Mammary Function Research Articles

Immune cell regulators in mouse mammary development and involution1

Dramatic changes in cell composition and function occur in the mammary gland during a pregnancy-lactation-involution cycle. We investigated the transcriptional changes associated with these biological events by using microarray analysis and identified the critical genes involved by using genetically modified mice. Two surprising findings arose from these studies. First, the microarray data showed that postlactational regression was associated with an acute phase inflammatory response, in addition to cell death. Conditional deletion of signal transducer and activator of transcription (Stat)3 or the nuclear factor-kappaB regulatory kinase inhibitor of kappa B kinase beta resulted in a failure of cell death induction during involution, an indication that these signaling pathways are essential mediators of the involution process. Both Stat3 and nuclear factor-kappaB have been shown to regulate acute phase gene expression in addition to apoptosis regulators. Four distinct transcriptional profiles are present in the first 4 d of involution, whereas there are 3 in lactation. At the peak of lactation (i.e., d 10 in mouse), more than 400 genes reach their maximum expression before declining dramatically in the first 12 h of involution. A reciprocal pattern was observed for more than 500 genes that were specifically upregulated within the first 12 h of forced involution. We are now investigating the role of a subset of these genes in involution. We also uncovered a role for genes normally associated with immune cell signaling in the differentiation of luminal mammary epithelial cells during pregnancy. Genetic deletion of the transcription factor Stat6 resulted in delayed development during pregnancy, and this phenotype was recapitulated in mammary tissue from IL-4 and IL-13 doubly deficient mice. Furthermore, we showed that mammary epithelial cells secrete T-cell regulatory cytokines. T-helper type 1 cytokines, such as interferon-gamma and IL-12a, are secreted by undifferentiated mammary epithelial cells, whereas T-helper type 2 cytokines, including IL-4 and IL-13, are secreted by differentiated cells. This unexpected finding demonstrates a role for immune cell signaling in mammary epithelial cell fate and function.

Notch Signaling Regulates Mammary Stem Cell Function and Luminal Cell-Fate Commitment

The recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.

ASAS Centennial Paper: Lactation biology for the twenty-first century

Knowledge of general aspects of mammary gland function, including metabolic pathways and hormonal regulation of mammary gland development and lactation, in livestock species was obtained several decades ago. As basic biological information of growth factor action, apoptotic mechanisms, and signal transduction events has exploded, the mouse became the model of choice for studying fundamental mechanisms regulating mammary function. A complete sequenced genome also has made the mouse amenable for studies of mammary gene network expression. Advances in molecular biology techniques currently allow researchers to genetically modify mice to either overexpress or completely lack specific genes, thereby studying their function in an in vivo setting. Furthermore, the use of mammary-specific promoters has allowed genes related to mammary gland function to be eliminated from the mammary gland in a developmental and tissue-specific manner. These studies have demonstrated the complexity that underlies mammary gland development and function in rodents and may provide insight into the mechanisms that ultimately allow the ruminant or swine mammary gland to function in a coordinated fashion throughout puberty, pregnancy, lactation, and involution. The challenge facing animal scientists is how to obtain similar information in much larger and expensive livestock. One possible approach is to manipulate gene expression in vitro using mammary cell culture models derived from domestic animals (e.g., genes can be "knocked down" using small interfering RNA approaches). Ultimately, major advances in understanding lactation biology may come from coupling basic mechanistic information with functional genomics, proteomics, and metabolomics approaches. A strong foundation in bioinformatics will also be required to optimize use of these new technologies. Stem cell biology also represents an exciting area in the next decade that holds promise for improving lactation efficiency. Strong training of our future scientists in these areas should facilitate livestock-focused mammary gland research that will allow basic information to be gained at unprecedented amounts, ultimately leading to optimization of livestock production.

HEXIM1 Regulates 17β-Estradiol/Estrogen Receptor-α–Mediated Expression of Cyclin D1 in Mammary Cells via Modulation of P-TEFb

Estrogen receptor alpha (ERalpha) plays a key role in mammary gland development and is implicated in breast cancer through the transcriptional regulation of genes linked to proliferation and apoptosis. We previously reported that hexamethylene bisacetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound ERalpha and bridges a functional interaction between ERalpha and positive transcription elongation factor b (P-TEFb). To examine the consequences of a functional HEXIM1-ERalpha-P-TEFb interaction in vivo, we generated MMTV/HEXIM1 mice that exhibit mammary epithelial-specific and doxycycline-inducible expression of HEXIM1. Increased HEXIM1 expression in the mammary gland decreased estrogen-driven ductal morphogenesis and inhibited the expression of cyclin D1 and serine 2 phosphorylated RNA polymerase II (S2P RNAP II). In addition, increased HEXIM1 expression in MCF-7 cells led to a decrease in estrogen-induced cyclin D1 expression, whereas down-regulation of HEXIM1 expression led to an enhancement of estrogen-induced cyclin D1 expression. Studies on the mechanism of HEXIM1 regulation on estrogen action indicated a decrease in estrogen-stimulated recruitment of ERalpha, P-TEFb, and S2P RNAP II to promoter and coding regions of ERalpha-responsive genes pS2 and CCND1 with increased HEXIM1 expression in MCF-7 cells. Notably, increased HEXIM1 expression decreased only estrogen-induced P-TEFb activity. Whereas there have been previous reports on HEXIM1 inhibition of P-TEFb activity, our studies add a new dimension by showing that E(2)/ER is an important regulator of the HEXIM1/P-TEFb functional unit in breast cells. Together, these studies provide novel insight into the role of HEXIM1 and ERalpha in mammary epithelial cell function.

Epidermal growth factor and parathyroid hormone-related peptide mRNA in the mammary gland and their concentrations in milk: effects of postpartum hypoxia in lactating rats.

The physiological adaptations of the neonatal rat to hypoxia from birth include changes in gastrointestinal function and intermediary metabolism. We hypothesized that the hypoxic lactating dam would exhibit alterations in mammary gland function leading to changes in the concentration of milk peptides that are important in neonatal gastrointestinal development. The present study assessed the effects of chronic hypoxia on peptides produced by the mammary glands and present in milk. Chronic hypoxia decreased the concentration of epidermal growth factor (EGF) in expressed milk and pup stomach contents and decreased maternal mammary gland EGF mRNA. The concentration of parathyroid hormone-related protein (PTHrp) was unchanged in milk and decreased in pup stomach contents; however, mammary PTHLH mRNA was increased by hypoxia. There was a significant increase in adiponectin concentrations in milk from hypoxic dams. Chronic hypoxia decreased maternal body weight, and pair feeding normoxic dams an amount of food equivalent to hypoxic dam food intake decreased body weight to an equivalent degree. Decreased food intake did not affect the expression of EGF, PTHLH, or LEP mRNA in mammary tissue. The results indicated that chronic hypoxia modulated mammary function independently of hypoxia-induced decreases in maternal food intake. Decreased EGF and increased adiponectin concentrations in milk from hypoxic dams likely affect the development of neonatal intestinal function.

Effects of uteroplacental insufficiency and reducing litter size on maternal mammary function and postnatal offspring growth

Human intrauterine growth restriction is often associated with uteroplacental insufficiency and a decline in nutrient and oxygen supply to the fetus. This study investigated the effects of uteroplacental insufficiency and intrauterine growth restriction (Restricted) or reducing litter size for normally grown pups (Reduced Litter) on maternal mammary development and function, milk composition, offspring milk intake, and their resultant effects on postnatal growth. Uteroplacental insufficiency was surgically induced by bilateral uterine vessel ligation on day 18 of gestation in the Wistar Kyoto rat. At birth, a group of sham control rats had their litter size reduced to five (Reduced Litter) to match that of the Restricted group. Cohorts of rats were terminally anesthetized on day 20 of gestation or day 6 of lactation, and a third group was studied throughout lactation. Restricted pups had a lower birth weight (by 16%) and litter size (by 36%) compared with controls, as well as reduced mammary parathyroid hormone-related protein content and milk ionic calcium concentrations associated with reduced total pup calcium. Restricted dams with lower circulating progesterone experienced premature lactogenesis, producing less milk per pup with altered composition compared with controls, further slowing growth during lactation. Reducing litter size of pups born of normal birth weight (Reduced Litter) was associated with decreased pup growth, highlighting the importance of appropriate controls. The present study demonstrates that uteroplacental insufficiency impairs mammary function, compromises milk quality and quantity, and reduces calcium transport into milk, further restraining postnatal growth.

The Eighth International Workshop on the Biology of Lactation in Farm Animals: Introduction

This volume represents the span of invited papers presented at the Eighth International Workshop on the Biology of Lactation in Farm Animals (BOLFA) held in Pirassununga, Brazil, August 21 to 23, 2006. That meeting was the first BOLFA workshop not held as a companion to a European Association for Animal Production (EAAP) or American Society of Animal Science (ASAS) meeting, and the first outside of North America or Europe. Despite these venue departures from the norm, the workshop format of interactive discussion on topics related to milk production in farm animal species was unchanged. This volume begins with a paper from Antonella Baldi’s group (Baldi et al., 2008) that provides a historical perspective of previous BOLFA workshops with an emphasis on nutritional factors that influence mammary function. It sets the stage for the later papers that consider aspects of milking management and mastitis treatment to improve milk yield in farm animal species. The next series of contributions focus on environmental and management influences on mammary function. Dahl (2008) summarizes recent developments in the study of photoperiod exposure during the dry period and develops a hypothesis that prolactin-mediated events influence mammary cell turnover and metabolic gene expression in the liver. These findings lead to a discussion of similarities between photoperiodic and thermal influences on prolactin-related responses, and the potential for a common pathway for environmental effects. Without competent milk ejection, lactation fails. More thorough understanding of what stressors lead to disruptions in the milk ejection reflex, or inhibit it completely, will allow for the development of new meth-

Mammary gland leptin in relation to lactogenesis in the periparturient dairy goat

The role of leptin in development of mammary gland secretory function was studied during the periparturient period in dairy goats. Changes in mammary leptin and leptin receptor (short cytoplasmic form) expression were evaluated by real-time RT-PCR and related to changes in milk and plasma leptin concentrations from 5 weeks pre-partum to 7 weeks post-partum. It was further investigated if systemic leptin concentration in the neonate is affected by milk leptin intake. We found no evidence of accumulation of leptin in colostrum pre-partum. Pre- and post-partum milk leptin concentrations were similar, but interestingly, leptin increased markedly post-partum to reach a peak 2 days after parturition ( P = 0.01). Plasma leptin concentrations were higher pre-partum than post-partum ( P < 0.05), decreased at parturition and arterio-venous differences for leptin across the mammary gland were at no time significantly different from zero. Plasma leptin does thus not appear to be involved in regulation of milk leptin. Both leptin and the short cytoplasmic form of its receptor were expressed in the mammary gland. The highest leptin receptor expression was observed pre-partum and decreased post-partum. Expression of leptin was relatively constant except for an increase in the last week prior to parturition. Our results can neither confirm nor exclude that increased leptin and/or leptin receptor synthesis is responsible for regulation of milk leptin in the early post-partum period. The physiological significance of the distinct peak in milk leptin 2 days post-partum needs to be understood. We did not find evidence that milk leptin can be absorbed, and thus play a role in systemic regulation, of the neonatal goat.

Breastfeeding After Reduction Mammaplasty Using Different Techniques

This study reviewed mammary glandular function and breastfeeding after reduction mammaplasty performed via four different surgical techniques. Patients who underwent this procedure were asked to answer questions concerning the birth of a child, natural breastfeeding, and the reasons why natural breastfeeding was not performed or was interrupted. Between 1992 and 2001, 368 reduction mammaplasties were performed in the Department of Plastic Surgery at the "La Sapienza" University of Rome. After reduction mammaplasty, 105 patients had a child and were enrolled in the study. Breastfeeding data were compared with data from hospital records at the time of surgery in terms of patient age, reduction mammaplasty technique, sensitivity of the nipple-areola complex after the operation, and proportion of the gland removed. Maternal breastfeeding was considered to have occurred if it lasted more than 3 weeks and was not accompanied by any nutritional supplements. Babies were breastfed by 60.7% of the patients who underwent a superior pedicle reduction mammaplasty, by 43.5% of those who underwent an inferior pedicle reduction mammaplasty, by 48% of those who underwent a medial pedicle reduction mammaplasty, and by 55.1% of those who underwent a lateral pedicle reduction mammaplasty. The findings demonstrate that conservative reduction mammaplasty techniques supported by medical and paramedical staff permit subsequent breastfeeding. In particular, the best outcomes resulted from superior pedicle reduction mammaplasty. Skilled execution of the surgical technique is mandatory to guarantee adequate vascularization and sensitivity of the nipple-areola complex and to spare as many of the glandular ducts and lobules as possible.

Activin and estrogen crosstalk regulates transcription in human breast cancer cells

Activin is a member of the transforming growth factor beta superfamily that regulates mammary cell function during development, lactation, and in cancer. Activin slows the growth of breast cancer cells by inducing G(0)/G(1) cell cycle arrest. Estrogen is a steroid hormone that stimulates the proliferation of mammary epithelial cells in development and oncogenesis. The crosstalk between estrogen and activin that regulates activin ligand expression, activin and estrogen signal transduction, and cell cycle arrest was investigated in this study. Estrogen antagonized activin-dependent production of plasminogen activator inhibitor 1 (PAI-1) mRNA, while activin repressed estrogen-dependent transcription of trefoil factor 1. The repression of estrogen signaling by activin was recapitulated using a simple estrogen response element-luciferase construct and was enhanced in the presence of overexpressed estrogen receptor alpha (ERalpha). In contrast, estrogen-mediated repression of activin signaling could not be recapitulated on a simple CAGA Smad-binding element but did inhibit the short PAI-1 promoter, p3TP-luciferase, especially when ERalpha was overexpressed. Repression of both estrogen- and activin-regulated transcription was found to be ligand induced and Smad3 dependent. In addition to transcriptional repression, estrogen also reduced the amount of activin B mRNA and protein produced by MCF7 breast cancer cells. These studies demonstrate the importance of activin and estrogen crosstalk during mammary cell growth and cancer initiation.

The Tammar Wallaby and Fur Seal: Models to Examine Local Control of Lactation

Mammary development and function are regulated by systemic endocrine factors and by autocrine mechanisms intrinsic to the mammary gland, both of which act concurrently. The composition of milk includes nutritional and developmental factors that are crucial to the development of the suckled young, but it is becoming increasingly apparent that milk also has a role in regulating mammary function. This review examines the option of exploiting the comparative biology of species with extreme adaptation to lactation to examine regulatory mechanisms that are present but not readily apparent in other laboratory and livestock species. The tammar wallaby has adopted a reproductive strategy that includes a short gestation (26 d), birth of an immature young, and a relatively long lactation (300 d). The composition of milk changes progressively during the lactation cycle, and this is controlled by the mother and not the sucking pattern of the young. Furthermore, the tammar can practice concurrent asynchronous lactation; the mother provides a concentrated milk high in protein and fat for an older animal that is out of the pouch and a dilute milk low in fat and protein but high in carbohydrates from an adjacent mammary gland for a newborn pouch young. This phenomenon suggests that the mammary gland is controlled locally. The second study species, the Cape fur seal, has a lactation characterized by a repeated cycle of long at-sea foraging trips (up to 28 d) alternating with short suckling periods of 2 to 3 d ashore. Lactation almost ceases while the seal is off shore, but the mammary gland does not progress to apoptosis and involution, most likely because of local control of the mammary gland. Our studies have exploited the comparative biology of these models to investigate how mammary function is regulated by endocrine factors, and particularly by milk. This review reports 3 major findings using these model animals. First, the mammary epithelial cell has an extraordinary intrinsic capacity for survival in our culture model, and the path to either function or death by apoptosis is actively driven. The second outcome is that the route to apoptosis is most likely regulated by specific milk factors. Finally, whey acidic protein, a major milk protein in some species, may play a role in normal mammary development, but that role in vivo may be limited to marsupials. Evolutionary pressure has led to changes in the structure of the protein with an accompanying change in function. Therefore, we propose that a loss of function of this protein in eutherians may relate to a reproductive strategy that is less dependent on lactation.

Regulation of Gene Expression in the Bovine Mammary Gland by Ovarian Steroids

It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent on progesterone. The effects of these steroid hormones on gene expression in the mammary gland are mediated primarily by their respective nuclear hormone receptors, which function as hormone-bound transcription factors. To gain insight into how estrogen and progesterone regulate mammary gland growth and function in cattle, we and others have characterized the expression patterns of their cognate nuclear hormone receptors in the bovine mammary gland throughout development, pregnancy, and lactation. This work has identified a lack of expression of estrogen receptor β and a greater abundance of progesterone receptor during lactation in the bovine mammary gland, compared with the rodent gland. We speculate that interactions among the estrogen receptor isoforms that regulate progesterone receptor expression may contribute to these species differences. Further, demonstrated expression of substantial quantities of estrogen receptor within the prepubertal bovine mammary fat pad, along with coordinated insulin-like growth factor-I expression, suggests that this tissue may stimulate parenchymal growth via an estrogen-responsive paracrine mechanism. In addition, the recent availability of bovine genomic sequence information and microarray technologies has permitted the study of global gene expression in the mammary gland in response to the steroid environment. We have identified more than 100 estrogen-responsive genes, of which the majority are novel estrogen gene targets. Estrogen-induced changes in gene expression were consistent with increased mammary epithelial cell proliferation, increased extracellular matrix turnover in parenchyma, and increased extracellular matrix deposition in the fat pad. A comparison of estrogen-responsive genes in the mammary glands of humans, mice, and cattle suggests considerable variation among species, as well as potential differences in regulatory elements in common estrogen receptor gene targets. Continuing studies using advanced molecular techniques should assist in elucidating the complex regulation of mammary function at the transcript level.

The tammar wallaby: A model to examine endocrine and local control of lactation

Marsupials, such as the tammar wallaby (Macropus eugenii), have adopted a reproductive strategy that is very different to eutherians. Both the rate of production and the composition of milk changes progressively during the lactation cycle to meet the nutritional demands of an altricial young. The tammar therefore provides a valuable model to study changes in milk composition, and in particular the genes that code for proteins secreted in the milk, to more accurately assess the role of gene products regulating either development of the young or mammary function.

Progesterone increases metabolic rate and progesterone withdrawal enhances epithelial integrity of mammary epithelium

Progesterone receptors (PR) were documented in a bovine mammary epithelial cell line (BME-UV), which allowed us to test the hypothesis that progesterone (PRG) has direct effects on mammary epithelial function. PR were identified in BME-UV cells at the mRNA and protein (Western blot) levels. Thus, BME-UV cells were grown as confluent monolayers in either no added hormones, PRG, prolactin (PRL), and PRG + PRL over a 14 day culture period. Parturition was simulated in vitro by using PRG-free medium for the final day in culture. PRG exposure caused an immediate and substantial increase in glucose utilization that was maintained for at least 10 days of continued PRG exposure. Small effects on transepithelial electrical resistance (Rte) were identified in the presence of these hormones. However, treatment including PRG and PRL for the first 13 days of culture, but lacking PRG for the final 24 hours prior to assay substantially increased Rte. The results demonstrate that PRG exposure is directly associated with an increase in mammary cell metabolism. PRG also ‘primed’ the epithelial cells for a rapid transition to greater Rte when PRG was withdrawn. This suggests that PRG increases metabolic activity of mammary epithelial cells during pregnancy and that the sudden reduction in circulating PRG levels at parturition signals the rapid closure of mammary epithelial tight junctions. [USDA NRI, RRO17686 NCRR NIH Support]

Mammary Response to Exogenous Prolactin or Frequent Milking During Early Lactation in Dairy Cows

Frequent milking of dairy cows during early lactation results in a persistent increase in milk yield; however, the mechanism underlying this effect is unknown. We hypothesized that increased exposure of the mammary gland to prolactin (PRL) mediates the milk yield response. Fifteen multiparous Holstein cows were assigned to 3 treatments for the first 3 wk of lactation: twice daily milking with (2× + PRL) or without (2×) supplemental exogenous PRL, or 4 times daily milking (4×). Mammary biopsies were obtained at 7 DIM, and rates of [3H]thymidine incorporation into DNA in vitro were determined. Mammary expression of suppressors of cytokine signaling (SOCS)-1, -2, and -3; the long form of PRL-receptor; and α-lactalbumin mRNA was measured by real-time reverse-transcription PCR. Incorporation of [3H]thymidine into DNA was not affected by frequent milking or PRL treatment; however, analysis of autoradiograms revealed that stromal cell proliferation was greater in 4× cows. Mammary expression of SOCS-1 was not affected by milking frequency or PRL treatment. Expression of SOCS-2 mRNA was increased with frequent milking or PRL treatment, whereas expression of SOCS-3 mRNA was reduced by frequent milking or exogenous PRL. Abundance of PRL-receptor mRNA was reduced, whereas α-lactalbumin mRNA was increased with PRL treatment. These results demonstrate that the bovine mammary gland is responsive to exogenous PRL during early lactation. In addition, differences in the response to frequent milking or exogenous PRL during early lactation indicate distinct effects of PRL and milk removal on the mammary function of dairy cows.

Prolactin affects leptin action in the bovine mammary gland via the mammary fat pad

One of the roles of the endocrine system is to synchronize mammary function. Hormones, such as estrogen, progesterone, and prolactin act directly on the mammary gland. Metabolic hormones, such as GH, glucocorticoids, insulin, and leptin are responsible for coordinating the body's response to metabolic homeostasis. Leptin has been shown to be an important factor in regulating the metabolic adaptation of nutrient partitioning during the energy-consuming processes of lactation. In the present study, we show that leptin is secreted from the mammary fat, and is regulated by prolactin. The expression of alpha-casein in a co-culture of epithelial cells and fat explants was enhanced by prolactin compared with that in epithelial cells cultured alone. Leptin antagonist abolished the effect of leptin on alpha-casein expression in mammary gland explants when exogenous leptin was not present in the medium. This finding supports our hypothesis that the antagonist abolishes the action of endogenous leptin secreted by the mammary adipocytes. These results lead us to the hypothesis that prolactin and leptin act in the bovine mammary gland, via mammary fat pad/adipocytes.

Leptin enhances STAT-3 phosphorylation in HC11 cell line: Effect on cell differentiation and cell viability

Leptin is produced in the mammary gland by the fat tissue or by the mammary epithelium. The aim of this study was to investigate the role of leptin on mammary epithelial cell differentiation and cell viability. This study was conducted using the mouse mammary epithelial cell line HC11. We show that leptin, synergizes with prolactin to increase β-casein gene expression during mammary epithelial cell differentiation. This was correlated with increased phosphorylation of the signal transducer and activator of transcription 3 (STAT-3). Inactivating the function of STAT-3 by expression of a short hairpin RNA demonstrated that the effect of leptin on β-casein expression is mediated by STAT-3. Secondarly, cells in which STAT-3 had been inactivated showed increased cell viability compared to controls and were resistant to the negative effect mediated by leptin. Further, leptin triggers apoptosis in mammary epithelial cells cultivated in non-differentiating conditions. Taken together, these results suggest that leptin, by activating STAT-3, may act as a paracrine factor modulating mammary epithelial cell function.

Steroid Receptor Coactivator 2 Is Critical for Progesterone-Dependent Uterine Function and Mammary Morphogenesis in the Mouse

Although the essential involvement of the progesterone receptor (PR) in female reproductive tissues is firmly established, the coregulators preferentially enlisted by PR to mediate its physiological effects have yet to be fully delineated. To further dissect the roles of members of the steroid receptor coactivator (SRC)/p160 family in PR-mediated reproductive processes in vivo, state-of-the-art cre-loxP engineering strategies were employed to generate a mouse model (PR(Cre/+) SRC-2(flox/flox)) in which SRC-2 function was abrogated only in cell lineages that express the PR. Fertility tests revealed that while ovarian activity was normal, PR(Cre/+) SRC-2(flox/flox) mouse uterine function was severely compromised. Absence of SRC-2 in PR-positive uterine cells was shown to contribute to an early block in embryo implantation, a phenotype not shared by SRC-1 or -3 knockout mice. In addition, histological and molecular analyses revealed an inability of the PR(Cre/+) SRC-2(flox/flox) mouse uterus to undergo the necessary cellular and molecular changes that precede complete P-induced decidual progression. Moreover, removal of SRC-1 in the PR(Cre/+) SRC-2(flox/flox) mouse uterus resulted in the absence of a decidual response, confirming that uterine SRC-2 and -1 cooperate in P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analysis disclosed the absence of significant ductal side branching and alveologenesis in the hormone-treated PR(Cre/+) SRC-2(flox/flox) mammary gland, reinforcing an important role for SRC-2 in cellular proliferative changes that require PR. We conclude that SRC-2 is appropriated by PR in a subset of transcriptional cascades obligate for normal uterine and mammary morphogenesis and function.

Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium

In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R(te)) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R(te) began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R(te). Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R(te), although total occludin was unchanged. Neither substitution of Na(+) with N-methyl-d-glucosamine (NMDG(+)) nor pharmacological inhibition of transcellular Na(+) transport pathways abrogated the effects of apical H-elec medium on R(te). Tumor necrosis factor alpha, but not interleukin-1beta nor interleukin-6, in the apical compartment caused a significant decrease in R(te) within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance.

Goat's alpha-s1 casein genotype influences its milk fatty acid composition and delta-9 desaturation ratios

A large number of studies about impacts of alpha-s1 (αs1) casein (CSN1S1) gene polymorphism on goat milk composition, including its milk fat concentration, secretion in mammary cells and transcriptome analysis, have been reported. Despite this knowledge, very little data is available on the fatty acid (FA) composition of goat milk according to the CSN1S1 genotype. The study reported here compares two groups of 33 and 38 goats with different CSN1S1 genotypes, associated respectively with low or high milk protein and fat concentrations, matched for lactation stage and diets. Results confirm the lower milk protein (−5.0 g/kg) and fat (−6.8 g/kg) concentrations in the Low genotype group, whereas milk yield and its lactose concentration were not changed. There were between-group differences in proportions in milk fat of at least 17 FA, particularly the C8–C12 saturated FA and palmitic, stearic, oleic, linoleic and rumenic acids. In addition, delta-9 desaturation ratios were higher in the Low CSN1S1 group for C10:0, C14:0, C17:0, C18:0, trans11-C18:1 and trans13-C18:1, which strongly suggests an effect on mammary desaturase activity, especially for C18:0 and trans11-C18:1. It is likely that higher delta-9 desaturation ratios for C10:0 to transC18:1 isomers in the Low genotype could compensate for the lower secretion of medium-chain FA (C8:0 to C12:0), thus contributing to maintenance of the melting point of milk fat in this genotype. The polymorphism in the CSN1S1 genotype has important impacts on goat milk fat, and its FA composition, possibly making the goat model of interest in the search for a better understanding of regulation of mammary function, in particular FA synthesis, desaturation and secretion, and how it could be related to protein synthesis and secretion.