Colin J. Wilde

Control of Milk Secretion and Apoptosis DuringMammary Involution

Lactation depends on regular suckling or milkingof the mammary gland. Without this stimulus, milksecretion stops and mammary involution is induced.Involution caused by abrupt cessation of milk removal is characterized by de-differentiation andapoptosis of mammary epithelial cells, the extent andtime course of the latter varying between species.Apoptosis is inhibited and milk secretion is restored by re-suckling, if milk stasis is of shortduration. Mammary involution and apoptosis also occurduring weaning, even in concurrently-pregnant animalswhen the interval between lactations is restricted, suggesting that tissue remodeling is essentialfor subsequent lactation. Declining milk production inruminants after peak lactation is also associated with,and probably results from, net cell loss by apoptosis. Involution and apoptosis arecontrolled by changes in systemic galactopoietic hormonelevels, and by intra-mammary mechanisms responsive tomilk removal. Milk stasis precipitated by litter removal or cessation of milking may involveintra-mammary control related to physical distension ofthe epithelium. Local control of apoptosis in rodentsduring weaning, and after peak lactation in dairyanimals, may be due to the actions of milk-bornesurvival factors or their inhibitors, and can bemanipulated by frequency of milk removal.

PROGRAMMED CELL DEATH DURING MAMMARY TISSUE INVOLUTION INDUCED BY WEANING,LITTER REMOVAL, AND MILK STASIS

Programmed cell death in mammary tissue was studied during natural weaning in lactating mice and after litter removal or milk stasis. All treatments stimulated mammary apoptosis, indicating that this process is an integral part of the tissues involution after lactation. Induction of apoptosis was slower in natural weaning than after litter removal but occurred earlier when mice were concurrently pregnant during natural weaning. Ipsilateral induction of apoptosis by milk stasis in teat‐sealed glands indicates that cell death is under local (i.e., intramammary) as well as endocrine regulation. Apoptosis detected by DNA laddering was associated with changes in expression of p53 and bax, two genes implicated in the regulation of cell death, and was accompanied by structural degeneration characteristic of mammary involution. Reciprocal changes in stromelysin mRNA, and that of its inhibitor TIMP‐2, suggested that this structural reorganisation was the result of coordinated changes in gene expression favouring proteolysis of the extracellular matrix.

Mammary cell changes during pregnancy and lactation

Abstract This review describes the changes in mammary secretory cell number and activity that occur during the lactation cycle of cows and goats. Magnetic resonance imaging has been used to measure mammary growth in goats in vivo and, in combination with determinations of cellular differentiation made in biopsy samples, this has allowed a complete picture of mammary development to be built up. Secretory tissue volume increases exponentially during gestation and early lactation through the combination of cellular hyperplasia (initially) and hypertrophy (later). Involution occurs gradually during declining lactation, due entirely to reduced cell number, and more rapidly after milking ceases. Secretory tissue volume is greater at the start of the second gestation than the first, and the rate of tissue growth is also greater during the later stages of this second gestation. Milk yield and secretory tissue volume are both greater in the second lactation than the first. The effects of new milk production strategies are described. Increased milking frequency enhances milk yield and also stimulates mammary cell activity and then number. Treatment with growth hormone (bovine somatotrophin or BST) also increases milk yield, and when the two treatments are applied together the yield effects are additive. BST accelerates the differentiative response to frequent milking and also increases secretory tissue mass.

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 animals 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 bats 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.

Mammary apoptosis and lactation persistency in dairy animals.

The decline in milk yield after peak lactation in dairy animals has long been a biological conundrum for the mammary biologist, as well as a cause of considerable lost income for the dairy farmer. Recent advances in understanding the control of the mammary cell population now offer new insights on the former, and a potential means of alleviating the latter. The weight of evidence now indicates that a change in mammary cell number, the result of an imbalance between cell proliferation and cell removal, is a principal cause of declining production. Further, it suggests that the persistency of lactation, the rate of decline in milk yield with stage of lactation, is strongly influenced by the rate of cell death by apoptosis in the lactating gland. Mammary apoptosis was first demonstrated during tissue involution after lactation, but has now been detected during lactation, in mammary tissue of lactating mice, goats and cattle. Those factors that determine the rate of cell death by apoptosis are as yet poorly characterized, but include the frequency of milking in lactating goats. Initial evidence suggests that nutrition also is likely to influence cell survival after peak lactation, an important factor being the degree of oxidative stress imposed by feed and the tissues ability to deal with, and prevent damage by, reactive oxygen species. Comparison of cows in calf or not pregnant during declining lactation also indicates a likely influence of reproductive hormones, with oestradiol and progesterone acting to preserve mammary ductal and alveolar integrity during the dry period, while allowing a degree of apoptosis and cell replacement. In each case, the molecular mechanisms controlling mammary cell survival (or otherwise) are as yet poorly defined. On the other hand, more persistent lactations are likely to benefit animal welfare through fewer calvings and by placing less emphasis on maximal production at peak lactation, and modelling of persistent lactation with longer calving intervals indicates their likely economic benefits. In these circumstances, there is considerable incentive to elucidate the determinants of mammary apoptosis, and the factors controlling the dynamic balance between cell proliferation and cell death in the lactating mammary gland.

Autocrine control in milk secretion.

The mammary gland is controlled at two levels in the body. The genetically controlled pattern of parental investment during lactation is modified by nutritional status and signalled to the mammary gland by the endocrine system (see Peaker 1989). The response to this strategic control of the rate of milk secretion is modulated by a tactical control mechanism operating within each mammary gland. The local intramammary mechanism responds to changes in the frequency or completeness of milk removal and acts to match the rate of milk secretion to the rate of milk removal by the young or, in dairy animals, by the milker. It is the local control of milk secretion by milk removal which has been uncovered in recent years, following the realization of the physiological significance of unilateral effects of frequent milking in goats (Linzell & Peaker 1971) and cows (Morag 1973) that is the subject of this brief review.

Feedback control of milk secretion from milk.

Extracellular storage allows biologically-active substances in milk to influence mammary function. Among these factors is one which regulates the rate of milk secretion acutely according to frequency or completeness of milk removal in each mammary gland. The active factor in goats milk has been identified by screening milk constituents for their ability to inhibit milk constituent secretion in tissue and cell culture bioassays, and found to be a novel milk protein. The proteins identified by bioassayin vitro, also inhibited milk secretion in lactating goats in a reversible, concentration-dependent manner. This protein, termed FIL (feedback inhibitor of lactation), acts by reversible blockade of constitutive secretion in the mammary epithelial cell. As the inhibitor is synthesized in the same epithelial cells, feedback inhibition is, therefore, an autocrine mechanism. FILs unusual mechanism of action also influences other aspects of mammary function. Acute disruption of mammary membrane trafficking is associated with downregulation of prolactin receptors and followed by a decrease in epithelial cell differentiation. Thus, in addition to acutely-regulating milk secretion, FIL may induce the adaptation in mammary cell differentiation which actsin vivo to sustain the secretory response to a sustained change in milk removal. In the long term, matching of milk output to demand is achieved by a change in mammary cell number. This developmental response is also local in nature. Whether it too is due to autocrine modulation by FIL of mechanisms influencing cell proliferation or survival, or elicited by another milk-borne factor, remains to be determined.

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 [32P]‐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. J. Cell. Physiol. 181:304–311, 1999.

Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling

Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.

Milk yield and mammary function in goats during and after once-daily milking.

Starting in week 5 of lactation, goats were milked unilaterally once daily for 4 1/2 weeks, then twice daily for 12 weeks and finally thrice daily for 2 weeks. The control gland was milked twice daily throughout. During less frequent milking, yield fell by 26%. There was a rapid and essentially complete recovery of milk yield on resumption of twice-daily milking, and five of the six goats subsequently responded to thrice-daily milking with a small increase in milk yield. Mammary function was assessed during each phase by determining enzyme activities and in vitro synthesis rates of lactose and casein in mammary biopsies. There was a trend towards reduced differentiation of secretory cells during infrequent milking, with very little recovery during twice-daily milking but complete recovery during thrice-daily milking. The differentiative state of the control gland remained unchanged throughout.