Induction of the G2/M transition stabilizes haploid embryonic stem cells.

The recent successful establishment of mouse parthenogenetic haploid embryonic stem cells (phESCs) and androgenetic haploid ESCs (ahESCs) has stimulated genetic research not only in vitro but also in vivo because of the germline competence of these cell lines. However, it is difficult to maintain the haploid status over time without a frequent sorting of the G1 phase haploid ESCs by fluorescence-activated cell sorting (FACS) at short intervals, because haploid cells tend to readily self-diploidize. To overcome this spontaneous diploid conversion, we developed a phESC culture condition using a small molecular inhibitor of Wee1 kinase to regulate the cell cycle by accelerating the G2/M phase transition and preventing re-entry into extra G1/S phase. Here, we demonstrate that, under this condition, phESCs maintained the haploid status for at least 4 weeks without the need for FACS. This method will greatly enhance the availability of these cells for genetic screening.

A Niche for GFRα1-Positive Spermatogonia in the Terminal Segments of the Seminiferous Tubules in Hamster Testes.

In invertebrate species such as flies and nematodes, germline stem cells are maintained in a niche environment, which is restricted to the terminal end of the tubular structure in the gonads. In mice, spermatogonial stem cells (SSCs), a subpopulation of Asingle GFRα1 (glial cell line-derived neurotrophic factor [GDNF] family receptor-α1)-positive spermatogonia, are widely distributed along the longitudinal axis in the convoluted seminiferous tubules, preferentially juxtaposed to the interstitial vasculature. However, whether this area is the only SSC niche is not known. In this study, we identified a valve-like terminal segment of the seminiferous tubules, the Sertoli valve (SV), adjacent to the rete testis as another niche for GFRα1-positive spermatogonia in hamsters. Here, we show that the SV epithelium is composed of the modified Sertoli cells that are still capable of proliferation and missing most spermatogenic activities in the adult stage. The SV epithelium constitutively expresses GDNF, a major niche factor for SSCs, and supports the stable proliferation and selective maintenance of an Asingle subpopulation of GFRα1-positive spermatogonia in hamsters. The SV region of hamster seminiferous tubules has features that are similar to the stem cell niche in invertebrate gonads. Therefore, we propose that the SV may be a novel niche for Asingle GFRá1-positive spermatogonia potentially including a SSC population, at the terminal segments of the seminiferous tubules in hamsters.

A novel Amh-Treck transgenic mouse line allows toxin-dependent loss of supporting cells in gonads.

Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.

Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects.

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

A germ cell-specific gene, Prmt5, works in somatic cell reprogramming.

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.

Aberrant IL-4 production by SOCS3-over-expressing T cells during infection with Leishmania major exacerbates disease manifestations.

Suppressor of cytokine signaling (SOCS) 3 is a major negative feedback regulator of signal transducer and activator of transcription 3-activating cytokines. Studies using T-cell-specific SOCS3-deficient mice indicate that the absence of SOCS3 in T cells results in exacerbation of disease progression after infection by Leishmania major due to skewing of the T(h)3 cell phenotype accompanied by hyper-production of IL-10 and transforming growth factor ß (TGF-ß). Here we show that transgenic mice over-expressing the SOCS3 gene in T cells (Lck-SOCS3 Tg mice) are also susceptible to infection by L. major. Forced expression of SOCS3 in T cells did not affect the production of the anti-inflammatory cytokines IL-10 and TGF-ß or that of the protective T(h)1 type cytokine IFN-γ, which is required for parasite clearance. CD4(+) T cells isolated from infected-Lck-SOCS3 Tg mice produced much higher levels of IL-4 when they were re-stimulated with L. major antigen in vitro. Exacerbation of disease progression in Lck-SOCS3 Tg mice was completely reversed by administration of a neutralizing antibody against IL-4. These data suggest that tight regulation of SOCS3 expression in T(h) cells is crucial for disease control during infection by L. major.

IL-1ß and TNFα-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes irreversible joint damage and significant disability. However, the fundamental mechanisms underlying how inflammation and joint destruction in RA develop and are sustained chronically remain largely unknown. Here, we show that signal transducer and activator of transcription 3 (STAT3) is the key mediator of both chronic inflammation and joint destruction in RA. We found that inflammatory cytokines highly expressed in RA patients, such as IL-1ß, tumor necrosis factor alpha and IL-6, activated STAT3 either directly or indirectly and in turn induced expression of IL-6 family cytokines, further activating STAT3 in murine osteoblastic and fibroblastic cells. STAT3 activation also induced expression of receptor activator of nuclear factor kappa B ligand (RANKL), a cytokine essential for osteoclastogenesis, and STAT3 deficiency or pharmacological inhibition promoted significant reduction in expression of both IL-6 family cytokines and RANKL in vitro. STAT3 inhibition was also effective in treating an RA model, collagen-induced arthritis, in vivo through significant reduction in expression of IL-6 family cytokines and RANKL, inhibiting both inflammation and joint destruction. Leukemia inhibitory factor expression and STAT3 activation by IL-1ß were mainly promoted by IL-6 but still induced in IL-6-deficient cells. Thus, our data provide new insight into RA pathogenesis and provide evidence that inflammatory cytokines trigger a cytokine amplification loop via IL-6-STAT3 that promotes sustained inflammation and joint destruction.

Genetic vasectomy-overexpression of Prm1-EGFP fusion protein in elongating spermatids causes dominant male sterility in mice.

Transgenic mice are vital tools in both basic and applied research. Unfortunately, the transgenesis process as well as many other assisted reproductive techniques involving embryo transfer rely on vasectomized males to induce pseudopregnancy in surrogate mothers. Vasectomy is a surgical procedure associated with moderate pain and must be carried out under full anaesthesia by qualified personnel. Eliminating the need for vasectomy would be beneficial from the economic and animal welfare point of view. Our aim was to develop a transgene-based alternative to the surgical vasectomy procedure. We generated several transgenic mouse lines expressing a Protamine-1 (Prm1) EGFP fusion protein under the transcriptional and translational regulatory control of Prm1. Male mice from lines showing moderate transgene expression were fully fertile whereas strong overexpression of the Prm1-EGFP fusion protein resulted in complete and dominant male sterility without affecting the ability to mate and to produce copulatory plugs. Sterility was due to impaired spermatid maturation affecting sperm viability and motility. Furthermore, sperm having high Prm1-EGFP levels failed to support preimplantation embryonic development following Intracytoplasmic Sperm Injection (ICSI). The "genetic vasectomy system" was further improved by genetically linking the dominant male sterility to ubiquitous EGFP expression in the soma as an easy phenotypic marker enabling rapid genotyping of transgenic males and females. This double transgenic approach represents a reliable and cost-effective "genetic vasectomy" procedure making the conventional surgical vasectomy methodology obsolete.

Transgenic over-expression of growth differentiation factor 11 propeptide in skeleton results in transformation of the seventh cervical vertebra into a thoracic vertebra.

Growth differentiation factor 11 (GDF11) is one of the significant genes that control skeletal formation. Knockout of GDF11 function causes abnormal patterning of the anterior/posterior axial skeleton. The mRNA of GDF11 is initially translated to a precursor protein that undergoes a proteolytic cleavage to generate the C-terminal peptide or mature GDF11, and the N-terminal peptide named GDF11 propeptide. The propeptide can antagonize GDF11 activity in vitro. To investigate the effects of GDF11 propeptide on GDF11 function in vivo, we generated transgenic mice that over-express the propeptide cDNA in skeletal tissue. The transgenic mice showed formation of extra ribs on the seventh cervical vertebra (C7) as a result of transformation of the C7 vertebra into a thoracic vertebra. The GDF11 propeptide transgene mRNA was detected in tail tissue in embryos and was highly expressed in tail and calvaria bones after birth. A high frequency of C7 rib formation was noticed in the transgenic mouse line with a high level of transgene expression. The anterior boundaries of Hoxa-4 and Hoxa-5 mRNA in situ expressions showed cranial shifts from their normal prevertebra locations in transgenic embryos. These results demonstrated significant effects of GDF11 propeptide transgene on vertebral formation, which are likely occurring through depressing GDF11 function and altered locations of Hoxa-4 and Hoxa-5 expression.

Abnormal DNA methylation of the Oct-4 enhancer region in cloned mouse embryos.

Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos--the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.

Mouse Sox17 haploinsufficiency leads to female subfertility due to impaired implantation.

Embryonic implantation comprises a dynamic and complicated series of events, which takes place only when the maternal uterine endometrium is in a receptive state. Blastocysts reaching the uterus communicate with the uterine endometrium to implant within a narrow time window. Interplay among various signalling molecules and transcription factors under the control of ovarian hormones is necessary for successful establishment of pregnancy. However, the molecular mechanisms that allow embryonic implantation in the receptive endometrium are still largely unknown. Here, we show that Sry-related HMG box gene-17 (Sox17) heterozygous mutant female mice exhibit subfertility due to implantation failure. Sox17 was expressed in the oviduct, uterine luminal epithelium, and blood vessels. Sox17 heterozygosity caused no appreciable defects in ovulation, fertilisation, blastocyst formation, and gross morphology of the oviduct and uterus. Another group F Sox transcription factor, Sox7, was also expressed in the uterine luminal and glandular epithelium relatively weakly. Despite uterine Sox7 expression, a significant reduction in the number of implantation sites was observed in Sox17 heterozygous mutant females due to haploinsufficiency. Our findings revealed a novel role of Sox17 in uterine receptivity to embryo implantation.

Targeted gene deletion of miRNAs in mice by TALEN system.

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.

Hematopoietic stem cells (HSCs) are maintained in a specific bone marrow (BM) niche in cavities formed by osteoclasts. Osteoclast-deficient mice are osteopetrotic and exhibit closed BM cavities. Osteoclast activity is inversely correlated with hematopoietic activity; however, how osteoclasts and the BM cavity potentially regulate hematopoiesis is not well understood. To investigate this question, we evaluated hematopoietic activity in three osteopetrotic mouse models: op/op, c-Fos-deficient, and RANKL (receptor activator of nuclear factor kappa B ligand)-deficient mice. We show that, although osteoclasts and, by consequence, BM cavities are absent in these animals, hematopoietic stem and progenitor cell (HSPC) mobilization after granulocyte colony-stimulating factor injection was comparable or even higher in all three lines compared with wild-type mice. In contrast, osteoprotegerin-deficient mice, which have increased numbers of osteoclasts, showed reduced HSPC mobilization. BM-deficient patients and mice reportedly maintain hematopoiesis in extramedullary spaces, such as spleen; however, splenectomized op/op mice did not show reduced HSPC mobilization. Interestingly, we detected an HSC population in osteopetrotic bone of op/op mice, and pharmacological ablation of osteoclasts in wild-type mice did not inhibit, and even increased, HSPC mobilization. These results suggest that osteoclasts are dispensable for HSC mobilization and may function as negative regulators in the hematopoietic system.

Abnormal distribution of chromosomes in the first division of nuclear transferred mouse embryos.

The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.