Testis-specific sulfoglycolipid, seminolipid, is essential for germ cell function in spermatogenesis

More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid, seminolipid. The sulfation of the molecule is catalyzed by a Golgi membrane-associated sulfotransferase, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice results in male infertility due to the arrest of spermatogenesis prior to the metaphase of the first meiosis. However, the issue of which side of the cell function-germ cells or Sertoli cells-is deteriorated in this mutant mouse remains unknown. Our findings show that the defect is in the germ cell side, as evidenced by a transplantation analysis, in which wild-type spermatogonia expressing the green fluorescent protein were injected into the seminiferous tubules of CST-null testis. The transplanted GFP-positive cells generated colonies and spermatogenesis proceeded over meiosis in the mutant testis. The findings also clearly show that the seminolipid is expressed on the plasma membranes of spermatogonia, spermatocytes, spermatids, and spermatozoa, as evidenced by the immunostaining of wild-type testes using an anti-sulfogalactolipid antibody, Sulph-1 in comparison with CST-null testes as a negative control, and that seminolipid appears as early as day 8 of age, when Type B spermatogonia emerge.     

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt mice

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt^+/− mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt^−/− males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt^+/− males sire offspring. The higher than 50% expression level of SGG in Cgt^+/− animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt^+/− mice were sufficient for normal spermatogenesis.     

The Lifetime of UDP-galactose:Ceramide Galactosyltransferase Is Controlled by a Distinct Endoplasmic Reticulum-associated Degradation (ERAD) Regulated by Sigma-1 Receptor Chaperones

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.     

Identification of a region of UDP-galactose:N-acetylglucosamine beta 4-galactosyltransferase involved in UDP-galactose binding by differential labeling

The location of regions in the primary structure of UDP-galactose:N-acetylglucosamine beta 4-galactosyl-transferase (GT) that are involved in binding UDP-galactose has been investigated by differential chemical modification with two different reagents in the presence and absence of UDP-galactose. Treatment with periodate-cleaved UDP and NaCNBH3 resulted in a loss of 80% of GT activity, which was largely prevented by UDP-galactose. Stoichiometry of labeling and peptide maps of the modified enzyme samples indicated partial labeling at many sites. A major site of reaction in the absence of UDP-galactose that was essentially unmodified in its presence was found to correspond to Lys341 in the cDNA sequence of GT. As a second approach, the reactivities of the amino groups of GT were compared in the presence and absence of saturating levels of UDP-galactose by trace acetylation with [3H]acetic anhydride. UDP-galactose binding was found to perturb the reactivities of a number of lysines in the C-terminal region of GT, the most pronounced effect being a reduction in the reactivity of Lys351. The two procedures thus identified a region between residues 341 and 351 as being associated with UDP-galactose binding. This region overlaps a small section in the sequence of GT that was previously noted to be similar to part of bovine alpha-1,3-galactosyltransferase (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Sequence comparisons indicate that extended regions at the C terminus of each enzyme encompassing this area may represent homologous UDP-galactose-binding domains.     

Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation

The niche is considered to play an important role in stem cell biology. Sertoli cells are the only somatic cells in the seminiferous tubule that closely interact with germ cells to create a favorable environment for spermatogenesis. However, little is known about how Sertoli cells develop to form the male germ line niche. We report here that Sertoli cells recovered and dissociated from testes of donor male mice can be microinjected into recipient testes, form mature seminiferous tubule structures, and support spermatogenesis. Sertoli cells from perinatal donors had a dramatically greater capacity for generating seminiferous tubules than those from adult donors. Furthermore, transplantation of wild-type Sertoli cells into infertile Steel/Steel(dickie) testes created a permissive testicular microenvironment for generating spermatogenesis and spermatozoa. Thus, our results demonstrate that the male germ line stem cell niche can be transferred between animals. In addition, the technique provides a novel tool with which to analyze spermatogenesis and might provide a mechanism for correcting fertility in males suffering from supporting cell defects.     

Analysis of spermatogenesis in senescence-accelerated mice

A comparative analysis of age-related dynamics of spermatogenesis has been performed in mutant mouse lines predisposed or resistant to accelerated senescence (SAMP1 and SAMR1 respectively). The results show that quantitative and morphohistological trends in the development of sperm cells and Sertoli cells in both lines are similar in both lines. Their comparison with data obtained in our previous studies (Zakhidov et al., 2001; Gordeeva et al., 2001) shows that sharp quantitative and qualitative changes in the structure of the spermatogenic system have occurred in senescence-accelerated mice of new generations, which confirms the fact of dynamic instability of the germinal lineage. The role of stem spermatogonial cells in restoration of spermatogenesis in animals reaching the critical age is discussed.     

Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.     

Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine had-1 mutant cell lines and construction of deletion mutants

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Enhancement of UDP-galactose:mucin galactosyltransferase activity by spermine

A microsomal preparation prepared from the mucosal lining of canine trachea catalyzed the transfer of galactose from its uridine diphosphate derivative to sialidase-treated ovine submaxillary mucin. Maximal incorporation occurred at 30 mm mn²⁺. When the concentration of mn²⁺ in the reaction mixture was reduced to 2.5 mm, approximately two-thirds of the enzymatic activity was lost, but full activity could be restored by the addition of 1 mm spermine. Under these conditions spermine did not affect the K_m for UDP-galactose, but lowered the K_m for sialidase-treated ovine submaxillary mucin and Mn²⁺ by a factor of 10. The effect of spermine was abolished with increasing concentrations of Mn²⁺, and in the absence of the metal, enzymatic activity was lost and could not be restored by the addition of spermine. Spermidine also stimulated activity at low levels of Mn²⁺, but to a lesser degree than spermine. A slight stimulatory effect was consistently derived from putrescine as well, while cadaverine, putreanine, and monoamines were ineffectual. Spermine had a similar effect on the enzymatic transfer of GalNAc to a protein core acceptor but had little or no effect on the enzymatic transfer of sialic acid to sialidase-treated ovine submaxillary mucin, galactose to N-acetylglucosamine, or fucose to sialidase-galactosidase-treated fetuin. Similar results were obtained with enzyme preparations prepared from canine submaxillary glands. Other polycationic compounds such as protamine, histone, and polylysine also stimulated enzymatic activity at suboptimal concentrations of mn²⁺.     

UDP-galactose inhibition of BALB/3T12-3 cell growth : Requirement for medium galactosyltransferase activity

Incubation of BALB/3T12-3 cells with uridine diphosphate galactose (UDP-gal) resulted in a concentration-dependent inhibition of cell growth when cells were cultured in calf serum-supplemented Dulbecco's modified Eagle medium (CS-DMEM). Cell growth was completely inhibited by 5 mM UDP-gal with an ID₅₀ of 0.75 mM. This inhibitory effect was reversible. Other nucleotide-sugars, as well as galactose, glucose, and galactose-1-phosphate had no effect on cell growth. UDP-gal had no effect on cell growth when cells were cultured in heat-inactivated calf serum containing DMEM (HICS-DMEM) suggesting that a serum enzyme activity was responsible for the inhibition observed in CS-DMEM. No significant difference could be detected by descending chromatography in the degradation of UDP-gal during 96 h of incubation in CS-DMEM and in HICS-DMEM. Furthermore, the potential breakdown products of UDP-gal had no effect on cell growth when added directly to 3T12 cultures. When cells were incubated with 5 mM UDP-gal+5 mM CDP-choline (a potent inhibitor of pyrophosphatase activity), complete inhibition of cell growth was still observed. However, if cells were incubated with 5 mM UDP-gal+UDP (which inhibited calf serum galactosyltransferase activity), no inhibition of cell growth was observed over that found for UDP alone, suggesting that galactosyltransferase and not pyrophosphatase activity mediated the effect of UDP-gal on cell growth. A direct effect of UDP-gal on cells was suggested by (a) normal growth of cells in UDP-gal-conditioned medium (preincubated with UDP-gal for 24 h followed by dialysis to remove UDP-gal); (b) 3-fold greater incorporation of [³H]galactose from UDP-[³H]gal into cells grown in CS-DMEM than in HICS-DMEM. These studies suggest that the inhibition of 3T12 cell growth by exogenous UDP-gal may be due to alteration of cell surface glycoconjugates by extracellular galactosyltransferase activity.     

Prostanoids in immunity: roles revealed by mice deficient in their receptors

Prostanoids including prostaglandins (PGs) and thromboxanes (TX) are a group of lipid mediators formed and released in response to various, often noxious, stimuli. While the roles of prostanoids in acute inflammatory responses are well known and have been extensively studied, it is generally believed that they play very little in immunity. This is partly because non-steroidal anti-inflammatory drugs that inhibit prostanoid synthesis have little effects on immune processes in vivo. Prostanoids exert their actions by acting on a family of G-protein-coupled receptors. They include PGD receptor, EP1, EP2, EP3 and EP4 subtypes of PGE receptor, PGF receptor, PGI receptor and TX receptor. We generated mice deficient in each of these prostanoid receptors individually, and examined their roles under various pathological conditions. These studies have revealed that prostanoids works at various sites or levels of immune responses and exert many, often opposing, actions. For example, using EP4-deficient mice, we found that stimulation of the PGE(2)-EP4 signaling in dendritic cells facilitates their migration and maturation, while the stimulation of the same pathway in T cells potently suppresses their activation and proliferation. The latter action is evident in PGE(2)-mediated suppression of T cell proliferation in the gut of mice subjected to dextran sodium sulfate-induced colitis, a model of inflammatory bowel disease. Here I summarize our findings obtained by these and other studies. These findings suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of certain immunological disorders.