Ploidy Classes Research Articles

−245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo

Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.

Relationship of megakaryocyte ploidy with platelet number and size in cats, dogs, rabbits and mice

Feline platelets are larger than platelets of many other species. The following parameters were examined in 13 normal cats in an attempt to determine a reason for the difference: platelet count, mean platelet volume (MPV), platelet mass and ploidy of mature megakaryocytes. Average ± SEM platelet count was 213 000 ± 15000/μl, MPV was 11.1 ± 0.2 fl, and platelet mass was 2.4 ± 0.2 × 106 fl/μl. The predominant ploidy classes of feline megakaryocytes were 32N (41.6%) and 64N (47.1%). These findings were compared to existing data from normal dogs, rabbits, and mice. These species exhibited progressively higher platelet counts (313 000 ± 28 000, 568 000 ±35000, and 1328 000 ± 79 000/μl, respectively) and progressively smaller MPVs (7.2 ± 0.2, 4.4 ± 0.1 and 3.9 ± 0.1 fl, respectively) than cats. Platelet mass was the same in cats, dogs and rabbits, but it was much higher in mice (5.2 ± 0.3 × 106 fl/μl) The modal megakaryocyte ploidy was 16N in mice and 32N in dogs and rabbits. The MPV was directly related to the ploidy of fully mature megakaryocytes except when comparing dogs and rabbits for which MPVs differed, but ploidy distributions did not. The findings suggested that ploidy of a megakaryocyte may be one of the determinants of the size and number of platelets it will produce during normal haemopoiesis.

Inheritance of parental genomes in progenies of Poa pratensis L. from sexual and apomictic genotypes as assessed by RAPD markers and flow cytometry

Moving gene(s) responsible for the apomictic trait into crop plants that naturally reproduce through a sexual process would open up new areas in plant breeding and agricultural systems. Kentucky bluegrass (Poa pratensis L.) is one of the most important forage and turf grasses in temperate climates. It reproduces through facultative aposporous parthenogenesis, but the reproductive behaviour ranges naturally from nearly obligate apomixis to complete sexuality. In addition to apomictic reproduction, sexual hybridization may take place. Selfing may also occur, and occasionally reduced egg cells may develop through parthenogenesis generating (poly)haploids. The inheritance of parental genomes was assessed in Kentucky bluegrass progenies by employing RAPD markers in combination with flow cytometry (FCM). Nine progenies from different crosses carried out between completely sexual and highly apomictic genotypes were evaluated in order to probe the reproductive behaviour of the mother plants and to distinguish the different classes of aberrant plants. Not only were maternals and balanced BII hybrids recorded, but so were (poly)triploid BIII hybrids, selfs, and (poly)haploids. The application of these techniques demonstrated that FCM analysis accurately distinguishes the n, 2n, and 3n ploidy levels of progenies, and that RAPD markers unequivocally recognize progenies of apomictic and hybrid origin. The occurrence of aneusomaty was documented in one of the selected sexual genotypes, whose crossed progeny plants manifested two distinct classes of ploidy. The nomenclature BI was adopted to refer to hybrids with a hypodiploid nuclear condition. On the whole, the FCM analysis confirmed most of the RAPD data. The combined evaluation of DNA markers and DNA contents proved to be an efficient screening tool for scoring maternal plants, assessing the genetic origin of aberrant plants, and quantifying the inheritance of parental genomes in Kentucky bluegrass. Hybrid populations from sexual×apomictic matings that segregate for the mode of reproduction represent a valuable basis for attempting to identify molecular markers linked to the apomixis gene(s).

Megakaryocyte ploidy and platelet changes in human diabetes and atherosclerosis.

Altered platelet morphology and function have been reported in patients with diabetes. They are likely to be associated with the pathological processes and increased risk of vascular disease seen in these patients. Mean platelet volume (MPV), platelet count, and megakaryocyte (MK) ploidy (DNA content) were measured in (1) nondiabetics with normal coronary arteries, (2) nondiabetics with coronary artery atherosclerosis, (3) diabetics without evidence of vascular complications, and (4) diabetics with vascular disease. The platelet count (+/- SD) was increased in all groups but only significantly in the diabetics with vascular disease (236 +/- 65 versus 250 +/- 54 versus 257 +/- 64 versus 295 +/- 90 [P < or = .05] x 10(9)/L, for groups, I, II, II, and IV, respectively). The MPV was significantly increased in patients with atherosclerosis (7.0 +/- 0.4 versus 8.0 +/- 1.2 [P < or = .05] versus 7.2 +/- 0.9 versus 8.1 +/- 0.9 [P < or = .05] IL). Geometric mean MK ploidy was significantly increased in all groups compared with controls (16 +/- 1.5 versus 18.7 +/- 1.8 [P < or = .05] versus 19.8 +/- 1.6 [P < or = .05] versus 20.1 +/- 2.7 [P < or = .05]). Furthermore, some patients with vascular disease and/or diabetes had a modal ploidy shift from 16 (the normal mammalian modal ploidy) to 32, with a concomitant reduction of MKs in the 8 and 16 ploidy classes. This shift was seen particularly in the diabetics with vascular disease (P = .007). Interleukin-6 (IL-6) levels were measured and were elevated in patients with atherosclerosis; the highest levels were found in the diabetic patients (0.7 +/- 0.9 versus 5.3 +/- 5.5 [P < or = .05] versus 2.5 +/- 2.8 versus 6.7 +/- 5.5 [P < or = .05] ng/L). In the diabetic patients with atherosclerosis, fibrinogen levels were also increased (2.85 +/- 0.76 versus 3.34 +/- 1.32 versus 2.43 +/- 1.50 versus 5.59 +/- 1.72 [P < or = .05] g/L). Furthermore, IL-6 levels correlated with MK ploidy (r = .45, P = .009) and fibrinogen levels (r = .5, P = .0001). This study demonstrates that patients with vascular disease, particularly diabetics, have an altered MK ploidy distribution, showing a shift toward higher ploidy in association with an increased platelet mass (count x volume). Changes in platelets in diabetes probably reflect MK changes, which themselves are a response to systemic change.

Romurtide, a synthetic muramyl dipeptide derivative, promotes megakaryocytopoiesis through stimulation of cytokine production in nonhuman primates with myelosuppression

The response of megakaryocytes and cytokines to the administration of romurtide, a synthetic muramyl dipeptidederiivative, was investigated in monkeys with myelosuppression by carboplatin-treatment. Romurtide increased the number of megakaryocytes and promoted the shift of megakaryocytes towards high ploidy class indicative of the promotion of the proliferation and maturation of megakaryocytes. The serum levels of interleukin-6, stem cell factor, and erythropoietin elevated significantly before the enhanced response of megakaryocytes induced by romurtide was observed. Romurtide also enhanced production of colony-stimulating factors (CSFs), such as granulocyte-CSF, macrophage-CSF, and granulocyte-macrophage CSF by monkey mononuclear cells. The stimulating effect of romurtide on the production of those cytokines and CSFs is likely to be responsible for the subsequent promotion of the proliferation and maturation of marrow megakaryocytes.

Effects of recombinant human interleukin-3 on maturation of megakaryocytic cell line, CMK

It is well known that interleukin-3 (IL-3) stimulates the proliferation of megakaryocyte colonies. On the other hand, it remains unclear whether IL-3 also promotes the maturation of megakaryocytes. In this report, we investigated the effects of IL-3 on megakaryocyte maturation in vitro using the human megakaryocytic cell line, CMK, which excludes the influence of bone marrow accessory cells. CMK cells were incubated both with and without 2–50 U/ml of recombinant human IL-3 (rhIL-3) and then analyzed for the effects on proliferation, cell size, DNA ploidy and the expression of platelet glycoproteins. We initially confirmed that CMK cells express IL-3 receptors (IL-3R) on the surface, and rhIL-3 stimulates the proliferation of CMK cells, showing their IL-3R function normally. With addition of rhIL-3 to the CMK cell culture system, increments in cell size, a significant shift toward high ploidy classes, and an increased expression of platelet glycoproteins CD29, CD41, CD42a, CD42b and CDw49f were newly observed. These findings suggest that rhIL-3 acts directly on human megakaryocytes and has the ability to directly promote both proliferation and maturation of megakaryocytes, without the help of accessory cells. Furthermore, rhIL-3 induced/enhanced the expression of mRNA for granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) in CMK cells. Therefore, the effects of rhIL-3 on the maturation of megakaryocytic cell lines demonstrated in this study may be partly mediated by the action of cytokines such as LIF and GM-CSF, which are induced by IL-3 in the megakaryocytic cell line.

A Single Injection of Pegylated Murine Megakaryocyte Growth and Development Factor (MGDF ) Into Mice Is Sufficient to Produce a Profound Stimulation of Megakaryocyte Frequency, Size, and Ploidization

AbstractDespite numerous studies investigating the action of c-mpl ligand, no reports have defined the in vivo changes in megakaryocytopoiesis in response to a single injection of this cytokine. Here we compare the kinetics of the megakaryocytopoietic response in C57Bl/6J mice administered 25 μg/kg or 250 μg/kg of pegylated (PEG) murine megakaryocyte growth and development factor (MGDF ) as a single intravenous injection. Megakaryocytes of mice treated with MGDF had normal ultrastructure, showing a typical distribution of the demarcation membrane system, α-granules, and other cytoplasmic organelles. Megakaryocyte ploidy, size, and frequency were markedly increased with both MGDF doses. Megakaryocyte ploidy was maximally increased from a modal value of 16N to 64N on day 3, with both doses of MGDF. Similarly, a comparable increase in megakaryocyte size occurred in the two MGDF groups. Increased megakaryocyte size was coupled to the increase in megakaryocyte ploidy, and no evidence for independent regulation of megakaryocyte size within individual ploidy classes was apparent. In contrast to megakaryocyte ploidy and size, the increase in megakaryocyte frequency was markedly different with the two doses of MGDF. The proportion of 2N and 4N cells was increased from a baseline of 0.035% to 0.430% by day 4 in mice treated with the higher dose of MGDF, but only to 0.175% in mice administered 25 μg/kg of MGDF. The marked increase in the pool of these immature megakaryocytes translated to a sustained elevation in the frequency of polyploid megakaryocytes (8N cells and greater). In contrast to the sustained increase in the frequency of polyploid cells, the level of polyploidization was downregulated on days 6 to 10, but normalized by day 14. We conclude that a single injection of MGDF is able to expand the megakaryocytic pool in a dose-dependent manner, which, with subsequent maturation, should lead to an increased rate of platelet production.

4 Megakaryocytes and platelets in myeloproliferative disorders

Increased megakaryocyte (MK) proliferation in bone marrow is a feature common to the three Ph-negative myeloproliferative disorders (MPDs), i.e. essential thrombocythaemia (ET), polycythaemia vera (PV), and myelofibrosis with splenic myeloid metaplasia (MMM), and to chronic myelocytic leukaemia (CML). Enlarged MKs with multilobulated nuclei and cell clustering in close proximity are the hallmark of all the Ph negative MPDs. Clonality of haematopoietic cells, based on X chromosome inactivation, can now be studied in a majority of female patients in all nucleated cell fractions as well as in platelets. Cytofluorometric studies have demonstrated a shift towards higher ploidy classes in PV and ET MKs which may be useful in discriminating between both primary and reactive thrombocytosis and CML patients which show a significant shift to lower MK ploidy values. The role of MK proliferation on the evolution of myelofibrosis common to MPDs has been firmly established. Implication of platelet-derived growth factor (PDGF) in myelofibrosis has already been demonstrated. More recently transforming growth factor beta (TGF beta) synthesized and secreted by MK has been implicated in fibroblasts stimulation. A significant increase in circulating colony-forming units of MKs (CFU-MK) has been repeatedly observed in MPDs as well as a spontaneous MK colony formation in a majority of ET patients. Hypersensitivity to thrombopoietin (TPO) in relation to a functional defect of the TPO-MPL pathway may play a major role in spontaneous MK growth. There is no currently available test of platelet functions able to predict the risk of occurrence of thrombotic or haemorrhagic complications in MPD patients. However, the role of platelet activation in the pathogenesis of ischaemic erythromelalgia has been established and a correlation between presenting haemorrhagic manifestations and platelet counts in excess of 1000 x 10(9)/l has been found.

Cytometric DNA analysis and prognostic biomarkers in breast carcinoma. Expression of P53 product in the different ploidy classes.

The aim of the study was to correlate the DNA Index (DI) and S‐phase fraction (SPF) values determined by multiparametric flow cytometry in breast cancer (mainly T1 and T2 stages) with several clinico‐pathologic variables and other biological parameters. For this purpose, a total of 136 breast cancers were submitted to flow cytometry and to several types of immunohistochemical analyses. Among clinico‐pathologic data we considered pT, pN and grade and among immunohistochemical markers, hormonal receptors, P53, c‐erbB‐2 and MIB‐1. We found that DNA aneuploidy was strongly correlated with high tumoral grade, absence of hormonal receptors, high proliferation, as shown by high MIB‐1 (≥36%) and SPF values (≥13.3%), and P53 positivity. Grouping the tumours according to their DI values, we observed a relative significantly higher correlation of the near‐triploid range carcinomas (DI 1.40–1.60) with immunohistochemical expression of P53 (p=0.0001). Near‐triploid DI was also associated with a high proliferative activity, expressed both by SPF (p=0.0001) and MIB‐1 reactivity (p=0.0001), high tumoral grade (p=0.0001) and presence of axillary metastases (p=0.03). These data suggest that DNA near‐triploid tumours in breast cancer may have a more aggressive behaviour in comparison with other DI classes.

Multiparameter absorption measurements in automated microscopy. Simultaneous quantitative determination of DNA and nuclear antigen.

To test a dual DNA-nuclear antigen staining method for multiparameter absorption image analysis. MCF 7 cells, grown on glass slides, served as a model to test the staining technique. For DNA, Feulgen-based CAS quantitative DNA staining, and for nuclear antigen, alkaline phosphatase-based immunocytochemical staining with CAS Red as the chromogen, were used. MIB-1, estrogen and progesterone receptors were used as examples of nuclear antigen staining. Measurements were performed with the DISCOVERY image analyzer. Scatterplots, in which the nuclear antigen content was plotted against the DNA content, were obtained. Immunostain-positive and -negative populations could be discriminated. These cells were visualized in image galleries. The DNA histograms of the positive and negative cells showed no change in coefficient of variation or integrated optical density ratio of the G0, G1 and G2 + M peaks as compared to single DNA staining. The intensity of the immunostain increased as compared to the single immunostaining result. This staining technique allows the simultaneous accurate measurement of costained DNA and antigen within the same nucleus. This opens the possibility for studies in which nuclear antigen expression is monitored during the cell cycle or in cells of different ploidy classes. Identified cells can also be visualized by presentation in an image gallery or by relocation on the slide. This can support the analysis of clinical samples, where cytometric data can be correlated with and confirmed by visual diagnosis.

Tumour heterogeneity of DNA cell cycle variables in breast cancer measured by flow cytometry.

Conflicting results have been reported concerning the prognostic value of DNA flow cytometric variables (DNA ploidy, DNA index, %S phase fraction) in breast cancer. Selection bias and differences in treatment may have contributed to these conflicting prognostic results. Differences in tissue processing, the number of nuclei measured, DNA histogram/cell cycle analysis, and intra-tumour heterogeneity may also have played a role. The aim of the present study was to assess intra-tumour heterogeneity of DNA flow cytometric variables in breast cancer. Fresh frozen specimens (n = 274) (0.3 x 0.3 x 0.3 cm) of 17 breast cancers and 167 slices, 50 microns thick, of 58 paraffin wax embedded blocks of 21 breast cancers were studied. All samples were prepared individually for DNA flow cytometry. DNA histograms were interpreted by semi-automated cell cycle analysis (MultiCycle) by two observers to avoid biased interpretation. An artificial averaged DNA histogram of each case was composed to simulate a sample prepared from whole tumour tissue. With regard to DNA ploidy, classified as diploid or aneuploid, the fresh frozen and paraffin wax embedded breast cancers showed intra-tumour heterogeneity in 53% and 38% of cases, respectively. For fresh frozen and paraffin wax embedded material, respectively, six samples had to be measured to detect the highest DNA ploidy class in 71% and 86% of cases. Averaged DNA histograms showed a loss of DNA aneuploidy in 36% and 6% of fresh frozen and paraffin wax embedded samples, respectively. High intra-tumour heterogeneity (wide ranges) was found for the %S phase fraction. Average %S phase fraction and average aneuploid %S phase fraction had the widest ranges at 9.5-31.6% and 0.0-62.7%, respectively. There was no correlation between the number of stemlines and intra-tumour %S phase variability on the one hand and tumour size and grade on the other. High intra-tumour heterogeneity for breast cancer was found for DNA ploidy, the DNA index and %S phase fraction as measured by flow cytometry, which may explain the conflicting prognostic results reported in the literature. To detect aneuploid cells, six samples may have to be prepared and measured separately. Measurement of these variables may be more reliable in paraffin wax sections because the thick slices provide a more representative sample. Prospective studies are required to determine whether the highest %S phase fraction value or the average value is more useful in the clinical context.

Dynamics of polyploidization and quantitative studies of Ag-NORs in the interphase nucleoli of cambial trophoblast cells in the developing rat placenta

SUMMARYParameters of silver impregnation of nucleoli of rat cambial trophoblast cells were evaluated at the 12th, 13th, and 14th prenatal day, i.e. in the period of their differentiation. The number of Ag-positive granules in the nucleoli has been found to vary from 10 to 120 at all the developmental stages studied, the modal number of the granules decreasing from 60–80 at the 12th day to 40–60 at the 14th day. At the transition to each next ploidy class (2c, 4c, 8c, 16c), the number of the Ag-positive granules and the area they occupy in the nucleoli increased but this increase was lesser than two-fold at all the stages studied. The area of the nucleoli, on the contrary, increased strictly parallel to the ploidy degree. The mean number and the total area of the argentophilic granules as well as the area of the nucleoli calculated for each ploidy class did not change in the course of the placenta development. This indicates an approximately equal level of activity of the nucleolar organizer regions at the...

Flow cytometric analysis of neoplastic nodules and hepatocellular carcinomas induced by ciprofibrate in the rat.

Alterations in DNA ploidy accompany hepatocellular carcinoma (HCC). However, changes in DNA content are also seen in regenerating liver and with increasing age. Thus, to investigate the role of DNA ploidy changes in development of HCC, flow cytometric DNA content determinations were done in a rat model system of peroxisome proliferator-induced HCC. Paraffin blocks of liver isolated from 18 Fisher 344 male rats fed ciprofibrate for 20 weeks (4), 40 weeks (4) or 20 months (10) were examined. Livers from age-matched control rats were also examined. From the 20 month ciprofibrate group, nine neoplastic nodules (NNs), 27 HCCs and four non-tumorous surrounding tissue controls (NTCs) were examined. Significant DNA tetraploid populations were seen in both the NNs and NTCs. A significant increase in the percentage of DNA diploid cells was observed in the NN samples. No significant difference in the percentage S-phase cells was seen. Emergence of cell populations with new DNA ploidy classes (8c or DNA aneuploid) as compared with NTCs was only seen in HCCs (7 of 27), and five of these seven were DNA aneuploid, as distinct from DNA tetraploid, populations. A total of 16 of 24 HCC samples that were adequate for cell cycle analysis had average percent S-phase greater than the mean of the NTCs plus three standard deviations. Although a direct role cannot be inferred, these results support the hypothesis that increases in the fraction of diploid cells is an important early event in the development of rat HCC and that further alterations in DNA ploidy and increased proliferative fraction accompany the development of HCC.

Biochemical, pathologic and morphometric alterations induced in male B6C3F1 mouse liver by short-term exposure to dichloroacetic acid

Dichloroacetic acid (DCA) is a complete hepatocarcinogen and tumor promoter in the male B6C3F1 mouse. Published reports indicate that the compound is non-genotoxic. This study examines possible non-genotoxic (epigenetic) mechanisms by which DCA elicits its carcinogenic response. Correlative biochemical, pathologic and morphometric techniques are used to characterize and quantify the acute, short-term response of hepatocytes in the male B6C3F1 mouse to drinking water containing DCA. Cellularity, [ 3H]ithymidine incorporation, DNA concentration, nuclear size, and binuclearity are evaluated in terms of level of exposure (0, 0.5 and 5 g/l) and length of exposure to DCA. The dose-related alterations in hepatocytes of animals exposed to DCA for 30 days or less indicate that shortterm exposure to DCA results in inhibition of mitoses, alterations in cellular metabolism and a shift in ploidy class. Thus, DCA carcinogenesis may involve cellular adaptations, development of drug resistance and selection of phenotypically altered cells with a growth advantage.

An improved method of mouse liver micronucleus analysis: an application to age-related genetic alteration and polyploidy study

The performance of a micronucleus test in liver cells in vivo requires two laborious procedures: stimulation of hepatocytes to division and dissociation of liver tissue into a single-cell suspension. We propose the method of inhalation treatment of mice with carbon tetrachloride to induce cell proliferation and alkaline dissociation of previously fixed tissue. The micronucleus incidence and ploidy classes in terms of cytophotometric DNA content were determined in liver of mice of three age groups (around 2.5, 5.0 and 7.0 months old) after CCl 4 treatment or partial hepatectomy. The data obtained show that both methods give the same results. The fraction of micronucleated hepatocytes was 0.69% at the age of 2.5 months; it increased to 8.5% and then to 13.5% at 5.0 and 7.0 months respectively. Simultaneously, the ploidy classes changed both with the aging of the animal and after induced liver regeneration. The percentage distribution of micronucleated cells by ploidy class showed that cells carrying micronuclei were the higher ploidies rather than the population in general. Since polyploid cells contain multiple molecular targets for genetic damage, the micronucleation index per genome unit was estimated. Then the real rate of accumulation of both intrinsic endogenous (and probably the exogenously induced) preclastogenic genetic alterations in hepatocytes during the adulthood of mice was evaluated to be 0.03% per diploid genome per day. This seems to be the first description of the phenomenon of liver cell aging in terms of micronuclear aberrations.

The prognostic significance of DNA flow cytometry in breast cancer: results from 881 patients treated in a single centre.

In this single-centre study of 881 patients, S-phase fraction (SPF) was shown to be a significant prognostic marker in terms of overall survival (OS), relapse-free survival (RFS) and survival after relapse (SAR). Further, SPF had independent prognostic significance when considering a range of other clinicopathological variables, namely tumour grade and stage, nodal status, patient age, tumour size, menstrual status and treatment details. For OS and RFS, SPF was the second strongest predictor of the clinical course of the disease after nodal status, and for SAR it was the strongest prognostic marker. SPF correlated positively with histological grade but was the stronger predictor of survival. The distribution of SPF values was markedly different for the two ploidy classes of tumour, with DNA aneuploid tumours having a significantly higher average SPF. However, SPF retained its independent prognostic ability when DNA diploid and aneuploid tumours were analysed separately, DNA ploidy itself also proved to be an independent prognostic marker but the survival difference between the two ploidy classes was much less than that seen for different levels of SPF. Tumours with several DNA aneuploid populations (multiploid tumours) tended to have a worse prognosis than other aneuploid tumours but this trend did not reach statistical significance. In this and other studies from this centre, SPF has proved to be a robust predictor of clinical outcome in carcinoma of the breast.

The physiology and biochemistry of diploid and triploid Manila clam ( Tapes philippinarum Adams & Reeve) larvae and juveniles

Triploidy was induced in fertilised eggs of the Manila clam [ Tapes philippinarum (Adams and Reeve)] at meiosis II. Growth rate, survival, food consumption, respiration rate and biochemical content of separate ploidy classes containing diploid or mainly (> 60%) triploid larvae and juveniles reared in the hatchery were compared and found to be similar. Newly formed D-larvae were 88–95 μm in length and became pediveligers after 15–17 days, when they had grown to 200–220 μm. Juveniles grew from 0.03 mg (mean dry weight) to 15 mg in 7 wk. Larval food consumption was described by the equation C= 0.577 × LW 0.587 (where C = organic weight of food cells consumed (μg·d −1) and LW = organic weight of larvae (μg). Juveniles consumed 0.76 μg·d −1 of food per μg organic weight. Lipid and carbohydrate reserves increased during larval growth to 17.5 and 7.5% of organic content, respectively. Lipid reserves were utilised during metamorphosis. In the juveniles, reserves remained constant at about 11% (lipid) and 7% (carbohydrate). The relationship between dry weight (W, mg) and oxygen consumption ( R, μg· d −1) of individual clams was described by R = 15.07 × W 0.638. Energy budgets for larval and juvenile Manila clams were calculated. Assimilation efficiency was high in the larvae (> 80%), and net growth efficiency increased with size and declining activity of the clams. The physiological and biochemical attributes of the clams were similar to those reported for other bivalve mollusc species. The results are discussed in relation to the apparent absence of recruitment of Manila clams in UK coastal waters.

A genetic and metabolic basis for faster growth among triploids induced by blocking meiosis I but not meiosis II in the larviparous European flat oyster, Ostrea edulis L.

This study establishes a genetic and metabolic basis to faster triploid growth in the oyster Ostrea edulis. Triploidy was induced using cytochalasin B, and image analysis of biopsied tissue employed to ensure similar ploidy of all animals within each class. Results indicate that lifetime growth in total dry tissue weight over 15 months was more than 60% faster ( p<0.001) in meiosis I triploids than in diploid siblings or meiosis II triploids, with no difference between meiosis II triploids and their diploid siblings. For six polymorphic enzyme loci, single-locus heterozygosity was consistently greatest in meiosis I triploids ( p<0.001), so that average multiple-locus heterozygosity in meiosis I triploids was 49% higher than in normal diploids, and 55% higher than in meiosis II triploids ( p<0.001). This suggests that faster growth resulted from increased allelic diversity, rather than the increased allelic quantity that results from the addition of one entire set of chromosomes among triploids generally. Results also confirm that the faster growth of meiosis I triploids resulted from reduced energy expenditure, associated with lower concentrations of RNA per unit total tissue protein, which infer reduced rates of whole-body protein turnover. Statistical analyses confirmed that differences in oxygen consumption and growth were associated with both ploidy class and average multiple-locus heterozygosity, indicating that performance in meiosis I triploids is not only improved as a result of reduced reproductive output, but also through the metabolic consequences associated with increased heterozygosity.

Ploidy-Dependent Growth and Binucleation in Cultured Rat Hepatocytes

The proliferative activity of rat hepatocytes, cultured in the presence of epidermal growth factor (EGF) and insulin, was examined by immunostaining of S-phase cells labeled with bromodeoxyuridine (BrdU) in culture. Proliferation rates of the different hepatocellular ploidy and nuclearity classes were measured by fluorescence image cytometry or by microscope counting of immunostained cells. Effects of EGF and insulin were largely additive, the binuclear cells being more growth factor-dependent (showing less growth in the absence of factors) than the mononuclear cells. A serial warm-washing procedure was used to remove excess BrdU from the culture medium, allowing the study of hepatocellular binucleation by a BrdU pulse-chase approach. A high rate of binucleation was detected (50%, possibly suggesting a quantal mechanism), indicating that the hormones induce a binucleating (polyploidizing) type of growth similar to that normally observed in the liver of growing rats. The highest proliferative activity (labeling index) in the hepatocyte cultures was found among the diploid cells, independent of the degree of mitogenic stimulation. The labeling index was inversely correlated with ploidy, suggesting that the ability of hepatocytes to proliferate decreases with increasing polyploidization.

Appearance of a Megakaryocyte Growth-Promoting Activity, Megapoietin, During Acute Thrombocytopenia in the Rabbit

Plasma obtained from rabbits made thrombocytopenic by the injection of antiplatelet serum (APS) stimulated megakaryocyte growth in an in vitro bone marrow culture. Although the number of megakaryocytes that grew was increased on average to 136%, the major effect was on megakaryocyte ploidy, which showed a shift in the modal ploidy class from 8N to 16N, an average change in the geometric mean ploidy from 8.7N to 11.8N, and the appearance of some 64N megakaryocytes. This in vitro stimulation of megakaryocyte number and ploidy was shown to be due to the appearance in the circulation of a positive activity, which we have named megapoietin. Levels of megapoietin in thrombocytopenic plasma could be quantitated by measuring the extent of megakaryocyte ploidization in vitro. The relationship over time of megapoietin to changes in the platelet count was then assessed in a series of rabbits made thrombocytopenic by APS injection. Although not elevated after 3 hours of thrombocytopenia, megapoietin increased after 8 hours to 48% of the maximal level, which occurred after 24 hours of thrombocytopenia. Levels of megapoietin were inversely and proportionally related to the platelet count during thrombocytopenia and the subsequent rebound thrombocytosis. During persistent thrombocytopenia, megapoietin levels remained maximally elevated. These results suggest that megapoietin may play a physiologic role in the feedback loop between the platelet and the bone marrow megakaryocytes.