Purification and characterization of macrophage migration inhibitory factor as a secretory protein from rat epididymis: Evidences for alternative release and transfer to spermatozoa

Background: The cytokine macrophage migration inhibitory factor (MIF). orig inally described as a T cell product, has recently been identified to media te cellular interactions in several endocrine organs. Western blots analysi s of rat epididymal homogenates using an anti-MIF antibody indicated the pr esence of substantial amounts of an immunoreactive protein with the apparen t M-r of 12 kDa. Our study aimed to characterize the molecular nature of th is immunoreactive factor. Materials and Methods: The purified 12 kDa protein and a cloned cDNA fragme nt were characterized by sequence analysis. Furthermore, expression pattern and localization of the 12 kDa protein were investigated using in situ hyb ridization, immunohistochemistry, immunoelectron microscopy, and western bl ots experiments on epididymal sections, isolated epididymal vesicles, and o uter dense fibers from spermatozoa. Results: The N-terminal amino acid sequence analysis over 10 amino acids re vealed a 100% homology of the 12 kDa protein to the N-terminus of the cytok ine MIF These data were confirmed by sequence analysis of a reverse transcr iption polymerase chain reaction (RT-PCR) amplified cDNA fragment from rat epididymis, which also showed complete homology to the MIF cDNA sequence. M IF protein and mRNA were localized in the epithelial cells of the epididymi s in a regional distribution manner, with the expression maximal in the cap ut. Immune cells were not labeled. MIF is the first classical cytokine iden tified to be expressed by the epididymal epithelial cells. Immunoelectron m icroscopy detected MIF immunoreactivity in the cytoplasm, with no reaction visible in the Golgi complex and the cisternae of the endoplasmic reticulum . At the apical cell surface, MIF accumulated in stereocilia and vesicles t hat were pinched off from the plasma membrane. MIF detection in vesicles is olated From epididymal secretion together with the lack of a N-terminal sig nal sequence for translocation in the endoplasmic reticulum strongly sugges ted a nonclassical secretion mode. Furthermore, MIF was identified as a new component of the outer dense fibers (ODF), a cytoskeletal element of the m id- and principal piece of the sperm tail. Conclusion: The cytokine MIF was identified in substantial amounts in the e pithelial cells of rat epididymis and in the outer dense fibers of rat epid idymal spermatozoa. Our results indicate a nonclassical secretion mode for MIF and suggest a cell-to-cell transfer of MIF via vesicles to the sperm ce lls.