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Originally published In Press as doi:10.1074/jbc.M410485200 on September 17, 2004

J. Biol. Chem., Vol. 279, Issue 49, 51266-51274, December 3, 2004
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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*

Gary E. Olson{ddagger}§, Virginia P. Winfrey{ddagger}, Subir K. NagDas{ddagger}, and Michael H. Melner{ddagger}

From the Departments of {ddagger}Cell and Developmental Biology and Obstetrics and Gynecology, Vanderbilt University, Nashville, Tennessee 37232

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.


Received for publication, September 13, 2004

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY607779.

* This work was supported by National Institutes of Health Grant HD20419. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232. Tel.: 615-322-0852; Fax: 615-343-4539; E-mail: gary.olson{at}vanderbilt.edu.


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