Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>10(14)-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.

CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.

Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation. The whole process, therefore, has never been reproduced in vitro in mammals, nor in any other species with a very few exceptions in some particular types of fish. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas-liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.

The modification of DNA by 5-methylcytosine (5mC) has essential roles in cell differentiation and development through epigenetic gene regulation. 5mC can be converted to another modified base, 5-hydroxymethylcytosine (5hmC), by the tet methylcytosine dioxygenase (Tet) family of enzymes. Notably, the balance between 5hmC and 5mC in the genome is linked with cell-differentiation processes such as pluripotency and lineage commitment. We have previously reported that the maternal factor PGC7 (also known as Dppa3, Stella) is required for the maintenance of DNA methylation in early embryogenesis, and protects 5mC from conversion to 5hmC in the maternal genome. Here we show that PGC7 protects 5mC from Tet3-mediated conversion to 5hmC by binding to maternal chromatin containing dimethylated histone H3 lysine 9 (H3K9me2) in mice. In addition, imprinted loci that are marked with H3K9me2 in mature sperm are protected by PGC7 binding in early embryogenesis. This type of regulatory mechanism could be involved in DNA modifications in somatic cells as well as in early embryos.

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites. To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 (paternally expressed 10), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.