The re-establishment of hypersensitive cells in the crypts of irradiated mouse intestine

Within 3-6 h of small doses of radiation (gamma-rays) the number of dead cells (apoptotic cells) in the crypts of the small intestine reaches peak values. These return to normal levels only after times later than 1 day. After higher doses elevated levels of cell death persist for longer times. The dead cells first occur most frequently at the lower positions of the crypt (median value for the distribution of apoptotic fragments is about cell position 6). At later times more dead cells are observed at higher positions. Two doses of radiation separated by various time intervals have been used to investigate when after irradiation the cell population susceptible to acute cell death is re-established. Dead cells were scored 3 or 6 h after the second dose. The yield of dead cells after two doses represents the sum of the dead cells produced by, and persisting from, the first dose and new apoptotic cells induced by the second dose. Since the temporal and dose-dependence aspects of the dead-cell yield after the first dose alone is known, the additional dead cells attributable to the second dose alone can be determined by subtraction. Within 1-2 days of small doses (0.5 Gy) the sensitive cells, recognized histologically as apoptotic cells, are re-established at the base of the crypt (around cell position 6). After higher doses (9.0 Gy) they are not re-established until about the fourth day after irradiation. Even in the enlarged regenerating crypts the sensitive cells are found at the same position at the crypt base. It has been estimated that the crypt contains five or six cells that are susceptible to low doses (0.5 Gy) (hypersensitive cells) and up to a total of only seven or eight susceptible cells that can be induced by any dose to enter the sequence of changes implicit in apoptosis. Between 4 and 10 days after an initial irradiation of 9.0 Gy the total number of susceptible cells increased from seven to eight to about 10 to 13 per crypt.     

The distribution of endocrine cells along the mouse intestine: a quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30-60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the bottom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3-4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

Intestinal crypt proliferation. II. Computer modelling of mitotic index data provides further evidence for lateral and vertical cell migration in the absence of mitotic activity

The position-dependent mitotic index before, and 1, 2 and 3 h after vincristine was scored. The accumulation of cells in mitosis leads to an increase in the mitotic index from 0.06 to 0.34 at crypt positions 8-12. Surprisingly, the leading edge of the position-related mitotic index distribution moves to higher crypt positions although cell division was stopped. In addition, the vertical clustering of mitotic figures in sections was recorded. The data were examined using a previously described computer crypt model. We conclude: the average mitotic phase duration is about 0.7 h (40 min) and varies little with cell position; the geometrical correction factor for overscoring mitoses in crypt sections is about 0.6-0.7 and adjacent cell columns can merge. Lateral cell displacement after mitosis, as predicted in a previous model analysis, would be a mechanism to counteract other forces that tend to reduce the crypt circumference. In the normal steady state merging and expansion processes would just balance each other. This would not follow if one mechanism was blocked. Thus we propose a new concept in which the crypt geometry would be dynamically determined by cell proliferative activity in connection with lateral positioning of new cells on one hand and contracting forces on the other hand.     

Scoring Mitotic Activity In Longitudinal Sections of Crypts of the Small Intestine

Abstract. Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. the correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. the results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestime before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor f_d for the mitotic index of 0.59 is obtained; (6) the correction factor f_T derived from the shape and position of the mitotic figures measured in 3 μm longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor f_mod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is f_D; for mouse small intestine it is 0.59.     

The effects of irradiation at different times of the day on rat intestinal goblet cells

Quantitative changes in jejunal goblet cells were studied in control and whole body irradiated rats using PAS-Alcian blue staining of crypt sections. A circadian dependence was observed when control animals were killed at different times during the light/dark cycle. Irradiation with 3 Gy produced a 2–3-fold increase within 36 h in goblet cells relative to controls, followed by a reduction to very low levels. There was a return to pre-treatment levels later than was observed for the columnar cells. The present results on the pattern of response of goblet cells and those of brush border enzyme activity are consistent with the hypothesis that ionizing radiation can influence differentiation. In fact during the first hours after irradiation an early induction of differentiation is evident while during the early repopulation phase columnar cells prevailed relative to the goblet cells. Only at later times were normal differentiation patterns seen. Groups of animals exposed to the same dose of radiation at different times of the day showed similar general patterns of behaviour even if the group irradiated at midnight showed a more marked and longer lasting injury.     

CIRCADIAN RHYTHMS OF PRESUMPTIVE STEM CELLS IN THREE DIFFERENT EPITHELIA OF THE MOUSE

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with ³H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.     

EARLY AND LATE INCORPORATION OF TRITIATED THYMIDINE INTO SKIN CELLS AND THE PRESENCE OF A LONG-LIVED G0-SPECIFIC PRECURSOR POOL

40 min after a single injection of 50 µCi of tritiated thymidine a 3 mm punch of DBA-1 mouse skin contains about 1000 dpm. This value remains constant for at least 48 hr after injection. 50 hair follicles contain about 40 dpm, and from these values the activity calculated to reside in the basal layer of a 3 mm punch of skin is 760 dpm. These values also remain constant with time after injection. Fresh punches of skin contain much more activity. The fixative-soluble fraction (the difference between fresh and fixed values) decays slowly with time. The values for DBA-2 mice are similar. Plucking the hair from the follicles appears immediately to increase the size of the fixative-soluble fraction and decrease the fixed tissue values to about 500 dpm per punch for whole skin and about 1 dpm per 50 follicles for DBA-1. Thus almost all the activity is restricted to the epidermis. The fixative-soluble fraction returns approximately to the unplucked value between 24 and 48 hr after plucking. However, during this period the fixed tissue values are rising rapidly as stimulated cells enter S. It appears that in both strains labeled material remains available for incorporation into stimulated cells for at least 48 hr after a single injection. The amount persisting appears to decrease with time. The whole-fixed skin, the hair follicles, and the epidermis all contain cells that are capable of becoming labeled after stimulation 8–48 hr after an injection. The label in question does not become incorporated into normal cycling skin or hair follicle cells. It is concluded that the DNA precursor pool is possibly connected with G₀ cells and that both the hair follicle and the basal layer of the epidermis contain these resting cells.     

Repeated injection (continuous labelling) experiments in mouse epidermis

Theoretical labelling index curves for epidermis have been generated under conditions of repeated tritiated thymidine injection. These curves take into account different injection intervals, circadian fluctuations in labelling and two different models for epidermal proliferation; one based on a homogeneous basal layer with “random” loss initially (later, loss was restricted to late G₁), and the other based on a programmed sequential aging of proliferative cells in a compartment derived from a minority class of stem cells. These curves have been compared with previously published experimental results and with results from some new experiments. Both models fit the data to some extent provided a mean value of T_c of about 140 h is assumed. However, the sequential aging model provides a slightly better overall fit. A further conclusion is that it is impossible to make any accurate statements on the epidermal growth fraction from repeated labelling data.     

Proliferative changes in the genital tissue of female mice during the oestrous cycle

Abstract. Changes in proliferation and number of epithelial cells of the murine genital tract, during the oestrous cycle, have been studied. A total of 47 animals in the pro-oestrous, metoestrous and dioestrous phases of the cycle were staged retrospectively on the basis of the genital tract histology. The average duration of the oestrous cycle in these aninals was 4 days, and half of this period was occupied by the pro-oestrous/oestrous phases. Significant cycles of growth were observed in the luminal uterine epithelium and in the basal epithelium of the cervix-vagina. Most of this growth occurred during the pro-oestrous phase, which lasted approximately 1 day. During this time the numbers of luminal epithelial cells in the uterus and suprabasal cells in the cervix-vagina increased 2–3 fold. This pattern of growth appeared partly synchronous and corresponded to the period when serum oestrogen levels are at their highest.
A corresponding and rapid reduction in the numbers of uterine luminal epithelial cells and suprabasal cells in the cervix and vagina was noted during the early metoestrous phase; and this occured during the period when serum oestrogens are at their lowest levels. No significant periodicity in the proliferation and numbers of the uterine gland epithelial cells was noted during the cycle. The kinetic role and function of the gland cells is discussed in relation to these data.     

Polymorphisms in the coding and noncoding regions of murinePgk-1 alleles

The mouse X-linkedPgk-1 gene encodes phosphoglycerate kinase. When transfected into human cells, thePgk-1^b allele causes the appearance of mouse PGK-1^b enzyme activity. We describe here cloning of mousePgk-1^a, an allele ofPgk-1 which encodes an enzyme, PGK-1^a, with distinct electrophoretic mobility. We constructed recombinants between the DNA encodingPgk-1^b andPgk-1^a and transfected these constructs into human to assess the electrophoretic characteristics of each recombinant. In this way the charge variation between the two proteins was localized to exons 4 or 5. Sequencing of these exons revealed a single base-pair difference between the two alleles at codon 155, which predicts the amino acids lysine and threonine in PGK-1^b and PGK-1^a, respectively. A number of other DNA sequence polymorphisms exist betweenPgk-1^b andPgk-1^a including part of an L1 repeated element unique toPgk-1^a. This work was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Deutsche Forschungsgemeinschaft, SFB 304.