Genetic variability among and within domestic Old and New World camels at the α-lactalbumin gene (LALBA) reveals new alleles and polymorphisms responsible for differential expression.

α-Lactalbumin (α-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for α-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species.

Correlation between estrogen plasma level and miRNAs in muscle of Piedmontese cattle.

A loss-of-function mutation of the myostatin gene has a very high prevalence in the Piedmontese cattle breed. The effect of such mutation is a double-muscle phenotype because of hypertrophy. However, differences in muscle mass development can still be detected in individuals of this breed. Such differences must be generated by other factors controlling skeletal muscle development. MicroRNAs are short noncoding RNA molecules that modulate gene expression at a post-transcriptional level. MicroRNAs have been demonstrated to be involved in skeletal muscle development, and some of them are controlled by steroid hormone signaling. Data on estrogen signaling are lacking, whereas more studies have been carried out on the effect of androgens. We aimed at identifying putative estrogen responsive miRNAs that might be involved in skeletal muscle development. At a slaughterhouse, we collected muscle samples from longissimus dorsi and blood samples. Blood 17ß-estradiol concentration was assessed, and RNA was extracted from muscle samples. The animals we sampled were divided into groups according to estrogen blood concentration, and through qPCR expression, levels of 7 muscle-related miRNAs were evaluated. We found that miR-26b (P < 0.01), miR-27a-5p (P < 0.05), miR-27b (P < 0.05), and miR-199a-3p (P < 0.01) were differentially expressed among experimental groups. Expression levels of miR-26b were reduced approximately 50% in samples with a low blood estrogen concentrations, and the other miRNAs showed a tendency to increase their expression levels when blood estrogen levels were higher. The variations of the circulating concentrations of estrogen in Piedmontese cattle might influence muscle mass development through miRNAs and thus contribute to individual variability in a breed with a high prevalence of a myostatin point mutation.

Mechanisms of modulation of the Egr gene family in mammary epithelial cells of different species.

In the adult female, within the estrous cycle, the mammary gland undergoes multiple rounds of growth, with increased cellular proliferation, and involution, with increased apoptosis. The increase in proliferation is elicited by endocrine (Estrogen, Progesterone), as well as locally produced (epidermal growth factor, insulin-like growth factor, etc) growth factors. Among the genes that are modulated during cellular proliferation, immediate early genes play a fundamental role, being rapidly upregulated and then downregulated within the G0/G1 phase of the cell cycle, allowing the progression to the subsequent phases. Egrs (1-4) are immediate early genes that encode for transcription factors that promote, within different cell types and depending on the strength and duration of the stimuli, several different responses like mitogenesis, differentiation, apoptosis or even anti-apoptosis. In this work we have studied the mechanisms of modulation of the Egr family, in mammary epithelial cells of different origin (bovine, canine, feline, murine). Following stimulation with growth medium, Egr mRNA expression showed a strong upregulation reaching a peak at 45-60min, that rapidly declined. Among several cytokines, particularly important for mammary morphogenesis, that we have tested (EGF, IGF-I, insulin, estrogen, progesterone), only EGF upregulated Egrs to levels close to those elicited by growth medium. In order to understand how the Egr transcription factors were regulated, we have inhibited Erk 1/2 and PI3K, molecules that drive two major intracellular signaling pathways. Inhibition of the Erk 1/2 pathway totally abolished Egr upregulation mediated by growth medium or EGF. On the other hand, the PI3K-Akt pathway played a minor role on Egr levels, with a strong inhibitory effect on cat GH2 cells only, that could be ascribed to reduced Erk phosphorylation following PI3K inhibition. Finally we showed that addition of growth medium also upregulated that the mammary luminal marker cytokeratin 18, but only in the murine NMuMG cell line. This is the first manuscript describing how the Egr transcription factors are expressed in mammary epithelial cells of domestic animals and which growth factors and signaling pathways modulate their expression.

Temporal correlation between differentiation factor expression and microRNAs in Holstein bovine skeletal muscle.

Satellite cells are adult stem cells located between the basal lamina and sarcolemma of muscle fibers. Under physiological conditions, satellite cells are quiescent, but they maintain a strong proliferative potential and propensity to differentiate, which underlies their critical role in muscle preservation and growth. MicroRNAs (miRNAs) play essential roles during animal development as well as in stem cell self-renewal and differentiation regulation. MiRNA-1, miRNA-133a and miRNA-206 are closely related muscle-specific miRNAs, and are thus defined myomiRNAs. MyomiRNAs are integrated into myogenic regulatory networks. Their expression is under the transcriptional and post-transcriptional control of myogenic factors and, in turn, they exhibit widespread control of muscle gene expression. Very little information is available about the regulation and behavior of satellite cells in large farm animals, in particular during satellite cell differentiation. Here, we study bovine satellite cells (BoSCs) undergoing a differentiation process and report the expression pattern of selected genes and miRNAs involved. Muscle samples of longissimus thoracis from Holstein adult male animals were selected for the collection of satellite cells. All satellite cell preparations demonstrated myotube differentiation. To characterize the dynamics of several transcription factors expressed in BoSCs, we performed real-time PCR on complementary DNA generated from the total RNA extracted from BoSCs cultivated in growth medium (GM) or in differentiation medium (DM) for 4 days. In the GM condition, BoSCs expressed the satellite cell lineage markers as well as transcripts for the myogenic regulatory factors. At the time of isolation from muscle, PAX7 was expressed in nearly 100% of BoSCs; however, its messenger RNA (mRNA) levels dramatically decreased between 3 and 6 days post isolation (P<0.01). MyoD mRNA levels increased during the 1st day of cultivation in DM (day 7; P<0.02), showing a gradual activation of the myogenic gene program. During the subsequent 4 days of culture in DM, several tested genes, including MRF4, MYOG, MEF2C, TMEM8C, DES and MYH1, showed increased expression (P<0.05), and these levels remained high throughout the culture period investigated. Meanwhile, the expression of genes involved in the differentiation process also miRNA-1, miRNA-133a and miRNA-206 were strongly up-regulated on the 1st day in DM (day 7; P<0.05). Analysis revealed highly significant correlations between myomiRNAs expression and MEF2C, MRF4, TMEM8C, DES and MYH1 gene expression (P<0.001). Knowledge about the transcriptional changes correlating with the growth and differentiation of skeletal muscle fibers could be helpful for developing strategies to improve production performance in livestock.

Bovine mammary stem cells: new perspective for dairy science.

Mammary stem cells provide opportunities for the cyclic remodelling of the bovine mammary gland. Therefore, understanding the character and regulation of mammary stem cells is important for increasing animal health and productivity. The exciting possibility that stem cell expansion can influence milk production is currently being investigated by several researchers. In fact, appropriate regulation of mammary stem cells could hopefully benefit milk yield, persistency of lactation, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and regulate the function of bovine mammary stem cells. However, research on mammary stem cells requires tissue biopsies, which represents a limitation for the management of animal welfare. Interestingly, different studies recently reported the identification of putative mammary stem cells in human breast milk. The possible identification of primitive cell types within cow's milk may provide a non-invasive source of relevant mammary cells for a wide range of applications. In this review, we have summarized the main achievements in this field for dairy cow science and described the interesting perspectives open to manipulate milk persistency during lactation and to cope with oxidative stress during the transition period by regulating mammary stem cells.

miRNAs highlights in stem and cancer cells.

MicroRNAs (miRNAs) are approximately 22 nucleotide endogenous RNA molecules which exert their functions by base pairing with messenger RNAs (mRNAs), thereby regulating protein-coding gene expression. In eukaryotic cells, miRNAs play important roles in regulating biological processes such as proliferation, differentiation, apoptosis, and stem cell self-renewal. miRNAs are encoded by the genome, and more than 1,000 human miRNAs have been identified so far. miRNAs are predicted to target -60% of human mRNAs and are expressed in all animal cells. Unique expression domains, targets, and gain- and loss-of-function phenotypes of particular miRNAs have important implications for directed to control differentiation of stem cell populations. Many cancers show variations in miRNA levels, and more specifically an overall downregulation, when compared to their normal counterparts. Therefore, miRNAs may be used as potential therapeutic agents to correct aberrant transcript levels found in the signaling pathways of cancer. This review examines the most recent acquisition on the role of miRNAs in regulating the cell cycle, with particular emphasis on their effects on cell proliferation and differentiation. The second part explores specifically the role of these factors in the physiological regulation of embryonic stem cells, of cellular reprogramming and their involvement in the activation of stem cells in adult tissues. In the third part, the article discusses some issues that relate to the role of miRNAs in the development of neoplastic diseases, focusing on aspects of the genetic and transcriptional alterations that determine the beginning and the development of tumor process, with emphasis on, looking to emphasize their involvement in the activation of adult cancer stem cells.

Positive effect of silymarin on cell growth and differentiation in bovine and murine mammary cells.

Silymarin, a naturally acknowledged hepatoprotector used in humans to treat liver diseases has been tested in murine (HC11) and bovine (BME-UV) mammary epithelial cell lines to evaluate a possible direct effect on cell growth and differentiation in mammary gland. Silymarin enhanced cell proliferation (p < 0.05) from 10 to 1000 ng/ml in association with growth factors, (up to 20%) or alone (up to 15%) versus controls. Furthermore, silymarin (100 ng/ml) was able to increase (p < 0.05) beta-casein gene expression alone or in association with prolactin (5 microg/ml). These effects may be related with protein kinase B (AKT) activation induced by silymarin treatment (p < 0.05) and/or by a dose-related inhibitory effect (p < 0.05) on caspase-3 activity related to a protective role in cell apoptosis. These data suggest that silymarin should be considered a candidate to support mammary gland activity during a lactogenetic state.

Epidermal growth factor and hepatocyte growth factor receptors collaborate to induce multiple biological responses in bovine mammary epithelial cells.

The aim of this work was to explore whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) could increase the biological responses of a mammary epithelial cell line of bovine origin when added simultaneously. We also investigated a possible molecular mechanism underlying this cooperation. The development of mammary gland requires several circulating and locally produced hormones. Hepatocyte growth factor and its tyrosine kinase receptor, mesenchymal-epithelial transition factor (MET), are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor and its ligands have also been implicated in the growth and morphogenesis of the mammary epithelium. Both EGF and HGF seem to exert a morphogenic program in this tissue; therefore, we hypothesized that these cytokines could act cooperatively in bovine mammary epithelial cells. We have already shown that the bovine BME-UV cell line, a nontumorigenic mammary epithelial line, expresses both MET and EGF receptor. Simultaneous treatment with HGF and EGF elicited an increase in proliferation, dispersion, degradation of extracellular matrix, and motility. Following EGF treatment, BME-UV mammary cells exhibited an increase in MET expression at both the mRNA and protein levels. Long-term treatment of BME-UV cells with HGF and EGF together increased the level of activation of the extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways when compared with HGF or EGF alone. These data outline a possible cooperative role of the EGF and HGF pathways and indicate that cross-talk between their respective receptors may modulate mammary gland development in the cow.