Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells

The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.     

Role of prolactin, growth hormone and insulin-like growth factor 1 in mammary gland involution in the dairy cow

Bovine mammary involution, an important process for subsequent lactations, is characterized by loss of epithelial cells by apoptosis, but its hormonal regulation is still not well defined. Prolactin (PRL) and growth hormone (GH) play a specific role on rat mammary gland apoptosis, through insulin-like growth factor 1 (IGF-1) and the IGF binding protein (IGFBP) system. The purpose of our investigation was to determine the possible role of PRL, GH, and IGF-1 on cell survival and on IGFBP-5 expression in the bovine mammary gland. Mammary gland explants were cultured in the presence of cortisol, 17beta-estradiol, progesterone, insulin, PRL, GH, and IGF-1 and with the same treatment but without PRL, GH or IGF-1, respectively. After 24 h of culture, we determined the level of apoptosis through evaluation of DNA laddering in the oligonucleosomal fraction and examined IGFBP-5 messenger RNA (mRNA) expression. The results show a high level of DNA laddering and an increase in IGFBP-5 mRNA content in mammary explants cultured in the absence of PRL, GH, or IGF-I with respect to explants treated with all hormones. Moreover, explants cultured in presence of PRL, GH, or IGF-I show a low level of DNA laddering and IGFBP-5 expression with respect to explants cultured without any hormones. These data demonstrate a relationship between levels of apoptosis and IGFBP-5 mRNA expression in the bovine mammary gland and confirm the involvement of this binding protein programmed cell death and its relationship with the main lactogenic hormones.     

Hormones, mammary growth, and lactation: a 41-year perspective

When I was a beginning graduate student 41 yr ago it had been established that estrogen caused mammary duct growth; a combination of estrogen and progesterone was required for lobule-alveolar development of the mammary glands; and prolactin and growth hormone were essential for mammary growth. In laboratory species exogenous prolactin, glucocorticoids, and estrogen would initiate secretion of milk provided the mammary glands had a well-developed lobule-alveolar system. It was not known with certainty that progesterone inhibited the process. For some species, prolactin and thyroxine had been shown to stimulate lactation, while glucocorticoids suppressed lactation. Definitive roles for growth hormone and insulin during lactation had not been established. Studies of hormonal control of mammary growth and function in cattle were few. In vitro methods to study hormonal regulation of the mammary glands were in their infancy. Quantitative measures of changes in mammary cell numbers and specific components of milk in response to hormones were rare. The concepts for quantification of hormone concentrations, hormone receptors, growth factors, and binding proteins in blood; hormonal regulation of nutrient partitioning; and hormonally induced mechanisms of action within mammary cells were waiting to be discovered. And eventually they were. However, lest we become too enamored with our current understanding of the hormones that control mammary growth and lactation, it remains a fact that the greatest physiological stimulus for milk yield is pregnancy, not some cocktail of exogenous hormones, growth factors, receptor agonists/antagonists, or gene therapies. Viva la mom!     

Hepatocyte growth factor exerts multiple biological functions on bovine mammary epithelial cells

The met proto-oncogene product Met is a member of the family of tyrosine kinase growth factor receptors, and hepatocyte growth factor/scatter factor (HGF/SF) has been identified as its only ligand. Bovine Met and HGF/SF have been recently cloned and their expression has been characterized in the mammary gland, but no data regarding the biological effects of this ligand/receptor couple in bovine mammary cells are yet available. We examined the role of HGF/SF and its receptor in a bovine mammary epithelial cell line (BME-UV). Expression of Met at the mRNA level in BME-UV mammary epithelial cells evaluated by real-time PCR was similar to the expression in MDCK cells, a widely used model for Met biology. Met expression in BME-UV at the protein level was confirmed by western blot. The analysis of some signal transductional pathways downstream from the Met receptor revealed that HGF/SF addition to BME-UV cells induced activation of the extracellular signal-regulated kinase ½ proliferative pathway and the Akt antiapoptotic pathway. The BME-UV cells treated with HGF responded with increased proliferation, cell scatter, and motility. Met activation by HGF induced degradation of the extracellular matrix and migration through matrigel coated transwells. Moreover, BME-UV cells included in a 3-dimensional matrix of collagen and treated with HGF developed tubular structures, reminiscent of the mammary gland ducts. These data indicate that HGF and Met might be important regulators of mammary gland growth, morphogenesis, and development in the bovine.     

Organization and growth of mammary epithelia in the mammary gland fat pad

Mammary gland development consists of a series of very highly ordered events involving interactions among a number of distinct cell types. An important aspect of mammary gland development is that the mammary gland consists of a fat pad of mesodermal origin into which epithelial cells of ectodermal origin proliferate. This proliferation of epithelial cells into the mammary fat pad is the subject of this review. The nature of the stroma into which epithelial cells proliferate is of considerable importance in determining the structure of the resulting gland. In mice, white adipose tissue appears to be required for normal mammary development. Transplantation of mammary epithelia to other types of stroma does not support epithelial growth or result in abnormal growth. To date, a synthetic substratum capable of mimicking white adipose tissue has not been developed. Although collagen gel cultures are generally considered superior to glass or plastic substratum in supporting near normal epithelial growth, the technique has not advanced to the point that the in vivo growth pattern is duplicated. Recent research on the generation of chimeric mammary tissue (by transplanting mammary epithelia from rats, cows, and women to the mammary fat pads of athymic nude mice) suggests that there are important species differences in the stromal requirements for mammary gland development. In particular, extensive and expansive growth of rat mammary tissue is observed in mouse mammary fat pads. However, the mouse mammary fat pad appears incapable of supporting expansive growth of bovine or human mammary epithelia. The reason for this difference is not clear. However, human and bovine mammary epithelia may require the presence of more fibrous (collagenous) tissue than rodent mammary epithelia for normal proliferation.     

Investigation of mouse mammary ductal growth regulation using slow-release plastic implants

Mammary ductal development in the mouse is now thought to depend on an interplay of locally produced (glandular) and systemic mammogens. A novel plastic implant material, ethylene vinyl acetate copolymer (Elvax 40P), capable of the slow-release of undenatured, bioactive molecules in situ, now enables treatment of small regions of the mammary gland for extended periods with hormones and growth factors. Here we describe results obtainable with this technique. Specifically, the classical mammogens, estrogen, growth hormone, and prolactin, as well as the nontraditional mammogens, epidermal growth factor and cholera toxin, were shown to stimulate ductal growth in zones around an implant in ovariectomized animals. The possibility that these observations reflect the existence of multiple mammogenic pathways is discussed.     

Spatial and temporal expression of insulin-like growth factor-I, insulin-like growth factor-II and the insulin-like growth factor-I receptor in the sheep fetal mammary gland

The mammary gland is an example of a tissue of epidermal origin that depends for the development of its characteristic morphology on underlying mesenchymal cells. The interaction between mesenchyme and epithelium appears to be mediated by polypeptide growth factors. In situ hybridization has been used to study, in the mammary gland of female sheep fetuses, the distribution of mRNA for the mammary mitogens, insulin-like growth factor (IGF)-I and IGF-II, and the IGF-I receptor, from 10 to 20 weeks of intrauterine life (term is approximately 22 weeks). At 10 weeks, secondary ducts had formed from the primary duct. By week 20, the gland had increased in volume and complexity, showing primitive lobules embedded in intralobular connective tissue disposed around main ducts. IGF-I and IGF-II mRNA were expressed in cells of the intralobular connective tissue underlying the epithelium, while the IGF-I receptor was expressed in epithelium. Quantitation by absorbance measurements showed that mRNA expression increased with pregnancy stage for IGF-I and IGF-II, but not significantly for the IGF-I receptor, and that IGF-II was more highly expressed than IGF-I. A role for the IGF system in mediating mesenchymal epithelial interactions in mammary development is indicated.     

A 100-Year Review: Mammary development and lactation

What is old is new again—and with respect to the study of the mammary development and function in dairy animals, the expression resonates. Many of the mammary and milk production questions raised in the early years of the Journal of Dairy Science apply today. To be sure, scientists have filled in many details regarding, for example, identification of hormones and growth factors important in the control of mammary growth, the onset of copious milk production at calving, and maintenance of lactation. Early years focused on identification and subsequent availability of classic mammogenic, lactogenic, and galactopoietic hormones (e.g., steroids, prolactin, and growth hormone). The advent of sensitive assays to measure concentrations of these hormones and, subsequently, myriad growth factors in blood, milk, and tissues, allowed creation of multiple hypotheses to explain mammary cell proliferation and regulation of function. It is also apparent that we understand many of the fundamentals of milk removal, milking frequency, milking management, and milk ejection for successful lactation. However, some questions remain. Are the principles that were identified when cows produced markedly less milk still valid for the high-producing cows of today and the future? What mechanism(s) explain the positive effects of early increased milking frequency on subsequent milk production? Can the persistency of lactation be improved (secretory cell number vs. secretory cell function) or does early management “program” future mammary development or productivity (epigenetics, immune responsiveness, other)? The explosion of tools and techniques (Southern and Northern blots, PCR, and the “-omics” revolution) has driven an almost overwhelming evaluation of cellular and molecular functions in the mammary gland and other tissues. One key may be the discovery of a “Rosetta stone” that will allow understanding of this mass of detailed information on gene expression, cell signaling, and so on. Many scientists can now better appreciate the difficulty of the dairy farmer seeking to process DHIA or Dairy Comp 305 data, milking data, weights, feeding reports, pedometer readings, or genomic evaluations to manage their operations.     

Variation among species in the endocrine control of mammary growth and function: the roles of prolactin, growth hormone, and placental lactogen

Prolactin, growth hormone, and placental lactogen form a family of structurally related hormones, which may have evolved from a common ancestral peptide. Prolactin and growth hormone are present in all mammals, but the biological activity associated with placental lactogen has been detected in only some groups. Attempts to detect placental lactogen using bioassay and radioreceptor assay are reported and have been unsuccessful in an insectivore (the shrew), a bat, an edentate (the armadillo), a lagomorph (the rabbit), several carnivores (dog, cat, ferret), perissodactyls (horse, zebra, rhino), and, within the artiodactyls, pigs. Placental lactogenic activity has been detected in primates (chimpanzee, orangutan), rodents (voles, Pinon mouse, guinea-pig, mara), and in numerous artiodactyls (llama, giraffe, several species of deer, antelope, gnu, gazelle, musk ox, cape buffalo, Barbary sheep, several sheep of the genus Ovis, goat, and cow). These results confirm and extend the work of others and are discussed in relation to the evolution of these hormones. In synergism with steroid and thyroid hormones, protein hormones of the prolactin and growth hormone family play a crucial role in stimulating the development of the mammary gland, the differentiation and function of mammary cells to secrete milk, and in the systemic adjustments in maternal metabolism in pregnancy and lactation. Studies in vitro have shown that mammary tissues from several species synthesize milk components in response to insulin plus adrenal corticoid plus prolactin. However, there are also species differences in minimal hormonal requirements for lactogenesis. In vivo, for example, rabbits will initiate or sustain lactation in response to prolactin alone, whereas sheep and goats require prolactin plus growth hormone plus adrenal corticoid plus thyroid hormone. Measurement of hormone concentrations in the plasma of pregnant animals shows considerable differences among species in the pattern of secretion of lactogenic hormones to bring about mammary development. A surge of prolactin secretion occurs at parturition but may not be essential in the initiation of lactation. The timing of progesterone withdrawal correlates well with lactogenesis in eutherian mammals, but species differ in the mechanisms at parturition which bring this about. Marsupials show a quite different pattern of suckling-induced lactation. In maintaining lactation the greatest contrast is between ruminants, in which growth hormone is of particular importance, and other mammals, in which reduction of prolactin secretion with bromocriptine rapidly suppresses milk synthesis and secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

IGFBP-5在乳腺发育过程中的生物学作用

细胞生长的调节主要由IGFs起作用,IGFBPs能够通过蛋白水解、翻译后修饰以及同细胞表面和细胞外基质的相互作用来加强或抑制IGFs的作用,从而调节细胞的增殖、存活和分化.IGFBPs也具有独立的作用方式.IGFBP-5与乳腺发育特别是乳腺退化关系密切,IGFBP-5的表达可能是细胞凋亡发生时的普遍事件,尤其是在乳腺退化的初始阶段.     

Genetic manipulation of the IGF-I axis to regulate mammary gland development and function

Insulin-like growth factor I (IGF-I) is known to regulate mammary gland development. This regulation occurs through effects on both cell cycle progression and apoptosis. Our laboratory has studied the IGF-I-dependent regulation of these processes by using transgenic and knockout mouse models that exhibit alterations in the IGF-I axis. Our studies of transgenic mice that overexpress IGF-I during pregnancy and lactation have demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation but has minimal effects during early lactation on milk composition or lactational capacity. In contrast, our analysis of early developmental processes in mammary tissue from mice carrying a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential anti-apoptotic effects. With both models, the effects of perturbing the IGF-I axis are dependent on the physiological state of the animal. The diminished ductal development that occurs in response to loss of the IGF-I receptor is dramatically restored during pregnancy, whereas the ability of overexpressed IGF-I to protect mammary cells from apoptosis does not occur if the mammary gland is induced to undergo forced involution. Data from our laboratory on the expression of IGF-signaling molecules in the mammary gland suggest that this effect of physiological context may be related to the expression of members of the insulin receptor substrate family.     

The cellular perspective on mammary gland development: stem/progenitor cells and beyond

Study of the mammary gland at the stem cell level is necessary for understanding mammary gland development. Knowledge of mammary gland development and growth is the first step toward formulating strategies to improve milk production. The success of these strategies requires an understanding of the dynamics of adult stem cells and their progeny in the development of the bovine mammary gland. The stem cell lineage pathway begins with adult stem cells and ends with the production of terminally differentiated cells. The progression of adult stem cells along the mammary gland stem cell lineage pathway requires the coordination of many events. One important event in this process is cell differentiation. This differentiation process evolves with a gradient appearance of cell organelles progressing from stem cells to terminally differentiated cells. To dissect differentiation, mechanisms that regulate stem cells to differentiate toward a particular cell fate must be identified. Ultrastructural characteristics assist in distinguishing cells in various stages of differentiation in the mammary gland cell lineage pathway. Cells in the lineage pathway can become either epithelial cells or myoepithelial cells. Epithelial cells function in the production and secretion of milk, whereas myoepithelial cells assist epithelial cells in milk secretion. This review focuses on current concepts regarding adult stem cells and the recent progress on bovine mammary gland stem/progenitor cell development and differentiation. Multistep strategies that incorporate manipulation of the mechanisms influencing lineage choices in the mammary gland will produce beneficial effects on mammary gland development and milk production.     

Approaches to the manipulation of mammary involution

Mammary involution is a gradual process that occurs following cessation of milking. Regression of mammary secretory tissue accompanies dramatic changes in secretion composition during the transition from lactation to involution. Conversely, rapid differentiation of secretory tissue and copious accumulation of colostrum occur as parturition approaches. The duration of the nonlactating period, mammary gland health, and secretory cell response to hormones influence subsequent lactational performance in most species. Manipulation of the bovine mammary gland in an attempt to hasten involution has been studied. The primary objective of these studies was to determine if hastened involution would decrease new intramammary infections during the early nonlactating period. Results of these studies have also led to a more fundamental understanding of events that occur during physiological transition of the mammary gland. Adequate regression, proliferation, and differentiation of mammary secretory epithelium during the nonlactating period of ruminants appear to be essential for maximal milk production during lactation. Factors that interfere with these mechanisms can adversely affect mammary function during the impending lactation. A greater understanding of these processes may provide new approaches for increasing milk production in dairy cattle.     

Quantitative estimates of mammary growth during various physiological states: a review

The parenchymal portion of the mammary gland is immature at birth and begins to grow at a faster rate than the whole body shortly before occurrence of puberty. This accelerated or allometric growth rate is maintained for several estrous cycles, then returns to a growth rate equal to general body growth. Allometric growth of the mammary gland returns at conception and continues in most species for a variable period after parturition. Elevated secretion of estradiol and progesterone throughout pregnancy drives the allometric mammary growth during pregnancy. However, mammary growth during lactation in cows is independent of ovarian secretions and prolactin. Mammary cell numbers during lactation eventually decline as milk production decreases. Concurrent pregnancy reduces mammary cell numbers during lactation, but during the dry period concurrent pregnancy markedly increases mammary cell numbers over those in nonpregnant animals. Dry periods that are short reduce the increments in mammary cell numbers, which normally occur during early stages of the next lactation. Because numbers of mammary epithelial cells are a major determinant of milk yield, understanding the mechanisms that stimulate mammary epithelial cell numbers has the potential to lead to new methods for increasing efficiency of milk production.     

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 tissue's 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.