Wakayama T, Koami H, Ariga H, Kobayashi D, Sai Y, Tsuji A et al.. Expression and functional characterization of the adhesion molecule spermatogenic immunoglobulin superfamily in the mouse testis. Biol Reprod 68: 1755-1763
Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80-130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 spermatids and was present in the elongating spermatids until spermiation. The immunoreactivity was localized primarily on the cell membrane. Consistent with the findings in adult testes, the analysis of the developing testes revealed that SgIGSF was expressed separately in the spermatogenic cells in earlier and later phases. Sertoli cells had no expression of SgIGSF, whereas both SgIGSF immunoprecipitated from the testis lysate and produced in COS-7 cells was shown to bind to the surface of Sertoli cells in primary culture. These results suggested that SgIGSF on the surface of spermatogenic cells binds to some membrane molecules on Sertoli cells in a heterophilic manner and thereby may play diverse roles in the spermatogenesis.
"Subsequent studies have found that Necl-2 protein is expressed on type A spermatogonia through to early pachytene spermatocytes, as well as step 5 to late spermatids , . Researchers have speculated that transcription of Necl-2 terminates in early pre-meiotic spermatocytes, while translation restarts in spermatids using mRNA that was synthesized in per-meiotic germ cells . In this study, we have unraveled the potential mechanism on how Necl-2 transcription is terminated and how Necl-2 protein at the cell-cell interface is internalized and degraded. "
[Show abstract][Hide abstract] ABSTRACT: Nectin-like molecule-2 (Necl-2), a junction molecule, is exclusively expressed by spermatogenic cells. It mediates homophilic interaction between germ cells and heterophilic interaction between Sertoli and germ cells. Knockout studies have shown that loss of Necl-2 causes male infertility, suggesting Necl-2-based cell adhesion is crucial for spermatogenesis. Transforming growth factor-βs (TGF-βs) are crucial for regulating cell junction restructuring that are required for spermatogenesis. In the present study, we aim to investigate the mechanism on how TGF-β1 regulates Necl-2 expression to achieve timely junction restructuring in the seminiferous epithelium during spermatogenesis. We have demonstrated that TGF-β1 reduces Necl-2 mRNA and protein levels at both transcriptional and post-translational levels. Using inhibitor and clathrin shRNA, we have revealed that TGF-β1 induces Necl-2 protein degradation via clathrin-dependent endocytosis. Endocytosis assays further confirmed that TGF-β1 accelerates the internalization of Necl-2 protein to cytosol. Immunofluorescence staining also revealed that TGF-β1 effectively removes Necl-2 from cell-cell interface. In addition, TGF-β1 reduces Necl-2 mRNA via down-regulating Necl-2 promoter activity. Mutational studies coupled with knockdown experiments have shown that TGF-β1-induced Necl-2 repression requires activation of Smad proteins. EMSA and ChIP assays further confirmed that TGF-β1 promotes the binding of Smad proteins onto MyoD and CCAATa motifs in vitro and in vivo. Taken together, TGF-β1 is a potent cytokine that provides an effective mechanism in controlling Necl-2 expression in the testis via Smad-dependent gene repression and clathrin-mediated endocytosis.
PLoS ONE 05/2013; 8(5):e64316. DOI:10.1371/journal.pone.0064316 · 3.23 Impact Factor
"In the testis, Cadm1 is expressed exclusively in spermatogenic cells but not in Sertoli cells. In spermatogenic cells, Cadm1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in step 7 and later spermatids, whereas it is not expressed in middle pachytene through to step 6 spermatids [23, 24, 26]. Cadm1 not only forms homophilic binding with the same molecule present on spermatogenic cells, but also heterophilic binding with another cell adhesion molecule poliovirus receptor (Pvr), that is present on Sertoli cells [23, 26]. "
[Show abstract][Hide abstract] ABSTRACT: The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells.
"P8340) were purchased from Sigma (St. Louis, MO). Anti-CADM1-cyto antibody was generated according to the method described by Wakayama et al. . This antibody recognizes the C-terminal 20 amino acids of CADM1. "
[Show abstract][Hide abstract] ABSTRACT: Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling.
Biochemical and Biophysical Research Communications 12/2011; 417(1):462-7. DOI:10.1016/j.bbrc.2011.11.140 · 2.30 Impact Factor
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