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), originally described as a T cell product, has recently been identified to mediate cellular interactions in several endocrine organs. Western blots analysis of rat epididymal homogenates using an anti-MIF antibody indicated the presence of substantial amounts of an immunoreactive protein with the apparent Mr of 12 kDa. Our study aimed to characterize the molecular nature of this immunoreactive factor.


METHODS
The purified 12 kDa protein and a cloned cDNA fragment were characterized by sequence analysis. Furthermore, expression pattern and localization of the 12 kDa protein were investigated using in situ hybridization, immunohistochemistry, immunoelectron microscopy, and western blots experiments on epididymal sections, isolated epididymal vesicles, and outer dense fibers from spermatozoa.


RESULTS
The N-terminal amino acid sequence analysis over 10 amino acids revealed a 100% homology of the 12 kDa protein to the N-terminus of the cytokine MIF. These data were confirmed by sequence analysis of a reverse transcription polymerase chain reaction (RT-PCR) amplified cDNA fragment from rat epididymis, which also showed complete homology to the MIF cDNA sequence. MIF protein and mRNA were localized in the epithelial cells of the epididymis in a regional distribution manner, with the expression maximal in the caput. Immune cells were not labeled. MIF is the first classical cytokine identified to be expressed by the epididymal epithelial cells. Immunoelectron microscopy 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 that were pinched off from the plasma membrane. MIF detection in vesicles isolated from epididymal secretion together with the lack of a N-terminal signal sequence for translocation in the endoplasmic reticulum strongly suggested a nonclassical secretion mode. Furthermore, MIF was identified as a new component of the outer dense fibers (ODF), a cytoskeletal element of the mid- and principal piece of the sperm tail.


CONCLUSIONS
The cytokine MIF was identified in substantial amounts in the epithelial cells of rat epididymis and in the outer dense fibers of rat epididymal spermatozoa. Our results indicate a nonclassical secretion mode for MIF and suggest a cell-to-cell transfer of MIF via vesicles to the sperm cells.
Eickhoff
R
R
Department of Anatomy and Cell Biology, Philipps University of Marburg, Germany.
Wilhelm
B
B
Renneberg
H
H
Wennemuth
G
G
Bacher
M
M
Linder
D
D
Bucala
R
R
Seitz
J
J
Meinhardt
A
A
D000818:Animals; D004822:Epididymis / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods; D008263:Macrophage Migration-Inhibitory Factors / Q000302:isolation & purification* / Q000502:physiology*; D008297:Male; D016253:Microscopy, Immunoelectron; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013094:Spermatozoa / Q000378:metabolism*
D008263:Macrophage Migration-Inhibitory Factors; D012333:RNA, Messenger