Reassembling gastrulation.

During development, a single cell is transformed into a highly complex organism through progressive cell division, specification and rearrangement. An important prerequisite for the emergence of patterns within the developing organism is to establish asymmetries at various scales, ranging from individual cells to the entire embryo, eventually giving rise to the different body structures. This becomes especially apparent during gastrulation, when the earliest major lineage restriction events lead to the formation of the different germ layers. Traditionally, the unfolding of the developmental program from symmetry breaking to germ layer formation has been studied by dissecting the contributions of different signaling pathways and cellular rearrangements in the in vivo context of intact embryos. Recent efforts, using the intrinsic capacity of embryonic stem cells to self-assemble and generate embryo-like structures de novo, have opened new avenues for understanding the many ways by which an embryo can be built and the influence of extrinsic factors therein. Here, we discuss and compare divergent and conserved strategies leading to germ layer formation in embryos as compared to in vitro systems, their upstream molecular cascades and the role of extrinsic factors in this process.

Bovine in-vitro produced embryos: Development of embryo proper and associated membranes from day 26 to 47 of gestation.

Based on in-vitro produced (IVP) bovine embryos, embryo proper and embryonic/fetal membranes were studied in 12 pregnancies from day 26 to 47. The embryos/fetuses displayed external as well as internal development of organs and structures according to the expectations from comparable in-vivo studies. However, the embryonic/fetal membranes were shorter than those reported for in-vivo-derived embryos/fetuses on days 26-35 of calculated age, whereas on days 41-47 they were of comparable lengths.

Impact of Mothers' Age on Telomere Length and Human Telomerase Reverse Transcriptase Expression in Human Fetal Membrane-Derived Mesenchymal Stem Cells.

Age-related cellular changes and limited replicative capacity of adult mesenchymal stem cells (MSCs) are few of the challenges confronting stem cell research. MSCs from human fetal membranes (hFM-MSCs), including placental, umbilical cord, and amniotic membrane, are considered an alternative to adult MSCs. However, the effect of mothers' age on hFM-MSC cellular properties is still not clearly established. This study aimed to evaluate the effect of mothers' age on hFM-MSC telomere length, telomerase activity, and proliferation ability in three different age groups: GI (20-29 years), GII (30-39 years), and GIII (≥40 years). hFM samples were collected from pregnant women ≤37 weeks after obtaining consent. hFM-MSCs were isolated and cultured to characterize them by flow cytometry and assess proliferation by MTT assay and doubling time. Telomere length and expression levels of human telomerase reverse transcriptase were assessed by quantitative real-time polymerase chain reaction (qRT-RCR). hFM-MSCs in the three age groups were spindle-shaped, plastic-adherent, and exhibited high proliferation rates and strong expression of hMSC markers. GI showed the longest telomere length in hMSCs in various FM regions, whereas GIII showed the highest level of telomerase expression. There was no difference in telomere length between GII and GIII, and both groups showed the same hMSC characteristics. In conclusion, although the hFM-MSCs derived from different fetal membranes maintained the MSC characteristics in all study groups, the hFM-MSCs of older mothers had shorter telomeres and higher telomerase activity and proliferation rate than did those derived from younger mothers. Thus, the hFM-MSCs of older mothers could be unsuitable for expansion in vitro or stem cell therapy. Determination of telomere length and telomerase expression level of hFM might help characterizing and understanding the biological differences of hFM-MSCs in different age groups.

Fetal membrane architecture, aging and inflammation in pregnancy and parturition.

Preterm birth is the single major cause of infant mortality. Short and long term outcomes for infants are often worse in cases of preterm premature rupture of the fetal membranes (pPROM). Thus, increased knowledge of the structure characteristics of fetal membranes as well as the mechanisms of membrane rupture are essential if we are to develop effective treatment strategies to prevent pPROM. In this review, we focus on the role of inflammation and senescence in fetal membrane biology.

Aquaporins in Fetal Development.

Water homeostasis during fetal development is of crucial physiologic importance. The successful formation and development of the placenta is critical to maintain normal fetal growth and homeostasis. The expression of several aquaporins (AQPs ) was found from blastocyst stages to term placenta and fetal membranes. Therefore, AQPs are proposed to play important roles in normal pregnancy, fetal growth, and homeostasis of amniotic fluid volume, and water handling in other organs. However, the functional importance of AQPs in fetal development remains to be elucidated.

Emil Selenka on the embryonic membranes of the mouse and placentation in gibbons and orangutans.

BACKGROUND: Emil Selenka made important contributions to embryology in marsupials, rodents and primates that deserve wider recognition. Here we review his work on early development of the mouse and placentation in the great apes. FINDINGS: Selenka was intrigued by germ layer theory, which led him to study inversion of the germ layers in the mouse and other rodents. He found it was growth of the ectoplacental cone that caused a downward shift in the position of the underlying ectoderm and endoderm, leading to an inside-outside inversion of these layers. In primates he made the important discovery that the embryos of gibbons and orangutans develop under a decidua capsularis. Thus all great apes, including humans, exhibit interstitial implantation; this is in contrast to other primates where implantation is superficial. CONCLUSIONS: Selenka's work was thorough and brilliantly illustrated. It was an important influence on his contemporaries and was well known to scientists of the following generation. Embryologists continue to advance our knowledge of fetal membranes and placentation in the mouse, but Selenka's work on gibbons is unique and our knowledge of orangutan placentation is restricted to his specimens.

Select nutrients, progesterone, and interferon tau affect conceptus metabolism and development.

Interferon tau (IFNT), a novel multifunctional type I interferon secreted by trophectoderm, is the pregnancy recognition signal in ruminants that also has antiviral, antiproliferative, and immunomodulatory bioactivities. IFNT, with progesterone, affects availability of the metabolic substrate in the uterine lumen by inducing expression of genes for transport of select nutrients into the uterine lumen that activate mammalian target of rapamycin (mTOR) cell signaling responsible for proliferation, migration, and protein synthesis by conceptus trophectoderm. As an immunomodulatory protein, IFNT induces an anti-inflammatory state affecting metabolic events that decrease adiposity and glutamine:fructose-6-phosphate amidotransferase 1 activity, while increasing insulin sensitivity, nitric oxide production by endothelial cells, and brown adipose tissue in rats. This short review focuses on effects of IFNT and progesterone affecting transport of select nutrients into the uterine lumen to stimulate mTOR cell signaling required for conceptus development, as well as effects of IFNT on the immune system and adiposity in rats with respect to its potential therapeutic value in reducing obesity.

Select nutrients in the uterine lumen of sheep and pigs affect conceptus development.

Interferon tau (IFNT) is the pregnancy recognition signal from ruminant conceptuses. IFNT also acts with P4 to induce expression of genes for transport of nutrients, such as glucose (Gluc) and arginine (Arg) into the uterine lumen to activate mechanistic mammalian target of rapamycin (MTOR) cell signaling that stimulates proliferation, migration, gene transcription and mRNA translation by conceptus trophectoderm (Tr). In ewes, Arg and Gluc increase significantly in the uterine lumen between Days 10 and 15 of pregnancy due to increased expression of transporters for Gluc (SLC2A1 and SLC5A1) and Arg (SLC7A2B) by uterine epithelia. Arg and Gluc stimulate proliferation, migration and mRNA translation by Tr. Arg increases expression of GTP cyclohydrolase 1 (GCH1) and IFNT mRNAs while Arg and Gluc increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1 mRNAs and proteins by Tr cells. GCH1 is required for synthesis of tetrahydrobiopterin, an essential cofactor for all NOS isoforms. Arg is metabolized to nitric oxide and polyamines that increase proliferation and migration of Tr cells. In pigs, Gluc, Arg, leucine (Leu) and glutamine (Gln) increase in the uterine lumen between Days 12 and 15 of pregnancy due to enhanced expression of transporters for Gluc and amino acids. Transporters for Gluc in porcine uterine LE (SLC2A1) and conceptus trophectoderm (SLC2A2) are abundant. Transporters for glutamate and neutral (SLC1A1, SLC1A4) and cationic (SLC7A1, SLC7A2, SLC7A7, SLC7A9) amino acids are expressed in uterine LE and SLC7A3 mRNA is expressed in conceptus Tr. Arg and Leu increase MTOR cell signaling and proliferation of pig Tr, as do Gluc and fructose. Azaserine, an inhibitor of hexosamine biosynthesis, inhibits effects of Gluc and fructose. Thus, select nutrients in the uterine lumen affect gene transcription and mRNA translation to affect conceptus development.

Genomic imprinting mechanisms in embryonic and extraembryonic mouse tissues.

Imprinted genes in mice and humans mainly occur in clusters that are associated with differential DNA methylation of an imprint control element (ICE) and at least one nonprotein-coding RNA (ncRNA). Imprinted gene silencing is achieved by parental-specific insulator activity of the unmethylated ICE mediated by CTCF (CCCTC-binding factor) binding, or by ncRNA expression from a promoter in the unmethylated ICE. In many imprinted clusters, some genes, particularly those located furthest away from the ICE, show imprinted expression only in extraembryonic tissues. Recent research indicates that genes showing imprinted expression only in extraembryonic tissues may be regulated by different epigenetic mechanisms compared with genes showing imprinted expression in extraembryonic tissues and in embryonic/adult tissues. The study of extraembryonic imprinted expression, thus, has the potential to illuminate novel epigenetic strategies, but is complicated by the need to collect tissue from early stages of mouse development, when extraembryonic tissues may be contaminated by maternal cells or be present in limited amounts. Research in this area would be advanced by the development of an in vitro model system in which genetic experiments could be conducted in less time and at a lower cost than with mouse models. Here, we summarize what is known about the mechanisms regulating imprinted expression in mouse extraembryonic tissues and explore the possibilities for developing an in vitro model.

Semiautonomous development of the extraembryonic membranes in the chicken embryo.

Based on an old paradigm that the extra-embryonic membranes develop semiautonomously from the embryo, it can also be postulated that subembryonic fluid (SEF) will be formed semiautonomously against embryonic growth, because the formation of SEF is mediated by the yolk sac membrane. In this study, we interfered in the development of SEF or the embryo. The acoustic resonance technique (which measures the resonant frequency of an excited egg) was used as a nondestructive tool to monitor the development of SEF. In the first experiment, in which the embryo was killed chemically with NaN3, it was proven that the formation of SEF continued, even when the embryo was killed after the initiation of the growth of the yolk sac membrane. In the second experiment, in which the development of SEF was inhibited chemically with amiloride, it was shown that the embryo developed further, although SEF formation was inhibited. In the last experiment, it was shown that the age of the flock affected the development of the embryo and the sudden decrease of the resonant frequency in a different way. However, some presetting conditions, such as storage, may affect both in a similar way. Our results further strengthen the idea that the formation of SEF develops semiautonomously against embryonic development by using the nondestructive acoustic resonance technique as an indirect method to monitor yolk sac membrane formation.

Quantitative and morphological assessment of early gestational sacs using three-dimensional ultrasonography.

OBJECTIVE: Our main objective was to determine the value of three-dimensional ultrasonography (3DUS) and Virtual Organ Computer-aided AnaLysis (VOCAL) in the evaluation of gestational sac volume and morphology during early pregnancy. METHODS: Twenty-eight normal early pregnancies were scanned approximately every 2 weeks using transabdominal (TAS) and transvaginal (TVS) sonography. The VOCAL technique was used to create computerized surface models to classify gestational sac shapes as discoid or ellipsoid. Serial sac volume changes were analyzed using repeated measures ANOVA. Bland-Altman plots determined examiner bias and limits of agreement (LOA) for sac volume measurements. Gestational sac volumes were compared between the two-dimensional (2D) ellipsoid and VOCAL techniques. Differences between volume measurements were tested using the two-tailed paired t-test with statistical significance at the P < 0.05 level. RESULTS: Each subject was examined at a mean +/- SD menstrual age of 7.9 +/- 0.6 weeks (Scan 1), 9.9 +/- 0.6 weeks (Scan 2), and 11.9 +/- 0.6 weeks (Scan 3). Sac volumes significantly increased over time from 22 +/- 11 mL at Scan 1, to 57 +/- 21 mL at Scan 2 and 116 +/- 35 mL at Scan 3 (P < 0.001). Predominant sac shapes were classified as ellipsoid (76.2%) or discoid (23.8%). Additional descriptors included: concave (60.7%), irregular (53.6%), or smooth (7.1%), with 19% of the overall group having more than one additional shape attribute. Clinically acceptable volume measurement bias and agreement were found for the following comparisons: (1) TAS versus TVS; (2) interobserver volume measurements; and (3) intraobserver volume measurements. The VOCAL technique yielded slightly greater sac volumes (64 +/- 45.4 mL) when compared to the 2D ellipsoid model (48.6 +/- 36.8 mL) (28.9 +/- 24.3% (95% limit of agreement range, - 18.7 to 76.5%), P < 0.001). CONCLUSIONS: Reproducible sac volume measurements can be obtained using VOCAL with either TAS or TVS. Early gestational sacs variably appear as discoid or ellipsoid structures with a concave indentation from the placenta. Sac volumes can be underestimated if an ellipsoid shape is assumed. Morphological and quantitative analysis of the gestational sac may provide baseline parameters for studying patients at risk for early pregnancy failure.

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.

Early placental insulin-like protein (INSL4 or EPIL) in placental and fetal membrane growth.

Early placental insulin-like protein (INSL4 or EPIL) is a member of the insulin superfamily of hormones, which is highly expressed in the placenta. We have confirmed this at term and shown it to be expressed by the maternal decidua. Although an abundance of locally acting growth factors are produced within the uterus during pregnancy, we hypothesized that INSL4 plays an important role in fetal and placental growth. We have demonstrated with cell lines and primary cells that it has a growth-inhibitory effect by causing apoptosis and loss of cell viability. We used primary amniotic epithelial cells for flow cytometry to show that INSL4 caused apoptosis, which was dose-related and significant (P < 0.05) at 50 ng/ml. This was confirmed by measurement of the nuclear matrix protein in the media. In comparison, relaxin treatment (up to 200 ng/ml) had no effect on apoptosis. The addition of INSL4 (3-30 ng/ml) also caused a loss of cell viability, although it had no effect on the numbers of cells at different phases of the cell cycle. Placental apoptosis is an important process in both normal placental development and in fetal growth restriction. Therefore, an in vivo clinical correlate was sought in fraternal twins exhibiting discordant growth. Expression of the INSL4 gene was doubled in the placenta of the growth-restricted twin compared to the normally grown sibling, suggesting that it may be linked to a higher level of apoptosis and loss of cell viability and, therefore, that it may contribute to fetal growth restriction.

Early maternal changes contributing to the formation of the chorioallantoic and yolk sac placentas in rat: a morphological study.

In order to determine the maternal changes contributing to the formation of the chorioallantoic and yolk-sac placentas, rat gestation sites were examined by light and electron microscopy on days 7 through 10 of pregnancy. On day 7, the implantation chamber showed different compartments and contained the blastocyst in the antimesometrial chamber. The epithelial lining of the implantation chamber disappeared at the antimesometrial chamber, transformed into disintegrated cells in the mesometrial chamber, and showed signs of the programmed cell death in the decidual crypt. On day 8, the mesometrial chamber lumen contained red blood cells and it was continuous with subepithelial sinusoids. The endothelial cells lining the mesometrial sinusoids also showed some characteristics of the sprouting type angiogenesis such as hypertrophy and cell proliferation. While the yolk-sac placental circulation was more obvious with participation of the giant trophoblasts at the antimesometrial pole of the conceptus on day 9, the antimesometrial cells showed autophagic degeneration after the formation of the chorioallantoic placenta on day 10. The contribution of the regional cell death and angiogenesis to form both of the two placentas are discussed.

Ontogeny of aquaporins 1 and 3 in ovine placenta and fetal membranes.

A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP(3)) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145-150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP(3)were found to be the trophoblast cells. Some AQP(3)was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP(1)was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP(3)expression is consistent with known ovine placental permeabilities to water, glycerol and urea.

Dynamic study of the extraembryonic vascular network of the chick embryo by fractal analysis.

Fractal analysis is widely used in many scientific fields, including the study of vascularization. It is a convenient method that defines the complexity of natural structures. The chorioallantoic membrane of the chick embryo is a standard experimental model for the study of vasculogenesis and angiogenesis. The aim of this investigation was to demonstrate that fractal geometry is more appropriate than any other method to describe and analyse the evolution of a vascular network, i.e. the extraembryonar vascular network of the chick embryo. We used an original methodology to evaluate the complexity of this network in the first stages of embryo development (day 3 until 6). We demonstrated an increase of fractal dimension, indicating an increasing complexity of the vascular tree, until an asymptotical value of about 1.70 at day 4. The fractal approach is more accurate than other usual semi-quantitative or quantitative methods evaluating the complexity of a growing vascular tree.

Development and structure of the placenta and fetal membranes of nonhuman primates.

This survey examines placental and fetal membrane structure, function, and development in nonhuman primates. It will focus on these aspects of fetal membranes in species exemplifying the two main types of primate placentation. The chorioallantoic placentas of most strepsirhine primates (e.g., lorises and lemurs), are diffuse, villous and epitheliochorial in structure. Villi are stout, usually unbranched and interdigitate with indentations in the uterine lining. Early in gestation trophoblast cells are columnar but with advancing gestational age considerable thinning of the interhemal membrane occurs. The villous tips have indented regions that appear to be involved in absorption of histotroph. Accessory placental structures known as areolae or chorionic vesicles occur in many strepsirhines. These are cup-shaped specializations that develop opposite the mouths of uterine glands and appear active in absorption of glandular secretions. In addition, most strepsirhines are characterized by a large yolk sac early in gestation, a transient choriovitelline placenta, and most have a large allantoic sac. In strepsirhines, amniogenesis occurs by folding. In contrast, the chorioallantoic placentas of most haplorhines are discoidal, villous, and hemochorial. The species emphasized in the review are macaque monkeys. The placental villi are covered by a layer of syncytiotrophoblast; the latter is active in maternal-fetal transport and hormone synthesis. In some haplorhines, extravillous trophoblast invades deeply into the endometrium and also into spiral arteries supplying the placenta. Other fetal membranes include 1) nonvillous chorion (chorion laeve); there is evidence supporting both an absorptive and synthetic role for trophoblast of the chorion laeve; 2) yolk sac, which is unusual in that a secondary yolk sac is formed. The yolk sac has both synthetic and absorptive functions in early gestation; and 3) amnion; amniogenesis in haplorhines occurs by cavitation.

Evaluation of normal gestational sac growth: appearance of embryonic heartbeat and embryo body movements using the transvaginal technique.

A cross-sectional transvaginal ultrasound study was conducted in 137 normal pregnancies with gestational ages ranging from 5-12 weeks. Several biometric measurements were obtained throughout pregnancy, including the three diameters of the gestational sac, the crown-rump length, and the yolk sac. In addition, the appearance of the embryo heartbeat and embryo body movements were evaluated. Linear relationships were found between the mean gestational sac diameters and gestational age (r = 0.911; P less than .00001) and between mean gestational sac growth and crown-rump length growth (r = 0.926; P less than .0001). A gestational sac could be identified at 5 weeks' gestation; embryo heartbeat was imaged when the mean gestational sac diameter measured 2 cm, and embryo body movements could be seen when the mean gestational sac diameter reached 3 cm. In the present study, embryo heartbeat was identifiable after 6 weeks and 4 days with a sensitivity of 100%, specificity of 93.1%, positive predictive value of 96.9%, and negative predictive value of 100%. The embryo body movements, which were absent before 7 weeks' gestation, were observed after 8 weeks' gestation with a sensitivity of 100%, specificity of 92.8%, positive predictive value of 94.3%, and negative predictive value of 100%. With identification by transvaginal sonographic evaluation, the following can serve as markers of normal embryo growth: a mean gestational sac diameter greater than 2 cm in the presence of the embryo heartbeat, or a mean sac diameter measurement greater than 3 cm in the presence of embryo movement.