AN ELECTROPHORETIC ANALYSIS OF THE FATE OF SPERM AND EGG HISTONES OF THE SEA URCHIN STRONGYLOCENTROTUS PURPURATUS AFTER FERTILIZATION, WITH AN ANALYSIS OF THE POSSIBLE ROLE THAT PROTEOLYTIC ENZYMES PLAY IN THE FATE OF THE GAMETE HISTONES BY Alan George Carroll A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1978 K)! k\ L119-) ABSTRACT AN ELECTROPHORETIC ANALYSIS OF THE FATE OF SPERM AND EGG HISTONES OF THE SEA URCHIN STRONGYLOCENTROTUS PERPURATUS AFTER FERTILIZATION, WITH AN ANALYSIS OF THE POSSIBLE ROLE THAT PROTEOLYTIC ENZYMES PLAY IN THE FATE OF THE GAMETE HISTONES BY Alan George Carroll Sea urchin sperm and eggs both contain cell specific histones in place of some of the histones found during later development. The embryonic histones Hl, HZA, and HZB are not found in sperm or eggs. The objective of this study was to determine if the egg and sperm specific histones are retained during development, or are lost after fertilization. A preliminary investigation was also made in this study on the possible role of proteases in any loss of gamete histones. In order to determine if egg and sperm histones are retained or lost, the histones of the egg, sperm, zygote, and later embryo were analysed by both acid-urea and SDS polyacrylamide gel electrophoreses. Egg and zygote histones were extracted from nuclei with acid. The histones were fractionated on a Panyim and Chalkley acid- urea polyacrylamide slab gel, a Laemmli SDS polyacrylamide Alan George Carroll slab gel, and, in the case of zygote histones, on a two- dimensional polyacrylamide gel. Comparison of the electrophoretic patterns of sperm, zygote, and embryonic histones showed that the sperm histones are lost by 40 minutes after fertilization. The egg histones, in con- trast, were not only retained, but were found in amounts indicating that additional egg histones were added to the sperm DNA and to the DNA synthesized in the first round of DNA replication. Evidence was presented to show that the histones of the egg and zygote were extracted from the chromatin and were not due to contamination. Because proteases have been implicated in the loss of histones in other systems, sperm and eggs of Lytechinus pictus and Strongylocentrotus purpuratus were tested for sperm histone protease activity. No histone protease activity could be detected in the sperm under the condi- tions tested. Some of the conditions tested were: the presence of 2-mercaptoethanol and Triton X-100; different pH; and various salt concentrations. In addition, sperm chromatin was dialysed against egg homogenate to determine if a protease could be activated by some small molecular weight egg component. No activity was detected in the sperm under any of these conditions. Sea urchin eggs were found to contain sperm histone protease activity both before and immediately after fertilization, but not at 60 minutes after fertilization. The protease activity Alan George Carroll in the fertilized egg was found to be inhibited by EDTA. The presence of sperm histone protease activity in the egg is consistent with the removal of the sperm histones after fertilization by a protease, but does not prove the latter mechanism. The role of the sperm and egg-zygote histones was discussed. It is suggested that one reason for the presence of the egg-zygote histones is to allow the orderly removal of the sperm histones. Under this hy- pothesis, the egg-zygote histones would immediately replace the sperm histones as they are lost from the sperm DNA. This hypothesis requires that the egg-zygote histones be resistant to the proteases active in the egg. Future experiments to test this hypothesis are suggested. DEDICATION I would like to dedicate this work to my parents, John and Margaret Carroll, who made it possible, in more ways than one. ii ACKNOWLEDGMENTS I would like to thank Dr. Hironobu Ozaki for his patience, help, and support which have helped make this dissertation possible. Thanks also to Dr. J. Butcher, who provided financial support when it was needed. I would like to thank all the members of my committee, Dr. Neal Band, Dr. John Shaver, Dr. John Boezi, and Dr. Allan Morris. Special thanks to Bill Eckberg and Connie Warner who helped get me over the rough spots. Supported in part by: NIH grant HD006683, and Biomedical Research Support grants to Dr. H. Ozaki; and Graduate Council Scholarships. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . 1 LITERATURE SURVEY . . . . . . . . . . . 4 Development of the Male Pronucleus and Zygote Nucleus in Sea Urchins . . . . . . . . 4 Histones . . . . . . . . . . . . . 6 Sea Urchin Sperm Histones . . . . . . . 9 Sea Urchin Sperm Chromatin . . . . . . . 15 Histones of Sea Urchin Eggs . . . . . . 18 Histones of Early Embryonic Stages of. Sea Urchins . . . . . . . . 21 Histones of Later Sea Urchin Embryos . . . . 26 Template Activity of Embryonic Sea Urchin Chromatin . . . . . . . . . . . . 29 Histone Protease Activity . . . . . . . 32 Protease of Sea Urchin Sperm . . . . . . 34 Proteases of Sea Urchin Eggs . . . . . . 35 STATEMENT OF THE PROBLEM . . . . . . . . . 40 MATERIAI‘S AND METHODS O o o o 6 o o o o o 42 Materials . . . . . . . . . 42 Sea Urchin Gametes and Embryos . . . . . . 42 Isolation of Nuclei from Eggs, Zygotes, and Blastulae . . . . . . . . . . . . 43 Extraction of Histones from Egg, Zygote, and Blastula Nuclei . . . . . . . . 46 Preparation of Sperm and Gastrula Histones . . 46 Electrophoresis . . . . . . . . . . . 50 RESULTS . . . . . . . . . . . . . . . 53 Identification of Sperm and Gastrula Histones . 53 iv Comparison of Sperm, Egg, Zygote, Blastula, Gastrula Histones . . . . . . . Histone Proteases in the Sea Urchin Egg and Zygote . . . . . . . . Histone Protease Activity in the Sea Urchin Sperm . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . Histones Are Present in Sea Urchin Eggs and Zygotes . . . . . . . Fate of Sperm Histones in the Zygote . . Histones of the Egg and Zygote . . . . Egg Protease . . . . . . . . . . Sperm Protease . . . . . . Function of Sperm, Egg: and Zygote Histones LIST OF REFERENCES . . . . . . . . . Page 58 74 91 100 100 104 106 116 120 123 132 TABLE 1. 2. LIST OF TABLES Names that have been Assigned to Histones Effect of pH on Endogenous Sperm Histone Protease . . . . . . . Effect of Salts and Soluble Sperm Fractions on Endogenous Sperm Histone Proteases Effect of Triton X-100, Soluble Sperm Fractions, and 2-mercaptoethanol (Z-ME) on Endogenous Sperm Protease vi Page 93 94 95 Figure 1. 10. LIST OF FIGURES Preparation of sperm chromatin for protease assays O O O O O O O O O O O O Electr0phoretic pattern of Strongylocentrotus purpuratus sperm histones 5n acid-urea, SDS and two-dimensional polyacrylamide gels . Electr0phoretic pattern of Strongylocentrotus purpuratus gastrula histones on acid-urea, SDS and two—dimensional polyacrylamide gels Identification of histone H1 of Strongylocentrotus purpuratus sperm and gastrula on SDS polyacrylamide gels . . Nuclei isolated from unfertilized eggs, zygotes, and mesenchyme blastulae of Strongylocentrotus purpuratus. Phase contrast . . . . . . . . . . . Electrophoresis of Strongylocentrotus purpuratus sperm, egg: 60 minute zygote, mesenchyme blastula, and gastrula histones on a Laemmli SDS polyacrylamide gel . . Scan of zygote histones from Figure 6 and a mixture of sperm and gastrula histones . Effect of phenylmethanesulfonyl fluoride (PMSF) on the SDS polyacrylamide gel pattern of 40-minute zygote histones of Strongylocentrotus purpuratus . . . . SDS polyacrylamide gel electrophoresis pattern of Strongylocentrotus purpuratus sperm, egg, zygote, and gastruIa histones, and heavy cytoplasmic components . . . Panyim and Chalkley acid-urea polyacrylamide gel pattern of Strongylocentrotus purpuratus sperm, egg, zygote, and gastrula histones . . . . . . . . vii Page 48 54 55 56 59 60 61 64 66 67 Figure Page 11. Electr0phoretic pattern of Strongylocentrotus purpuratus zygote histones on acid-urea, SDS, and two-dimensional polyacrylamide gels . . . . . . . . . . . . . 69 12. Scan of the egg and zygote histones from Figure 6 . . . . . . . . . . . 72 13. Effect of EDTA on the protease activity of a homogenate of 25-minute postinsemination zygotes of Lytechinus pictus . . . . . 75 14. Effect of EDTA on the protease activity of a homogenate of 25-minute postinsemination zygotes of Strongylocentrotus purpuratus . 77 15. Effects of EDTA and cysteine on the protease activity of homogenates of unfertilized eggs and 11 minute postinsemination zygotes of Eytechinus pictus . . . . . 79 l6. Histone protease activity cannot be elicited in Lytechinus pictus Sperm chromatin by small molecules in the egg . . . . . 82 17. Effect of soluble sperm fractions on the protease activity of homogenates of unfertilized eggs and 60 minute postinsemination zygotes of Strongylocentrotus purpuratus . . . . 84 18. Lack of inhibition of the protease activity of unfertilized egg homogenates by homogenates of 60 minute postinsemination zygotes of Strongylocentrotus purpuratus . 87 19. Effect of time and temperature of incubation on the protease activity of unfertilized egg homogenates of Strongylocentrotus purpuratus . . . . . . . . . . . 89 20. Absence of histone or hemoglobin protease activity in the sperm of Strongylocentrotus purpuratus . . . . . . . . . . . 98 viii INTRODUCTION In somatic cells, histones show relatively little variability. Indeed, the most evolutionarily stable proteins known are the arginine-rich histones. It seems likely that the function of the histones is so specific that changes in the structure of the histone destroys its ability to function. In sperm, however, nuclear basic proteins show a wide variety of forms. The most basic of the sperm basic proteins are the protamines, which may contain as much as 95% arginine. Various basic proteins with characteristics intermediate between protamines and histones are found in many species (Bloch, 1969). Bloch (1969) and Subirana (1975) have suggested that these sperm-specific basic proteins may function to condense the sperm chromatin into a compact nucleus and may also have other functions. They suggest that these functional requirements may be filled in different ways, thereby allowing the great variation in sperm basic proteins. Whatever the reason that sperm basic proteins are unlike the somatic histones, the evolutionary stability shown by some of the somatic histones would seem to require that the structurally different sperm basic proteins be removed or lost for proper function of the somatic chromatin. l Evidence shows that sperm-specific basic proteins are lost after fertilization in some cases. All these studies have been done on Species which have protamines in the sperm. The best evidence is an autoradiographic study of fertilization in the mouse which showed that 3H-arginine labeled sperm protamines are not found after fertilization (Ecklund and Levine, 1975; Kopecny and Pavlock, 1975). Sea urchin sperm contain basic proteins which are similar to histones rather than protamines. Whether these basic proteins are removed after fertili- zation has not been demonstrated. Johnson and Hnilica (1970) mentioned that sperm histones were absent in the fertilized egg of the sea urchin Strongylocentrotus purpuratus but their data were not presented. In addi- tion, Johnson and Hnilica claimed that no histones are present before the 32 cell stage. However, as discussed below, a number of other workers have found histones earlier, even in the unfertilized egg. The histones of the unfertilized egg of Strongylocentrotus purpuratus have some components similar to late embryonic histones, but, as in the sperm of Strongylocentrotus purpuratus, some of the histones seem to be unique to the egg (Evans and Ozaki, 1973). The question may therefore be asked, what happens to the histones of the egg and sperm of the sea urchin after fertilization? To answer this question, histones from sperm, unfertilized eggs, fertilized eggs, blastula, and gastrula were extracted and compared by polyacrylamide gel electrOphoresis to determine if sperm or egg histones could be detected after fertilization. If either sperm or egg histones are not detectable after fertilization, their disappearance may be due to proteolytic degradation. Removal of histones is believed to occur in the reactivation of avian erythrocyte nuclei in heterokaryons (Appels, Bolund, and Ringertz, 1974). The erythrocyte nucleus is metabolically inactive, highly condensed, and contains a specific histone, the H5. Reactivation of the erythrocyte nucleus is blocked by protease inhibitors (Darzynkiewicz, Chelmicka—Szorc, and Arnason, 1974a, 1974b). Similarly, bull and rabbit sperm contain a protease that will digest the protamine under the prOper conditions and may be responsible for the loss of the protamines from the male chromatin after fertili- zation (Marushige and Marushige, 1975; Zirkin and Chang, 1977). As discussed below, proteases have been reported for sea urchin eggs and sperm, but whether histone protease activity is present in them has not been specifically examined. Therefore, sperm and eggs of Strongylocentrotus purpuratus and Lytechinus pictus were examined for histone protease activity. LITERATURE SURVEY Development of the Male Pronucleus and Zygote Nucleus in Sea Urchins The Sperm nucleus of the sea urchin starts the formation of the pronucleus soon after it enters the egg. The nuclear envelope of the sperm nucleus begins to break down soon after penetration of the egg. As the membrane is breaking down, the condensed chromatin of the sperm nucleus begins to disperse. Chromosome decondensation begins at the periphery of the nucleus and continues inward until the chromatin appears as a delicate mesh of entangled filaments. As the Sperm nucleus is decon- densing, the pronuclear membrane is forming around the chromatin, incorporating portions of the original sperm nuclear membrane. In Arbacia punctulata these changes may be completed by six minutes after insemination at 20°C. After the formation of the pronuclear membrane and decondensation of the chromatin, the two pronuclei meet and fuse to form the zygote nucleus. During the entire period of male pronucleus formation, there are no morphological changes in the female pronucleus (Longo and.Anderson, 1968; Longo, 1976). The first round of DNA synthesis occurs at about tile time the pronuclei fuse, 30-40 minutes after 4 insemination in Strongylocentrotus purpuratus (Hinegardner, Rao, and Feldman, 1964). Male pronucleus formation appears to depend on cytoplasmic factors that are formed or released after the time of germinal vesicle breakdown. Longo (1978) insemi- nated immature sea urchin eggs of the species Arbacia I punctulata. No changes were seen in the structure of the sperm nucleus or the sperm nuclear membrane in eggs with germinal vesicles. Complete pronuclear formation did not occur unless the egg had completed meiosis and entered the pronuclear stage. Hiramoto (1962) did not find pronuclear formation or any other changes in the sperm when he injected whole Sperm into mature sea urchin eggs. The difference in results between the two workers is probably due to the way the sperm are introduced into the egg. Longo (1978) inseminated the eggs in the normal manner while Hiramoto injected the sperm into the egg. It is probable that changes occur in the sperm, especially the sperm membranes, during fertiliZation that are neces- sary for further development. The formation of the male pronucleus does not depend on the breakdown of the cortical granules since the cortical reaction was not present in the pronuclear stage eggs (Longo, 1978; see also Longo and Anderson, 1970) . Separate analysis (Kunkle, Magun, and Longo, 1978) has established that the g; vitro decondensation of acid washed sperm nuclei involves the uptake of egg proteins when sperm nuclei are added to egg cytosol that includes the protease inhibitor phenylmethanesulfonyl fluoride. If the nuclei are not washed with acid, which removes the histones, no decondensation takes place. If Kunkle 23.21; (1978) incubated unwashed sperm nuclei in cytosol prepared according to the method of Brewer (1975), which does not involve protease inhibitors, the nuclei did decondense. The different results obtained in the presence and absence of inhibitors suggests that proteases are involved in the loss of sperm histones. Histones Histones are small basic proteins associated with DNA in the chromatin of somatic cells and some gametes. Protamines are small proteins, more basic than the histones, that are associated with the DNA and are found only in mature spermatozoa. Because of the many names that have been assigned to histones, a list of the most frequent names are listed in Table 1 (from Bradbury, 1975a). In this thesis, each individual author's desig- nation will be given followed by the CIBA symposium nomenclature in parenthesis where a clear assignment can be made. When such an assignment cannot be made, the terndnology of the cited author only will be used or the TABLE l.--Names that have been assigned to histones. Class CIBA Name Other Names lysine-rich H1 I, Fl, KAP slightly lysine-rich HZA IIbl, F2a2, ALG slightly lysine-rich HZB 11b2, F2b, KSA arginine-rich H3 III, F3, ARK arginine-rich H4 IV, F2al, GRK erythrocyte specific H5 V, F2c, KAS (lysine-rich) likely designation will be put in parentheses with a question mark, e.g. (H4?). 0f the histones, H3 and H4 Show very little vari- ability between species and even phyla (Delange and Smith, 1971; Elgin and Weintraub, 1975), while H1 shows great variability, both between Species and within species (Appels and Wells, 1972; Ruderman and Gross, 1974; Elgin and Weintraub, 1975). Histones H2A and HZB show an intermediate degree of variability (Strickland gt 31., 1977a, 1977b). Histones are known to be invOlved in the packaging of DNA into chromatin. In the current picture of chromatin structure, DNA is complexed with histones to form nucleosomes. Nucleosomes consist of globular bodies or cores connected by a linker region composed of DNA, either naked or associated with histone H1. The core is composed of two molecules each of histones H2A, HZB, H3 and H4, plus the associated DNA (Kornberg, 1974; Olins and Olins, 1974; Van Holde gt 21., 1974; Bradbury, 1975b; Felsenfeld, 1978), although there is some dis- agreement on this (Goldblatt, Bustin, and Sperling, 1978). The length of DNA enclosed in the core, plus the length of DNA in the linker region is the repeat length. Recent work has shown that the repeat length is different in different species and sometimes in different tissues, ranging from 154 base pairs (bp) in a fungus to 241 bp in a sea urchin sperm. This difference in repeat length is due to the length of the DNA linker, since the amount of DNA in the core is essentially constant at 140 bp (Compton gt 31., 1976; Morris, 1976; Spadafora gt al., 1976). Morris (1976) and Noll (1976) have suggested that the length of the linker region is due to the size and charge of the H1 which is believed to cover the linker DNA. This hypothesis has been disputed by Wilhelm gt 31. (1977). In addition to possibly determining the length of the linker region, histone H1 is believed to play a role in chromosome condensation and decondensation (Bradbury g£_§l., 1975a, 1975b; Puigdomenech gt 21., 1976). At one time, histones were thought to be Specific regulators of gene activity, but at present this role seems to be filled by nonhistone proteins although histones may be nonspecific repressors (Stein, 1974; Paul and Gilmour, 1975; Tsai gt'gl., 1976). Sea Urchin Sperm Histones The presence of histones in the sperm of sea urchins was first reported by Mathews in 1897, in Arbacia. Kossel and Edlbacher (1915) demonstrated histones in Echinus acutus and Paracentrotus lividus sperm by chemical analysis. In 1926, Kossel and Stuadt analysed histones from Echinus esculentus and found that they contained 22- 24% arginine, 7% histidine and 14% lysine. A more com- plete amino acid analysis was reported by Hultin and Herne (1949) on the sea urchins Arbacia lixula, Brissopsis lyrifera, and Echinocardium cordatum. They found only 11 amino acids present in these sperm with arginine, lysine, alanine, and glycine predominating. Due to the small number of amino acids present, Hultin and Herne considered the basic proteins of sea urchin Sperm to be intermediate between protamines and true histones. Six years later, Hamer (1955) published an analysis of the Sperm of Arbacia punctulata and found 16 amino acids present in the basic proteins rather than just 11 as Hultin and Herne (1949) reported. Hamer thought that the difference in the results was due to the different analytical methods used rather than a species difference. Vendrely and Vendrely published a review of histones and protamines in 1966, which included the amino acid compositions of Paracentrotus lividus and Echinocyanus pusillus basic sperm proteins. vendrely and Vendrely, like Hultin and Herne (1949), considered the 10 basic proteins of the sea urchin sperm to be of inter- mediate character. The first published electrOphoretic pattern of histones from sea urchin sperm wasknrRepsis (1967) who separated the acid-soluble proteins of Lytechinus variegatus sperm as well as those from fertilized eggs, blastula and plutei. The polyacrylamide gel patterns showed a great many proteins in the acid extracts of all stages. The basic proteins of the sperm, however, bore little resemblence to the acid-soluble proteins of other stages. Subirana and Palau (1968) studied both the amino acid composition and the electrophoretic patterns of Sphaerechinus granularis, Arbacia lixula and Paracentrotus lividus. When they compared the histones from Sphaerechinus granularis with those of Astroidea (Astropecten aurantiacus), Ophiuroidea (Qphiotrix fragilis), Holothoroidea (Holothurea polli), and calf thymus, they found that animals within a class have very similar histone patterns but there are notable differences between classes when examined by electrophoresis. However, two of the fastest moving bands, their gamma and delta bands, were present in all Species including calf. They refer to echinoderm sperm basic proteins as histone-like. Con- tinuing their work on Arbacia lixula, Palau, Ruiz- Carrillo, and Subirana (1969) fractionated whole sperm 11 histone into 5 fractions by starch gel electrophoresis, and obtained the amino acid composition of each fraction. They named these fractions ¢l (H1), ¢2al (H4), ¢2a2 (H2A), ¢2b (H2B), and ¢3 (H3) according to the calf thymus fraction each resembled. Sperm histones ¢l (H1) and ¢2b (HZB) showed the greatest differences from the corre— sponding calf histone, while ¢3 (H3) and ¢2al (H4) were similar in both Species. A similar characterization was carried out by Paoletti and Huang (1969) on the histones of Arbacia punctulata. They reported that their slightly lysine-rich fractions delta and epsilon resembled calf thymus FIIbl (HZA) and F2b2 (H2B). The arginine-rich fractions of sperm, alpha and beta, closely resembled the arginine-rich histones of calf thymus. They said that their sperm histone gamma resembled the erythrocyte histone F2c or V (H5). An amino acid analysis of the total histone extracted from Strongylocentrotus purpuratus and Lytechinus pictus with 0.1 M glycine was done by Messineo (1969). The histone was precipitated with 0.1 mM trichloroacetic acid or 0.1 mM HCl. According to Messineo, all the histones were precipitated by this acid treatment. Thaler £3 31. (1970) compared the histones of Arbacia punctulata Sperm to gastrula histones and calf thymus histones. The whole sperm amino acid composition 12 differed widely from the gastrula histone but differed little from the calf thymus histone, mainly in an increased proportion of glutamic acid. Senshu (1971) fractionated the histones of Strongylocentrotus purpuratus sperm by electrOphoresis and CM-cellulose chromotography with ethanolic formic acid. He found 5 fractions: 51 (H2A, H4), 82 (H3), S3a (H2B), S3 (H2B), and S4 (H1). dsc Senshu also found a highly basic fraction, S4 (Hl), SC unique to Sperm instead of the typical H1. Benttinen and Comb (1971) compared morula, blastula, gastrula, and Sperm histones of Lytechinus variegatus by electrophoresis. They found that of the five sperm bands, only two, their bands 3 and 5, were Shared with embryos. Ozaki (1971) separated sperm histones of Strongylocentrotus purpuratus into 4 bands by electrophoresis, the Slowest of which was soluble in 5% perchloric acid. An amino acid analysis of the perchloric acid soluble histone and the corresponding gastrula histone, the fl (H1), showed the sperm histone contained less lysine, aspartic acid and glutamic acid, but more alanine and much more arginine. Easton and Chalkley (1972) studied the histones of Arbacia punctulata by fractionation on acid-urea polyacrylamide gels. They were able to identify histones of all 5 types. The sperm histones f3 (H3) and f2al (H4) had identical mobility to calf thymus and embryonic f3 (H3) and f2al (H4), while histones fl (H1), f2a2 (H2A), and f2b (HZB) had mobilities 13 different from their embryonic or calf thymus counter- parts. Johnson £3 21- (1973) fractionated the histones of Strongylocentrotus purpuratus into 7 fractions by electrOphoretic and chemical means. They found by amino acid analysis, that the histones of Strongylocentrotus purpuratus sperm closely resemble the histones of Arbacia punctulata sperm and that two of the sperm histones are similar to the £3 (H3) and f2al (H4) histones of the embryo. Seale and Aronson (1973) found two electrophoretic bands that were unique to sperm in Strongylocentrotus purpuratus. Ruiz-Carrillo and Palau (1973) found that the £1 (H1), f2a2 (H2A), and f2b (HZB) histones of Arbacia lixula have different electrophoretic mobilities in sperm and embryos and that the amino acid composition of the £1 (H1) is different in sperm and embryo. They also found that the amount of each histone was different in sperm, blastula, and gastrula. Evans and Ozaki (1973) found that egg, sperm, and blastula stages of Strongylocentrotus purpuratus have some histones in common but other histones that are stage Specific. Wouters-Tyrou,Sautiere, and Biserte (1974) subjected two histones of Psammechinus miliaris Sperm, the ALG (HZA) and the GRK (H4) to amino acid composition analysis. They found that the GRK (H4) histone was very similar to calf 14 thymus except that cysteine was present in the sea urchin histone. The ALG histone (H2A), however, was different in the amounts of 10 amino acids. Brandt gt El: (1974) sequenced the 50 N-terminal amino acids of histone f3 (H3) from male gonads of Parechinus angulosus, shark erythrocytes, chick erythrocytes, and male gonads of the mollusc Patella. Amino acid composition analysis showed that the £3 (H3) of all the animals is the same and differs from the calf thymus only in having one more serine and one less cysteine. The sequence analysis showed that all the £3 (H3) histones in the four species tested are the same in the 50 N-terminal positions. In a companion paper (Strickland gt 31., 1974), they showed that the f2b (H28) of the sea urchin has similarities to both the f2b (HZB) and the f2al (H4) of calf thymus and that the sea urchin f2b (H2B) is actually two closely related proteins. They prOposed the names F2b-lsu and F2b-2 , 811 but later referred to them as H23 and (l)Parechinus H (Strickland 23 31,, 1977a, 1977b). 2B(2)Parechinus Wouters-Tyrou, Sautiere, and Bisert (1976) found that the structure of the H4 from Psammechinus miliaris gonad differs from calf thymus H4 only in a substitution of cysteine for threonine at position 73. The amino acid sequences of HZB(1) and H2B(2) from Parechinus angulosus 15 have also recently been worked out. These forms of the H23 resemble each other but are more basic and larger than the calf thymus H2B (Strickland gt al., 1977a, 1977b). Sea Urchin Sperm Chromatin Bernstein and Mazia (1953a) investigated some of the properties of deoxyribonucleOproteins (DNP) of sea urchin sperm. They extracted DNP from sea urchin sperm with distilled water and found that the mass ratio of DNA to protein depended on the number and length of extractions. In a later paper (1953b), Bernstein and Mazia studied the solubility of Strongylocentrotus purpuratus sperm DNP in different concentrations of salt. They found that sea urchin sperm DNP is soluble in very low salt, insoluble in NaCl up to 0.8 M, and soluble above 0.8 M. They attributed this solubility in high salt to the dissociation of the DNA and histones. When they decreased the salt concentration from 1.0 M, the DNP precipitated and would not go back into solution in very low salt. From this they suggested that the structure of the DNP was not regained. Hamer (1955), working with Arbacia punctulata, found that the prOportionS of DNA, histone, and acid-insoluble proteins was 27%, 21%, and 52% (1:0.77:l.9). Messineo (1962) extracted DNP from Strongylocentrotus purpuratus in 0.1 M glycine and reported 16 29% DNA, and 71% protein. It is likely that the proteins of Hamer (1955) and Messineo (1962) included many proteins not of nuclear origin Since they both studied extracts of whole sperm. Paoletti and Huang (1969) reported a mass ratio of DNA, basic protein, and nonbasic protein of l:1.2:0.2 for Arbacia punctulata DNP prepared by homogenization of sperm in saline-EDTA followed by homogenization in 0.01 M Tris, pH 8. In addition to a mass analysis, they obtained a melting curve of the sea urchin DNP which showed a two- step pattern with Tm's of 66°C and 84°C versus a Tm for DNA of 47°C in 2.5 X 10-4 M EDTA. The melting curve of DNP showed a reduction of Tm as histones were selectively removed by increasing the salt concentration. Paralleling the change in Tm was a change in template activity. Sea urchin sperm DNP has only 2% of the template activity of purified DNA, but as the histones are removed with salt the template activity approaches that of naked DNA. The removal of different histones occurs at different salt concentrations. Their histone fractions gamma (H1 or H5) and delta (HZA) are removed in 0.6 M -l.5 M salt, while the histones epsilon (HZB) and the arginine-rich alpha and beta are removed in 1.5 M - 4.0 M NaCl. Ozaki (1971) investigated the properties of sea urchin Sperm chromatin in Strongylocentrotus purpuratus. 17 He found that the chromatin of the sperm had about 2% of the template activity of the naked DNA, much less than previously found for embryonic chromatin (Marushige and Ozaki, 1967). He also found a biphasic melting curve for sperm chromatin with Tm's of 70°C and 84°C with the Tm of naked DNA 51°C in 2.5 x 10"4 M EDTA. This also differed from the pattern of embryonic chromatin which Marushige and Ozaki (1967) found to be monOphasic. The ratio of DNA, histones and nonhistone proteins for the Sperm chromatin was 1:1.02:0.13. Johnson and Hnilica (1970) prepared Arbacia punctulata Sperm chromatin by a different procedure and found that the chromatin had 0.08% of the template activity of naked DNA. They did not use the same method used by Ozaki (1971) so the template activity of the two preparations cannot be directly compared. Gineitis, Stankevicinte, and Vorob'ev (1976) found a DNA, histone, nonhistone ratio of l:1.09:0.18 in Strongylocentrotus droebachiensis sperm. Kinoshita (1976) treated sperm chromatin with heparin and found the melting temperature changed so that it was between that of untreated chromatin and pure DNA. Unlike Paoletti and Huang (1969) or Ozaki (1971), Kinoshita found that the untreated sperm chromatin had a Simple one step melting curve with a Tm of 84°C versus a Tm of 61°C for pure DNA in dilute saline-citrate, while 18 the other authors reported a two step curve with Tm's of about 50°C for DNA and 70°C and 84°C for sperm chromatin in 2.5 x 10'4 M EDTA. Studies of the structure of Paracentrotus lividus chromatin by Spadafora and Geraci (1975) have revealed a nucleosome repeat length of 200 bp. In 1976, they reported that the chromatin of Arbacia lixula sperm has a nucleosome core containing 140 bp of DNA with a total repeat length of 241 bp (Spadafora gt gt., 1976a), which is the longest repeat length reported for any animal (Compton gt gt., 1976). Studies of the role of histone H1 in Paracentrotus lividus by nuclear magnetic resonance have shown that the H1 histone has a function similar to that of the H1 in calf thymus; both are involved in the expansion and contraction of chromatin (Puigdomenech §£_§;.. 1976). Histones of Sea Urchin Eggs The presence or absence of histones during the earliest stages of sea urchin development has been a matter of controversy. Histones or histone-like proteins were first extracted from unfertilized eggs by Taleporos (1959). Taleporos extracted whole unfertilized eggs of Strgngylocentrotus purpuratus with acid and found proteins he called histones and protamines in amounts equal to those needed for embyogenesis. Backstrom (1975) reported 19 that nuclei of unfertilized eggs of Paracentrotus lividus will stain faintly with fast green stain which reacts with basic proteins. 0rd and Stocken (1968) compared histones extracted from nuclei of unfertilized eggs of Arbacia punctulata, Paracentrotus lividus, and Echinus esculentus to calf thymus histones. Electrophoresis showed that the histones of all four animals were similar, except that the sea urchins had little or no fl (H1). Thaler, Cox, and Villee (1970) extracted acid-soluble proteins from unfertilized eggs. They found that the electrophoretic pattern of egg histones tended to resemble the calf thymus F1 (H1) and F3 (H3), while embryonic histones tended to move with the mobility characteristic of F2a2 (H2A) and F2b (HZB). An amino acid analysis of the total egg and early gastrula histones showed that the egg had more lysine, aspartic acid, and serine, but less alanine and much less arginine than the gastrula. Hnilica and Johnson (1970) isolated acid-soluble proteins from nuclei of Strongylocentrotus purpuratus, Sphaerechinus granulosus, and Paracentrotus lividus. They said that they were unable to detect histones after electrOphoresis of the acid-soluble proteins. Amino acid analysis of the acid-soluble proteins indicated more acidic amino acids than expected for histones. In another paper (Johnson and Hnilica, 1970), they investi- gated the template activity of unfertilized egg nuclei 20 and unfertilized egg nuclei and had been exposed to trypsin. Unlike sperm chromatin and blastula nuclei, and like the 32-64 cell stage nuclei, egg nuclei Show no increase in template activity after trypsin treatment. They interpreted this to mean that egg nuclei and 32-64 cell stage nuclei do not contain histones since histones are known to be sensitive to trypsin. Evans and Ozaki (1973) demonstrated, however, that the unfertilized egg nucleus of Strongylocentrotus purpuratus does contain histones. They showed that the electrophoretic mobility of some of the egg histones was the same as some of the sperm and blastula histones, but other histones were unique to the egg. They also showed in a careful study that the histone bands could not be the result of contamination with ribosomes or yolk granules, the most likely source of contamination. Cognetti, Spinelli, and Vivoli (1974) investigated histone synthesis during oogenesis and found that acid- soluble proteins are made which have the same electro- phoretic mobility as blastula histones. Furthering their study of histone synthesis, Cognetti gt_gt. (1977) extracted a newly synthesized protein with the electro- phoretic mobility of histone H2B from Strongylocentrotus purpuratus oocytes. Amino acid analysis of this protein band Showed good similarity to histone HZB. 21 Histones of Early Embryonic Stgges of Sea Urchins The presence of histones in cleavage stage sea urchin embryos has also been disputed. Orengo and Hnilica (1970) reported that arginine-rich histones are synthesized and transported to the nucleus at the 4-8 cell stage in Strongylocentrotus pugpuratus, while newly synthesized, slightly lysine-rich f2 (HZA and HZB?) histones are not incorporated into the nucleus until the early blastula stage. Newly synthesized lysine- rich histones are incorporated into the nucleus still later at hatching. Hnilica and Johnson (1970) extracted acid-soluble proteins from morula, blastula and gastrula stages of Strongylocentrotus pgrpuratus, Sphaerechinus granulosus, and Paracentrotus lividus. They did not consider the acid-soluble proteins of the morula stage to be histones on the basis of electrophoretic mobilities and amino acid composition. In 1971, Johnson and Hnilica again found that typical histones were not present in Strongylocentrotus pgrpuratus in unfertilized eggs, fertilized eggs, or at the 32-64 cell stage. They said typical histones are being made at the 32-64 cell stage, but a complete complement of histones does not appear in the nucleus until the blastula stage. Seale and Aronson (1973) reported that the 4-cell stage of Strongylocentrotus purpuratus contains only the F2a (H2A?) and F2b (H28?) 22 histones and three other unidentified acid~soluble proteins. At the 16-cell stage, histone Fl (H1) appeared as did histone F3 (H3), while the 3 extra bands become less prominent. No mention was made of histone F2al (H4). Other workers have found histones during early development. Backstrom (1965) showed that nuclei of sea urchin embryos will stain faintly with fast green from the zygote stage to the 16-cell stage. After the 16-ce11 stage the intensity of the stain increases. Comb and Silver (1966) investigated the synthesis of basic proteins from fertilization to gastrulation in Lytechinus variegatus and found that proteins with chromatographic properties similar to calf thymus histones were being made. Repsis (1967) published an electrophoretic pattern of acid-soluble proteins isolated from.whole nuclei of fertilized Lytechinus variegatus eggs. At least 13 bands were present. 0rd and Stocken (1968) claimed that histones from nuclei of fertilized eggs of Arbacia punctulata, Paracentrotus lividus, and Echinus esculentus are similar in electrophoretic mobility to calf thymus histones, except that the sea urchin zygotes contained little or no F1 (H1). Unfortunately, they did not Show the electrophoretic patterns of sperm or embryonic histones for comparison. In 1970, 0rd and Stocken 23 (1970a) again stated that they had extracted histones from fertilized eggs of Echinus esculentus and gave the amino acid composition of the acid-soluble nuclear proteins. The basic/acidic amino acid ratio was 0.82 in an undialysed sample and 0.55 in a dialysed sample. Later that year 0rd and Stocken (1970b), analysed histones of Echinus esculentus obtained by a different procedure and obtained different results. This time the basic/acidic ratio was 1.1 for early cleavage stages and 1.4 for gastrula histones. There were some differences in the amino acid compositions of the cleavage stages versus the gastrula stage histones: most notably, the cleavage stages contained less alanine and more arginine, aspartic acid, and glutamic acid. They suggest that these results are consistent with a change in the relative proportions of the histones during differentiation, and an increase in the amount of F1 (H1). They did not present an electr0phoretic pattern showing the changes during development. Benttinen and Comb (1971) found that all of the classes of histones were present in the nuclei of Lytechinus variegatus at the 32-ce11 stage. They said that the individual histones present change markedly between the 32-ce11 stage and the blastula stage. Ruderman and Gross (1974) reported that histones of all 24 classes are made and incorporated into the nucleus at the 2-cell stage in Arbacia punctulata although they found that the synthesis of the F2a1 (H4) is very low. They also found that different varieties of the histone Fl (H1) are made at morula and gastrula stages in Arbacia punctulata, Lytechinus pictus, and Strongylocentrotus purpuratus. Cohen, Newrock, and Zweidler (1975) not only found that histones are synthesized from fertilization in Strongylocentrotus purpuratus, but they have presented data showing that there are several forms of the histones H2A and H2B synthesized during specific stages of develop- ment. They believe that the new forms of H2A which appear at the 16-cell stage, 100-300 cell stage and at blastula, and the new HZB which appears at the blastula stage, have different primary structures and are not just modified forms of the same proteins. All the different forms are conserved during development. Arceci, Senger, and Gross (1976) found a full complement of histones at the loo-cell stage of Lytechinus pictus. Kano and Mano (1976) found cyclic synthesis of acid-soluble proteins with electrOphoretic and ion exchange chromatographic properties similar to histones in acid extracts of whole Hemicentrotus pulcherrimus embryos. The cycles of synthesis begin before the first cleavage and almost parallel the times of DNA synthesis. 25 They did not make clear whether all the histones were being made at the one cell stage. Arceci and Gross (1977) found that proteins with the electrophoretic mobilities of histones were synthesized in the zygote of Arbacia punctulata. At least four of the five classes of histones could be detected in the acid extracts of whole zygotes. Unlike the findings of Kano and Mano (1976), synthesis of these proteins did not seem to be cyclic. It should be pointed out that the only published patterns of zygote stage, acid soluble, nuclear proteins are those of Repsis (1967), 0rd and Stocken (1968), and Johnson and Hnilica (1971). All other electrophoretic patterns are of later stages or are proteins extracted from whole eggs. The patterns shown by Repsis (1967) show a large number of bands. This suggests that sub- stantial contamination was present. Repsis noted that the nuclei were not washed after isolation. The pattern of 0rd and Stocken (1968) are called into question by the different values obtained for EChinus esculentus histones in 1970 (0rd and Stocken, 1970a, 1970b). In addition, 0rd and Stocken (1968) did not present Sperm or embryonic histone patterns for comparison. Johnson and Hnilica (1971) stated that their electroPhoretic patterns of acid soluble nuclear proteins did not Show histones but rather contaminants. Thus no reliable 26 comparison of zygote histones to the histones of other stages exists in the literature. Histones of Later Sea Urchin Embryos Backstrom (1965) demonstrated histones in sea urchin embryos with fast green stain and by biochemical techniques. Repsis (1967) separated the acid-soluble nuclear proteins of Lytechinus variggatus blastula and pluteus stages into more than 10 fractions by electro- phoresis. Marushige and Ozaki (1967) fractionated histones of Strongylocentrotus pugpuratus by electrophoresis and found three bands. Vorobyev, Geneitis, and Vinograndoza (1969) found histones in blastula and gastrula stages of Strongylocentrotus drobachiensis. All histones were present, but the amount of arginine-rich histones was reported to diminish during later stages. The amino acid composition of total histones were given for blastula and gastrula. Orengo and Hnilica (1970) reported that typical histones were present in sea urchin blastula and early prism stages of Strgngylocentrotus purpuratus. The histones were fractionated by chromatography on Amberlite CG 50 and an amino acid analysis performed on the various fractions. Hnilica and Johnson (1970) separated the histones of Paracentrotus lividus blastula and pluteus, Sphaerechinas granulosus gastrula, and Strongylocentrotus purpuratus blastula and pluteus. In addition, they did 27 an amino acid analysis on some of the blastula, gastrula, and pluteus histone fractions of Strongylocentrotus purpuratus. 0rd and Stocken (1970b) reported the amino acid composition of the total gastrula histones of Echinus esculentus. Subirana EE.§l: (1970) investigated the amino acid composition of the lysine-rich histone of Arbacia lixula. Thaler gt gt. (1970) reported the amino acid composition of whole histones from Arbacia punctulata gastrula. They found a lysine/arginine ratio of 0.6 compared to 8.4 for egg. Vorobyev gt gt. (1969) found a lysine/arginine ratio of 1.98 for Strongylocentrotus droebachiensis gastrula, and Hill gt gt. (1971) had a value of 1.78 for Strongylocentrotus purpuratus gastrula. The reason for the difference between Thaler gt_gt. and the other workers is not apparent. Benttinen and Comb (1971) reported that between the blastula and gastrula stages of Lytechinus variegatus, the arginine-rich histones become more prominent as judged by stained polyacrylamide gels. Easton and Chalkley (1972) found that Arbacia punctulata histones Fl (H1), Fla (Hla), and F2b (H2B) have different electrophoretic mobilities from the corresponding calf thymus histones. The also reported that the relative amount of histone F2b (H2B) decreased between blastula and gastrula stages while one component of the F1 (H1) became more prominent. A change in the 28 types of F1 (H1) was also reported by Seale and Aronson (1973) who found that one variety of F1 (H1) was not made after 20-30 hours of development but was conserved. They reported that the relative proportions of the other histones was constant. Ruiz-Carrillo and Palau (1973) found that the relative amount of the histone F2a2 (H2A) increases between the blastula and gastrula stages of Arbacia lixula, while the relative amount of histone F3 (H3) decreased. An amino acid analysis was performed on whole histones of gastrula, whole histones of blastula, and 4 of the 5 histone fractions of blastula. They were unable to purify the F2b (H28). Johnson gt gt. (1973) fractionated the histones of Strongylocentrotus purpuratus plutei by electrophoresis. They performed an amino acid analysis on the various fractions. The sea urchin histones were similar to calf thymus histones except that two forms of the F1 (H1) were present and both contained more glutamic acid than the calf thymus H1. Ruderman and Gross (1974) and Ruderman, Baglioni, and Gross (1974) confirmed the synthesis of stage specific Fl (81) in Arbacia pgnctulata, Lytechinus pictus, and Strongylocentrotus purpuratus. Poccia and Hinegardner (1975) observed the synthesis of stage-specific f1 (H1) and noted changes in the subfractions of f2b (H28) in Lytechinusgpictus. They did not indicate whether they thought the f2b (H28) changes 29 were due to a different protein Species or changes in posttranslational modifications. Arceci gt gt. (1976) showed that the two forms of H1 in Lytechinus pictus are synthesized on two different mRNAs, one maternal and one embryonic. Geneitis gt gt. (1976) reported that the amounts of histones H3 and H4 remained constant while H1 and H2A increased and H28 decreased from blastula to gastrula stages of Strongylocentrotus droebachiensis. Template Activity of Embryonic Sea Urchin Chromatin Marushige and Ozaki (1967) were the first to investigate the biochemical properties of sea urchin embryonic chromatin. They found that the amount of histone associated with the DNA decreased from 1.04 (histone/DNA) at the blastula stage to 0.86 at the pluteus stage of Strongylocentrotus purpuratus. The ratio of nonhistone proteins to DNA increased from 0.48 to 1.04. This change in the amount of protein associated with DNA is not reflected in the melting temperature of the chromatin which is 72°C at both blastula and pluteus stages, versus 51°C for DNA when melted in 2.5 x 10"4 M EDTA. The difference in protein content of the chromatin may be reflected in the template activity of the blastula and gastrula chromatin which has 10% and 20% of the template activity of purified sperm DNA as measured tg_vitro with exogenous RNA polymerase. 30 Johnson and Hnilica (1970) compared the template activity of egg, 64 cell, mesenchyme blastula, gastrula, and pluteus stage nuclei of Strongylocentrotus putpuratus with exogenous RNA polymerase. The percentage of template activity of each stage compared to purified sperm DNA was: egg, 1.5%; 64 cell stage, 2%; mesenchyme blastula, 2.4%; gastrula, 2%; and pluteus, 6.1%. These values are markedly different from those of Marushige and Ozaki (1967). This is probably due to different conditions of £2.Yi££2 RNA synthesis and the use of different templates: chromatin (Marushige and Ozaki, 1967) and nuclei (Johnson and Hnilica, 1970). The findings of Marushige and Ozaki (1967) were supported and extended by Cetsanga gt_gt. (1970). They found that the RNA made $2.YEE£2 showed stage related specificity when hybridized to filter bound DNA. Kinoshita (1976) compared the melting curves of early and late blastula stages of Hemicentrotus pulcherrimus. Kinoshita found that the curve changed from essentially a one step curve to a two step curve. This is different from the results of Marushige and Ozaki (1967) who reported a one step curve at both blastula and gastrula stages. These two groups, however, obtained their curves under different conditions. Kinoshita suggested that the two step curve reflects a 31 partial relaxation of the chromatin, which he relates to changes in template activity. Di Mauro, Finotti, and Ponponi (1977) investigated the template activity of blastula stage chromatin of Paracentrotus lividus. This study differed from all previous studies of template activity of sea urchin chromatin because it measured the number of initiation sites on the chromatin for RNA transcription rather than the amount of RNA synthesized. They found that blastula chromatin has 1% as many initiation sites as found on purified DNA. Immers, Markman, and Runnstrom (1967) found that the staining properties of the sea urchin nucleus changes during the blastula stage. Before midblastula, the nucleus did not stain with Hale stain, which is believed to stain unneutralized phosphate groups of DNA. After the midblastula stage, the nucleus stained if it was first treated with trypsin. After gastrulation the nucleus stained without pretreatment. Runnstrom (1967) found that the chromatin changed from an electron dense, tightly coiled mass to a less dense, more dispersed mass after the blastula stage when examined by electron microscopy. These workers thought that the changes they observed might be connected with changes in the template activity of the chromatin. 32 Hill gt gt. (1971) analysed the histones of Strongylocentrotus pgrpuratus on long acid-urea polyacrylamide gels and found that the histones of blastula, gastrula, and pluteus exhibit microheterogeneity, and changes in the relative amounts of histones in the different classes. Johnson, Wilhelm, and Hnilica (1973) showed that the acetylation patterns of sea urchin histones are very similar to those of vertebrate histones. Sures and Gallwitz (1975) found that two forms of acetyltransferases are present in the eggs of Arbacia lixula, but none are present in the Sperm. Burdick and Taylor (1976) said they found a correlation between acetylation of Arbacia punctulata histones and gene activation. Gineitis gt gt. (1976) found that the number of subfractions of H2A, H28, and H3 remains the same at blastula and gastrula stages of Strgngylocentrotus droebachiensis. Spadafora gt gt. (1976) reported that the subunit structure of gastrula chromatin of Arbacia lixula is similar to that of all other eukaryote sources but has a repeat length of 218 bp which longer than usual. Histone Protease Activity Histone protease activity has been found in the nucleus or chromatin of many species: calf thymus (Furlan and Jericijo, 1967; Bartley and Chalkley, 1970; 33 Kurecki, Toczko and Chmielewska, 1971; Fornells and Subirana, 1977), rat liver (Garrels, Elgin and Bonner, 1972; Nooden, Van Den Broek and Sevall, 1973; Chae gt_gt., 1975), avian erythroid cells but not erythrocytes (Harlow and Wells, 1975; Carter and Chae, 1976), mouse and bull testes chromatin (Chauviere, 1977) , and rat thymus, kidney, testes,brain, rabbit bone marrow, and Ehrlich ascites cells (Carter and Chae, 1976). Histone proteases have also been found in the cytoplasm of rat liver (DeLumen and Tappel, 1973), rat kidney (Paik and Lee, 1970), leukocytes (Davies gt gt., 1971), tadpole liver (Paik and Lee, 1971), tissues and embryos of Xenopus (Destree, d'Adelhart-Toorop, and Charles, 1972), and regenerating newt limbs (Procacci, Procacci and Pease, 1974) . Histone protease of uncertain intracellular origin has been reported in calf thymus (Phillips and Johns, 1959; Panyim, Jensen and Chalkley, 1968). Protease activity against protamines has been found in bull sperm nuclei (Marushige and Marushige, 1975), and in rabbit sperm (Zirkin and Chang, 1977). In general, these proteases are reported to be trypsin or chymotrypsin-like, with Optimal pH in the acid or neutral range. Activity of the proteases is normally greatest against H1 and H3 histones in chromatin, with the other histones more resistant. An exception to this rule is a protease found in calf thymus which has a 34 specificity for histone H2A (Eickbush, Watson, and Moudrinakis, 1976). Several functions have been suggested for histone proteases. Several workers (Furlan, Jericijo, and Suhar, 1968; Garrels gt gt., 1972; Chong, Garrard, and Bonner, 1974; Kurecki and Toczko, 1974) have suggested that histone proteases may be involved in gene expression, but offered no suggestion of how they might work. Others have suggested that proteases may be used in the transi- tion from histones to protamines during spermatogenesis (Marushige and Dixon, 1971; Marushige, Marushige, and WOng, 1976). Marushige gt gt. (1976) showed that a combination of protelytic degradation and acetylation of histones could replace calf thymus histones with protamines in a model experiment. Marushige and Marushige (1975) and Zirkin and Chang (1977) proposed that the protamine degrading enzyme they found in bull and rabbit sperm functions to remove protamines after fertilization. Protease of Sea Urchin Sperm Protease activity has also been reported in extracts of sea urchin sperm. Lundblad has extracted proteases from Paracentrotus lividus and Arbacia lixula Sperm.with distilled water, acetic acid, or dilute salt. At least two proteases could be extracted with pH optima 35 in the range of 4, and 7-10. Activity was shown against gelatin, hemoglobin, polylysine, polyglutamic acid, and casein (Lundblad, 1949, 1950, 1954c; Lundblad and Johansson, 1968). Lundblad suggests that the protease activity is located in the acrosome but has no evidence to support this suggestion (Lundblad and Johansson, 1968). The presence of proteolytic activity in the sea urchin acrosome was indicated by the results of Stambaugh and Buckley (1972). They mixed sea urchin sperm and fluorescein labeled soybean trypsin inhibitor. They found that the acrosomal region and only the acrosomal region showed fluorescence. Harris gt gt. (1977) extracted the sperm of Arbacia punctulata by freeze— thawing and found protease activity which, according to inhibitor analysis, was chymotrypsin-like. None of the workers on the proteases of sea urchin sperm has used histones as a substrate. None of the workers has considered the possibility of a nuclear origin of the Sperm proteases. Proteases of Sea Urchin Eggg' The proteases of sea urchin eggs have been studied by many workers. The most extensive work has been done by Lundblad and his coworkers. In his paper of 1954 (Lundblad, 1954a), Lundblad presented his scheme for the sequential appearance of three neutral proteases. All three 36 proteases, El, 82, and 83, are inactive before fertiliza- tion. Entry of the sperm at fertiliation activates protease 82, which Lundblad says is likely to be a cortex enzyme. The activity of 82 peaks very rapidly and dis- appears. As the activity of 82 is peaking, the cysteine dependent enzymes 81 and 83 are activated. The activity of El and 83 rises rapidly as the activity of 82 falls. The activity of El and 83 then falls, so that by 10 minutes after fertilization in Echinocardium cordatum, Paracentrotus lividus, and Arbacia lixula, little activity is left. These three proteases have different pH Optima: 81, pH 6.7; 82, pH 7; and 83 pH 7.6-7.8. Lundblad suggested that enzyme 82, the first to appear, is degraded by the cysteine acti- vated enzymes 81 and 83 (Lundblad, 1954b). In 1972, Lundblad, Borga, and 8kstrom reviewed the work of the pre— vious two decades and further refined the data on the neutral proteases. The optimal pH for the three enzymes was found to be 6.5, 7.25, and 7.8 for El, 82, and 83, with hemoglobin as a substrate. Lundblad gt_gl. (1972) said that enzyme 82 is a trypsin-like protease based on inhibi- tion of activity with soybean trypsin inhibitor. They also found that 82 is dependent on the presence of calcium. The sequential activation of the neutral proteases was reaffirmed. In addition to the neutral proteases, Lundblad also found and characterized cathepsin-like proteases 37 active around pH 4, which Showed no change in activity at fertilization (Lundblad, Immers, and Schilling, 1966; Lundblad and Schilling, 1968). In other papers, Lundblad described the isolation of the proteases,and the effects of activators and inhibitors (Lundblad, 1949, 1950, 1952; Lundblad and Hultin, 1954; Lundblad and Lundblad, 1953, 1962; Lundblad and Runnstrom, 1962; Lundblad, Schilling, and von Zeipel, 1969). Many other workers have also studied the proteases of the sea urchin egg and zygote. Gustafson and Hasselberg (1951) found a constant level of pH 4 hemoglobinase activity throughout development in five Species of urchins. Gross (1952) found calcium activated, pH 6.6 protease activity in unfertilized eggs of Arbacia punctulata. Maggio (1957) found protease activity only at pH 5.4-5.9 in Paracentrotus lividus. This activity increased after fertilization and underwent a decrease in activity by 30 minutes. Mano (1966) described a protease, active at pH 8, which is sensitive to soybean trypsin inhibitor, and which is present after fertili- zation but not before in Hemicentrotus pulcherrimus. In addition, he found pH 4.3 and 6.7 activity which was present both before and after fertilization. Later, Mano and Nagano (1970) said that the pH 8 protease activity is found in the 8,000-15,000 g pellet and, 38 like Lundblad's neutral proteases, declines in activity soon after fertilization. Grossman and Troll (1970) described a trypsin-like protease present in Arbacia punctulata, which shifts from the 700 g pellet before fertilization to the supernatant after fertilization. This was confirmed by Grossman gt gt. (1971). Unlike Lundblad and others, Krischer and Chambers (1970) found pH 8 protease activity in Lytechinus variegatus which did not Show a marked change in activity at fertilization. This activity is chymotrypsin-like and is inhibited by EDTA, although a minor, poorly studied, trypsin-like component is present. Lundblad 22.21- (1972) suggested that the chymotrypsin-like enzyme iS either the 81 or E3, and the trypsin-like component is the 82. Some of the proteolytic enzymes are present in the cortical granules of the sea urchin egg. Studies have established that these enzymes are involved in the detachment of bound sperm and the establishment of the fertilization membrane (Vacquier, Tenger, and Epel, 1973; Shapiro, 1975). Because cortical granule breakdown does not seem to have an effect on sperm decondensation and formation of the male pronucleus (Longo and Anderson, 1970; Longo, 1978), the cortical granule proteases are probably not involved in these processes. 39 It has been suggested that some of the proteases of the sea urchin egg are present in cytOplasmic vacuoles which contain maternal messenger RNA and are involved in the activation of protein synthesis by the activation of masked messenger RNA (Mano and Nagano, 1970). These proteases may also be involved in the activation of other metabolic functions after fertilization, but this has not been proven (Mano, 1966; Runnstrom, 1966; Mano and Nagano, 1970; Grossman gt gt., 1971; Lundblad gt gt., 1972). STATEMENT OF THE PROBLEM Sea urchin sperm and eggs each contain histones which are different from those found at the blastula stage (Evans and Ozaki, 1973). Because the histones present in the zygote and early cleavage stages have not been adequately characterized, it is not known whether the sperm and egg specific histones are retained and diluted after fertilization, or are lost from the zygote to be completely replaced by embryonic histones. In order to determine which of the two alternatives applies to sea urchin development, the histones of Strongylocentrotus purpuratus zygotes that had completed pronuclear fusion and the first round of DNA synthesis were compared to sperm, egg, and embryonic histones of the same Species by electrOphoresis on acid-urea, SDS, and two-dimensional polyacrylamide gels. The results showed that sperm histones are lost after fertilization. Since proteolytic enzymes have been implicated in the removal of histones and protamines in other systems, and Since protease activity has been reported in sea urchin eggs, zygotes, and sperm; the eggs, zygotes, and sperm of the sea urchins Lytechinus 40 41 pictus and Strongylocentrotus purpuratus were tested for histone hydrolase activity. MATERIALS AND METHODS Materials Two species of sea urchins were used: Lytechinus pictus and Strongylocentrotus purpuratus. They were obtained from Controlled Environments, Bellvue, Washington, and from Pacific Bio-Marine Supply Co., Venice, California. Trypsin, Type 1, lot # 20C-8030, 12 BAEE units/mg; phenylmethylsulfonyl fluoride (PMSF), lot # 64C-0335; 3-amino-l,2,4-triazole, lot # 55C-0160; and Bovine hemoglobin, Type II, lot # 60C-80321, were obtained from Sigma Chemical Co., St. Louis, Missouri. The stock solution of PMSF was prepared by dissolving it in isopr0pyl alcohol at a concentration of 25 mg/ml (142 mM). Artifical sea water was made according to Herbst (1904) except that the chlorinity was lowered to 18.57°/oo. Sea Urchin Gametes and Embryos Gametes were collected by intracoelomic injections of isotonic KCl. Eggs were collected in artificial sea water and filtered through zoxx Danitex silk screen to remove large debris. For nuclear isolation experiments, the eggs were dejellied by repeated passage through the 42 43 20XX Danitex. Sperm were collected dry. Eggs were fertilized and embryos cultered at 15°C until used. Only cultures showing 95% or better fertilization were used. Isolation of Nuclei from Eggs, Zygotes, and Blastulae Unfertilized egg nuclei of Strongylocentrotus purpuratus were obtained by the method of Hinegardner (1962) as modified by Evans (1972) and Evans and Ozaki (1973), with additional modifications. Packed, dejellied eggs (10-25 ml) were washed repeatedly with 8 volumes of ice cold 1.5 M dextrose by centrifugation at 500 x g for 2 minutes. Eggs were washed until they showed signs of cytolysis. The packed eggs were then lysed by the addition of 5 volumes of 2 mM MgCl at 5°C. After 4-5 2 minutes, when a majority of the eggs had lysed, an equal volume of ice cold 2M sucrose containing 6 mM MgCl was 2 added. The mixture was then swirled to disperse the cytolysed eggs. The lysate was centrifuged at 750 x g for 15 minutes. The sediment was resuspended in l M sucrose, 4 mM MgC12, and layered on a discontinuous sucrose gradient. The gradient was composed of 4 m1, 2.375 M sucrose; 8 m1, 2 M sucrose; 3 ml, 1.75 M sucrose; 3 ml, 1.5 M sucrose; and 3 ml, 1.25 M sucrose. The samples were then centrifuged in a Spinco SW 25.1 rotor at 20,000 RPM for 45 minutes. The purified nuclei at 44 the 2.375 - 2.0 M interface were collected and diluted with an equal volume of 2 mM MgC12. Fertilized Strongylocentrotus purpuratus egg nuclei were isolated from eggs 40 and 60 minutes after fertili- zation. By this time the zygotes have completed DNA synthesis (Hinegardner gt_gt., 1964) in preparation for cleavage about an hour later. Fertilization membranes were removed from zygotes by insemination in the presence of the catalase inhibitor 3-amino-l,2,4-triazole which prevents hardening of the fertilization membrane (Foerder and Shapiro, 1977). About 20 m1 of packed, dejellied eggs in 500 ml of articicial sea water containing 1 mM inhibitor were fertilized with 2 drops of dry sperm diluted to 10 ml. The sperm was removed by washing the fertilized eggs with artificial sea water that did not contain the inhibitor. At 30 minutes after insemination, the zygotes were lightly packed and forced rapidly through zoxx Danitex to strip the unhardened membranes from the zygotes. The zygotes were allowed to settle in 15°C artificial sea water. The free membranes in the supernatant fluid were removed by decentation. This step was repeated until the zygotes were free of membranes. Membrane removal is better than 95%. The inhibitor has no visible effect on the development of treated, fertilized eggs up to the pluteus stage. At 40 to 60 minutes after insemi- nation, the zygotes were washed in ice cold sea water. The 45 zygote nuclei were then isolated by the same procedure as unfertilized egg nuclei, except that lysis took 10-15 minutes at 5°C in 2 mM MgCl and the 2 M sucrose added 2! after lysis contained only 2 mM MgCl instead of 6 mM 2 MgClZ. The number of egg or zygote nuclei prepared by the above procedure was estimated at this point. One drop of the nuclear suspension was placed on a microsc0pe slide so that the entire area under the cover slip was filled. The number of nuclei in 100-110 fields, equally Spaced in a grid pattern, was counted. The average number of nuclei per field was determined by dividing the total number of nuclei by the number of fields. The average number of nuclei/field was multiplied by 3,180 (the total area of the cover slip divided by the area of one micro- scopic field) to obtain the total number of nuclei on the microscope Slide. This number was then multiplied by the number of drops/m1 to obtain the number of nuclei/ml. Nuclei from mesenchyme blastulae of Stroggylocen— trotus purpuratus were also isolated by this method with modifications. Embryos were grown for 24 hours at 15°C. The blastulae were washed in 1.5 M dextrose four times and then lysed in 2 mM MgCl as above. After 4.5 minutes 2 at 5°C in 2 mM MgC12, an equal volume of ice-cold 2 M 46 sucrose, 2mM MgCl2 was added. Since the embryos did not readily disperse, they were homogenized with five strokes of a tight fitting Dounce homogenizer. From this point on, the nuclei were treated the same as the egg and zygote nuclei. Extraction of Histones from Egg, Zygote, and Blastula Nuclei Histones were extracted from egg, zygote, and blastula nuclei according to Evans (1972) and Evans and Ozaki (1973). The purified nuclei were concentrated at 750 x g for 15 minutes. The pellet was washed 3-4 times with 0.5 ml of saline-EDTA (75 mM NaCl, 24 mM EDTA, pH 8.0) by centrifugation at 12,000 x g for 10 minutes. The nuclei were then washed twice with 10 mM Tris, pH 8.0, at 17,000 x g for 15 minutes. These washes remove proteins of the nuclear sap and any adhering ribosomes. Histones were extracted from the nuclear pellet with 0.2 N HCl for 30 minutes at 0°C. Acid insoluble proteins and DNA were pelleted by centrifugation at 12,000 x g for 10 minutes. The histones in the supernatant were stored at 3°C until use. Preparation of Sperm and Gastrula Histones Sperm chromatin for the assay of proteolytic activity was prepared from Strongylocentrotus pugpuratus or Lytechinus pictus sperm according to the method of 47 Ozaki (1971) as shown in Figure l. The method of Ozaki was followed except that the sucrose centrifugation step and the following dialysis were sometimes deleted. If the sucrose centrifugation and dialysis steps were omitted, the chromatin was referred to as crude chromatin; if these steps were included, the product was purified chromatin. Chromatin was stored at 3°C until use. Chromatin stored at 3°C showed no degradation after 6 months. Chromatin concentration figures are based on the optical density of the chromatin at 260 nm. The centrifugation supernatants following the homogenization steps were saved and were stored at 3°C as the soluble Sperm fractions (SSF). Egg homogenate for assay of proteolytic activity was prepared by washing either fertilized or unfertilized eggs in isotonic NaCl-KCl, 19:1, followed by homogeni- zation in a tight fitting Dounce homogenizer. The homogenate was used fresh or was frozen at -20°C until used. A stock solution of bovine hemoglobin for the protease assay was prepared according to Anson (1938) by dissolving 0.2 g hemoglobin and 3.6 g urea in about 5 ml distilled water with 0.8 ml of N NaOH. After the hemoglobin was completely dissolved, 0.01 ml of M Tris, pH 8, was added. The solution was brought to pH 8 with 48 l gran sperm l sperm washed in saline-EDTA, pH 8 l sperm hanogenized in saline-EDTA, pH 8 Waring blender, semi-micro 100 volts, 1 minute 50 volts, 2 minutes I Iumgenate centrifuged 12,0009, 10 minutes -———- SSF l l pellet Imogenized in saline-EDTA, pH 8 Waring blender, sani-micro 45 volts, 30 seconds I hatogenate centrifuged 12,0009, 10 minutes -— SSF 2 I pellet hmogenized in 10 11M Tris, pH 8 Waring blender, semi-micro 30 volts, 2 minutes I harngenate centrifuged 12,0009, 10 minutes-——SSF 3 I pellet resuspended in 10 11M Tris, pH 8———Sheared, Sorvall I W, full layered on 1.7 M sucrose, speed, 5 minutes 10HMTris, 1mm, p118 I crude chrcmatin Centrifuged 22,000 RPM, 4 hours Spinoo SW 25.1 rotor supernatant discarded pellet dialysed against 10 nM Tris, pH.8 Figure l.--Preparatim of sperm chrunatin for protease assays. 49 HCl and the volume was adjusted to 10 ml. The final concentration of the stock solution of hemoglobin was 20 mg/ml hemoglobin, 6 M urea, 10 mM Tris, pH 8. The protease assay was performed by mixing sperm chromatin, salts, buffers, and any other components of the assay mixture as indicated in the various figures and tables. The OD/ml of sperm chromatin and the percent of egg homogenate is the final concentration in the assay mixture. After the samples were incubated at the temperatures and times indicated in the figures and tables, an equal volume of 0.8 N HZSO4 was added. After 30 minutes at 0°C the samples were centrifuged at 6,000 x 9 for 20 minutes. Four volumes of 95% ethanol were added to the supernatant which was then stored at -20°C overnight. The precipitate was centrifuged down at 1,200 x 9 for 20 minutes and washed in ethanol, ethanol-ether, and ether. After air drying, the histone was dissolved in 0.9 N acetic acid, 15% sucrose at a concentration of approximately 1 mg/ml based on preincubation Optical density. The samples were not dialysed to remove salt because the amount of salt present in the samples did not have any effect on electrophoresis. 50 Electrophoresis Electr0phoreses were conducted by the acid-urea method of Panyim and Chalkley (1969), the SDS method of Laemmli (1970), and a two-dimensional system modified from O'Farrell (1975). Acid-urea polyacrylamide gel electrOphoresis (Panyim and Chalkley, 1969) was performed in cylindrical gels, 6 mm x 90 mm, or in slab gels 100 x 130 x 0.75 mm. The gels contained 15% recrystallized acrylamide, 0.2% bisacrylamide, and 2.5 M urea. Electrophoresis was at 2 mA/tube or 15 mA/slab until the tracking dye, methyl green, reached the bottom of the gel. SDS polyacrylamide gel electrophoresis (Laemmli, 1970) was performed only on slab gels. The acrylamide concentrations of Laemmli were adjusted to 15% acrylamide, 0.4% bisacrylamide in the separating gel (90 x 130 x 0.75 mm), and 5% acrylamide, 0.13% bisacrylamide in the stacking gel (10 x 130 x 0.75 mm). Electr0phoresis was at 15 mA until the tracking dye, bromphenyl blue, was 1 cm from the bottom of the gel. The two-dimensional electroPhoresis was based on the method of O'Farrell (1975). The first separation was in a Panyim and Chalkley acid-urea polyacrylamide gel, 2 mm in diameter and 9 cm long. The second separation was in a Laemmli SDS polyacrylamide slab gel. After the 51 first electrophoresis, the cylindrical acid-urea polyacrylamide gel was removed by breaking the glass tube. The gel was then equilibrated with Laemmli sample buffer without glycerol by several changes of the buffer solution until the indicator dye, bromphenol blue, remained blue. The equilibrated gel was placed over the Laemmli slab gel as shown in O'Farrell (1975) and was held in place with 1% agarose made up in Laemmli sample buffer without glycerol. Electr0phoretic conditions were the same as for Laemmli single dimension gel electrophoresis. Samples of protein to be run on Laemmli SDS polyacrylamide gels were neutralized with NaOH if in acid. If necessary, the samples were partially evaporated under a stream of nitrogen to a concentration of about 1 mg protein/ml. An equal volume of double strength Laemmli sample buffer was added to aqueous samples. Laemmli sample buffer contains: 62.5 mM Tris, pH 6.8; 2% SDS; 5% 2-mercaptoethanol; 10% glycerol; and 0.001% bromphenol blue. All samples were heated to lOO‘C for 1.5 minutes. Samples of 1-5 ul were analysed. Dried proteins to be run on Panyim and Chalkley acid-urea polyacrylamide gels were dissolved in 0.9 N acetic acid, 15% sucrose, and 0.05% methyl green. Aqueous protein samples were reduced in volume if neces- sary by evaporation under a stream of nitrogen. Sucrose 52 and methyl green were than added to the sample if required. Samples of 1-5 ul were analyzed on slab gels; larger amounts were analyzed on cylindrical gels. Slab gels were stained with Coomassie Brilliant Blue R-250 according to the method of Fairbanks, Steck, and Wallach (1971) as modified by Ames (1974). Stained gels were photographed with a #15 Kodak Wratten filter on Plus-X film. Cylindrical gels were stained with 0.1% Amido Black 108, 40% methanol, 7% acetic acid overnight and destained in 40% methanol, 7% acetic acid. Gels were scanned on a Gilford model 2400 spectrophotometer at 600 nm (Coomassie Brilliant Blue) or 610 nm (Amido Black). RESULTS Identification of Sperm and Gastrula Histones The identify of Sperm and gastrula histone bands on Panyim and Chalkley acid-urea gels and Laemmli SDS gels was determined by mobility, staining properties, formation of dimers, and solubility. The electrOphoretic patterns of Sperm histones on acid—urea (a), SDS (b), and two-dimensional (c) polyacrylamide gels are shown in Figure 2. The electrophoretic patterns of gastrula histones on acid-urea (a), SDS (b), and two-dimensional (c) polyacrylamide gels are shown in Figure 3. Analysis by two-dimensional gels allowed a histone identified in one electrOphoretic system to be located in the other gel system. Labels on the gel patterns (1, 2A, 28, 3, and 4) identify the histones H1, H2A, H28, H3, and H4. Histone H1 was identified by~its solubility in 5% perchloric acid (Johns, 1964) as shown in Figure 4. The H1 of sperm is composed of one protein while the H1 of gastrula is composed of three proteins. Histone 3 was identified by its ability to form dimers (Fambrough and Bonner, 1968). Both the oxidized (Box) and reduced (Bred) forms of the sperm 83 are seen 53 1.021; -!nas 3A 3 . I -u ‘ _C 54 ’lhod Figure 2.--Electrophoretic pattern of Strongylocentrotus pugpuratus sperm histones on acid-urea, SDS and two-dimensional polyacrylamide gels. a. Panyim and Chalkley acid-urea gel, origin at top. b. Laemmli SDS gel, origin at tOp c. Two-dimensional gel, first separation, acid-urea (left to right), second separa- tion, SDS (top to bottom). Numbers identify the various histones. 55 a 5 c - .. 3' 5351 ‘Il’l r: -“ (IIIDS cp:3 -m- ij :4 GIIDJt agliia“ IE4 Figure 3.--Electr0phoretic pattern Of Strongylocentrotus pgrpuratus gastrula histones on acid-urea, SDS and two-dimensional polyacrylamide gels. a. Panyim and Chalkley acid-urea gel, origin at top b. Laemmli SDS gel, origin at tOp c. Two-dimensional gel: first separation, acid-urea (left to right); second separa- tion, SDS (tOp to bottom). Numbers identify the various histones. 56 II: 3 CI- -3 I 2A,!I --. - 2A Figure 4.--Identification of histone 81 of Strongylocen- trotus purpuratus sperm and gastrula on SDS polyacrylamide gels. a. Hl enriched fraction of gastrula histones b. 81 depleted fraction of gastrula histones c. Residual sperm histones after extraction with 5% perchloric acid. Gastrula histones were fractionated by chromatography on CM cellulose. 57 in Figure 20, a two-dimensional polyacrylamide gel pattern, even though the second separation is performed in an SDS system which containse1reducing agent. The sperm H3 was oxidized $2.Z$E£2.by the addition of a drop of 8202 (Jocelyn, 1972). The identical mobility of the bands identified as H3 in the sperm and gastrula in both SDS and acid-urea gels (Figures 6 and 10), is additional evidence that this band is the H3 since the 83 is a highly conservative protein in the evolutionary sense (Brandt gt gt., 1974). The H3 of gastrula is one protein which migrates as three on an acid-urea gel (Figure 3a) due to post-synthetic modification. The other protein with the same mobility in both sperm and gastrula, on both acid-urea and SDS gels as seen in Figures 6 and 10, must be the highly conservative H4 (DeLange and Smith, 1971; Wouters-Tyrou 2E.El°v 1976). This histone is modified to form a doublet in the gastrula as seen in Figure 3a. Histone 28 was identified on acid-urea gel patterns of gastrula histones by its prOperty of staining blue- black, instead of blue, with Amido Black (Easton and Chalkley, 1972; Cohen and Gotchel, 1971). Histone 28 of sperm was named in accordance with the Arbacia punctulata sperm pattern of Easton and Chalkley (1972). 0n SDS gels (Figure 2b), the H28 of Strongylocentrotus purpuratus sperm consists of two separate proteins. This 58 is similar to the H28 of Parechinus angulosus which was shown to consist of two distinct proteins (Strickland SE.El-v 1974, 1977a, 1977b). The remaining band on the acid-urea gel and the SDS gel of both sperm and gastrula was identified as the H2A by elimination. Comparison of Sperm, Egg, Zygote, Blastula, and Gastrula Histones Sperm, egg, zygote, blastula, and gastrula histones were compared by electrophoresis. The histones of egg, zygote and blastula were extracted from isolated nuclei (Figure 5). The histones of sperm and gastrula were extracted from purified chromatin. The Laemmli SDS polyacrylamide gel pattern of sperm (S), egg (8), 60 minute zygote (Z), 24-hour mesenchyme blastula (B), and gastrula (G) histones is shown in Figure 6. Sperm-specific histones H1 and H28 are not present in the zygote. This is confirmed by a scan of zygote histones and a mixture of sperm and gastrula histones in a 1:3 ratio (Figure 7). (This ratio (1:3) is the expected ratio of Sperm histones to other histones if the sperm histones had been retained in the zygote after nuclear fusion and DNA replication. The Sperm H1 and H28 are clearly visible in the sperm-gastrula mixture but not in the zygote. Thus, if the sperm histones had 59 BLASTULA Figure 5.--Nuclei isolated from unfertilized eggs, zygotes, and mesenchyme blastulae of Strongylocentrotus purpuratus. Phase contrast. 60 as!!! 3-- aim--3 2A -a—--n,u 4-C~-¢----4 8 I l I 0 Figure 6.--Electrophoresis of Strongylocentrotus purpuratus sperm, egg, 60 minute zygote, mesenchyme blastula, and gastrula histones on a Laemmli SDS polyacrylamide gel. S, sperm; 8, egg; Z, 60 minute zygote; B, blastula; G, gastrula. Sperm histones are identified on the left of the pattern; gastrula histones are identified on the right of the pattern. Origin is at the top. 61 Figure 7.--Scan of zygote histones from Figure 6 and a mixture of sperm and gastrula histones. Scan of zygote histones from Figure 6 (upper curve) and a mixture of one part sperm histones and 3 parts gastrula histones. Peaks D and 8 of zygote are egg-zygote specific histones. Peak H2 of the sperm gastrula mixture contains the sperm H2A and the gastrula H2A and H28. Origin at left. 62 Z YGOTE H3 0 t; H4 SPERM PLUS GASTRULA SPERM “SVRUlA SPERM HI H1 H2. H3 H2 H4 63 been retained in the post—replication nucleus, they must have been lost or removed from the DNA. The possibility of sperm histone degradation during nuclear isolation may be discounted for two reasons. First, there is some evidence that histone protease activity is not present in the zygote at this time as discussed below. Second, isolation of the nuclei in the presence of a protease inhibitor does not change the pattern. The histone pattern of Figure 8 shows histones from 40-minute post-insemination zygotes. The nuclei were obtained from zygotes lysed in the presence of 0.5 mM PMSF. PMSF was also included in the sucrose solution the nuclei were resuspended in prior to purification on the sucrose gradient. PMSF is an irreversible inhibitor of trypsin and chymotrypsin (Fahrney and Gold, 1963), and has been used to inhibit histone protease activity in other systems (Nooden, gt gt., 1973; Ballal, Goldberg, and Busch, 1975; Chae gt gt., 1977). Because of the inclusion Of the above amount of protease inhibitor, it is unlikely that the loss of sperm histones was due to a protease acting during nuclear isolation. In order to determine if the egg and zygote patterns of Figure 6 are due to nuclear proteins or to cytoplasmic contamination, a portion of centrifuged egg lysate was extracted with acid instead of being centri- fuged on the sucrose gradient. The electrophoretic 64 (II! - 1 .v-‘l ' 28' 3" .03 2A¢v O‘C-OZAZI 4. ' a». 4 Figure 8.--Effect of phenylmethanesulfonyl fluoride (PMSF) on the SDS polyacrylamide gel pattern of 40- minute zygote histones of Strongylocentrotus purpuratus. The protease inhibitor PMSF (0.5mM) was added to zygotes during lysis in low magnesium and during loading of the nuclei on the sucrose gradient prior to nuclear purification. S, sperm; 2, 40-minute zygote; G, gastrula; A, aldolase; 0, ovalbumin; 8, bovine serum albumin; C, chymotrypsinogen. Origin at top. 65 pattern of this material (C) is shown in Figure 9 along with the patterns of sperm (S), egg (8), 60-minute zygote (Z) and gastrula (G) histones. No significant cytoplasmic bands have the mobility of the histones although there are many high molecular weight bands. Figure 6 also shows that while egg and zygote his- tones have the same mobility on an SDS gel, they are not entirely the same as blastula or gastrula histones. The differences between egg and zygote histones and later embryonic histones is better shown in Figure 10, an acid- urea polyacrylamide gel pattern of sperm (S), egg (8), 40-minute zygote (Z), and gastrula (G) histones. It is clear from the acid-urea polyacrylamide gel pattern in Figure 10, that the egg and zygote do not contain proteins with the same mobility as the 81, HZA, or H28 of gastrula. Both egg and zygote, however, have bands with the mobility of some of the Sperm and gastrula H3 and H4 bands. The SDS polyacrylamide gel pattern of egg and zygote histones (Figure 6) shows that, in the SDS system, the egg and zygote histones also have bands with the mobility of histones 3 and 4. Figure 11¢, a two- dimensional polyacrylamide gel pattern of 60-minute zygote histones, shows that the same protein has the mobility of the H3 in both gel systems, and the same protein has the mobility of H4 in both gel systems. As noted in the discussion, it seems likely that the egg and zygote 66 _ '3 I Lad I ! as m- ‘ 2A In- ”I ‘- “M‘ "M S I I G C Figure 9.--SDS polyacrylamide gel electrophoresis pattern of Strongylocentrotus pugpuratus sperm, egg, zygote, and gastrula hIstones, and heavy cytoplasmic components. S, Sperm; 8, egg; Z, 60 minute zygote; G, gastrula; C, acid extract Of the material put on the sucrose gradient, this fraction includes nuclei and other heavy cytoplasmic components. .........-.......‘.- .4..- > I b-d c: . I I.- -1 23,3..- .. 3% ~ L3 :3 x , -2: 4 4 ..:4 . . , I S I 1 O Figure 10.--Panyim and Chalkley acid-urea polyacrylamide gel pattern of Strongylocentrotus purpuratus sperm, egg, zygote, and gastrula hiStones. S, sperm; 8, egg; Z, 40 minute zygote; G, gastrula. Bands A-F are egg-zygote Specific histones. Numbered bands identify other his- tones. Origin at top. 68 contain the same H3 and H4 as found in the sperm and gastrula. Analysis of the remaining zygote histones may be made by reference to Figure 11. Figure 11 shows the electrophoretic pattern of zygote histones on Panyim and Chalkley acid-urea (a), Laemmli SDS (b), and two- dimensional (c) polyacrylamide gels. The bands are labeled A-F according to mobility on the acid-urea gel. Bands corresponding to histones H3 and H4 are labeled 3 and 4. The correspondence of bands 3, 4, D, 8, and F in the two systems can be seen in Figure 11c. Correlation of bands A, B, and C in the acid-urea system (Figure 11a) to bands in the SDS system (Figure 11b) in not possible. These bands are present in all four samples of zygote histones and in the one sample of egg histones run on acid-urea polyacrylamide gels, but no bands appear on the SDS gels at positions expected from the two-dimensional gel. The most likely explanation for this discrepancy is that the bands A, B, and C are aggregates or complexes of the his- tones 3, 4, D, E, and F. This will be considered further in the discussion. Another facet of Figure 11 which needs explanation is hand F. Band F has approximately the same mobility as the sperm H2A on both SDS gels (Figure 6) and acid- urea gels (Figure 10). A protein with the mobility of band F also appears in the egg: which has not come in c b H o——-- '*A H: U? .___... 69 .94 Figure ll.--Electrophoretic pattern of Strongylocentrotus purpuratus zygote histones on acid-urea, SDS, and two-dimensional polyacrylamide gels. a. b. C. Panyim and Chalkley acid-urea gel, origin at top Laemmli SDS gel, origin at top Two-dimensional gel: first separation, acid-urea (left to right); second separa- tion, SDS (tOp to bottom). The acid-urea gel pattern is of 40 minute zygote histones, the SDS and two-dimensional patterns are of 60 minute zygote histones. Letters and numbers identify the various histones. 70 contact with Sperm, and in the gastrula where it would have been diluted out if of sperm origin (Figure 10). It is possible that band F is a minor cOmponent of egg, zygote, and gastrula histones as well as a major component of sperm histones. A similar suggestion may be made for histone D which appears in sperm, blastula, and gastrula as well as in egg and zygote (Figure 6). As noted above and Shown in the acid-urea pattern of Figure 10, the zygote nucleus does not contain embryonic histones, 1,2A, or 28. The zygote histones of Figure 10 were prepared from zygotes 40 minutes after insemination. At the temperature of development, 15°C, the pronuclei fuse at about 30 minutes after insemination and nuclear DNA synthesis occurs at 30-40 minutes postinsemination (Hinegardner gt gt., 1964). These zygotes therefore contain a tetraploid amount of DNA and histones compared to the haploid amount of the egg. If the zygote does in fact contain four times as much histone as the egg, addi- tional egg type histone must have been added to the newly replicated DNA because only egg histones are present. The amount of histone in the egg and in the zygote can be estimated from Figure 6, the SDS polyacrylamide gel pattern of sperm, egg, zygote, blastula, and gastrula histones. The egg histone shown in Figure 6 was derived from 6.4 x 106 nuclei; the zygote pattern was derived from 1.25::106 nuclei. Approximately five times more 71 egg nuclei were used to extract histones. The four histone peaks of the gel shown in Figure 6 were scanned (Figure 12), then the peaks were cut from the scan and weighted. Approximately 1.5 times as much histone is present in the egg pattern. On a per nucleus basis, the zygote con- tained 3.3 times as much histone as the egg, indicating that additional egg type histone must have been added during DNA replication. In all other experiments, the staining intensity of the histones, as judged visually, was roughly prOportional to the amount of chromatin expected in the sample. Further examination of Figure 12 shows that the histones are not present in the same pro- portions before and after fertilization. The extraction of five times as many egg nuclei as zygote nuclei for Figure 6 may also explain the large amount of proteins with mobility less than histones. By extracting five times as many nuclei, each contaminating protein would be present in five times greater amounts, and should thus be more prominent. In summary, it has been shown that spermrspecific histones H1 and H28 are not present in the 40 or 60 minute zygote but that egg type proteins are present, and even more egg type histones have been added during DNA replication. 72 Figure 12.--Scan of the egg and zygote histones from Figure 6. Egg histones, upper curve; zygote histones, lower curve. Origin at the left. 74 Histone Proteases in the Sea Urchin Egg and Zygote The histones of sea urchin Sperm are not found in the zygote. These histones may have been removed by a protease. Sperm, unfertilized eggs, and fertilized eggs of various ages were therefore assayed for possible histone protease activity. Protease activity against sperm histones was found in Lytechinus pictus unfertilized eggs (Figure 15) and in zygotes at 11 (Figure 15), 25 (Figure 13), and 35 minutes after fertilization. Histone protease activity was also found in the eggs of Strongylocentrotus purpuratus before fertilization (Figures 17 and 19) and at 25 minutes after fertilization (Figure 14) but not at 60 minutes after fertilization (Figure 17). The presence of protease activity is indicated by the disappearance of sperm histones, particularly the H1, or by the appearance of extra bands (P) which are products of the digestion. The activity of the fertilized egg protease is reduced in the presence of EDTA as Shown in Figures 13, 14, and 15. Unlike the fertilized egg activity, the protease activity of the unfertilized egg does not seem to be reduced by EDTA as indicated by the appearance of similar amounts of degradation products (P) in the absence or the presence of EDTA (Figure 15, a and c). The unfertilized egg protease activity does seem to be reduced by the 75 Figure 13.--Effect of EDTA on the protease activity of a homogenate of 25—minute postinsemination zygotes of Lytechinus pictus. zygote homogenate (1.3%) plus sperm chromatin with 8 mM Ca++ and 21 mM Mg++. Unincubated control. ..... zygote homogenate (1.3%) plus sperm chromatin with 5 mM EDTA, pH 8, added. Incubated 43 hours at 24°C. ----- zygote homogenate (1.3%) plus 3 erm chromatin with 8 mM Ca++ and 21 mM Mg +. Samples were incubated 43 hours at 24°C. All samples contained 10 mM Tris (pH 8), 0.2 M NaCl. Samples were fractionated on an acid-urea polyacrylamide gel, origin on the left. 76 a....... v. 0000000000000000000000 J $.00... 77 Figure l4.--Effect of EDTA on the protease activity of a homogenate of 25-minute postinsemination zygotes of Strongylocentrotus purpuratus. .... egg homogenate (1.6%) plus Sperm chromatin with 2 mM EDTA. egg homogenate (1.6%) plus sperm chromatin with 5 mM Ca+ and 14 mM Mg +. Samples were incubated 17.5 hours at 24°C. 10 mM KCl, and 5.1 OD/ml purified Sperm chromatin. Samples were fractionated on an acid-urea polyacrylamide gel, origin on the left. 78 4 1 28,3 2A 79 Figure 15.--Effects of EDTA and cysteine on the protease activity of homogenates of unfertilized eggs and 11 minute postinsemination zygotes of Lytechinus pictus. a. unfertilized egg homogenate (3%) plus sperm chromatin with no additions. b. unfertilized egg homogenate (3%) plus sperm chromatin with 17 mM cysteine added. c. unfertilized egg homogenate (3%) plus sperm chromatin with 3.3 mM EDTA, pH 8, added. d. homogenate of demembranated zygotes (3%) plus sperm chromatin with no additions. e. homogenate of demembranated zygotes (3%) plus sperm chromatin with 17 mM cysteine added. f. homogenate of demembranated zygotes (3%) plus sperm chromatin with 3.3 mM EDTA, pH 8, added. 9. homogenate of membranated zygotes (3%) plus sperm chromatin with no additions. h. homogenate of membranated zygotes (3%) plus Sperm chromatin with 17 mM cysteine added. 1. homogenate of membranated zygotes (3%) plus sperm chromatin with 3.3 mM EDTA, pH 8, added. j. sperm chromatin without additions. Sperm were incubated 20 hours at 35°C. All samples contained 10 mM Tris (pH 8.0), 0.25 M NaCl, 13 mM KCl, and 9 OD/ml crude chromatin. Samples were fractionated on an acid-urea polyacrylamide gel, origin at the top. P, degradation products; 1, 2A, 28, 3, and 4 designate the various histones as in the other figures. 80 '"HHUUUUIUU gzuflihilllflflfl 2‘“ 01.00 010.000 400.0063 ‘°’O.o. ..!Ooo"“" 81 presence of cysteine, however (Figure 15b). Cysteine has little or no effect on fertilized egg protease activity (Figure 15e). The protease activity of Lytechinus pictus zygotes at 11 minutes after fertilization is not due to cortical granule proteases trapped between the plasma membrane and the fertilization membrane because mechanical removal of the fertilization membrane followed by two washes of the egg in buffer before homogenization did not remove the protease activity (Figure 15, d-e). Lytechinus pictus sperm chromatin assayed under the same conditions but without egg homogenate did not show any histone protease activity (Figure 16a, f, k). When the Sperm chromatin was dialysed against egg homogenate (Figure 16c, h, m), no degradation of histones occurred. The same result was seen if an unfertilized egg homogenate of Strongylocentrotus purpuratus was used. This indicates that there is no inactive protease in the sperm that can be activated by small molecular weight components in the egg. The use of purified chromatin, crude chromatin, or crude chromatin combined with soluble sperm fractions all gave the same results. Addition of soluble sperm fractions to an egg homogenate-sperm chromatin incubation mixture resulted in an increase in digestion as seen in Figure 17. When Strongylocentrotus purpuratus zygotes were homogenized 82 Figure 16.--Histone protease activity cannot be elicited in Lytechinus pictus Sperm chromatin by small molecules in the egg. a-e. purified chromatin f-j. crude chromatin k-o. crude chromatin plus the combined soluble Sperm fractions. Samples were incubated at 35°C for 20.5 hours. All samples contained 10 mM Tris (pH 8), 0.25 M NaCl, 13 mM KCl. Samples were fractionated on an acid-urea polyacrylamide gel, origin at the tOp. (C) control sperm chromatin (12.6 OD/ml) incubated without egg homogenate. (T) trypsin (5 ug) added to sample after incubation. (H) unfertilized egg homogenate (2.5%) and Sperm chromatin (12.6 OD/ml) mixed together. (D) sperm chromatin in dialysis tubing dialysed against unfertilized egg homogenate. :00!— U U 83 84 Figure 17.--Effect of soluble Sperm fractions on the protease activity of homogenates of unfertilized eggs and 60 minute postinsemination zygotes of Strongylocentrotus purpuratus. a. b. unfertilized egg homogenate (2.5%) and purified chromatin (8 OD/ml). unfertilized egg homogenate (2.5%) and crude chromatin (10.8 OD/ml). unfertilized egg homogenate (2.5%) and crude chromatin (10.8 OD/ml), with combined soluble Sperm fractions. 60 minute zygote homogenate (2.5%) and purified chromatin (8 OD/ml). 60 minute zygote homogenate (2.5%) and crude chromatin (10.8 OD/ml). 60 minute zygote homogenate (2.5%) and crude chromatin (10.8 OD/ml), with combined soluble sperm fractions. fresh unfrozen unfertilized egg homogenate (2.5%) and crude chromatin (10.8 OD/ml). freeze-thawed (2x) unfertilized egg homogenate (2.5%) and crude Sperm chromatin (10.8 OD/ml). crude chromatin (10.8 OD/ml) with no additions. All samples were incubated 25 hours at 24°C. All samples contained 10 mM Tris (pH 8), 0.25 M NaCl, and 13 mM KCl. Samples were fractionated on an acid-urea polyacrylamide gel, origin at the tap. 85 EiliLst an -u- -‘ nus-000000 gnu .6.‘“ . '00' 2A 86 60 minutes after fertilization, no degradation of sperm histones occurred, with or without added soluble sperm fractions (Figure 17). If the 60-minute zygote homogenate is added to an unfertilized egg homogenate and the combi- nation incubated with sperm chromatin, there is only a slight loss of activity (Figure 18). Figure 18 also shows the same result if hemoglobin is used as a substrate instead of chromatin. These results can be explained at least two ways. An inhibitor may be present at 60 minutes preventing protease activity. This inhibitor would have to have no effect on the unfertilized egg protease. An alternate explanation is that, at 60 minutes, the histone protease enzyme has itself been degraded. The effect of temperature on unfertilized egg protease activity is shown in Figure 19. Sperm chromatin and egg homogenate were incubated at 15°C, 24°C,cn:35°C for 7.5 hours, 21 hours, or 30 hours. It is apparent that as the time or temperature is increased, the degree of degradation is increased. The increase in digestion from 21 hours to 30 hours at 35°C indicates that the protease activity is stable at 35°C for more than 21 hours. Interpretation of this experiment is complicated by the presence of degradation in the control chromatin (Figure 19a) that was incubated at 25°C for 30 hours with- out homogenate. However because of an error this chromatin 87 Figure 18.--Lack of inhibition of the protease activity of unfertilized egg homogenates by homogenates of 60 minute postinsemination zygotes of Strongylocentrotus purpuratus. a. sperm chromatin without additions. b. unfertilized egg homogenate (2.5%) plus sperm chromatin. c. zygote homogenate (2.5%) plus sperm chromatin. d. unfertilized egg homogenate (2.5%) and zygote homogenate (0.5%) plus sperm chromatin. e. unfertilized egg homogenate (2.5%) and zygote homogenate (1.0%) plus sperm chromatin. f. unfertilized egg homogenate (2.5%) and zygote homogenate (1.5%) plus sperm chromatin. g. unfertilized egg homogenate (2.5%) plus hemoglobin. . h. zygote homogenate (2.5%) plus hemoglobin. i. unfertilized egg homogenate (2.5%) and zygote homogenate (0.5%) plus hemoglobin. j. unfertilized egg homogenate (2.5%) and zygote homogenate (1.0%) plus hemoglobin. k. unfertilized egg homogenate (2.5%) and zygote homogenate (1.5%) plus hemoglobin. 1. hemoglobin with no additions. Samples were incubated 24 hours at 35°C. All samples contained 10 mM Tris (pH 8), 0.4 M NaCl, 21 mM KCl and either 20 OD/ml crude Sperm chromatin or 2mg/ml hemoglobin. Samples were fractionated on a SDS polyacrylamide gel. Hb, hemoglobin; 1, 2A, 28, 3, and 4 designate the various histones as in the other figures. 88 - i m ’ 5v 89 Figure 19.--Effect of time and temperature of incubation on the protease activity of unfertilized egg homogenates of Strongylocentrotus purpuratus. Time Temperature hr °C a. 30 24 Sperm chromatin, no additions b. 7.5 15 sperm chromatin and egg homogenates c. 7.5 24 sperm chromatin and egg homogenates d. 7.5 35 sperm chromatin and egg homogenates e. 21 15 Sperm chromatin and egg homogenates f. 21 24 sperm chromatin and egg homogenates g. 21 35 sperm chromatin and egg homogenates h. 30 15 Sperm chromatin and egg homogenates i. 30 24 sperm chromatin and egg homogenates j. 30 35 sperm chromatin and egg homogenates k. 30 24 sperm chromatin and egg homogenates: l. 30 24 sperm chromatin and egg homogenates *egg homogenate centrifuged at 12,000 x g for 10 minutes, pellet resuspended to original volume. * *12,000 x 9 egg homogenate supernatant. All samples contained 10 mM Tris (pH 8), 0.4 M NaCl, 21 mM KCl, 10 OD/ml crude sperm chromatin, and 3% egg homogenate. Samples were fractionated on an acid-urea polyacrylamide gel. 90 UHHHH H" ”3”“ Mt: H zlAkfiUHUflH .00” annuuuuu. ”a: 4 .0- "33 toll 91 probably did contain homogenate. The appearance of a low mobility band in all the samples, a-f, but not in sperm chromatin without homogenate, as in Figure 16c, is evidence that this was the case. However, the degradation of histones at both 21 hours (Figure 191) and 30 hours (Figure 19j) at 35°C is more extensive than the "control" chromatin degradation. Thus, there is an increase in digestion at 35°C between 21 and 30 hours, and the conclu- sion that the homogenate protease is stable for more than 21 hours at 35°C is valid. The unfertilized egg protease activity is found in both the supernatant and the pellet after centrifugation at 12,000 x g for 10 minutes as Shown in Figure 19, k and 1. More activity is found in the supernatant (1) than in the pellet (k). It should be noted that the low mobility egg homo- genate band seen in Figure 19 does not have the same mobility as bands A, B, and C of egg histones (Figure 10). The egg homogenate band has a relative mobility of 0.31 compared to 0.25 for band A, 0.42 for band B, and 0.47 for band C. Histone Protease Activity in the Sea Urchin Sperm While sperm histones can be degraded by egg proteases, histone protease activity cannot be consistently found in the sperm of Strongylocentrotus purpuratus. 92 The effect of pH and added salts is shown in Tables 2 and 3. No digestion was observed at any pH tested between 6.0 and 8.0 (Table 2). No digestion was detected when concentrations of Na+, K+, Mg++, and Ca++ were varied, with or without the addition of the soluble sperm frac- tions (Table 3). Some degradation was found when Triton x-100 and the soluble sperm fractions were added to crude chromatin (Table 4). In experiment 1, the digestion occurred with Triton X-100 and fractions 1 and 2. In experiment 2, digestion occurred with fraction 1 with or without Triton X-100, and with fraction 3 but only if Triton X-100 was present. In experiment 3 degradation took place with Triton and fraction 3, but did not occur if fractions 1 and 2 were also added. No digestion was seen in experi- ments 4 or 5, in which samples were run in duplicate. This discrepancy may be due to contamination. In experi- ment 4, the incubation tubes were heated to 158°C for two hours before use and extra care was taken to prevent other contamination. No degradation was seen in this experiment or the following experiment. When sperm chromatin was assayed for protease activity in the presence of the reducing agent 2-mercaptoethanol and the soluble sperm fractions, with or without Triton X-100, no di- gestion was observed (Table 4, experiments 1, 6, and 7). 93 TABLE 2.--Effect of pH on endogenous Sperm histone protease. Purified NaCl KCl MgC12 CaCl2 Chromatin pH mM mM mM mM OD/ml 6.01 200 20 10 3.5 2.0 6.01 200 20 -- -- 2.0 7.02 200 20 10 3.5 2.0 7.02 200 20 -- -- 2.0 7.52 200 20 10 3.5 2.0 7.52 200 20 -- -- 2.0 8.02 200 20 10 3.5 2.0 8.02 200 20 -- -- 2.0 lso mM phosphate buffer. 2100 mM Tris buffer. Incubation: 26 hours, 14°C, 0.5 m1 total volume. No Degradation Observed in Any Sample. 94 TABLE 3.--Effect of salts and soluble sperm fractions on endogenous sperm histone proteases. Soluble Sperm NaCl KCl MgCl2 CaCl2 Fractions Chromatin mM mM mM mM 0.2 ml/ml OD/ml 200 20 10 3.5 -, 1, 2, 3 2.0c 200 20 -- -- -, 1, 2, 3 2.0c 200 5 10 3.5 -, 1, 2, 3 12.0cl 200 5 1 17.5 -, 1, 2, 3 12.0o1 200 5 -- -- -, 1, 2, 3 12.0c1 50 5 50 5 -, 1, 2, 3 12.4ol 50 5 -- -- -, 1, 2, 3 12.4cl 50 200 13 5 ---- 10.4p 25 100 6.5 2.5 ---- 10.4p 12.5 50 3.25 1.25 ---- 10.4p 20 -- 1 35 -, 1, 2, 3 2.0c -- -- so 5 -, 1, 2, 3 12.4c1 -- -- -- -- ---- 10.4p -- -- -- —- -, 1, 2, 3 2.0c -_ __ -- -- -, 1, 2, 3 12.0ol 1 Incubated 19 hours at 24°C, all other samples incubated 24 hours at 24°C. All samples contained 10 mM Tris (pH 8.0). Total volume 0.5 ml. c = crude chromatin, p = purified chromatin. No Degradation Observed in Any Sample. 95 TABLE 4.--Effect of Triton X-100, soluble sperm fractions, and 2-mercaptoethanol (2-ME) on endogenous sperm protease. Soluble Sperm Crude Exp. 2-ME Fractions Chromatin Time No. mM Triton 0.2 ml/ml OD/ml hr Effect 1 - - 1 8.0 30 - - — 2 8.0 30 - - - 3 8.0 30 - - - - 8.0 30 - - + l 8.0 30 ++ - + 2 8.0 30 ++ - + 3 8.0 30 - - + - 8.0 30 - 88 + 1 8.0 30 - 88 + 2 8.0 30 - 88 + 3 8.0 30 - 88 + - 8.0 30 - 2 - - l 9.2 14.5 +++++ - - 2 9.2 14.5 - - - 3 9.2 14.5 - - - - 9.2 14.5 -+ - + l 9.2 14.5 +++++ - + 2 9.2 14.5 - - + 3 9.2 14.5 -+ - + - 9.2 14.5 + 3 - - 1: 13.4 21 - - - 1 13.4 21 - - - 1 13.4 21 - - - 2 13.4 21 - - - 3 13.4 21 - - - - 13.4 21 - - - l, 2, 3 13.4 21 - - + 1 13.4 21 - - + 2 13.4 21 - - + 3 13.4 21 +++++ - + - 13.4 21 - - + l, 2, 3 13.4 21 - 4 - - 1 13.2 20.5 - — - 2 13.2 20.5 - - - 3 13.2 20.5 - - - - 13.2 20.5 - - + 1 13.2 20.5 - - + 2 13.2 20.5 - TABLE 4.--Continued. 96 Soluble Sperm Crude Exp. 2-ME Fractions Chromatin Time No. mM Triton 0.2 ml/ml OD/ml hr Effect - + 13.2 20.5 - - + - 13.2 20.5 - 5 - - l 9.4 19 - - - 2 9.4 19 - - - 3 9.4 19 - - - - 9.4 19 - - + 1 9.4 19 - - + 2 9.4 19 - - + 3 9.4 19 - - + - 9.4 19 - 6 88 - l 1.0 15.5 - 88 - 2 1.0 15.5 - 88 - 3 1.0 15.5 - 88 - - 1.0 15.5 - 7 88 - 1 12.4 19 - 88 - 2 12.4 19 - 88 - 3 12.4 19 - 88 - - 12.4 19 - *Same supernatant as experiment 2. All samples run in 0.5 ml, 10 mM Tris, pH 8.0, at 24°C. Triton - 0.2% Triton X-100, 0.1% sucrose, 0.2 mM MgCl 2. 97 As already shown in Figure 16, no latent protease activity is present in the sperm that can be released or activated by small molecules present in the egg homogenate. Just as sperm histone protease activity is not present in the sperm of Strongylocentrotus putpuratus, neither does the sperm contain pH 8 protease activity against hemoglobin as shown in Figure 20. Crude sperm chromatin and hemoglobin were incubated with (b) or without (j) combined soluble sperm fractions at 25°C for 24 hours. No digestion occurred as judged by samples h and p which had trypsin added. An experiment performed with Lytechinus pictus gave the same result. 98 Figure 20.--Absence of histone Or hemoglobin protease activity in the Sperm of Strongylocentrotus purpuratus. a. b. c. d. e. f. g. h. i. j. k. 1. sperm chromatin plus hemoglobin, unincubated. sperm chromatin plus hemoglobin, incubated 24 hours. sperm chromatin, unincubated. Sperm chromatin, incubated 24 hours. hemoglobin, unincubated. hemoglobin, incubated 24 hours. hemoglobin plus 5 ug trypsin, incubated 10 minutes. Sperm chromatin and hemoglobin plus 5 pg trypsin, incubated 10 minutes. sperm chromatin and combined soluble sperm fractions plus hemoglobin, unincubated. Sperm chromatin and combined soluble sperm fractions plus hemoglobin, incubated 24 hours. sperm chromatin and combined soluble Sperm fractions, unincubated. sperm chromatin and combined soluble sperm fractions, incubated 24 hours. hemoglobin and combined soluble sperm fractions, unincubated. hemoglobin and combined soluble sperm fractions, incubated 24 hours. hemoglobin and combined soluble sperm fractions, plus 5 ug trypsin, incubated 10 minutes. . sperm chromatin and combined soluble sperm fractions, with hemoglobin and 5 pg trypsin, incubated 10 minutes. All samples contained 10 mM Tris (pH 8). Samples with chromatin contained 40 OD/ml crude chromatin; samples with hemoglobin contained 2 mg/ml hemoglobin. Samples were fractionated on a SDS polyacrylamide gel. Hb, hemoglobin; 1, 2A, 28, 3, and 4 designate the various histones. 99 1.... u.- 20.... ”-- 3“ u.- 2‘ ---- ---- A 4 a... .-..u."5 DISCUSSION Histones Are Present in Sea Urchin Eggs and Zygotes Among those who disputed the existence of histones in very early development, only Johnson and Hnilica (1970, 1971) presented electrophoretic patterns of the acid- soluble proteins extracted from eggs or zygotes. Johnson and Hnilica (1970) showed the electrOphoretic pattern of acid-soluble nuclear proteins from unfertilized eggs of Strongylocentrotus purpuratus. Several bands were present in the region of sperm and blastula histones. When they washed their nuclei with Triton X-100 according to Thaler gt gt. (1970), no hands were found. Johnson and Hnilica (1970) stated that the bands did not, therefore, represent typical histones and that the loss of the bands after detergent treatment of the nuclei indicated that the bands were due to cytOplasmic contamination. A further reason for their 1970 rejection of the presence of histones was that trypsin treatment of egg nuclei did not result in a large increase of template activity as treatment of sperm or blastula nuclei did. Since typical histones are known to be sensitive to trypsin, histones must therefore not be present. In a later paper (Johnson 100 101 and Hnilica, 1971), they repeated the claim that typical histones are not present in the egg, zygote or cleavage stage nucleus. They instead found a protein similar to the F3 (H3) in mobility and two prominent bands of lower mobility. They stated that since the zygote lacked histones, sperm histones must have been lost. In contrast to Johnson and Hnilica (1970, 1971), Evans and Ozaki (1973) found histones in the sea urchin egg. Evans and Ozaki showed that the proteins were histones according to three criteria. (1) The proteins were acid extracted from micrOSCOpically pure nuclei which were washed to remove nuclear sap and adhering ribosomes; therefore the proteins meet the criterion of histones by solubility. (2) Some of the histones thus extracted had mobilities either Similar to or identical with the mobility of some of the somatic histones. (3) The electrophoretic pattern of the egg histones was characteristic of the nucleus and could not be derived from ribosomes or yolk granules, the most likely source of contamination. ‘Because the histones shown in the electrOphoretic patterns of the present work were prepared in the same manner as those of Evans and Ozaki, the same arguments apply to these patterns. These arguments may be strength- ened by some of the present results. The second argument 102 of similar mobility may be expanded from one gel system to three. All of the egg-zygote histones, discounting the probable aggregates A, B, and C, have mobilities in the range of sperm and somatic histones. Two of the egg- zygote histones have the mobility of the sperm and somatic H3 and H4, as discussed later. The third argument that the histones have mobility different from likely contaminants has also been shown to be true by electrophoresis on SDS polyacrylamide gels (Figure 9). A fourth argument against the pattern being due to contamination may be added. In all experiments the amount of histone run on a gel was based on the amount of chromatin extracted, not the number of nuclei extracted. Thus, in Figure 6, the amount of protein in the 4 histone bands is similar although 5 times more egg than zygote nuclei were extracted. If the 4 histone bands were cytOplasmic or nuclear sap contaminants, the amount of protein in the histone bands should be much higher in the egg than in the zygote. But, in fact, the amount of histone extracted is roughly proportional to the amount of chromatin extracted. Thus the proteins must be of chromosomal origin. This argument is further strengthened by the appearance of a large amount of contaminating proteins in the egg pattern of Figure 6. A larger amount of contamination would be expected in 103 the egg because of the large number of nuclei extracted. Despite the obvious increase in contamination, the strength of the egg histone bands is still roughly prOportional to the amount of chromatin. This increase in contamination might explain the larger amount of histones D and E in the egg relative to bands 3 and 4 (Figure 12), possibly some contaminant has the mobility of the histones D and E. The lack of bands with the mobility of D and E in Figure 9, an acid extract of heavy cytOplasmic components, makes this unlikely how- ever. In summary, the proteins presented as egg-zygote histones in this thesis are histones because: (1) They are acid extracted from purified nuclei in amounts indicating chromosomal origin; (2) All of the proteins have mobilities similar to histones while two of the proteins have the same mobility as sperm and somatic histones in three electrophoretic systems; (3) Heavy cytoplasmic components which would be expected to con- taminate nuclei have different mobilities on acid-urea (Evans and Ozaki, 1973) and SDS gel systems; and (4) The presence of contamination does not increase the amount of histone present. 104 Fate of §perm Histones in the Zygote Acid-urea and SDS polyacrylamide gel patterns of Strongylocentrotus purpuratus zygote histones do not show the sperm-specific histones H1 and HZB. The lack of these histones cannot be due to proteolytic degradation during nuclear isolation, because the presence of an irreversible protease inhibitor, PMSF (Farney and Gold, 1963), during nuclear isolation has no effect on the histone pattern. Another indication that sperm histones were not degraded during preparation of the zygote histones is that the proteins extracted from zygotes show no signs of degradation products. The presence of many high molecular weight contaminants from egg nuclei extracted without PMSF (Figure 5) further suggests that protease activity during isolation is not a problem. The finding that zygotes do not contain any protease activity at 60 minutes after fertilization when the zygote histones of Figures 6 and 9, were extracted also supports the conclusion that the Sperm-specific histones were lost from the DNA before the nuclei were isolated. The complete loss of the sperm HZA is not as certain as the loss of the sperm H1 and H23. A minor band with the mobility of the sperm HZA is present in the acid-urea pattern of zygote histones (Figure 10). However, a band with the mobility of the sperm HZA also appears in the egg which had no contact with the sperm, 105 and in the gastrula where it would have been diluted out if of sperm origin. Easton and Chalkley (1972) also found a band with the mobility of the sperm f2a2 (H2A) in Arbacia punctulata prism histones. It is likely that this egg, zygote, and gastrula band is a normal, minor component of the histones of all stages. There is thus no reason to believe that this histone, which is present in small amounts, is of sperm origin although this cannot be ruled out. Similarly, the zygote band labeled D in Figure 11, is present in Sperm, blastula, and gastrula (Figure 6), and may also be a normal minor component. Evidence that proteases are involved in the histone removal is indirect. As shown in this thesis, sperm histone protease activity is present in the egg and early zygote. Kunkle, Magun, and Longo (1978) have shown that sperm nuclei will not decondense if added to egg homo- genate that contains the protease inhibitor PMSF. If the histones are removed with acid Or salt before the sperm nuclei are added to the homogenate, the nuclei do decondense. When Kunkle gt El! (1978) used homogenate prepared by a different method without protease inhibitors, the untreated nuclei did decondense. The lack of decon- densation in the presence of PMSF suggests that proteases are involved in the decondensation which is probably when 106 sperm histones are lost. It is possible, however, that some other enzyme was affected by the PMSF. An experiment by Penn 33 a1. (1976), however, seems to indicate that proteases are not involved. They incubated fertilized eggs of Strongylocentrotus purpuratus and bytechinus pictus in various inhibitors of protease activity. One inhibitor (TLCK) was found to delay each cleavage but had no other observable effect. This sug- gests that it had no effect on decondensation of the sperm. However, the inhibitor was added between one minute and 10 minutes after fertilization. It is probable that insufficient inhibitor entered the egg soon enough to prevent decondensation, which probably is finished 5-15 minutes after fertilization. Thus the results of Penn gt El- (1976) are not sufficient to disprove the involvement of protease in sperm histone removal. Histones of the Egg and Zygote The egg histone patterns presented in this thesis are consistent with the patterns of Evans and Ozaki (1973). Evans and Ozaki used a different acid-urea polyacrylamide gel electrophoresis system and found that some of the histones of the egg have the mobility of blastula and sperm histones, but the egg also has unique components. 107 Evans and Ozaki (1973) found a fast band with the mobility of the sperm and blastula F2a1 (H4) but no egg band with the mobility of the F3 (H3). The apparent lack of an egg component with the mobility of the sperm and blastula H3 might have been due to microheterogeneity of the histone. The acid-urea polyacrylamide gel patterns of the present work (Figure 10) shows that such microheterogeneity exists in both the H3 and H4. This heterogeneity is due to postsynthetic modifications such as acetylation and phosphorylation (0rd and Stocken, 1968; Johnson et_§1., 1973; Wouters-Tyrou gt 31., 1976). The SDS polyacrylamide gel patterns of Figures 6, 8, and 9, are not affected by such modifications because the separation is primarily by the size of the molecule. These figures show that bands with the mobility of both the H3 and H4 are present in all stages. Since the acid- urea pattern of Figure 10 is also consistent with the presence of H3 and H4 at all stages of development, it may be tentatively concluded that the H3 and H4 are, in fact, common to sperm, egg, zygote, blastula, and gastrula. Evans and Ozaki (1973) reported the presence of a very slow moving band in the egg. The figure showing their egg pattern shows this band and a band with still lower mobility. These bands probably correspond to bands A, B, and C in the acid-urea gel pattern of Figure 11a, 108 which do not appear on the SDS gel of Figure 11b. These bands do, however, appear in the pattern of the two- dimensional gel of Figure 11c. The most likely reason that these bands appear in the acid-urea gel but not the SDS gel is that bands A, B, and C are aggregates or complexes of histones 3, 4, D, E, and F. Their presence on the two-dimensional gel may be exPlained if the denaturing conditions were not sufficient to dissociate the histone complex after infiltration of the acid-urea gel with SDS sample buffer between the first and second dimension separations. Evidence that the denaturing conditions were not sufficient to dissociate complexes or dimers is the presence of both oxidized and reduced forms of the sperm H3 in the two-dimensional gel of Figure 2c. It is likely, therefore, that the absence of bands A, B, and C in the SDS gel of Figure 11b is because they are aggregates or multimers which were broken down to their component histones in the SDS system. Because these bands did not break down on the two-dimensional gel, it is not possible to state which histones are involved in the complex. In addition to Evans and Ozaki (1973), there have been only three papers published with electrophoretic patterns of egg or zygote histones (not counting Johnson and Hnilica (1970, 1971), who claimed no histones exist 109 in the egg and zygote): Repsis, 1967; 0rd and Stocken, 1968; and Thaler 2E.§l°r 1970. Repsis found that the electrOphoretic pattern of the acid-soluble nuclear proteins of fertilized eggs of Bytechinus variegatus is similar to the pattern of blastula and pluteus, but, as reported by Evans and Ozaki (1973) and in this thesis, there are some differences between zygote and later stages. Repsis did not characterize any of the bands and noted that since the nuclei were not washed, acid soluble cytoplasmic proteins may also have been extracted. This may be the reason so many bands, 13 or more, are present in the gel pattern. The histones of the sperm migrated in a completely different part of the gel. Because of this, Repsis stated that the zygote contains only maternal acid-soluble proteins. An electrophoretic pattern of acid-soluble nuclear proteins of Arbacia punctulata, Paracentrotus lividus and Echinus esculentus zygotes was published by 0rd and Stocken (1968). They reported that all classes of histones except for the F1 (H1) are present in sea urchin zygotes. Interpretation of their patterns in terms of changes between sperm and zygote or zygote and later stages cannot be made because only zygote and rat thymus patterns are shown. The sea urchin zygote histone patterns do show six or more bands in one group and 110 additional bands with very low mobility. 0rd and Stocken (1970b), using purified nuclei from Echinus esculentus, reported that the fastest major electrophoretic band (H4?) was prominent only in extracts from embryos, not cleavage stages. They found that a band tentatively identified as the F3 (H3) was present during early cleavage. Two slow moving bands were also present in the 1-2 cell stage. A band with the mobility of the F1 (H1) became increasingly evident as develOpment progressed. However the patterns cannot be compared because no electrOphoretic patterns were presented by 0rd and Stocken; rather reference was made to the patterns published earlier (0rd and Stocken, 1968) which showed zygote histones prepared by a different method. Thaler gt_§1. (1970) examined histones extracted from unfertilized eggs of Arbacia punctuala. They found that the egg and embryo patterns are different. They said that the egg histones migrated predominately in the low mobility zone of the gel with F1 (H1) and F3 (H3), whereas the embryo has most of the histones in a region of higher mobility. Inspection of the published gel patterns shows that some of their egg histones have the same mobility as embryonic histones and some bands have different mobility. There is some question about the work of Thaler gt_al, however. Evans and Ozaki (1973), 111 who performed a similar type of electrophoretic frac- tionation on histones of Strongylocentrotus purpuratus found that the embryonic electrophoretic patterns were very different from those of Thaler gt El; Since the sperm and embryo histone patterns of Arbacia punctulata (Easton and Chalkley, 1972) are the same as those of Strongylocentrotus purpuratus (this thesis), the results of Thaler gt 21° are called into question. Acid-soluble chromosomal proteins synthesized by Arbacia punctulata during the first cell division were examined by Ruderman and Gross (1974). They found incorporation of labeled lysine into all five classes of histones although incorporation into the F2a1 (H4) was very low. They stated in the discussion, that their results do not support the existence of juvenile or atypical histones and that their data demonstrate that newly synthesized histones of all five classes are associated with the chromatin as early as the first cell division. These claims appear to be in contradiction to the zygote patterns shown in this thesis. According to the present work, no typical Hl, H2A, or H2B are detectable in the zygote by staining methods but atypical histones are present. These apparent contradictions may not be real. Ruderman and Gross only examined the histones made at the time of first cleavage. If the egg-zygote histones were 112 made before this time and stored, they could not have been detected by Ruderman and Gross. On the other hand, the presence of a small amount of the embryonic histones H1, H2A, and H2B could not be detected in the zygote by the present staining method but may be detectable by the use of radioactive label techniques. It may be pointed out that the two-cell embryos extracted by Ruderman and Gross have completed at least one more round of DNA synthesis than the zygotes studied in this thesis (Arceci and Gross, 1977), and would thus have more histones to detect. The conclusion that newly synthesized histones are associated with the chromatin by first cleavage is open to question. Ruderman and Gross extracted the chromatin by the method of Benttinen and Comb (1971). This method purifies chromatin through sucrose from a homogenate of whole egg, not nuclei. It is probable that histones in the cytoplasm would adhere to the chromatin during chromatin preparation. Because of this possible contamination, it cannot be known for certain that the radioactive proteins were, in fact, associated with the chromatin in the nucleus. These questions could be removed by repeating the experiments of Ruderman and Gross using methods which involve nuclear purification before chromatin isolation. 113 In a related paper, Arceci and Gross (1977) fractionated the total newly synthesized protein of Arbacia punctulata zygotes in SDS polyacrylamide gradient gels. This gel system gives a pattern very similar to that of Figure 6. When proteins are labeled during the first period of DNA synthesis, 20-35 minutes after fertilization, a small peak in the position of the zygote histone D is found. Much larger peaks are found in the positions of the H3, H4, and the combined H2A, HZB, E, and F peak. A large amount of protein was being synthesized in the general region of the H1 so that any peak from H1 synthesis would have been obscured. If the zygotes were labeled later than 35 minutes after fertili- zation, the peak in the area of egg-zygote histone D is no longer detectable. These results suggest that the egg-zygote histones are not made after fertilization and thus must be stored histone. The actual amount of histone synthesized was not measured by Arceci and Gross, so it is not known whether the new histone could be detected by staining if it was immediately associated with the DNA. While Ruderman and Gross (1974) denied the presence of juvenile or atypical histones during cleavage, they did find stage specific forms of histone Fl (H1). They found morula, batched blastula, and gastrula specific forms. 114 Cohen gt 31, (1975) found that stage specific forms of the H2A and H2B also exist in Strongylocentrotus purpuratus by studying newly synthesized histones. They labeled embryos of the appropriate stage, then allowed further development in the presence of cold amino acid. The histones were extracted at a much later stage. They found one histone-like protein which was synthesized from fertilization to the 16 cell stage, but was conserved after the 16 cell stage. Stage specific forms of the H2A and H2B were also found. One form of each was synthesized from fertilization to the mesenchyme blastula stage and conserved from then on. These histone variants could only be fractionated on an acid-urea polyacrylamide gel containing Triton X-100 and could not be fractionated on the standard Panyim and Chalkley acid-urea gel. Thus Ruderman and Gross (1974) could not have detected them. The Panyim and Chalkley gels of the present work, however, clearly show the presence of egg-zygote histone variants. It is thus unlikely that the egg-zygote variants are the same as the stage specific forms of HZA and HZB found by Cohen §E_§1. (1975). Cohen 23 31., like Ruderman and Gross (1974) and Arceci and Gross, only looked at newly synthesized histones and thus could not have detected the zygote histones if they were made before the labeling period. 115 Cognetti et 21. (1974) studied the synthesis of acid-soluble proteins during oogenesis in Paracentrotus lividus. They extracted 3H-lysine labeled, whole oocytes with acid. They then subjected the acid-soluble proteins to coelectroPhoresis with 16 cell stage or gastrula histones. They found that some oocyte proteins had the same mobility as the histones of the gastrula and the histones of the 16 cell stage. Similar results were found if the proteins were fractionated on Amberlite CG 50. However, since there was very high incorporation into many other fractions and since no other tests for histones were done, their conclusion that they found histone synthesis in the oocyte is tenuous. Cognetti et_§l. (1977) continued the study of histone synthesis during oogenesis of Strongylocentrotus purpuratus. They again fractionated acid-soluble proteins from whole oocytes by polyacrylamide gel electrophoresis and again found a great many bands. They then cut out a band that had the mobility of the gastrula F2b (H2B) and compared the amino acid composition to the gastrula histone. They found that the amino acid composition of the oocyte band was similar to that of the gastrula histone. If the amino acid composition of the gastrula F2b (H2B) of Cognetti 33 a1. (1977) is compared to the F2b-like histone of Johnson et_gl. (1973) from the pluteus stage of the same species, however, the agreement is not good. Cognetti 116 33 31. found 31 mole % glycine while Johnson gt_al. found only 7.0 mole % glycine in the F2b-like protein, and only 11.4 mole % glycine in any pluteus histone. Similarly, Cognetti gt_al. reported 15.2% alanine, 10.6% lysine, and 11% serine (recalculated without glycine), while Johnson gt_al. reported 13% alanine, 15% lysine, and 9.2% serine (recalculated without glycine). These discrepancies make questionable the conclusion that F2b (H2B) is synthesized in the oocyte. Thus the work of Cognetti gt 31. (1974, 1977) does not support or contradict the suggestion that the egg-zygote histones are made before fertilization. Egg Protease The pH 8 protease activity described in this thesis is similar to the pH 8 protease activity of Mano (Mano, 1966; Mano and Nagano, 1970), the activity of Krischer and Chambers (1970), and the E2 activity of Lundblad (Lundblad, 1954a; Lundblad g£_§1., 1972). Mano (Mano, 1966; Mano and Nagano, 1970) reported the presence of pH 8, trypsin-like protease activity in fertilized eggs of Hemicentrotus pulcherrimus but not in unfertilized eggs. The present work found pH 8 activity in both unfertilized and fertilized eggs. This difference is probably due to homogenization: low salt with calcium in Mano's work; isotonic salt without calcium in the 117 present work. Krischer and Chambers (1970) found that much of their protease activity was lost if calcium was present in their isotonic salt solution during homogeni- zation of unfertilized or fertilized eggs. Mano reported that the activity of the fertilized egg was much greater at 10 minutes after fertilization than at 60 minutes after fertilization, which is in agreement with the results reported in this thesis. Krischer and Chambers (1970) homogenized un- fertilized and fertilized eggs of Bytechinus variegatus in an isotonic salt solution. They found protease activity in the range of pH 7 to pH 12. From substrate analysis they established that most of the activity was chymotrypsin-like, but a small amount of trypsin-like activity was also present. The unfertilized egg protease activity described in this thesis was active for more than 20 hours at 35°C; their activity was stable at pH 7.4 for at least 20 hours at 0°C. The protease activity of Krischer and Chambers and the fertilized egg protease activity described in this thesis are decreased by 2-5 mM EDTA and 0.5-1.5 mM EDTA respectively. The unfertilized egg protease activity described in this thesis was not reduced by 3.3 mM EDTA, suggesting that it is a different enzyme than that of the fertilized egg or that described by Krischer and Chambers. Krischer and Chambers did not 118 test for the presence of protease activity at any time later than 15 minutes after fertilization. At 15 minutes postfertilization they did not see any loss of activity. Unlike the protease activity described in this thesis, their protease activity was found only in the material that sedimented at 10,000 x 9. However, they used fresh homogenate whereas the homogenate used in this thesis was first frozen. The protease in the supernatant may have been solublized by freezing and thawing. From the available evidence, it appears that the fertilized egg protease activity described in this thesis is like the protease activity of Krischer and Chambers. Lundblad (Lundblad, 1954a; Lundblad g£_al., 1972) found a calcium-requiring protease, E2, with an optimal pH of 7.2 with hemoglobin as a substrate, but also active at pH 8. This enzyme was most easily extracted from the fertilized egg but some activity could be detected in the unfertilized egg. The activity of this enzyme increased sharply at fertilization.- Soon after fertili- zation the activity of E2 declined and was replaced by the activity of proteases E1 and E3, which do not require calcium but do require cysteine or some other reducing agent. The activity of these enzymes also declined after a short time. The fertilized egg enzyme activity described in the present work is like the E2 activity because the activity is reduced in the presence of EDTA. 119 In the unfertilized egg, the protease activity is un- affected by EDTA but is slightly reduced by cysteine. This suggests that an enzyme other than Lundblad's El, E2, or E3 is present. It is apparent that the protease activation scheme proposed by Lundblad (1954a) does not explain the histone protease results found in this thesis. According to Lundblad, the EDTA-sensitive activity should be gone by about 10 minutes after fertilization. Before this time, the addition of cysteine should reduce the EDTA-sensitive activity while stimulating the EDTA-insensitive activity of the El and E3 enzymes. However, I have shown that EDTA-sensitive activity is present in both Strongylocentrotus purpuratus and Bytechinus pictus at 25 minutes after fertilization (Figures 13 and 14). This is contrary to what Lundblad observed. It is possible that the histone protease activity described in this thesis does not follow the pattern of protease activation described by Lundblad because of Species differences, differences in the way the activity is extracted, or differences in the sub- strates used, histones rather than hemoglobin, casein, or gelatin. Another possibility is that the histone protease activity is due to enzymes entirely different from those described by Lundblad. Only additional work can determine whether the enzymes involved in the 120 degradation of sperm histones are the same as those described by Lundblad, Mano, or Krischer and Chambers. Sperm Protease Protease activity against sperm histones was not found in sea urchin sperm. This lack of protease activity is in contrast to the work of Lundblad (Lundblad, 1949, 1950, 1954c; Lundblad and Johansson, 1968) and Harris (Harris et 31., 1977), who found protease activity in extracts of sea urchin sperm and to the work of Stambaugh and Buckley (1972) who found binding of soy bean trypsin inhibitor to whole sea urchin sperm. It is possible that sperm protease activity was lost during preparation of the sperm chromatin and fractions. The sperm in the present work were first washed in saline-EDTA. If the protease was located on the surface of the sperm, it could have been washed away or inactivated at this step. Next the sperm were homogenized in saline-EDTA. A protease could have been solublized at this time; however, the saline-EDTA soluble material was added to the chromatin during incu- bation but no protease activity was observed. It may be that a protease was present in this fraction but was inactivated by the saline-EDTA, or was present in amounts too small to be detected. These same possibilities apply to the 0.01 M Tris supernatant fractions. Any protease 121 present in the final chromatin could also have been inactivated during preparation. Lundblad (Lundblad, 1954c; Lundblad and Johansson, 1968) in contrast, extracted whole sperm in such solvents as distilled water, M KCl, 0.1 M acetic acid, and 0.15 M LiCl for periods ranging from 17 hours to 5 days. He found that it was necessary to use lyophilized sperm or fresh sperm that had been freeze-thawed to obtain good activity. The sperm were washed, sedimented and frozen in sea water. The thawed or lyophilized sperm were extracted immediately, without additional washing. Likewise, Harris §E_§l. (1977) extracted protease activity from sperm that had been frozen and thawed. The sperm tested for protease activity in this thesis were either fresh or frozen in glycerol. In either case, the sperm were washed in saline-EDTA immediately before the first homogenization. This difference in the washing of sperm before extraction or homogenization suggests that a protease may have been lost during the initial saline-EDTA washes of the whole sperm. If the protease was bound to the outside of the sperm, or was located in a vesicle that is sensitive to EDTA or low osmolarity, the saline-EDTA wash would result in a loss of the protease. The sea water washes of Lundblad would have no effect. The presence of a protease located in a vesicle or on the 122 sperm plasma membrane is consistent with the work of Stambaugh and Buckley (1972). They dried whole sea urchin sperm on a slide and added labeled soy bean trypsin inhibitor. The soy bean trypsin inhibitor was localized by immunological techniques in the acrosomal region of the Sperm. Harris et El- (1977) described chymotrypsin-like activity in Arbacia punctulata sperm, which they suggested was part of the fertilization product released into the surrounding sea water during sea urchin fertilization. This again suggests a surface or vesicle protease that is easily dissociated from the sperm. Marushige and Marushige (1975) and Zirkin and Chang (1977) both found that the addition of reducing agents and Triton X-100 to bull or rabbit sperm nuclei resulted in the liberation or activation of protease activity. This protease activity resulted in the loss of the sperm protamines. Marushige and Marushige found that this protease activity was localized in the bull sperm chromatin. No such protease activity was found in sea urchin chromatin when it was incubated with 2- mercaptoethanol with or without Triton X-100. While the sea urchin chromatin and its fractions were without protease activity, the addition of the soluble Sperm fractions (SSF) to the egg homogenate- sperm chromatin incubation mixture resulted in increased 123 histone protease activity (Figure 17). One possible reason for this effect is that the SSF contain some factor that activates additional enzyme molecules or increases the activity of existing enzyme molecules. An alternate possibility is that something in the SSF complexes with some protease inhibitor in the egg or sperm resulting in more protease activity. A third possibility is that some factor in the SSF interacts with the sperm chromatin to make the sperm histones more available to the egg protease. The evidence is insuffi- cient to decide between these three, or other, possibili- ties. It must be stated that the lack of histone protease activity in sea urchin sperm has not been proven. It has simply been Shown that there is no histone protease activity under the conditions tested. It may well be that under different conditions of extraction or incu- bation histone protease activity will be found. Whether histone protease activity actually exists in the sperm is not necessary for the hypothesis that the sperm- Specific histones are removed by a protease, because such a protease clearly exists in the egg. Function of Sperm, Egg! and Zygote Histones While it is not possible to assign functions to the egg-zygote histone bands of Figure 6 or Figure 10, 124 or to the egg bands of Evans and Ozaki (1973), it may be speculated that these stage-specific histones serve the function of the H2A and HZB of later embryos. Although the presence of nucleosomes in sea urchin eggs and zygotes has not been investigated, it is probable that the chromatin of the sea urchin egg and zygote has the usual nucleosome structure. Since the nucleosome core is made up of four different histone molecules in equal numbers (Felsenfeld, 1978) it is likely that all four of the egg-zygote histones are in the nucleosome core. If the common egg, zygote, sperm, and embryo bands are in fact the H3 and H4, the histones D and E of egg and zygote would fill the role of the HZA and H2B. Because only four main bands seem to be present, the other histone, the H1, which has been associated with the linker region of DNA, would seem to be absent. However, stage-specific forms of the H1 have been found during later stages of sea urchin development (Easton and Chalkley, 1972; Seale and Aronson, 1973; Johnson gt_al., 1973; Ruderman and Gross, 1974; Poccacia and Hinegardner, 1975; Arceci £5 21., 1976). This suggests that a stage-specific form for egg and zygote also exists. It may be that an egg or zygote H1 is very loosly bound and was lost during washing or it may be that it is difficult to extract with 0.2 N HCl. It is possible, of course, that one 125 of the histones seen on the gel is filling the function of the H1. Why should the sperm have histones that are different from those of the egg, zygote, and embryo? One possible function of the sperm-specific basic proteins is the packaging of the chromatin into a small volume giving rise to the streamlined shape of the sperm. Bloch (1969) rejected the necessity of sperm-specific basic protein for condensation of the sperm nucleus, because he could find no correlation between the type of basic protein and the degree of condensation. Subirana (1975), however, found a relationship between the basicity of the sperm proteins and the degree of condensation. Thus Rana pipiens Sperm, which contains somatic histones plus one spermrspecific component, is less condensed than mammalian sperm which contains highly basic protamines. Subirana therefore concluded that one role of the sperm-specific proteins is the condensation of the chromatin. A second possible function is the repression of the genome. There is no question that, in the sea urchin, the sperm histones repress transcription relative to naked DNA or embryonic chromatin (Paoletti and Huang, 1969; Ozaki, 1971). It is difficult to say, however, whether the lack of transcription is due solely to the 126 presence of histones or to the lack of nonhistone proteins combined with the presence of histones. The third possible function is that the sperm have special basic proteins to protect the DNA from damage. Bloch (1969) rejected this hypothesis on the grounds that there is no correlation between the condi- tions the sperm are exposed to and the type of basic nuclear proteins in the Sperm. Subirana (1975) pointed out that, because of the highly condensed state of the chromatin, the DNA will naturally be better protected than DNA in less condensed chromatin. Therefore, protection of the DNA is, as a result of the condensation of the chromatin, brought about by the presence of the Spermrspecific basic proteins. The fourth hypothesis (Bloch, 1969) is that the Sperm basic proteins erase the developmental history of the sperm, allowing the Sperm genome to become totipotent. Bloch rejected this as a cause of spermespecific basic proteins because both 5222! which contains somatic histones, and Triturus, which contains protamines, have totipotent cleavage nuclei. The fifth possibility (Bloch, 1969) is that the sperm proteins may have some relationship to early develop- mental patterns. The loss of protamines in the mouse (Kopecny and Pavlock, 1975; Ecklund and Levine, 1975) 127 and sperm histones in sea urchins (this thesis) after fertilization rules out any effect of sperm basic proteins on later development. The last hypothesis cited by Bloch (1969) is that the different Sperm basic proteins are present not because they have some special function, but rather because their function may be filled many ways, permitting the evolution of many forms of Sperm-specific proteins. It seems likely that this is true. The sperm basic proteins, like somatic histones, certainly function in the packaging of the DNA, but obviously this is done in a number of ways. Since variation is permitted, variation exists. It would be interesting to determine if all sperm chromatin which has nucleosome structures, which sea urchin sperm does (Spadafora st 31., 1976), also has the standard H3 and H4, and conversly, if all sperm lacking H3 and H4 also lack nucleosomal orginization as the protamine containing mouse sperm does (Kierszenbaum and Tres, 1975). Why the egg and zygote should have Special histones is not known. It is a safe assumption that they function in the packaging of the DNA. The most recent work has indicated that the unfertilized egg nucleus is more active in transcription than the blastula nucleus which has embryonic histones (Dworkin and Infants, 1978). Therefore simple repression of the genome does 128 not occur as it does in the Sperm. The suggestion that sperm histones are involved in the developmental fate of the embryo could apply to the egg histones. Felsenfeld (1978) indicates that old histones remain together as the chromatin is replicated. It is not known whether this applies to short stretches of a few nucleosomes or much longer stretches of chromatin. It is conceivable that different blastomeres could contain different types of histones. There is, however, absolutely no indication that this is so. I would like to propose a different reason for the presence of egg-zygote specific histones. Sperm histones are lost from the DNA after fertilization and are replaced by zygote histones. This can be done if the enzymes involved do not affect the histones that replace the sperm histones. There are two ways to do this: proteases that affect the sperm histones only; or special histones that are not affected by a more general protease. I believe that the latter is the mechanism used in the sea urchin. There are several lines of evidence, all circumstantial, which support this conclusion. There are several proteases in the egg as established by various workers as previously discussed. Lundblad 35 31. (1972) and Mano (1966) have suggested that these enzymes are involved in the activation 129 of the egg, which could also apply to the sperm nucleus. These enzymes have a broad Spectrum of activity, in- cluding sperm histones and hemoglobin as shown in this thesis. Given this amount of protease activity, it is likely that the egg-zygote histones would be degraded unless they are resistant to protease activity. An indication that they are resistant is given in the work of Johnson and Hnilica (1970). They added trypsin to isolated egg nuclei and found no increase in transcription. When they added trypsin to gastrula nuclei the rate of transcription increased. This suggests that the egg histones are not affected by trypsin which degrades Sperm and embryonic histones. Staining experiments by Immers gt a1. (1967) also suggest that the histones of sea urchin egg and zygote are resistant to trypsin. They found that trypsin did not increase the staining of egg and zygote nuclei with Hale stain but did increase the stainability of gastrula nuclei. Hale stain reacts with unneutralized phosphate groups of DNA. The lack of staining after trypsin treatment in the egg suggests that the egg basic nuclear proteins are resistant to trypsin. The resistance of egg-zygote histones to protease activity could be due to an amino acid sequence which does not contain those amino acid pairs cleaved by the 130 proteases. Another way the histones could be resistant to proteases is adopting a conformation which is resistant to the proteases. An example of this is histone H1. Studies of histone proteases have shown that H1 is rapidly degraded when complexed with DNA in the chromatin. When the H1 is free in solution, however, it is resistant to the action of the histone protease (Bartley and Chalkley, 1970; Kurecki §£_§1., 1971). Thus the histones of the egg and zygote may have a conformation that makes them resistant to proteolytic degradation. A different explanation for the loss of sperm histones and their replacement by egg histones is due to turnover. It may be that both sperm and egg histones are actively degraded, but the egg-zygote histones con- stantly replace degraded histones, both sperm and egg, from a pool of preexisting or newly synthesized egg- zygote histones. Since, as previously discussed, the egg-zygote histones do not seem to be made after fertili- zation, any egg-zygote histones degraded must be replaced by stored histone. The evidence for both of these hypotheses, egg- zygote protease resistance and egg-zygote replacement, is insufficient to prove either one. The egg-zygote protease resistance hypothesis is easily testable, how- ever. 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