Unfertilized Hamster Eggs Research Articles

The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor.

The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.

Thimerosal causes calcium oscillations and sensitizes calcium-induced calcium release in unfertilized hamster eggs

Calcium-induced-calcium-release (CICR) was assayed in unfertilized golden hamster eggs by injecting Ca 2+ and monitoring Ca 2+-dependent hyper-polarizing responses (HRs) and Ca 2+-sensitive fluo-3 fluorescence. Incubating eggs in the sulfhydryl reagent thimerosal caused [Ca 2+] 1 oscillations as monitored by Ca 2+-dependent HRs and decreased approximately 10-fold the Ca 2+ injection current required to generate an HR and cause a large intracellular Ca 2+ increase. Thimerosal also enhanced the sensitivity of eggs to Ca 2+ injection in a calcium-free medium. The effects of thimerosal on CICR were prevented by dithiothreitol and were not mimicked by injecting inositol 1,4,5-triphosphate. The data suggest that thimerosal may be an alternative agent for studying CICR in caffeine-insensitive cells.

Zona-induced acrosome reaction of hamster spermatozoa.

It is well established that the zonae pellucidae of mature unfertilized eggs have the ability to induce the acrosome reaction of capacitated spermatozoa. To determine if this capacity of the zona is species-specific, hamster spermatozoa were allowed to attach to the zonae of homologous and heterologous eggs and examined for the acrosome reaction. The zonae of eggs from six different species were tested and the zona of hamster egg was found to have the strongest capacity to induce the acrosome reaction of hamster spermatozoa, followed by human and rat zonae. The zonae and mouse, guinea pig, and domestic fowl eggs were incapable of inducing the acrosome reaction of hamster spermatozoa. The acrosome reaction-inducing ability of the hamster zona was found to increase during maturation in the ovary. The zona of mature unfertilized hamster eggs maintained their acrosome reaction-inducing ability even after aldehyde fixation or storage in a highly concentrated solution of ammonium sulfate.

A delayed all-or-none hyperpolarisation induced by a single Ca action potential in hamster eggs.

A transient change of membrane potential and resistance could be evoked after a long latency (ca. 9 s) by a single calcium action potential in some unfertilized hamster eggs. The estimated reversal potential for the delayed response was close to EK supporting the conclusion that K channels were opened indirectly by the Ca2+ influx through voltage-gated channels. A second action potential elicited after the first did not induce a similar response. A number of treatments (insertion of a Ca2+ pipette, application of Na+-free solution, La3+ or high external pH) likely to raise [Ca2+]i also induced similar large changes of potential and resistance after which a single action potential failed to evoke a large delayed response. The evidence indicates that a small rise in [Ca2+]i activates a slow process leading to a further large increase in [Ca2+]i.

Scanning electron microscope study of in vitro prepenetration gamete interactions

AbstractHamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs, and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. It was found that sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin‐treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, we found that the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatozoa. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona‐reacted and in trypsin‐treated hamster eggs inseminated in vitro with homologous spermatozoa.

Retention of Biologic Characteristics of Zona Pellucida in Highly Concentrated Salt Solution: The Use of Salt-Stored Eggs for Assessing the Fertilizing Capacity of Spermatozoa

When unfertilized hamster eggs are placed in highly concentrated solutions of neutral salts (e.g., 2 M ammonium sulfate), the egg proper, or vitellus, shrinks, creating a large perivitelline space; the zona pellucida remains unchanged in appearance under the light microscope. When these eggs are inseminated, many spermatozoa attach to and penetrate the zona. The specificity as well as several physical and chemical characteristics of the zona seem to remain unchanged during storage of the eggs in salt solutions. The properties of the human zona pellucida which allow the attachment and penetration of human spermatozoa are also retained in concentrated salt solutions. Shipment of salt-stored human eggs at ambient temperature to any part of the world is feasible and inexpensive. The present study suggests that salt-stored eggs can be used as substitutes for fresh living eggs in the preliminary assessment of fertilizing capacity of spermatozoa when collection of a large number of fresh unfertilized eggs, particularly in humans, is not practical.

Detrimental effect of visible light on meiosis of mammalian eggs in vitro.

Short wavelength visible light (less than 470-480 nm) emmitted from ordinary light sources is detrimental to unfertilized hamster eggs in that prolonged exposure to the light disturbs the completion of normal meiosis after the eggs are penetrated by spermatozoa. The fluorescent light commonly used in modern laboratories is more harmful than the light from incandescent lamps. In experiments involving the handling of eggs in vitro, minimal exposure to the light or the use of appropriate filters (e.g., red cellophane sheets) is recommended.

Behavior of nuclei of testicular, caput and cauda epididymal spermatozoa injected into hamster eggs.

The behavior of isolated nuclei of testicular and cauda and caput epididymal spermatozoa of the hamster injected into mature unfertilized hamster eggs was studied. Decondensation of nuclei within the egg cytoplasm was observed regardless of the type of spermatozoa used and whether the eggs were activated or not. In activated eggs the decondensed nuclei of testicular and cauda but not caput epididymal spermatozoa transformed into pronuclei. This failure of caput epididymal spermatozoa nuclei to transform into pronuclei may be due to a factor in the nuclei which prevents such a transformation. It is possible that this factor may be inactive or absent in the nuclei of testicular spermatozoa or may be inactivated by another factor while spermatozoa are present in the cauda epididymis.

Immunological interference with fertilization.

Rabbit antisera against mouse and hamster ovaries and sheep antisera against rabbit ovary have been used to demonstrate ovary specific antigens in these three species of mammals. That one or more of the specific antigens are located in the zona pellucida which surrounds the egg was indicated in immunofluorescence studies and by the effects antibodies have on the zona. After exposure to the antibody a precipitate forms on the zonae of preovulatory, unfertilized and fertilized eggs and all embryonic stages up to the time when the zona is shed. The precipitate was always seen near the outer surface of the zona suggesting that the active sites of the antigen(s) are located in this region. Trypsin and pronase which usually dissolve the zona were ineffective on the precipitated outer portion but did dissolve the inner, unprecipitated region. Species- and tissue-specificity and chemical nature of the zona are discussed. Important roles for the zona antigen(s) in a number of early events in reproduction were indicated by the effects of antizona antibodies have on fertilization and implantation. Pretreatment of unfertilized hamster eggs with zona precipitating antibodies was most effective in blocking sperm attachment to the zona. When the antibody was added subsequent to in vitro insemination, but before the sperm had passed through the outer region of the zona, sperm penetration was blocked. Mouse embryos treated with zona precipitating antibody and cultured in vitro failed to escape from the zonae, implying that implantation could be inhibited by antibody. Passive immunization of female mice with antizona antibodies and precipitattion of the zonae of hamster embryos in transfer experiments reduced the number of implantation sites. The identification of zona antigens and the effects of zona precipitating antibodies on early reproductive events encourage further studies on the zona in reference to immunological control of fertility.

Histochemical demonstration of phosphorylase and UDPG-glycogen transferase in fertilized and unfertilized mammalian eggs.

Phosphorylase and UDPG-glycogen transferase in fertilized and unfertilized hamster eggs at various times were examined histochemically. When eggs were stained with iodine after incubation in substrate solution for phosphorylase, blue granules of newly formed polysaccharide spread evenly in the cytoplasm but not in the polar bodies, while red-purple granules appeared with similar distribution when treated for UDPG-glycogen transferase. Often there were differences among blastomeres of cleaved eggs in the intensity of both phosphorylase and UDPG-glycogen transferase.Loss of fertilizability was not proved to be accompanied by the decrease of enzyme activities. It was also found that the activities of these enzymes were not significantly different between fertilized and unfertilized eggs at the same age.In fertilized hamster eggs, phosphorylase activity was constant up to the 8-cell stage, but disappeared completely by blastocyst stage. The activity of UDPG-glycogen transferase decreased gradually throughout the stages, and disappeared at the blastocyst stage. A similar picture was seen in rabbit eggs.