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Extracellular vesicles from early pregnant uterine fluids trigger expression of implantation-related markers in ovine endometrium. 3. Cellfit annual meeting 2019

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Remerciements à Isabelle Hue (INRA, Jouy-en-Josas) et à l'Unité Commune d’Expérimentation Animale (UCEA, INRA, Leudeville) for animal management. Remerciements à Eric Pailhoux et Maëlle Pannetier (INRA, Jouy-en-Josas) for providing us FOXL2-pSG5 and pSG5 plasmids. Remerciements à Kaïs Al-Gubory et Philippe Bolifraud (INRA, Jouy-en-Josas) Remerciements à ICE platform (Claudia Bevilacqua and Nicolas Crapart, INRA, Jouy-en-Josas) for access to the bioanalyzer; International audience; Intensive selection for milk production has led to reduced reproductive efficiency in high-producing dairy cattle. The impact of intensive milk production on oocyte quality as well as early embryo development has been established but few analyses have addressed this question at the initiation of implantation, a critical milestone ensuring a successful pregnancy and normal post-natal development. Our study aimed to determine if contrasted maternal metabolism affects the previously described sensory properties of the endometrium to the conceptus in cattle. Following embryo transfer at Day 7 post-oestrus, endometrial caruncular (CAR) and intercaruncular (ICAR) areas were collected at Day 19 from primiparous postpartum Holstein-Friesian cows that were dried-off immediately after parturition (i.e., never milked; DRY) or milked twice daily (LACT). Gene quantification indicated no significant impact of lactation on endometrial expression of transcripts previously reported as conceptus-regulated (PLET1, PTGS2, SOCS6) and interferon-tau stimulated (RSAD2, SOCS1, SOCS3, STAT1) factors or known as female hormone-regulated genes (FOXL2, SCARA5, PTGS2). Compared with LACT cows, DRY cows exhibited mRNA levels with increased expression for FOXL2 transcription factor and decreased expression for oxidative stress-related genes (CAT, SOD1, SOD2). In vivo and in vitro experiments highlighted that neither interferon-tau nor FOXL2 were involved in transcriptional regulation of CAT, SOD1 and SOD2. In addition, our data showed that variations in maternal metabolism had a higher impact on gene expression in ICAR areas. Collectively, our findings prompt the need to fully understand the extent to which modifications in endometrial physiology drive the trajectory of conceptus development from implantation onwards when maternal metabolism is altered.

Theme: Early Development and Pregnancy; Extracellular vesicles (EVs) are crucial for intercellular communications and they may play important role in the delivery of molecular messages during the pre-implantation period of pregnancy. EVs have been isolated from uterine luminal fluid (ULF) in human and sheep. Recent data have provided evidence that EVs contained in ovine ULF can penetrate endometrial epithelial cells after a 6-days infusion in vivo. Nevertheless, embryo implantation involves rapid and dynamic changes in molecular interactions with the endometrium. Our present work aims to determine whether EVs collected from ULF interact with endometrial epithelial cells and modify cell physiology after a short time of in vitro and in vivo incubation conditions.Primary cultures of endometrial epithelial cells were derived from ovine uteri on day 12 post-oestrus. EVs were purified from ovine ULFs on Day 14 of pregnancy (2 days before conceptus implantation). The presence of EVs in ULF was confirmed by transmission electron microscopic observation. ULF EVs were labeled with lipophilic PKH26 fluorescent dye and then incubated with primary cultures of epithelial cells during 30 min to 24h. Confocal microscopy analyses revealed an uptake of EVs as early as 30 minutes after incubation, followed by a progressive increase of intracellular fluorescence up to 6 hours.For the in vivo study, ovine ULF EVs isolated on Day 14 of pregnancy were labeled with PKH26 fluorescent dye and infused into the uterine lumen of cyclic ewes on Day 12 post-oestrus. After 24h, numerous epithelial cells from the luminal layer and superficial glands exhibited an intensive fluorescence signal, whereas deep endometrial glands displayed few fluorescent cells. No signal was detectable in the stroma. The impact of EVs on endometrial function was investigated by quantifying transcript expression of a selection of endometrial genes. First data pointed out that expression of two genes, including the Myxovirus-Resistance Protein (MX1) was up-regulated following EVs uptake by the endometrial epithelium. This work provides first evidence that ovine EVs from pregnant ULF can (i) enter in endometrial epithelial cells within 30 min in vitro or 24h in vivo (ii) modulate expression of endometrial gene expression known to be critical for embryo implantation. These results suggest a critical role for EVs in the preparation of endometrium when implantation iniates.

Once-daily milking is known to modify cell number and activity in the bovine mammary gland. However, in goats, the effect of once-daily milking on mammary cell death is controversial. To assess the effect of once-daily milking on mammary transcriptome and cell turnover and the effect of the duration ofonce-daily milking, 10 goats producing 2.5 kg of milk per day at 100 DIM were divided into 2 groups. All goats were twice daily milked during a pre-experimental period of 2 wk. Then, the goats were once daily milked for the 3 following weeks. Mammary biopsy samples were collected at d −1 and 7 after the start of once-daily milking for one group of 5 goats and at d −1 and 21 for a second group of 5 goats. Cell apoptosis and proliferation rates were analyzed in mammary tissue by immunohistological analyses after TUNEL and PCNA staining, respectively. Ribonucleic acid was extracted from mammary tissues. A transcriptomic analysis using the Agilent Bovine 4x44k microarrays has been performed to compare the effect of once-daily milking on mammary transcript profiles. Data were normalized and statistical significant raw P-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure. A similar reduction in milk yield (−19%) wasobserved in both groups of goats during once-daily milking compared with twice-daily milking. Cell apoptosis was higher during once-daily milking than during twice-daily milking for both groups of goats (P < 0.05) whereas cell proliferation did not vary (P = 0.27). The transcriptomic analysis showed a differential gene expression of 4,039 transcripts, 2,238 and 1,801 transcripts up- and downregulated, respectively, by once-daily milking compared with twice-daily milking. More than 1,000 transcripts were commonly regulated between the 2 groups of goats. IPA analysis showed that these transcripts were part of networks associated with DNA replication, cellular growth and proliferation, and cell-to-cell signaling and interaction in both groups of goats. RT-qPCR analysis of 11 genes confirmed the differential gene expression with a downregulationof genes involved in milk synthesis and an upregulation of genes involved in cellular cycle and apoptosis. These results showed that once-daily milking induce cell turnover in goat mammary tissue, with a small impact of time.

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Alterations to the metabolic endocrine environment during early life are crucial to mammary gland development. Among these environmental parameters, the initial nutritional event after birth is the consumption of milk, which represents the first maternal support provided to mammalian newborns. Milk is a complex fluid that exerts effects far beyond its immediate nutritional value. The present study, therefore, aimed to determine the effect of the nutritional changes during the neonatal and prepubertal periods on the adult mammary phenotype. Newborn rabbits were suckled by dams fed a high-fat/high-sugar obesogenic (OD) or a control (CON) diet and then subsequently fed either the OD or CON diets from the onset of puberty and throughout early pregnancy. Mammary glands were collected during early pregnancy (Day 8 of pregnancy). Rabbits fed with OD milk and then subjected to an OD diet displayed an abnormal development of the mammary gland: the mammary ducts were markedly enlarged ( < 0.05) and filled with abundant secretory products. Moreover, the alveolar secretory structures were disorganized, with an abnormal aspect characterized by large lumina. Mammary epithelial cells contained numerous large lipid droplets and exhibited fingering of the apical membrane and abnormally enlarged intercellular spaces filled with casein micelles. Leptin has been shown to be involved in modulating several developmental processes. We therefore analyzed its expression in the mammary gland. Mammary leptin mRNA was strongly expressed in rabbits fed with OD milk and subjected to an OD diet by comparison with the CON rabbits. Leptin transcripts and protein were localized in the epithelial cells, indicating that the increase in leptin synthesis occurs in this compartment. Taken together, these findings suggest that early-life nutritional history, in particular through the milking period, can determine subsequent mammary gland development. Moreover, they highlight the potentially important regulatory role that leptin may play during critical early-life nutritional windows with respect to long-term growth and mammary function.

International audience

International audience; Milking frequency influences milk production in cows by regulating milk synthesis and secretion. This in turn involves a number of genes that are differentially expressed. In this study, we hypothesized that milking frequency changes the milk yield through altered gene expression by modifying the methylation at CpGs dinucleotides located within regulatory regions. We focused on a candidate regulatory region of one casein gene (CSN1). Eight heifers were studied over three periods. Period 1: twice daily milking for 5 days of both udders of each heifer. Period 2: differential milking for 7 days: right and left udders were milked twice and once a day, respectively. Period 3: both udders returned to twice daily milking. Milk yield was recorded every day over the three periods. Biopsies from left & right udders were taken at the the end of period 2. Global DNA methylation was evaluated using the LUMA assay. DNA methylation of three CpGs located within a distal regulatory region CSN1 was analyzed using pyrosequencing after bisulfite treatment of genomic DNA. The mRNA expression levels have been quantified by RNASeq. Significance of results was studied with the Wilcoxon test. Global methylation of the once daily milking group (75.9%) is higher compared with the twice daily milking group (74.6%) but only significant if one of the eight heifers, which behaved differently from the others, was excluded (P=0.04). The DNA methylation of all three CpG in the once daily milking group is higher that that of twice daily milking group (P=0.02, P=0.07, and P=0.02, respectively). Once daily milking increases DNA methylation at a regulatory region of the CSN1 gene and at the global level in the bovine mammary gland, to a certain extent. These modifications are related to transcription of the casein genes and to some ncRNA.

International audience; The nuclear organization of mammary epithelial cells has been shown to be sensitive to the three-dimensional microenvironment in several models of cultured cells. However, the relationships between the expression and position of genes have not often been explored in animal tissues. We therefore studied the localization of milk protein genes in the nuclei of luminal mammary epithelial cells during lactation as well as in two non- expressing cells, i.e. hepatocytes and the less differentiated embryonic fibroblasts. We compared the position of a cluster of co-regulated genes, encoding caseins (CSN), with that of the whey acidic protein (WAP) gene which is surrounded by genes displaying different expression profiles. We show that the position of the CSN cluster relative to various nuclear compartments is correlated with its activity. In luminal cells, the CSN cluster loops out from its chromosome territory and is positioned in the most euchromatic regions, and frequently associated with elongating RNA polymerase II rich zones. In hepatocytes and embryonic fibroblasts, the cluster is found preferentially closer to the nuclear periphery. Interestingly, we had previously observed a very peripheral position of the CSN locus in the nuclei of HC11 mammary epithelial cells weakly expressing milk protein genes. We thus show that cultured cell lines are not fully representative of the nuclear organization of genes in a complex and highly organized tissue such as the mammary gland and propose that the spatial positioning of the locus is important to ensuring the optimum control of CSN gene activity observed in the mammary tissue.

National audience

National audience; Though virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We used the mammary epithelial cell line HC11 to investigate the first steps of the formation of cytoplasmic lipid droplets and to evaluate the potential connections between the lipid and protein synthetic pathways. Cytoplasmic lipid droplets originate from the endoplasmic reticulum and display specific proteins on their surface such as ADRP (adipose differentiation-related protein), TIP47 (tail-interacting protein of 47 kDa) and Cav-1 (caveolin-1). Here we show that unsaturated fatty acids (oleate and linoleate) induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of ADRP but not that of TIP47 or Cav-1. Unexpectedly, the cellular store of b-casein, accumulated under the lactogenic action of prolactin and glucocorticoids, was decreased following cell treatment with unsaturated fatty acids. In contrast, saturated fatty acids (stearate and palmitate) had no significant effect on either cytoplasmic lipid droplet proliferation or cellular level of b-casein. We show that the effect of unsaturated fatty acids on the level of b-casein is posttranslational, requires protein synthesis to occur and is blocked by lysosome inhibitors. We also observe that proteasome inhibitors potentiate b-casein degradation, indicating that it can destroy some unknown cytosolic protein(s) implicated in the process that negatively controls b-casein levels. Finally, our data suggest that the degradation of b-casein occurs via the autophagic pathway.

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Chantier qualité GA; The mammary gland undergoes extensive remodeling between the beginning of pregnancy and lactation; this involves cellular processes including cell proliferation, differentiation, and apoptosis, all of which are under the control of numerous regulators. To unravel the role played by miRNA, we describe here 47 new ovine miRNA cloned from mammary gland in early pregnancy displaying strong similarities with those already identified in the cow, human, or mouse. A microarray study of miRNA variations in the adult ovine mammary gland during pregnancy and lactation showed that 100 miRNA are regulated according to three principal patterns of expression: a decrease in early pregnancy, a peak at midpregnancy, or an increase throughout late pregnancy and lactation. One miRNA displaying each pattern (miR-21, miR-205, and miR-200b) was analyzed by qRT-PCR. Variations in expression were confirmed for all three miRNA. Using in situ hybridization, we detected both miR-21 and miR-200 in luminal mammary epithelial cells when expressed, whereas miR-205 was expressed in basal cells during the first half of pregnancy and then in luminal cells during the second half. We therefore conclude that miR-21 is strongly expressed in the luminal cells of the normal mammary gland during early pregnancy when extensive cell proliferation occurs. In addition, we show that miR-205 and miR-200 are coexpressed in luminal cells, but only during the second half of pregnancy. These two miRNA may cooperate to maintain epithelial status by repressing an EMT-like program, to achieve and preserve the secretory phenotype of mammary epithelial cells.