Endomitotic Cycle Research Articles

SETD2 Regulates Nuclear Division to Achieve Polyploidization in Megakaryocyte

Megakaryocytes (MKs) undergo a fascinating endomitosis process to become naturally large multilobulated, polyploid nuclei cells that give rise to platelets. Two key steps involving in this process are the controlled failure in cytokinesis (cytoplasm division) and karyokinesis (nuclear division). The cytokinesis defect has been reported to be regulated by factors related to abnormal actomyosin ring and cleavage furrow formation such as RhoA, MYH9 and Rac1. In contrast, it remains largely unknown how nuclear division defect is regulated during MK endomitosis. Using a mouse model and an in vitro differentiation system, we found SETD2 expression markedly declined when MKs underwent polyploidization. Knockdown of SETD2 increased the high ploidy of primary bone marrow (BM) murine MKs, as well as MKs derived from cultured human cord-blood CD34+ cells and MEG-01 cells. Furthermore, the higher ploidy of MKs was accompanied by the increase of platelet formation both in the mouse model and the in vitro differentiation system. With forced overexpression of SETD2, the proportion of high ploidy MKs (≥8N) was remarkably decreased. These data suggest that SETD2, an α-tubulin methyltransferase, negatively regulates polyploidization during megakaryopoiesis. To investigate how SETD2 regulates endomitosis, we employed live-cell imaging to monitor the whole process of endomitosis at the single-cell level by observing the spindle geometry. We found SETD2 deficient MKs were capable of completing the endomitotic cycle, allowing cells to reach a higher ploidy in a significantly shorter time compared to control cells (2.9 hours vs. 4.9 hours). In comparison, overexpression of SETD2 significantly increased endomitosis cycle time compared to control conditions (5.2 hours vs. 4.3 hours). These results show that SETD2 deficiency accelerates the endomitotic cell cycle. Moreover, loss of SETD2 increased the percentage of cells within the presence of nucleoplasmic bridges (NPBs), a thin DNA link referred as a characteristic structure for karyokinesis defect. These results suggest that SETD2 may regulate endomitosis through facilitating the defect of karyokinesis. During karyokinesis, microtubules localized near spindle poles are need to be depolymerized as force-generating apparatus to separate nuclei. Thus, we measured the amount of polymerized and depolymerized microtubule forms in the presence and absence of SETD2. We found that loss of SETD2 decreased the proportion of the depolymerized form of tubulin in spindles, while SETD2 overexpression increased depolymerized microtubule forms. These data suggest that loss of SETD2 promotes the defect of karyokinesis by reducing depolymerized microtubule state. Mechanistically, as SETD2 could trimethylate α-tubulin at lysine 40 (α-tubK40me3), we found that the levels of α-TubK40me3 near spindle poles were inversely correlated with the degree of karyokinesis defect and MK polyploidization. Moreover, SETD2-deficiency resulted in an absence of α-TubK40me3 with an increase of the MK ploidy. And this ploidy increase was largely reversed by overexpressing a mutant tubulin protein that mimics the α-tubK40me3. Together, these data indicate that SETD2-mediated α-TubK40me3 regulates MK polyploidization by influencing microtubule dynamics. In summary, our study reveals that SETD2 is an important, novel regulator of MK endomitosis by regulating α-TubK40me3 to influence microtubule dynamics. These findings help fill current knowledge gaps in the mechanism of the karyokinesis defect during MK endomitosis. Targeting SETD2 may improve high ploidy MKs and platelet production that could promote the regeneration of platelets in vitro and help treat megakaryocytic dysplasia diseases with abnormal polyploidization in the future.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even>64N) and extremely large cell (40-60 µm). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Mass measurements during lymphocytic leukemia cell polyploidization decouple cell cycle- and cell size-dependent growth

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2 Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.

Defective endomitosis during megakaryopoiesis leads to thrombocytopenia in Fanca−/− mice

AbstractFanconi anemia (FA) is an inherited chromosomal instability syndrome that is characterized by progressive bone marrow failure. One of the main causes of morbidity and mortality in FA is a bleeding tendency, resulting from low platelet counts. Platelets are the final products of megakaryocyte (MK) maturation. Here, we describe a previously unappreciated role of Fanconi anemia group A protein (Fanca) during the endomitotic process of MK differentiation. Fanca deficiency leads to the accumulation of MKs with low nuclear ploidy and to decreased platelet production. We show, for the first time, that Fanca−/− mice are characterized by limited number and proliferative capacity of MK progenitors. Defective megakaryopoiesis of Fanca−/− cells is associated with the formation of nucleoplasmic bridges and increased chromosomal instability, indicating that inaccurate endoreplication and karyokinesis occur during MK polyploidization. Sustained DNA damage forces Fanca−/− MKs to enter a senescence-like state. Furthermore, inhibition of the Rho-associated kinase, a regulator of cytokinesis, improves the polyploidization of Fanca−/− MKs but greatly increases their genomic instability and diminishes their differentiation potential, supporting the notion that accumulation of DNA damage through endomitotic cycles affects MK maturation. Our study indicates that Fanca expression during endomitosis is crucial for normal megakaryopoiesis and platelet production.

Differential regulation of MCM7 and its intronic miRNA cluster miR-106b-25 during megakaryopoiesis induced polyploidy

Megakaryocytes exit from mitotic cell cycle and enter a phase of repeated DNA replication without undergoing cell division, in a process termed as endomitosis of which little is known. We studied the expression of a DNA replication licensing factor mini chromosome maintenance protein 7 (MCM7) and its intronic miR-106b-25 cluster during mitotic and endo-mitotic cycles in megakaryocytic cell lines and in vitro cultured megakaryocytes obtained from human cord blood derived CD34+ cells. Our results show that contrary to mitotic cell cycle, endomitosis proceeds with an un-coupling of the expression of MCM7 and miR-106b-25. This was attributed to the presence of a transcript variant of MCM7 which undergoes nonsense mediated decay (NMD). Additionally, miR-25 which was up regulated during endomitosis was found to promote megakaryopoiesis by inhibiting the expression of PTEN. Our study thus highlights the importance of a transcript variant of MCM7 destined for NMD in the modulation of megakaryopoiesis.

DNA replication events during larval silk gland development in the silkworm, Bombyx mori

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200–400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.

Endomitosis in tapetal cells of some Cymbidiums (Orchidaceae)

True endomitosis in the anther tapetum of the three Cymbidium species viz. C. aloifolium, C. devonianum and C. tigrinum is described. In these Cymbidiums, most tapetal cells go through endomitosis instead of either normal mitosis or so called “inhibited” mitosis. The nuclear membrane does not disappear, but during metaphase the chromosomes are considerably condensed far more than in normal mitosis, and nucleolus persists throughout the endomitotic cycle. Endomitosis may not be unusual to the tapetal cells of these peculiar Cymbidiums but may have a wider application and explain many of the cytological phenomena occurring in the tapetal cells of other plants. Further, physical localization of 45S rRNA genes during endomitotic divisions may help to confirm their atypical activities as well as provide insight into the course and degree of tapetum polyploidization. Analysis of DNA-methylation levels is also recommended for understanding the role of nucleolus for spindle formation during endomitosis.

Role of RhoA-Specific Guanine Exchange Factors in Regulation of Endomitosis in Megakaryocytes

Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mks), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often overexpressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-4N) endomitotic cycle requires GEF-H1 downregulation, whereas subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mks. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mks lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.

Endomitotic Megakaryocytes that Form a Bipolar Spindle Exhibit Cleavage Furrow Ingression Followed by Furrow Regression

Megakaryocyte (MK) differentiation is marked by the development of progressive polyploidy, due to repeated incomplete cell cycles in which mitosis is aborted during anaphase, a process termed endomitosis. We have postulated that anaphase in endomitotic MKs diverges from diploid mitosis at a point distal to the assembly of the midzone, possibly involving impaired cleavage furrow progression. To define the extent of furrow initiation and ingression in endomitosis, we performed time-lapse imaging of MKs expressing yellow fluorescent protein (YFP)-tubulin and monitored shape change as they progressed through anaphase. We found that in early endomitotic cells that have a bipolar spindle, cleavage furrows form that can undergo significant ingression, but furrows regress to produce polyploid cells. Compared to cells that divide, cells that exhibit furrow regression have a slower rate of furrow ingression and do not furrow as deeply. More highly polyploid MKs undergoing additional endomitotic cycles also show measurable furrowing that is followed by regression, but the magnitude of the shape change is less than seen in the early MKs. This suggests that in the earliest endomitotic cycles when there is formation of a bipolar spindle, the failure of cytokinesis occurs late, following assembly and initial constriction of the actin/myosin ring, whereas in endomitotic MKs that are already polyploid there is secondary inhibition of furrow progression. This behavior of furrow ingression followed by regression may explain why midbody remnants are occasionally observed in polyploid MKs. This finding has important implications for the potential mechanisms for cytokinesis failure in endomitosis.

Expression of MTPG-24, a Peptide Derived from a Protein Regulating Dynamic Changes in Cortical Actin, Induces Endomitotic Replication in Megakaryocytic Cells.

Prior to the fragmentation of their cytoplasm leading to platelet formation, differentiating megakaryocytes (MKs) increase their ploidy and cellular volume by undergoing repeated rounds of DNA replication without concomitant cell division, a process known as endomitosis. There is now accumulating evidence that endomitotic MKs develop as a result of aberrant regulation of the cleavage furrow formation, the site where dynamic changes in actin, myosin and membrane structure mediate the process of cytokinesis during animal cell division. Here we report that transfection of human megakaryocytic cell lines with vectors expressing a 24mer-peptide (MTPG-24) resulted in remarkable changes in morphology, resulting in cells with the appearance of mature polyploid megakaryocytes. MTPG-24 is derived from a protein involved in regulating actin-based structures and motility which possesses a putative site for PKA phosphorylation. Despite its hydrophilic primary structure, MTPG-24 fused to a fluorescent tag was detected closely associated to the plasma membrane where it accumulated in a punctuate pattern, in addition to co-localizing with a Golgi marker. Interestingly, in each recombinant clone, cells with different ploidy levels developed among normally dividing ones, suggesting that transit from a mitotic cycle to an endomitotic cycle seemed to only occur in cells having completed a given number of cell cycles. These effects were obtained independently of the nature of the tag fused to the peptide (Hemagglutinin, Orange protein, GFP) but were not observed with a control peptide in which the amino acids of MTPG-24 were disposed in an arbitrarily sequence. Because it possibly interferes with some effector molecules required for cleavage furrow membrane assembly in late anaphase, MTPG-24 provides thus a new tool for understanding the molecular basis of endomitosis. Furthermore, since there is a correlation between DNA content and the numbers of platelets released by MK, we are currently testing whether this peptide could be used to increase the ploidy level of CD34+ human cord blood-derived MKs which have a low propensity to become highly polyploid.

Stathmin Prevents the Transition from a Normal to an Endomitotic Cell Cycle during Megakaryocytic Differentiation

Physiological polyploidy is a characteristic of several cell types including themegakaryocytes (MK) that give rise to circulating blood platelets. MK achieve polyploidy byswitching from a normal to an endomitotic cell cycle characterized by the absence of late mitoticstages. During an endomitotic cycle, the cells enter into mitosis and proceed normally throughmetaphase and early anaphase. However, late anaphase, telophase and cytokinesis are aborted. Thisabortive mitosis is associated with atypical multipolar mitotic spindles and limited chromosomesegregation. Stathmin is a microtubule-depolymerizing protein that is important for the regulation ofthe mitotic spindle and interfering with its expression disrupts the normal mitotic spindle and leadsto aberrant mitotic exit. As cells enter mitosis, the microtubule depolymerizing-activity of stathminis switched-off, allowing microtubules to polymerize and assemble into a mitotic spindle.Reactivation of stathmin in the later stages of mitosis is necessary for the disassembly of the mitoticspindle and the exit from mitosis. Previous studies had shown that stathmin expression isdownregulated as MK become polyploid and inhibition of its expression in K562 cells increasestheir propensity to become polyploid. In this report, we describe our studies of the mechanism bywhich stathmin plays its role in MK polyploidization. We show that stathmin overexpressionprevents the transition from a mitotic cycle to an endomitotic cycle as determined by a decrease inthe number of multipolar mitotic spindles. These observations support a model in whichdownregulation of stathmin expression in megakaryocytes and other polyploid cells may be acritically important factor in endomitosis and polyploidy.

Visualization of Chromosome Territories in Interphase Nuclei of Ovarian Nurse Cells in Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.

Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation.

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.

Shifts in postembryonic somatic ploidy levels in Daphnia pulex

Although the presence of polyploid cells in somatic tissues of the cladoceran Daphnia has been reported, the nature of ploidy level shifts through life has not been investigated. This study examined the effects of growth and age on ploidy level variation across 10 instars in Daphnia pulex. The transition from mitotic to endomitotic cycles occurred during embryogenesis in all tissues, but the timing of it appeared to be tissue-specific. Lower than expected DNA estimates for the 4C and 8C ploidy classes suggested an underreplication of the genome during the first two endomitotic cycles, while complete replication seemed to occur in subsequent cycles. Polyploid cells underwent from four to seven endomitotic cycles over the 13 postembryonic instars, with ploidy levels reaching a maximum of 2048C in the labrum. The number of polyploid cells in all tissues, except the digestive tract, remained uniform beyond the fourth instar. Three patterns of ploidy level increments were observed in the various tissues. Polyploid cells in the epipodite and rostrum exhibited age-related shifts, reaching asymptotic levels, while those in the digestive tract similarly reached asymptotic levels but then re-entered the mitotic cycle to enable tissue growth. Finally, ploidy level shifts in both the labrum and the epidermis were affected by age and food levels.

Disproportionate numbers of rDNA loci in diploid and polyploid testis nuclei of gerris najas detected by fluorescence in situ hybridization

The replication state of rDNA in testes nuclei undergoing polyploidization by classical-type endomitosis was investigated in Gerris najas (Heteroptera) by means of fluorescence in situ hybridization. The number of just one rDNA locus per haploid genome was determined by in situ hybridization on meiotic nuclei. Additionally, DNA measurements of spermatids and testes nuclei were performed. Although regular duplication levels of nuclear DNA were found within the limits of the accuracy of the method, these did evidently not apply to the ribosomal genes. The comparison of the number of rDNA signals with the DNA content of 106 testis nuclei revealed drastic variations of the number of rDNA loci between individual nuclei with similar DNA content. Polyploid nuclei of the testis epithelium showed too low numbers of rDNA loci in relation to those expected from the levels of ploidy, while cyst cell nuclei displayed increased numbers of rDNA loci. The results indicate that the ribosomal genes are either underreplicated, or in part eliminated, during the endomitotic cycles of epithelium cell nuclei, but amplified in the cyst cell nuclei, probably already at their diploid stage.

Increased GPIIB/IIIA expression and altered DNA-ploidy pattern in megakaryocytes of diabetic BB-rats.

Hyperactive platelets contribute to angiopathic complications in diabetes mellitus. It is unclear whether the increased platelet function is a primary pathogenetic factor in diabetes or follows vascular injury. Increased platelet size and numbers of glycoprotein receptors on diabetic platelets suggest that thrombopoiesis is altered in diabetes mellitus. For further support of this hypothesis we studied whether megakaryocytes are changed with regard to the DNA-ploidy pattern and the GPIIB/IIIA expression in 10 acute diabetic (AD) and 24 insulin treated diabetic (ITD) BB rats in comparison with 22 diabetes resistant (ND) BB rats. In the AD group megakaryocyte size (P = 0.035) and the modal DNA-ploidy distribution dropped (P = 0.0001) concomitant with increased TNF-alpha activity (P = 0.001). GPIIB/IIIA expression and the peripheral platelet status were unchanged. After 4 weeks of insulin substitution metabolic parameters (glucose, cholesterol, triglycerides) were lowered, but remained still elevated. As compared to the AD group the modal DNA-ploidy pattern reversed, but the relative percentage of 64n megakaryocytes increased 2.3-fold and GPIIB/IIIA expression increased 1.6-fold. Simultaneously, the peripheral platelet count and size increased. From these results we conclude that alterations of the megakaryocyte compartment occur at early onset of diabetes. These changes could reflect a response to increased systemic cytokine production during inflammatory islet cell destruction. The peripheral platelet thrombotic potency increased with insulin treatment. This was associated with an increase of 64n-megakaryocytes with upregulated GPIIB/IIIA expression and could reflect a mitogenic effect of insulin upon the endomitotic cycle of the megakaryocytes.

Megakaryocyte endomitosis: A review

During the early stages of their differentiation, megakaryocytes become polyploid through repeated DNA replication and endomitotic cycles without cytokinesis. The molecular basis for the process of polyploidization remains unknown. This review summarizes available information on the cell biology of the process of endomitosis.

Arrest of micronuclear DNA replication during genomic exclusion in Tetrahymena produces haploid strains.

Diploid cells of Tetrahymena thermophila were crossed to strain A*V, whose micronucleus is defective, to induce the unilateral transfer of gametic nuclei from the diploid cells to the A*V cells (round I of genomic exclusion). These haploid nuclei presumably undergo one endomitotic cycle and then become diploid with a G1 (2C) DNA content. However, further DNA replication from 2C to 4C was transiently arrested until the pairs separated. When endomitosis was blocked by treatment with cycloheximide during 6-8 hours of conjugation, the exconjugants of round I of genomic exclusion remained haploid. Competence for diploidization is apparently limited to some period of time after nuclear transfer. Blocking of diploidization during round I of genomic exclusion can be used as an efficient way to induce haploid strains in Tetrahymena.

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail, Planorbarius corneus

The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with 3H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.

Polymorphism of males inFormica exsecta Nyl. (Hym.: Formicidae)

Males ofFormica exsecta from two alpine valleys were found to belong to two significantly different size classes, called micraner and macraner. Nests contained either one or the other or both male types. Tot test whether both male types were haploid or one was diploid, chromosome numbers in brain cells from prepupae were counted and the relative DNA value of single nuclei from adult brains was determined. Most of the metaphase plates in brains from micraner as well as from macraner turned out to be haploid. The rest of the metaphases showed a 2n or 4n chromosome set. Workers had diploid brain cells together with some 4n cells. A difference between micraner and macraner was the percentage of cells with more than n chromosomes. All macraner had 90% or more haploid cells in their brain while the percentage of haploid cells in micraner could be much lower, as low as 59%. Only micraner showed chromosome numbers higher than 2n. DNA measurements gave principally the same result. Both male types exhibited the same low DNA value, lower than worker brain cells. In agreement with the chromosome countings, macraner had only one class of cells with a higher DNA value. In addition to the DNA values which are thought to represent the chromosome numbers n and 2n, lower values were found in macraner which are interpreted as degenerating nuclei. Both male types contained sperm. The presented results show that inF. exsecta differences in male size are not induced by a haploid-diploid mechanism. All males were haploid. However, the frequency of endomitotic cycles, the doubling of the chromosome number without subsequent cell division, was lower in macraner than in micraner.