Low Expression Of CR16Changes The Structure And Function Of Sertoli Cells In Mouse

Objective To observed the CR16expression changes by using chemicallysynthesized small interfering RNA (siRNA) transfected mouse Sertoli cell line TM4cells, screening the effective interference sequence, provided an experimental basisfor the subsequent study.Methods Designing three different siRNA sequence (si-CR16sequence1、2and3)and determination the proper transfection concentration after investigating theCR16mRNA target sequence on the basis of the expression of the endogenous CR16in TM4through the fluorescence-labeled siRNA (Cy3-CR16-siRNA). Threetargeting CR16siRNA were synthesized and transfected into TM4cells with themediation of Lipofectamine2000, a blank control group (untransfected), reagentscontrol group (supplemented only Lipofectamine2000) and the negative controlgroup.(turn stained negative siRNA, N-control siRNA). CR16mRNA and proteinexpression were respectively detected at48h and72h after RNA transfectionrespectively by using real-time quantitative PCR (qRT-PCR) and Western-Blot.Results The endogenous expression of CR16was detected in TM4cells. When TM4cells were transfected with different concentration of Cy3-CR16-siRNA, thefluorescence intensity was observed progressively increasing with the increasedconcentration of transfection. After20nM Cy3-siRNA transfected into TM4cellsonly emitted slight red fluorescence, while those with50nM、100nM and150nMemitted brighter fluorescence. So50nM was selected as the effectively transfectedconcentration. Three si-CR16sequences were transfected into TM4cells, the CR16expression level were decreased significantly compared with the blank control group(P <0.01), espeically the si-CR16sequence3, while there has no significant changeof the CR16mRNA expression in si-CR16sequence1and2group.Conclusion This section is successfully screened out for effective CR16mRNAtarget sequence the interference sequence si-RNA3, and the best transfectionconcentration50nM were screened by fluorescent transfection. The expression ofCR16and CR16mRNA were both reduced after CR16transfected. Objecive To observe the CR16and N-WASP localization and quantitativeexpression in TM4cells of the blank group、reagent group、negative control groupand the interference group, to further explore both the relevance and the role of thefunction in TM4cells.Methods Using immunocytochemistry, double immunofluorescence, WB (westernblot) and reverse transcription-polymerase chain reaction (reverse transcriptpolymerase chain reaction, RT-PCR) detection the exression of CR16protein andthree mRNA of CR16mRNA N-WASPmRNA and the Arp2/3mRNA in the TM4cells. Results Double immunofluorescence shows CR16and N-WASP co-expressed inTM4cells, mainly expressed in the Sertoli cell cytoplasm、cell membrane and theconnection between Sertoli cells and the Spermatogenic cells, after RNA interferencethe expression of CR16was significantly weaker than the control group, thisinterference effect is more apparent with time extended. The CR16mRNA expressionafter CR16interference at72h (0.2468±0.034) and48h (0.0986±0.026) cell groupwas weaker than the group at24h(0.2856±0.032), but there was not statisticallydifference(P>0.05) between72h and24h. While the CR16mRNA expression at24h、48h and72h comparied with each other has significant difference(P <0.01). ThreemRNA expression after interference in three periods comparied with the blank grouphave statistically significant differences (P <0.01). The CR16mRNA expression at48h interference group (0.0986±0.026) was significantly weaker than the blankgroup (1.000±0.001)(P <0.01). At the48h and the72h after different times of thetransfection, the same kind mRNA exression was statistically significant different (P<0.05), and the48h CR16mRNA expression orresponding withN-WASPmRNA(0.1896±0.028) and Arp2/3mRNA (0.2088±0.035). N-WASPmRNAat24h and48h, both have significant difference comared with72h after transfection(P <0.01). The difference of Arp2/3mRNA between48h(0.2088±0.035) and72h(0.2357±0.042) was statistically significant (P <0.05), meanwhile compared with24h、48h and72h respectively, shows a significant difference (P <0.01).Western Blotanalysis shows the similar changes with three mRNA expression in TM4cells,48hafter transfection three proteins and mRNAs expression also presents a good timeeffect and achieve to the best inhibition level.Conclusion The expression of the protein and mRNA were significantly lowercomparied with control group, and the N-WASP and Arp2/3expression are consistentwith CR16. Immunocytochemistry results show that CR16showed low expression at48h after RNA interference, Speculate that it presumably related with the TM4cellsactivity suppressed, three proteins expression consistant with the the three mRNA expression levels, double immunofluorescent labeling results further confirm thatCR16and N-WASP expressed in testis Sertoli cells forming a CR16/N-WASPcomplex. Objective By studying the morphology and function of sertoli cell after RNAinterference, preliminary explore the relationship between CR16and spermtogenesis,exploring the role and mechanism of CR16in spermtogenesis.Method Using HE staining, R250cell skeleton staining and transmission electronmicroscopy (TEM) to observe the general structure and cytoskeleton changes inSertoli cells in different treated groups, observation the β-tubulin expressionchanges by immunofluorescence.Results HE staining shows, there has no significant morphological changes in blankgroup, reagent group, negative sequence group and interference group, the TM4morphological integrity was unspoiled in microscope. R250cytoskeleton stainingshows that cytoskeletal changes of interference group compared with the controlgroup, the reagent group and negative sequence groups, connections between cellsfracture increased, a large number of vacuoles in the cytoplasm, electron microscopeshows interference group has more fat droplets and autophagic vacuoles, endoplasmicreticuluming, microtubules and microfilaments fracturing, nuclear chromatincondensation in the interference group, the nuclear membrane is rough, cell surfaceapophysis disappeared. The microtubule protein fluorescent displays that the β-microtubule protein was decreased after CR16interference, especially at theconnection between the cell body and cells, but cytoplasmicβ-tubulin expression did not change significantly, and β-tubulin expression also did not change significantlyamong the blank group, the reagent group and the NC sequence group.Conclusion The TM4cytoskeleton structure of interference group damageSeriously, cell membrane and nuclear membrane is not smooth, appearing apoptosisand autophagic vacuoles, microtubules and microfilaments fracturing, the connectionsbetween cells swelling, β-microtubule expression decreased significantly at theconnections between cells. Prompt that the normal expression of CR16has animportant role for keeping cytoskeleton of the TM4cells. Speculating that the lowexpression of CR16may reduce the polymerization of cytoskeleton actin and the Arp2/3, in turn affecting the morphology and function of the Sertoli cells, and resulting inabnormal spermatogenesis (especially late sperm maturation and sperm migrationmovement).