Dominic Poccia

Remodeling of Nucleoproteins during Gametogenesis, Fertilization, and Early Development

Publisher Summary This chapter reviews the transformation of nucleoprotein types during gametogenesis, fertilization, and early development, outlines chromatin structural changes that accompany these nucleoprotein transitions, and discusses some functions of the nucleoproteins and histone variants associated with gamete, zygote, and early embryonic nuclei. As a result of nucleoprotein transitions during spermatogenesis, most sperm nuclei enter egg cytoplasm with proteins which may, to varying degrees, be incompatible with subsequent chromosomal activities such as replication, transcription, recombination, and mitotic condensation. Transitional proteins might be utilized before or after fertilization for effective histone replacement or might serve specific structural role during meiosis or pronuclear decondensation. Although fundamental conservation of histone sequence and structure remains the rule in chromosome construction, variation on histone forms, including their extreme replacements by other types of nucleoproteins, continue to offer insight into nucleoprotein function.

Towards a total macromolecular analysis of sea urchin embryo chromatin

Abstract A new and relatively gentle procedure has been developed for the resolution of sea urchin embryo chromatin into its components. In this procedure non-histones and nucleic acids are never exposed to pH outside the range 7.7 to 9.1 and histones are only transitorily exposed to pH 2. Approximately thirty non-histones and eleven histones have been resolved. Within each of these classes, several quantitative changes in individual protein species have been observed to occur with embryonic development. The RNA of the chromatin has a DNA-like base composition consistent with its being largely nascent heterogeneous nuclear RNA.

Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm

Abstract The eggs of Xenopus laevis are capable of initiating spindle formation and cleavage in response to microinjection of partially purified components of sea urchin sperm. High activity was assayed from a sperm head fraction obtained after removal of the plasma and nuclear membranes, acrosome, midpiece mitochondrion, and tail. Chromatin, the basal plate, and the distal centriole comprised the components of the head fraction. Disruption of the chromatin did not impair activity and purified chromatin lacked activity, suggesting the centriole and basal plate as the active materials. Low doses of active material induced apparently normal cleavage at 90 min after injection, with 16% of the eggs reaching the late blastula stage. High doses of active material induced precocious multiple cleavage, with some eggs cleaving into 3–10 segments within 20 min after injection. These eggs contained numerous spindles and asters in the animal hemisphere, as judged by light microscopy of stained sections. Microinjection of eggs is presented as a semi-quantitative bioassay for agents initiating spindle formation and cleavage.

Transitions in histone variants of the male pronucleus following fertilization and evidence for a maternal store of cleavage-stage histones in the sea urchin egg☆

Abstract A system has been developed for the biochemical study of male pronuclear histones following fertilization. Pronuclei are isolated in high yield and purity from polyspermically fertilized sea urchin eggs. Acid extracts of the pronuclei or chromatin are analyzed by two-dimensional gel electrophoresis. During male pronuclear decondensation, sperm-specific (Sp) H1 is completely replaced by the cleavage-stage (CS) H1 variant and two sperm H2B species are more gradually replaced by two closely migrating species (O, P) perhaps modifications of the Sp2Bs. Histones CS2A and CS2B begin to accumulate on the male chromatin predominantly during DNA synthesis, as does a subspecies of H3 (3″). By the time of chromosome condensation, the histone complement is chiefly CS1; CS2A and Sp2A; CS2B and O, P; three H3s and two H4s. Controls indicate that these patterns are not artifacts of cytoplasmic or maternal pronuclear contamination or modification of sperm histones during isolation. The histone transitions occur even if protein synthesis is inhibited, suggesting a maternal pool of CS histones. Such a store can be demonstrated directly in acid extracts of whole eggs. At least 30 haploid equivalents of CS histones are stored per egg. No α, β, γ, or δ variants appear on the chromatin in the first cycle. This system should allow, for the first time, detailed biochemical study of male pronuclear chromatin.

Phosphorylation of sea urchin sperm H1 and H2B histones precedes chromatin decondensation and H1 exchange during pronuclear formation

Immediately following fertilization in the sea urchin, sperm-specific histones Sp H1 and Sp H2B are phosphorylated. Then, in parallel with chromatin decondensation, nearly all phosphorylated Sp H1 is lost from the pronuclear chromatin, with the concurrent assimilation of the egg phosphoprotein CS H1. Chemical cleavage of in vivo labeled Sp H1 and Sp H2B shows that serine phosphorylation occurs in the unusually long N-terminal region of these proteins. These regions contain tandemly repeated tetra- and pentapeptide units each containing serine, proline, and two basic amino acids. It is proposed that sperm chromatin decondensation may require prior phosphorylation of these unusual N-terminal regions, whose function in the mature sperm may be to condense or stabilize its highly compact chromatin.

Packaging and unpackaging the sea urchin sperm genome

Abstract Two species of histones in sea urchin sperm (Sp H1 and Sp H2B) are chimeric molecules whose highly basic amino-terminal domains are dephosphorylated at the last stage of sperm cell differentiation, and rephosphorylated immediately following fertilization. The phosphorylated regions consist largely of repeating tetrapeptides with two basic residues flanking Ser-Pro residues (‘SPKK’ motifs) and are predicted to have β-turn secondary structures. Alteration of the charge and structure of the SPKK sites may play a role in the unusually dense DNA packaging of the mature sperm chromatin. The motif resembles the target site of cell-cycle-associated cdc2 kinases and is found in several other proteins whose nucleic acid affinities may be altered during the cell cycle.

Transitions in histone variants during sea urchin spermatogenesis

Transitions in the histone complement of nuclei during sea urchin spermatogenesis were investigated by two-dimensional gel electrophoresis. Nuclei were isolated from male gonads of individuals differing in degree of maturity. Unlike protamines, the sea urchin sperm-specific histone variants Sp H1 and Sp H2B appear early in spermatogenesis, well before spermatid differentiation, as the predominant representatives of their classes. Both proteins are phosphorylated from their first appearance until the last steps of spermiogenesis, when the highly condensed late spermatid nuclei become spermatozoan nuclei. Phosphorylation of serine occurs mostly (Sp H1) or entirely (Sp H2B) on the N-terminal portions of these molecules. We conclude that phosphorylated sperm-specific histone variants in the sea urchin function in spermatocytes during meiosis and are the major histones present during replication and transcription in some spermatogonia as well. We propose that the dephosphorylation of Sp H1 and Sp H2B in late spermatids is not primarily responsible for spermatid chromatin condensation but instead may act to stabilize the chromatin of the spermatozoon or aid in the final shaping of the sperm nucleus.

Phosphatidylinositol metabolism and membrane fusion

Membrane fusion underlies many cellular events, including secretion, exocytosis, endocytosis, organelle reconstitution, transport from endoplasmic reticulum to Golgi and nuclear envelope formation. A large number of investigations into membrane fusion indicate various roles for individual members of the phosphoinositide class of membrane lipids. We first review the phosphoinositides as membrane recognition sites and their regulatory functions in membrane fusion. We then consider how modulation of phosphoinositides and their products may affect the structure and dynamics of natural membranes facilitating fusion. These diverse roles underscore the importance of these phospholipids in the fusion of biological membranes.

Remodeling of sperm chromatin following fertilization: nucleosome repeat length and histone variant transitions in the absence of DNA synthesis

Within the first cell cycle following fertilization the average nucleosomal repeat length of sea urchin male pronuclear chromatin declines by 30-40 base pairs to a value typical of that found in the embryo. This decline occurs after a lag of about 30 min postfertilization, and is accompanied by replication of the male chromatin and accumulation of cleavage-stage (CS) core histone variants. When replication is inhibited by greater than 95% with aphidicolin, the decline in repeat length still occurs, although it is slightly retarded. The decline in repeat length also occurs when protein synthesis is blocked by greater than 98% and DNA synthesis by 60-70% with emetine. The adjustment of nucleosome repeat length therefore can occur in vivo without extensive movement of replication forks across the length of the chromatin, or normal progression of the cell cycle, and appears to require no proteins synthesized postfertilization. Blocking of DNA synthesis or protein synthesis also does not prevent the normal histone variant transitions involved in male pronuclear chromatin remodeling. Although their accumulation is slowed, CS core variants eventually become the predominant male pronuclear histones in their classes when replication is inhibited. Since a shortening of the average nucleosomal repeat length of approximately 10-20% is not sufficient to account for this large acquisition of CS variants, some of the sperm (Sp) core histones are probably displaced from the replication-blocked pronucleus. Therefore, accumulation of CS H2A and CS H2B are temporally correlated with the repeat length transition, whereas replication, normal progression of the cell cycle, and the early histone transitions involving SpH1 and SpH2B are not.

Developmental changes in chromatin proteins of the sea urchin from blastula to mature larva

Abstract Chromatin proteins of the sea urchin Lytechinus pictus from blastula to fully grown feeding larva were analyzed by polyacrylamide-gel electrophoresis. Two subspecies of histone protein changed during development. Histone f1g increased progressively relative to f1m and within a few days after the larvae began to feed constituted >99% of the f1 fraction. Two slower-moving subfractions of histone f2b disappeared in the same interval, being replaced by a single fastermoving species. The electrophoretic profile of nonhistone protein changed quantitatively and qualitatively with the largest change, a relative decrease of high molecular weight bands and a new pattern of middle molecular weight bands, also occurring several days after feeding began. The profiles remained virtually the same during subsequent development though the cell number increased at least tenfold. The onset of feeding thus seems to be correlated with major changes in chromatin proteins.