Lymphocyte subsets involved in tumor-activated nonspecific feedback suppression

Cells from the spleen, lymph nodes, and peritoneum of DBA/2 mice bearing a subcutaneous tumor mediate nonspecific suppression of an in vitro antibody response to sheep red blood cells (SRBC) when cocultured with a normal T-cell subset(s). The spleen cells from the tumor-bearing mouse required for the suppression bear the Lyt 1 and Ala 1 surface markers characteristic of "inducer" T cells and activated cells, respectively. The activity of this cell population is also sensitive to irradiation. The normal T-cell subset which cooperates in the suppression bears the Qa-1 surface antigen which has been associated with suppressor cell precursors in several systems but lacks detectable surface Lyt 1 and 2 markers. Suppression of antibody responses in spleen cell cultures from tumor-bearing mice alone could also be elicited, but only when increased numbers of cells were cultured. These data are consistent with the theory that a tumor-activated, Lyt 1+ T-cell subset has the capacity to nonspecifically suppress immune responses by activating a Qa-1+ subset(s) of T suppressor cells, perhaps via feedback signals.     

On lysosomal fragility and induction of liver hexose-monophosphate dehydrogenases in the fasted-refed rat.

Young adult male rats were fasted for 3 days, then fed a glucose-rich diet, ad libitum. At the end of the fasting period, the specific activity of liver glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was decreased to 60% of control (nonfasted) levels. After 24 to 72 h of refeeding, the specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increased seven- and twofold, respectively. During the fasting period, the liver lysosome fragility increased, as judged by increased release of bound acid phosphatase and β-N-acetylglucosammidase activity during standard homogenization. Three hours after feeding a carbohydrate-rich diet, a further increase in liver lysosomal fragility was observed that returned to control values prior to the induction of the dehydrogenases. Similarly, the susceptibility of liver lysosomes from fasted rats to increased fragility by the intraperitoneal injection of glucose or galactose was also observed. Prior starvation was not a requisite for labilization of lysosomal membranes by injected glucose, but induction of the pentose phosphate shunt dehydrogenase was not observed.In a group of 6-week old male rats fed a commercial pellet diet throughout, the injection of insulin caused no change in liver lysosomal fragility, though hypoglycemia resulted. Similar animals made diabetic by treatment with Streptozotocin and diabetic rats given insulin, showed no change in liver lysosmal fragility based on the percentage of free to total activities of β-N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, and Cathespin D. However, when adult female rats were fasted for 24 h, then injected with sufficient insulin to produce hypoglycemia, liver lysosomal fragility, based on the release of β-N-acetylglucosaminidase during homogenization, increased nearly threefold. These studies demonstrate that stimulated lysosomal fragility can be initiated by refeeding fasted animals a carbohydrate-rich diet, by intraperitoneal injections of fasted rats with glucose or galactose, or by administering insulin alone to fasted rats. However, hyperglycemia induced by diabetogenic doses of Streptozotocin, or hypoglycemia induced in well-fed animals by insulin injection failed to elicit an enhanced liver lysosomal fragility. Whether induction of the enzymes of lipogenesis by rat liver is dependent upon a prior lysosomal membrane labilization remains to be determined.     

Studies on the mechanisms of induction of N-nitrosodimethylamine demethylase by fasting,acetone, and ethanol

Previous work has shown that induction of a high-affinity NADPH-dependent nitrosodimethylamine demethylase (NDMAd) in liver microsomes occurs in rats due to fasting, ethanol consumption, and streptozotocin-induced diabetes. Several lines of observations suggest that this is due to the induction of specific cytochrome P-450 isozymes. Induction of P-450 species by ethanol has also been observed by other investigators. Since each of the above altered metabolic states has in common elevated levels of ketone bodies, the possible role of acetone, a known inducer of NDMAd, in the induction of the demethylase activity was investigated. Levels of endogenous acetone in fasted rats correlated (r = 0.72) with a three- to fourfold increase in NDMAd activity. However, a dose-response experiment showed endogenous levels of acetone to be capable of causing at most 40% of the induction in fasted rats. This suggests that other ketone bodies or factors may have contributed to the induction. The induction of NDMAd by ethanol was enhanced by alcohol dehydrogenase inhibitors pyrazole and acetaldehyde oxime, suggesting that ethanol, rather than its metabolites, was responsible for the induction.     

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [³⁵S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.     

Enzyme induction by daunorubicin in neonatal heart cells in culture

The effect of the antineoplastic agent daunorubicin on beating heart cells from neonatal rats was investigated with respect to cell damage and induction of enzymes possibly involved in drug metabolism. Of the enzymes assayed DT-diaphorase and glutathione-S-transferase showed a two-to-four fold increase in activity: higher concentrations of daunorubicin inactivated glutathione-S-transferase. Daunorubicin toxicity increased in the presence of dicoumarol, a specific inhibitor of DT-diaphorase. These results indicate that both DT-diaphorase and glutathione-S-transferase may be involved in the metabolism of daunorubicin.     

The induction of forward gene mutation and gene conversion in yeasts by treatment with cyclophosphamide in vitro and in vivo

The present paper assesses the most suitable conditions for metabolic activation with yeasts in vitro, at least as far as cyclophosphamide (Cy) is concerned. These include treatment time, incubation temperature, the amounts of S9 and cofactors. Particular attention is devoted to the use of various solvents, showing that their use can considerably affect the mutagenic response of the chemical being tested. It also examines the effects of enzyme inducers (by using S9 from rats and mice) such as phenobarbital (PB) and 5,6-benzoflavone (BF) administered separately or together. The metabolizing capability of other organs such as the lungs and kidneys is also determined. All these data are compared with Cy genotoxicity (in vivo) evaluated by the intrasanguineous host-mediated assay and by recovering the yeast target cells from the liver, lungs and kidneys. The most striking effects are that, in vitro, PB greatly enhances Cy genotoxicity, whilst in vivo it substantially reduces it.     

Coordinate induction of multiple mRNAs specific for rat liver phenobarbital-inducible cytochromes P-450

Utilizing two-dimensional gel electrophoresis, the polypeptide composition of a purified microsomal cytochrome P-450 preparation isolated from phenobarbital-treated Long-Evans rats obtained from Charles River Laboratories has been examined. The purified protein consists of three polypeptides with nearly identical subunit molecular weights (approximately 52,000) but differing in net charge. These three polypeptides can be detected in liver microsomes isolated from phenobarbital-treated rats by immunoblot analysis but are virtually absent in microsomes isolated from untreated rats. All three polypeptides appear to be products of distinct mRNAs since they can be immunoprecipitated from rabbit reticulocyte lysates programmed with poly(A+)-RNA isolated from phenobarbital-treated rats. The amount of functional mRNA specific for the P-450 polypeptides increases dramatically in response to an acute administration of phenobarbital; however, in untreated rats the amount of functional mRNA was below the level of detection by the translational assay. These data are consistent with the very low level of the phenobarbital-inducible cytochromes P-450 in liver microsomes isolated from untreated rats. Finally, the data indicate that all three cytochrome P-450 mRNAs increase rapidly in response to phenobarbital administration and are regulated coordinately.     

Silver staining test of nucleolar suppression in the Lilium hybrid ‘Black Beauty’

Nucleolar organizing regions in mitotic chromosomes from Lilium longiflorum, L. henryi and L. speciosum, have been silver banded. Nucleoli in interphase cells were also silver stained. Silver banding and nucleolar counting in Lilium cv ‘Black Beauty’, a hybrid between L. speciosum and L. henryi, indicate that nucleolar suppression is not occurring.     

Glucose requirement for induction by sodium butyrate of the glycoprotein hormone alpha subunit in HeLa cells

Butyric acid produces multiple effects on mammalian cells in culture, including alterations in morphology, depression of growth rate, increased histone acetylation, and modified production of various proteins and enzymes. The latter effect is exemplified by the induction in HeLa cells of the glycoprotein hormone alpha subunit by millimolar concentrations of the fatty acid. This report demonstrates that increased subunit accumulation in response to sodium butyrate is strikingly dependent on the presence of glucose (or mannose) in the growth medium. In contrast, basal levels of subunit synthesis are only marginally affected when the culture medium is supplemented with one of a variety of hexoses. An increase in the accumulation of HeLa alpha does not occur in medium containing pyruvate as the energy source, and sustained induction requires the simultaneous and continued presence of both glucose and butyrate. The effects of butyrate on HeLa cell morphology and subunit induction can be separated, since the latter is glucose-dependent while the former is not. Failure of butyrate to induce alpha in medium containing pyruvate does not result from restricted subunit secretion, since the levels of intracellular alpha are not increased disproportionately relative to those in the medium. The hexoses which support induction of HeLa alpha (glucose greater than or equal to mannose greater than galactose greater than fructose) are identical to those which have been shown previously to stimulate the glucosylation of lipid-linked oligosaccharides and enhance the synthesis of certain glycoproteins. Labeling of various glycosylation intermediates with [3H]mannose indicates that in glucose medium there is a decrease in the level of radioactivity associated with both dolicholpyrophosphoryl oligosaccharide and cellular glycoproteins and a concomitant increase in the fraction of label recovered in secreted glycoproteins. Butyrate also causes a decrease in [3H]mannose-labeled cellular glycoproteins and an increase in tritiated extracellular glycoproteins, particularly in glucose medium. Likewise, glucose stimulates the incorporation of [3H]glucosamine into immunoprecipitable alpha subunit relative to the bulk of HeLa-secreted glycoproteins, and this is further enhanced by butyrate. However, as demonstrated by lectin chromatography of conditioned media, a nonglycosylated subunit does not accumulate in pyruvate medium, either in the absence or presence of butyrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of cobalt chloride and 3-amino-1,2,4-triazole on the induction of cytochrome P-450 synthesis by phenobarbitone in rat liver

Administration of cobalt chloride and 3-amino-1,2,4-triazole leads to a suppression of phenobarbitone-mediated increase in total cytochrome P-450 as well as cytochrome P-450b contents of the liver. This suppression is due to a decrease in the content of the protein species which is the result of a decrease in its rate of synthesis as measured in vivo and in vitro. Cobalt chloride as well as 3-amino-1,2,4-triazole treatments lead to a decrease in the translatability of cytochrome P-450b RNA without affecting total protein synthesis. It is a possibility that a small pool of heme regulates the RNA levels for the cytochrome P-450 species.     

Inhibition of the UV induction of sister-chromatid exchanges in ICR-2A frog cells by pretreatment with gamma-rays

Nuclear toxicity of several known carcinogens and fecal fractions obtained from 10 healthy individuals was investigated in the colonic nuclear aberration (NA) assay using an intrarectal administration. Two known colon carcinogens, MNNG and DMAB, and a carcinogen of organs other than the colon, B(a)P, induced NA in a dose-related manner. Chromatographic fractions of feces from 10 donors were tested for their ability to produce NA. The dichloromethane fraction for several was active and yielded a significantly positive response which was dose-related. Our study demonstrated that the feces of some healthy individuals contain compound(s) which damage colonic nuclei in a similar manner to that seen with some known carcinogens.     

The effects of insulin and glucose on the induction and intracellular translocation of delta-aminolevulinic acid synthase

The administration of insulin and glucose to young Sprague-Dawley rats (125-150 g) resulted in changes in the intracellular distribution and in the turnover rates of delta-aminolevulinic acid synthase (ALAS) activity in the mitochondria and the cytosol. When starved, allylisopropylacetamide (AIA)-induced rats were injected with either insulin or glucose, the percentage of the total ALAS activity found in the cytosol increased from 27% in control animals to 33-40% in insulin-treated and 50% in glucose-treated rats. Similar increases of the ALAS activity in the cytosol were observed after insulin treatment of noninduced, starved animals. Glucose administration also repressed 25-40% of the AIA induction of ALAS as previously reported; however, this effect apparently was not a result of elevated insulin levels, since there was no observed repression of AIA induction after insulin administration. The effects of insulin and glucose on the turnover rates of ALAS activity in the mitochondria and in the cytosol were investigated by observing changes in the half-lives of ALAS activity in the two intracellular compartments. Administration of both insulin and glucose resulted in an increased half-life of ALAS activity in the cytosol from 20.8 to over 100 min, while the mitochondrial half-life was not significantly changed. When insulin was given to either fed, AIA-induced or to starved, noninduced rats, the half-life of the cytosolic ALAS increased from about 14 to 40 min. In contrast to the starved, induced animals, the mitochondrial ALAS half-life in starved, noninduced animals decreased 50%. These results suggest that insulin and glucose treatment may inhibit the translocation of ALAS from the cytosol into the mitochondrial matrix.     

The similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicate that the alkaloid is the principal toxin responsible for the induction of locoism

A neurological condition resembling that observed in hereditary mannosidosis occurs in animals ingesting spotted locoweed and plants of the genus Swainsona. Swainsonine has been isolated from these plants and has been suggested to be the primary causative agent in inducing the pathological condition. This alkaloid has also been found to increase tissue acid alpha-D-mannosidase levels in rats while lowering liver Golgi mannosidase II levels. In the present study, the effects of locoweed and swainsonine were directly compared for the first time, with the pig as experimental animal. Both increased most lysosomal acid glycosidase activities in most tissues, decreased liver Golgi mannosidase II levels, increased plasma hydrolase levels, and greatly increased tissue oligosaccharide, especially Man5GlcNAc2 and Man4GlcNAc2. These results indicate that swainsonine is the agent in locoweed responsible for the enzymatic and oligosaccharide changes. The behavior of the animals was also similarly affected by swainsonine and locoweed.     

Effect of albumin on binding and recovery of enzymes in affinity chromatography on Cibacron Blue

Bovine serum albumin appears to improve the specificity of Cibacron Blue F3GA in affinity chromatography of enzymes which interact with nucleotides. The action of bovine serum albumin may rest in its ability to selectively mask affinity sites in the dye, which are not specific for the nucleotide-binding region of the enzyme, while not seriously impairing binding nor its elution by nucleotides. Thus, the elution of Chlorella nitrate reductase from a Blue Sepharose chromatographic column by its coenzyme, NADH, fails, unless the column is first treated with bovine serum albumin. Such treatment also improves the recovery of some other nucleotide-binding enzymes tested.     

Phosphodiesterase induction in Dictyostelium discoideum by inhibition of extracellular phosphodiesterase activity

Adenosine 3′,5′-monophosphate (cAMP) is a chemoattractant in Dictyostelium discoideum; it also induces phosphodiesterase activity. Recently it was shown (M. H. Juliani, J. Brusca, and C. Klein, (1981)Develop. Biol.83, 114–121) that N⁶-(aminohexyl)adenosine 3′,5′-monophosphate (hexyl-cAMP) effectively induced phosphodiesterase activity, while this compound was chemotactically inactive and did not effectively bind to the cell surface receptor for cAMP. It was suggested that hexyl-cAMP and cAMP induce phosphodiesterase activity via a chemoreceptor-independent mechanism. In another recent report (P. J. M. Van Haastert, R. C. Van der Meer, and T. M. Konijn (1981)J. Bacteriol.147, 170–175) investigation of induction of phosphodiesterase by several cAMP derivatives revealed that phosphodiesterase induction and chemotaxis had similar cyclic nucleotide specificity. Based on this result it was suggested that cAMP induces phosphodiesterase activity via activation of the chemotactic receptor. In this report we show that hexyl-cAMP transiently inhibits extracellular and cell surface phosphodiesterase. This transient inhibition of the inactivating enzyme and the permanent release of small amounts of cAMP by the cells leads to a transient increase of extracellular cAMP levels. Hexyl-cAMP does not inhibit beef heart phosphodiesterase, and is not degraded by this enzyme. Addition of hexyl-cAMP to a cell suspension containing beef heart phosphodiesterase does not result in an accumulation of extracellular cAMP, and phosphodiesterase induction is absent. We conclude that hexyl-cAMP inhibits phosphodiesterase activity which leads to the accumulation of cAMP; consequently cAMP binds to the chemotactic cAMP receptor resulting in the induction of phosphodiesterase activity.     

Colchicine inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase in cultured avian liver explants

Chicken embryo liver explants cultured in chemically defined medium in the absence of serum provide a unique system to probe into the mechanism of insulin induction of lipogenic enzymes. Colchicine at concentrations of 0.2 and 1 microM in the culture medium caused inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase by 50 and 90%, respectively. As measured by immunochemical techniques, the inhibition of the induction of these two enzyme systems resulted from the decreased content of the delta 9-terminal desaturase component of the stearoyl-CoA desaturase and the fatty acid synthetase. Colchicine, however, had no effect on the general protein synthesis, nor did it affect the malic enzyme, which is induced by triiodothyronine but not by insulin. Also, colchicine had no influence on the binding of 125I-insulin to isolated plasma membrane. Pretreatment of liver explants with insulin for 0.5-1 h and subsequent incubation in insulin-free media for 48 h resulted in induction of the desaturase and fatty acid synthetase. However, inclusion of colchicine in the media for 3 h subsequent to the treatment with insulin completely abolished the inductive effect of insulin, suggesting that colchicine affects events occurring subsequent to insulin binding to the cell surface membranes. Since lumicolchicine, an inactive isomer of colchicine, had no effect on insulin action, it is suggested that the inhibition of insulin induction of the desaturase and synthetase is related to the depolymerizing action of colchicine. Therefore, in eliciting long-term responses to insulin, microtubular integrity of the cell may be required for the transfer of a putative from cell surface insulin receptor to intracellular sites.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Inhibitors of prostaglandin synthesis block the induction of staphylococcal enterotoxin B-activated T-suppressor cells

A variety of arachidonic acid metabolites possess the ability to modulate immune cell function. Various inhibitors of arachidonic acid metabolism were compared with regard to their effects on T-suppressor (Ts) cell function. Using staphylococcal enterotoxin B (SEB) to activate Lyt-2+ Ts cells, it was shown that indomethacin and 5,8,11,14-eicosatetraynoic acid (ETYA) inhibit the induction phase, but not the expression phase, of suppressor cell activity. Agents which inhibit thromboxane synthetase or lipoxygenase activities (imidazole, nordihydroguaiaretic acid, and pyrogallol) were not found to affect Ts cell induction. Since inhibitors of prostaglandin synthesis are thought to induce lower levels of cyclic adenosine monophosphate, an attempt to overcome the indomethacin inhibition of Ts cell induction by modulating cyclic adenosine monophosphate levels was made. It was found that theophylline and isoproterenol are not able to overcome the inhibition by indomethacin of Ts cell activity. These results strongly suggest that induction of Ts cells by SEB is dependent on the synthesis of products of the prostaglandin synthetase pathway.     

Cellular events in tolerance IX: Maintenance of immunological tolerance in the presence of normal B-cell precursors and in the absence of demonstrable suppression

The cellular events involved in immunological tolerance to fluoresceinated sheep gammaglobulin (FL-SGG) were analyzed at the level of hapten-specific B cells. One single iv injection of FL-SGG induced tolerance as measured by challenge with thymus-dependent (FL-KLH) or thymus-independent (FL-Ficoll) antigens in vivo or thymus-independent (FL-LPS) antigen in vitro. As noted earlier, unresponsiveness was maintained until 6–8 weeks after tolerance induction. Limiting-dilution precursor analysis demonstrated a reduction in B-cell precursors on Day 7 after tolerogen treatment; precursor frequencies returned to control levels by 3–4 weeks. This recovery of precursors in the presence of stable tolerance was not due to suppressor activity. Rather, results show that tolerant hapten-specific B cells are clonally anergic and display a reduced burst size in response to antigen. Hence, unresponsiveness is maintained in the presence of apparently normal precursor levels by an intrinsic defect in antigen-specific B cells.     

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.