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...

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder-free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round-shaped cells resembling immature oocytes. Mvh(+) cells formed clumps by co-aggregation with differentiation-supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency.

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.

Mice carrying two t complementary haplotypes (tw5/tw32) are totally sterile. Their spermatozoa have poor motility and fertilize neither zona-intact nor zona-free oocytes, even though they are structurally indistinguishable from control (wild-type) spermatozoa. However, when injected directly into oocytes, these infertile spermatozoa are able to participate in normal development. This suggests that infertility of tw5/tw32 male (spermatozoa) is more likely to be due to poor sperm-oocyte interaction than to genetic incompetence of sperm nuclei.

Detection of protein^protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein^protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins. ß