Outer Dense Fiber Proteins Are Dominant Postobstruction Autoantigens in Adult Lewis Rats1

Abstract

Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Autocatalytic Phosphorylation of CDK2 at the Activating Thr160

“…A Coomassie-stained gel band was processed by standard procedures. 28 In short, CDK2 was reduced, alkylated and digested with trypsin in-gel to produce peptides suitable for sequencing by mass spectrometry. Sequencing was accomplished using a ThermoElectron LTQFT mass spectrometer.…”

Spermatocyte/Spermatid-specific Thioredoxin-3, a Novel Golgi Apparatus-associated Thioredoxin, Is a Specific Marker of Aberrant Spermatogenesis

“…These results indicate that Sptrx-3 is a post-obstruction sperm autoantigen, at least in some animals. Because vasectomy mimics immunization of rats with spermatozoa (46), the postvasectomy sera reacted with multiple other protein spots (39,45). Study of a limited set of postvasectomy sera showed that two sera stained spots that comigrated with those bound by anti-SPTRX-3 serum (Fig.…”

Cloning and Characterization of a Novel Sperm-Associated Isoantigen (E-3) with Defensin- and Lectin-Like Motifs Expressed in Rat Epididymis1

“…For 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) of rat epididymal fluid proteins and sperm extracts, isoelectric focusing (IEF) in the first dimension was performed using gel and ampholine (isoelectric point [pI] of 3.5-6.5) compositions described previously [17]. Sperm extracts containing 150-200 g of protein or 200-250 g of solubilized epididymal fluid proteins were separated on 2-D (IEF and SDS-PAGE) gels and transferred to nitrocellulose paper [29].…”

“…The resulting postobstruction sera and hyperimmune sera reacted on 1-D or 2-D Western blots with subsets of sperm proteins, which were thereby identified as autoantigens, isoantigens, or both [16]. Microsequencing analysis subsequently revealed the outer dense fiber proteins odf1 and odf2 [17], sperm mitochondria-associated cysteine-rich protein (SMCP) [18], and the testis-abundant protein termed asparaginase-like protein (ALP) [19] as major rat sperm autoantigens. Less attention has been given to the antigenicity of epididymal proteins, although the abundant epididymal protein DE has been characterized as an isoantigen and an autoantigen in rats [20].…”