Cell adhesion molecules are glycoproteins, expressed on all cell surfaces. They are crucial for normal tissue architecture formation and maintenance 1. Adhesion molecules play vital roles in numerous cellular processes. Some of these include embryogenesis, cell growth and differentiation, immune response, and wound repair 2–4.
Many studies have reported that there are three major subclasses of cadherins: E (epithelial), P (placental), and N (neural). Each one has distinct adhesive specificities and tissue distribution 5–7. Another study dealing with the mechanisms of epithelial cell–cell adhesion, some authors clarified that cadherins are calcium-dependent adhesion molecules. They participate in adherens junctions, thus establishing homophilic and heterophilic bonds between adjacent cells 8.
E-cadherin (E-cad), the founder member of the cadherin superfamily, is considered the master organizer of the epithelial phenotype 9–12. Cadherins are considered to play a major role in establishing and maintaining stable intercellular adhesion in the majority of tissues 13. They play an essential mechanical role in binding the cell to its environment, either to the extracellular matrix or to another cell 14,15. Other major roles include embryonic morphogenesis and tissue rearrangement, determination of cell phenotype, cell migration, and dissemination of tumor cells 16.
In their developmental review, Masso-Welch et al. 17 reported that the rat mammary gland is an excellent model for examining the interactions of multiple cell types in response to hormonal and growth factor stimulation. They added that the mammary gland is a dynamic tissue that undergoes epithelial proliferation, lobular morphogenesis, secretory activity, followed by regression, and involution during pregnancy, lactation, and weaning. In addition, in another study on mouse mammary gland, the authors clarified that the postnatal differentiations in the mammary gland epithelium may contribute toward its susceptibility to carcinogenesis 18.
In the animal research field on cell adhesion molecules, it is difficult to obtain stable immunostaining results in formalin-fixed mammary glandular tissue. The lack of the histological studies on the pattern of expression of immunohistochemical staining of E-cad and its quantification morphometrically during the different stages of histogenesis of the mammary gland led to the idea of this work in perspective. Thus, the aim of the current study was to show the histological changes and the pattern of expression of E-cad adhesion molecule microscopically and morphometrically in the female rat mammary gland during different stages of pregnancy, lactation, and weaning compared with the nonpregnant (resting) gland.
Materials and methods
The adult female rats were mated as follows: one fertile male albino rat was introduced into a cage with two females. The animals remained together overnight. Next morning, the presence of spermatozoa in the vaginal smear of female animals was assessed. Pregnancy was confirmed by the presence of the vaginal mucous plug, to be considered the first day of gestation. The lactation stage started from day 1 of delivery. The involution stage was considered to start after weaning (21 days after delivery).
Thirty-five adult female Wistar albino rats (3–4 months old) were purchased from the animal house of the King Fahd Medical Research Center (KFMRC). The female rats were divided into the following groups (five rats each):
Group I [nonpregnant (resting)], group II (P7), and group III (P14): rats were sacrificed at day 7 and day 14 of pregnancy. Group IV (L7) and group V (L14): rats were sacrificed at day 7 and day 14 of lactation. Group VI (W7) and Group VII (W14): rats were sacrificed at day 7 and day 14 after weaning.
The animals were housed in large environmentally controlled cages (25°C, 12-h light /12-h dark cycles). Commercial food and tap water were supplied ad libitum. All the procedures followed the ethical rules of animal house of KFMRC, Jeddah, which were in accordance with the guidelines of the Canadian Council on Animal Care.
Animals of different groups were anesthetized and their right inguinal mammary glands were dissected. The trimmed strips obtained from each mammary gland were immediately immersed in 10% neutral-buffered formalin. Then, they were introduced into tissue-embedding cassettes, dehydrated, cleared, and embedded in paraffin wax. Four-micrometer-thick paraffin sections were cut using a rotator microtome. The prepared serial sections were stained by H&E for assessment of the general structure of the gland 19.
Four-micrometer-thick paraffin sections were mounted onto poly-l-lysine-coated slides, and subjected to an immunohistochemical procedure using the Avidin–Biotin detection system following the manufacturer’s instructions. The antibody used was Novocastra Lyophilized mouse monoclonal antibody E-Cadherin (NCL-E-Cad), dilution 1:50. The immunohistochemical procedure was carried out using an automatic immunostainer (Ventana Bench Mark XT; Ventana Inc., Tucson, Arizona, USA). In each analysis, positive controls were used consisting of human fibroadenoma samples previously shown to stain with this antibody. Tris-buffered saline in place of the primary antibody was used as a negative control. The reaction was visualized with the standard 3,3 diaminobenzidine (DAB). Sections were counterstained with hematoxylin for morphological orientation 20. Sections were examined and photographed using a light microscope (BX51; Olympus Optical Corporation, Tokyo, Japan) fitted with an Olympus digital camera (DP20).
Morphometrical and statistical study
The expression of E-cad was evaluated according to two parameters. The first was the semiqualitative expression of the marker as a positive (+) or a negative (−) reaction. The positive reaction was considered when all borders showed a dark brown color. The negative reaction was considered when the basal border lost the positive expression. The second parameter of expression was the pattern of staining, which was assessed as membranous when positive expression was localized to the borders and cytoplasmic when it was intracytoplasmic. Morphometrically, the number of positive and negative cells in the luminal epithelial cells was determined in five consecutive microscopic fields (×1000) of three serial sections from five blocks of each group. The color density of the positive immunostaining was expressed as an area fraction (%). This was measured in five consecutive fields (×1000) of three serial sections from each block. The recorded results were expressed as mean±SD. The morphometric analysis was carried out on digital light microscopic images that had been captured by a digital camera Olympus (DP 20) on an Olympus BX51 light microscope. Image analysis procedures were performed using the Image Pro-plus Program (version 6), (Georgia, USA). The statistical analysis of the results of all groups was carried out using the SPSS (16) program. The results of different groups were compared with those of group I using a Student t-test. A significant difference was considered at P value of 0.05 or less.
Group I (nonpregnant)
The H&E-stained sections of the non-pregnant resting rat mammary gland showed a group of collapsed ducts together with aggregation of epithelial cells forming uncanalized terminal end bulbs. Most of the sections showed abundant adipose connective tissue (CT) stroma with small blood vessels on the expense of the glandular tissue. Most of the ducts were lined with a single layer of cubical epithelial cells surrounded by myoepithelial cells. Scattered mast cells were observed near the blood vessels (Fig. 1a and b).
Microscopic examination of immunostained sections showed a strong positive E-cad reaction of epithelial lining the ducts. The positive dark brown linear reaction was limited to the membrane borders of the lining epithelial cells. Few cells appeared with a faint cytoplasmic positive reaction together with negatively reacted myoepithelial cells (Fig. 2a and b). Morphometrically, the mean percentage of cytoplasmic reaction did not exceed a mean of 7% in the examined sections compared with 78% of the membranous reaction. Cells with a negative E-cad reaction did not exceed 15% (Table 1 and Histogram 1). The density area fraction of E-cad expression had a mean of 6.6% (Table 2 and Histogram 2).
Group II (P7)
The H&E-stained sections of the early pregnant rat showed a group of ducts with a marked increase in the budding alveoli forming the lobules. A large number of blood vessels together with marked cellularity especially mast cells were observed adjacent to the developing alveoli, consistent with reduced adipose CT stroma. The partially canalized ducts were lined by a single layer of cubical epithelium with a pale vacuolated cytoplasm. Also, most of the cells lining the alveoli appeared vacuolated (Fig. 3a and b).
The immunostained sections showed a positive dark brown E-cad reaction localized to all the membranous borders of epithelial cell lining of both ducts and alveoli outlining the cells. Some cells showed negative E-cad expression on their basal membranes. Others showed a granular cytoplasmic positive reaction. The myoepithelial cells showed no reaction (Fig. 4a and b). There was a statistically nonsignificant change in the percentage of the positive cytoplasmic cells (6%) and positive membranous cells (83%), together with a nonsignificant decrease in the negative luminal cells (11%) compared with group I (Table 1 and Histogram 1). The density of the immunostained area fraction showed a nonsignificant change from resting gland, mean 6.8% (Table 2 and Histogram 2).
Group III (P 14)
The sections of rat mammary gland of late pregnancy showed marked proliferation of the alveolar part of the lobules at the expense of the adipose tissue. The ducts appeared canalized and filled with secretion. The congested blood capillaries were observed in most of the examined sections (Fig. 5a).
The interlobular ducts were lined by stratified cubical epithelium. They were encircled by myoepithelial cells and thick collagenous CT. The epithelial cubical cell lining of the alveoli and intralobular ducts appeared pale and vacuolated. Some alveoli showed evident secretions. The stroma was highly vascular, together with mast cells (Fig. 5b).
The immunostained sections of late pregnant rat mammary gland showed a faint positive E-cad reaction in the alveolar parts. This faint reaction was apparent along the cell membranes of both alveoli and ducts but not continuous along the cellular borders. The basal borders of some cells together with the myoepithelial cells showed a negative expression of E-cad. A weak cytoplasmic reaction was observed or recorded (Fig. 6a and b). Morphometrically, the majority of cells (93%) were E-cad negative, with no cytoplasmic reaction, and the membranous positive reaction was only 7%, with a statistically significant difference compared with the nonpregnant group (Table 1 and Histogram 1). Also, there was a significant decrease (P<0.05) in the immunostained density area fraction (3.9%) compared with the resting mammary gland (Table 2 and Histogram 2).
Group IV (L7)
The H&E-stained sections of the early lactating group showed marked expansion of the glandular tissue, mainly alveoli, at the expense of the stromal adipose CT. There were minimal interlobular CT septa, with many blood capillaries adjacent to the lactating alveoli. The dilated alveoli appeared full of secretions and lined by a single layer of cubical cells with a dark basophilic cytoplasm. Points of discontinuity were observed among some of these alveoli (Fig. 7a and b).
The immunostained sections appeared with a strong positive E-cad reaction localized to the basolateral membranes of the alveolar epithelial cell lining. There were areas of negative E-cad expression along the basal membranes of some alveolar cells as well as the myoepithelial cells. Some of the epithelial lining appeared with a granular positive cytoplasmic reaction (Fig. 8a and b). In morphometric analysis, the cells showed a statistically significant decrease in the membranous reaction (40%) and a nonsignificant change in the cytoplasmic reaction (6%) compared with nonpregnant rats (group I), together with a significant increase in the negative luminal reaction (54% of the cells) (Table 1 and Histogram 1). Also, there was a significant decrease (P<0.05) in the immunostained density area fraction (4.2%) in comparison with the nonpregnant mammary gland (Table 2 and Histogram 2).
Group V (L 14)
At a later stage of lactation, 14 days from its onset, the sections of rat mammary gland showed highly distended alveoli with milk secretion and marked sac formation. There was a marked decrease in stromal CT, together with the appearance of large blood vessels (Fig. 9a). Some alveoli showed protrusion of their epithelial cell lining into the lumen. The cytoplasm of these cells was observed to be vacuolated and the cell debris was extruded into the alveolar lumen. The myoepithelial cells were difficult to be distinguished around the alveoli (Fig. 9b).
The immunostained sections of late lactating rat mammary gland showed a positive E-cad expression limited to the basolateral membranes of cells lining the ducts and alveoli. Some alveoli showed a linear pattern of positive E-cad expression outlining the basal membranes of lining cells. The negative expression along their apical membranes was evident. Few cells showed a positive granular cytoplasmic reaction (Fig. 10a and b). The morphometric results indicated that 68% of cells showed a negative E-cad reaction, 28% showed a positive membranous reaction, and only 4% showed a positive cytoplasmic reaction with statistical significance compared with group I (Table 1 and Histogram 1). This stage showed a significant decrease (P<0.05) of the immunostained density area fraction (4.5%) in relation to the resting mammary gland (Table 2 and Histogram 2).
Group VI (W7)
The H&E-stained rat mammary gland of group VI (W7) showed mainly collapsed interlobular ducts surrounded by marked disorganized involuted alveoli, which were infiltrated by numerous mononuclear cells. The lobules were separated by abundant fatty CT stroma (Fig. 11a). The involuted alveolar epithelial cells showed dense basophilic nuclei. Apparently, they were replaced by a group of eosinophilic multinuclear giant cells, mast cells, and other mononuclear cells (Fig. 11b).
The expression pattern of the E-cad in the early stage of involution was highly positive along the membranes of epithelial cells of regressed alveoli and ducts. The dark brown E-cad expression appeared mostly in the membranous borders of lining cells. The reappearance of a positive apical E-cad reaction was a characteristic feature. Some stromal cells with a positive granular E-cad expression were observed (Fig. 12a and b). Morphometric analysis showed a statistically significant positive membranous reaction (20%), cytoplasmic reaction (4%), and negative reaction (76%) of cells (Table 1 and Histogram 1). The measured density of the positive reaction showed a nonsignificant alteration from the nonpregnant gland with a mean area fraction of 5.3% (Table 2 and Histogram 2).
Group VII (W 14)
During late weaning, rat mammary sections showed collapsed interlobular ducts surrounded by extensive stromal CT rich in fat cells. Few epithelial cell aggregations were observed representing the involuted alveoli with remnant secretion. Myoepithelial cells were observed surrounding the lining epithelium. The presence of mast cells was a persistent feature (Fig. 13a and b).
The rat mammary gland sections showed a strong positive E-cad expression restricted to the membranes of epithelial cells lining of the ducts and the involuted alveoli. The basal membranes of some epithelial cells showed a weak immunostained reaction, whereas the myoepithelial cells appeared negatively reacted. The positive E-cad expression appeared as brown granules in large stromal cells (Fig. 14a and b). Morphometrically, the mean percentage of positive cytoplasmic reaction (4%) and positive membranous reaction (20%) was statistically significant compared with group I. The luminal cells showed a significant negative E-cad reaction, mean 76% (Table 1 and Histogram 1). However, there was a nonsignificant decrease in the immunostained density area fraction (5.6%) compared with the resting gland of group I (Table 2 and Histogram 2).
In the current study, the pattern of expression and distribution of E-cad were studied in rat mammary gland during the different stages of its development through the fertile period. To our knowledge, this is one of the first studies that combined the qualitative assessment of the E-cad expression pattern with the quantitative findings using image analysis, in the rat mammary tissue during the various stages of its normal differentiation.
The present work showed that, the mammary gland of nonpregnant rats had abundant adipose CT stroma with an evident decrease in the glandular elements represented by collapsed ducts and uncanalized terminal end bulbs. These findings correlated with those of Knudsen and Wheelock 21, who described the mammary gland of the young virgin female to be formed of ducts that branch and end in terminal end buds.
The immunohistochemical assessment of the current work showed that the borders of the lining epithelial cells of the ducts of the nonpregnant rats had a strong positive E-cad expression with a mean area fraction of 6.6%. The membranous pattern of E-cad staining in these cells was observed as continuous brown staining of cell–cell borders. The mentioned morphological description has been reported previously in human beings studies 22–24, in cats 25, and in dogs 26,27.
The apical localization of E-cad observed in the current study can be attributed to the concentration of actin underneath the plasma membrane, forming a ring-like structure. This suggestion was supported by Desrivieres et al. 28, who reported that actin underneath the plasma membrane simulated staining of the E-cad cell adhesion molecule, which is a component of adherens junctions in terminally differentiated epithelium. They added that reorganization of the actin cytoskeleton at the sites of cell–cell contacts accompanies cellular differentiation. Also, Knudsen and Wheelock 21 had reported that cadherins are single-pass transmembrane proteins whose extracellular domain promotes cell–cell adhesion, whereas the intracellular domain interacts with cytoplasmic proteins, such as catenins, which link classical cadherins directly and indirectly to the actin cytoskeleton. Maintenance of adherens junctions is also dependent on the dynamic actin cytoskeleton. Disruption of many actin regulators leads to a loss of junctional integrity, indicating a close interplay between junction dynamics and the actin cytoskeleton 29.
Recently, it has been reported that endocytosis of E-cad plays a major role in regulation of the local concentration of E-cad in cellular membranes together with cellular trafficking and motion within the cell surface. Thus, this regulation is a fundamental process for cell–cell interactions and integrity of the epithelial tissues 30.
The myoepithelial cells of the studied groups did not show immunostaining reactivity. This finding was similar to that reported in studies of human, mice, and feline mammary glands studies 25,31,32. This negative reaction of myoepithelial cells was explained by Daniel et al. 32, who reported that myoepithelial cells are attached to the basement membrane and express P-cadherin, but not E-cad. In contrast, other studies have reported that E-cad existed in both luminal and myoepithelial cells in the normal mammary glands of human beings 23,33, mice 34 and dogs 27. However, others have reported that myoepithelial cells showed inconsistent and weak cytoplasmic positivity in the normal gland as well as in mammary tumors 26.
The marked structural changes during pregnancy observed in the current study were associated with a strong positive dark brown E-cad reaction of the epithelial cell lining that was localized to all the membranous cell borders of both ducts and alveoli. These findings were reported by other studies 21,32. Morphometrically, in early pregnancy, most of the cells showed a significant membranous positive reaction (83%), with a 6% positive cytoplasmic reaction. This positive reaction was reversed by late pregnancy, where 93% of cells showed a significant negative reaction with no cytoplasmic reaction. Also, the density area fraction of E-cad expression showed a significant decrease during late pregnancy. In contrast, other authors have reported that there was no change in E-cad expression up to day 16 in the mammary gland of pregnant mice 11. These results may be because of the qualitative assessment used in the mentioned study.
Significant cytoplasmic expression was recorded in pregnancy and lactation. However, the density area percent was significantly altered only during late pregnancy and lactation. Thus, these findings reflected the vulnerability of this duration to the occurrence of any abnormal cellular changes. Therefore, this indicates the importance of investigating both the pattern and the degree of E-cad expression by epithelial cells, especially the degree, during different periods. In contrast to the current conclusion, other authors have reported that the cytoplasmic pattern was more prevalent in malignancy, especially solid carcinomas 26. The authors recommended the use of the E-cad pattern of expression, rather than its grade, as a stronger parameter to differentiate between benign and malignant tumors. However, some studies have shown normal expression of E-cad in some carcinomas 35, but this normal E-cad expression described in the studies mentioned was attributed to abnormal-functioning cadherin molecules. E-cad staining observed in some poorly differentiated tumor cells could strengthen the hypothesis that the intercellular adhesion systems in some carcinomas showed marked changes in their components 35.
Other studies on human breast and cervical carcinomas have attributed the cytoplasmic immunostaining pattern to failure in the translocation or in the anchorage of E-cad to the cell membrane 36,37. However, Bringuier et al. 38 considered E-cad cytoplasmic staining a functional defect of the E-cad cytoplasmic tail, which could not bind the intracellular catenins. Therefore, the quantitative assessment of the presence of E-cad, which was carried out in the present work, should be considered a valuable predictive tool in addition to the qualitative assessment of pathological conditions.
Furthermore, some studies have reported that the changes observed in the pattern of expression of E-cad, and in the number of epithelial cells stained in the mammary neoplasms, provide further evidence that this cell adhesion molecule plays an important role as a tumor suppressor 25,39.
In this work, the recorded a negative expression of E-cad during late pregnancy and lactation, indicated by a significant increase in negative cell percentage together with a significant decrease in its intensity reflecting marked changes in hormonal levels during these periods in the quantity and quality of the E-cad produced. Therefore, the importance of expression of E-cad, as a component of junctional complex, ensured its role in the normal structure of the mammary gland. The current suggestion is in agreement with Knudsen and Wheelock 21, who reported the important roles of the E-cad complex in the normal mammary gland signaling in mice. This was reported after their study using gene manipulation of cadherins. In addition, a previous study on mutant mice using the conditional gene inactivation scheme observed that the mutant mammary gland developed normally up to 16–18 days of pregnancy and then changed markedly around parturition. They reported that the lack of E-cad affected the terminal differentiation program of the lactating mammary gland 11.
The localization of a positive E-cad reaction to the basolateral boundaries of the epithelial cell lining as well as the faint or the negative reaction of the basal membranes of some cells in the present study can be attributed to the presence of different cadherin subclasses. This suggestion was supported by researchers who reported that all cadherin subclasses are concentrated at the boundary between cells. Localization decreased or even disappeared at the boundary between cells expressing different cadherin subclasses. They may interact with each other by lower affinities, thus resulting in weaker or no cadherin accumulation in the intercellular boundary 13.
During the weaning period of the current investigation, the involuted lobules appeared regressed, with the presence of multinuclear giant cells. There was a significant decrease in the percentage of positive cells (membranous) together with a significant increase in negative cells in combination with a non-significant decrease in the density area fraction of E-cad immunostaining. These results can be attributed to the actual decrease in the glandular tissue after involution. In contrast, other articles have reported that during epithelial to mesenchymal transition, intercellular contacts between epithelial cells were lost because of decreased E-cad expression and disruption of the adherens junction complex as cells became less adhesive and potentially more motile 40,41.
The appearance of a positive E-cad reaction in the stromal cells in this research suggests its phagocytic function. This explanation could be confirmed by the emphasis in the literature on the clearance of apoptotic cells, especially macrophages 42. In addition, the current results showed an apparent progressive increase in mast cell numbers during pregnancy and weaning. These findings were consistent with those of a developmental study on the mouse mammary gland 43. The authors reported that mast cells are capable of normal degranulation, and have activated granule-associated proteases to facilitate normal mammary gland pubertal development. The marked decrease in the fibrofatty stroma at the expense of proliferated glandular tissue during lactation may be the reason for the rarely observed mast cell in the present study.
In summary, the E-cad was mainly localized in the membranes of the epithelial cell lining, not in the myoepithelial cells. The significant changes in the pattern and degree of expression were observed mainly during pregnancy and lactation. These observations indicate that E-cad expression is altered according to the hormonal status of the females during their fertile period. These vulnerable periods should encourage scientists to design valuable experimental research that aims to help oncologists in the design and choice of the most effective and appropriate therapies and prognostic markers.
This project was funded by the Deanship of Scientific Research (DSR) King Abdulaziz University (KAU), under Grant No (477/248/1431). The authors, therefore, thank DSR for technical and financial support.
Conflicts of interest
There are no conflicts of interest.
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