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1.
Anim Sci J ; 81(6): 694-8, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21108690

ABSTRACT

In dairy cows, hydrogen peroxide (H2O2) produced from a low-molecular-weight compound in milk from inflamed quarters was lower than that in milk from un-inflamed quarters. In milk of delivery grade, characteristics of H2O2 production in milk with high electrical conductivity (EC) were examined in this study. Milk samples were collected from a total of 230 cows at 1-month intervals, and the EC of skimmed milk was determined. Based on the highest and the lowest EC of a cow's quarter milk, the inter-quarter difference of ≥0.6 mS/cm (mean + t0.01 SE) was taken as a high EC. Milk with high EC was found in 52 quarters. In cows with milk of high EC, H2O2 production in milk with normal EC was higher than that in milk with high EC in the same animal but was lower than that in the control population. In milk with high EC, the decrease of H2O2 production correlated with the increase in EC. The production of H2O2 decreased in particular when the inter-quarter difference exceeded 0.8 mS/cm. In milk collected from the same quarter 1 month before, EC changed from normal to high, and H2O2 production decreased. In milk from the other three quarters, EC remained normal and H2O2 production remained unchanged. We concluded that milk with high EC appeared in low H2O2 -producing cows. The results suggest that the degree of decrease in H2O2 production reflects the extent of quarter abnormality.


Subject(s)
Electric Conductivity , Hydrogen Peroxide/metabolism , Milk/metabolism , Animals , Cattle , Female , Milk/physiology , Molecular Weight
2.
FASEB J ; 23(8): 2514-20, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19276171

ABSTRACT

The innate immune system plays an important role in protecting organs that are continuous with the outer surface of the body from bacterial infection. The antibacterial factors involved in this system have been sought in exocrine glands, particularly in the mammary glands. Because milk produced in the mammary glands is enriched in various nutrients, supporting the proliferation of bacteria, mammary glands appear to be at the greatest risk of bacterial infection and proliferation. Here, we show that mouse milk contains L-amino acid oxidase (LAO), a lactating mammary gland-specific protein that displays antibacterial activity in vitro through the production of hydrogen peroxide from free amino acids. We produced LAO-disrupted mouse lines to define the physiological properties and importance of the protein in vivo. The LAO-knockout mice were healthy and had normal mammary gland development; however, the antibacterial activity normally observed in milk from wild-type mice was absent from the milk of knockout mice. The content of free amino acids targeted by LAO was very low in wild-type milk, whereas these amino acids were abundant in LAO-knockout milk. Knockout mice exhibited weak resistance to an intramammary bacterial challenge compared to their wild-type counterparts. Further, preadministration of wild-type milk whey reduced the severity of bacterial infection in LAO-knockout mice. These results demonstrate that milk LAO protects the mammary gland against bacterial infection, and this antibacterial effect may be due to the generation of hydrogen peroxide by using free amino acids abundantly present in milk.


Subject(s)
Immunity, Innate , L-Amino Acid Oxidase/immunology , Mammary Glands, Animal/enzymology , Mammary Glands, Animal/immunology , Animals , Bacterial Infections/enzymology , Bacterial Infections/immunology , Bacterial Infections/prevention & control , Female , L-Amino Acid Oxidase/deficiency , L-Amino Acid Oxidase/genetics , Lactation/immunology , Lactation/metabolism , Mammary Glands, Animal/microbiology , Mastitis/enzymology , Mastitis/immunology , Mastitis/prevention & control , Mice , Mice, Inbred C57BL , Mice, Knockout , Milk/chemistry , Milk/enzymology , Milk/immunology , Staphylococcal Infections/enzymology , Staphylococcal Infections/immunology , Staphylococcal Infections/prevention & control , Staphylococcus aureus/immunology , Staphylococcus aureus/pathogenicity
3.
Differentiation ; 77(2): 181-7, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19281778

ABSTRACT

Mammary glands exhibit a series of developmental states that are typified by proliferation, differentiation, and involution. Here, we demonstrate that a microRNA (miRNA), miR-101a, plays an important role in the process of mammary gland development. We used miRNA microarray analysis to show that some miRNAs exhibit changes in their expression during mouse mammary gland epithelial cell (HC11) differentiation, which corresponds to the time when these cells acquire the milk-producing phenotype. In particular, we observed an increase of miR-101a expression throughout differentiation and involution in mammary gland tissue, as well as in HC11 cells. Overexpression experiments revealed that miR-101a suppressed the expression of beta-casein mRNA, a milk protein, and marker of cell differentiation, but its suppression was not mediated by transcriptional or direct post-transcriptional regulation of beta-casein mRNA. Overexpression of miR-101a also inhibited HC11 cell proliferation that could influence the differentiation state of the mammary gland. We speculate that a direct target of miR-101a is cyclooxygenase-2 (Cox-2) mRNA because there was an inverse relationship between these two genes during mammary gland development. Indeed, Cox-2 protein expression was suppressed by the overexpression of miR-101a, and the luciferase activity of reporter constructs containing the Cox-2 3'UTR was also suppressed by miR-101a overexpression. As Cox-2 has been shown to mediate cell proliferation, it is possible that the inhibition of HC11 cell proliferation by miR-101a might be mediated by Cox-2. Taken together, these results suggest that miR-101a regulates cell proliferation via altering Cox-2 expression, which is critical for controlling mammary gland development.


Subject(s)
Cyclooxygenase 2/metabolism , Gene Expression Regulation, Enzymologic , Mammary Glands, Animal/growth & development , Mammary Glands, Animal/metabolism , MicroRNAs/metabolism , Animals , Blotting, Western , Cell Line , Cell Proliferation , Female , Mice , Mice, Inbred ICR , Polymerase Chain Reaction
4.
J Dairy Res ; 75(3): 335-9, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18680618

ABSTRACT

Mastitis is the most frequent and prevalent production disease in dairy herds in developed countries. Based on a milk somatic cell count (SCC) of either >300,000 or <200,000 cells/ml in this study, we defined the quarter as either inflamed or uninflamed, respectively. The electrical conductivity (EC) of milk was used as an indicator of udder epithelial cell damage. We determined the amount of H2O2 produced by utilizing a small molecular weight compound in milk, and examined the characteristics of H2O2 production and EC in milk from inflamed and uninflamed quarters. In cows with milk of delivery grade (control population), H2O2 production and EC were 3.6+/-1.3 nmol/ml and 5.4+/-0.4 mS/cm (mean+/-sd), respectively. In 37 inflamed quarter milk samples, the production of H2O2 was 1.9+/-1.0 nmol/ml and was significantly smaller than that in the control population (P<0.01). Production of H2O2 was moderately but significantly correlated with EC (r<-0.71). In 20 cows with inflamed quarters, the production of H2O2 in milk from inflamed quarters was significantly smaller than that in milk from uninflamed quarters (P<0.01). In 18 out of 20 cows, milk from inflamed quarters showed the smallest H2O2 production among all tested quarters in each cow. We conclude that inflammation caused a decrease in H2O2 production in milk. In this study, we present parameters for evaluating the lactoperoxidase/H2O2/thiocyanate antibacterial defence system in bovine milk.


Subject(s)
Hydrogen Peroxide/metabolism , Milk/cytology , Milk/physiology , Animals , Cattle , Cell Count , Electric Conductivity , Female , Hydrogen Peroxide/analysis , Inflammation/veterinary , Mastitis, Bovine/diagnosis , Molecular Weight
5.
J Dairy Res ; 75(3): 257-61, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18423073

ABSTRACT

Using an ultrafiltration membrane (molecular cut-off, 3,000), low molecular weight compounds in bovine milk were collected (YM-3 filtrate). A hydrogen peroxide (H2O2)-like substance was generated in the YM-3 filtrate. This substance was undetected at 0 h, but increased in a time-dependent manner, peaking after 2 h of incubation at 38 degrees C. After incubating the YM-3 filtrate with catalase and lactoperoxidase, the signal showing the presence of this substance disappeared. The substance was quantified using one chemiluminescence and three colorimetric H2O2 detection systems. In all systems, their estimates were within the same range. The amount of substance, as estimated by the chemiluminescence H2O2 detection system, was correlated with that estimated by the other three colorimetric systems (r=0.98, 0.95 and 0.87). The substance was eluted at the same position as H2O2 by gel filtration on Superdex 30. Thus, the substance had the same characteristics as H2O2. An H2O2-generating substance in either the YM-3 filtrate or whey had a molecular mass of about 600. In this study, we clarify that bovine milk is capable of generating H2O2 by utilizing a low molecular weight compound. Thus, we present a new type of H2O2-supplying system in bovine milk.


Subject(s)
Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Milk/chemistry , Animals , Cattle , Chromatography, Gel , Dairying , Female , Hydrogen Peroxide/analysis , Molecular Weight , Temperature , Time Factors
6.
Exp Cell Res ; 313(13): 2937-45, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17512931

ABSTRACT

The expression of many genes, such as beta-casein, c-myc, and cyclin D1, is altered by lactogenic hormone stimulation during mammary epithelial cell differentiation. Here, we demonstrate that post-transcriptional regulation plays an important role to establish gene expression required to initiate milk production as well as transcriptional control. AUF1 protein, a member of the AU-rich element (ARE)-binding protein family, plays a role in ARE-mRNA turnover by regulating mRNA stability and/or translational control. Cytoplasmic localization of AUF1 protein is critically linked to function. We show that as the mammary gland differentiates, AUF1 protein moves from the cytoplasm to the nucleus. Moreover, in mammary gland epithelial cells (HC11), stimulation by lactogenic hormone decreased cytoplasmic and increased nuclear AUF1 levels. Direct binding of AUF1 protein was observed on c-myc mRNA, but not beta-casein or cyclin D1 mRNA. AUF1 downregulation in HC11 cells increased the expression of beta-casein mRNA and decreased the expression of c-myc mRNA by lactogenic hormone. Conversely, overexpression of AUF1 inhibited these effects of lactogenic hormone stimulation in HC11 cells. These results suggest that AUF1 participates in mammary gland differentiation processes under the control of lactogenic hormone signals.


Subject(s)
Cell Differentiation , Heterogeneous-Nuclear Ribonucleoprotein D/metabolism , Mammary Glands, Animal/cytology , Animals , Antigens, Surface/analysis , Antigens, Surface/metabolism , Caseins/genetics , Caseins/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , ELAV Proteins , ELAV-Like Protein 1 , Female , Heterogeneous Nuclear Ribonucleoprotein D0 , Heterogeneous-Nuclear Ribonucleoprotein D/analysis , Mammary Glands, Animal/chemistry , Mammary Glands, Animal/metabolism , Mice , Mice, Inbred ICR , Prolactin/metabolism , Prolactin/pharmacology , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/analysis , RNA-Binding Proteins/metabolism
7.
Biol Reprod ; 75(4): 508-17, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16775227

ABSTRACT

The signal transducer and activator of transcription 5 (STAT5) is an essential factor in the signal transduction pathways for a number of cytokines that regulate the growth and differentiation of mammalian cells. In this study, we investigated the STAT5 signaling pathway in mouse embryos, to elucidate the mechanism of cytokine signal transduction that regulates preimplantation development. The results of the RT-PCR analysis showed that both STAT5A and B were expressed throughout preimplantation development. Immunocytochemistry revealed that the STAT5A/B proteins were located in the nucleus from the early 1-cell stage to the blastocyst stage. STAT5 activation appeared to be regulated by Janus kinases (JAKs) and SRC family kinases (SFKs), since inhibitors of these kinases inhibited the localization of STAT5 proteins to the nucleus. The JAK inhibitor Ag490 reduced both the developmental rate of the embryos and the expression levels of the downstream genes of the JAK-STAT5 signaling pathway. These findings suggest that STAT5 proteins function in preimplantation development by mediating the signals from cytokines.


Subject(s)
Embryonic Development/physiology , STAT5 Transcription Factor/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Blastocyst/metabolism , Blastocyst/physiology , Embryonic Development/drug effects , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Developmental , Indoles/pharmacology , Male , Mice , Mice, Inbred ICR , Mice, Inbred Strains , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , STAT5 Transcription Factor/genetics , Signal Transduction , Sulfonamides/pharmacology , Tyrphostins/pharmacology , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolism
9.
Biochim Biophys Acta ; 1759(3-4): 132-40, 2006.
Article in English | MEDLINE | ID: mdl-16714065

ABSTRACT

The maintenance of mRNA stability has emerged as a mechanism of post-transcriptional control. We demonstrate that beta-casein mRNA stability is dictated by the poly(A) tail and sequences in the 3'-UTR. An in vitro mRNA decay assay revealed that beta-casein mRNA with a long poly(A) tail had higher stability than that with a short poly(A) tail. The addition of poly(A) homopolymer and 3'-UTR cRNA as competitor induced rapid degradation of beta-casein mRNA. The interaction between full-length beta-casein mRNA and poly(A) homopolymer was inhibited by the addition of the 3'-UTR cRNA in the lysates, which indicates that one region of the 3'-UTR associates with the poly(A) tail through an RNA-protein interaction. The putative AU-rich element (ARE) is present at nt 897-905; deletion and mutation analysis showed that the ARE site was required for maintaining the stability of the beta-casein 3'-UTR. In the immunoprecipitation analysis, the poly(A)-binding protein (PABP) and the RNA-binding protein HuR were pulled down by 3'-UTR cRNA, and the absence of the ARE site reduced the binding of these proteins. These experiments further revealed that the rapid degradation of beta-casein mRNA was induced by incubation with HuR- and PABP-depleted RRLs. Collectively, our results suggest that beta-casein mRNA is protected from degradation by virtue of the structural interaction between the 3'-UTR and poly(A) tail via a protein complex of HuR and PABP.


Subject(s)
3' Untranslated Regions/genetics , Antigens, Surface/metabolism , Caseins/genetics , Poly(A)-Binding Proteins/chemistry , Poly(A)-Binding Proteins/metabolism , RNA Stability/genetics , RNA-Binding Proteins/metabolism , Animals , Antigens, Surface/chemistry , Carrier Proteins/metabolism , ELAV Proteins , ELAV-Like Protein 1 , Mice , Mutation/genetics , NIH 3T3 Cells , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry
10.
Mol Reprod Dev ; 73(7): 850-8, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16596627

ABSTRACT

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.


Subject(s)
Cell Adhesion , Chemokines, CXC/metabolism , Embryo Implantation , Endometrium/metabolism , Sheep/embryology , Trophoblasts/metabolism , Animals , Extracellular Matrix/metabolism , Female , Fibronectins/metabolism , Interferon Type I/pharmacology , Interferon-gamma/pharmacology , Pregnancy , Pregnancy Proteins/pharmacology , Receptors, CXCR3 , Receptors, Chemokine/metabolism
11.
Biol Reprod ; 71(1): 89-96, 2004 Jul.
Article in English | MEDLINE | ID: mdl-14998905

ABSTRACT

Jak2, which is a member of the Janus tyrosine kinase family, plays essential roles in cytokine signal transduction and in the regulation of cell growth and gene expression. To investigate the involvement of Jak2 in the regulation of early preimplantation development, we examined the expression of Jak2 in mouse embryos. Reverse transcription-polymerase chain reaction assays revealed that the relative amount of Jak2 mRNA was highest in unfertilized oocytes, gradually decreased until the four-cell stage, and remained at low levels until the blastocyst stage. Immunocytochemistry showed that Jak2 was localized predominantly to the female pronucleus in one-cell embryos. The immunofluorescence signal was very weak or undetectable in the male pronucleus. In unfertilized oocytes and one-cell embryos at M phase, Jak2 was localized to the chromosomes. After cleavage to the two-cell stage, the intensity of the immunofluorescence signal decreased in the nucleus while the embryos were in late G2. This decrease was independent of DNA synthesis because it was not affected by inhibition of DNA replication. However, inhibition of protein synthesis repressed the disappearance of Jak2 from the nucleus. These results suggest a novel function for Jak2 in the regulation of early preimplantation development.


Subject(s)
Cell Nucleus/metabolism , Cleavage Stage, Ovum/metabolism , Oocytes/physiology , Oogenesis/physiology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Blastocyst/enzymology , Cells, Cultured , Female , Fluorescent Antibody Technique , Janus Kinase 2 , Male , Mice , Microscopy, Confocal , Oocytes/enzymology , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tissue Culture Techniques , Tissue Distribution
12.
J Biol Chem ; 278(31): 29048-56, 2003 Aug 01.
Article in English | MEDLINE | ID: mdl-12756249

ABSTRACT

For a pregnancy to be established, initial apposition and adhesion of the blastocyst to maternal endometrium must occur in a coordinated manner; however, a key factor(s) that mediates the trophoblast cell migration and attachment to the apical surface of the endometrium has not been identified. In this study, we examined the effect of an endometrial chemokine, interferon-gamma-inducible protein 10 kDa (IP-10), on conceptus migration to the endometrial epithelium. We first studied endometrial IP-10 mRNA expression, which was localized in the subepithelial stromal region, and detected the protein in the uterine flushing media during early pregnancy. Expression of IP-10 mRNA by the endometrium of cyclic animals was stimulated by the addition of a conceptus factor interferon-tau (IFN-tau). Immunofluorescent analysis revealed that IP-10 receptor, CXCR3, was localized in the trophoblast cells, to which biotinylated-recombinant caprine IP-10 (rcIP-10) bound. Chemotaxis assay indicated that rcIP-10 stimulated the migration of trophoblast cells, and the effects of rcIP-10 were neutralized by the pretreatment with an anti-IP-10 antibody. Adhesive activity of trophoblast cells to fibronectin was promoted by rcIP-10, and the effect was inhibited by the use of anti-IP-10 antibody. Further adhesion experiments demonstrated that binding of trophoblast cells to fibronectin was completely inhibited by a peptide of the Arg-Gly-Asp (RGD) sequence, which binds to integrins alpha5beta1, alphaVbeta1, alphaVbeta3, and alphaVbeta5, whereas non-binding peptide containing Arg-Gly-Glu (RGE) had minimal effects. More importantly, rcIP-10 promoted the adhesion of trophoblast cells to primary cells isolated from endometrial epithelium. Furthermore, rcIP-10 stimulated the expression of integrin alpha5, alphaV, and beta3 subunit mRNA in trophoblast cells. These findings suggest that endometrial IP-10 regulates the establishment of apical interactions between trophoblast and epithelial cells during early gestation.


Subject(s)
Blastocyst/physiology , Chemokines, CXC/physiology , Gestational Age , Animals , Biotinylation , Cell Adhesion , Cell Movement , Chemokine CXCL10 , Chemokines, CXC/analysis , Chemokines, CXC/genetics , Endometrium/chemistry , Epithelial Cells/physiology , Female , Fibronectins/metabolism , Fluorescent Antibody Technique , Gene Expression/drug effects , Goats , Integrins/genetics , Interferon Type I/pharmacology , Pregnancy , Pregnancy Proteins/pharmacology , RNA, Messenger , Receptors, CXCR3 , Receptors, Chemokine/analysis , Receptors, Chemokine/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Trophoblasts/cytology , Trophoblasts/physiology , Uterus/chemistry
13.
Mol Reprod Dev ; 65(1): 23-9, 2003 May.
Article in English | MEDLINE | ID: mdl-12658630

ABSTRACT

Studies of ovine interferon-tau (oIFNtau) gene regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, have been carried out, but a definitive mechanism for its spatial-temporal transcription has not been elucidated. Recently, specific binding regions for transcription factors AP-1 and Ets-2 on the oIFNtau gene were identified; however, a molecular mechanism by which these factors regulate oIFNtau gene transcription has not been characterized. In the present study, we investigated the potential relationship between AP-1 and Ets-2, and their association with a coactivator, cAMP-response element binding protein-binding protein (CBP), on oIFNtau gene transcription in a transient transfection system using human choriocarcinoma JEG3 cells. The oIFNtau gene promoter/enhancer (-654 to + 1 bases, wild type)-luciferase reporter construct (pGL3-654) or its mutant at the AP-1 or Ets-2 site was cotransfected with CBP (pRc/RSV-CBP) construct along with c-jun, c-fos, and/or Ets-2 expression plasmid. CBP enhanced transcription of the wild type oIFNtau-reporter construct; however, this coactivator had no effect on the oIFNtau-reporter construct with mutated AP-1 or Ets-2 site. Cotransfection of CBP with c-jun and/or Ets-2, but not with c-fos, further increased oIFNtau gene transactivation although amounts of c-jun and c-fos expression, resulting from expression vectors, were similar. In addition, CBP inhibitor adenovirus 12S E1A (E1A), but not the mutant of E1A without CBP binding domain (Delta2-36), suppressed oIFNtau gene transcription. These observations suggest that c-jun and Ets-2 are the most probable binding partners for CBP in the potentiation of oIFNtau gene transcription. Mol. Reprod. Dev. 65: 23-29, 2003.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins , Gene Expression Regulation, Developmental , Interferon Type I/genetics , Pregnancy Proteins/genetics , Repressor Proteins , Transcription Factors , Transcription, Genetic , Choriocarcinoma/metabolism , Humans , Interferon Type I/metabolism , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinases/metabolism , Pregnancy Proteins/metabolism , Proto-Oncogene Protein c-ets-2 , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription Factor AP-1/metabolism , Transcriptional Activation
14.
Biol Reprod ; 68(4): 1413-21, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12606423

ABSTRACT

Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-gamma-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-tau on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-alpha, IFN-gamma, and IFN-tau in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-tau. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-tau stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-tau regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.


Subject(s)
Endometrium/cytology , Endometrium/immunology , Interferon-gamma/pharmacology , Interleukin-18/physiology , Amino Acid Sequence/genetics , Animals , Cell Movement/drug effects , Cloning, Molecular , Endometrium/drug effects , Female , In Vitro Techniques , Interferon Type I/genetics , Interferon Type I/pharmacology , Interferon-gamma/genetics , Interleukin-18/genetics , Interleukin-18/metabolism , Interleukin-18/pharmacology , Molecular Sequence Data , Monocytes/physiology , Pregnancy , Pregnancy Proteins/genetics , Pregnancy Proteins/pharmacology , RNA, Messenger/metabolism , Receptors, CXCR3 , Receptors, Chemokine/genetics , Sheep , Tissue Distribution
15.
J Reprod Dev ; 49(4): 323-8, 2003 Aug.
Article in English | MEDLINE | ID: mdl-14967925

ABSTRACT

Protein tyrosine phosphatase (PTP) expression was examined in preimplantation mouse embryos. We previously reported that SHP-2, LAR, PTPT9, SHP-1, and mRPTPB were expressed in preimplantation mouse embryos. Here, we examined changes in the expression levels of these PTPs during preimplantation development. cDNA was obtained by reverse transcription of embryo mRNA, amplified with 10 PCR cycles, and then subjected to real-time fluorescence-monitored PCR. Experiments with an mRNA dilution series revealed that the data obtained matched the quantities of mRNA used. The measurements obtained with real-time fluorescence-monitored PCR showed that the expression of each PTP mRNA changed dynamically, and that each had a different expression pattern. This suggests that PTPs are involved in the regulation of growth and differentiation during preimplantation development.


Subject(s)
Embryonic and Fetal Development/genetics , Protein Tyrosine Phosphatases/genetics , Animals , Base Sequence , DNA, Complementary/genetics , Embryonic Development , Embryonic and Fetal Development/physiology , Female , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, Inbred ICR , Polymerase Chain Reaction/methods , Pregnancy , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism
16.
Endocr J ; 49(2): 195-205, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12081239

ABSTRACT

Interferon-tau (IFNtau), produced by the trophectoderm of ruminant ungulates, binds to the type I IFN receptor (IFNAR) located at the uterine endometrium in a paracrine manner. Since IFNtau attenuates the secretory pattern of an endometrial luteolysin, prostaglandin F2alpha, IFNtau has been considered as a conceptus factor implicated in the process of maternal recognition of pregnancy. Here we report the presence of IFNAR subunit (IFNAR1) in ovine conceptuses during the period of peri-implantation development and demonstrate that 125I-human (h) IFNalpha binds to membrane preparations from ovine corpus luteum and conceptus. Using an antibody against hIFNAR1, immunohistochemical analysis revealed that IFNAR1 protein was present in day 14 and 16 conceptuses (day 0 = day of estrus) and luminal and glandular epithelia of the endometrium. Conceptus membrane proteins analyzed by western blot with the same antibody displayed immunoreactive bands at 95, 60 and 55 kDa while endometrial membrane proteins showed bands at 200, 95 and 55 kDa. Northern blot analysis revealed that IFNAR1 mRNA was present in days 15-19 conceptuses and day 18-19 allantoic membranes. Receptor binding studies indicated that 125I-hIFNalpha binding to day 16, but not earlier, conceptus membrane proteins could be displaced with hIFNalpha or ovine IFNtau. Based on Scatchard analysis, day 16 conceptus membranes contained 28 fmol IFNAR/mg protein with a dissociation constant of 300 pM. Cross-linking experiments demonstrated that 125I-hIFNalpha-receptor complex migrated at 120 kDa, indicating that the receptor component(s) was approximately 100 kDa. These data provide evidence that although the binding does not occur until day 16, ovine conceptuses possess IFNAR1 near or at the time of implantation, suggesting that IFNtau, a factor produced by the trophectoderm of ruminant ungulates, could act on the conceptus in an autocrine manner. In addition to functioning as an antiluteolytic factor, therefore, IFNtau may have a direct effect on conceptus development.


Subject(s)
Embryo Implantation/physiology , Extraembryonic Membranes/metabolism , Interferon-alpha/metabolism , Pregnancy, Animal/metabolism , Receptors, Interferon/metabolism , Sheep/physiology , Animals , Blotting, Northern/veterinary , Blotting, Western , Embryo Implantation/immunology , Endometrium/immunology , Endometrium/metabolism , Extraembryonic Membranes/immunology , Extraembryonic Membranes/physiology , Female , Immunohistochemistry/veterinary , Interferon-alpha/immunology , Interferon-alpha/physiology , Male , Membrane Proteins , Pregnancy , Pregnancy, Animal/immunology , Pregnancy, Animal/physiology , RNA/chemistry , RNA/genetics , Receptor, Interferon alpha-beta , Receptors, Interferon/genetics , Receptors, Interferon/immunology , Sheep/immunology , Sheep/metabolism , Time Factors
17.
J Biol Chem ; 277(21): 19080-6, 2002 May 24.
Article in English | MEDLINE | ID: mdl-11907037

ABSTRACT

l-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with l-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His other 11 amino acids tested and produced H(2)O(2) in a dose- and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N- and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H(2)O(2) using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.


Subject(s)
Amino Acid Oxidoreductases/metabolism , Milk/enzymology , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/isolation & purification , Animals , Base Sequence , Chromatography, Gel , Cloning, Molecular , DNA , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , L-Amino Acid Oxidase , Mice , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
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