Oxidative stress and the role of novel thiol compounds at fertilization

A new class of thiols, the 1-methyl-4-mercaptohistidines, has been found in high concentrations in invertebrate eggs. This family, called the ovothiols, has unusual redox properties, including the ability to confer a CN- -resistant NAD(P)H oxidase activity on ovoperoxidase, the enzyme that catalyzes the physiological crosslinking of the fertilization envelope with dityrosine residues. Ovothiol has a redox potential of 44 mV positive to glutathione and thus is maintained in the reduced state in eggs by reduced glutathione, without the need for an ovothiol reductase. We propose that high concentrations of reduced ovothiol are present in eggs to protect them from the oxidative stress caused by the respiratory burst of fertilization.     

Temporal-spatial expression of two Paracentrotus lividus cell surface proteins

The temporal expression of two cell surface proteins, called BEP1 and BEP4, during Paracentrosus lividus embryonic development was studied. These proteins are found in both monomeric and dimeric forms in egg and embryos and we have established that their specific form is related to their being in the cytoplasm or on the cell surface. The spatial distribution of BEP1 and BEP4 proteins in eggs and embryos was established by whole mount immunohistochemistry. These proteins are located in the animal part of unfertilized and fertilized eggs; thereafter they are much less represented in structures derived from the vegetal cells of the embryo such as the micromeres of the 16 cell stage, the primary mesenchyme of blastula and the gut of gastrula. At the prism stage BEP1 and BEP4 proteins are present to some ectodermal parts and thereafter, at the pluteus stage, to the oral region.     

Stability of alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.     

Association of 7 SL RNA and an SRP-like particle with polysomes and endoplasmic reticulum in the developing sea urchin embryo

We have identified the sea urchin cognate of the mammalian signal recognition particle (SRP). This particle contains the diagnostic 7 SL small RNA, sediments at a similar velocity to that reported for the mammalian particle, and is found associated with the ER and polysomes. We have examined its subcellular localization during embryogenesis in order to determine whether it could serve in a translational regulatory capacity for a subset of the stored maternal mRNAs. In these studies the 7 SL RNA was used as a marker for the particle, since we determined that the 7 SL RNA exists exclusively within the SRP-like particle at all developmental stages. The relative distribution of the SRP among cytoplasmic structures changes dramatically during development. This represents an actual change in subcellular localization because the 7 SL RNA level remains nearly constant per embryo until the pluteus stage, when it increases slightly. In eggs, the SRP exists almost entirely free in the cytoplasm as an 11 S particle. Very soon after fertilization and throughout development there is an increase in the association of the particle with rapidly sedimenting structures, until by the pluteus stage greater than 90% of the SRP exists in a bound state. The nature of the associations is complex, and the bound structures include, at least in part, ribosomes, polysomes, and microsomes. The SRP is associated with microsomal membranes in gastrula (36 hr) but not in blastula (12 hr) or earlier embryos. Using the criteria of sensitivity to Triton X-100, we determined that 16% of the SRP in a 10,000g cytoplasmic fraction was bound to membranes in a microsomal (endoplasmic reticulum)-containing fraction in the gastrula. In contrast, less than 1% was membrane associated in the blastula. The SRP was also found in a ribosome-polysome fraction in 12-, 36-, and 48-hr embryos, but not in eggs. Finally, a small but significant portion of the SRP was found associated with monosomes in cleavage stage embryos. The possible role the SRP could play in the elongation arrest of stored maternal messages for secreted proteins is discussed.     

Glutathione transferase activity during Bufo bufo development

High levels of glutathione transferase activity were measured during the development of the embryos of Bufo bufo including unfertilized eggs. After stage 4 glutathione transferase activity gradually decreased until stage 25 when the minimum was reached. No change in the number of isozymes was noted during development according to isoelectric focusing analysis performed on the cytosolic fractions of selected stages.     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.     

Enzymes of DNA biosynthesis in developing sea urchins. Changes in ribonucleotide reductase, thymidine, and thymidylate kinase activities.

The pattern of ribonucleotide reductase, thymidine kinase, and thymidylate kinase activities during development of Paracentrotus lividus eggs and the effect of actinomycin on these enzymatic activities have been studied. Ribonucleotide reductase activity is detectable, though at a low level, in the unfertilized egg; the activity increases sharply soon after fertilization and reaches a peak at the morula stage. Thereafter it decreases and remains at a lower level than that of the unfertilized egg. Actinomycin, at a concentration sufficient to inhibit messenger RNA (mRNA) synthesis does not affect the level of enzymatic activity, indicating that preexisting maternal mRNA is used for the synthesis of this enzyme. Thymidine kinase is present at a low level in the egg; it increases sharply after the hatching blastula until the pluteus stage. Actinomycin does not affect the enzyme activity from fertilization until blastula but prevents the increase in enzyme activity that is observed between blastula and pluteus. Thymidylate kinase activity shows an increase after fertilization, followed by fluctuations throughout development with a considerable decrease at the blastula stage and at the end of gastrulation. Actinomycin has no effect on the activity of thymidylate kinase regardless of when the drug is added to the embryo suspension. Possible regulatory mechanisms of DNA synthesis in sea urchin embryos are discussed: The presence in the unfertilized egg of the most important enzymes controlling the cellular flow of DNA precursors and the availability of dTTP suggest that the block in DNA synthesis observed in the unfertilized egg is due to some particular mechanism that is switched on at fertilization.     

Lipid composition of gametes and embryos of the sea urchin Strongylocentrotus intermedius at early stages of development

The lipid composition of sea urchin gametes and embryos was examined in detail by micro thin-layer chromatography (tlc) and gas-liquid chromatography (glc). Lipids of unfertilized eggs contain 53.7% triglycerides, 33.2% phospholipids, and 9.4% cholesterol, while spermatozoa lipids consist of 65.0% phospholipids, 15.5% cholesterol, and no triglycerides. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), diphosphatidylglycerol (DPG), and lysophosphatidylcholine (LPC) were identified among the phospholipids of both eggs and spermatozoa. The major part of egg and embryo PE was present as plasmalogen. After fertilization and the first cleavage, phospholipid content decreased from 33.2 to 29.4%, but the amount of phospholipids returned to the 33.2% level by the blastula stage and reached 39.7% by the pluteus stage. Lipid class composition showed no qualitative changes during development, but concentrations of PE, PS, LPC, and cholesterol increased, while those of PC, PI, and triglycerides decreased during the process. The principal fatty acids of neutral and polar lipid fractions are 14:0, 16:0, 18:1, 18:4, 20:1, 20:4, and 20:5. Their relative content underwent some changes during development.     

Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilized oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilized eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilization. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilized oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilization but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.     

Microtubule formation from maternal tubulins during sea urchin embryogenesis: measurement of soluble and insoluble tubulin pools

The mass of tubulin protein in developing embryos of the sea urchin Lytechinus pictus was measured using a radiodilution immunoassay based on densitometric analysis of immunoprecipitated tubulins resolved electrophoretically. The tubulins constitute an average of 360 +/- 35 pg per egg, or 0.66% of the total protein, and there is no significant change in their concentration during embryogenesis. The masses of soluble and polymerized tubulin were measured for extracts prepared under conditions that stabilize microtubules. In eggs, a maximum of 14% of the tubulin is insoluble, and this increases throughout embryogenesis to 67% at pluteus stage (72 hr). The concentration of tubulin in eggs is at least 500 micrograms/ml, well above the critical concentration for tubulin assembly in vitro, yet microtubules have not been observed in eggs. The mass of newly synthesized tubulin, estimated from the mass of tubulin mRNA per embryo, accounts for a small fraction of the total tubulin by the end of gastrulation but for over half of the tubulin by the 72-hr pluteus stage. These observations are consistent with a model in which the declining level of unpolymerized tubulin controls the stability of tubulin mRNa, providing an autogenous regulation of the ontogenetic pattern of tubulin synthesis during sea urchin embryogenesis (Gong and Brandhorst, Development 102: 31-43).     

Fluorometric quantitation of cellular and nonprotein thiols

A microfluorometric assay for thiols has been developed using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM). The technique may be used to quantitate either cellular or plasma thiols over a range of 0.01 to 3.0 nmol and may be used with as few as 1-3 X 10(5) cells giving highly proportional and reproducible results. Values for nonprotein thiols obtained with this assay agree well with previous reports on glutathione (GSH) levels for both lymphocytes and plasma. Readings are determined with the aid of an automated fluorescence microplate reader which allows up to 96 samples, including standards, to be read at the same time. Cellular thiols accessible after lysis were also quantitated before and after treatment of intact cells with various thiol-reactive chemicals. Interestingly, HgCl2, bromoethanesulfonic acid, and N-ethylmaleimide differentially modified protein and nonprotein thiol levels.     

Chromatin-associated proteins of the developing sea urchin embryo. II. Acid-soluble proteins

Of the five well-characterized histories, only the slightly lysine-rich histories F2a and F2b are present in sea urchin embryos before the 16-cell stage. At the 16-cell stage, the arginine-rich (F3) and lysine-rich (Fla) histones appear and all the major histones are then present in the same relative proportions until the pluteus stage except for a second lysine-rich protein, Flb, which is first detected at 12 to 16 hours of development and increases to the pluteus stage. From 16 cells to pluteus at 70 hours, all the histones are labeled by a 30-minute incubation with radioactive lysine, with the exception of the lysine-rich histone Fla which does not incorporate label after 20 to 30 hours of development and Fib which is labeled only after 20 to 30 hours. Fla is conserved, however, to the pluteus stage.The total acid-soluble protein content of chromatin remains constant to 22 hours of development. During the period of 22 to 45 hours, there is a slight loss of protein followed by a rapid loss from 45 to 70 hours such that at 70 hours only 20% remains.     

TOTAL CELL NUMBER AND NUMBER OF THE PRIMARY MESENCHYME CELLS IN WHOLE, 1/2 AND 1/4 LARVAE OF CLYPEASTER JAPONICUS*

Total cell number and number of the primary mesenchyme cells of 1/2 and 1/4 larvae were counted at several developmental stages after hatching in comparison with those of a whole larva, using Clypeaster japonicus as material. To obtain partial larvae, blastomeres were isolated at the 2- or 4-cell stage in Ca-free sea water and cultured in natural sea water at around 23°C. Isolated blastomeres cleaved as in situ, namely, as a part of an embryo. Although each partial embryo tended to spread into a plate, it acquired spherical shape prior to hatching of control whole embryo and developed normally in terms of both developmental rate and morphogenesis. Total cell number of a whole larva was about 620 just after hatching and increased almost linearly until i t reached 1850 at the pluteus stage. A half and quarter larvae contained roughly 1/2 and 1/4, respectively, of the number of cells of whole larva through all stages counted. Numbers of the primary mesenchyme cells in the partial larvae, however, tended to be slightly larger than a half or a fourth of that in whole larva. In whole larva, 35, 50, 56 and 58 was counted at the mesenchyme blastula, early gastrula, late gastrula and pluteus stage, respectively.     

Expression of Cyclophilin during the Embryonic Development of the Sea Urchin

The spatial and temporal expression pattern of cyclophilin (Cyp) was examined during the embryonic development of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus using Western blot analysis and indirect immunofluorescence microscopy. In this study, affinity-purified anti-human Cyp A antibody was used as the primary antibody. Western blot analysis revealed that a single 17.5 kDa immunoreactive band of Cyp was present in unfertilized eggs, in embryos during several stages of development, and in ovaries and testes of adult sea urchins. Cyp was also recognized in unfertilized eggs and embryonic sea urchin cells by indirect immunofluorescence microscopy, but its concentrations within the embryonic tissues varied significantly during embryogenesis. Expression of Cyp during the cleavage stage was thought to be attributable to maternal Cyp products, with zygotic expression appearing after gastrulation. Cyp expression appears to increase depending on the Cyp concentration in the vegetal and apical plates and primary mesenchyme cells in mesenchyme blastulae, and in the oral ectodermal ridge, gut and skeletogenetic mesenchyme cells in pluteus larvae. These results suggest that widespread embryonic distribution and an increased Cyp content occur during the gastrulation in sea urchin development.     

Maternal Exogastrula-Inducing Peptides (EGIPs) and Their Changes during Development in the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing activity was examined in eggs and embryos of the sea urchin Anthocidaris crassispina at various stages. During fractionation on a column of DEAE-cellulose, the exogastrula-inducing activity was found in the flow-through fraction at all developmental stages. In particular, the activity present in the flow-through fraction of unfertilized eggs represents the presence of maternal exogastrula-inducing peptides (EGIPs). The flow-through fractions from the column of DEAE-cellulose were applied to a column of Sephadex G-100 and the activities in the eluate were assayed. The active low-molecular-weight fraction was obtained in all cases with the exception of pluteus larvae, extracts of which contained another active fraction. Immunoblots of protein samples from eggs and embryos probed with antiserum against EGIP-D indicated that there is a major immunoreactive protein that migrates with an apparent molecular weight of about 6 kDa in all cases with the exception of pluteus larvae, and that there are two major immunoreactive proteins that migrate with apparent molecular weights of 6 kDa and 35 kDa, respectively, in pluteus larvae.     

Changes in plasma thiol levels induced by different phases of treatment in breast cancer; the role of commercial extract from black chokeberry

Different low-molecular-weight thiols, including glutathione, cysteine, and cysteinylglycine are physiological free radical scavengers. On the other hand, homocysteine may play a role as an oxidant. The aim of our present study was to establish in vitro the effects of the commercial extract of Aronia melanocarpa (Aronox^?) on the amount of selected low-molecular-weight thiols and the activity of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in plasma obtained from patients with invasive breast cancer during different phases of treatment [before or after the surgery and patients after different phases of chemotherapy (doxorubicin and cyclophosphamide)] and from healthy subjects. Patients were hospitalized in Department of Oncological Surgery and Department of Chemotherapy, Medical University of Lodz, Poland. The level of low-molecular-weight thiols was determined by high-performance liquid chromatography. We observed that in the presence of the Aronia extract changes in amount of thiols in plasma from breast cancer patients (at all tested groups) were significantly reduced. Our results showed that tested commercial extract reduced modifications of antioxidative enzymes activity in plasma from patients during different phases of treatment, but this effect was not statistical significant. Our results suggest that the Aronia extract supplementation in breast cancer patients has a beneficial effect on thiols concentration in plasma. Plasma, as reported in this work, could be used as an experimental model to evaluate the beneficial action of plant supplements, including phenolic extracts on thiols or other molecules during different phases of treatment.     

Ovothiols, a family of redox-active mercaptohistidine compounds from marine invertebrate eggs

We have previously reported a novel thiol compound, 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine, or ovothiol, present at high concentration in the eggs of the sea urchin Strongylocentrotus purpuratus [Turner, E., Klevit, R., Hopkins, P. B., & Shapiro, B. M. (1986) J. Biol. Chem. 261, 13056-13063]. Here we report two related compounds, 1-methyl-N alpha-methyl-4-mercaptohistidine, or ovothiol B, from the scallop Chlamys hastata, and 1-methyl-4-mercaptohistidine, or ovothiol A, from the starfish Evasterias troschelii. These two compounds, as well as the S. purpuratus compound now designated ovothiol C, were isolated from eggs or ovarian tissue by S-carboxymethylation with [3H]iodoacetic acid, ion-exchange chromatography and ion-pairing high-pressure liquid chromatography. The structures of S-(carboxymethyl)ovothiols A and B were determined by 1H NMR, and that of ovothiol A was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. In the ovary of each species, the predominant methylation form of ovothiol accounts for at least 80% of the total 4-mercaptohistidine. The ovothiol concentration of the ovary far exceeds that of the testis or somatic tissues. The ovothiol C content of unfertilized S. purpuratus eggs is 1.14 mumol/10(6) eggs, equivalent to approximately 4.3 mM average concentration; the glutathione (GSH + GSSG) content is 0.9 mumol/10(6) eggs. In this species, high ovothiol levels persisted for the first 2 weeks of embryonic development. Ovothiol and glutathione account for virtually all of the trichloroacetic acid soluble-SH groups in the egg; these results are compared to several previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

CHANGES IN ACTIVITIES OF CREATINE KINASE, ARGININE KINASE AND THEIR MULTIENZYME FORMS DURING EMBRYONIC AND LARVAL DEVELOPMENT OF SEA URCHINS

The presence and the changes of CPK and APK have been studied during larval development through metamorphosing of Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudo-centrotus depressus . While no CPK activity was found in the unfertilized eggs and the embryos of early developmental stages, APK was quite active throughout these stages. At the late 8-armed pluteus stage just prior to metamorphosis, CPK first became active. Electrophoretically this CPK was identical with one of three CPK forms of sperm, tube feet and esophagus but not with two CPK forms of lantern muscle. APK in the unfertilized eggs and early embryos was electrophoretically separated into two distinct molecular forms, one of which disappeared during the late larval stages. The persisting form of larval APK was identical with a single APK form present in the adult muscular tissues.