Investigation into a role for the primitive streak in development of the murine allantois

Despite its importance as the source of one of three major vascular systems in the mammalian conceptus, little is known about the murine allantois, which will become the umbilical cord of the chorio-allantoic placenta. During gastrulation, the allantois grows into the exocoelomic cavity as a mesodermal extension of the posterior primitive streak. On the basis of morphology, gene expression and/or function, three cell types have been identified in the allantois: an outer layer of mesothelial cells, whose distal portion will become transformed into chorio-adhesive cells, and endothelial cells within the core. Formation of endothelium and chorio-adhesive cells begins in the distal region of the allantois, farthest from the streak. Over time, endothelium spreads to the proximal allantoic region, whilst the distal outer layer of presumptive mesothelium gradually acquires vascular cell adhesion molecule (VCAM1) and mediates chorio-allantoic union. Intriguingly, the VCAM1 domain does not extend into the proximal allantoic region. How these three allantoic cell types are established is not known, although contact with the chorion has been discounted. In this study, we have investigated how the allantois differentiates, with the goal of discriminating between extrinsic mechanisms involving the primitive streak and an intrinsic role for the allantois itself. Exploiting previous observations that the streak contributes mesoderm to the allantois throughout the latter's early development, microsurgery was used to remove allantoises at ten developmental stages. Subsequent whole embryo culture of operated conceptuses resulted in the formation of regenerated allantoises at all time points. Aside from being generally shorter than normal, none of the regenerates exhibited abnormal differentiation or inappropriate cell relationships. Rather, all of them resembled intact allantoises by morphological, molecular and functional criteria. Moreover, fate mapping adjacent yolk sac and amniotic mesoderm revealed that these tissues and their associated bone morphogenetic protein 4 (BMP4) did not contribute to restoration of allantoic outgrowth and differentiation during allantoic regeneration. Thus, on the basis of these observations, we conclude that specification of allantoic endothelium, mesothelium and chorio-adhesive cells does not occur by a streak-related mechanism during the time that proximal epiblast travels through it and is transformed into allantoic mesoderm. Rather, all three cell-types are established by mechanisms intrinsic to the allantois, and possibly include roles for cell age and cell position. However, although chorio-adhesive cells were not specified within the streak, we discovered that the streak nonetheless plays a role in establishing VCAM1's expression domain, which typically began and was thereafter maintained at a defined distance from the primitive streak. When allantoises were removed from contact with the streak, normally VCAM1-negative proximal allantoic regions acquired VCAM1. These results suggested that the streak suppresses formation of chorio-adhesive cells in allantoic mesoderm closest to it. Together with previous results, findings presented here suggest a model of differentiation of allantoic mesoderm that invokes intrinsic and extrinsic mechanisms, all of which appear to be activated once the allantoic bud has formed.     

Brachyury is required for elongation and vasculogenesis in the murine allantois

Mouse conceptuses homozygous for mutations in brachyury (T) exhibit a short, misshapen allantois that fails to fuse with the chorion. Ultimately, mutant embryos die during mid-gestation. In the 60 years since this discovery, the role of T in allantoic development has remained obscure. T protein was recently identified in several new sites during mouse gastrulation, including the core of the allantois, where its function is not known. Here, using molecular, genetic and classical techniques of embryology, we have investigated the role of T in allantoic development. Conceptuses homozygous for the T(Curtailed) (T(C)) mutation (T(C)/T(C)) exhibited allantoic dysmorphogenesis shortly after the allantoic bud formed. Diminution in allantoic cell number and proliferation was followed by cell death within the core. Fetal liver kinase (Flk1)-positive angioblasts were significantly decreased in T(C)/T(C) allantoises and did not coalesce into endothelial tubules, possibly as a result of the absence of platelet endothelial cell adhesion molecule 1 (Pecam1), whose spatiotemporal relationship to Flk1 suggested a role in patterning the umbilical vasculature. Remarkably, microsurgical perturbation of the wild-type allantoic core phenocopied the T(C)/T(C) vascularization defect, providing further support that an intact core is essential for vascularization. Last, abnormalities were observed in the T(C)/T(C) heart and yolk sac, recently reported sites of T localization. Our findings reveal that T is required to maintain the allantoic core, which is essential for allantoic elongation and vascular patterning. In addition, morphological defects in other extraembryonic and embryonic vascular organs suggest a global role for T in vascularization of the conceptus.     

Expression of trophoblastic interferon genes in sheep and cattle.

The trophoblastic interferons ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1, respectively) have been implicated as mediators of maternal recognition of pregnancy in sheep and cattle. The objective of this study was to describe the onset and duration of gene expression for oTP-1 and bTP-1 in preimplantation ovine and bovine conceptuses by in situ hybridization and Northern analysis. Sections from paraffin-embedded ovine conceptuses, collected on Days 10, 11, 12, 13, and 15 of gestation (n = 1, 3, 3, 2, 2), and bovine conceptuses, collected on Days 12/13, 15/16, and 19 (n = 2, 4, 5), were hybridized to specific [35S]-labeled cDNA probes. Two different probes, one encompassing bases 442-918 and representing both coding and 3'-untranslated regions, and a second 3'-specific probe (bases 650-912) were used to detect oTP-1 mRNA. At all stages examined, oTP-1 mRNA was confined to trophectoderm of ovine conceptuses. Consistent with earlier studies, expression increased markedly at Day 13. oTP-1 mRNA was detected at low levels in seven of seven ovine conceptuses prior to Day 13 when the longer probe was employed. With the 3'-specific probe, however, oTP-1 mRNA was detected in only one of the seven ovine conceptuses prior to Day 13. Thus, although low amounts of oTP-1 mRNA may be present in ovine conceptuses prior to Day 13, massive induction of this mRNA occurs on Day 13 coincident with the initiation of maternal recognition of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conceptus development in large white and prolific Chinese Meishan pigs

Large White (LW) and Meishan (MS) gilts were killed on Days 8, 10, 11, 12, 14 and 30 of gestation. Mean diameters (mm) for MS and LW conceptuses, respectively, were: Day 8, 0.45 and 0.69; Day 10, 2.7 and 1.9; Day 11, 5.3 and 2.7, with the differences among days being affected by breed (P less than 0.01). Variation in diameter among conceptuses from LW gilts was greater (P less than 0.01) than that for MS gilts on Days 8-11, respectively: Day 8, 20 and 46%; Day 10, 29 and 38%; and Day 11, 22 and 44%. Conceptuses had elongated in 3 of 5 MS and 1 of 4 LW gilts on Day 11, 6 and 6 MS and 2 of 4 LW gilts on Day 12 and all gilts of both breeds on Day 14. These results indicate that conceptuses of MS gilts develop more rapidly and more uniformly between Days 8 and 14 of gestation. Overall, embryonic survival for Days 8-12 for gilts not having elongated conceptuses was 90.2% for MS and 73.2% for LW gilts (P less than 0.01). On Day 30 of gestation, embryonic survival was also higher (P less than 0.01) for MS (89%) than LW (55%) gilts. However, embryonic weight, crown-rump length, placental length, allantoic fluid volume, amniotic fluid volume, as well as total glucose, fructose and protein in allantoic fluid were not affected by breed. Placental weight was greater (P less than 0.01) for LW gilts. Uterine development at Day 30 of gestation, based on total length and weight of uterine horns, width of uterine horns, total endometrial surface area and total endometrial weight was greater (P less than 0.01) for LW gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal and physical changes associated with bovine conceptus development.

Conceptus (placental membranes, fetal fluids and fetus) development was characterized between Days 27 and 111 of gestation. Progestagens, oestrone, oestradiol, oestrone sulphate and prostaglandins (PG) F were measured in maternal plasma and allantoic and amniotic fluids. Protein concentrations are described for fetal fluids. The early increase in placental membrane weight from 1.12 g (27 days) to 58.45 g (50 days) was associated with oestrogen production presumably of conceptus origin. Oestrogens increased significantly in allantoic and amniotic fluids throughout the period studied with oestrone being the primary free oestrogen, rising from 2 pg/ml (Day 33) to 144 ng/ml by 111 days in allantoic fluid. Changes in plasma oestrogens of the maternal circulation were not detected until after Day 70 at which time oestrone concentration was greater than that of oestradiol. Fetal fluid concentrations of progestagens, oestrone sulphate and PGF were not related to maternal plasma levels and a sequestration of these hormones by the allantois is postulated.     

A comparison of the anti-luteolytic activities of recombinant ovine interferon-alpha and -tau in sheep

Interferon tau (IFNT) is secreted by the trophectoderm of ruminant conceptuses during the peri-implantation period and serves an anti-luteolytic function. The question as to whether IFNT is superior as an anti-luteolytic agent to closely related Type I IFNs, such as IFN alpha (IFNA), which have a different function, remains unanswered. Thus, the aim of this study was to determine whether equivalent antiviral (AV) units of ovIFNA and ovIFNT are equipotent in extending estrous cycle length. Four distinct ovIFNA mRNA (ovIFNA1-4) were cloned from ovine lymphocytes. Recombinant ovine IFNs (ovIFNT4 and ovIFNA1) were prepared in the yeast Pichia pastoris. The AV activity of the purified IFNs was determined on a bovine cell line (MDBK) and on transformed ovine luminal uterine epithelial cells. Indwelling uterine catheters were fitted into crossbred ewes on Day 3 postestrus (Day 0 = estrus). Between Days 10 and 18 postestrus, ewes received twice-daily infusions of 0.7 x 10(7) IU of either ovIFNA1 or T4, plus serum albumin. Control ewes received serum albumin only. Daily blood samples were collected for progesterone determination, and ewes were monitored twice daily for estrus. Both ovIFNA (P = 0.04) and ovIFNT (P = 0.01) caused estrous cycle extension in nonpregnant ewes compared to controls when administered at equivalent AV doses. In conclusion, the uniqueness of IFNT as an anti-luteolytic agent most likely resides in its unique expression pattern rather than its special biopotency.     

Porcine conceptuses secrete an interferon during the preattachment period of early pregnancy

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.     

A correlative study of the allantois in pig and rabbit highlighting the diversity of extraembryonic tissues in four mammalian species,including mouse and man

Despite its conserved role in placenta and umbilical cord formation, the mammalian allantois shows remarkable diversity in size and form as well as in the timing of its appearance and attachment to the chorion. In the mouse, the common allantoic diverticulum is lacking; instead, the allantoic core domain is defined as a progenitor center for allantoic development. In this study, the allantoises of the pig and the rabbit as two nonrodent mammals of increasing significance in biomedical research are compared (1) morphologically using high resolution light and electron microscopy and (2) molecularly using brachyury mRNA expression as a mesodermal marker. Multiple small allantoic diverticula in the rabbit contrast with a single large cavity filling the entire allantois of the pig, but neither pig nor rabbit allantois expresses brachyury . The mesothelium on the allantois surface shows regional variability of cell contacts and microvilli, while blood vessels appear randomly around the allantoic diverticula in a mesodermal layer of variable thickness. Primordial germ cell‐like cells are found in the allantois of the pig but not of the rabbit. To understand further the relevance of this developmental and morphological diversity, we compare the allantois development of pig and rabbit with early developmental landmarks of mouse and man. Our findings suggest that (1) tissue interaction between endoderm and mesoderm is important for allantoic development and vascular differentiation in species with a rudimentary allantoic diverticulum, (2) allantoic mesothelium plays a specific role in chorioallantoic attachment, allantoic differentiation and vascularization, and (3) there is a pronounced diversity in the extraembryonic migratory pathways of primordial germ cells among mammals. Finally, the phylogenetically basal characteristics of the pig allantois are suggestive of a functional similarity in mammals with a large allantois before placentation and in (aplacental) sauropsids with a chorioallantoic membrane well‐adjusted to material exchange function.     

Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.     

Retinol-binding protein: a major secretory product of the pig conceptus

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.     

Characterization of the interleukin-1beta system during porcine trophoblastic elongation and early placental attachment

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1beta (IL-1beta) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1beta, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1beta gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1beta increased during the process of trophoblastic elongation on Day 12. Uterine IL-1beta content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1beta gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1beta and IL-1Rant gene expression were lowest during Days 10-15 of the estrous cycle and pregnancy. The temporal expression of IL-1beta during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1beta synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.     

Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism.

F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.     

The carnivore pregnancy: the development of the embryo and fetal membranes

The aim of this research was to compare the morphological aspects during the development of pregnancy in dogs and cats, distinguishing features of the fetal membranes, such as yolk sac evolution and differentiation of hemangioblasts, and the degree of elaboration of the amnion and allantois. Canine and feline placentae from 20, 24, 35, 45 and 55 d of pregnancy were perfusion-fixed for histological investigation and vascular corrosion casts were produced. The casts were prepared for scanning electron microscopy (SEM) and the embryo and fetal membrane development was analyzed. The growth patterns of the conceptuses were compared with the organization of the placentation process, and changes of the morphology during pregnancy were recorded. In feline placentae, an incomplete zonary shape was present in 62.5% out of 60 studied cases. This was located distal to the insertion of the umbilical cord. In the lamellar zone, the interhemal membrane or placental barrier resembled endotheliochorial conditions, and the maternal-fetal microvascular blood flow interrelationship was of simple crosscurrent type. Dogs have a zonary placenta, completely surrounding the fetus, and complex lamellar organization of maternal and fetal tissues. At the border, two marginal hematomes with green colouration delimited the central placental girdle. The yolk sac consisted of one large sacculation with an inverted "T" shape and an enormous number of blood vessels; it had hemangioblast cells in contact with the epithelium. The amnion was avascular in early stages, but became vascularized by blood vessels of the internal allantoic membrane in later stages of pregnancy by intrinsic relation.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Purification and immunolocalization of ovine placental retinol-binding protein.

A retinol-binding protein (RBP), synthesized and secreted by ovine allantois in vitro, was purified from culture medium. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical molecular masses of about 23,000 Da as determined by two-dimensional PAGE under reducing conditions. Thirty-one of the first 34 N-terminal amino acids of the purified protein were sequenced and shown to have complete homology with bovine placental and bovine plasma RBP. The ultraviolet absorption spectrum and fluorescence excitation and emission spectra of the purified ovine placental RBP indicated the presence of bound retinol. Metabolic labeling studies demonstrated that the protein was synthesized by placental membranes. Using antiserum to bovine placental RBP, ovine placental RBP was immunolocalized in trophectoderm of 13-day-old blastocysts and trophectodermal cells of the chorion, endodermal cells lining the allantois, and ectodermal cells lining the amnion of 23-, 45-, and 53-day-old conceptuses. Results from this study suggest that ovine placental membrane epithelia synthesize and secrete RBP. Transport, storage, and metabolism of retinol mediated by placental RBP may be essential for normal embryonic development during pregnancy.