Biomedical subjects
B J Gulyas
Publications and source records attributed to B J Gulyas.
Cortical reaction and zona hardening in mouse oocytes following exposure to ethanol.
Mouse oocytes were treated with 8% ethanol for 3-6 min. The rate and pathways of parthenogenetic activation, occurrence of cortical reaction, and zona solubility changes were assessed in alcohol-treated eggs. The incidence of parthenogenetic activation was greatest (91%) after 3-4-min exposure, and it was reduced (84%) after 5-6-min exposure to alcohol. Also, the rate of haploid single pronucleate parthenogenones decreased and the rate of fragmented ova increased with increase time of exposure to ethanol. Ultrastructural observations showed occurrence of cortical reaction, disappearance and subsequent reappearance of short microvilli. A slight damage occurred to the ER in alcohol-exposed ova. The zona dissolution assay utilizing alpha-chymotrypsin demonstrated decreased solubility of the zonae pellucidae after exposure to alcohol. The zona dissolution t50 increased from 0.5-2.5 min in nontreated unfertilized oocytes to about 4 h in activated ova. The t50 of in vivo fertilized eggs was 4 1/2 h. Empty zonae exposed to alcohol lysed at the same rate as nontreated control zonae did. The results indicate that activation of mouse oocytes with alcohol initiates completion of meiosis and triggers the cortical reaction, which results in subsequent hardening of the zona pellucida.
Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: effects on fertilization and early development.
During development and maturation, mammalian oocytes are surrounded by the zona pellucida which in the mouse is comprised of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. Previously, monoclonal antibodies to ZP-2 have been isolated. The isolation and characterization of monoclonal antibodies specific for ZP-3, the zona protein with sperm receptor activity are now reported. Following passive immunization, these monoclonal antibodies localize to the intraovarian zonae pellucidae and their presence precludes both in vivo and in vitro fertilization of subsequently ovulated eggs. Monoclonal antibodies specific for either ZP-2 or ZP-3 also completely block in vitro fertilization at relatively low concentration ranging from 0.4 to 75 micrograms/ml. The contraceptive effect requires the presence of the zona and appears to inhibit the penetration of the zona pellucida by sperm rather than by blocking the sperm binding site. Neither antibody interferes with in vitro development from the two-cell to the blastocyst stage or with subsequent hatching from the enveloping zona pellucida.
Fusion of oocytes and development of oocyte fusion products in the mouse.
Utilizing fusion techniques we demonstrated that 57-72% of meiotic mouse oocytes could be fused with polyethylene glycol. Activated oocyte fusion products (OFP) completed second meiosis and supported early embryonic development to the blastocyst stage (12%). Furthermore, when oocytes from MF1 strain that demonstrate a developmental block at the 2-cell stage when cultured from the zygote stage, were fused with oocytes of an F1 hybrid not demonstrating this block, the OFP overcame the 2-cell block and developed to blastocysts. Of the 20 blastocysts from OFPs that were transferred to pseudopregnant recipients 11 implanted and 1 F1 OFP developed to the 14-somite stage.
Response of monkeys to porcine zona pellucida as detected by a solid-phase radioimmunoassay.
The immunological response of cynomolgus monkeys (Macaca fascicularis) to immunization was evaluated utilizing collagenase-isolated pig zona pellucida. Six weeks after initial immunization a high serum titer of antibody was exhibited. Serum antibody titers demonstrated a noticeable decline 5 months after booster injections were discontinued. The assay method used is rapid and is capable of detecting antibody in serum dilutions of 1:78,000, as compared to 1:125,000 with the indirect fluorescence assay.
Identification of mammalian sperm surface antigens: II. Characterization of an acrosomal cap protein and a tail protein using monoclonal anti-mouse sperm antibodies.
Monoclonal anti-mouse sperm antibodies have been produced by fusing mouse myeloma cells with spleen cells from rats immunized with epididymal sperm of C3H mice, Immunoprecipitation and immunoperoxidase techniques showed that one such monoclonal antibody, AMS IV-33, recognized a 200 000 dalton protein localized on the acrosomal cap of the sperm cell. Two other monoclonal antibodies AMS IV-54 and -76, reacted with a 68 000 dalton component on the surface of the sperm tail. Both antigenic targets were species specific and were present in about equal amounts on sperm from several different strains of mice. The tail protein was sperm specific, whereas the antibody reacting with the acrosomal cap protein also appeared to react somewhat with antigens present in other mouse tissues.
Identification of mammalian sperm surface antigens. I. Production of monoclonal anti-mouse sperm antibodies.
Surface antigens of mammalian sperm were studied by use of monoclonal antibodies (MAs). Six hybridoma cell lines were obtained by fusion of mouse myeloma cells with spleen cells from rats immunized with unwashed, epididymal sperm from C3H mice. Quantitative assessment of antibody binding, using a solid phase, antibody-protein A assay, indicated that four MAs bound to integral, sperm surface antigens; two others bound to nonintegral sperm antigens or epididymal fluid components. Immunofluorescence studies showed specific binding of individual MAs to localized regions: acrosome, midpiece, and midpiece and tail. All of these MAs inhibited sperm-egg binding, and those to the midpiece and/or tail immobilized sperm cells. The monoclonal antibodies provide probes for immunochemical characterization of sperm antigens and for elucidation of the role of the antigens in sperm.
An improved method for processing single cells for electron microscopy utilizing agarose.
An improved method is presented for processing single cells for electron microscopy. Agarose, which has a low (30 degrees C) gelling temperature, was used as an initial embedding medium for single cells (spermatozoa and oocytes) and dissociated cell preparations (luteal cells and spleen cells). Dispersed cells of corpus luteum, spleen, and epididymal spermatozoa were placed in 1.5% agarose after aldehyde fixation. These fixed cells, embedded in agarose, were packed into a dense pellet by centrifugation, postfixed, then embedded in Epon. Mammalian eggs were not centrifuged; instead, they were embedded in agarose discs. Cells embedded in agarose were cooled below 30 degrees C to allow for gelling, then processed for electron microscopy. Because agarose has a low gelling temperature, some heat-labile substances were preserved, as demonstrated by retention of peroxidase activity using the DAB histochemical method. The agarose embedding procedure is both rapid and facile, and has proven to be of value in the handling of fragile single cells for electron microscopic studies.
Visualization of binding and internalization of a horseradish peroxidase-hCG conjugate by monkey luteal cells.
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Progesterone production by dispersed monkey (Macaca mulatta) luteal cells after exposure to trypsin.
In an attempt to justify use of trypsin to achieve more thorough dispersion of luteal cell clumps in vitro, progesterone (P) production by collagenase dispersed monkey luteal cells from the mid-luteal phase corpus luteum (CL) was examined in vitro either after 10 min, or continuous (3h) exposure to trypsin (TR). In the first experiment, cells were pre-incubated in TR, then incubated at 37 degrees C for 3h with human chorionic gonadotropin (hCG) after the addition of soybean-trypsin inhibitor (STI). Pre-incubation of luteal cells with TR had no effect on the level of P production under basal conditions. Cells that were preincubated with TR responded to hCG stimulation with increased progesterone secretion (P less than 0.01) in a fashion similar to untreated cells. P production in response to hCG was independent of TR concentration over the range of 0.05% to 0.2% during the pre-incubation period. However, continuous exposure (3h) of cells to TR significantly depressed (P less than 0.01) basal P secretion and inhibited the response to hCG. We conclude that TR had no effect on the biopotency of hCG per se, but probably the over-exposure to TR had an adverse effect on the LH/hCG receptors. Addition of STI after a 10 min pre-incubation with TR, prevented these deliterious effects, thereby permitting the use of TR to improve the completeness of luteal cell dissociation.
Cortical granules of mammalian eggs.
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Synthesis of progesterone and estradiol by monkey luteal cells in culture: Effects of insulin, thyroxine, cortisol, and cholesterol with and without hCG.
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Mammalian sperm-egg recognition and binding in vitro. I. Specificity of sperm interactions with live and fixed eggs in homologous and heterologous inseminations of hamster, mouse, and guinea pig oocytes.
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Degeneration of mouse oocytes in response to polycyclic aromatic hydrocarbons.
Destruction of mouse oocytes in primordial and small primary follicles in response to treatment with 3-methylcholanthrene (MC) was studied at the ultrastructural level. Four-week old C57Bl/6N (B6) strain mice received a single injection of 80 mg/Kg MC in corn oil intraperitoneally. Controls received only corn oil. Ovaries from animals were prepared for light and electron microscopic examination at specified intervals after treatment. The number of primordial follicles remained constant in control animals. In contrast, their number decreased significantly (P less than 0.01) by three days, and they were depleted by seven days after MC treatment. Subtle degenerative modifications were noted in the ooplasm of primordial follicles two days after treatment. These changes consisted of vesiculation of mitochondrial cristae, increased electron density of the mitochondrial matrix, myelin structures in lipid droplets and in mitochondria. More advanced stages of degeneration of primordial follicles were characterized by further vesiculation or disappearance of mitochondrial cristae, chromatin clumping, and increased density of the ooplasm. Small primary follicles had undergone similar initial degeneration as primordial follicles. In more advanced stages of degeneration nuclear and cytoplasmic contents condensed, endoplasmic reticulum, Golgi complex and mitochondria swelled, small vesicles and multivesicular bodies appeared. In the most advanced stages of degeneration of small primary follicles it appeared that small portions of the oocyte were engulfed by the surrounding follicular cells. It is concluded that exposure of B6 mice to a single dose of MC results in atresia of oocytes in primordial and small primary follicles. Ultrastructurally, these degenerating oocytes of treated mice looked much like the spontaneously atretic oocytes in untreated animals.
Progesterone synthesis and fine structure of dissociated monkey (Macaca mulatta) luteal cells maintained in culture.
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Association of microperoxisomes with the endoplasmic reticulum in the granulosa lutein cells of the Rhesus monkey (Macaca mulatta).
Aldehyde fixed tissue from monkey (Macaca mulatta) corpus luteum was incubated in alkaline 3,3inch-diaminobenzidine (DAB), and prepared for electron microscopic histochemical observations. The association of microperoxisomes with the granular (GER) or agranular (AER) endoplasmic reticulum was reconstructed from serially sectioned tissues and by tilting of specimens in the microscope. Out of 107 microperoxisomes, 106 were directly associated with the AER. Two different forms of attachment were found between microperoxisomes and the AER and that of the microperoxisome are confluent. In the second, lingulate type of connection, a blunt-end structure either is inserted into an invagination of the AER, or penetrates into the lumen of the AER. The lumen of the lingula is confluent with the microperoxisome, but not with the AER. In addition to these connections, fine thread-like structures were observed extending between AER and adjacent microperoxisomes.
Fetal or maternal hypophysectomy in rhesus monkeys (Macaca mulatta): effects on the development of testes and other endocrine organs.
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Effects of fetal or maternal hypophysectomy on endocrine organs and body weight in infant rhesus monkeys (Macaca mulatta): with particular emphasis on oogenesis.
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