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INVOLUTION OF MOUSE MAMMARY GLANDS DURING WHOLE ORGAN CULTURE OCCURS VIA APOPTOSIS OF EPITHELIAL TISSUE
Cell Biology International, 1996, Vol. 20, No. 11, 763–767
INVOLUTION OF MOUSE MAMMARY GLANDS DURING WHOLE ORGAN CULTURE OCCURS VIA APOPTOSIS OF EPITHELIAL TISSUE T. M. CASEY1, H. CHEN2, K. PLAUT*1 and J. F. CHIU2 Department of Animal and Food Sciences and 2Department of Biochemistry, University of Vermont Burlington, VT 05405, U.S.A.
1
Accepted 23 August 1996
Apoptosis was measured in mammary glands during whole organ culture, to determine whether regression resulting from hormone withdrawal results in epithelial cell death as in vivo involution. Glands were evaluated for morphology and DNA degradation prior to whole organ culture, after lobulo-alveolar development and 2, 4, or 6 days after hormone withdrawal. The data indicated that mammary regression during whole organ culture mimics in vivo involution ? 1996 Academic Press Limited and results in part from apoptosis of epithelial tissue. K: apoptosis; epithelial tissue; mouse; mammary glands
INTRODUCTION Mammary gland differentiation is regulated and maintained by a complex interaction between hormonal stimulation and the extracellular matrix. Involution is a dynamic transitional period of mammary development resulting in vivo from the cessation of the suckling stimulus. Involution is marked by a decrease in galactopoietic hormone levels and an increased expression of tissue remodelling enzymes and programmed cell death, apoptosis (Walker et al., 1989; Strange et al., 1992). The molecular mechanisms regulating this transitional period have yet to be elucidated. Mouse mammary whole organ culture (WOC) is a defined in vitro system that has been used to investigate cyclic mammary gland development (Ichinose and Nandi, 1966). WOC maintains the mammary cells in their natural environment while addition and removal of factors can be readily manipulated. During WOC, glands are hormonally stimulated to undergo lobulo-alveolar differentiation and express â-casein mRNA (Ganguly et al., 1981). Withdrawal of all hormones except insulin causes mammary glands to regress in a manner similar to in vivo involution (Ganguly et al., 1981). Atwood et al. (1995) determined that apoptosis occurred in mammary tissue differentiated and involuted in WOC. *To whom correspondence should be addressed. 1065–6995/96/110763+05 $25.00/0
Lactogenic hormones have been shown to have both positive and negative effects on the growth of other types of tissues. In particular, lymphocytes and lymph tissue are very sensitive to glucocorticoid-induced apoptosis (Witorsch et al., 1993; Evans-Storms and Cidlowski, 1995). However, Witorsch et al. (1995) demonstrated that the lactogenic hormone prolactin can inhibit glucocorticoid induced apoptosis and induce mitosis in rat Nb2 lymphoma cells when cultured in prolactin alone. The objective of this study was to determine the type of mammary tissue apoptosis occurred in during involution of glands in WOC, and if the presence of mammary lymph nodes affected mammary involution in a positive or negative way. In this report, it is demonstrated that apoptosis occurs in the epithelium during involution of mammary glands in WOC with and without mammary lymph nodes, and the presence or absence of lymph nodes does not appear to effect the involution of glandular epithelium. MATERIALS AND METHODS Whole organ culture Animal experimentation was conducted under the regulations of Interdisciplinary Principles and Guidelines for the Use of Animals in Research, ? 1996 Academic Press Limited
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Cell Biology International, Vol. 20, No. 11, 1996
Fig. 1. Mouse mammary glands were cultured for 5 days in Ins, Prl, Ald, Hyd, and EGF to (a) induce full lobulo-alveolar development. Glands were then involuted in insulin alone. Following hormone withdrawal for: (b) 2 days, much of lobulo-alveolar development still maintained; (c) 4 days, lobulo-alveoli clearly lost; and (d) 6 days; almost complete involution, demonstrating a progressive loss of glandular epithelium. Hematoxylin stained whole mounts. Bar=0.835 mm.
Testing, and Education. Virgin BALB/c mice, 26–28 days old (Charles River Laboratories, Quebec, Canada) were treated with estrogen and progesterone for 9 days by s.c. implantation of a 10-mg pellet (1:1000:1000/estrogen: progesterone:cholesterol) (Plaut, 1993). Animals were euthanized and abdominal #4 mammary glands were removed. Lymph nodes were removed from half of the glands in each treatment and the glands were subjected to WOC for differentiation and involution as described previously (Ichinose and Nandi, 1966; Plaut, 1993). Briefly, glands were cultured in Waymouth’s 752/1 media supplemented with insulin (5.0 ìg/ml), aldosterone (0.10 ìg/ml), hydrocortisone (0.10 ìg/ml), prolactin (1.0 ìg/ml) and epidermal growth factor (60 ng/ml) for 5 days to achieve lobulo-alveolar differentiation. Subsequently, to initiate in vitro mammary involution, glands were cultured with media supplemented with insulin (5.0 ìg/ml) alone. Glands were collected on day 0 prior to WOC, after 5 days of WOC, and 2, 4, or 6 days after hormone withdrawal. Morphology was evaluated in whole mounts of hematoxylin stained glands (Vonderhaar and Greco, 1975).
Electrophoresis of DNA DNA from two glands in each treatment was isolated according to the method described by Fukuda et al. (1993) with minor modifications. Following overnight incubation of the glands in DNA extraction buffer (10 m Tris-HCl, 100 m EDTA, 0.5% SDS, 100 ìg/ml RNase A), DNA was precipitated with 1/10 volume 3 sodium acetate and 100% ethanol. DNA was resuspended in TE buffer (10 m Tris-HCl, pH 8.0, 1 m EDTA), extracted with phenol/chloroform/isoamyl alcohol, and reprecipitated. Twenty ìg of DNA was electrophoresed on a 1.4% agarose gel. The gel was stained with ethidium bromide and DNA bands were visualized with a UV transilluminator and photographed. In situ detection of DNA fragmentation Glands were fixed with 10% buffered formalin, dehydrated, and paraffin embedded for sectioning. Tissues were sectioned (6 ìm) and left unstained for the terminal transferase (TdT) assay. The TdT
Cell Biology International, Vol. 20, No. 11, 1996
765
assay was performed as described by Gavrieli et al. (1992). Positive controls were treated with DNase I. Negative controls for TdT and biotinylated dUTP were prepared by treating slides with biotinylated dUTP or TdT alone, respectively. The number of epithelial cells containing fragmented DNA was determined by counting positively stained nuclei in three random fields of 100 cells each. RESULTS AND DISCUSSION Prior to WOC, the glands contained terminal end buds and were undifferentiated (data not shown). On day 5 of WOC, glands were fully differentiated (Fig. 1(a)). As glands began to involute, lobuloalveolar structures were lost. As the days of involution progressed, the glandular structures were regressed to alveolar buds and ducts (Fig. 1(b)–(d)). The gland resembled mammary glands taken from animals after cessation of lactation (Vonderhaar, 1988). Electrophoretic analysis of DNA isolated from mammary glands at day 0 revealed intact high molecular weight DNA (Fig. 2, lane 1). Oligonucleosomal length DNA fragments were detected in tissue from all other treatment groups, regardless of presence of lymph nodes (Fig. 2). These data indicated that DNA fragmentation, which is characteristic of apoptosis, occurred in mouse mammary tissue during WOC. The terminal transferase assay was used to determine the type of mammary cell undergoing apoptosis during WOC. DNA fragmentation was not detected in any cell type in uncultured mammary glands. This is consistent with the lack of a DNA ladder seen after electrophoresis. In situ labelling of 3* ends of DNA fragments revealed DNA degradation occurred in the lymph tissue and not in epithelium in fully differentiated glands at day 5 of WOC (Fig. 3(a),(b); Table 1). The high rate of cellular death in the lymph tissue may be due to the glucocorticoids, since lymphocytes and lymph tissue are very sensitive to glucocorticoid-induced apoptosis (Witorsch et al., 1993; Evans-Storms and Cidlowski, 1995). In the present study, prolactin was not able to protect the lymph tissue from glucocorticoid induced apoptosis, as demonstrated by Witorsch et al. (1993). It is unclear whether apoptosis in the mammary lymph node during WOC occurs because of the concentration of the corticosteroids relative to prolactin in the culture or represents an unknown paracrine effector secreted by epithelial cells, which effects lymph tissue.
Fig. 2. DNA isolated from mouse mammary tissue was electrophoresed on 1.4% agarose gel. Glands developed and regressed during WOC exhibit oligonucleosomal ladder pattern typical of tissue undergoing apoptosis regardless of presence of lymph node. DNA from 123 bp ladder (L); DNA from mammary glands prior to WOC (lane 1); developed for 5 days in WOC with lymph node (lane 2) and without lymph node (lane 3); and regressed in WOC by hormone withdrawal for 2 days with lymph node (lane 4) and without lymph node (lane 5); 6 days with lymph node (lane 6) and without lymph node (lane 7).
During involution caused by hormone withdrawal in WOC, apoptotic cells were located in both epithelial tissue and lymph nodes of the mammary glands. As the days after hormone withdrawal progressed from day 2 to day 6, the number of apoptotic cells in the epithelium also increased, revealing a substantial amount of DNA degradation during mammary gland involution (Fig. 3, Table 1). Red-stained apoptotic nuclei were identified in alveolar structures on day 6 involution at higher magnification (Fig. 3(f)). This is consistent with the structural changes seen in whole mounts of mammary glands in WOC (Fig. 1), and the changes observed during in vivo involution (Strange et al., 1992). The rate of involution appears to be delayed for 2 days during WOC as compared with the rate of involution observed in vivo. Apoptosis during in vivo involution of mouse mammary glands initiated on day 10 of lactation peaked 4 days after weaning pups as demonstrated by formation of oligonucleosomal ladders during gel electrophoresis (Strange et al., 1992) and in situ labelling of apoptotic nuclei (Quarrie et al., 1995). Based on in situ labelling of
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Cell Biology International, Vol. 20, No. 11, 1996
Fig. 3. In situ labelled 3* ends of DNA fragments in 6-ìm sections of mouse mammary tissue, demonstrate the increased rate of apoptosis in epithelial tissue following hormone withdrawal in WOC. Red-stained tissue indicates the cells contain fragmented DNA. Tissues were counterstained with hematoxylin. Glands developed in WOC for 5 days in the presence of Ins, Prl, Ald, Hyd, and EGF do not exhibit apoptotic nuclei in: (a) adipose (A) and epithelial (E) tissue; but apoptotic nuclei are clearly evident in: (b) lymph nodes (L) of these glands. Following hormone withdrawal there is a marked increase in the number of apoptotic nuclei in epithelial of glands regressed for (c) 2 days; (d) 4 days; (e) 6 days; (f) 6 days, higher magnification of alveoli, reveals apoptotic nuclei of epithelial tissue. Bar=0.227 mm.
apoptotic nuclei, peak apoptosis occurred 6 days after hormone withdrawal in WOC. This discrepancy may be due to a neuroendocrine or local factor related to the suckling stimulus or milk stasis that increase the rate of in vivo involution but are not present in the WOC system. A possible candidate for the greater rate at which in vivo involution occurs is feedback inhibitor of lactation protein,
which may be present in glands developed and involuted in vivo, but not glands developed in WOC (Wilde et al., 1995). Our findings support the data of Atwood et al. (1995) that apoptosis occurs in mammary glands during WOC. In addition, we were able to demonstrate with the terminal transferase assay that involution of mouse mammary glands in WOC occurs
Cell Biology International, Vol. 20, No. 11, 1996
767
Table 1. Effect of hormone withdrawal during whole organ culture on percentage of mammary epithelial cell death Day of development Day 0 Day 5 Days after hormone withdrawal Day 2 Day 4 Day 6
Percent epithelial cell death 0 0 0.33&0.57 0.33&0.57 43 &3.6
via apoptosis of epithelial cells as in vivo, regardless of the presence of lymph nodes. Apoptosis did not occur in the epithelial cells nor adipocytes of fully differentiated glands. These data indicate that WOC conditions specifically maintain and affect the mammary epithelium. However, they may not maintain cells of hematopoietic origin as demonstrated by cell death in lymph nodes on all days of WOC. During involution of glands in WOC, apoptosis markedly increased as evidenced by epithelial cell death and the loss of morphological integrity. These data indicate mammary gland involution can be mimicked in vitro using mouse mammary WOC. WOC provides an excellent means to investigate involution and apoptosis in a defined hormonal system, which maintains the mammary cells in their natural environment. ACKNOWLEDGEMENT This work was supported by the Vermont Agricultural Experiment Station. REFERENCES A C, I M, V B, 1995. Involution of mouse mammary glands in whole organ culture: a model for studying programmed cell death. Biochem Biophys Res Comm 207: 860–867.
E-S RB, C JA, 1995. Regulation of apoptosis by steroid hormones. J Steroid Biochem Mol Biol 53: 1–8. F K, M K, C J, 1993. Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 777 tissue: Apoptosis or necrosis? Amer J Pathol 142: 945–953. G N, G R, M N, C L, B MR, 1981. Simultaneous occurrence of pregnancy like lobulo-alveolar morphogenesis and casein-gene expression in a culture of whole mammary gland. In Vitro 17: 55–62. G Y, S Y, B-S SA, 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493–497. I RR, N S, 1966. Influence of hormones on lobulo-alveolar differentiation of mouse mammary glands in vitro. J Endocrinol 35: 331–340. P K, 1993. Role of epidermal growth factor and transforming growth factors in mammary development and lactation. J Dairy Sci 76: 1526–1538. Q LH, A C, W CJ, 1995. Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling. Cell Tissue Res 281: 413–419. S S, 1992. Programmed cell death: Concept mechanism and control. Biol Rev 67: 287–319. S R, L F, S S, B A, F RR, 1992. Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115: 49–58. V BK, 1988. Regulation of development of normal mammary gland by hormones and growth factors. In: Lippman ME, Dickson RB, eds. Breast Cancer: Cellular and Molecular Biology. Kluwer Academic Publishers, Boston, 251–265. V BK, G AE, 1975. Lobulo-alveolar development of mouse mammary glands is regulated by thyroid hormones. Endocrinology 104: 409–418. W NI, B RE, K J, 1989. Cell death by apoptosis during involution of the lactating breast in mice and rats. American J Anat 185: 19–32. W CJ, A C, B-F LM, P M, 1995. Autocrine control of milk secretion: from concept to application. In: Wilde CJ, Peaker M, Knight CH, eds. Intercellular Signalling in the Mammary Gland. Plenum Press, New York, 227–238. W RJ, D EB, LV HA, H N, T JK, 1993. Comparison of glucocorticoid-induced effects of prolactin-dependent and autonomous rat Nb2 lymphoma cells. Proc Soc Exp Biol Med 203: 454–460.
INVOLUTION OF MOUSE MAMMARY GLANDS DURING WHOLE ORGAN CULTURE OCCURS VIA APOPTOSIS OF EPITHELIAL TISSUE T. M. CASEY1, H. CHEN2, K. PLAUT*1 and J. F. CHIU2 Department of Animal and Food Sciences and 2Department of Biochemistry, University of Vermont Burlington, VT 05405, U.S.A.
1
Accepted 23 August 1996
Apoptosis was measured in mammary glands during whole organ culture, to determine whether regression resulting from hormone withdrawal results in epithelial cell death as in vivo involution. Glands were evaluated for morphology and DNA degradation prior to whole organ culture, after lobulo-alveolar development and 2, 4, or 6 days after hormone withdrawal. The data indicated that mammary regression during whole organ culture mimics in vivo involution ? 1996 Academic Press Limited and results in part from apoptosis of epithelial tissue. K: apoptosis; epithelial tissue; mouse; mammary glands
INTRODUCTION Mammary gland differentiation is regulated and maintained by a complex interaction between hormonal stimulation and the extracellular matrix. Involution is a dynamic transitional period of mammary development resulting in vivo from the cessation of the suckling stimulus. Involution is marked by a decrease in galactopoietic hormone levels and an increased expression of tissue remodelling enzymes and programmed cell death, apoptosis (Walker et al., 1989; Strange et al., 1992). The molecular mechanisms regulating this transitional period have yet to be elucidated. Mouse mammary whole organ culture (WOC) is a defined in vitro system that has been used to investigate cyclic mammary gland development (Ichinose and Nandi, 1966). WOC maintains the mammary cells in their natural environment while addition and removal of factors can be readily manipulated. During WOC, glands are hormonally stimulated to undergo lobulo-alveolar differentiation and express â-casein mRNA (Ganguly et al., 1981). Withdrawal of all hormones except insulin causes mammary glands to regress in a manner similar to in vivo involution (Ganguly et al., 1981). Atwood et al. (1995) determined that apoptosis occurred in mammary tissue differentiated and involuted in WOC. *To whom correspondence should be addressed. 1065–6995/96/110763+05 $25.00/0
Lactogenic hormones have been shown to have both positive and negative effects on the growth of other types of tissues. In particular, lymphocytes and lymph tissue are very sensitive to glucocorticoid-induced apoptosis (Witorsch et al., 1993; Evans-Storms and Cidlowski, 1995). However, Witorsch et al. (1995) demonstrated that the lactogenic hormone prolactin can inhibit glucocorticoid induced apoptosis and induce mitosis in rat Nb2 lymphoma cells when cultured in prolactin alone. The objective of this study was to determine the type of mammary tissue apoptosis occurred in during involution of glands in WOC, and if the presence of mammary lymph nodes affected mammary involution in a positive or negative way. In this report, it is demonstrated that apoptosis occurs in the epithelium during involution of mammary glands in WOC with and without mammary lymph nodes, and the presence or absence of lymph nodes does not appear to effect the involution of glandular epithelium. MATERIALS AND METHODS Whole organ culture Animal experimentation was conducted under the regulations of Interdisciplinary Principles and Guidelines for the Use of Animals in Research, ? 1996 Academic Press Limited
764
Cell Biology International, Vol. 20, No. 11, 1996
Fig. 1. Mouse mammary glands were cultured for 5 days in Ins, Prl, Ald, Hyd, and EGF to (a) induce full lobulo-alveolar development. Glands were then involuted in insulin alone. Following hormone withdrawal for: (b) 2 days, much of lobulo-alveolar development still maintained; (c) 4 days, lobulo-alveoli clearly lost; and (d) 6 days; almost complete involution, demonstrating a progressive loss of glandular epithelium. Hematoxylin stained whole mounts. Bar=0.835 mm.
Testing, and Education. Virgin BALB/c mice, 26–28 days old (Charles River Laboratories, Quebec, Canada) were treated with estrogen and progesterone for 9 days by s.c. implantation of a 10-mg pellet (1:1000:1000/estrogen: progesterone:cholesterol) (Plaut, 1993). Animals were euthanized and abdominal #4 mammary glands were removed. Lymph nodes were removed from half of the glands in each treatment and the glands were subjected to WOC for differentiation and involution as described previously (Ichinose and Nandi, 1966; Plaut, 1993). Briefly, glands were cultured in Waymouth’s 752/1 media supplemented with insulin (5.0 ìg/ml), aldosterone (0.10 ìg/ml), hydrocortisone (0.10 ìg/ml), prolactin (1.0 ìg/ml) and epidermal growth factor (60 ng/ml) for 5 days to achieve lobulo-alveolar differentiation. Subsequently, to initiate in vitro mammary involution, glands were cultured with media supplemented with insulin (5.0 ìg/ml) alone. Glands were collected on day 0 prior to WOC, after 5 days of WOC, and 2, 4, or 6 days after hormone withdrawal. Morphology was evaluated in whole mounts of hematoxylin stained glands (Vonderhaar and Greco, 1975).
Electrophoresis of DNA DNA from two glands in each treatment was isolated according to the method described by Fukuda et al. (1993) with minor modifications. Following overnight incubation of the glands in DNA extraction buffer (10 m Tris-HCl, 100 m EDTA, 0.5% SDS, 100 ìg/ml RNase A), DNA was precipitated with 1/10 volume 3 sodium acetate and 100% ethanol. DNA was resuspended in TE buffer (10 m Tris-HCl, pH 8.0, 1 m EDTA), extracted with phenol/chloroform/isoamyl alcohol, and reprecipitated. Twenty ìg of DNA was electrophoresed on a 1.4% agarose gel. The gel was stained with ethidium bromide and DNA bands were visualized with a UV transilluminator and photographed. In situ detection of DNA fragmentation Glands were fixed with 10% buffered formalin, dehydrated, and paraffin embedded for sectioning. Tissues were sectioned (6 ìm) and left unstained for the terminal transferase (TdT) assay. The TdT
Cell Biology International, Vol. 20, No. 11, 1996
765
assay was performed as described by Gavrieli et al. (1992). Positive controls were treated with DNase I. Negative controls for TdT and biotinylated dUTP were prepared by treating slides with biotinylated dUTP or TdT alone, respectively. The number of epithelial cells containing fragmented DNA was determined by counting positively stained nuclei in three random fields of 100 cells each. RESULTS AND DISCUSSION Prior to WOC, the glands contained terminal end buds and were undifferentiated (data not shown). On day 5 of WOC, glands were fully differentiated (Fig. 1(a)). As glands began to involute, lobuloalveolar structures were lost. As the days of involution progressed, the glandular structures were regressed to alveolar buds and ducts (Fig. 1(b)–(d)). The gland resembled mammary glands taken from animals after cessation of lactation (Vonderhaar, 1988). Electrophoretic analysis of DNA isolated from mammary glands at day 0 revealed intact high molecular weight DNA (Fig. 2, lane 1). Oligonucleosomal length DNA fragments were detected in tissue from all other treatment groups, regardless of presence of lymph nodes (Fig. 2). These data indicated that DNA fragmentation, which is characteristic of apoptosis, occurred in mouse mammary tissue during WOC. The terminal transferase assay was used to determine the type of mammary cell undergoing apoptosis during WOC. DNA fragmentation was not detected in any cell type in uncultured mammary glands. This is consistent with the lack of a DNA ladder seen after electrophoresis. In situ labelling of 3* ends of DNA fragments revealed DNA degradation occurred in the lymph tissue and not in epithelium in fully differentiated glands at day 5 of WOC (Fig. 3(a),(b); Table 1). The high rate of cellular death in the lymph tissue may be due to the glucocorticoids, since lymphocytes and lymph tissue are very sensitive to glucocorticoid-induced apoptosis (Witorsch et al., 1993; Evans-Storms and Cidlowski, 1995). In the present study, prolactin was not able to protect the lymph tissue from glucocorticoid induced apoptosis, as demonstrated by Witorsch et al. (1993). It is unclear whether apoptosis in the mammary lymph node during WOC occurs because of the concentration of the corticosteroids relative to prolactin in the culture or represents an unknown paracrine effector secreted by epithelial cells, which effects lymph tissue.
Fig. 2. DNA isolated from mouse mammary tissue was electrophoresed on 1.4% agarose gel. Glands developed and regressed during WOC exhibit oligonucleosomal ladder pattern typical of tissue undergoing apoptosis regardless of presence of lymph node. DNA from 123 bp ladder (L); DNA from mammary glands prior to WOC (lane 1); developed for 5 days in WOC with lymph node (lane 2) and without lymph node (lane 3); and regressed in WOC by hormone withdrawal for 2 days with lymph node (lane 4) and without lymph node (lane 5); 6 days with lymph node (lane 6) and without lymph node (lane 7).
During involution caused by hormone withdrawal in WOC, apoptotic cells were located in both epithelial tissue and lymph nodes of the mammary glands. As the days after hormone withdrawal progressed from day 2 to day 6, the number of apoptotic cells in the epithelium also increased, revealing a substantial amount of DNA degradation during mammary gland involution (Fig. 3, Table 1). Red-stained apoptotic nuclei were identified in alveolar structures on day 6 involution at higher magnification (Fig. 3(f)). This is consistent with the structural changes seen in whole mounts of mammary glands in WOC (Fig. 1), and the changes observed during in vivo involution (Strange et al., 1992). The rate of involution appears to be delayed for 2 days during WOC as compared with the rate of involution observed in vivo. Apoptosis during in vivo involution of mouse mammary glands initiated on day 10 of lactation peaked 4 days after weaning pups as demonstrated by formation of oligonucleosomal ladders during gel electrophoresis (Strange et al., 1992) and in situ labelling of apoptotic nuclei (Quarrie et al., 1995). Based on in situ labelling of
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Cell Biology International, Vol. 20, No. 11, 1996
Fig. 3. In situ labelled 3* ends of DNA fragments in 6-ìm sections of mouse mammary tissue, demonstrate the increased rate of apoptosis in epithelial tissue following hormone withdrawal in WOC. Red-stained tissue indicates the cells contain fragmented DNA. Tissues were counterstained with hematoxylin. Glands developed in WOC for 5 days in the presence of Ins, Prl, Ald, Hyd, and EGF do not exhibit apoptotic nuclei in: (a) adipose (A) and epithelial (E) tissue; but apoptotic nuclei are clearly evident in: (b) lymph nodes (L) of these glands. Following hormone withdrawal there is a marked increase in the number of apoptotic nuclei in epithelial of glands regressed for (c) 2 days; (d) 4 days; (e) 6 days; (f) 6 days, higher magnification of alveoli, reveals apoptotic nuclei of epithelial tissue. Bar=0.227 mm.
apoptotic nuclei, peak apoptosis occurred 6 days after hormone withdrawal in WOC. This discrepancy may be due to a neuroendocrine or local factor related to the suckling stimulus or milk stasis that increase the rate of in vivo involution but are not present in the WOC system. A possible candidate for the greater rate at which in vivo involution occurs is feedback inhibitor of lactation protein,
which may be present in glands developed and involuted in vivo, but not glands developed in WOC (Wilde et al., 1995). Our findings support the data of Atwood et al. (1995) that apoptosis occurs in mammary glands during WOC. In addition, we were able to demonstrate with the terminal transferase assay that involution of mouse mammary glands in WOC occurs
Cell Biology International, Vol. 20, No. 11, 1996
767
Table 1. Effect of hormone withdrawal during whole organ culture on percentage of mammary epithelial cell death Day of development Day 0 Day 5 Days after hormone withdrawal Day 2 Day 4 Day 6
Percent epithelial cell death 0 0 0.33&0.57 0.33&0.57 43 &3.6
via apoptosis of epithelial cells as in vivo, regardless of the presence of lymph nodes. Apoptosis did not occur in the epithelial cells nor adipocytes of fully differentiated glands. These data indicate that WOC conditions specifically maintain and affect the mammary epithelium. However, they may not maintain cells of hematopoietic origin as demonstrated by cell death in lymph nodes on all days of WOC. During involution of glands in WOC, apoptosis markedly increased as evidenced by epithelial cell death and the loss of morphological integrity. These data indicate mammary gland involution can be mimicked in vitro using mouse mammary WOC. WOC provides an excellent means to investigate involution and apoptosis in a defined hormonal system, which maintains the mammary cells in their natural environment. ACKNOWLEDGEMENT This work was supported by the Vermont Agricultural Experiment Station. REFERENCES A C, I M, V B, 1995. Involution of mouse mammary glands in whole organ culture: a model for studying programmed cell death. Biochem Biophys Res Comm 207: 860–867.
E-S RB, C JA, 1995. Regulation of apoptosis by steroid hormones. J Steroid Biochem Mol Biol 53: 1–8. F K, M K, C J, 1993. Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 777 tissue: Apoptosis or necrosis? Amer J Pathol 142: 945–953. G N, G R, M N, C L, B MR, 1981. Simultaneous occurrence of pregnancy like lobulo-alveolar morphogenesis and casein-gene expression in a culture of whole mammary gland. In Vitro 17: 55–62. G Y, S Y, B-S SA, 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119: 493–497. I RR, N S, 1966. Influence of hormones on lobulo-alveolar differentiation of mouse mammary glands in vitro. J Endocrinol 35: 331–340. P K, 1993. Role of epidermal growth factor and transforming growth factors in mammary development and lactation. J Dairy Sci 76: 1526–1538. Q LH, A C, W CJ, 1995. Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling. Cell Tissue Res 281: 413–419. S S, 1992. Programmed cell death: Concept mechanism and control. Biol Rev 67: 287–319. S R, L F, S S, B A, F RR, 1992. Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115: 49–58. V BK, 1988. Regulation of development of normal mammary gland by hormones and growth factors. In: Lippman ME, Dickson RB, eds. Breast Cancer: Cellular and Molecular Biology. Kluwer Academic Publishers, Boston, 251–265. V BK, G AE, 1975. Lobulo-alveolar development of mouse mammary glands is regulated by thyroid hormones. Endocrinology 104: 409–418. W NI, B RE, K J, 1989. Cell death by apoptosis during involution of the lactating breast in mice and rats. American J Anat 185: 19–32. W CJ, A C, B-F LM, P M, 1995. Autocrine control of milk secretion: from concept to application. In: Wilde CJ, Peaker M, Knight CH, eds. Intercellular Signalling in the Mammary Gland. Plenum Press, New York, 227–238. W RJ, D EB, LV HA, H N, T JK, 1993. Comparison of glucocorticoid-induced effects of prolactin-dependent and autonomous rat Nb2 lymphoma cells. Proc Soc Exp Biol Med 203: 454–460.