Molecular cloning and localization of a CEACAM2 isoform, CEACAM2-L, expressed in spermatids in mouse testis.

Carcinoembryonic antigen (CEA) family, a subgroup of the immunoglobulin (Ig) superfamily, is divided into two sub-families: the CEA-related cell adhesion molecules (CEACAM) and the pregnancy-specific glycoproteins. The isoform CEACAM2 is expressed in mouse testis; in this study, we identified a novel isoform of Ceacam2, Ceacam2-Long (Ceacam2-L). CEACAM2-L is different from CEACAM2 in that it has much longer cytoplasmic tail region. Ceacam2-L starts to appear faintly in mouse testis after 3 weeks of postnatal development, and its expression level increased after 5 weeks. Immunoblot analysis confirmed the expression of CEACAM2-L in the seminiferous epithelium of mouse testis. Immunohistochemical data showed that CEACAM2-L was not observed on spermatogonia, spermatocytes, round spermatids, or Sertoli cells, but was seen at the plasma membrane of elongating spermatids in contact with extended cytoplasmic processes of Sertoli cells. CEACAM2-L was not detected at the head region of elongating spermatids, where the apical ectoplasmic specialization is constructed. These data suggest that CEACAM2-L might be a novel adhesion molecule contributing to cell-to-cell adhesion between elongating spermatids and Sertoli cells within the seminiferous epithelium.

Testis-specific cell adhesion molecule, CEACAM6-L, forms homophilic interaction at the cell adhesion site in vitro.

Ceacam6-L (carcinoembryonic antigen-related cell adhesion molecule 6-long) has recently been isolated from rat testis by differential display technique followed by RT-PCR and DNA sequence analysis. CEACAM6-L is a member of the immunoglobulin (Ig) superfamily and composed of an Ig-like domain, three IgCAM domains, a transmembrane region, and a short intracellular domain. We previously reported that CEACAM6-L was localized at the interface between Sertoli cells and elongating/elongated spermatids and might be an adhesion molecule contributing to apical ectoplasmic specialization in testis. In this study, we investigate the adhesive capacities and the complex structures of CEACAM6-L, using COS-7 cells as a study model. Transfection and immunoprecipitation experiments showed that CEACAM6-L expressed in the cells was distributed to the plasma membrane and interacted homophilically between transfected COS-7 cells. A chemical cross-linking experiment and a binding assay with recombinant CEACAM6-L proteins suggested that CEACAM6-L could form trans-tetra complexes, constructed by cis-homodimers at the adhesion site between COS-7 cells expressing CEACAM6-L. We hypothesized that CEACAM6-L expressed in both germ cells and Sertoli cells forms homophilic trans-tetra complexes between these cells in the seminiferous epithelium.

Identification of CEACAM6 as an intermediate filament-associated protein expressed in Sertoli cells of rat testis.

Ceacam6 (carcinoembryonic antigen-related cell adhesion molecule 6 gene) has recently been isolated by differential display followed by RT-PCR and DNA sequence analyses. Ceacam6 is a member of an immunoglobulin superfamily and encodes a protein of 266 amino acid residues possessing one immunoglobulin (Ig)-like domain. RT-PCR analysis showed that Ceacam6 was dominantly expressed in rat testis and its expression level prominently increased after 6 wk of postnatal development in testis. Immunohistochemical analyses using the anti-CEACAM6 antibody revealed that CEACAM6 colocalized with intermediate filaments (vimentin) in Sertoli cells and interstitial cells. The association between CEACAM6 and vimentin was observed throughout postnatal development in rat testis. Transfection experiments performed in COS-7 cells suggested that overexpression of CEACAM6 brought about aggregation of vimentin filament around nuclei with which CEACAM6 colocalized and that the N-terminus region of CEACAM6, including the Ig-like domain, seemed to be required for association with vimentin filaments. Interaction between CEACAM6 and vimentin in rat testis and transfected COS-7 cells was confirmed by immunoprecipitation. Our observations strongly suggested that CEACAM6 might be a novel intermediate filament-associated protein involved in regulation of vimentin architecture in Sertoli cells.

Characterization and subcellular localization of Tektin 3 in rat spermatozoa.

Mammalian sperm flagella have filament-forming Tektin proteins (Tektin 1-5) reported to be involved in the stability and structural complexity of flagella. Male mice null for Tektin3 produce spermatozoa with reduced forward progression and increased flagellar structural bending defects. The subcellular localization of Tektin3 (TEKT3) in spermatozoa, however, has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT3 in flagella of rat spermatozoa, we performed extraction studies followed by immunoblot analysis, immunofluorescence microscopy, and immunogold electron microscopy. Extraction of sperm flagella from the cauda epididymis resulted in complete removal of axonemal tubulins, while TEKT3 was resistant to extraction with the same S-EDTA (1% SDS, 75 mM NaCl, 24 mM EDTA, pH 7.6) solution, suggesting that TEKT3 might be present in the peri-axonemal component and not directly associated with axonemal tubulins. Resistance to S-EDTA extraction might be due to disulfide bond formation during epididymal maturation since concentrations of DTT greater than 5 mM drastically promoted release of TEKT3 from flagella. Immunofluorescence microscopy and pre-embedding immunoelectron microscopy revealed that TEKT3 was predominantly associated with the surface of mitochondria and outer dense fibers in the middle piece. In addition, TEKT3 was found to be present at the equatorial segment region of the acrosome membrane in sperm heads. TEKT3 might not only work as a flagellar constituent required for flagellar stability and sperm motility but also may be involved in acrosome-related events, such as the acrosome reaction or sperm-egg fusion.

Immunolocalization of spetex-1 at the connecting piece in spermatozoa of the musk shrew (Suncus murinus).

Spetex-1, which has been isolated by differential display and rat cDNA library screening as a haploid spermatid-specific gene, encodes a protein with two coiled-coil motifs that locates at both the segmented column in the connecting piece and outer dense fibers-affiliated satellite fibrils in rat sperm flagella. Orthologs of Spetex-1 are identified in many animal species, including human, chimpanzee, macaque, cow, dog, African clawed frog, green spotted puffer, and zebrafish. In this study, we used RT-PCR in combination with 5' and 3' RACE (Rapid Amplification of cDNA End) technique to isolate Spetex-1 ortholog of the musk shrew (Suneus murinus), which yielded a full-length Suncus Spetex-1 gene containing an open reading frame of 1,908 base pairs encoding a protein of 636 amino acids with the predicted molecular mass of 72,348 Da. Suncus Spetex-1 has two coiled-coil motifs at 118-184 and 242-276 amino acid residues, which is a characteristic shared by mammalian Spetex-1 proteins. To examine the subcellular localization of Spetex-1 in Suncus spermatozoa, we produced the anti-Suncus Spetex-1 antibody and carried out immunocytochemistry. In spite of that the primary structure of Suncus Spetex-1 is basically similar to that of rat and mouse Spetex-1, confocal laser scanning microscopy and immunoelectron microscopy revealed that Spetex-1 was restricted to the segmented column and capitulum in the connecting piece of Suncus spermatozoa and was not detected in other parts of flagella, suggesting a diversity of Spetex-1 localization in mammalian spermatozoa.

Subcellular localization of Tektin2 in rat sperm flagellum.

Tektins are evolutionarily conserved filament-forming proteins localized in flagella and cilia, and have been reported to be involved in the stability and structural complexity of axonemal microtubules. Five mammalian Tektins (Tektin1-5) have been reported. Of these, Tektin2 (TEKT2) has been found to be required for normal flagellum structure and function. Tekt2-null sperm display flagellum bending and reduced motility, probably due to disruption of the dynein inner arm. However, the subcellular localization of TEKT2 in spermatozoa has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT2 in flagella of rat spermatozoa, we performed confocal laser scanning microscopy, extraction of flagella followed by immunoblot analysis, and immunogold electron microscopy. Extraction of sperm flagella by SDS-EDTA resulted in complete extraction of axonemal tubulins, while TEKT2 was only partially released from flagella, suggesting that TEKT2 might be present in the peri-axonemal component, not directly associated with axonemal tubulins. Confocal laser scanning microscopy and pre-embedding immunoelectron microscopy revealed that TEKT2 is associated with the surface of outer dense fibers (ODFs). TEKT2 may function as an ODF-affiliated molecule required for flagellum stability and sperm motility.

Characterization of Spetex-1, a new component of satellite fibrils associated with outer dense fibers in the middle piece of rodent sperm flagella.

Spetex-1, which has been isolated by differential display as a haploid spermatid-specific gene, encodes a protein with two coiled-coil motifs located in the middle piece of flagella in rodent spermatozoa. The middle piece of flagella is composed of axoneme and peri-axonemal elements including outer dense fibers (ODFs) and satellite fibrils. Pre-embedding immunoelectron microscopy clearly demonstrated that Spetex-1 is located at satellite fibrils associated with ODFs in the middle piece of flagella of rat spermatozoa. Extraction of Spetex-1 from spermatozoa by SDS or urea required dithiothreitol, suggesting crosslinking by disulfide bond is involved in the assembly of satellite fibrils containing Spetex-1. We identified putative Spetex-1 orthologs in many animal species, and both cysteine residues and coiled-coil motifs were well conserved in mammalian orthologs of Spetex-1. When Spetex-1 was co-transfected into COS-7 cells with myc-tagged Tektin4, another filamentous protein associated with ODFs, the two molecules were co-localized in various sizes of aggregates in the cells. These data suggested that Spetex-1, a new component of satellite fibrils, might be involved in the structural stability of the sperm flagellar middle piece and functions in co-operation with Tektin4.

Bactericidal/Permeability-increasing protein is associated with the acrosome region of rodent epididymal spermatozoa.

To elucidate the molecular mechanisms involved in sperm maturation during epididymal transit, we intended to isolate secretory molecules that are region-specifically expressed along the epididymis and secreted into the lumen of epididymal ducts. By using differential display screening and DNA sequence analyses, we isolated a rat bactericidal/permeability-increasing protein (BPI) possessing a signal sequence at its N-terminal, which was expressed in the caput region of epididymis, but not in the caudal region. Reverse transcription polymerase chain reaction analysis and in situ hybridization showed that rat BPI messenger RNA (mRNA) was highly expressed in caput epididymal epithelium and that its expression level was developmentally up-regulated. Confocal laser scanning microscopy with the anti-BPI antibody revealed that in both rats and mice, BPI protein was detected on granulelike structures in the lumen of both caput and cauda epididymal ducts, as well as at the sperm surface covering the acrosome region in spermatozoa freshly isolated from epididymis. Acrosome reaction induced by calcium ionophore A23187 in vitro brought about the disappearance of BPI on mouse spermatozoa. These data suggested that BPI, which is synthesized in caput epididymis and secreted into the lumen, is associated with not only the granulelike structures, but also the sperm surface covering the acrosome region, and that BPI bound to the acrosome region is extinguished by acrosome reaction. Possibly BPI bound to the sperm surface covering the acrosome region in rodent spermatozoa is involved in sperm maturation or fertilization.

Intron retention generates a novel isoform of CEACAM6 that may act as an adhesion molecule in the ectoplasmic specialization structures between spermatids and sertoli cells in rat testis.

By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigen-related cell adhesion molecule 6 (Ceacam6) and its novel spliced variant Ceacam6-Long (Ceacam6-L) from rat testis. Ceacam6-L mRNA was generated by retention of 67 nucleotide-length third intron in Ceacam6 gene. Ceacam6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that Ceacam6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII-VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis.