Effect of suboptimal nutrition during lactation on milk protein gene expression in the rat

Human milk provides infants with proteins that aid in the prevention of infections and facilitate the digestion and absorption of other nutrients. Maternal diet is not believed to affect the protein concentration of breast milk. However, the maternal factors that regulate the expression of genes for specific milk proteins are not well characterized. We hypothesized that nutrition could be one of the factors. We fed Sprague-Dawley rats five diets representing common nutrient deficiencies and energy deficiency during pregnancy and lactation: low-zinc (Zn; 7 microg/g), low-iron (Fe; 6 microg/g), low-protein (12.5% albumin), pair-fed control diet (lactation only, 20% less kcal) and control diet (Zn, 25 microg/g; Fe, 100 mug/g; protein, 21%) ad libitum. At day 10 of lactation, the mammary gland was removed for RNA extraction. Northern blots of mRNA from the different groups were performed by hybridization with beta-casein and whey acidic protein (WAP) cDNA probes. The expression of beta-casein mRNA in rat mammary gland was significantly (P<.005) increased in the pair-fed group when compared to the control group. The expression of WAP mRNA was also significantly (P<.005) increased in the pair-fed group as well as in the low-Fe group when compared to the control group. The concentration of beta-casein in milk was significantly higher for the low-zinc and the pair-fed groups only. The concentration of WAP in milk was not different among groups. These results suggest that compromised maternal nutrition can affect the expression of two individual milk proteins and may have functional implications with regard to bioactive proteins in milk.     

Variation of the poly(A) size classes in the rat mammary gland during the lactation cycle

The sizes of the poly(A) tracts associated with rat mammary RNA were determined at several time points in the lactation cycle. The poly(A) tracts in the lactating gland displayed two predominant size class peaks at 80-85 and 45-47 residues. The 9S whey protein mRNA and the 15S casein mRNA purified from the 12 day lactating mammary gland both contained poly(A) tracts displaying a similar size distribution. The 45 residue tracts were a characteristic of lactation; they were not found at 8 days of pregnancy and only small amounts of these shorter poly(A) tracts were found in the 16 day pregnant gland. The poly(A) tracts of the involuted gland displayed the same size characteristics as those of late pregnancy. At all the developmental stages that were examined, the fraction of 45 residue poly(A) tracts was always proportional to the total poly(A) content of the mammary cells.     

Changes in the yield, and carbohydrate, lipid and protein content of milk during lactation in the rat

The milk yield and composition was studied during the first three lactations of a group of rats. Milk yield increased steadily throughout the three lactations, but was somewhat lower during the first than subsequent lactations. Protein concentration was similar during all three lactations and varied little with stage of lactation. In contrast the lactose concentration, which was reasonably constant for the first 8 days post partum, increased thereafter two-fold by the end of the period studied in all three lactations. However, the N-acetyl-neuraminyl lactose concentration showed somewhat reciprocal changes. Considerable variations in the triacylglycerol concentration was found during the first lactation but few changes were observed during subsequent lactations. The free fatty acid concentration was at all times low and showed no significant changes during or between lactations. At most stages of lactation in raw milk, the major fatty acids are palmitate, oleate and linoleate. However, as lactation progresses there is an increase in the proportion of medium-chain saturated fatty acids and a corresponding decrease in the proportion of long chain unsaturated fatty acids in milk fat. Clearly the composition of milk is not invariable but changes both during and between lactations. Such changes may be expected to have some influence on the metabolism of the offspring.     

Enzymic changes in rabbit and rat mammary gland during the lactation cycle

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.     

Assaying the polyadenylation state of mRNAs

The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.     

Variation of the cholesterol content in breast milk during 10 days collection at early stages of lactation

More and more research is done concerning nutritional programming. Human milk nutrients which are consumed by infants can influence their health in later life. High level of cholesterol in human milk paradoxically lowers the cholesterol concentration in blood in adults. During the course of human lactation the cholesterol concentration decreases from 31 mg/100cm(3) (colostrum) to 16 mg/100 cm(3) (mature milk). According to Scopesi et al., 2002, Clin Nutr 21: 379-384, cholesterol concentration in mature milk ranged from 6.5 to 18.4 mg/100 cm(3). The aim of the study was to assess the variations in breast milk cholesterol content during 10 day collection at early lactation. 48 samples of human milk were analyzed. Mean age of women was 31 years. Women were collecting samples during 10 days of an early lactation stage (1-3 months after delivery). An Attenuated Total Reflectance Fourier Transformed Infrared (FTIR-ATR) method for easy and rapid determination of cholesterol in human milk was elaborated. Cholesterol content assessed by the FTIR method ranged from 3.36 to 12.98 mg/100 cm(3). Results indicate that milk cholesterol concentration during 10 consecutive days of early lactation is highly variable. Cholesterol content depends on an individual. Therefore it is suggested that not only the period of lactation but also mother's diet, age, season and place of residence are important factors determining cholesterol content.     

Quantitation of milk proteins and their mRNAs in rat mammary gland at various stages of gestation and lactation.

The content of alpha-lactalbumin and three species of caseins, 42K, 29K, and 25K, have been measured along with the levels and activities of their mRNAs in the rat mammary gland. Changes in these values were followed during gestation and lactation. An increment of 3- to 4-fold over the virgin level was observed for both alpha-lactalbumin and 42K casein during the 1st day of gestation. From this point on, the level of 42K remained unchanged during the 1st week of gestation and increased thereafter. After the increment of the 1st day, the alpha-lactalbumin content decreased rapidly during the 2nd day of gestation, continued to decrease more slowly until the 12th day, and then started to increase thereafter. During the 2nd and 3rd week of gestation. the amounts of alpha-lactalbumin within the gland increased continuously but not uniformly and caseins accumulated rapidly with a tendency to plateau around the 13th to 16th day of gestation. The relative proportions remained, respectively, 42K greater than 29K greater than 25K greater than alpha-lactalbumin until parturition. At the onset of lactation, both alpha-lactalbumin and casein content increased sharply, the relative proportion for caseins changed to 42K greater than 25K greater than 29K greater than alpha-lactalbumin and remained so throughout the lactation period. alpha-Lactalbumin and casein mRNA activity, as judged by the wheat germ translational system, remained unchanged during the 1st week of gestation, then showed a steady but not uniform increase from the 7th day of gestation until parturition. These activities increased sequentially during lactation, alpha-lactalbumin reaching a plateau by the 1st week, caseins between the 1st and 2nd week, and other mRNAs by the end of the 2nd week of lactation. By the 21st day of lactation, the activity of all mRNA had declined. The levels of alpha-lactalbumin mRNA and 16 S doublet casein mRNA sequences measured with the cDNA probes increased by about 8-fold for alpha-lactalbumin mRNA and 6-fold for casein mRNA during the 1st week of gestation. These levels declined slightly early in the 2nd week and then continued to increase until parturition with a shoulder in the levels around the 13th to 16th day. During lactation, these levels increased until the 8th to 12th day and from then on declined. The content of alpha-lactalbumin and caseins, as well as the measurement of sequences and activities of their mRNAs, showed that in the rat mammary gland these differentiated functions are already expressed at the onset of gestation. Both concentration and activity of mRNA are out of phase with protein levels during the 1st week of gestation but they remain in phase thereafter.     

Prolactin heterogeneity in the serum and milk during lactation

Prolactin heterogeneity in serum and milk were separated using Sephadex G-100. Three components were present in serum from lactating women with the following proportions: “void volume” -13.4%, “big” - 26.4%, and “little” - 60.3%. Milk from the same subjects did not contain “big” prolactin. Over 90% of the prolactin found in milk was “little” prolactin. The “little” prolactin in milk may not be similar to the “little” prolactin in the serum.     

Regulated polyadenylation of clam maternal mRNAs in vitro

During meiotic maturation of Spisula oocytes, maternal mRNAs undergo changes in translation and in the length of their poly(A) tails. In general, those mRNAs that are translationally activated, i.e., unmasked become polyadenylated, while deactivated mRNAs lose their poly(A) tails. The activated class of mRNAs encode ribonucleotide reductase, cyclins A and B and histone H3, while the proteins that stop being made include tubulin and actin. Previously, we demonstrated that mRNA-specific unmasking can be brought about in vitro by preventing the interaction of protein(s) with central portions of the 3′ noncoding regions (masking regions) of ribonucle-otide reductase and cyclin A mRNAs. In this report, we show that clam egg extracts are capable of sequence-specific polyadenylation of added RNAs since the 3′ untranslated regions (UTRs) of ribonu-cleotide reductase and histone H3 mRNAs are polyadenylated, while that of actin mRNA is not. In contrast, oocyte extracts, as in vivo, are essentially devoid of polyadenylation activity. We present an initial characterisation of the cis-acting sequences in the 3′ UTR of ribonucleotide reductase mRNA required for polyadenylation. The results suggest that the sequences for cytoplasmic polyadenylation are more complex and extensive than those determined in vertebrates and that they may partly overlap with the masking regions. © 1993 Wiley-Liss, Inc.     

Changes of beta-casomorphin content in human milk during lactation

Milk is the best, complete food important for the development and nourishment of a neonate. Except for nutrients, milk contains biologically active opioid peptides derived from beta-casein, named beta-casomorphins (BCMs), which can exert effects in the gastrointestinal tract as well as in the whole body of neonates. The content of beta-casomorphins in human milk during maturation phases has not been studied so far. The aim of this study was to determine the content of beta-casomorphin-5 and -7 in human milk in different phases of lactation. A significantly higher concentration of both beta-casomorphins was found in colostrum than in mature milk. The concentration of beta-casomorphin in milk collected in the second month of lactation was similar to the level obtained in the fourth month of lactation. The content of beta-casomorphins in human milk was observed with the period of lactation. The level of opioid peptides may depend on the function of these peptides in neonate's body and may be associated with the maturation process.     

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)

Background

Lactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby.

Results

Results showed that high levels of miRNA secreted in milk and allowed the identification of differentially expressed milk miRNAs during the lactation cycle as putative markers of mammary gland activity and functional candidate signals to assist growth and timed development of the young. Comparative analysis of miRNA distribution in milk and blood serum suggests that milk miRNAs are primarily expressed from mammary gland rather than transferred from maternal circulating blood, likely through a new putative exosomal secretory pathway. In contrast, highly expressed milk miRNAs could be detected at significantly higher levels in neonate blood serum in comparison to adult blood, suggesting milk miRNAs may be absorbed through the gut of the young.

Conclusion

The function of miRNA in mammary gland development and secretory activity has been proposed, but results from the current study also support a differential role of milk miRNA in regulation of development in the pouch young, revealing a new potential molecular communication between mother and young during mammalian lactation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1012) contains supplementary material, which is available to authorized users.     

Gelsolin expression in sheep milk somatic cells during lactation

The identification of genes involved in phenotypes related to milk quality is important for both economic and health aspects in livestock production. The aim of this study was to assess the level of gelsolin gene expression in two breeds of dairy sheep – Sarda and Gentile – with pronounced differences in quantitative and qualitative milk traits. Gelsolin, a type of actin-modulating proteins is involved in the processes of actin remodeling during cell growth and apoptosis; therefore a role of this protein in mammary changes during lactation was here hypothesized. Individual milk samples were collected three times during lactation from 26 ewes of the two breeds. The differential gene expression of gelsolin in the two breeds and the three lactation times was estimated by quantitative PCR on RNA extracted from milk somatic cells. Correlations of gelsolin gene expression with milk yield and quality and days of lactation were also estimated. The results showed that gelsolin gene expression was significantly higher in the Sarda compared to the Gentile at each lactation stage, in agreement with the longer lactation duration and the higher daily milk yield of the first breed. Significant correlations of gelsolin gene expression were found with milk fat content in Sarda breed (−0.46, P<0.05). Gelsolin expression analysis confirmed the link between gelsolin gene function and milk fat content of sheep.     

Jennet milk production during the lactation in a Sicilian farming system

In Italy, the interest for jennet milk production has recently developed. An 18-month-long experiment was carried out on a jennet farm near Milo (CT), where 24 jennets, which derived from the Ragusana breed, were tested for milk yield and composition over an entire lactation period. The jennets were fed with hay and concentrate in a large paddock. From the 28th post-foaling day to the end of the lactation, the jennets were machine-milked twice a day with an in-between milking interval of 5 h. The milk amount from each jennet was recorded every 3 weeks and individual samples were collected and analyzed for fat, protein, casein, non-proteic nitrogen, lactose and somatic cell count. This study showed that jennets at Sicilian latitudes are not seasonal polyestrous. The daily milk yield, the length of lactation and the milk characteristics varied depending on the foaling season. The total average milk production was 490 ± 36 kg in 295 ± 12 post-foaling days, considering two milking records per day. During the lactation, milk yield decreased constantly from 1.98 to 1.28 kg/jennet per day. When looking at the jennet milk quality during lactation, the percentage of fat and protein decreased, while the lactose percentage increased, according to a tendency apparently unique for equines when compared to the ruminants. When looking at the productive season, spring generally gave the best qualitative and quantitative results. Based on these results, jennet milk yield and quality could be improved; furthermore, jennet milk production may turn out to be a profitable business.     

Effects of genistein or soy milk during late gestation and lactation on adult uterine organization in the rat

In utero and lactational exposure to estrogenic agents has been shown to influence morphological and functional development of reproductive tissues. Thus, consumption of dietary phytoestrogens, such as isoflavones, during pregnancy and lactation could influence important periods of development, when the fetus and neonate are more sensitive to estrogen exposure. In this study, reproductive outcomes after developmental exposure to isoflavones were examined in Long-Evans rats maternally exposed to isoflavones via a commercial soy beverage or as the isolated isoflavone, genistein. Most reproductive endpoints examined at birth, weaning, and 2 months of age were not significantly modified in pups of either sex after lactational exposure to soy milk (provided to the dams in place of drinking water) from birth until weaning. However, soy milk exposure induced a significant increase in progesterone receptor (PR) in the uterine glandular epithelium of the 2-month-old pups. In pregnant dams treated with genistein (GEN; 15 mg/kg body weight) by gavage, from Gestational Day 14 through weaning, PR expression in the uterine glandular epithelium from 2-month-old GEN-treated females (postexposure) was also significantly increased. Diethylstilbesterol (DES) also stimulated uterine PR expression only in the glandular but not luminal epithelial cells. However, unlike DES, in utero/lactational exposure to GEN did not increase expression of the proliferation marker, proliferating cell nuclear antigen (PCNA), in the luminal epithelial cells of the 2-month-old rat uteri. These experiments demonstrate that developmental exposure to dietary isoflavones, at levels comparable to the ranges of human exposure, modify expression of the estrogen-regulated PR in the uterus of sexually mature rats weeks after exposure ended. Since the PR is essential for regulating key female reproductive processes, such as uterine proliferation, implantation, and maintenance of pregnancy, its increased expression suggests that soy phytoestrogen exposure during reproductive development may have long-term reproductive health consequences.     

Lactoperoxidase and thiocyanate contents of goats' milk during lactation

The lactoperoxidase level in milk from 10 goats throughout a 150 d lactation period was 1.55 units/ml, with a range of 0.05-3.55 units/ml for invidiual samples. Samples obtained 0–24 h after kidding exhibited the lowest mean concentration (0.50 units/ml). Mean thiocyanate content was 4.03 ppm, with a range of 0.67-11.17 ppm for indidual samples.     

Alteration of the N-glycome of bovine milk glycoproteins during early lactation

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1) , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.     

Leptin treatment during lactation increases transfer of iodine through the milk

We have previously shown that protein restriction during lactation is associated with changes in iodine secretion into the milk and that a pup's serum leptin concentration was increased at the end of lactation. So, here we evaluate whether leptin treatment during lactation affects iodine transfer through the milk to the pups. Lactating rats were divided into two groups: the leptin (Lep) group, single injected with recombinant rat leptin (8 microg/100g of body weight, daily for 3 consecutive days), and the control (C) group that received the same volume of saline. We studied iodine transfer to the pups through the milk on Days 4, 12 and 21 of lactation. In those days, the dams were separated from their pups for 4 h. Then, the mothers received an injection of 131I (2.22x10(4) Bq ip) and the pups were allowed to nurse for 2 h. The animals were sacrificed 2 h later. Leptin, total serum T3 and total serum T4 concentrations were higher (P<.05) in pups of Lep mothers only on Day 4, suggesting a higher transfer of leptin through the milk at this period, probably with a direct stimulatory effect on thyroid hormone secretion. In other periods, however, even without a detectable increase in a pup's serum leptin concentration, maternal leptin administration increased the pup's thyroid iodine uptake (Day 12, 39%; Day 21, 34%), probably caused by a higher transfer of iodine through the milk, since they had a higher gastric content of 131I on Days 12 (31%) and 21 (128%).     

Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.