A career in lactation biology.

This introduction summarizes some of the contributions made by woman in the field of lactation biology and endocrine regulation of lactation. In particular, it highlights the career of Dr. Karen Plaut, who at the time of writing this introduction was Dean of the College of Agriculture at Purdue University. She was a trailblazer for women in lactation biology following in the footsteps on some influential women in the field. She describes some of the accomplishments made by women in the field and some of the opportunities for the future.

Circadian clocks and their role in lactation competence.

Circadian rhythms are 24 h cycles of behavior, physiology and gene expression that function to synchronize processes across the body and coordinate physiology with the external environment. Circadian clocks are central to maintaining homeostasis and regulating coordinated changes in physiology in response to internal and external cues. Orchestrated changes occur in maternal physiology during the periparturient period to support the growth of the fetus and the energetic and nutritional demands of lactation. Discoveries in our lab made over a decade ago led us to hypothesize that the circadian timing system functions to regulate metabolic and mammary specific changes that occur to support a successful lactation. Findings of studies that ensued are summarized, and point to the importance of circadian clocks in the regulation of lactation competence. Disruption of the circadian timing system can negatively affect mammary gland development and differentiation, alter maternal metabolism and impair milk production.

Effect of high-fat diet on secreted milk transcriptome in midlactation mice.

High-fat diet (HFD) during lactation alters milk composition and is associated with development of metabolic diseases in the offspring. We hypothesized that HFD affects milk microRNA (miRNA) and mRNA content, which potentially impact offspring development. Our objective was to determine the effect of maternal HFD on secreted milk transcriptome. To meet this objective, 4 wk old female ICR mice were divided into two treatments: control diet containing 10% kcal fat and HFD containing 60% kcal fat. After 4 wk on CD or HFD, mice were bred while continuously fed the same diets. On postnatal day 2 (P2), litters were normalized to 10 pups, and half the pups in each litter were cross-fostered between treatments. Milk was collected from dams on P10 and P12. Total RNA was isolated from milk fat fraction of P10 samples and used for mRNA-Seq and small RNA-Seq. P12 milk was used to determine macronutrient composition. After 4 wk of prepregnancy feeding HFD mice weighed significantly more than did the control mice. Lactose and fat concentration were significantly ( P < 0.05) higher in milk of HFD dams. Pup weight was significantly greater ( P < 0.05) in groups suckled by HFD vs. control dams. There were 25 miRNA and over 1,500 mRNA differentially expressed (DE) in milk of HFD vs. control dams. DE mRNA and target genes of DE miRNA enriched categories that were primarily related to multicellular organismal development. Maternal HFD impacts mRNA and miRNA content of milk, if bioactive nucleic acids are absorbed by neonate differences may affect development.

Does the circadian system regulate lactation?

Environmental variables such as photoperiod, heat, stress, nutrition and other external factors have profound effects on quality and quantity of a dairy cow's milk. The way in which the environment interacts with genotype to impact milk production is unknown; however, evidence from our laboratory suggests that circadian clocks play a role. Daily and seasonal endocrine rhythms are coordinated in mammals by the master circadian clock in the hypothalamus. Peripheral clocks are distributed in every organ and coordinated by signals from the master clock. We and others have shown that there is a circadian clock in the mammary gland. Approximately 7% of the genes expressed during lactation had circadian patterns including core clock and metabolic genes. Amplitude changes occurred in the core mammary clock genes during the transition from pregnancy to lactation and were coordinated with changes in molecular clocks among multiple tissues. In vitro studies using a bovine mammary cell line showed that external stimulation synchronized mammary clocks, and expression of the core clock gene, BMAL1, was induced by lactogens. Female clock/clock mutant mice, which have disrupted circadian rhythms, have impaired mammary development and their offspring failed to thrive suggesting that the dam's milk production was not adequate enough to nourish their young. We envision that, in mammals, during the transition from pregnancy to lactation the master clock is modified by environmental and physiological cues that it receives, including photoperiod length. In turn, the master clock coordinates changes in endocrine milieu that signals peripheral tissues. In dairy cows, it is clear that changes in photoperiod during the dry period and/or during lactation influences milk production. We believe that the photoperiod effect on milk production is mediated, in part by the 'setting' of the master clock with light, which modifies peripheral circadian clocks including the mammary core clock and subsequently impacts milk yield and may impact milk composition.

Lactation Biology Symposium: circadian clocks as mediators of the homeorhetic response to lactation.

The transition from pregnancy to lactation is the most stressful period in the life of a cow. During this transition, homeorhetic adaptations are coordinated across almost every organ and are marked by changes in hormones and metabolism to accommodate the increased energetic demands of lactation. Recent data from our laboratory showed that changes in circadian clocks occur in multiple tissues during the transition period in rats and indicate that the circadian system coordinates changes in the physiology of the dam needed to support lactation. Circadian rhythms coordinate the timing of physiological processes and synchronize these processes with the environment of the animal. Circadian rhythms are generated by molecular circadian clocks located in the hypothalamus (the master clock) and peripherally in every organ of the body. The master clock receives environmental and physiological cues and, in turn, synchronizes internal physiology by coordinating endocrine rhythms and metabolism through peripheral clocks. The effect of the circadian clock on lactation may be inferred by the photoperiod effect on milk production, which is accompanied by coordinated changes in the endocrine system and metabolic capacity of the dam to respond to changes in day length. We have shown that bovine mammary epithelial cells possess a functional clock that can be synchronized by external stimuli, and the expression of the aryl hydrocarbon receptor nuclear translocator-like gene, a positive limb of the core clock, is responsive to prolactin in bovine mammary explants. Others showed that 7% of genes expressed in breasts of lactating women had circadian patterns of expression, and we report that the diurnal variation of composition of bovine milk is associated with changes in expression of mammary core clock genes. Together these studies indicate that the circadian system coordinates the metabolic and hormonal changes needed to initiate and sustain lactation, and we believe that the capacity of the dam to produce milk and cope with metabolic stresses in early lactation is related to her ability to set circadian rhythms during the transition period.

Effects of transforming growth factor-ß on mammary remodeling during the dry period of dairy cows.

Mammary remodeling in dairy cows involves coordinated changes in stromal and epithelial tissue. Tissue remodeling is characterized by changes in cell proliferation, activation of fibroblasts into myofibroblasts, and changes in extracellular matrix content. Transforming growth factor ß-1 (TGF-ß1) increases differentiation of fibroblasts to myofibroblasts, regulates expression of extracellular matrix proteins and proteases, and has cell-type dependent effects on proliferation. The objective of this study was to determine whether TGF-ß1 treatment of mammary tissue from cows in late lactation and the dry period affects cell proliferation, expression of matrix metalloproteinase-3 (MMP-3) and fibronectin (FN), and the differentiation of fibroblasts into myofibroblasts that express smooth muscle α actin (SMA). Tissue was biopsied from 7 Holstein cows at 4 time points: late lactation, 1 wk after dry-off, 3 wk before expected calving, and 1 wk before expected calving. Explants of biopsied tissue were incubated for 2h in Waymouth's medium containing insulin, hydrocortisone, and 0 or 5 ng of TGF-ß1/mL; a subset of cultures was also incubated with bromodeoxyuridine to measure epithelial and stromal cell proliferation. Tissues were fixed, embedded in paraffin, sectioned, and stained by immunohistochemistry. Stage at biopsy had an overall effect on rate of epithelial and stromal cell proliferation, and TGF-ß1 treatment increased rate of bromodeoxyuridine incorporation more than 2-fold in both cell types at 1 wk after dry-off. The number of fibroblasts expressing SMA was 19% higher in the intralobular stroma at 1 wk after dry-off compared with that at 1 wk before expected calving, and the percentage of activated fibroblasts tended to be higher in tissue incubated with TGF-ß1. Biopsy stage had an overall effect on percentage area of epithelium expressing FN and MMP-3. Incubation with TGF-ß1 had no effect on percentage intralobular stroma area expressing FN or MMP-3. Effects of TGF-ß1 treatment were most apparent at 1 wk after dry-off, indicating that the first week of dry period may be an ideal target for testing effects in vivo.

Characterization of mammary stromal remodeling during the dry period.

During the dry period between successive lactations, the mammary gland of dairy cows undergoes extensive remodeling that is marked by phases of involution and mammogenesis. Changes in the mammary epithelium during the dry period have been well characterized; however, few studies have examined the changes that occur in stromal tissue. The objective of this study was to characterize changes that occur in mammary stroma during the dry period. Mammary biopsies were taken from 9 multigravid Holstein cows in late lactation, at 1 wk after dry-off, 3 wk before expected calving date, and 1 wk before expected calving date. Tissue was fixed in formalin, embedded in paraffin, and cut into 5-mum sections. Sections were stained with hematoxylin and eosin or with immunohistochemistry for expression of smooth muscle alpha actin (SMA), fibronectin, stromelysin-1 (MMP-3), transforming growth factor-beta1 (TGF-beta1), and TGF-beta receptor 2 (TGF-betaR2). Images of tissues were captured with light microscopy, and imaging software was used to measure intralobular stromal area, number of activated fibroblasts, as identified by expression of SMA, and percentage of intralobular stromal area expressing fibronectin, MMP3, TGF-beta1, and TGF-betaR2. Analyses of variance were conducted and statistical differences were based on the least squares means of biopsy stage. Number of activated fibroblasts was greater at 1 wk dry than at 1 wk before calving (2,720 vs. 1,800 cells/mm(2)), percentage intralobular stromal area was greater at 1 wk dry (32%) and 3 wk before calving (37%) than at 1 wk before calving (25%), and TGF-beta1 expression decreased 15% from late lactation to the dry period. The percentages of stromal area expressing fibronectin, MMP-3, and TGF-betaR2 and the percentage of myofibroblasts were not different across biopsy stages. These results support the concept that stromal expression of transforming growth factor-beta1 and fibroblast proliferation may be important for remodeling during the dry period.

Mammary epithelial cells treated concurrently with TGF-alpha and TGF-beta exhibit enhanced proliferation and death.

Transforming growth factor-alpha (TGF-alpha) stimulates while TGF-beta inhibits mammary epithelial cell growth, suggesting that when cells are treated concurrently with the growth factors their combined effects would result in no net growth. However, combined treatments stimulate proliferation and cellular transformation in several cell lines. The objective of this paper was to describe the effect of long-term (6 days) concurrent TGF-alpha and TGF-beta treatment on normal mammary epithelial cell growth pattern, morphology, and gene expression. Growth curve analysis showed that TGF-alpha enhanced while TGF-beta suppressed growth rate until Day 4, when cells entered lag phase. However, cells treated concurrently with both growth factors exhibited a dichotomous pattern of growth marked by growth and death phases (with no intermittent lag phase). These changes in growth patterns were due to a marked induction of cell death from Day 2 (16.5%) to Day 4 (89.5%), resulting in the transition from growth to death phases, even though the combined treated cultures had significantly more (P < 0.05) cells in S phase on Day 4. TGF-beta stimulated epithelial to mesenchyme transdifferentiation (EMT) in the presence of TGF-alpha, as characterized by increased expression of fibronectin and changes in TGF-beta receptor binding. Expression patterns of genes that regulate the cell cycle showed significant interaction between treatment and days, with TGF-beta overriding TGF-alpha-stimulated effects on gene expression. Overall, the combined treatments were marked by enhanced rates of cellular proliferation, death, and trans-differentiation, behaviors reminiscent of breast tumors, and thus this system may serve as a good model to study breast tumorigenesis.

Tissue-specific variation in expression of prolactin receptor gene subtypes in hypergravity-exposed rats.

Prolactin (PRL) effects are mediated by membrane receptors (PRLR) of which the long (PRLR-L) and short form (PRLR-S) predominate. Our objective was to compare the distribution pattern of PRLR-L and PRLR-S transcripts and their ratio in adipose (AD), liver (LV), mammary (MG) and pituitary (PG) tissues of stationary (SC, n = 8) and hypergravity (HG, n = 8) exposed periparturient rats. Pregnant rats were exposed to 2 g force from day 11 of gestation (G11) through post partum day 1 (P1). PRLR-L mRNA expression compared to PRLR-S was greater (P < 0.001) in AD, MG and PG but was lower (P < 0.001) in LV in both HG and SC animals at P1. The ratio of PRLR-L/PRLR-S mRNA in the AD, LV, MG and PG was not different between HG and SC rats. In summary, these data reveal that the hypergravity-induced downregulation of PRLR is not directly triggered by deranged distribution of PRLR isoforms.

Exogenous TGF-beta1 promotes stromal development in the heifer mammary gland.

The objectives of this study were to determine the local effects of transforming growth factor-beta1 (TGF-beta1) on mammary epithelial and stromal cell proliferation and expression of the TGF-beta1 responsive genes c-myc and fibronectin. A single slow-release plastic pellet containing 5 microg of TGF-beta1 and 20 mg of BSA was implanted in the parenchyma of the right rear quarter of the mammary gland of 9-mo-old prepubertal heifers. A control pellet containing 20 mg of BSA was implanted in the left rear quarter of each heifer. All heifers were treated with bromodeoxyuridine (BrdU) at 4, 12.5, and 22 h after the pellets were implanted to label proliferating cells. Two hours after the last BrdU injection, the animals were euthanatized, and their mammary glands were recovered. Proliferation of mammary stromal cells was significantly higher in TGF-beta1-treated quarters than in BSA-treated, control quarters (3.5 vs. 1.8% BrdU-positive cells). This result coincided with a lack of significant effect of TGF-beta1 on proliferation of mammary epithelial cells and apoptosis. By quantitative reverse transcriptase-polymerase chain reaction, we found that c-myc gene expression was unchanged after TGF-beta1 treatment, but fibronectin gene expression was increased 3-fold in TGF-beta1-treated quarters compared with BSA-treated, control quarters. Thus, we concluded that TGF-beta1 selectively acts on the stromal compartment of the bovine mammary gland by increasing cell proliferation and gene expression of the extracellular matrix protein fibronectin.

Persistency of adenoviral-mediated lysostaphin expression in goat mammary glands.

Gene therapy has great potential to enable synthesis of protein molecules in targeted cells of an animal. One application may be the production of antibacterial enzymes by the mammary gland as a means of preventing or treating mastitis. We have previously demonstrated that goat mammary cells are capable of producing lysostaphin, an antistaphylococcal enzyme, after being transduced in vivo with a recombinant adenoviral vector containing a modified lysostaphin gene (Ad-lys). The current study examined duration of expression, and antibody response to lysostaphin and the adenoviral vector. Following intramammary infusion into nonlactating goats (n = 4), recovery of transducible adenoviral vector in mammary secretions persisted for 11 d. Transducible vector was not detected in serum, saliva, urine, or feces. Peak lysostaphin concentrations (< 20 microg/mL) in mammary secretions of infused udders were detected approximately 1 wk postinfusion, and generally returned to undetectable levels after an additional 1 to 2 wk. The poor persistency of expression was likely due to the very potent immune response to both the adenovirus and the expressed lysostaphin. Serum IgG antibodies recognizing the adenoviral vector developed within 7 d of the infusion, and titers rose dramatically to greater than 1:1 x 10(5). Similar titers of serum IgG antibodies to lysostaphin developed in 3 goats, with more moderate titers in the fourth goat. The antibody response to lysostaphin was delayed by approximately 4 d in comparison to the response to the adenovirus. Serum IgG antibody profiles were reflected in mammary secretions. No IgA antibodies to adenovirus or lysostaphin were detected in sera or mammary secretion. We demonstrate that while the lysostaphin gene can be introduced to the mammary gland using an adenoviral-mediated gene transfer technique, the strong immune response that it provokes makes the approach unsuitable for combating mastitis.

Association between gravitational force and tissue metabolism in periparturient rats.

Recently, interest in mammalian reproduction and offspring survival in altered gravity has been growing. Because successful lactation is critical for mammalian neonate survival, we have been studying the effect of gravity metabolism. We have shown an exponential relationship between glucose metabolic rate in mammary tissue of periparturient rats and an increase in gravity load. In this study we showed that changes in mammary metabolic rate due to gravity force were accompanied by a decrease in glucose metabolism in adipose tissue and by a reduced size of adipocytes. We assume that these changes are likely due to changes in prolactin or leptin levels related to altered gravity load.

Effects of hypergravity on mammary metabolic function: gravity acts as a continuum.

Mammary metabolic activity in pregnant rats is significantly increased in response to spaceflight. To determine whether changes in mammary metabolism are related to gravity load, we exposed pregnant rats to hypergravity and measured mammary metabolic activity. From days 11-20 of gestation (G), animals were centrifuged (20 rpm; 1.5, 1.75, or 2.0 x gravity) or were maintained at 1 G. On G20, five rats from each group were removed from the centrifuge and euthanized. The remaining dams (n = 5/treatment) were housed at 1 G until parturition. After 2 h of nursing by the pups, the postpartum dams were euthanized (G22). Glucose oxidation to CO2 and incorporation into lipids was measured. Mammary glands from dams euthanized on G20 revealed a strong negative correlation between metabolic rate and increased G load. Approximately 98% of the variation in glucose oxidation and 94% of the variation in glucose incorporation into lipids can be accounted for by differences in G load. Differences in metabolic activity disappeared in the postpartum dams. When we combined previous data from the microgravity with hypergravity environments and plotted the ratio of mammary metabolic rate vs. G load, there was a significant exponential relationship (r2 = 0.99). These data demonstrate a remarkable continuum of response across the microgravity and hypergravity environments and support the concept that gravitational load influences mammary tissue metabolism.

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland.

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Glucocorticoids maintain the extracellular matrix of differentiated mammary tissue during explant and whole organ culture.

Mouse mammary whole organ culture (WOC) and explant culture of lactating tissue were used to investigate the mechanism by which glucocorticoids maintain secretory epithelium following lobuloalveolar development. The relative number of mammary epithelial cells expressing glucocorticoid receptors did not change with the loss of secretory epithelium during involution as demonstrated with competitive binding assays and immunohistochemistry for the glucocorticoid receptor. Furthermore, glucocorticoids did not inhibit AP-1 binding activity. However, Northern analysis demonstrated that genes associated with the breakdown of the extracellular matrix were not expressed in tissues cultured with glucocorticoids, in contrast to their upregulation during involution of mammary tissue cultured with insulin alone. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression was lowest in tissue cultured in the presence of glucocorticoids and increased 2.3-, 3.4-, and 9-fold when tissues were involuted in the presence of insulin (Ins) alone, Ins and hydrocortisone (Hyd) with 0. 005 mg/ml, or 0.01 mg/ml collagenase IV, respectively. These data indicate that glucocorticoids maintain mammary differentiation in part by inhibiting the turnover of basement membrane.

The effects of spaceflight on mammary metabolism in pregnant rats.

The effects of spaceflight on mammary metabolism of 10 pregnant rats was measured on Day 20 of pregnancy and after parturition. Rats were flown on the space shuttle from Day 11 through Day 20 of pregnancy. After their return to earth, glucose oxidation to carbon dioxide increased 43% (P < 0.05), and incorporation into fatty acids increased 300% (P < 0.005) compared to controls. It is unclear whether the enhanced glucose use is due to spaceflight or a response to landing. Casein mRNA and gross histology were not altered at Day 20 of pregnancy. Six rats gave birth (on Day 22 to 23 of pregnancy) and mammary metabolic activity was measured immediately postpartum. The earlier effects of spaceflight were no longer apparent. There was also no difference in expression of beta-casein mRNA. It is clear from these studies that spaceflight does not impair the normal development of the mammary gland, its ability to use glucose, nor the ability to express mRNA for a major milk protein.

Progesterone stimulates DNA synthesis and lobulo-alveolar development in mammary glands in ovariectomized mice.

The objective of this study was to determine whether sustained progesterone (P) use in the absence of estrogen could influence mammary development in mice. Three-week-old intact or ovariectomized mice were primed with subcutaneous (s.c.) cholesterol (C), estrogen (E), P, or estrogen and progesterone (E/P) together. Nine days after priming, mammary glands were removed and incubated as a whole organ in media supplemented with various combinations of lactogenic hormones. After 5 days in whole organ culture, glands were removed and end buds, alveolar buds and lobulo-alveoli were quantified. Glands from mice primed with C or E developed significantly less lobulo-alveoli than glands from mice primed with P or E/P. While the development was greater in animals treated with E/P compared to those treated with P, it was clear that P in the absence of E could still induce lobulo-alveolar development. We have shown in this paper that P, in the absence of E, can stimulate cell proliferation during priming. Subsequently, the P primed glands can differentiate in response to lactogenic hormones.

Effects of steroid hormone treatment on mammary development in prepubertal heifers.

The objective of this study was to determine the effects of steroid hormone implantation in heifer calves on the ability of mammary tissue to develop subsequently in organ culture. Twenty-four calves were paired by date of birth and assigned to groups (eight calves/group). At 4, 7, or 10 mo of age, calves were implanted subcutaneously (s.c.) with pellets containing cholesterol or cholesterol, 17 beta-estradiol, and progesterone for 9 or 18 d. The calves were euthanized and uteri and mammary glands were removed and weighed. Slices of mammary parenchymal tissue were incubated for 5 d at 37 degrees C in a 50% O2, 5% CO2 humidified atmosphere in Waymouth's 752/liter medium supplemented with insulin (5.0 micrograms/ml) or lactogenic hormone complex insulin (5.0 micrograms/ml), aldosterone (0.1 microgram/ml), hydrocortisone (0.1 microgram/ml), and prolactin (1.0 microgram/ml) in the presence or absence of epidermal growth factor (EGF) (0.06 microgram/ml) to promote lobulo-alveolar development. Tissue sections were stained and mounted on slides for morphologic and histologic analysis or prepared to evaluate expression of beta-casein mRNA. There were no morphologic differences in slices from calf mammary tissues despite age, steroid hormone priming, or hormones used in tissue culture. The 4-mo-old calves required steroid priming followed by incubation of the tissue slices with the lactogenic complex with or without epidermal growth factor to induce cytological changes associated with lactogenesis but did not express beta-casein mRNA. At 7 mo of age, steroid hormone priming was not necessary for induction of alveolar formation and secretion. Incubation of the tissue slices from 7-mo-old calves with the lactogenic complex was sufficient to induce alveolar formation and secretion. However, beta-casein mRNA was not expressed. At 10 mo of age, exposure of tissue from calves to the lactogenic hormones caused histologic changes reminiscent of the ability to secrete milk regardless of hormone priming. However, estrogen and progesterone priming was necessary before incubation of the tissue slices with the lactogenic hormones to induce beta-casein mRNA expression. When epidermal growth factor was added to the lactogenic hormone complex, beta-casein mRNA expression decreased. These data support the concept that there is a sequential development of responsiveness of the mammary gland to various hormones. By 10 mo of age, prepubertal heifers reach a stage of maturity where steroid hormone priming followed by incubation of tissue slices with the lactogenic hormones is sufficient to induce both structural and functional differentiation.

Estrogen affects development of alveolar structures in whole-organ culture of mouse mammary glands.

In order to determine if estrogen affects lobulo-alveolar development, and to determine if whole organ culture of mouse mammary glands is an appropriate model of estrogen effects on mammary development, mouse mammary glands were cultured in the presence or absence of phenol red and estrogen. Lobulo-alveolar development did not occur in vitro when phenol red was eliminated from culture medium. Lobulo-alveolar development was stimulated by 17 beta-estradiol in a dose dependent manner over a physiological range. These results support the hypothesis that estrogen plays a critical role in alveolar development in the mammary gland. In this report an in vitro model of mammary development is identified in which the effects of estrogen are morphologically described and quantified.