Cellular localization of sphingomyelin synthase 2 in the seminiferous epithelium of adult rat testes

Abstract

Sphingomyelin synthase 2 (SMS2) is an enzyme that catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol, and it is crucial to cellular lipid metabolism. Using the technique of subtraction hybridization, we have isolated a full-length cDNA encoding SMS2 from rat testes, which shared 93 and 87% identity at the nucleotide level with SMS2 in mice and humans respectively. A specific polyclonal antibody was prepared against a 20 amino acid peptide of NH 2 -FSWPLSWPPGCFKSSCKKYS-COOH near the C-terminus of SMS2. Studies by RT-PCR and immunoblotting have shown that the expression of SMS2 was limited to late round spermatids and elongating spermatids, but it was not detected in late elongate spermatids and Sertoli cells. Furthermore, SMS2 was shown to associate with the developing acrosome beginning in late round spermatid through elongating spermatids (but not late elongate spermatids) and the cell membrane in studies using fluorescent microscopy and immunohistochemistry. These data were further confirmed by studies using immunogold electron microscopy. The expression of SMS2 in the seminiferous epithelium is stage-specific with its highest expression detected in the acrosome region in late round spermatids from stages VIII-IX, and also in the acrosome in elongating spermatids with diminished intensity in stages X-V; however, it was not found in the acrosome in elongate spermatids in stages VI-VIII. Collectively, these results suggest that SMS2 may play a crucial role in the lipid metabolism in acrosome formation and the plasma membrane restructuring from late round spermatids to early elongating spermatids.

Both Sphingomyelin Synthases SMS1 and SMS2 Are Required for Sphingomyelin Homeostasis and Growth in Human HeLa Cells

“…The human genome contains two of these genes, named SMS1 and SMS2. The corresponding enzymes fulfill essentially all criteria attributed previously to SM synthase and reside in organelles where SM synthesis is known to occur, namely the Golgi (SMS1) and plasma membrane (SMS2) (15,17). In line with these findings, Yamaoka et al (4) reported the expression cloning of human SMS1 using a mouse lymphoid cell line with severely reduced SM synthase activity and susceptibility to methyl-␤-cyclodextrin-induced cell death.…”

“…Together with the finding that SMS2 primarily resides at the plasma membrane, these observations indicate that SMS2 operates as the major if not only cell surface-associated SM synthase in HeLa cells. Because SMS2 is expressed in essentially all major tissues (15,17,36) and in a variety of mammalian cell lines (this study), we anticipate that the presence of a cell surface-associated SM synthase activity is a general feature of mammalian cells.…”

Sphingomyelin Synthase 2 Deficiency Attenuates NFκB Activation

“…Consistent with our observation, studies of SMS2 function in sperm cell also suggest that SMS2 is important for reconstruction of plasma membrane structure. 30 SMS2 deficiency could alter signal transduction mediated by lipid raft-associated receptors. As reported, the interaction of SM and cholesterol drives the formation of plasma membrane rafts, 5 and the relative proportions of both SM and cholesterol appear critical for the stability and function of lipid rafts.…”

Differentiation-related changes in lipid classes with long-chain and very long-chain polyenoic fatty acids in rat spermatogenic cells

“…In the seminiferous epithelium of rat testis, stage-specifi c expression of a form of SMS2 was localized to late round spermatids and early elongating spermatids and not present in late elongated spermatids or Sertoli cells ( 39 ). This enzyme was suggested to play a role in acrosome formation and the plasma membrane restructuring that occurs between late round spermatids and early elongating spermatids.…”