A Comparative Analysis of Expression and Processing of the Rat Epididymal Fluid and Sperm-Bound Forms of Proteins D and E1

Abstract

The mammalian epididymis secretes numerous proteins important for sperm maturation. Among these are proteins D and E, which belong to the CRISP family (cysteine-rich secretory proteins) and are the product of the Crisp-1 gene. These proteins have been the focus of a number of studies and have been implicated in sperm/egg fusion. Protein D and protein E have been purified to apparent homogeneity in several laboratories. Polyclonal antibodies raised against each protein typically cross-reacted with both proteins, suggesting that they were immunologically similar, if not identical. Our laboratory has previously reported the generation of a monoclonal antibody (mAb 4E9) that recognizes only protein E. Using mAb 4E9, the localization of protein E was shown to be domain specific on the sperm surface and there is processing of the protein in the fluid, with only the lowest molecular weight form associating with sperm. Subsequent purification and amino acid sequencing of protein D confirmed that proteins D and E are nearly identical and differ only by presence of the 4E9 epitope on protein E. Here we report the generation of antibodies to regions of amino acid sequence identity in proteins D and E. Using these antibodies, we demonstrate that protein D associates with the sperm head and that a portion of this protein may be proteolytically processed. In addition, we demonstrate that the proteolytic processing of protein E occurs in the carboxy terminal region of this protein. The data also suggest that a portion of protein D may also undergo processing, similar to that of protein E. Finally, we use these antibodies to demonstrate that proteins D and E are differentially expressed by the epididymal epithelium. Taken together, these data suggest that proteins D and E may have individual roles in sperm function.

Allurin: Exploring the Activity of a Frog Sperm Chemoattractant in Mammals

“…One possibility is that these domains are important for cell-surface binding and localization so as to target ICR domain actions. Evidence for this capacity comes from the fact that Crisp 1 binds to the mouse sperm surface in the epididymis and remains bound to the sperm in the female tract (Cohen et al 2000 ;Da Ros et al 2004 ;Roberts et al 2002Roberts et al , 2008. Its localization on the sperm and its binding lifetime are dependent on its glycosylation state.…”

“…Of additional interest is that the "E" form of Crisp 1, during production in the epididymis, is proteolytically processed in a manner that eliminates the ICR domain, leaving a truncated Crisp 1 that bears a lot of similarity to allurin, albeit highly glycosylated (Roberts et al 2002 ). This point raises the possibility that proteolytic processing of a full-length Crisp protein in the ovarian follicle could lead to the allurin-like protein detected in mouse follicular fl uid that we have hypothesized might bind to sperm and act as a chemoattractant (see Fig.…”

The role of cysteine-rich secretory proteins in male fertility

“…However, CRISP1 is produced as two isoforms, proteins D and E, which are differentially localized within the rat epididymis 47 Protein D is more abundant than E (70% of total rat CRISP1), and is synthesized by the principal cells of all regions of the epididymis and localizes to the sperm head. [47][48][49][50] Protein E is produced only in the corpus and proximal cauda epididymis. 47 Although originally localized in the dorsal region of the sperm head, as rat spermatozoa undergo the acrosome reaction, protein E migrates to the equatorial segment.…”

Ultrastructure of the epithelium lining of cauda epididymidis in mongrel dogs

“…Thus, mammalian spermatozoa leaving the testis undergo a series of structural, functional and biochemical changes during their descent through the epididymis by they gain the ability to move and to fertilize eggs. The mammalian epididymis synthesizes and secretes numerous proteins into its lumen (Roberts et al 2002). Maturation of spermatozoa takes place in the proximal and middle regions of the epididymis in most species.…”