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Biomedical subjects

B S Dunbar

Publications and source records attributed to B S Dunbar.

At least 19 recordsLinked to original sources
PubMed1995

Immunization of monkeys with recombinant complimentary deoxyribonucleic acid expressed zona pellucida proteins.

OBJECTIVE: To evaluate the effects of immunization with zona pellucida (ZP) proteins produced by recombinant complementary DNA (cDNA) technology for the elicitation and antibodies that inhibit sperm binding without altering ovarian function in the nonhuman primate. DESIGN: Controlled nonhuman primate study. SETTING: Controlled environment with individual housing of monkeys in facility approved by National Institutes of Health (NIH) guidelines. PARTICIPANTS: Monkeys housed and treated according to NIH regulations. INTERVENTIONS: Monkeys immunized and boosted at regular intervals with ZP proteins produced using recombinant cDNA techniques. MAIN OUTCOME MEASURE: Urinary estrogen, P, serum antibody levels, sperm-ZP binding, and ovarian morphology. RESULTS: Monkeys immunized with a recombinant rabbit 75-kd ZP protein expressed from a partial cDNA in the pEX bacteria expression system produce antibodies that interfere with ovarian follicular development and ovarian cyclicity. On the contrary, monkeys immunized with a recombinant rabbit 55-kd ZP protein develop antibodies that inhibit homologous sperm binding but do not affect ovarian follicular development or subsequent ovarian hormonal cyclicity. CONCLUSION: Monkey antibodies to the rabbit 75-kd ZP recombinant protein can be generated that inhibit ovarian cyclicity as desired for animal sterilization vaccines. Antibodies to the 55-kd ZP recombinant protein inhibit homologous monkey sperm binding to the ZP without altering ovarian endocrine function or morphology as is desired for human immunocontraception.

Animals↗
PubMed1995

Immunogenicity enhancement of recombinant rabbit 55-kilodalton zona pellucida protein expressed using the baculovirus expression system.

In the present study we have used a molecular approach to evaluate the immunogenicity and antigenicity of glycosylated and non-glycosylated recombinant rabbit 55-kDa zona pellucida (ZP) protein. The 55-kDa cDNA was expressed in insect (Sf9) cells through use of a baculovirus expression system to obtain nonfusion glycosylated recombinant ZP protein (BV-55). SDS-PAGE and immunoblot analysis demonstrated that the recombinant protein is expressed as two forms having relative molecular masses of 70 kDa and 80 kDa. Because cells treated with tunicamycin produce predominantly the 70-kDa form, this heterogeneity is presumed to be due to differential glycosylation. Further studies using lectin blot and immunoblot analyses showed that the BV-55 protein has both N-linked and O-linked oligosaccharides. However, this glycosylation is distinct from that of the native 55-kDa ZP protein, since it was not recognized by a monoclonal antibody associated with lactosaminoglycan-type carbohydrate epitopes in native ZP proteins. Immunogenicity studies demonstrated that antibodies against the BV-55 protein are developed by female rabbits and guinea pigs and that these antibodies recognize epitopes associated with native, enzyme-deglycosylated as well as nonglycosylated recombinant forms of the rabbit 55-kDa ZP protein. In contrast, recombinant protein expressed in bacteria did not elicit antibodies in either rabbits or guinea pigs. These results demonstrate that expression of the 55-kDa recombinant protein in the baculovirus expression system enhances its immunogenicity.

Animals↗
PubMed1994

The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology, and developmental expression.

Many studies of the molecular and biochemical aspects of mammalian fertilization have focused on the interaction of the spermatozoa with the zona pellucida (ZP). The zona pellucida, a unique extracellular matrix surrounding the mammalian oocyte, is formed during ovarian follicular development. Following ovulation of the mature ovum, the spermatozoa must bind to and penetrate this matrix before the fertilization process is completed and the male and female genetic information combine. Although numerous models for this interaction have been proposed, the complete process has yet to be elucidated. The precise mechanisms by which these interactions occur also vary markedly among different mammalian species, making it more difficult to establish a unified model. To a great extent, the study of the molecules involved in these interactions have been limited because small numbers of female gametes are available for these studies. The recent development of techniques to isolate large numbers of zonae pellucidae as well as advances in immunological and molecular biology techniques have permitted the detailed characterization of ZP proteins. Although there is a paucity of information on the post-translational modification and extracellular processing of these molecules which result in matrix formation, a number of properties have been elucidated allowing better correlation between the structure and function of different ZP proteins among species. This review reflects these studies in relation to protein nomenclature and the molecular complexity of ZP antigens.

Animals↗
PubMed1994

Initial characterization of equine inhibin.

Inhibin has been characterized from a number of mammals; however, it has not been extensively studied in horses. Western blot analysis was used to examine the size heterogeneity of equine inhibin alpha- and beta-subunits. The distribution of equine inhibin activity from the initial sizing column (S-200, 25 x 94 cm) indicated that the majority of equine inhibin activity was present as larger-molecular-size forms. When the large forms were analyzed by Western blot in nonreducing conditions, alpha-subunit bands were detected at 40,000 M(r), 56,000 M(r), 80,000 M(r), and 90,000 M(r); beta a reactive bands were identified at 56,000 M(r) (strong) and 90,000 M(r) (faint). Western blot analysis of the lower-molecular-weight inhibins on one-dimensional (1D) SDS-PAGE gels revealed one inhibin band at 32,000 M(r). In reduced 1D-PAGE gels, a doublet alpha-subunit band was found at 18,000 M(r), and one beta a band was found at 14,000 M(r). The 18,000 M(r) equine alpha-subunit was present in three distinct spots in the isoelectric focusing (IEF) dimension of two-dimensional (2D)-PAGE, and closely overlapped those of porcine inhibin alpha-subunit. In conclusion, inhibin is present in good yield in equine follicular fluid. A higher proportion of the total activity is present in higher-molecular-weight forms than with porcine inhibin. Inhibin was detected at 90,000 M(r), 56,000 M(r), and 32,000 M(r). Alpha-subunit-only bands at 40,000 M(r) and 80,000 M(r) were detected. The lower-molecular-weight form of equine inhibin is similar to porcine inhibin in size and pattern on 2D-PAGE.

Animals↗
PubMed1994

Characterization of antigenicity and immunogenicity patterns of native and recombinant zona pellucida proteins in the white-tailed deer (Oidocoileus virginianus).

The effectiveness of zona pellucida antigens in immunizing white-tailed deer to reduce fertility was evaluated by analysing the constituent deer zona pellucida proteins and their immunogenicity. Does were immunized with porcine zona pellucida antigens. The antibodies were characterized using immunohistochemical and immunoblot analysis, in which zona pellucida proteins were separated by one-dimensional and two-dimensional PAGE. Deer anti-porcine zona pellucida antibodies were found to recognize all the major proteins of the porcine zona pellucida. These antibodies also recognized several proteins of deer zona pellucida, indicating that it is possible to break immune tolerance in the deer using such a protocol. The antibodies were also found to recognize peptides of 55 and 75 kDa that were produced by expressing cDNA clones containing antigens of major glycoproteins of rabbit zona pellucida. Furthermore, antibodies against rabbit zonae pellucidae recognized antigens in zonae of paraffin-embedded deer ovaries. Taken together, these experiments demonstrate the crossreactive nature of a number of zona pellucida epitopes found in deer and in several other species. They also illustrate the immunogenicity possible in such an immunization protocol, and provide valuable probes for the investigation of follicular development in this and other species.

Animals↗
PubMed1993

Identification and structural characterization of the 75-kDa rabbit zona pellucida protein.

A cDNA (rc75) encoding a 75-kDa rabbit zona pellucida (ZP) glycoprotein (R75) has been cloned and sequenced. The predicted amino acid sequence consists of 676 amino acids including seven potential N-glycosylation sites. The cDNA hybridizes to a 2.4-kilobase mRNA in ovary that is not detectable in other rabbit tissues. The R75 mRNA was also found to be expressed during the early stages of rabbit ovarian development (2-6 weeks of age) when the ovary contains primordial, primary, and early secondary follicles. The deduced amino acid sequence of rc75 has 77% similarity to the mouse ZP2 protein but no similarity to mouse ZP3. R75 also contains regions with 35-55% similarity to a previously cloned rabbit 55-kDa ZP protein. Monte Carlo simulation comparison confirmed that R75 has a significant probability of homology with mouse ZP2 and the rabbit 55-kDa ZP protein. Antibodies were developed against a fragment of R75 (rc75a cDNA) expressed in the pEX expression vector. These antibodies were used to confirm that the expressed protein contained epitopes found in the native rabbit ZP glycoprotein. These data suggest that some, but not all, ZP proteins may be conserved among different species and that within a species ZP proteins may share similar regions.

Aging↗
PubMed1993

Developmental expression of the rabbit 55-kDa zona pellucida protein and messenger RNA in ovarian follicles.

The events associated with the early stages of oocyte activation and granulosa cell (GC) differentiation during ovarian follicular development have been investigated using immunological and cDNA probes. Because zona pellucida (ZP) proteins provide excellent markers for oocyte activation during early stages of ovarian development, a cDNA encoding a full-length ZP protein with calculated molecular weight of 57,185 (rc55) and a specific antibody to the corresponding native rabbit ZP protein (R55) were developed. Immunohistochemical analysis of ovaries demonstrated that R55 was localized in follicles of animals 2 weeks and older. R55 was localized in the oocytes of primordial follicles and the oocytes and GCs of primary follicles but was undetectable in the GCs of large antral follicles. GCs were isolated from 6-week-old rabbit ovaries and cultured in defined media to demonstrate expression of rabbit ZP proteins by GCs in vitro. Rabbit ZP proteins were localized in the cytoplasm of cultured GCs if these cells were treated with monensin, a compound that blocks glycoprotein secretion. Northern blot analysis with the cDNA probe rc55 revealed that cultured GCs contained a mRNA for R55 and that there is stage-specific expression of the R55 mRNA in developing rabbit ovaries. Immunoblot analysis demonstrated that R55 is detectable at 4 weeks and accumulates in 6-, 8-, and 12-week ovaries which contain developing primary and secondary follicles. Collectively, these data illustrate developmental expression of R55 in oocytes and GCs of rabbit ovarian follicles.

Animals↗
PubMed1993

Molecular cloning and expression of an abundant rabbit ovarian protein with 20 alpha-hydroxysteroid dehydrogenase activity.

An abundant 37-kDa protein, which comprises up to 30% of the soluble proteins of the ovary, has been found to have 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD) activity. The steroidogenic enzyme 20 alpha HSD regulates the conversion of progesterone to 20 alpha-hydroxyprogesterone in many mammalian species. Complimentary DNA clones encoding a unique and abundant 20 alpha HSD were isolated from a mature rabbit ovary library using guinea pig antisera generated to the purified 37-kDa protein and from a 5' EcoRI fragment from the initial positive clone. A full-length cDNA clone of 1217 basepairs encoding a 323-amino acid protein with an estimated mol wt of 37 kilodaltons was obtained. Amino acid sequence data indicate a similarity to human chlordecone reductase, bovine lung prostaglandin F synthase, human aldose reductase, human aldehyde reductase, and frog lens rho-crystallin, placing rabbit ovarian 20 alpha HSD in the aldo-keto reductase family of proteins. Northern blot analysis demonstrated a 1.2-kilobase mRNA in the interstitial tissue of mature rabbit ovaries and, to a lesser extent, in corpora luteal tissue. 20 alpha HSD was expressed in bacteria as a recombinant protein and was shown to possess enzymatic activity, preferring NADP as a cofactor. These studies demonstrate that an abundant ovarian protein belonging to the superfamily of NADP-dependent aldo-keto reductases has 20 alpha HSD activity. This is the first example of an abundant crystallin-related protein with known enzymatic activity in a tissue other than the lens.

20-Hydroxysteroid Dehydrogenases↗
PubMed1992

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.

Animals↗
PubMed1992

Localization of a carbohydrate antigen associated with growing oocytes and ovarian surface epithelium.

We used a monoclonal antibody (PS1) to a carbohydrate antigen to study the development of the oocyte and follicle during early stages of differentiation in several mammalian species. This antigen has been shown to localize within the cytoplasm of oocytes in primordial follicles as well as in growing oocytes. It is also localized within distinct layers of the zona pellucida (ZP) of developing follicles. Although this antibody was made against a specific ZP glycoprotein, the antigen also appears to be abundant in cells of the ovarian surface epithelium (OSE). The localization of this carbohydrate moiety has been observed in ovaries of rabbits of different ages as well as in the ovarian surface epithelium of other mammalian species including cat, cynomolgus monkey, baboon, and human. These studies demonstrate that there is an abundant carbohydrate antigenic determinant which is associated with both the mammalian oocyte and the ovarian surface epithelium but which is not apparent in other ovarian cell types or in non-ovarian secretory epithelium. This antibody probe should provide a valuable tool for studying the development and differentiation of the ovary, since this antigen is associated with two highly differentiated but distinct cell types.

Animals↗
PubMed1992

Characterization of equine zona pellucida glycoproteins by polyacrylamide gel electrophoresis and immunological techniques.

This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubilized, and then analysed by 1D- and 2D-PAGE. Silver stained 2D-PAGE of the EZP revealed the presence of three EZP glycoprotein families of apparent molecular mass ranges of 93-120 kDa, 73-90 kDa and 45-80 kDa. Immunoblot analysis of EZP glycoproteins resolved by 2D-PAGE using rabbit antisera against pig zonae pellucidae (R alpha HSPZ) confirmed the presence of three EZP glycoprotein families and established the existence of common epitopes between equine and porcine ZP glycoproteins. Further immunodetection using 2D-PAGE-separated glycoproteins illustrated that the 45-80 kDa family is recognized by the monoclonal antibody R5, developed against the porcine ZP glycoprotein of molecular mass 55-120 kDa. Guinea-pig antiserum against endo-beta-galactosidase-treated rabbit ZP 55 kDa glycoprotein (R55K), which specifically recognizes the rabbit ZP glycoprotein with the lowest molecular mass, also recognized the EZP 45-80 kDa glycoprotein family. Guinea-pig polyclonal antisera developed against total heat-solubilized rabbit ZP (GP alpha HSRZ) recognized the 73-90 kDa EZP glycoprotein family exclusively. After heat solubilization and treatment of EZP with endo-beta-galactosidase to remove polylactosaminoglycans, silver stained 1D-PAGE again demonstrated the presence of three glycoproteins with apparent molecular masses of 60, 75 and 90 kDa. The partially deglycosylated 60 kDa equine glycoprotein is recognized on immunoblot by the monoclonal antibody R5; the 75 kDa EZP glycoprotein is recognized by GP alpha HSRZ; and all three EZP glycoproteins separated by 1D-PAGE are recognized by R alpha HSPZ. These data add further support to the concept of cross-species zona pellucida glycoprotein antigenicity.

Animals↗
PubMed1990

Mouse phenylalanine hydroxylase. Homology and divergence from human phenylalanine hydroxylase.

The laboratory mouse represents an important model for the study of phenylalanine metabolism and the pathochemistry of phenylketonuria, yet mouse phenylalanine hydroxylase (PAH) has not been extensively studied. We report the cloning and sequencing of a mouse PAH cDNA, the expression of enzymic activity from the mouse PAH cDNA clone and the identification of mouse PAH and human PAH by two-dimensional PAGE of liver samples. These data confirm the expected homology of mouse PAH and human PAH and suggest differences in the primary sequence and the phosphorylation state of the two enzymes.

Amino Acid Sequence↗
PubMed1990

Identification of extracellular proteins in the rat cumulus oophorus.

We have examined the proteins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte-cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postovulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW approximately 81,000 and 100,000). The other two proteins were more basic and occurred at MW approximately 90,000 and 150,000. Our results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.

Animals↗
PubMed1990

Effects of extracellular matrix on the expression of specific ovarian proteins.

A unique ovarian follicle cell culture system has been established to analyze the effects of extracellular matrix (ECM) on early granulosa cell differentiation. Primary and early secondary follicles isolated from ovaries of sexually immature rabbits were grown on poly-D-lysine or Englebreth-Holm-Swarm basement membrane biomatrix substrata (EHS) in serum-free, hormonally defined medium. Granulosa cells from these follicles were examined for growth pattern characteristics and for secretory protein synthesis by two-dimensional (2D) PAGE. Whereas some proteins were synthesized by cells on either matrix, the expression of other secreted proteins was markedly affected by the ECM used. Secretion of zona pellucida (ZP) proteins was demonstrated by ELISA assays and immunoblots of one-dimensional (1D) and 2D-PAGE separations of secreted proteins probed with monoclonal and epitope-selected antibodies. Expression of two ZP proteins was altered by ECM: 55-kDa endo-beta-galactosidase (EBGD)-treated ZP glycoprotein (55-kDaEBGD) was secreted by cells grown on either ECM, but a greater amount of 75-kDaEBGD was secreted by cells grown on poly-D-lysine. These studies are the first to show that granulosa cells from early-stage follicles express ZP proteins in vitro in the absence of oocytes, although proper post-translational modification may not occur. They also demonstrate the dramatic effect of ECM on the expression of these and other secretory proteins.

Animals↗