UBC Theses and Dissertations
Biology of the histones and protamines in trout testis Louie, Andrew James
Trout testes maturation is characterized by the complete replacement of the histones by the protamines. A study of the different cell types involved in spermatogenesis was initiated. Cells from testes at different stages of development were resolved according to their size by sedimentation on serum albumin gradients. The temporal order of appearance of different cell types was noted. DNA determinations and analysis of basic proteins indicated that (a) large cells (spermatogonia and early primary spermatocytes), (b) early, middle, and late spermatids, and (c) mature sperm were resolvable. The rates of DNA synthesis, histone synthesis, and his-tone phosphorylation bear a 1:1:1 relationship to each other in the large cells. Protamine synthesis begins in middle spermatids, while histones are progressively lost during the transition from middle to late spermatids. Very little histone phosphorylation was found in spermatids, indicating that this process is not involved in the removal of histones during the replacement process. The duration of each of the early, middle, and late spermatid stages was about 1 week, a total of 3 weeks being required for spermiogenesis in trout. During development the testis weight increases expo- nentially 500 to 1000 fold then decreases as spermatozoa are lost. About 10 to 11 successive spermatogonial cell divisions (12 to 13 total cell divisions) occur during development. A kinetic model for the appearance and disappearance of the different cell types was derived from the chronological and geometric relationship of the different cell types. The protamines undergo a series of phosphorylations and dephosphorylations during spermatid development. At least 6 modified bands of unsubstituted protamine were resolved by gel electrophoresis. The temporal relationship of the bands was elucidated by following the fate of newly synthesized protamine labeled with [³H] arginine. Label was not found in an unsubstituted protamine until 5 to 10 days after synthesis. The phosphorylation and dephosphorylation of protamine appear to be obligatory and unidirectional. A large number of pools (at least 6) is postulated to account for the long lapse between synthesis of protamine and appearance of label in unmodified protamine characteristic of mature spermatozoa. The controlled phosphorylation of protamine may be important in the correct binding of protamine to DNA and removal of his tones, while dephosphorylation of protamine may be involved in the progressive condensation of spermatid chromatin. Phosphorylation of protamine is not involved in the removal of protamine from ribosomes or its transport into the nucleus . Substantial quantities of highly phosphorylated protamines suitable for in vitro studies of the interaction of phosphoprotamine with DNA were prepared. There are at least 2 series of phosphoprotamines, each with 0, 1, 2, and 3 phosphates per molecule, and differing in the number of seryl residues. The metabolism of the histones was examined. The 5 major histone fractions were separated by Bio-Gel P-10 chromatography; gel electrophoresis of each fraction resolved phos-phorylated from unphosphorylated species. The levels of phospho-histone were low (~5%) in histone IIb₂ and III, and high in histone IV (~30%) and I (40 to 50%) . These different levels suggest that phosphorylation may have different functions in each histone. Labeling studies showed that newly synthesized histone IV undergoes an obligatory and sequential series of acetylations and deacetylations, which may be involved in the correct binding of newly synthesized histone IV to DNA. After an initial lag, newly synthesized histone I is sequentially phosphorylated and dephosphorylated. This cycle of phosphorylation and dephosphorylation is repeated in the next cell cycle. On the other hand, histone Ilb₁ is rapidly phosphorylated shortly after synthesis and then dephosphorylated. After a short lag (correct binding to DNA) histone IV is phosphorylated and then slowly dephosphorylated. Previously formed ("old") histones Ilb₁ and IV do not appear to be appreciably phosphorylated in the succeeding cell cycle. These data suggest that phosphorylation and dephosphor-ylation may have a role in the correct binding of newly synthesized histone Ilb₁ to DNA. On the other hand, phosphorylation and dephosphorylation of "new" and "old" histone I may have an active role in regulating the compactness of chromosomes. A scheme for the regulation of chromosome coiling during the cell cycle was proposed, in which molecules of histone I in extended chromosomes are highly phosphorylated while those in condensed (super coiled) chromosomes are un-phosphorylated. The phosphorylation of "old" histone I may serve to uncoil the chromosomal fibre during telophase and about the DNA replication fork during S phase; the phosphorylation of newly synthesized histones I and IV may be necessary to maintain the diffuse state of euchromatin. The dephosphorylation of I could be part of a mechanism involved in the condensation of interphase chromosomes into metaphase chromosomes during mitosis and meiosis.
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