title: Region-specific expression and secretion of the fibrinogen-related protein, fgl2, by epithelial cells of the hamster epididymis and its role in disposal of defective spermatozoa.
abstract: The cauda epididymidis functions in the storage and protection of mature, fertile spermatozoa. We previously identified a region-specific secretory glycoprotein (termed HEP64) of the hamster proximal cauda epididymidis that specifically bound and coated the nonviable, but not the viable, spermatozoa within the epididymal lumen. In this study we employed expression screening of a hamster epididymal cDNA library to obtain the full-length sequence of HEP64 and to identify it as the fibrinogen-like protein fgl2. Northern blot analysis demonstrated that fgl2 mRNA is highly expressed by the proximal cauda epididymidis in comparison to other hamster tissues examined, and, in situ hybridization analysis of the epididymis revealed that fgl2 mRNA exhibited a region- and principal cell-specific expression pattern. Immunohistochemistry confirmed the association of fgl2 with abnormal spermatozoa in the cauda epididymidis and revealed smaller fgl2-containing particles. Immunoelectron microscopy revealed that fgl2 was distributed throughout an amorphous, "death cocoon," complex assembled onto abnormal spermatozoa and that the smaller fgl2 aggregates consisted of the amorphous material with embedded sperm fragments, organelles, and membrane vesicles. A protocol was developed to isolate an enriched death cocoon fraction. SDS-PAGE and microsequence analyses revealed that the Mr 64,000 fgl2 monomer was assembled into two disulfide-linked oligomers of Mr 260,000 and 280,000. These data demonstrate that the epididymis possesses a specific mechanism to identify and envelop defective spermatozoa with a protein complex containing the fibrinogen-like protein fgl2. We propose that this represents an important protective mechanism not only to shield the viable sperm population from potentially deleterious enzymes released by dying spermatozoa but also to prevent the release of sperm proteins that could initiate an immune response if they escaped the epididymal environment.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee 37232, USA. gary.olson@vanderbilt.edu
authors.forename: Gary E
authors.initials: GE
authors.lastname: Olson
authors.forename: Virginia P
authors.initials: VP
authors.lastname: Winfrey
authors.forename: Subir K
authors.initials: SK
authors.lastname: NagDas
authors.forename: Michael H
authors.initials: MH
authors.lastname: Melner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D005340:Fibrinogen / Q000096:biosynthesis* / Q000737:chemistry; D015723:Gene Library; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D016253:Microscopy, Immunoelectron; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism* / Q000648:ultrastructure; D014018:Tissue Distribution
chemical_list: D018076:DNA, Complementary; C490637:Fgl2 protein, mouse; D012333:RNA, Messenger; D012313:RNA; D005340:Fibrinogen