Quantitation of calmodulin in the mammary gland of the lactating sow

It has been suggested that calmodulin, a calcium-binding protein, has a functional role during milk secretion. High levels of calmodulin are present during lactation in rat mammary glands and a substantial increase has been observed in the bovine mammary gland prior to parturition. In the sow, regressed glands involute while suckled glands remain highly active even though they are under the same hormonal influence. In this study, tissue samples were taken from suckled and regressed glands of the same sow at both peak and late lactation. Calmodulin and total protein were measured in tissue homogenate supernatants. Residual milk was apparent in regressed glands during mid lactation but not in the same glands by late lactation. Calmodulin levels in tissue were the same for both suckled and regressed glands. There was a slight but non-significant increase in the tissue calmodulin level from peak to late lactation. Protein levels declined significantly from mid to the late stage of lactation. There was no change in protein level between the suckled and regressed glands. Calmodulin may be responsible for casein phosphorylation and/or the mediation of prolactin action on the gland. The precise regulatory mechanisms relating hormonal control to calmodulin levels during lactation need further investigation.     

Preparation of an epithelium-free mammary fat pad and subsequent mammogenesis in ewes

The objective of this study was to develop a surgical procedure for the preparation of an epithelium-free mammary fat pad (cleared mammary fat pad; CFP) in ewes. At 7 to 10 d of age, ewe lambs (n = 43, mean BW 9.2 +/- .2 kg at 14 d) were sedated and one mammary gland was locally anesthetized. An incision circumscribing the base of the teat was made and blunt dissection was performed through the extraneous mammary fat pad tissue to enable the parenchyma and teat to be wholly removed. Failure to completely remove the epithelium enabled it to regenerate and grow into the mammary fat pad. Mean diameter of the parenchymal rudiment at 7 to 10 d of age was 8.9 +/- .5 mm (range of 5 to 16 mm). The excision site was closed with wound clips and recovered lambs returned to their dams. The contralateral mammary gland remained intact, allowing it to undergo normal development. Live weight gain was unaffected by this procedure. Ewes were subsequently slaughtered in groups at various stages of prepuberty, puberty, gestation, and lactation. Of 39 operated glands recovered, only one demonstrated epithelial outgrowth within the CFP. Parenchyma within the contralateral, intact gland underwent phases of rapid growth in prepuberty, puberty, and late gestation and was capable of milk synthesis after steroid induction or parturition. Change in weight of the CFP paralleled that of the intact mammary gland to 100 d of pregnancy. Sham CFP surgery was performed on four additional ewes wherein the parenchyma was completely excised and immediately replaced. Sham-operated epithelium populated the mammary fat pad and synthesized milk that could be expressed from the teat. A CFP in sheep will be a useful model for future investigations into the local growth regulatory mechanisms associated with ruminant mammogenesis.     

Aberrant development of mammary glands, but precocious expression of beta-casein in transgenic females ubiquitously expressing whey acidic protein transgene

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.     

乳腺中脂肪细胞的转化和更迭

雌性动物的乳腺组织是由胚胎时期的外胚层发育而来,出生时只有少量导管和皮下基质结构,而后在性成熟、妊娠和泌乳期乳腺发育达到峰值,这一特点使乳腺成为出生后唯一可以重复再生的器官。在乳腺发育到退化的循环中,乳腺的上皮细胞、基质白色脂肪细胞、棕色脂肪细胞和肌上皮细胞经历了一系列转化和更迭,脂肪细胞的动态转化反映了乳腺的功能变化。研究乳腺细胞的转化和更迭与母畜的泌乳直接相关,对延续母畜的生产效率有重要意义。本文就乳腺中脂肪细胞转化方面的最新研究进展进行综述,为深度揭示乳腺发育过程中细胞更迭的机制提供前沿研究信息。     

Changes in tissue composition associated with mammary gland growth during lactation in sows.

Twenty-four primiparous sows were used to determine the extent of mammary gland growth during lactation. Litter size was set to nine or 10 pigs immediately after birth. Sows were slaughtered in groups representing d 0 (within 12 h after farrowing), 5, 10, 14, 21, and 28 of lactation. Sows were provided 17.5 Mcal ME and 65 g of lysine per day during lactation. Mammary glands were collected at slaughter and trimmed of skin and extraneous fat pad. Each gland was weighed, cut in half to measure cross-sectional area, and ground for chemical analysis. Dry matter content, dry fat-free tissue (DFFT) content, protein content, amino acids composition, ash content, and DNA content were measured. Only glands known to have been suckled were included in these data. Wet and dry tissue weight; cross-sectional area; and the amount of DFFT, tissue protein, and amino acids in each suckled mammary gland increased (P < .05) during lactation to a peak on d 21. Fat percentage of each suckled gland declined (P < .05) and the percentage of protein and DFFT increased (P < .05) as lactation progressed. These results suggest that hypertrophy occurred in the tissue during lactation. There was a linear increase in the amount and percentage of DNA during lactation (P < .05), suggesting hyperplasia of the mammary tissue. Mammary tissue growth continues in suckled glands during lactation in sows, with gland wet weight increased by 55% and total gland DNA increased by 100% between d 5 and 21 of lactation.     

Effect of nutrient intake on mammary gland growth in lactating sows

Sixty-one primiparous sows were used to determine the response of mammary gland growth to different energy and protein intakes during lactation. After birth, litter size was set to 9 or 10 pigs. Sows were slaughtered at selected times up to 30 d of lactation. Individual sows were fed one of four diets that were combinations of different amounts of energy and protein (3.0 Mcal ME and 8.0 g lysine/kg diet; 3.0 Mcal ME and 16.2 g lysine/kg diet; 3.5 Mcal ME and 6.4 g lysine/kg diet; or 3.5 Mcal ME and 13.0 g lysine/kg diet). Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was weighed, cut in half to measure cross-sectional area, ground, and stored at -20 degrees C for chemical analysis. Frozen, ground tissue was used to determine dry matter, dry fat-free tissue (DFFT), total tissue protein, ash, and DNA content. Only glands known to have been suckled were included in this data set. Response surface regression was used for statistical analysis. The percentage of protein, fat, ash, and DNA in each suckled mammary gland was affected only by total energy intake (P<.05). The percentage of dry tissue and fat decreased as the total energy consumed during lactation increased, whereas the percentage of protein and DFFT increased as total energy intake increased. There were quadratic effects (P<.05) of both total energy and protein intake on wet weight, dry weight, protein amount, DFFT amount, and DNA amount of each suckled mammary gland during lactation. This study shows that mammary gland growth is affected by nutrient intake during lactation. The weight of suckled mammary glands and the amount of mammary tissue protein, DFFT, and total DNA were maximal on d 27.5 of lactation when sows had consumed an average of 16.9 Mcal of ME and 55 g of lysine per day during lactation. Provision of adequate amounts of nutrients to sows during lactation is important for achieving maximal growth of mammary glands and maximal milk production.     

Effects of intra-mammary bacterial infection with coagulase negative staphylococci and stage of lactation on shedding of epithelial cells and infiltration of leukocytes into milk: comparison among cows, goats and sheep

The effects of mammary gland bacterial infection and stage of lactation on leukocyte infiltration into the mammary gland were compared among cows, goats and sheep. Animals were at two stages of lactation: mid or late. In mid-lactation animals, bacterial-free glands and coagulase negative Staphylococcus (CNS)-infected glands were compared. In late lactation only uninfected glands were studied. Of mid-lactation bacteria-free animals, goats had the highest number of leukocytes and % polymorphonuclears (PMNs), whereas sheep had the lowest and leukocytes number in cows were intermediate between sheep and goats. Based on %PMN, two cell clusters were found in sheep, which overlapped with the parallel cell clusters of cows and goats, but with a slightly higher number of leukocytes in each cell cluster. At late lactation, goats had higher values for %PMN and leukocyte numbers in comparison to cows, which had a similar cellular profile to sheep. The cellular immune response to CNS infection was similar for the three animal species, although the number of cells was different, while the basal cell level at mid-lactation and especially at the end of lactation was species specific.     

Mammary-derived growth inhibitor in lactation and involution.

Presence of mammary-derived growth inhibitor (MDGI) in mammary tissue of lactating and involuted cows was investigated. Eighteen lactating, non-pregnant high-producing Holstein cows were randomly assigned to 3 experimental groups of 6 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group were slaughtered at 2-3 d, and the others at 4-8 d following sudden cessation of milking. Cessation of milking occurred at approximately 300 d in lactation. Western blot analysis revealed the presence of MDGI in the cytosolic and microsomal fractions of mammary tissue homogenates. High levels of MDGI were detected in mammary tissue obtained from lactating non-pregnant cows. A dramatic reduction in MDGI was observed in early involution (2-3 or 4-8 d following cessation of milking). These data suggest that a relationship exists between MDGI levels and the physiological status of the gland. Lack of MDGI may play a role during the processes of mammary involution and development prior to parturition.     

Apoptosis and oxidative stress of infiltrated neutrophils obtained from mammary glands of goats during various stages of lactation

OBJECTIVE: To examine apoptosis in infiltrated neutrophils during involution of mammary glands and compare them with those obtained during late and peak lactation, and to measure oxidative stress and activities of antioxidant enzymes and determine involvement of free radicals in apoptosis of infiltrated neutrophils. SAMPLE POPULATION: Neutrophils from mammary gland secretions of 8 goats at 4 stages (late and peak lactation and 1 and 2 weeks after end of lactation). PROCEDURE: DNA fragmentation was evaluated to characterize apoptosis. Concentration of thiobarbituric acid reactive substances (TBARS) was used to evaluate oxidative stress. Activities of superoxide dismutase and glutathione peroxidase were determined. RESULTS: Neutrophils from secretions obtained after end of lactation of all goats and from late-lactation milk of some goats underwent prominent apoptosis, whereas neutrophils from peak lactation secretions did not. Higher lipid peroxidation and lower antioxidant enzyme activities in neutrophils during involution were observed, compared with those during late and peak lactation. A significant negative correlation existed between TBARS concentrations and antioxidant enzyme activities during the nonlactating period. CONCLUSIONS AND CLINICAL RELEVANCE: Apoptosis is a feature of infiltrated neutrophils during involution of mammary glands in goats. This feature may allow prompt resorption and clearance of infiltrated neutrophils without damaging surrounding tissues. Increased oxidative stress in infiltrated neutrophils from secretions obtained after end of lactation is probably related to a deficiency in antioxidant enzyme activities. Understanding the relationship between apoptosis and oxidative stress will lead to new strategies for manipulating involution and reducing tissue damage.     

Mammary gland growth as influenced by litter size in lactating sows: impact on lysine requirement

Twenty-eight primiparous sows were used to determine the effect of litter size on the growth of mammary glands and nursing pigs during lactation. Litter size was set to 6, 7, 8, 9, 10, 11, or 12 pigs by cross-fostering immediately after birth. Four sows were allotted to each litter-size group. Sows were allowed to consume a daily maximum of 13.6 Mcal ME and 46.3 g of lysine during lactation. Sows were slaughtered on d 21 (20.6+/-1.1) of lactation. Mammary glands were collected at slaughter and trimmed of skin and the extraneous fat pad. Each gland was separated, weighed, and ground for chemical analysis. Dry matter, dry fat-free tissue (DFFT), crude protein, ash, and DNA contents were measured. Only glands known to have been nursed were included in the data set. Wet and dry weights and the amounts of DFFT, protein, DNA, ash, and fat in individual nursed mammary glands linearly decreased (P<.05) as litter size increased. Percentages of DFFT, protein, and DNA were quadratically affected (P<.05) by litter size on d 21 of lactation. Total mammary wet and dry weights and total DFFT, protein, DNA, fat, and ash amount of all nursed mammary glands of each sow were increased as litter size increased (P<.05). Changing litter size from 6 to 12 pigs resulted in 2,098, 432, 253, 227, 4.4, 178, and 20 g increases in the amounts of total mammary wet weight, dry weight, DFFT, protein, DNA, fat, and ash, respectively, on d 21 of lactation. Litter weight gain was 18.1 kg greater in sows with 12 pigs than in sows with 6 pigs. Sows with a larger litter size had a greater increase in total mass of mammary gland tissue and litter weight but had lower growth of individual nursed mammary glands and individual pigs than sows with the smaller litter size. The need for nutrients to support additional mammary gland and litter growth as litter size increases should be considered when estimating nutrient requirements for lactating sows. Sows need an additional .96 g lysine per day to account for mammary gland growth for each pig added to a litter.     

Research Progress on the Development of Mammary Glands

Mammary gland is a kind of exocrine glands and a dynamic organ.Structure of mammary gland changes throughout the female reproductive cycles.Development of mammary gland occurs in the defined stages that are closely linted to sexual development and reproduction, namely embryonic, prepubertal and pubertal stages, pregnancy, lactation and involution.In this review, the roles of epithelial-stromal (mesenchymal) interactions in embryonic and postnatal mammary development were summarized, elaborated the regulations of different kinds of hormones on the mammary development after pubertal stage, and described the discovery of mammary stem cell populations (MaSC) and selection markers of mice and human.The objective of this work was to learn the basic processes and potential regulation mechanisms of the mammary development to provide some help for the researchers in the field of mammary gland.     

The effect of insulin and relaxin upon mitosis (in vitro) in mammary tissue from pregnant and lactating pigs

The rate of cellular proliferation in the mammary glands of pigs during late gestation and lactation was assessed by measuring the incorporation of ³H-thymidine (T₁) into the DNA of mammary gland explants in vitro. The T₁ showed a linear response over the first 9 hr in vitro, and was not affected by the addition of 500 ng insulin/ml medium. From day 100 to parturition the T₁ rose, reached a peak at 2 d after parturition and declined during lactation to the lowest levels seen at day 21 of lactation.

The inclusion of 0–1000 ng relaxin/ml medium on T₁ at 24–72 hr in vitro had no effect in stimulating T₁ in mammary tissue explants taken from either pregnant or lactating pigs.     

乳腺发育的研究进展

乳腺属于外分泌腺的一种,在雌性动物生殖周期中呈动态性变化。乳腺的发育可根据动物性别发育和繁殖时期的不同分为胚胎期、青春期前期、青春期、妊娠期、泌乳期和退化期。作者总结了上皮-基质(间质)之间相互作用对胚胎时期及出生后动物个体乳腺发育的影响,详细阐述了乳腺不同发育阶段不同种类激素所发挥的调控作用,并针对小鼠、人乳腺干细胞的发现及标记物进行简要描述。本综述旨在清晰、深入地揭示乳腺发育的基本过程及潜在的调控机理,为进一步了解乳腺相关方面的研究工作提供参考资料。     

Local immunity in the mammary gland.

The mammary glands of pregnant and non-pregnant sheep were stimulated by infusion of killed Staphylococcus aureus, and the lymphoid cell response delineated with a panel of monoclonal antibodies. Seven days after antigen infusion, the mammary glands of both pregnant and non-pregnant sheep displayed a striking feature, characterised by the presence of numerous CD45R+ MHC class II+ B cells in the periductal connective tissues. These cells were seen to be clustering around blood capillaries with very prominent endothelial cell lining. Some CD5+ CD4+ lymphocytes were scattered among the B-cell clusters, whereas a few CD8+ lymphocytes were seen mainly at the periphery of the B-cell clusters. Fourteen days after antigen infusion, numerous plasma cells were observed, most of them being of the IgA isotype. Seven days after parturition (approximately 40 days after antigen infusion) the number of lymphocytes and plasma cells in the infused glands had declined dramatically. These data indicate that B cell and helper T-cell interaction can take place at the local sites of antigen stimulation in the mammary gland.     

Growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution of the bovine mammary gland: relation to secretion composition

Mammary secretions from 12 Holstein dairy cows were collected to evaluate growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution and during physiologic transitions of the mammary gland. Mammary secretions obtained during late lactation poorly inhibited growth of E coli and K pneumoniae. However, as involution progressed, mammary secretions increasingly inhibited growth of both coliform mastitis pathogens. Greatest inhibition of E coli and K pneumoniae growth was observed when mammary glands were fully involuted. Growth inhibition remained high until 7 days before parturition, and then it decreased significantly (P less than 0.05) to that observed during late lactation. Inhibition of coliform mastitis pathogen growth was associated with high concentrations of lactoferrin and immunoglobulin G, decreased citrate concentration, and a low citrate to lactoferrin molar ratio. These data suggested that differences in susceptibility or resistance to new intramammary infection with coliform mastitis pathogens during the nonlactating period may be attributable, in part, to marked changes in mammary secretion composition that develop during physiologic transitions of the mammary gland. Resistance of the fully involuted mammary gland to coliform infection may be associated with high concentrations of natural protective factors.     

Effects of coumestrol administration to maternal mice during pregnancy and lactation on immunoblogulin A‐secreting cells in mammary glands

Mortality and morbidity of neonates continue to be major problems in humans and animals, and immunoblogulin A (IgA) provides protection against microbial antigens at mucosal surfaces. The present study was conducted to clarify the effects of coumestrol administration to maternal mice during pregnancy and lactation on IgA antibody‐secreting cells (ASC) in mammary glands in lactating mice. From 6.5 to 16.5 days post coitus and 1 to 13 days post partum (dpp), maternal mice were administered coumestrol at 200 μg/kg body weight/day. Coumestrol administration increased the number of IgA ASC and the messenger RNA expression of IgA C‐region and vascular cell adhesion molecule‐1 in mammary glands of maternal mice at 14 dpp, but coumestrol administration had no effect on the number of IgA ASC in the ileum. Coumestrol administration increased serum IgA concentration in maternal mice at 14 dpp, but IgA concentrations in serum, stomach contents, intestine and feces of neonatal mice were not affected by treatment. These results imply that coumestrol administration to maternal mice during pregnancy and lactation is effective in increasing the numbers of IgA ASC in mammary glands during lactation owing to the activated messenger RNA expressions of IgA C‐region and vascular cell adhesion molecule‐1 in mammary gland.     

Endocrinology of milk production

The physiology of lactation includes development of the mammary gland from the foetal to the adult stage, further development during pregnancy and onset of lactation, with the accompanying metabolic and behavioural adaptation. At the onset of pregnancy the endocrine system undergoes dramatic changes. The growth of the mammary gland is stimulated by growth hormone and prolactin, adrenocortical steroids, oestrogens and progesterone, and that of the gastrointestinal (GI) tract by gastrin, CCK and secretin. The onset of lactation is accompanied by increases in the blood volume, cardiac output, mammary blood flow and blood flow through the GI-tract and liver, aiming to provide the udder with nutrients and hormones for regulation of milk synthesis. Food intake and distribution of nutrients to the mammary gland are partially regulated by hormones as well as the repartitioning of nutrients away from body stores towards the udder. To improve milk production, administration of growth hormone has been practised, but also much discussed. Besides central mechanisms, local mechanisms within the mammary gland regulate initiation of lactation, maintenance, regulation of blood flow and mammary gland cell apoptosis. Most of the milk in a filled dairy cow udder is stored in the alveolar compartments. The milk ejection reflex must be activated to gain access to the udder milk, i.e. oxytocin contracts the myoepithelial cells. Recent studies show that vasopressin may also elicit milk ejection. More efficient oxytocin release is achieved if the cows are fed during milking. Beyond milk let down, oxytocin influences maternal behaviour and metabolism. Furthermore, it has been indicated that suckling or milking activates a vagal reflex, which may link the milk production to the endocrine system of the GI-tract. The question has been raised whether the mammary gland is a supporting or consuming organ.