Understanding the Inguinal Sinus in Sheep (Ovis aries)-Morphology, Secretion, and Expression of Progesterone, Estrogens, and Prolactin Receptors.
ABSTRACT: Post-parturient behavior of mammalian females is essential for early parent-offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.
Project description:These trials were carried out to determine firstly the effect of diet and type of pregnancy on the transcriptional expression of genes involved in angiogenesis and cell turnover/lactogenesis inside the sheep mammary gland from late gestation to late lactation. Eighteen Ile de France sheep, 8 twin- and 10 single-bearing ewes were alloted into two groups according to their diet, either based on ad libitum naturalized pasture or red clover hay plus lupine from day -45 pre-partum until day +60 post-partum. Samples from diets and mammary glands were collected at day -10 pre partum (time 1), day +30 (time 2) and day +60 post-partum (time 3) and analyzed by qRT-PCR. Additionally, samples from longissimus dorsi muscle were taken from lambs twice, at weaning and 45 days later, to determine the effect of the maternal treatment with regard to diet and type of pregnancy, on the mRNA expression of genes involved in lipid metabolism. The data was processed using the lme4 package for R, and SPSS Statistics 23.0 for Windows®. The results showed that the group of twin-bearing ewes fed red clover showed a higher expression of genes involved in angiogenesis before lambing and in cell turnover/lactogenesis during late lactation, explained by a lamb survival mechanism to delay apoptosis as a way to keep a secretory cells population and boosted by the diet quality, assuring a longer milk production potential during late lactation. Regarding lambs, apparently the maternal diet would influence the transcriptional expression of lipogenic enzymes in the longissimus dorsi muscle after weaning, but further studies are necessary to validate these results. In summary, Twin-bearing ewes fed red clover performed best at increasing the expression of genes associated with angiogenesis and cell turnover/lactogenesis in the mammary gland.
Project description:Much of our knowledge of the drivers of immune variation, and how these responses vary over time, comes from humans, domesticated livestock or laboratory organisms. While the genetic basis of variation in immune responses have been investigated in these systems, there is a poor understanding of how genetic variation influences immunity in natural, untreated populations living in complex environments. Here, we examine the genetic architecture of variation in immune traits in the Soay sheep of St Kilda, an unmanaged population of sheep infected with strongyle gastrointestinal nematodes. We assayed IgA, IgE and IgG antibodies against the prevalent nematode Teladorsagia circumcincta in the blood plasma of > 3,000 sheep collected over 26 years. Antibody levels were significantly heritable (h2 = 0.21 to 0.57) and highly stable over an individual's lifespan. IgA levels were strongly associated with a region on chromosome 24 explaining 21.1% and 24.5% of heritable variation in lambs and adults, respectively. This region was adjacent to two candidate loci, Class II Major Histocompatibility Complex Transactivator (CIITA) and C-Type Lectin Domain Containing 16A (CLEC16A). Lamb IgA levels were also associated with the immunoglobulin heavy constant loci (IGH) complex, and adult IgE levels and lamb IgA and IgG levels were associated with the major histocompatibility complex (MHC). This study provides evidence of high heritability of a complex immunological trait under natural conditions and provides the first evidence from a genome-wide study that large effect genes located outside the MHC region exist for immune traits in the wild.
Project description:This study estimated genetic parameters for ewe reproductive traits [number of lambs born (NLB) and weaned (NLW) per ewe lambing] and fecal egg counts (FEC) during the peri-parturient rise (PPR) for use in genetic evaluation of Katahdin sheep. Data included NLB and NLW for 23,060 lambings by 9,295 Katahdin ewes, 1,230 PPR at lambing (PPR0) for 750 ewes, 1,070 PPR at approximately 30 d postpartum (PPR30) for 611 ewes, BW at birth, weaning, and (or) post-weaning for 12,869 lambs, and FEC at weaning and (or) post-weaning for 4,676 lambs. Direct additive, permanent environmental, and residual (co)variances were estimated in univariate and bivariate animal models. Fixed effects included effects of ewe management group and ewe age for all traits, and, for PPR, a continuous effect of days between lambing and measurement. Effects of litter size on PPR0 and number of lambs suckled on PPR30 were included in univariate models but excluded from bivariate models for PPR and NLB or NLW. Heritability estimates in univariate models for NLB, NLW, PPR0, and PPR30 were 0.09 ± 0.01, 0.06 ± 0.01, 0.35 ± 0.06, and 0.24 ± 0.07, respectively. Estimates of permanent environmental variance as a proportion of total phenotypic variance were 0.02 ± 0.01 for NLB, 0.03 ± 0.01 for NLW, 0.05 ± 0.06 for PPR0, and 0.13 ± 0.07 for PPR30. Direct additive, phenotypic, permanent environmental, and residual correlations between NLB and NLW were 0.88 ± 0.03, 0.74 ± 0.004, 0.54 ± 0.15, 0.74 ± 0.003, respectively; corresponding correlations between PPR0 and PPR30 were 0.96 ± 0.07, 0.46 ± 0.03, 0.98 ± 0.50, 0.18 ± 0.05, respectively. The additive genetic correlation (rd) between ewe reproductive traits and PPR ranged from 0.12 to 0.18. Estimates of rd between lamb BW and subsequent ewe NLB and NLW ranged from 0.07 to 0.20, and those between PPR and lamb BW ranged from -0.03 to 0.29. The rd between ewe reproductive traits and lamb FEC ranged from 0.27 to 0.40, and those between PPR and lamb FEC ranged from 0.56 to 0.77. Correlations between maternal additive effects on BW and direct additive effects on PPR were low (-0.08 to 0.10), and those between maternal additive effects on BW and direct additive effects on ewe reproductive traits were variable (-0.36 to 0.11). We conclude that FEC in growing lambs and peri-parturient ewes are controlled by similar genes and that modest, but manageable, genetic antagonisms may exist between FEC and ewe productivity.
Project description:Clinical mastitis in sheep has gravely restrained production performance for a long time. Knowledge of mechanisms of its pathogenesis and resistance in meat sheep mammary gland with clinical mastitis are not yet understood, especially for clinical mastitis caused by natural infection. In this work, RNA-sequencing was firstly used to screen the differentially expressed genes (DEGs) in clinical mastitic mammary tissues (CMMTs) when compared with healthy mammary tissues (HMTs) from meat sheep flocks. We identified 420 DEGs including 316 upregulated and 104 downregulated genes in CMMTs. Gene ontology annotation revealed these DEGs were mainly engaged in immune response and inflammation response. Pathway enrichment showed they were primarily enriched in pathways relevant to inflammation, immune response and metabolism. Alternative splicing analysis showed most common differential splicing genes in CMMTs and HMTs were implicated in immune response. Immunostaining for three immune response-related proteins encoded by DEGs were mainly observed in mammary epithelium from both CMMTs and HMTs, and their positive signals were more intensive in CMMTs than those in HMTs. These findings provide experimental basis and reference for further researching the molecular genetic mechanisms, particularly immune defence mechanisms, of sheep mammary gland during clinical mastitis.
Project description:Clinical mastitis is still an intractable problem for sheep breeding. The natural immunologic mechanisms of the mammary gland against infections are not yet understood. For a better understanding of the disease-associated proteins during clinical mastitis in meat sheep, we performed two-dimensional electrophoresis (2-DE)-based comparative proteomic analyses of mammary tissues, including from healthy mammary tissues (HMTs) and from mammary tissues with clinical mastitis (CMMTs). The 2-DE results showed that a total of 10 up-regulated and 16 down-regulated proteins were identified in CMMTs when compared to HMTs. Of these, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses revealed that most proteins were associated with immune responses or metabolisms. The results of qRT-PCR and Western blot for randomly selected four differentially expressed proteins (DEPs) including superoxide dismutase [Mn] (SOD2), annexin A2 (ANAX2), keratin 10 (KRT10) and endoplasmic reticulum resident protein 29 (ERP29) showed that their expression trends were consistent with 2-DE results except ANXA2 mRNA levels. This is an initial report describing the 2-DE-based proteomics study of the meat sheep mammary gland with clinical mastitis caused by natural infection, which provides additional insight into the immune and metabolic mechanisms during sheep mastitis.
Project description:Milk protein gene expression in mammary epithelial cells is regulated by the action of the lactogenic hormones insulin, glucocorticoids and prolactin. The mammary gland factor, MGF, has been shown to be a central mediator in the lactogenic hormone response. The DNA binding activity of MGF is hormonally regulated and essential for beta-casein promoter activity. We have used Red A Sepharose- and sequence-specific DNA affinity chromatography to purify MGF from mammary gland tissue of lactating sheep. Proteins of 84 and 92 kDa were obtained, proteolytically digested and the resulting peptides separated by reverse phase high pressure liquid chromatography. The 84 and 92 kDa proteins yielded very similar peptide patterns. The amino acid sequence of two peptides was determined. The sequence information was used to derive oligonucleotide probes. A cDNA library from the mRNA of mammary gland tissue of lactating sheep was screened and a molecular clone encoding MGF was isolated. MGF consists of 734 amino acids and has sequence homology with the 113 (Stat113) and 91 kDa (Stat91) components of ISGF3, transcription factors which are signal transducers of IFN-alpha/beta and IFN-gamma. Two species of MGF mRNA of 6.5 and 4.5 kb were detected in mammary gland tissue of lactating sheep. Lower mRNA expression was found in ovary, thymus, spleen, kidney, lung, muscle and the adrenal gland. MGF cDNA was incorporated into a eukaryotic expression vector and cotransfected with a vector encoding the long form of the prolactin receptor into COS cells. A strong MGF-specific bandshift was obtained with nuclear extracts of COS cells induced with prolactin. Treatment of activated MGF with a tyrosine-specific protein phosphatase resulted in the loss of DNA binding activity. Prolactin-dependent transactivation of a beta-casein promoter-luciferase reporter gene construct was observed in transfected cells.
Project description:The minimal 5' regulatory region of the sheep beta-lactoglobulin gene (BLG), as defined in transgenic mice, was used to identify nuclear factors which may be involved in milk protein gene expression in the lactating mammary gland. This 406bp promoter region was dissected into short, overlapping, double-stranded oligonucleotides to facilitate identification of the bound proteins. A variety of sites, for both known and previously undescribed DNA-binding proteins, are occupied in vitro. Some of these factors were investigated in detail. Two forms of nuclear factor I (NFI), which have different recognition site affinities, are present in nuclear extracts from lactating mammary gland and bind to at least 5 sites in this BLG control element. In addition, a factor (milk protein binding factor, MPBF) which is specific to extracts from both mouse and sheep lactating mammary gland binds to 3 BLG promoter sites and may be a milk protein gene transcription factor.
Project description:Mammary gland development is fueled by stem cell self-renewal and differentiation. External cues from the microenvironment coupled with internal cues such as post-transcriptional regulation exerted by microRNAs regulate stem cell behavior and fate. Here, we have identified a miR-205 regulatory network required for mammary gland ductal development and stem cell regeneration following transplantation into the cleared mammary fat pad. In the postnatal mammary gland, miR-205 is predominantly expressed in the basal/stem cell enriched population. Conditional deletion of miR-205 in mammary epithelial cells impairs stem cell self-renewal and mammary regenerative potential in the in vitro mammosphere formation assay and in vivo mammary reconstitution. miR-205 null transplants display significant changes in basal cells, basement membrane, and stroma. NKD1 and PTPA, which inhibit the Wnt signaling pathway, and AMOT, which causes YAP cytoplasmic retention and inactivation were identified as miR-205 downstream mediators. These studies also confirmed that miR-205 is a direct ?Np63 target gene that is critical for the regulation of basal cell identity. Stem Cells 2018;36:1875-15.
Project description:Small-Tailed Han (STH) sheep are known for their high fecundity, but the survival of lambs is compromised and influences the commercial return from farming these sheep, with this being attributed in part to starvation from insufficient milk production by the ewes. In this study, the transcriptome profiles of the mammary gland of lactating and non-lactating STH ewes were investigated using paired-end RNA sequencing (RNA-Seq). An average of 14,447 genes were found to be expressed at peak-lactation in the STH sheep, while 15,146 genes were expressed in non-lactating ewes. A total of 4,003 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the DEGs were associated with a wide range of cellular components, biological processes and metabolic pathways, including binding activities, signaling pathways, cellular structures, and immune responses. The most highly expressed genes at peak-lactation included CSN2, LGB, LALBA, CSN1S1, CSN1S2, and CSN3, and the 10 most highly expressed genes accounted for 61.37% of the total Reads Per Kilobase of transcript, per Million mapped reads (RPKM). The most highly expressed genes in the mammary gland of non-lactating ewes included IgG, THYMB4X, EEF1A1, IgA, and APOE, and the 10 most highly expressed genes accounted for only 12.97% of the total gene RPKM values. This suggests that the sheep mammary gland undergoes a substantial development in milk protein synthesis infrastructure and promotion of protein transportation during lactation.
Project description:Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA with >200 nucleotides in length. Some lncRNAs have been proven to have clear regulatory functions in many biological processes of mammals. However, there have been no reports on the roles of lncRNAs in ovine mammary gland tissues. In the study, the expression profiles of lncRNAs were studied using RNA-Seq in mammary gland tissues from lactating Small-Tailed Han (STH) ewes and Gansu Alpine Merino (GAM) ewes with different milk yield and ingredients. A total of 1894 lncRNAs were found to be expressed. Compared with the GAM ewes, the expression levels of 31 lncRNAs were significantly up-regulated in the mammary gland tissues of STH ewes, while 37 lncRNAs were remarkably down-regulated. Gene Ontogeny (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the target genes of differentially expressed lncRNAs were enriched in the development and proliferation of mammary epithelial cells, morphogenesis of mammary gland, ErbB signaling pathway, and Wnt signaling pathway. Some miRNA sponges of differentially expressed lncRNAs, reported to be associated with lactation and mammary gland morphogenesis, were found in a lncRNA-miRNA network. This study reveals comprehensive lncRNAs expression profiles in ovine mammary gland tissues, thereby providing a further understanding of the functions of lncRNAs in the lactation and mammary gland development of sheep.