Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Abstract

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)-insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll(R) density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB-insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS-insoluble material was collected and quantitatively dissolved in 8 M urea. SDS-gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti-cytokeratin antibodies detected two urea-soluble, SDS-insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45-kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1-18 and CK 20), KL1, was the only antibody that reacted with the 66-kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope-like structures, we suggest that these intermediate filaments play an important structural or tension-bearing role in sperm flagella.

Voltage-dependent Anion-selective Channels VDAC2 and VDAC3 Are Abundant Proteins in Bovine Outer Dense Fibers, a Cytoskeletal Component of the Sperm Flagellum

“…Biochemical and immunological analyses have revealed that bovine ODF contain sperm keratins (31). Presently it is thought that the ODF mainly consists of cytoskeletal proteins and lacks membranes.…”

“…For the enrichment of ODF fractions, spermatozoa from about 50 ejaculates were used. ODF were purified by a previously published protocol (30,31) Purified bovine ODF were resuspended in SDS-sample buffer. The SDS-insoluble and -soluble proteins were separated by centrifugation for 15 min at 12,000 ϫ g. The supernatant was subjected to preparative SDS-PAGE.…”

Electron microscopic observations of human sperm whole‐mounts after extraction for nuclear matrix and intermediate filaments (NM‐IF)

“…Without using specific molecular probes, it is difficult to hypothesize which proteins compose the extraction-resistant cell remnant, but our electrophoretic result shows that they correspond to major protein bands in the profile of unextracted spermatozoa. Other authors have identified intermediate filament-like proteins in perinuclear theca (Kierszenbaum et al, 2003), outer dense fibres (Tres & Kierszenbaum, 1996;Hinsch et al, 2003) and fibrous sheath (Eddy et al, 1991;Jassim, 1991;Tres & Kierszenbaum, 1996;Catalano et al, 2001). In fact, the Odf proteins described as major constituents of outer dense fibres can be regarded as a group of keratins (Kierszenbaum, 2002).…”

“…Other authors have identified intermediate filament‐like proteins in perinuclear theca (Kierszenbaum et al. , 2003), outer dense fibres (Tres & Kierszenbaum, 1996; Hinsch et al. , 2003) and fibrous sheath (Eddy et al.…”

Cross comparison of seminal plasma proteins from cattle and buffalo (Bubalus bubalis)

“…Keratin type II cytoskeletal 5 is present in the sperm flagellum (Calvin, ; Hinsch, Boehm, Groeger, Mueller‐Schoesser, & Hinsch, ) and contributes to its dense fibre composition (Lindemann & Kanous, 1),whose function is to maintain cell structure (Calvin, ; Hinsch et al, ).…”