Giant pharyngeal gland nuclei of Ascaris: unusual cytology suggests a novel pathway for transport of ribosomal proteins to the nucleus.

We describe the unusual giant pharyngeal gland nuclei of the parasitic roundworm Ascaris lumbricoides suum and attempt to reconcile the relationships among its uncommon, and in some instances unique, collection of constituents. The nuclei were studied by light and electron microscopy, Feulgen cytophotometry, nuclear size analysis and histochemical methods, including those for detection of DNA, RNA, acidic and basic proteins, lipids and carbohydrates. A highly active nucleus is revealed: an extensive system of intranuclear annulate lamellae (IAL); membrane-bounded, acidophilic bodies which contain non-basic proteins; an abundance of free nucleoli; and an exceedingly large chromocenter containing a core of DNA surrounded by nucleoli (an apparent nucleolar organizing region, NOR). The relationships among the various nuclear constituents suggest that the acidophilic bodies consist of nucleolus-related proteins imported from the cytoplasm to the NOR, and that the annulate lamellae mediate this transport. Incidental findings include rounded cytoplasmic invaginations into the nucleus, thread-like structures with dense cores and surrounding small granules found among concentrations of nucleoli, and bundles of 12 nm filaments closely associated with these nucleoli. The significance of such huge, morphologically complex and highly polyploid nuclei, derived from chromatin-diminished progenitor cells early in development, and the possible interrelationship of these phenomena, remain obscure.

An islet of Langerhans located within the epithelium of a human pancreatic duct.

An unusual structure resembling an islet of Langerhans was described in the pancreatic ductal epithelium of a presumably normal human male. Further studies to establish the identities of both islet and duct were performed. The original hematoxylin and eosin section served for histological description, as well as for nuclear volume analysis to characterize levels of beta cell polyploidy. The section was restained with Gomori's Chrome Alum Hematoxylin Phloxine to ascertain the presence of islet-like cells and to quantitate the ratio of beta- to non-beta cells. The section was then restained by the Feulgen technique to confirm proportionality between nuclear volume and DNA content. An apparently normal islet in the same section served as control. The combined observations were consistent with the interpretation that the structure is indeed an islet situated within the ductal epithelium. The almost complete absence of polyploid beta cells, however, and the high ratio of non-beta cells:beta cells suggested that it was a young islet. A similarly high proportion of non-beta cells in the control islet, as well as the frequent occurrence of ducts within, or in close proximity to, other islets, suggested that the entire islet organ was under some form of proliferative stimulus at time of resection. Alterations of the ductal epithelium and the secretory contents suggested that the duct was also in the process of transformation. Although the islet had apparently arisen within the ductal epithelium, the ultimate source of the progenitor cells could not be determined from these studies. The relationship of the ductal islet to other well known forms of islet regeneration is discussed.

Excess glucose intake induces accelerated beta-cell polyploidization in normal mice: a possible deleterious effect.

Normal nondiabetic homozygous and heterozygous littermates of db/db diabetes-prone mice (seven/group) were fed a 5% solution of glucose as their sole source of liquid for 10 wk. Controls (seven/group) drank tap water, and both groups received stock diet ad libitum. Body weights, tail lengths, food and fluid consumption were recorded throughout the study, and plasma and urine glucose were measured during wk 10. The total caloric intake, including the glucose solution drunk by some of the mice, was not significantly different among the four groups. No differences in plasma or urine glucose were detected. Total-body dry weight, water and lipid were measured, and pancreata were analyzed for beta-cell polyploidy by a combination of Feulgen cytophotometry and nuclear size analysis. The percentage of polyploid beta-cells was significantly higher in the animals that drank glucose than in those that drank water and was independent of both genotype and growth indices attributable to genotype. The greater polyploidization was interpreted as reflecting premature aging of the beta-cell population. It was hypothesized that such glucose-induced premature aging in animals with a genetically restricted potential for beta-cell proliferation could contribute to the precipitation of overt diabetes.

Are CNS neurons polyploid? A critical analysis based upon cytophotometric study of the DNA content of cerebellar and olfactory bulbar neurons of the bat.

A cytophotometric study of the nuclear DNA content of bat cerebellar and olfactory bulbar neurons was performed with particular attention to corrections for distributional error and non-specific light loss and to selection of appropriate control nuclei for the establishment of reliable haploid and diploid DNA values. Feulgen stained sections were measured with an integrating, scanning microdensitometer for correction of distributional error. The values thus obtained were further corrected in 3 different ways to subtract the contribution of background absorbance at 546 nm. Bat haploid (1c) DNA values were derived from spermatozoa, and diploid (2c) values from pancreatic acinar cells, hepatocytes and non-hepatocyte liver cells. Microglia of olfactory bulb also had 2c values. After correction, all neurons measured, except Purkinje cells, had DNA values more closely approximating the tetraploid (4c) than the diploid (2c) level. Purkinje neurons had mean DNA values closer to 2c than to 4c, but inherent technical difficulties in measuring these very large, light staining nuclei has created less confidence in the values obtained than for those of the other neurons. This uncertainty, plus the apparent existence of two populations of Purkinje neurons, one 2c and the other hyperdiploid, suggested by the DNA distribution curves, make it difficult to eliminate the possibility of polyploidization of Purkinje neurons. A critical analysis of various cytophotometric, radioautographic and biochemical approaches to the problem of CNS neuron polyploidization has revealed potentially serious flaws in many of them, rendering virtually impossible interpretation of the numerous contradictory results in the literature. Standardization of the cytophotometric technique and improvement of the radioautographic approach seem to be prerequisite to the resolution of the existing dilemma.

Polyploidy in islets of normal and diabetic humans.

This study was designed as a pilot project to determine whether the increased polyploidization of pancreatic B cells in diabetic mice reported from our laboratory is also characteristic of human diabetes. Nuclei of hematoxylin and eosin stained islets cells were traced by camera lucida and their volumes determined by semiautomatic particle size analysis. Previous studies have confirmed that nuclear volume can be used as an index of polyploidy, since, in mouse and human islets, the nuclear DNA content is directly proportional to the nuclear volume. Five insulin independent and three insulin dependent human diabetic patients and their age and sex matched controls were studied. The percentages of polyploid nuclei in insulin independent diabetic islets were elevated significantly over those in control subjects in four of five cases; the exception was an 85 year old male who was diagnosed as a diabetic only two months prior to death. Of the three insulin dependent diabetics, one, whose islets appeared otherwise normal, had a significantly greater percentage of polyploid nuclei than the controls; the other two, whose islets were markedly hyalinized, exhibited percentages of polyploid nuclei within normal ranges. The implications of relative percentages of polyploid nuclei in normal and diabetic islets and the possible relationship with previous studies of genetically diabetic mice are discussed.

Changes in polyploidization of exocrine pancreas in db/db diabetic and normal mice.

Polyploidy in tele-insular and peri-insular exocrine pancreas of C57BL/KsJ-db/db diabetic mice and normal littermate controls was quantitated, because the diseased state has been shown to produce an increase in the percentage of polyploid islet B-cells. Three diabetic and 3 normal animals were sacrificed at 4.5, 7, 9.5, 12, 14.5, and 17 weeks of age. Semi-automatic nuclear size analysis on Feulgen stained sections separated the nuclei into polyploid classes, with the following results: 1) In both normal and diabetic mice, percentages of polyploid cells in peri-insular and tele-insular regions differed significantly from one another, and from that of the islet B-cells. Per-cent polyploidy was highest in the peri-insular regions, and lowest in the islets. 2) In the tele-insular regions, per cent polyploidy did not differ between normals and diabetics at 4.5 weeks. By 17 weeks, polyploidy in the diabetics was essentially unchanged, while that of the normal animals had risen and become significantly elevated over that of both the 4.5 week normal and the 17 week diabetic animals. 3) In peri-insular regions, per cent polyploidy did not differ significantly between normals and diabetics throughout the period studied.

Development of polyploidy in B-cells of normal and diabetic mice.

This study was designed to clarify the relationship between pancreatic B-cell polyploidization and the progress of the diabetic syndrome in genetically diabetic (C57BL/Ks-db/db) and normal control mice (C57BL/KsJ) of matched age groups. Nuclear volume was confirmed to be a proper index of the polyploid class of the B-cell by correlation with Feulgen-DNA content as measured by microdensitometry. Nuclei of B-cells, identified by aldehyde fuchsin positive cytoplasmic granules, were traced by camera lucida and their volumes determined by semiautomatic particle size analysis. Six age groups were studied: 4.5, 7, 9.5, 12, 14.5 and 17 weeks. The major conclusions are: 1) The percentage of tetraploid nuclei in normal mice is consistently between 1.0 and 2.0% from 4.5 to 14.5 weeks of age and increases to approximately 3.0% at 17 weeks of age; however, further studies are required to determined the significance of this increase; 2) in all age groups studied, percentages of polyploid nuclei are significantly greater in diabetic than in control mice; 3) the percentage of tetraploid nuclei in diabetic animals is elevated 220% over controls at 4.5 weeks of age, remains constant until 12 weeks (while other parameters such as blood glucose level and body weight continue to rise) and increases significantly between 12 and 14.5 weeks of age. Implications of both the increased polyploidy observed at the onset of disease symptoms, and the dramatic increase occurring during the later stages of the disease, are discussed.

Polyploidy in the pancreas of the normal and diabetic mutant mouse.

The DNA content of endocrine and exocrine pancreatic nuclei of normal C57BL/KsJ and diabetic mutant C57BL/Ks-db/db mice was measured by Feulgen microdensitometry. The exocrine and endocrine pancreatic nuclei of the 4-week-old normal, 12-week-old normal, and 4-week-old (prehyperglycaemic) diabetic mutant mice contained diploid and tetraploid cells, while the 12-week-old (established hyperglycaemic) mutant contained diploid, tetraploid, and octaploid nuclei. The polyploidy in the endocrine pancreas of all these mice was confined to the B-cells, while the A-cells were always diploid.

The ultrastructure of polyploid B-cells in the islets of normal mice.

Light and electron microscopic studies of diploid, tetraploid and octaploid B-cells in the islets of normal C57BL/KsJ mice revealed that polyploid cells were characterized by a wider range of granulated states than diploid B-cells. The maximum granule densities were similar for polyploid and diploid cells; however, some polyploid cells were almost devoid of granules, while the least granulated diploid cells contained intermediate granule densities. The tetraploid cell also appeared to be characterized by an increased mitochondrial stage which suggests compensation for the greater degree of degranulation. These observations were confirmed by morphometric analysis. Two interpretations of the apparent polyploidy are discussed; that polyploid B-cells may be more responsive to insulin releasing stimuli than diploid B-cells and that tetraploid cells may only be diploid cells in the G2 phase of the mitotic cycle.