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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 1st and 2nd lactations. Lactation rank induces 981 differential methylations of cytosines (DMCs) in mammary tissue. Inflammation in 1st lactation compared to inflammation in 2nd lactation results in the identification of 964 DMCs. When comparing inflammation in 1st vs. 2nd 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Somatic cells are present in the milk throughout lactation and consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Our objective was to determine the efficacy of using somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. Over a 4-wk interval, cells were isolated from daily morning milk samples and samples taken 30 min after milking. They were characterized by direct cell counts and by flow cytometry analysis after immunostaining with antibodies directed against cytokeratin and CD45, a common leukocyte antigen. Epithelial cell counts within the morning milk ranged from 15 to 45% of total milk somatic cells. After-milking samples contained twice as many cells as did morning milk samples. The RNA was extracted from the somatic cells of both types of milk samples with equivalent efficiency (a mean of 1.2 [micro]g RNA/mL of milk). Four mRNA variants of the [alpha]-S1 casein gene were detected by Northern blot analysis and the amount of each mRNA in milk cells was related to protein concentration in milk. The comparison between mRNA from the mammary gland and from congruently collected milk cells showed that relative amounts of mRNA for each milk-protein ([alpha]-S1 and [kappa]-casein and [alpha]-lactalbumin) were conserved. In a third experiment, daily milk cell RNA preparations were extracted to assess the effect of growth hormone (GH) on mammary gene expression; four goats were separated into two groups in order to perform a switch-back design consisting of three treatment weeks: Control, GH-Control or GH-Control-GH. In this study, treatment of goats with GH increased milk yields by 5%. Throughout the control and GH treatments, the expression of the three milk-protein genes studied were highly and significantly correlated (r = 0.949 and r = 0.958, P < 0.001 for, respectively, [alpha]-S1 and [kappa]-casein and for [alpha]-S1 casein and [alpha]-lactalbumin). During GH treatment, the three milk-protein mRNA abundances increased with the same pattern. In conclusion, the opportunity to use milk somatic cells for RNA preparation and analysis provides a significant improvement over the use of biopsy samples in assessing gene activity in the mammary gland and allows easy and repetitive sampling without damaging mammary tissue. Furthermore, we propose that this method could be used to investigate the transcriptional status of the mammary gland of an animal in relation to its genotype, nutritional and pathologic status, and under influence by hormonal factors. Key Words: Goats, Mammary Tissue, Messenger RNA, Somatotropin

Abstract Background As a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes. Results Three hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of intra-uterine growth retardation (IUGR). Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals. Conclusion We could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5.10-5), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.

Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer (SCNT) is still doomed by a high incidence of pregnancy failure. In cattle, most gestation losses initially take place during the peri-implantation period. Then, abnormal placentation associated with fetal overgrowth (abnormal offspring syndrome) occurs in late pregnancy. As a consequence, IETS recommendations include regular ultrasound evaluation of SCNT pregnancies, with qualitative ultrasound evaluation of the placentomes, fetal movements and fluids being proposed. The objective of this study was to evaluate the use of new, quantitative parameters for the assessment of feto-placental development in AI and SCNT bovine pregnancies. Twenty-two heifers of 4 different breeds were used as SCNT recipients and 11 Holstein heifers were used as AI controls (C). All SCNT fetuses were produced as previously published in the laboratory, using the same fibroblast donor. Animals were scanned every 2 weeks from Day 150 using a Voluson-i (GE Medical Systems) with a transabdominal multifrequency probe (2.2 to 6.5 MHz), allowing automatic 3D volume and Doppler acquisition. Each time, 7 placentomes were scored from 0 to 3 according to echogenicity, general appearance and degree of oedema and their 2D surface was measured. Fetal intercostal width (ICW) was measured in a coronal view just behind the heart. Doppler velocimetry indices, pulsatility index (PI) and resistance index (RI) of one of the two fetal umbilical arteries were obtained and the diameters of the umbilical vessels were measured (UD). The 3D power Doppler was performed to obtain vascular index (VI), flow index (FI) and vascular flow index (VFI). Data were analysed by ANOVA with GraphPad Prism®. So far, 27 heifers have either delivered (n = 10 SCNT and 7 C) or pregnancies have been terminated because of abnormal offspring syndrome (n = 10 SCNT). One thousand five hundred and 197 placentomes have been analysed for 2D and 3D analyses, respectively. The mean placentome score is significantly higher (less normal) in SCNT compared with C (P < 0.0001) but does not vary according to gestational age. The placentome surface is also significantly larger in SCNT vs C (P < 0.0001) but also in SCNT that did not go to term vs those that were alive at birth (P < 0.002). Fetal ICW and UD are consistently larger in SCNT vs C (P < 0.0001) at all stages of pregnancy and there is a significant correlation (r2 = 0.81) between ICW at 15 days before term and birth weight. The PI and RI were not different between SCNT and C. Intra-operator reproducibility of 3D analyses was very high (intra-class correlation coefficient: 80 to 95% for a 95% confidence interval). There was no significant difference for VI, FI, or VFI between SCNT and C. Ongoing work taking into account pregnancy outcomes indicates that placentome scores are useful indicators of pregnancy outcome in SCNT pregnancies. In contrast, the abnormal vascularization observed by histology in SCNT placentomes does not appear to be accompanied by abnormal placental blood flow when analysed using quantitative 3D Doppler. This project received financial support from ANR (ref. PCS-09-GENM-022). [ABSTRACT FROM AUTHOR]