Abstract
The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.
Highlights
The epididymis is responsible for sperm concentration, transport and storage, and promotes maturation by adding various proteins to the sperm surface [1,2,3]
Capacitated or acrosome-reacted spermatozoa obtained by treatment with a nanomolar concentration of the calcium ionophore A23187 [27] were distributed in three samples: control group I - incubated for 3 h in M16-bovine serum albumin (BSA) medium alone, control group II - incubated in medium containing 10% rabbit normal serum, and test group - incubated in medium containing 10% monoclonal antibodies (mAb) TRA 54
Affinity (Con A) chromatography of caput epididymis homogenates yielded two peaks of unbound material, of which only fractions 1 and 2 of the first peak reacted with mAb TRA 54; no additional proteins were eluted with the glucose gradient, indicating that under these conditions there was little protein interaction with the column (Fig. 2A, B)
Summary
The epididymis is responsible for sperm concentration, transport and storage, and promotes maturation by adding various proteins to the sperm surface [1,2,3]. Sperm maturation depends on the expression and secretion of proteins and glycoproteins by the epididymal epithelium, from the caput towards the cauda. Immunohistochemistry revealed that the TRA 54-reactive molecule is located in the supranuclear cytoplasm of caput epididymal epithelial cells and in luminal sperm. Overall, these results suggest that the molecule recognized by mAb TRA 54 is produced and released by epididymal epithelial cells and subsequently binds to the sperm surface as these cells move down the epididymal duct. We used a combination of column chromatography, SDS-PAGE with in situ digestion and mass spectrometry to identify the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. Database searches and molecular modeling were used to identify the protein, and fertilization assays in vitro were used to examine the involvement of this protein in egg fertilization
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