Human embryonic stem (ES) cells are predicted to be a valuable source for producing ES-derived therapeutic spare tissues to treat diseases by controlling their growth and differentiation. To understand the regulative mechanisms of their differentiation in vivo and in vitro, ES cells derived from nonhuman primates could be a powerful tool. We established four ES cell lines from cynomolgus monkey (Macaca fascicularis) blastocysts produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The ES cells were characterized by the expression of specific markers such as alkaline phosphatase and stage-specific embryonic antigen-4. They were successfully maintained in an undifferentiated state and with a normal karyotype even after more than 6 months of culture. Pluripotential competence was confirmed by the formation of teratomas containing ectoderm-, mesoderm-, and endodermderivatives after subcutaneous injection into SCID mice. Differentiation to a variety of tissues was identified by immunohistochemical analyses using tissue-specific antibodies. Therefore, we established pluripotent ES cell lines derived from monkeys that are widely used as experimental animals. These lines could be a useful resource for preclinical stem cell research, including allogenic transplantation into monkey models of disease.

Ca2؉ , which enters cardiac myocytes through voltagedependent Ca 2؉ channels during excitation, is extruded from myocytes primarily by the Na ؉ /Ca 2؉ exchanger (NCX1) during relaxation. The increase in intracellular Ca 2؉ concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na ؉ /Ca 2؉ exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na ؉ /Ca 2؉ exchange activity was detected in null mutant hearts. The Na ؉ -dependent Ca 2؉ exchange activity as well as protein content of NCX1 were decreased by ϳ50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na ؉ -free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na ؉

Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products. An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing abnormal elongated spermatids in the lumina of seminiferous tubules. We cloned the disrupted locus of kisimo and identified a novel testis-specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse. The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-1⑀, suggesting that THEG protein functions as a regulatory factor in protein assembly. Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility.The integration of foreign DNA into the mouse germ line by retroviral infection or microinjection can result in insertional disruption of endogenous genes with important roles in development (1-3). Foreign DNA insertion provides an approach to the cloning of disrupted host loci. By using the introduced DNA as a probe to screen genomic libraries from mutant animals, it has been possible in a few instances to isolate clones that contain DNA flanking the exogenous integrated material and, thus, include portions of the interrupted gene (4, 5). We have produced a series of 12 transgenic mouse lines with the human phosphodiesterase 5A (PDE5A) 1 gene (6). As the mice were bred, it became evident that many males of one line were sterile and that the sterility arose from a defect in spermatogenesis (we named this mutant kisimo for a Japanese goddess of easy delivery). Because the sterility segregated with the hemizygous transgene and occurred in the absence of the detectable expression of the transgene, we concluded that the abnormal phenotype was due to mutagenesis by insertion of the transgene. In this study, to clone the junctions between the inserted transgene and adjoining cellular DNA, we used thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) (7) and defined a genomic locus important for spermatogenesis. The process of spermatogenesis in the mouse has been well characterized at the morphological level. The spermatogenic process can be subdivided into three main phases. Spermatogonia, the germinal stem cells, undergo mitosis to produce additional spermatogonia, a portion of which develop into primary spermatocytes. The spermatocytes enter meiosis and proceed through two cell divisions to give rise to haploid round spermatids. These, in turn, undergo a complex morphological transformation involving nuclear condensation and elongation resulting in the production of mature spermatozoa. However, at the molecular level, relatively little is known about the control of cellular differentiation and the architectural changes during spermatogenesis.In this study, we found that an insertion of foreign DNA results in abnormal male ...

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The present study was performed to determine whether testicular toxicity can be detected of the administration of adriamycin (ADR), an anticancer drug, for 2 weeks. ADR was intravenously administered to Slc:SD male rats at 1 mg/kg once a week for 4 or 2 weeks, and 2 mg/kg once a week for 2 weeks. Suppression of body weight gain was observed in the 1 mg/kg/week (4 w) and 2 mg/kg/week (2 w) groups. The testes weights in these groups were also lower than those of the control group. On histological examination, a decrease in spermatogonia was observed in the 1 mg/kg/week (4 w) and 2 mg/kg/week (2 w) groups. No abnormalities were detected in the control and 1 mg/kg/week (2 w) groups. Stage analysis of seminiferous tubules was performed for control and 1 mg/kg/week (2 w) groups. In the 1 mg/kg/week (2 w) group, the numbers of spermatogonia in stages II-III, V and XII-XIII were significantly decreased. The numbers of preleptotene spermatocytes in stage VII and zygotene spermatocytes in XII-XIII were also significantly reduced in the same group. These findings suggest that the testicular toxicity of ADR can be detected after 2 weeks administration at a sufficient high dosage, if simplified morphometrical analysis is used.