Effects of supplementing pregnant heifers with methionine or melatonin on the anatomy and other characteristics of their lateral hind claws.

Three groups of four primiparous Holstein-Friesian heifers were fed throughout pregnancy either a control diet or that diet supplemented with either 5 to 6 g per day of rumen-protected intestinally available methionine or 25 mg melatonin. They were euthanased three days after calving. The dietary supplements had no effect on the impression hardness or the concentrations of cysteine and methionine in samples of claw horn collected from a range of sites, or on the areas of erosion in the sole and heel. Significant differences were recorded for the hardness of the horn in the order wall >sole >heel. These differences were associated with higher concentrations of cysteine and lower concentrations of methionine in samples of horn from the dorsal wall than in samples from the prebulbar region of the sole. There were no significant differences attributable to the dietary supplements in the soft tissue anatomy of the solear dermis and epidermis.

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.

[How to recognize POAD? The diagnosis of peripheral occlusive arterial disease in the physician's office]. / Diagnostik der arteriellen Verschlusskrankheit: Mit Stethoskop, Doppler und Laufband der pAVK auf den Fersen.

Peripheral occlusive arterial disease (POAD) encompasses all stenotic and occlusive changes affecting the aorta and arteries supplying the extremities, and in 90% of the cases is due to atherosclerosis. In the majority of patients the lower extremities are involved, andonly every third patient has symptoms. Apart from its significance as an independent disease entity, POAD is also an important coincidence marker, in particular for coronary heart disease. For the diagnostic clarification of POAD, a stepped strategy is recommended, including clinical function testing, such as an exercise treadmill test. Doppler sonography and determination of the Doppler pressure index (ankle-brachial index) make possible a noninvasive accurate staging. Color-coded duplex sonography and imaging procedures provide further information, and are indispensable for establishing the indication for interventional measures.

Effects of time of year and reproductive state on the proliferation and keratinisation of bovine hoof cells.

Cell proliferation and protein synthesis (keratinisation) were measured in vitro in hoof biopsy samples taken from two groups of seven heifers, the first calving in the winter and the second in the summer. Both parameters were significantly higher in summer than in winter irrespective of the heifers' reproductive state. The mean (se) measure of the rate of protein synthesis was 199 (27) dpm/microg DNA/hour in summer and 4 (1) dpm/microg DNA/hour in winter, and the equivalent values for cell proliferation (measured as DNA synthesis) were 375 (56) dpm/microg DNA/hour and 17 (4) dpm/microg DNA/hour. Changes around parturition depended on the time of the year. In the winter-calving heifers, the rates of proliferation and keratinisation increased significantly after calving from 22.3 (7.2) to 70.4 (16.6) and from 2.1 (0.7) to 12.4 (2.8) dpm/microg DNA/hour, respectively. In the summer-calving heifers, proliferation decreased from 388.2 (91.0) to 66.7 (9.6) dpm/microg DNA/hour but the rate of keratinisation did not change. Lesion scores and locomotion scores deteriorated after parturition, especially in the winter-calving group. The hooves were harder in summer than winter but their hardness was not affected by the heifers' reproductive state.

Lactation failure in crossbred Sahiwal Friesian cattle.

Milk producers in Malaysia make extensive use of crossbred Sahiwal Friesian dairy cattle. These animals have, however, been found susceptible to lactation failure. A survey of cows in an experimental herd of F1 Sahiwal Friesian animals indicated that, in 30% of animals, milk yield decreased to negligible levels within the first 8 weeks post partum. Lactation failure was associated with a progressive increase in the amount of residual milk left in the udder after normal milking. By week 3 of lactation, residual milk volume was significantly greater than that in animals that, based on previous lactation history were not susceptible to lactation failure, and accounted for up to 30% of milk available at the morning milking. The cellular consequences of residual milk accumulation were evident in the activities of acetyl-CoA carboxylase, fatty acid synthetase and galactosyltransferase, key enzyme markers of cellular differentiation, which decreased in glands undergoing lactation failure and were lower than values measured in tissue of control cows. Mammary cell number, estimated by tissue DNA content, was also reduced in animals undergoing lactation failure. These indices of mammary development indicate that lactation failure is the result of premature involution in susceptible animals. Premature involution is a predictable consequence of progressive milk stasis in failing lactation, and attributable to an increase in autocrine feedback by inhibitory milk constituents. The progressive increase in residual milk is, on the other hand, unlikely to be attributable to impaired mammary development. Measurements of milk storage during milk accumulation showed no differences between control and lactation failure cows in the distribution of milk between alveolar and cisternal storage compartments. We conclude that lactation failure in Sahiwal Friesian cows is due to a failure of milk removal, and probably the result of an impaired milk ejection reflex rather than to the glands' milk storage characteristics.

Synthesis and distribution of cytokeratins in healthy and ulcerated bovine claw epidermis.

Keratinization of the epidermal cells of the bovine claw generates the horn that gives the tissue its mechanical strength. Disruption of keratinization is likely to have a detrimental effect on the strength and integrity of the horn, and could lead to solar lesions and lameness. As part of a wider investigation of the cell biological causes of lameness in dairy animals, we have compared keratin synthesis and distribution in healthy bovine claw tissue with those in hooves with solar ulcers. Protein synthesis was measured by [35S]-labelled amino acid incorporation in claw tissue explant cultures. [35S]-labelled protein synthesis was higher in tissue from diseased claws than in healthy claws, and highest at the ulcer site. The identity of proteins synthesised in vitro did not differ between healthy and diseased tissue. DNA synthesis indicative of cell proliferation was also elevated in diseased tissue. Immunoblotting after one- or two-dimensional electrophoresis showed cytokeratins (CK) 4, 5/6, 10 and 14 to be amongst those expressed in healthy claw tissue. The relative abundance of these keratins was not altered in healthy regions of ulcerated hooves, nor at the ulcer site, but CK16, not usually found in healthy tissue, was detected in the sole of diseased claws. CK5/6 and CK14 were shown by immunohistochemistry to be present in the basal epidermis of healthy tissue, whereas CK10 was found in supra-basal layers. In healthy tissue from ulcerated claws, this distribution was unaltered, but at the site of solar ulcers, CK5/6 and CK14 were each found in both basal and supra-basal epidermis. The study suggests that solar ulceration of the bovine claw is not associated with gross alteration in the keratin composition of the tissue, but causes abnormal distribution of cytokeratins, perhaps as a result of loss of positional cues from the basement membrane. Ulceration did, however, stimulate cell repair involving epidermal protein synthesis (including keratins), and keratinocyte proliferation.

Apoptotic cell death, bax and bcl-2 expression during sheep mammary gland involution.

Involution in ovine mammary tissue was studied by light and electron microscopy, and bax and bcl-2 protein distribution was examined by immunohistochemistry from the last day of lactation until the 8th day of drying off. The mammary gland alveoli were examined and the area of glandular epithelium was evaluated morphometrically. Regression of mammary gland epithelium by apoptosis was first identified 2 days after the end of lactation, and increased until day 8. Bax protein was detected throughout this period and was highest on the eighth day. A weak positive reaction for bcl-2 was observed only on days 1 and 8 after cessation of lactation. It is concluded that sheep mammary gland involution involves cell death by apoptosis and that bcl-2 gene family members are involved in the process.

Influence of microenvironment on mammary epithelial cell survival in primary culture.

Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix form multicellular structures termed mammospheres, in which cells and matrix become arranged around a central luminal space. In the presence of lactogenic hormones, cells within mammospheres become polarized, form tight intercellular junctions, and secrete milk proteins vectorially into the luminal space. This study examined the mechanism of lumen formation. Histological examination of developing mammospheres showed that cavitation was associated spatially and temporally with the appearance of fragmented nuclear material in apoptotic bodies, and with the presence of cells positively labeled by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [(32)P]-deoxynucleotide end-labeled genomic DNA by electrophoresis and autoradiography showed DNA laddering indicative of apoptosis. A transient increase in laddering coincided with both lumen formation and the presence of TUNEL-positive cells. Lumen formation, DNA laddering, and detection of TUNEL-positive cells were all accelerated when matrix composition was altered. They were also impaired coordinately when caspase inhibitor was present during the first two days of culture. Therefore, lumen formation in mammosphere cultures is due to selective apoptosis of centrally located cells. Mammosphere cavitation was accompanied by redistribution of matrix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also associated with a transient increase in insulin-like growth factor binding protein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propose that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocrine apoptotic factors.

Identification of cell types in the developing goat mammary gland.

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.

Modulation of mammary development and programmed cell death by the frequency of milk removal in lactating goats.

1. Unilateral changes in mammary cell number are elicited when one gland is milked more or less frequently than the contralateral gland in lactating goats. These changes were investigated using histochemical and immunocytochemical markers of mammary cell types, and the degree of mammary apoptosis was determined by end-labelling of fragmented DNA. 2. Histological analysis confirmed that unilateral cessation of milking initiated involution and cell loss preferentially in the unmilked gland. The presence of fragmented DNA and morphological characteristics consistent with apoptosis demonstrated that these changes in mammary cell number in unmilked glands were, in part, the result of programmed alveolar cell death. 3. De-differentiation of the remaining secretory cells to ductal epithelial cells occurred with an increase in staining of cytokeratin markers and decreased staining by peanut lectin and casein antisera. 4. Differential once- and thrice-daily milking of lactating goats was also associated with unilateral changes in mammary cell number and milk yield. Milk yield and alveolar size were reduced after 4 weeks of infrequent milking. The latter was due to the increased loss of secretory cells by apoptosis, as indicated by a higher degree of fragmented DNA laddering. 5. After 10 weeks of differential milking, a homogeneous secretory morphology, albeit with smaller alveoli, was maintained in thrice-daily milked glands. Once-daily milked glands possessed a heterogeneous composition of terminal structures, resulting in the simultaneous presence of secretory and involuting alveoli as well as resting ductules. 6. The differences in programmed cell death and mammary morphology between unmilked and twice-daily milked glands, and between once- and thrice-daily milked glands, suggests that mammary apoptosis is subject to modulation by intra-mammary mechanisms sensitive to the frequency of milk removal.

Control of milk secretion and apoptosis during mammary involution.

Lactation depends on regular suckling or milking of the mammary gland. Without this stimulus, milk secretion stops and mammary involution is induced. Involution caused by abrupt cessation of milk removal is characterized by de-differentiation and apoptosis of mammary epithelial cells, the extent and time course of the latter varying between species. Apoptosis is inhibited and milk secretion is restored by re-suckling, if milk stasis is of short duration. Mammary involution and apoptosis also occur during weaning, even in concurrently-pregnant animals when the interval between lactations is restricted, suggesting that tissue remodeling is essential for subsequent lactation. Declining milk production in ruminants after peak lactation is also associated with, and probably results from, net cell loss by apoptosis. Involution and apoptosis are controlled by changes in systemic galactopoietic hormone levels, and by intra-mammary mechanisms responsive to milk removal. Milk stasis precipitated by litter removal or cessation of milking may involve intra-mammary control related to physical distension of the epithelium. Local control of apoptosis in rodents during weaning, and after peak lactation in dairy animals, may be due to the actions of milk-borne survival factors or their inhibitors, and can be manipulated by frequency of milk removal.

Influence of torpor on milk protein composition and secretion in lactating bats.

In the pipistrelle bat (Pipistrellus pipistrellus), the metabolic load of lactation is not met to any significant extent by increased food intake or mobilization of body reserves, and aerial foraging accounts for most of the animal's energy expenditure even during lactation. Energy conservation must, therefore, play a critical role in maintaining lactation. The principal mechanism for energy conservation appears to be the bat's ability to enter torpor, but this may itself interrupt milk synthesis and secretion unless the pipistrelle mammary gland is adapted to counteract its effect. The effect of torpor on mammary tissue function was studied in mammary tissue explant cultures prepared in weeks 1-3 of lactation, when milk water yield was 0.20, 0.88, and 0.30 mL/d respectively. Protein synthesis measured by incorporation of radiolabeled amino acids was 44% lower (P < 0.001) in bat tissue explants cultured at ambient temperature (22 degrees C) compared with 37 degrees C. The reduction was similar to that observed in mouse mammary tissue (57%) and was unaffected by stage of lactation. Analysis of explant protein after [35S]methionine labelling showed the majority of proteins synthesised in culture to be milk proteins; it also demonstrated that the decrease in protein synthesis at ambient temperature was a general phenomenon: synthesis of both secretory and intracellular mammary proteins was reduced at the lower culture temperature. The results suggest that bat mammary tissue has no mechanism to counteract the effect of reduced body temperature and that periods of lactational torpor are likely to cause a pronounced diurnal variation in the rate of milk secretion.

Effect of endocrine and paracrine factors on protein synthesis and cell proliferation in bovine hoof tissue culture.

Laminitis is a major cause of lameness in dairy cattle, and is widely attributed to a defect in the horny tissue that gives the hoof its mechanical strength. Defective horn is associated with, and may be preceded by, impaired keratin deposition in the hoof epidermis. The cause of abnormal keratin deposition is not easily identified but, like epidermal keratinization in other tissues, is likely to be controlled by hormones and the paracrine action of locally produced growth factors. The hormonal regulation of keratin synthesis and cell proliferation in the bovine hoof was studied using tissue explants in organ culture. As the highest incidence of laminitis is in early lactation, the study focused on insulin, cortisol and prolactin, three hormones implicated in lactogenesis and galactopoiesis. Incubation of tissue explants for 24 h in medium containing insulin (10-5000 ng/ml) stimulated protein synthesis measured by incorporation of 35S-labelled amino acids. Histochemical examination showed that insulin binding co-localized with the site of protein synthesis. Insulin also stimulated DNA synthesis, an index of cell proliferation, which was measured by incorporation of [3H]methyl thymidine. Cortisol (10-5000 ng/ml) decreased protein synthesis, whereas prolactin (10-5000 ng/ml) had no significant effect on protein or DNA synthesis. Epidermal growth factor (10-200 ng/ml), a potent inhibitor of keratinization in other tissues, stimulated protein synthesis compared with untreated controls. Epidermal growth factor binding was located microscopically to the germinal and differentiating epidermal layers. SDS-PAGE and fluorography showed that the population of proteins synthesized in the presence of any hormone or growth factor combination did not differ from that in untreated controls and included the keratins involved in horn deposition. The results show that bovine hoof keratinization is under endocrine and growth factor control, and suggest that systemic changes in lactogenic hormones may act to inhibit keratin deposition.

Autocrine inhibition of milk secretion in the lactating tammar wallaby (Macropus eugenii).

The lactating tammar wallaby progressively alters the rate of secretion and composition of its milk to provide appropriate nutrition for the developing offspring, whose needs are signalled by changes in the pattern and efficiency of its sucking. Tammars are also capable of asynchronous concurrent lactation, when the mother provides a dilute milk for a newborn young permanently attached to the teat (phase 2A of lactation), and a concentrated milk from an adjacent mammary gland for a young-at-heel (phase 3). The relationship between suckling behaviour and milk secretion, and the ability of adjacent glands to function independently, suggests that milk secretion is controlled locally, within each mammary gland, by a mechanism sensitive to frequency and completeness of milk removal. To determine if tammar milk contains a factor able to control milk secretion, milk fractions have been screened in tissue and cell culture bioassays. A 6-30 kDa fraction of phase 3 whey was found to inhibit milk constituent synthesis and secretion in vitro, and inhibitory activity was associated with two discrete fractions obtained by anion exchange chromatography, which contained protein bands migrating anomalously at 66 kDa and 63 kDa in SDS-PAGE. These bands were recognised in Western blotting by antiserum raised against a bovine autocrine inhibitor of milk secretion. By the same criteria, milk secreted in phase 2B of tammar lactation, when milk secretion is low and suckling intermittent but less vigorous than phase 3, also contained a feedback inhibitor of milk secretion. The results indicate that, as in dairy animals, marsupial milk secretion is under local control through feedback inhibition by a milk protein, and raise the possibility that autocrine feedback may influence the transition from phases of low milk secretion (phase 2A, 2B) to a high rate in the final third phase of lactation.

Autocrine regulation of protein secretion in mouse mammary epithelial cells.

Milk secretion is under autocrine control by an inhibitory milk protein, named FIL (feedback inhibitor of lactation). Lactating mammary acini and epithelial cells cultured on reconstituted basement membrane (EHS matrix) with lactogenic hormones were used to study the characteristics of autocrine inhibition. FIL inhibited milk protein secretion in lactating acini, but not in epithelial cells on EHS matrix. The latter's insensitivity to FIL was due to formation of multicellular structures termed mammospheres, in which cell surrounded a central luminal space. Cell polarization, and the formation of tight intercellular junctions prevented FIL access to the apical cell surface, which faced the mammosphere lumina. When apical access was permitted either by incomplete mammosphere formation or EGTA treatment, FIL inhibited mammosphere protein secretion to the same extent as in lactating acini. The study shows that autocrine inhibition by FIL occurs specifically through interaction with the apical surface of the mammary epithelial cell, and suggests the presence of a FIL receptor on this, but not the basolateral cell membrane.

Local control of mammary development and function.

For the mother, lactation represents the final stage of an investment in her genetic material. Like any investment it is costly and, hence, it needs to be carefully controlled. To her offspring, lactation means survival, so it must happen at any cost. This apparent conflict is rationalized by the mother devolving some control to the offspring while retaining ultimate sanction herself. Part of this results from overt and more subtle influences of the presence of young on the mother's endocrine system, but an equally important part operates at each mammary gland to ensure that output is appropriate to the needs of the young, and no more. The young exert influence by removing milk, while the mother retains control by responding on an hour to hour basis to the presence of milk in the gland. Local control is inevitably most evident where secretion itself is concerned, but also operates to influence lactogenesis, gland development and, eventually, gland involution. This paper will review local control of mammary function, emphasising the important role played by an autocrine inhibitory protein, the feedback inhibitor of lactation.

Local control of the mammary gland.

Studies on increasing the frequency of milking in dairy animals have led to the uncovering of the mechanism by which tactical control of the rate of milk secretion is achieved locally within each mammary gland, against a strategic, systemic control by the hormones that maintain all glands in the secretory condition. Experiments in vivo established that the response is local, and were compatible with the hypothesis that milk contains an inhibitor of its own secretion which accumulates during storage within the lumen of the mammary gland and which acts in an autocrine manner on the secretory cells. Isolation of a protein, initially from goats' milk, called FIL (feedback inhibitor of lactation) has enabled, and is enabling, further studies to be done from the whole-animal down to the molecular level. Examples at the whole-animal level are: the effects of immunization against FIL on the rate of secretion; the concentration of FIL and the kinetics of its formation and breakdown; the importance of the internal structure of the mammary gland and the capacities of the alveolar and ductular storage regions in determining feedback inhibition; differences between individuals and species influencing the degree of control exerted by FIL in matching supply of milk to demand by the young. Other local control mechanisms at the onset and cessation of lactation, including mammary distension, are also discussed.

Autocrine regulation of milk secretion.

Mammary development and the rate of milk secretion are regulated by frequency and completeness of milk removal. This regulation occurs through chemical feedback inhibition by a milk constituent. Novel, immunologically related milk proteins able to perform this function have been isolated from caprine, bovine and human milk, based on their ability to inhibit milk constituent synthesis in mammary tissue and cell cultures, and to decrease temporarily milk secretion when added to milk stored in the mammary gland. Inhibition is concentration-dependent, suggesting that milk accumulation and removal is accompanied by cyclical changes in inhibitor accretion and depletion in milk. Feedback inhibition is an autocrine mechanism: the caprine inhibitor, termed FIL (feedback inhibitor of lactation) is synthesized by mammary epithelial cells in primary culture. Inhibition is by reversible blockade of the secretory pathway, an effect which, by down-regulating cell-surface hormone receptors, has longer-term consequences on epithelial cell differentiation. Treatment of goat mammary epithelial cell cultures with caprine FIL initially decreased milk protein secretion and subsequently reduced milk protein messenger RNA abundance. Thus the actions of a single milk constituent can bring about both the effect of milking frequency on milk secretion rate and a sequential modulation of cellular differentiation which acts to sustain the secretory response. Long-term regulation, through changes in galactopoietic hormone receptors, also provides an efficient mechanism for integrating acute intramammary regulation of lactation with strategic endocrine control of mammary tissue development.

Programmed cell death in bovine mammary tissue during lactation and involution.

Cessation of milk removal causes mammary tissue involution, which in rodents is characterized by extensive tissue degeneration and loss of the majority of luminal epithelial cells by apoptosis. In contrast, bovine mammary tissue shows little histological evidence of tissue remodelling between lactations. In this study, we combined histology with molecular biology to examine the cellular and molecular changes in bovine mammary tissue on cessation of milking. Oligonucleosomal laddering of genomic DNA extracted from lactating tissue indicated that a proportion of cells were dying by apoptosis. This was confirmed by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labelling of apoptotic cells in lactating tissue sections (TUNEL). One week after cessation of milking, alpha-lactalbumin and alpha S1-casein messenger RNA (mRNA) abundance had decreased by 99 and 85%, respectively, whereas lactoferrin mRNA had increased 20-fold. Drying off was also accompanied by an increase in oligonucleosomal laddering of genomic DNA, and by an increase in the proportion of TUNEL-positive cells, which were localized preferentially in regions where alveolar structure had deteriorated. Therefore, termination of lactation was associated with partial loss of the mammary cell population and dedifferentiation of the remainder.