Developmental Cell11,125–132,July,2006ª2006Elvier Inc.DOI10.1016/j.devcel.2006.05.010
Short Article In Vitro-Differentiated Embryonic
Stem Cells Give Ri to Male Gametes
that Can Generate Offspring Mice
Karim Nayernia,1,7,*Jessica Nolte,1
Hans W.Michelmann,2Jae Ho Lee,1
Kristina Rathsack,1Nadja Drunheimer,1
Arvind Dev,1Gerald Wulf,3Ingrid E.Ehrmann,4
疏的意思David J.Elliott,4Vera Okpanyi,5Ulrich Zechner,5 Thomas Haaf,5Andreas Meinhardt,6
and Wolfgang Engel1
1Institute of Human Genetics
2Department of Obstetrics and Gynecology
3Department of Hematology and Oncology
考研总分University of Go¨ttingen
37073Go¨ttingen
Germany
4Institute of Human Genetics
International Centre for Life
University of Newcastle upon Tyne
Newcastle upon Tyne,NE17RU
United Kingdom
5Institute of Human Genetics
University of Mainz
51131Mainz
Germany
6Department of Anatomy and Cell Biology
University of Giesn
35378Giesn
Germany
Summary
Male gametes originate from a small population of spermatogonial stem cells(SSCs).The cells are be-lieved to divide infinitely and to support spermatogen-esis throughout life in the male.Here,we developed a strategy for the establishment of SSC lines from em-bryonic stem(ES)cells.The cells are able to undergo meiosis,are able to generate haploid male gametes in vitro,and are functional,as
sanishown by fertilization after intracytoplasmic injection into mou oocytes.Result-ing two-cell embryos were transferred into oviducts, and live mice were born.Six of ven animals devel-oped to adult mice.This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and develop-ment.Our approach provides an accessible in vitro model system for studies of mammalian gametogene-sis,as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.
Introduction
In xually reproducing animals,all gametes of either x ari from primordial germ cells(PGCs).In mam-mals,PGCs ari from the proximal epiblast,a region of the early embryo that also contributes to thefirst blood lineages of the embryonic yolk sac(Lawson and Hage,1994;Zhao and Garbers,2002).In the mou, PGCs migrate through the dorsal mentery and enter the developing fetal gonad at the genital ridge;this oc-curs between E10.5and E12.5.Once they arrive in the genital ridge,the PGCs in males are enclod by Sertoli cells and become gonocytes(McLaren,2000).The gon-ocytes proliferate for a few days and then arrest in G0/G1 pha until birth.Within a few days after birth,the gon-ocytes resume proliferation to initiate spermatogenesis and to produce male gametes.Recent studies have demonstrated that embryonic stem(ES)cells differenti-ate into germ ce
lls and more mature gametes(Hubner et al.,2003;Geijn et al.,2004;Toyooka et al.,2003),al-though significant unanswered questions remain about the functionality of the cells.The derivation of germ cells from ES cells in vitro provides an invaluable assay both for the genetic disction of germ cell development and for epigenetic reprogramming,and it may one day facilitate nuclear transfer technology and infertility treatments.
In the prent study,we demonstrate that in vitro male gametes derived from ES cells are functional and are able to produce viable offspring.
Results and Discussion
To examine the functionality of ES cell-derived male gametes,we developed a new strategy for the establish-ment of male germline stem cells,spermatogonial stem cells(SSCs),from ES cells.First,we produced two fu-sion genes,Stra8-EGFP and Prm1-DsRed,harboring coding regions of enhanced greenfluorescent protein (EGFP)and redfluorescent protein(DsRed)under con-trol of promoter regions of murine Stra8(Oulad-Abdel-ghani et al.,1996)and protamine1(Prm1)genes(Zam-browicz et al.,1993),respectively(Figure1A).Both reporter genes contain the neomycin phosphotransfer-a II gene(NEO),which is driven by the SV40early pro-moter and enhancer for positive lection(Figure1A)
. Previously,it was demonstrated that1.4kb of the50flanking region of the Stra8gene(Nayernia et al.,2004) and0.5kb of the50flanking region of the Prm1gene (Zambrowicz et al.,1993)are able to direct reporter gene expression specifically in premeiotic and haploid male germ cells,respectively.ES cells were transfected with the Stra8-EGFP fusion gene and were cultured for2 months in the prence of G418to lect the cells that were positive for the Stra8-EGFP construct.The culture, transfection,and lection of cells were performed ac-cording to previously described protocols(Nayernia et al.,2004).
Previous studies have shown that a cocktail of soluble growth factors,including retinoic acid(RA),are able to sustain the survival and lf-renewal of mou germ cells in the abnce of somatic cell support.After germ cell adhesion to an acellular substrate,such growth factors and compounds were able to prevent the occurrence
*Correspondence:karim.nayernia@ncl.ac.uk
7Prent address:Institute of Human Genetics,International Centre for Life,University of Newcastle upon Tyne,Newcastle upon Tyne, NE17RU,United Kingdom.
of significant levels of apoptosis in germ cells;stimulate their proliferation;and,when LIF was omitted f
direct是什么rom the cocktail,allow most of them to enter into and progress through meiotic propha I (Farini et al.,2005;Koshimizu et al.,1995;Bowles et al.,2006).Furthermore,RA is known to promote the developmental progression of spermatocytes through early stages of meiosis (Leid et al.,1992;Akmal et al.,1997),though the molecular mechanisms underlying the effects remain to be eluci-dated.We therefore cho to u RA in our efforts to di-rect functional spermatogenesis in vitro.
After RA induction (final concentration of 1025M),ex-pression of Stra8-EGFP was detected in about 60%of all cells (data not shown).The positive cells were lected by using fluorescence-activated cell sorting (FACS)and were cultured for an additional 2months under non-induced conditions.Since transgene silencing is a com-mon problem in ES cells,it is important to note that the cells survived in medium containing G418and remained GFP positive.It can therefore be concluded that the transgene is expresd.To examine whether the -lected cells maintain the differentiation properties after RA induction,the cells were subjected to a cond FACS sorting.Expression of Stra8-EGFP was detected in 90%of all cells (data not shown),indicating the main-tenance of the differentiation potential of the sorted cells.The cells were transfected with the Prm1-DsRed fusion construct,and positive cell colonies were lected by PCR with primers specific for the DsRed coding region.Using this strategy,two cell lines,SSC7and SSC12,were established.Both cell lines express E
GFP without RA induction,which is an indication of the permanent activation of the Stra8promoter as well as differentiation toward male germ cells (data not shown).The cells formed embryoid body-like structures (Figure 1B)and expresd mou vasa homolog (Mvh)protein (Fig-ure 1C).Mvh is specific for differentiating germ cells from the late migration stage to the postmeiotic stage (Toyooka et al.,2000).After RA induction for 72hr,the SSC7and SSC12cell lines are able to produce DsRed-positive (red)cells that aro from EGFP-positive cells (Figure 1D).Red cells are relead into the medium and can be collected from the supernatant (Figure 1E).Using live pha contrast microscopy,the motility of cells in the supernatant of the SSC7and SSC12cell lines can be demonstrated after 72hr of RA induction (Movie S1;e the Supplemental Data available with this article online).The formation of tail-like structures could be obrved in some red cells (Figure 1F).Expression of DsRed protein is an indication that EGFP-positive
cells
Figure 1.Strategy for the Establishment of ES Cell-Derived Male Germline Stem Cell Lines
(A)Schematic reprentation of the Stra8-EGFP and Prm1-DsRed reporter genes harboring a 1.4kb
and 0.5kb promoter region of Stra8and the mou protamine 1(Prm1)gene,respectively.The regions are able to direct reporter gene expression specifically in premeiotic and haploid male germ cells,respectively.Both reporter genes contain neomycin phosphotransfera II gene (NEO ),which is driven by the SV40early promoter and enhancer (SV40).
(B)HE staining of an embryoid-like cell colony.
slammer(C)Immunostaining of an embryoid body-like cell colony with the anti-Mvh antibody shows differentiation of PGCs (arrow).
(D)Fluorescent images of Stra8-EGFP (green)and Prm1-DsRed (red)proteins in the ES-derived SSC12cell line after 72hr of RA induction.The merged image shows different cells with green and red fluorescence,indicating activation of Stra8and Prm1promoters in different cell types.DAPI:fluorescent image showing DNA localization.
red169(E)The merged fluorescent image of the supernatant from SSC12cells after 72hr of RA induction showing only red cells,which is an indication for relea of Prm1-DsRed-positive cells into the medium.
(F)Magnification of a red cell from supernatant showing positive immunoreaction with a sperm tail-specific protein,PHGPx (arrow).Bars in (B),(C),and (E)are 50m m,bars in (D)are 100m m,and bars in (F)are 10m m.
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differentiated into haploid male germ cells and activated the Prm1promoter region.
Characteristics of ES Cell-Derived Male Gamete Differentiation
The cells were characterized by determining the expres-sion of different markers for PGCs,premeiotic,meiotic,and postmeiotic male germ cells by RT-PCR analysis.RT-PCR showed expression of markers for the early stages of male germ cell development (Oct4,fragilis ,stella ,Mvh ,and Rnf17)(Saitou et al.,2002;Scholer et al.,1990;Tanaka et al.,2000;Wang et al.,2001)in un-treated and RA-treated SSC7and SSC12cells (Figures 2A and 2B).Furthermore,expression of Ddx4,which is a marker for late germ cells,was found (Figure 2).This gene is not expresd in ES cells (Zwaka and Thomson,2005).Expression of PGC-specific genes in SSC7and SSC12cells indicates that a subpopulation of the cells can be characterized as PGCs.Immunohistochemical analysis with antibodies against Hsp-90a ,Dazl,Rbmy (Elliott et al.,1996),Piwil2,and Stra8further confirmed this po
stulate (Figure 2C).It is worth noting that in-cread expression of c-Kit after RA treatment (Figures 2A and 2B)indicates that both cell lines are able to differ-entiate to spermatogonia.
To investigate whether SSC cell lines undergo meiosis in vitro,we determined the DNA content of the cells by flow cytometric analysis.As shown in Figure 3A,w 30%of SSC cells undergo meiosis and produce a haploid cell population (1C in Figure 3A)after 72hr of RA induction.Without RA induction,w 3%of SSC cells undergo meiosis (data not shown).Furthermore,we an-alyzed the formation of the synaptonemal complex in SSC cell lines after RA induction by using electron mi-croscopy.The formation of the synaptonemal complex in SSC cell lines after RA induction is additional evidence for meiotic differentiation (Figure 3D).After 24hr of in-duction,the cells are able only to differentiate to early spermatids,but not to late spermatids,which were shown by markers specific for the stages (Figure S1;e the Supplemental Data available with this article online).
To analyze postmeiotic differentiation on the molecu-lar level,we examined expression of different stage-specific genes in RA-treated and untreated SSC7and SSC12cells.Using RT-PCR,we analyzed expression of synaptomal complex protein 3(SCP3)and acrosin (Acr ),two markers for meiotic differentiation (Nayernia et al.,1996;Yuan et al.,1998),and transition protein 2(Tp2)and Prm1,two postmeiotic markers.Expression of SCP3and Acr was obrved after RA induction (1026M,72hr,Figu
re 2B).Tp2is a basic chromosomal protein that functions as an intermediate in the replace-ment of histones by protamines,and its mRNA is first detectable in step 7,round spermatids (Kistler et al.,1996).As shown by RT-PCR,both cell lines were positive for Tp2and Prm1after 72hr of induction by RA (Fig-ure 2B),and the data provide evidence for postmeiotic differentiation of the SSC cell lines.Furthermore,the formation of acrosome,the occurrence of
nuclear
Figure 2.ES Cell-Derived Male Germ Cells Showing Premeiotic,Meiotic,and Postmeiotic Markers
monstrum(A)RT-PCR analysis of SSC7and SSC12cell lines under nondifferentiating conditions shows only expression of PGC (Oct4,fragilis [Frgl ],stella ,mou vasa homolog [Mvh ],and Ddx4)and premeiotic-specific genes (Stra8,Rbm ,Rnf17,and c-Kit [Vincent et al.,1998]).
(B)RT-PCR analys of SSC7and SSC12cells after treatment with RA (72hr)show induction of meiotic (synaptonemal complex protein 3[Scp3],acrosin [Acr ],and Dmc1[Tarsounas et al.,1999])and postmeiotic (transition protein 2[Tp2]and protamine 1[Prm1])gene expression.T,testis;C,nontemplate control.
(C)Immunohistochemical analysis and formation of sperm structures.In immunohistochemical analysis,the expression of premeiotic-specific proteins,Hsp-90a ,Dazl,Rbmy,Piwil2,and Stra8,is demonstrated (red).Meiosis was demonstrated by using immunostaining with an antibody against hnRNPGT,which is expresd specifically in pachytene spermatocytes (green).The formation of an acrosome-like structure is shown by antibodies against outer acrosomal membrane (OAM)and Acr (red).Condensation of the nucleus is demonstrated by immunohistochemical analysis by using Tp2.DSRed is located in the cytoplasm of the cells with condend nuclei.1+2,primary and condary antibodies;2,only condary antibody ud as a negative control.
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condensation,and the formation of a tail-like structure in the SSC12-derived postmeiotic cells was confirmed by immunocytochemical analysis with three specific an-tibodies to Acr and outer acrosomal membrane protein (OAM),to Tp2,and to midpiece-specific protein PHGPx (phospholipid hydroperoxide glutathione peroxida),respectively (Figures 1F and 2C).DsRed protein was de-tected in the cells with condend nuclei (Figure 2C).
gotta go my own way
During spermiogenesis,structural and biochemical modifications of chromatin are responsible for the final nuclear maturation of spermatozoa.Chromatin conden-sation is achieved through replacement of somatic-type histones first by transition proteins and finally by prot-amines.Several staining procedures have been ud to analyze the structural and biochemical modifications of chromatin in spermatozoa.Aniline blue staining can discriminate between stages of spermatid differentia-tion.Early stages have nuclei containing lysine-rich his-tones that will react by taking up the aniline blue stain,whereas the protamine-rich nuclei of mature spermato-zoa,with abundant arginine and cysteine,remain un-
stained.Using aniline blue staining,we obrved an increa of unstained cells from 3%in untreated cells to 56%in cells relead to the medium (Figure S2).This result is an indication of the modification of chroma-tin of SSC cells during in vitro differentiation.
DNA methylation is an epigenetic regulator of gene expression and acts as an important molecular mark un-derlying the parental-specific expression of genes sub-ject to genomic imprinting.Several studies have now demonstrated that the erasure of at least methylation imprints occurs in the germline (Surani,2001).To obtain conclusive evidence that the SSC7and SSC12cell lines were indeed germ cells,we analyzed whether the cells manifested erasure of epigenetic imprints,which i
s a unique property of germ cells.We analyzed the methyl-ation status of the imprinting control region (ICR)of H19,the differentially methylated region 2(DMR)of Igf2r ,and DMR1of the small nuclear ribonucleoprotein N (Snrpn )genes (Lucifero et al.,2002)in independent clones of the SSC7and SSC12cells.The analysis was carried out at different CpG sites by a bisulfite quencing as-say.The H19ICR and the Igf2r DMR2showed the ex-pected decrea of methylation after RA induction,whereas an unexpected increa of methylation was ob-rved for the Snrpn DMR1(Figure 3).The findings suggest that after RA induction the grandparental meth-ylation imprints are efficiently erad at many,but not all,alleles of two of the three analyzed genes.In super-natant cells,the Igf2r DMR2was completely unmethy-lated at all analyzed sites in SSC12,and the Snrpn DMR1was more or less completely unmethylated at the analyzed sites in both SSC7and SSC12(Figure 3E).The methylation profiles are consistent with previ-ously published methylation profiles in haploid male germ cells (Lucifero et al.,2002),indicating that at least some methylation imprints were correctly reprog-rammed in all or the majority of cells in the supernatant.In contrast,the Igf2r DMR2displayed hemizygous meth-ylation patterns in the supernatant of SSC7.Similarly,the supernatants of both SSC7and SSC12showed hemimethylated patterns for the H19ICR (Figure 3E).Functionality of ES Cell-Derived Male Gametes
The testis cell transplantation technique provides ac-cess to the mammalian germline and has been ud in experimental animal models to study stem cell/niche biology and germline development (Dobrinski,2005).To investigate SSC capacity and the further develop-ment of SSC7and SSC12cell lines in vivo,cells were transplanted into one of the testes of germ cell-depleted recipient mice.The other testis rved as an internal control.Histological analysis of testes after 4months showed the appearance of spermatogenesis-like struc-tures (Figure S3A)and sperm in the lumen (Figure S3B)of two of ten transplanted mice.Colonization of SSC7and SSC12cells was evaluated by histological ction-ing.Each slide was viewed at a magnification of 4003for the analysis.In five mice,teratoma were detected.No regeneration of spermatogenesis was obrved in non-transplanted control testes (Figure S3C)controlled by rial histological analysis.However,all sperm were im-motile or showed reduced motility (data not shown).To examine the origin of detected postmeiotic cells,the ctions were evaluated by fluorescence microscopy,
Figure 3.Identification of Haploid Male Germ Cells by FACS (A)The SSC12cell line shows w 30%haploid cells (1C)after 72hr of RA treatment.
(B)Testis cell suspension shows populations of cells that are in the haploid stage (1C),G0/G1pha (2C),and G2/M pha (4C).(C)No haploidization was detected in feeder layer cells.
(D)The formation of synaptonemal complex (Page and Hawley,2004)in SSC12cells after RA treatment identified by electron mi-croscopy.Chromatin (red arrows)and the lateral element (green ar-rows)are shown.
(E)Profiles of H19,Igf2r,and Snrpn methylation in noninduced (n),induced (i),after FACS,and supernatant (s)red cells of SSC7and SSC12cell lines.The percentages of unmethylated (red bars)and methylated (blue bars)CpG sites are given on the y axis.
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and red spermatids (Figure S3B)were detected in the lumen,which is evidence that sperm are derived from transplanted cells.Furthermore,using PCR analysis on genomic DNA isolated from sperm,it was shown that sperm contain the Prm1-DsRed transgene (Figure S3D).This finding indicates that SSC7and SSC12cells can be partially differentiated into postmeiotic germ cells and can develop into sperm in vivo.
Intracytoplasmic injection of in vitro-generated red cells (haploid cells)into unfertilized oocytes of wild-type females was carried out to examine the oocyte-ac-tivating competence of male gametes and the develop-ment of preimplantation embryos.Polar body extrusion,pronuclear formation,and nor
mal features of preimplan-tation embryos (Figure 4A)indicate that in vitro ES-de-rived male gametes are able to activate the unfertilized oocytes and initiate early embryonic development.We succeeded in obtaining progeny by transferring the two-cell embryos into the oviducts of pudopregnant females (Figure 4B).Out of 65embryos transferred into oviducts,12animals were born.The results of the PCR analysis were confirmed by Southern blot analysis by using a DsRed-specific probe (Figure 4D).To examine expression of the Prm1-DsRed transgene,testis c-tions were evaluated by fluorescence microscopy.Red spermatids were detected in the testis of one of the transgenic progeny (Figure S4).One animal carried both transgene alleles (Stra8-EGFP and Prm1-DsRed),and six animals carried only the Prm1-DsRed transgene (Figure 4C).The offspring were either smaller or larger than controls and died prematurely (5days to 5months after birth).Some offspring were not transgenic.Pre-sumably,this is related to the likelihood of transgene loss (subquent to the relaxation of lection),recom-bination during meiosis,and/or allele gregation.
To support our conclusion that disturbed establish-ment of male germline-specific methylation imprints in ES-derived gametes leads to abnormal methylation pat-terns and phenotypic abnormalities in the offspring,bisulfite quencing assays were applied to spleen tis-sues of three animals conceived by ICSI with ES-derived haploid cells.Interestingly,the H19ICR and the Snrpn DM
R1showed strong hypomethylation in spleen tissues of mice 3/3(number/designation)and 3/6,which both displayed overgrowth,whereas the Igf2r DMR2was hypermethylated in the spleen of mou 3/3(Figure S5).In contrast,the DMR1of Snrpn was strongly hyperme-thylated in the spleen of the growth-retarded mou,2/2(Figure S5).Thus,similar methylation abnormalities were obrved in the two mice with overgrowth,and op-posite abnormalities were en in the mou with growth retardation.The spleen of the wild-type mou showed normal methylation patterns for all three analyzed re-gions.Although similar to somatic cell nuclear transfer technologies,ES-derived gamete differentiation may in-terfere with the esntial reprogramming events in ga-metogenesis,and a certain percentage of ES-derived sperm could display correct methylation imprints re-quired for normal embryonic development.
Conclusions
The results indicate that our established ES cell-de-rived lines SSC7and SSC12can produce functional gametes in vitro that are able to fertilize oocytes and support full-term development to embryos and adult animals.Thus,male gametes produced by our strategy were able to initiate and to support embryonic and post-natal development after ICSI.Previously,different groups showed the establishment of spermatogonial cell lines from testis (Feng et al.,2002;Kubota et
al.,
Figure 4.Functionality of ES Cell-Derived Male Gametes
(A)Development of preimplantation embryos derived from intracytoplasmic injection (ICSI)of Prm1-DsRed-positive haploid cells into the CD1or NMRI oocytes.
(B)Full-term development of transferred two-cell embryos derived from ICSI with Prm1-DsRed-positive haploid cells.The red arrow shows an agouti mou (5days after birth)that carried both transgenic alleles,Stra8-EGFP and Prm1-DsRed;the green arrow demonstrates a white mou (7days after birth)that carried only the Prm1-DsRed re-porter gene.
(C)Genomic PCR with DNA isolated from tail biopsies shows ven mice with the Prm1-DsRed transgene and a mou with the dou-ble transgene,Stra8-EGFP and Prm1-DsRed.Genomic PCR with primers specific for the TP2gene rves as a positive control for PCR.+,PCR with Stra8-EGFP and the Prm1-DsRed reporter gene.2,nontemplate negative control.SSC12/1,SSC12/2,and SSC12/3:ICSI experiments with haploid germ cells of the SSC12cell line.hateyou
(D)Southern blot analysis with genomic DNA isolated from tail biopsies of the born mice.The blots w
ere hybridized with DsRed-and EGFP-specific transgene probes,as well as with a loading control probe for the endoge-nous gene BrunoL1.
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2004;Ogawa et al.,2004;Hofmann et al.,2005).How-ever,only the cell lines established by Feng et al.(2002) showed differentiation to spermatocytes and sperma-tids in vitro(Feng et al.,2002).Recently,two groups demonstrated the derivation of male gametes from ES cells(Toyooka et al.,2003;Geijn et al.,2004).How-ever,full-term development of embryos produced by ES-derived gametes was not shown.Furthermore,in contrast to our strategy,haploid gametes were ob-tained after3weeks of induction with RA.We estab-lished SSC cell lines that undergo meiosis and produce male gametes after only72hr.This result indicates that our cell lines sustain differentiation characteristics of SSCs.
Although we succeeded in obtaining progeny from ES cell-derived male gametes,technical improvements are necessary.Improvement of oocyte microinjection is im-portant to obtain more two-cell embryos.In this study, we ud210oocytes for microinjection;65oocytes developed to2-cell embryos,and7animals that carried transgene alleles were born.The fertilization rate might have been
affected by damage during oocyte injection or by the heterogeneity of microinjected male gametes. To demonstrate the expression of Prm1-DsRed in trans-genic mice,we prepared testes of positive animals and monitored them for DsRed-positive cells.In one trans-genic line,we obrved DsRed-positive spermatids (Figures S4A and S4B).This is an indication for specific expression of the Prm1-DsRed fusion transgene in testis.
Ourfindings suggest that development of male germ cells form ES cells is a cell-autonomous process that is controlled by the microenvironment generated in dif-ferent developmental stages.Our report,together with the recent demonstration of oocyte and sperm genera-tion from ES cells(Hubner et al.,2003;Toyooka et al., 2003;Geijn et al.,2004;Clark et al.,2004),provides new possibilities for investigating germ cell develop-ment,epigenetic reprogramming,and germline gene modification.
Experimental Procedures
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Fusion Gene Constructs
Construction of the Stra8-EGFP construct is previously described (Nayernia et al.,2004).For construction of the Prm1-DsRed fusion gene,the50flanking region of the mou Prm1gene(Zambrowicz et al.,1993),the+652/+2fragment(+1indicates the start of transla-tion),was a
mplified by genomic PCR with the50-GTCTAGTAATGTC-CAACACCT-30and50-GGTGCTGGCTTGGCCGGGAGC-30primers and was cloned into the pDsRed plasmid(BD Bioscience). Isolation of Recombinant ES Cell-Derived Germ Cells
Mou embryonic stem cell line R1(XY)(provided by Dr.A.Nagy, University of Toronto,Canada)was cultured in an undifferentiated state on a feeder layer of mitomycin C-inactivated mou embryonic fibroblasts with Dulbecco’s modified Eagle’s medium(DMEM, GIBCO-BRL)supplemented with15%FCS,2mM L-glutamine (GIBCO-BRL),50m M b-mercaptoethanol(b-ME;Promega),13non-esntial amino acids(NEAA;GIBCO-BRL),and103U/ml LIF as de-scribed previously(Joyner,2000).Linearized plasmid DNA(30m g) was electroporated into ES cells.Colonies resistant to G418(400 m g/ml)were lected.Resistant colonies were tested by PCR,and colonies that contain the Stra8-EGFP construct were lected and cultured in an undifferentiated state.Cultures were proliferated in the above-described medium for an additional2months(four pas-sages)and were then frozen.Thereafter,cells were cultured on a feeder layer of mitomycin C-inactivated mou embryonicfibro-blasts with basic ES cell medium.To induce differentiation,medium was changed to medium containing retinoic acid(RA)(Sigma)at a final concentration of1025M,and the cells were cultured for10 days.Positive cells(60%)were sorted by FACS.Briefly,cells were dissoci
ated with0.25%trypsin/EDTA,neutralized with DMEM with 10%FCS,washed twice with PBS,and then resuspended in PBS containing0.5%BSA.Approximately23106cells/ml in PBS/BSA were ud for sorting.Theflow cytometry was performed on a FAC-Star Plus(Becton Dickinson)equipped with dual488nm argon and 633nm helium neon lars.Sorted cells were cultured in RA-free medium.After8–10weeks(4passages),medium was changed with medium supplemented with RA(1026M),and after12hr, GFP-positive cells(90%)were sorted by FACS.Thereafter,the cells were cultured in basic medium supplemented with LIF onfibroblast feeder layers and transfected with the Prm1-DsRed construct.Pos-itive cell colonies were lected after PCR analysis.Two cell lines were established and designated as SSC7and SSC12.For differen-tiation,the cells were cultured on gelatine-coated dishes,without LIF.
RNA Isolation and RT-PCR
Total RNA was isolated,and cDNA synthesis was carried out with oligo-dT primers as described(Nayernia et al.,2004).RT-PCR ampli-fication was performed by using specific primers for EGFP,Stra8, Tex18,Dazl,Rbm,Oct4,c-kit,Scp3,Pgk2,and Tp2.RT-PCR was achieved after35–40cycles of94ºC,30s;50ºC–62ºC,30s;72ºC, 45s,depending on the primer ts.Primer quences are available as Supplemental Data.
Aniline Blue Staining
A total of10m l of the cell sample was spread onto glass slides and allowed to dry at room temperature.Smears werefixed in3%glutar-aldehyde(12.5ml glutaraldehyde25%in87.5ml PBS)for30min at room temperature.Slides were then stained with5%w/v aniline blue in PBS(pH3.5).Each slide was then washed with PBS and left to dry in the air.Stained and unstained cells were counted and examined under the microscope at6003magnification. Immunocytochemistry and Fluorescence Microscopy
Adherent cells werefixed in4%paraformaldehyde(PFA)in PBS(pH 7.4)for1hr at ambient temperature.Fixed cells were rind in PBS (pH7.4)and subquently incubated overnight with primary anti-bodys specific to integrin a6,integrin b1,heat shock protein90a (Hsp90a),c-kit,Oct4,Tp2,or outer acrosomal membrane(OAM)pro-tein.All primary antibodies,except tho for Tp2,Acr,Rbm, hnRNPG-T,Dazl,Piwil2,Stra8,and OAM,were purchad from Santa Cruz.Other antibodies were provided in collaboration with other rearch groups(e Supplemental Data).Cells were rind three times and incubated in the appropriate Cy3-,FITC-,or AP-con-jugated condary antibody(Sigma).All incubations were per-formed in PBS(pH7.4),5%BSA,and0.1%Triton X-100.For nuclear stainings,fixed cells were incubated for5min with DAPI(40,60-dia-midino-2-phenylindole)(Vec
tor)dye.Microscopy was performed on a Zeissfluorescence microscope.
Transplantation into Seminiferous Tubules of Testes
Recipients ud were129/Sv and FVB mice.At6–8weeks of age,the recipient mice were injected i.p.with busulfan(40mg/kg body weight),which destroys endogenous spermatogenesis.Recipients were ud for transplantation R4weeks after injection.Cultured SSC7and SSC12cells were harvested by using0.25%trypsin plus 1mM EDTA,and the cells were suspended in DMEM culture me-dium.The cell concentration for transplantation was10–303106 cells/ml.Approximately10m l of the cell suspension was trans-planted via rete testis into miniferous tubules of one testis.The other testis rved as an internal control.Ten animals were trans-planted,and ten animals were ud as control.As an external con-trol,busulfan-treated,nontransplanted males were ud.After4 months,recipient mice were killed,and testes were examined by immuncytochemistry,image cytometry,and RT-PCR analysis.All of the experimental procedures complied with national regulations for the Care and U of Laboratory Animals(similar to the U.S. National Rearch Council guidelines).
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