Knock-in embryonic stem (ES) cells, in which GFP or lacZ was expressed from the endogenous mouse vasa homolog (Mvh), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during in vitro differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells in vitro. In vitro germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering. G erm-line cells are responsible for transmitting genetic information and for reproducing totipotency from generation to generation. Pluripotent stem cell lines, embryonic stem (ES) cells, and embryonic germ cells are established from cells of the germ cell lineage. Therefore, germ cell specification must be linked to the maintenance of pluripotency, as well as to cell fate commitment leading to gametogenesis.Unlike many animal species, in which the germ line is predetermined by maternal factors, germ cell specification in mammals takes place at the onset of gastrulation, after implantation of the embryo. In the mouse, primordial germ cells (PGCs) are first distinguished at the base of the allantois in gastrulating embryos at embryonic day (E) 7.25 (1). Lineage studies of epiblast cells show that mouse PGCs are specified by inductive interactions at the onset of gastrulation (2, 3). Genetic analyses using targeted mutations have revealed that bone morphogenic protein (BMP) 4 and -8b, soluble growth factors belonging to the transforming growth factor ␤ superfamily that are produced by extraembryonic ectoderm close to the boundary with the proximal epiblast, are required for the generation of PGCs from epiblast cells (4,5). Moreover, primary cultures of epiblast fragments from embryos at E5.5-E6.0 generate migrating PGCs when they are cocultured with extraembryonic ectoderm (6), and culturing of whole epiblasts from E6.0 embryos on feeder cells expressing both BMP4 and BMP8b gives rise to PGCs (7). These results reveal that BMPs derived from the extraembryonic ectoderm play crucial roles in PGC determination in the proximal epiblast.Despite such developments, it is not yet known how the founder population of PGCs is segregated from other pluripotent epiblast cells that form somatic cells. To approach this question, we examined the production of germ cells by an established pluripotent ES cell line. ES cells can form all cell lineages when introduced into host blastocysts and give rise to various somatic cell lineages in culture. However, it is not known whether they can generate the germ cell lineage in culture in the absence of the morphogenetic events associated with gastrulation. It has been difficult to addr...

Restricted expression of a mouse Vasa homolog gene (Mvh) expression is first detected in primordial germ cells (PGCs) after colonization of the genital ridges. Subsequently,Mvh is maintained until postmeiotic germ cells are formed. Here, we demonstrate that male mice homozygous for a targeted mutation of Mvh exhibit a reproductive deficiency. Male homozygotes produce no sperm in the testes, where premeiotic germ cells cease differentiation by the zygotene stage and undergo apoptotic death. In addition, the proliferation of PGCs that colonize homozygous male gonads is significantly hampered, and OCT-3/4 expression appears to be reduced. These results indicate that the loss ofMvh function causes a deficiency in the proliferation and differentiation of mouse male germ cells.

Germ cell-specific ATP-dependent RNA helicase, the product of the mouse vasa homolog (Mvh), has been shown to play an essential role in the development of the male germ cell. In male Mvh knockout mice, premeiotic germ cells arrest at the zygotene stage. To investigate the role of MVH protein in the progression of meiosis, we searched for genes encoding partners that interact with MVH in testicular germ cells. Using the yeast two-hybrid system, we found that MVH interacts with mouse RanBPM, a Ran-GTP binding protein involved in microtubule nucleation. RanBPM is predominantly expressed in the testis, especially in maturating spermatocytes. Within the cell, RanBPM and MVH are closely associated with perinuclear RNA-protein complexes and chromatoid bodies. The interaction of MVH with RanBPM points to a functional relationship between translational regulation and the microtubule nucleation during meiosis. Mol. Reprod. Dev. 66: 1-7, 2004.