Abstract
Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm.
Highlights
Mammalian spermatogenesis, which occurs in the seminiferous tubules within testis, is the process of continuous production of sperm throughout adulthood life in most mammals
In-silico analysis was performed for predicted Cilia and Flagella Associated Proteins (CFAP) extracted from HUGO database [18]
We found 59 proteins predicted as Cilia and Flagella Associated Proteins (CFAP)
Summary
Mammalian spermatogenesis, which occurs in the seminiferous tubules within testis, is the process of continuous production of sperm throughout adulthood life in most mammals This process is divided into three phases, mitosis, meiosis, and post-meiosis, which ends up with the release of the immotile sperm into the seminiferous lumen, the so-called testicular sperm [1]. The exact mechanism of Ca2+ ion signaling participating in the regulation of flagellar beating, as well as the acrosome reaction of mammalian spermatozoa, is so far unclear. To understand this signaling, identifying the calcium-binding proteins (CaBPs) that express in sperm are necessary
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