Ploidy Stages Research Articles

Tetrasporophytic bias coupled with heterozygote deficiency in antarctic plocamium sp. (florideophyceae, rhodophyta).

Meiosis and syngamy generate an alternation between two ploidy stages, but the timing of these two processes varies widely across taxa, thereby generating life cycle diversity. One hypothesis suggests that life cycles with long-lived haploid stages are correlated with selfing, asexual reproduction, or both. Though mostly studied in angiosperms, selfing and asexual reproduction are often associated with marginal habitats. Yet, in haploid-diploid macroalgae, these two reproductive modes have subtle but unique consequences whereby predictions from angiosperms may not apply. Along the western Antarctic Peninsula, there is a thriving macroalgal community, providing an opportunity to explore reproductive system variation in haploid-diploid macroalgae at high latitudes where endemism is common. Plocamium sp. is a widespread and abundant red macroalga observed within this ecosystem. We sampled twelve sites during the 2017 and 2018 field seasons and used ten microsatellite loci to describe the reproductive system. Overall genotypic richness and evenness were high, suggesting sexual reproduction. Eight sites were dominated by tetrasporophytes, but there was strong heterozygote deficiency, suggesting intergametophytic selfing. We observed slight differences in the prevailing reproductive mode among sites, possibly due to local conditions (e.g., disturbance) that may contribute to site-specific variation. It remains to be determined whether high levels of selfing are characteristic of macroalgae more generally at high latitudes, due to the haploid-diploid life cycle, or both. Further investigations of algal life cycles will likely reveal the processes underlying the maintenance of sexual reproduction more broadly across eukaryotes, but more studies of natural populations are required.

An Individual-Based Model of the Red Alga Agarophyton chilense Unravels the Complex Demography of Its Intertidal Stands

Algal demographic models have been developed mainly to study their life cycle evolution or optimize their commercial exploitation. Most commonly, structured-aggregated population models simulate the main life cycle stages considering their fertility, growth and survival. Their coarse resolution results in weak predictive abilities since neglected details may still impact the whole. In our case, we need a model of Agarophyton chilense natural intertidal populations that unravels the complex demography of isomorphic biphasic life cycles and be further used for: (i) introduction of genetics, aimed at studying the evolutionary stability of life cycles, (ii) optimizing commercial exploitation, and (iii) adaptation for other species. Long-term monitoring yield 6,066 individual observations and 40 population observations. For a holistic perspective, we developed an Individual-Based Model (IBM) considering ploidy stage, sex stage, holdfast age and survival, frond size, growth and breakage, fecundity, spore survival, stand biomass, location and season. The IBM was calibrated and validated comparing observed and estimated sizes and abundances of gametophyte males, gametophyte females and tetrasporophytes, stand biomass, haploid:dipoid ratio (known as H:D or G:T), fecundity and recruitment. The IBM replicated well the respective individual and population properties, and processes such as winter competition for light, self-thinning, summer stress from desiccation, frond breakage and re-growth, and different niche occupation by haploids and diploids. Its success depended on simulating with precision details such as the holdfasts’ dynamics. Because “details” often occur for a reduced number of individuals, inferring about them required going beyond statistically significant evidences and integrating these with parameter calibration aimed at maximized model fit. On average, the population was haploid-dominated (H:D > 1). In locations stressed by desiccation, the population was slightly biased toward the diploids and younger individuals due to the superior germination and survival of the diploid sporelings. In permanently submerged rock pools the population was biased toward the haploids and older individuals due to the superior growth and survival of the haploid adults. The IBM application demonstrated that conditional differentiation among ploidy stages was responsible for their differential niche occupation, which, in its turn, has been argued as the driver of the evolutionary stability of isomorphic biphasic life cycles.

Influence of nutrients on ploidy-specific performance in an invasive, haplodiplontic red macroalga.

Worldwide, macroalgae have invaded near-shore marine ecosystems. However, their haplodiplontic life cycles have complicated efforts to predict patterns of growth and spread, particularly since most theoretical predictions are derived from diplontic taxa (i.e., animals). To complete one revolution of the life cycle, two separate ploidy stages, often including separate haploid sexes, must pass through development and reproduction. In the case of the invasive, red macroalga Agarophyton vermiculophyllum, during the invasion of soft-sediment estuaries throughout the Northern Hemisphere, diploid tetrasporophytes came to dominate all free-floating populations and haploid gametophytes were consistently lost. The ecological hypothesis of nutrient limitation might contribute to an explanation of this pattern of tetrasporophytic dominance in free-floating populations. Under this hypothesis, gametophytes should outperform tetrasporophytes under nutrient limited conditions, but tetrasporophytes should be better able to exploit optimal or even abundant nutrient conditions, such as in eutrophic estuaries. We sampled tetrasporophytes, male gametophytes, and female gametophytes from two sites each located on either side of the Delmarva Peninsula that separates the Chesapeake Bay from the Atlantic Ocean. We subjected apices excised from multiple thalli from each life cycle stage to a nutrient-enriched and a nutrient-poor seawater treatment and assessed growth and survival. While nutrient addition increased growth rates, there was no significant difference among ploidies or sexes. Gametophytes did, however, suffer higher mortality than tetrasporophytes. We discuss how nutrient-dependent differences in growth and survival may contribute to observed patterns of tetrasporophytic dominance in soft-sediment A.vermiculophyllum populations.

Maintenance of Complex Life Cycles Via Cryptic Differences In The Ecophysiology Of Haploid And Diploid Spores Of An Isomorphic Red Alga1.

Recognition of the wide diversity of organisms that maintain complex haploid-diploid life cycles has generated interest in understanding the evolution and persistence of such life cycles. We empirically tested the model where complex haploid-diploid life cycles may be maintained by subtle/cryptic differences in the vital rates of isomorphic haploid-diploids, by examining the ecophysiology of haploid tetraspores and diploid carpospores of the isomorphic red alga Chondrus verrucosus. While tetraspores and carpospores of this species did not differ in size or autofluorescence, concentrations of phycobiliproteins of carpospores were greater than that of tetraspores. However, tetraspores were more photosynthetically competent than carpospores over a broader range of photosynthetic photon flux densities (PPFDs) and at PPFDs found at both the depth that C.verrucosus is found at high tide and in surface waters in which planktonic propagules might disperse. These results suggest potential differences in dispersal potential and reproductive success of haploid and diploid spores. Moreover, these cryptic differences in ecological niche partitioning of haploid and diploid spores contribute to our understanding of some of the differences between these ploidy stages that may ultimately lead to the maintenance of the complex haploid-diploid life cycle in this isomorphic red alga.

Transcriptional and Spatial Heterogeneity of Mouse Megakaryocytes at Single-Cell Resolution

Megakaryocytes (MKs) have long been described solely as platelet progenitors. However, recent studies show that MKs also act as an essential component of the bone marrow niche to maintain hematopoietic stem cell function, and combat infection by engulfing and presenting antigens. However, it is not known whether these diverse programs are executed by a single cell population or distinct subsets of cells. We performed single-cell RNA sequencing (scRNA-seq) to dissect the heterogeneity of MKs. To overcome the difficulty in obtaining the rare (0.1% in BM) and oversized (up to 65μm) highly polyploid, fragile MKs, we developed an efficient isolation strategy by combining fluorescence-activated cell sorting (FACS) sorting, manual selection of highly viable cells, and FISH verification of ploidy. We obtained 920 CD41+ highly-purified, bone-marrow derived, murine MKs spanning each ploidy stages (2N-32N) for scRNA-seq with modified Smart-seq2 protocol. On average, we detected over 6800 expressed genes and 250,000 transcripts in each MK. All cells expressed classical markers such as Pf4 and Itga2b (CD41). Four cell clusters were identified using an unsupervised clustering method. Cells in Cluster 1 expressed mature MK markers CD42 and CD61, were enriched for hemostasis and platelet activation expression signatures, and consist of ≥8N cells, suggesting these MKs may represent platelet generating MKs. Cells in cluster 2 had lower CD42 and CD61 expression, were low ploidy (≤8N), and had higher expression of inflammation-related genes, including Ctss and Itgam ("inflammatory response-associated MKs"). Cells in Cluster 3 were enriched for DNA replication and DNA strand elongation (GO terms) and were in all ploidy stages ("MKs in polyploidization stage"). Cluster 4, most of which were high-ploidy, expressed high levels of CD42, CD61, TGF-β, and IGF1: factors regulating HSC behavior ("HSC niche cells"). Furthermore, we identified cell population-specific surface markers and transcription factors (TFs) for each of the 4 clusters. Then, immunostaining using antibodies against corresponding markers were performed to confirm the presence of respective MK subpopulations in the bone marrow. Our analyses suggest that defining MK stages by ploidy and traditional markers CD42 and CD61 alone may result in a genetically and developmentally heterogenous population of MK. Rather, MKs at various stages may be more specifically identified by these gene signatures. MKs with different functions are known to have specific spatial distribution in the bone marrow (BM) niche. To test whether MKs with distinct expression signatures are uniquely localized within BM, we performed immunofluorescence staining on BM sections using antibodies against cell population-specific marker genes. Remarkably, we observed that Cluster 1 cells directly contacted blood vessels while most of Cluster 4 cells resided within one cell diameter of HSCs. The unique spatial distribution of cluster 1 and 4 population are in consistency with their respective transcriptomic signatures, and support that platelet generation and HSC maintenance are carried out by two distinct MK subpopulations. We further investigated the potential intrinsic relationship of these four Clusters during megakaryopoiesis. Developmental time courses were reconstructed using Monocle analysis, demonstrating that polyploidization (Cluster 3) occurs at the early stage of MK development with subsequent differentiation toward three orientations. While MKs with low ploidy appear to have two distinct cell fates (immuno-modulation or polyploidization), MKs with high ploidy (≥8N) differentiate towards populations associated with platelet production or stem cell regulation. In summary, our study provides the first in vivo transcriptomic profile of megakaryopoiesis and a potential map of megakaryocyte heterogeneity at the single-cell resolution. MKs may be classified into different functional subpopulations irrespective to their developmental stage and degree of ploidy. These observations suggest that megakaryopoiesis does not occur merely in a stepwise process, but is dynamic and adaptive to locations in the BM and biological needs. Disclosures No relevant conflicts of interest to declare.

Megakaryopoiesis and Platelet-Innate Immune Cell Interactions Are Developmentally Regulated

Megakaryocyte endoreplication, maturation and platelet production have all been reported to be affected by developmental stage (Elagib, Exp Hematol 2018). Additionally, we have recently found that P-selectin expression and function are altered in fetal and neonatal murine platelets (Stolla, Blood Adv 2019), suggesting that both megakaryopoiesis and platelet function are developmentally regulated. The developmental regulation of megakaryopoiesis can have important implications regarding platelet production in premature and full-term neonates, platelet regeneration following cord blood transplantation, and the in vitro generation of platelets from iPS cells (Elagib, Exp Hematol 2018). To further explore differences between fetal and adult megakaryocytes, we analyzed primary murine megakaryocytes in the fetal liver and adult bone marrow, and found that fetal megakaryocytes have lower average ploidy as well as increased cell size and initial accumulation of surface GP1bbeta expression at 2N and 4N stages. These latter findings indicate that cytoplasmic maturation begins at lower ploidy stages in fetal versus adult murine megakaryocytes. Consistent with findings in cultured human cord blood versus adult blood-derived progenitors (Liu, Blood 2015), we found lower ploidy and earlier cytoplasmic maturation in cultures of fetal versus adult megakaryocytes. These findings suggest the existence of a cell intrinsic developmental program of megakaryocyte maturation. Global gene expression studies of cultured megakaryocytes derived from fetal liver versus adult bone marrow progenitors revealed differential expression of Gata1, Cyclin D1, Cyclin D2, and Igf2bp3, supporting their proposed roles in fetal versus adult megakaryopoiesis (Klusmann, Genes Dev 2010; Liu, Blood 2011; Elagib, J Clin Invest 2017). Igf1R transcripts were increased in fetal megakaryocytes consistent with their sensitivity to IGF signaling (Klusmann, Genes Dev 2010). In addition, we found increased expression of Lin28b, a negative regulator of Let7 miRNAs, as well as multiple downstream targets of Let7 miRNAs in fetal liver-derived megakaryocytes. Induction of Lin28b expression in adult mice (Zhu, Cell 2011) led to increased expression of Gata1, Cyclin D1, Igf2bp3 and IGF1R in bone marrow-derived megakaryocytes. Interestingly, adult megakaryocyte average ploidy was decreased and the sensitivity to IGF1R inhibition was increased by Lin28b induction, suggesting that the Lin28b axis regulates key aspects of fetal versus adult megakaryopoiesis. The largest category of upregulated genes in adult versus fetal megakaryocyte transcriptomes was immune-related genes, including PPBP, a neutrophil chemotactic cytokine, and beta2M, which we have recently shown to mediate platelet-monocyte interactions (Hilt, JCI Insight 2019). Consistent with the transcriptomic data, neonatal platelets express less MHCI on their surface (an indirect indication of less platelet beta2M protein expression) and release less PPBP post-activation when compared to adult platelets. Activated neonatal platelets induce less neutrophil migration and activation when compared to activated adult platelets. In addition, the releasates from activated neonatal versus adult platelets differentially altered the balance of proinflammatory versus proreparative signaling in monocytes. Finally, the induction of Lin28b expression in adult mice resulted in platelets that express less MHCI, release less PPBP, and induce less neutrophil chemotaxis. Taken together, our studies support the hypothesis that megakaryocyte maturation and resulting platelet-immune cell interactions are developmentally regulated, in part, through a Lin28b-mediated program. Disclosures Palis: Rubius Therapeutics: Consultancy.

Invasion of novel habitats uncouples haplo-diplontic life cycles.

Baker's Law predicts uniparental reproduction will facilitate colonization success in novel habitats. While evidence supports this prediction among colonizing plants and animals, few studies have investigated shifts in reproductive mode in haplo-diplontic species in which both prolonged haploid and diploid stages separate meiosis and fertilization in time and space. Due to this separation, asexual reproduction can yield the dominance of one of the ploidy stages in colonizing populations. We tested for shifts in ploidy and reproductive mode across native and introduced populations of the red seaweed Gracilaria vermiculophylla. Native populations in the northwest Pacific Ocean were nearly always attached by holdfasts to hard substrata and, as is characteristic of the genus, haploid-diploid ratios were slightly diploid-biased. In contrast, along North American and European coastlines, introduced populations nearly always floated atop soft-sediment mudflats and were overwhelmingly dominated by diploid thalli without holdfasts. Introduced populations exhibited population genetic signals consistent with extensive vegetative fragmentation, while native populations did not. Thus, the ecological shift from attached to unattached thalli, ostensibly necessitated by the invasion of soft-sediment habitats, correlated with shifts from sexual to asexual reproduction and slight to strong diploid bias. We extend Baker's Law by predicting other colonizing haplo-diplontic species will show similar increases in asexuality that correlate with the dominance of one ploidy stage. Labile mating systems likely facilitate colonization success and subsequent range expansion, but for haplo-diplontic species, the long-term eco-evolutionary impacts will depend on which ploidy stage is lost and the degree to which asexual reproduction is canalized.

Post-mortem degradation of the muscle tissue in diploid and triploid turbots (Scophthalmus maximus L.)

Structural and ultrastructural muscle changes were studied throughout post-mortem degradation in diploid (2N) and triploid (3N) 22-month-old turbots (Scophthalmus maximus L.). Textural changes were also studied. Muscle degradation was fast in 2N and 3N turbot, as indicated by detachment of myofibrils from the sarcolemma and swollen organelles from day one post-mortem onwards. Interfibrillar and intermyofibrillar spaces, disruptions in sarcolemma-endomysium and autophagic vacuoles increased after 3 days in both fish groups. The percentage of interfibrillar detachments increased from 3 to 7 days in 2N and 3N, remaining constant until 14 days post-mortem, whereas the autophagic processes were more marked after 7 days. Post-mortem muscle alterations were stronger in the triploid group. Thus, the autophagic mechanisms and the percentage of interfibrillar detachments were higher in triploid than in diploid turbot. Hardness was not affected by ploidy or post-mortem stages.

Effects of Anagrelide on Megakaryopoiesis and Platelet Production

Possible pathomechanisms regarding the thromboreductive effect of anagrelide (ANA) include decrease in the megakaryocyte cell mass or life span of platelets, interference with maturation, and stimulation of apoptosis and proliferation. Finally, a fibrogenic effect has been reported in patients with high-risk essential thrombocythemia (ET). Based on scrutinized evaluations, including immunohistochemistry and morphometry performed on representative bone marrow (BM) biopsies, ANA was found to exert a significant influence on the endoreduplicative activity of megakaryopoiesis, with an arrest of maturation at lower ploidy stages causing a predominance of precursors. This result confirms and greatly extends former investigations that also failed to document a decrease in quantity or enhancement of apoptosis. Moreover, a comparative study on the putative mutagenic capacity of hydroxyurea (HU) versus ANA treatment reveals that both agents generate a left-shifting of megakaryocytes. However, HU creates conspicuously expressed maturation defects consistent with dysplastic changes of megakaryopoiesis, and therefore supports concerns about the possible leukemogenic potential of this drug. On the other hand, in follow-up examinations of BM specimens, ANA failed to show a stimulation of myelofibrosis in ET, provided diagnosis was established according to the World Health Organization criteria.

Flow cytometry in renal cell carcinoma

– The most important prognostic factors in renal carcinoma are the stage and grade. World literature is full of information on new markers, the analysis of DNA with the cytofluorimetric method being foremost, as it allows the cell lines in the neoplasm to be assessed and at the same time the ploidy and cell cycle stages to be studied. We have found an excellent correlation between the ploidy and cell grade. After a preliminary study we can conclude that the method is easy to perform and to reproduce. The procedure is also interesting for retrospective studies which allow the pathologist to select the blocks to be examined, thereby avoiding those widely contaminated by hemorrhages or necrosis.

Myelodysplastic syndromes: immunohistochemical and morphometric evaluation of proliferative activity in erythropoiesis and endoreduplicative capacity of megakaryocytes.

An immunohistochemical and morphometric analysis was performed on bone marrow trephine biopsies in 40 patients with primary myelodysplastic syndromes (MDS) to evaluate the proliferative activity in erythropoiesis and the endoreduplicative capacity of megakaryocytes. Control groups included normal bone marrow and marrow from cases presenting with pernicious anaemia. Double-immunostaining was applied with a monoclonal antibody (PC10) directed against proliferating cell nuclear antigen (PCNA), followed by antibodies against glycophorin C (Ret40f) or platelet glycoprotein IIIa (Y2/51-CD61) for the identification of the erythroid and megakaryocytic cell lineage. Comparison with normal bone marrow showed a reduction of erythropoiesis accompanied by an increase in atypical (micro-) megakaryocytes. Erythroid precursors displayed significant enhancement of PCNA-immunostaining. Megakaryocytes showed no increase in the relative frequency of PC10-positive cells (PCNA-labelling index). In pernicious anaemia, predominance of macrocytic-megaloblastoid erythropoiesis was associated with a striking increase in PCNA-labelling. Cell kinetic studies in this disorder revealed an abnormal arrest, particularly in S-phase which generates an over-expression of PCNA. Similar conditions were believed to be present in MDS with secondary folate deficiency. This mechanism explains the relatively high rate of positively-reacting pro- and erythroblasts which is not invariably accompanied by an increase in cell proliferation. Determination of megakaryocyte size and PCNA-staining capacity resulted in a significant increase in PC10-positive cells among micromegakaryocytes. Our findings on this cell lineage are in keeping with the assumption of a block in endoreduplicative activity at higher ploidy levels, associated with an apparently not-deregulated endomitosis in small-sized megakaryocytes of lower ploidy stages.

Pretherapeutic flow cytometric dna investigations in radiotherapy patients with maxillo-facial carcinomas

In order to analyze the possible meaning of cellular DNA content and cell cycle phases for the radiosensitivity and the prognosis of human malignant tumors, flow cytometric measurements have been performed in biopsies of 131 patients with histologically proven squamous cell carcinomas of the maxillo-facial region. In two-thirds of the patients ( 88 131 ; 67%), aneuploid tumor cell lines have been found, only 33% ( 43 131 ) had a diploid DNA distribution pattern. The average DNA index (DI) of the aneuploid carcinomas was 3.4 ± 0.6 (normal nonmalignant tissue DI = 2.0). The frequency of S-phase cells, which represents the ‘proliferative activity’, was between 4.8 and 63.2%, regardless of the ploidy stages. The aneuploid carcinomas had about twice as many S-phase cells (mean 23.7 ± 11.8%) than diploid tumors (mean 12.7 ± 4.8%). Mean survival for patients with diploid carcinoma and aneuploid carcinoma was 12 and 9.5 months, respectively. Concerning the relationship of S-phase frequency and survival times in our material there was a high negative statistical correlation (Spearman-Rank test) in patients with diploid carcinomas. A high S-phase fraction resulted in short survival times. No correlation was found in the aneuploid carcinomas: patients with tumors in high S-phase values in their biopsies showed no difference in prognosis in comparison to tumors with lower S-phase fractions.

Wirkung einer zweiten CCl4- Intoxikation auf die CCl4-geschädigte Leber der Maus

The effect of a second CCl4 intoxication on the CCl4-pretreated mouse liver is investigated. After a single dose of 1 ml/kg CCl4 injected intraperitoneally all CCl4-sensitive hepatocytes are destroyed. These CCl4-sensitive cells, localized in the centrolobular area, represent 55% of the total liver volume. During the 10 day period of regeneration the liver parenchyma is completely resistant to any further CCl4 intoxication, even at doses 3–5 fold lethal to normal mice. Shortly after regeneration is completed the liver regains its CCl4 sensitivity. As in the single CCl4 intoxication cell damage is confined to the centrolobular area and is characterized by a breakdown of protein synthesis and a subsequent necrosis. Time course and extent of the damage are completely identical with that after a single CCl4 intoxication. Continuous infusion of3H-thymidine leads to a 100% labeling of the CCl4-undamaged peripheral liver cells 4 days after a single CCl4-injection. Only part of these S-phases is necessary for the replacement of necrotic cells by normal mitoses. The larger part of the S-phases results in nuclei of higher ploidy stages. Considering both processes, mitosis and polyploidization, as a formation of new cells, the liver after regeneration from CCl4 intoxication must be regarded as consisting completely of new cells. By the end of regeneration the liver consists only of CCl4-resistent cells. The sensitivity to CCl4 regained in the centrolobular region after regeneration demonstrates that the genetic information for induction of enzymes capable of toxic conversion of CCl4 must also exist in peripheral liver cells and their descendants. However, this information is only activated in centrolobular liver cells, i.e. its activation depends on the localization of the cell within the lobule.

Cell proliferation in the "preleukaemic" phase of acute leukaemia. A cytophotometric and autoradiographic study.

In a 13-year-old patient with acute leukaemia the leukaemic blast cells, the nucleated red cells and the megakaryocytes were studied by cytophotometrie determination of the DNA content and autoradiographic labelling with ³H-TdR <i>in</i><i>vitro</i>. In the preleukaemic phase a striking proliferation defect in the early polychromatic erythroblasts was observed, consisting of an accumulation of cells in G₁ and a decreased proportion of cells in S. In the megakaryocytes low DNA values from 2c-8c as compared to 4c-32c in normal megakaryocytopoiesis, and a decreased number of labelled cells between the ploidy stages was observed, indicating a severely restricted polyploidization capacity. These results suggest that in the preleukaemic stage a basic proliferation defect of acute leukaemic is becoming apparent in the so-called non-leukaemic cell systems, leading to peripheral mono- or pancytopenia.

Polyploidization of megakaryocytes in normal humans, in patients with idiopathic thrombocytopenia and with pernicious anaemia.

Summary. Polyploidization of human megakaryocytes was studied in bone marrow aspirates from three normal individuals, three patients with idiopathic thrombocytopenic purpura (ITP) and three patients with pernicious anaemia (PA). The cells were labelled in vitro with tritiated thymidine and analysed by a combined cytophotometric and autoradiographic method. Cells were divided into types I, II and III.Normal megakaryocytes showed ploidy stages from 4c to 32c, the 4c stage occurring only in type I. The labelling indices showed that in normal megakaryocytes polyploidization takes place in type I and II and not in type III cells. This study provided evidence for alternation of DNA synthesis and rest periods. It is postulated that rhythmical polyploidization in megakaryocytopoiesis takes place. The progressive increase in the proportion of cells in successive resting periods (G‐phases) suggests that high ploidy cells have longer resting periods than low ploidy cells.In ITP, megakaryocyte morphology, labelling indices and distribution of DNA content were normal. Assuming a normal ploidy composition in ITP, the megakaryocytic hyperplasia observed in typical cases may be explained by an increased influx from the unrecognizable progenitor cell.In PA, an arrest of megakaryocytic DNA synthesis was observed comparable to that found in megaloblastic erythropoiesis.

Maturation of rat megakaryocytes studied by microspectrophotometric measurement of DNA.

SummaryThe amount of DNA in the nuclei of individual megakaryocytes of male Sprague-Dawley rats was measured micro-spectrophotometrically in marrow smears stained by the Feulgen reaction. When the DNA values were arranged in a frequency distribution, the megakaryocytes fell into groups with the modal DNA content of successive groups increasing approximately by a factor of 2. Ploidy of the megakaryocyte groups was determined by comparison with DNA values of diploid cells. Frequencies of 4N, 8N, 16N, and 32N megakaryocytes were, respectively, 1.6%, 10.5%, 71.2%, and 17.1%. The results indicate synchronous division of the nucleus of polyploid megakaryocytes. The relative numbers of cells in ploidy stages may indicate the relative length of time spent in the stages. An hypothesis of megakaryocyte maturation is proposed. The method may prove useful in studies of megakaryocyte maturation and regulation.ADDENDUM: In a recent microspectrophotometric study, de Leval observed a frequency distribution pattern of DNA...