DNA ploidy and proliferative activity of human pulmonary epithelium

DNA ploidy and distribution has been determined in normal and abnormal bronchial, bronchiolar and alveolar epithelium from 22 patients, aged between 0 and 85 years, 9 of whom had received chemotherapy for malignant disease. The DNA ploidy was diploid in all the specimens examined. The S + G2/M fraction was significantly greater in diseased than normal bronchial trees. In the bronchial epithelium, mean values ± the standard deviation (SD) were 5.5 + 2.2% vs 1.1±0.6%, in bronchiolar epithelium 4.6 ± 1.6% vs 1.0 ± 0.9% and in alveolar epithelium 4.6 ± 1.6% vs 0.8 ± 0.5%. The highest S + G2/M value of 8.9% was obtained from inflamed bronchial epithelium. Polyploid cells up to the octaploid range occurred infrequently but their incidence was slightly increased to between 0.16% and 0.9% in diseased lungs and in patients who had received chemotherapeutic drugs. It was concluded that (1) non-cancerous pulmonary epithelium is diploid, that (2) pulmonary epithelium shows steady-state renewal at all ages and polyploid cells are rare under normal conditions and that (3) the S + G2/M fraction increases up to approximately 10% in reactive proliferative states.     

DNA ploidy in seminal vesicle cells. A potential diagnostic pitfall in urine cytology

OBJECTIVE: To verify that abnormal DNA ploidy in urine cytology can occasionally be attributed to contamination by seminal vesicle cells. STUDY DESIGN: In the first part of this study, we analyzed the DNA content of six urine cytology specimens containing seminal vesicle cells. In the second part, we evaluated 21 Feulgen-stained prostate core biopsies containing seminal vesicle-type epithelium using a CAS-200 system. DNA index, proliferative activity (S + G2M) and degree of hyperploidy (> 5C) were determined in each case. RESULTS: All six urine cytology specimens were diploid, with all but one containing hyperploid cells (range, 0-16%; mean, 6.3%). Seminal vesicle cells from prostate biopsies showed a broad range of ploidy abnormalities. Ten cases (48%) showed an aneuploid peak, two cases (9%) showed a tetraploid peak, and nine cases (43%) showed only a diploid peak. All but one case showed both an elevation in proliferative activity (mean S + G2M, 24.2%) and some hyperploid cells (mean, > 5C; 4.5%). CONCLUSION: Seminal vesicle cells, although rarely seen in urine cytology, can cause abnormal DNA ploidy measurements. Morphologic criteria remain vital to an accurate cytologic diagnosis.     

Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser-assisted microdissection and real-time PCR detect anti-inflammatory effect of perfluorocarbon

The aim of this study was to identify cell types involved in the anti-inflammatory effect of ventilation with perfluorocarbon in vivo. Fifteen anesthetized, surfactant-depleted piglets received either aerosolized perfluorocarbon (Aerosol-PFC), partial liquid ventilation (rLV) at functional residual capacity (FRC) volume (FRC-PLV), or intermittent mandatory ventilation (control). After laser-assisted microdissection of different lung cell types, mRNA expression of IL-8 and ICAM-1 was determined using TaqMan real-time PCR normalized to hypoxanthine phosphoribosyltransferase (HPRT). IL-8 mRNA expression (means +/- SE; control vs. Aerosol-PFC) was 356 +/- 142 copies IL-8 mRNA/copy HPRT mRNA vs. 3.5 +/- 1.8 in alveolar macrophages (P <0.01); 208 +/- 108 vs. 2.7 +/- 0.8 in bronchiolar epithelial cells (P <0.05); 26 +/- 11 vs. 0.7 +/- 0.2 in alveolar septum cells (P <0.01); 2.8 +/- 1.0 vs. 0.8 +/- 0.4 in bronchiolar smooth muscle cells (P <0.05); and 1.1 +/- 0.4 vs. 0.2 +/- 0.05 in vascular smooth muscle cells (P <0.05). With FRC-PLV, IL-8/HPRT mRNA expression was significantly lower in macrophages, bronchiolar epithelial, and vascular smooth muscle cells. ICAM-1 mRNA expression in vascular endothelial cells remained unchanged. Predominantly, alveolar macrophages and bronchiolar epithelial cells were involved in the inflammatory pulmonary process. The anti-inflammatory effect of Aerosol-PFC was most pronounced.     

Kinetics of mucous cells in bronchial and bronchiolar epithelia during aerogenic elicitation with aspergillary proteins. A quantitative cytologic study.

During a prolonged exposure of rabbits to aerosols with asperigillary antigens, the content of bronchial and bronchiolar epithelia in mucus-secreting cells was quantified. In normals, the mucous cells do not exceed 3.2 per cent in bronchial epithelium and 0.44 per cent in bronchiolar one. After exposure, these proportions rapidly increase, after a primary discharge of goblet cells. The mucous cells become a majority after 7 exposures in bronchi and after 21 in bronchiolar epithelium. The maturation is more rapid in bronchioli than in bronchi. The secretion of neutral and acid mucopolysaccharides is early, concomitant in the same bronchus, and predominantly located in the impingement zones, especially the bifurcations of bronchial tree. The mitoses are extremely rare in exposed bronchi, but the epithelial and mesenchymal alveolar cells present a high mitotic index (1.14%) 24 hours after the second exposure to antigens.     

Distribution of antioxidant enzymes in developing human lung, respiratory distress syndrome, and bronchopulmonary dysplasia.

We studied cell-specific protein expression of all the major antioxidant enzymes (AOEs) and related proteins, such as copper-zinc superoxide dismutase (CuZnSOD), manganese SOD (MnSOD), extracellular SOD (ECSOD), catalase, the heavy and light chains of gamma-glutamylcysteine synthetase (gamma-GCS-l and gamma-GCS-h, also called glutamate cysteine ligase), the rate-limiting enzyme in glutathione synthesis, hemeoxygenase-1 (HO-1), and thioredoxin (Trx), in developing human lung, respiratory distress syndrome, and bronchopulmonary dysplasia by immunohistochemistry. Generally, after 17 weeks of gestational age, MnSOD was predominantly expressed in bronchial epithelium, alveolar epithelium, and macrophages, CuZnSOD was expressed in bronchial epithelium, ECSOD was expressed in bronchial epithelium, vascular endothelium, and the extracellular matrix, catalase was expressed in bronchial epithelium and alveolar macrophages, gamma-GCS-h was expressed in bronchial epithelium and endothelium, and gamma-GCS-l was expressed in bronchial epithelium. Trx was restricted to bronchial epithelium and to a lesser extent to alveolar macrophages, and HO-1 found in alveolar macrophages. Basically, the expression of these enzymes was similar in normal and diseased lung. It can be concluded that various AOEs and related proteins differ in their distribution and expression in lung before term, but generally it seems that infants are better adapted to high oxygen tension than might be expected.     

Ploidy of endothelium in high-grade astrocytomas

To determine the ploidy and proliferative activity of the endothelium in high-grade astrocytomas, the nuclear DNA content of 11 high-grade astrocytomas, including two gliosarcomas, was measured by cytophotometry. This technique allowed comparison of the endothelial population with the astrocytic population. In all cases, the endothelium was diploid, with an average of 24.6% of cells in the S + G2M phases of the cell cycle. In contrast, the astrocytic population displayed marked DNA abnormalities. The two gliosarcomas had a marked difference in proliferative activity of the endothelium with 4% and 40% of cells, respectively, in the S + G2M phases. These data indicate that the vast majority of endothelial cells compromising the vascular hypercellularity observed in high-grade astrocytomas are in the normal cell cycle whereas in many cases the malignant astrocytes are not. The nuclear DNA content of gliosarcomas appears to be similar to other high-grade astrocytomas.     

Establishment of a diploid reference value for DNA ploidy analysis by image cytometry in mouse cells.

OBJECTIVE: To establish a diploid reference value for DNA ploidy analysis of mouse cells (Mus musculus) by image cytometry using the CAS 200, an analysis system suitable for DNA content studies in human cells. STUDY DESIGN: To establish this standard, we used spleen imprints from 26 normal animals. A minimum of 150 lymphocytes present in each imprint was counted. The mean DNA content (pg/cell) of the G0/G1 peak and the DNA index observed in all samples were statistically analyzed. Cytospins with peritoneal cells from the same animals were then analyzed with this reference DNA value to confirm the diploid range. RESULTS: The DNA diploid reference value was determined by the mean DNA content of all spleen samples, which was 6.42 +/- 0.234 pg/cell, and the diploid range, defined as the diploid value +/- 10%, was 5.78-7.06 pg/cell. All the peritoneal samples showed a DNA diploid histogram, with a mean value for the G0/G1 peak DNA content of 6.742 +/- 0.15. CONCLUSION: The diploid reference value found in this study differs from those reported for other species, including the human being, and should be used in further studies of mouse pathology.     

Cation selective channel in fetal alveolar type II epithelium.

A cation selective channel was identified in the apical membrane of fetal rat (Wistar) alveolar type II epithelium using the patch clamp technique. The single channel conductance was 23 +/- 1.2 pS (n = 16) with symmetrical NaCl (140 mM) solution in the bath and pipette. The channel was highly permeable to Na+ and K+ (PNa/PK = 0.9) but essentially impermeant to chloride and gluconate. Membrane potential did not influence open state probability when measured in a high Ca2+ (1.5 mM) bath. The channel reversibly inactivated when the bath was exchanged with a Ca(2+)-free (less than 10(-9) M) solution. The Na+ channel blocker amiloride (10(-6) M) applied to the extracellular side of the membrane reduced P(open) relative to control patches; P(control) = 0.57 +/- 0.11 (n = 5), P(amiloride) = 0.09 +/- 0.07 (n = 4, p less than 0.01), however, amiloride did not significantly influence channel conductance (g); g(control) 19 +/- 0.9 pS (n = 5), 18 +/- 3.0 pS (n = 4). More than one current level was observed in 42% (16/38) of active patches; multiple current levels (ranging from 2 to 6) were of equal amplitude suggesting the presence of multiple channels or subconductance states. Channel activity was also evident in cell attached patches. Since monolayers of these cells absorb Na+ via an amiloride sensitive transport mechanism we speculate that this amiloride sensitive cation selective channel is a potential apical pathway for electrogenic Na+ transport in the alveolar region of the lung.     

Immunohistochemical localization of epidermal growth factor receptor (EGF-R) in normal and diseased newborn lung tissues

The distribution of EGF receptors (EGF-R) was examined in normal, hyaline membrane diseased and pneumonic newborn lung tissues by immunohistochemical methods under the light microscope. The PAP technique with polyclonal antibodies was performed to demonstrate the EGF receptor localisation in these tissues. Strong EGF-R reactivity was observed on bronchiolar epithelium and type I and type II alveolar cells in normal newborn lung tissues; whereas, poor reactivity was observed in alveolar macrophages. On the other hand, strong immunoreactivity was detected in type I alveolar cells and alveolar macrophages in hyaline membrane disease, but no reactivity was present in type II alveolar cells. The strongest immunoreactivity was observed in alveolar macrophages of newborn pneumonic lung tissues. In conclusion, the most meaningful form of reactivity was observed in normal newborn lung tissues of airway track and respiration area. This result is related with the maturation of the lungs after birth.     

Effect of lung volume on ventilation distribution

To examine the effect of preinspiratory lung volume (PILV) on ventilation distribution, we performed multiple-breath N2 washouts (MBNW) in seven normal subjects breathing 1-liter tidal volumes over a wide range of PILV above closing capacity. We measured the following two independent indexes of ventilation distribution from the MBNW: 1) the normalized phase III slope of the final breaths of the washout (Snf) and 2) the alveolar mixing efficiency during that portion of the washout where 80-90% of the lung N2 had been cleared. Three of the subjects also performed single-breath N2 washouts (SBNW) by inspiring 1-liter breaths and expiring to residual volume at PILV = functional residual capacity (FRC), FRC + 1.0, and FRC - 0.5, respectively. From the SBNW we measured the phase III slope over the expired volume ranges of 0.75-1.0, 1.0-1.6, and 1.6-2.2 liters (S0.75, S1.0, and S1.6, respectively). Between a PILV of 0.92 +/- 0.09 (SE) liter above FRC and a PILV of 1.17 +/- 0.43 liter below FRC, Snf decreased by 61% (P less than 0.001) and alveolar mixing efficiency increased from 80 to 85% (P = 0.05). In addition, Snf and alveolar mixing efficiency were negatively correlated (r = 0.74). In contrast, over a similar volume range, S1.0 and S1.6 were greater at lower PILV. We conclude that, during tidal breathing in normal subjects, ventilation distribution becomes progressively more inhomogeneous at higher lung volumes over a range of volumes above closing capacity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells

Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.     

Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung

125I-Human calcitonin gene-related peptide (hCGRP) binding sites were localized in human and guinea pig lungs by an autoradiographic method. Scatchard analysis of saturation experiments from slide-mounted sections of guinea pig lung displayed specific 125I-hCGRP binding sites with a dissociation constant (Kd) of 0.72 +/- 0.05 nM (mean +/- S.E.M., n = 3) and a maximal number of binding sites (Bmax) of 133.4 +/- 5.6 fmol/mg protein. In both human and guinea pig lung, autoradiography revealed that CGRP binding sites were widely distributed, with particularly dense labeling over bronchial and pulmonary blood vessels of all sizes and alveolar walls. Airway smooth muscle and epithelium of large airways was sparsely labeled but no labeling was found over submucosal glands. This localization corresponds well to the reported pattern of CGRP-like immunoreactive innervation. The findings of localization of CGRP binding sites on bronchial and pulmonary blood vessels indicate that CGRP may be important in the regulation of airway and pulmonary blood flow.     

Chronic bronchial allergic inflammation increases alveolar liquid clearance by TNF-alpha -dependent mechanism

Bronchial inflammation in allergic asthma is associated with active exudation from the bronchial tree into the interstitial space of both mucosa and submucosa. The aim of this study was to evaluate epithelial and endothelial permeability as well as alveolar fluid movement in a model of chronic allergic inflammation in Brown-Norway rats sensitized and challenged with ovalbumin (OA). Control groups were challenged with saline solution (C), and rats were immunized by OA but not challenged (Se). Lung sections showed a marked inflammatory infiltrate associated with perivascular and peribronchiolar edema in OA. To measure alveolar liquid clearance, a 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces. Alveolar-capillary barrier permeability was evaluated by intravascular injection of 1 microCi of (131)I-labeled albumin. Endothelial permeability was significantly increased in OA, from 0.08 +/- 0.01 in the C group to 0.19 +/- 0.03 in OA group (P < 0.05). Final-to-initial protein ratio was also statistically higher in OA (1.6 +/- 0.05) compared with C (1.38 +/- 0.03, P = 0.01) and Se groups (1.42 +/- 0.03, P = 0.04). Administration of anti-tumor necrosis factor-alpha antibodies within the instillate significantly decreased this ratio (1.32 +/- 0.08, P = 0.003 vs. OA). To conclude, we demonstrated a tumor necrosis factor-alpha-dependent increase in alveolar fluid movement in a model of severe bronchial allergic inflammation associated with endothelial and epithelial leakage.     

Ischemic cardiomyopathy in pigs with two-vessel occlusion and viable, chronically dysfunctional myocardium

A chronic left anterior descending coronary artery (LAD) stenosis leads to the development of hibernating myocardium with severe regional hypokinesis but normal global ventricular function after 3 mo. We hypothesized that two-vessel occlusion would accelerate the progression to hibernating myocardium and lead to global left ventricular (LV) dysfunction and heart failure. Pigs were instrumented with a fixed 1.5-mm constrictor on the proximal LAD and circumflex arteries. After 2 mo, there were no overt signs of right-heart failure and triphenyl tetrazolium chloride infarction was trivial (1.4 +/- 0.1% of the LV). Compared with shams, regional function [myocardial systolic excursion (DeltaWT); 2.1 +/- 0.3 vs. 4.6 +/- 0.4 mm, P < 0.05] and resting perfusion (0.90 +/- 0.13 vs. 1.32 +/- 0.09 ml small middle dot min(-1) small middle dot g(-1), P < 0.05) were reduced, consistent with hibernating myocardium. Pulmonary systolic (45.9 +/- 3.3 vs. 36.5 +/- 2.2 mmHg, P < 0.05) and wedge pressures (19.1 +/- 1.6 vs. 11.2 +/- 0.9 mmHg, P < 0.05) were increased with global ventricular dysfunction (ejection fraction 43 +/- 2 vs. 50 +/- 2%, P < 0.05). Early LV remodeling was present with increased cavity size and mass. Reductions in sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban were confined to the dysfunctional LAD region with no change in calsequestrin. Thus combined stenoses of the LAD and circumflex arteries accelerate the development of hibernating myocardium and result in compensated heart failure.     

Comparison of cytologic and acridine-orange flow-cytometric detection of malignant cells in human body cavity fluids

Flow cytometrically (FCM) derived DNA and RNA profiles were studied in acridine orange (AO)-stained body cavity fluid (BCF) specimens obtained from 78 patients with various solid tissue and hematologic malignancies. The ploidy (DNA index), RNA content (RNA index), proliferative activity (% S + G2M) and DNA and RNA scattergram patterns were tested "double-blind" against the cytologic scoring of specimens as malignant, benign or reactive. It was determined that expression of an "abnormal" RNA index (greater than or equal to 2.8) and an elevated proliferative activity (% S + G2M greater than or equal to 7.4) was dependent on the presence of malignancy; 21 of 22 specimens having those abnormal indices had DNA aneuploidy and were cytologically scored as positive. The AO FCM sensitivity and specificity for detecting malignant cells (when measured against cytology scoring) were 61% and 90%, respectively, using the "abnormal" RNA index and % S + G2M cut-offs together with the cellular DNA aneuploidy marker. By supplementing the cytologic scoring with AO FCM DNA and RNA features, the sensitivity for detecting malignant cells was 94%, as compared to 72% for cytology alone. Two specimens gave false-positive FCM results: a tuberculous effusion with a tetraploid subpopulation and a reactive mesothelial proliferation that was diploid and negative cytologically. Scoring for malignancy based on the visual pattern of the DNA and RNA FCM scattergrams, while showing good correlation for aneuploid specimens, in some cases failed to identify diploid disease. The results demonstrate the usefulness of FCM DNA and RNA analysis for supplementing cytologic examination of BCF specimens for the purpose of detecting malignant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Method of separating mouse platelets by discontinuous sucrose-gradient centrifugation

A discontinuous sucrose gradient was employed in the separation of mouse blood platelets using a modified Booyse method. The platelets of male CD-1 mice aged 8 to 12 weeks were divided into five distinct populations (A, B, C, D & E). Distribution of light to heavy platelets patterns in 10 normal CD-1 mice was demonstrable at; A (S.G. 1.188), as 14.8 +/- 5.6%; B (S.G. 1.199), 44.0 +/- 4.6%; C (S.G. 1.207), 24.1 +/- 3.4%; D (S.G. 1.214), 13.0 +/- 3.6%; and E (S.G. 1.221), 4.0 +/- 1.5%.     

Hepatocyte ploidy in normal young rat

The aim of the present study was to examine the relation between hepatocyte size and ploidy in Sprague-Dawley rat liver. Therefore, subpopulations of hepatocytes of various sizes were separated from the isolated crude hepatocyte population either mechanically or by using centrifugal elutriation. Hepatocyte size was determined on scanning electron microscopy photographs. Ploidy of hepatocytes was assessed by flow cytometry. The crude hepatocyte population was very heterogeneous in sizes, with diameters ranging from 8 to 39 microm. Hepatocyte ultrastructure was well preserved as demonstrated by transmission electron microscopy. The distribution of hepatocytes within the ploidy classes was the following: 19.6+/-3.6% diploid, 56.2+/-3.2% tetraploid and 3.4+/-0.6% octoploid mononucleated cells. Thus approximately 79% of hepatocytes appeared mononucleated. The binucleated hepatocytes (21%) had two diploid nuclei (18.7+/-2.9%) or two tetraploid nuclei (2.1+/-0.6%). A similar distribution of hepatocytes into ploidy classes was obtained in subpopulations of hepatocytes of various sizes. Our findings suggest that distribution into ploidy classes is not strictly correlated with hepatocyte size. In accordance with previous observations, our results on hepatocyte ploidy from periportal or perivenous origin using digitonin perfusion, is in favour of the existence of ploidy zonation within the rat hepatic lobule.     

Regeneration of the liver of Chinese hamster Cricetulus griseus

Using cytofluorimetry and interferometry, hepatocyte DNA, dry mass and distribution of hepatocyte ploidy classes were measured in hamsters Cricetulus griseus in 1 month after partial hepatoctomy. Ploidy of normal liver hepatocyte was 2.35 +/- 0.03 (mean +/- SD) c. Modal ploidy class was presented by mononuclear hepatocytes with diploid nuclei (82.4 +/- 1.3 %). Hepatocyte dry mass was 605.2 +/- 4.8 pg. One month after partial hepatectomy the distribution of ploidy classes and dry mass of hepatocyte did not change. A similar hepatectomy in mice resulted in significant polyploidization of liver parenchyma: the middle level of hepatocyte ploidy increased by 32% and mononuclear octaploid cells, the number of which increased 5-fold, composed modal ploidy class in place of 4cx2-hepatocytes predominated in control mice. The number of 8cx2-hepatocytes in the liver of mice creased by more than 5-fold. Thus, in contrast with mice, in hamsters Cricetulus griseus an increase in the liver mass followed partial hepatectomy depended completely on hepatocyte proliferation.     

Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.