The role of N-glycans in spermatogenesis

Many proteins, in particular those in the plasma membranes, are glycosylated with carbohydrates, which are grouped into O-glycans and N-glycans. O-glycans are synthesized step by step by glycosyltransferases, whereas N-glycans are synthesized by en-bloc transfer of the so-called high-mannose-type oligosaccharide from lipid-linked precursor to polypeptide. The high-mannose-type N-glycans are then modified by processing alpha-mannosidases. Alpha-mannosidase IIx (MX) was identified as the gene product of processing alpha-mannosidase II (MII)-related gene. MX apparently plays subsidiary role for MII in many cell types, as N-glycan patterns of MX null mouse tissues are not altered significantly. Surprisingly MX null male mice are infertile due to a failure of spermatogenesis. This review provides a brief overview of the in vivo role of N-glycans which are revealed by the gene knockout mouse approach, and introduce our studies on the MX gene knockout mouse. The MX gene knockout experiments unveiled a novel function of a specific N-glycan, which is N-acetylglucosamine-terminated and has a fucosylated triantennary structure, in the adhesion between germ cells and Sertoli cells. The study of MX is a good example of how the in vivo roles of an apparently redundant gene product are determined by the gene knockout approach.     

Change of cyclin D2 mRNA expression during murine testis development detected by fragmented cDNA subtraction method

Using subtractive hybridization and polymerase chain reaction, we developed a differential cloning system, the fragmented cDNA subtraction method, that requires only small amounts of materials. The cloning system was used to isolate several cDNA fragments expressed more abundantly in the premeiotic day 3 post-natal mouse testis than in the adult mouse testis. The isolated cDNA fragments included cDNA encoding the murine cyclin D2. Northern blot and in situ hybridization analyses revealed that, during testis development, cyclin D2 expression was most abundant in the neonatal proliferating Sertoli cells. Those type A spermatogonia that were thought to divide mitotically also expressed cyclin D2 mRNA. Other spermatogenic cells, such as mitotically arrested gonocytes in neonatal testis and meiotically dividing germ cells in adult testis as well as adult Sertoli cells, were negative for the cyclin D2 signal. Adult W/W ^v mutant mice lacking germ cells expressed cyclin D2 mRNA in terminally differentiated Sertoli cells. Elimination of germ cells other than the undifferentiated type A spermatogonia by treating wild-type mice with an anti-c- kit monoclonal antibody did not result in the expression of cyclin D2 in Sertoli cells. These results demonstrate that there are lineage- and developmental-specific expression patterns of cyclin D2 mRNA during mouse testis development. At the same time, it is suggested that primitive type A spermatogonia affect the cyclin D2 expression of Sertoli cells.     

The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages.     

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.     

Unusual N-glycan structures in alpha-mannosidase II/IIx double null embryos identified by a systematic glycomics approach based on two-dimensional LC mapping and matrix-dependent selective fragmentation method in MALDI-TOF/TOF mass spectrometry

alpha-Mannosidase IIx (MX) is an enzyme closely related to alpha-mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid- to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T. O., Nakagawa, H., Wong, N. K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M. N. (2006) Essential and mutually compensatory roles of alpha-mannosidase II and alpha-mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983-8988). In the present study, we determined minor but unusual N-glycan structures found in MII/MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. (2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097-6101). Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls, and wild-type mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.     

A testis-specific regulator of complex and hybrid N-glycan synthesis

Database analyses identified 4933434I20Rik as a glycosyltransferase-like gene expressed mainly in testicular germ cells and regulated during spermatogenesis. Expression of a membrane-bound form of the protein resulted in a marked and specific reduction in N-acetylglucosaminyltransferase I (GlcNAcT-I) activity and complex and hybrid N-glycan synthesis. Thus, the novel activity was termed GlcNAcT-I inhibitory protein (GnT1IP). Membrane-bound GnT1IP localizes to the ER, the ER-Golgi intermediate compartment (ERGIC), and the cis-Golgi. Coexpression of membrane-anchored GnT1IP with GlcNAcT-I causes association of the two proteins, inactivation of GlcNAcT-I, and mislocalization of GlcNAcT-I from the medial-Golgi to earlier compartments. Therefore, GnT1IP is a regulator of GlcNAcT-I and complex and hybrid N-glycan production. Importantly, the formation of high mannose N-glycans resulting from inhibition of GlcNAcT-I by GnT1IP markedly increases the adhesion of CHO cells to TM4 Sertoli cells. Testicular germ cells might use GnT1IP to induce the expression of high mannose N-glycans on glycoproteins, thereby facilitating Sertoli–germ cell attachment at a particular stage of spermatogenesis.     

Cell type-autonomous and non-autonomous requirements for Dmrt1 in postnatal testis differentiation

Genes containing the DM domain, a conserved DNA binding motif first found in Doublesex of Drosophila and mab-3 of Caenorhabditis elegans, regulate sexual differentiation in multiple phyla. The DM domain gene Dmrt1 is essential for testicular differentiation in vertebrates. In the mouse, Dmrt1 is expressed in pre-meiotic germ cells and in Sertoli cells, which provide essential support for spermatogenesis. Dmrt1 null mutant mice have severely dysgenic testes in which Sertoli cells and germ cells both fail to differentiate properly after birth. Here we use conditional gene targeting to identify the functions of Dmrt1 in each cell type. We find that Dmrt1 is required in Sertoli cells for their postnatal differentiation, and for germ line maintenance and for meiotic progression. Dmrt1 is required in germ cells for their radial migration to the periphery of the seminiferous tubule where the spermatogenic niche will form, for mitotic reactivation and for survival beyond the first postnatal week. Thus Dmrt1 activity is required autonomously in the Sertoli and germ cell lineages, and Dmrt1 activity in Sertoli cells is also required non-autonomously to maintain the germ line. These results demonstrate that Dmrt1 plays multiple roles in controlling the remodeling and differentiation of the juvenile testis.     

The Enzymatic Activity of Cu/Zn Superoxide Dismutase Does Not Fluctuate in Mouse Spermatogenic Cells Despite mRNA Changes

In the mammalian testis, multiple mRNAs encoding the copper zinc superoxide dismutase (SOD-1) are expressed in postmeiotic male germ cells. Here we relate SOD-1 mRNA levels to SOD-1 protein and enzyme activity levels in mouse spermatogenic cells. Although the sizes and relative amounts of the multiple SOD-1 mRNAs vary as male germ cells enter meiosis and proceed into the postmeiotic stages of spermatogenesis, the amount of SOD-1 protein and enzyme activity does not fluctuate significantly, suggesting a precise control of SOD-1 activity in male germ cells.     

Impaired fertility and spermiogenetic disorders with loss of cell adhesion in male mice expressing an interfering Rap1 mutant

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.     

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.     

Excess type I interferon signaling in the mouse seminiferous tubules leads to germ cell loss and sterility

Type I (α and β) interferons (IFNs) elicit antiproliferative and antiviral activities via the surface receptor IFNAR. Serendipitous observations in transgenic mice in 1988 strongly suggested that IFNα/β overexpression in the testis disrupts spermatogenesis. Here, we compare a new mouse strain transgenic for IFNβ (Tg10) and a sister strain lacking the IFNAR1 subunit of IFNAR (Tg10-Ifnar1(-/-)), both strains expressing the transgene in the testis. The main source of IFNβ RNA was the spermatid population. Importantly, the Tg10 mice, but not the double mutant Tg10-Ifnar1(-/-), showed altered spermatogenesis. The first IFNAR-dependent histological alteration was a higher apoptosis index in all germ cell categories apart from non-dividing spermatogonia. This occurred 3 weeks after the onset of IFNβ production at postnatal day 20 and in the absence of somatic cell defects in terms of cell number, expression of specific cell markers, and hormonal activities. Several known interferon-stimulated genes were up-regulated in Tg10 Sertoli cells and prepachytene germ cells but not in pachytene spermatocytes and spermatids. In concordance with this, pachytene spermatocytes and spermatids isolated from wild-type testes did not display measurable amounts of IFNAR1 and phosphorylated STAT1 upon IFNβ challenge in vitro, suggesting hyporesponsiveness of these cell types to IFN. At day 60, Tg10 males were sterile, and Sertoli cells showed increased amounts of anti-Mullerian hormone and decreased production of inhibin B, both probably attributable to the massive germ cell loss. Type I interferon signaling may lead to idiopathic infertilities by affecting the interplay between germ cells and Sertoli cells.     

N-glycosylation affects the adhesive function of E-Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-435

E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.     

Inverse correlation between the extent of N-glycan branching and intercellular adhesion in epithelia. Contribution of the Na,K-ATPase beta1 subunit

The majority of cell adhesion molecules are N-glycosylated, but the role of N-glycans in intercellular adhesion in epithelia remains ill-defined. Reducing N-glycan branching of cellular glycoproteins by swainsonine, the inhibitor of N-glycan processing, tightens and stabilizes cell-cell junctions as detected by a 3-fold decrease in the paracellular permeability and a 2-3-fold increase in the resistance of the adherens junction proteins to extraction by non-ionic detergent. In addition, exposure of cells to swainsonine inhibits motility of MDCK cells. Mutagenic removal of N-glycosylation sites from the Na,K-ATPase beta(1) subunit impairs cell-cell adhesion and decreases the effect of swainsonine on the paracellular permeability of the cell monolayer and also on detergent resistance of adherens junction proteins, indicating that the extent of N-glycan branching of this subunit is important for intercellular adhesion. The N-glycans of the Na,K-ATPase beta(1) subunit and E-cadherin are less complex in tight renal epithelia than in the leakier intestinal epithelium. The complexity of the N-glycans linked to these proteins gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell-cell adhesion-induced increase in expression of GnT-III (stops N-glycan branching) and a decrease in expression of GnTs IVC and V (promote N-glycan branching) as detected by real-time quantitative PCR. Consistent with these results, partial silencing of the gene encoding GnT-III increases branching of N-glycans linked to the Na,K-ATPase beta(1) subunit and other glycoproteins and results in a 2-fold increase in the paracellular permeability of MDCK cell monolayers. These results suggest epithelial cells can regulate tightness of cell junctions via remodeling of N-glycans, including those linked to the Na,K-ATPase beta(1)-subunit.     

Immunohistochemical study of protein 4.1B in the normal and W/W(v) mouse seminiferous epithelium.

Cell-cell adhesion is crucial not only for mechanical adhesion but also for tissue morphogenesis. Protein 4.1B, a member of the protein 4.1 family named from an erythrocyte membrane protein, is a potential organizer of an adherens system. In adult mouse seminiferous tubules, protein 4.1B localized in the basal compartment, especially in the attaching region of spermatogonia and Sertoli cells. Protein 4.1B localization and appearance were not different in each spermatogenic stage. Developmentally, protein 4.1B was not detected at postnatal day 3 (P3), was diffusely localized at P15, and was found in the basal compartment during the third week. By double staining for protein 4.1B and F-actin, their localizations were shown to be different, indicating that protein 4.1B was localized in a region lower than the basal ectoplasmic specialization that formed the Sertoli-Sertoli junction. By electron microscopy, immunoreactive products were seen mainly on the membranes of Sertoli cells. In the W/W(v) mutant mouse, the seminiferous epithelium had few germ cells. Protein 4.1B and beta-catenin were not detected, although the basal ectoplasmic specialization was retained. These results indicate that protein 4.1B may be related to the adhesion between Sertoli cells and germ cells, especially the spermatogonium.     

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood-testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150(Glued), EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.