Impaired Lactation Research Articles

Maternal concentrations of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) and duration of breastfeeding

Perfluorooctanoate (PFOA) has been associated with impaired lactation in mice. We examined whether maternal perfluorooctanesulfonate (PFOS) and PFOA concentrations correlated with duration of breastfeeding among women. We randomly selected 1400 pregnant women from the Danish national birth cohort (1996-2002) and measured PFOS and PFOA concentrations in early pregnancy by using high performance liquid chromatography/tandem mass spectrometry. Self-reported data on the duration of any and exclusive breastfeeding were collected twice during telephone interviews around 6 and 18 months after the birth of the child. The duration of breastfeeding decreased with increasing concentrations of pregnancy PFOS and PFOA among multiparous women, for whom the adjusted odds ratios (OR) for weaning before 6 months of age were 1.20 (95% CI 1.06-1.37) per 10 ng/ml increase in PFOS concentrations and 1.23 (95% CI 1.13-1.33) per 1 ng/ml increase in PFOA concentrations. No consistent association was found for primiparous women. These findings suggest that PFOA and PFOS may reduce the ability to lactate, but could equally reflect reverse causation since no association was seen in primiparous women.

Impact of Anxiety in the Puerperium on Breast‐feeding Outcomes: Role of Parity

: Studies in animals indicate that stress and anxiety can depress lactation, but there is much less information available concerning humans. We aimed to determine whether maternal anxiety in puerperium, potentially exacerbated by the primiparity inexperience, has a negative impact on breast-feeding outcomes. : The state-trait anxiety inventory Y form (Spielberger, 1983) was administered to a total of 204 mothers, 101 primiparae, and 103 pluriparae on the third to fourth day postpartum. : We found that primiparae had state anxiety (T) scores significantly higher than pluriparae (44.57 +/- 5.85 vs 43.28 +/- 7.10, P = 0.03). Increased state anxiety levels impaired breast-feeding success. In addition, in the logistic regression analysis model used to assess factors predictive of breast-feeding longer than 3 months, maternal state anxiety was the most significant risk factor (odds ratio 0.99; 0.88-0.98, P < 0.01). : In the puerperium, anxiety, potentially exacerbated by primiparae inexperience, is associated with impaired lactation. Alleviating maternal anxiety could be beneficial for stimulating breast-feeding in more vulnerable women.

Uteroplacental insufficiency causes a nephron deficit, modest renal insufficiency but no hypertension with ageing in female rats

In rats, uteroplacental insufficiency induced by uterine vessel ligation restricts fetal growth and impairs mammary development compromising postnatal growth. In male offspring, this results in a nephron deficit and hypertension which can be reversed by improving lactation and postnatal growth. Here, growth, blood pressure and nephron endowment in female offspring from mothers which underwent bilateral uterine vessel ligation (Restricted) on day 18 of pregnancy were examined. Sham surgery (Control) and a reduced litter group (Reduced at birth to 5, equivalent to Restricted group) were used as controls. Offspring (Control, Reduced, Restricted) were cross-fostered on postnatal day 1 onto a Control (normal lactation) or Restricted (impaired lactation) mother. Restricted-on-Restricted offspring were born small but were of similar weight to Control-on-Control by postnatal day 35. Blood pressure was not different between groups at 8, 12 or 20 weeks of age. Glomerular number was reduced in Restricted-on-Restricted offspring at 6 months without glomerular hypertrophy. Cross-fostering a Restricted pup onto a Control dam resulted in a glomerular number intermediate between Control-on-Control and Restricted-on-Restricted. Blood pressure, along with renal function, morphology and mRNA expression, was examined in Control-on-Control and Restricted-on-Restricted females at 18 months. Restricted-on-Restricted offspring did not become hypertensive but developed glomerular hypertrophy by 18 months. They had elevated plasma creatinine and alterations in renal mRNA expression of transforming growth factor-beta(1), collagen IV (alpha1) and matrix matelloproteinase-9. This suggests that perinatally growth restricted female offspring may be susceptible to onset of renal injury and renal insufficiency with ageing in the absence of concomitant hypertension.

Prenatal growth restriction and postnatal growth restriction followed by accelerated growth independently program reduced bone growth and strength

Low birth weight increases the risk of developing adult onset cardiovascular and metabolic diseases. Recently being born small has also been identified as a risk factor for adverse bone growth, development and adult fracture risk. Evidence also suggests that accelerated growth in offspring of normal birth weight, following periods of slowed growth, can also independently program adult diseases. The aim of this study was to determine the relative roles of prenatal and postnatal growth restriction on adult bone characteristics and strength. Bilateral uterine vessel ligation (Restricted) or sham surgery (Control) was performed on gestational day 18 in WKY rats to induce fetal growth restriction. Control, Reduced (reduced Control litter size to match Restricted) and Restricted pups were cross-fostered onto different Control (normal lactation) or Restricted (impaired lactation) mothers 1 day after birth. Femur length, dimensions, strength, mineral content and density were quantified using DXA and pQCT analysis. Markers of bone turnover were measured in offspring at 6 months. Restricted pups were born lighter than Controls with males, not females, remaining smaller than Control-on-Control at 6 months (P<0.05). Pups born of normal weight from a reduced litter suckling on a Restricted mother (Reduced-on-Restricted) grew slowly during lactation then quicker after weaning compared to Controls (P<0.05). Cortical bone mineral content, dimensions and strength were lower in Restricted-on-Restricted and Reduced-on-Restricted offspring compared to Controls with lower density in Reduced-on-Restricted females (P<0.05). The stress strain index of bone bending strength remained lower in the Restricted male offspring when body weight adjustments were made. Cross-fostering Restricted females, but not males, onto mothers with normal lactation (Restricted-on-Control) restored growth and bone parameters to Controls (P<0.05). Being born small, or postnatal growth restriction for normal birth weight offspring followed by accelerated growth, programs bone content and strength deficits. Deficits were corrected by improving postnatal nutrition for females born small, highlighting sex specific programming outcomes and impact of postnatal nutrition. These findings suggest a link between growth restriction and adult bone health with additional studies needed to further explore this link in humans.

Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk

The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1-5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.

Dioxin Exposure and Shortened Duration of Lactation in Seveso

ISEE-325 Objective: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant, has been shown to disrupt multiple endocrine systems in animals. Impaired mammary gland development, leading to impaired lactation has been reported in TCDD-treated mice. Higher maternal levels of other endocrine disrupting compounds have been associated with shortened duration of lactation. No epidemiologic studies have examined the potential effects of TCDD exposure on lactation. On July 10, 1976, as a result of a chemical explosion, residents of Seveso, Italy, experienced the highest levels of TCDD in a human population. Twenty years later, we initiated the Seveso Women's Health Study (SWHS), a retrospective cohort study of the reproductive health of the women. We examined the relationship of TCDD with initiation and duration of lactation for the first postexplosion pregnancy. Material and Methods: The SWHS cohort comprises 981 women who were aged 0 to 40 years in 1976 and resided in the most contaminated areas. Individual TCDD exposure was measured in archived sera collected soon after the explosion by high-resolution mass spectrometry. For each live birth after the explosion, women were asked whether they breast-fed and, if yes, how many months. Duration of lactation for the first live birth after the explosion was examined as a continuous dependent variable using multiple linear regression. Results: In total, 252 (64%) women initiated breast-feeding, and the average duration of lactation was 4.6 (±4.3) months. More highly exposed women were nonsignificantly more likely to initiate breast-feeding (OR per 10-fold increase in TCDD = 1.4, 95% CI: 0.9–2.0). However, duration of lactation (in months) was nonsignificantly decreased for each 10-fold increase in TCDD (β = −0.34, 95% CI: −0.96 to 0.27). Conclusions: These results suggest that TCDD exposure, like other endocrine disrupting compounds, may affect women's duration of lactation. Multivariate results will be presented and interpreted in light of study advantages and limitations.

Normal Lactational Environment Restores Nephron Endowment and Prevents Hypertension after Placental Restriction in the Rat

Uteroplacental insufficiency in the rat restricts fetal growth, impairs mammary development, compromising postnatal growth; and increases adult BP. The roles of prenatal and postnatal nutritional restraint on later BP and nephron endowment in offspring from mothers that underwent bilateral uterine vessel ligation (restricted) on day 18 of pregnancy were examined. Sham surgery (control) and a group of rats with reduced litter size (reduced; litter size reduced at birth to five, equivalent to restricted group) were used as controls. Offspring (control, reduced, and restricted) were cross-fostered on postnatal day 1 onto a control (normal lactation) or restricted (impaired lactation) mother. BP in male offspring was determined by tail cuff at 8, 12, and 20 wk of age, with glomerular number and volume (Cavalieri/Physical Dissector method) and renal angiotensin II type 1 receptor (AT(1)R) mRNA expression (real-time PCR) determined at 6 mo. Restricted-on-restricted male offspring developed hypertension (+16 mmHg) by 20 wk together with a nephron deficit (-26%) and glomerular hypertrophy (P < 0.05). In contrast, providing a normal lactational environment to restricted offspring improved postnatal growth and prevented the nephron deficit and hypertension. Reduced-on-restricted pups that were born of normal weight but with impaired growth during lactation subsequently grew faster, developed hypertension (+16 mmHg), had increased AT(1A)R and AT(1B)R mRNA expression (P < 0.05), but had no nephron deficit. Our study identifies the prenatal and postnatal nutritional environments in the programming of adult hypertension, associated with distinct renal changes. It is shown for the first time that a prenatally induced nephron deficit can be restored by correcting growth restriction during lactation.

Socs 3 modulates the activity of the transcription factor Stat3 in mammary tissue and controls alveolar homeostasis

Signal transducer and activator of transcription 5 and 3 (Stat5 and Stat3) control pregnancy-mediated mammary development and involution-dependent remodeling, respectively. Suppressor of cytokine signaling 3 (Socs3) has been implicated in the modulation of both Stat3 and Stat5 activity. To explore the biology of Socs3 in mammary tissue, the gene was deleted using Cre-mediated recombination. Deletion of the Socs3 gene from mammary stem or early progenitor cells did not grossly alter pregnancy-mediated mammary development but resulted in impaired lactation due to attenuated proliferation. Loss of Socs3 from differentiated luminal cells did not interfere with glandular function during lactation, but resulted in accelerated tissue remodeling upon weaning. Loss of Socs3 led to enhanced and precocious Stat3 activation. Thus, Socs3 serves as a modulator of Stat3 activity to ensure controlled proliferation and apoptosis in pregnancy and involution, respectively.

Alterations in Skin and Stratified Epithelia by Constitutively Activated PPARα

Peroxisome proliferator-activated receptor (PPAR)alpha is a pleiotropic regulator in many cell types and has recently been implicated in skin homeostasis. To determine the role of PPARalpha in skin physiology, transgenic mice were generated using the tetracycline Tet-off regulatory system to target constitutively activated PPARalpha to the epidermis and other stratified epithelia by the bovine keratin K5 promoter. Expression of the transgene during early development resulted in postnatal lethality within 2 days after birth. A thin epidermis, few hair follicles, and abnormal development of the tongue were observed in neonatal transgenic mice. Early mortality was not observed when transgenic PPARalpha expression was diminished by administration of doxycycline (dox) to the mothers. The alterations noted in neonatal mice were not observed in adult mice upon re-expression of the PPARalpha transgene by withdrawing dox. Attenuated hyperplasia of interfollicular epidermis after topical application of the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was observed in adult mice expressing the PPARalpha transgene. In addition, expression of the PPARalpha transgene in mammary gland during pregnancy resulted in abnormal development of this organ and impaired lactation. Further investigations using primary keratinocytes revealed that expression of the transgene in keratinocytes resulted in increased differentiation and decreased proliferation, which may contribute to the observed phenotype in the transgenic mice. Thus, these results indicate that PPARalpha plays an important role in the development of stratified epithelia including skin, tongue, and mammary gland.

Potential Role for Human Cytochrome P450 3A4 in Estradiol Homeostasis

Previously, a human CYP3A4-transgenic (Tg-CYP3A4) mouse line was reported to exhibit enhanced metabolism of midazolam by cytochrome P450 3A4 (CYP3A4) expressed in small intestine. Here we show that expression of CYP3A4 and murine cyp3a and cyp2b was both age and sex dependent. CYP3A4 was expressed in the livers of male and female Tg-CYP3A4 mice at 2 and 4 wk of age. Since 6 wk, CYP3A4 was undetectable in male livers, whereas it was constitutively expressed in female livers at decreased levels (3- to 5-fold). Pregnenolone 16alpha-carbonitrile markedly induced hepatic CYP3A4 expression, and the level was higher in females than males. Induction of intrinsic murine cyp3a and cyp2b was also sex dependent. Tg-CYP3A4 females were found to be deficient in lactation, leading to a markedly lower pup survival. The mammary glands of the Tg-CYP3A4 lactating mothers had underdeveloped alveoli with low milk content. Furthermore, beta-casein and whey acidic protein mRNAs were expressed at markedly lower levels in Tg-CYP3A4 pregnant and nursing mouse mammary glands compared with wild-type mice. This impaired lactation phenotype was associated with significantly reduced serum estradiol levels in Tg-CYP3A4 mice. A pharmacokinetic study revealed that the clearance of iv administrated [(3)H]estradiol was markedly enhanced in Tg-CYP3A4 mice compared with wild-type mice. These results suggest that CYP3A4 may play an important role in estradiol homeostasis. This may be of concern for treatment of pregnant and lactating women because CYP3A4 gene expression and enzymatic activity can be potentially modified by CYP3A4 inhibitors or inducers in medications, supplements, beverages, and diet.

The Transareolar Incision for Breast Augmentation Revisited

Of the various possible incisions for breast augmentation, the transareolar access has gained only limited popularity. The potential side effects of this incision are said to be altered nipple sensation, impaired lactation, an increased rate of infections with capsular fibrosis, well visible scar formation with hypopigmentation, and the need for an additional access in case a breast ptosis correction should prove necessary at a later date. The purpose of this retrospective study was to judge advantages and limitations of transareolar breast augmentation, and to verify whether the reluctant attitude toward this surgical approach is justified. A sample of 18 patients with a transareolar, retropectoral breast augmentation was selected for a retrospective evaluation. The suitability of the technique in general was examined together with early postoperative complications, sensory changes, and late complications on the basis of an evaluation system for cosmetic surgical results. The study showed that only women with an areolar diameter of 3.5 cm or more without pronounced breast ptosis were suitable for the transareolar access. No early infections were noted. The rate of capsular fibrosis was 11%. Two years after breast augmentation, 16 women (89%) judged their breast sensation to be normal, but objective assessment showed that mean pressure and vibration sensation were moderately compromised in all parts of the breast. The scars were of good quality, with very little hypopigmentation. With appropriate patient selection, respecting the advantages and limitations, the transareolar incision has its definite place among the different incisions for breast augmentation.

Loss of connexin 26 in mammary epithelium during early but not during late pregnancy results in unscheduled apoptosis and impaired development

Gap junctions are intercellular channels that are formed by the protein family of connexins (Cxs). In mammary tissue, Cx26 and Cx32 are present in the secretory epithelium and Cx43 is localized in the myoepithelium. The expression of Cx26 and Cx32 is induced during pregnancy and lactation, respectively, thus suggesting unique roles for them in the functional development of the gland. The requirement for these connexins was explored using several strains of genetically altered mice: mice with an inactivated Cx32 gene, mice in which the Cx43 gene had been replaced with the Cx32 gene (Cx43KI32 mice) and mice in which the Cx26 gene was specifically ablated in mammary epithelium at different stages of development using Cre-loxP-based recombination. Normal mammary development was obtained in Cx32-null mice and in Cx43KI32 mammary tissue. In contrast, loss of Cx26 in mammary epithelium before puberty resulted in abrogated lobulo-alveolar development and increased cell death during pregnancy, which was accompanied by impaired lactation. Loss of Cx26 in mammary epithelium during the later part of pregnancy did not adversely interfere with functional mammary development. These results demonstrate that the presence of Cx26 is critical during early stages but not during the end of pregnancy when the tissue has completed functional differentiation. Cx26 is considered a tumor suppressor gene and Cx26-null mammary tissue was evaluated after five pregnancies. No hyperproliferation or hyperplasia was observed, suggesting that Cx26 does not function as a tumor suppressor.

Cell surface beta1,4-galactosyltransferase function in mammary gland morphogenesis: insights from transgenic and knockout mouse models.

Development and morphogenesis are profoundly influenced by cell-cell and cell-extracellular matrix (ECM) interactions that are governed by cell surface receptor association with specific ligands. One such receptor is the long isoform of beta1,4-galactosyltransferase I (GalT I), a small proportion of which is targeted to the plasma membrane. Surface-expressed GalT I binds to specific glycoside residues on multiple extracellular ligands, and GalT I binding to specific ligands mediates cell-cell as well as cell-matrix interactions for a variety of cells, including mammary epithelia. Significant insight into surface GalT I function in mammary gland development and morphogenesis has been gained through the analysis of mouse transgenic and knockout models of surface GalT I misexpression. Overexpression of cell surface GalT I leads to impaired lactation as a result of reduced branching and differentiation and elevated apoptosis, while deleting surface GalT I enhances branching and differentiation and reduces apoptosis. These phenotypes can be attributed in large part to altered cell-ECM interactions. The current and future challenges are to use these mouse models to dissect the molecular mechanisms that govern surface GalT I function as a receptor in the normal mammary gland, as well as to assess the potential for surface GalT I misexpression to contribute to disease.

Targeted expression of the dominant-negative prolactin receptor in the mammary gland of transgenic mice results in impaired lactation.

The F3-short form of the rat PRL receptor (F3-SPRLR) form acts as a dominant negative inhibitor in vitro. We have developed a transgenic mouse model in which the rat F3-SPRLR was expressed in mammary epithelium under the control of the mouse mammary tumor virus promoter. Two lines of mice were characterized and shown to express the transgene in the mammary gland. No developmental abnormalities or differences from wild-type littermates were observed on the basis of size, activity, or fertility. Mice with a low level of transgene expression had a mammary phenotype similar to the wild type. However, mice overexpressing the transgene (levels much higher than those of the endogenous long PRLR transcript) had impaired mammary gland differentiation and lactation. In these mice, whole-mount and histological analyses demonstrated normal ductal development, but severely reduced lobuloalveolar outgrowth. signal transducer and activator of transcription-5 phosphorylation and expression of beta-casein and whey acidic protein gene were decreased. In vivo bromodeoxyuridine incorporation at midpregnancy showed that the reduction in mammary development was not due to an inhibition of ductal growth and side-branching. This model demonstrates for the first time in vivo a function of the SPRLR and a local and targeted effect of PRL on the mammary gland that are essential for its function, but not for its development.

Activating Transcription Factor 4 Overexpression Inhibits Proliferation and Differentiation of Mammary Epithelium Resulting in Impaired Lactation and Accelerated Involution

The basic leucine zipper containing activating transcription factors (ATFs) modulates the expression of growth-regulating genes. In this study, we sought to determine specifically the consequences of ATF4 expression on mammary gland development in transgenic mice. Overexpression of ATF4 severely impaired normal development of the mammary gland, which was associated with reduced proliferation and differentiation of mammary alveolar epithelium and up-regulation of p21(WAF1) and p27(Kip1). In addition, there was also impaired lactation accompanied by decreased expression of alpha-lactoalbumin, whey acidic protein, and beta-casein, possibly because of the down-regulation of STAT5a tyrosine phosphorylation. Mammary gland involution in ATF4-transgenic mice was accelerated, compared with wild type littermates by whole mount analysis. In addition, day 18 of lactation in transgenic mice was phenotypically equivalent to day 3 of involution in wild type mice, as determined by the TUNEL assay and expression of Bax. The concentration of the proapoptotic molecule caspase-3 was increased during lactation in ATF4-transgenic animal. Mammary glands from ATF4-transgenic mice also showed significant nuclear translocation of activated STAT3 and up-regulation of one of its target genes, insulin-like growth factor-binding protein-5, which is thought to facilitate apoptosis by sequestering insulin-like growth factor. Together, these findings suggest that ATF4 may play a role during mammary gland development and that down-regulation of ATF4 may be important for the onset of involution in the mammary gland.

Critical analysis of potential body temperature confounders on neurochemical endpoints caused by direct dosing and maternal separation in neonatal mice: a study of bioallethrin and deltamethrin interactions with temperature on brain muscarinic receptors.

The present investigation was conducted to understand better possible confounding factors caused by direct dosing of neonatal mice during the pre-weaning developmental period. By direct dosing, pups might encounter thermal challenges when temporarily removed from their 'natural habitat'. Typically, this leads to a cold environment and food deprivation (impaired lactation) and modulation of the toxic potency of the substance administered. Growth retardation as a consequence of such behavioural changes in pups makes it increasingly difficult to differentiate specific from non-specific mechanisms. Neonatal NMRI mice were dosed daily by gavage (0.7 mg kg(-1) body wt.) from postnatal day (PND) 10-16 with S-bioallethrin, deltamethrin or the vehicle. Then the pups, including their non-treated foster dams, were subjected temporarily for 6 h day to a hypo-, normo- or hyperthermic environment, which was followed by normal housing. The measured temperatures in the environmental chambers were ca. 21, 25 and 30 degrees C, respectively. Thus, temperatures in the hypo- and normothermic groups are comparable to the temperatures commonly present in testing laboratories, whereas the hyperthermic condition is that temperature typically present in the 'natural habitat' of pups. A deviation from the normal behaviour of both pups and dams was observed in the hypo- and normothermic groups. In these groups the rectal temperatures of pups were markedly decreased, especially in the early phase of the study (PND 10-12). Neonates that received either test substance displayed changes in body weights and brain weights at terminal sacrifice (PND 17) when subjected temporarily to a non-physiological environment. An enormous influence of environmental temperature on the density of muscarinic receptors in the crude synaptosomal fraction of the cerebral cortex was ascertained. In summary, these results demonstrate that the direct dosing of thermolabile neonatal mice by gavage is subject to significant artefacts that render the interpretation of findings from such studies difficult. It appears that if direct dosing of neonatal pups is mandated, and inhalation is a relevant route of exposure, the combined inhalation exposure of dams with their litters is an alternative procedure that does not cause disruption of the 'natural habitat' of pups. However, owing to their higher ventilation, under such conditions the pups may receive dosages at least double those of the dams.

Growth hormone, acting in part through the insulin-like growth factor axis, rescues developmental, but not metabolic, activity in the mammary gland of mice expressing a single allele of the prolactin receptor.

The heterozygous prolactin (PRL) receptor (PRLR(+/-)) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. PRL and GH, acting through the IGF system, have interactive effects to enhance epithelial cell survival. Thus, we propose that a reduction in the expression of the PRLR may lead to increased IGFBP-5 expression (proapoptotic) and that GH may rescue mammary development by increasing IGF-I, an important mitogen and survival factor for the mammary epithelium. Mammary IGF-binding protein-5 (IGFBP-5) concentrations and plasmin activity in PRLR(+/-) mice were increased on d 2 postpartum, indicative of increased cell death and extracellular matrix remodeling. After GH treatment, a restoration of mammary alveolar development and a reduction in the activities of IGFBP-5 and plasmin were observed. Despite the severely impaired mammary development in PRLR(+/-) mice, both mRNA and protein expression for caseins and acetyl-coenzyme A (acetyl-CoA) carboxylase and acetyl-CoA caboxylase-alpha mRNA increased at parturition, although not to the extent in wild-type animals. Surprisingly, GH treatment actually led to a further decrease in milk protein and acetyl-CoA carboxylase-alphaexpression when expressed per cell. This was confirmed by the smaller alveolar size, the relative paucity of milk in the mammary glands of GH-treated animals, and the inability of their pups to gain weight. In a subsequent study IGFBP-5 was administered to wild-type mice and produced a 45% decrease in mammary DNA content, a 30% decrease in parenchymal tissue, and impaired lactation. These results suggest that GH can improve mammary development in PRLR(+/-) mice, but that it fails to enhance metabolic activity. This may be due to the maintenance by GH/IGF-I of a proliferative, rather than a differentiative, phenotype.

The aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt) are dispensable for normal mammary gland development but are required for fertility.

The aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt) are transcription factors that play a role in the detection of and adaptation to environmental signals. AhR-null mice are viable but show impaired lactation. Deletion of the Arnt gene from the mouse genome results in embryonic lethality. To determine the role of Arnt in mammary development and function, we inactivated the Arnt gene in mammary epithelium using Cre-loxP recombination. Inactivation of the Arnt gene during pregnancy did not disrupt alveolar development or the ability of dams to nurse their litters. In contrast, dams in which the Arnt gene had been inactivated during puberty and in ovaries were subfertile, exhibited retarded mammary development, and impaired mammary function. To distinguish defects autonomous to mammary epithelium from indirect effects controlled by ovarian hormones, we transplanted Arnt-null and AhR-null mammary epithelium into wild-type mice and evaluated development after one pregnancy. Normal mammary structures were observed in the absence of Arnt and AhR, demonstrating that neither transcription factor is necessary for mammary development.

Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development.

Targeted mice lacking functional EGF or amphiregulin (AR) were derived and bred to the TGFalpha-knockout to generate mice lacking various combinations of the three ligands. In contrast to EGF receptor (EGFR) knockout mice, triple null mice lacking half of the EGFR ligand family were healthy and fertile, indicative of overlapping or compensatory functions among EGF family members. Nevertheless, pups born to triple null dams frequently died or were runted, suggesting a mammary gland defect. Comparison of individual and combinatorial knockouts established that specific loss of AR severely stunted ductal outgrowth during puberty, consistent with dramatic expression of AR transcripts in normal developing ducts. Surprisingly, loss of all three ligands did not significantly affect cellular proliferation, apoptosis, or ERK activation within terminal end buds. Following pregnancy, most AR single null females, but few triple null females could nurse their young, revealing collaborative roles for EGF and TGFalpha in mammopoiesis and lactogenesis. In triple null glands, alveoli were poorly organized and differentiated, and milk protein gene expression was decreased. Additionally, Stat5a activation was frequently reduced in AR single and combinatorial nulls in association with impaired lactation. Collectively, our results provide genetic confirmation of a requirement for EGFR signaling throughout the development of the mouse mammary gland, and reveal stage-dependent activities for different EGFR ligands. Finally, the additional loss of growth factors from pups nursed by triple null dams further worsened their survival and growth, establishing functions for both maternal- and neonatal-derived growth factors.

A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation.

The mutant mouse waved-2 (wa-2) is strikingly similar to transforming growth factor alpha-deficient mice generated by gene targeting in embryonic stem cells. We confirm that wa-2 is a point mutation (T-->G resulting in a valine-->glycine substitution at residue 743) in the gene encoding the epidermal growth factor (EGF) receptor. wa-2 fibroblastic cells lack high-affinity binding sites for EGF, and the rate of internalization of EGF is retarded. Although the tyrosine kinase activity of wa-2 EGF receptors is significantly impaired, NIH 3T3 cells lacking endogenous EGF receptors but overexpressing recombinant wa-2 EGF receptor cDNA are mitogenically responsive to EGF. While young and adult wa-2 mice are healthy and fertile, 35% of wa-2 mice born of homozygous wa-2 mothers die of malnutrition because of impaired maternal lactation.