【作者】 黄东晖；

【导师】 熊承良；

【作者基本信息】 华中科技大学 ， 妇产科学， 2007， 博士

【摘要】 精子发生是个复杂、多步骤的过程,可分为三个过程:精原细胞的增殖更新、精母细胞的成熟分裂和精子细胞变态为精子三个阶段。目前发现在曲细精管中uPAR主要表达在精子细胞,可能与精子的排放有关。Tong Zhang等应用原位杂交技术检测发现猕猴睾丸支持细胞在精子发生的特定阶段(VII～VIII期)表达uPA mRNA;uPAR mRNA由睾丸生精细胞产生,但是不同发育阶段及不同生殖细胞表达的差异表明uPA/uPAR系统可能在精子发生过程中的特定阶段起作用。Vassalli认为支持细胞这种时期特异性表达的uPA与生殖细胞uPAR结合后可能是通过激活uPAR介导的信号传导通路作为生长因子而起作用。但是uPAR是通过激活何种信号传导通路来调节精子的发生过程目前尚不清楚。uPAR是否还通过信号传导来刺激生精细胞的发生及分化功能呢?因此,uPAR在精子发生中的作用机制尚待进一步研究。然而由于睾丸组织结构和功能的复杂性,一般情况下很难在整体情况下对生殖细胞的生理生化功能和与其他细胞的相互作用进行深入研究。生精细胞培养技术的建立为深入地了解生精细胞发育过程,研究生精细胞自身及与其它生精细胞的关系,提供了一种新的、强有力的工具。RNA干扰(RNAi)是一种由双链小干扰RNA(siRNA)引发的转录后基因沉默(PTGS)新型机制,可导致靶基因mRNA的降解及特异性基因沉默信号的扩增。目前siRNA已经成为选择性沉默体外培养的哺乳细胞基因表达的一个有力工具。本实验首先应用实时RT-PCR和Western Blot检测出生后不同发育阶段大鼠睾丸中uPAR基因及蛋白表达的变化,然后建立的生精细胞体外培养体系,最后应用siRNA沉默技术阻断体外培养生精细胞uPAR的基因表达,为研究uPAR在精子发生中作用机制及信号传导通路的研究提供新的重要研究手段和技术平台。一、uPAR在大鼠睾丸第一精子发生波中的表达变化目的:为探讨uPAR在大鼠精子发生中的作用,研究在第一个精子发生波中大鼠睾丸组织uPAR的mRNA表达及蛋白表达变化。方法:将SD大鼠按生后年龄分组,以出生当天为d0,取出生后d0、d5、d10、d15、d21、d28、d35、d42、d49、d56大鼠睾丸组织,用实时荧光定量分析方法检测各年龄组大鼠uPAR mRNA表达,并用Western印迹法检测各年龄组大鼠uPAR蛋白表达。结果:大鼠睾丸组织uPAR mRNA表达与蛋白表达表现出相似的趋势:刚出生时表达水平较高,后逐渐下降,约在d15天达最低,到d28时开始增加,到d35时到达高峰,随后d42下降,后保持一定的水平。双变量回归相关分析显示uPAR mRNA表达与蛋白表达为中度正相关。结论:大鼠睾丸uPAR表达在第一精子发生波中出现两个高峰,第一高峰在刚出生时,这个时期生殖母细胞向基底部迁移的时期,这在精子以后的发生中起着至关重要的作用,说明uPAR与精子发生的启动有着密切联系。第二高峰是在第5周升高明显,第5周为精子变形并开始向管腔排放的时期,这说明uPAR可能参与精子排放过程中的组织重塑及精子变态过程。二、大鼠睾丸生殖细胞培养体系的建立大鼠睾丸生殖细胞培养体系的建立是体外研究睾丸精子发生过程中各种调控因素的的重要工具。本实验通过建立支持细胞及生精细胞两种培养体系,为进一步研究uPAR在精子发生中的作用奠定基础。(一)大鼠睾丸支持细胞的分离纯化和鉴定目的培养高纯度的大鼠睾丸支持细胞,并应用检测ABP mRNA原位杂交方法鉴定分离培养的支持细胞。方法选用18～22天龄SD雄性大鼠睾丸,采用0.25%胰蛋白酶、0.1%透明质酸酶、0.1%胶原酶三酶依次消化法分离支持细胞,放于32℃5%CO2的培养箱培养,48小时后用20mmol Tris—HCl低渗处理培养细胞。培养一周后应用伊红染色、吖啶橙荧光染色、Feulgen染色对所培养支持细胞进行鉴定,同时应用原位杂交检测ABP mRNA方法鉴定分离培养的支持细胞。结果培养一周后所获培养的支持细胞纯度达95%以上。分离培养的支持细胞ABP mRNA表达阳性,其形态结构特征与用其它方法鉴定为支持细胞的形态结构特征一致。结论采用三酶连续消化及低渗处理法分离培养的支持细胞纯度高,而且应用原位杂交方法检测ABP mRNA是一种新的、特异、有效的鉴定支持细胞及其功能的方法。(二)大鼠睾丸支持细胞/生精细胞共培养系统的建立目的建立生精细胞体外分化培养体系,为uPAR在精子发生中作用的体外研究提供一个很好的研究模型。方法取20～22天龄SD雄性大鼠睾丸,去被膜及血管,放在预冷的Hank’s液中洗两次,然后用加1 mg/ml胶原酶的F12/DMEM液32℃消化15～20min。用眼科剪将睾丸剪碎成非常小的片断,再重复用1 mg/ml胶原酶消化5～10min。细胞用内含10%胎牛血清及各种营养因子的HamF12/ DMEM培养液悬浮,约按1*106CELL/cm2的密度接种于双室培养槽或放有盖玻片的六孔板中,在32℃、5%CO2条件下培养。相差显微镜下动态观察细胞生长、形态等变化,及定期应用伊红染色、Brdu染色法对所培养生精细胞/支持细胞进行染色鉴定。结果在生精细胞/支持细胞共培养体系中,双室培养2周后,部分生精细胞胞体一端有鞭毛出现,培养4周后,培养体系中仍有一定数量的生精细胞附着在支持细胞上,部分生精细胞上可见鞭毛。单室培养的生精细胞在培养第4-5天开始出现明显脱落,培养1周后大多数生精细胞发生脱落死亡,生精细胞上未见鞭毛出现,但在培养体系中可见次级精母细胞及精子细胞。结论:应用双室培养,生精细胞可存活达一月之久,而且部分生精细胞尾部出现鞭毛,从形态上发生了减数分裂及精子变态过程。三、应用siRNA沉默共培养生精细胞uPAR的基因表达目的利用小分子干扰RNA(siRNA)技术沉默共培养生精细胞uPAR的基因表达,为uPAR在精子发生中的作用研究提供一个很好的研究工具。方法siRNA混合鸡尾酒是根据ShortCut RNAi Kit手册来制备。简单的说,首先通过RT-PCR应用加有T7启动子的特异性引物来合成DNA模板,然后DNA模板通过体外转录过程产生双链RNA,最后通过ShortCut RNaseIII酶消化来制备siRNAs混合物。在培养48小时用Transfect转染试剂分别将15nM、30nM siRNA混合物转染到共培养的生精细胞,48小时应用实时荧光定量RT-PCR方法检测共培养生精细胞uPAR的基因表达。该试验设立两个对照组:空白对照(未加siRNA及转染试剂)及阴性对照组(只加转染试剂,无siRNA)。结果15nM及30nM siRNA转染组uPAR mRNA表达量均明显低于阴性对照组及空白对照组(p<0.05),其中以30nMsiRNA转染组更为明显;相对于阴性对照组,15nM及30nM siRNA转染组uPAR的基因表达抑制率分别为63.5%及76.7%。结论应用ShortCut RNase III法制备的siRNAs鸡尾酒能有效地抑制共培养生精细胞uPAR基因的表达。五、结论在本研究中我们发现uPAR在第一精子发生波中表达最高峰为出生后35天,此时为圆形精子转变为伸长精子,并开始向管腔排放的时期。在我们的共培养体系中,来自20～22天大小的大鼠睾丸组织的生精细胞在培养二周后能分化成精子细胞及变形成为伸长型精子。而这些变化相应于体内uPAR的表达出现高峰期所发生的变化。因此在本培养体系中,应用siRNA沉默uPAR的表达,可以为研究uPAR在精子发生特定时期中的作用及机制提供一个很好的研究工具,如其信号传导通路等。此研究模型的建立也有助于减数分裂及精子变态过程的其它调控因素的研究。更多还原

【Abstract】 Spermatogenesis is a complex differentiation process that consists of three major phases: a proliferation phase of spermatogonial cells by mitosis, a meiotic phase of spermatocytes in which recombination of genetic materials and reductive division occur and a transformation phase which produces mature spermatozoa from haploid spermatids. Tong Zhang et al investigated the expression and localization of mRNAs for urokinase PA(uPA), uPA receptor(uPAR) in monkey testes by using in-situ hybridization, the results demonstrated that uPA mRNA was expressed stage-specifically in Sertoli cells of adult testis, and uPAR mRNA was localized in germ cells of mature testis. The expression levels differed among different germ cells and at different developmental stages indicated uPA/uPAR system may play an important role in the specifical stage of spermatogenesis. The stage-specifically expressed uPA in Sertoli cells may bind to its receptor on germ cells and act as serine protease, or as growth factor through activation of signal transduction pathways by uPA receptor. It is still unclear which signal transduction pathways can be activated by uPAR in spermatogenesis. Can uPAR modulate growth and differentiation of spermatogenic cell by its signal transduction pathway? So the role of uPAR and its mechanism in spermatogenesis is worthy of further study.Because of complex of structure and function of testitular, the physiology and biochemistry function of spermatogenic cell is difficult to further research. Establish of spermatogenic cell culture system can provide a powerful tool to study the relationship between spermatogenic cell and other cell and investigate gene regulation in spermatogenesis. RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. siRNA pairs with its cognate mRNA, leading to degradation of target mRNA and amplification of gene-specific silencing signals. siRNA has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. In present study, firstly gene and protein expression of uPAR in rat testis at first wave of spermatogenesis was investigated by real time RT-PCR and Western Blot respectively, secondly germinal cell culture system was set up, finally siRNA cocktails were used to silience the gene expression of uPAR in the co-cultured germinal cells. This research provide a new study method and technology platform to investigate the role and signal pathways of uPAR in spermatogenesis.1.Expression of uPAR in rat testis at first wave of spermatogenesisObjective: To elucidate the role of urokinase-plasminogen activator receptor (uPAR) system in rat spermatogenesis,gene and protein expression of uPAR in rat testis at first wave of spermatogenesis was investigated. Method: The day of birth was designated as postnatal d0 ,the male rat were divided into 10 groups according to different postnatal stages, that is, d0, d5, d10, d15, d21, d28, d35, d42, d49, d56. The gene and protein expression of uPAR in rat testis was quantitated by Real-time polymerase chain reaction (PCR) and western blot methods separately. Result: Gene and protein expression of uPAR had similar tendency in testis of rats at first wave of spermatogenesis: Express level of uPAR situate at high relatively at postnatal d0, declined to a lowest level at postnatal d15, then increased obviously at postnatal d28 and reached peak at postnatal d35, then declines at postnatal d42 and retained low afterwards. Regression and correlation analysis showed that there was a moderately positive correlation between the gene and protein expression levels of uPAR. Discussion:Expression of uPAR exhibited two peak in testis of rats at first wave of spermatogenesis: first peak was at short after birth, when gonacyte migrated to the basilar part of seminiferous tubules and was crucial to initiate spermatogenesis, which indicated uPAR had intimate relationship whit onset of spermatogenesis. Second peak was at postnatal d35 when round spermatids transformed to elongate spermatids and began to spermiation, which indicated uPAR may be involved in spermiation and/or spermiogenesis. 2.Establish of germinal cell culture system of rat testisEstablishment of germinal cell culture system of rat testis is an important instrument to investigate the regulator involved in spermatogenesis. This experiment is to set up both sertoli / spermatogenic cells coculture system for further research of role of uPAR in spermatogenesis.2.1 Isolation、purification and identification of sertoli cells from rat testisObjective To obtain highly pure cultured sertoli cells from rat testis and identificate cultured cell by in situ hybridization for ABP mRNA. Methods Testis from 18～22-day-old SD rats were removed and decapsulated, then chopped and sequentially digested with three enzymes at 37℃: first with 0.25% trypsin for 15 minutes, then with 0.1% hyaluronidase for 30 minutes, last with 0.1% collagenase V for 2～3 hours. The isolated cells were incubated at 32℃in a humidified atmosphere of 5% CO2. To increase the purity of the Sertoli cells, cultured cells were subjected to hypotonic shock(treatment) with 20 mmol Tris—HCl after 48 hours incubation. After a week incubation, cultured cells were identificated by HE staining、AO fluorescence staining、Feulgen staining, at the same time by in situ hybridization with digoxin-labeled rat ABP cDNA probe. Conclusion Over 95% cultured cells were sertoli cells. Most of cultured cells expressed ABP mRNA and their structural features were the same as sertoli cells identificated by other methods. Results Highly pure sertoli cell can obstained by sequentially digested with three enzymes and hypotonic shock. As a specifical secreted protein by sertoli cells in testes, ABP mRNA detected by in situ hybridization is a new、specific、effective identification method of sertoli cells from testis. Meanwhile as an index of changes in sertoli cell function, this method can be used to study cultured sertoli cell function.2.2 Coculture of Rat Sertoli/Spermatogenic Cells Object To further study the role of uPAR in spermatogenesis in vitro, rat Sertoli/Spermatogenic cells coculture system was established. Method: The testis were removed from 20～22 day old SD rats and decapsulated. The tissues were washed twice with ice-cold Hank’s solution, were digested at 32℃in F12/DMEM containing 1 mg/ml collagenase at 32℃for 15～20 min, then were cut into small fragments and by 1 mg/ml collagenase again for 5～10 min. The cells were resuspended in culture medium supplemented various nutrition factors and 10% fetal calf serum, and seeded at about 106 cells/cm2 in bicameral chambers or culture plates covered with glass. Coculture was carried out at 32℃in a water-saturated atmosphere of 95% air and 5% CO2. The growth and morphology of coculture cells were monitored daily under contrast phase microscope, and were identificated by HE staining、Brdu staining at specific day. Results In bicameral culture system, flagella were seen emerging from one end of some spermatogenic cells after two-week coculture and a certain number of spermatogenic cells still attached on the surface of sertoli cell after four-week cultue while some of them has a short flagellum at one end. In unicameral culture system, spermatogenic cell began obviously to fall off the surface of sertoli cell after 4～5 day culture, there are only a few spermatogenic cells attached the surface of sertoli cell after one week coculture. But the second spermatocyte and spermatid were observed except for flagellum in this unicameral culture system. Conclusion: In bicameral culture system, the spermatogenic cells survived as long as four week, and some of them generated flagella at one end, which indicated that the process of meiosis and spermiogenesis could taken place in this culture system.3. Inhibit uPAR gene expression by siRNA cocktails in co-cultred spermatogenic cellsObjective To futher study the role of uPAR in spermatogenesis, uPAR gene expression were inhibit by siRNA cocktails in co-cultred spermatogenic cell. Methods siRNA cocktails were generated according to shortcut RNAi Kit handbook. Biefly,suitable DNA templates were generated by RT-PCR using specific primers with appended T7 promoters, then the DNA template produces double-stranded RNA by in vitro transcription, finally digested by ShortCut RNase III digestion to produces a mixture of siRNAs. 15nM or 30nM siRNA cocktails were transfected in co-cultured spermatogenic cell using RNAiFect Transfection Reagent at 48 hours of culture. After 48 hours transfected, expression of uPAR mRNA were ananysized by real time RT-PCR.. The study consisted of two control groups; blank control group treated with neither siRNA nor transfection reagent and negative control group treated only with transfection reagent. Results The expression level of uPAR mRNA with 15nM and 30 nM of siRNA cocktails were significantly lower than those of negative control and blank control group(p<0.05); The inhibition rate silencing with 15nM and 30 nM of the siRNA cocktails were 63.5% and 76.7% separately compared with negative control group. Conclution siRNA cocktail producted by shortcut Rnase III can efficially inhibited the gene expression of uPAR in co-cultured spermatogenic cell.ConclusionIn present study, expression of uPAR reached peak at postnatal d35 in testis of rats at first wave of spermatogenesis, when round spermatids transformed to elongate spermatids and began to spermiation. In our co-culture system, spermatogenic cell from 20-22 day old rat testis differentiated into spermatids and transformed to elongate spermatid during two-week co-culture. These changes were corresponding to those in vivo when expression of uPAR reached the peak. Hence, silencing uPAR gene expression with siRNA cocktails on this culture system can provide a powerful tool to further study the role and mechanism of uPAR involved in specific stages of spermatogenic cell development, such as its signal transduction in spermatogenesis. In the meantime, this model can also be helpful to research for other regulator invovled in meiosis and spermiogenesis.更多还原