Assessment of egg quality in atlantic salmon,Salmo salar,treated with testosterone—II. Amino acids

• 1.1. Atlantic salmon (Satmo salar) were treated with Silastic pellet implants containing testosterone (200 μg/g body weight) four times in a year. Eggs stripped from control (sham implantation) and testosterone-treated fish were fertilized and comparisons of free and total amino acid compositions made until first feeding.
• 2.2. Despite having eggs which were smaller in diameter, lighter in weight and lower in total amino acid contents, alevins from testosterone-treated fish were heavier in wet weight and larger in body length, and exhibited enhanced free amino acid contents at first feeding.
• 3.3. The qualitative composition of total amino acids in eggs from treated and control fish did not differ.
• 4.4. Total amino acid pool of eggs and alevins declined during development, but an increase in the free amino acid pool was noticed through development. The increase in free amino acid pool was higher in eggs and alevins from treated fish than controls, perhaps due to enhanced mobilization of the free amino acid pool.

     

The effect of light intensity on the nightly movements of juvenile Atlantic salmon alevins away from the redd

Atlantic salmon ( Salmo salar ) alevins move away from the redd predominantly at night time, suggesting that light level is the controlling factor releasing activity. However, the exact relationship between Light level and alevin activity is unknown, so a series of artificial redds was used to monitor alevin movements under various night time incident light levels. Five experimental redds and two controls were run over a period of 3 years. Combining the results gave a significant negative correlation between the numbers of salmon alevins moving away from redds on light nights and light level. This behaviour was interpreted as a negative photoresponse since the frequency of alevin movement away from the redd was dependent on the light intensity. Two possible explanations as to why alevins may react in this way to light level were considered. Firstly, the behaviour could have evolved to ensure that alevins only move away from the redd when they are least likely to be caught by a predator or secondly, retinal developmental differences between alevins could have led to the observed behaviour.     

DNA synthesis and content and the accumulation of total protein and hemoglobin during the differentiation of primary erythroid cells in chickens

Using cytophotometric and autoradiographic methods, it was shown that on days 2-3 of embryogenesis primary erythroid cells (PEC) divided actively. The distribution of erythroblasts (EB) according to their DNA content is not, however, typical of a proliferating population: it contains an unusually large number of 4c cells resulting from the cell cycle arrest at the G2 phase. It is established that reticulocytes (RC) do not divide and are arrested at G1 or G2 phases, since they do not incorporate 3H-thymidine after their formation is complete and their DNA contents are strictly confined to either 2c or 4c. All types of PEC include a large number of cells containing H2c DNA which is due either to the cell cycle arrest at the S phase, or to the formation of accessory nuclei. All PECs have much higher contents of hemoglobin and total protein than do adult hen erythrocytes (EC). Hemoglobin and total protein contents of H2c and accessory nuclei containing cells are much higher than those in 2c-cells. We have calculated that adult birds and embryos contain the same amount of hemoglobin per gram of weight, but the quantity of red blood cells in the former is ten times higher. A conclusion is drawn that proliferation and cytodifferentiation regulation mechanisms are directed, in primary erythropoiesis, to intense hemoglobinization of the cells, and, in adult erythropoiesis, to increasing their number. In both the cases homeostatic regulation of erythropoiesis works.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of egg size on yolk utilization and growth of rainbow trout alevins (Oncorhynchus mykiss Walbaum)

Changes in body weight and yolk weight were studied in rainbow trout alevins derived from large eggs (diameter >4 mm) and small eggs (egg diameter <4 mm) from the same female. Maximal body weight was reached later, and was higher, for alevins derived from large than from small eggs. The yolk was absorbed more quickly by the alevins of small eggs. Percentage of body water increased in alevins during fasting up to a limit of 91%. Although the limit was the same for both groups of alevins, it was reached more quickly by alevins from small eggs. Relative water content of the yolk did not seem to be influenced by egg size. These results suggest that original egg size had both a quantitative and qualitative effect on the early development of alevins.     

Differentiation pathways of primary erythroid cells in chickens

Proliferation and differentiation processes of chick embryo primary erythroid cells (PEC) were studied. A novel differentiation pathway was discovered by which cells of proerythroblastic and erythroblastic stages are blocked in G1 or G2 phases, to develop then directly into reticulocytes, i.e. terminally differentiated non-dividing cells with high hemoglobin contents differing in shape from erythrocytes. These cells appear in blood two days earlier than erythrocytes, then they co-exist with the latter and are eliminated in parallel with them. This pathway leads to a rapid enrichment of PEC with hemoglobin. A fraction of PEC forms accessory nuclei, which, as it is shown here, contain an extra quantity of DNA. Compared to the diploid ones, such cells reveal increased hemoglobin contents which enabled us to assume that they may have amplified the globin genes. The above-mentioned pecularities of cytodifferentiation may be presumably an adaptation to oxygen supply of growing embryos which are known to stay in hypoxia. A comparison of these results with results of our earlier study on experimental anemia makes it possible to suppose that pecularities of these two types of cytodifferentiation may be based on similar or, perhaps, analogous mechanisms of regulation.     

Comparison of absorption measurements of DNA stain content by utilizing video and scanning image cytometers

After staining with the Feulgen reaction, the DNA stain contents of 155 mouse bone marrow cells and 22 adjacent chicken erythrocytes were measured by absorption image cytometry by utilizing two different systems--a scanning cytometer and a video cytometer. In the scanning cytometer (M85 microdensitometer, Vickers Instruments, Malden, MA), a spot of light was scanned across the cell. In the video cytometer (TAS Plus, E. Leitz, Rockleigh, NJ), the microscope field, which may contain several nuclei, was imaged onto a Plumbicon video camera. With each system, cells were scanned, digitized into their elementary pixels, and analyzed to determine their integrated absorbance. Comparison of the DNA stain contents of the same G0/G1 bone marrow cells and chicken erythrocytes, as measured by video and scanning cytometry, showed that both techniques gave comparable results; scanning cytometry is more precise. The coefficients of variation of the measurements for the G0/G1 bone marrow cells and for the chicken erythrocytes were 5.9% and 7.0%, respectively, when measured by video cytometry at the absorption peak (584 nm), compared to 4.1% and 3.5%, respectively, for the same cells when measured by scanning cytometry off the absorption peak (615 nm). The video-based measurements were relatively lower than the scanning measurements for darkly stained cells; this suggests that glare and other optical errors which increase with stain darkness caused greater systematic errors in the video cytometer than they did in the scanning cytometer.     

Reappraisal of nuclear DNA content of rodlet cells compared to several cell types from some freshwater teleosts, using two methods of microdensitometry

Nuclear DNA contents of rodlet cells from Catostomus commersoni, Semotilus atromaculatus and Cyprinus carpio were compared with nuclear DNA of erythrocytes and larger cells of the same species, using scanning microdensitometry and averaging microdensitometry. This study reappraises the work of Barber & Westermann (1983), which employed averaging microdensitometry only, and compared rodlet cell nuclear DNA only with erythrocyte DNA. In addition, this work considers sources of error in both methods of microdensitometry, and comments upon the use of microdensitometry of either method as a mechanism for making distinctions among the DNA contents of cells of different types. The results of the present work consistently indiate no significant differences within species between nuclear DNA content of rodlet cells and larger teleost cells, using either method of microdensitometry. Because of the lack of statistically significant difference in DNA content between nuclei of rodlet cells and those of known teleost cells, it has been concluded that the rodlet cell itself is probably of teleost origin. However, the method indicates nothing about the origin of the rodlets, which have also been shown to contain DNA, but are Feulgen-negative.     

Changes in the nuclear polyamine content of chick erythrocytes during embryonic development.

The polyamine content of the circulating erythrocyte population in the embryonic chick was studied during its development. Total cellular polyamine content fell dramatically between 5 and 7 days of development, paralleling the decrease in metabolic activity exhibited by these cells. Nuclei were isolated from the erythrocytes by a non-aqueous technique, which not only eliminated the polyamine loss that occurred with aqueous isolation, but also prevented redistribution of the polyamines from the cytoplasm. Nuclear spermidine and spermine contents decreased markedly between 5 and 6 days of development from 31 to 10 pmol/microgram of DNA and from 33 to 18 pmol/microgram of DNA respectively. Thereafter the spermine content remained constant, but the spermidine content continued to decline. Good correlations between spermidine and RNA contents were observed in both cells and nuclei, and similarly between spermine and RNA contents in cells, but no such correlation was observed between spermine and RNA in nuclei.     

Cardiovascular responses of eggs, embryos and alevins of Atlantic salmon and rainbow trout to nitric oxide donors, sodium nitroprusside and isosorbide dinitrate, and inhibitors of nitric oxide synthase, N-nitro-l-arginine methyl ester and aminoguanidine, following short- and long-term exposure

Atlantic salmon embryos and alevins Salmo salar that had been exposed to isosorbide dinitrate (ISDN) for 4 weeks, on transfer to fresh water, showed an increase in heart rate. Unexposed embryos and alevins showed a decrease in heart rate following transfer to 100 μmol l⁻¹ ISDN for 4 h. This is in contrast to adult rainbow trout and higher vertebrates where tachycardia occurred in response to nitric oxide (NO) donors. The decreased heart rate in response to ISDN was inhibited by 2 mg 1⁻¹ methylene blue, indicating that NO activates cardiovascular events via guanylyl cyclase and cyclic guanidine monophosphate. Heart rate of rainbow trout alevins Oncorhynchus mykiss exposed to 100 μmol l⁻¹ aminoguanidine responded with a slowly developed but significant bradycardia over 10 min as did those reared in aminoguanidine for 4 weeks then transferred to fresh water. A potentiated increase in heart rate on exposure to the NO donor sodium nitroprusside (SNP), occurred within 1 min in salmon alevins reared in l -nitro-arginine methyl ester ( l -NAME) for 4 weeks, indicating up-regulation of NO receptors. The evidence for down-regulation of SNP-reared alevins exposed to l -NAME was less well defined. The results suggest that both salmonid embryos and alevins have a functional l -arginine-NO pathway and that NO has a physiological role in control of cardiovascular events.     

Effect of N-methyl-N-nitrosourea on avian erythrocyte nuclei reactivation.

Avian erythrocytes are terminally differentiated cells but they can be reactivated by fusion with actively metabolising cells. We have examined the effects of treating the erythrocytes with a carcinogenic methylating agent, N-methyl-N-Nitrosourea (MNU), on the process of reactivation of adult and embryonic nuclei. We have found that the rate of nuclear enlargement is slightly lower in nuclei from MNU-treated cells than from control cells and that there is a marked delay of about 24 h in the appearance of nucleoli in both adult and embryonic cells. This is not due to an effect of MNU on ribosomal (r)DNA: the number of rDNA genes appears to be similar in treated and control cells. Also, the number of rDNA genes appears to be similar in adult and embryonic cells and in unreactivated and reactivated embryonic nuclei: thus, differences in reactivation rate between adult and embryonic cells, observed by us and others, can not be attributed to a gross difference in their ribosomal DNA contents, and reappearance of nucleoli on reactivation can not be due to an amplification of rDNA (i.e., to recovery of such genes if lost on terminal differentiation). We suggest that MNU, although a monofunctional alkylating agent, may cause increased association--possibly cross-linkage--between DNA and protein in chromatin, thereby hindering access of host cell reactivating proteins, especially to the nucleolar regions.     

DNA ploidy analysis and cytologic examination of sorted cell populations from human breast tumors

Two different flow cytometric procedures were applied on cell samples from human breast tumors. One procedure involved DNA ploidy analysis on suspensions of isolated nuclei. The mean ploidy ratios of 27 benign breast lesions to chicken erythrocytes and rainbow trout erythrocytes were found to be 2.66 +/- 0.03 and 1.25 +/- 0.02, respectively. From the 45 stemlines found in a series of 43 carcinomas, 12 were diploid, 13 hyperdiploid and 20 near-tetraploid. No association was found between the lymph node status and the DNA ploidy level. The second procedure involved sorting fixed cells from DNA "windows" for the preparation of permanent cytologic specimens. The sorted cells appeared to be shrunken, but the morphologic quality was similar to that of imprint specimens from the same tumors, permitting discrimination between various types of normal cells and tumor cells. The combined use of both flow cytometric procedures may lead to greater insight into the relationship between the cytologic and cytogenetic heterogeneity of breast carcinomas.     

A comparison of internal standards for plant cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

Differential regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells with various proliferative potentials

The regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells of various proliferative potential was studied. The following regularities were found: 1) Both immortalized and non-immortalized cells can efficiently reactivate DNA synthesis in erythrocyte nuclei. 2) Erythrocytes drastically inhibit the entry of non-malignant culture cell nuclei into the S-period, not acting upon DNA synthesis. 3) The inhibitory action is characteristic of erythrocytes from different stages of chicken ontogenesis (from 5-day-old embryos to the adult bird). 4) Malignant cells are completely refractory to the inhibitory action of erythrocytes. The ability of erythrocytes to inhibit the onset of replication in heterokaryons may be connected with the mechanisms of maintaining these terminally differentiated cells in a non-proliferating state.     

Direct hybridization to DNA from small numbers of flow-sorted nucleated newborn cells

A technique is described that allows direct hybridization to the DNA of cells flow sorted onto nitrocellulose filters, which obviates an intervening DNA isolation step. The feasibility of this technique for studying small numbers of cells is demonstrated with human cord blood, which has a high proportion of nucleated cells. The cells are stained with fluorescein-conjugated anti-HLe-l, a monoclonal antibody that recognizes mature leucocytes. Anti-HLe-l-positive cells are all nucleated, and a controlled, precise number of them may be sorted directly onto a nitrocellulose membrane. In cord blood, a small percentage of anti-HLe-l-negative cells are nucleated erythrocytes, which may also serve as a source of DNA. Studies were performed on male or female newborn cells flow sorted onto nitrocellulose membranes and hybridized with either a non-specific human repeat DNA probe or a Y chromosome-specific probe. Importantly, the sex of the newborn could be determined at the DNA level from as few as 50 sorted cord blood leucocytes or 5,000 HLe-l-negative cells. Since nucleated erythrocytes are common in fetal blood but rarely found in the peripheral circulation of adults, the method has potential application for the determination of fetal sex from analysis of flow-sorted nucleated erythrocytes present in the maternal circulation during pregnancy.     

DNA degradation during maturation of erythrocytes - molecular cytogenetic characterization of Howell-Jolly bodies

Howell-Jolly bodies (HJBs) are small DNA-containing inclusions of erythrocytes and are often present after splenectomy. The genetic composition of HJBs is unknown at present. We isolated individual erythrocytes that had inclusion bodies from five splenectomized patients and performed DNA amplification using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) with subsequent reverse painting on normal male metaphase spreads. We also measured the sizes of HJBs in erythrocytes from a splenectomized patient using an inverted microscope. Two-dimensional positions of HJBs were projected onto a virtual erythrocyte. The average size of HJBs was 0.73 +/- 0.17 microm (range 0.4-1.1 microm). Inside the erythrocyte the HJBs were found to be equally distributed. Small HJBs contained DNA from one or two centromeres and larger HJBs contained DNA from up to eight different centromeres. Centromeric DNA from chromosomes 1/5, 7, 8, and 18 was most frequently observed. Signals from the centromeric regions of chromosomes 3, 4, 9, and 10 were not observed. Signals from euchromatic regions were detected in a few cases. We hypothesize that in addition to enucleation and nucleus fragmentation DNA degradation during maturation of erythrocytes preferentially eliminates euchromatic DNA. Similarities between these processes and those described for embryonic stem cells suggest that most stem cells are able to degrade DNA in a genetically controlled manner.     

Genetic variation of resistance to mercury poisoning in steelhead (Oncorhynchus mykiss) alevins

Newly hatched steelhead alevins were obtained from the factorial breeding of 24 male and 10 female steelhead trout, Oncorhynchus mykiss. Each set of offspring were in a separate cell. They were tested for resistance to intoxication by methylmercuric chloride (CH3HgCl) in water at a nearly constant mercury concentration of 8 microg l(-1). High mortality (81% of the tested alevins) occurred within 2 weeks. Resistance to intoxication, as measured by the time to death, as well as by the survival rate, shared high paternal and maternal variation with negligible interaction. Heritability of time to death was 0.59 +/- 0.17; heritability of survival (all-or-none trait) was lower (0.26 +/- 0.09). Mercury in dead alevins increased with time to death, exhibiting a large environmental variation and (comparatively) negligible genetic influence. At the end of the bioassay, the mercury content in survivors varied widely (3-21 microg g(-1) wet weight). The content was greater than, but correlated with that of dead alevins from the same cells, and it showed little relation with survival rate. Thus, it seems that resistance to poisoning implies a tolerance to high levels of mercury rather than a limitation of its accumulation.     

A Comparison of Internal Standards for Plant Cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

Lipid peroxidation and antioxidant system in the blood of cancerous patients with metastasis

Free-radical-mediated damages may play an important role during metastasis. To investigate their relevance in the metastatic process MDA levels, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, and selenium, zinc and copper contents were determined in plasma and erythrocytes from 20 cancerous patients with metastasis and 30 age-matched controls. Significantly higher concentrations of MDA in plasma as well as in erythrocytes were found comparing to the control group. In both plasma and erythrocytes, GPX activity and selenium and zinc levels were significantly lower in patients than in controls. However, SOD activity in erythrocytes and copper levels in both plasma and erythrocytes were significantly higher in patients. The impaired antioxidant system may favor accumulation of free radicals which may induce the process of metastasis. On the other hand, it is possible that the antioxidant system is impaired as a consequence of abnormality in the antioxidative metabolisms due to the cancer process.     

Comparison of nuclear DNA content of rodlet cells and erythrocytes in some freshwater teleosts

DNA of rodlet cells and erythrocytes from three species of freshwater teleosts, Semotilus atromaculatus atromaculatus, Catostomus commersoni and Cyprinus carpio , was stained with the Feulgen reaction and examined by microdensitometry. Rodlet cells showed nuclear DNA content significantly different from erythrocytes of the same species, but the difference was less than a factor of C, assuming that erythrocytes reflect the normal 2C genome of somatic cells. In two species, S. atromaculatus and C. carpio , the rodlet cell nuclei contained less DNA than the erythrocytes; in C. commersoni they contained more. The identity of the rodlet cell is unknown; the results of these experiments lead to the rejection of the hypothesis that rodlet cells and erythrocytes of a species have the same DNA content, i.e. that the rodlet cell is a normal somatic component of fish tissue.     

Use of diploid and triploid trout erythrocytes as internal standards in flow cytometry.

DNA content determination requires the use of standards. Vindelov has shown the need to use two standards. Chicken and trout erythrocytes are commonly used, but they are not ideal standards. On the one hand, their DNA contents rarely frame the studied sample DNA content, and, on the other hand, as their base compositions are different in terms of A + T/G + C, their relative indices change according to the stains used. Use of triploid trout erythrocytes instead of chicken erythrocytes allows elimination of these two drawbacks; however, diploid trout must be differentiated from triploid trout. The present paper shows that an anatomic malformation is found with the triploid trout and so justifies the use of paired diploid and triploid trout as standards to measure nuclear DNA content.