The Research Of Reproductive Ability And Autophagy Mechanism In Mouse Parthenogenetic Haploid Embryonic Stem Cells

Stem cells self-renew and differentiate into various cell types under certain conditions,and could be a good model for development and disease studies.The genome of most stem cells in the mammals are diploid,which containing double sets of chromosomes,if one copy of genome is disrupted,the other copy can compensate.However,the diploid status makes mutation screens more complicated and limits efforts to identify the function of specific genes.In genetic transmission,mammalian diploid stem cells almost never develop independently into individual because the genetic development and epigenetic regulation of diploid cells are notably different from that of germ cells.For haploid germ cells,one of the major limits is self proliferation failure.The generation of functional haploid cells with high reproduction efficiency,either by differentiation of pluripotent stem cells or in vitro culturing of the embryonic genital ridges,was not achieved.At 2011,two research groups of Elling and Leeb independently reported ground-breaking work on establishing mouse haploid ES cells showing that h ES cells can be used as an ideal tool for genetic analyses and producing genetic models for recessive traits.Haploid embryonic stem(h ES)cells are a group of cells containing only one set of chromosome and resembling characteristic with normal embryonic stem cells,which owns the ability of unlimited proliferation in vitro,and giving rise to a wide range of functioning cell of all the tissues and organs.Recent researches demonstrate that hEScells have been derived from parthenogenetic(phESCells)and androgenetic mouse embryos(ahESCells)at the present.In the past two years,abundant data and converging evidence idolized hEScells as an ideal tool for genetic analyses and homozygous mutant animal production.However,the production of live transgenic mice derived from h ES cells via intracellular injection is inefficient,and the survival rate of new-born pups is very low resulting from the epigenetic defects of the hEScells.Hence,the aim of this study was to screening the appropriate culturing method to improve the efficiencies and stability of ph ES Cells,to provide the experimental framework for a system that simulates the in vitro replication of mammalian germ cells via ph ES cells improving oocyte regulatory functions and female fertility,and to explore the inside mechanism about readily diploidizing and autophagy of ph ES Cells.The main results in this paper were as follows:1.Effective establishing pathways of C57BL/6J mouse ph ES Cells1.1 Optimization of activated and Culturing methods of C57BL/6J mouse mature oocytesActivation of unfertilized oocytes is the key for establishing haploid parthenogenetic embryonic stem cell lines.Generally,fertilization causes the calcium ion oscillations in the oocyte and then contributes to the embryonic development,which could be replicated via treatment with strontium chloride(Srcl2)or alcohol substitutes in unfertilized oocytes.To this end,we optimized the selected activator and culture medium by designing 18 groups of activator and 5 groups of culture medium.Results were as follows: At a time point 16 hr post-injection(h CG),cumulus-free MII phase oocytes(C57BL/6 background)were collected from female mice.Results indicated that when oocytes were treated with 10% ethyl alcohol treatment for 10 min followed by 5 m M strontium chloride with M2 treatment for 2 h as the oocyte activator,the extrusion of second polar body would be inhibitted.After the production of the morula and blastula rate developing from activated parthenogenetic haploid embryos,we chose M16 as the optimal culture medium because it showed higher rate of overcoming the two-cell block and generation of morula and blastocyst were produced.At last,186 haploid parthenogenetic morulae were obtained and then 87 outgrowths embryos were generated.1.2 Derivation and haploid trends of ph ES cellsDuplicate sets of chromosomes is beneficial for normal celluar development,but the haploid state of ph ES cells frequently makes conversion into spontaneous diploid status during passages.To ensure the haploid state and the proliferation of the ph ES cells,a sorting of haploid ESCs by fluorescence activated cell sorting(FACS)at short intervals and chromosome spreads by Giemsa staining over time are required for culturing ph ES cells.Results were as follows: After sucked and blown in 0.125% tryptase,dispersed single ph ES cells were collected and then plated into feeder-coated well(mouse foetus fibroblast cell layer treated with mitomycin C)with ES cell 2i medium(DMEM/F12 medium with 1?m PD0325901,3?m CHIR99021 and 1?m LIF factor)and further culture 4 to 7 days to observe ph ES cells colonies;Fluorescence-activated cell sorting(FACS)for DNA content was performed after staining with Hoechst 33342 should be used to enrich haploid ES cells every 5 passages for ensuring the haploid.13 ph ES cells line(named respectively HAP-1…13)was obtained and subsequently expanded for over 20 passages.After early rounds of sorting,ph ES cells showed better haploid DNA content and karyotype along with typical cellular and colonial morphology of ESCs,but ph ES cells lines underwent spontaneous diploidization and heteroploidiasation at the high passages.2.Pluripotency evaluation and differentiation potential of ph ES cells2.1 Pluripotency Evaluation of ph ES cells at the molecular levelph ES cells colonier obtained from HAP1 ph ES cells line with relatively stable haploid state were collected and plated on the mitomycin C-treated MEFs feeder layer at the third passages for culturing and identified of pluripotent nature.Results indicated that ph ES cells owned typical cellular and colonial morphology of ESCs;RT-PCR and western blotting detected that the ph ES cells lines expressed typical pluripotency marker genes Oct4,Nanog and Sox-2;Positive Oct-4 and SSEA-1 immunocytochemistry staining was observed in ph ES cells;Additionally,positive signals were observed for pluripotency marker alkaline phosphatase(AP)in ph ES cells,OD values for ph ES cells treated with the AP Quantitative Analysis Kit had no significant differences compared with those of normal ES cells(p>0.05),further illustrating that AP expression in ph ES cells closely resembled that of normal ES cells.2.2 Differentiation Potential of ph ES Cells in vivo and in vitroWe next examined the differentiation ability of the ph ES cells both in vitro and in vivo.First,we assayed for differentiation of the stem cells into nerve cells after treatment with retinoic acid(RA),a test for the differentiation potential of the ph ES cells in vitro.Secondly,we injected the ph ES cells colonies into severe-combined-immune-deficient mice to test the differentiation potential of the ph ES cells in vivo.The results showed that the ph ES cells readily underwent nerve cell formation and expressed nestin(a neurocyte marker)after treatment with 100 ?M RA.The ph ES cells had the ability to differentiate into noble cells(nerve cells)in vitro over successive passages.The ph ES cells could form teratomas containing derivatives of all three germ layers.Teratoma dissection slices from paraffin sectioning showed all three germ layers: the endoderm(gland),mesoderm(fat and muscle)and ectoderm(nerve and epithelia)layers were identified by H&E staining.These results indicated that the ph ES cells were capable of differentiation into several lineages of all three germ layers in vivo and in vitro.2.3 Development Potential and Genetic Transmission of ph ES CellsTo confirm the development and genetic ability of the established ph ES cells to contribute into adult mice,the parthenogenetic haploid ES cells line(C57BL/6 background)carrying a chicken ?-actin gene promoter-driven enhanced green fluorescent protein transgene(EGFP)marker were established by the previous method.One ph ES cells line expressin EGFP(EGFP-ph ES cells)was obtained from 61 EGFP positive haploid parthenogenetic morulae.We then injected freshly FACS-sorted G0-or G1-phase EGFP-positive ph ES cells into KM mouse blastocysts(white-coloured coats)to test the ability of form chimaeras.After cultured for 12 h in vitro,chimaera embryos were transplanted into the oviducts of pseudopregnant KM female mice.The results showed that EGFP expression were detected in at embryonic day 6.5,10.5,19.5(E6.5,E10.5,E19.5),respectively meaning substantial contributions of ph ES cells to chimeric embryo formation and adult mice production.In addition,evidence for the developmental potential and genetic transmission of ph ES cells to adult chimeric mice was also provided by their coat-colour chimerism.Howerer,the GFP-positive cells extracted from the EGFP ph ES cell derived foetus(E10.5)underwent extensively diploidization,indicating that no haploid ES cells could be detected during the embryonic development.3.Effects of Tet3 expression about reproductive potential of ph ES cells3.1 Expression profile of Tet3 in ph ES cellsA large number of scientists are striving for deriving functional germ cells in vitro.Previous works suggest that ph ES cells own the ablility to “fertilize” in the enucleated oocytes,support embryo dvelopment and produce fertile mice after supplement with parental genome,but the efficiency was very low and the survival rate of new-born pups is very low.Therefore,we assumed that if the difference in special homologous chromosomes and epigenetic modification(especially gene imprinting)of ph ES cells from normal oocutes resulted in individual developmental barriers.Here in an attempt to address this question,we generated ph ES cells with the goal of a higher offspring yield and better survival rate via in vitro related genetic modification.The molecular characteristics of regulating zygotic genome reprogramming gene in ph ES cells were identified by RT-PCR.As a result,ph ES cells showed significantly downregulation in the m RNA level expression of DNA dioxygenases Tet3(tet methylcytosine dioxygenase family 3),which convert 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hm C)with respect to those in control oocytes;These were further confirmed by immunostaining and western blotting of Tet3 in ph ES cells;In our study,ph ES cells line was treated with 100 ?M retinoic acid(RA),a reagent often used to prime ES cells to neuron.The variation trend of Tet3 levels among original oocytes,haploid embryos,ph ES cells and ph ES cells differentiation were similar as in the normal diploid ES cells.As Tet3 was upregulated in ph ES cells differentiation,we speculated that Tet3 activity was urgently demanding in ph ES cells to improve reproductive potential,which might be regulated just like the supposed “passive demethylation” of the maternal pronucleus stimulating 5m C/5hm C methylation pattern.3.2 Construction of Tet3 Overexpression vector in ph ES CellsTo address the importance of Tet3 in genome reprogramming of ph ES cells for development and reproductive potential,we utilized Tet3 overexpression vector and the lentiviral transfection system to overexpress Tet3 followed by cell proliferation assay and cell differentiation detection.Results were as follows: Representative western blotting and immunofluorescence was performed in Tet3 overexpressed ph ES cells and 5hm C amounts were significantly higher(p<0.01)than those in ES cells and ph ES cells at 48 h cultured after transfected plasmid.It was demonstrated that Tet3 overexpression had no negative effect on affect cell proliferation and differentiation of ph ES cells as a role in embryonic development similar with other related genes.3.3 Tet3 overexpression in ph ES cells interacts with generation of healthy offspringTo test whether Tet3-overexpressed ph ES cells could replace the maternal pronucleus in supporting embryonic development,we performed an intracytoplasmic ph ES cell injection followed by sperm injection(ICPSI)experiment.Firstly,we collected MII oocytes from KM female mice(kunming background with white coat colour)and then removed the maternal spindle to generate enucleated oocytes.Secondly,we injected parthenogenetic haploid single cells dispersed from Tet3-overexpressed ph ES cell colonies(C57BL/6 background with black coat colour,two HAP cells line and one EGFP ph ES cells line)into enucleated oocytes for reconstructing a new oocyte with fertility and a novel maternal genome.Thirdly,the mature sperm(kunming background)was supplied via ICSI transporting into the reconstructed oocyte after activating the reconstructed oocytes.Results were as follows: 5hm C immunofluorescence indicated the ICPSI reconstructed embryos exhibited a genomic methylation pattern very similar to the ICSI embryos and natural fertilized cytula as the maternal pronuclei in the new reconstructed cytula;The ICPSI embryos further developed to morulae in M16 medium,and the GFP transgene was correctly detected in corresponding embryos at each embryonic age;A total of 76 reconstructed morulae from 3 randomly selected Tet3-overexpressed ph ES cells lines were transplanted into 3 pseudopregnant mice(KM background).Finally,11 full-term pups by ICPSI were harvested inheriting the GFP transgene.Among them,2 ICPSI pups were healthy and survived with full-term developmental and reproduction ability,whereas the female offspring adult mice mated with inbreeding male mice and could give birth to healthy third-generation pups with obvious cross-fertilized coat-colour;Additionally,we also observed that the ICPSI and ICPSI with Tet3 expressed pups had almost normal methylation pattern of differentially methylated regions(DMRs)of Igf2,H19 and Ifg2 R,similar with normal ICSI pups by means of bisulfite sequencing analysis.Together,all the results demonstrated that Tet3 overexpression supported full-term development with stronger reproductive ability and better genetic capacity in ph ES cells replacing oocytes accompanied by paternal pronucleus to produce full-term healthy offspring for contributing to the germ line.4.Identification of the mechanism of autophagy and readily diploidizing in ph ES cells4.1 SIRT1 regulates autophagy in ph ES cellsThe haploid state of ph ES cells frequently makes conversion into spontaneous diploid status during differentiation or high passages,and ph ES cells after diploidization are congenitally more stable and developmental potential.Nevertheless,the mechanism for readily diploidizing of ph ES cells remains unclear right now.As previous researches,the repressed cell proliferation and abnormal karyotype of ph ES cells indeed suggested that the age-related pathologies might result from cellular stress conditions during high passage.Several studies have identified autophagy is a protective or cell-death inhibitor under higher oxidative stress conditions induced by 1m M H₂O₂.Here in an attempt to address the question,we generated ph ES cells with the goal of evaluating connections between induction of autophagy and spontaneous diploidization in haploid ESCs.Based on the likely activation of autophagy by certain concentration of 1m M H₂O₂,age-related reactive oxygen species(ROS)generation increased mitochondrial function and oxidative stress,which was highly linked to autophagy and apoptosis in haploid embryonic stem cells.As shown,age-related oxidative stress and autophagic cell death in ph ES cells at high passage were similar with 1m M H₂O₂ treatment in cell morphology;Flow cytometry analysis of DNA contents confirmed that ph ES cells at high passage and treated with 1m M H₂O₂ both exhibited the previously reported self-diploidization;After quantitative real time-PCR and western blot assay,we found that SIRT1 was highly expressed in ph ES cells at high passage and treated with H₂O₂.To further determine a role for SIRT1 in ph ES cells after treatment with exogenous H₂O₂,lentivirus carrying small hairpin RNA(shRNA) constructing against SIRT1(sh SIRT1)and Nicotinamide(short as NAM,noncompetitive inhibitor of SIRT1)were used.With the knockdown or inhibition of SIRT1,the shifting balance between autophagy and apoptosis in H₂O₂-treated cells was broken in gene and protein level,whereas decreased oxidative stress-induced autophagy and increased activation of apoptosis were observed in Sirt1-knockdown ph ES cells after H₂O₂ stress was specific to deletion of SIRT1.Western bloting showed that the expression of autophagy-associated proteins were downregulated significantly.We further characterized H₂O₂-treated control cells showed decreased phosphorylation of p70/S6 kinase and m TOR compared to the H₂O₂-treated Sirtl-knockdown cells.The above information indicated that in ph ES cells SIRT1 perhaps was essential for induction of autophagy at least in part by suppressing the m TOR pathway.4.2 SIRT1 impacted P53 transcriptional activity and apoptosis in ph ES cellsSince its discovery,the role of P53 in various cellular mechanisms has expanded as a transcriptional factor modulating an increasing number of genes involved in cell cycle arrest,autophagy and apoptotic pathway.To further investigate the mechanism underlying the regulation of P53 by SIRT1 in ph ES cells,RT-PCR,western bloting and co-immunoprecipitation assays(Co IP assays)were performed.Our results demonstrated that SIRT1 interacted with H3 and P53 for deacetylation and further methylated of H3K9 in ph ESCs under oxidative stress.Thus P53 was still subject to SIRT1 regulation leading to rhythmic H3K9 deacetylation and methylated after H₂O₂ stress.SIRT1 bound to H3K9 within the P53 promoter region,and resulting in H3K9 expression by decreased acetylation and increased tri-methylation,thereby inhibited P53 at the transcriptional level.When SIRT1 present under oxidative stress,SIRT1 indirectly methylates H3K9 in nucleus and deacetylates P53 in cytoplasm to inhibited P53 and abrogated apoptosis;On the other hand,when SIRT1 absent under oxidative stress,P53 is induced by acetylation of H3K9 and further promotes apoptosis through caspase-3 pathways.4.3 SIRT1 indirectly suppressed Tet3 expression in ph ES cellsAs previously shown,Tet3 contributed to 5-methylcytosine(5m C)-derived oxidation for 5-hydroxymethylcytosine(5hm C)and replication-dependent dilution upon the demethylation process of H3K9 in mouse zygotes and enhancing expressed levels of Tet3 in ph ES cells produce better fertile mice and more transmitgenetic modifications to the offspring.Here,the expression of Tet3 in H₂O₂-treated ph ES cells after sh SIRT1 and/or NAM treatment was all markedly upregulated.SIRT1-knockdown showed no obvious effects on m RNA expression of two different upstream genes(Cul4a and DDB1)of Tet3.Next,the in vitro binding characteristic to Tet3 of SIRT1 was not almost observed under H₂O₂ treatment.Western blot assay revealed that the expression of Tet3 was rescued after H3K9me2 suppressed in SIRT1-knockdown cells.To further prove the mechanism,we forcedly expressed Tet3 in ph ES cells.The expression of SIRT1 and H3K9me2 were not remarkably changed after Tet3 overexpressed in 1m M H₂O₂ treated cells.Similarly,no more 5hm C staining signals were observed than 1m M H₂O₂ treated cells,which indicated overexpressed-Tet3 indeed not associated with permitting rapid 5m C oxidation.These data clearly demonstrated that SIRT1 could indirectly decrease the expression of Tet3 through accelerating dimethylation of H3K9 at high passage or under oxidative stress.4.4 SIRT1 mediated autophagy was associated with further diploidization in ph ES cellsAfter analysis of DNA content,we found that SIRT1-knockdown ph ES cells retained a better haploid state and higher ratio of the haploid cells than control,indicating that the delection of SIRT1 slowed down diploidization.Previous studies have reported that ph ES cells gain a diploid karyotype when they differentiate.After ph ES cells injected into severe-combined-immune-deficient mice for 6 weeks,immunohistochemistry staining of teratoma dissection slices showed that there was a greater degree of high expression of SIRT1 at different passages of ph ES cells than that in diploid ESCs.Above all,the diploidization of ph ES cells at high passage might correlate with SIRT1 as an important role in regulating autophagy,rather than apoptosis.Conclusions: Our resulting mouse parthenogenetic haploid embryonic stem cells had stable proliferation capacity,expressed a normal epigenetic phenotype,differentiated into three germ layer cells,and contributed to the germ line as the typical characteristics of embryonic stem cells.Optimizing the activation and culturing methods can contribute to the haploid rate and stability in ph ES cells;Also,we provide more evidence that ph ES cells with Tet3 overexpressed forcedly can be readily utilized for producing fertile offspring and improving reproductive potential;In addition,SIRT1 regulates autophagy in haploid embryonic stem cells via reducing m TOR pathway,simultaneously depresses P53 activity and Tet3 expression via targeting H3K9;It is important that SIRT1 mediated autophagy may be the key of diploidization of haploid embryonic stem cells at high passage.