Effects of oral ß-cryptoxanthin administration on the transcriptomes of peripheral neutrophil and liver tissue using microarray analysis in post-weaned Holstein calves.

We investigated the effects of oral administration of ß-cryptoxanthin (ß-CRX), a precursor of vitamin A synthesis, on the transcriptomes of peripheral neutrophils and liver tissue in post-weaned Holstein calves with immature immunity. A single oral administration of ß-CRX (0.2 mg/kg body weight) was performed in eight Holstein calves (4.0 ± 0.8 months of age; 117 ± 10 kg) on Day 0. Peripheral neutrophils (n = 4) and liver tissue (n = 4) were collected on Days 0 and 7. Neutrophils were isolated by density gradient centrifugation and treated with the TRIzol reagent. mRNA expression profiles were examined by microarray and differentially expressed genes were investigated using the Ingenuity Pathway Analysis software. The differentially expressed candidate genes identified in neutrophils (COL3A1, DCN, and CCL2) and liver tissue (ACTA1) were involved in enhanced bacterial killing and maintenance of cellular homoeostasis respectively. The changes in the expression of six of the eight common genes encoding enzymes (ADH5 and SQLE) and transcription regulators (RARRES1, COBLL1, RTKN, and HES1) were in the same direction in neutrophils and liver tissue. ADH5 and SQLE are involved in the maintenance of cellular homoeostasis by increasing the availability of substrates, and RARRES1, COBLL1, RTKN, and HES1 are associated with the suppression of apoptosis and carcinogenesis. An in silico analysis revealed that MYC, which is related to the regulation of cellular differentiation and apoptosis, was the most significant upstream regulator in neutrophils and liver tissue. Transcription regulators such as CDKN2A (cell growth suppressor) and SP1 (cell apoptosis enhancer) were significantly inhibited and activated, respectively, in neutrophils and liver tissue. These results suggest that oral administration of ß-CRX promotes the expression of candidate genes related to bactericidal ability and regulation of cellular processes in peripheral neutrophils and liver cells in response to the immune-enhancing function of ß-CRX in post-weaned Holstein calves.

Evidence for existence of insulin-like factor 3 (INSL3) hormone-receptor system in the ovarian corpus luteum and extra-ovarian reproductive organs during pregnancy in goats.

Insulin-like factor 3 (INSL3), initially described as a male hormone, is expressed in female reproductive organs during the estrous cycle and pregnancy but its function has not yet been established. This study explores the function of INSL3 in pregnant Saanen goats by characterizing the expression dynamics of INSL3 and its receptor, relaxin family peptide receptor 2 (RXFP2) and by demonstrating specific INSL3 binding in reproductive organs, using molecular and immunological approaches and ligand-receptor interaction assays. We demonstrate that the corpus luteum (CL) acts as both a source and target of INSL3 in pregnant goats, while extra-ovarian reproductive organs serve as additional INSL3 targets. The expression of INSL3 and RXFP2 in the CL reached maximum levels in middle pregnancy, followed by a decrease in late pregnancy; in contrast, RXFP2 expression levels in extra-ovarian reproductive organs were higher in the mammary glands but lower in the uterus, cervix and placenta and did not significantly change during pregnancy. The functional RXFP2 enabling INSL3 to bind was identified as an ~ 85 kDa protein in both the CL and mammary glands and localized in large and small luteal cells in the CL and in tubuloalveolar and ductal epithelial cells in the mammary glands. Additionally, INSL3 also bound to multiple cell types expressing RXFP2 in the uterus, cervix and placenta in a hormone-specific and saturable manner. These results provide evidence that an active intra- and extra-ovarian INSL3 hormone-receptor system operates during pregnancy in goats.

Characterisation of bovine embryos following prolonged culture in embryonic stem cell medium containing leukaemia inhibitory factor.

In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos invitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in invivo embryos, hypoblast cells formed just inside the trophoblast cells of the invitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in invitro-cultured embryos, levels of both genes were significantly lower in the invitro than invivo embryos. Similar to invivo embryos, binucleate cell clusters seen in Day 23invitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells invivo.

Nfkbiz regulates the proliferation and differentiation of keratinocytes.

Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) inhibitor zeta (Nfkbiz) is a nuclear inhibitor of NF-κB (IκB) protein that is also termed as molecule possessing ankyrin repeats induced by lipopolysaccharide, interleukin-1-inducible nuclear ankyrin repeat protein, or IκBζ. We found previously that disrupting the Nfkbiz gene resulted in atopic dermatitis-like lesions in mice, suggesting an important role for Nfkbiz in the skin. In this study, we examined the cellular function of Nfkbiz in keratinocytes. Immunohistochemical analyses for Ki-67 revealed that Nfkbiz-/- keratinocytes were hypoproliferative. In skin from Nfkbiz-/- mice, the expression of the keratinocyte differentiation markers K10 and filaggrin were reduced, although that of K14 was unchanged. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that the frequency of apoptosis was comparable between control and Nfkbiz-/- keratinocytes. Interestingly, the subcellular localization of the NF-κB subunits and the transcriptional activity of NF-κB were not changed in Nfkbiz-/- keratinocytes. These findings indicate a novel possible role of Nfkbiz in controlling the proliferation and differentiation of epidermal keratinocytes through NF-κB-independent mechanisms.

Fematrin-1 is involved in fetomaternal cell-to-cell fusion in Bovinae placenta and has contributed to diversity of ruminant placentation.

During placentation, mammals employ different strategies for nourishing and supporting fetuses. Members of the Bovidae family, consisting of cloven-hoofed ruminants, utilize multiple maternal attachment points on the placenta, known as cotyledons, and hybrid cells, named trinucleate cells or syncytial plaques, made up of a fusion of fetal trophoblasts and maternal endometrial cells to provide essential hormones and maintain long gestation periods. These hybrid cells are unique to the Bovidae, as fetomaternal borders are clearly separated by syncytiotrophoblasts or epithelial cells in the placenta of other mammals. Recently, it was reported that Syncytin-Rum1 was inserted into ruminant genomes, including cattle and sheep, and was possibly involved in fetomaternal cell-to-cell fusion in both species. However, Syncytin-Rum1 alone is insufficient to explain the morphological diversity of the fetomaternal hybrids between Bovinae and Caprinae (i.e., trinucleate cells in Bovinae and syncytial plaques in Caprinae). Here we report that the bovine endogenous retrovirus K1 (BERV-K1) envelope, which we term Fematrin-1, was specifically expressed in binucleated trophoblasts throughout gestation in cattle and induced fusion with bovine endometrial cells in vitro at a significantly higher level than Syncytin-Rum1 under physiological conditions. Fematrin-1 was found to be integrated into intron 18 of FAT tumor suppressor homolog 2 (FAT2) about 18.3 to 25.4 million years ago and has been subject to purifying selection through the evolution of Bovinae. Phylogenetically, Fematrin-1 is distinct from Syncytin genes found in other mammalian species that form syncytiotrophoblasts. Our results suggest that the newly acquired endogenous retroelement has contributed to generating placentation diversity through ruminant evolution.

SOLD1 is expressed in bovine trophoblast cell lines and regulates cell invasiveness.

BACKGROUND: Secreted protein of Ly-6 domain 1 (SOLD1), a secretory-type member of the Ly-6 superfamily, is expressed in both fetal and maternal tissues throughout gestation. SOLD1 mRNA is expressed in the endometrium and in trophoblast mononucleate and binucleate cells, suggesting it plays an important role not only in placental architecture at early gestation, but also in remodeling the endometrium at late gestation. Here, we investigate the expression of SOLD1 mRNA and protein in trophoblast cell lines. In addition, we examine the effect of SOLD1 on the invasive ability of trophoblast cells. METHODS: We measured SOLD1 gene expression in thirteen bovine trophoblast (BT) cell lines by using quantitative reverse transcription PCR (qRT-PCR). SOLD1 protein levels were examined in two cell lines, BT-C and BT-K, by using Western blotting and immunocytochemistry. In addition, we measured the invasive activity of BT cells in the presence or absence of anti-bovine SOLD1 antibodies. RESULTS: At variable levels, SOLD1 was expressed in all thirteen cell lines; however, expression remained below that of proximal fetal membrane tissue. SOLD1 protein, which was approximately 28 kDa in size, was detected in perinuclear area of the cytoplasm in BT cells. Treatment with anti-bovine SOLD1 antibody had a dose-dependent suppressive effect on the invasiveness of BT-K cell lines. CONCLUSIONS: The present study is the first to investigate SOLD1 expression in vitro, in trophoblastic cell lines. Our data suggested that SOLD1 is involved in the regulation of the trophoblast invasiveness. Therefore, SOLD1 may play an active and crucial role in mediating communication at the fetomaternal interface.

Differential neutrophil gene expression in early bovine pregnancy.

BACKGROUND: In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT)-stimulated gene expression in peripheral blood leukocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. METHODS: PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q) PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (MX) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analyzed in each fraction through day 28 of gestation using qPCR. RESULTS: Microarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and with density gradient separation. CONCLUSIONS: The expression profiles of ISG15, MX1, MX2, and OAS1 could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of these genes in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination.

Dynamics of CD3⁺ T-cell distribution throughout the estrous cycle and gestation in the bovine endometrium.

T cells are the dominant lymphocytes in the endometrium and are considered to play a crucial role in implantation and in the maintenance of gestation through cytokine production and immune regulation. The mechanisms underlying immunoregulation at the feto-maternal interface are still obscure for this complex system. Understanding the role of T cells is a key factor in understanding the endometrial immune system. In this study, the distribution of endometrial CD3⁺ T cells in bovines was examined by immunohistochemical analysis. The estrous cycle and gestation was divided into 4 stages, and the number of CD3⁺-positive T cells was counted in each stage. CD3⁺ cells were found in the endometrium in significant numbers throughout the estrous cycle and were mostly located in the subepithelial area. The number of CD3⁺ cells significantly increased in the early and mid-luteal phases but decreased after implantation with the progression of gestation. No T cells were found in the placentome or specifically in the tissues near the fetus, including the trophoblastic area. In addition, very few T cells were found in stromal regions close to the myometrium of the endometrium. These findings suggest that downregulation of bovine endometrial CD3⁺ T-cell functions is closely related to the successful maintenance of gestation in a spatiotemporal manner.

Tenascin-C Expression in Equine Tendon-derived Cells During Proliferation and Migration.

In vitro cell studies might be a useful tool for studying tendon pathology, but no suitable in vitro models exist for tendon disorders. The purpose of this study was to confirm whether cell scratch culture using tendon-derived fibroblasts can provide a suitable in vitro tendon disorder model. Extracellular matrix components were examined immunohistochemically in tendon tissue, and then their related gene expression levels were analyzed by conventional reverse transcription polymerase chain reaction (RT-PCR) and/or quantitative real-time RT-PCR in tissues and cells. Collagen type I (Col I), collagen type III (Col III), tenascin-C (TN-C) and cartilage oligomeric matrix protein (COMP) were detected in tendon tissue sections, and RT-PCR confirmed their expression in tendon tissue and cells. Cells that had been cultured from explanted tendon tissue maintained the characteristics of in vivo tendon cells. The combination of TN-C and COMP might be a useful marker of tendon cells because they display more tendon-specific expression than Col I and III. In particular, the significant increase of TN-C mRNA expression in the scratch wound assay, at 12 hr after scratching, concomitant with the regeneration of the cell sheet, indicates its crucial role in tendon cell proliferation and migration. Thus, TN-C appears to be a key factor in tendon wound healing. In vitro cell scratch assays using tendon cells appear to mimic the repair of tendon tissue after injury.

Interferon-Stimulated Gene Expression in Peripheral Blood Leucocytes as a Convenient Prediction Marker for Embryo Status in Embryo-Transferred Japanese Black Cows during the Peri-Implantation Period.

Pregnancy diagnosis during early gestation is important for cattle reproduction. The expression of interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) was studied in embryo-transferred (ET) Japanese Black cattle. ISGs in PBLs-ISG15, MX1, MX2, and OAS1-were detected in multiple ovulation ET cattle using a real-time quantitative polymerase chain reaction, and receiver operating characteristic (ROC) curve analysis was performed. Gestational status was predicted using the average ISG levels during the normal estrous cycle (AVE) and the Youden index from the ROC curve analysis as cutoff values. The ISG15, MX1, and MX2 levels were significantly higher in pregnant cattle (n = 10) than in non-pregnant cattle (n = 23) on gestation day 21, whereas the levels of all ISGs were similar between non-pregnant and non-pregnant cattle with late embryonic death (n = 7). ISG15, MX1, and MX2 appropriately predicted the gestational status of ET cows. The statistical evaluation of the diagnostic accuracy in ET cows on day 21 of gestation presented higher values of sensitivity, specificity, accuracy, and positive predictive values of ISG15, MX1, and MX2 using the Youden index than using the AVE. Therefore, ISG15, MX1, and MX2 are excellent biomarkers of gestational status during the peri-implantation period in ET cattle.

The role of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of bovine endometrial cell functions.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein that stimulates the production of several matrix metalloproteinases (MMPs) for tissue remodeling. Previously, we detected EMMPRIN in the bovine endometrium, and it is mainly expressed in the luminal and glandular epithelium whereas MMPs are expressed in the underlying stroma. From this expression pattern, we hypothesized that EMMPRIN may regulate stromal MMPs in endometrial cell functions. To test this hypothesis, a coculture of epithelial and stromal cells was performed using a transwell system. In the coculture, epithelial cells were cultured on the insert membrane and stromal cell on the surface of well plates. Expression of stromal MMP-2 and MMP-14 was significantly higher in coculture with epithelial cell. Further, with the addition of anti-EMMPRIN antibody into the epithelial cell compartment, the expression of stromal EMMPRIN and MMP-2 and MMP-14 was decreased. To identify the active site of EMMPRIN for the augmentation of MMPs, EMMPRIN synthetic peptides that correspond to the extracellular loop domain-I (EM1, EM2, EM3, and EM4) were added into the epithelial cell compartment, and only EM2 at a higher dose interfered with EMMPRIN-mediated expression of MMP-14. Next, we examined the effects of progesterone and/or estrogen on the expression of EMMPRIN, MMP-2, and MMP-14. Progesterone (300 nM) significantly stimulated the expression of EMMPRIN but had no effects on any of the MMPs. These results suggest that EMMPRIN derived from epithelial cells regulates MMPs in the endometrium under progesterone-rich conditions and may thereby modulate bovine endometrial cell functions during gestation.

Identification of novel endogenous betaretroviruses which are transcribed in the bovine placenta.

Sequences of retroviral origin occupy approximately 10% of mammalian genomes. Various infectious endogenous retroviruses (ERVs) and functional retroviral elements have been reported for several mammals but not cattle. Here, we identified two proviruses, designated bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2, containing full-length envelope (env) genes in the bovine genome. Phylogenetic analysis revealed that they belong to the genus Betaretrovirus. By reverse transcription (RT)-PCR, both BERV-K1 and -K2 env mRNAs were detected in the placenta and cultured bovine trophoblast cells. Real-time RT-PCR analysis using RNAs isolated from various bovine tissues revealed that BERV-K1 env mRNA was preferentially expressed in the placenta. Moreover, we also found the expression of doubly spliced transcripts, named the REBK1 and REBK2 genes. Both the REBK1 and REBK2 proteins have motifs for a putative nuclear localization signal and a nuclear export signal. REBK1 and REBK2 fused with green fluorescent proteins were localized mainly in the nuclei when they were expressed in bovine and porcine cells. In the env and 3' long terminal repeats of BERV-K1 and -K2, we found regulatory elements responsible for the splicing and transport of viral RNAs and/or translation of the env genes. Although we have not identified the expressed Env proteins in bovine tissues, these data suggest that both BERV-K1 and BERV-K2 express Env proteins and that these proteins may have physiological functions in vivo.

Bovine trophoblastic cell differentiation and binucleation involves enhanced endogenous retrovirus element expression.

BACKGROUND: Endogenous retrovirus (ERV) envelope (env) genes are involved in the differentiation of trophoblastic cells in humans and mice. However, there is limited information about their roles in ruminant trophoblastic cells. Thus, we attempted to explore the possible roles of ERV elements in the binucleation of bovine trophoblastic cells using in vitro bovine trophoblastic (BT) cell lines. METHODS: In this study, blastocysts and elongated embryos were obtained from Japanese Black cows, and endometrial and fetal membrane tissues were collected from day 17 to 37 of gestation. The gene expression levels of four ERV elements, bERVE (bovine endogenous retrovirus envelope element-like transcript) -A, bERVE-B, BERV (bovine endogenous retrovirus) -K1 env, and BERV-K2 env, were analyzed in the fetal and endometrial tissue and cultured BT cell lines using quantitative RT-PCR. On-Matrigel gel and on-collagen gel culturing were used to induce binucleate cell (BNC) formation in the BT cell lines. How the culture conditions affected the expression of BNC-specific genes and ERV elements was examined by quantitative RT-PCR and immunocytochemistry. RESULTS: bERVE-A, bERVE-B, BERV-K1 env, and BERV-K2 env were expressed in almost all BT cell lines; however, only bERVE-A and BERV-K1 env were detected in trophoblastic tissues during the peri-implantation period. In the on-Matrigel cultures, the expression levels of BNC-specific genes and molecules were enhanced in the BT cells. The expression levels of bERVE-A and BERV-K1 env were also increased in the BT cells during on-Matrigel culturing. The BT cell expression levels of these ERV elements were consistent with those of BNC-specific genes during on-Matrigel culturing (P < 0.01). CONCLUSIONS: These results suggest that bERVE-A and BERV-K1 env are involved in the expression of BNC-specific genes and the progression of bovine trophoblastic cell binucleation, as their expression levels increased during periods of increased BNC-specific molecule expression, which is strongly suggestive of the development of BNC from mononucleate trophoblastic cells. The on-Matrigel culture system is a convenient in vitro tool for studying bovine trophoblastic cell lineages.

Bone morphogenetic protein 4 accelerates the establishment of bovine trophoblastic cell lines.

Trophoblastic cells play a crucial role in implantation and placentogenesis. A large proportion of the failures of conception in cows occur in the peri-implantation period, which are known as early embryo losses. In exploring this critical phenomenon, trophoblastic cell lines can provide substantial information. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stock in the long term. In this study, 12 new cell lines were established using bone morphogenetic protein 4 (BMP4). BMP4 stimulated embryonic cells to enter the trophoblastic cell lineage but there were no significant differences between intact and BMP4-treated groups. Only one out of 49 embryos developed trophoblastic cells in the intact group. Finally, 12 cell lines were maintained for around 30 passages, and they retained trophoblastic characteristics and expressed bovine trophoblastic genes: placental lactogen, interferon-τ, pregnancy-associated glycoprotein 1, and prolactin-related protein 1. Although the gene expression patterns were different among cell lines and depended on the cells, there was no significant relationship between the expression intensities of genes and the treatment dose of BMP4. All of them expressed bovine POU domain class 5 transcription factor 1 and caudal-type homeobox 2. The expression of these genes was confirmed by quantitative RT-PCR and immunohistochemical detection. These results suggest that BMP4 is involved in the raising of trophoblast cell lines from early embryonic cells and the newly developed cell lines can provide different types of bovine trophoblastic cells with different cell lineages. This may constitute a significant new tool for the examination of trophoblastic differentiation.

Single oral ß-cryptoxanthin administration increases its serum concentration and enhances peripheral blood neutrophil function in Holstein cattle.

We investigated the effect of oral administration of ß-cryptoxanthin (ß-CRX) on its serum concentration and peripheral neutrophil functions by the chemiluminescence (CL) response in Holstein cattle. A single oral administration of ß-CRX was performed for serum ß-CRX concentration (0, 0.05, 0.1, or 0.2 mg/kg body weight [BW]) and for peak CL response of peripheral neutrophils (0.2 mg/kg BW). The serum ß-CRX concentration was peaked on 2 days after, similar to peak CL response on 3 days after ß-CRX administration. Therefore, a single oral administration of ß-CRX (0.2 mg/kg BW) induces higher serum concentration and concurrently enhances bactericidal ability of peripheral neutrophils in Holstein cattle.

Cloning and expression of SOLD1 in ovine and caprine placenta, and their expected roles during the development of placentomes.

BACKGROUND: The Ly-6 (Ly-6/uPAR) superfamily members share the Ly-6 domain defined by distinct disulfide bonding patterns between 8 or 10 cysteine residues. They comprise membrane- and secretory-type proteins. We recently reported the gene and protein characterization of the bovine secreted protein of Ly-6 domain 1 (SOLD1). Bovine SOLD1 is expressed in trophoblast mononucleate cells (TMCs) and is localized in the cotyledonary mesenchyme. Here, we compared the expression and functionality of SOLD1 among the ruminants. We examined mRNA expression by chorionic fibroblasts as a measure of one of the SOLD1 functions. RESULTS: Ovine and caprine SOLD1 mRNAs have 303 bp open reading frames and encode for deduced SOLD1 proteins with 100 amino acids, including a 22-aa-long signal peptide at the N-terminal. Both of the SOLD1 amino acid sequences have high similarities with the bovine sequence. Both SOLD1 mRNAs were also expressed in TMCs of cotyledons and intercotyledonary membranes. The mature SOLD1 proteins were localized in the mesenchymal villi of cotyledons after secretion. Bovine, ovine and caprine SOLD1 affected gene expression in mesenchymal fibroblasts in vitro; nucleoredoxin expression was upregulated and BCL2-like 13 was downregulated. Thus, we suggest that SOLD1 acts as a modulator of cell proliferation and apoptosis. CONCLUSION: Expressing cells and protein localization of SOLD1 coincided among the three ruminants. SOLD1 participated in regulating nucleoredoxin and BCL2-like 13 expression in chorionic fibroblasts. SOLD1 is produced specifically in the cotyledons and intercotyledonary membranes in ruminants and appears to be involved in the construction of the ruminant placenta.

Transforming growth factor beta family expression at the bovine feto-maternal interface.

BACKGROUND: Endometrial remodelling is necessary for implantation in all mammalian species. The TGF beta super-family plays a crucial role in this event in humans and mice. However, the role of TGF beta super-family members during implantation is still unclear in ruminants. In the present study, the spacio-temporal expression of TGF beta super-family members including activin was explored in bovine trophoblasts and endometrial tissue during the peri-implantation period in order to elucidate whether it is essential for promoting cell proliferation at the implantation site. METHODS: Gene expression in the fetal membrane and endometrium of the gravid and non-gravid horn around Day 35 of gestation were analyzed with a custom-made oligo-microarray in cattle. The expression of activin and its related genes was also analyzed with quantitative RT-PCR. Activin-like activity in trophoblastic tissue and BT-1 cells was examined using a fibroblast cell proliferation test and Western blotting. RESULTS: The expression of various TGF beta super-family related genes including activin was detected in trophoblasts and the endometrium in cattle. The most intensive activin expression was found in the gravid horn endometrium, and rather intense expression was detected in the non-gravid trophoblastic tissue. Extracts from the fetal membrane including trophoblasts and purified activin both stimulated fibroblast proliferation effectively, and activin was immunologically detected in BT-1 cells, which have trophoblastic features. CONCLUSIONS: Specific expression of the activin gene (gene name: inhibin beta A) was found in the gravid horn endometrium during peri-implantation. An activin-like molecule, which was derived from the endometrium and trophoblasts, stimulated the proliferation of fibroblast cells. These results suggested that as in other species, the activity of TGF beta super-family members including activin-like molecules plays a pivotal role in endometrial remodelling, which is an essential process in implantation and placentogenesis during the peri-implantation period in cattle.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle.

BACKGROUND: Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. METHODS: In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis. RESULTS: EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium. CONCLUSION: EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions.

Gene expression profiles in bovine granulocytes reflect the aberration of liver functions.

Liver performs several important functions; however, predicting its functions is difficult. Methods of analyzing gene expression profiles, for example, microarray, provide functional information of tissues. Liver and peripheral blood leukocytes (PBLs) were collected from Holstein cows subjected to two different physiological conditions (non-pregnant and pregnant), and PBLs were fractionated by gradient cell separation. RNA from PBLs and liver were applied to oligo-DNA microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). It revealed a group of stable bovine liver genes under constant physiological conditions. When they applied to physiological conditions including non-pregnant and pregnant, the profiles of some genes in liver were consistent with those in PBLs. Microarray data subjected to a principal component analysis (PCA) showed that the hepatic gene expression profiles were more consistent with those of granulocytes than mononuclear cells. The relationship of gene profiles in liver with granulocytes was confirmed by RT-qPCR and hierarchical cluster analysis. Gene profiles of granulocytes were more reliable than those of mononuclear cells, which reflected liver functions. These results suggest that the genes expressed in PBLs, particularly granulocytes, may be convenient bioindicators for the diagnosis of clinical disorder and/or detecting aberration of liver functions in cows subjected to different physiological conditions.

Use of a prediction method for early pregnancy status utilizing receiver operating characteristic curve analysis of peripheral blood leukocyte interferon-stimulated genes in Japanese-Black cattle.

A prediction method for early pregnancy status (pregnant or non-pregnant) in cattle that can be used within 3 weeks after insemination is desired. Interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) have been examined as prediction molecules for determination of pregnancy status. Relative abundances of ISG15 and MX2 gene transcripts in PBLs were suitable biomarkers for the prediction of pregnancy status when there were assessments of Holstein cattle. In the present study, it was determined whether ISG biomarkers are applicable for predicting gestation in Japanese-Black (JB) cattle and evaluation of the applicability of receiver operating characteristic (ROC) analysis procedures for this purpose. There was assessment of the reliability of using average ISG values in PBLs collected during the estrous cycle (AVE) as a cutoff compared to the Youden index cutoff values. Application of AVE to assessment of pregnancy status in JB cattle indicated there was reliable predictions for pregnancy status when using ISG15 and MX2 values on day 21 after insemination, which coincided with the time of assessment in the previous study with Holstein cattle. The area under the curve values of the ROC curves confirmed the reliability of using ISGs to predict pregnancy from days 18 to 21 after insemination. Comparing AVE with Youden index values, there was confirmation of the accuracy of AVE for predicting gestation. The average mRNA transcript abundance values of ISG15 and MX2 may serve as excellent pregnancy biomarkers for cattle within 3 weeks of insemination.