药物诱导大鼠隐睾模型比较及T3干预Flutamide诱导隐睾NCAM表达研究
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摘要
目的：
比较邻苯二酸二丁酯(DBP)、己烯雌酚(DES)、氟他胺(Flu)宫内诱导SD鼠隐睾模型的组织病理变化,选择与临床隐睾病理改变接近的模型。观察T3调控SD鼠隐睾生殖母细胞(Gonocyte, Go)发育及NCAM表达。
方法：
①SD孕鼠,于受孕(gestational day GD)12-21d给予DES、Flu皮下注射,DBP灌胃,建立大鼠隐睾模型；收集50个临床隐睾活检组织。
②免疫组化定性、定位检测睾丸NCAM的表达。
③T3 15μg·kg-1·d-1皮下注射Flu诱导PD1仔鼠,PD20隐睾鼠,观察干预后隐睾发生率、组织学改变,以及NCAM在的表达。
结果：
①Flu诱导大鼠隐睾发生率为42.2%,睾丸位于腹股沟,尿道下裂发生率93.3%。睾丸体积、脏器系数明显小于正常对照组(P<0.01)。Flu隐睾管腔化延迟；PD20、PD80隐睾管腔中央仍可见未迁移的Go,PD80曲细精管中无精子生成；平均曲细精管直径(MSTD)和面积(MSTA)与正常相比有显著性差异(P<0.01)。
②DBP诱导大鼠隐睾发生率为为42.5%,隐睾位于腹腔,尿道下裂发生率25.0%。睾丸体积、脏器系数明显小于正常(P<0.01),曲细精管中无精子生成,生精细胞稀少,部分管腔仅有支持细胞。
③DES高剂量可致SD孕鼠流产或死亡,低剂量1 .5μg·kg-1·d-1不能诱导出隐睾模型。仔鼠发育与正常接近,成年后均具有生殖能力。睾丸体积、脏器系数、MSTD、MSTA与正常组无显著性差异(P>0.05),管腔中央可见大量的精子生成。
④50个临床隐睾中,NCAM表达分布模式有4种：(1)曲细精管内阴性表达、间质细胞阳性表达42%,为生理发育规律表达；(2)曲细精管内阳性表达、间质细胞阳性表达26%；(3)曲细精管内阳性表达、间质细胞阴性表达4%；(4)曲细精管内阴性表达、间质细胞阴性表达28%。
⑤DES、DBP大鼠曲细精管NCAM表达与正常组一致,PD1管腔内Go均有阳性表达,PD10管腔内Go迁移至基底膜,NCAM表达明显减弱至消失。PD20、PD80隐睾及下降睾丸曲细精管内NCAM阴性表达。Flu诱导PD10、PD20、PD80隐睾管腔NCAM阳性表达。
⑥T3 15μg·kg-1·d-1干预Flu诱导PD1仔鼠,成年后隐睾发生率为25%(4/16),较未干预组的隐睾发生率42.9%(12/28)降低,并可促进曲细精管管腔化,隐睾曲细精管内NCAM阴性表达。
⑦Flu诱导的PD13仔鼠睾丸NCAM的mRNA表达均明显高于正常对照组(P<0.05),T3干预后,NCAM的mRNA表达明显低于非T3干预组(P<0.05),并与正常对照组无明显差异(P>0.05)。
结论：
①孕期宫内诱导隐睾模型中,Flu诱导的隐睾组织中存在NCAM曲细精管阳性,Go残留,该模型更适宜于研究临床隐睾Go残留及发育障碍。
②Flu仔鼠出生后早期T3干预,可以降低隐睾发生率；促进曲细精管管腔化,下调NCAM表达。降低或消除隐睾曲细精管Go残存。
Objectives:
To establish rat models of cryptorchidism in-utero exposing to dibutyl phthalate (DBP)、diethylstilbestrol (DES)、Flutamide (Flu), and compare pathological difference. Explore the effect of T3 regulate the development of Gonocyte (Go) and the expression of NCAM in the Flu-induced cryptorchidism SD rats.
Methods:
①Pregnant SD rats were treated DES、Flu by subcutaneous injections and DBP by intragastric administration from gestational day (GD) 12 to 21 to establish cryptorchidism.50 testis biopsies of clinical cryptorchidism were collected.
②The expression of NCAM in the testes were detected by Immunohistochemistry and real RT-PCR.
③Postnatal day (PD) 1 and PD20 cryptorchidism SD rat induced by Flu were treated T3 15μg·kg-1·d-1 by subcutaneous injections. Incidence of the cryptorchidism、histology and the expression of NCAM were included in the research.
Results:
①The testes of Flu-induced located in the groin, and the incidence of cryptorchidism and hypospadias were 42.2% and 93.3% respectively. The volume and organ coefficient of the testes were significant lower than that of the normal (P<0.01). Lumen of seminiferous tubules in the Flu-induced cryptorchidism were delayed to be present. Gonocyte remained in the center of cryptorchidism seminiferous tubules on PD20 and PD80, and no spermatozoas could be seen on PD80. There was significant difference in mean seminiferous tubular diameters (MSTD) and area (MSTA) compared to the normal (P<0.01).
②The incidence of cryptorchidism in DBP-induced rat and hypospadias were 42.5% and 25.0% respectively. Testes of the cryptorchidism were located in the abdominal cavity, and no spermatozoas could be seen in the seminiferous tubules. The volume and organ coefficient were significant lower than that of the normal (P<0.01). There were few spermatogenic cells and no spermatozoas could be seen in the seminiferous tubules, and only a few Sertoli cells found in some tubules.
③High dose of DES would lead the pregnant rat to abortion or death, while DES 1.5μg·kg-1·d-1 could not induce cryptorchidism. There was no significant difference between DES induced testis and the normal in the volume、organ coefficient、MSTD and MSTA of the testes (P>0.05). Abundant of spermatozoas could be seen in the tubules.
④There were 4 types of the NCAM expression in the 50 biopsies testis from human cryptochidism. (1) 42% of them NCAM negative expression in seminiferous tubules but positive in interstitial cells, which were physiological expression during maturation; (2) 26% of them NCAM positive expression in seminiferous tubules and in interstitial cells; (3) 28% of them NCAM positive expression in seminiferous tubules and negative in interstitial cells; (4) 4% of them NCAM negative expression in seminiferous tubules and in interstitial cells.
⑤The expression of NCAM in the DES, DBP and normal group were similar. It could be seen positive expression on the Gonocyte membrane in the PD1 seminiferous tubules. Gonocyte already migrated to the basement membrane on PD10, and the expression of NCAM became weak or even disappear. NCAM negatively expressed in PD20 and PD80 cryptorchidism seminiferous tubules. NCAM positively expressed in Flu-induced cryptorchidism seminiferous tubules on PD10, PD20 and PD80. After Flu-induced offsprings treated by T3 15μg·kg-1·d-1, NCAM negatively expressed in seminiferous tubules of cryptorchidism.
⑥The incidence of cryptorchidism in the group of Flu-induced offsprings treated by T3 15μg·kg-1·d-1 from PD1 was 25% (4/16), which was lower than that of Flu group. And T3 maybe contribute to the presence of lumen and down-regulate NCAM expression in Flu-induced cryptorchidism.
⑦NCAM mRNA expression in Flu-induced testes on PD13 was up-regulate than that of the control (P<0.05). NCAM mRNA expression in Flu-induced testes treated by T3 15μg·kg-1·d-1 was significant down-regulate than that without T3 (P<0.05), and there was no significant difference compare with the normal testes (P>0.05).
Conclusions:
①Among the experimental cryptorchidism models induced by DES, DBP and Flu in-utero exposure, only in Flu-induced testes can be found the NCAM positive expression and remained Gonocyte in the center of seminiferous tubules, which adapte to research the maldevelopment of the Gonocyte in clinical cryptorchidism.
②The incidence of Flu-induced cryptorchidism can be decreased by T3 after birth. T3 may contribute to the presence of lumen and down regulate the expression of NCAM, and reduce or eliminate the remainded Gonocytes in crytorcidism seminiferous tubules.
比较邻苯二酸二丁酯(DBP)、己烯雌酚(DES)、氟他胺(Flu)宫内诱导SD鼠隐睾模型的组织病理变化,选择与临床隐睾病理改变接近的模型。观察T3调控SD鼠隐睾生殖母细胞(Gonocyte, Go)发育及NCAM表达。
方法：
①SD孕鼠,于受孕(gestational day GD)12-21d给予DES、Flu皮下注射,DBP灌胃,建立大鼠隐睾模型；收集50个临床隐睾活检组织。
②免疫组化定性、定位检测睾丸NCAM的表达。
③T3 15μg·kg-1·d-1皮下注射Flu诱导PD1仔鼠,PD20隐睾鼠,观察干预后隐睾发生率、组织学改变,以及NCAM在的表达。
结果：
①Flu诱导大鼠隐睾发生率为42.2%,睾丸位于腹股沟,尿道下裂发生率93.3%。睾丸体积、脏器系数明显小于正常对照组(P<0.01)。Flu隐睾管腔化延迟；PD20、PD80隐睾管腔中央仍可见未迁移的Go,PD80曲细精管中无精子生成；平均曲细精管直径(MSTD)和面积(MSTA)与正常相比有显著性差异(P<0.01)。
②DBP诱导大鼠隐睾发生率为为42.5%,隐睾位于腹腔,尿道下裂发生率25.0%。睾丸体积、脏器系数明显小于正常(P<0.01),曲细精管中无精子生成,生精细胞稀少,部分管腔仅有支持细胞。
③DES高剂量可致SD孕鼠流产或死亡,低剂量1 .5μg·kg-1·d-1不能诱导出隐睾模型。仔鼠发育与正常接近,成年后均具有生殖能力。睾丸体积、脏器系数、MSTD、MSTA与正常组无显著性差异(P>0.05),管腔中央可见大量的精子生成。
④50个临床隐睾中,NCAM表达分布模式有4种：(1)曲细精管内阴性表达、间质细胞阳性表达42%,为生理发育规律表达；(2)曲细精管内阳性表达、间质细胞阳性表达26%；(3)曲细精管内阳性表达、间质细胞阴性表达4%；(4)曲细精管内阴性表达、间质细胞阴性表达28%。
⑤DES、DBP大鼠曲细精管NCAM表达与正常组一致,PD1管腔内Go均有阳性表达,PD10管腔内Go迁移至基底膜,NCAM表达明显减弱至消失。PD20、PD80隐睾及下降睾丸曲细精管内NCAM阴性表达。Flu诱导PD10、PD20、PD80隐睾管腔NCAM阳性表达。
⑥T3 15μg·kg-1·d-1干预Flu诱导PD1仔鼠,成年后隐睾发生率为25%(4/16),较未干预组的隐睾发生率42.9%(12/28)降低,并可促进曲细精管管腔化,隐睾曲细精管内NCAM阴性表达。
⑦Flu诱导的PD13仔鼠睾丸NCAM的mRNA表达均明显高于正常对照组(P<0.05),T3干预后,NCAM的mRNA表达明显低于非T3干预组(P<0.05),并与正常对照组无明显差异(P>0.05)。
结论：
①孕期宫内诱导隐睾模型中,Flu诱导的隐睾组织中存在NCAM曲细精管阳性,Go残留,该模型更适宜于研究临床隐睾Go残留及发育障碍。
②Flu仔鼠出生后早期T3干预,可以降低隐睾发生率；促进曲细精管管腔化,下调NCAM表达。降低或消除隐睾曲细精管Go残存。
Objectives:
To establish rat models of cryptorchidism in-utero exposing to dibutyl phthalate (DBP)、diethylstilbestrol (DES)、Flutamide (Flu), and compare pathological difference. Explore the effect of T3 regulate the development of Gonocyte (Go) and the expression of NCAM in the Flu-induced cryptorchidism SD rats.
Methods:
①Pregnant SD rats were treated DES、Flu by subcutaneous injections and DBP by intragastric administration from gestational day (GD) 12 to 21 to establish cryptorchidism.50 testis biopsies of clinical cryptorchidism were collected.
②The expression of NCAM in the testes were detected by Immunohistochemistry and real RT-PCR.
③Postnatal day (PD) 1 and PD20 cryptorchidism SD rat induced by Flu were treated T3 15μg·kg-1·d-1 by subcutaneous injections. Incidence of the cryptorchidism、histology and the expression of NCAM were included in the research.
Results:
①The testes of Flu-induced located in the groin, and the incidence of cryptorchidism and hypospadias were 42.2% and 93.3% respectively. The volume and organ coefficient of the testes were significant lower than that of the normal (P<0.01). Lumen of seminiferous tubules in the Flu-induced cryptorchidism were delayed to be present. Gonocyte remained in the center of cryptorchidism seminiferous tubules on PD20 and PD80, and no spermatozoas could be seen on PD80. There was significant difference in mean seminiferous tubular diameters (MSTD) and area (MSTA) compared to the normal (P<0.01).
②The incidence of cryptorchidism in DBP-induced rat and hypospadias were 42.5% and 25.0% respectively. Testes of the cryptorchidism were located in the abdominal cavity, and no spermatozoas could be seen in the seminiferous tubules. The volume and organ coefficient were significant lower than that of the normal (P<0.01). There were few spermatogenic cells and no spermatozoas could be seen in the seminiferous tubules, and only a few Sertoli cells found in some tubules.
③High dose of DES would lead the pregnant rat to abortion or death, while DES 1.5μg·kg-1·d-1 could not induce cryptorchidism. There was no significant difference between DES induced testis and the normal in the volume、organ coefficient、MSTD and MSTA of the testes (P>0.05). Abundant of spermatozoas could be seen in the tubules.
④There were 4 types of the NCAM expression in the 50 biopsies testis from human cryptochidism. (1) 42% of them NCAM negative expression in seminiferous tubules but positive in interstitial cells, which were physiological expression during maturation; (2) 26% of them NCAM positive expression in seminiferous tubules and in interstitial cells; (3) 28% of them NCAM positive expression in seminiferous tubules and negative in interstitial cells; (4) 4% of them NCAM negative expression in seminiferous tubules and in interstitial cells.
⑤The expression of NCAM in the DES, DBP and normal group were similar. It could be seen positive expression on the Gonocyte membrane in the PD1 seminiferous tubules. Gonocyte already migrated to the basement membrane on PD10, and the expression of NCAM became weak or even disappear. NCAM negatively expressed in PD20 and PD80 cryptorchidism seminiferous tubules. NCAM positively expressed in Flu-induced cryptorchidism seminiferous tubules on PD10, PD20 and PD80. After Flu-induced offsprings treated by T3 15μg·kg-1·d-1, NCAM negatively expressed in seminiferous tubules of cryptorchidism.
⑥The incidence of cryptorchidism in the group of Flu-induced offsprings treated by T3 15μg·kg-1·d-1 from PD1 was 25% (4/16), which was lower than that of Flu group. And T3 maybe contribute to the presence of lumen and down-regulate NCAM expression in Flu-induced cryptorchidism.
⑦NCAM mRNA expression in Flu-induced testes on PD13 was up-regulate than that of the control (P<0.05). NCAM mRNA expression in Flu-induced testes treated by T3 15μg·kg-1·d-1 was significant down-regulate than that without T3 (P<0.05), and there was no significant difference compare with the normal testes (P>0.05).
Conclusions:
①Among the experimental cryptorchidism models induced by DES, DBP and Flu in-utero exposure, only in Flu-induced testes can be found the NCAM positive expression and remained Gonocyte in the center of seminiferous tubules, which adapte to research the maldevelopment of the Gonocyte in clinical cryptorchidism.
②The incidence of Flu-induced cryptorchidism can be decreased by T3 after birth. T3 may contribute to the presence of lumen and down regulate the expression of NCAM, and reduce or eliminate the remainded Gonocytes in crytorcidism seminiferous tubules.
引文
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[13]Hutson JM, Hasthorpe S. Abnormalities of testicular descent [J]. Cell Tiss Res. 2005,322:155-8.
[14]Bullock BC, Newbold RR, McLachlan JA. Lesions of testis and epididymis associated with prenatal diethylstilbestrol exposure [J]. Environ Health Perspect. 1988,77:29-31.
[15]Yamamoto M, Shirai M, Sugita K, et al. Effects of maternal exposure to diethylstilbestrol on the development of the reproductive system and thyroid function in male and female rat offspring [J]. J Toxicol Sci.2003,28(5):385-94.
[16]Okur H, Muhtaroglu S, Bozkurt A, et al. Effects of prenatal flutamide on testicular development, androgen production and fertility in rats [J]. Urol Int. 2006,76(2):130-3.
[17]Mizuno K, Hayashi Y, Kojima Y, et al. Influence for testicular development and histological peculiarity in the testes of flutamide-induced cryptorchid rat model [J]. Int J Urol.2007,14(1):67-72.
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[19]Kleymenova E, Swanson C, Boekelheide K, et al.Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact [J]. Biol Reprod.2005,73(3):482-90.
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[5]Sarina Schrager, Beth E. Potter, Diethylstilbestrol Exposure [J]. Am Fam Physician.2004,69(10):2395-400.
[6]Nomura T, Kanzaki T. Induction of urogenital anomalies and some tumors in the progeny of mice receiving diethylstilbestrol during pregnancy [J]. Cancer Res. 1977,37(4):1099-104.
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[10]王炜,魏光辉,邓永继等.邻苯二甲酸二-(2-乙基)己酯致小鼠隐睾睾丸和附睾的组织病理学改变[J].中华男科学.2004,10(11)：807-810,814。
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[12]Bergh A. Experimental models of cryptorchidism. In:Keel B, Abney T, editors. The cryptorchid testis [J]. CRC Press, Boca Raton.1989,15-34.
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[14]Bullock BC, Newbold RR, McLachlan JA. Lesions of testis and epididymis associated with prenatal diethylstilbestrol exposure [J]. Environ Health Perspect. 1988,77:29-31.
[15]Yamamoto M, Shirai M, Sugita K, et al. Effects of maternal exposure to diethylstilbestrol on the development of the reproductive system and thyroid function in male and female rat offspring [J]. J Toxicol Sci.2003,28(5):385-94.
[16]Okur H, Muhtaroglu S, Bozkurt A, et al. Effects of prenatal flutamide on testicular development, androgen production and fertility in rats [J]. Urol Int. 2006,76(2):130-3.
[17]Mizuno K, Hayashi Y, Kojima Y, et al. Influence for testicular development and histological peculiarity in the testes of flutamide-induced cryptorchid rat model [J]. Int J Urol.2007,14(1):67-72.
[18]Shultz VD, Phillips S, Sar M, et al. Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate [J]. Toxicol Sci.2001,64(2):233-42.
[19]Kleymenova E, Swanson C, Boekelheide K, et al.Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact [J]. Biol Reprod.2005,73(3):482-90.
[20]van Straaten HW. Lack of primary defect in maldescended testis of the neonatal pig [J]. Biol Reprod.1978,19(5):994-8.
[21]Hughes IA, Acerini CL. Factors controlling testis descent [J]. Eur J Endocrinol. 2008,159 Suppl 1:S75-82.
[22]Yin Y, Stahl BC, DeWolf WC, Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis [J]. J Androl.2002,23:64-70.
[23]Ishikawa T, Kondo Y, Goda K, Fujisawa M. Overexpression of endothelial nitric oxide synthase in transgenic mice accelerates testicular germ cell apoptosis induced by experimental cryptorchidism [J]. J Androl.2005,26:281-8.
[24]Nishiyama H, Danno S, Kaneko Y, Itoh K, Yokoi H, Fukumoto M, et al. Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature [J]. Am J Pathol.1998,152:289-96.
[25]Hayashi T, Yoshida S,Yoshinaga A,Ohno R, Ishii N, Yamada T. HtrA2 is up-regulated in the rat testis after experimental cryptorchidism [J]. Int J Urol. 2006,13:157-64.
[26]Han XB, Zhou XC, Hu ZY, Zhang ZH, Liu YX. Cloning and characterization of temperature-related gene TRS1 [J]. Arch Androl.2002,48:273-8.
[27]Kumagai J, Fukuda J, Kodama H, Murata M, Kawamura K, Itoh H, et al. Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis[J]. Eur J Biochem.2000,267:3073-8.
[28]Bergh A, Damber JE, Ritzen M. Early signs of Sertoli and Leydig cell dysfunction in the abdominal testes of immature unilaterally cryptorchid rats [J]. Int J Androl.1984,7:389-408.
[29]Hagen"as L, Ritzen M, Svensson J, Hansson V. Temperature dependence of Sertoli cell function [J]. Int J Androl.1978,2:449.
[30]Bergh A. Morphological signs of a direct effect of experimental cryptorchidism on the Sertoli cells in rats irradiated as fetuses [J]. Biol Reprod.1981,24:145-52.
[31]Danno S, Itoh K, Matsuda T, Fujita J. Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of cryptorchid testis [J]. Am J Pathol.2000, 156:1685-92.
[32]Durham LA 3rd, Grogan WM. Characterization of multiple forms of cholesteryl ester hydrolase in the rat testis [J]. J Biol Chem.1984,259:7433-8.
[33]Bergh A. Experimental models of cryptorchidism [J]. CRC Press, Boca Raton. 1989,15-34.
[34]Suomi AM, Main KM, Kaleva M, Schmidt IM, Chellakooty M, Virtanen HE, et al. Hormonal changes in 3-month-old cryptorchid boys [J]. J Clin Endocrinol Metab. 2006,91:953-8.、
[35]Bergh A, Damber JE. Vascular controls in testicular physiology. In:De Kretser DM, editor. Cellular and molecular mechanisms in male reproduction [J]. NewYork:Academic Press.1993,439-68.
[36]Jegou B, Peake RA, Irby DC, de Kretser DM. Effects of the induction of experimental cryptorchidism and subsequent orchidopexy on testicular function in immature rats [J]. Biol Reprod 1984.30,179-87.
[37]Overbeek PA, Gorlov IP, Sutherland RW, Houston JB, Harrison WR, Boettger-Tong HL, et al. A transgenic insertion causing cryptorchidism in mice [J]. Genesis.2001,30:26-35.
[38]Lugg JA, Penson DF, Sadeghi F, Petrie B, Freedman AL, Gonzalez-Cadavid NF, et al. Prevention of seminiferous tubular atrophy in a naturally cryptorchid rat model by early surgical intervention [J]. J Andrology.1996,17:726-32.
[39]Jegou B, Laws AO, de Kretser DM. The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats [J]. J Reprod Fertil.1983,69: 137-45.
[40]Colenbrander B, van StraatenHW,WensingCJ. Gonadotrophic hormones and testicular descent [J]. Arch Androl.1978,1:131-7.
[41]Bergh A, Rooth P, Widmark A, Damber JE. Treatment of rats with hCG induces inflammation-like changes in the testicular microcirculation [J]. J Reprod Fert. 1987,79:135-43
[42]Kaleva M, Arsalo A, Louhimo I, Rapola J, Peerentupa J, Henrikisen K, et al. Treatment with human chorionic gonadotropin for cryptorchidism:clinical and histological effects [J]. Int J Androl.1996,19:293-98.
[43]Rudolfsson SH, Wikstrom P, Jonsson A, Collin O, Bergh A. Hormonal regulation and functional role of vascular endothelial growth factor A in the rat testis [J]. Biol Reprod.2004,70:40-7.
[44]Assmus M, Svechnikov K, von Euler M, Setchel B, Sultana T, Zetterstr "om C, et al. Single subcutaneous administration of chorionic gonadotropin to rats induces a rapid and transient increase in testicular expression of pro-inflammatory cytokines [J]. Pediatric Res.2005,57:896-901.
[45]Li X, Strauss L, Makela S, Streng T, Huhtaniemi I, Santti R, et al. Multiple structural and functional abnormalities in the p450 aromatase expressing transgenic male mice are ameliorated by a p450 aromatase inhibitor [J]. Am J Pathol.2004,164:1039-48.
[46]Kumagai A, Kodama H, Kumagai J, Fukuda J, Kawamura K, Tanikawa H, et al. Xanthine oxidase inhibitors suppress testicular germ cell apoptosis induced by experimental cryptorchidism [J]. Mol Hum Reprod.2002,2:118-23.
[47]DeFoor WR, Kuan CY, Pinkerton M, Sheldon CA, Lewis AG Modulation of germ cell apoptosis with a nitric oxide synthase inhibitor in a murine model of congenital cryptorchidism [J]. J Urol.2004,172:1731-5.