Molecular cloning and characterization of Izumo1 gene from bovine testis
© Kim; licensee BioMed Central. 2015
Received: 3 December 2014
Accepted: 19 March 2015
Published: 9 April 2015
A well-characterized sperm specific protein of the Member of immunoglobulin superfamily, IZUMO1, has crucial role in fertilization by mediating sperm binding to the egg plasma membrane in the mouse. However little is known about IZUMO1 in bovine. Here, we describe the molecular cloning and expression analysis of bovine IZUMO1 (bIZUMO1). RT-PCR and Western blot analysis of the bovine tissues indicated that bIZUMO1 was specifically expressed in the testis and sperm, Furthermore, the result of our biotinylation assay from ejaculated bovine sperm strongly suggest the assumption that bIZUMO1 is localized on the cell surface. These data imply the potential role of bovine IZUMO1 in mammalian fertilization.
The germ specific IgSF (Member of immunoglobulin superfamily) family of glycoprotein comprises one of the largest families of male infertility factors essential for spermatogenesis and sperm-egg interaction. It tightly regulates several aspects of biological processes, including degradation of the cumulus mass, sperm-zona pellucida (ZP) of egg and fusion of the egg plasma membrane after penetration of sperm into zona pullucida [1-3]. Intriguingly, many infertility cases exist anti sperm antibodies in either the male or female partner of an infertile couple, as cause of dysfunctions above mentioned mechanisms . In 2005, the Okabe research group identified the mouse IZUMO1 protein, originally known as OBF13, as a novel member of the immunoglobulin superfamily (IgSF), containing a single immunoglobulin (Ig) domain in the extracellular domain. They found that it is expressed on the inner acrosomal membrane of mature sperm, suggesting that the Ig domain is exposed on the sperm surface after the acrosome reaction [5-7]. Although mouse IZUMO1 is absolutely essential for gamete fusion, its expression pattern in different species remains unknown . In particular, research on bovine reproduction could provide a concrete basis on the molecular biology underlying animal fertilization, and thereby contribute to the understanding of specific gene functions related to human reproduction.
As IZUMO1 is critical for sperm–egg fusion in mice, understanding its expression and function in different animals may be of great relevance. IZUMO1 is encoded by a single-copy gene on mouse chromosome 7, but until now, IZUMO1 had not yet been identified in bovine. In the present study, we cloned and characterized the bovine IZUMO1 (bIZUMO1) gene and subsequently, generated a specific polyclonal antibody against IZUMO1 to identify its expression pattern and subcellular localization.
Fresh bovine tissues were purchased from local slaughterhouse (Daegu, Korea). The animal tissues in this study were used under the Guidelines of the Institutional Animal Care and Use Committee of Catholic University of Daegu.
Total RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR)
Primer sequences for characterization of bovine IZUMO1
Sense primer (5′ to 3′)
Anti sense primer (5′ to 3′)
The PCR products obtained from the testis tissue of bovine was cloned and sequenced. In brief, after electrophoresis in a 1% agarose gel, the desired PCR bands were excised with a razor blade. Gel fragments were purified using a gel extraction kit (Qiagen, USA) according to manufacturer’s guidelines. Purified DNA fragments were cloned into pGEM-T-easy, and propagated in Escherichia coli DH5α. After isolation of plasmid DNA, inserts were sequenced with vector-specific T7 and SP6 sequencing primers (Promega, USA) on a 3730XL DNA analyzer (Applied Biosystems, USA).
IZUMO1 amino acid sequence homology among bovine, mouse and human
Ig domain (%)
Preparation of protein extracts
Various bovine tissues were chilled on ice for 2 h and subjected to a lysis buffer consisting of 20 mM Tris–HCl, pH 7.4, 1% Triton X-100 (TX-100), 150 mM NaCl, and 1% protease inhibitor cocktail (Sigma-Aldrich) for the extraction of proteins . After centrifugation at 10,000 g for 10 min at 4°C, proteins retained in the supernatant were analyzed.
Western blot analysis
Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon-P membranes (Millipore, USA). The blots were blocked with 2% skim milk followed by incubation with primary antibodies for 2 h and subsequently, with horseradish peroxidase-conjugated secondary antibodies for 1 h. Then, the immunoreactive proteins were detected using an ECL western blotting detection kit (Amersham Biosciences, Little Chalfnot, UK).
Construction of expression vector and transfection into HEK293 cells
An expression vector of bIZUMO1 was constructed in pEGFP N1 vector (Clontech, Mountain View, USA). The primers 5′-CTCGAGGCCACCATGGATTATCTGCCTGGCCACCT-3′ and 5′-GGATCCAGCAGCTCGACTGCCAGAGCTGAAC-3′ were used to amplify the entire bIZUMO1 ORF from bovine testis cDNA. The amplified DNA was then digested with XhoI and BamHI and sub-cloned into the pEGFP N1 vector. After we confirmed the integrity of the reading frame and cloning sites of the expression vector by DNA sequencing, the plasmid vector was transfected into HEK293 cells . Briefly, HEK293 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. The cells were transiently transfected with the bIZUMO1 expression vector using ViaFect (Promega) according to the manufacturer’s protocols (Promega). Forty-eight hours after transfection, the transfected cells were washed 3 times in phosphate-buffered saline (PBS) and lysed in 1% Triton-100 (TX-100) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). Western blotting was performed using 1:300 dilutions of anti-bIZUMO1 antibody, followed by incubation with a 1:3000 dilution of horseradish peroxidase-labeled goat anti-rabbit IgG. ECL detection of bands was performed as described in the previous section.
Biotinylation of bovine sperm surface
Biotinylation of bovine sperm (2.5 × 107/ml) were kept at room temperature for 1 h in PBS containing 1 mM sulfo-NHS-LC biotin (Pierce). The biotinylated sperm samples were washed twice with PBS and lysed with the above protein lysis buffer. Proteins were subjected to SDS-PAGE under reducing conditions followed by Western blot analysis .
Results and discussion
Isolation and characterization of bovine IZUMO1 (bIZUMO1)
The sequences of the bovine IZUMO1 showed 55.1% and 64.0% similarity to the mouse and human, respectively. Although the sequences of the N-terminus were highly conserved across species, the C-terminus sequences were divergent; there was very little homology between the sequences of the cytoplasmic tail domains (Figure 3 and Table 2). We found that there are three types of C-terminus regions in various IZUMO1 homologs: those with a larger C-terminal region, such as in the mouse and human; those with a somewhat shorter C-terminal, such as in humans; and those where the C-terminal consists of approximately 20 amino acid, such as in the bovine and pig (Figure 3). Thus, one of the main phylogenetic groups of IZUMO1 across species is the group where a single amino acid residue occupies the cytoplasmic tail region.
Given that the extracellular domains were highly conserved for IZUMO1 across species, it is likely that bIZUMO1 also functions in relation to the fusion of sperm and egg; however, the biochemical function(s) of the various types of C-terminal cytoplasmic domains remain to be defined.
Presence of IZUMO1 in bovine sperm
Subcellular localization of IZUMO1 in ejaculated bovine sperm
In summary, two major observations were made in the present study. First, we demonstrated the existence of IZUMO1 in bovine sperm. Interestingly, the C-terminal sequence of IZUMO1 is quite different among other species, suggesting that is may have a distinct function. Second, we showed that bIZUMO1 is expressed on the surface of sperm as a homodimeric complex. Inter-IZUMO1 complex formation was recently described during mouse IZUMO1 immunoprecipitation experiments, and it was suggested that the protein forms a homodimeric complex in mouse sperm [13,14]. Indeed, most immunoglobulin superfamily members have been reported to exert their function in various biological processes, such as germ cell migration, transmigration of leucocytes and viral invasion, through homophilic and heterophilic interactions [15,16]. As shown Figure 6, the results from Western blot analysis under non-reducing conditions suggested that bIZUMO1 was involved in homophilic interactions, but it may also interact with other unidentified sperm membrane proteins. This is relevant because although the physiological role of IZUMO1 in the fusion between the sperm and unfertilized egg is relatively well understood, little is known about how its structure is linked with this function. Our observations suggest that the complex formation of IZUMO1 may be necessary for retaining the conformation of the protein, which in turn, might be contributing to sperm-egg fusion in some way. Future investigations on this aspect can help gain clearer insights into the molecular mechanisms underlying fertilization in species of interest.
This study was supported in research grant from Catholic University of Daegu in 2012, Republic of Korea.
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