Inhibition Of UPAR Gene Expression By SiRNA Cocktails In Co-cultured Spermatogenic Cells

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.