15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet.
Lipoxygenases (LOXs) are a family of enzymes capable of peroxidizing phospholipids. A member of the LOX family of enzymes, 15-LOX, participates in the degradation of mitochondria and other organelles within differentiating red blood cells, the reticulocytes. The present study provides biochemical and immunocytochemical evidence for the presence of 15-LOX in the sperm cytoplasmic droplet (CD). Testicular, epididymal and ejaculated spermatozoa were evaluated for the presence of 15-LOX using an affinity-purified immune serum raised against a synthetic peptide corresponding to the C-terminal sequence of rabbit reticulocyte 15-LOX. Western blotting revealed an appropriate single band of approximately 81 kDa in boar spermatozoa but not in boar seminal plasma. When ejaculated boar spermatozoa were subjected to separation on a 45/90% Percoll gradient, 15-LOX co-migrated with the immotile sperm and cellular debris/CD fractions, but not with the motile sperm fraction containing morphologically normal spermatozoa without CDs. Varied levels of 15-LOX were expressed in ejaculated sperm samples from boars with varied semen quality. By immunofluorescence, prominent 15-LOX immunoreactivity was found within the residual body in the testis and within the CDs from caput, corpus and cauda epididymal and ejaculated spermatozoa. Components of the ubiquitin-dependent proteolytic pathway, which is thought to facilitate both spermiogenesis and reticulocyte organelle degradation, were also detected in the sperm CD. These included ubiquitin, the ubiquitin-conjugating enzyme E2, the ubiquitin C-terminal hydrolase PGP 9.5, and various 20S proteasomal core subunits of the alpha- and beta-type. The 15-LOX and various components of the ubiquitin-proteasome pathway were also detected in sperm CDs of other mammalian species, including the human, mouse, stallion and wild babirusa boar. We conclude that 15-LOX is prominently present in the mammalian sperm CD and thus may contribute to spermiogenesis, CD function or CD removal.
Fischer
K A
KA
Division of Animal Sciences, Department of Obstetrics and Gynecology, University of Missouri, Columbia 65211-5300, USA.
Van Leyen
K
K
Lovercamp
K W
KW
Manandhar
G
G
Sutovsky
M
M
Feng
D
D
Safranski
T
T
Sutovsky
P
P
D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000032:analysis* / Q000235:genetics; D015153:Blotting, Western / Q000379:methods; D003593:Cytoplasm / Q000201:enzymology*; D004822:Epididymis; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008322:Mammals / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000201:enzymology; D013094:Spermatozoa / Q000648:ultrastructure*; D013552:Swine; D013737:Testis
D012333:RNA, Messenger; D001093:Arachidonate 15-Lipoxygenase