Expression of Lysostaphin in Milk of Transgenic Mice Affects the Growth of Neonates

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35±0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 ± 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 ± 0.33 g) and nontransgenic (0.91 ± 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.Authors Abhijit Mitra and Kathleen S. Hruska contributed equally to this work     

Mania‐ and anxiety‐like behavior and impaired maternal care in female diacylglycerol kinase eta and iota double knockout mice

Genome‐wide association studies linked diacylglycerol kinase eta and iota to mood disorders, including bipolar disorder and schizophrenia, and both genes are expressed throughout the brain. Here, we generated and behaviorally characterized female mice lacking Dgkh alone, Dgki alone, and double Dgkh/Dgki‐knockout (dKO) mice. We found that fewer than 30% of newborn pups raised by dKO females survived to weaning, while over 85% of pups survived to weaning when raised by wild‐type (WT) females. Poor survival under the care of dKO mothers was unrelated to pup genotype. Moreover, pups from dKO dams survived when fostered by WT dams, suggesting the poor survival rate of dKO‐raised litters was related to impaired maternal care by dKO dams. Nest building was similar between WT and dKO dams; however, some dKO females failed to retrieve any pups in a retrieval assay. Pups raised by dKO dams had smaller or absent milk spots and reduced weight, indicative of impaired nursing. Unlike WT females, postpartum dKO females showed erratic, panicked responses to cage disturbances. Virgin dKO females showed behavioral signs of anxiety and mania, which were not seen in mice lacking either Dgkh or Dgki alone. Our research indicates that combined deletion of Dgkh and Dgki impairs maternal behavior in the early postpartum period, and suggests female dKO mice model symptoms of mania and anxiety.     

Passive immunization of lactating mice with stanniocalcin-1 antiserum reduces mammary gland development, milk fat content, and postnatal pup growth

During pregnancy and lactation in rodents, stanniocalcin-1 (STC-1) production by the ovaries is upregulated markedly and released into the circulation. The mammary glands are one target of this systemically delivered hormone. The purpose of this study was to lower serum levels of STC-1 in lactating mice through passive immunization so as to monitor the effects on mammary gland function and postnatal pup growth. Passive immunization significantly reduced circulating hormone levels, and pup growth was significantly compromised (30%), even though control and experimental litters had ingested equal amounts of milk. When mammary glands were analyzed, the alveolar area was significantly reduced in antibody-treated mothers. An analysis of milk composition revealed no changes in lactose, protein, or electrolyte levels but an approximately 40% reduction in triglyceride levels. The latter was due to a significant reduction in mammary gland lipoprotein lipase activity and led to a significant buildup of triglycerides in the serum. Body fat content was also significantly reduced in pups from antibody-treated mothers, whereas pup fecal fat content was increased. In mothers, passive immunization also caused significant behavioral effects, in particular, increased locomotor and hindleg rearing activities. Collectively, the results suggest that systemically derived STC-1 has important effects on mammary gland development and the transfer of serum-based triglycerides into milk. Locomotor effects suggest that STC-1 also has a role in maternal behavior.     

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.     

Induction of Size-Dependent Breakdown of Blood-Milk Barrier in Lactating Mice by TiO2 Nanoparticles

This study aims to investigate the potential nanotoxic effects of TiO₂ nanoparticles (TNPs) to dams and pups during lactation period. TiO₂ nanoparticles are accumulated in mammary glands of lactating mice after i.v. administration. This accumulation of TiO₂ NP likely causes a ROS-induced disruption of tight junction of the blood-milk barrier as indicated by the loss of tight junction proteins and the shedding of alveolar epithelial cells. Compared to larger TNPs (50 nm), smaller ones (8 nm) exhibit a higher accumulation in mammary glands and are more potent in causing perturbations to blood-milk barrier. An alarming finding is that the smaller TNPs (8 nm) are transferred from dams to pups through breastfeeding, likely through the disrupted blood-milk barrier. However, during the lactation period, the nutrient quality of milk from dams and the early developmental landmarks of the pups are not affected by above perturbations.     

Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin

Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.     

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5-dihydrothiazole in female mice

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development, was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.     

3,5,3'-Triiodothyronine administered to rat dams during lactation increases milk yield and triglyceride concentration and hastens pups growth

It is known that lactation induces a mild hypothyroid state in rats and other mammals while thyroid hormone administration increases milk secretion in ruminants. The aim of this study was to investigate the effects of a moderate dose of 3,5,3'-triiodothyronine (T3), administered to rat dams during lactation on pups' growth and milk yield and composition. Primiparous Wistar rats with litters adjusted to 10 pups per dam received either tap water or T3 (75 microg/kg x day) in their drinking water from parturition till weaning. Food and water intake of dams and body weight of dams and pups were measured daily. In other groups of rats with similar treatments, milk yield of dams, macronutrient milk composition, and mammary arteriovenous differences for triglycerides (TG) and glucose were also determined. Dams treated with T3 ingested more food and their pups gained more weight than controls. Milk yield, milk TG concentration and glucose extraction by mammary glands were also higher in T3 treated dams. The results show that compensation of the mild hypothyroidism of the lactating rat may contribute to an increase in milk production and lipid levels, leading to an increase in growth of pups.     

Lack of ceruloplasmin expression alters aspects of copper transport to the fetus and newborn, as determined in mice

Copper transport and accumulation were studied in virgin and lactating C57BL/6 mice, with and without expression of ceruloplasmin (Cp), to assess the importance of Cp to these processes. One hour after i.p. injection of tracer ⁶⁴Cu, liver and kidney accounted for 80% of the radioactivity, and mammary gland 1%, while in lactating Cp+/+ mice 2–4 days post partum, uptake by mammary gland was 9-fold higher and that of liver and other organs was decreased, with ⁶⁴Cu rapidly appearing in milk. Parallel studies in Cp−/− mice (siblings from same colony) gave virtually identical results. However, their milk contained less ⁶⁴Cu, and actual copper contents determined by furnace atomic absorption were less than half those for milk from normal dams. Liver copper concentrations of pups born to Cp−/− dams also were half those of pups from wild type dams. Copper in pup brains was unaffected; but iron concentrations were reduced. We conclude that absence of Cp, while not affecting entry of exchangeable copper from the blood into the mammary gland, does have a significant effect on the availability of this metal to the newborn through the milk and in the form of stores accumulating in gestation.     

Effect of chronic treatments with ghrelin on milk secretion in lactating rats

Ghrelin, a novel acylated peptide and endogenous ligand for growth hormone (GH) secretagogue receptor, was originally isolated from rat and human stomachs. In addition to its GH-releasing activity, ghrelin plays an important role in many physiological functions, including food intake, gastric acid secretion, neonatal development, and so on. In this study, the effect of daily treatment with ghrelin on milk production was investigated in lactating rats and the development of the pups was monitored. Daily subcutaneous injection of ghrelin into nursing dams for 8 days from parturition caused a significant increase in milk yield and litter weight gain. When litters nursed by ghrelin-treated and saline-treated dams were interchanged on day 4 of lactation, the growth curves were reversed. Daily injections of ghrelin also increased plasma GH levels. Northern blot analysis revealed that daily injection of ghrelin significantly increased mammary casein mRNA expression. In addition, RT-PCR analysis showed that a ghrelin receptor was present in the mammary glands of lactating rats. These results suggest that ghrelin may play an important role in milk production in lactating dams.     

The influence of reproductive experience on milk energy output and lactation performance in the grey seal (Halichoerus grypus)

Although evidence from domestic and laboratory species suggests that reproductive experience plays a critical role in the development of aspects of lactation performance, whether reproductive experience may have a significant influence on milk energy transfer to neonates in wild populations has not been directly investigated. We compared maternal energy expenditures and pup growth and energy deposition over the course of lactation between primiparous and fully-grown, multiparous grey seal (Halichoerus grypus) females to test whether reproductive experience has a significant influence on lactation performance. Although there was no difference between primiparous females in milk composition and, thus, milk energy content at either early or peak lactation primiparous females had a significantly lower daily milk energy output than multiparous females indicating a reduced physiological capacity for milk secretion. Primiparous females appeared to effectively compensate for lower rates of milk production through an increased nursing effort and, thus, achieved the same relative rate of milk energy transfer to pups as multiparous females. There was no difference between primiparous and multiparous females in the proportion of initial body energy stores mobilised to support the costs of lactation. Although primiparous females allocated a greater proportion of energy stores to maternal maintenance versus milk production than multiparous females, the difference was not sufficient to result in significant differences in the efficiency of energy transfer to pups. Thus, despite a lower physiological capacity for milk production, primiparous females weaned pups of the same relative size and condition as multiparous females without expending proportionally more energy. Although reproductive experience does not significantly affect the overall lactation performance of grey seals, our results suggest that increases in mammary gland capacity with reproductive experience may play a significant role in the age-related increases in neonatal growth rates and weaning masses observed in other free-ranging mammals.     

Novel development of mammary glands in the nursing transgenic mouse ubiquitously expressing WAP gene.

Although whey acidic protein (WAP) has been suggested to have some biological functions, its true function has not yet been clearly elucidated. We have generated transgenic mice ubiquitously and highly expressing the WAP gene. The pups born from one female among these transgenic mice showed low growth or died during nursing. This transgenic founder showed novel development of the mammary glands, and demonstrated normal parturition and nursing behavior. The mammary glands showed low-distended ductal structures, and poor development of lobulo-alveolar and acinous formations despite normal nursing, while mammary ducts were rather large in comparison with those of normal lactating females. Although this founder was found to be mosaic for transgenesis, it was shown to be a useful animal model for investigating WAP function.     

Dopamine receptor D2 deficiency reduces mouse pup ultrasonic vocalizations and maternal responsiveness

Dopamine signalling facilitates motivated behaviours, and the D2 dopamine receptor (D2R) is important in mother–infant interactions. D2R antagonists disrupt maternal behaviour and, in isolated rat pups, reduce ultrasonic vocalizations (USVs) that promote maternal interaction. Here, we examined the effects of genetic D2R signalling deficiency on pup‐dam interaction with Drd2 knockout (D2R KO) mice. Using heterozygous (HET) cross littermates, the effect of pup genotype on isolation‐induced USVs was quantified. Independent of parental genotype, D2R‐deficient pups emitted fewer USVs than wild type (WT) littermates in a gene dose‐dependent manner. Using reciprocal D2R KO‐WT crosses, we examined how parental genotype affects pup USVs. Heterozygous pups from D2R KO dams produced fewer USVs than HET pups from WT dams. Also, exposure to USV‐emitting pups increased plasma prolactin levels in WT dams but not in D2R KO dams, and KO dams showed delayed pup retrieval and nest building. These findings indicate the importance of the interaction between pup and dam genotypes on behaviour and further support the role of D2R signalling in maternal care.     

Intestine, bone, and mammary gland contributions to maternal plasma calcium increase after abrupt weaning.

The response of maternal plasma calcium concentration to the abrupt and permanent removal of the suckling pups on Day 13 of lactation was investigated. Maternal plasma calcium did not change at 6 hr or 12 hr following pup removal. At 18 hr and 24 hr after weaning, the maternal plasma calcium concentration increased in mothers consuming either 0.47% calcium or 0.02% calcium diets. At 24 hr after weaning, the plasma calcium increase in mothers consuming low dietary calcium was 55% that of mothers consuming adequate dietary calcium. The contribution of the mammary gland to the plasma calcium increase in rats consuming the low dietary calcium was investigated by removing the mammary glands. Following mammary gland removal, plasma calcium increased 50% compared with mothers that had intact mammary glands. The data suggest that intestinal absorption of calcium and bone calcium mobilization remain stimulated by the lactation process for at least 24 hr after removal of the nursing pups.     

Maternal Obesity Reduces Milk Lipid Production in Lactating Mice by Inhibiting Acetyl-CoA Carboxylase and Impairing Fatty Acid Synthesis

Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.     

In a hypergravity environment neonatal survival is adversely affected by alterations in dam tissue metabolism rather than reduced food intake.

Exposure of rat dams to hypergravity during pregnancy is associated with increased pup mortality, reduced food intake, and decreased rates of glucose oxidation and lipogenesis in mammary tissue. We hypothesized that increased pup mortality is due to changes in maternal metabolism and not to reduced food intake of dams. Effects of hypergravity on rate of glucose oxidation and lipogenesis in mammary, liver, and adipose tissue were measured in rat dams centrifuged at 2.0 G [hypergravity (HG)], kept at 1.0 G (control), or fed to match the intake of HG rats (pair fed) from gestation day 11 (G11) until G21 or postpartum day 3 (P3). Body weight, percent body fat, metabolizable energy, and nitrogen balance were significantly less in HG dams compared with controls (P<0.05); however, these factors were not different between HG and pair-fed dams. By P3, 100% of control and pair-fed pups survived, while only 49% of HG pups survived. At G21, rates of glucose oxidation and lipogenesis in mammary and adipose tissue were less in HG than in control and pair-fed dams (P<0.1 and P<0.05). In liver, at G21, the rate of lipogenesis was greater in HG than control and pair-fed dams (P<0.01); at P3, lipogenesis was greater in control than HG and pair-fed dams (P<0.05). Gene expression of ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase increased in liver from pregnancy to lactation in control and pair-fed dams but not HG dams. Thus reduced food intake and body mass due to hypergravity exposure cannot explain the dramatic decrease in HG pup survival.     

Nursing Behavior in Mexican Free-tailed Bat Maternity Colonies

The nursing behavior of Mexican free-tailed bats (Tadarida brasiliensis mexicana) was examined in two large maternity cave roosts during six nursing seasons. After depositing pups in large creches, adults return to these creches two or more times daily to nurse. Densities, movements, and roosting associations of pups in creches were documented, and a night-vision device and infrared-sensitive video system were used to observe female-pup reunions and nursing affiliations. Densities of young (< 15 d) pups in creches averaged 4000 pups/m². Pups moved independently of one another between milk meals. Stable roosting associations among pups were absent, and females evidently could not predict where a given pup would be relocated. Upon returning to the creche, females searched for and relocated pups using vocal and olfactory cues. Experiments with marked individuals demonstrated that females nursed the same pups on subsequent nights. These same experiments indicated that female/pup recognition was mutual and that the pup participated in reunions by moving toward its putative mother. However, pups also attempted to suckle from other females and 35 instances of apparently successful milk “theft” were observed. Observed milk theft was insufficient to account for the frequency of alloparental nursing that was estimated independently using genotype tests. The high densities of bats within roosts and the lack of stable roosting associations among individuals argue against selection for shared nursing via kin selection and/or reciprocity. Errors in pup recognition, milk-dumping by females, and adoption by mothers whose own pup had died may occur, but high pup survival to weaning suggests that adoption is rare. The estimate of alloparental nursing obtained from genotype tests may be inflated as a result of increased opportunities for milk theft caused by our disturbance of adults. Current evidence suggests that energetic costs to females of alloparental nursing and searching for pups are compensated by mutualistic benefits obtained from aggregative roosting.     

High fat diet alters lactation outcomes: possible involvement of inflammatory and serotonergic pathways

Delay in the onset of lactogenesis has been shown to occur in women who are obese, however the mechanism altered within the mammary gland causing the delay remains unknown. Consumption of high fat diets (HFD) has been previously determined to result decreased litters and litter numbers in rodent models due to a decrease in fertility. We examined the effects of feeding a HFD (60% kcal from fat) diet versus a low-fat diet (LFD; 10% kcal from fat) to female Wistar rats on lactation outcomes. Feeding of HFD diet resulted in increased pup weights compared to pups from LFD fed animals for 4 d post-partum. Lactation was delayed in mothers on HFD but they began to produce copious milk volumes beginning 2 d post-partum, and milk yield was similar to LFD by day 3. Mammary glands collected from lactating animals on HFD diet, displayed a disrupted morphologies, with very few and small alveoli. Consistently, there was a significant decrease in the mRNA expression of milk protein genes, glucose transporter 1 (GLUT1) and keratin 5 (K5), a luminobasal cell marker in the mammary glands of HFD lactating animals. Expression of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin (5-HT) biosynthesis, and the 5-HT(7) receptor (HTR7), which regulates mammary gland involution, were significantly increased in mammary glands of HFD animals. Additionally, we saw elevation of the inflammatory markers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α). These results indicate that consumption of HFD impairs mammary parenchymal tissue and impedes its ability to synthesize and secrete milk, possibly through an increase in 5-HT production within the mammary gland leading to an inflammatory process.     

Suppression of lactation by pregnancy-dependent mammary tumors in GR/A mice

Pregnancy-dependent mammary tumors (PDMT) in GR/A mice appear during pregnancy, disappear soon after parturition, and appear again during subsequent pregnancies. The retardation of pup growth, an indication of the level of milk production, was also observed with the advance of lactation numbers in this strain. This study was performed to elucidate the relationship between PDMT and lactational performance. At the end of the second pregnancy, mice were divided into two groups according to the presence of PDMT [PDMT(-) and PDMT(+) groups]. Although all PDMT disappeared within a day after parturition, the weight and growth of pups on Day 12 of lactation were significantly less in the PDMT(+) group than in the PDMT(-) group. Associated with this, the DNA and RNA contents of the mammary glands were apparently lower in the former than in the latter, although the differences were not statistically significant. There was little difference in mammary RNA/DNA ratio between groups. No difference was also observed between groups in endocrine organ weights, mother body weights, morphology of the mammary glands, adrenals and ovaries and plasma prolactin and progesterone levels. These results suggest that PDMT suppression of lactation is principally due to the retardation of mammary gland growth. Furthermore, no significant correlations were obtained between the size of PDMT and the parameters for mammary gland function. The data suggest that the development of PDMT per se is important for the retarded mammary gland growth.