Sheep Mammary Gland Research Articles

Expression of amphiregulin in the sheep mammary gland

The reverse transcription–polymerase chain reaction (RT–PCR) was used to amplify, from sheep mammary gland total RNA, a 280 bp sequence of amphiregulin cDNA. Cloned and sequenced, it corresponded to the 78 amino acids of the major secreted form of amphiregulin, showing 81, 70 and 69% identity with human, rat and mouse amphiregulin, respectively. Expression of amphiregulin was detected by RT–PCR in the mammary gland at several developmental stages (fetal, lamb, early and late pregnant and lactating ewes) and in isolated myoepithelial cells. By Western blotting with an antiserum to human amphiregulin, two molecular weight forms, 27 and 51 kDa were detected in sheep mammary gland microsomal preparations, in a mammary gland extract after heparin affinity chromatography and in a medium conditioned by mammary epithelial cells. By immunocytochemistry, amphiregulin was detected in the cytoplasm and nuclei of luminal epithelial cells, myoepithelial cells and in intralobular stroma. An autocrine/paracrine role in sheep mammary growth is indicated.

Construction and analysis of an hn-cDNA library derived from the p-arm of pig chromosome 12.

Our aim is to find unidentified genes on specific pig chromosomes or chromosome fragments. Our approach has involved the construction of a heterogeneous nuclear complementary (hn-c) DNA library of the p-arm of pig Chromosome (Chr) 12, the only pig chromosome present in the pig x hamster hybrid cell line 8990. Total RNA was extracted from the cells and first-strand synthesis of hn-cDNA carried out with random and oligo dT primers. Pig hn-cDNA was isolated by amplification of first-strand synthesized hn-cDNA with primers specific for Short Interspersed Repeat Elements (SINEs) via the polymerase chain reaction (PCR). Hn-cDNAs were size selected and cloned in E. coli XL-1 blue cells with PCR-Script as the vector. The library consisted of 6000 clones. Clone inserts were amplified by PCR with vector-specific primers, and randomly picked inserts greater than 600 bp were sequenced. Homology searches were carried out with the FASTA search program on the GenEmbl database. Thirty clones were sequenced, and of these three showed strong homologies to GenEmbl sequences: (1) to sheep, mouse, human, and rat mammary gland factor (MGF); (2) to MLN-50, a gene that is amplified in human familial breast cancer and is present on human Chr 17; the latter is homologous to pig chromosome 12; (3) to a family of unassigned overlapping human ESTs. Of the other sequenced clones, seven were over 80% homologous with pig SINE sequences; three were over 75% homologous to human LINE sequences; six displayed open reading frames over a mean distance equivalent to 50 amino acids, although these showed no significant similarities with sequences in the databases. Using this approach, we have been able to identify several new genes on the p-arm of pig Chr 12. This is the first report of gene isolation from a library derived from a pig chromosome fragment.

Expression of gene‐gun injected plasmid DNA in the ovine mammary gland and in lymph nodes draining the injection site

Abstract A jet‐injection based gene‐gun delivery system has been evaluated as a means to transiently transfect the lactating mammary gland in vivo. The model expression plasmid contained the human growth hormone (hGH) structural gene driven by the human cytomegalovirus immediate early gene 1 promoter/enhancer region (CMV). Expression from plasmid DNA jet‐injected into lactating mammary glands of sheep was at a level sufficient to allow detection by Northern blot analysis when tissue was obtained 48 hours post‐transfection. In contrast, mRNA expression following DNA transfer by needle and syringe was detectable by RT‐PCR, but not by Northern blot analysis. Furthermore, specific mRNA was detected by RT‐PCR in lymph nodes draining the mammary gland injection sites. Jet‐injection of CMV‐hGH into either muscle or mammary gland resulted in the development of serum antibodies to hGH. The ability to transiently transfect lactating mammary tissue in vivo may circumvent the difficulties encountered with in vitro cu...

Truncated β-lactoglobulin transgenes are expressed in the kidney

The major milk whey protein of ruminants is β-lactoglobulin. Ovine β-lactoglobulin-encoding gene expression is restricted to the sheep mammary gland. This report describes the expression profile of a truncated β-lactoglobulin transgene which, although not expressed in the mammary gland, is expressed in the kidney in the majority of lines generated. The high frequency of ectopic kidney expression may relate to the ability of the larger β-lactoglobulin transgenes to be expressed in a position-independent manner in the mammary gland of transgenic mice.

Regulation of mammary gland factor/Stat5a during mammary gland development.

The rat homolog of sheep mammary gland factor (MGF)/Stat5 has been isolated and used to study the regulation of Stat5 during mammary gland development and PRL regulation in COS cells transfected with Stat5a and the PRL receptor. Two alternatively spliced isoforms, designated Stat5a1 and Stat5a2, were identified, the latter encoding a carboxy-terminal truncated protein. A polyclonal antibody to a carboxy-terminal peptide of Stat5a1 was generated and used to measure the level of this isoform during mammary gland development and after PRL induction in COS cells transiently transfected with Stat5a and the long form of the PRL receptor. Surprisingly, Stat5a mRNA and protein were readily detected both in virgin rats and after mammary gland involution. The levels of Stat5a increased during pregnancy, were highest in late pregnancy, and then, unexpectedly, decreased during lactation, the time at which the highest levels of milk protein gene expression are observed. Electrophoretic mobility shift assays using the specific anti-Stat5a1 antisera demonstrated that Stat5a1 comprises part of the heterogeneous, PRL-inducible, protein-DNA complex associated with the beta-casein GAS site. Immunocytochemical analysis detected considerable cytoplasmic and some nuclear staining for Stat5a1 during late pregnancy and predominantly nuclear staining during early lactation. The lack of correspondence of Stat5a gene expression and beta-casein gene expression suggests that Stat5 activation may facilitate the interaction of other factors binding within composite response elements identified recently in the milk protein gene promoters that are then responsible for the stable expression of milk protein genes in terminally differentiated mammary epithelial cells.

Identification and purification of human stat proteins activated in response to interleukin-2

A key cytokine Induced during the Immune response is IL-2. Following T cell activation, the genes encoding IL-2 and the various chains of Its receptor are transcriptionally induced. In turn, secreted IL-2 serves to stimulate the proliferation and differentiation of T lymphocytes. Several recent studies have implicated Jak kinasea in the signaling pathway Induced by IL-2. Following this lead, we set out to identify transcription factors Induced In response to IL-2. Human peripheral blood lymphocytes were observed to contain several IL-2-induclbie DNA binding activities. Similar activities were also observed in a transformed human lymphocyte line, termed YT. We have purified these activities and found that the principal IL-2-inducible component bears significant relatedness to a prolactin-induced transcription factor first identified in sheep mammary gland tissue. We hypothesize that activation of this protein, designated hStat5, helps govern the biological effects of IL-2 during the Immune response.

Transforming growth factor-alpha: receptor binding and action on DNA synthesis in the sheep mammary gland.

Microsome factions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-alpha (TGF-alpha). Binding was dependent on microsomal protein concentration, time and temperature. It showed the characteristics of an epidermal growth factor (EGF) receptor, being displaced by TGF-alpha and EGF, but not by insulin or IGF-I. The non-linear curve fitting program LIGAND was used to determine affinity and number of binding sites. A single class of high-affinity binding sites was found. The apparent dissociation constant (Kd) was similar in all physiological states (2.43 +/- 0.27 mol/l x 10(-10), n = 23). Numbers of binding sites were lower in late-pregnant (20 weeks) and lactating sheep (14.07 +/- 2.45 fmol/mg protein, n = 10) than in non-pregnant, 10- or 15-week pregnant sheep (43.04 +/- 5.93 fmol/mg protein, n = 13). DNA synthesis by mammary alveolar epithelial cells cultured on collagen gels was increased twofold by TGF-alpha (maximum response at 10 micrograms/l; 1.8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-alpha on day 3 of culture, but the response was delayed to day 4-5 of culture in cells from other physiological states. Dose-response was not significantly affected. TGF-alpha and IGF-I produced an additive effect on DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase variation of slime production in Staphylococcus aureus: implications in colonization and virulence

Two methods commonly used for slime detection in coagulase-negative staphylococci (tube biofilm formation and colony morphology in Congo red agar) were used to study 144 ruminant mastitis Staphylococcus aureus strains. Slime production was detected in 21 strains. A majority of cells (85%) in slime-producing (SP) strains and a minority of cells (5%) in non-slime-producing (NSP) strains showed a condensed exopolysaccharide matrix (slime) surrounding the bacterial cell wall, as revealed by electron microscopy and immunofluorescence. In vivo slime production was also detected immunohistochemically after experimental infection of the mammary gland in sheep. Upon repeated subcultures in Congo red agar, NSP variants were obtained from four ovine and four bovine SP strains at a frequency ranging from 0.5 x 10(-4) to 10(-4). Because SP variants could not be obtained from NSP strains within this range or at a higher frequency, they were obtained by the tube biofilm formation (requiring repeated subculturing of NSP strains in tryptic soy broth containing 2% glucose for subsequent recovery of colonies adherent to the walls of the culture tubes). In experimental challenge, the SP variant showed a significantly higher colonization capacity than did the NSP variant of the same strain used (P < 0.001). However, the NSP variant had a higher virulence than did the SP variant (P < 0.001). These results may help to explain the different roles of S. aureus slime production cell types (SP and NSP) coexisting in disease.

Characteristics of ruminant mammary epithelial cells grown in primary culture in serum-free medium.

Cells were obtained from the mammary glands of sheep and cows by collagenase-hyaluronidase digestion. Characterization of cells as epithelial was by reaction with a monoclonal antibody to cytokeratin. A subpopulation of spindle-shaped or stellate cells reacted with a monoclonal antibody to desmin and may be related to myoepithelial cells. The development is described of a simple serum-free culture system for these cells on gels of rat tail (type 1) collagen. A commercial medium (M199) was used, buffered with Hepes and with bovine serum albumin as the sole protein supplement, plus fibronectin for the first 18 h only as an attachment factor. The cell cultures showed stimulated DNA synthesis in response to mitogens on attached gels and also responded as floating cultures to lactogenic hormones with production of alpha-lactalbumin.

Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue.

We used 35S-labeled cRNA probes to localize the sites of alpha-lactalbumin, alpha-S1-casein, and lactoferrin mRNA synthesis in sheep and forcibly weaned cattle mammary tissue. Expression of alpha-lactalbumin was absent in three of four "virgin" glands studied, present in some alveoli of "pregnant" glands but not in others, despite a similar histological appearance. In the early lactating gland, expression was high in those alveoli with few fat globules in their cells and lumen and was absent in alveoli with abundant fat globules. These observations suggest either that alpha-lactalbumin gene expression is linked to the long-term secretory activity of cells and falls once cells are resting or regressing, or that there are cyclical variations in expression, or that in the lactating gland some groups of epithelial cells are synthesizing alpha-lactalbumin and some are synthesizing fat. Expression patterns of alpha-S1-casein were similar to those of alpha-lactalbumin. Lactoferrin, in contrast, was expressed almost exclusively in the "fatty alveoli" of both species. Our results show that dramatic variations in milk gene expression can occur throughout the mammary gland of sheep and cattle and that at no stage of pregnancy, lactation, or involution can the gland be considered metabolically homogeneous.

Differential migration of T and B cells during an acute inflammatory response

Inflammation in the liver and mammary glands of sheep caused by challenge infection with Taenia hydatigena or infusion of killed Staphylococcus aureus, respectively were characterized by the recruitment of both T and B cells. The patterns of migration of these two major lymphocyte subpopulations were distinctly different. While T cells seemed to migrate out of existing, flat endothelium-lined blood vessels resulting in a diffuse distribution at the sites of inflammation, B cells were characteristically present as clusters of tightly packed cells at restricted sites in the inflamed tissue. Within these B cell clusters distinct capillary vessels lined with plumb endothelial cells were always present suggesting that they were the sites of intense migration of B cells originating from the draining lymph nodes. These results indicate differential regulation of adhesion molecules on B and T cells and/or their ligands on endothelium during acute inflammatory reactions.

Pathological changes in the lungs and mammary glands of sheep and their relationship with maedi-visna infection.

Maedi-visna, a multisystemic disease of adult sheep, was first described in Spain in 1984. To get an idea of the seroprevalence of the disease locally and to estimate the number of seropositive animals with lesions, samples of blood, lungs and mammary glands were taken from 124 randomly selected sheep killed in the main slaughterhouse of Zaragoza. In the agar gel immunodiffusion test, 74 (59.7 per cent) of the sheep were positive and 50 were negative. Among the 74 seropositive animals, 19 (25.6 per cent) had no lesions in any organ, 12 (16.2 per cent) had lesions in the lungs only, 15 (20.2 per cent) had lesions in the mammary glands and 28 (37.8 per cent) had lesions in both organs. In the lungs hyperplasia of lymphoid follicles was more evident than an interstitial infiltrate but in the mammary glands this relationship was not observed. Even when the lesions occurred in both organs, they did not show the expected proportion in terms of either type or severity. Among the 50 seronegative sheep, eight (16 per cent) showed maedi-like lesions, formed exclusively by the hyperplasia of lymphoid follicles.

Biochemical Alterations in the Blood, Milk and Tissues of Sheep Mammary Gland After Experimental Mycoplasmal Mastitis

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Evidence for tertiary structure in natural single stranded RNAs in solution

Binding isotherms (20 degrees C) of ethidium bromide to a number of tRNA species at various ionic strengths indicate that i) the number ni of intercalation sites is high 7 to 11 per molecule, in the low salt form III, but small, 2 to 1, at high Mg2+ or Na+ when form I predominates. ii) modification of tRNA at strategic positions for 3D folding prevents full expression of intercalation restriction iii) maximal restriction is obtained at salt concentrations higher than needed for full conversion to form I. It is inferred that restriction, which is not observed with bihelical RNA (or DNA), requires the native tRNA 3D structure but also some physical coupling between the region of 3D folding and bihelical arms. Ribosomal RNAs, some viral RNAs, mRNA from sheep mammary gland as well as the random copolymers Poly UG, Poly AUG, Poly AUCG all exhibit intercalation restriction. Hence 3D folding of the polyribonucleotide chains appears to be a feature common to single-stranded RNAs when free in solution under physiological conditions.

Local intra-arterial infusion of growth hormone into the mammary glands of sheep and goats: effects on milk yield and composition, plasma hormones and metabolites.

Lactating goats and sheep were fitted with catheters in the external pudendal arteries supplying both mammary glands. Saline was infused continuously into one artery whereas the other artery received continuous infusions, over successive 4-day periods, of either saline or growth hormone (GH)-doses increasing twofold between successive periods from 100 to 400 micrograms/day in goats and 400 to 3200 micrograms/day in sheep. Local infusion of GH at up to 1600 micrograms/day in sheep did not affect milk yield or composition nor peripheral plasma concentrations of GH, insulin, glucose, urea and non-esterified fatty acids (NEFA). Infusion of GH at 3200 micrograms/day in sheep increased peripheral plasma concentrations of GH, tended to increase milk yield and peripheral plasma NEFA but there were no changes in peripheral plasma insulin, glucose and urea. It is concluded that GH does not exert direct effects on the mammary glands of sheep and goats in situations where the hormone is administered over short periods.

Pathology of acute experimental Actinobacillus seminis mastitis in ewes.

Sequential pathological changes were studied for 9 days after inoculating Actinobacillus seminis in the mammary gland of sheep. The inoculated mammary glands became enlarged (2 to 4 times normal), turgid or consolidated and contained creamy or greenish-yellow viscid contents. A seminis was isolated from all inoculated udders at necropsy. Microscopically, the reaction in the udder to A. seminis may be divided into 4 overlapping phases; acute purulent, subacute purulent, necrotising, and proliferative. It was concluded that A. seminis was pathogenic for the ovine mammary gland, the clinical and pathological findings were nonspecific, and that A. seminis could survive within ovine mammary tissue for at least 9 days after inoculation.

Specific antibody-containing cells in the mammary gland of non-lactating sheep after intraperitoneal and intramammary immunisation

Ewes were immunised intraperitoneally with ovalbumin and Brucella abortus in Freund's complete adjuvant, followed seven days later by intramammary immunisation in which ovalbumin was presented to one mammary gland and Brucella abortus to the other. Mammary tissue taken after a further seven days contained more antigen-specific plasma cells than ewes given intraperitoneal or intramammary immunisation alone. These cells were found predominantly in the specifically immunised gland and only a few were found in the contralateral gland. Most of these cells were of the IgG1 isotype. There was also an increase in the total number of IgG1- and IgG2-containing cells in mammary gland tissues of these ewes, indicating a non-specific response to immunisation. Following either intraperitoneal or intramammary immunisation there was also a significant increase in the number of antigen-specific IgA cells in the lamina propria of the jejunum. The gut response following intramammary immunisation alone was abrogated by chronic drainage of intestinal lymph but not mammary lymph. This suggests that antigen may relocate from the mammary gland to the intestine where an IgA response is generated from gut associated lymphoid tissue. These data provide evidence for interaction between the gut and mammary gland of sheep in response to antigen.

Endocrine Activity of the Mammary Gland: Oestrogen and Prostaglandin Secretion by the Cow and Sheep Mammary Glands During Lactogenesis

SUMMARY Mammary venous and carotid arterial plasma concentrations of oestradiol-17β and prostaglandin Fα (PGFα), and mammary secretion concentrations of lactose were determined in three Jersey cows and four Friesland sheep from seven days pre-partum until two days post partum. Significant mammary secretion of oestradiol-17β and PGFα into the venous drainage was observed in both cows and sheep pre-partum, supporting the hypothesis postulated for the goat that the mammary gland is an endocrine organ. The timing of the onset of copious milk secretion, coincident with an increased mammary secretion concentration of lactose (lactogenesis), differed between cows and sheep in relation to parturition. However, the temporal pattern of changes in the mammary secretion of oestradiol-17β and PGFα, in relation to lactogenesis, was similar in the two species; a cessation of mammary secretion of PGFα and a transient peak in the mammary secretion of oestradiol-17β occurring immediately prior to the onset of copious milk secretion. This relationship is similar to that observed previously in the goat, and indicates common mechanisms for the control of lactogenesis in these three ruminants which have differing endocrinological profiles associated with the maintenance of pregnancy and control of parturition.

Cell composition as assessed from osmolality and concentrations of sodium, potassium and chloride: Total contributions of other substances to osmolality and charge balance

1. 1. From published data for various tissues on intracellular Na, K and Cl were calculated the net anionic charge on all other substances present and also the total contribution of these to osmolality, assuming osmotic equilibrium with extracellular fluid. 2. 2. These parameters were compared for muscle and nerve of animals differing widely in osmolality and also for other mammalian cells. 3. ]3. Cell volume regulation in some euryhaline species was considered: it is only partly due to ninhydrin-positive materials. 4. 4. In sheep mammary glands K seems to be sequestered with lactose and anion. 5. 5. Changes in mammalian muscle due to adrenalectomy, hypophysectomy, K deficiency, myotonia, acid-base imbalance and treatment with deoxycorticosterone or insulin were discussed.

A freeze-fracture study of tight junction structure in sheep mammary gland epithelium during pregnancy and lactation.

Freeze fractures of the tight junctions at the apices of sheep mammary secretory cells showed that the junction at 72 d of pregnancy was significantly wider than at any later stage. The number of ridges in the junction only increased significantly between 122 and 142 d of pregnancy. The initially even distribution of intramembrane particles across the tight junction changed gradually until in the fully lactating animal there were far more particles on the lateral surface of the plasmalemma. Possible correlations between these changes, the alterations in permeability of the mammary epithelium, and the differences in hormone levels are briefly discussed.