Lipid-protein interactions in abnormal egg yolk: a comparison of the major lipoprotein fractions from normal eggs and from the eggs of hens fed methyl sterculate.
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A study of interaction between gametes in the mouse, using live and glutaraldehyde-fixed eggs, showed that, while in live eggs the binding is a two-step process ("early" and "late" binding), the process is one step when the spermatozoa interact with fixed eggs. The second point that emerged from this study is that uncapacitated and capacitated spermatozoa bind the zonae pellucidae of live and fixed eggs in the same way, but only the capacitated spermatozoa bound to live eggs undergo a complete acrosome reaction and penetrate the zona pellucida. Moreover, it has been shown that binding to fixed eggs, just as to live eggs, is Ca2+-dependent, and it can be reversed by EGTA. Fixed eggs thus are a good model to study only one step of the sperm-egg interaction removed from all the other events of the fertilization process.
A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.
The possible role of the egg nucleus in regulating the behavior of sperm nuclei and the cycling of maturation-promoting factor (MPF) was investigated in the physiologically polyspermic eggs of the newt Cynops pyrrhogaster. Many sperm entered all areas of the egg, but only one sperm pronucleus, the principal sperm pronucleus, moved to the center of the animal hemisphere to form a zygote nucleus with the egg pronucleus. All sperm and egg pronuclei synthesized DNA, but the zygote nucleus completed the synthesis of DNA 0.5-1 hr earlier than the accessory sperm nuclei. Entrance into M phase by the accessory sperm nuclei was delayed to a greater and greater extent with increasing distance of these nuclei from the zygote nucleus. When DNA in the egg nucleus was damaged by uv irradiation, not only were both prophase and M phase in the zygote nucleus prolonged, but also the MPF cycle was delayed. Some accessory sperm nuclei in the animal hemisphere escaped degeneration to form additional bipolar spindles, so that delayed multipolar cleavage occurred. The MPF activity in the vegetal hemisphere was less than 25% of that found in the animal hemisphere at M phase in normally fertilized eggs. The levels of immunologically detectable proteins that contained the sequence PSTAIR in vegetal hemispheres were less than 25% of those in animal hemispheres. These results indicate that the egg nucleus of the Cynops egg is involved in the control of the activation of MPF and that the accessory sperm nuclei in the vegetal hemisphere degenerate as a result of the lack of components that are indispensable for entry into M phase.
The biochemical and ultrastructural changes in the envelope of the Xenopus laevis egg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M., Methods Cell Biol. 36, 231-247, 1991; Larabell, C. A., and Chandler, D. E., J. Electron Microsc. Tech. 17, 294-318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm-egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J., J. Cell Biol. 136, 1099-1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64 sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure-function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors during X. laevis fertilization.
By a simple model involving the state variables size and storage, it is possible to describe a wide variety of observations on the feeding, growth, energy storage and reproduction of animals. The model is based on the assumption that reproduction, growth as well as maintenance depend on the stored energy only and not directly on feeding. If an egg is thought of as a non-feeding animal, the model predicts the respiration ontogeny and growth of the embryo inside the egg. These predictions seem to hold well for published data on the development of eggs of fish and ratite, precocial and altricial birds. The latter two are known to follow different respiration ontogenies, but both are described well, differing only in one (compound) parameter value. The model explains why the incubation times of eggs of different species tend to increase linearly with egg size to the power 1/4, and why kiwis and petrels, which lay relatively large eggs, have to brood them much longer than larger birds with eggs of the same size. Conversely, it explains why the small eggs of the (parasitic) European cuckoo, hatch earlier than the still smaller eggs of their tiny hosts. Furthermore, it has been shown how the maintenance rate constant, which frequently appears in the microbial literature, can be obtained from measurements on the respiration and weight ontogeny in embryos, so linking independent lines of research. Application of the model shows an increase of the maintenance rate constant from bacteria, crustaceans, up to fish and birds, and a decrease from bacteria to green algae, suggesting lines of evolutionary development.
Eggs of several insect species are protected against natural enemies by noxious components. However, almost nothing is known about the fate of these defensive substances during egg development nor their site of biosynthesis. The eggs of several leaf beetle species of the taxon Galerucini contain components that are unusual in insects: 1,8-dihydroxylated anthraquinones and anthrones that deter predators such as ants and birds. These components, i.e., the anthrones dithranol and chrysarobin, and the anthraquinones chrysazin and chrysophanol, are not sequestered from host plants. We asked whether the amounts of these components in the overwintering eggs of Galeruca tanaceti change from deposition to larval hatching. Gas chromatography-mass spectroscopy (GC-MS) analyses of eggs revealed a significant decrease in total amounts of dithranol and chrysophanol from egg deposition in autumn to the next spring 5 months later. Thus, these results do not provide any hint of active anthraquinone biosynthesis within eggs. Instead, the anthrones and anthraquinones that must be incorporated by the female into the eggs seem to be degraded to some extent either by the embryo or endosymbionts. GC-MS analyses showed that parasitization of eggs had some effects on the quantities of anthrones and anthraquinones.
A series of experiments was designed to identify factors in a sperm microinjection system that could influence egg viability and decondensation of sperm nuclei after microinjection. Egg viability and sperm decondensation rates were not different among eggs microinjected with rodent sperm. The microinjection of ram sperm required a larger diameter needle for injection, which resulted in low egg viability and sperm decondensation in the first 3 mo of the study but improved greatly after 9 mo of technical experience. The degree of technical experience (3 vs 9 mo) also improved (P<0.05) egg viability after microinjection with rodent sperm; however, the rate of sperm decondensation remained unaffected. Altering the dimensions of the injection needle from a tapered needle barrel to a more uniform needle barrel increased egg viability from 61 to 96% and sperm decondensation from 3 to 27%. The use of medium 199 for incubating microinjected eggs further increased (P<0.05) the percentage of eggs containing decondensed sperm nuclei (52%) compared to eggs incubated in Holmes defined medium (28%). By altering the dimensions of the injection needle, by selecting an appropriate incubation medium, and by gaining technical experience in microinjection, the efficiency of a sperm microinjection system was improved for both rodents and domestic animals.
The effects of vitamin D3 sources, egg production and egg cycle on the genomic expression of calbindin (Mr 28,000) in the intestine and egg shell gland (ESG) of quail were characterized by Northern blot and solution hybridization, using synthetic oligonucleotide probe. In vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-fed quail, onset of egg production induced duodenal and ESG calbindin mRNA and calbindin synthesis. Duodenal calbindin mRNA was slightly higher during the period of shell calcification as compared with the period during which shells were not formed (ESG inactivity). ESG calbindin mRNA was markedly higher during the period of shell calcification than of ESG inactivity. Increasing dietary intake of [3H]1 alpha-hydroxyvitamin D3 increased the duodenal, but not ESG, content of 1,25-(OH)2D3 and calbindin. Duodenal calbindin and its mRNA were absent in vitamin D-deficient quail and were not affected by egg laying. ESG calbindin in the vitamin D-deficient quail was not affected by egg laying, but calbindin mRNA increased in the vitamin D-deficient birds during shell calcification. The results suggest that: (a) intestinal calbindin mRNA and calbindin are induced and/or regulated, either directly or indirectly, by 1,25-(OH)2D3; (b) intestinal calbindin and its mRNA are further induced at the onset of egg laying by an additional stimulator besides 1,25-(OH)2D3; (c) 1,25-(OH)2D3 is required for the expression of the latter stimulator; (d) ESG calbindin mRNA and calbindin are induced in egg-laying birds by a stimulator associated with the egg cycle; and (e) the induction of ESG calbindin mRNA does not need vitamin D metabolites, but 1,25-(OH)2D3 is required for the translation of the mRNA.
BACKGROUND INTRODUCTION: A key step leading to fertilization is the binding of sperm to the egg plasma membrane. When a mammalian sperm reaches the egg plasma membrane, fertilinbeta, an extracellular sperm membrane protein, is believed to bind to an egg plasma membrane receptor triggering fusion. We set out to identify the fertilinbeta binding partner on the egg plasma membrane. RESULTS: We synthesized an 125-labeled peptide with the consensus Asp-Glu-Cys-Asp (DECD) sequence of fertilinbeta's disintegrin domain. This peptide contains a benzophenone photoaffinity probe and inhibits sperm-egg fusion. Upon photoactivation in the presence of whole mouse eggs, a single polypeptide was covalently labeled. This polypeptide has been identified by immunoprecipitation as an alpha6 integrin complexed with beta1 integrin. CONCLUSIONS: Our experiments establish that small peptides containing the consensus DECD sequence of sperm fertilinbeta bind specifically to an alpha6beta1 integrin receptor on the egg membrane. We conclude that fertilinbeta binds directly to the alpha6beta1 integrin on the egg surface and this partnership mediates sperm-egg fusion.
The process of egg formation in the body cavity of a phytoseiid mite, Phytoseiulus persimilis, was observed to examine fertilization of male eggs. After insemination, one of the ova at the periphery of the ovary began to expand, taking up yolk. Two pronuclei appeared in the expanded egg, located dorsally in the ovary, and yolk granules were formed gradually. After the egg became filled with yolk granules the two pronuclei fused. The egg moved via the narrow entrance at the ventral region into the oviduct, where the eggshell was formed. When the eggshell was complete, and while embryogenesis proceeded, the egg was deposited. In the meantime some ova began to expand sequentially and two joining pronuclei appeared in expanding eggs. The joining pronuclei in the first egg proved male diploidy. This is additional evidence of pseudo-arrhenotoky in this phytoseiid mite species, since the first eggs developed into males.
Some freshwater turtles appear unable to produce eggs large enough to achieve the balance between size and number of eggs predicted by optimal egg size theory. We present evidence that pelvic girdle structure constrains egg size and thus offspring size in females of smaller-bodied species (Chrysemys picta and Deirochelys reticularia). The constraint is demonstrated by the correspondence of slopes of the increase of the pelvic aperture and egg width with increasing body size. This constraint appears to be relaxed in a larger-bodied species (Pseudemys scripta), in which the increase in pelvic aperture relative to body size is greater than the increase in egg width. This type of structural constraint on a reproductive trait should not occur unless there is strong selection on pelvic architecture for other functions, such as locomotion, support, and limb retraction, that prevent expansion of the pelvic aperture. Although other explanations may exist for other groups of organisms that appear to vary egg size, the large variation in egg size associated with body size in some species of turtles can be reconciled with optimal egg size theory only if a pelvic constraint model is accepted.
The interaction between essential dietary components and changes in tissue nutrient reserves, egg quality and egg composition, were studied from 60 d before and during the spawning of Sparus aurata broodstock. Fish were given isonitrogenous (550 g/kg dry weight) and isolipidic (100 g/kg dry weight) diets, based on protein and lipid extracts of squid meal. Diets differed in the levels of n-6 (10-30 mg/g dry weight) and n-3 (0-10 mg/g dry weight) essential fatty acids. The effects of these diets on biochemical and fatty acid composition of body tissues, and the subsequent effects on egg composition and egg viability were measured. Dietary essential fatty acids were mostly incorporated into the liver, ovaries, digestive tract and associated adipose tissues. The lipid composition of these tissues reached an equilibrium with dietary lipid composition within 15 d of feeding on any given diet. Muscle and gill cartilage tissues did not show any significant changes in their biochemical and fatty acid composition, even after 60 d feeding. Egg viability decreased significantly within 10 d of feeding the broodstock with a diet deficient in n-3 highly unsaturated fatty acids (n-3 HUFA). The levels of n-3 HUFA in both polar and neutral fractions of egg lipid were directly correlated with their levels in the broodstock diet. When the total amount of egg n-3 HUFA dropped below 17 mg/g dry weight, egg viability and larvae hatching rate decreased by 53% and 47% respectively. These results suggest that the biochemical composition of organs involved in S. aurata reproduction are highly sensitive to the nutritional value of the diet, which affects egg and larval quality rapidly.
The negative relationship between offspring number and offspring size provides a classic example of the role of trade-offs in life history theory. However, the evolutionary transitions in egg size and clutch size that have produced this negative relationship are still largely unknown. Since body size may affect both of these traits, it would be helpful to understand how evolutionary changes in body size may have facilitated or constrained shifts in clutch and egg size. By using comparative methods with a database of life histories and a phylogeny of 222 genera of cichlid fishes, we investigated the order of evolutionary transitions in these traits in relation to each other. We found that the ancestral large-bodied cichlids first increased egg size, followed by a decrease in both body size and clutch size resulting in the common current combination of a small-bodied cichlid with a small clutch of large eggs. Furthermore, lineages that deviated from the negative relationship between clutch and egg size underwent different transitions in these traits according to their body size (large bodied genera have moved towards the large clutch/small egg end of the continuum and small bodied genera towards the small clutch/large egg end of the continuum) to reach the negative relationship between clutch size and egg size. Our results show that body size is highly important in shaping the negative relationship between clutch size and egg size.