm was significantly decreasing(P<0.05). Lesion of mitochondrion in spermatogenic cell and Sertoli's cell was took a bad turn. Twenty-one days after X-ray exposure, the weight of testis continued significantly descent(P<0.05). The quantity of spermatogenous cell was slowly elevated, but others were still in a lower level. Rimmed vacuoles was found in cells through electron microscope. Disruption of cell membrane and outflow of organell. The quantity
of sperm continued decreasing and more malformation sperm were found. Level of FSH was elevated. Forty-two days after X-ray exposure, comparing with the weight of twenty-one days, the weight of testis was no significant change(P>0.05). The quantity of Sperma
近年来，由于环境污染和电离辐射等原因，男性生殖健康问题如不育、流产、死胎、先天性畸形等而就诊的患者有不断增多趋势。电离辐射包括X射线，中子，质子，α或β粒子，γ射线。人们接触较多的是X射线。据国外流行病学调查，长期接触放射线的男性，生殖健康问题的发生率较普通人群高。如何降低此发生率一直是人们关注的问题。抑制素B是精子发生的血清标记物，和精子的生成状态密切相关。正常男性的抑制素B值为135.6pg／ml。血清抑制素B的测定简便。那么，它对放射线工作者睾丸生精功能状态的检测是否敏感有效呢?线粒体作为细胞三羧酸循环和氧化磷酸化进行的场所，是提供能量物质ATP的主要细胞器。线粒体损伤，细胞代谢无法正常进行，线粒体是细胞内最易受损伤的一个敏感细胞器。电镜下观察线粒体功能情况可以作为评判细胞早期损伤的一个指标。恒和猴在一定剂量的放射线照射后，睾丸活检可见曲细精管的扩张，生精细胞数量的明显减少，支持细胞变形，排列紧密。但未见以抑制素B来评估放射性睾丸生精功能损伤的文献报道。
浙江大学硕士学位论文
研究目的
分析血清抑制素B和辜丸生精功能之间的关系，了解放射线对皋丸生精细胞、
支持细胞和间质细胞的线粒体的损害。为从事放射线职业的男性工作者，特别是
有生育要求的，提供检测肇丸生精功能状态的有效简便方法。探索修复辜丸生精‘
功能的途径。
研究方法
10一12周的ICR雄性小鼠40只，随机分为对照组和实验组各20只。实验组小
鼠2%戊巴比妥钠0.1 sml腹腔内注射麻醉后固定于放射床，以x一射线3.SGy单剂
量快速照射辜丸，分别于X射线照射后2天，7天，21天，42天分批处死小鼠，
眼球取血，高速离心取血清，以ELISA双抗体夹心法测定血清抑制素B值及放射
免疫法测定血清FSH，LH，T等性激素值;同时解剖出辜丸，计辜丸的重量。光
镜标本以 10%福尔马林固定后切片，染色:电镜标本以2.5%的戊二醛固定后切片，
染色。光镜观察肇丸结构并对翠丸生精细胞包括精原细胞、精母细胞、精子细胞、
精子，支持细胞，间质细胞进行计数，电镜观察生精细胞及支持细胞的超微结构
形态及细胞各超微结构的改变，细胞内线粒体功能情况。
结果
翠丸X一射线照射后2天，小鼠辜丸重量无明显减轻，光镜下辜丸结构未发生
明显变化，电镜下可发现生精细胞及支持细胞的线粒体功能受到了损害，同时血
清抑制素B值下降(P<0.05)。肇丸X一射线照射后7天，小鼠辜丸重量显著减轻
(P<0 .05)，光镜下肇丸精子细胞及精子数量显著减少(P
翠丸重量继续显著减轻(P<0.05)，光镜下精原细胞数量回升，其他生精细胞仍维持
于低水平。电镜下各细胞边缘空泡状改变，细胞膜破裂，细胞器外流。精子数量
继续减少，出现较多的畸形精子，血清FSH水平升高。辜丸X一射线照射后42天，
小鼠翠丸重量与照射后21天比较无显著差异(P>0 .05)。光镜下精子细胞及精子仍
显著下降(P<0 .05).电镜下见线粒体功能开始恢复，间质细胞增生，精子数量极
少。
浙江大学硕士学位论文
结论
x--射线照射小鼠攀丸可引起生精细胞及支持细胞的损伤，间质细胞对X射线
不敏感，在照射后一段时间可出现增生;线粒体是细胞内最易受损伤的一个敏感
细胞器。辜丸重量及血清FsH可在一定程度上反映辜丸的生精功能，但准确性与
及时性受限制。抑制素B是一个能早期反映生精功能受损的血消指标。
BACKGROUND
Problem of reproduction health of masculinity such as infertility, abortion, fetal deatru
congenital malformation has a tendency to be increased because of environmental pollution and ionization radiation in recent years. lonization radiation involves X-ray , neutro, obligation, a or (3 particle, y-ray. The most frequently that people contact is X-ray. An epidemiological study from abroad said that incidence of problem of reproduction health in man who are contact X-ray for a long-term is more higher than common people. So people pay close attention to how to reduce the incidence. Inhibin B is a serum markers of spermatogenesis. The level of inhibin B in normal man is 135. 6pg/ml . Assay of Inhibin B is not difficult. So we want to know whether inhibin B is a sensitive marker for reflecting the functional status of spermatogenesis in man who has a working in X-ray enviroment. As a locus of tricarboxylic acid cycle and oxidative phosphorylation, Mitochondrion is a main organell that provid ATP. Metabolism of cells can not procee in normal when mitochondrion are injuries. Mitochondrion is athe most
sensitive organell for injuries. Observing the functional condition of mitochondrion through electron microscope can be used as a maker of earlier period injures. After exposure to a dosage of X-ray i n monkeys, testicle biopsy showed dilataltion of seminiferous tubule, significantly decreasing of quantity of spermatogenic cells, deformation of Sertoli's cells. Up to now, there is no study reported that inhibin B act as a maker to evaluate radioactivity injuries of spermatogenesis.
OBJECTIVE
To learn alteration of the function of mitochondria after exposure to X-ray and to explore the status and role of inhibinB on screening injuries of spermatogenesis in mice. MATERIAL AND METHODS
10-12weeks age ,40 male mice were randomly divided into X-ray group and control group. Mice were given 2% soluble pentobarbitone 0.15ml through intraperitoneal injection .The dose of X-ray was 3.5Gy and was given mono-dosage. The mice were killed in batch in days of 2, 7, 21, 42 after X-ray exposure. Taking blood from eyeball and high speed centrifugation to obtain blood serum. Enzyme-linked immunoadsordent assay inhibin B and radio- Immunization assay the levels of FSH, LH, T. with dissection of testis and calculation the weight of testis. The sample for light microscope was fixed with 10% formaldehyde solution by turns with filleting and staining ; the sample for electron microscope was fixed with 2.5% glutaral by turns with filleting and staining. Observing structure of testis with light microscope and counting Sertoli's cell - interstitial cell and spermatogenic cell which include spermatogenous cell, spermatocyte, spermatid, Sperm. Observing the ultrastructure of spermatogenic cell and Sertoli's cell with electron microscope and pay attention to the function of mitochondrion at the same time. RESULTS
Two days after X-ray exposure, the weight of testis was not descent and no significant change was found by light microscope. But lesion of mitochondrion in spermatogenic cell and Sertoli's cell was found by electron microscope. The tite of inhibin B was descent at the same time(P<0.05). Seven days after X-ray exposure, the weight of testis was significantly descent(P<0.05). The quantity of spermatid and sperm was significantly decreasing(P<0.05). Lesion of mitochondrion in spermatogenic cell and Sertoli's cell was took a bad turn. Twenty-one days after X-ray exposure, the weight of testis continued significantly descent(P<0.05). The quantity of spermatogenous cell was slowly elevated, but others were still in a lower level. Rimmed vacuoles was found in cells through electron microscope. Disruption of cell membrane and outflow of organell. The quantity
of sperm continued decreasing and more malformation sperm were found. Level of FSH was elevated. Forty-two days after X-ray exposure, comparing with the weight of twenty-one days, the weight of testis was no significant change(P>0.05). The quantity of Sperma
引文
1. Roman E, Doyle P, Ansell P et al. Health of children born to medical radiographers. Occup Environ Med. 1998 Sep;55(9):647-648
2. Shakhatreh FM, Reproductive health of male radiographers. Saudi Med J 2001 Feb,22(2): 150-152
3. Bohring C, Krause W. Serum levels of inhibin B in men with different causes of spermatogenic failure. Andrologia, 1999 May;31 (3): 137-141.
4. Andersson AM Muller J skakkeb ek NE .Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels [J].J Clin Endo Metab, 1998 Dec;83(12).4451-4458.
5. Vergouwen RP, Huiskamp R, Bas RJ et al. Radiosensitivity of testicular cells in the fetal mouse. Radiat Res 1995 Jan; 141 (1):66-73
6. Vergouwen RP, Huiskamp R, Bas RJ et al. Radiosensitivity of testicular cells in the prepubertal mouse. Radiat Res. 1994 Sep;139(3):316-326
7. Rooij DG, Van de Kant HJ, Dol R, Wagemaker G et al. Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey. Biol Reprod 2002 Feb;66(2):486-494
8． 王兰，孙海峰，李春源．镉对长江华溪蟹精子发生的影响．动物学报2002 48 (5)：677-84．
9. Nicopoulou-Karayianni K, Koligliatis T, Donta-Bakogianni C. et al. Radiation absorbed doses at compact bone-titanium interfaces in diagnostic radiography: a Monte Carlo approach. Dentomaxillofac Radiol. 2003 Sep;32(5):327-32.
10. Artamonov MV, Zhukov OD, Horid＇ko TM et al. Effect of N-stearoylethanolamine and ionizing radiation on lipid components of the liver and heart microsomes in ratsUkr Biokhim Zh. 2003 Jul-Aug;75(4):81-90
11. Frydelund-larsen L, Krausz C, Leffers H et al. J Clin Endoerinol Metab 2002 Dec;87(12):5618-5624.
12. Bohring C, Schroeder-Printzen l, Weidner W ea al. Serum levels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men azzospermia who are undergoing testicular sperm extraction. Fertil Steril 2002 Dec;78(6)1195-1198,
13. Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Retprod. 1998 Apr: 13(4):920-926.
14. Von Eckardstein S, Simone M, Bergmann M et al. Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J Clin Endocrinol Metab. 1999 Jul;84(7):2496-2501
15. Vergouwen RP, Huiskamp R, Bas RJ et al. Radiosensitivity of testicular cells in the mouse. Radiat Res. 1995 Jan;141(1):66-73.
2. Shakhatreh FM, Reproductive health of male radiographers. Saudi Med J 2001 Feb,22(2): 150-152
3. Bohring C, Krause W. Serum levels of inhibin B in men with different causes of spermatogenic failure. Andrologia, 1999 May;31 (3): 137-141.
4. Andersson AM Muller J skakkeb ek NE .Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels [J].J Clin Endo Metab, 1998 Dec;83(12).4451-4458.
5. Vergouwen RP, Huiskamp R, Bas RJ et al. Radiosensitivity of testicular cells in the fetal mouse. Radiat Res 1995 Jan; 141 (1):66-73
6. Vergouwen RP, Huiskamp R, Bas RJ et al. Radiosensitivity of testicular cells in the prepubertal mouse. Radiat Res. 1994 Sep;139(3):316-326
7. Rooij DG, Van de Kant HJ, Dol R, Wagemaker G et al. Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey. Biol Reprod 2002 Feb;66(2):486-494
8． 王兰，孙海峰，李春源．镉对长江华溪蟹精子发生的影响．动物学报2002 48 (5)：677-84．
9. Nicopoulou-Karayianni K, Koligliatis T, Donta-Bakogianni C. et al. Radiation absorbed doses at compact bone-titanium interfaces in diagnostic radiography: a Monte Carlo approach. Dentomaxillofac Radiol. 2003 Sep;32(5):327-32.
10. Artamonov MV, Zhukov OD, Horid＇ko TM et al. Effect of N-stearoylethanolamine and ionizing radiation on lipid components of the liver and heart microsomes in ratsUkr Biokhim Zh. 2003 Jul-Aug;75(4):81-90
11. Frydelund-larsen L, Krausz C, Leffers H et al. J Clin Endoerinol Metab 2002 Dec;87(12):5618-5624.
12. Bohring C, Schroeder-Printzen l, Weidner W ea al. Serum levels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men azzospermia who are undergoing testicular sperm extraction. Fertil Steril 2002 Dec;78(6)1195-1198,
13. Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Retprod. 1998 Apr: 13(4):920-926.
14. Von Eckardstein S, Simone M, Bergmann M et al. Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J Clin Endocrinol Metab. 1999 Jul;84(7):2496-2501
15. Vergouwen RP, Huiskamp R, Bas RJ et al. Radiosensitivity of testicular cells in the mouse. Radiat Res. 1995 Jan;141(1):66-73.