This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Proceedings of the National Academy of Sciences of the United States of America, Sep 1, 1988
The mouse sperm receptor, a glycoprotein called ZP3, is synthesized and secreted by growing oocyt... more The mouse sperm receptor, a glycoprotein called ZP3, is synthesized and secreted by growing oocytes. It is present in more than a billion copies in the unfertilized egg's extracellular coat, or zona pellucida. We have cloned and characterized a region of the mouse (CD-1) genome that spans 10 kilobases of the ZP3 locus. The genomic clones described encompass the entire ZP3 coding region, which contains eight exons. The exons were identified, mapped, and sequenced, yielding the entire primary structure of the ZP3 polypeptide chain (424 amino acids; Mr, 46,300), which includes a 22-amino acid signal sequence. In addition, sequencing of genomic clones has revealed some unusual features of ZP3 mRNA and a region just downstream of the ZP3 gene. All mammalian eggs are surrounded by a relatively thick extracellular coat, called the zona pellucida, that participates at several steps in the fertilization pathway (1, 2). The mouse egg zona pellucida is =7 zm thick, contains z3 ng of protein, and is composed of three glycoproteins, called ZP1 (Mr, =200,000), ZP2 (Mr, =120,000), and ZP3 (Mr, =83,000), that associate with one another through noncovalent interactions to form an insoluble network of crosslinked filaments (1-4). During fertilization of ovulated eggs, ZP3, one of the glycoproteins, serves as both sperm receptor and inducer of the acrosome reaction (3, 5-7). The sperm receptor activity of ZP3 is attributable solely to certain of its 0-linked oligosaccharides (3, 8), whereas the acrosome-reaction-inducing activity of ZP3 is attributable to its polypeptide chain as well (3, 9). Thus, ZP3 plays structural and functional roles in the zona pellucida. ZP1, ZP2, and ZP3 are synthesized and secreted exclusively by growing and fully grown mouse oocytes (3, 10-13). ZP3 is synthesized as a Mr 44,000 polypeptide chain to which either three or four high mannose.type, N-linked oligosaccharides are added cotranslationally, giving rise to precursors with molecular weights of 53,000 and 56,000, respectively (12). The oligosaccharides are then processed to complextype, presumably in the Golgi, and 0-linked oligosaccharides are added, giving rise to the mature, M, 83,000 form of ZP3 Abbreviation: nt, Nucleotide(s).
Contents. List of Contributors. Preface (P.M. Wassarman). The In Vivo Function of Mullerian-Inhib... more Contents. List of Contributors. Preface (P.M. Wassarman). The In Vivo Function of Mullerian-Inhibiting Substance During Mammalian Sexual Development (Y. Mishina and R.R. Behringer). The Role of the dpp-Group Genes in Dorsoventral Patterning of the Drosophila Embryo (C.Rushlow and S. Roth). The Terminal Gene Hierarchy of Drosophila and the Genetic Control of Tissue Specification and Morphogenesis (M.L.R. Yip and H.D. Lipshitz). Anterior-Posterior Polarization and Mesoderm Inducing Factors in the Pregastrula Mouse Embryo: Comparison to Chick and Frog Embryos (R.F. Bachvarova). Human Y Chromosome Function in Male Germ Cell Development (P.H. Vogt). Index.
Human In Vitro Fertilization: Origins and History. Human In Vitro Fertilization: Clinical Aspects... more Human In Vitro Fertilization: Origins and History. Human In Vitro Fertilization: Clinical Aspects. Human In Vitro Fertilization: Technical Advances. Human Oocyte and Early Embryo Developmental Potential: Extrinsic and Intrinsic Influences. Application of In Vitro Fertilization to Domestic Animals. Molecular Genetics of the Zona Pellucida: Implications for Immunologic Perturbations of Fertilization. Sperm Antigens and Immunologic Interference of Fertilization. Index.
Biochimica et Biophysica Acta (BBA) - Enzymology, 1969
I. Crystalline lobster-muscle glyceraldehyde-3-phosphate dehydrogenase (Dglyeeraldehyde-3-phospha... more I. Crystalline lobster-muscle glyceraldehyde-3-phosphate dehydrogenase (Dglyeeraldehyde-3-phosphate : NAD + oxidoreductase (phosphorylating), EC 1.2.I.I2) possesses 7.3-1-0.3 cysteine residues per mole of enzyme (14o ooo g), i.e. approx. 2 cysteine residues per polypeptide chain, which are extremely reactive toward organic mercurials ; 8 of the 20 cysteine residues of the enzyme are totally unreactive toward organic mercurials. 2. Enzyme carboxymethylated at each of the 4 'active site' cysteine residues possesses 7.4-+-0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate and cannot be distinguished from native enzyme on the basis of these titrations. 3. Enzyme treated with iodosobenzoic acid and containing an intramolecular disulphide bridge at the 'active center' possesses 4.0 _~ 0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate, i.e. approx. 4 less than native enzyme. 4. Under normal conditions, lobster-muscle glyceraldehyde-3-phosphate dehydrogenase is 90-95% inactivated concomitant with the formation of 8 moles of mercaptide per mole of enzyme (14o ooo g) ; inhibition takes place in a linear manner with respect to mercurial concentration. 5. In the presence of a large molar excess of NAD +, the formation of 4 moles of mercaptide per mole of enzyme (14o ooo g) has no inhibitory effect and must be attributed to reaction of a 'nonessential' sulphydryl group.
All eggs have an extracellular coat (EC) that plays unique roles during reproduction and developm... more All eggs have an extracellular coat (EC) that plays unique roles during reproduction and development. ECs are designed to protect eggs and support their growth, regulate fertilization of eggs, and protect early embryos. ECs of mammalian and nonmammalian eggs consist of only a few proteins that are closely related to one another. All these proteins possess regulatory elements and a structural domain responsible for processing and assembly of the proteins into ECs. They also possess regions responsible for their functional roles during and after fertilization. Our essay addresses these and other aspects of EC biology.
Advances in Experimental Medicine and Biology, 1979
The nature, intracellular distribution, and role of proteins synthesized during meiotic maturatio... more The nature, intracellular distribution, and role of proteins synthesized during meiotic maturation of mouse oocytes in vitro have been examined. Proteins synthesized during the initial stages of maturation are concentrated within the nucleus (germinal vesicle) and become intimately associated with the condensing chromosomes. Inhibition of protein synthesis during this period does not prevent germinal vesicle dissolution or chromosome condensation, but meiotic progression is blocked reversibly at the circular bivalent stage. A protein is synthesized during meiotic maturation of the mouse oocyte which exhibits several of the characteristics of the very lysine-rich histone, Fi; this and other histones are phosphorylated during the initial stages of maturation. These results are discussed in relation to studies of meiotic maturation of oocytes from non-mammalian species and chromosome condensation in both oocytes and mitotic cells.
Ciba Foundation Symposium 145 - Carbohydrate Recognition in Cellular Function, 2007
The mouse sperm receptor, called ZP3, is a glycoprotein (83,000 Mr) that consists of a 44,000 Mr ... more The mouse sperm receptor, called ZP3, is a glycoprotein (83,000 Mr) that consists of a 44,000 Mr polypeptide chain (402 amino acids), three or four N-linked oligosaccharides, and an undetermined number of O-linked oligosaccharides. There are more than 10(9) copies of ZP3 present throughout the mouse egg extracellular coat, or zona pellucida. As a prelude to fertilization, each acrosome-intact sperm binds in a relatively species-specific manner to tens-of-thousands of copies of ZP3 at the surface of the zona pellucida. Binding to ZP3 induces sperm to undergo the acrosome reaction (membrane fusion) and, consequently, enables them to penetrate through the zona pellucida and to reach, and then fuse with, egg plasma membrane (fertilization). Purified ZP3, as well as a specific class of ZP3-derived O-linked oligosaccharides (3900 Mr), exhibit sperm receptor activity in vitro. The oligosaccharides, which represent a relatively low percentage of total ZP3 O-linked oligosaccharides, account for the glycoprotein's sperm receptor activity in vitro (i.e., recognition and binding). Furthermore, either enzymic removal or modification of certain sugars that constitute these oligosaccharides results in destruction of sperm receptor activity. These and other findings strongly suggest that during mammalian fertilization carbohydrates play a fundamental role in species-specific sperm-egg interactions.
Publisher Summary This chapter describes zona pellucida (ZP), which is a thick extracellular coat... more Publisher Summary This chapter describes zona pellucida (ZP), which is a thick extracellular coat that surrounds the plasma membrane of all mammalian eggs. It highlights mouse egg ZP structure. Either three or four glycoproteins constitute the ZP of eggs from a wide variety of mammals. ZP is well organized with primary and secondary sperm receptors (two different ZP glycoproteins), arranged periodically along ZP filaments. During the process of fertilization in mammals, sperm must bind to and penetrate through the ZP to reach and fuse with egg plasma membrane. The ZP is responsible for both species specificity of sperm binding and establishment of a slow block to polyspermy following gamete fusion. The mouse egg ZP is a relatively thick translucent extracellular coat that contains ∼3–4 ng of protein and is extremely porous, permitting relatively large macromolecules and even small viruses to pass through it. The unfertilized mouse egg ZP is composed of three glycoproteins: ZP1, ZP2, and ZP3. These glycoproteins appear either as very broad bands following one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), or as a very complex set of spots following high resolution two-dimensional SDS–PAGE.
Society of Reproduction and Fertility supplement, 2007
For sperm to fertilize eggs, they must bind to and penetrate the zona pellucida (ZP) that surroun... more For sperm to fertilize eggs, they must bind to and penetrate the zona pellucida (ZP) that surrounds the plasma membrane of all mammalian eggs. The ZP first appears during oocyte growth and increases in thickness as oocytes increase in diameter. The ZP is an extracellular matrix composed of long, crosslinked filaments. In mice, three glycoproteins, called mZP1-3, are synthesised and secreted by growing oocytes and assembled into a thick (-6.5 microm) extracellular coat over a 2-3 week period. Recently, we identified several regions of nascent ZP glycoproteins that affect their secretion and incorporation into the ZP (assembly) by growing oocytes. Among these are the ZP domain, the consensus furin cleavage site (CFCS) and the C-terminal propeptide (CTP) with its transmembrane domain (TMD), external hydrophobic patch (EHP), charged patch (CP), conserved cysteine (Cys) residue, and short cytoplasmic tail (CT). Particularly important is the ZP domain, a approximately 260 amino acid regio...
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1976
DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endon... more DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.
New Approaches in Eukaryotic DNA Replication, 1983
Our objective has been to understand, at the molecular level, the replication of eukaryotic chrom... more Our objective has been to understand, at the molecular level, the replication of eukaryotic chromosomes as a prerequisite to investigating the regulation of chromosome replication and its relationship to cell proliferation. Chromosomes from eukaryotic cells, simian virus 40 and polyoma virus consist of a series of nucleosomes, each containing about 200bp of DNA coiled around an octamer consisting of two each of the four core histones that gives chromatin its stable repeating structure. Additional structure is provided by histone H1 and non-histone chromosomal proteins. However, chromatin is not homogenous. The chromatin structure of quiescent genes differs from that of active genes, and chromosomal regions differ in their higher-ordered structure. For example, chromatin can be euchromatic or heterochromatic, 10nm thick fibers or 30nm thick coils, folded into loops or not folded. Therefore, replication of eukaryotic chromosomes, in contrast to prokaryotic chromosomes, requires accurate duplication of the structure of both chromatin and DNA. Furthermore, both chromatin structure and DNA sequence are likely involved in controlling replication by modifying the interaction of proteins with DNA. For example, nonuniformly spaced clusters of replication origins are activated at different times during S phase, but the same origin is never activated twice, and some origins that are active in embryonic cells are not used in somatic cells9. Once a replicon is activated, replication proteins must be directed to replication forks and termination of replication must result in complete separation of sibling chromatids to allow normal mitosis.
Fertilization is the process by which sperm and eggs unite to form a zygote, the true beginning o... more Fertilization is the process by which sperm and eggs unite to form a zygote, the true beginning of a new individual. Among mammals, sperm first make contact with eggs at the surface of the egg extracellular coat, or zona pellucida (ZP). This contact can lead to species-specific binding of sperm to eggs via sperm receptors located in the ZP. Bound sperm then undergo changes that enable them to penetrate the ZP and fuse with egg plasma membrane. Fusion results in activation of eggs, and development of the organism ensues [1–5].
During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head... more During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.
Synthesis of histone H4 by mouse oocytes and unfertilized eggs has been examined by using a modif... more 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 (R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979, Develop. Biol. 68,341-359; 73,120-133). 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.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Proceedings of the National Academy of Sciences of the United States of America, Sep 1, 1988
The mouse sperm receptor, a glycoprotein called ZP3, is synthesized and secreted by growing oocyt... more The mouse sperm receptor, a glycoprotein called ZP3, is synthesized and secreted by growing oocytes. It is present in more than a billion copies in the unfertilized egg's extracellular coat, or zona pellucida. We have cloned and characterized a region of the mouse (CD-1) genome that spans 10 kilobases of the ZP3 locus. The genomic clones described encompass the entire ZP3 coding region, which contains eight exons. The exons were identified, mapped, and sequenced, yielding the entire primary structure of the ZP3 polypeptide chain (424 amino acids; Mr, 46,300), which includes a 22-amino acid signal sequence. In addition, sequencing of genomic clones has revealed some unusual features of ZP3 mRNA and a region just downstream of the ZP3 gene. All mammalian eggs are surrounded by a relatively thick extracellular coat, called the zona pellucida, that participates at several steps in the fertilization pathway (1, 2). The mouse egg zona pellucida is =7 zm thick, contains z3 ng of protein, and is composed of three glycoproteins, called ZP1 (Mr, =200,000), ZP2 (Mr, =120,000), and ZP3 (Mr, =83,000), that associate with one another through noncovalent interactions to form an insoluble network of crosslinked filaments (1-4). During fertilization of ovulated eggs, ZP3, one of the glycoproteins, serves as both sperm receptor and inducer of the acrosome reaction (3, 5-7). The sperm receptor activity of ZP3 is attributable solely to certain of its 0-linked oligosaccharides (3, 8), whereas the acrosome-reaction-inducing activity of ZP3 is attributable to its polypeptide chain as well (3, 9). Thus, ZP3 plays structural and functional roles in the zona pellucida. ZP1, ZP2, and ZP3 are synthesized and secreted exclusively by growing and fully grown mouse oocytes (3, 10-13). ZP3 is synthesized as a Mr 44,000 polypeptide chain to which either three or four high mannose.type, N-linked oligosaccharides are added cotranslationally, giving rise to precursors with molecular weights of 53,000 and 56,000, respectively (12). The oligosaccharides are then processed to complextype, presumably in the Golgi, and 0-linked oligosaccharides are added, giving rise to the mature, M, 83,000 form of ZP3 Abbreviation: nt, Nucleotide(s).
Contents. List of Contributors. Preface (P.M. Wassarman). The In Vivo Function of Mullerian-Inhib... more Contents. List of Contributors. Preface (P.M. Wassarman). The In Vivo Function of Mullerian-Inhibiting Substance During Mammalian Sexual Development (Y. Mishina and R.R. Behringer). The Role of the dpp-Group Genes in Dorsoventral Patterning of the Drosophila Embryo (C.Rushlow and S. Roth). The Terminal Gene Hierarchy of Drosophila and the Genetic Control of Tissue Specification and Morphogenesis (M.L.R. Yip and H.D. Lipshitz). Anterior-Posterior Polarization and Mesoderm Inducing Factors in the Pregastrula Mouse Embryo: Comparison to Chick and Frog Embryos (R.F. Bachvarova). Human Y Chromosome Function in Male Germ Cell Development (P.H. Vogt). Index.
Human In Vitro Fertilization: Origins and History. Human In Vitro Fertilization: Clinical Aspects... more Human In Vitro Fertilization: Origins and History. Human In Vitro Fertilization: Clinical Aspects. Human In Vitro Fertilization: Technical Advances. Human Oocyte and Early Embryo Developmental Potential: Extrinsic and Intrinsic Influences. Application of In Vitro Fertilization to Domestic Animals. Molecular Genetics of the Zona Pellucida: Implications for Immunologic Perturbations of Fertilization. Sperm Antigens and Immunologic Interference of Fertilization. Index.
Biochimica et Biophysica Acta (BBA) - Enzymology, 1969
I. Crystalline lobster-muscle glyceraldehyde-3-phosphate dehydrogenase (Dglyeeraldehyde-3-phospha... more I. Crystalline lobster-muscle glyceraldehyde-3-phosphate dehydrogenase (Dglyeeraldehyde-3-phosphate : NAD + oxidoreductase (phosphorylating), EC 1.2.I.I2) possesses 7.3-1-0.3 cysteine residues per mole of enzyme (14o ooo g), i.e. approx. 2 cysteine residues per polypeptide chain, which are extremely reactive toward organic mercurials ; 8 of the 20 cysteine residues of the enzyme are totally unreactive toward organic mercurials. 2. Enzyme carboxymethylated at each of the 4 'active site' cysteine residues possesses 7.4-+-0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate and cannot be distinguished from native enzyme on the basis of these titrations. 3. Enzyme treated with iodosobenzoic acid and containing an intramolecular disulphide bridge at the 'active center' possesses 4.0 _~ 0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate, i.e. approx. 4 less than native enzyme. 4. Under normal conditions, lobster-muscle glyceraldehyde-3-phosphate dehydrogenase is 90-95% inactivated concomitant with the formation of 8 moles of mercaptide per mole of enzyme (14o ooo g) ; inhibition takes place in a linear manner with respect to mercurial concentration. 5. In the presence of a large molar excess of NAD +, the formation of 4 moles of mercaptide per mole of enzyme (14o ooo g) has no inhibitory effect and must be attributed to reaction of a 'nonessential' sulphydryl group.
All eggs have an extracellular coat (EC) that plays unique roles during reproduction and developm... more All eggs have an extracellular coat (EC) that plays unique roles during reproduction and development. ECs are designed to protect eggs and support their growth, regulate fertilization of eggs, and protect early embryos. ECs of mammalian and nonmammalian eggs consist of only a few proteins that are closely related to one another. All these proteins possess regulatory elements and a structural domain responsible for processing and assembly of the proteins into ECs. They also possess regions responsible for their functional roles during and after fertilization. Our essay addresses these and other aspects of EC biology.
Advances in Experimental Medicine and Biology, 1979
The nature, intracellular distribution, and role of proteins synthesized during meiotic maturatio... more The nature, intracellular distribution, and role of proteins synthesized during meiotic maturation of mouse oocytes in vitro have been examined. Proteins synthesized during the initial stages of maturation are concentrated within the nucleus (germinal vesicle) and become intimately associated with the condensing chromosomes. Inhibition of protein synthesis during this period does not prevent germinal vesicle dissolution or chromosome condensation, but meiotic progression is blocked reversibly at the circular bivalent stage. A protein is synthesized during meiotic maturation of the mouse oocyte which exhibits several of the characteristics of the very lysine-rich histone, Fi; this and other histones are phosphorylated during the initial stages of maturation. These results are discussed in relation to studies of meiotic maturation of oocytes from non-mammalian species and chromosome condensation in both oocytes and mitotic cells.
Ciba Foundation Symposium 145 - Carbohydrate Recognition in Cellular Function, 2007
The mouse sperm receptor, called ZP3, is a glycoprotein (83,000 Mr) that consists of a 44,000 Mr ... more The mouse sperm receptor, called ZP3, is a glycoprotein (83,000 Mr) that consists of a 44,000 Mr polypeptide chain (402 amino acids), three or four N-linked oligosaccharides, and an undetermined number of O-linked oligosaccharides. There are more than 10(9) copies of ZP3 present throughout the mouse egg extracellular coat, or zona pellucida. As a prelude to fertilization, each acrosome-intact sperm binds in a relatively species-specific manner to tens-of-thousands of copies of ZP3 at the surface of the zona pellucida. Binding to ZP3 induces sperm to undergo the acrosome reaction (membrane fusion) and, consequently, enables them to penetrate through the zona pellucida and to reach, and then fuse with, egg plasma membrane (fertilization). Purified ZP3, as well as a specific class of ZP3-derived O-linked oligosaccharides (3900 Mr), exhibit sperm receptor activity in vitro. The oligosaccharides, which represent a relatively low percentage of total ZP3 O-linked oligosaccharides, account for the glycoprotein's sperm receptor activity in vitro (i.e., recognition and binding). Furthermore, either enzymic removal or modification of certain sugars that constitute these oligosaccharides results in destruction of sperm receptor activity. These and other findings strongly suggest that during mammalian fertilization carbohydrates play a fundamental role in species-specific sperm-egg interactions.
Publisher Summary This chapter describes zona pellucida (ZP), which is a thick extracellular coat... more Publisher Summary This chapter describes zona pellucida (ZP), which is a thick extracellular coat that surrounds the plasma membrane of all mammalian eggs. It highlights mouse egg ZP structure. Either three or four glycoproteins constitute the ZP of eggs from a wide variety of mammals. ZP is well organized with primary and secondary sperm receptors (two different ZP glycoproteins), arranged periodically along ZP filaments. During the process of fertilization in mammals, sperm must bind to and penetrate through the ZP to reach and fuse with egg plasma membrane. The ZP is responsible for both species specificity of sperm binding and establishment of a slow block to polyspermy following gamete fusion. The mouse egg ZP is a relatively thick translucent extracellular coat that contains ∼3–4 ng of protein and is extremely porous, permitting relatively large macromolecules and even small viruses to pass through it. The unfertilized mouse egg ZP is composed of three glycoproteins: ZP1, ZP2, and ZP3. These glycoproteins appear either as very broad bands following one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), or as a very complex set of spots following high resolution two-dimensional SDS–PAGE.
Society of Reproduction and Fertility supplement, 2007
For sperm to fertilize eggs, they must bind to and penetrate the zona pellucida (ZP) that surroun... more For sperm to fertilize eggs, they must bind to and penetrate the zona pellucida (ZP) that surrounds the plasma membrane of all mammalian eggs. The ZP first appears during oocyte growth and increases in thickness as oocytes increase in diameter. The ZP is an extracellular matrix composed of long, crosslinked filaments. In mice, three glycoproteins, called mZP1-3, are synthesised and secreted by growing oocytes and assembled into a thick (-6.5 microm) extracellular coat over a 2-3 week period. Recently, we identified several regions of nascent ZP glycoproteins that affect their secretion and incorporation into the ZP (assembly) by growing oocytes. Among these are the ZP domain, the consensus furin cleavage site (CFCS) and the C-terminal propeptide (CTP) with its transmembrane domain (TMD), external hydrophobic patch (EHP), charged patch (CP), conserved cysteine (Cys) residue, and short cytoplasmic tail (CT). Particularly important is the ZP domain, a approximately 260 amino acid regio...
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1976
DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endon... more DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.
New Approaches in Eukaryotic DNA Replication, 1983
Our objective has been to understand, at the molecular level, the replication of eukaryotic chrom... more Our objective has been to understand, at the molecular level, the replication of eukaryotic chromosomes as a prerequisite to investigating the regulation of chromosome replication and its relationship to cell proliferation. Chromosomes from eukaryotic cells, simian virus 40 and polyoma virus consist of a series of nucleosomes, each containing about 200bp of DNA coiled around an octamer consisting of two each of the four core histones that gives chromatin its stable repeating structure. Additional structure is provided by histone H1 and non-histone chromosomal proteins. However, chromatin is not homogenous. The chromatin structure of quiescent genes differs from that of active genes, and chromosomal regions differ in their higher-ordered structure. For example, chromatin can be euchromatic or heterochromatic, 10nm thick fibers or 30nm thick coils, folded into loops or not folded. Therefore, replication of eukaryotic chromosomes, in contrast to prokaryotic chromosomes, requires accurate duplication of the structure of both chromatin and DNA. Furthermore, both chromatin structure and DNA sequence are likely involved in controlling replication by modifying the interaction of proteins with DNA. For example, nonuniformly spaced clusters of replication origins are activated at different times during S phase, but the same origin is never activated twice, and some origins that are active in embryonic cells are not used in somatic cells9. Once a replicon is activated, replication proteins must be directed to replication forks and termination of replication must result in complete separation of sibling chromatids to allow normal mitosis.
Fertilization is the process by which sperm and eggs unite to form a zygote, the true beginning o... more Fertilization is the process by which sperm and eggs unite to form a zygote, the true beginning of a new individual. Among mammals, sperm first make contact with eggs at the surface of the egg extracellular coat, or zona pellucida (ZP). This contact can lead to species-specific binding of sperm to eggs via sperm receptors located in the ZP. Bound sperm then undergo changes that enable them to penetrate the ZP and fuse with egg plasma membrane. Fusion results in activation of eggs, and development of the organism ensues [1–5].
During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head... more During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.
Synthesis of histone H4 by mouse oocytes and unfertilized eggs has been examined by using a modif... more 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 (R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979, Develop. Biol. 68,341-359; 73,120-133). 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.
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Papers by Paul Wassarman