In dairy cows, diet is one factor that can affect their milk production and composition. However, the effect of feed restriction on milk miRNome has not yet been described. Indeed, milk is the body ...fluid with the highest RNA concentration, which includes numerous microRNA. Its presence in the four different milk fractions, whole milk, fat globules, mammary epithelial cells and extracellular vesicles, is still poorly documented. This study aimed to describe the effects of different feed restrictions on the miRNome composition of different milk fractions. Two feed restrictions were applied to lactating dairy cows, one of high intensity and one of moderate intensity. 2,896 mature microRNA were identified in the different milk fractions studied, including 1,493 that were already known in the bovine species. Among the 1,096 microRNA that were sufficiently abundant to be informative, the abundance of 1,027 of them varied between fractions: 36 of those were exclusive to one milk fraction. Feed restriction affected the abundance of 155 microRNA, with whole milk and milk extracellular vesicles being the most affected, whereas milk fat globules and exfoliated mammary epithelial cells were little or not affected at all. The high intensity feed restriction led to more microRNA variations in milk than moderate restriction. The target prediction of known microRNA that varied under feed restriction suggested the modification of some key pathways for lactation related to milk fat and protein metabolisms, cell cycle, and stress responses. This study highlighted that the miRNome of each milk fraction is specific, with mostly the same microRNA composition but with variations in abundance between fractions. These specific miRNomes were affected differently by feed restrictions, the intensity of which appeared to be a major factor modulating milk miRNomes. These findings offer opportunities for future research on the use of milk miRNA as biomarkers of energy status in dairy cows, which is affected by feed restrictions.
Mastitis is among the main reasons women cease breastfeeding. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, the effect of ...inflammation on the mammary gland is not completely understood. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation after in vivo intramammary challenges and the differences in DNA methylation between 1
st
and 2
nd
lactations. Lactation rank induces 981 differential methylations of cytosines (DMCs) in mammary tissue. Inflammation in 1
st
lactation compared to inflammation in 2
nd
lactation results in the identification of 964 DMCs. When comparing inflammation in 1
st
vs. 2
nd
lactations with previous inflammation history, 2590 DMCs were identified. Moreover, Fluidigm PCR data show changes in the expression of several genes related to mammary function, epigenetic regulation, and the immune response. We show that the epigenetic regulation of two successive physiological lactations is not the same in terms of DNA methylation and that the effect of lactation rank on DNA methylation is stronger than that of the onset of inflammation. The conditions presented here show that few DMCs are shared between comparisons, suggesting a specific epigenetic response depending on lactation rank, the presence of inflammation, and even whether the cells had previously suffered inflammation. In the long term, this information could lead to a better understanding of the epigenetic regulation of lactation in both physiological and pathological conditions.
Abbreviations: RRBS, reduced representation bisulphite sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; MaSC, mammary stem cell; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process
Mastitis is among the main reasons women cease breastfeeding, which leads to them supplementing breast milk with artificial formula. In farm animals, mastitis results in significant economic losses ...and the premature culling of some animals. Nevertheless, researchers do not know enough about the effect of inflammation on the mammary gland. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation (4 h post-injection of lipopolysaccharide). We analysed the expression of some genes related to mammary gland function, epigenetic regulation, and the immune response. The analysis focused on three comparisons: inflammation during the first lactation, inflammation during second lactation with no history of inflammation, and inflammation during second lactation with previous inflammation. We identified differentially methylated cytosines (DMCs), differentially methylated regions (DMRs), and some differentially expressed genes (DEGs) for each comparison. The three comparisons shared some DEGs; however, few DMCs and only one DMR were shared. These observations suggest that inflammation is one of several factors affecting epigenetic regulation during successive lactations. Furthermore, the comparison between animals in second lactation with and without inflammation, with no inflammation history during first lactation showed a different pattern compared to the other conditions in this experiment. This indicates that inflammation history plays an important role in determining epigenetic changes. The data presented in this study suggest that lactation rank and previous inflammation history are equally important when explaining mammary tissue gene expression and DNA methylation changes.
Abbreviations: RRBS, reduced representation bisulfite sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process
The concept of milk as a healthy food has opened the way for studies on milk components, from nutrients to microRNAs, molecules with broad regulatory properties present in large quantities in milk. ...Characterization of these components has been performed in several species, such as humans and bovine, depending on the stages of lactation. Here, we have studied the variation in milk microRNA composition according to genetic background. Using high throughput sequencing, we have characterized and compared the milk miRNomes of Holstein and Normande cattle, dairy breeds with distinct milk production features, in order to highlight microRNAs that are essential for regulation of the lactation process. In Holstein and Normande milk, 2,038 and 2,030 microRNAs were identified, respectively, with 1,771 common microRNAs, of which 1,049 were annotated and 722 were predicted. The comparison of the milk miRNomes of two breeds allowed to highlight 182 microRNAs displaying significant differences in the abundance. They are involved in the regulation of lipid metabolism and mammary morphogenesis and development, which affects lactation. Our results provide new insights into the regulation of molecular mechanisms involved in milk production.
Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is ...an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.
MicroRNA (miRNA) are negative regulators of gene expression, capable of exerting pronounced influences upon the translation and stability of mRNA. They are potential regulators of normal mammary ...gland development and of the maintenance of mammary epithelial progenitor cells. This study was undertaken to determine the role of miR-30b on the establishment of a functional mouse mammary gland. miR-30b is a member of the miR-30 family, composed of 6 miRNA that are highly conserved in vertebrates. It has been suggested to play a role in the differentiation of several cell types.
The expression of miR-30b was found to be regulated during mammary gland development. Transgenic mice overexpressing miR-30b in mammary epithelial cells were used to investigate its role. During lactation, mammary histological analysis of the transgenic mice showed a reduction in the size of alveolar lumen, a defect of the lipid droplets and a growth defect of pups fed by transgenic females. Moreover some mammary epithelial differentiated structures persisted during involution, suggesting a delay in the process. The genes whose expression was affected by the overexpression of miR-30b were characterized by microarray analysis.
Our data suggests that miR-30b is important for the biology of the mammary gland and demonstrates that the deregulation of only one miRNA could affect lactation and involution.
MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Until ...now their implication in the mammary gland biology was suggested by few studies mainly focusing on pathological situations allowing the characterization of miRNAs as markers of breast cancer tumour classes. If in the normal mammary gland, the expression of known miRNAs has been studied in human and mice but the full repertoire of miRNAs expressed in this tissue is not yet available.
To extend the repertoire of mouse mammary gland expressed miRNAs, we have constructed several libraries of small miRNAs allowing the cloning of 455 sequences. After bioinformatics' analysis, 3 known miRNA (present in miRbase) and 33 new miRNAs were identified. Expression of 24 out of the 33 has been confirmed by RT-PCR. Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them. No correlation could be established between their expression pattern and evolutionary conservation. Six of them appear to be mouse specific. In several cases, multiple potential precursors of miRNA were present in the genome and we have developed a strategy to determine which of them was able to mature the miRNA.
The cloning approach has allowed improving the repertoire of miRNAs in the mammary gland, an evolutionary recent organ. This tissue is a good candidate to find tissue-specific miRNAs and to detect miRNA specific to mammals. We provide evidence for 24 new miRNA. If none of them is mammary gland specific, a few of them are not ubiquitously expressed. For the first time 6 mouse specific miRNA have been identified.
RSPO1
is a newly discovered gene involved in sex differentiation. Two goat BAC clones encompassing the
RSPO1
gene (
gRSPO1
) were injected into mouse oocytes and several transgenic lines derived. ...Both clones induced
gRSPO1
over-expression in various tissues, including male and female gonads, with no obvious phenotype and normal sex-ratios. Introgression of the
gRSPO1
transgene into a mouse
RSPO1
knockout genotype resulted in the rescue of the fertility and the disappearance of the masculinized gonadic features of the females, demonstrating the functionality of the goat protein in a mouse context. On the contrary, over-expression of
gRSPO1
within a
mSRY
or a
gSRY
-XX genotypes did not interfere with the
SRY
-induced male phenotype.
DNAJC2 protein, also known as ZRF1 or MPP11, acts both as chaperone and as chromatin regulator. It is involved in stem cell differentiation and its expression is associated with various cancer ...malignancies. However, the role of Dnajc2 gene during mouse embryogenesis has not been assessed so far. To this aim, we invalidated Dnajc2 gene in FVB/Nj mice using the CrispR/Cas9 approach. We showed that this invalidation leads to the early post-implantation lethality of the nullizygous embryos. Furthermore, using siRNAs against Dnajc2 in mouse 1-cell embryos, we showed that maternal Dnajc2 mRNAs may allow for the early preimplantation development of these embryos. Altogether, these data demonstrate for the first time the requirement of DNAJC2 for early mouse embryogenesis.
•Invalidation of DNAJC2 leads to early post-implantation lethality of mouse embryos.•Dnajc2 is expressed throughout early mouse embryogenesis.•Maternal Dnajc2 mRNAs allow early preimplantation mouse embryonic development.
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