| BackgroundThere is nearly 100 years controversy whether or not ovary stem cell can replenish primordial follicle pool in postnatal higher vertebrate.Recent studies showed that female mammals still could produce germ cells after birth,It imply that there are certain numbers of Ovarian germline stem cells（OGSCs）or Female germline stem cells（FGSCs）to exist in the postnatal mammalian ovary.This kind of germline stem cells（GSCs）is generally located in the ovarian surface epithelium（OSE）.In the whole reproductive cycle of female mammals,OGSCs can provide new oocyte to supply the primordial follicle pools and slow down the consumption and exhaustion follicle by constant proliferation and differentiation.Hence,the exploration of OGSCs proliferation and differentiation can provide new thought to comprehend ovarian function and offer a new straegy to therapy premature ovarian failure or recover infertility phenomenon.Although OGSCs was discovered in the postnatal mammalian ovary,the exact number,location,and proliferation ability of OGSCs are still unknown.Especially,with the ovary aging,how does the number and proliferation ability of OGSCs changed?What is the molecular mechanism to regulate the proliferation and differentiation of OGSCs?These queries are worthy of research in depth.As one of the classic signal pathways,the Notch signal pathway mainly regulates the cellular proliferation and differentiation,it plays a key role in cellular development and cell fate determination.In the regulation of stem cells,the main function of the Notch signal pathway includes maintaining constant self-renewal and regulating differentiation.The research results of invertebrate drosophila showed that the Notch signal pathway has taken part in the process of drosophila ovarian stem cells formation,it can promote the proliferation and prohibit differentiation of OGSCs.In addition,it was also found that the proliferation ability of ovarian stem cells will weaken along with the ovary aging.Interestingly,meanwhile,the activity of the Notch signal is decreased too,and the lifetime of ovarian stem cells was prolonged when the activity of the Notch signal was increased.Therefore,the Notch signaling pathway plays an important role in regulating the proliferation and differentiation of stem cells,especially the drosophila ovarian stem cells.In the ovaries of mice,Notch signaling pathway was discovered to participate in the formation of primordial follicle,which indicated the Notch signaling pathway also regulates the mammal ovarian function.However,there’s still not report about whether the Notch signaling pathway can regulate the proliferation and differentiation of mice OGSCs,and if there is some relationship between the Notch signaling pathway and OGSCs.Section 1 The proliferative ability of OGSCs in the ovarian surface epithelium alters along with the reproductive age and the correlation with the Notch signaling pathway.Objectives:It’s considered that OGSCs in the ovary are mainly located in the OSE.Here,the research sought to clarify how the proliferation ability changes of OGSCs along with reproductive aging of mouse ovary,and whether this change is due to the change of Notch signal pathway activity.Methods:1.Animal:laboratory mice;female mice of various reproductive ages originated from KunMing which were fed with a free diet in normal state;the laboratory mice meet with approbation of Animal and Ethics Committee of Nanchang University.2.Real time PCR and Western blot detection:The mice ovaries of 3D、2M、12M、20M were extracted,respectively.Then OSE tissue and cells were collected under the stereomicroscope as soon as possible by using cell brush.The total RNA and protein were collected,Real time PCR and Western blot were used to detect various reproductive ages the expression level of the germline stem cells makers Mvh、Oct4,the Notch signaling pathway important molecular Notchl and key target molecular Hes1、Hes5.3.Hematoxylin-sosin（HE）Staining and Immunohistochemistry（IHC）detection:The mice ovaries of 3D、2M、20M were extracted and ovarian appendage were removed as clean as possible,respectively.Then ovaries were fixed by 4%formaldehyde solution overnight.The routine HE staining and IHC were used to determine the expression levels of Mvh、Oct4、Notchl、Hesl and Hes5 in the OSE of various reproductive ages ovaries.4.Dual-Immunofluorescence（Dual-IF）test:The mice ovaries of 3D、2M、20M were extracted,respectively.After the paraffin sections were dewaxed and antigen retrieval,serum blocking and other operations,Dual-IF was used to detect the co-expression of Mvh/Notchl,Mvh/Hes1 and Mvh/BrdU in OSE of various reproductive ages ovaries.5.Data Process and Statistic analysis:All data were analyzed by SPSS18.0 software package.The quantitative data were demonstrated with means±standard deviation（x±s）,and a mean square;student’s t test was used in the comparison between groups.p<0.05 was considered to be significant,and p<0.01 and p<0.001 were considered extremely significant.Results:1.The expression tendency of gremline stem cell markers and key Notch pathway molecules in the mouse OSE:the Real time PCR and Western blot results indicated that the mRNA and protein level of Mvh and Oct4 were the highest in 3D mice and were extremely down-regulated in 2M mice.There was little Mvh and Oct4 expression in 12M and 20M mice（p<0.05）.The key components of the Notch pathway（Notchl,Hesl and Hes5）were significantly reduced along with the increasing reproductive age.However,compared with Mvh and Oct4,expression of these Notch pathway molecules was still observed at 12M and 20M（p<0.05）.2.HE staining and IHC detected the expression of gremline stem cell markers and key Notch molecules in the OSE:the HE staining results indicated that the OSE of 3D and 2M mice are integrated,and the cell arrangement is more compact.The cell nucleus is blue and the cytoplasm is red,the OSE of 20M is incomplete,and the cell arrangement is loose.The IHC results showed that MVH and OCT4 expression was highest in the OSE at 3D,a higher level was observed at 2M,and there was less or no staining at 20M.In addition,we detected the expression of Notch signaling components,the results showed that the expression levels of NOTCH1,HES1 and HES5 were localized in the OSE in all three age stages,but compared to the results at 3D,there was an obvious decrease in expression at 2M and 20M.3.Germline stem cell markers and Notch components are co-expressed in the OSE:We adopted Dual-IF to detect the co-expression between germline stem cell markers and Notch molecules in the OSE.The Dual-IF results showed that MVH（green）and NOTCH1（red）are co-expressed in the cortical layer of ovaries in a pattern that showed the strongest expression at 3D,clearly decreased expression at 2M and almost undetectable expression at 20M.Similar co-expression patterns appeared for both MVH（green）and HES1,BrdU（red）.Conclusions:1.There are a certain number of OGSCs exist in the OSE in postnatal mice,its number was reduced or the ability of proliferation was weakened with the increase of reproductive age of mice.It is embodied that relative major OGSCs were found in the OSE of postnatal mice and there still exists a certain number of OGSCs after reproductive maturity,but hardly OGSCs were found in the OSE of aged mice.2.The Notch signaling pathway molecules expressed in the OSE,and the expressive level will decrease along with mouse reproductive aging.3.In the OSE,there exists a co-expression between germline stem cells markers and Notch signaling molecules.Furthermore,there is a concordance expressive tendency for both components,which are weakened along with reproductive aging.Therefore,the results indicated that Notch signaling pathway are related to the proliferation ability of OGSCs.Section 2 The effect on the proliferation ability of OGSCs and follicular development after alter the Notch signaling pathway activity in miceObjectives:This experiment intend to observe the effects on proliferation of OGSCs in OSE,OGSCs after primordial culture and follicular development through altering Notch signaling pathway activity,and further exploring the function of Notch signaling in regulating the proliferation of the OGSCs.Methods:1.The culture of the ovary in vitro and construction of the infertility mice model The ovaries of various reproductive-aged mice were intact extracted under the stereomicroscope as soon as possible.Those ovaries were washed with the pre-cooling D-hanks and placed in the transwell room and cultured with the Waymonth system supplemented 10%FBS in 37℃,5%C02 condition.The 6-8W female mice were treated with busulfan and cyclophosphamide to create an infertility mice model via intraperitoneal injection.2.Construct over-expressed lentivirus vector of Notch signal pathway:According to the intracellular NICD sequence of Notch 1（Genbank:NM008714.3）,using cDNA of mice as template to amplify the total length of the sequence of NICD.Lentivirus expressed vector、pGag/Pol、pRev、pVSV-G four plasmid co-transfect into 293T cells to package the over-expressed lentivirus particle.The particle was named LV-N1ICD and measured the titration.Finally,the virus was determined efficiency by infected 293T cells and stored in-80℃.3.Primary isolation and culture of OGSCs:approximately 20 ovaries from 5-7D mice were isolated under the stereomicroscope.Then ovaries were cut into pieces as soon as possible and digested with the collagenase and pancreatic enzyme.Cells precipitate was collected by centrifuge.The germ cell specific marker Mvh was taked as primary antibodies to enrich OGSCs via immunomagnetic bead sorting technology.Finally,OGSCs were cultured on the feeding layer STO cells which pre-treated with mitomycin C.4.Real time PCR and Western blot detected the germline stem cells marker Mvh and Oct4 expression after Notch pathway activity had been increased or decreased in ovaries culture in vitro:the ovaries of 5-7D mice were collected and randomly divided into 4 groups:Group DMSO,Group DAPT 10μtM,Group DAPT 20μM and Group DAPT 40μM.The OSE of ovaries were collected by cell brush after treated with DAPT for 48h,and total RNA and total protein of OSE were extracted.The Real time PCR and Western Blot were used to detect the expressive level of marker molecular Mvh,Oct4,Hesl and Hes5 in OSE.The ovaries of 5-7D mice were collected and randomly divided into group LV-Control and group LV-N1ICD,Total RNA of OSE were extracted after treated for 48h,the expressive level of marker molecules Mvh、Oct4、Hes1、Hes5 were detected by Real time PCR.5.Real time PCR detected the germline stem cells marker Mvh and Oct4 expression after Notch pathway activity had been increased or decreased in OGSCs:OGSCs were subcultured and divided into group DMSO、group DAPT 10μM;group LV-Control、group LV-N1ICD,Total RNA of OGSCs were extracted after treated for 48h,the expressive level of marker molecules Mvh、Oct4、Hes1、Hes5 were detected by Real time PCR.6.Cells dual-IF detection:The OGSCs were fixed with Carnoy fixative and processed with routine IF operations,primary antibodies Mvh/Hes1,Mvh/Hey2,Mvh/Oct4 were added to detect the co-expression between germ stem cells marker and Notch pathway key molecules,respectively.7.HE staining and tissue Daul-IF detected the follicular development and the Mvh,Oct4 expression after Notch pathway activity had been increased or decreased in OSE:the ovaries of 5-7D mice were collected and randomly divided into 3 groups:Group DMSO,Group DAPT 20μM and Group DAPT 40μM.After treated with DAPT for 48h,ovaries were fixed by 4%paraformaldehyde overnight,and cut into pieces after paraffin embedding.The follicles development of whole ovary was observed by HE staining,the co-expressed level of Mvh/Oct4 in OSE was detected by Daul-IF.8.Living microinjection for mice model:normal 6-8W female mice were divided into group DMSO and group DAPT,the skin and muscle of mice were scissored off after anesthesia in aseptic conditions,then ovaries were exposed and microinjected.Infertile female mice models were divided into group LV-Control and LV-N1ICD,the same method were used to microinject mice model.Skin and muscle were seamed after injecting and fed in normal condition.9.ELISA（indirect method）detected the concentration change of FSH、P、E2 in mice serum:the mice were microinjected DAPT or LV-N1ICD respectively,and the concentration of FSH、P、E2 were detected at 7D and 14D after microinjection.10.Mated with wild male mice:the mice were microinjected DAPT or LV-N1ICD respectively,and the offsprings number were counted at 20D、50D and 80D after mated.11.IHC detection:the ovaries were isolated from the No.8 mircoinjection mice,HE staining was used to observe the follicle development of Group DAPT,group LV-N1ICD after injected 7D、14D,respectively.IHC was used to detect the Mvh and Oct4 expression level of Group DAPT,group LV-N1ICD in OSE after injected 7D.12.Alkaline phosphatase（ALP）detection:The OGSCs were subcultured and divided into 2 groups:group DMSO,group DAPT lOuM;the same method,OGSCs were also divided into group LV-Control and group LV-N1ICD.All cells groups were fixed with 4%formaldehyde after cultured for 48h,and observed the result under light microscope after the cells were stained by staining solution.Results:1.LV-N1ICD was successfully constructed:the packaged LV-N1ICD infected 293Tcells for 48h.The Real time PCR and Western blot were used to detect the target gene Hesl and Hes5 of Notch signaling pathway.Compared with the LV-Control,the expression level of Hesl and Hes5 were significantly increased.It is indicated that LV-N1ICD can activate the Notch signaling pathway effectively.2.Primarily isolated and cultured gremline stem cells:the isolated cells are ovoid and smaller than 20μm diameter,the results of Real time PCR,ALP technology and Dual-IF showed that this type of cells characteristics with OGSCs,the results indicated that this type of cells is actual OGSCs.3.Notch components and germline stem cells marker are co-expressed in OGSCs:Dual-IF results showed that Mvh/Hes1、Mvh/Hey2 are co-expressed in the OGSCs in vitro culture.4.Inhibition of Notch signaling decreased the expression of germline stem cell markers:the Real time PCR and Western blot results showed that the mRNA and protein level of Mvh and Oct4 in OSE was decreased along with the increasing DAPT concentration;Meanwhile,the Notch pathway two key target genes Hesl and Hes5 were a successive decrease with increasing dose of DAPT（p<0.05）.5.Inhibition of Notch signaling diminished the number of follicles:5-7D ovaries of group DMSO、group DAPT 20μM and group DAPT 40μM were treated with DAPT for 48h.The HE staining results shows that the number of primordial follicles was obviously decreased in group 20μM,and sharply diminished in group 40μM.In addition,for 6-8w mice ovaries which were treated with DAPT via microinjected in vivo,the HE staining resulted showed that ovaries follicles were decreased.6.Ovaries were infected with LV-N1ICD:there is not obviously increase for Mvh and Oct4 mRNA level in 5-7 mice OSE after infected LV-N1ICD;for infertility mice model,there are follicles structure to be observed by HE staining after microinjected LV-N1ICD in vivo.7.The concentration changes of FSH、P、E2 in mice serum:for group DAPT,there are no statistical significance for the concentration change of FSH and P,but the concentration of E2 was decreased（P<0.01）;unfortunately,for group LV-V1ICD,there are no statistical significance for the concentration change of FSH、P and E2.8.The offsprings number of mated with male mice:for group DAPT,the offsprings number of 2 group were descended（P<0.05）;however,for group LV-N1ICD,there are no offsprings to produce.9.Attenuating Notch sigaling decreased the activity of germline stem cells in OSE:5-7D mouse ovaries were divided into three groups:the group DMSO,the group DAPT 20μM and the group DAPT 40μM.After treated with DAPT for 48h,Dual-IF results showed that the expression level of Mvh and Oct4 in OSE were descended along with the increasing of DAPT concentration.10.IHC results showed that compared with group DMSO,the Mvh and Oct4 expression level of group DAPT was decreased after microinjection 7D in mice OSE;but there is no change for Mvh and Oct4 between group LV-Control and group LV-N1ICD in OSE.11.Inhibiting/activating Notch signaling can decrease/increase the expression level of Mvh、Oct4、Hes1、Hes5 in OGSCs:OGSCs were treated with DAPT for 48h,the results showed that the mRNA expression level of Mvh and Oct4 was obviously decreased;the level of Mvh and Oct4 was increased to some extent after treated OGSCs with LV-N1ICD for 48h.In addition,the mRNA of Hes1、Hes5 was showed corresponding descend or raise.12.Inhibiting/activating Notch signaling can decrease/increase the activity ALP:OGSCs were treated with DAPT or LV-N1ICD for 48h,the results showed that group DAPT activity of ALP was decreased,group LV-N1ICD was increased compared with group control,respectively.Conclusions:1.LV-N1ICD was successfully constructed and packaged,which can effectively activate the Notch signaling pathway.2.OGSCs were successfully isolated via primary culture method,which can culture in vitro and maintain the characters germline of stem cells.3.There is co-expression between the Notch molecules and germ stem cells marker in OGSCs.The activity of ALP in OGSCs will alter along with Notch signaling change,Notch signaling is related with the activity of OGSCs.4.Notch signaling has regulated the activity of germline stem cells in OSE.And the number of primordial follicles is also regulated by Notch signaling,but neo-oogenesis is still to be proved when Notch signaling was activated in infertility mice model.5.Our experimental results showed that the proliferative ability of OGSCs is related with Notch signaling,Notch signaling pathway maybe is one of the key pathways to regulate the proliferative ability of OGSCs. |
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