Gland Involution Research Articles

Cripto: roles in mammary cell growth, survival, differentiation and transformation.

Cripto-1 (Cr-1) protein, encoded by the teratocarcinoma-derived growth factor gene (TDGF-1), is highly correlated with transformation in breast cancer. Eighty-two percent of breast carcinomas express Cr-1 whereas it is undetected in normal human breast tissue. We confirmed and extended findings that Cr-1 protein is expressed during the pregnancy and lactating stages of normal murine mammary glands but is barely detectable in glands from virgin animals and is undetectable in involuted glands. Cr-1 was found to be expressed in CID 9 cells, a line of mammary epithelial cells derived from 14.5 day pregnant mice and we have used these cells to investigate the roles of this gene. Exogenous mouse Cr-1 expression from a retroviral vector caused CID 9 cells to grow at an increased rate and to increased cell densities compared to parental and control cells. CID 9 cells overexpressing Cr-1 did not differentiate efficiently. Infection of CID 9 cells with a Cr-1 antisense vector caused these cells to change in morphology, to grow slowly, to undergo apoptosis at a higher rate and to achieve lower saturation densities but the cells were still capable of differentiating. We concluded that Cr-1 is an autocrine growth factor for normal breast cells, that when over-expressed stimulates excessive cell proliferation at the expense of differentiation. In transplantation studies, Cr-1 over-expression stimulated the growth and survival of mammary cells, but did not stimulate tumorigenesis in vivo.

The regulatory role of transforming growth factor-beta in activation of milk mononuclear cells.

Immunoregulatory role of milk mononuclear cells and cytokines during the involution of the mammary gland. Bioassays were used to determine the levels of transforming growth factor beta (TGF-beta) and interleukin-2 (IL-2) and their effects on milk mononuclear cells (MMCs). MMCs collected from involuting glands were less responsive to Con A stimulation than peripheral blood mononuclear cells (PBMCs), and purified huTGF-beta 1 inhibited the activation of both MMCs and PBMCs by Con A. Furthermore, secretions collected over a period of approximately 4 weeks into the involution period contained high levels of active TGF-beta and extremely high levels of latent TGF-beta, MMCs stimulated with Con A produced higher levels of IL-2 than did the PBMCs of the same animals, and bovine milk TGF-beta and huTGF-beta 1 significantly inhibited the IL-2 production by MMCs. Mammary secretions of involuting glands did not contain any detectable levels of IL-2, and they inhibited the growth of the IL-2-dependent bovine lymphoblastoid T cell line (BLTC), even in the presence of 10 U/ml of rBo IL-2. The findings suggest that TGF-beta is a major immunoregulatory factor in the involuting mammary gland.

Lactation stage-dependent changes of lymphocyte subpopulations in mammary secretions: inversion of CD4+/CD8+ T cell ratios at parturition.

Determination of lactation stage-dependent changes in levels of lymphocyte subpopulations in milk. Flow cytometric assay was used to identify and assay lymphocyte subpopulations in bovine milk at different stages of lactation. Lymphocyte subpopulations in mammary secretions of dairy cows change during the lactation cycle. In involuting glands (dry gland), approximately 80-90% of lymphocytes were CD2+ T cells. The proportion of CD2+ T cells, however, decreased to approximately 50% at the colostral stage an fluctuated between 50 to 60% in normal (mature) milk. Throughout the lactation stages, less than 5% were B cells as identified by the monoclonal antibodies against CD21 and MHC class II antigens. Subset analysis showed, however, that the proportion of CD5+ T cells decreased from 90% in involuting gland secretions to 75% in colostrum (peripartum stage), and to approximately 40-50% in the normal (mature) milk, CD4+ T cells constituted between 45 to 55% of lymphocytes in the dry gland secretion but decreased drastically at parturition and maintained at the level below 20% throughout normal lactation. In contrast, the proportion of CD8+ T cells in the dry gland secretion was low, between 30 to 40%, but increased steadily, in an inversely-related manner with that of CD4+ T cells, to approximately 40-50% at parturition and maintained at approximately 30-40% during the normal lactation stage thereafter. Two-color immunofluorescence study revealed further that practically all of the CD8+ cells in dry gland secretions were CD2+, and approximately 40% of them were CD5-. Throughout the lactation cycle, WC1+ gamma delta T cells comprised only 2 to 5% of lymphocytes in mammary secretions. T lymphocyte subpopulations change dynamically during stages of the lactation cycle. The selective migration of T lymphocyte subpopulations to and from the mammary gland, and their functional roles in the immune competence and regulation of the dam and sucklings remain to be elucidated.

Elevation of Lactoferrin Gene Expression in Developing, Ductal, Resting, and Regressing Parenchymal Epithelium of the Ruminant Mammary Gland

Accumulation of lactoferrin mRNA in mammary tissue from virgin, pregnant, lactating, and involuting ewes and cows was localized using 35S-labeled cRNA probes. Expression of lactoferrin was low in the glands of virgin animals. In the glands of animals in early pregnancy, very high expression occurred in the ducts and immature alveoli, but expression tended to decrease as the alveoli matured. In the lactating and involuting gland, expression was generally low or absent in actively secreting alveoli and high in alveoli that had an accumulation of vesicles in the lumen and secretory epithelium, which was indicative of stasis. Occasionally, expression of lactoferrin was seen in cells that appeared to be secretory, particularly in involuting glands. Lactoferrin mRNA was expressed not only at different sites from other milk protein genes, such as α-lactalbumin and αs1-casein, but also during different stages of mammary development, supporting the view that the expression of lactoferrin is regulated differently from that of other milk proteins. For all ewes and cows, lactoferrin mRNA was detected in the epithelial ducts of the mammary parenchyma and the teat in a gradient that increased in ducts nearer the teats. The expression of lactoferrin in the ductal epithelium close to the teat was consistent with the antibacterial role of lactoferrin.

Somatic cell counts in milk of Welsh-Mountain, Dorset-Horn and Chios ewes throughout lactation

During four experiments the somatic cell counts (SCC) of 2642 milk samples were determined. SCC were studied in the milk of healthy Welsh-Mountain and Dorset-Horn ewes (experiment I), healthy Chios ewes (experiment II), Welsh-Mountain ewes subjected to intramammary inoculation with Staphylococcus simulans (experiment III) or Dorset-Horn ewes similarly inoculated (experiment IV). SCC of less than 1.0 × 10 6 cells ml −1 were found in 98.1% of samples from confirmed healthy mammary glands and in 14.2% of samples from glands with confirmed subclinical mastitis. Milk samples obtained in the evening had up to 40% higher SCC than those obtained in the morning. SCC increased as lactation advanced. Breed and parity of ewes did not affect milk SCC. In involuting glands of ewes, whose lambs had been abruptly weaned 2 months after lambing, SCC were more than 1.0 × 10 6 cells ml −1. Macrophages were the predominant type among SCC. It is concluded that the value of 1.0 × 10 6 cells ml −1 is the upper limit of SCC in healthy ewes' milk and that non-infectious reasons affect SCC.

Insulin-like growth factor-I and insulin-like growth factor binding protein-3 inhibit involution of the mammary gland following lactation: Studies in transgenic mice

The role of the insulin-like growth factor system on mammary gland development and involution following pregnancy and lactation was studied. Transgenic mice expressing rat IGF-I and human IGFBP-3 specifically in the mammary gland tissue, were created using the whey acidic protein gene. Mammary gland development was normal in transgenic animals expressing either rIGF-I or hIGFBP-3. In contrast, involution of the gland was delayed in both groups of transgenic mice. Specifically, the number of apoptotic cells was less in the involuting glands of transgenic mice compared with control animals. These results confirm the hypothesis that the IGF system regulates mammary gland function.

Two-Dimensional Electrophoretic Analysis of Proteins of Bovine Mammary Gland Secretions Collected During the Dry Period

Proteins in mammary gland secretions, collected from Holstein cows during the dry period, were analyzed by preparative isoelectric focusing, followed by SDS-PAGE. Protein profiles changed throughout the dry period. Intact casein bands were present throughout the dry period, but in reduced proportions from d 7 after dry-off through d 7 prepartum. Breakdown fragments of casein were particularly apparent in secretions from d 7 to 21 of the dry period. A fragment of β-CN (–13 kDa) was identified in secretions collected on d –1 prior to dry-off through d 21 of the dry period. β-Lactoglobulin generally focused in a limited number of fractions, but, in the sample at d 21 prepartum, β-LG focused over a broad pH range. Lactoferrin was found in all fractions after isoelectric focusing, but specific degradation products of lactoferrin were apparent only at certain times during the dry period. This method is a valuable approach for separation of proteins from the complex mixtures found in mammary secretions during the dry period. A number of peptides identified by this method may be generated from breakdown of milk proteins in the mammary gland during the dry period and could have a role in the involuting gland.

Mucosal Pathology of the Nose and Sinuses: A Study in Experimental Maxillary Sinusitis in Rabbits Induced by Streptococcus Pneumoniae, Bacteroides Fragilis, and Staphylococcus Aureus

Unilateral maxillary sinusitis was experimentally induced in New Zealand White rabbits with Streptococcus pneumoniae serotype 3, Bacteroides fragilis NCTC 9343, and Staphylococcus aureus V8 in order to study possible differences in the inflammatory response of the sinus and nasal mucosa at different time-intervals during a 12-week period of infection. The initial sinus mucosal response, most pronounced in pneumococcal sinusitis, was characterized by leukocytosis, epithelial desquamation, and squamous cell metaplasia. Tissue reactions at later intervals included fibrosis of lamina propria, gland involution, polyp formation, and bone remodelling, and were most pronounced in S. aureus and B. fragilis sinusitis. The nasal mucosa was altered with a redistribution of goblet cells, development of polyps in the ethmoidal region, involution of Bowman's glands and locally, areas of degenerated olfactory sensory epithelium. These findings endorse that the degree of local pathology depends on the infecting microorganism's specific pathogenetic factors. However, local tissue factors guiding the cellular inflammatory proliferative and regenerative processes are also of fundamental importance for the type of pathological changes occurring in an infected nasal or sinus mucosa.

Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution.

Extracellular matrix (ECM) plays an important role in the maintenance of mammary epithelial differentiation in culture. We asked whether changes in mouse mammary specific function in vivo correlate with changes in the ECM. We showed, using expression of beta-casein as a marker, that the temporal expression of ECM-degrading proteinases and their inhibitors during lactation and involution are inversely related to functional differentiation. After a lactation period of 9 d, mammary epithelial cells maintained beta-casein expression up to 5 d of involution. Two metalloproteinases, 72-kD gelatinase (and its 62-kD active form), and stromelysin, and a serine proteinase tissue plasminogen activator were detected by day four of involution, and maintained expression until at least day 10. The expression of their inhibitors, the tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor-1, preceded the onset of ECM-degrading proteinase expression and was detected by day two of involution, and showed a sharp peak of expression centered on days 4-6 of involution. When involution was accelerated by decreasing lactation to 2 d, there was an accelerated loss of beta-casein expression evident by day four and a shift in expression of ECM-remodeling proteinases and inhibitors to a focus at 2-4 d of involution. To further extend the correlation between mammary-specific function and ECM remodeling we initiated involution by sealing just one gland in an otherwise hormonally sufficient lactating animal. Alveoli in the sealed gland contained casein for at least 7 d after sealing, and closely resembled those in a lactating gland. The relative expression of TIMP in the sealed gland increased, whereas the expression of stromelysin was much lower than that of a hormone-depleted involuting gland, indicating that the higher the ratio of TIMP to ECM-degrading proteinases the slower the process of involution. To test directly the functional role of ECM-degrading proteinases in the loss of tissue-specific function we artificially perturbed the ECM-degrading proteinase-inhibitor ratio in a normally involuting gland by maintaining high concentrations of TIMP protein with the use of surgically implanted slow-release pellets. In a concentration-dependent fashion, involuting mammary glands that received TIMP implants maintained high levels of casein and delayed alveolar regression. These data suggest that the balance of ECM-degrading proteinases and their inhibitors regulates the organization of the basement membrane and the tissue-specific function of the mammary gland.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of goitrogenesis, involution, and goitrogenic rechallenge on the clonogenic cell content of the rat thyroid.

A fraction of enzymatically monodispersed rat thyrocytes from untreated animals clonally proliferate into thyroid follicular units following transplantation into the subcutaneous fat pads of syngeneic recipients. During the induction of experimental goiters in rats either with 3-amino-1,2,4-triazole/iodine sufficient diet or KClO4/Remington low iodine diet, the clonogenic fractions of cells from aminotriazole goiters decreased to 1.9 x 10(-4) and KClO4 goiters to 9.8 x 10(-5) as compared to 5.8 x 10(-3) for cells from age-matched controls during the growth phase of goitrogenesis. With continued aminotriazole treatment after thyroid hyperplasia had ceased, the clonogenic fraction increased to 2.0 x 10(-3) while continued KClO4 treatment had little further effect. The changes in the clonogenic fraction induced by both regimens were reversed during involution; goitrogenic rechallenge of involuted glands led to changes in the clonogenic fraction similar to that noted during the initial challenge. The clonogenic fractions of cells from aminotriazole goiters were greater than that of cells from KClO4 goiters at all time points examined despite similar TSH levels in situ. We conclude that the rat thyroid contains a hierarchy of cells with different proliferative capacities and that the clonogenic thyrocytes possess many of the attributes of a stem-cell.

Aluminum metabolism and bone histology after kidney transplantation: a one-year follow-up study.

Parathyroid activity, bone aluminum (Al) metabolism, bone histology, and clinical bone disease were studied in 55 successfully grafted kidney recipients at transplantation (Tx) and after 1 yr of immunosuppression with a low dose corticosteroid and a high dose cyclosporin-A regimen. Symptoms of Al-related bone disease disappeared after Tx. Serum Al and stainable bone Al decreased. The rate of Al removal from the bone surfaces was independent of graft function (creatinine range, 62-415 mumol/L) and bone turnover. Osteoblast activity and bone formation rate increased, while mineralization lag time normalized. Indices of bone resorption remained elevated, indicating persisting hyperparathyroidism. Eighteen percent of the recipients had posttransplant hypercalcemia, most likely caused by incomplete involution of hyperplastic parathyroid glands, while 53% had normocalcemic hyperparathyroidism related to impaired graft function. Cortical thickness decreased, while cancellous bone volume remained stable after Tx; both indices correlated significantly at follow-up with their respective values at Tx. None of 46 radiologically examined recipients had aseptic bone necrosis 1 yr after Tx.

Antibodies to iron-regulated outer membrane proteins of coliform bacteria isolated from bovine intramammary infections

Expression of iron-regulated outer membrane proteins (OMP) by Escherichia coli and Klebsiella pneumoniae initially isolated from bovine intramammary infections (IMI) was investigated. Additionally, the presence of antibodies in bovine serum and mammary secretion directed against the iron-regulated OMP was examined. Outer membrane proteins were separated by sodium-dodecyl polyacrylamide electrophoresis. Detection of immunoglobulin G directed against OMP was by immunoblotting. All Gram-negative bacteria expressed iron-regulated OMP when grown in skim milk or trypticase soy broth plus iron chelator, α-α′-dipyridyl. Immunoglobulin G directed against the iron-regulated OMP, as well as the major OMP and several other proteins, was detected in serum and milk of lactating cows with or without Gram-negative bacterial IMI. Antibody against the iron-regulated OMP was detected also in colostrum, secretion from the involuted gland, and in newborn calf serum 4 days after ingesting colostrum.

Mammary epithelial cells, extracellular matrix, and gene expression.

The mammary gland is an appropriate system with which to study mechanisms that control gene expression during both growth and differentiation. It is one of the few tissues with a developmental potential after birth, since following the onset of pregnancy, epithelial cells in the mammary gland proliferate and differentiate into milk-secreting cells. A schematic cross- section of one of the ten mammary glands of the mouse shows the gross changes in structure that occur during progression of the gland in pregnancy (figure 1, upper panel), from the virgin state (V) to lactation (L). During pregnancy (P), certain epithelial cells lining the ducts (which themselves are embedded in stromal tissue) proliferate and form alveolar buds. By the time of parturition, cells in the alveoli become secretory and occupy the entire gland. This structural development is accompanied by the onset of lactogenic function, in which expression of milk proteins begins during pregnancy and culminates with the secretion of milk after parturition [1]. The steady-state levels of the mRNA for β—casein (figure 1, lower panel) illustrate this type of progression with no expression in the virgin state, some during pregnancy, and the highest levels at lactation. After weaning when the gland involutes, β—casein expression is down regulated (figure 1, lower panel, lane I).KeywordsMammary GlandMammary Epithelial CellMilk ProteinAlveolar StructureWhey Acidic ProteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Involution and redevelopment of the mammary gland in goats. Histological-histochemical studies after a lactation time of 8-10 months

7 chamois-coloured mountain goats were used to investigate histologically and histochemically the processes of involution and redevelopment of the mammary gland after a lactation period of 8 to 10 months. Tissue specimens were obtained by incision biopsy at drying-off two months prepartum and afterwards in intervals of 8-16 days up to parturition. The findings of this investigation were compared with results of two former investigations, in which involution and proliferation of the gland were studied separately, thus precluding an overlapping of the two processes. At drying-off, after an 8 to 10 month-lactation, sections indicative of active lactation occurred concomitantly with sections at various stages of involution, as well as early stages of redeveloping alveoli. At 16 days post drying-off, the sections indicating lactation at first examination were in a stage of maximum involution and transformation. Thus, the time required for involution was reduced by half compared to drying-off at peak lactation. At 32 days post drying-off, stages of proliferation predominated with only a few involuting glands. Specimens obtained thereafter contained only redeveloping glands. The histological and histochemical differentiation between areas of involution and those of proliferation may present difficulties during the mid-portion of the dry period. There appears to be an association between the length of the time interval from drying-off to parturition and the rate of tissue transformation in the caprine mammary gland; the rate is increased when the duration of the dry period is reduced.

Morphometric analysis of involuting bovine mammary tissue after 21 or 42 days on non-suckling.

Mammary gland involution was morphologically evaluated 21 or 42 d after prevention of suckling of one udder half in 10 crossbred beef cows. Parenchymal tissue was taken from lower, middle and upper zones of each quarter from the teat to the ventral body wall. Udder halves, trimmed of extraparenchymal tissue, were weighed and used for DNA determination. DNA content was reduced 50 and 64% after 21 and 42 d of involution. However, the percentage of tissue occupied by epithelium was similar in suckled and nonsuckled glands. Well-differentiated cells, typical of suckled glands, were rarely observed in nonsuckled glands. Alveolar structure was evident in nonsuckled glands, but the number of cells per alveolar cross-section was reduced (30 vs 22). Unlike in suckled glands, there was a marked gradation in classification of epithelial cells across zones in involuting glands. For example, nearly 10% of the epithelium was well-differentiated in the tissue from the upper zone, whereas no well-differentiated cells were found in the lower zones. Regression of the mammary parenchyma does not occur uniformly through the udder, so use of single biopsy to study involution should be avoided. Presence of alveoli after 42 d indicates that redevelopment of the udder with subsequent lactations is less dramatic than suggested from study of other species.

Microvasculature of normal and involuted mouse thymus. Light- and electron-microscopic study.

The spatial arrangement of blood vessels in the thymus of normal and hydrocortisone-injected mice was studied by light and electron microscopy. The thymus is supplied by one thymic artery which branches into arterioles as it enters the parenchyma. These, in turn, feed capillary networks in the cortex and in the medulla. Cortical networks at the periphery of the lobule form loops which return blood to postcapillary venules at the corticomedullary junction and in the medulla. There is no subcapsular venous drainage. The overall distribution of blood vessels in the involuted thymus is essentially the same as in the normal thymus but the pattern becomes irregular and the vessels are tortuous. The endothelium of the postcapillary venules is flat and surrounded by a wide perivascular space containing many lymphocytes. This space is delimited by basal laminae, on the one side by that of the abluminal surface of the venular endothelium, on the other side by that of a thin, sheet-like epithelial layer formed by cytoplasmic processes of reticular cells. The perivascular space is in continuity with the surrounding interstitial space via gaps in its epithelial sheet. It does not form continuous longitudinal channels along the venules, but is interrupted by epithelial trabeculae. There is no obvious difference in structure between the postcapillary venules and their perivascular space of normal and those of involuted thymuses. Lymphocytes are intercalated in the venular endothelium particularly in involuted glands. They are present in the perivascular space and in gaps of the outer epithelial sheet. These findings suggest that the postcapillary venules and the perivascular spaces may function as pathways for the migration of thymic lymphocytes into or out of the blood circulation.

Distribution of lysosomal enzymes within the non-secretory epithelium of the bovine mammary gland.

The levels of N-acetyl-beta-glucosaminidase (NAGase) and acid phosphatase were analysed in tissue from the teat and lactiferous sinus epithelia of dairy cows at different stages of lactation. Enzyme levels were lower in the epithelia of the teat sinus than of the lactiferous sinus. For both enzymes, levels were highest in tissue from post-partum glands, lowest in those from dry and mid-lactation glands. In involuting glands the levels were high, but not as high as the post-partum glands.

Changes in Triglyceride Fatty Acid Composition of Mammary Secretions During Involution

Changes in fatty acid composition were determined for fat triglycerides from mammary secretions of 5 Holstein cows during the first 31 d of the nonlactating period. Proportions of short-chain (4:0 to 8:0) and medium-chain (10:0 to 14:0) fatty acids declined by about 50% during the first 3 d of involution, whereas proportions of stearic (18:0) and oleic acids (18:1) increased during the same period. Little change in proportions of those fatty acids occurred after d 3 of involution. Palmitic acid (16:0) was unchanged in proportion during involution. Fatty acid composition of mammary secretion triglycerides changed rapidly in the early nonlactating period, which may reflect a specific decline in de novo fatty acid synthesis in the involuting gland.

In Vitro Growth of Mastitis Pathogens in Mammary Secretions of the Dry and Peripartum Periods

In vitro growth of Escberichia coli, Streptococcus uberis, and Staphylococcus aureus was measured in skimmed, cell-free secretions of bovine mammary glands collected on d 0, 7, 14, and 28 of the dry period; 12, 8, and 4 d before expected calving; and on d 0 and 14 postpartum. There were marked and nearly inverse changes in growth of Escherichia coli and Streptococcus uberis over the experimental period. Escherichia coli grew well in secretions of d 0 of the dry period and d 0 and 14 postcalving but poorly or not at all in secretions of the mid-dry period. In contrast, Streptococcus uberis grew well in secretions of the involuted gland and poorly in samples from day of dry off and day of calving. Growth of Staphylococcus aureus changed relatively little over the dry period, but there was reduction in growth at d 7 of the dry period. Changing ability of the secretion to support bacterial growth may influence changing susceptibility to infection, but for Streptococcus uberis, poor correspondence between changes in growth supported by the secretion and changes in the incidence of naturally occurring infection over the dry period indicates that other factors may be more important.

Direct toxic effect of Lodide in excess on Lodine-deficient thyroid glands: Epithelial necrosis and inflammation associated with lipofuscin accumulation

Involution of thyroid hyperplasia (induced by a low iodine diet and a goitrogen, propylthiouracil, PTU) was obtained in mice by administering a high or a moderate dose of iodide (HID or MID, respectively). In HID involuting glands, vasoconstriction was observed after 12 hr whereas necrosis and inflammation were very abundant as early as after 6 hr and maximal after 48 hr. They were not prevented by papaverine by which vasoconstriction was inhibited, but were inhibited by the continuation of PTU by which iodide oxidation and organification were inhibited. Lipofuscin inclusions in thyroid and inflammatory cells were always associated with necrosis. On the contrary, when involution was induced by MID or by HID + triidothyronine (T 3), or by T 3 alone, neither necrosis nor inflammation occurred and apoptosis was the only mode of cell deletion. No lipofuscin inclusion occurred. Our results demonstrate that iodide in excess, after being oxidized or organified, is directly toxic for iodine-deficient thyroid cells. The presence of lipofuscin suggests that its toxicity is mediated by lipid peroxidation, a consequence of production of free radicals in excess.