DNA synthesis in rat heart cells after injury and the regeneration of myocardia

The regenerative responses of the myocardia of post-natal rats of different age groups (1, 2, 3 and 4 weeks old) to an injury made by a clinical electricator were studied. DNA synthesis and the ultrastructural organization of the cardiac myocytes of the injured myocardia were examined for an evaluation of the potential for regeneration of the developing myocardia. The maximum labeling index of cardiac myocytes was observed in 1-week-old rats showing 8% labeled myocytes 3 days after injury as opposed to 3.2, 2.2 and 0.2% indices in 2-, 3- and 4-week-old rats respectively, 3 days after injury. In subsequent days after injury the labeling indices declined considerably in all age group hearts, and attained values less than 1% labeled myocytes 30 days after injury with the lowest labeling index in the oldest age group heart. When DNA synthesis in uninjured myocardial tissue adjacent to the injured tissue was examined, it was found to be significantly lower than it was in the injured tissue. However, both injured and adjacent uninjured tissue attained a peak in the labeling indices 3 days after injury, with the exception of 3- and 4-week-old uninjured tissue. The overall incorporation of ³H-thymidine into the DNA of heart cells as revealed by scintillation counts showed that the rate of incorporation of the isotope in younger hearts was significantly higher than in the older hearts. Non-muscle cells contributed significantly to the rise of scintillation counts in hearts of all age groups.Ultrastructural analyses of 1- to 4-week-old hearts showed that 24 hr after injury, injured areas of myocardia were heavily crowded with macrophages that surrounded damaged myocytes. Later on, fibroblasts and other non-muscle cells predominated the injury sites along with fibrous connective tissue. Scattered regenerating cardiac myocytes were frequently observed in the injury sites of 1- and 2-week-old hearts 3 days after injury. Myocytes were rare in the corresponding regions of 3- and 4-week-old hearts. Instead abundant non-muscle cells and fibrous connective tissue were predominant. In the fourth and final week of this study, the repaired areas of myocardia in 1- and 2-week-old rats contained more myocytes than those of the 3- and 4-week-old rats, and the repaired zone of the 1-week-old heart contained more myocytes than the repaired areas of the other age groups. These findings suggest that the mammalian myocardia possess an age-dependent potential for regeneration that involves the healing of injury sites with contractile and connective tissues.     

Growth Fraction and Cycle Duration of Hepatocytes In the Three-Week-Old Rat

The proliferation of hepatocytes in the liver of 3-week-old rats has been investigated by autoradiographic methods. This investigation is a continuation of earlier work on the same topic (Schultze & Maurer, 1972; 1973). 21 days after birth, 102 rats received a single injection of ³H-TdR. the percentage of labelled mitoses was then determined 1 hr later and at various times throughout the interval up to 12 days after application of ³H-TdR. In agreement with earlier work, a first peak of labelled mitoses was found 7 hr after ³H-TdR injection. the area under the peak indicates an S phase duration of 8 hr. In addition a second very broad peak of labelled mitoses was found between 2 and 12 days after pulse labelling. the analysis of the results leads to the conclusion that the hepatocytes of the 3-week-old rat have a growth fraction close to 1 and a doubling time of 6–7 days. This is at variance with earlier results of Post, Huang & Hoffman (1963) and Grisham (1969) who had derived a value of 21.5 hr for the duration of the cell cycle and a value of only 0. 1–0.2 for the growth fraction of the hepatocytes.     

STUDIES ON LYMPHOCYTES

Some of the time parameters of the cell cycle in bovine thoracic duct lymphocytes have been estimated by analysing labeled mitoses curves, and by double labeling. The two methods gave similar estimates of T_s. Thus, T_s measured directly from labeled mitoses curves varied from 4 to 6 hr, while the estimates from double labeling were 4.8 and 4.5 hr. T _G measured directly from labeled mitoses curves was 5 hr, and estimates of T_G from the values of T_s ranged from 6.3 to 7.7 hr. The present data confirm the short generative cycle of large thoracic duct lymphocytes. Extracorporeal irradiation of the lymph (ECIL) had no detectable effect on the cell cycle or the fractional production rate of the lymphocytes. However, the calculated absolute production was reduced following ECIL, due to a decrease in the absolute number of cells present. The grain count over mitoses after ECIL was approximately one-half that found before ECIL.     

KINETICS OF PHYTOHEMAGGLUTININ STIMULATED LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKEMIA

The DNA synthesis pattern and several kinetic parameters of in vitro PHA stimulated normal and CLL lymphocytes were determined. The DNA synthesis peak of CLL lymphocytes occurred 2–3 days later than that of normal lymphocytes. The generation time, estimated by the labeled mitoses method, was found to be 28 hr and 20 hr for CLL and normal lymphocytes respectively. This difference was mainly due to longer S and G_t periods. It was also shown that both CLL and normal lymphocytes divide several times. These data were confirmed by the chromatid labeling pattern and by the halving of the grains and the double labeling techniques. By combining continuous and pulse labeling the growth fraction of CLL lymphocytes was found to be progressively increasing, because of the recruitment of new cells in cycle, from the third day of culture. Therefore the delayed peak of DNA synthesis of CLL lymphocytes was caused by a longer cell cycle and by a longer pre-replicative phase.     

CELL POPULATION KINETICS OF TRANSPLANTED AND METASTATIC LEWIS LUNG CARCINOMA

The growth rate of Lewis lung carcinoma, either as primary subcutaneous tumors or as spontaneous lung metastases, decreases with increasing age or mass. The length of the median cell cycle of the primary tumors increased from 17.6 hr at Day 5 to 25.9 hr at Day 21 with a concomitant increase in experimental doubling time from 2.5 to 9.6 days. The length of the median cell cycle of the metastatic lung nodules was 15.2 hr in the mice bearing 21-day primary tumors. Two mathematical models were used to analyse the per cent labeled mitoses data and the results are given for comparison. The labeling index of the primary tumors decreased in a nearly linear manner with time postimplant and the percentage of cells labeled in the metastatic nodules was higher than in the primary tumors when measured in the same hosts.     

CELL CYCLE KINETICS IN AN IN VITRO TUMOR MODEL

Cell cycle kinetic parameters of multicell spheroids in vitro have been estimated using thymidine labeling techniques and autoradiography. Both the mitotic index and thymidine labeling index decreased in larger spheroids, whereas the duration of the cell cycle, as determined by two independent methods utilizing labeled mitoses or labeled cells, was essentially independent of spheroid size or age. These results suggest that the tumor-like growth exhibited by spheroids is the result of a decreasing growth fraction and a large apparent cell loss, rather than a general elongation of the cell cycle.     

THE KINETICS OF CELL PROLIFERATION IN THE TRACHEOBRONCHIAL EPITHELIA OF RATS WITH AND WITHOUT CHRONIC RESPIRATORY DISEASE

Labelling indices of the tracheobronchial epithelia of conventionally-derived rats with chronic respiratory disease (CRD) and minimal-disease rats without CRD have been determined. The duration of the DNA synthesis phase (t_s) computed from the percentage of mitoses labelled at various intervals of time after injection of tritiated thymidine was 7 hr: t_G2 was 3.5 hr. Using the measured value of t_s and the labelling indices, the mean turnover times of the tracheobronchial epithelia in three groups of six 5-week-old conventionally-derived rats were calculated to be 11.2, 14.6 and 22.4 days, while in similar groups of 5-week-old minimal-disease rats the turnover times were found to be 24.3, 36.5 and 41.6 days. The majority of cell divisions in the tracheobronchial epithelium of these minimal disease rats were probably required for growth rather than renewal. The mean turnover time of this tissue in 5-week-old Syrian hamsters was 73 days. The cells of the rat tracheobronchial epithelium have been classed as basal or superficial, depending on their shape and proximity to the basement membrane. The mean turnover time of the basal cells in 5-week-old minimal-disease rats was 11.7 days calculated from labelling indices. The migration method of Brown & Oliver (1968) gave a similar value for the basal cells in minimal-disease rats, and a value of 9.5 days for the basal cells in a group of conventionally-derived rats. The mean turnover time in the latter was only 5.4 days if two rats with tracheobronchitis were included. Consideration of the slow rate of fall in mean grain count over labelled cells at intervals of time after labelling and the calculated turnover times suggests that the proliferative fraction of the basal cell population is close to unity. Well-labelled cells were still present in both basal and superficial populations in the minimal-disease rats at 10 days after labelling. The marked effects of CRD on cell proliferation in this epithelium are emphasized and the significance of this in relation to published work is discussed.     

The effects of short-term manipulation of thyroid hormone status coinciding with primary wool follicle development on fleece characteristics in Merino sheep

Thyroidectomy surgery performed late in gestation results in perturbations in wool follicle development in foetal sheep, showing the importance of thyroid hormones for wool follicle development. The aim of this study was to determine the influence of transient manipulation of thyroid hormone status at a time corresponding with foetal primary wool follicle initiation. Pregnant Merino ewes (n = 12 per treatment) were treated daily between gestational days 55 and 64 with control (vehicle), exogenous thyroxine (T4) or propylthiouracil (PTU), an inhibitor of T4 synthesis, and conversion to the active form of the thyroid hormone (triiodothyronine). There were no significant differences in birth weight, gestational lengths and birth coat scores of the resultant lambs. The total primary and secondary follicle densities were significantly lower in lambs exposed to exogenous T4 compared with other treatments (P < 0.05). However, the T4 group displayed a higher proportion of mature secondary follicles (reflected by increased mature secondary follicle densities and mature secondary/primary follicle ratios) than the other treatment groups (P < 0.05). The skin morphology of the lambs differed 12 months later, with the T4 group having significantly higher total follicle densities compared with the PTU group, largely attributed to increased mature and total secondary follicle densities. However, this increase in wool follicle densities did not translate to differences in the fleece yields and weight, fibre diameter, staple lengths or any other fibre parameters. This study showed that transient manipulation of thyroid hormone status during foetal primary follicle initiation does have long-term consequences on the morphology of wool follicles, in particular the maturity of secondary wool follicles.     

PROLIFERATION OF LIVER EPITHELIA IN THREE-WEEK-OLD RATS

The proliferative activity of liver epithelia in 3-week-old rats was studied auto-radiographically using ³H-thymidine. Only a single peak of labelled mitoses (late pro- to anaphases) at 7 hr was found in the per cent labelled mitoses curve after injection of ³H-thymidine. A second peak at about 32 hr described by Post, Huang & Hoffman (1963) and Post & Hoffman (1964, 1965) as well as Grisham (1969) and a cycle time of about 22 hr derived from the distance between the two peaks could not be confirmed by the present work.
According to the present experiments the cycle time of parenchymal liver cells in 3-week-old rats must range between 50 hr (with a growth fraction of 0·25) and 7·1 days (with a growth fraction of 1·0). The present results do not support the existence of a growth fraction of only 0·1 as assumed by Post et al. (1963).     

Nonresponsiveness of the thyroid gland to goitrogen in the early neonatal period in the rat: light- and electron-microscopic observations.

The thyroid response of fetal and neonatal rats to propylthiouracil (PTU) as a goitrogen was studied with observation of the thyroid glands by light and electron microscopy. On day 19 of gestation and on days 1, 3, 5 and 8 after birth, fetal and neonatal rats were given a subcutaneous injection of PTU and were autopsied 2 days later. PTU induced conspicuous goiters in fetal rats but did not in neonatal rats aged up to day 5 after birth. Beyond that age, PTU again induced goiters. Histologically, the follicular cell height in goitrous thyroid glands was significantly increased. Ultrastructurally, follicular cells in goitrous thyroid glands often had colloid droplets and lysosomes. It seems that nonresponsiveness of the thyroid glands in early neonatal rats to goitrogen is due to a temporary decline of the pituitary activity of thyrotropin secretion. About 5 days or more after birth, the pituitary-thyroid system begins to operate again in response to goitrogen.     

Rat C-6 Glioma Cells Maintained In an Organ Culture System: A Study of Kinetic Parameters

Rat C-6 glioma cells were grown on a sponge foam matrix in an organ culture system and the cell cycle parameters, including the growth fraction (GF), were assessed after autoradiography. the zones of growth consisted of a compact upper layer (UL) at the gaseous interface, a central necrotic layer and a deeper lower layer (LL) which invaded the matrix. the fraction of continuously labeled mitoses (FCLM) was similar in both the UL and LL cells. the derivatives of the FCLM curves obtained in three experiments gave an average modal T_G2 of 5 hr. A mathematical model relating GF, T_G2, T_C and labeling index as a function of time, LI(t), was devised for cells in a steady state exposed continuously to tritiated thymidine and was applied to data obtained from UL cells. A mean GF of 9% (range: 8–10%) and a mean cell cycle time (T_C') of 27 hr (range: 13–47 hr) were obtained. the mean T_S was calculated to be 11 hr (range: 8–16 hr) by the method of grain counts per mitotic figure or grain index (GI). Knowledge of T_S permitted alternative calculation of the cell cycle time from the equation T_S/T_C= LI(0)/GF: this gave a mean cell cycle time (T_C) of 29 hr (range: 20–45 hr). Except for the GF, the cell kinetics were comparable to those of the same cell line grown in monolayer culture. the GF in the in vitro system described is in the lower range reported in some human malignant gliomas in vivo.     

Simultaneously Continuous Separation of Glucose,Maltose, and Maltotriose Using a Simulated Moving-Bed Adsorber

The purpose of this study was to find whether the regulation of urea synthesis was mediated through the activation of N-acetylglutamate synthesis by ornithine when the thyroid status was manipulated. Experiments were done on three groups of rats, given 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) without triiodothyronine (T₃) treatment, treated with PTU + T₃, or neither PTU nor T₃ (control). Urinary excretion of urea and the liver concentrations of ornithine and N-acetylglutamate in rats given PTU + T₃ were significantly lower than in rats given PTU alone. The liver concentration of N-acetylglutamate was correlated with the liver concentration of ornithine (r = 0.920, p < 0.001). Ornithine administration (0.5 mmol/100g body wt) elevated the liver concentration of N-acetylglutamate in all three groups. The results suggest that the greater concentration of ornithine in the hypothyroid (PTU alone) rats is likely to increase the N-acetylglutamate concentration in liver and stimulate urea synthesis.     

Renewal of the gonads in Styela clava (Urochordata: Ascidiacea) as revealed by autoradiography with tritiated thymidine

DNA-synthesizing cells in the gonads of the ascidian Styela clava were labeled with tritiated thymidine and detected with autoradiography. In the testis, spermatogonia and primary spermatocytes are labeled after 1 hr. Labeled spermatozoa occur in the lumen of the testis follicles after 10 days and in the sperm ducts after 20 days. In the ovary, only germ cells (oogonia and pre-leptotene primary oocytes) and follicle cells are labeled after 1 hr. By 60 days, oocytes with basophilic cytoplasm (15–65 μ in diameter) are labeled; test cells embedded in larger eosinophilic oocytes (150 μ in diameter) are also labeled. Germ cells give rise to both oocytes and follicle cells. Through continued cell division, follicle cells give rise to test cells.     

Neonatal Persistent Exposure to 6‐Propyl‐2‐thiouracil,a Thyroid‐Disrupting Chemical,Differentially Modulates Expression of Hepatic Catalase and C/EBP‐β in Adult Rats

Persistent exposure of rats to 6‐propyl‐2‐thiouracil (PTU) from birth resulted in decreases in plasma thyroid hormone (TH) levels and hepatic expression of catalase and CCAAT enhancer binding protein β (C/EBP‐β). Catalase promoter region (–185 to +52) that contains binding sites for C/EBP‐β showed an augmentation in the methylation level along with a change in methylation pattern of CpG islands in response to PTU treatment. PTU withdrawal on 30 days of birth restored TH levels and C/EBP‐β to control rats in adulthood. Although catalase expression was restored to some extent in adult rats in response to PTU withdrawal, a permanent change in its promoter CpG methylation pattern was recorded. The results suggest that downregulation of adult hepatic catalase gene in response to persistent neonatal PTU exposure may not solely be attributed to thyroid‐disrupting properties of PTU. It is possible that besides thyroid‐disrupting behavior, PTU may impair expression of hepatic catalase by altering methylation pattern of its promoter.     

Inhibitory effect of large doses of propylthiouracil and methimazole on an increase of thyroid radioiodine release in response to thyrotropin.

Thyroidal radioiodine release increased shortly after a single injection of small doses of PTU, while moderate doses of MMI produced a similar increase of thyroidal radioiodine release with a latency of 7-9 hr. Large doses of PTU and MMI failed to augment thyroidal radioiodine release for at least 29 to 34 hr after the initial administration of goitrogens, although plasma TSH increased significantly because of goitrogen administration. An increase of thyroid hormone release in response to exogenous TSH was depressed by PTU and MMI in rats and mice treated with T4. Since this depression of TSH action only continued for a short period in spite of continuous administration of goitrogens, and since final thyroidal radioiodine release rate was similar to that produced by small doses of PTU, the effects mentioned were not simply due to general toxic action of goitrogens. It is suggested that large doses of PTU and MMI not only block thyroid hormone synthesis but also interfere with the action of TSH on thyroid hormone secretion.     

COMPARATIVE PROLIFERATIVE KINETICS OF CELLS FROM NORMAL HUMAN EPIDERMIS AND BENIGN EPIDERMAL HYPERPLASIA (PSORIASIS) IN VITRO

Proliferation kinetics of epidermal cells from normal human skin and lesions of psoriasis (benign epidermal hyperplasia) were studied in vitro. Epithelial out-growths were obtained from skin explants and the cell cycle studied using the conventional method of following two successive curves of labeled mitoses after an initial pulse with ³H thymidine. The length of T_c was 59 hr and 53.5 hr respectively for normal and psoriatic cells. The shorter T_c for psoriasis was due to a shorter duration of S. The growth fraction was 66% and 74% for normal and psoriatic cells respectively as determined by continuous labeling with ³H thymidine. Under the conditions of the present experiments, therefore, normal and psoriatic epidermal cells showed no significant difference in proliferative capacity.     

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

Therapeutic Potential of Date Palm Pollen for Testicular Dysfunction Induced by Thyroid Disorders in Male Rats

Hyper- or hypothyroidism can impair testicular function leading to infertility. The present study was designed to examine the protective effect of date palm pollen (DPP) extract on thyroid disorder-induced testicular dysfunction. Rats were divided into six groups. Group I was normal control. Group II received oral DPP extract (150 mg kg^-1), group III (hyperthyroid group) received intraperitoneal injection of L-thyroxine (L-T4, 300μg kg^-1; i.p.), group IV received L-T4 plus DPP extract, group V (hypothyroid group) received propylthiouracil (PTU, 10 mg kg^-1; i.p.) and group VI received PTU plus DPP extract. All treatments were given every day for 56 days. L-T4 or PTU lowered genital sex organs weight, sperm count and motility, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone (T), testicular function markers and activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). Moreover, L-T4 or PTU increased estradiol (E2) serum level, testicular oxidative stress, DNA damage and apoptotic markers. Morphometric and histopathologic studies backed these observations. Treatment with DPP extract prevented LT4- or PTU induced changes. In addition, supplementation of DPP extract to normal rats augmented sperm count and motility, serum levels of LH, T and E2 paralleled with increased activities of 3β-HSD and 17β-HSD as well as testicular antioxidant status. These results provide evidence that DPP extract may have potential protective effects on testicular dysfunction induced by altered thyroid hormones.     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.     

The synthesis and turnover of spermidine and spermine in mouse brain

Following the administration to mice of radiolabeled putrescine by intraventricular injection, changes in the specific radioactivity of putrescine, spermidine, and spermine have been measured. Putrescine decline was biphasic, being more rapid over the first 12 hr(t _1/2=5 hr) than over the remainder of the 48-hr period (t _1/2=11 hr) that significant labeling was detected. Spermidine was rapidly labeled during the decline in putrescine radioactivity and maximum incòrporation of label occurred at 18 hr. Subsequently, spermidine specific activity declined with a half-life of 22 days. Spermine synthesis was slower, with maximum labeling occurring after 4 days. Spermine turnover, measured at a time when spermidine radioactivity had substantially declined, was extremely slow (t _1/2=92 days). The data supports the view that putrescine is a precursor of spermidine which in turn is required for spermine synthesis.