ABSTRACT
We recently demonstrated that conceptus-derived interferon tau (IFNT), responsible for maternal recognition in cattle, acts on the uterus in a dose- and time-dependent manner by upregulating key interferon-stimulated genes (ISGs) in the endometrium. In high producing dairy cows, postpartum uterine infection is a major factor influencing fertility and pregnancy outcome. Lipopolysaccharide (LPS), an endotoxin of Gram-negative bacteria such as Escherichia coli, generates an altered uterine environment by inducing excessive inflammation at the maternal-conceptus interface. Thus, we aimed to investigate whether the endometrial response to IFNT is altered in the presence of LPS. Endometrial explants were isolated from uteri collected at a local abattoir from Holstein Friesian cows (n = 8) during the mid-luteal stage of the estrous cycle, and cultured in RPMI medium for 24 h in 5 % CO2 in humidified air without (control), or with IFNT (100 ng/mL), a single Day 15 conceptus, LPS (1 µg/mL), both IFNT and LPS, or both a Day 15 conceptus and LPS. Incubation with IFNT and a Day 15 conceptus up-regulated (P < 0.05) well-known classical ISGs (ISG15, OAS1, MX1 and MX2) as well as other candidate ISGs (CMPK2, IFI35, TRIM38 and TNFSF10) and down-regulated expression of IL1B in endometrial explants. Incubation with LPS increased (P < 0.05) abundance of NFKB1 (a key transcription factor involved in inflammatory and immune response), TNFA, IL1B and IL6 (pro-inflammatory cytokines), IL10 (anti-inflammatory cytokine), IL8, CXCL1, CXCL3 and CCL2 (chemokines), and, to a lesser extent, classical ISGs in endometrial explants. However, LPS did not alter endometrial response to IFNT, irrespective of IFNT concentration (1, 10 or 100 ng/mL). Results suggest that the expression of ISGs, up-regulated by conceptus-derived IFNT, is not altered in the endometrium in the presence of LPS; however, the increased expression of inflammation-related genes induced by LPS indicate an altered endometrial immune response that may be associated with compromised pregnancy establishment or pregnancy failure.
Subject(s)
Endometrium , Interferon Type I , Lipopolysaccharides , Pregnancy Proteins , Female , Animals , Cattle , Endometrium/metabolism , Endometrium/drug effects , Lipopolysaccharides/pharmacology , Pregnancy Proteins/metabolism , Pregnancy Proteins/genetics , Pregnancy Proteins/pharmacology , Interferon Type I/metabolism , Interferon Type I/genetics , Gene Expression Regulation/drug effects , PregnancyABSTRACT
Failure by the developing conceptus to secrete sufficient interferon tau (IFNT), required for maternal recognition of pregnancy (MRP), at the appropriate time is related to early pregnancy loss in cattle. We aimed to test the hypothesis that there is a dose- and time-dependent relationship between IFNT and the endometrial expression of key interferon-stimulated genes (ISGs) involved in the signalling cascade leading to MRP in cattle. Candidate genes were identified first through a bioinformatic approach, where integrated transcriptomic data from two previous studies were analyzed to identify endometrial genes induced by IFNT. Next, expression of selected candidate genes was investigated in vitro in endometrial explants. Endometrial explants collected from cows (n = 8) in the late luteal phase of the estrous cycle were cultured in medium without (control) or with recombinant ovine IFNT (1, 10, 100 ng/mL) for 6 h. Simultaneously, endometrial explants were cultured in medium containing 100 ng/mL IFNT for different time periods (15 min, 30 min, 1 h, 3 h, 6 h). Gene expression was analyzed by RT-qPCR. We identified 54 endometrial genes responding to IFNT and to some degree to the conceptus, from which five ISGs (CMPK2, BPNT1, IFI35, TNFSF10 and TRIM38) were further selected for the dose- and time-dependent experiments. Classical ISGs (ISG15, OAS1, MX1 and MX2) were up-regulated (P < 0.05) in endometrium by 1 ng/mL IFNT. However, other selected ISGs (CMPK2, BPNT1, IFI35, TNFSF10 and TRIM38) were induced only by higher concentrations (10 and 100 ng/mL) of IFNT (P < 0.05). In terms of duration of exposure, IFNT at 100 ng/mL induced a significant (P < 0.05) increase in ISG15 and CMPK2 expression after 1 h incubation, while all other studied ISGs in the endometrium were upregulated when cultured for 3 or 6 h, but did not affect expression when the duration of culture was for 1 h or less. These results suggest that IFNT acts on the uterus in both a dose- and time-dependent manner in cattle and that timely exposure of the endometrium to sufficient IFNT is essential for appropriate signalling to ensure successful pregnancy establishment.
Subject(s)
Cattle Diseases , Interferon Type I , Sheep Diseases , Pregnancy , Female , Cattle , Animals , Sheep , Abortion, Veterinary , Interferon Type I/genetics , Interferon Type I/pharmacology , Interferon Type I/metabolism , Endometrium/metabolism , Gene Expression , Cattle Diseases/metabolismABSTRACT
Mature granulated trophoblast binucleate cells (BNC) have been found in all ruminant placentas examined histologically so far. BNC are normally fairly evenly distributed throughout the fetal villus and all their granules contain a similar variety of hormones and pregnancy associated glycoproteins (PAGs). Only the Giraffe is reported to show a different BNC protein expression, this paper is designed to investigate that. Gold labelled Lectin histochemistry and protein immunocytochemistry were used on deplasticised 1 µm sections of a wide variety of ruminant placentomes with a wide range of antibodies and lectins. In the Giraffe placentomes, even though the lectin histochemistry shows an even distribution of BNC throughout the trophoblast of the placental villi, the protein expression in the BNC granules is limited to the BNC either in the apex or the base of the villi. Placental lactogens and Prolactin (PRL) are present only in basally situated BNC: PAGs only in the apical BNC. PRL is only found in the Giraffe BNC which react with many fewer of the wide range of antibodies used here to investigate the uniformity of protein expression in ruminant BNC. The possible relevance of these differences to ruminant function and evolution is considered to provide a further example of the versatility of the BNC system.
Subject(s)
Giraffes , Placenta , Animals , Female , Lectins/metabolism , Placenta/metabolism , Pregnancy , Prolactin/metabolism , Ruminants/metabolism , Trophoblasts/metabolismABSTRACT
Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post-partum. This study investigated the effects of pre- and post-partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post-partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post-partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi- and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post-partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post-partum patterns of IGF-1, insulin, ß-hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid-related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post-partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post-partum dairy cows.
Subject(s)
Cattle/physiology , Estrous Cycle/physiology , Gene Expression Regulation/physiology , Ovary/metabolism , Peripartum Period/physiology , Uterus/metabolism , Animals , Female , Food Deprivation , Reverse Transcriptase Polymerase Chain Reaction/veterinaryABSTRACT
In the present study, we examined the lysophosphatidic acid (LPA) pathway in the ovine uterus during the estrous cycle and early pregnancy. With the use of quantitative reverse transcription PCR, expression of LPAR1 and LPAR3 was analyzed. Both receptors were present in the ovine uterus. Immunolocalization showed that LPAR1 was mainly present in the stroma of the ovine endometrium, whereas LPAR3 was mostly restricted to epithelial compartments. In luminal and glandular epithelia, LPAR1 and LPAR3 levels were affected by pregnancy status, day, or the day-by-status interaction, whereas in stroma the receptors were not modified. Analysis of the whole endometrium from ovariectomized ewes showed that the expression of LPAR3 but not LPAR1 was regulated by the administration of progesterone. However, the examination of receptors at cellular levels showed that progesterone increases LPAR1 and LPAR3 in glandular epithelium and, in a minor extent, in endometrial stroma. Emerging evidence suggests that LPA is an essential component in the estrous cycle and early pregnancy regulation. We demonstrated that LPA induced stress fiber formation in ovine uterine epithelial cells, suggesting that LPA may be involved in cytoskeleton reorganization occurring cyclically in ovine uterus.
Subject(s)
Estrous Cycle/physiology , Progesterone/pharmacology , Receptors, Lysophosphatidic Acid/biosynthesis , Sheep/physiology , Uterus/physiology , Animals , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Epithelial Cells , Female , Immunohistochemistry/veterinary , Pregnancy , Progesterone/metabolism , RNA/chemistry , RNA/genetics , Random Allocation , Real-Time Polymerase Chain Reaction/veterinary , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolism , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sheep/metabolism , Uterus/metabolismABSTRACT
This study documents the expression of prostacyclin (PGI2) synthase (PTGIS) and PGI2 receptors in the trophoblast and uterus of the ewe at the time of maternal recognition of pregnancy (i.e. days 7, 9, 12, 14 and 17). The membrane receptor for PGI2 (PTGIR) and the nuclear receptors, i.e. peroxisome proliferator-activated receptors (PPAR) and their heterodimer partners the retinoid X receptors (RXR), were analysed. In the endometrium, PTGIS transcript and protein were expressed at day 9 of pregnancy and levels declined from days 12 to 17. Immunohistochemistry and in situ hybridization indicated that PTGIS was mainly located in the luminal epithelium of the endometrium. Endometrial PTGIR, PPARA, PPARG and RXRG expression was regulated during the peri-implantation period whereas PPARD, RXRA and RXRB were consistently expressed. In the trophoblast, PTGIS transcript levels rose as development progressed and peaked at day 17. PTGIR and PPARA transcripts peaked before day 12 and then declined and became nearly undetectable by day 17, whereas PPARD and PPARG transcript levels rose steadily from days 12 to 17. Because the PPARs and the RXRs display different expression profiles, we suggest that different heterodimers may form and support distinct functions as development proceeds. Our results also underline the importance of PTGIS and PPARD in the trophoblast and PTGIR in the uterus, suggesting that PGI2 is of both uterine and trophoblastic origin and is involved in a complex signalling pathway at around the time of implantation in the ewe.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Embryo Implantation/physiology , Endometrium/chemistry , Gene Expression Regulation, Developmental , Intramolecular Oxidoreductases/genetics , Receptors, Epoprostenol/genetics , Trophoblasts/chemistry , Animals , Base Sequence , Blotting, Western/methods , Cattle , Cytochrome P-450 Enzyme System/analysis , DNA Probes/genetics , Female , Humans , Immunohistochemistry/methods , In Situ Hybridization/methods , Intramolecular Oxidoreductases/analysis , Molecular Sequence Data , Peroxisome Proliferator-Activated Receptors/analysis , Peroxisome Proliferator-Activated Receptors/genetics , Pregnancy , Receptors, Epoprostenol/analysis , Retinoid X Receptors/analysis , Retinoid X Receptors/genetics , Reverse Transcriptase Polymerase Chain Reaction , SheepABSTRACT
cDNA arrays have proven to be useful tools to screen gene expression in many animal species including livestock species. A collaborative program was launched to construct a ruminant cDNA collection, representative of three tissues: Muscle, Embryo and Mammary gland, named MEM. This collection gathers clones mainly arising from 3 non-normalised cDNA libraries: a directed bovine muscle library, a 14-day-old bovine embryo library and a goat lactating mammary library. It is made up of 1896 clones (637 muscle, 882 embryo and 377 mammary cDNAs), selected after sequencing and bioinformatic analyses. Amplification products yielded from these clones as well as controls were printed onto Nylon membranes to generate macroarrays. Hybridisation with relevant cDNA targets allowed checking the location of about 50 cDNAs and the specificity of each sub-set of the repertoire. Macroarrays were hybridised with radiolabelled cDNA complex targets from five different tissues (muscle, embryo, mammary gland, adipose tissue and oocyte). Both somatic and germinal complex targets gave valid hybridisation signals with 45 to 80% of the printed probes. This specific cDNA collection now provides a powerful tool for transcriptomic studies with the ultimate objective to better understand physiological and metabolic functions in ruminants. It will be subsequently included into a forthcoming larger collection.
Subject(s)
Gene Expression Profiling/methods , Meat , Milk Proteins/genetics , Muscle Proteins/genetics , Oligonucleotide Array Sequence Analysis , Reproduction/genetics , Ruminants/genetics , Animals , Cattle/genetics , DNA Probes , Databases, Genetic , Embryo, Mammalian/chemistry , Female , Gene Library , Goats/genetics , Male , Mammary Glands, Animal/chemistry , Muscle, Skeletal/chemistry , RNA/analysis , Reproducibility of Results , Sheep/genetics , Transcription, GeneticABSTRACT
Following hatching, pre-elongated conceptuses undergo elongation by intense proliferation, until implantation. We investigated the changes in gene expression associated with these physiological events using human cDNA arrays containing 2370 known genes. Comparison of pre-elongated, elongated, and implanting trophoblasts allowed the determination of 313 expressed genes, 63 of which were differentially regulated. These were classified into four functional families. Pre-elongated trophoblasts were characterized by preferential expression of genes involved in protein trafficking, whereas only latter developmental stages expressed cell signaling genes and receptors. Among the 63 developmentally regulated genes, four exhibited the highest levels of expression (TMSB10, CTNNA1, NMP1, and CX3CL1). Each of these also represents a functional family and display a specific expression pattern. One of them, CX3CL1 (CX3C chemokine, also known as fractalkine), is a chemokine that seems to have potential importance in trophoblast development, and which deserves further clarification of its role in implantation.
Subject(s)
Embryo Implantation/genetics , Gene Expression Regulation, Developmental , Oligonucleotide Array Sequence Analysis , Sheep/genetics , Trophoblasts/physiology , Animals , Endometrium/physiology , Female , Gene Expression Profiling , Genetic Testing , Multigene Family , Reverse Transcriptase Polymerase Chain Reaction , Sheep/embryologyABSTRACT
IGF system expression has been largely explored in the bovine follicular wall whereas it remains poorly studied in the COC. Using semi-quantitative RT-PCR and Western blot analysis, we have investigated spatial and temporal expression of IGF-1, IGFR-1, IGFBP-2, IGFBP-4, as well as gonadotropin receptors in bovine COC during oocyte maturation. In addition, we have compared changes in the IGF system and FSHR expression during in vitro maturation in TCM199 alone or in the presence of 10 ng/ml of EGF. The transcripts for IGFR-1 and IGFBP-2 were detected in cumulus and germinal cells whereas IGF-1, IGFBP-4 and FSHR mRNA were restricted to cumulus cells. Topography of the IGF system and gonadotropin receptor expression within COC were unaffected by the maturation step. In contrast, levels of IGFBP-2 and FSHR expression decreased (P < 0.05) in matured COC. Under defined culture conditions, IGFBP-2 and FSHR mRNA expression remained at a high level in TCM199 alone and were significantly reduced (P < 0.05) in the presence of 10 ng/ml EGF after a 24 h period of in vitro maturation. In conclusion, our results demonstrate a cell-specific pattern of IGF system member gene expression within bovine COC suggesting interaction between the somatic and germinal compartments. In addition, synchronized changes in the pattern of COC IGFBP-2 and FSHR expression during oocyte maturation suggest possible synergistic actions between IGF-1 and FSH.
Subject(s)
Cattle , Oocytes/growth & development , Oocytes/metabolism , Ovarian Follicle/metabolism , Receptors, FSH/genetics , Somatomedins/genetics , Animals , Blotting, Western , Epidermal Growth Factor/pharmacology , Female , Gene Expression , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor Binding Protein 4/genetics , Insulin-Like Growth Factor I/genetics , Ovarian Follicle/chemistry , RNA, Messenger/analysis , Receptor, IGF Type 1/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means +/- SEM) both at the germinal vesicle and metaphase II stages (2.8 +/- 0.06 pmol vs. 2.6 +/- 0.07 pmol and 2.9 +/- 0.1 pmol vs. 2.3 +/- 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 +/- 2.1 % vs. 36.7 +/- 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 +/- 14 104, ranging from 2.0 x 10(3) to 1.2 x 10(6)) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.
Subject(s)
Adenosine Triphosphate/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Fertilization in Vitro/veterinary , Oocytes/physiology , Animals , Base Sequence , Cattle , Female , Haplotypes , In Vitro Techniques , Molecular Sequence DataABSTRACT
Aspirin consumption has been reported to be able to reduce colorectal cancer risk in humans and in animal models of colon carcinogenesis. Although the mechanism involved in such an effect is not yet clear, both prostaglandin-dependent and -independent effects have been proposed. Using HT-29 Glc(-/+)cells, which originate from a human colon adenocarcinoma, we demonstrated in this study a dose-dependent effect of millimolar concentration of aspirin on cell growth that was concomitant with a rapid accumulation of the cells in the G0/G1 phase, followed by an accumulation in the G2/M phase and by a minor increase in the proportion of cells undergoing nuclear condensation. Cell membrane integrity and cell release into the culture medium were not affected by this treatment. The aspirin effects were apparently unrelated to prostaglandin biosynthesis inhibition, since although these cells were found to express high levels of cyclooxygenase 1 (COX-1) and low levels of COX-2 proteins, they did not produce any measurable net amounts of prostaglandins, based on both utilization of radiolabelled arachidonic acid and the radioimmunoassay of prostaglandins E2 and F2 alpha. In contrast, we identified polyamine biosynthesis as a cellular target of aspirin, since the treatment of HT-29 Glc(-/+) cells with aspirin reduced the flux of L-ornithine through ornithine decarboxylase, an effect that could not be explained by an acute action of the drug on the ornithine decarboxylase catalytic activity. Since polyamine biosynthesis is strictly necessary for HT-29 cell growth, our data suggest that reduced flux through ornithine decarboxylase may participate in the antiproliferative activity of aspirin towards colonic tumoral cells. It is concluded that in HT-29 Glc(-/+) cells that are not functional for prostaglandin production, aspirin can affect cell growth, cell cycle, and polyamine biosynthesis without affecting cell membrane integrity.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anticarcinogenic Agents/pharmacology , Aspirin/pharmacology , Prostaglandins/metabolism , Putrescine/biosynthesis , Adenocarcinoma , Caco-2 Cells , Cell Cycle/drug effects , Cell Membrane/drug effects , Colonic Neoplasms , Cyclooxygenase 1 , Cyclooxygenase 2 , HT29 Cells , Humans , Isoenzymes/biosynthesis , Membrane Proteins , Ornithine/metabolism , Polyamines/metabolism , Prostaglandin-Endoperoxide Synthases/biosynthesisABSTRACT
Oocyte maturation is accompanied by differentiation of surrounding cumulus cells. These cells produce hyaluronic acid (HA) and its storage in intercellular spaces results in expansion of the cells. The cumulus cells also accumulate cyclooxygenase-2 (cox-2) during maturation. Both expansion and cox-2 storage are regulated by FSH and EGF. The aim of this study was to determine whether oocyte meiotic resumption is involved in the regulation of cumulus differentiation or not. We investigated the effects of roscovitine, a reversible inhibitor of meiosis resumption of cattle oocytes on EGF induced expansion and cox-2 expression at the transcript and protein levels respectively (RT-PCR and Western blot), in cumulus oocyte complexes (COCs) and cumulus complexes alone (CCs). EGF induced expansion and cox-2 expression in both COCs and CCs. These effects were prevented by roscovitine, whether in the presence or in the absence of oocyte. However, the oocyte was essential for the reversibility of inhibition by roscovitine. In conclusion, our results indicate that i) oocyte secreted-factors are not essential for cumulus expansion, and ii) roscovitine mediated inhibition of meiotic resumption also respects the functionality of the surrounding somatic cells.
Subject(s)
Enzyme Inhibitors/pharmacology , Isoenzymes/genetics , Oocytes/metabolism , Ovarian Follicle/drug effects , Prostaglandin-Endoperoxide Synthases/genetics , Purines/pharmacology , Animals , CDC2 Protein Kinase/antagonists & inhibitors , Cattle , Cyclooxygenase 2 , Female , Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/metabolism , RoscovitineABSTRACT
The mechanisms that lead to the onset of human parturition are still unknown, although selected critical factors have been identified. To investigate the changes in myometrial gene expression associated with parturition, we used two macroarrays each containing 1176 different complementary human cDNA clones. Methods involving hierarchical clustering and conventional statistical analysis allowed us to generate a profile of genes expression at three stages of late pregnancy: preterm (29 wk amenorrhea); full term, not in labor (38 wk amenorrhea); and full term in labor (39 wk amenorrhea). Only 4% of the genes investigated were differentially expressed between the preterm and term groups (P < 0.05). These genes could be clustered as groups of either down-regulated or up-regulated transcripts. The changes in transcript abundance were particularly marked between the preterm and term stages of gestation, whereas the differences between term not in labor and term in labor were less pronounced. The parturition was characterized by a massive down-regulation of a large panel of developmental, cell adhesion molecule and proliferation-related genes, along with the up-regulation of inflammatory, contraction and apoptosis associated genes. We propose that the mechanisms of parturition consist primarily in the arrest of the processes of myometrial development, a step that might be essential to allow the uterus to recover appropriate contractile function before delivery.
Subject(s)
Gene Expression Regulation/physiology , Labor Onset/physiology , Labor, Obstetric/physiology , Myometrium/metabolism , Parturition/physiology , Adult , Cell Division/genetics , Cell Division/physiology , DNA Probes , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Female , Humans , In Situ Hybridization , Inflammation/genetics , Pregnancy , RNA, Messenger/biosynthesis , RNA, Messenger/isolation & purificationABSTRACT
Prostaglandins could be involved in various aspects of final differentiation of ovarian follicles. Prostaglandins are generated by the cyclooxygenase (cox) pathway. Until now, the expression pattern of isoforms cox-1 and cox-2 of cyclooxygenase in bovine cumulus-oocyte complexes (COCs) was unknown. Using immunodetection procedure, we demonstrated in the present study that cox-2 was expressed by cumulus cells during in vivo and in vitro maturation. Time course induction of cox-2 expression was investigated during in vitro maturation using Western blot analysis. Specific signal of cox-2 was markedly evidenced from 6 hr of culture and increased to reach a maximal level at 24 hr of culture. In vitro, cox-2 expression in COCs was associated with increased concentrations of PGE(2) and PGF(2alpha) in the maturation medium. In addition, the effects of culture conditions on cox-2 expression was considered using RT-PCR and Western-blot analysis. We demonstrated that the addition of 10 ng/ml of EGF to TCM199 clearly increased the expression level of cox-2 mRNA and protein. Higher levels of in vitro cox-2 expression was associated with greater rates of cumulus expansion and oocytes at metaphase II at 24 hr of culture. In conclusion, our present results suggest that cox-2 expression in cumulus cells may be involved in differentiation of COCs that occurs during oocyte maturation.
Subject(s)
Isoenzymes/metabolism , Oocytes/growth & development , Oogenesis , Ovarian Follicle/cytology , Ovarian Follicle/enzymology , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Arachidonic Acid/metabolism , Blotting, Western , Cattle , Cell Culture Techniques/methods , Chromatography, High Pressure Liquid , Cyclooxygenase 1 , Cyclooxygenase 2 , Dinoprost/metabolism , Dinoprostone/metabolism , Female , Immunohistochemistry , Isoenzymes/genetics , Oocytes/metabolism , Oogenesis/genetics , Oogenesis/physiology , Ovarian Follicle/drug effects , Prostaglandin-Endoperoxide Synthases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Radioimmunoassay , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
The main sulfated proteins secreted by rabbit mammary gland tissue had M(r) of approximately 67 000, 63 000 and 23 000, and one component which most likely corresponded to proteoglycans had a diffuse electrophoretic mobility (M(r)200 000). The sulfate groups in the 67-63 kDa proteins were mostly linked to carbohydrates. These proteins and the 23 kDa protein were co-purified and identified to heavy chains of immunoglobulin A (IgA) and J chain, respectively. Sulfation of alpha-chains also occurred in rat mammary and rabbit lacrimal glands. We conclude that polymeric IgA which are produced by plasma cells and released in secretion fluids after transcytosis through epithelia are sulfated.
Subject(s)
Immunoglobulin A, Secretory/chemistry , Immunoglobulin A/chemistry , Mammary Glands, Animal/metabolism , Milk Proteins/chemistry , Amino Acid Sequence , Animals , Epithelial Cells/metabolism , In Vitro Techniques , Lacrimal Apparatus/metabolism , Lactation , Rabbits , Rats , Rats, Inbred F344 , Sulfates/chemistry , Sulfur RadioisotopesABSTRACT
Ovine endometrium showed transient expression of high concentrations of the inducible isoform of cyclooxygenase, cyclooxygenase 2 (COX-2), whereas the constitutive isoform, cyclooxygenase 1 (COX-1), was expressed at much lower concentrations and did not change. In this study, the pattern of prostaglandin synthesis in endometrial luminal cells was investigated in relation to their COX-2 content. Endometrial cells from cyclic or pregnant ewes at days 9, 12, 14 and 16 were isolated and analysed for the presence of COX-1 and COX-2 proteins using western blot analysis. Freshly isolated cells were incubated with 0.5 microCi [3H]arachidonic acid ml-1. Radioactive cyclooxygenase metabolites were analysed by reverse-phase HPLC. Luminal cells produced mainly PGF2 alpha, PGE2, PGD2 and 13,14-dihydro-15-keto PGF2 alpha and to a lesser extent 6-keto PGF1 alpha, thromboxane B2 and 13,14-dihydro-15-keto PGE2. The production of PGE2 and PGD2 was proportional to the cellular concentration of COX-2. PGE2 and PGD2 release was low on day 9 when COX-2 was not expressed, whereas high concentrations of PGE2 and PGD2 were synthesized on days 12-14 when COX-2 was highly expressed, reaching 100 ng microgram-1 cellular protein. In contrast, the basal production of PGF2 alpha did not appear to be related to COX-2 concentration and was greatest on day 16. Moreover, the release of PGF2 alpha was maintained at steady state values between days 9 and 14 by the production of 13,14-dihydro-15-keto PGF2 alpha. Although PGF2 alpha output was lower at day 16 of pregnancy compared with the oestrous cycle, no difference was observed in the pattern of prostaglandin synthesis between pregnant and non-pregnant ewes.
Subject(s)
Endometrium/metabolism , Isoenzymes/metabolism , Luteal Phase/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Prostaglandins/metabolism , Sheep/metabolism , Analysis of Variance , Animals , Arachidonic Acids/metabolism , Blotting, Western , Cells, Cultured , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Cyclooxygenase 2 , Dinoprostone/metabolism , Endometrium/enzymology , Estrus/metabolism , Female , Prostaglandin D2/metabolismABSTRACT
Two proteins (17 and 22-24 kDa) produced by day 17 goat conceptuses were purified from in vitro culture media. Analysis of their N-terminal amino acid sequences and of their antiviral activity confirmed that both proteins belonged to the interferon tau family characteristic of ruminant conceptuses. The two molecules were glycosylated (22-24 kDa) or nonglycosylated (17 kDa) isoforms of the same protein. The time course of secretion was plotted and immunoblotting of the protein contents of uterine flushings from day 13 to day 21 of pregnancy was performed. The nonglycosylated isoform (17 kDa) was first detected on day 16; both isoforms were present at day 17 and, thereafter during pregnancy, the two proteins were not present in uterine flushings. Immunohistochemistry was used to show that the goat interferon tau was present in the trophoblastic cells as early as day 14 and until day 17. However, immunostaining was not uniform along the conceptus; labelling was greater at the abembryonic pole than at the embryonic pole. By day 18, as implantation proceeded, goat interferon tau was no longer detected. These results confirmed that the goat conceptus secretes interferon tau during the period of maternal recognition of pregnancy but its rapid decrease suggests that other factors need to be present by day 18 to take over its role in the maintenance of luteal function.
Subject(s)
Embryo, Mammalian/metabolism , Goats/physiology , Interferon Type I/isolation & purification , Pregnancy Proteins/isolation & purification , Pregnancy, Animal/physiology , Animals , Embryonic Development , Female , Immunoblotting , Immunohistochemistry , Interferon Type I/analysis , Isomerism , Pregnancy , Pregnancy Proteins/analysis , Trophoblasts/chemistryABSTRACT
In this study we investigated expression of the two isoforms of the prostaglandin-forming enzyme, cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2), in sheep embryos. Using Western blot and immunohistochemical analyses, we demonstrated that Cox-2 was highly expressed in embryos from Day 8 to Day 17 of development whereas Cox-1 was undetectable during this time. The expression of Cox-2 was developmentally regulated. It was maximal between Days 14 and 16. There was a 30-fold increase in Cox-2 content per protein extract between Day 10 and Day 14, corresponding to a 50,000-fold increase in the whole embryo. The expression of Cox-2 declined after Day 16 to become undetectable by Day 25 of pregnancy. Cox-2 was localized in the trophoblastic cells and was not detected in the inner cell mass. The [3H]arachidonic acid metabolites synthesized by Cox-2-rich conceptuses were analyzed by HPLC after short-term embryo culture. Day 14 conceptuses released mainly cyclooxygenase metabolites and to a lesser extent lipoxygenase derivatives. Cyclooxygenase products were 6-keto-prostaglandin (PGF)1alpha 18.2% (+/- 4.2), thromboxane-B2 22.51% (+/- 15.9), PGF2alpha 21% (+/- 11), PGE2 14.5% (+/- 7.4), and PGD2 2.7% (+/- 2.6). Taken together, these results suggest an important role for the Cox-2-dependent cyclooxygenase metabolites during embryo development.
Subject(s)
Embryo Implantation/physiology , Embryo, Mammalian/enzymology , Isoenzymes/biosynthesis , Prostaglandin-Endoperoxide Synthases/biosynthesis , Amino Acid Sequence , Animals , Arachidonic Acid/metabolism , Blotting, Western , Cyclooxygenase 1 , Cyclooxygenase 2 , Eicosanoids/biosynthesis , Female , Immunohistochemistry , Molecular Sequence Data , Placenta/cytology , Placenta/enzymology , Pregnancy , Sheep , Uterus/cytology , Uterus/enzymologyABSTRACT
In this study we investigated the expression of the two cyclooxygenases, cox-1 and -2, in sheep uterine tissues during the estrous cycle and early pregnancy. We identified the cox-2 isoform in the ovine uterus by Western blot and demonstrated that the two cyclooxygenases exhibited different patterns of expression. Cox-1 was expressed at steady state levels in the endometrium during the estrous cycle and comparable stages of pregnancy. In contrast, cox-2 was highly and transiently expressed from days 12-15 of the estrous cycle and declined thereafter to undetectable levels. Endometrium from early pregnant ewes showed a similar pattern of cox-2 expression, although there was a slower decrease beyond day 15. Immunohistochemical studies demonstrated that cox-1 was localized in both epithelial and stromal cells, whereas cox-2 was localized solely in the luminal epithelium and to a lesser extent in the superficial glands. Treatment of ovariectomized ewes with steroids indicated that expression of cox-1 remained at constant levels whatever the treatment. In contrast, endometrial cox-2 was highly induced by a 10-day progesterone treatment. Estradiol slightly increased cox-2 expression but only after progesterone priming. Collectively these results suggest that the developing ability of the uterus to synthesize PGs is due to the induction of cox-2.
Subject(s)
Endometrium/enzymology , Estrus/physiology , Isoenzymes/metabolism , Pregnancy, Animal/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Blotting, Western , Cyclooxygenase 1 , Cyclooxygenase 2 , Dinoprost/metabolism , Estradiol/pharmacology , Female , Immunohistochemistry , Isoenzymes/analysis , Ovariectomy , Pregnancy , Progesterone/pharmacology , Prostaglandin-Endoperoxide Synthases/analysis , SheepABSTRACT
This review considers the potential reduction of embryo mortality in vitro and in vivo in ruminants. Data on cytokines provided by different fields of reproductive immunology and biology were collated. Because of the crucial importance of the local interactions between the embryo and its dam, the expression of growth-factor and cytokine genes was analysed in the embryo proper, trophoblast, oviduct and endometrium by reverse transcriptase polymerase chain reaction in sheep and in cattle during the pre- and periimplantation periods. Many deleterious cytokines, such as tumour necrosis factor-alpha, interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and beneficial cytokines, such as transforming growth factor-beta, leukaemia inhibiting factor, colony-stimulating factor-1 (CSF-1), granulocyte-macrophage CSF, IL-1, IL-3, IL-4, IL-6, IL-10 and IFN-tau appeared to be involved in embryo survival in ruminants and other species. Their administration is efficient in a murine experimental model (CBA/J x DBA/2) of embryonic and fetal mortality. For instance, recombinant ovine IFN-tau (roIFN-tau) injected at the moment of implantation drastically reduces embryonic mortality in this model. In ruminants, roIFN-tau and recombinant bovine IFN-tau are very efficient in maintaining progesterone luteal secretion in cyclic animals. The involvement of IFN-tau in the mechanisms of maternal pregnancy recognition are particularly detailed in relation to inhibition of 13,14 dihydro-15-keto-prostaglandin F2 alpha (PGFM) pulses and oxytocin uterine receptivity. A synthetic model of the anti-luteolytic effects of IFN-tau on the endometrial cell is proposed. Finally, the particular potential of serum pregnancy-specific proteins (PSPs: PSPB, PSP60, pregnancy-associated glycoprotein) for monitoring embryo survival, with examples given for cattle and sheep is underlined.