Male gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed 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 development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.

EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. Presently, the nature of EPOregulated survival genes has been investigated through transcriptome analyses of highly responsive, primary bone marrow erythroblasts. Two proapoptotic factors, Bim and FoxO3a, were rapidly repressed not only via the wild-type EPOR, but also by PY-deficient knocked-in EPOR alleles. In parallel, Pim1 and Pim3 kinases and Irs2 were induced. For this survival gene set, induction failed via a PY-null EPOR-HM allele, but was restored upon reconstitution of a PY343 STAT5-binding site within a related EPOR-H allele. Notably, EPOR-HM supports erythropoiesis at steady state but not during anemia, while EPOR-H exhibits near wildtype EPOR activities. EPOR-H and the wild-type EPOR (but not EPOR-HM) also markedly stimulated the expression of Trb3 pseudokinase, and intracellular serpin, Serpina-3G. For SERPINA-3G and TRB3, ectopic expression in EPOdependent progenitors furthermore significantly inhibited apoptosis due to cytokine withdrawal. BCL-XL and BCL2 also were studied, but in highly responsive Kit pos CD71 high Ter119 neg erythroblasts, neither was EPO modulated. EPOR survival circuits therefore include the repression of Bim plus IntroductionIn response to anemia, erythropoietin (EPO) is expressed by interstitial kidney and fetal liver cells via hypoxia-inducible transcription factor pathways. 1,2 As a secreted monomeric sialoglycoprotein, EPO then targets developing erythroblasts, and is essential for red cell formation during definitive bone marrow and fetal liver erythropoiesis. [3][4][5][6][7][8] Prospective roles for EPO in promoting primitive red cell formation in yolk sac also have recently been described. 9 Beyond this, recombinant EPO has been demonstrated in ischemia and other cell damage models to provide cytoprotective effects for injured renal, cardiac, retinal, and neuronal tissues. 10,11 Taken together, these considerations have heightened interest in the specific nature of key EPO action mechanisms, especially those associated with progenitor cell survival.EPO's prime effects are mediated via interactions with its dimeric single-transmembrane receptor (EPOR). [3][4][5][6][7][8]12 These interactions appear to evoke EPOR conformational events, 13 which are relayed to an upstream Janus kinase, JAK2 14 (and JAK2 likewise may preassemble with EPOR dimers at a juxtamembrane box1 domain). 15,16 JAK2, as activated via a Y1007 phosphorylation loop, 17 next stimulates 2 separable signal transduction pathways. First, JAK2 interestingly can support steady-state erythropoiesis via EPOR-PY-independent routes that, in part, may involve MEK1,2 and ERK1,2 stimulation. 18 Second, JAK2 also mediates the phosphorylation of 8 conserved EPOR cytoplasmic PY sites, which can then form a scaffold for the binding of up to 20 SH2-or PTB-domain encoding signal transduction factors and molecular adaptors. [6][7][8][19][20][21] Among conserve...

Purpose of review-In 1985-1989 EPO, its receptor (EPOR) and JAK2 were cloned; established to be essential for definitive erythropoiesis; and initially intensely studied. Recently, new impetus, tools, and model systems have emerged to re-examine EPO/EPOR actions -and are addressed in this review. Impetus includes indications that EPO affects significantly more than standard erythroblast survival pathways; the development of novel erythropoiesis stimulating agents (ESA's); increasing evidence for EPO/EPOR cytoprotection of ischemically injured tissues; and potential EPO-mediated worsening of tumorigenesis.Recent findings-are reviewed in four functional contexts: i) (Pro)erythroblast survival mechanisms; ii) New candidate EPO/EPOR effects on erythroid cell development, plus new EPOR responses; iii) EPOR down-modulation, and trafficking; iv) Novel ESA's. Summary-As

Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit ؊ CD71 high Ter119 ؊ "stage E2" proerythroblast pool first is described, which (unlike its Kit ؉ "stage E1" progenitors, or maturing Ter119 ؉ "stage E3" progeny) proved to selectively expand ϳ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E13E23E3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices. IntroductionErythropoiesis in mouse and humans is ontogenically compartmentalized. Primitive yolk sac hematopoietic progenitor cells initially give rise to nucleated red cells (which can later enucleate) and also colonize the aorta-gonad-mesonephros and umbilical cord. [1][2][3] In fetal liver (which may be seeded by yolk sac and aorta-gonadmesonephros hematopoietic progenitor cells), red cell formation from committed erythroid progenitors becomes erythropoietin (EPO) dependent. 4 Fetal liver erythropoiesis also relies on stromal cell interactions. Examples include erythroblast KIT and Eph4 binding to their stromal ligands (KIT-L and Ephrin-B2) 4-6 as well as fetal liver erythroblast ␣4, ␤1 integrin effects. 7,8 In perinatal and adult life, erythropoiesis (and hematopoiesis) shift to the bone marrow (BM) compartment. In humans, this remains the prime erythropoietic tissue, although under atypical conditions (eg, spherocytosis or inhibited vascular endothelial growth factor signaling) spleen and liver can become erythropoietic sites. 8,9 In mouse, splenic erythropoiesis additionally can be readily induced, and experimentally such stress erythropoiesis can be a useful barometer of a compromised erythron. 10,11 BM erythropoiesis is less studied in part because of low frequencies of erythroid progenitors and limited tissue per se. Genetic evidence also indicates that erythroblast development in BM differs in at least several basic ways from splenic, fetal liver, and yolk sac erythropoiesis. Examples include roles for Hedgehog plus BMP4 interplay in splenic but not BM...