Metabolic adaptations in goat mammary tissue during pregnancy and lactation

Metabolic adaptations of goat mammary tissue during pregnancy and lactation were monitored in serial biopsies of the tissue. Changes in the synthetic capacity of secretory cells were studied by combining measurements of enzyme activities with short-term culture of mammary explants to measure lactose, casein and total protein synthesis. By these criteria, the main phase of mammary differentiation began in late pregnancy and was essentially complete by Week 5 of lactation, coinciding with the achievement of peak milk yield. While milk yield declined after Week 5, the activities of key enzymes expressed per mg DNA and the rates of lactose and casein synthesis in mammary explants were maintained over a considerable period. The results suggest that changes in the synthetic capacity of epithelial cells may account for much of the rise in milk yield in early lactation, but are not responsible for the declining phase of milk production characteristic of lactation in ruminants.     

Seasonal changes in daily torpor patterns of free-ranging female and male Daubenton’s bats (Myotis daubentonii)

Daily torpor can provide significant energy and water savings in bats during cold ambient temperatures and food scarcity. However, it may reduce rates of foetal and juvenile development. Therefore, reproductive females should optimize development by minimizing times in torpor. To test this hypothesis, the use of torpor by female and male free-ranging Daubenton’s bats (Myotis daubentonii) during reproduction (gestation, lactation, and post-lactation period) was investigated in 1998 and 1999. Temperature-sensitive radio transmitters were attached to the bats to measure skin temperature. Simultaneously, ambient temperature was recorded. While both sexes became torpid during daytime, male bats used daily torpor (>6°C below individual active temperature) significantly more often during reproductive period (mean: 78.4 % of day time in May and 43 % in June) than females. Female bats went into daily torpor, particularly in late summer when juveniles were weaned (mean: 66.6 % of daytime). Lowest skin temperatures occurred in a female bat with 21.0°C during post-lactation. Skin temperatures of male bats fluctuated from 16.8°C in torpor to 37.2°C during times of activity. There was a significant effect of reproductive period on skin temperature in females whereas mean ambient temperature had no significant effect. However, mean ambient temperature affected mean skin temperatures in males. Our findings indicate that female Daubenton’s bats adopt their thermoregulatory behaviour in particular to optimize the juvenile development.     

Periodic fluctuations in the pulmonary surfactant system in Gould's wattled bat (Chalinolobus gouldii)

Pulmonary surfactant is a mixture of phospholipids, neutral lipids, and proteins that controls the surface tension of the fluid lining the lung. Surfactant amounts and composition are influenced by such physiological parameters as metabolic rate, activity, body temperature, and ventilation. Microchiropteran bats experience fluctuations in these parameters throughout their natural daily cycle of activity and torpor. The activity cycle of the microchiropteran bat Chalinolobus gouldii was studied over a 24-h period. Bats were maintained in a room at constant ambient temperature (24 degrees C) on an 8L : 16D cycle. Diurnal changes in the amount and composition of surfactant were measured at 4-h intervals throughout a 24-h period. The C. gouldii were most active at 2 a.m. and were torpid at 2 p.m. Alveolar surfactant increased 1.5-fold immediately after arousal. The proportion of disaturated phospholipid remained constant, while surfactant cholesterol levels increased 1.5-fold during torpor. Alveolar cholesterol in C. gouldii was six times lower than in other mammals. Cholesterol appears to function in maintaining surfactant fluidity during torpor in this species of bat.     

Effects of evaporative cooling on the regulation of body water and milk production in crossbred Holstein cattle in a tropical environment

The aim of this study was to determine how evaporative cooling modifies body function with respect to water metabolism and other variables relevant to milk synthesis in crossbred cattle. The study was conducted on two groups of 0.875HF:0.125RS crossbred Holstein cattle (87.5%) housed in an open-sided barn with a tiled roof (non-cooled animals) and in a close-sided barn under an evaporative cooling system (cooled animals). The maximum ambient temperature and relative humidity for the non-cooled group were 33 degrees C and 61%, with the corresponding values for the evaporatively cooled barn being 28 degrees C and 84%, respectively. The temperature humidity index (THI) of under non-cooled conditions was higher (P < 0.05) than that in the cooled barn. Rectal temperatures and respiration rates of non-cooled animals were higher (P < 0.05) than those of cooled animals. Daily dry matter intake (DMI) of cooled animals was higher while water intakes were lower (P < 0.05) than those of non-cooled animals. The mean absolute values of plasma volume, blood volume, and extracellular fluid (ECF) of cooled animals were significantly higher (P < 0.05) than those of non-cooled animals throughout all stages of lactation. Milk yields of cooled animals were higher by 42%, 36% and 79% on average than those of non-cooled animals during early-, mid- and late-lactation, respectively. The decline in milk yields as lactation advances was markedly apparent in late-lactating non-cooled animals, while no significant changes in milk composition at different stages of lactation were observed in either group. Mean arterial plasma concentrations, arteriovenous concentration differences (A-V differences) and the extraction ratio across the mammary gland for acetate, glucose and triglyceride of cooled animals were not significantly different compared with values for non-cooled animals. No differences were seen in plasma hormonal levels for triiodotyronine (T(3)) and insulin-like growth factor-1 (IGF-1), but plasma cortisol and thyroxine (T(4)) levels tended to be lower in non-cooled animals. This study suggests that low cooling temperature accompanied by high humidity influences a galactopoietic effect, in part through increases in ECF, blood volume and plasma volume in association with an increase in DMI, which partitions the distribution of nutrients to the mammary gland for milk synthesis. Cooled animals were unable to maintain high milk yield as lactation advances even though a high level of body fluids was maintained during long-term cooled exposure. The decline in milk yield, coinciding with a decrease in net energy for lactation as lactation advances, could be attributed to a local change within the mammary gland.     

Pten对奶牛乳腺上皮细胞活力及乳蛋白合成的影响

Pten作为抑癌基因,参与调控细胞生长、粘附、凋亡以及其它细胞活动.目前,国内外关于Pten在奶牛乳腺发育过程中表达及调节的研究鲜有报道.为了揭示Pten的表达与奶牛乳腺发育与泌乳之间的关系,本研究应用qRT-PCR技术检测Pten在不同泌乳时期和不同乳品质的奶牛乳腺组织中的表达差异,进而应用脂质体转染方法,通过siRNA介导的RNA干扰技术改变Pten基因在奶牛乳腺上皮细胞中的表达量,CASY法检测细胞活力,用ELISA试剂盒检测细胞分泌β-酪蛋白的含量,采用qRT-PCR、Western 印迹等技术检测Pten对奶牛乳腺上皮细胞中乳蛋白相关信号通路基因表达的影响.结果显示,泌乳期高乳品质奶牛乳腺组织中Pten表达水平显著低于泌乳期低乳品质及干乳期奶牛;Pten基因沉寂后,细胞活力提高,β-酪蛋白质量浓度增加,CSN2、AKT、MTOR、STAT5表达量增加.研究表明,Pten可通过抑制细胞活力和乳蛋白分泌而影响泌乳.     

Bovine mammary explant versus primary cell cultures: Effect of bovine somatotropin and insulinlike growth factor-I on DNA content and protein synthesis

Summary Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 ± 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulinlike growth factor-I, and somatotropin + insulinlike growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G₀G₁ phase was increased by: (a) insulinlike growth factor-I in explant cells; (b) somatotropin, insulinlike growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulinlike growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G₀G₁ or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more β-casein than primary cells but secretion trends for β-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.     

Environmental conditions, rather than season, determine torpor use and temperature selection in large mouse-eared bats (Myotis myotis)

We tested whether food availability, thermal environment and time of year affect torpor use and temperature selection in the large mouse-eared bat (Myotis myotis) in summer and winter. Food-deprived bats were torpid longer than bats offered food ad libitum. Bats placed in a gradient of low (0 degrees C-25 degrees C) ambient temperatures (T(a)) spent more time in torpor than bats in a gradient of high (7 degrees C-43 degrees C) T(a)'s. However, we did not observe seasonal variations in the use of torpor. Moreover, even when food deprived in winter, bats never entered prolonged torpor at T(a)'s characteristic of their natural hibernation. Instead, bats preferred shallow torpor at relatively high T(a), but they always maintained a difference between body and ambient temperatures of less than 2 degrees C. Calculations based on respirometric measurements of metabolic rate showed that food deprived bats spent less energy per unit of time in torpor than fed individuals, even when they entered torpor at higher T(a)'s. We conclude that T(a) likely serves as a signal of food availability and daily torpor is apparently an adaptation to unpredictable changes in food availability, such as its decrease in summer or its increase in winter. Thus, we interpret hibernation to be a second step in the evolution of heterothermy in bats, which allows survival in seasonal environments.     

Bovine lactoferrin in involuting mammary tissue

Bovine lactoferrin in involuting mammary tissue was identified by immunohistochemistry and tissue explant culture. Immunoreactive lactoferrin was associated with mammary epithelial cells. Immunostaining for lactoferrin increased during involution, in contrast to declining immunostaining of epithelia for the milk-specific protein β-lactoglobulin. Immunostaining for lactoferrin also was observed at the basal region of alveolar epithelia, perhaps in association with basement membrane components. Lactoferrin was preferentially synthesized in involuting mammary tissue compared with lactating tissue. Synthesis of lactoferrin in the involuting mammary gland occurs despite the apparent decline in synthesis of milk-specific proteins.     

转基因小鼠乳腺上皮细胞的体外培养及其对催乳素反应的研究

实现转基因生物乳腺反应器对外源蛋白的高效表达是目前生物制药亟待解决的难题。催乳素对泌乳期乳蛋白的合成与分泌具有重要的调控功能。通过转基因小鼠乳腺上皮细胞模型的建立,研究催乳素如何调控乳蛋白的表达,为提高乳腺反应器高效表达外源蛋白提供技术及理论支撑。应用机械破碎及胶原酶消化法,经差速贴壁纯化,成功培养含人转铁蛋白基因的小鼠乳腺上皮细胞,细胞上清液中检测到人转铁蛋白表达。细胞经牛催乳素诱导后人转铁蛋白的表达水平明显升高。利用转基因小鼠乳腺上皮细胞模型,可以进行催乳素和环境因素等对乳腺上皮细胞合成及分泌蛋白能力影响的研究。     

Activity patterns of pipistrelle bats (Pipistrellus pipistrellus) in Oxfordshire

A maternity colony of pipistrelle bats ( Pipisfrellus pipistrellus ), in Oxfordshire, was monitored between 1 March 1989 and 6 October 1989. An infra-red 'automatic bat counter' was installed at the roost, to record the number of bats entering and leaving each minute throughout the night. Air temperature, light intensity at sunset, cloud cover, wind speed and rain were recorded on each night of monitoring. Insect abundance was estimated on 18 nights.
The nightly activity pattern was found to be unimodal in pregnancy, bimodal during lactation and unimodal post-weaning. The mean time that each bat spent outside the roost ranged from 103–483 min, with a mean of 321 min.
Ambient air temperature and length of night were significant factors affecting mean time spent outside the roost. The percentage of the night which the bats spent away from the roost ranged from 22 to 88%, with a mean of 64%. There was a significant positive correlation between ambient air temperature and percentage of the night spent away from the roost. Insect abundance showed no significant correlation with the time that bats spent outside the roost. Wind and rain had no apparent effect on time spent outside the roost.     

The Dietary Protein/Carbohydrate Ratio Differentially Modifies Lipogenesis and Protein Synthesis in the Mammary Gland,Liver and Adipose Tissue during Gestation and Lactation

During gestation and lactation, a series of metabolic changes that are affected by the diet occurs in various organs of the mother. However, little is known about how the dietary protein (DP)/carbohydrate (DCH) ratio regulates the expression of metabolic genes in the mother. Therefore, the purpose of this work was to study the effect of consuming different percentages of DP/DCH, specifically 10/73, 20/63 and 30/53%, on the expression of genes involved in lipogenesis and protein synthesis in the mammary gland, liver and adipose tissue during gestation and lactation in dams. While the amount of weight gained during gestation was similar for all groups, only dams fed with 30/53% DP/DCH maintained their weight during lactation. In the mammary gland, the expression of the genes involved in lipogenesis, specifically SREBP1 and FAS, was dramatically increased, and the expression of the genes involved in protein synthesis, such as mTOR1, and the phosphorylation of its target protein, S6K, were also increased throughout pregnancy and lactation, regardless of the concentration of DP/DCH. In the liver and adipose tissue, the expression of the genes and proteins involved in lipid metabolism was dependent on the proportion of DP/DCH. The consumption of a low-protein/high-carbohydrate diet increased the expression of lipogenic genes in the liver and adipose tissue and the amount of lipid deposition in the liver. Conversely, the consumption of a high-protein/low-carbohydrate diet increased the expression of genes involved in amino acid oxidation in the liver during gestation. The metabolic adaptations reflected by the changes in the expression of metabolic genes indicate that the mammary gland has a priority for milk synthesis, whereas the adaptations in the liver and adipose tissue are responsible for providing nutrients to the mammary gland to sustain milk synthesis.     

Hypothermia versus torpor in response to cold stress in the native Australian mouse Pseudomys hermannsburgensis and the introduced house mouse Mus musculus

This study compared torpor as a response to food deprivation and low ambient temperature for the introduced house mouse (Mus musculus) and the Australian endemic sandy inland mouse (Pseudomys hermannsburgensis). The house mouse (mass 13.0+/-0.48 g) had a normothermic body temperature of 34.0+/-0.20 degrees C at ambient temperatures from 5 degrees C to 30 degrees C and a basal metabolic rate at 30 degrees C of 2.29+/-0.07 mL O2 g(-1) h(-1). It used torpor with spontaneous arousal at low ambient temperatures; body temperature during torpor was 20.5+/-3.30 degrees C at 15 degrees C. The sandy inland mouse (mass 11.7+/-0.16 g) had a normothermic T(b) of 33.0+/-0.38 degrees C between T(a) of 5 degrees C to 30 degrees C, and a BMR of 1.45+/-0.26 mL O2 g(-1) h(-1) at 30 degrees C. They became hypothermic at low T(a) (T(b) about 17.3 degrees C at T(a)=15 degrees C), but did not spontaneously arouse. They did, however, survive and become normothermic if returned to room temperature (23 degrees C). We conclude that this is hypothermia, not torpor. Consequently, house mice (Subfamily Murinae) appear to use torpor as an energy conservation strategy whereas sandy inland mice (Subfamily Conilurinae) do not, but can survive hypothermia. This may reflect a general phylogenetic pattern of metabolic reduction in rodents. On the other hand, this may be related to differences in the social structure of house mice (solitary) and sandy inland mice (communal).     

Proteomic analysis of microsomes from lactating bovine mammary gland

Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. We have carried out a proteomic analysis of mammary tissue to discover proteins that affect lipid metabolism. Unfractionated microsomes from lactating bovine mammary tissue were analyzed using one-dimensional SDS-PAGE with RPLC-ESI-MS/MS. This approach gave 703 proteins including 160 predicted transmembrane proteins. Proteins were classified according to their subcellular localizations and biological functions. Over 50 proteins were associated with cellular uptake, metabolism, and secretion of lipids, including some enzymes that have been previously associated with breast cancer and potential therapeutic targets. This database develops a proteomic view of the metabolic potential of mammary gland that can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation, and to further dissection of normal and pathological processes in mammary tissue.     

A novel whey protein synthesized only in late lactation by the mammary gland from the tammar (Macropus eugenii).

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.     

Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii)

Lactating tammars can provide two different milks simultaneously from adjacent glands to a young newborn (phase 2 of lactation) and an older animal at heel (phase 3 of lactation). Evidence that the two glands are controlled independently is shown by the capacity of mammary explants from these glands to synthesize different whey proteins and DNA and RNA at different rates. Prolactin is essential for the maintenance of milk synthesis, but its role in differential responses of the individual mammary glands remains to be established. Potential mechanisms for the control of milk synthesis are discussed.     

The Relationship between Cutaneous Water Loss and Thermoregulatory State in Kuhl's Pipistrelle Pipistrellus kuhlii, a Vespertillionid Bat

Abstract Total evaporative water loss is the sum of respiratory water loss (RWL) and cutaneous water loss (CWL) and constitutes the main avenue of water loss in bats. Because bats fly and have large surface-to-volume ratios, they potentially have high rates of RWL and CWL. Most species of small insectivorous bats have the ability to reduce their body temperature (T(b)) at rest, which substantially reduces energy expenditure and water loss. We hypothesized that bats reduce evaporative water loss during bouts of deep hypothermia (torpor) by decreasing RWL and CWL. We measured T(b), RWL, CWL, and resting metabolic rate (RMR) in Kuhl's pipistrelle Pipistrellus kuhlii, a small insectivorous bat. In support of our hypothesis, we found that RWL decreased with decreasing RMR. We found that CWL was lower in torpid individuals than in normothermic bats; however, bats in deep torpor had similar or higher CWL than bats in shallow torpor, suggesting that they exert a less effective physiological control over CWL when in deep torpor. Because insectivorous bats spend most of their lives in torpor or hibernation, the regulation of CWL in different heterothermic states has relevant ecological and evolutionary consequences.     

Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S₁-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.     

Metabolism of aminoacyl-p-nitroanilides by rat mammary tissue

We have examined the metabolism of aminoacyl-p-nitroanilides by rat mammary tissue isolated from rats during late pregnancy, peak lactation and late lactation. The rate of hydrolysis depended upon the chemical nature of the aminoacyl-p-nitroanilide compound and the physiological state of the donor animals. Thus, mammary tissue isolated from rats during late pregnancy and peak lactation hydrolysed aminoacyl-p-nitroanilides in the order L-met-p-nitroanilide=L-leu-p-nitroanilide>L-lys-p-nitroanilide>gamma- glu-p-nitroanilide. The order of activity was the same for mammary tissue taken from rats during late lactation except that L-lys-p-nitroanilide was hydrolysed at the same rate as the neutral aminoacyl-p-nitroanilides. Mammary tissue from peak lactating rats also hydrolysed alpha-L-glu-p-nitroanilide and alpha-L-asp-p-nitroanilide but to a lesser extent than the other compounds tested. The anionic aminoacyl-p-nitroanilides were able to trans-stimulate D-aspartate efflux from mammary tissue explants and the perfused mammary gland via the high-affinity anionic amino acid carrier. The clearance of gly-L-phe by the perfused mammary gland was markedly inhibited by L-phe. The results suggest that mammary tissue expresses a variety of dipeptidases at the basolateral aspect of the mammary epithelium which are capable of hydrolysing peptides extracellularly. These enzymes may be important for providing amino acids for milk protein synthesis and/or inactivating signal peptides.     

Hibernation by a free-ranging subtropical bat (Nyctophilus bifax)

Knowledge about torpor in free-ranging subtropical bats is scarce and it is widely believed that low and stable ambient temperatures are necessary for prolonged torpor. We present temperature-telemetry data from free-ranging male (n = 4) and female (n = 4) subtropical vespertilionid bats, Nyctophilus bifax (~10 g), exposed to pronounced daily fluctuations of ambient temperature. All bats used torpor on every day in winter and both males and females exhibited multi-day torpor bouts of up to 5.4 days. Although females were larger than males, patterns of torpor were similar in both sexes. Torpor use was correlated with prevailing weather conditions and, on days when bats remained torpid, maximum ambient temperature was significantly lower than on days when bats aroused. Moreover, the duration of interbout normothermic periods at night increased with increasing average nightly ambient temperature. Skin temperature of torpid bats varied by 10.2 ± 3.6°C day⁻¹ (n = 8, N = 47) and daily minimum skin temperature was positively correlated with the daily minimum ambient temperature. Our study shows that prolonged torpor is an important component of the winter ecology of a subtropical bat and that torpor and activity patterns of N. bifax predominantly reflect prevailing weather conditions.     

Metabolic proteomics of the liver and mammary gland during lactation

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by β-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and β-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than β-oxidation.