Molecular Cloning And Characterization Of Izumo1 And CD9 From Sheep And Cashmere Goat And Preliminary Study On Their Interaction

Mammalian fertilization consists of an intricate cascade of cell-cell interactions that culminate in the fusion of sperm and egg membranes and formation of a zygote.A number of molecules with putative roles in sperm-oocyte fusion have recently been identified with genomic and proteomic techniques. However,the mechanisms by which acrosome-reacted spermatozoa bind to receptor molecules on the oocyte plasma membrane and the subsequent fusion with the oolemma remain unknown.It has been established in knockout mice that Izumol,a novel member of immunoglobulin superfamily(IgSF) located on the sperm,and CD9,a member of tetraspanin superfamily(TM4SF) located on the oocyte,are crucial for sperm-egg fusion.Sheep and goat are a major source of food,wool and cashmere,but little research on the sperm-egg fusion has been reported in these livestocks.Since the mechanisms of sperm-egg fusion are important for us to understand the regulatory aspects of caprine and ovine fertilization and reproduction,we cloned and characterized the Izumol and CD9 genes in sheep and chashmere goat,and expressed their recombinant proteins in E.coli.To demonstrate the expression pattern of Izumol in testis and its localization in sperms,we generated ascitic polyclonal antibodies against caprine and ovine Izumol proteins and synthesized DIG-tagged Izumol RNA probes in vitro.We also attempted to study the interactions between caprine Izumol and CD9 using BiFC assay in human Hep-G2 cells.Alignment of the cloned 4600 bp sequence of sheep Izumol gene with the 4598 bp of goat Izumol gene as well as other animals' Izumol gene revealed 99.9% identity between sheep and goat Izumol,and they both share high homology with their counterparts in cattle,mouse,rat and human.Extensive cDNA cloning captured a canonical open reading frame(ORF) of 963 bp which made of eight exons and three(del 69,del 182 and del 217) alternative splicing isoforms in goat and two(del 69 and ins 30) in sheep Izumol.Bioinformatics analysis showed that the canonical caprine and ovine Izumol cDNA both encode a peptide of 320amino acides(aa) which shares similar structure with their murine orthologue in a feature that there are a signal peptide at the N-terminus(1-22aa),a transmembrane domain at the C-terminus(302-319aa),and an extracellular immunoglobulin-like(Ig-like) region with a putative N-linked glycosylation site(N²⁰⁵-N-S) in the middle (161-252aa).All of those isoforms are derived from alternative splicing at the typical GT-AG sites resulting in the Ig-like domain partially truncated or completely deleted.Alignment of Izumol protein sequences among 15mammalian species displayed several highly conserved regions,including LDC and YRC motifs with cysteine residues for potential disulfide bridge formation,CPNKCG motif upstream of the Ig-like domain,GLTDYSFYRVW motif upstream of the putative N-linked glycosylation site,and a number of scattered cysteine residues.These conservation features are very informative to pinpoint the key motifs related to Izumol function. The C-terminal regions,however,are more variable across species.The molecular phylogenetic tree constructed using Izumol cDNA sequences is consistent with their morphological taxonomy,for which sheep and goat are in the same cluster and are closely related to cattle.We also cloned CD9 cDNA of 1.1kb in length from sheep and cashmere goat. Both of them have an ORF of 681 bp encoding a predicted protein of 226 amino acids with a typical TM4SF structure.The deduced peptide consists of a putative N-linked glycosylation site(N-X-S/T)(N⁵⁰),four transmembrane domains,two extracellular domains and a CCG motif which is a hallmark of the TM4SF.The sheep and goat CD9 proteins share 99.1%identity and are highly homologous to their orthologues in cattle,mouse and human.Alignment of CD9 protein sequences among 26 mammalian species revealed extensive homologies within the same family and a highly conserved LWLR motif in the small extracellular loop(SEL) in all species.The SFQ(173-175) motif within the large extracellular loop(LEL), which was reported as a key motif for mediating sperm-egg fusion,is found only in mouse and rat.Although CD9 is generally annotated as a glycoprotein,we found that the N-linked glycosylation sites are missing in some species and are thus less conserved as expected.These results present new clues for CD9's function in sperm-egg fusion.Molecular phylogenetic analysis based on CD9 cDNA sequences indicated that goat and sheep are closely related to cattle,which is consistent with their morphological taxonomy.We expressed the recombinant sheep CD9 and goat Izumol proteins in E.coli and succeeded in generating ascitic polyclonal antibodies against the goat Izumol protein.Immunohistochemical analysis showed that Izumol proteins present in the meiotic male germ cells and are stored in the nucleus.Concomitant with spermatid maturation,Izumol proteins contract to a narrow area in elongated spermatids and finally concentrate in the equatorial segment of the mature sperm head where sperm-egg fusion is initiated.RNA in situ hybridization analysis with DIG-tagged anti-sense Izumol riboprobes indicated that the goat Izumol gene is mainly transcribed in the primary spermatocytes.Finally,we constructed BiFC expression vectors BiFC-VC155-CD9 and BiFC-VN173-Izumol and transiently cotransfected them into human hepatocellular line Hep-G2 to examine the binding ability of Izumol to CD9 in cytoplasm. However,that only a small number of fluorescent cells were observed was not conclusive for the binding activity between caprine CD9 and Izumol proteins.