In an effort to study the molecular basis of the determination processes of the mammalian germ cell lineage, we have tried to isolate a mouse gene homolog to vasa, which plays an essential role as a maternal determining factor for the formation of Drosophila germ cell precursors. By reverse transcriptase PCRs of mouse primordial germ cell cDNAs using family-specific primers, we obtained a gene (Mvh) encoding a DEAD-family protein that showed a much higher degree of similarity with the product of the Drosophila vasa gene (vas) than previously reported mouse genes. In adult tissues, Mvh tnscripts were exclusively detected in testicular germ cells, in which Mvh protein was found to be localized in cytoplasm of spermatocytes and round spermatids including a perinuclear granule. The protein was also expressed in germ cells colonized in embryonic gonads but was not detected in pluripotential embryonic cells such as stem cells and germ cells. These results suggest the possibility that the Mvh protein may play an important role in the determination events of mouse germ cells as in the case of Drosophila vasa.In mice, germ cell precursors, termed primordial germ cells (PGCs), are generated in the epiblast. They consist of putative pluripotential cells that can be first identified as a small cluster of alkaline phosphatase-positive cells in the extraembryonic mesoderm at 7.25 days postcoitum (dpc). Then they move down to the embryonic mesoderm at the posterior end of the primitive streak (8.0 dpc), migrate through the hindgut endoderm, and colonize the developing genital ridges (10.5-11.5 dpc), in which they are destined to form functional germ cells (1, 2). Despite these detailed morphological observations, molecular mechanisms regulating this developmental pathway still remain unclear.Unlike mammals, it has been well established that several maternal factors are involved in germ cell determination of many animal species. In particular, in Drosophila it has been shown that the pole plasm localized at the posterior pole ofthe oocyte contains determining substances for the abdomen and the germ lineage. After fertilization, only the nuclei that migrate into the pole plasm are destined to form the germ-line progenitors (pole cells). Genetic identification ofgenes whose function is required for pole cell formation has revealed at least eight maternally active genes, cappucino, spire, staufen, oskar, vasa, valois, mago-nashi, and tudor (3, 4). Among these genes, vasa (vas) is one of the best characterized. Homozygous mutant vasa females produce no eggs. In ectopic formation of pole cells induced by mislocalization of oskar mRNA to the anterior pole of the oocyte, only vasa and tudor were required for ectopic pole cell formation (5-10). The gene vas encodes a DEAD (Asp-Glu-Ala-Asp)-family protein of putative RNA helicases, which is found to be present as a component of both the polar granules at the posterior end of the oocyte and the nuage structure in the germ cells, and zygotic expression is also restricted to the ge...

Background: Germline-speci®c differential DNA methylation that persists through fertilization and embryonic development is thought to be thè imprint' distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are ®rst erased in primordial germ cells (PGCs) and then re-established during oocyte growth.

Although seminolipid has long been suspected to play an essential role in spermatogenesis because of its uniquely abundant and temporally regulated expression in the spermatocytes, direct experimental evidence has been lacking. We have tested the hypothesis by examining the testis of the UDP-galactose:ceramide galactosyltransferase-deficient mouse, which is incapable of synthesizing seminolipid. Spermatogenesis in homozygous affected males is arrested at the late pachytene stage and the spermatogenic cells degenerate through the apoptotic process. This stage closely follows the phase of rapid seminolipid synthesis in the wild-type mouse. These observations not only provide the first experimental evidence that seminolipid is indeed essential for normal spermatogenesis but also support the broader concept that cell surface glycolipids are important in cellular differentiation and cell-to-cell interaction.Seminolipid (3-sulfogalactosyl-1-alkyl-2-acyl-sn-glycerol) is the principal glycolipid in spermatozoa of mammals comprising, for example, approximately 3% of total lipids and more than 90% of total glycolipids in boar spermatozoa (1-3). During spermatogenesis, seminolipid is synthesized rapidly in the early phase of spermatocyte development and maintained in subsequent germ cell stages (4 -6). This developmentally regulated rapid synthesis suggested a specific and possibly essential function of seminolipid in spermatogenesis (7) but experimental evidence has been lacking. Firm evidence in support of the speculation would have important bearing to the general concept that cell surface glycoconjugates are important in cellular differentiation, and cell-to-cell interaction (8).Seminolipid is synthesized by sulfation of its precursor, galactosylalkylacylglycerol (GalEAG) 1 . GalEAG is synthesized by UDPgalactose:ceramide galactosyltransferase (CGT, EC 2.4.1.62), which, besides GalEAG, also synthesizes the major myelin galactolipid, galactosylceramide (GalCer), galactosylsphingosine (psychosine), and galactosyldiacylglycerol (GalAAG) (9, 10). The CGTdeficient mice recently generated by gene-targeting do not synthesize any of these products and subsequent derivatives of the products (11)(12)(13)(14). Thus, the CGT-deficient mouse is an ideal experimental model to examine the consequences of lack of seminolipid to spermatogenesis. This report describes the first definitive evidence that deficient seminolipid biosynthesis indeed causes devastating disruption of the normal spermatogenetic process. EXPERIMENTAL PROCEDURESMice-The mice heterozygous for the disrupted Cgt gene (11) were originally supplied by Dr. B. Popko and maintained by backcrossing to C57BL/6N. Genotype was determined according to Coetzee et al. (11). WBB6F1 Kit W/W-v and WBB6F1 Mg f Sl/Sl-d mutant mice were purchased from Japan SLC, Inc., and C57BL/6N inbred mice were purchased from CLEA Japan, Inc. Isolation of Testicular Germ Cells-Testicular germ cells were isolated from decapsulated testes of sexually mature male C57BL/6N mice (15).RT-PCR Analysis-RNA...

An in vivo gene transfer technique for living mouse testes was used to develop a novel transient expression assay system for transcriptional regulatory elements of spermatogenic specific genes. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation resulted in an efficient and convenient assay system for gene expression during spermatogenesis. The transfer of the firefly luciferase reporting gene driven by the Protamine-1 (Prm-1) enhancer region revealed a significant increase in the activity of the reporter enzyme. Histochemical studies of the transfer of the lacZ gene driven by the Prm-1 enhancer showed specific lacZ expression only in haploid spermatid cells in adult testes, corresponding with the expression pattern of endogenous Prm-1. We were able to detect long-lasting transgene expression in the transfected spermatogenic cells. A group of spermatogenic differentiating cells maintained the transfected lacZ expression after more than 2 mo of transfection, suggesting that spermatogenic stem cells and/or spermatogonia could also incorporate foreign DNA and that the transgene could be transmitted to the progenitor cells derived from a transfected proliferating germ cell.

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