Remodeling of the metabolome during early frog development

A rapid series of synchronous cell divisions initiates embryogenesis in many animal species, including the frog Xenopus laevis. After many of these cleavage cycles, the nuclear to cytoplasmic ratio increases sufficiently to somehow cause cell cycles to elongate and become asynchronous at the mid-blastula transition (MBT). We have discovered that an unanticipated remodeling of core metabolic pathways occurs during the cleavage cycles and the MBT in X. laevis, as evidenced by widespread changes in metabolite abundance. While many of the changes in metabolite abundance were consistently observed, it was also evident that different female frogs laid eggs with different levels of at least some metabolites. Metabolite tracing with heavy isotopes demonstrated that alanine is consumed to generate energy for the early embryo. dATP pools were found to decline during the MBT and we have confirmed that maternal pools of dNTPs are functionally exhausted at the onset of the MBT. Our results support an alternative hypothesis that the cell cycle lengthening at the MBT is triggered not by a limiting maternal protein, as is usually proposed, but by a decline in dNTP pools brought about by the exponentially increasing demands of DNA synthesis.     

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development.     

Sea urchin spectrin in oogenesis and embryogenesis: a multifunctional integrator of membrane-cytoskeletal interactions

Using indirect immunofluorescence microscopy on semithin cryosections of maturing ovarian tissue, eggs, and developing embryos, we have mapped the cellular distribution and dynamic redistribution of spectrin in oogenesis and early embryogenesis. During oogenesis, spectrin is initially found in the cortex of oogonia and previtellogenic oocytes, and later accumulates in the cytoplasm of vitellogenic oocytes on the surfaces of cortical granules, pigment granules/acidic vesicles, and yolk platelets. Following egg activation, spectrin undergoes a rapid redistribution coincident with three major developmental events including: (1) restructuring of the cell surface, (2) translocation of pigment granules/acidic vesicles to the cortex during the first cell cycle, and (3) amplification of the embryo's surface during the rapid cleavage phase of early embryogenesis. The synthesis and storage of spectrin during oogenesis appears to prime the egg with a preestablished pool of membrane-cytoskeletal precursor for use during embryogenesis. Results from this study support the hypothesis that spectrin may function as a key integrator and modulator of multiple membrane-cytoskeletal functions during embryonic growth and cellular differentiation.     

Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes

Ribonuclease protection assays have been used to quantitatively assess changes in steady-state levels of specific mRNAs during oogenesis and early embryogenesis in mice. The mRNAs encode ZP3 (a glycoprotein that serves as a sperm receptor), LDH-B (heart-type lactate dehydrogenase), and MOM-1 (a protein of unknown function). MOM-1 and LDH-B are expressed in a variety of adult mouse tissues and midgestation embryos, whereas ZP3 expression is restricted completely to oocytes. All three mRNAs are expressed by growing mouse oocytes and accumulate to unusually high levels in fully grown oocytes as compared to somatic cells; 240,000, 200,000 and 74,000 copies mRNA per fully grown oocyte for ZP3, LDH-B and MOM-1, respectively. Steady-state levels of LDH-B and MOM-1 mRNA undergo a modest decline (approximately 20-40%) during ovulation when fully grown oocytes become unfertilized eggs and, in general, mirror the reported change in poly(A)+RNA levels during this period of development. On the other hand, the level of ZP3 mRNA declines dramatically (approximately 98%) during ovulation, from approximately 240,000 copies per oocyte to approximately 5000 copies per unfertilized egg, and ZP3 mRNA is undetectable in fertilized eggs (less than 1000 copies per fertilized egg). MOM-1 mRNA is expressed at relatively low levels in morulae (approximately 2000 copies per embryo) and blastocysts (approximately 5000 copies per embryo), whereas ZP3 mRNA remains undetectable (less than 1000 copies per embryo) at these stages of preimplantation development. These findings are discussed in the context of overall gene expression during oocyte growth, meiotic maturation and early embryogenesis in mice.     

Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype

The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis.     

Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-->3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.     

Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli

Intracellular pool sizes of deoxyribonucleoside triphosphates (dNTPs) are highly regulated. Unbalanced dNTP pools, created by abnormal accumulation or deficiency of one nucleotide, are known to be mutagenic and to have other genotoxic consequences. Recent studies in our laboratory on DNA replication in vitro suggested that balanced accumulation of dNTPs, in which all four pools increase proportionately, also stimulates mutagenesis. In this paper, we ask whether proportional dNTP pool increases are mutagenic also in living cells. Escherichia coli was transformed with recombinant plasmids that overexpress E. coli genes nrdA and nrdB, which encode the two protein subunits of aerobic ribonucleotide reductase. Roughly proportional dNTP pool expansion, by factors of 2- to 6-fold in different experiments, was accompanied by increases in spontaneous mutation frequency of up to 40-fold. Expression of a catalytically inactive ribonucleotide reductase had no effect on either dNTP pools or mutagenesis, suggesting that accumulation of dNTPs is responsible for the increased mutagenesis. Preliminary experiments with strains defective in SOS regulon induction suggest a requirement for one or more SOS functions in the dNTP-enhanced mutagenesis. Because a replisome extending from correctly matched 3'-terminal nucleotides is almost certainly saturated with dNTP substrates in vivo, whereas chain extension from mismatched nucleotides almost certainly proceeds at sub-saturating rates, we propose that the mutagenic effect of proportional dNTP pool expansion is preferential stimulation of chain extension from mismatches as a result of increases in intracellular dNTP concentrations.     

Embryogenesis in the Presence of Blockers of Mechanosensitive Ion Channels

Certain developmental events are thought to be controlled by mechanical tension, but the nature of the transduction mechanism for sensing and responding to tension changes is unknown. A good candidate for such a sensing system would be stretch-activated (SA) ion channels, a type of mechanosensitive (MS) ion channel found in many preparations including the oocytes or embryos of ascidians, fish, and amphibians. To test the hypothesis that SA channel activation is important for early embryogenesis, we treated amphibian and ascidian eggs and embryos with inhibitors of MS ion channels. Xenopus laevis eggs and embryos were treated with gadolinium (Gd³⁺) concentrations up to 100 times the K_d for SA channel inhibition. Boltenia villosa eggs and embryos were exposed to three agents (Gd³⁺, tubocurarine, and gallamine) which are known to block SA channels in other organisms. None of these drugs interfered with morphogenesis in a manner that would suggest SA channel activity is critical to early embryogenesis.     

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Rapid changes in deoxynucleoside triphosphate pools in mammalian cells treated with mutagens

In this communication we describe the rapid increase in cellular deoxynucleoside triphosphate (dNTP) concentrations in Chinese Hamster cell line V79 after exposure to known mutagens. With this cell line an expansion of dATP and dTTP pools was detected; changes in dCTP were not large; changes in dGTP were either not significant or too low to quantitate. This situation may reflect the existence of imbalances in dNTP pools at the DNA replication fork. The expansion of dATP and dTTP pools occurred within 2 to 4 hours after exposure of cultured cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Ultraviolet light (UV), mitomycin C, and cytosine arabinoside also caused similar dNTP pool changes.     

Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse

Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

Soluble tubulin complexes, gamma-tubulin, and their changing distribution in the zebrafish (Danio rerio) ovary, oocyte and embryo

Tubulin dynamics, i.e., the interchange of polymeric and soluble forms, is important for microtubule (MTs) cellular functions, and thus plays essential roles in zebrafish oogenesis and embryogenesis. A novel finding in this study revealed that there were soluble pools of tubulins in zebrafish oocytes that were sequestered and maintained in a temporary "oligomeric" state, which retained assembling and disassembling potential (suggested by undetected acetylated tubulin, marker of stable tubulin), but lacked abilities to assemble into MTs spontaneously in vivo. Using differential centrifugation, gel chromatography and DM1A-probed western blot, soluble alpha-tubulin was found to be associated with large molecular weight complexes (MW range to over 2 MDa) which were reduced in amount by the blastula stage, especially in some batches of embryos, with a concomitant decrease in soluble tubulin. Complexes (MW range less than 2 MDa) then increased in the gastrula with an increase in soluble alpha-tubulin. Two different anti-gamma-tubulin monoclonal antibodies, GTU 88 and TU 30, revealed the existence of soluble gamma-tubulin in both zebrafish oocytes and embryos, which also decreased by the blastula stage and increased in the gastrula stage. Soluble alpha-tubulin and gamma-tubulin extracted from zebrafish ovaries, oocytes and embryos co-localized in fractions on three different columns: S-200 Sephacryl, DEAE and Superose-6b. The soluble tubulin complexes were competent to assemble into MTs in vitro induced by taxol, and gamma-tubulin was co-localized with assembled MTs. These soluble tubulin complexes were stable during freeze-thaw cycles and resisted high ionic interaction (up to 1.5 M NaCl). Furthermore, some ovarian soluble alpha-tubulin could be co-immunoprecipitated with gamma-tubulin, and vice versa. Two antibodies specific for Xenopus gamma-tubulin ring complex proteins (Xgrip 109 and Xgrip 195) detected single bands from ovarian extracts in western blots, suggesting the existence of Xgrip 109 and Xgrip 195 homologues in zebrafish. These findings, together with recent work on gamma-tubulin ring complexes in oocytes, eggs and embryos of other species, suggest that soluble gamma-tubulin-associated protein complexes may be involved in regulating tubulin dynamics during zebrafish oogenesis and embryogenesis.     

p70(S6K) controls selective mRNA translation during oocyte maturation and early embryogenesis in Xenopus laevis

In mammalian cells, p70(S6K) plays a key role in translational control of cell proliferation in response to growth factors. Because of the reliance on translational control in early vertebrate development, we cloned a Xenopus homolog of p70(S6K) and investigated the activity profile of p70(S6K) during Xenopus oocyte maturation and early embryogenesis. p70(S6K) activity is high in resting oocytes and decreases to background levels upon stimulation of maturation with progesterone. During embryonic development, three peaks of activity were observed: immediately after fertilization, shortly before the midblastula transition, and during gastrulation. Rapamycin, an inhibitor of p70(S6K) activation, caused oocytes to undergo germinal vesicle breakdown earlier than control oocytes, and sensitivity to progesterone was increased. Injection of a rapamycin-insensitive, constitutively active mutant of p70(S6K) reversed the effects of rapamycin. However, increases in S6 phosphorylation were not significantly affected by rapamycin during maturation. mos mRNA, which does not contain a 5'-terminal oligopyrimidine tract (5'-TOP), was translated earlier, and a larger amount of Mos protein was produced in rapamycin-treated oocytes. In fertilized eggs rapamycin treatment increased the translation of the Cdc25A phosphatase, which lacks a 5'-TOP. Translation assays in vivo using both DNA and RNA reporter constructs with the 5'-TOP from elongation factor 2 showed decreased translational activity with rapamycin, whereas constructs without a 5'-TOP or with an internal ribosome entry site were translated more efficiently upon rapamycin treatment. These results suggest that changes in p70(S6K) activity during oocyte maturation and early embryogenesis selectively alter the translational capacity available for mRNAs lacking a 5'-TOP region.     

Pool sizes of the deoxynucleoside triphosphates in the sea urchin egg and developing embryo.

The four deoxynucleoside triphosphate pools in unfertilized eggs of L. pictus and S. purpuratus were measured and found to be very large, ranging from 10^?3 to 10^?2 pmoles per egg. The high levels of the individual dNTP pools are sufficient for one to eight rounds of DNA synthesis. During the first division cycle these pools fluctuate with the highest levels being attained prior to DNA synthesis. The pools then decrease just preceding or during the S period. There is a large reduction in the total cellular dNTP in later stages of development when DNA synthesis is reduced relative to the cleavage stages.     

Synthesis of Mitochondrial DNA Precursors during Myogenesis,an Analysis in Purified C2C12 Myotubes

During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.     

The pattern of damage to the oogenetic series of cells after a single feeding of cis-diamminedichloroplatinum to Habrobracon females

A single meal of cis-diamminedichloroplatinum (DDP) fed to virgin braconid wasps decreased drastically the number of eggs derived from oogonia. In contrast, most of the larger oocytes completed oogenesis even after a dose which shortened average lifespan to 1/3 its normal length. Temporary infecundity resulted from the destruction of the germarial cells which produced the 32 cystocytes per follicle (1 oocyte and 31 trophocytes) by mitosis. As determined by egg hatchability, oogonia were the most vulnerable cell type to DDP in the ovariole sequence. Therefore, oogonial vulnerability to DDP was demonstrated by failure to complete both gametogenesis and embryogenesis. In combination with gamma radiation, DPP reduced egg production and hatchability below the values obtained from either agent used alone. However, the decreases were moderate, as expected from additivity of effect. A difference from the results from either radiation or several types of alkylating agents appeared in the proportion of early embryonic deaths. Deaths during cleavage predominated in every daily sample of eggs treated with DDP as oocytes. Usually only the eggs laid the first 2 days after treatment are characterized by a large number of 'stage 1' deaths.     

Silencing of Maternal Heme-binding Protein Causes Embryonic Mitochondrial Dysfunction and Impairs Embryogenesis in the Blood Sucking Insect Rhodnius prolixus

The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.     

Reactions of deoxyribonucleotide synthesis system to irradiation and their modification by radioprotectors

The paper covers the problem on reactions of deoxyribonucleotide (dNTP) synthesis system in blood-forming organs of animals induced by irradiation. The synthesis of dNTP is a rate-limiting stage for DNA synthesis. Cellular requirements for dNTP pools during DNA synthesis are related with ensuring of the accuracy of DNA copying during replication and repair. It has been shown that organism defence mechanisms against irradiation include the following stages: 1. The prompt SOS-activation of dNTP synthesis 30 min later after irradiation, playing the important role in protecting of cell's genetic apparatus from damage. 2. The inhibition of dNTP synthesis within 3-24 h after irradiation resulting to the imbalance of four dNTP and the decrease of their pools. As result of that, the abnormal repair is observed due to depurinations, errors of base incorporations and "misrepair". 3. The restore of dNTP synthesis occurred 2 days later after irradiation. The increase of dNTP pools promotes the increase of DNA synthesis rate as well as proliferative activity of cells. Confirming the fact that the alterations in dNTP pools play essential role in the production of DNA lesions became an important step in understanding of the multistage process leading to radioprotection. To get high and balanced pools of dNTP needed for the increase in the volume of repair of DNA lesions the radioprotectors with high efficiency relative to the survival test were used in experiments. They induced the elevated dNTP synthesis in bone marrow and spleen during the time when the irradiation alone caused the essential prolonged suppression of dNTP synthesis as well as DNA and protein synthesis in organs of nonprotected animals. It has been shown that substances with antioxidant and antiradical activity induced the dNTP synthesis, too. In vivo regulatory factors of dNTP synthesis have been studied to elucidate the mechanisms of getting of high and balanced dNTP pools by using of different substances.