Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein

In this investigation, the interaction of mouse sperm with unfertilized eggs and embryos, solubilized zonae pellucidae isolated from eggs and embryos, and purified zona pellucida glycoproteins ZP1, 2, and 3 (J. D. Bleil, and P. M. Wassarman, (1980b) Dev. Biol. 76, 185-202) has been examined in vitro by light and electron microscopy. The experiments described were carried out in order to determine the temporal sequence of events during sperm-egg interaction in vitro and to identify the component(s) of zonae pellucidae responsible for inducing mouse sperm to undergo the acrosome reaction. "Pulse-chase" analysis of the sequence of sperm-egg interactions revealed that mouse sperm first "attach" loosely and then "bind" tightly to the unfertilized egg's zona pellucida. Binding of sperm to egg zonae pellucidae is followed by induction of the acrosome reaction. Induction of the acrosome reaction can be mediated by the zona pellucida, since solubilized zonae pellucidae isolated from unfertilized eggs were found to be just as effective as the calcium ionophore A23187 in inducing the reaction in vitro. Furthermore, ZP3 purified from zonae pellucidae isolated from unfertilized eggs, but not from two-cell embryos, was also just as effective as either solubilized zonae pellucidae from eggs or ionophore A23187 in inducing the acrosome reaction. ZP1 and 2 from both eggs and embryos, and ZP3 from embryos, had little effect on the extent of the acrosome reaction as compared to control samples. The results of these and other experiments (J. D. Bleil, and P. M. Wassarman, (1980b) Cell 20, 873-882) strongly suggest that, at least in vitro, mouse sperm recognize and bind to ZP3 of egg zonae pellucidae, and that such binding leads to the induction of the acrosome reaction. Modification of ZP3 following fertilization eliminates sperm binding to zonae pellucidae and, consequently, induction of the acrosome reaction is precluded.     

Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs and preimplantation embryos. Fertilization results in transformation of the zona pellucida (“zona reaction”), such that additional sperm are unable to bind to the zona pellucida of fertilized eggs and embryos, and sperm that had partially penetrated the zona pellucida of eggs prior to fertilization are prevented from further penetration after fertilization. The failure of sperm to bind to fertilized mouse eggs and embryos is attributable to modification of the sperm receptor, ZP3, an 83,000-molecular weight glycoprotein present in zonae pellucidae isolated from both eggs and embryos [Bleil, J. D., and Wassarman, P. M. (1980). Cell, 20, 873–882]. In this investigation, ZP2, the major glycoprotein found in mouse zonae pellucidae [Bleil, J. D., and Wassarman, P. M. (1980). Develop. Biol., 76, 185–202] was analyzed by gel electrophoresis under a variety of conditions in order to determine whether or not it undergoes modification as a result of fertilization. Under nonreducing conditions, ZP2 present in solubilized zonae pellucidae that were isolated individually from mouse oocytes, eggs, and embryos migrates on SDS-polyacrylamide gels with an apparent molecular weight of 120,000. However, under reducing conditions, ZP2 from embryos, but not from oocytes or unfertilized eggs, migrates with an apparent molecular weight of 90,000 and has been designated ZP2_f. The evidence presented suggests that modification of ZP2 following fertilization involves proteolysis of the glycoprotein, but that intramolecular disulfide bonds prevent the release of peptide fragments. It is shown that the same change in ZP2 can be generated in vitro by artificial activation of unfertilized mouse eggs with the calcium ionophore A23187, thus eliminating the possibility that a sperm component is responsible for the modification of ZP2 following fertilization. These results suggest that some of the changes in the biochemical and biological properties of zonae pellucidae, observed following fertilization or activation of mouse eggs, result from modification of the major zona pellucida glycoprotein, ZP2.     

Subnormal zona pellucida change in parthenogenetic mouse embryos

Parthenogenetic mouse embryos were obtained by the method of electrical stimulation of eggs in vivo(Tarkowski et al., 1970), and their developmental retardation and limited viability were confirmed. Very early deviations from normalcy seemed likely in these embryos, and we chose to investigate their “zona reaction,” as this is one of the earliest events identified (Braden, et al., 1954) in normal fertilization. The change, ordinarily triggered by sperm penetration of the egg, decreases the permeability of the zona pellucida to supernumerary sperms, and has been attributed (Austin and Braden, 1956) to products released by cortical granules.An indirect assay for the state of the zona pellucida is presented. It is based on the observation (Mintz, 1970) that pronase, other proteolytic enzymes, and the normal uterine zonalytic factor lyse zonas of fertilized eggs more slowly than those of unfertilized eggs. Comparative zona lysis times in pronase are thus employed as a test for the degree of zona change after parthenogenetic activation relative to that after activation by sperm.The zonas of parthenogenetic embryos in stages ranging from 2 to 14 cells varied in their lysis times in pronase and overlapped with those of unfertilized and fertilized egg zonas. As a population, the zonas of the parthenogenones had intermediate lysis times. Thus, in the strains tested, electrical shock evokes only a partial zona reaction and, in this respect, is not an adequate substitute for sperm penetration.A working hypothesis for future testing is that the subnormal zona change and retarded development may both be due to inadequate release of products from cortical granules, under these conditions of artificial activation of the mouse egg.     

Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay

The heads of mouse spermatozoa obtained 5 min after release from the excised caudae epididymides showed a characteristic fluorescence pattern in the presence of the fluorophore chlortetracycline (CTC). There was uniform fluorescence over the entire head with about half the sperm population showing a brighter line of fluorescence across the equatorial segment; this fluorescence pattern was designated “F.” After 90-min incubation in culture medium (CM) containing 2% (w/v) bovine serum albumin, most of the sperm heads showed a dark band of nonfluorescence over the equatorial and postequatorial segment, while the anterior portion of the head showed bright fluorescence. This fluorescence pattern was designated “B.” The time course for the disappearance of pattern F matched the time course of the appearance of pattern B, with a half-time of 30 min. The transformation was complete in 90 min. At longer times of incubation in CM, the percentage of spermatozoa showing pattern B declined; fluorescence over the entire head was lost, characteristic of the pattern for acrosome-reacted sperm (P. M. Saling and B. T. Storey (1979). J. Cell Biol.83, 544–555). Mouse sperm showing pattern B were able to undergo the acrosome reaction, either spontaneously or by induction with acid-solubilized zonae pellucidae from mouse eggs (H. M. Florman and B. T. Storey (1982). Dev. Biol.91, 121–130). The latter reaction was blocked by its specific inhibitor 3-quinuclidinyl benzilate (QNB). Mouse sperm showing pattern F could not be induced to undergo the acrosome reaction by exposure to solubilized zonae. This implies that the change from fluorescence pattern F to fluorescence pattern B corresponds with changes in the sperm which make them susceptible to undergo the acrosome reaction. This change occurs during the time interval previously determined to be needed for capacitation of mouse sperm in vitro in CM (M. Inoue and D. P. Wolf (1975). Biol. Reprod.13, 340–346). These results imply that spermatozoa showing CTC fluorescence pattern B can be considered to be capacitated and that a functional definition for capacitation is the acquired ability to undergo the acrosome reaction rapidly when treated with acid-solubilized zonae pellucidae. The CTC fluorescence assay provides for the first time a means to monitor the time course of epididymal mouse sperm capacitation in vitro.     

Program of early development in the mammal: Synthesis and intracellular migration of histone H4 during oogenesis in the mouse

Synthesis of histone H4 by mouse oocytes and unfertilized eggs has been examined by using a modified high-resolution two-dimensional gel electrophoresis procedure capable of resolving basic proteins (M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol.73, 103–119). Histones were separated on such gels and observed rates of incorporation of [³⁵S]methionine into histone H4 were converted into absolute rates of synthesis by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and unfertilized eggs Schultz et al., 1979a, Schultz et al., 1979b. Histone H4 was synthesized at all stages of oogenesis examined, and accounted for 0.07, 0.05, and 0.04% of total protein synthesis in growing oocytes, fully grown oocytes, and unfertilized eggs, respectively. During oocyte maturation the absolute rate of histone H4 synthesis decreased by about 40%, as compared to a 23% decrease in the rate of total protein synthesis during the same period. These measurements indicate that enough histone is synthesized during oogenesis in the mouse to support two to three cell divisions. Examination of the intracellular location of newly synthesized proteins in fully grown oocytes revealed that histone H4 was highly concentrated in the nucleus (germinal vesicle), whereas total protein and tubulin were not. Nearly 50% of the histone H4 synthesized during a 5-hr period was located in the oocyte's germinal vesicle, as compared to 1.9 and 0.9% for total protein and tubulin, respectively. These results are compared with those obtained using oocytes and eggs from nonmammalian animal species.     

An approach to a physico-chemical model of olfactory stimulation in vertebrates by single compounds

During recent years, numerous attempts have been made to correlate both quantitative (Davies &; Taylor, 1959; Engen, 1962; Beck, 1964; Engen, Cain &; Rovee, 1968; Cain, 1969; Dravnieks &; Laffoit, 1970; Laffort, 1969a,b) and qualitative (Davies, 1965; Amoore &; Venstrom, 1965; Döving, 1966a,b; Wright &; Michels, 1964; Leveteau &; MacLeod, 1969) odorous properties of single compounds to their molecular properties. These attempts have been only partially successful.In the present paper we will try to explain the several odorous properties of single compounds on the basis of the non-specific properties of odorants involved in solubility.This model is a first approach, and although it gives statistically highly significant relations, it is not as accurate as those advanced with respect to the physical and sensory dimensions of stimuli in the fields of vision and audition.We will first give the present definitions of the most suitable physicochemical parameters, and then advance quantitative and qualitative models for single compounds. Quantitative odorous properties are: odour threshold, rate of change of odour intensity with odorant concentration in the suprathreshold region, and the somewhat controversial upper odour intensity. Qualitative properties refer to odour character.     

Enzymatic dissection of the functions of the mouse egg's receptor for sperm

During the course of sperm-egg interaction in mice, zona pellucida glycoprotein ZP3 (approximately equal to 80 kDa) serves as both receptor for sperm (J. D. Bleil and P. M. Wassarman, 1980c, Cell 20, 873-882) and inducer of the acrosome reaction (J. D. Bleil and P. M. Wassarman, 1983, Dev. Biol. 95, 317-324). In this investigation, small ZP3 glycopeptides (approximately equal to 1.5-6 kDa), obtained by extensive digestion of the purified glycoprotein with insoluble Pronase, were assayed for both sperm receptor and acrosome reaction-inducing activities. While ZP3 glycopeptides were virtually as effective as intact ZP3 in inhibiting binding of sperm to eggs in vitro ("receptor activity"), unlike intact ZP3, they failed to induce sperm to undergo the acrosome reaction. The latter was determined by indirect immunofluorescence using a monoclonal antibody directed against the acrosomal cap region of sperm. These results suggest that the sperm receptor activity of ZP3 is dependent only on its carbohydrate components, whereas acrosome reaction-inducing activity is dependent on the polypeptide chain of ZP3 as well.     

The role of migration in the genetic structure of populations in temporally and spatially varying environments II. Island Models

The Island Model introduced by Sewall Wright (1951) has proven to be a useful construction for studying the interaction of genetic drift, population subdivision, and mutation. Interest in the model has recently increased because of its relevance to certain questions involving the rate of differentiation of sub-populations under the neutral allele hypothesis (e.g., Smith, 1970; Latter, 1973). It is perhaps the only realistic population structure in which the test for neutrality proposed by Lewontin and Krakauer (1973) is valid (Lewontin and Krakauer, 1975). If data from natural populations is to be compared to the predictions of the Island Model, it is desirable to have an alternative model with the same migration pattern but with natural selection operating. In this paper one such model will be introduced where the stochastic element comes from random fluctuations in the environment rather than from genetic drift. The model is a direct extension of the one in the previous paper in this series (Gillespie, 1975) which dealt with a population which is subdivided into two patches with restricted migration between them.     

Does a functional relationship exist between the polymerization and catalytic activity of beef liver glutamate dehydrogenase?

The recent work of Cohen &; Benedek (1976) and Cohen et al. (1975, 1976) on the apparent interdependence of beef liver glutamate dohydrogenase catalytic activity and degree of polymerization is examined in the light of previously published equilibrium and kinetic results. It is shown that some of the hypotheses central to the Cohen &; Benedek (1976) model are in contradiction with existent data. Consideration of all available information leads to the conclusion that effector-induced depolymerization may simply be an incidental side reaction in the events leading to inhibition.     

Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptors with human homologs

The zona pellucida surrounding ovulated mouse eggs contains three glycoproteins, two of which (ZP2 and ZP3) are reported sperm receptors. After fertilization, the zona pellucida is modified ad minimus by cleavage of ZP2, and sperm no longer bind. Crosstaxa sperm binding is limited among mammals, and human sperm do not bind to mouse eggs. Using transgenesis to replace mouse ZP2 and/or ZP3 with human homologs, mouse lines with human-mouse chimeric zonae pellucidae have been established. Unexpectedly, mouse, but not human, sperm bind to huZP2 and huZP2/huZP3 rescue eggs, eggs fertilized in vitro with mouse sperm progress to two-cell embryos, and rescue mice are fertile. Also unanticipated, human ZP2 remains uncleaved after fertilization, and mouse sperm continue to bind early rescue embryos. These observations are consistent with a model in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

Kinship and covariance

Price's (1970) covariance theorem can be used to derive an expression for gene frequency change in kin selection models in which the fitness effect of an act is independent of the genotype of the recipient. This expression defines a coefficient of relatedness which subsumes r(Wright, 1922), b(Hamilton, 1972), ρ (Orlove &; Wood, 1978), and R(Michod &; Hamilton, 1980). The new coefficient extends the domain of Hamilton's rule to models in which the average gene frequency of actors differs from that of recipients.     

The fine structure of Ameson pulvis (Microspora,Microsporida) and its implications regarding classification and chromosome cycle

Nosema pulvisPerez, 1905, Ameson pulvis (Perez) Sprague, 1977, in muscles of the crabs Carcinus maenas and C. mediterraneus from the coast of France, was observed with the electron microscope. It was found to be structurally similar to the type species A. michaelis (Sprague, 1970). Sprague, 1977, having moniliform sporogonial plasmodia, unikaryotic sporoblasts, and hirsute sporulation stages. It is treated as distinct from A. michaelis because it has slightly smaller spores (by comparison with syntype material of A. michaelis) and appears to have fewer coils in the polar filament. The results require the removal of the genus Ameson from the family Nosematidae Labbé, 1899, where Sprague (1977) had placed it under the erroneous supposition that its sporoblasts are diplokaryotic. Ameson is transferred to family Unikaryonidae Sprague, 1977. Ameson is distinguished from PereziaLéger and Duboscq, 1909, shown by Ormieres et al. to have a similar developmental pattern, by presence of appendages on its sporulation stage. A. nelsoni (Sprague, 1950), the third, and only other species of Ameson, lacks the appendages and is transferred to genus Perezia.     

Theory of radiation dose-survival curves for the case where organism lethality is defined as loss of reproductive capacity. II. Application to cells and physical interpretation

The general theory of survival curves (Craig, 1971) is applied to the case of cells and sub-cellular organisms with a physical interpretation via gene or chromosome damage as the terminal lesion.It is indicated how the proposed terminal lesion is consistent with the salient features of cellular response to radiation and analytical expressions for reactivity and sensitivity in terms of a damage, or mutation, cross section are obtained.The probability of a complex cross section and conditions under which it reduces to simple approximations are discussed and the influence of various factors on the cross section are indicated.Acceptable fits are obtained to the data of Barendsen, Beusker, Vergroesen &; Budke (1960), McCulloch &; Till (1962) and Puck &; Marcus (1956) with simple forms of cross section.     

Box-Jenkins forecasting in ecology: An answer to poole

This answering of Poole, 1978, Poole, 1976 aims at rounding off our exchange of views, without losing the readership from an excess of toing and froing between the four contributions. So my final rejoinder only attempts at treating the general points raised by Poole (1978), rather than taking issue with all the minutiae, which would require too many quotes of quotes and counterquotes. The main nub of contention remains as to whether or not statistical fits can be meaningfully interpreted biologically.     

Human sperm do not bind to rat zonae pellucidae despite the presence of four homologous glycoproteins

The specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.     

Warfare and hominid brain evolution.

This paper reviews literature on the evolutionary effects of warfare upon the hominid brain. Alexander &; Tinkle (1968) and Bigelow (1969) are found to be the first to propose that warfare was the principle evolutionary pressure that created the novel substance of the human brain, and that it acted at least from the early Pleistocene. These writers are distinguished from Darwin (1871), Keith (1947) and Wilson (1975) who saw warfare influencing the development of the brain only in historical or near-historical times.The warfare hypothesis of Alexander &; Tinkle is found to be an excellent explanation of the evolution of the human brain, but to be unsatisfactory from a biological viewpoint because they do not explain how warfare evolved in the first place, nor do they attempt to account for the apparent absence of warfare as a behavioral adaptation in species other than some eusocial insects.This author underpins the warfare hypothesis, arguing that it evolved as a necessary consequence of the circumstances of early hominids. Proficient tool use gave domination over predators and opened up new food resources, thereby diminishing two population controls. A population explosion resulted and, at critical densities, when starvation threatened, warfare was the genetically most successful behavioral adaptation. Alternative hypotheses are shown to be inadequate. Finally, the author asks why such an important hypothesis has been ignored for almost a decade.     

An autoradiographic analysis of the time of appearance of neurons in the developing chick neural retina

The pattern of incorporation of [³H]thymidine into the chick neural retina has been used to establish the time and order in which different classes of neuroepithelial cells withdraw from the cell cycle and initiate migration and differentiation.The posterior pole of the retina is the first to form during development. In this region most neuroepithelial cells complete mitotic activity between the third and sixth day of incubation. Presumptive ganglion cells initiate the withdrawal process, and they are soon followed by the neuroepithelial precursors of amacrine, horizontal, and receptor cells. Bipolar cell precursors are the last to begin and the last to complete cell cycle activity. It is worthy of note, however, that, in any given region of the retina, neuroepithelial cells of all types cease mitosis in close, overlapping succession.These results are in reasonable agreement with those previously published on the chick retina by Fujita and Horii (1963), and other investigators on the mouse (Mus), killifish (Fundulus), and toad (Xenopus). The present data are also consistent with those proposals of Angevine (1970), Jacobson, 1968a, Jacobson, 1968b, Jacobson, 1970, and others that relate the cessation of mitotic activity of neuroepithelial cells to the determination of neuronal size, axon length, and the specification of neuronal connections.     

Dinucleotide fold proteins. Interaction of arabinose binding protein with Cibacron Blue 3G-A.

Cibacron Blue 3G-A, the dye moiety of Blue dextran-Sepharose, has been shown to not specifically bind a protein with a dinucleotide fold-like supersecondary structure, L-arabinose binding protein from Escherichia coli. This shows that Cibacron Blue 3G-A is not suitable as a definitive probe for the dinucleotide fold as suggested earlier (Thompson et al., 1975; Stellwagen, 1977). An explanation for the large predominance of proteins containing this protein supersecondary structure that bind to this dye is presented.