Cystatin-Related Epididymal Spermatogenic Aggregates in the Epididymis

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
Journal of Andrology (Impact Factor: 2.47). 07/2011; 32(6):679-85. DOI: 10.2164/jandrol.111.012963
Source: PubMed


Cystatin-related epididymal spermatogenic (CRES) is the defining member of a reproductive subgroup within the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. CRES is synthesized and secreted by the initial segment of the epididymis and is present in the sperm acrosome, suggesting roles in sperm maturation and fertilization. We have previously demonstrated that CRES is present within the epididymal lumen as monomeric (14 and N-glycosylated 19-kd forms) as well as sodium dodecyl sulfate-sensitive and sodium dodecyl sulfate-resistant high-molecular mass complexes. We have also shown that recombinant CRES protein will self-aggregate and form amyloid structures in vitro, raising the possibility that CRES might also form amyloid in vivo. Amyloid is a large protein aggregate with a specific cross-β sheet structure, and its presence is usually associated with disease. This review discusses protein aggregation in the epididymis and provides a brief overview of amyloid formation, including recent studies in other organ systems identifying examples of amyloid that are nonpathologic and carry out biologic functions (ie, functional amyloid). Studies that were carried out to determine if amyloid is present in the epididymal lumen and if CRES is associated with these structures are also described. The presence of CRES amyloid in the mouse epididymal lumen and the absence of pathology suggest either the presence of mechanisms to neutralize the cytotoxicity associated with pathologic amyloid or that CRES is a new example of a functional amyloid with roles in epididymal function.

Download full-text


Available from: Henning Von Horsten, Jun 26, 2015
  • Source
    • "CRES killed E. coli by permeabilizing and disrupting E. coli membranes, and inhibited macromolecular synthesis to block bacterial growth, consistent with previous findings [27], [29]. Cornwall et al demonstrated the CRES protein can self-aggregate and form amyloid structures in vitro and in vivo [41], [42]. They reported that the amyloid structures containing CRES were a component of the normal mouse epididymal lumen and exerted no apparent cytotoxic effects on spermatozoa [42]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cystatin-related epididymal spermatogenic (CRES) protein, a member of the cystatin superfamily of cysteine protease inhibitors (also known as CST8), exhibits highly specific, age-dependent expression in mouse testis and epididymis. The CRES protein possesses four highly conserved cysteine residues which govern the overall conformation of the cystatins through the formation of two disulfide bonds. Previous studies have revealed that other cystatin family members, such as cystatin 3 and cystatin 11, show antibacterial activity in vitro. This prompted us to investigate the potential antimicrobial activity of the CRES protein. Colony forming assays and spectrophotometry were used to investigate the effects of recombinant CRES protein on Escherichia coli (E. coli) and Ureaplasma urealyticum (Uu), respectively, in vitro. After incubation of E. coli with CRES recombinant protein fused with glutathione-S-transferase (GST), a substantial decrease in colony forming units was observed, and the effect was dose and time dependent. Furthermore, it took longer for Uu to grow to plateau stage when incubated with GST-CRES recombinant protein compared with the control GST. The antibacterial and Anti-Uu activities were not impaired when the cysteine residues of CRES protein were mutated, indicating that the antimicrobial effect was not dependent on its disulfide bonds. Functional analysis of three CRES polypeptides showed that the N-terminal 30 residues (N30) had no antimicrobial activity while N60 showed similar activity as full-length CRES protein. These results suggest that the active center of CRES protein resides between amino acid residues 31 and 60 of its N-terminus. Mechanistically, E. coli membrane permeabilization was increased in a dose-dependent manner, and macromolecular synthesis was inhibited on treatment with GST-CRES. Together, our data on the antimicrobial activities of CRES protein suggest that it is a novel and innate antimicrobial protein which protecting the male reproductive tract against invading pathogens.
    Preview · Article · Nov 2012 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cystatin Related Epididymal Spermatogenic protein (CRES, also CST8) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14- and 12- kDa) and glycosylated isoforms (19- and 17- kDa). The role of CRES remains enigmatic and is dependent on localization of its isoforms, which is the objective of this study. Our initial approach was to investigate testicular origins of these isoforms by immunohistochemistry and immunogold electron microscopy. We confirmed previous reports that CRES was expressed in the cytoplasm of elongating spermatids from steps 8-16. However, we noticed that the pattern of testicular expression was reminiscent of outer dense fiber (ODF) and fibrous sheath (FS) proteins. Western blot analysis of extracts of cauda epididymal sperm revealed a non-ionic detergent insoluble 14 kDa CRES isoform. To further pinpoint and confirm CRES localization we separated sperm heads and tails and extracted the tails with progressively harsher protein solubilizing agents. Western blots of these sequential extracts, designed to progressively remove the mitochondrial sheath and the ODF or FS, detected a CRES-immunoreactive 14 kDa band associated with the accessory fibers of the tail. Immunogold labeling was concentrated over growing ODFs in the testes and persisted in spermatozoa. This study discovers a CRES isoform which assembles as part of the ODF during the elongation and maturation phases of spermiogenesis and is retained as a covalently bound component of the ODF in spermatozoa.
    Preview · Article · Dec 2012 · Biology of Reproduction
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glycosaminoglycans (GAGs) have crucial roles in cell-cell interaction and communication. The communication between sperm and egg during fertilization is the finest example of intercellular communication involving a protein-carbohydrate recognition system. GAGs, especially heparin, are implicated in various processes, such as capacitation, acrosome reaction (AR), and sperm nuclei decondensation by interacting with a wide range of proteins, leading to fertilization. Seminal plasma (SP) comprises of multiple proteins that bind to heparin and related GAGs. Heparin binding proteins (HBPs) originating from secretions of the male accessory sex glands are known to play a vital role during fertilization events. They interact with GAGs present in the female genital tract and enhance the subsequent zona pellucida-induced AR, and thus have been correlated with fertility in some species. Several carbohydrate and zona pellucida-binding proteins, many of which belong to the spermadhesin family, are identified as HBPs. Many studies have been documented about the potential physiological role of some HBPs in various steps of fertilization. However, there is insufficient knowledge about functions executed by various HBPs and their exact mechanism and pathways. An in-depth knowledge of HBPs and their role in fertilization is of fundamental importance to resolve biological pathways and protein interactions at the molecular level. This review surveys some of the relevant findings supporting the potential role of heparin and HBPs in reproduction. It also describes consensus heparin binding sites emerging from known literature on HBPs that interact with heparin.
    Full-text · Article · Feb 2013 · Current Protein and Peptide Science
Show more