Mutation of SOCS2 induces structural and functional changes in mammary development.

Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or control mice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare.

Specific Milk Composition of miR-30b Transgenic Mice Associated with Early Duodenum Maturation in Offspring with Lasting Consequences for Growth.

BACKGROUND: Milk composition is complex and includes numerous components essential for offspring growth and development. In addition to the high abundance of miR-30b microRNA, milk produced by the transgenic mouse model of miR-30b-mammary deregulation displays a significantly altered fatty acid profile. Moreover, wild-type adopted pups fed miR-30b milk present an early growth defect. OBJECTIVE: This study aimed to investigate the consequences of miR-30b milk feeding on the duodenal development of wild-type neonates, a prime target of suckled milk, along with comprehensive milk phenotyping. METHODS: The duodenums of wild-type pups fed miR-30b milk were extensively characterized at postnatal day (PND)-5, PND-6, and PND-15 using histological, transcriptomic, proteomic, and duodenal permeability analyses and compared with those of pups fed wild-type milk. Milk of miR-30b foster dams collected at mid-lactation was extensively analyzed using proteomic, metabolomic, and lipidomic approaches and hormonal immunoassays. RESULTS: At PND-5, wild-type pups fed miR-30b milk showed maturation of their duodenum with 1.5-fold (P < 0.05) and 1.3-fold (P < 0.10) increased expression of Claudin-3 and Claudin-4, respectively, and changes in 8 duodenal proteins (P < 0.10), with an earlier reduction in paracellular and transcellular permeability (183 ng/mL fluorescein sulfonic acid [FSA] and 12 ng/mL horseradish peroxidase [HRP], respectively, compared with 5700 ng/mL FSA and 90 ng/mL HRP in wild-type; P < 0.001). Compared with wild-type milk, miR-30b milk displayed an increase in total lipid (219 g/L compared with 151 g/L; P < 0.05), ceramide (17.6 µM compared with 6.9 µM; P < 0.05), and sphingomyelin concentrations (163.7 µM compared with 76.3 µM; P < 0.05); overexpression of 9 proteins involved in the gut barrier (P < 0.1); and higher insulin and leptin concentrations (1.88 ng/mL and 2.04 ng/mL, respectively, compared with 0.79 ng/mL and 1.06 ng/mL; P < 0.01). CONCLUSIONS: miR-30b milk displays significant changes in bioactive components associated with neonatal duodenal integrity and maturation, which could be involved in the earlier intestinal closure phenotype of the wild-type pups associated with a lower growth rate.

In silico identification of variations in microRNAs with a potential impact on dairy traits using whole ruminant genome SNP datasets.

MicroRNAs are small noncoding RNAs that have important roles in the lactation process and milk biosynthesis. Some polymorphisms have been studied in various livestock species from the perspective of pathology or production traits. To target variants that could be the causal variants of dairy traits, genetic variants of microRNAs expressed in the mammary gland or present in milk and localized in dairy quantitative trait loci (QTLs) were investigated in bovine, caprine, and ovine species. In this study, a total of 59,124 (out of 28 millions), 13,427 (out of 87 millions), and 4761 (out of 38 millions) genetic variants in microRNAs expressed in the mammary gland or present in milk were identified in bovine, caprine, and ovine species, respectively. A total of 4679 of these detected bovine genetic variants are located in dairy QTLs. In caprine species, 127 genetic variants are localized in dairy QTLs. In ovine species, no genetic variant was identified in dairy QTLs. This study leads to the detection of microRNA genetic variants of interest in the context of dairy production, taking advantage of whole genome data to identify microRNA genetic variants expressed in the mammary gland and localized in dairy QTLs.

Effect of feed restriction on dairy cow milk production: a review.

In the dairy cow, negative energy balance affects milk yield and composition as well as animal health. Studying the effects of negative energy balance on dairy cow milk production is thus essential. Feed restriction (FR) experiments attempting to reproduce negative energy balance by reducing the quantity or quality of the diet were conducted in order to better describe the animal physiology changes. The study of FR is also of interest since with climate change issues, cows may be increasingly faced with periods of drought leading to a shortage of forages. The aim of this article is to review the effects of FR during lactation in dairy cows to obtain a better understanding of metabolism changes and how it affects mammary gland activity and milk production and composition. A total of 41 papers studying FR in lactating cows were used to investigate physiological changes induced by these protocols. FR protocols affect the entire animal metabolism as indicated by changes in blood metabolites such as a decrease in glucose concentration and an increase in non-esterified fatty acid or ß-hydroxybutyrate concentrations; hormonal regulations such as a decrease in insulin and insulin-like growth factor I or an increase in growth hormone concentrations. These variations indicated a mobilization of body reserve in most studies. FR also affects mammary gland activity through changes in gene expression and could affect mammary cell turnover through cell apoptosis, cell proliferation, and exfoliation of mammary epithelial cells into milk. Because of modifications of the mammary gland and general metabolism, FR decreases milk production and can affect milk composition with decreased lactose and protein concentrations and increased fat concentration. These effects, however, can vary widely depending on the type of restriction, its duration and intensity, or the stage of lactation in which it takes place. Finally, to avoid yield loss and metabolic disorders, it is important to identify reliable biomarkers to monitor energy balance.

Epigenetics: New Insights into Mammary Gland Biology.

The mammary gland undergoes important anatomical and physiological changes from embryogenesis through puberty, pregnancy, lactation and involution. These steps are under the control of a complex network of molecular factors, in which epigenetic mechanisms play a role that is increasingly well described. Recently, studies investigating epigenetic modifications and their impacts on gene expression in the mammary gland have been performed at different physiological stages and in different mammary cell types. This has led to the establishment of a role for epigenetic marks in milk component biosynthesis. This review aims to summarize the available knowledge regarding the involvement of the four main molecular mechanisms in epigenetics: DNA methylation, histone modifications, polycomb protein activity and non-coding RNA functions.

RumimiR: a detailed microRNA database focused on ruminant species.

The ever-increasing use of next-generation sequencing technologies to explore the genome has generated large quantities of data in recent years. Numerous publications have described several thousand sequences of microRNAs, all species included. A new database (RumimiR) has been created from the literature to provide a detailed description of microRNAs for three ruminant species: cattle, goats and sheep. To date, 2887, 2733 and 5095 unique microRNAs from bovine, caprine and ovine species, respectively, are included. In addition to the most recent reference genomic position and sequence of each microRNA, this database contains details about the animals, tissue origins and experimental conditions mentioned in the publications. Identity to human or mouse microRNA is also indicated. The RumimiR database allows data filtering by selecting microRNAs on the basis of defined criteria such as animal status or tissue origin. For ruminant studies, RumimiR supplements the widely used miRBase database, by using complementary criteria to allow browsing and filtering, and integrates all newly described published sequences. The principal goal of this database is to provide easy access to all the ruminant microRNAs described in the literature.

Puberty is a critical window for the impact of diet on mammary gland development in the rabbit.

BACKGROUND: Nutritional changes can affect future lactation efficiency. In a rabbit model, an obesogenic diet initiated before puberty and pursued throughout pregnancy enhances mammary differentiation, but when started during the neonatal period can cause abnormal mammary development in early pregnancy. The aim of this study was to investigate the impact of an unbalanced diet administered during the pubertal period only. RESULTS: Consuming an obesogenic diet at puberty did not affect either metabolic parameters or certain maternal reproductive parameters at the onset of adulthood. In contrast, at Day 8 of pregnancy, epithelial tissue showed a lower proliferation rate in obesogenic-diet fed rabbits than in control-diet fed rabbits. Wap and Cx26 genes, mammary epithelial cell differentiation markers, were upregulated although Wap protein level remained unchanged. However, the expression of genes involved in lipid metabolism and in alveolar formation was not modified. CONCLUSION: Taken together, our results demonstrate that the consumption for 5 weeks of an obesogenic diet during the pubertal period initiates mammary structure modifications and affects mammary epithelial cell proliferation and differentiation. Our findings highlight the potentially important role played by unbalanced nutrition during critical early-life windows in terms of regulating mammary epithelial cell differentiation and subsequent function in adulthood.

Defects of the endoplasmic reticulum and changes to lipid droplet size in mammary epithelial cells due to miR-30b-5p overexpression are correlated to a reduction in Atlastin 2 expression.

During lactation, mammary epithelial cells secrete fat in the form of milk fat globules that originate from intracellular lipid droplets. These droplets may form de novo from the endoplasmic reticulum or be derived from existing lipid droplets; they then either grow because enzymes of triacylglycerol synthesis relocate from the reticulum to their surface, or due to fusion and fission with other droplets. The overexpression of miR-30b-5p in the developing mouse mammary gland impairs lactation, which includes an increase in lipid droplet size. This study was performed to understand the origin of this defect affecting lipid droplets observed in transgenic mice. Electron microscopy analyses revealed a fragmented and discontinued tubular network of endoplasmic reticulum in the mammary epithelial cells of transgenic mice. The milk fatty acid composition was modified, with lower levels of medium-chain saturated fatty acids and a proportional increase in long-chain monounsaturated fatty acids in transgenic versus wild-type mice. Further, investigations of microRNA targets revealed a significant downregulation of ATLASTIN 2 (a GTPase described as playing a key role in lipid droplet formation) due to miR-30b-5p overexpression. Our results suggest that the increase in lipid droplet size observed in the mammary epithelial cells of transgenic mice might result from changes to lipid droplet formation and secretion because of direct modifications to Atl2 expression and indirect changes to endoplasmic reticulum morphology resulting from the overexpression of miR-30b-5p.

Sunflower oil supplementation affects the expression of miR-20a-5p and miR-142-5p in the lactating bovine mammary gland.

Oil supplementation in dairy cattle diets is used to modulate milk fat composition, as well as the expression of mammary lipogenic genes, whose regulation remains unclear. MiRNAs are small non-coding RNA considered as crucial regulators of gene expression, offering clues to explain the mechanism underlying gene nutriregulation. The present study was designed to identify miRNAs whose expression in the cow mammary gland is modulated by sunflower oil supplementation. MiRNomes were obtained using RNAseq technology from the mammary gland of lactating cows receiving a low forage diet, supplemented or not with 4% sunflower oil. Among the 272 miRNAs characterized, eight were selected for RT-qPCR validations, showing the significant down-regulation of miR-142-5p and miR-20a-5p by sunflower supplementation. These two miRNAs are predicted to target genes whose expression was reported as differentially expressed by sunflower supplementation. Among their putative targets, ELOVL6 gene involved in lipid metabolism has been studied. However, a first analysis did not show its significant down-regulation, in response to the over-expression of miR-142-5p, of miR-20a-5p, or both, in a bovine mammary epithelial cell line. However, a clearer understanding of the miRNA expression by lipid supplementation would help to decipher the regulation of lactating cow mammary gland in response to nutrition.

Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy.

R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1's partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-ß signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function.

Food Deprivation Affects the miRNome in the Lactating Goat Mammary Gland.

BACKGROUND: Nutrition affects milk composition thus influencing its nutritional properties. Nutrition also modifies the expression of mammary genes, whose regulation is not fully understood. MicroRNAs (miRNA) are small non coding RNA which are important post-transcriptional regulators of gene expression by targeting messenger RNAs. Our goal was to characterize miRNA whose expression is regulated by nutrition in the lactating goat mammary gland, which may provide clues to deciphering regulations of the biosynthesis and secretion of milk components. METHODOLOGY/PRINCIPAL FINDINGS: Using high-throughput sequencing technology, miRNomes of the lactating mammary gland were established from lactating goats fed ad libitum or deprived of food for 48 h affecting milk production and composition. High throughput miRNA sequencing revealed 30 miRNA with an expression potentially modulated by food deprivation; 16 were down-regulated and 14 were up-regulated. Diana-microT predictive tools suggested a potential role for several nutriregulated miRNA in lipid metabolism. Among the putative targets, 19 were previously identified as differently expressed genes (DEG). The functions of these 19 DEG revealed, notably, their involvement in tissue remodelling. CONCLUSION/SIGNIFICANCE: In conclusion, this study offers the first evidence of nutriregulated miRNA in the ruminant mammary gland. Characterization of these 30 miRNA could contribute to a clearer understanding of gene regulation in the mammary gland in response to nutrition.

Annotation of the goat genome using next generation sequencing of microRNA expressed by the lactating mammary gland: comparison of three approaches.

BACKGROUND: MicroRNAs (miRNA) are small endogenous non-coding RNA involved in the post-transcriptional regulation of specific mRNA targets. The first whole goat genome sequence became available in 2013, with few annotations. Our goal was to establish a list of the miRNA expressed in the mammary gland of lactating goats, thus enabling implementation of the goat miRNA repertoire and considerably enriching annotation of the goat genome. RESULTS: Here, we performed high throughput RNA sequencing on 10 lactating goat mammary glands. The bioinformatic detection of miRNA was carried out using miRDeep2 software. Three different methods were used to predict, quantify and annotate the sequenced reads. The first was a de novo approach based on the prediction of miRNA from the goat genome only. The second approach used bovine miRNA as an external reference whereas the last one used recently available goat miRNA. The three methods enabled the prediction and annotation of hundreds of miRNA, more than 95% were commonly identified. Using bovine miRNA, 1,178 distinct miRNA were detected, together with the annotation of 88 miRNA for which corresponding precursors could not be retrieved in the goat genome, and which were not detected using the de novo approach or with the use of goat miRNA. Each chromosomal coordinate of the precursors determined here were generated and depicted on a reference localisation map. Forty six goat miRNA clusters were also reported. The study revealed 263 precursors located in goat protein-coding genes, amongst which the location of 43 precursors was conserved between human, mouse and bovine, revealing potential new gene regulations in the goat mammary gland. Using the publicly available cattle QTL database, and cow precursors conserved in the goat and expressed in lactating mammary gland, 114 precursors were located within known QTL regions for milk production and composition. CONCLUSIONS: The results reported here represent the first major identification study on miRNA expressed in the goat mammary gland at peak lactation. The elements generated by this study will now be used as references to decipher the regulation of miRNA expression in the goat mammary gland and to clarify their involvement in the lactation process.

No effect of an elevated miR-30b level in mouse milk on its level in pup tissues.

Recent reports have shown that ingested microRNAs may be transferred to blood, accumulate in tissues and exert canonical regulation on endogenous transcripts. In spite of several attempts to replicate these findings, they have not been confirmed and several questions remain. By using a transgenic mouse model presenting a high level of miR-30b in milk, the horizontal delivery of this microRNA via oral ingestion was studied in pups. Our findings demonstrated that, although very high levels of miR-30b were found in milk and in stomach contents of the pups, we did not detect an increase in miR-30b in tissues of pups fed by transgenic females compared to pups fed by wild-type females.

Characterisation and comparison of lactating mouse and bovine mammary gland miRNomes.

BACKGROUND: The mammary gland is a dynamic organ that undergoes important physiological changes during reproductive cycles. Until now, data regarding the characterisation of miRNA in the mammary gland have been scarce and mainly focused on their abnormal expression in breast cancer. Our goal was to characterise the microRNA (miRNA) involved in mechanisms regulating the mammary function, with particular focus on the lactation stage. METHODOLOGY/PRINCIPAL FINDINGS: Using high-throughput sequencing technology, the exhaustive repertoires of miRNA expressed (miRNome) in mouse and bovine mammary glands during established lactation were identified, characterized and compared. Furthermore, in order to obtain more information on miRNA loading in the RNA-induced silencing complex (RISC), the miRNome was compared with that obtained from RNA associated with the AGO2 protein (AGO2-miRNome) in mouse lactating mammary gland. This study enabled the identification of 164 and 167 miRNA in mouse and bovine, respectively. Among the 30 miRNA most highly expressed in each species, 24 were common to both species and six of them were preferentially highly expressed in lactating than non-lactating mammary gland. The potential functional roles of these 24 miRNA were deduced using DIANA-miRPath software, based on miRNA/mRNA interactions. Moreover, seven putative novel miRNA were identified. Using DAVID analysis, it was concluded that the predicted targets of two of these putative novel miRNA are involved in mammary gland morphogenesis. CONCLUSION/SIGNIFICANCE: Our study provides an overview of the characteristics of lactating mouse and bovine mammary gland miRNA expression profiles. Moreover, species-conserved miRNA involved in this fundamental biological function were identified. These miRNomes will now be used as references for further studies during which the impact of animal breeding on the miRNA expression will be analysed.

Preeclampsia-like symptoms induced in mice by fetoplacental expression of STOX1 are reversed by aspirin treatment.

Preeclampsia (PE) is a common human-specific pregnancy disorder defined by hypertension and proteinuria during gestation and responsible for maternal and fetal morbimortality. STOX1, encoding a transcription factor, was the first gene associated with PE as identified by positional cloning approaches. Its overexpression in choriocarcinoma cells mimics the transcriptional consequences of PE in the human placenta. Here, we created transgenic mouse strains overexpressing human STOX1. Wild-type female mice crossed with transgenic male mice reproduce accurately the symptoms of severe PE: gestational hypertension, proteinuria, and elevated plasma levels of soluble fms-like tyrosine kinase 1 and soluble endoglin. Placental and kidney histology were altered. Symptoms were prevented or alleviated by aspirin treatment. STOX1-overexpressing mice constitute a unique model for studying PE, allow testing therapeutic approaches, and assessing the long-term effects of the preeclamptic syndrome.

Overexpression of miR-30b in the developing mouse mammary gland causes a lactation defect and delays involution.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

Prion protein and Shadoo are involved in overlapping embryonic pathways and trophoblastic development.

The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various Prnp transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the Prnp-knockout-Sprn-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various Prnp and Sprn genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in Eutherians and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.

Expression of the prion-like protein Shadoo in the developing mouse embryo.

The prion-like protein Shadoo has been suggested to compensate for the lack of PrP in Prnp-knockout mice, explaining their lack of extreme phenotype. In adult mice, both PrP and Shadoo have shown overlapping expression patterns and shared functions. Their expression in the mouse embryo has also been suggested to be complementary, as invalidation of both genes results in embryonic lethality. The developmental expression profile of PrP has been described from post-implantation stages up until birth. However the spatial expression pattern of Shadoo in the developing mouse embryo is not known. We previously described the expression profile of the prion-like protein Shadoo in adult mice using Sprn reporter mice (Sprn-GFP and Sprn-LacZ). Here we used these mice to describe the developmental expression of Shadoo between 10.5 and 14.5 dpc. The observed pattern in specific embryonic cell lineages and in extra-embryonic tissues is consistent with the previously reported phenotype resulting from its knockdown.

Generation of Sprn-regulated reporter mice reveals gonadic spatial expression of the prion-like protein Shadoo in mice.

The protein Shadoo (Sho) is a paralogue of prion protein, and encoded by the gene Sprn. Like prion protein it is primarily expressed in central nervous system, and has been shown to have a similar expression pattern in certain regions of the brain. We have generated reporter mice carrying a transgene encompassing the Sprn promoter, exon 1, intron 1 and the 5'-end of exon 2 driving expression of either the LacZ or GFP reporter gene to study the expression profile of Shadoo in mice. Expression of the reporter genes was analysed in brains of these transgenic mice and was shown to mimic that of the endogenous gene expression, previously described by Watts et al. [1]. Consequently, the Sprn-LacZ mice were used to study the spatial expression of Sho in other tissues of the adult mouse. Several tissues were collected and stained for ß-gal activity, including the thymus, heart, lung, liver, kidney, spleen, intestine, muscle, and gonads. From this array of tissues, the transgene was consistently expressed only in specific cell types of the testicle and ovary, suggesting a role for Shadoo in fertility and reproduction. These mice may serve as a useful tool in deciphering the regulation of the prion-like gene Sprn and thus, indirectly, of the Shadoo protein.