Bovine pituitary, kidney, uterine and mammary gland extracts contain bovine mammary epithelium growth factors that synergise with IGF-I and fetal calf serum: indication for involvement of GTP-binding proteins

Bovine mammary undifferentiated epithelial cells from young female calves, cultured in three-dimensional collagen gels in serum-free medium exhibited ultrastructural organization that resembled the in vivo situation. Extracts of bovine pituitary, kidney, uterus and mammary gland, stimulated cell proliferation in a dose-dependent manner. This mitogenic activity strongly synergised with the existant growth factors (GFs) in FCS and with IGF-I, while the addition of EGF had only minor effect. No synergistic manifestation was found with cholera toxin but pertussis toxin inhibited the growth-promoting activity of all four extracts. Other experiments indicated that this mitogenic activity does not result from prolactin, growth hormone or fibroblast growth factor. The present and former results, in which synergism between IGF-I and cholera toxin was demonstrated, suggest therefore, that the mitogenesis of normal mammary epithelial cells regulated by several tissue derived growth factors, consists of at least two pathways which are distinct from those activated by EGF and IGF-I. One of these pathways indicates involvement of pertussis toxin-sensitive GTP-binding proteins, and the other, activation of cholera toxin-sensitive adenylate cyclase.     

Partial purification and characterization of proteins with growth promoting activities from ovine mammary gland secretions.

Developmental regulation of growth promoting activities in mammary secretions of pregnant Awassi ewes was defined, and growth factors contained in these secretions were partially purified and characterised. Mammary secretions from pregnant ewes enhanced fibroblast cell (AKR-2B) and mammary cell (CID-9 cell strain) proliferation to levels comparable to that induced by 10% Foetal calf serum. Major milk proteins in mammary secretions collected from pregnant ewes one month prior to lambing up to one week after lambing, were resolved by SDS-PAGE, while gelatinases were resolved by zymography. Gelatinase activity was noted prior to P134 and decreased thereafter to reach a minimum during lactation. This decrease was concomitant with the onset of casein production. It is during this critical developmental period that highest growth promoting activity in mammary secretions was detected.Secretions with highest growth promoting activity were fractionated by ion exchange and gel filtration chromatography. Two heat-resistant, trypsin/chymotrypsin sensitive, growth-promoting activities were characterised. The first, designated ovine mammary derived growth factor-1 (oMDGF-1), had around a 30 kDa molecular weight and eluted at 0.65 M NaCl gradient on cation ion exchange chromatography. The second, oMDGF-2, eluted under gel filtration conditions at a molecular weight of 50 kDa and 150 kDa. oMDGF-1 induced changes in Connexin 43, but not in beta-casein mRNA expression by CID-9 mammary cells.In conclusion, growth factor activities in ewe mammary secretions peak during gestation at a period that overlaps maximal gelatinase expression and precedes milk protein synthesis. The factors modulate mammary cell function and may play a role in mammary gland development.     

Effect of mammary secretions on functions of polymorphonuclear leukocytes in pigs

OBJECTIVE: To determine the effects of porcine mammary secretions on polymorphonuclear (PMN) leukocyte function and to relate concentrations of estradiol-17beta and cortisol in mammary secretions to PMN cell function. SAMPLE POPULATION: Mammary secretions from 10 healthy sows and blood PMN leukocytes from 27 healthy sows. PROCEDURE: Mammary secretions were collected within 24 hours after parturition (colostrum) and 12 to 13 days later (milk). Chemoattractant properties were assessed by use of a cell migration assay. Phagocytic capacity of PMN cells in colostrum and milk was assessed by recording chemiluminescence following phagocytosis of Escherichia coli or zymosan. Estradiol-17beta and cortisol concentrations were determined by use of radioimmunoassays. RESULTS: Chemoattractant properties of colostrum and milk were significantly greater than that of zymosan-activated serum. However, chemoattractant properties did not differ significantly between the 2 types of secretions. The capacity of PMN cells in colostrum to phagocytose either zymosan or E. coli was less, compared with cells in milk, and the ability of cells in either type of mammary secretion to phagocytose E. coli was greater than the ability to phagocytose zymosan. Concentrations of estradiol-17beta and cortisol were greater in colostrum, compared with milk. No clear relation was evident between PMN cell activity and hormone concentrations in mammary secretions. CONCLUSIONS AND CLINICAL RELEVANCE: Although chemoattractant properties of colostrum and milk did not differ, the phagocytic capacity of PMN cells in colostrum was significantly less than that of cells in milk. This may predispose sows to coliform mastitis during the early postparturient period.     

Colostral growth factors. Possible role in bovine udder epithelial cell regeneration.

Growth of the secretory epithelium during prepartum time, and for a short period after calving, is under hormonal control by estrogen, progesterone and prolactin. The mechanism(s) by which these hormones act is not known but colostrum and milk have been shown to contain different growth promoting substances. In an attempt to unravel these relationships the effect of bovine colostrum on cellular proliferation in vitro have been characterized. Colostral thermostable factors not present in milk nor associated with fat, potently induce the proliferation associated enzyme, ornithine decarboxylase, in fibroblast cell lines. However, mammary epithelial cells appear to proliferate in response to different colostral heat sensitive factor(s) that await further characterization.     

Course of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in mammary secretions of the goat during end-pregnancy and early lactation

The epidermal growth factor (EGF) plays a crucial role in mammogenesis in many species. In ruminants, studies are limited, as EGF does not occur in peripheral plasma and specific analytical systems do not exist. Therefore a heterologous radioimmunoassay based on rhEGF was set up to monitor EGF in mammary gland secretions from goats during end-pregnancy and early lactation. IGF-I was measured with an established radioimmunoassay. Samples were collected from 13 goats for 25 days ante-partum and 25 days post-partum. Mammary gland secretions were obtained ante-partum by removing a small amount of the udder secretions (control half) or milking (stimulated half). Post-partum normal milk samples were collected. Blood samples were drawn by jugular venipuncture for the same period. EGF was found to occur in different molecular weight forms in the mammary glands. For routine measurements these proteins were extracted with acetone and not further separated. IGF-I and EGF concentrations in mammary secretions and similarly IGF-I in blood were high ante-partum and decreased slightly towards birth. IGF-I but not EGF is found in the peripheral plasma. Whereas IGF-I concentrations in blood were quite constant post-partum, IGF-I and EGF dropped in mammary secretions close to the detection limits. The decrease was more pronounced in the stimulated half than in the control half. The data support a synergistic role for EGF and IGF-I for mammogenesis. Both factors are further influenced by the milking stimulus and thus the functional state of the udder.     

Colostral and milk insulin-like growth factors and related substances: mammary gland and neonatal (intestinal and systemic) targets

The identification of hormones and regulatory factors in colostrum and milk has led to intensive investigations on their roles in the development and maintenance of the mammary and neonatal tissues. Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in transgenic mice influence mammary biology gland towards the end of lactation. In the bovine, IGFBP-3 is the major IGFBP in mammary secretions. In addition to binding IGFs, IGFBP-3 also binds to lactoferrin (Lf). Secreted IGFBP-3 re-enters mammary epithelial cells and with the presence of a nuclear localization sequence, IGFBP-3 and Lf enter the nucleus. Nuclear IGFBP-3 affects apoptotic signaling through the retinoic-x-receptors, while Lf affects apoptotic events through unknown mechanisms. Such interactions likely influence mammary development and involution. Furthermore, ingested colostral bioactive factors can exert regulatory functions in neonates. Intestinal receptors for IGFs and insulin are modified by age and/or diet. Feeding IGF-I had no effect, but colostrum extracts had small intestinal effects (stimulation of proliferation and villus size), suggesting that several factors, rather than one single bioactive factor were responsible. Systemic changes of metabolic and endocrine profiles in neonates depend on composition, amounts, time and duration of feeding colostrum. Early postnatal colostrum intake is not only important for the provision and absorption of immunoglobulins. Thus, in neonatal calves the lack of colostrum intake during the first 24h after birth results in a low immunoglobulin G, beta-carotene and Vitamin A status that persists for weeks and plasma patterns of fatty acids, essential amino acids and the glutamine/glutamate ratios are affected. In calves oral administration of IGF-I had no and feeding of colostrum whey extracts had only minor effects on metabolic and endocrine traits. Thus, mammary secretions influence regulatory functions of mammary and neonatal tissues.     

Antibody titres to lamb rotavirus in colostrum and milk of vaccinated ewes.

Ewes were vaccinated two to three weeks prior to mating with a formalin-treated preparation of lamb rotavirus. The colostrum and milk produced by vaccinated ewes after the subsequent pregnancy were shown to contain significantly higher titres of antibody to the virus than did mammary secretions from non-vaccinates. The virus neutralising antibody activity was associated with IgG in both colostrum and milk. However, IgG concentrations in the mammary secretions of vaccinates and non-vaccinates did not differ. It is suggested that vaccination of the dam may be of value in protecting the suckled neonatal lamb against rotavirus infection.     

A study of cells in the mammary secretions of sows

The morphology and some of the in vitro functional properties of the cells in the mammary secretions of sows have been examined. A mean cell yield of 1 × 10⁷ cells/ml was obtained from sow colostrum but during the first week post-partum the yield decreased approximately 10 fold. The polymorphonuclear leucocyte was the predominant cell type in colostrum and milk and was associated with varying proportions of lymphocytes, macrophages and epithelial cells. The phagocytes of sow milk ingested heat-killed yeast, although the phagocytic index for milk macrophages was low compared with autologous neutrophils and alveolar macrophages. Milk whey provided an effective opsonising medium for yeast ingestion. Intra-mammary immunisation of sows with ovalbumin induced antigen-reactive lymphocytes in both peripheral blood and milk.     

生长因子EGF、bFGF对猪孤雌胚体外发育的影响

在胚胎发育的不同阶段，分别在培养基中添加EGF和bFGF，研究EGF和bFGF对猪孤雌胚体外发育的作用。结果表明：在1细胞阶段添加EGF或bFGF，添加EGF能够显著提高孤雌胚的卵裂率（P〈0．05）；2～4细胞阶段添加EGF和bFGF，添加EGF组和添加bFGF组的囊胚率都显著高于对照组（P〈0．05），而添加bFGF组的囊胚率和囊胚细胞数都略高于对照组和添加EGF组。说明EGF和bFGF有利于猪孤雌胚的体外发育，而且，bF-GF能够通过提高囊胚细胞数而提高猪孤雌胚的质量。     

Recent progress of porcine milk components and mammary gland function

As the only nutritional source for newborn piglets,porcine colostrum and milk contain critical nutritional and immunological components including carbohydrates,lipids,and proteins(immunoglobulins).However,porcine milk composition is more complex than these three components.Recently,scientists identified additional and novel components of sow colostrum and milk,including exosomes,oligosaccharides,and bacteria,which possibly act as biological signals and modulate the intestinal environment and immune status in piglets and later in life.Evaluation of these nutritional and non-nutritional components in porcine milk will help better understand the nutritional and biological function of porcine colostrum and milk.Furthermore,some important functions of the porcine mammary gland have been reported in recent published literature.These preliminary studies hypothesized how glucose,amino acids,and fatty acids are transported from maternal blood to the porcine mammary gland for milk synthesis.Therefore,we summarized recent reports on sow milk composition and porcine mammary gland function in this review,with particular emphasis on macronutrient transfer and synthesis mechanisms,which might offer a possible approach for regulation of milk synthesis in the future.     

In vitro studies on the antimicrobial effects of colostrum and milk from vaccinated and unvaccinated pigs on Escherichia coli.

Unsupplemented porcine colostrum and milk exhibited a powerful bactericidal effect for porcine strains of E coli incubated in vitro at 37 degrees C. This activity was independent of complement but was susceptible to acid pH, to the presence of soluble iron and to the selective immunoprecipitation of IgG, IgA and IgM. Manifestation of bactericidal activity required bacteria in an active state of metabolism and the length of incubation was an important factor in demonstrating the quality of the anticoli activity, ie, proliferation-inhibitory, bacteriostatic or bactericidal. Whey obtained by acid precipitation or by the application of rennin was devoid of bactericidal activity but was capable of slowing down proliferation of E coli. There was no correlation between lysozyme and anticoli activity although the complete removal of lysozyme by adsorption on to bentonite reduced bactericidal titres. With very few exceptions the highest bactericidal titres were recorded for colostrum, but even 28 days post partum about one half of 22 undiluted milk samples exhibired bactericidal activity.     

Cytokine activity in bovine mammary gland secretions during the periparturient period.

The presence of cytokine activity in periparturient bovine mammary secretions was evaluated. Mammary secretions were modified for use in biological assays for interleukin-2 (IL-2) like and antiviral activity. The level of IL-2 like activity in mammary gland secretions was lower during the last week of gestation when compared to levels detected approximately two weeks prepartum. Antiviral titers gradually increased as parturition approached. Results from Western blots indicated that the antiviral activity observed in prepartum secretions may be due to tumor necrosis factor (TNF). Interferons (IFN) were not detected in the colostrum samples.     

Protective factors in mammary gland secretions during the periparturient period in the mare

Changes in mare mammary secretion composition were characterized prior to and after foaling. Eighteen mares were used for collection of mammary secretions from 23 days prepartum through 44 days postpartum. Concentrations of lactose, total protein, IgG and lysozyme (activity assay) were determined. Considerable variabilty was observed among mares. Mean lactose concentrations by mare were lower (P<.05) in the prepartum period compared with the postpartum period. Mean total protein and immunoglobulin G concentrations by mare were higher (P<.05) in the prepartum period compared with the postpartum period. Mean lysozyme activities by mare tended to be lower (P<.08) during the prepartum period compared with the postpartum period. Mammary secretion protein profiles, characterized by SDS-PAGE, were consistent with changes in concentrations of specific proteins measured in the secretions. Dynamic changes occurring in mammary secretions during the transition from prepartum accumulation of colostrum to postpartum production of milk include factors like IgG and lysozyme which have protective roles in the neonatal foal.     

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.     

Epidermal growth factor-like activity in mares' milk.

Epidermal growth factor (EGF)-like activity was measured in mares' colostrum and milk by radioreceptor assay. Milk samples were collected from 22 mares 1 or more times during early lactation. Samples of colostrum were taken after parturition and before the foal first suckled (presuckle), within 6 hours after the foal first suckled (postsuckle), and on days 1, 2, 4, and 8 of lactation. In the 5 mares from which milk samples were obtained at each sampling time, presuckle colostral mean EGF-like activity (17.8 ng/ml) was greatest (P less than 0.05). The mean values for EGF-like activity at all other sampling times were not significantly different from each other (postsuckle colostrum, 9.7 ng/ml; day 1, 9.6 ng/ml; day 2, 8.5 ng/ml; day 4, 8.0 ng/ml; day 8, 7.8 ng/ml).     

A simple, rapid method for differential cell counts in porcine mammary secretions.

The use of the fluorescent dye acridine orange for making differential cell counts in mammary secretions from sows was investigated, and the variations in cell type during the lactation period were also studied. In untreated samples of mammary secretions polymorphonuclear leucocytes, mononuclear phagocytes, lymphocytes and epithelial cells could be identified with certainty, and the methodological error was small (1.80 to 2.22 per cent). The mammary secretions could be stored at 4 degrees C for six hours without any effect on the differential count. Washing the secretions decreased the percentage of polymorphonuclear leucocytes and increased the percentage of epithelial cells. The polymorphonuclear leucocytes predominated in colostrum (58.0 to 65.5 per cent) and epithelial cells predominated in milk (60 to 89 per cent). Polymorphonuclear leucocytes were the predominant phagocytes in all mammary secretions (7.6 to 65.5 per cent).     

Secretory component and IgA expression by epithelial cells in sow mammary gland and mammary secretions

Secretory component (SC) and IgA expression of epithelial cells were studied in the mammary tissue and mammary secretions of sows. In mammary tissue, SC was not detected until day 105 of gestation. From the time of delivery (day 115) to the time of established lactation, the proportion of epithelial cells containing sc rose from 20 per cent to nearly 100 per cent. There was no IgA in alveolar epithelial cells until day 105 of gestation; on day 115, IgA positive epithelial cells were present in 10 per cent of the alveoli, which increased to 80 per cent during lactation. Epithelial cells represented more than 20 per cent of the total cells in colostrum, and predominated over leucocytes in milk. In colostrum, these epithelial cells (9 to 15 μm) showed weakly positive membrane, sc, contained cytoplasmic SC and had a limited capacity for in vitro proliferation. Ten per cent of epithelial cells contained intracytroplasmic IgA. In milk, the epithelial cells were larger (15 to 40 μm) with a higher expression of both membrane and intracytoplasmic sc; 66 per cent of these cells expressed intracytoplasmic IgA. These data showed that the capacity of mammary epithelium to process IgA to secretory IgA was complete at the end of mammary gland organisation, and established that the epithelial cells of milk contribute to the transfer of IgA to neonates.     

乳腺分泌物中体细胞的研究进展

不同物种的乳腺分泌物中含有的细胞成分被称为体细胞,其中包括淋巴细胞、白细胞、巨噬细胞和上皮细胞。物种、乳腺感染情况、不同生理阶段和饲养管理条件等因素均会影响乳中的体细胞数量和细胞类型。近年来,乳中体细胞得到了人们的关注和深入研究,显示出广阔的应用前景。人们利用从初乳和常乳中得到的乳腺上皮细胞已经成功进行了乳腺细胞的原代培养和建立了乳腺细胞系,为乳生成、被动免疫转移和乳腺癌的研究提供了良好平台。体细胞中提取的RNA代表了乳腺组织的基因表达,因此为研究乳腺组织的基因表达提供了方便、良好的来源。     

Composition and physiological functions of the porcine colostrum

The first secretion, 24-h post parturition of the mammary glands of sows, known as colostrum, is high in protein and low in lactose and fat. As a consequence of an insufficient ingestion of colostrum, more than 50% of piglets fail to reach weaning and die. The composition and some functions of colostrum have been previously reported. For example, colostrum carbohydrates consist of mainly lactose. Lipids in the colostrum are mostly triacylglycerols, but <1% is fatty acids, which may act as homeostasis regulators. Similarly, proteins are found mostly as casein and whey, the latter being ≥80% immunoglobulins. Colostrum-derived immunoglobulins and bioactive proteins such as azurocidin help the immune system of the piglet fend off infections. In addition, leukocytes and exosomes are other minor but nonetheless equally crucial bioactive components in the porcine colostrum. Modern pig farming has achieved increases in pig productivity and litter size, but this has been accomplished in detriment of the health and the survival rate of piglets. Therefore, porcine colostrum is now even more important in pig farming. In the present review, we discuss the current knowledge on the composition and physiological functions of the porcine colostrum and briefly propose future research directions.     

Inhibition of lacteal leukocyte phagocytosis by colostrum, nonlactating secretion, and mastitic milk

The Fc receptors on leukocytes obtained from normal milk, colostrum, nonlactating gland secretion, and mastitic milk were detected by rosette formation, using sensitized erythrocytes. The percentage of leukocytes bearing Fc receptors from normal milk was significantly (P less than 0.01) greater than that from other secretions. Fc receptors were found primarily on polymorphonuclear leukocytes from normal milk, mastitic milk, and colostrum. However, in nonlactating gland secretion obtained 6 weeks after milking was completed, Fc receptors were predominantly on macrophages. The low percentage of leukocytes bearing Fc receptors obtained from colostrum, nonlactating gland secretion obtained 7 days after milking was completed, and mastitic milk was associated with the presence of a blocking factor in these secretions, which specifically attached to Fc receptors. These secretions significantly (P less than 0.01) blocked the Fc receptors on leukocytes from normal milk and on other cells bearing FC receptors. The presence of Fc receptors on leukocytes obtained from normal milk was related to a high percentage of phagocytizing leukocytes through Fc receptors and a large number of phagocytized bacteria (phagocytic activities). In contrast, the low percentage of leukocytes bearing Fc receptors from colostrum, from nonlactating gland secretion (7 days after milking), and from mastitic milk was associated with depressed phagocytic activities. Preincubation of leukocytes from normal milk with whey from colostrum, nonlactating gland secretion (7 days after milking), and mastitic milk significantly (P less than 0.01) inhibited phagocytosis. This effect was associated with the blocking of Fc receptors by these secretions. Possible mechanisms for and implications of these findings are discussed.