Placental Cotyledons Research Articles

Computational modeling of tumor metastases in bone – investigation of the vicious cycle concept in osteolytic bone lesions

The aim of this study was to examine the vasodilatory effects of parathyroid hormone-related protein (PTHrP) (1–34) and parathyroid hormone (PTH) (1–34) on the human fetal-placental circulation utilising an in vitro placental perfusion model. In all experiments, the vasculature of an isolated human placental cotyledon was pre-constricted with the throm☐ane A2 mimetic U46619. A single dose of PTHrP (1–34) or PTH (1–34) (1.7–300 n m) was then infused into the fetal-placental circulation of the cotyledon. In other experiments, cotyledons were repeatedly infused with PTHrP (1–34) or PTH (1–34) (51.3 n m). Vasodilatory responses were significantly reduced in response to repeated exposure to PTHrP (1–34) and PTH (1–34) (P<0.001), indicating that this peptide desensitizes the fetal-placental vasculature. PTHrP (1–34) and PTH (1–34) equipotently stimulated a significant vasodilatation of the fetal-placental circulation (P<0.0001). The PTHrP receptor antagonist [Asn10, Leu11]PTHrP (7–34) (102 nM) was infused in U46619-constricted placentae in the presence and absence of PTHrP (1–34) (10.2 nM). The PTHrP antagonist alone had no significant effect in the fetal-placental circulation. The antagonist significantly attenuated the response to PTHrP (1–34) (P<0.015). Based on the data obtained in this study it is suggested that locally produced PTHrP (1–34) may be involved in the regulation of normal human fetal-placental vascular tone in an autocrine and/or paracrine fashion.

Maternal and fetal fatty acid composition in ovine muscle tissues

In species characterized by a cotyledonary placenta, as sheep, the relative contribution of maternally derived and placenta synthesized fatty acids is not fully understood. For this reason, the FA composition of mother muscle and the deposition of FA in the fetal muscle were studied by gas-chromatography. Five pregnant Sarda ewes were slaughtered at approximately 145 days of pregnancy. Semitendinosus, semimembranosus and femoral biceps muscles were immediately removed from ewes and fetuses. Data were analyzed by a paired t-test, to detect differences in FA composition between fetus and mother tissues. Results showed that FA profile of fat muscle differed markedly between fetus and mother. The intramuscular fat content were 6.38% and 11.79% on DM basis in fetus and mother muscle, respectively. Linoleic (LA; 18:2n-6), and linolenic (ALA; 18:3n-3) acid were found at smaller concentrations in fetus (0.77 and 0.01 mg/100 mg total FA for LA and ALA, respectively) than in maternal muscle (5.55 and 1.04 for LA and ALA, respectively). On the contrary, a higher proportion of their long-chain polyunsaturated metabolites, like arachidonic (AA; 20:4n-6) and docoexadienoic (DHA; 22:6n-3) acid in fetus compared to mother tissue (AA, 2.32 vs 1.30; DHA, 0.83 vs 0.09 mg/100 mg total FA) indicates a preferential fetal accumulation of those FA which are important for fetuses growth and central nervous system development. The c9,t11 Conjugated linoleic acid (CLA) isomer concentration in mother tissue was 0.72 mg/100 mg FA. It was found also in fetus muscle (0.11 mg/100 mg total FA) suggesting a transplacental fatty acid passage to fetal tissues or a desaturation activity on vaccenic acid (VA; 18:1 t11) in the placental or in the fetal tissue. Surprisingly, the c9,t11-CLA was not the most abundant CLA isomer found in fetus fat. Others CLA isomers, separated but not identified, were found in concentration of 0.17, 0.15 and 0.53 mg/100 mg of FA. Concentrations of these CLA isomers were lower in mother tissue (0.12, 0.05 and 0.10 mg/100 mg of FA). The concentration of VA was 0.46 and 0.90 in fetus and mother muscle, respectively. Correlation analysis between FA profile of mothers and fetuses evidenced a close relationship only for odd-numbered long-chain fatty (r = -0.72 for C15:0 and r = -0.88 for C17:0). The results suggested a different FA metabolism in the muscle tissue of mother and fetus in dairy sheep.

The Diverse Effects of Vasopressors on the Fetoplacental Circulation of the Dual Perfused Human Placenta

We studied the effects of 5 vasopressors on fetal arterial perfusion pressure (FAP) in vitro using the dual perfused, single isolated cotyledon, human placental model. In 29 separate experiments, epinephrine (75 mg), norepinephrine (75 mg), ephedrine (50 mg), phenylephrine (2 mg), and methoxamine (40 mg) were introduced into the 250-mL reservoir serving the maternal perfusion circuit to determine the effect of each drug on FAP. The duration of drug exposure for each placental cotyledon was approximately 180 minutes. After 180 minutes, FAP (mean +/- sd) increased significantly with ephedrine from 64 +/- 3 to 172 +/- 71 mm Hg (P < 0.001) and with phenylephrine from 81 +/- 4 to 132 +/- 11 mm Hg (P = 0.003). No changes in FAP were seen with epinephrine, norepinephrine, and methoxamine. In the dual perfused, single isolated cotyledon, human placental model, exposure of the maternal circulation to ephedrine and phenylephrine caused an increase in FAP, whereas exposure to norepinephrine, epinephrine, and methoxamine did not. The pharmacodynamic mechanisms underlying these differences have yet to be explained. Thus, the clinical implications of the findings are as yet unclear.

Continuous infusion of 17-hydroxyprogesterone caproate into either the fetoplacental or intervillous circulation of a placental cotyledon attenuates vasoconstriction of the fetoplacental arteries by thromboxane mimetic U46619

The objective of the study was to determine whether pretreatment of fetal or maternal placental vasculature with 17-hydroxyprogesterone caproate (17-P) attenuates the vasoactive effect of the thromboxane mimetic U46619. Two cotyledons were obtained from each placenta studied. For the first 5 placentas, the fetal artery of 1 cotyledon from each pair was infused with 17-P. After 30 minutes, a bolus dose of U46619 was administered to both cotyledons. An identical procedure was carried out on the next 5 placentas except that 17-P was infused into the intervillous space. The pressure excursion caused by bolus administration of U46619 was less in the cotyledons infused with 17-P, both in the 5 cases in which the fetal vasculature was infused with 17-P (P = .0035) and in the 5 cases in which the maternal vasculature was infused with 17-P (P = .038). Pretreatment of either the fetal or maternal circuits of the placenta with 17-P attenuates U46619-mediated fetoplacental vasoconstriction.

Feto-maternal interactions in pregnancies: Placental microparticles activate peripheral blood monocytes

Normal pregnancy is associated with a systemic maternal inflammatory reaction, including the activation of peripheral blood monocytes. This reaction is exaggerated in pre-eclampsia, a severe placenta-dependent disorder of pregnancy specific to humans. It has been suggested that placental syncytiotrophoblast membrane microparticles (STBM), which are released into the peripheral blood, may contribute to the maternal response. The aim of this study was to investigate the inflammatory properties of STBM generated by four different approaches on primary human monocytes in vitro. Cellular viability, phenotype and functional response were analysed. STBM isolated by mechanical dissection and STBM generated from villous explant cultures incubated in hypoxic conditions had only minor influences on the monocytic phenotype and failed to induce a proinflammatory response. By contrast, STBM washed from the maternal side of a placental cotyledon and STBM shed by explants cultured in air up-regulated cell surface expression of the adhesion molecule CD54 and induced the production of interleukin (IL)-8, IL-6 and IL-1β. Cytokine production was time- and dose-dependent. Our study, therefore, suggests that monocyte activation in normal pregnancy and pre-eclampsia may be induced by STBM released by the placenta. The higher amounts of STBM circulating in maternal blood in pre-eclampsia might lead to the excessive maternal inflammatory reaction.

Antibody-Dependent Transplacental Transfer of Malaria Blood-Stage Antigen Using a Human Ex Vivo Placental Perfusion Model

Prenatal exposure to allergens or antigens released by infections during pregnancy can stimulate an immune response or induce immunoregulatory networks in the fetus affecting susceptibility to infection and disease later in life. How antigen crosses from the maternal to fetal environment is poorly understood. One hypothesis is that transplacental antigen transfer occurs as immune complexes, via receptor-mediated transport across the syncytiotrophoblastic membrane and endothelium of vessels in fetal villi. This hypothesis has never been directly tested. Here we studied Plasmodium falciparum merozoite surface protein 1 (MSP1) that is released upon erythrocyte invasion. We found MSP1 in cord blood from a third of newborns of malaria-infected women and in >90% following treatment with acid dissociation demonstrating MSP1 immune complexes. Using an ex vivo human placental model that dually perfuses a placental cotyledon with independent maternal and fetal circuits, immune-complexed MSP1 transferred from maternal to fetal circulation. MSP1 alone or with non-immune plasma did not transfer; pre-incubation with human plasma containing anti-MSP1 was required. MSP1 bound to IgG was detected in the fetal perfusate. Laser scanning confocal microscopy demonstrated MSP1 in the fetal villous stroma, predominantly in fetal endothelial cells. MSP1 co-localized with IgG in endothelial cells, but not with placental macrophages. Thus we show, for the first time, antibody-dependent transplacental transfer of an antigen in the form of immune complexes. These studies imply frequent exposure of the fetus to certain antigens with implications for management of maternal infections during pregnancy and novel approaches to deliver vaccines or drugs to the fetus.

The role of organic cation transporters (OCTs) in the transfer of metformin in the dually perfused human placenta

Objectives Our aim was to investigate the mode of placental transfer of metformin in term human placenta with special reference to involvement of the organic cation transporters (OCTs). Study design Twenty-nine placentas were obtained after delivery and a 2-h non-recirculating perfusion of a single placental cotyledon was performed to study maternal-to-fetal and fetal-to-maternal transport of metformin, which is a substrate for OCTs by using antipyrine as a reference of passive diffusion transfer compound. Cimetidine was used as an inhibitor for OCT-dependent active transfer. Results Maternal-to-fetal transfer of metformin and antipyrine were 3.7% and 10.0%, respectively, and fetal-to-maternal transfers were 15.5% and 42.3%, respectively. Cimetidine did not have any effect on the transfer of metformin. Fetal-to-maternal transfer of metformin was significantly higher than maternal-to-fetal transfer ( P < 0.05) in perfusions performed with or without cimetidine. Conclusions A higher transfer rate of metformin was detected in fetal-to-maternal than maternal-to-fetal direction, but a similar difference was observed with antipyrine. Inhibition of OCTs did not have a significant effect on the placental transfer of metformin. Although the existence of other active transporting systems cannot be ruled out, the influence of OCT-dependent active transport system on the placental pharmacokinetics of metformin is unlikely significant.

Antagonism of the Sphingosine 1-Phosphate (S1P) Signaling Pathway in Pregnant Ewes Alters Placentome Architecture, Amino Acid Transport, and Fetal Growth.

Angiogenesis is a critical factor in the process of placentation. In ruminants, angiogenesis occurs primarily in placentomes, formed by interdigitation and branching morphogenesis of placental cotyledons and maternal endometrial caruncles, which supply hematotrophic nutrition to the developing conceptus. We hypothesized that the lysosphingolipid sphingosine-1-phosphate (S1P) stimulates angiogenesis within developing sheep placentomes through activation of the S1P receptors (S1P1-S1P5) and that their antagonism would alter placentomal angiogenesis and subsequent uteroplacental nutrient exchange during early to mid pregnancy. In Study 1, passage 4-5 primary ovine uterine artery endothelial cells (oUAEC) from 3 late pregnant sheep were seeded as monolayers onto three dimensional (3D) collagen matrices containing angiogenic growth factors and S1P. S1P dose-dependently (1–1000 nM) enhanced endothelial cell invasion compared to VEGF and FGF2 alone. Using RT-PCR to detect S1P1-S1P5, only S1P1 and S1P3 receptors were found to be expressed in oUAEC. Antagonism of these receptors with FTY720 and VPC 23019 blocked S1P-induced endothelial cell invasion in vitro. In Study 2, we examined potential physiological mechanisms for actions of endogenous S1P in the control of ovine placental angiogensis. Ewes with a single fetus were given daily intravenous injections of FTY720 (n=6; 1.5 mg/day) or vehicle (n=5) from Days 30 to 60 prior to collection of tissues on Day 60. FTY720 treatment altered the histoarchitecture of the placentome as evidenced by decreased stromal area of caruncular crypts and diminished thickness of connective tissue forming the placentomal capsule. Concomitant with these effects on interdigitation of uterine and placental tissues, the total volume of cotyledonary tissue per placentome was greater in FTY720-treated ewes. Immunofluorescence localization of the intermediate filament proteins, cytokeratin and vimentin indicated no overt change in synctial epithelia, whereas stromal tissue thickness and density were decreased with FTY720 treatment. Immunofluorescence localization of alpha-smooth muscle actin and the endothelial marker, von Willebrand Factor, in placentomes revealed that FTY720 treatment increased diameter of the lumen, thickness of the tunica muscularis of vessels, but decreased the total number of vessels in caruncles as compared to controls. FTY720 treatment also affected transport of amino acids across the placentae and fetal development as evidenced by decreases in methionine levels in allantoic fluid (P<0.05), aspartate and glutamate levels in amniotic fluid (P<0.005), and crown-rump length of fetuses (P<0.05), however, total fetal weight was not affected (P>0.05). Results of this study indicates that antagonism of the S1P signaling pathway using FTY720 interfered with placentomal development, including angiogenesis, transport of micronutrients from the uterus to the placenta and fetal growth. We conclude that S1P is a key regulatory molecule responsible for mediating development of the angiogenic network in placentomes of sheep that provide hematatrophic support for the developing conceptus. (Research supported by National Research Initiative Competitive Grants Program, Cooperative State Research, Education, and Extension Service, USDA award 2008-35203-18830 to KAD and AHA Scientist Development Grant #053020N to KJB) (poster)

The endometrial–myometrial junction: a fresh look at a busy crossing

The endometrial–myometrial junction: a fresh look at a busy crossing

Successful diagnostic and therapeutic intrauterine fetal interventions by natural orifice transluminal endoscopic surgery (with videos)

Despite advances in the development of fetal surgery, morbidity and mortality are substantial. A natural orifice transluminal endoscopic surgery (NOTES)-guided approach to the gravid uterus may offer a less-invasive technique. To assess the feasibility of NOTES for diagnostic and therapeutic intrauterine fetal interventions. Survival and nonsurvival experiments on pregnant sheep. Nonsurvival experiments performed in 2 pregnant sheep (80-110 days' gestation). A third ewe underwent NOTES and survived for 4 weeks. Transgastric (nonsurvival ewes) and transvaginal (1 nonsurvival and the survived ewe) peritoneoscopy was performed after standard needle-knife entry into the peritoneal cavity. Endoscopic access to the gravid uterus was assessed. EUS-guided, transuterine injection of saline solution into the fetal cardiac ventricle and vessels was attempted in all. Feasibility of NOTES- and EUS-guided intervention in a pregnant animal model, visibility of fetal parts via EUS compared with transabdominal US. Entry into the peritoneal cavity was achieved in each ewe. Access to and complete visualization of the gravid uterus were successful once within the abdominal cavity. Visualization of the fetal parts and the placental cotyledons by EUS was achieved in all animals. EUS-guided amniocenteses and transuterine intracardiac and intravascular injection of saline were successful. There were no complications or preterm delivery after the procedures. Animal model. NOTES is technically feasible in the pregnant ewe. Intraperitoneal EUS via a NOTES approach provides excellent access and visualization of the intrauterine cavity and fetal parts.

Progesterone levels in cesarean and normal delivered term placentas

One of the most important hormones synthesized by the placenta during pregnancy is progesterone. The regulating mechanisms of progesterone synthesis and the mechanism responsible for the spontaneous onset of labor in women are still not fully understood. Progesterone is thought to have been involved in human parturition. The objective of this study was to compare the levels of progesterone in the human placentas, at the end of the gestation (37-41 weeks) in vaginal versus cesarean deliveries, and to evaluate the pattern of progesterone accumulation, instantly following its synthesis by the human placenta at the end of the pregnancy. Progesterone levels in human placental tissue were determined by immunochemiluminescent analysis, following tissue homogenization. Progesterone secretion and accumulation pattern in the placental tissue was demonstrated using the ex vivo, closed, dual perfusion system of isolated human placental cotyledon. Immunochemiluminescent analysis of progesterone levels in human normal and cesarean-delivered placentas showed that placentas following normal vaginal delivery store higher concentrations of progesterone, and produce progesterone more intensively. Results obtained from 120-min perfusions (of vaginal and cesarean-delivered placentas) showed that progesterone tended to accumulate in the maternal rather than the fetal compartment. These data indicate that progesterone levels continuously rise till the end of pregnancy, with no apparent drop in progesterone levels during the labor process. In addition, progesterone is released from the syncytiotrophoblast preferably into the maternal component of the placental tissue.

Different promoter usage for CYP19 gene expression in buffalo ovary and placenta

Aromatase is the key enzyme for estrogen biosynthesis and is coded by CYP19 gene. The expression of CYP19 gene is regulated in tissue-specific manner by alternate use of different promoters. In this study, we have analyzed tissue-specific expression and their regulation of CYP19 gene in preovulatory and postovulatory stages of buffalo ( Bubalus bubalis) ovary and placenta. RT-PCR analysis showed that the CYP19 gene expression was significantly ( p < 0.05) higher in granulosa cells of large follicles as compared to the other tissues. The transcript analysis and transcriptional start site identified by 5′-RLM RACE for CYP19 expression indicated different transcriptional start sites within the different sized follicles during folliculogenesis. Sequence analysis showed that the transcription start site of transcript isolated from buffalo granulosa cells of small follicles was 37 bases upstream of the transcript isolated from granulosa cells of large follicles. However, both the transcripts were found to be derived from proximal promoter II. Difference in the transcriptional start site indicates the different promoter sequence usage in granulosa cells of different sized follicles. Further, in silico analysis of the difference in promoter sequence based on the 5′-UTRs isolated from granulosa cells of small follicles (151 bases) and large follicles (114 bases) showed that consensus sequence for certain important trans-elements (viz., TATA binding protein, E2F and CAAT binding protein) would lie in the promoter sequence isolated from the granulosa cells of large follicles. These transcription factors may be involved in regulation of CYP19 gene expression in ovary, either directly or indirectly. The difference in the size of 5′-UTR among the granulosa cells of ovary reflects the possible mechanism for the differential regulation of CYP19 gene during development. The transcripts isolated from buffalo corpus luteum and placental cotyledons were having same 5′-UTR comprises of 168 bases and found to be derived from PI.1. Estimates of CYP19 gene transcript concentration in the different tissues revealed that the CYP19 gene expression in granulosa cells is predominantly regulated by PII and to a minor extent by PI.1. However, PI.1 is almost exclusively responsible for CYP19 gene expression in placenta and residual expression in corpus luteum. In order to understand the complex CYP19 gene regulation in these tissues, further studies are needed to elucidate the activity of different promoters and define regulatory elements for binding of transcription factors.

Molecular mapping deep within a living human organ: analysis of microvessel function on the timescale of seconds and with sub-micrometre spatial resolution

Visualising vascular endothelial cell function in individual blood microvessels allows elucidation of molecular interactions at the vascular wall, the first barrier between blood-borne therapeutic agent and its target. Functional analysis in situ requires sub-micrometer spatial resolution and tagged molecules generating contrast in living blood vessels. Light microscopy fulfills these requirements, particularly if fluorescent tags deliver the contrast. However, vascular arborisations in living organs defy morpho-functional analysis, filling tissues with closely meshed three-dimensional networks which are inaccessible to optical imaging. We protocol here successful morpho-functional analysis of microvascular processing in a living organ, the human placental cotyledon. Fluorescence-tagged tracer was positionally fixed by snap-freezing, frozen sections were cut, freeze-dried and heat-fixed. A brief histochemical procedure then labelled all vascular elements in the sections, providing fluorescence contrast in two colour channels. Mosaic monochromatic images acquired in both channels delivered high-resolution maps of centimeter-wide tissue areas. Quantitative analysis of the images' greyscale histograms defined objectifiable, reproducible thresholds, used to reduce the images to colour-coded wide-area functional maps tracking placental vascular processing of the tagged molecules. Rapid positional fixing of tracer with reduction of images to maps was combined with ultrastructural tracking to elucidate vascular processing at scales of nanometres and seconds.

Tolerance of Human Placental Tissue to Severe Hypoxia and Its Relevance for Dual Ex Vivo Perfusion

In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20–30min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage.There seem to be parallels between “depressed metabolism” seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by “partial metabolic arrest” in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress.There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin.A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the artificial reperfusion, may lead to an improvement of the ex vivo perfusion technique.

49: Continuous infusion with with 17-Hydroxyprogesterone Caproate (17-P) into the fetal or maternal arterial circuits of the ex vivo placental cotyledon model attenuates vasoconstriction by thromboxane and angiotensin-II

49: Continuous infusion with with 17-Hydroxyprogesterone Caproate (17-P) into the fetal or maternal arterial circuits of the ex vivo placental cotyledon model attenuates vasoconstriction by thromboxane and angiotensin-II

711: Placental transfer of the long acting insulin analogue glargine in the ex-vivo dually perfused placental cotyledon model

711: Placental transfer of the long acting insulin analogue glargine in the ex-vivo dually perfused placental cotyledon model

Carrier‐mediated transport of metformin across the human placenta determined by using the ex vivo perfusion of the placental cotyledon model

Metformin is a polar positively charged compound. The aim of the study was to characterize its permeability across the human placenta using the ex vivo placental perfusion model. Selected cotyledons from term placentas were cannulated and dually perfused. Metformin (10 mg/L, 1000 mg/L) and a permeability reference, antipyrine (50 mg/L), were added to the maternal circulation. Samples from maternal and fetal compartments were analyzed for metformin and antipyrine by high performance liquid chromatography (HPLC). The permeation of metformin was also studied using the parallel artificial membrane permeation assay (PAMPA) that was designed to predict passive transcellular permeability of drugs. In this study, 15 complete placental perfusion experimental set-ups were performed. The mean percent transport increased as metformin concentrations were raised and it was 11 +/- 1.32 and 16.92 +/- 0.98 for 10 mg/L and 1000 mg/L, respectively. The transport rate of metformin across the placenta was asymmetric yet, an active efflux against the gradient concentration could not be observed. Using the PAMPA assay, we confirmed that metformin does not cross by passive diffusion. The data suggest that metformin permeability across the placenta is mediated by a carrier that transport cationic compounds bi-directionally, with a higher transfer rate from the fetal to the maternal side.

Identification of multiple pregnancy-associated glycoproteins (PAGs) purified from the European bison (Eb; Bison bonasus L.) placentas

This paper describes the first identified chorionic PAGs in the European bison (Eb), named EbPAGs, predominantly expressed during early and mid-pregnancy (45–120 day post-coitum; dpc). Many EbPAGs were extracted from various cotyledonary tissues, precipitated, chromatographed (DEAE and VVA: Vicia villosa agglutinin), electrophoresed (1D- and 2D-PAGE), analysed by heterologous (cross-species) Western blotting and then micro-sequenced by Edman degradation. Finally, twelve selected VVA-purified isoforms (Ip 3.7–7.4) were entirely characterised. Nine identified NH 2-terminal micro-sequences were found to be PAGs. On 45 dpc, three identified forms were named: EbPAG 67 A kDa (RGSNLTHPLRNIGDLFYVGN), EbPAG 55 B kDa (RGSNLTHPL) and EbPAG 50 C kDa (SQISLRGSNLTI). On 60 dpc, the next three forms were named: EbPAG 71 D kDa (RGSNLTIHPLRNIIDLFYVG), EbPAG 55 E kDa (RGSNLTHPLRNI) and EbPAG 50 F kDa (SQISLRGS). On 120 dpc, three other forms were named: EbPAG 71 G kDa (RGSNLTHPLRNIRDLFYVG), EbPAG 60 H kDa (RGSNLTTHPLRNIKDLVVYM) and EbPAG 50 I kDa (SGSNLTTV). These EbPAG ( A–I) sequences are unique, as they are not identical to any other PAGs purified previously in related species of the Bovidae family. However, the EbPAGs (A–I forms) have some sequence resemblance to internal sequences of various full-length polypeptide PAG precursors ( in silico translated from cloned cDNAs) identified in domestic cattle. Three other novel native isoforms (J1, J2 and K): EbUPG 45 kDa J1 (SKDNYKNYIPLIVPFAT), EbUPG 45 kDa J2 (SKDNQKNYIPLIVPFAT) and EbUPG 76 kDa K (SPEFTV), were temporarily named ‘unknown placental glycoproteins’ (UPGs), due to their efficient VVA-purification (specific for glycoproteins only) and a lack of considerable consensus to previously sequenced placental glycoproteins in the Bovidae family. This is the first study identifying NH 2-terminals of multiple/diverse EbPAGs and some EbUPGs purified from the synepitheliochorial cotyledonary placenta of the endangered Bison bonasus (Red List).

Excess Estrogen Sulfoconjugation Causes a Weak Sign of Delivery in Somatic Cell Clone Recipient Cows.

This study was conducted to elucidate a factor causing a weak sign of delivery and a prolonged gestation, which are frequently observed in cows carrying somatic clone fetus, in spite of the maturity of fetal size or the occurrence of large offspring syndrome. Till date, several investigators have suggested morphological and functional abnormalities of the placenta in clone fetus in the various gestational stages. However, the roles for the placenta in the final process of clone pregnancy, namely placental estrogen synthesis and regulation of estrogen activity with sulfoconjugation, have not been fully understood. In IETS (2007 in Kyoto, Japan), we reported that prepartum rise of estrone and estradiol-17b concentrations in clone pregnancy was subtle, but estrone-sulfate concentration in clone pregnancy was higher than that in cows carrying fetus derived from in vivo embryo. In this study, we analyzed mRNA expression levels of estrogen-related genes, estrogen sulfotransferase (SULT1E1), estrogen sulfatase (STS) and aromatase (CYP19), in placental tissues, cotyledon (COT) and caruncle (CAR). Placentomes were obtained immediately after purtrition from cows, which delivered clone and in vivo calves after induction of parturition (gestation length was 284 to 289 days). Correlation analyses between mRNA abundances in placental tissues and maternal estrogen concentrations at parturition were performed using the data obtained from clones. Localization of gene expression in placentome was examined with in situ hybridization (SULT1E1 and STS) and immunohistochemistry (CYP19). Number of binucleate cells (BNC), existing in placentome, was counted from sections stained with hematoxylin-eosin. mRNA abundance of SULT1E1 was tended to be high in COT compared with CAR, clone pregnancy showed significantly higher (p<0.05) expression level than in vivo pregnancy. In situ hybridization signal of SULT1E1 was detected in binucleate cells. SULT1E1 mRNA abundance was negatively and positively correlated with maternal estrone (p<0.05) and estrone-sulfate (p<0.05) at parturition, respectively. mRNA abundances of STS and CYP19 were significantly higher in CAR and COT, respectively. In situ hybridization and immunohistochemistry showed that BNC and carunclar epithelial cells expressed both genes. Expression levels of STS and CYP19 mRNA were similar between clone and in vivo pregnancy, and were positively correlated (p<0.05) with maternal estradiol-17b at parturition of clones. Population of BNC in placentome did not differ between clone (vaginally delivered clone; 18.8}1.4/0.1mm2, cesarean section delivered clone; 18.6}1.7/0.1mm2) and in vivo (18.9}1.8/0.1mm2) pregnancy. Therefore, it can be surmised that increased SULT1E1 mRNA abundance in clone placental tissue was due to an increased transcription of the gene, rather than to an increased proportion of SULT1E1 producing BNC. The results obtained here suggested that excess estrogen sulfoconjugation in placenta was reasonable for the confusion of the ratio of unconjugated (active) and sulfoconjugated (inactive) estrogens. The hormonal abnormality would cause the weak sign of delivery and the prolonged gestation in cows carrying clone.

Trophoblast Differentiation in the Ovine Placenta: Insights into Transcription Factors and Regulatory Pathways.

Placental morphogenesis in ruminants, humans and rodents involves the growth, development and differentiation of mononuclear trophectoderm cells (MTC) into giant binucleate cells (BNC) as well as multinucleated syncytiotrophoblasts (STB). For example in sheep, MTC express IFNT only from Days 10 to 25, while BNC, which begin to differentiate on Day 14, and STB express CSH1 but not IFNT. In rodents, trophoblast differentiation and gene expression is governed by temporal and cell-specific changes in transcription factors, but little is known about this process in ruminants. In Study One, candidate transcription factors identified previously in humans, rodents and cattle (CDX2, EED, ETS2, ID2, GCM1, TEAD3, TCF12, TFAP2A, TFAP2B, TFAP2C) as well as IFNT and CSH1 were studied in the Day (D) 13-20 conceptus and D40, D80 and D120 placenta (intercotyledonary and cotyledonary). IFNT mRNA was observed in most MTC in D13-20 conceptuses but not in MTC of the D40-120 placenta; CSH1 was first observed in BNC of D16 conceptuses and in BNC and STB of the D18-120 placenta. EED, ID2 and TFAP2B were not detected in the trophectoderm, but EED and ID2 were observed in the endometrial epithelia. ETS2, TCF12 and TEAD3 were observed in both the conceptus trophectoderm and endometrium. CDX2, GCM1, TFAP2A and TFAP2C appeared trophectoderm-specific. CDX2 and GCM1 mRNAs were observed specifically in the BNC and STB, whereas TFAP2A and TFAP2C were in MTC, BNC, and STB. Thus, CDX2, GCM1, TFAP2A and TFAP2C may play a critical role in trophoblast growth and differentiation in the ovine conceptus. In Study Two, microarray analysis was conducted using total RNA isolated from D16 conceptuses, D45 intercotyledonary placenta, and D45 placental cotyledons (n=4 ewes/day/tissue) and a comprehensive 24K bovine oligonucleotide array. As expected, IFNT was 132-fold higher (P<0.05) in the D16 conceptus than D45 placental tissues, and CSH1 was 70-fold higher (P<0.05) in the D45 placenta. Gene ontology analysis found that transcriptional regulators (CDCA7, EGR1, HTATIP2, KLF5, MAZI, MYBL2, PPP1R13B, TP53BP2), cyclins (CCNC, CCNY), and chromatin modification factors (CUL4A, MYST4, JARID1B, PHF12, SSRP1, SUDS3) were higher in D16 conceptuses than D45 placental tissues. TGFB superfamily-related, IGF-related genes, and Eph/Ephrin-related genes were also higher in the D16 conceptus. EGR1 and KLF5 interact with TP53 that is key factor in the cell cycle, and TP53BP2, PPP1R13B and SSRP1 enhance TP53 activity. The Eph/Ephrin family is regulated by TP53 and plays a critical role in vascular development and trophoblast differentiation in rodents. In D45 placenta, vasculogenesis/angiogenesis-related genes (AGTR1, AGTR2, ANGPT1, CTGF FGF2, SPP1, TNFRSF12A, TNFSF15, VWF) were higher than D16 conceptus. TGFB superfamily-related genes (BMP1, BMP5, BMP7, FSTL1, KLF11, LTBP1, LTBP2, LTBP4, TGFB1) were also higher in D45 placenta. In contrast to Day 16 conceptus, transcription factor AP1-related genes (JUNB, JUN, JUND, FOSL1) were higher in Day 45 placenta. AP1 regulates the transcription of many genes including TGFB1. These results suggest that TGFB superfamily signaling and AP1 regulate placental development. These studies suggest species-specific differences in transcription factor expression during trophoblast differentiation and provide new insights into regulatory networks and pathways governing ovine placental morphogenesis. (Supported by NIH HD052745)