Uninucleate Trophoblast Cells Research Articles

Placentation and hormonal maintenance of pregnancy in the impala (Aepyceros melampus)

IntroductionThe impala is a widely distributed African ungulate. Detailed studies of the placenta and ovaries in impala undertaken in the 1970s did not address the endocrine functions of the placenta. MethodsThe uteri of 25 pregnant impala estimated to be between 49 and 113 days of the 190 day gestation were examined grossly, histologically and immunohistochemically. ResultsA single corpus luteum was present in either maternal ovary but the conceptus was always situated in the right uterine horn. The fetal membranes extended to the tips of both uterine horns. The amnion was in intimate contact with, but not fused to, the allantochorion. Placentation was typically ruminant with fetal macrocotyledons attached to the rows of maternal caruncles. The fetal villi were highly branched, especially in the centre of each placentome where the attenuated maternal epithelium lining the placental crypts was absent in some places. Both the corpus luteum and the uninucleate trophoblast cells of the interplacentomal allantochorion stained strongly for 3-β hydroxysteroid dehydrogenase, and progestagen concentrations in allantoic and amniotic fluids increased significantly as gestation progressed, with a tendency to do likewise in maternal serum. Binucleate trophoblast cells stained positively for bovine placental lactogen, but neither the placenta nor the maternal corpus luteum showed evidence of oestrogen synthesis. DiscussionDespite exhibiting the same basic type of placentation, both the gross and histological structure of the impala placenta, along with its immunohistochemical properties, demonstrates that great variation exists across ruminant placentas.

Trophoblast cell differentiation in the bovine placenta: differentially expressed genes between uninucleate trophoblast cells and trophoblast giant cells are involved in the composition and remodeling of the extracellular matrix and O-glycan biosynthesis

BackgroundIn the bovine placenta, intimate fetomaternal contacts are restricted to discrete placentomes. Here, widely branched fetal chorionic villi interdigitate with corresponding maternal caruncular crypts. The fetal trophoblast epithelium covering the chorionic villi consists of approximately 80% uninucleate trophoblast cells (UTCs) and 20% binuclear trophoblast giant cells (TGCs). The weakly invasive TGCs migrate toward the caruncle epithelium and eventually fuse with individual epithelial cells to form short-lived fetomaternal hybrid cells. In this way, molecules of fetal origin are transported across the placental barrier and released into the maternal compartment. The UTC/TGC ratio in the trophoblast remains almost constant because approximately as many new TGCs are produced from UTCs as are consumed by the fusions. The process of developing TGCs from UTCs was insufficiently understood. Therefore, we aimed to detect differentially expressed genes (DEGs) between UTCs and TGCs and identify molecular functions and biological processes regulated by DEGs.ResultsWe analyzed gene expression patterns in virtually pure UTC and TGC isolates using gene arrays and detected 3193 DEGs (p < 0.05; fold change values < − 1.5 or > 1.5). Of these DEGs, 1711 (53.6%) were upregulated in TGCs and 1482 (46.4%) downregulated. Gene Ontology (GO) analyses revealed that molecular functions and biological processes regulated by DEGs are related to the extracellular matrix (ECM) and its interactions with cellular receptors, cell migration and signal transduction. Furthermore, there was some evidence that O-glycan biosynthesis in TGCs may produce sialylated short-chain O-glycans (Tn antigen, core 1 O-glycans), while the synthesis of other O-glycan core structures required for the formation of complex (i.e., branched and long-chain) O-glycans appears to be decreased in TGCs.ConclusionThe differentiation of UTCs into TGCs particularly regulates genes that enable trophoblast cells to interact with their environment. Significant differences between UTCs and TGCs in ECM composition indicate reduced anchoring of TGCs in the surrounding matrix, which might contribute to their migration and their weakly invasive interaction with the maternal endometrium. Furthermore, increased expression of sialylated short chain O-glycans by TGCs could facilitate the modulation of maternal immune tolerance.

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.

Estrogen-specific sulfotransferase (SULT1E1) in bovine placentomes: inverse levels of mRNA and protein in uninucleated trophoblast cells and trophoblast giant cells.

The bovine trophoblast produces significant amounts of estrogens. In maternal and fetal blood, estrogens occur predominantly in sulfonated forms, which are unable to bind to estrogen receptors (ESRs). However, estrogens may act as local factors in ESR-positive trophoblast cells or in the adjacent caruncular epithelium, which in addition to ESR highly expresses steroid sulfatase. Estrogen sulfonation is catalyzed by the cytosolic enzyme SULT1E1. Previous studies clearly indicated the trophoblast as the primary site of estrogen sulfonation. However, investigations into the cellular localization of SULT1E1 yielded conflicting results. In situ hybridization studies detected SULT1E1 mRNA only in trophoblast giant cells (TGCs), whereas in immunohistochemical experiments the SULT1E1 protein was virtually restricted to uninucleated trophoblast cells (UTCs). The aim of this work was to resolve this conflict by analyzing SULT1E1 expression in isolated UTCs and TGCs. Highly enriched pools of UTCs and TGCs were obtained from four bovine placentas (Days 118-130 of gestation) using an optimized fluorescence-activated cell sorting procedure. UTC and TGC pools were analyzed by quantitative RT-PCR and Western blot experiments to measure the amounts of SULT1E1 transcript and protein, respectively. In contrast to previously published results, both SULT1E1 transcript and SULT1E1 protein were clearly present in the UTC and TGC pools. However, some evidence indicated a higher transcript concentration in TGCs and a higher amount of protein in UTCs. Thus, our results resolve the conflicting results on the localization of SULT1E1 from earlier studies and suggest that posttranscriptional mechanisms play an important role in the control of SULT1E1 expression during TGC differentiation.

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.

Expression of steroidogenic proteins in bovine placenta during the first half of gestation

The aim of the present study was to determine the occurrence and localisation of the principal steroidogenic proteins in bovine placenta from Day 50 to Day 120 of pregnancy. Immunohistochemistry revealed that, at all stages investigated, bovine steroidogenic acute regulatory protein (StAR), cytochrome P45011A1 and hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1 proteins were found principally at the fetomaternal interdigitations: the chorionic villus and maternal septum. Moreover, caruncular epithelial cells and uninucleate trophoblast cells were the principal cells detected that were positive for the three markers. Western blot analysis showed that only caruncular tissue expressed all three steroidogenic markers; in contrast, cotyledons only expressed StAR and cytochrome P45011A1. Immunoblot results showed a complementary pattern of StAR and cytochrome P45011A1 expression between caruncles and cotyledons at different stages. These observations suggest that, in early pregnancy, the maternal compartment contributes significantly to bovine placental steroidogenesis, particularly for the synthesis of progesterone. Furthermore, the variation in StAR and cytochrome P45011A1 expression between caruncular and cotyledonary tissues across gestation suggests that placental steroidogenesis requires cell-to-cell communication between maternal and fetal cells.

Expression of SULT1E1 protein in bovine placentomes: Evidence for localization in uninucleated trophoblast cells

The bovine placenta produces considerable amounts of pregnancy-associated estrogens, predominantly estrone sulfate, which does not interact with classical nuclear estrogen receptors. Thus, bovine placental estrogens may rather act as local regulatory factors than as hormones in a classical sense. To obtain information on the local availability of free estrogens in bovine placentomes, the expression pattern of the estrogen specific sulfotransferase SULT1E1 was characterized using antisera against the bovine and human enzyme, respectively. In western blot both antisera detected a band of the expected molecular weight (approx. 33 kDa) in placentomes and fetal liver. In immunohistochemistry the two antisera yielded virtually identical results. In placentomes distinct staining was restricted to the cytosol of uninucleated trophoblast cells (UTC). The staining pattern in UTC, immature and mature trophoblast giant cells (TGC) is consistent with a down-regulation of SULT1E1 during TGC differentiation. During early and midgestation staining intensity in UTC was higher in the trophoblast covering the chorionic plate and basal parts of stem villi compared to other regions of the villous tree, whereas during late gestation and at parturition an almost ubiquitous distinct staining of UTC was found. Correspondingly, relative SULT1E1-mRNA levels as measured by quantitative real-time RT-PCR increased significantly during late gestation ( p = 0.0043). Comparative measurements showed that SULT1E1-mRNA levels in adult bovine organs were considerably lower compared to placentomes and fetal liver. The results suggest that the activities of free estrogens produced in bovine TGC are curtailed by SULT1E1 expressed in UTC and in fetal liver. Bovine pregnancy-associated estrogens produced in trophoblast giant cells are predominantly sulfonated in uninucleated trophoblast cells.

Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention

Matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) are balancing extracellular matrix (ECM) formation and degradation. The latter is believed to be an important aspect for the detachment of fetal membranes postpartum when loosening the feto-maternal connection which is a prerequisite to avoid placental retention a common disease in cows leading to considerable economic loss. Membrane-type (MT) MMPs have been suggested as potential activators controlling ECM remodelling. In particular, MT1-MMP (MMP-14) is able to degrade ECM substrates and activate MMP-2 through binding TIMP-2 at the cell surface. Since the connection between the trophoblast and the maternal caruncular epithelium is supported by integrin receptors bound to ECM, we hypothesize that impaired modulation of the ECM by TIMPs/MMPs participates in the aetiology of bovine retained fetal membranes. To analyse this involvement, placentomes were collected from cows after term parturition and timely release of fetal membranes (n = 4) and cows with retained fetal membranes after various treatments for the induction of parturition using progesterone antagonist (aglepristone), PGF2α analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TIMP-2 was examined by real-time-PCR, immunohistochemistry, Western blot and zymography. The relative mRNA expression levels of MMP-14 remained unchanged, while the expression levels of TIMP-2 and MMP-2 partly increased in animals with induced parturition and retention of fetal membranes compared to animals without placental retention. MMP-14 protein was expressed in cells of the uninucleated trophoblast, the fetal mesenchyme and maternal stroma. TIMP-2 was present exclusively in trophoblast giant cells, while MMP-2 could be detected in uninucleated trophoblast cells and the fetal mesenchyme. The presence of the activated enzyme was confirmed by zymography. In conclusion, MMP-14, MMP-2 and TIMP-2 are co-localized in the fetal compartment and therefore could influence the timely release of fetal membranes in cattle.

EGF stimulates proliferation in the bovine placental trophoblast cell line F3 via Ras and MAPK

In the bovine placenta, multinucleate trophoblast giant cells (TGC), evolving from uninucleate trophoblast cells, are crucial for feto-maternal interaction as they show endocrine activity and the ability to migrate and fuse with caruncular epithelial cells. In contrast to caruncular epithelial cells, the isolation and culture of bovine trophoblast cells is complicated because they cease to express their specific products, like placental lactogen (PL), during prolonged culture. In the present study, we aimed to establish a bovine cotyledonary trophoblast cell line targeting our long term goal to develop an in vitro model for the bovine placenta. Therefore, the functional activity of important signalling pathways was tested. Primary trophoblast cells were isolated from a bovine cotyledon of a male fetus and successfully subcultured and cryopreserved. The obtained cell line, termed F3, showed epithelial morphology and characteristic binuclear giant cells in small numbers through all passages. The trophoblastic origin of F3 cells was verified by amplification of a Y-chromosome specific DNA-sequence and the presence of PL mRNA. Immunofluorescence demonstrated that F3 cells were continuously positive for zonula occludens-2 (ZO-2), cytokeratin and vimentin, whereas they expressed the TGC specific marker PL only in the first two passages. F3 cell growth was accelerated in medium supplied with epidermal growth factor (EGF). EGF-stimulated proliferation was mediated through activation of Ras and the phosphorylation of mitogen-activated protein kinase (MAPK) 42 and 44. In conclusion, the F3 cell line shows several in vivo characteristics of bovine cotyledonary trophoblast cells. The response to EGF stimulation indicates that EGF plays a role during bovine placentation, and illustrated that F3 cells may provide a valuable tool for further mechanistic studies elucidating the feto-maternal interplay.

Placental Steroids in Cattle: Hormones, Placental Growth Factors or By-products of Trophoblast Giant Cell Differentiation?

The bovine placenta produces large amounts of steroids, mainly estrone (E1) and progesterone (P4). Specific features of bovine placental steroidogenesis are 1) the expression of all enzymes needed for the production of estrogens from cholesterol in the trophoblast 2) an only marginal and temporal contribution to peripheral maternal P4 levels restricted to a period between approx. days 150 - 240 of gestation 3) the predominance of sulfoconjugated over free E1 and 4) a complementary setting of steroidogenic enzymes in the two morphologically discriminable trophoblast cell types, the uninucleated trophoblast cells (UTC) and the trophoblast giant cells (TGC). In cattle so far no definite information is available on the specific biological roles of placental estrogens and P4. However, the detection of estrogen receptors and progesterone receptors in the placentomes suggests a role primarily as local regulators of caruncular growth, differentiation and functions. Inconsistent with a function as a caruncular growth factor is the strong evidence that in cattle placental estrogens enter the maternal compartment almost completely as estrone sulfate (E1S), which is not active at classical nuclear receptors. On the other hand, E1S may be converted locally to free active estrogens via the action of steroid sulfatase (StS), which has been detected in specific parts of the bovine caruncular epithelium. Alternatively or in addition, StS expression in the caruncular epithelium may serve the utilization of sulfated neutral steroid precursors (e.g. pregnenolone sulfate or cholesterol sulfate) supplied with maternal blood, thus providing free substrates for further metabolization in the adjacent trophoblast. The down-regulation of P450scc and P450c17 and the up-regulation of 3beta-HSD and aromatase during the differentiation of TGC from UTC in parallel with the up-regulation of ER beta and estrogen sulfotransferase in maturing TGC suggests a function of placental estrogens primarily as autoor intracrine regulators during this process and assigns to conjugated placental estrogens a role as inactivated by-products of TGC differentiation intended for excretion. Collectively, despite some evidence from recent studies for putative roles of placental steroids in cattle their exact functions in the bovine species remain still undefined.

Dynamics and genetics of PrPSc placental accumulation in sheep

Placentae from scrapie-affected ewes are an important source of contamination. This study confirmed that scrapie-incubating ewes bearing susceptible genotypes could produce both abnormal prion protein (PrPSc)-positive and -negative placentae, depending only on the PRP genotype of the fetus. The results also provided evidence indicating that scrapie-incubating ARR/VRQ ewes may be unable to accumulate prions in the placenta, whatever the genotype of their progeny. Multinucleated trophoblast cells appeared to play a key role in placental PrPSc accumulation. PrPSc accumulation began in syncytiotrophoblasts before disseminating to uninucleated trophoblasts. As these result from trophoblast/uterine epithelial cell fusion, syncytiotrophoblast cells expressed maternal and fetal PrPC, whilst uninucleated trophoblast cells only expressed fetal PrPC. In ARR/VRQ scrapie-infected ewes, expression of the ARR allele by syncytiotrophoblasts appeared to prevent initiation of PrPSc placental deposition. The absence of prions in affected ARR/VRQ sheep placentae reinforces strongly the interest in ARR selection for scrapie control.

The Pattern of Glucocorticoid and Estrogen Receptors May Explain Differences in Steroid Dependency of Intrauterine Prostaglandin Production at Parturition in Sheep

We have recently described two distinct pathways of intrauterine prostaglandin (PG) synthesis: a cortisol-dependent/estradiol-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an estradiol-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2(2alpha). We hypothesized that the differential effects of cortisol and estradiol on intrauterine PGH synthase-II (PGHS-II) expression and PG production may be because of the tissue specific expression of the glucocorticoid and estradiol receptors (GR and ER, respectively) within the intrauterine tissues. In addition, we suggest that these two pathways of PG production are linked through the expression of P450(C17hydroxylase) (P450(C17)) and subsequent increase in placental estradiol synthesis. To test the hypotheses, we infused singleton, chronically catheterized fetal sheep beginning at day 125 of gestation (term 147 to 150 days) with (1) cortisol (0.45 mg/mL; n = 5); (2) cortisol and 4-hydroxyandrostenedione, a P450(aromatase) inhibitor (4-OHA: 1.44 mg/h; n = 5); (3) saline (n = 5); or (4) saline and 4-OHA (n = 5). PGHS-II, ER alpha, ER beta, and GR alpha were localized using immunohistochemistry. ER alpha, ER beta, P450(C17), and GR alpha protein expressions were determined by Western blot analysis. Data were analyzed by analysis of variance (ANOVA) (P < or =.05). Fetal cortisol infusion in the presence or absence of a rise in placental estrogen synthesis increased placental expression of GR alpha; both PGHS-II and GR alpha localized to the uninucleate trophoblast cells of the placentome and were excluded from the maternal stroma and binucleate cells. Both forms of ER were excluded from the trophoblast tissue of the placentome. ER alpha, ER beta, and PGHS-II showed a similar pattern of distribution within the luminal epithelium of the endometrium; there were no alterations in the level of the ER in the presence of cortisol +/- 4-OHA. Placental P450(C17) protein expression was increased in the presence of a rise in fetal cortisol independent of changes in placental estrogen synthesis. We concluded that the differential effects of cortisol and estradiol on intrauterine PGHS-II expression and PG production may be due to the tissue-specific expression of the GR and ER within the intrauterine tissues. Glucocorticoid effects on trophoblast PG production may be mediated in a positive feed-forward manner. We further suggest that either cortisol or a cortisol-stimulated intermediate, like PGE2, increased P450(C17) expression, leading to a rise in placental estradiol synthesis and triggering maternal intrauterine tissue PG production.

Reciprocal expression of 17α-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis

No definitive information is yet available on the steroidogenic capacity of the two morphologically distinct cell types forming the bovine trophoblast, the uninucleated trophoblast cells (UTCs) and the trophoblast giant cells (TGCs). Hence, in order to localise 17alpha-hydroxylase-C17,20-lyase (P450c17) on a cellular level and to monitor its expression as a function of gestational age, placentomes from pregnant (days 80-284; n = 19), prepartal (days 273-282; 24-36 h prior to the onset of labour; n = 3) and parturient cows (n = 5) were immunostained for P450c17 using an antiserum against the recombinant bovine enzyme. At all stages investigated, P450c17 was exclusively found in the UTCs of chorionic villi (CV), where staining was ubiquitous between days 80 and 160, but was largely restricted to primary CV and the branching sites of secondary CV between days 160 and 240. Thereafter, a distinct ubiquitous staining reoccurred in the UTCs of all CV in late pregnant, prepartal and parturient animals. Using an antiserum against human aromatase cytochrome P450 (P450arom), specific cytoplasmic staining was observed in TGCs. In placentomes from pregnant cows, staining intensity was higher in mature compared with immature TGCs and was more pronounced in the trophoblast covering big stem villi compared with the trophoblast at other sites of the villous tree. In placentomes of a parturient cow, specific staining was only found in mature TGCs that survived the normal, but substantial, prepartal decline in TGC numbers. These results clearly showed that bovine UTCs and TGCs exhibit different steroidogenic capacities, constituting a 'two-cell' organisation for oestrogen synthesis. P450c17 expression appears to be quickly down-regulated and P450arom is up-regulated when UTCs enter the TGC differentiation pathway.

Expression of Cyclooxygenase-II (COX-II) and 20α-Hydroxysteroid Dehydrogenase (20α-HSD)/Prostaglandin F-synthase (PGFS) in Bovine Placentomes: Implications for the Initiation of Parturition in Cattle

The chain of events leading to prepartal luteolysis in cattle is not yet fully understood. Prostaglandin F 2α (PGF 2α) seems to be a major factor involved. However, only little information is available about the underlying regulatory mechanisms. Consequently, the expression of cyclooxygenase-II (COX-II) and 20α-hydroxysteroid dehydrogenase (20α-HSD), an enzyme recently shown to be most likely responsible for the production of luteolytic PGF 2α in the endometrium of cyclic cows, was investigated in bovine placentomes. Immunohistochemical methods were applied to placentomes from 17 pregnant cows between days 100 and 284, from three cows during the prepartal progesterone decrease (days 273–282) and from five parturient cows. COX-II was found in uninucleated trophoblast cells (UTC) from day 100 until parturition. However, between days 100 and 235 expression was only weak to moderate, focal and mainly restricted to the chorionic plate and adjacent basal parts of chorionic stem villi. In placentomes from a 270 and a 284 day pregnant cow, in which the prepartal decline of progesterone levels had not started yet, staining had substantially increased and extended to secondary and tertiary chorionic villi. In prepartal and parturient cows strong to intense staining was present in UTC all over the villous tree. Real time RT-PCR confirmed an extensive pre- and intrapartal rise of COX-II expression in bovine placentomes with a 70–100-fold increase of COX-II-mRNA levels. 20α-HSD/PGFS was widely expressed in UTC of chorionic villi. Like COX-II it was down-regulated in UTC differentiating into trophoblast giant cells. Immunostaining pattern did not change remarkably during the period under investigation, and 20α-HSD/PGFS-mRNA levels increased only 2.6-fold in the prepartal phase. Thus, in UTC PGF 2α may be produced via COX-II and 20α-HSD/PGFS, but only COX-II may be substantially involved in the control of a putative prepartal placentomal output of luteolytic PGs, whereas 20α-HSD/PGFS seems to be expressed in a more constitutive manner.

Estrogen Receptor β (ERβ) Is Expressed Differently From ERα in Bovine Placentomes

The functions of placental estrogens in cattle are still poorly understood. In order to investigate a putative role as local regulators of placental growth, differentiation and functions via estrogen receptor beta (ERbeta), the expression of this ER isoform was determined in bovine placentomes obtained from cows at midgestation (days 110-150; n=7), late gestation (days 180-280; n=9), from prepartal cows (sampling immediately after the prepartal decline in maternal progesterone blood levels became obvious; n=3) and from cows at normal term (n=5) on the protein and mRNA level. By means of immunohistochemistry using a monoclonal antibody against human ERbeta (clone PPG5/10), nuclear signals were found in numerous cell types of the fetal (cotyledon) and maternal (caruncle) component of the placentomes with highest intensities in mature trophoblast giant cells (TGC), fetal and maternal vascular cells and caruncular stromal cells, in which the percentage of positive cells increased from 58.9+/-2.3 and 59.1+/-3.3 at mid- and late gestation to 65.1+/-4.9 and 69.4+/-4.2 in prepartal and parturient cows, resp. (p<0.0001). Clear staining of uninucleated trophoblast cells was only observed in prepartal and parturient animals. Conventional RT-PCR confirmed ERbeta expression in caruncles and cotyledons at all phases under investigation. With semi-quantitative real-time RT-PCR higher levels of ERbeta-specific mRNA were measured in cotyledons compared to caruncles (p<0.01). The results show that in contrast to ERalpha, which is only expressed in the caruncle, ERbeta is also extensively expressed in the cotyledon and suggest a role of ERbeta primarily in TGC differentiation and placental angiogenesis and/or vascular functions.

Developmental changes in localization of steroid synthesis enzymes in camelid placenta

The uninucleate trophoblast of epitheliochorial camelid placenta produces multinucleate giant cells starting between day 30 and day 35 of pregnancy. The giant cells are found scattered along the trophoblast at similar frequency throughout gestation. Light microscope immunocytochemistry indicates that the four steroid synthesis enzymes (cholesterol side chain cleavage, 3beta-hydroxysteroid de-hydrogenase, 17alpha-hydroxylase and aromatase) are present in all uninucleate trophoblast cells between day 14 and day 30 of pregnancy, but are found only in the giant cells once they are established, and that this localization persists until term. The giant cells show massive amounts of smooth endoplasmic reticulum and numerous small mitochondria, again as has been shown in other steroid-producing cells. As progesterone from the corpus luteum is necessary throughout gestation in camels, the capacity for oestrogen production by the trophoblast presumably has an important role, but one limited mostly to the immediate vicinity of the fetomaternal interface.

Developmental changes in localization of steroid synthesis enzymes in camelid placenta.

The uninucleate trophoblast of epitheliochorial camelid placenta produces multinucleate giant cells starting between day 30 and day 35 of pregnancy. The giant cells are found scattered along the trophoblast at similar frequency throughout gestation. Light microscope immunocytochemistry indicates that the four steroid synthesis enzymes (cholesterol side chain cleavage, 3beta-hydroxysteroid de-hydrogenase, 17alpha-hydroxylase and aromatase) are present in all uninucleate trophoblast cells between day 14 and day 30 of pregnancy, but are found only in the giant cells once they are established, and that this localization persists until term. The giant cells show massive amounts of smooth endoplasmic reticulum and numerous small mitochondria, again as has been shown in other steroid-producing cells. As progesterone from the corpus luteum is necessary throughout gestation in camels, the capacity for oestrogen production by the trophoblast presumably has an important role, but one limited mostly to the immediate vicinity of the fetomaternal interface.

Increased expression of major histocompatibility complex (MHC) class I transplantation antigens in bovine trophoblast cells before fusion with maternal cells

The mammalian fetus is potentially at risk from maternal immune attack because it can express paternally inherited polymorphic antigens, including those encoded by the major histocompatibility complex (MHC). The aim of this study was to investigate in more detail MHC class I upregulation by binucleate trophoblast cells in the bovine placenta. A method was developed to isolate binucleate cells by enzymatic disaggregation and density gradient centrifugation of bovine placental cotyledons. In cytospin preparations, 25-30% of purified binucleate cells stained positively with antibodies that recognize bovine MHC class I. The same antibodies were used to immunoprecipitate radiolabelled class I molecules from lysates of binucleate cells and fetal peripheral blood mononuclear cells. The protein species isolated from the two types of cell were similar in size and degree of glycosylation. PCR amplification of cDNA generated from binucleate cells and subsequent sequence analysis demonstrated transcription of MHC class I mRNA species similar to those found in fetal peripheral blood mononuclear cells, and there was no evidence of genetic imprinting of paternally inherited alleles. These results indicate that binucleate cells upregulate expression of MHC class I as they differentiate from MHC-negative uninucleate trophoblast cells. This finding has important implications for the immunological status of the fetus, as binucleate trophoblast cells are destined to cross to the maternal side of the placenta where they fuse with maternal cells. The immunological function of the resulting antigenically mixed fetomaternal hybrid minisyncytia is unknown.

Increase in 15-hydroxyprostaglandin dehydrogenase activity in the ovine placentome at parturition and effect of oestrogen

Type 1 NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is the key enzyme for metabolism of active primary prostaglandins to inactive forms in gestational tissues. The present study examined the activity and immunolocalization of PGDH in the ovine placenta, fetal membranes and uterus over the latter half of pregnancy, and its potential regulation by oestradiol. Placenta, fetal membranes and myometrium were collected from sheep with known single insemination dates on days 70, 100 and 135 of gestation and in active labour demonstrated by electromyographic activity. In addition, chronically catheterized fetuses were infused with oestradiol (100 microgram kg(-1) per 24 h) (n = 5) or saline vehicle into the fetus from day 120 to day 125. PGDH activity measured in placental extracts remained constant from day 70 to day 135 of gestation, and then significantly (P < 0.05) increased by 300% in active labour. Immunoreactive PGDH was localized in the placentome at all stages and was present predominantly in the fetal component of the placentome in uninucleate, but not in binucleate, trophoblast cells. Similarly, in the fetal membranes PGDH immuno-reactivity was present in the uninucleate trophoblast but not in the binucleate cells of the chorion. PGDH immunostaining was also present in the endometrial luminal epithelium, in the smooth muscle of the myometrium, and the glandular epithelium of the cervix. Infusion of oestradiol into the fetal circulation from day 120 to day 125 of gestation had no effect on placental PGDH activity. Immunohistochemistry was used to localize oestrogen receptor alpha in intrauterine tissues to investigate further the failure of oestradiol to increase PGDH activity. Immunoreactive oestrogen receptor alpha was not present in the fetal component of the placenta, although it was expressed in adjacent maternal-derived cells. It is concluded that (1) PGDH activity increases in late gestation; (2) PGDH is expressed in uninucleate trophoblast cells in the ovine placenta and fetal membranes, and also in the maternal endometrial epithelium and stroma, myometrium and cervix; (3) oestrogen receptor alpha is not expressed in fetal cells in the placenta or fetal membranes; and (4) the increase in PGDH activity is not regulated by oestradiol administered to the fetus.

Temporal Expression of Prostaglandin H Synthase Type 2 (PGHS-2) and P450C17in Ovine Placentomes with the Natural Onset of Labour

Labour in the sheep is preceded by increased tissue and plasma prostaglandin (PG) concentrations, and PGs could potentially contribute to the regulation of P450C17in placental tissue. Therefore, we determined the cellular localization and temporal pattern of expression of P450C17and prostaglandin H synthase type 2 (PGHS-2), the primary PG synthetic enzyme, in intrauterine tissues from three groups of pregnant ewes at term; animals not in labour (NIL;n=5; 140–145 days of gestation), animals in early labour (EL;n=6; 143–149 days) and animals in active labour (L;n=6; 145–149 days). Allocation of animals into the three groups was based on continuous monitoring and assessment of myometrial contractile activity (EMG) and changes in the intrauterine pressure (IUP). Levels of mRNA encoding PGHS-2 and P450C17 were determined by in situ hybridization. Localization and levels of immunoreactive (ir-) P450C17and ir-PGHS-2 protein were determined by immunohistochemistry and Western blotting. PGHS-2 mRNA and ir-PGHS-2 were already elevated in placentomes of NIL animals and did not increase further with the progression of labour, whereas P450C17 mRNA increased progressively with labour, and ir-P450C17 rose significantly only in animals in active labour. The rise in P450C17 expression corresponded temporally to a progressive increase in maternal plasma concentration of oestradiol. We suggest that the temporal relationship and subsequent co-localization of PGHS-2 and P450C17proteins in the uninucleate trophoblast cells of the placentomes are consistent with the possibility that placental PGs could act to enhance placental output of oestrogen leading to labour and delivery.