APOPTOSIS IN THE MAMMARY GLAND

MORPHOLOGICAL CHANGES DURING PROGRAMMED CELL DEATH (PCD) IN THE INVOLUTING MOUSE MAMMARY GLAND

SUMMARY

Tissue homeostasis is a result of a coordinated regulation of proliferation and elimination of cells. Developmentally regulated removal of cells is mostly controlled by programmed cell death (PCD). We studied the morphology of apoptotic cells by confocal laser scanning microscopy and by transmission electron microscopy in the mouse mammary gland during involution. The gland is characterized by a proliferation of epithelial cells during pregnancy which are mostly removed during post-lactational involution. Apoptosis starts at about day 1 after weaning, it peeks at day 3 and decreases thereafter. DNA fragmentation was measured by in situ terminal transferase labeling which revealed a striking prevalence of labeling along the periphery of the the nucleus. Only a weak staining was found in the central area of most apoptotic nuclei. This may be due to a specific absence of DNA fragmentation in the central area of apoptotic nuclei or an inaccessibility of the chromatin as a specific consequence of cell death. For cells in culture a duration of cell death of about one hour was reported. A rough estimation based on the relative abundance of apoptotic cells at different time points during involution (as estimated by terminal transferase positive nuclei) revealed a similar duration. Ultrastructurally, involution is accompanied by dramatic morphological changes that initially take place in the cytoplasm. Subsequently, nuclear changes become apparent. Apoptotic cells contain condensed and fragmented chromatin and extensive indentations of the nuclear membrane. At a late stage of apoptosis condensed chromatin becomes encapsulated into apoptotic bodies which are phagocytosed by neighboring cells.

INTRODUCTION

Pregnancy induces a massive development of mammary alveolar structures which at birth embody the differentiated secretory epithelium required for milk production. After cessation of lactation a collapse of the lobulo-alveolar structures occurs which is paralleled by a reductive remodeling of the gland. This process that is termed involution is characterized by a proteolytic degradation of the extracellular matrix and a loss of secretory epithelial cells mainly by programmed cell death (Strange et al., 1992; Jaggi et al, 1996).

GENERAL ROLE OF PROGRAMMED CELL DEATH

Programmed cell death (PCD) represents an efficient way to physiologically eliminate supernumerary or undesired cells without the occurrence of inflammatory events that are generally induced by necrotic cell death. PCD is a major regulatory feature of embryological development (e.g. neurogenesis or limb development), establishment of immune-self tolerance, immune effector cell killing and regulation of cell viability by hormones and growth factors. The study of programmed cell death got considerable attention over the last few years, when it became apparent that its deregulation may contribute to neoplastic transformation and viral pathogenesis (Schwartz and Osborne, 1993).

CHARACTERIZATION OF APOPTOSIS

PCD can be separated into two phases: commitment of a cell to enter the death pathway and the actual death process (apoptosis). Many different stimuli that may trigger the suicide of a cell have been identified in different cell systems, e.g. DNA-damage, inappropriate expression of genes that stimulate cell cycle progression, withdrawal (or addition) of hormones or growth factors and viral infections. As most of these apoptosis-triggering factors interfere with the cell cycle it has been proposed that PCD primarily reflects a cell cycle perturbation (Evan and Littlewood, 1993; Marti et al., 1995).
Apoptosis, the actual death process, is characterized by a number of morphological changes. The process starts with a loss of cell junctions and other specialized plasma membrane structures such as microvilli. At the same time the nuclear chromatin condenses and marginates into one or several large masses at the nuclear periphery. The nucleolus disaggregates and the nuclear membrane generally adopts complex internal folds. Subsequently, the nucleus may split into several distinct fragments. It has been suggested that apoptosis corresponds to an aberrant mitosis. Chromatin condensation and disassembly of the nuclear lamina are involved in both processes and may though exploit the same molecular mechanisms. While the nucleus condenses a contraction of cytoplasmic volume occurs which is associated with a loss of fluid. Membranes bleb and the cell transiently adopts a deeply convoluted outline before it finally breaks into discrete membrane-bounded apoptotic bodies. These apoptotic bodies are phagocytosed by neighboring cells (Wyllie et al., 1980; Kerr et al., 1987; Lazebnik et al., 1993).
The apoptotic process itself is relatively short, in vitro it was shown to last only about one hour. In contrast the cells may rest for variable periods in a "latent" phase ranging from commitment to apoptosis to the actual apoptosis itself. This "latent" stage may last from a few hours to several days. As the cells do not synchronously enter apoptosis and as the apoptotic phase is only brief compared to the latent phase, it is very difficult to examine biochemical changes underlying the morphological changes of apoptosis. A hallmark of apoptosis is the characteristic oligonucleosomal fragmentation of chromosomal DNA which is observed in many, although not all cases of PCD. In situ it is possible to visualize apoptotic cells by coupling a detectable nucleoside to free 3’ends of the partially fragmented DNA. This reaction, sometimes referred to as tunel assay, is performed by exogenously added terminal transferase (Gavrieli et al., 1992).

APOPTOSIS IN THE MAMMARY GLAND

The aim of our study was to get deeper insight into the distribution and kinetics of the fragmentation process during PCD of mammary epithelial cells. We studied the distribution of DNA (as detected by propidium iodide staining) and of DNA-fragmentation (as detected by the terminal transferase reaction) by laser scanning microscopy within the nuclei of apoptotic cells. Confocal scanning microscopy allowed examinations at a higher resolution than would have been possible with a conventional microscope. Furthermore, we found the laser scanning microscope to be especially suitable for characterizing various sources of artifacts and limitations of the terminal transferase assay in the in situ detection of fragmentation.

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Fig. 1A	Fig. 1B	Fig. 1C	Fig. 1D	Fig. 1E

Involution of the mammary gland was induced by forced weaning five to seven days after parturition. PCD in the involuting mammary gland was monitored by in situ terminal transferase reactions on 26 um thick paraffin sections derived from mammary glands at lactation, days 1, 2 and 3 of involution (Figures 1A, 1B, 1C and 1D respectively). Shown are large overviews where positive nuclei are detected as white spots. DNA fragmentation increases from a very low level at lactation and day 1 of involution to a substantial number at day 2 of involution reaching a relative maximum at day 3 of involution. Thereafter the relative number of terminal transferase positive nuclei decreases. Figure 1E shows a tissue slice which was pretreated with DNAse I before the tunel assay, hence resulting in a positive signal in essentially all nuclei.

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Fig. 2A	Fig. 2B

The distribution of DNA fragmentation was investigated in more detail by laser scanning microscopy (BIORAD MRC600). In the secretory epithelium we predominantly found two classes of terminal transferase positive nuclei. Representatives of these classes are shown in Figures 2A and 2B. Nuclei were double stained with propidium iodide (PI) which intercalates with DNA and stains all nuclei (red staining) and with terminal transferase which selectively stains apoptotic nuclei containing fragmented DNA (green staining). Apoptotic nuclei (Figure 2A) have a very compact appearance, as judged by their size and intense staining (compare with normal nuclei in the vicinity). The terminal transferase predominantly labels these nuclei only at their borders suggesting that DNA fragmentation is perinuclear, or more likely that the transferase enzyme or the labeling reagents cannot penetrate this condensed structure. This effect is characteristic for apoptotic nuclei as a homogenous labeling was seen in DNAseI treated nuclei (not shown). Most of the terminal transferase positive nuclei condense their collapsed chromatin into only one nuclear mass, in some instances we also found nuclei which condensed their chromatin into more than one aggregate. A different appearance was found in the other group of nuclei (Figure 2B). This group probably represents fragmented nuclei or apoptotic bodies. Note that the compactness of the unfragmented nuclei still persists in some fragments while some other debris are strongly terminal transferase positive and contain relatively little PI stainable DNA.

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Fig. 3A	Fig. 3B

We performed a three dimensional reconstruction of the terminal transferase labeled region of the two different groups of nuclei. The image processing was performed on a Silicon Graphics workstation with Imaris software. In Figure 3A a nucleus is shown which is virtually sliced just above the equator. The terminal transferase labeling distributes in a cup-like structure, showing that the halo of terminal transferase labeled DNA seen in a projective view originates from a selective staining of the perinuclear area. Figure 3B shows spread fragments of a disintegrating nucleus. Apoptotic bodies are phagocytosed by their neighboring cells.

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Fig. 4A	Fig. 4B

Morphological changes during early involution were monitored by transmission electron microscopy. The studied specimens were embedded in Epon and double stained with lead citrate and uranyl acetate. Figure 4A (lactation) and Fig 4B (1 day of involution) demonstrate that striking changes occur in this initial - mostly reversible - phase of involution. A massive reduction of the cytoplasmic mass was observed. Concomitantly, there is a large reduction in the height of the epithelium. A strong vacuolarisation takes place in the secretory epithelial cells, which disappears by day 3 of involution. Alveolar lumen is indicated by an asterix.

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Fig. 5

Figure 5 shows a secretory luminal cell at day 3 of involution which is in an early stage of apoptosis. Compacting of chromatin at the margins of the nucleus has occurred. This epithelial cells typically segregate from the ductal lumen (asterix) toward the basal lamina (arrow heads).

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Fig. 6

At day three of involution, but not at lactation, we often found cells whose nuclei have an appearance similar to that shown in Figure 6. The notable feature of these nuclei is their indentations which, depending on the plane of section, may lead to a nucleus which seems to be divided into several fragments. This stage may represent a rather early state of PCD in the mammary gland. However, in these cells the distribution a of the heterochromatin resembles that observed in normal epithelial cell nuclei of the mammary gland.

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Fig. 7A	Fig. 7B

At the final stage of apoptosis, dying cells in the mammary gland mostly dissociate into several fragments which are part of apoptotic bodies. Apoptotic bodies are phagocytosed by neighbouring cells (Figure 7A; the apoptotic body is indicated by arrow heads). More rarely, apoptotic cells are extruded into the ductal lumen (Figure 7B).

Our results reveal that secretory epithelial cells of the involuting mouse mammary gland die by programmed cell death. The cells probably enter a "latent", most likely transcriptionally active stage before they undergo actual cell death by apoptosis.

REFERENCES

Evan, G.I. and Littlewood, T.D. (1993). The role of c-myc in cell growth. Curr. Opin. Genet. Dev. 3, 44-49.

Gavrieli, Y., Sherman, Y., and Ben-Sasson, S.A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493-501.

Jaggi, R., Marti, A., Ke, G., Feng, Z., and Friis, R. (1996). Regulation of a physiological apoptosis: mouse mammary involution. J. Dairy Sci. (in press).

Kerr, J.F.R., Searle, J., Harmon, B.V., and Bishop, C.J. (1987). Apoptosis. In Perspectives on mammalian cell death. C.S. Potten, ed. (Oxford: Oxford University Press), pp. 93.

Lazebnik, Y.A., Cole, S., Cooke, C.A., Nelson, W.G., and Earnshaw, W.C. (1993). Nuclear Events of Apoptosis Invitro in Cell-Free Mitotic Extracts - A Model System for Analysis of the Active Phase of Apoptosis. J. Cell Biol. 123, 7-22.

Marti, A., Feng, Z., Jehn, B., Djonov, V., Chicaiza, G., Altermatt, H.J., and Jaggi, R. (1995). Expression and activity of cell cycle regulators during proliferation and programmed cell death in the mammary gland. Cell Death Diff. 2, 277-283.

Schwartz, L.M. and Osborne, B.A. (1993). Programmed cell death, apoptosis and killer genes. Immunology Today 14, 582-590.

Strange, R., Li, F., Saurer, S., Burkhardt, A., and Friis, R.R. (1992). Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115, 49-58.

Wyllie, A.H., Kerr, J.F.R., and Currie, A.R. (1980). Cell death: The significance of apoptosis. Int. Rev. Cytol. 68, 251-306.

Note:
This information has been contributed by Claudio Vallan, Zhiwei Feng and Rolf Jaggi.
We would like to acknowledge Beat Haenni, Bettina De Breuyn and Peter Burri from the Institute of Anatomy, University of Bern, Switzerland, for their contributions. If information from this page is used in publications, the Mammary Gland Web site should be cited.