Acute phase protein expressions in secretory and cistern lining epithelium tissues of the dairy cattle mammary gland during chronic mastitis caused by staphylococci.

BACKGROUND: Mastitis is the most common disease in dairy cattle and the costliest for the dairy farming industry, as it lowers milk yield and quality. Mastitis occurs as a result of interactions between microorganisms and the individual genetic predispositions of each animal. Thus, it is important to fully understand the mechanisms underlying these interactions. Elucidating the immune response mechanisms can determine which genetic background makes an animal highly resistant to mastitis. We analyzed the innate immune responses of dairy cows naturally infected with coagulase-positive staphylococci (CoPS; N = 8) or coagulase-negative staphylococci (CoNS; N = 7), causing persistent mastitis (after several failed treatments) vs. infection-free (i.e., healthy [H]; N = 8) dairy cows. The expressions of the acute phase protein genes serum amyloid A3 (SAA3), haptoglobin (HP), ceruloplasmin (CP) genes in the tissues most exposed to pathogens- mammary gland cistern lining epithelial cells (CLECs) and mammary epithelial cells (MECs)-were analyzed. RESULTS: We found constitutive and extrahepatic expressions of the studied genes in both tissue types. HP expression in the MECs of the CoPS-infected group was higher than in the H group (p ≤ 0.05). Moreover, higher SAA3 expression in the CoPS and CoNS groups than in the H group (p = 0.06 and 0.08, respectively) was found. No differences between SAA3 and HP in CLECs were revealed, regardless of the pathogen type. However, higher expression of CP (p ≤ 0.05) in the CoPS group than in the H group was noted. CONCLUSIONS: The expressions of selected acute phase proteins were similar between CLECs and MECs, which means that CLECs are not only a mechanical barrier but are also responsible for the biological immune response. Our findings agree with the results of other authors describing the immunological response of MECs during chronic mastitis, but the results for CLECs are novel.

Technical note: Selecting the best references in gene expression experiments in liver of cows receiving glucogenic supplements during the transition period.

Measuring gene expression is a commonly used method to monitor the reaction of cells and tissues to changing nutritional or physiological conditions. Selection of appropriate reference genes is a crucial point in gene expression experiments using real-time PCR techniques. Expression of the "ideal" reference gene should not be affected by the experimental treatments or physiological state of the tissue, organ, or the whole organism. Many programs are available from which to choose the most stable reference gene. In this study, 4 algorithms--ΔCt, BestKeeper, NormFinder, and geNorm--were used to assess the expression stability of 5 candidate reference genes: ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S9 (RPS9), ribosomal protein L32 (RPL32), and TATA-box-binding protein (TBP), for use in an experiment aimed at measuring gene expression in the liver of cows fed glucogenic supplements in the transition from pregnancy to lactation. The results demonstrated that RPS9 and RPL32 were the most stably expressed in the liver under the conditions of the present experiment; the least stably expressed was ACTB.

Allelic gene expression imbalance of bovine IGF2, LEP and CCL2 genes in liver, kidney and pituitary.

Allelic expression imbalance (AEI) is an important genetic factor being the cause of differences in phenotypic traits that can be heritable. Studying AEI can be useful in searching for factors that modulate gene expression and help to understand molecular mechanisms underlying phenotypic changes. Although it was commonly recognized in many species and we know many genes show allelic expression imbalance, this phenomena was not studied on a larger scale in cattle. Using the pyrosequencing method we analyzed a set of 29 bovine genes in order to find those that have preferential allelic expression. The study was conducted in three tissues: liver, pituitary and kindey. Out of the studied group of genes 3 of them-LEP (leptin), IGF2 (insulin-like growth factor 2), CCL2 (chemokine C-C motif ligand 2) showed allelic expression imbalance.

A note on the organization and expression of ß-defensin genes in Polish goats.

Nucleotide (cds) and amino acids sequences of the caprine ß2-defensin genes were in silico compared to search for the sequence variation and for the LAP gene sequences in the goat genome and for the presence of LAP gene transcripts in goat tissues. The comparison of the exon sequences revealed that the first 64 amino acids are identical in both LAP and ß1-defensin. However, the GBD-1 prepropeptide is shorter by 18 amino acids due to the presence of the stop codon UAA at position 209-211 in GBD-1 mRNA. The LAP gene, which was found, so far, only in Indian goat breeds, is absent in the genome of Polish dairy goats. The introns of the caprine ß1- and ß2-defensin genes were, for the first time, sequenced; their sequences showed 99.6 % identity, differing in six nucleotide positions.

Transcriptomic index of skeletal muscle of beef breeds bulls.

In the present study cDNA microarray (18263 probes) were used for analysis of bovine skeletal muscle (m.semitendinosus) transcriptome in 12-month-old bulls of four cattle breeds: Holstein-Friesian (HF), Limousine (LIM), Hereford (HER) and Polish Red (PR), aiming to identify the genes, which expression is common for beef breed bulls. The number of transcripts significantly different from HF bulls muscle amounted to 393, 462 and 638 for LIM, HER and PR, respectively. As a result of this study the transcriptomic index was proposed, being the set of 48 genes expressed similarly in beef breed bulls in comparison to HF bulls. Classification of genes according to molecular function of their protein products has shown the highest number of genes encoding proteins involved in nucleic acid binding (10), regulatory proteins (6), kinases (4) and signaling molecules (3). Classification according to biological processes revealed the highest number of genes involved in protein metabolism i modification (14), signal transduction (5), cell cycle (4), intracellular protein traffic (4), nucleoside, nucleotide and nucleic acid metabolism (4), apoptosis (3), cell structure and motility (3), and cellular transport (3). Since the role of the most genes included to the transcriptomic index has not been described yet in bovine skeletal muscle, obtained results may be very useful in revealing new candidate genes to search a new criteria of animal selection in beef production.

Comparison of skeletal muscle transcriptional profiles in dairy and beef breeds bulls.

A cDNA microarray (18 263 probes) was used for transcriptome analysis of bovine skeletal muscle (m. semitendinosus) in 12-month-old bulls of the beef breed Limousin (LIM) and the typical dairy breed Holstein-Friesian (HF, used as a reference). We aimed to identify the genes whose expression may reflect the muscle phenotype of beef bulls. A comparison of muscle transcriptional profiles revealed significant differences in expression of 393 genes between HF and LIM. We classified biological functions of 117 genes with over 2-fold differences in expression between the examined breeds. Among them, 72 genes were up-regulated and 45 genes were down-regulated in LIM vs. HF. The genes were involved in protein metabolism and modifications (22 genes), signal transduction (15), nucleoside, nucleotide and nucleic acid metabolism (13), cell cycle (9), cell structure and motility (9), developmental processes (9), intracellular protein traffic (7), cell proliferation and differentiation (6), cell adhesion (6), lipid, fatty acid and steroid metabolism (5), transport (5), and other processes. For the purpose of microarray data validation, we randomly selected 4 genes: trip12, mrps30, pycrl, and c-erbb3. Real-time RT-PCR results showed similar trends in gene expression changes as those observed in microarray studies. Basing on results of the present study, we proposed a model of the regulation of skeletal muscle growth and differentiation, with a principal role of the somatotropic pathway. It may explain at least in part the development of muscle phenotype in LIM bulls. We assume that the growth hormone directly or indirectly (through IGF-1) activates the calcium-signaling pathway with calcineurin, which stimulates myogenic regulatory factors (MRFs) and inhibits early growth response gene. The inhibition results in indirect activation of MRFs and impaired activation of TGF-beta1 and myostatin, which finally facilitates terminal muscle differentiation.

Effect of new SNP within bovine prolactin gene enhancer region on expression in the pituitary gland.

A new single nucleotide polymorphism was revealed using PCR-SSCP and sequencing methods within the bovine prolactin distal promoter region described as a functional enhancer. The A-->G transition at position -1043 abolishes the recognition site for Hsp92II restriction endonuclease, allowing for PCR-RFLP genotyping. The application of real-time PCR revealed that the prolactin gene expression level in the pituitary was higher in cattle with the AA genotype than in those with the GG genotype. EMSA analysis, however, showed increased nuclear protein binding to the sequence variant with G, suggesting a possible inhibition event, in which the transcription factors Pit1, Oct1, and YY1 could be involved.

The polymorphism in the beta4-defensin gene and its association with production and somatic cell count in Holstein-Friesian cows.

The aim of this study was to find a polymorphism of the bovine beta4-defensin gene and search for its association with milk yield and composition and with the somatic cell count in milk. The data were from the years 1999 to 2004 on 212 Holstein-Friesian (HF) dairy cows, descended from 70 sires. Based on the sequence of the bovine beta4-defensin gene (GenBank no. AF008307) the primers were designed for the amplification of the 924-bp or 393-bp long fragments. The 924-bp long fragment was sequenced and the sequence was compared with that available in the GenBank. Ten putative nucleotide sequence polymorphisms were found in the intron of the bovine beta4-defensin gene. One of them, a C-->T transition at position 2239, that creates a new NlaIII (Hin1II) restriction site, was genotyped with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a cohort of 212 HF cows. The CC genotype was the most common (72%). The heterozygous CT genotype was found in 26% of the genotyped cows and four cows (2%) were TT homozygotes. In order to determine the relationship between the polymorphism of the beta4-defensin gene and milk production traits a multi-trait repeatability test-day animal model was used. The Derivative-free Multivariate analysis program was used for computation. The differences between estimates for genotypes were checked using Student's t-test. The model included the animal genotype, year-season of calving and parity as fixed effects and the animal additive genetic effect and permanent environmental effect of individual cows as well as dates of the tests as random effects. Significant associations were found between the RFLP-NlaIII and milk fat, protein and lactose contents. Also, a significant effect was shown of the defensin genotype on the somatic cell count in the milk.

Polymorphism of 5'-region of the bovine growth hormone receptor gene.

Genes coding for growth hormone (GH) and GH receptor (GHR) are candidates for quantitative trait markers in farm animals. This work describes a search for nucleotide sequence polymorphisms within the 5'-region of the bovine GHR gene. Two new single nucleotide polymorphisms were found: restriction fragment length polymorphisms (RFLPs) at a Fnu4HI/TseI site (C/T transition at position -1104), and at a Sau96I site (C/T transition at position -262). The Fnu4HI/TseI polymorphic site is located within the 1.2-kbp LINE-1 retrotransposon upstream of the P1 promoter, while the Sau96I RFLP locates in the P1 promoter for exon 1A. The appearance of the Sau96I RFLP was studied in representatives of two bovine species, Bos taurus and Bos indicus. An absolute correlation was observed between Sau96I genotype and the insertion/deletion of LINE-1.

Stimulation of luteinizing hormone subunit gene expression by pulsatile intracerebroventricular microinjection of galanin in female rats.

Although galanin, which exerts its effects both at the hypothalamic and pituitary level, has been implicated as an important neuroendocrine regulator of hypothalamic-pituitary-gonadal axis activity, there is a lack of data concerning its involvement in the regulation of gonadotropin subunit gene expression. To elucidate whether galanin can influence luteinizing hormone (LH) subunit mRNA content, as well as affect gonadotropin-releasing hormone (GnRH) receptor activity, a model based on pulsatile (one pulse per hour over 5 h) galanin (1 nM) microinjections directly into the third cerebral ventricle of ovariectomized (OVX) and/or oestrogen/progesterone-pretreated rats was used. Furthermore, to determine galanin effects on GnRH-induced LH subunit mRNA synthesis, a cocktail of 1 nM GnRH and 1 nM galanin was coadministered in a pulsatile manner to OVX/steroid primed rats. Subsequently, to obtain data concerning the role of galanin receptors in the regulation of pituitary alpha (common to LH, follicle-stimulating hormone, thyroid-stimulating hormone) and LHbeta subunit gene expression, OVX/oestrogen/progesterone rats received microinjections of 1 nM of the receptor antagonist galantide and 1 nM of galanin. In this case, both substances were administered separately, with a 30 min lag, according to which each galantide pulse always preceded a galanin pulse. Northern-blot analysis revealed that intracerebroventricular pulsatile galanin injections were effective in stimulation of both alpha and LHbeta subunit mRNA levels and that this effect was apparently steroid-dependent. Moreover, galanin also up-regulated GnRH receptor functional parameters (affinity and maximum binding capacity) but was ineffective in potentiating GnRH-induced accumulation of both subunit mRNAs. The results from the study also indicate that galanin acts through its own receptor(s) because a receptor antagonist, galantide, significantly reduced the stimulatory effect exerted by galanin on the expression of both LH subunit genes in vivo.

Differential induction of transcription factors and expression of milk protein genes by prolactin and growth hormone in the mammary gland of rabbits.

Previously we demonstrated that administration of lactogenic hormones - prolactin (PRL) and growth hormone (GH) - to pregnant rabbits differentially induces expression of casein and whey proteins in the mammary gland. Now we extend these observations to transcription factors (TFs) that are responsive for differential induction of milk protein genes. Analysis of correlation between the number of putative TF binding sites in 5'-upstream sequences and the levels of induction of milk protein genes allowed preselection of the TFs involved. An electrophoretic mobility shift assay with nuclear proteins derived from rabbit mammary glands showed changes in the patterns of Stat5, MAF, NF1 and Oct1 DNA-protein binding during progression of pregnancy and transition to lactation. Administration of lactogenic hormones - PRL or GH - to early-pregnant rabbits induced DNA-protein complexes similar to those formed by nuclear proteins from the mammary glands of lactating (Stat5, MAF, NF1) or late-pregnant (Oct1) animals. Induction of milk protein genes by PRL was several-fold greater than that by GH. However, PRL and GH similarly induced MAF DNA-protein complexes, thus suggesting that the amount of MAF factor in the mammary gland can be limiting for expression of these genes. Our study for the first time provided the evidence that in the mammary gland both PRL and GH can induce DNA-binding activity of transcription factors other than Stats.

Polymorphism of molecular-genetic systems in the Polish red cattle.

The object of this study was to investigate gene polymorphism in the Polish Red (PR) cattle, a local Polish breed included in the FAO conservation programme. Milk protein genes and other genes with possible effect on production traits were analysed. Frequencies of different gene variants were compared with those in the Polish Friesian cattle. The following gene polymorphisms were analysed with PCR/RFLP technique: milk protein genes--kappa-casein and beta-lactoglobulin, growth hormone (GH), PitI (a transcription factor) and leptin. Moreover, SSCP analysis was performed of myostatin (MSTN) gene at the site previously shown to cause muscle overgrowth in Belgian Blue double-muscled cattle. A significant difference was found in this study between kappa-casein A and B allele frequency in PR and Friesian cattle. No such differences were found in the frequency of A and B alleles of beta-lactoglobulin, L and V alleles of GH, A and B alleles of PitI, and A, B and C alleles of leptin gene. In the analysed group of the Polish Red cattle three animals were found with the rare AI genotype of -lactoglobulin. No such genotype was identified in analysed Friesians. Moreover, 8 PR animals were identified carrying a mutation in MSTN gene, possibly identical to that causing the double-muscled phenotype in some breeds of meat cattle.

Genetic variation in stimulated GH release and in IGF-I of young dairy cattle and their associations with the leucine/valine polymorphism in the GH gene.

Genetic variations in plasma GH concentrations before and following thyrotropin-releasing hormone (TRH) stimulation and in IGF-I concentrations were studied in 11-mo-old Polish Friesian cattle (104 heifers and 110 bulls). A possible association between stimulated GH release, IGF-I, and the polymorphism in the GH gene causing substitution of leucine-Leu to valine-Val at amino acid position 127 of the protein was also investigated. The GH concentrations were determined in serial plasma samples collected every 15 min from 15 min before to 135 min after intravenous administration of 0.15 microg TRH/kg live weight. The analysis was performed on three variables: baseline (mean of samples at -15 and 0 min), peak (sample at 15 min after injection) and rate (peak minus sample at 60 min, divided by 45 min). The IGF-I concentrations were measured in plasma samples taken before the TRH stimulation. Additionally, first lactation records from the 75 cows earlier tested for GH release and IGF-I were used to study a possible association of milk production traits with GH genotypes. The data were analyzed by multivariate mixed linear models. The heritability of IGF-I reached a higher value (0.35) than variables baseline, peak, and rate (0.02, 0.14, and 0.14, respectively). The GH variables were positively genetically correlated with each other (0.22 to 0.93), whereas they had negative genetic correlations with IGF-I (-0.26). The Val/Val genotypes reached the highest peak value compared with other GH genotypes (P > 0.01), whereas the Leu/Leu genotypes had the highest IGF-I concentrations (P < or = 0.05). Moreover, the Leu/Val heterozygotes were superior to others in milk and protein yields, whereas the Leu/Leu homozygotes reached the highest fat yield (P > or = 0.01). We conclude that GH peak, GH rate, and IGF-I are heritable traits in young dairy cattle and are affected by the Leu/Val polymorphism in the GH gene.

Inhibition of ornithine decarboxylase by alpha-difluoromethylornithine induces apoptosis of HC11 mouse mammary epithelial cells.

The effect of a-difluoromethylornithine (DFMO) on the apoptosis of HC11 mouse mammary epithelial cells was investigated. The involvement of reactive oxygen species (ROS) and Bcl-2 protein in the mechanism of apoptosis induced by ornithine decarboxylase (ODC) inhibition was also assessed. DFMO (0.1, 1 and 5mM) induced apoptosis of HC11 cells in dose- and time-dependent manner. Apoptosis manifests itself with morphological features like: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis (putrosis). The decrease in the nuclear DNA contents appearing as the hypodiploidal peak sub-G1 in the DNA histogram was not dependent on the presence of prolactin (5 microg/ml) in DFMO-treated cultures. Apoptosis induced by ODC inhibition was associated with a rapid increase in ROS concentration in HC11 cells observed within 1 h after DFMO treatment. The down-regulation of Bcl-2 as a decrease in cell number expressing bcl-2 and a lowered Bcl-2 protein content in cells expressing this protooncogene was also noted. The administration of putrescine (50 microM) lowered the number of early-apoptotic, late-apoptotic and necrotic cells. Moreover, it increased the number of cells expressing bcl-2. In conclusion, the disturbance of cellular polyamine homeostasis by inhibition of their synthesis enhances mammary epithelial cell susceptibility to apoptosis. It may occur in the mammary gland at the end of lactation, when the depletion of circulating lactogenic hormones and activation of intra-mammary apoptogenic factors expression take place.

LH/hCG receptors in the porcine uterus--a new evidence of their presence in the cervix.

High-affinity LH/hCG binding sites have been characterized in bovine, lepine, murine, human uteri and porcine myometrium and endometrium. In the present studies we analyzed these receptors in the porcine cervix. Radioreceptor ligand assays were performed with cell membrane preparations of the cervix which were analyzed for binding sites specificity, capacity and affinity. Corpus luteum and myometrium were used as positive control tissues. In the cervix there was little competition for receptor occupancy between hCG and porcine FSH (1.2%) or bovineTSH, porcine GH and porcine PRL (0.1%, 0.1% and < 0.001%; respectively) but porcine LH could completely inhibit the binding of [125I] hCG. There was not binding for LH/hCG in crude membrane preparations of kidney or skeletal muscle. The concentration (fmol/mg protein) of cervical LH/hCG receptor did not vary significantly during particular phases of the estrous cycle, except the early luteal phase (Days 6-7) when the level of LH receptors was very low (p < 0.05). The affinity of uterine LH/hCG binding sites in the cervix and the myometrium was not different from the affinity of LH/hCG binding sites in luteal cells. The porcine cervix as well as the myometrium contains a 75- and 48-kDa immunoreactive LH/cCG receptor proteins similar to corpus luteum. Southern blot of RT-PCR products performed to enhance the specificity and sensitivity of LH receptor transcripts determination in uterine tissues revealed that expected fragments of 740 and 470 bp were present in myometrium and corpus luteum. The cervix showed only 740 bp fragment. In situ hydridization showed the expression of mRNA for LH receptor in the epithelium of the cervix. Immunoreactive staining for LH/hCG receptors was also observed only in epithelial cells of the cervical tissue. Our studies are probably the first evidence demonstrating the specific LH/hCG binding sites in female cervix.

Stimulated growth hormone (GH) release in Friesian cattle with respect to GH genotypes.

The effects of bovine growth hormone (GH) polymorphism at the amino acid position 127 (substitution of leucine to valine) on milk and meat production traits have been reported; however, the physiological background of this influence has not yet been recognised. The aims of this study were to estimate allele frequencies of the growth hormone gene in a population sample of Friesian cattle, and to characterise the TRH-induced GH release with respect to GH genotypes. The analysis covered data on 214 Polish Friesians, aged 11 months, used to identify GH genotypes by the PCR-RFLP technique. Frequencies of leucine (Leu) and valine (Val) alleles were 0.69 and 0.31, respectively. The GH release was analysed after thyrotropin releasing hormone (TRH) stimulation in blood samples collected over a period of 2.5 h. There was a lack of significant difference in the overall characteristics of GH release in the blood of Friesian cattle with different GH genotypes (P > 0.05). Nevertheless, the Val/Val homozygotes had higher GH baselines both within heifers and bulls (14.1 +/- 2.8 and 14.6 +/- 2.0 ng.mL-1, respectively) than others. Moreover, males of the Val/Val genotypes showed the highest peak amplitude of GH release (55.5 +/- 8.1 ng.mL-1) in comparison to all other animals. The results presented provide evidence for the lack of difference in stimulated GH release with respect to GH genotypes in dairy cattle.

Antiapoptotic action of prolactin is associated with up-regulation of Bcl-2 and down-regulation of Bax in HC11 mouse mammary epithelial cells.

The effect of prolactin on apoptosis and the expression of bcl-2 and bax in HC11 mouse mammary epithelial cells were investigated. Flow cytometric analysis of Bcl-2 level (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated monoclonal anti-IgG1 antibody as a negative control), number of apoptotic cells and cell cycle phases (DNA stained with DAPI) was performed. Bax transcript was measured using the RT-PCR method with GAPDH serving as a reference gene. Administration of prolactin (5 microg/ml) in the presence of insulin stimulated differentiation of mammary epithelial cells, which manifested in stopping cells at G0/G1 phase, cell swelling and increase of cell number with enhanced protein content. Moreover, prolactin highly significantly reduced the extent of apoptosis of HC11 cells during 48 h of incubation. Nevertheless, the apoptotic cell number rose with increased time length of cell culture, probably due to the resulting high cell density and EGF withdrawal from t he incubation medium. The antiapoptotic effect of prolactin was associated with up-regulation of bcl-2 expression, shown as an increase in cell numbers expressing this protooncogene and elevated Bcl-2 content in these cells. A negative relationship (r=-0.87, p< or =0.001) between the number of apoptotic cells and those expressing bcl-2 was also found. Prolactin administration lowered Bax transcript by 68.8% and 70.7% after 3 and 6h, respectively. In conclusion, the results presented indicate that stimulation of bcl-2 expression with simultaneous suppression of bax may be key events in the mechanism of antiapoptotic action of prolactin in HC11 mammary epithelial cells.