Biomedical subjects

K M Ensrud

Publications and source records attributed to K M Ensrud.

9 recordsLinked to original sources

PubMed1999

Cloning and characterization of the rat Crisp-1 gene.

Rat androgen-regulated acidic epididymal glycoprotein (AEG), also known as Protein DE, is a product of the Crisp-1 gene. Protein DE is secreted into the epididymal lumen and binds to sperm heads during their transit through the epididymis. In experiments reported here, the rat Crisp-1 gene has been cloned and its structure determined. The rat Crisp-1 gene spans 38kb and contains nine exons encoding an 1120bp epididymal Protein DE mRNA. The boundaries of the protein-coding exons are structurally organized similar to the mouse Crisp-1 gene, except for the 5' untranslated sequence, which is encoded by one exon in the mouse Crisp-1 gene and two exons in the rat gene. All the introns are flanked by AG/GT consensus splice sequences. Crisp-1 is a single-copy gene as shown by the presence of single bands by Southern blot analysis and PCR using rat genomic DNA as template. Recognition sites for steroid hormone receptors are present in the 5' flanking region and in intron 1, consistent with the known regulation of Protein DE expression by androgens. RT-PCR experiments demonstrate three splice variant mRNAs involving the non-coding exon 2. The Crisp-1 gene also produces an mRNA without an exon 1 sequence by utilizing a transcription start site in intron 1, 5' of the start of exon 2. All forms of the Crisp-1 mRNA are predicted to encode Protein DE.

Alternative Splicing↗

PubMed1997

The 26 kD protein recognized on rat cauda epididymal sperm by monoclonal antibody 4E9 has internal peptide sequence that is identical to the secreted form of epididymal protein E.

MAb 4E9, raised against a detergent extract of rat cauda epididymal sperm, recognizes a 26 kD glycoprotein that is found on the plasma membrane of the sperm tail in cauda, but not caput, sperm (Moore et al., 1994). It also recognizes an epididymis-secreted protein that has been shown to be protein E (Xu and Hamilton, 1996). It is felt that the secreted protein becomes associated with sperm, but there has been no biochemical evidence of molecular identity between the secreted and membrane proteins. In this report, the membrane form of the antigen has been purified by reverse phase HPLC. Cyanogen bromide cleavage of the purified protein yielded 3 peptides that were purified, also by reverse phase HPLC. One of the peptides yielded an unambiguous sequence of 34 amino acids that is identical to an internal peptide of the protein found in epididymal fluid. This is the first report showing sequence identity between an epididymis-secreted protein and a protein of the sperm plasma membrane.

Amino Acid Sequence↗

PubMed1995

Identification of osteopontin (OPN) mRNA and protein in the rat testis and epididymis, and on sperm.

We have identified a bone cell adhesion molecule, osteopontin, in the rat testis and epididymis by Northern analysis, RT-PCR, Western immunoblot analysis and immunocytochemistry. A polyclonal antibody raised against rat epididymal fluid proteins was used to detect fusion proteins produced by a testis lambda gt11 cDNA library. Sequence analysis of one of four positive cDNA clones, designated as pREP5, revealed identity with the rat osteopontin (OPN) cDNA. The partial cDNA clone pREP5 encompasses 64% of the 1,457 residues reported by Oldberg et al. (1986; Proc Natl Acad Sci USA 83:8819-8823). Immunoblot analysis with a monoclonal antibody against OPN detects the presence of immunoreactive polypeptides in rat testis homogenates as well as in epididymal fluid and sperm extracts. Immunocytochemical localization to the basal and adluminal region of the seminiferous tubule suggests that OPN could be a Sertoli cell product. Indeed, Northern blot analysis of testicular cell preparations demonstrated positive hybridization to Sertoli cell-enriched RNA, but not to RNA isolated from interstitial cell preparations or to isolated germ cell RNA preparations. OPN is also detected in the rat epididymis and on epididymal spermatozoa. This is the first report on the presence of OPN mRNA and protein in rat testis and epididymis and on the presence of OPN on the surface of epididymal spermatozoa. The characterization of this protein in other tissue suggests that OPN could play a role in testicular cell adhesion during spermatogenesis and/or epididymal maturation, although other potential functions in the male reproductive tract are discussed.

Animals↗

PubMed1994

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis.

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin I lectin column. Epididymal fluid antigens have apparent M(rs) of 38-26 kD, whereas the membrane-associated form of the molecule has an M(r) of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secreted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm.

Animals↗

PubMed1994

Putative rat sperm lipid-binding protein: isolation and partial characterization.

Previous work has identified a prominent 22-24-kD protein that is present in rat male reproductive tissues, including epididymis and testis (Brooks, 1985; Jones and Brown, 1987; Moore et al., 1987). Using a monoclonal antibody (designated mAb-B109) against this 24-kD antigen (referred to as B109), we have isolated the protein using a combination of chromatofocusing and electroelution from SDS-PAGE gels, and reverse phase HPLC. B109 (pI = 4.8) is amino-terminal blocked. To obtain internal amino acid sequences, the isolated protein was cleaved either with cyanogen bromide in 70% formic acid or with TLCK-treated chymotrypsin. With cyanogen bromide treatment, two peptides, 17.8 kD and 11.9 kD, were isolated and partial amino acid sequences obtained. Chymotryptic peptides were isolated by reverse-phase HPLC and two were chosen for sequence analysis. A computer search for sequence homology through the protein identification resource (PIR) matched B109 to a basic 21-kD cytosolic protein (pI = 7.4) found in bovine brain (> 80% homology). When peptide sequence differences obtained in the present study were substituted into the 21-kD cytosolic protein sequence obtained from the PIR using Intelligenetics software, the calculated pI dropped from 7.4 to 5.8, suggesting that pI differences between the bovine and rat molecules are the result of amino acid substitutions in the testis protein and not tissue-specific posttranslational processing. It has been postulated that the 21-kD bovine brain protein is associated with phospholipid transport, although the function of B109 is unknown.

Amino Acid Sequence↗

PubMed1989

The major maturation glycoprotein found on rat cauda epididymal sperm surface is linked to the membrane via phosphatidylinositol.

The experiments reported here further characterize a approximately 26[3H] kD cell surface glycoprotein that can be detected on rat cauda epididymal sperm using the galactose oxidase/NaB[3H]4 technique (1). When labeled sperm are treated with PI-PLC the 26[3H] kD is completely released from the cell. The released molecule can be recovered undegraded from incubation supernatant. Release by PI-PLC converts the hydrophobic, membrane-anchored form into a hydrophilic molecule as assessed by partition studies using Triton X114. Isoelectric focusing studies using both untreated (control) and PI-PLC treated samples shows that there is charge heterogeneity with two major peaks at pls of approximately 5.0 and approximately 4.5. We also show for the first time that the molecule persists on ejaculated cells.

Animals↗

PubMed1982

Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymphoma.

Using a new high-resolution technique for chromosomal analysis, we have successfully studied biopsy specimens of lymph nodes from 42 of 44 patients with non-Hodgkin's lymphoma and have categorized them using the new international histologic formulation and immunologic markers. Abnormalities of the clonal chromosomes were detected in all 42 patients. Three recurrent chromosomal aberrations were found to correlate with certain histologic types: a translocation between chromosomes 18 and 14 in 16 of 19 patients with follicular lymphomas (small cleaved cell, mixed cell, and large cell); a translocation between chromosomes 8 and 14 in 5 of 6 patients with small noncleaved-cell (non-Burkitt's) or large-cell immunoblastic lymphoma; and a trisomy 12 in 4 of 11 patients with small-cell lymphocytic lymphoma. Our findings suggest that characteristic chromosomal defects occur in certain lymphoma subtypes and that high-resolution chromosomal analysis promises to become an important tool in improving our basic understanding of lymphoid cancers.

Adolescent↗

PubMedDate not supplied

Expression of crisp-1 mRNA splice variants in the rat epididymis, and comparative analysis of the rat and mouse crisp-1 gene regulatory regions.

The rat Crisp-1 gene encodes Protein DE (acidic epididymal glycoprotein; AEG), a glycoprotein secreted by the epididymal epithelium that associates with maturing sperm and has been implicated in the process of sperm-egg fusion. Previous characterization of the Crisp-1 messenger RNA in the rat epididymis has demonstrated the presence of 3 splice variants (Klemme et at, 1999). This study was undertaken to determine if expression of the Crisp-1 splice variants in the rat epididymis is region-specific and correlates with the region-specific pattern of synthesis of the D and E forms of the Crisp-1 protein. Expression of each of the splice variants was shown by RNase protection assays to be under the control of androgens, but they are not differentially regulated either within the epididymal segments or along the length of the organ. The reported structure of the mouse Crisp-1 gene does not include an exon that is equivalent to the rat exon 1, suggesting that the rat splice variants cannot exist in the mouse and may be specific to the rat. Furthermore, the mouse transcription start site is situated in a different region of the gene than in the rat. In this study, a comparison of the mouse and rat genes in the region flanking the mouse exon 1 and the rat exon 2 (within the rat intron 1) shows greater than 80% sequence identity, including the conservation of several putative androgen receptor binding sites. In addition, the rat gene is shown to have a corrupted TATA box in intron 1 that corresponds to the TATA box located in the mouse gene. These observations explain the preferential transcription for the mouse gene in this region, while the predominant start site for the rat gene is 5' of the upstream exon 1. Although an exon corresponding to the rat exon 1 has not been found in the mouse gene, reverse transcription-polymerase chain reaction experiments using mouse epididymal RNA suggest that such an exon exists in the mouse gene and is transcribed at low frequency.

Alternative Splicing↗

PubMedDate not supplied

Use of neonatal tolerization and chemical immunosuppression for the production of monoclonal antibodies to maturation-specific sperm surface molecules.

Mammalian sperm acquire functional maturity as they move from the caput to the cauda epididymidis. Changes occur in the protein/glycoprotein composition of the sperm plasma membrane during this time, and may be essential to the maturation process. The production of monoclonal antibody (Mab) probes to the maturation-specific molecules has been difficult since new proteins comprise a minor portion of total membrane proteins. This report describes a protocol for enhancing the production of Mabs to maturation specific molecules. By injecting neonatal mice with caput epididymal sperm plasma membranes, in combination with chemical immunosuppression at adulthood, the mice were made tolerant to the antigens expressed on the caput sperm membranes. Subsequent immunization with cauda epididymal sperm plasma membranes allowed the production of Mabs to the maturation-specific moieties without the necessity for extensive antigen purification procedures. The majority of the resulting Mabs recognize cauda, not caput, epididymal sperm plasma membranes as determined by enzyme-linked immunosorbent assay (ELISA), immunocytochemistry on unfixed cells, and Western blot analyses, even though the protein profile from caput epididymal sperm plasma membranes is very similar to that from cauda membranes. The five Mabs described also recognize cauda fluid antigens, suggesting that the maturational changes on the sperm plasma membranes arise from interactions with the epididymal fluid. Use of the tolerization/immunosuppression protocol has provided Mab tools to assist in the study of sperm maturation during epididymal transit.

Animals↗