小鼠精原干细胞体内增殖和分化阶段睾丸组织基因的差异表达
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摘要
研究背景：精原干细胞是一群具有高度自我更新能力和分化潜能的成体干细胞,是精子发生的起始。揭示精原干细胞增殖和分化的调控机制是促进精原干细胞体外培养和移植以及精子发生机制研究的重要基础。由于精原干细胞在生精细胞中的含量极低且缺乏特异性的表面标志,以及精子发生存在不同步性(在小鼠中精子发生的12个阶段同时存在于曲细精管生精上皮),这两方面因素使得直接研究精原干细胞在体内增殖和分化的分子调控机制非常困难。研究显示烷化剂白消安对生精细胞的杀伤作用具有剂量和物种相关性。若能利用白消安杀灭大部分精原干细胞,那么,为了恢复干细胞池的稳定,精子再生初期精原干细胞的更新会得到加强而其分化会被延缓,精原干细胞体内增殖与分化的相对同步就可能再这一过程中得到实现。
目的：探讨建立昆明小鼠精子再生模型的白消安适宜剂量,通过该剂量建立的较理想的精子再生模型实现精子再生初期精原干细胞增殖与分化的相对同步。在此基础上,采用基因芯片检测精原干细胞增殖和分化阶段小鼠睾丸组织基因表达谱的差异,初步探讨精原干细胞增殖和分化的分子调控机制。
方法：8-10周龄雄性昆明白小鼠按区组随机分为实验组(168只)和对照组(8只)。实验组小鼠接受白消安腹腔注射,根据不同给药剂量进一步分为4组：A、B两组各36只,分别接受10mg/kg和20mg/kg白消安单次注射；C、D两组各48只,分别接受10mg/kg和15mg/kg白消安间隔24d二次注射。对照组小鼠接受单次等量溶剂腹腔注射。于给药结束后1、2、3、4、6和8周,分别将各实验组小鼠按期颈椎脱臼处死(组内等量),对照组小鼠在注射后1周一次性处死。取出小鼠双侧睾丸。一侧睾丸采用HE染色观察曲细精管的组织形态学变化和免疫组化法检测C、D组和对照组生精细胞Ki-67表达;对侧睾丸组织采用电镜观察超微结构。通过前述研究判断建立精子再生模型的白消安适宜剂量和模型分期。以此适宜剂量白消安再次建立精子再生模型,分别选取精原干细胞处于增殖和分化阶段的睾丸组织标本,Trizol一步法提取组织总RNA并进行纯化和荧光标记。将经前述反应得到的标记DNA于42℃在芯片杂交仪上与36kMouse Genome Array杂交过夜。用双通道激光扫描仪扫描杂交芯片,对各芯片进行片间线性校正和片内归一化处理。对筛选得到的差异表达基因进行GO和KEGG信号通路分析。参照www. sabiosciences. com/gene_array_product/HTML/OMM-405. html.筛选差异表达的干细胞相关基因。实时定量PCR验证Kit、bFGF和Oct4表达。免疫组化检测Oct4和Thy-1在生精上皮的表达。
结果：第一周C、D两组生精上皮的损伤程度介于A、B两组之间。第二周,C、D两组生精上皮仅基底膜部残留以单个型精原细胞(As型)为主精原细胞和Sertoli细胞,D组生精上皮部分Sertoli细胞空泡变性;第三周,C、D两组As型精原细胞均增多;第四周,C、D两组均出现分化精原细胞和精母细胞；6-8周,两组均出现精子发生,C组生精上皮结构逐步恢复正常,而D组曲细精管直径和生精上皮厚度仍显著低于对照组。C、D两组精原细胞Ki-67阳性率在第一、二周显著低于对照组,在第三周极度增高,第四周开始下降,6-8周与对照组无显著差异,第二、三周D组精原细胞Ki-67阳性率均低于C组。基因芯片试验结果显示911个基因在精原干细胞增殖和分化阶段的睾丸组织中表达存在差异,其中,上调608个(增殖期／分化期),下调303个。这些差异表达基因分别涉及生物学过程、分子功能和分子组成。84个信号通路功能改变具有统计学意义(P<0.05),包括Notch和Wnt信号通路。与干细胞相关的差异基因有56个,上调40个,下调16个。部分干细胞的阳性标记物(如Cd9, Stra8, Itgb1,Oct4和Thyl)和部分生长因子(如Fgf2, Csfl和Pdgfa)上调。免疫组化染色结果显示Oct4抗原表达于曲细精管基底膜的精原细胞且在精原干细胞增殖期表达显著高于分化期。
结论间隔24天10mg/kg白消安二次腹腔注射是建立小鼠精子再生模型的理想剂量。该模型可实现精子发生的相对同步：二次给药后3周主要为精原干细胞增殖期,4周为分化期,6-8周为精子发生恢复期。小鼠精原干细胞增殖和分化过程的调控涉及许多基因(分属不同信号通路)的差异表达,进一步研究这些基因(和通路)功能有助于揭示精原干细胞增殖和分化的调控机制。
Background:Spermatogonia stem cells (SSCs), the origin of spermatogenesis, are a cluster of adult stem cells which have the potential of both self-renewal and differentiation. To uncover the mechanism behind SSCs proliferation and differentiation is a prerequisite to the progress in SSCs culturing and transplantation and regulation in spermatogenesis. The extremely low content of SSCs in germ cells and the concurrence of all stages of spermatogenesis in seminiferous tubules constitute two main obstacles to this mechanism research in vivo. Busulfan, an alkylating agent, can kill germ cells in a manner of dose and species dependant. If busulfan evanishes most SSCs, doubtless, SSCs' self-renewal will be strengthened and their differentiation suspended in the initial of spermatogenesis, from which synchronization of SSCs proliferation and differentiation could be achieved.
Objective:To establish a model of spermatogenesis recovery and achieve the relative synchronization of the SSCs proliferation and differentiation in male Kunming mice, and base on that, to detect the difference in gene expression patterns of mouse testis during SSCs proliferation and differentiation and probe preliminarily into the molecular regulation mechanism in SSCs proliferation and differentiation.
Methods:Adult male Kunming mice were randomly treated with busulfan injection intraperitoneally at different doses or types (A:single dose of 10mg/kg; B: single dose of 20mg/kg; C:two doses of lOmg/kg,24 days apart; D:two doses of 15mg/kg,24 days apart). Mice that received single injection of 50% DMSO (10ml/kg) were used as control. Testes were dissected out 1w,2w,3w,4w,6w and 8w after treatment. Testes of mice in control group were all taken out one week after the treatment. The expression of Ki-67 in germ cells was detected by immunohistochemistry in two doses of busulfan treatment groups and control group. A mice model of spermatogenesis regeneration was reestablished with the suitable busulfan dose determined by the aforementioned study. A 36k Mouse Genome Array was used to detect the differential gene expression profiles between stages of SSCs proliferation and differentiation. Bioinformatics analysis was conducted in GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway to describe the potential roles that may play in regulation of spermatogonial stem cells behavior. Oct4 and Thy-1 expression patterns in spermatogonia were detected by immunohistochemistry.
Results:The damage effects in seminiferous epithelium by two doses of 10mg/kg or 15mg/kg busulfan injection were between single dose of 10mg/kg and 20mg/kg. Two weeks after the second injection, only fewer dispersed spermatogonia (mainly As type) survived in close contact with the basal portions of adjacent Sertoli cells in group C and D, vacuolization of Sertoli cells appeared in some seminiferous tubulues in group D. In week 3, the number of As spermatogonia increased. In week 4, differentiated spermatogonia and spermatocytes appeared. In week 6-8, spermatogenesis regeneration took place and morphology of seminiferous epithelium got back to normal gradually in group C, while diameter of seminiferous tubulues and thickness of seminiferous epithelium in group D were significantly lower than control group. In week 1 and 2, the Ki-67 positive rates of spermatogonia in group C and D were extremely lower than control group. They rebounded rapidly to apex in week 3, descended in week 4 and restored to the control level in week 6 and 8. In week 2 and 3, Ki-67 positive rates of spermatogonia in group C were significantly higher than group D.911 differential expression genes were identified by gene arrays in mice testes, consisting of 608 up-regulated and 303 down-regulated in SSCs proliferation stage vs. SSCs differentiation stage. The differential expression genes were classified by their biological process, molecular function and cellular component, respectively. Alterations with statistical significance (P<0.05) appeared in 84 KEGG signal pathways, including Notch and Wnt signaling pathways which have been proved to be important for stem cell maintenance.56 differential expression genes were selected as genes related to stem cells, among which 40 genes were up-regulated, including some stem cell biomarkers (such as Cd9, Stra8, Itgbl, Oct4 and Thy1) and some growth factors (such as Fgf2, Pdgfa and Csfl). Oct4 antigen was expressed in spermatogonia located in the basal portions. The positive rate of Oct4 in spermatogonia in SSCs proliferation stage was higher than that in SSCs differentiation stage.
Conclusions:24 days apart two doses of 10mg/kg busulfan treatment could establish an ideal mice model of spermatogenesis recovery. Relative synchronization of spermatogenesis could be achieved in this model. After the administration, the 3rd week was mainly proliferation stage of spermatogonial stem cells, the 4th week was mainly differentiation stage, and week 6 to 8 was stage of spermatogenesis regeneration. The regulation of SSCs proliferation and differentiation involved in many genes expressed differentially locating in various signal pathways. This study provided a basis for the elucidation of the molecular mechanism behind self-renewal and differentiation of spermatogonial stem cells in vivo.
目的：探讨建立昆明小鼠精子再生模型的白消安适宜剂量,通过该剂量建立的较理想的精子再生模型实现精子再生初期精原干细胞增殖与分化的相对同步。在此基础上,采用基因芯片检测精原干细胞增殖和分化阶段小鼠睾丸组织基因表达谱的差异,初步探讨精原干细胞增殖和分化的分子调控机制。
方法：8-10周龄雄性昆明白小鼠按区组随机分为实验组(168只)和对照组(8只)。实验组小鼠接受白消安腹腔注射,根据不同给药剂量进一步分为4组：A、B两组各36只,分别接受10mg/kg和20mg/kg白消安单次注射；C、D两组各48只,分别接受10mg/kg和15mg/kg白消安间隔24d二次注射。对照组小鼠接受单次等量溶剂腹腔注射。于给药结束后1、2、3、4、6和8周,分别将各实验组小鼠按期颈椎脱臼处死(组内等量),对照组小鼠在注射后1周一次性处死。取出小鼠双侧睾丸。一侧睾丸采用HE染色观察曲细精管的组织形态学变化和免疫组化法检测C、D组和对照组生精细胞Ki-67表达;对侧睾丸组织采用电镜观察超微结构。通过前述研究判断建立精子再生模型的白消安适宜剂量和模型分期。以此适宜剂量白消安再次建立精子再生模型,分别选取精原干细胞处于增殖和分化阶段的睾丸组织标本,Trizol一步法提取组织总RNA并进行纯化和荧光标记。将经前述反应得到的标记DNA于42℃在芯片杂交仪上与36kMouse Genome Array杂交过夜。用双通道激光扫描仪扫描杂交芯片,对各芯片进行片间线性校正和片内归一化处理。对筛选得到的差异表达基因进行GO和KEGG信号通路分析。参照www. sabiosciences. com/gene_array_product/HTML/OMM-405. html.筛选差异表达的干细胞相关基因。实时定量PCR验证Kit、bFGF和Oct4表达。免疫组化检测Oct4和Thy-1在生精上皮的表达。
结果：第一周C、D两组生精上皮的损伤程度介于A、B两组之间。第二周,C、D两组生精上皮仅基底膜部残留以单个型精原细胞(As型)为主精原细胞和Sertoli细胞,D组生精上皮部分Sertoli细胞空泡变性;第三周,C、D两组As型精原细胞均增多;第四周,C、D两组均出现分化精原细胞和精母细胞；6-8周,两组均出现精子发生,C组生精上皮结构逐步恢复正常,而D组曲细精管直径和生精上皮厚度仍显著低于对照组。C、D两组精原细胞Ki-67阳性率在第一、二周显著低于对照组,在第三周极度增高,第四周开始下降,6-8周与对照组无显著差异,第二、三周D组精原细胞Ki-67阳性率均低于C组。基因芯片试验结果显示911个基因在精原干细胞增殖和分化阶段的睾丸组织中表达存在差异,其中,上调608个(增殖期／分化期),下调303个。这些差异表达基因分别涉及生物学过程、分子功能和分子组成。84个信号通路功能改变具有统计学意义(P<0.05),包括Notch和Wnt信号通路。与干细胞相关的差异基因有56个,上调40个,下调16个。部分干细胞的阳性标记物(如Cd9, Stra8, Itgb1,Oct4和Thyl)和部分生长因子(如Fgf2, Csfl和Pdgfa)上调。免疫组化染色结果显示Oct4抗原表达于曲细精管基底膜的精原细胞且在精原干细胞增殖期表达显著高于分化期。
结论间隔24天10mg/kg白消安二次腹腔注射是建立小鼠精子再生模型的理想剂量。该模型可实现精子发生的相对同步：二次给药后3周主要为精原干细胞增殖期,4周为分化期,6-8周为精子发生恢复期。小鼠精原干细胞增殖和分化过程的调控涉及许多基因(分属不同信号通路)的差异表达,进一步研究这些基因(和通路)功能有助于揭示精原干细胞增殖和分化的调控机制。
Background:Spermatogonia stem cells (SSCs), the origin of spermatogenesis, are a cluster of adult stem cells which have the potential of both self-renewal and differentiation. To uncover the mechanism behind SSCs proliferation and differentiation is a prerequisite to the progress in SSCs culturing and transplantation and regulation in spermatogenesis. The extremely low content of SSCs in germ cells and the concurrence of all stages of spermatogenesis in seminiferous tubules constitute two main obstacles to this mechanism research in vivo. Busulfan, an alkylating agent, can kill germ cells in a manner of dose and species dependant. If busulfan evanishes most SSCs, doubtless, SSCs' self-renewal will be strengthened and their differentiation suspended in the initial of spermatogenesis, from which synchronization of SSCs proliferation and differentiation could be achieved.
Objective:To establish a model of spermatogenesis recovery and achieve the relative synchronization of the SSCs proliferation and differentiation in male Kunming mice, and base on that, to detect the difference in gene expression patterns of mouse testis during SSCs proliferation and differentiation and probe preliminarily into the molecular regulation mechanism in SSCs proliferation and differentiation.
Methods:Adult male Kunming mice were randomly treated with busulfan injection intraperitoneally at different doses or types (A:single dose of 10mg/kg; B: single dose of 20mg/kg; C:two doses of lOmg/kg,24 days apart; D:two doses of 15mg/kg,24 days apart). Mice that received single injection of 50% DMSO (10ml/kg) were used as control. Testes were dissected out 1w,2w,3w,4w,6w and 8w after treatment. Testes of mice in control group were all taken out one week after the treatment. The expression of Ki-67 in germ cells was detected by immunohistochemistry in two doses of busulfan treatment groups and control group. A mice model of spermatogenesis regeneration was reestablished with the suitable busulfan dose determined by the aforementioned study. A 36k Mouse Genome Array was used to detect the differential gene expression profiles between stages of SSCs proliferation and differentiation. Bioinformatics analysis was conducted in GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway to describe the potential roles that may play in regulation of spermatogonial stem cells behavior. Oct4 and Thy-1 expression patterns in spermatogonia were detected by immunohistochemistry.
Results:The damage effects in seminiferous epithelium by two doses of 10mg/kg or 15mg/kg busulfan injection were between single dose of 10mg/kg and 20mg/kg. Two weeks after the second injection, only fewer dispersed spermatogonia (mainly As type) survived in close contact with the basal portions of adjacent Sertoli cells in group C and D, vacuolization of Sertoli cells appeared in some seminiferous tubulues in group D. In week 3, the number of As spermatogonia increased. In week 4, differentiated spermatogonia and spermatocytes appeared. In week 6-8, spermatogenesis regeneration took place and morphology of seminiferous epithelium got back to normal gradually in group C, while diameter of seminiferous tubulues and thickness of seminiferous epithelium in group D were significantly lower than control group. In week 1 and 2, the Ki-67 positive rates of spermatogonia in group C and D were extremely lower than control group. They rebounded rapidly to apex in week 3, descended in week 4 and restored to the control level in week 6 and 8. In week 2 and 3, Ki-67 positive rates of spermatogonia in group C were significantly higher than group D.911 differential expression genes were identified by gene arrays in mice testes, consisting of 608 up-regulated and 303 down-regulated in SSCs proliferation stage vs. SSCs differentiation stage. The differential expression genes were classified by their biological process, molecular function and cellular component, respectively. Alterations with statistical significance (P<0.05) appeared in 84 KEGG signal pathways, including Notch and Wnt signaling pathways which have been proved to be important for stem cell maintenance.56 differential expression genes were selected as genes related to stem cells, among which 40 genes were up-regulated, including some stem cell biomarkers (such as Cd9, Stra8, Itgbl, Oct4 and Thy1) and some growth factors (such as Fgf2, Pdgfa and Csfl). Oct4 antigen was expressed in spermatogonia located in the basal portions. The positive rate of Oct4 in spermatogonia in SSCs proliferation stage was higher than that in SSCs differentiation stage.
Conclusions:24 days apart two doses of 10mg/kg busulfan treatment could establish an ideal mice model of spermatogenesis recovery. Relative synchronization of spermatogenesis could be achieved in this model. After the administration, the 3rd week was mainly proliferation stage of spermatogonial stem cells, the 4th week was mainly differentiation stage, and week 6 to 8 was stage of spermatogenesis regeneration. The regulation of SSCs proliferation and differentiation involved in many genes expressed differentially locating in various signal pathways. This study provided a basis for the elucidation of the molecular mechanism behind self-renewal and differentiation of spermatogonial stem cells in vivo.
引文
1. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A.1994; 91:11303-11307.
2. Brinster RL, Zimmerman JW. Spermatogenesis following male germ cell transplantation. Proc Natl Acad Sci U S A.1994; 91:11298-11302.
3. Brinster CJ, Ryn BY, Avarbock MR, Karagenc L, Brinster RL. Restoration of fertility by germ cell transplantation requires efficient recipient preparation. Biol Reprod.2003; 69:412-420.
4. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Remodeling of the postnatal mouse testis is accomplished by dramatic changes in stem cell number and niche accessibility. Proc Natl Acad Sci U S A.2001; 98:6186-6191.
5. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Gem line stem cell competition in postnatal mouse testes. Biol Reprod.2002; 66:1491-1497.
6. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat Med 2000; 6:29-34.
7. Boettger-Tong HL, Johnston DS, Russell LD, Griswold MD, Bishop CE. Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis. Biol Reprod 2000; 63:1185-1191.
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9. Tegelenbosch, RAJ, de Rooij, DG. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutat Res, 1993; 290,193-200.
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11. Loh YH, Wu Q, Chew JL et al. The Oct-4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet,2006; 38:431-440. 29.
12. Ogawa T, Ohmura M, Tamura Y, et al. Derivation and morphological characterization of mouse spermatogonial stem cell lines. Arch Histol Cytol,2004; 67:297-306.30.
13. Nagano M, McCarrey JR, Brinster RL. Primate spermatogonial stem cells colonize mouse testes. Biol Reprod 2001; 64 (5):1409-1416.31.
14. Nagano M, Patrizio P, Brinster RL. Long-term survival of human spermatogonial stem cells in mouse testes. Fertil Steril 2002; 78 (6):1225-1233.
15. Oatley JM, Avarbock MR, Telaranta AI, et al. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci U S A.2006 Jun 20;103(25):9524-95.
16. Moisan AE, Foster RA, Betteridge KJ,et al. Dose-response of RAG2-/-/gammac-/-mice to busulfan in preparation for spermatogonial transplantation. Reproduction. 2003;126(2):205-16
17. Jiang FX. Behaviour of spermatogonia following recovery from busulfan treatment in the rat. Anat Embryol.1998,198:53-61
18. Bucci LR and Meistrich ML Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations. Mutat Res.1987,176:259-268.
19. Kim J-H, Jung-Ha H-S, Lee H-T and Chung K-S. Development of a positive method of male stem cell mediated gene transfer in mouse and pig. Mol Reprod Dev. 1997,46:515-526
20. Stellflug J, Green J and Leathers C. Antifertility effect of busulfan and Procarbazine in male and female coyotes. Biol Reprod.1985,33:1237-1243
1. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A.1994; 91:11303-11307.
2. Brinster RL, Zimmerman JW. Spermatogenesis following male germ cell transplantation. Proc Natl Acad Sci U S A.1994; 91:11298-11302.
3. Brinster CJ, Ryn BY, Avarbock MR, Karagenc L, Brinster RL. Restoration of fertility by germ cell transplantation requires efficient recipient preparation. Biol Reprod.2003; 69:412-420.
4. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Remodeling of the postnatal mouse testis is accomplished by dramatic changes in stem cell number and niche accessibility. Proc Natl Acad Sci U S A.2001; 98:6186-6191.
5. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Gem line stem cell competition in postnatal mouse testes. Biol Reprod.2002; 66:1491-1497.
6. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat Med 2000; 6:29-34.
7. Jiang FX. Behaviour of spermatogonia following recovery from busulfan treatment in the rat. Anat Embryol.1998,198:53-61
8. Moisan AE, Foster RA, Betteridge KJ,et al. Dose-response of RAG2-/-/gammac-/-mice to busulfan in preparation for spermatogonial transplantation. Reproduction. 2003;126(2):205-16
9. Bucci LR and Meistrich ML Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations. Mutat Res.1987,176:259-268.
10. Kim J-H, Jung-Ha H-S, Lee H-T and Chung K-S. Development of a positive method of male stem cell mediated gene transfer in mouse and pig. Mol Reprod Dev. 1997,46:515-526
11. Stellflug J, Green J and Leathers C. Antifertility effect of busulfan and Procarbazine in male and female coyotes. Biol Reprod.1985,33:1237-1243
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