Identification and characterization of a bovine sperm acrosomal matrix protein and its mechanism of interaction with acrosomal hydrolases

Abstract

Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads is comprised of 54, 50, 45, and 38–19kDa polypeptides. The objective of this study is to identify and to characterize the 45kDa (OMC45) polypeptide and to define its role in binding acrosomal hydrolases and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI–TOF–TOF yielded 8 peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X–100–permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti–OMC45 and anti–TEKT3. The OMC45 polypeptide was solubilized by RIPA (radio-immunoprecipitation assay) buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti–OMC45 immunoprecipitation pellet. An identical blot stained with anti–TEKT3 exhibited the presence of TEKT3 polypeptide in the anti–OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide; that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N–linked and O–linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N–acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex.

Translocation of Tektin 3 to the equatorial segment of heads in bull spermatozoa exposed to dibutyryl cAMP and calyculin A

“…Nagdas et al () recently identified a 45‐kDa acrosomal matrix protein, localized at the acrosomal cap of bovine epididymal spermatozoa, as TEKT3; however, we detected TEKT3 as a 55‐kDa protein residing at the post‐acrosomal region of bovine spermatozoa (Fig. ).…”

“…The equatorial subsegment is also hypothesized to be a site where membrane fusion is initiated between sperm and egg at fertilization (Jones et al, 2008). Nagdas et al (2015) recently identified a 45-kDa acrosomal matrix protein, localized at the acrosomal cap of bovine epididymal spermatozoa, as TEKT3; however, we detected TEKT3 as a 55-kDa protein residing at the postacrosomal region of bovine spermatozoa (Fig. 1).…”

“…conceivable source of these differences is the use of ejaculated frozen bull spermatozoa (this study) versus epididymal spermatozoa (Nagdas et al, 2015).…”

FSIP2plays a role in the acrosome development during spermiogenesis

“…These downregulated proteins make contact with each other to develop complex interacting networks, which widely participate in vesicle formation and transport, sperm head organisation, fertilisation, acrosome assembly and sperm axoneme assembly (figure 5C). According to the literature, nine of these proteins, including ZPBP1, SPACA1, SPINK2, CSNK2A2, DPY19L2, CCDC62, CCIN, TEKT2 and TEKT3, are explicitly related to the formation of the acrosome 17–24. Additionally, H1-7 is associated with spermatid nucleus differentiation25; SPAM126and ACR27 are involved with the binding of sperm to zona pellucida and TEKT5 participates in sperm flagella 28.…”

Site of Mammalian Sperm Acrosome Reaction

“…Because IVF bypasses all such interactions with the female reproductive tract, the results from IVF experiments do not cover all aspects of naturally occurring fertilization. Nonetheless, IVF is still a promising approach for discovering new molecular models for sperm-ZP interactions (Visconti and Florman 2010;Redgrove et al 2012;Nagdas et al 2015). A recent study using gene rescue experiments clearly demonstrated that human spermatozoa could recognize the human zona pellucida protein ZP2 (Avella et al 2014).…”