Placentomal Tissues Research Articles

Comparison of bison and elk susceptibility to experimental challenge with Brucella abortus strain 2308.

Brucellosis is endemic in bison and elk in Yellowstone National Park and surrounding areas. A comparative study was conducted using data from naive (n = 82 and 67, respectively) and Brucella abortus strain RB51 (RB51) vaccinated (n-99 and 29, respectively) bison and elk experimentally challenged with virulent B. abortus strain during pregnancy. The incidence of abortion, fetal infection, uterine or mammary infection, or infection in maternal tissues after experimental challenge was greater (p < 0.05) in naïve and vaccinated bison when compared to similar groups in elk. Vaccinated bison had lower (p < 0.002) abortion rates and recovery of Brucella from fetal or uterine/mammary tissues when compared to naïve bison. Vaccinated elk had reduced (p < 0.01) rates of maternal infection, but rates of abortion and fetal or uterine/mammary infection did not differ (p > 0.05) from naïve elk. Naïve and vaccinated bison had greater (p < 0.05) Brucella colonization in placentomes, and parotid and supramammary lymphatic tissues when compared to elk. In elk or bison that aborted, mean colonization in placentome tissues were typically more than 5 logs higher than in animals that did not abort. The results of our study suggest differences in disease pathogenesis between these two wildlife reservoirs of B. abortus.

Increasing the Understanding of Nutrient Transport Capacity of the Ovine Placentome.

Placental nutrient transport capacity influences fetal growth and development; however, it is affected by environmental factors, which are poorly understood. The objective of this study was to understand the impact of the ovine placentome morphological subtype, tissue type, and maternal parenteral supplementation of arginine mono-hydrochloride (Arg) on nutrient transport capacity using a gene expression approach. Placentomal tissues of types A, B, and C morphologic placentome subtypes were derived from 20 twin-bearing ewes, which were infused thrice daily with Arg (n = 9) or saline (Ctrl, n = 11) from 100 to 140 days of gestation. Samples were collected at day 140 of gestation. Expression of 31 genes involved in placental nutrient transport and function was investigated. Differential expression of specific amino acid transporter genes was found in the subtypes, suggesting a potential adaptive response to increase the transport capacity. Placentomal tissues differed in gene expression, highlighting differential transport capacity. Supplementation with Arg was associated with differential expressions of genes involved in amino acid transport and angiogenesis, suggesting a greater nutrient transport capacity. Collectively, these results indicate that the morphological subtype, tissue type, and maternal Arg supplementation can influence placental gene expression, which may be an adaptive response to alter the transport capacity to support fetal growth in sheep.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

Establishment of Repertoire of Placentome-Associated MicroRNAs and Their Appearance in Blood Plasma Could Identify Early Establishment of Pregnancy in Buffalo (Bubalus bubalis).

Precise early pregnancy diagnosis in dairy animals is of utmost importance for an efficient dairy production system. Not detecting a dairy animal pregnant sufficiently early after the breeding results to extending the unproductive time of their milk production cycle and causes substantial economic loss for a dairy producer. At present, the most conventional and authentic pregnancy confirmation practice in cows and buffaloes is rectal palpation of the reproductive organs at Days 35–40 after insemination, which sometime leads to considering an animal as false pregnant. Other alternative methods available for early pregnancy diagnosis lack either accuracy or reproducibility or require elaborate instrumentation and laboratory setup not feasible to practice at farmers’ doorstep. The present study was aimed at establishment of the microRNA (miRNA) repertoire of the placentome in buffaloes, which could capture the event of the cross talk between a growing embryo and a dam, through fetal cotyledons and maternal caruncles, and thus could hint at the early pregnancy establishment event in ruminants. Total RNA was isolated from buffalo placentome tissues during early stages of pregnancy (at Day < 25 and Days 30–35), and global small RNA analysis was performed by using Illumina single-end read chemistry and Bubalus bubalis genome. A total of 2,199 miRNAs comprising 1,620 conserved and 579 non-conserved miRNAs were identified. Stringent functional miRNA selection criteria could predict 20 miRNAs worth evaluating for their abundance in the plasma of pregnant, non-pregnant, cyclic non-bred, and non-cyclic prepubertal animals. Eight of them (viz., miR-195-5p, miR-708-3p, miR-379-5p, miR-XX1, miR-XX2, miR-130a-3p, miR-200a-3p, and miR-27) displayed typical abundance patterns in the plasma samples of the animals on Day 19 as well as Day 25 post-insemination, thus making them ambiguous candidates for early pregnancy detection. Similarly, higher abundance of miR-200a-3p and miR130a-3p in non-pregnant animals was indicative of their utility for detecting the animals as not pregnant. Most interestingly, miR-XX1 and miR-XX2 were very characteristically abundant only in pregnant animals. In silico target prediction analysis confirmed that these two miRNAs are important regulators of cyclooxygenase-2 (COX-2) and cell adhesion molecule-2 (CADM-2), both of which play a significant role in the implantation process during feto-maternal cross talk. We interpret that circulatory miR-XX1 and miR-XX2 in blood plasma could be the potential biomarkers for early pregnancy detection in buffaloes.

PSII-7 Uterine artery hemodynamics and placentome quantification during maternal nutrient restriction in beef cattle

Abstract Proper fetal nutrition via adequate uteroplacental blood flow is critical for maximizing fetal development and reducing mortality. However, our understanding of adaptations during maternal nutrient restriction is incomplete. The purpose of this study was to determine the effect of nutrient restriction during mid to late gestation on utero-placental hemodynamics and placentome morphometrics in gestating beef cattle. Animals (n = 10) were randomly assigned one of 2 treatments: Control (CON; 100% NRC Requirements) vs Restricted Feed (NR; 60% of NRC Requirements) from 140 to 240 days of gestation. Hemodynamic parameters were collected by Doppler ultrasonography 1 week prior to slaughter from the ipsilateral and contralateral uterine arteries, after which, total uterine artery blood flow was calculated. Six animals (n = 3 per treatment) were slaughtered on day 240 of gestation (Group 1) and the remaining 4 animals were slaughtered on 265 ± 15 days (Group 2). Placentome tissues were harvested for weight and number determination. Placentome number decreased (P = 0.01) in nutrient restricted (49 ± 6) versus control (83 ± 6) fed dams. There was an interaction between treatment and group in which ipsilateral diameter increased (P &amp;lt; 0.05) in group 2 NR animals versus group 2 CON animals while there was no difference between NR and CON in group 1. There was no difference (P = 0.30) in total uterine artery blood flow in fetal weight, which averaged 212 ± 37 mL/min*kg in nutrient restricted versus 153 ± 34 mL/min*kg in control fed dams. In summary, these data indicate limited alterations in uterine artery blood flow and fetal weight in nutrient restricted dams during the last half of gestation. The decrease in placentome number with no difference in placentome weight could indicate larger placentomes in nutrient restricted dams were able to compensate for the reduction in total number.

Morphological Characterization of Basally Located Uninucleate Trophoblast Cells as Precursors of Bovine Binucleate Trophoblast Giant Cells

Binucleate trophoblast giant cells (TGCs) are one characteristic feature of the ruminant placenta. In cows, the frequency of TGCs remains constant for most of the duration of pregnancy. As TGCs are depleted by their fusion with uterine epithelial cells, they need to be constantly formed. It is still unclear whether they develop from stem cells within the trophectoderm or whether they can arise from any uninucleate trophoblast cell (UTC). Within the latter, generally accepted theory, a basally located uninucleate cell (BUC) without contact to the feto-maternal interface would represent a transient cell between a UTC and a TGC. So far, no evidence for the existence of such transient cells or for the presence of stem cells has been shown. The aim of the present study is to morphologically characterize the early stages of TGC development. Placentomal tissue of 6 pregnant cows from different gestational stages (gestational days 51–214) was examined for BUCs, UTCs, and TGCs either in serial sections (light and transmission electron microscopy, TEM, n = 3), in single sections (TEM, n = 2), or by serial block face-scanning electron microscopy (n = 1). These investigations revealed the occurrence of BUCs, as well as young TGCs showing contact with the basement membrane (BM), but without apical contact to the feto-maternal interface. The study morphologically defines these 2 cell types as early stages of TGC development and shows that binucleation of TGCs can precede detachment from the BM.

Bovine placentomal heparanase and syndecan expression is related to placental maturation

IntroductionImpaired placental maturation has been associated with retention of fetal membranes, which is a major reproductive disease in cattle. This maturation includes alterations in all tissue compartments of the placenta, specifically of epithelial and stroma cells and extracellular matrix. It is believed to be controlled by hormones, adhesion molecules and proteolytic enzymes. To investigate if the proteolytic enzyme heparanase and its substrates, the syndecans (SDCs) could be involved in the release of fetal membranes, their expression in bovine placentomes was analyzed. MethodsPlacentomes were taken from gestational day 35 until term, directly after spontaneous parturition, after preterm caesarean section, and after chemically induced parturition. Heparanase and SDCs were localized by immunohistochemistry and the respective mRNAs were quantified by qRT-PCR. Heparanase expression was additionally quantified by Western blot. ResultsHeparanase, SDC1 and SDC4 displayed significant changes in expression and localization depending on gestational progress and mode of parturition. All three proteins showed an expression at the end of gestation, together with an altered, predominant localization in fetal and maternal epithelia. After physiological parturition, the placentomal tissue stained weaker for all syndecans. This change in staining pattern could not be observed after induced preterm parturition. SDC2 expression did not change during the course of gestation. DiscussionThe changing placental expression patterns of heparanase, SDC1 and SDC4 indicate that these molecules might be involved in fetomaternal communication and placental maturation in cattle. The matrix degrading properties of heparanase could assist in a timely reduction of fetomaternal adhesion and thus promote separation of the membranes after parturition.

Hot topic: 16S rRNA gene sequencing reveals the microbiome of the virgin and pregnant bovine uterus

We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome by 16S rRNA gene sequencing of the virgin and pregnant bovine uterus. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyledonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using aseptic techniques. The DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced using Illumina MiSeq technology (Illumina Inc., San Diego, CA). Operational taxonomic units (OTU) were generated from the sequences using Qiime v1.8 software, and taxonomy was assigned using the Greengenes database. The effect of tissue on the microbial composition within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons of 16S rRNA gene were generated in all samples, supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average, 53, 199, 380, 382, 525, and 13,589 reads annotated as 16, 35, 43, 63, 48, and 176 OTU in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uterus of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria, and they accounted for approximately 40, 35, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue (external surface of the cervix) was significantly different from that of the amniotic fluid, intercotyledonary placenta, and placentome tissues. Interestingly, many bacterial species associated with postpartum uterine disease (i.e., Trueperella spp., Acinetobacter spp., Fusobacteria spp., Proteus spp., Prevotella spp., and Peptostreptococcus spp.) were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA gene sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity, and that pregnancies are established and maintained in the presence of a uterine microbiome.

Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells

Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate “BNC”. Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering.

Is assisted reproductive technology associated with findings of placenta in monochorionic-diamniotic twins?

Alteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after somatic cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies.Genotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin, Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and somatic cloning pregnancies.The expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta.In bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes.These observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy.

Spatial and temporal changes of Decorin, Type I collagen and Fibronectin expression in normal and clone bovine placenta

IntroductionAlteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after somatic cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies. MethodsGenotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin, Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and somatic cloning pregnancies. ResultsThe expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta. DiscussionIn bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes. ConclusionsThese observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy.

Androgen receptor is widely expressed in bovine placentomes and up-regulated during differentiation of bovine trophoblast giant cells

ObjectivesIn cattle no biological role has been definitely identified for placental estrogens and progesterone. However, in the bovine trophoblast androgens may also be produced and have local effects. Thus, the aims of this study were to identify androgen receptor (AR) expressing cells and to monitor testosterone tissue concentrations in bovine placentomes throughout gestation. MethodsPlacental AR expression was characterized at the mRNA and protein level applying conventional and real-time RT-qPCR, western blot and immunohistochemistry. Testosterone was measured by radioimmunoassay. ResultsAR-mRNA was qualitatively detected from day 50 of gestation until term. Mean relative gene expression levels were constant between day 100 and late gestation. A slight non-significant increase was observed in the prepartal period. With immunohistochemistry distinct nuclear signals were predominantly observed in invasive trophoblast giant cells (TGC) from day 80 until term. In mature TGC of the trophoblast, immature TGC and uninucleated trophoblast cells, stromal cells of the chorionic villi, caruncular epithelial and stromal cells immunoreactive score values were low at early and midgestation but increased significantly (p < 0.01) during late gestation and remained high until parturition. With western blot in placentomal tissue a specific band of approximately 110 kDa was detected as it was the case in epididymis used as a positive control. Testosterone concentrations increased from 0.70 ± 0.29 pmol/g wet tissue between days 60–220 to 4.22 ± 1.29 pmol/g during late gestation (p < 0.001). DiscussionThe results are consistent with androgens as active products of bovine placental steroidogenesis. The substantial up-regulation of AR expression during TGC differentiation suggests that androgens may be related to this process.

Pathogenesis of retained foetal membranes (RFM) is influenced by apoptosis and degradation of extracellular matrix in bovine placentomal tissue

Pathogenesis of retained foetal membranes (RFM) is influenced by apoptosis and degradation of extracellular matrix in bovine placentomal tissue

Developmental changes in antioxidant enzymatic defences against oxidative stress in sheep placentomes

Early pregnancy is susceptible to oxidative stress, and thus characterisation of antioxidant systems and pro- and anti-apoptotic pathways would improve understanding of placental development and function. We aimed, therefore, to determine the activities of the antioxidant enzymes, copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), catalase (CAT), glutathione (GSH) peroxidase (GPX) and GSH reductase (GSR); and to quantify the expression of BAX and MCL1 proteins in relation to the developmental changes in antioxidant defences in sheep placentomes sampled on days 35, 55 and 80 of pregnancy. Placentome progesterone content was analyzed to determine steroidogenic capacity. Malondialdehyde (MDA) and protein carbonyl were quantified in placentomes as biomarkers of lipid peroxidation and protein damage respectively. Placentome tissues demonstrated significantly increased content of progesterone and MDA at day 80 of pregnancy and protein carbonyl as early as day 50 of pregnancy. Progesterone and MDA contents were not different between days 35 and 55 of pregnancy. While SOD1 and CAT activities did not alter significantly, SOD2 activity decreased from days 35 to 55. GPX activity increased from days 35 to 55 and increased further to day 80 of pregnancy. GSR activity increased from days 35 to 55 of pregnancy. BAX protein expression decreased, while MCL1 increased from days 35 to 55 and 80 of pregnancy. The increased GPX activity was associated with a decrease in the BAX/MCL1 protein expression ratio. Changes in the antioxidant enzymatic defences could be a part of placentome adaptation to reactive oxygen species-induced oxidative stress at specific early developmental stages of pregnancy.

280 INDISTINGUISHABLE TRANSCRIPTIONAL PROFILES BETWEEN IN VIVO- AND IN VITRO-PRODUCED BOVINE FETUSES

During the past several decades, in vitro fertilization (IVF) has been increasingly used in animal production and human infertility treatment. In vitro production (IVP) has been shown to cause reduced developmental competence, aberrant gene expression, and developmental abnormalities. Our objective was to determine how in vitro procedures influence global gene expression during fetal development. To this end, we analyzed the gene expression profiles of liver and placentome tissue samples (n = 18) from IVP and in vivo-derived fetuses at Days 90 and 180 of gestation (n = 5 IVP and n = 4 in vivo-derived pregnancies for each day of gestation). Standard in vitro maturation and fertilization protocols were employed. Putative zygotes were co-cultured with bovine oviductal epithelial cells to the blastocyst stage. In vivo embryos were collected 7 days after AI by nonsurgical uterine flushing. Blastocyst-stage IVP and in vivo embryos were transferred to synchronized recipients and monitored until collection at Day 90 or 180. The pregnancy rate at Day 90 was 12% and 27% for IVP and in vivo pregnancies, respectively (Bertolini et al. 2004 Reproduction 128, 341-354). To conduct expression profiling, total RNA from each tissue sample and a standard reference was indirectly labeled with Cy3 and Cy5, respectively, and hybridized in duplicate to custom, bovine 13 K oligonucleotide microarrays. After Loess normalization, a two-way (origin and day) ANOVA model (GeneSpring 7.3.1) was used to identify differentially expressed genes in each tissue. The P-values were adjusted for multiple comparisons using a 5% false discovery rate (FDR). The expression of 11 candidate genes was confirmed independently by quantitative RT-PCR. Surprisingly, in both the liver and placentome tissues, no differential gene expression was detected between the IVP and in vivo fetuses at Day 90 and 180. This was observed even when the FDR was relaxed to 10% and 20%. A total of 879 genes (523 genes ≥ 1.5-fold) were differentially expressed during liver development from 90 to 180 days of gestation. Conversely, no differential gene expression was detected in the placentomes during this developmental period. Our findings show that during early and mid gestation, surviving IVP fetuses had normal patterns of gene expression. It is possible that embryos with less severe perturbations may survive with their gene expression normalized as development proceeds. Additionally, initial changes in gene expression caused by IVP may affect subsequent development, but do not necessarily persist throughout gestation. Present addresses: L. Jiang, Columbia University, New York, NY, USA; S. L. Marjani, Yale University, New Haven, CT, USA; M. Bertolini, University of Fortaleza, CE, Brazil. This work was supported by USDA grants to X.Y, H.A.L., and X.C T.

Effect of Periconceptional Undernutrition in Sheep on Late Gestation Expression of mRNA and Protein From Genes Involved in Fetal Adrenal Steroidogenesis and Placental Prostaglandin Production

In sheep, maternal periconceptional undernutrition precociously activates fetal hypothalamic-pituitary-adrenal function in a high proportion of animals, resulting in preterm birth. We investigated whether the effects of periconceptional undernutrition were mediated through genes encoding enzymes involved in adrenal steroidogenesis and in placental production and metabolism of steroids and prostaglandins. Singleton-bearing ewes were fed ad libitum (control, N) throughout gestation or undernourished from 60 days before until 30 days after mating. Fetal adrenal and placentome tissues were collected on gestational day 131. Fetal adrenal P450C17 protein expression was significantly increased with undernutrition. Placental prostaglandin endoperoxide synthase-2 expression was not different between groups. In undernourished pregnancies, there were significant positive correlations between fetal plasma adrenocorticotrophic hormone and cortisol concentrations and between fetal plasma adrenocorticotrophic hormone concentrations and placental prostaglandin endoperoxide synthase-2 messenger RNA (mRNA) and protein. These studies suggest that periconceptional undernutrition affects fetal adrenal P450C17, consistent with an elevation in plasma cortisol, and this occurs prior to activation of placental prostaglandin endoperoxide synthase-2 expression at gestational day 131.

DNA methylation and chromatin accessibility of the proximal Cyp19 promoter region 1.5/2 correlate with expression levels in sheep placentomes

Placental oestrogens play an important role as local regulators of placental growth and differentiation during gestation, and toward term they are also involved in the preparation of parturition. They are synthesized within the fetal cotyledons of placentomes by aromatase cytochrome P450 (P450arom; EC 1.14.14.1), the product of the Cyp 19 gene. The first step of regulation of P450arom expression, and hence enzyme activity and oestrogen production, takes place at the level of Cyp 19 transcription, which is driven by a proximal promoter region, P1.5/2, in the sheep placenta. The aim of the present study was to find out if different Cyp 19 expression levels, which previously had been observed in ovine placentome tissues, correlate with the tissue-specific chromatin structure of the promoter. To this end, we investigated the chromatin structure across the P1.5/2 region in caruncles and cotyledons from 100 and 125 days pregnant ewes, and in term placentae, respectively, by analyzing the DNA methylation and the accessibility to restriction digestion. Our data show that: (1) cotyledonal DNA was significantly lower methylated than caruncular DNA; (2) methylation of cotyledonal DNA was low at 100 and 125 days of pregnancy, and increased to a significant higher level in term placentae; and (3) concurrently, cotyledonal chromatin became inaccessible to restriction digestion at term of gestation. The results imply that DNA methylation and chromatin accessibility of the P1.5/2 promoter region correlate with expression levels of the Cyp 19 gene.

Binucleate trophoblast giant cells in the water buffalo (Bubalus bubalis) placenta

The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.

Functional Significance and Cortisol Dependence of the Gross Morphology of Ovine Placentomes During Late Gestation1

The gross morphological appearance of ovine placentomes is known to alter in response to adverse intrauterine conditions that increase fetal cortisol exposure. The direct effects of fetal cortisol on the placentome morphology, however, remain unknown, nor is the functional significance of the different placentome types clear. The present study investigated the gross morphology of ovine placentomes in relation to placental nutrient delivery to sheep fetuses during late gestation and after experimental manipulation of the fetal cortisol concentration. As fetal cortisol levels rose naturally toward term, a significant decrease was observed in the proportion of the D-type placentomes that had the hemophagous zone everted over the bulk of the placentomal tissue. When the prepartum cortisol surge was prevented by fetal adrenalectomy, there were proportionately more everted C- and D-type placentomes and fewer A-type placentomes with the hemophagous zone inverted into the placentome compared with those of intact fetuses at term. Raising cortisol concentrations by infusion before term reduced the incidence of D-type placentomes and lowered the proportion of individually tagged placentomes that became more everted during the 10- to 15-day period between tagging and delivery. Cortisol, therefore, appears to prevent hemophagous zone eversion in ovine placentomes during late gestation. The distribution of placentome types appeared to have no effect on the net rates of placental delivery of glucose and oxygen to the fetus under normal conditions. When fetal cortisol levels were raised by exogenous infusion, however, placental delivery of glucose, but not oxygen, to the fetus, measured as umbilical uptake, was reduced to a greater extent in fetuses with a higher proportion of C- and D-type placentomes. The gross morphology of the ovine placentomes is, therefore, determined, at least in part, by the fetal cortisol concentration and may influence placental nutrient transfer when fetal cortisol concentrations are high during late gestation. These findings have important implications for the placental control of fetal growth and development, particularly during adverse intrauterine conditions.

Effects of implantation and early pregnancy on the expression of cytokines and vascular surface molecules in the sheep endometrium

Successful implantation is a highly coordinated process involving changes in cytokines, adhesion molecules, hormones, enzymes and growth factors. This study examines the expression of key cytokines and vascular surface molecules in the pregnant uterus of sheep around the time of implantation. Uterine tissues and uterine washings were collected from non-pregnant and pregnant sheep at 17–19 days post-coitus (dpc), 26–27 and 34–36 dpc. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of caruncular/placentomal tissues revealed that cytokines IL-2, IL-4 and IL-8, which were not detected in non-pregnant uterus, were induced more strongly at 26–27 dpc than at other stages of pregnancy tested. Cytokines LIF, IL-6, IL-10, TNF-α were also most highly expressed at 26–27 dpc, expression of them being lower at other time-points during early pregnancy and non-pregnancy. The cytokines IL-1β, IFN-γ and TGF-β were expressed in all non-pregnant and pregnant tissues examined. Enzyme-linked immunosorbent assay (ELISA) performed on uterine washings clearly detected the presence of IL-1α protein at 26–27 and 34–36 dpc. Immunohistochemistry revealed that expression of vascular adhesion molecule VCAM-1 in endometrial endothelium was strongly induced at 26–27 dpc in the pregnant endometrium. Expression of CD5 on vascular endothelium was not induced in placentomal tissues until 26–27 dpc and was further increased by 34–36 dpc. These results demonstrate a dynamic change in a wide range of cytokines during early stages of pregnancy, with a critical period around 26–27 dpc. In addition, at 26–27 dpc, expression of the surface/adhesion molecules, CD5 and VCAM-1, is induced on vascular endothelium of the sheep endometrium, possibly as a direct consequence of the changed cytokine environment, and may be involved in directing the changes in leucocyte migration observed during pregnancy.