Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium.

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.

Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants.

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation and likely contribute to the remarkable diversity of placental structures. Independent capture events have been identified in primates, rodents, lagomorphs, and carnivores, where they are involved in the formation of a syncytium layer at the fetomaternal interface via trophoblast cell-cell fusion. We searched for similar genes within the suborder Ruminantia where the placenta lacks an extended syncytium layer but displays a heterologous cell-fusion process unique among eutherian mammals. An in silico search for intact envelope genes within the Bos taurus genome identified 18 candidates belonging to five endogenous retrovirus families, with one gene displaying both placenta-specific expression, as assessed by quantitative RT-PCR analyses of a large panel of tissues, and conservation in the Ovis aries genome. Both the bovine and ovine orthologs displayed fusogenic activity by conferring infectivity on retroviral pseudotypes and triggering cell-cell fusion. In situ hybridization of placenta sections revealed specific expression in the trophoblast binucleate cells, consistent with a role in the formation--by heterologous cell fusion with uterine cells--of the trinucleate cells of the cow and the syncytial plaques of the ewe. Finally, we show that this gene, which we named "Syncytin-Rum1," is conserved among 16 representatives of higher ruminants, with evidence for purifying selection and conservation of its fusogenic properties, over 30 millions years of evolution. These data argue for syncytins being a major driving force in the emergence and diversity of the placenta.

Changes in WNT signaling-related gene expression associated with development and cloning in bovine extra-embryonic and endometrial tissues during the peri-implantation period.

We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses.

Comparative implantation and placentation.

In all placental mammals, the establishment of an intimate contact between the embryo and the mother follows a succession of common critical steps whose chronology and timing may considerably vary from species to species. These processes present a great diversity based on the anatomy and the histology of the uterus, the developmental stage of the embryo at the time of implantation, and the endocrine and molecular interactions between the uterine and the embryonic tissues. This paper provides an overview of the general mechanical, endocrinological and cellular aspects involved in implantation in mammals with an emphasis on domestic species.

Myogenesis is delayed in bovine fetal clones.

We have recently reported that maturation of the skeletal muscle is delayed in cloned calves during their first year postnatally. This delay could originate from perturbations in fetal myogenesis. The aim of this study was to evaluate the developmental characteristics of muscle in clones versus animals derived from conventional reproduction. We have characterized the anatomical and biochemical properties of the Semitendinosus muscle of clones versus controls at day 60 and day 260. We have analyzed the contractile and metabolic properties of muscle fibers by measuring the abundance of myosin heavy chain (MyHC) isoforms and activities of metabolic enzymes (LDH, PFK, COX, CS, ICDH), respectively. The spatial repartition of some components of the extracellular matrix (collagen types I, IV, VI, chondroitin-6-sulfate, decorin, and tenascin-X) was also studied. At day 60 we found lower numbers and structural organization of fibers, and a delay in the setup of the extracellular matrix. IGF-2 transcript abundance was lower in clones than in their controls. There was no difference in the expression of VEGF (a growth factor regulating vascularization and myogenesis) and its receptor. At day 260 the muscles of fetal clones have not reached the same degree of differentiation than controls as shown by their lower energy metabolisms and their MyHC pattern. These results show for the first time that disturbances in myogenesis occur early in fetal life in cloned cattle.

Cellular organization of the trophoblastic epithelium in elongating conceptuses of ruminants.

We present a computational approach to analyze the cellular organization during the elongation process of the ovine conceptus. First, we selected a set of mathematical descriptors to quantify cell geometry and cell neighborhood within the external epithelial layer of the conceptus: the trophoblast. Second, we established a hybrid image segmentation framework, and analyzed the extracted features with statistical tools to describe and compare the spatio-temporal dynamics of cellular organization within this epithelium. The main results indicated that the average geometry and neighborhood of the trophoblast cells are relatively stable from a sampling position to another and from a stage of development to another. Further, their elongation axes are randomly distributed. The cellular organization fits the Poisson's process. Moreover, no clustering structures or grid-like regular point patterns are found inside the studied cell population. This suggests that the trophoblast elongation observed in ruminants is not due to the geometrical change of cell shape but might be the consequence of cell addition associated with peculiar plans of cell division or intercalation.

Cellular features of the extra-embryonic endoderm during elongation in the ovine conceptus.

The extraembryonic endoderm of the elongating ovine conceptus was analyzed by scanning and transmission electron microscopy and by whole mount actin staining and immunofluorescence. Morphological and functional differences between the visceral endoderm (VE), the founding cell layer, and the parietal endoderm (PE) are presented. During the elongation process, the PE differentiated to fusiform multinucleated cells aligned parallel to the elongation axis of the conceptus, whereas the VE cells retained the aspect of typical epithelial cells. The multinucleated PE cells however, expressed cellular and nuclear markers typical of endodermal and polarized epithelial cells. The proteins of the extracellular matrix, laminin, and fibronectin, were specifically expressed in the PE. The presence of pairs of nuclei linked by mid-bodies positively stained with tubulin antibodies, indicated that the syncytial differentiation of the PE was due to karyokinesis which was not followed by cytokinesis rather than by cell fusion.

The placenta of bovine clones

Background: Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer (SCNT) in various mammals has increased significantly. The incidence of pregnancy failure and fetal death, however, is still very high, whatever the species, and impairs the commercial development of this technology, even in the bovine species where the success rates are highest compared to other species. Review: In cattle, most gestation losses are initially due to abnormal implantation and poor placental development leading to fetal demise during the early post-implantation period (30 to 70 days of pregnancy). Thereafter, in continuing pregnancies, losses usually occur in the last third of gestation and affect about 25% of the on-going pregnancies, with very large differences according to phenotype. These are currently referred to as the Large Offspring Syndrome (LOS), Large Placenta Syndrome or Abnormal Offspring Syndrome. In all cases, the placenta appears to be central to the onset of the pathology, with placentomegaly and hydrallantois being the most common features. Clinically, transabdominal ultrasound monitoring of fetal and placental development as well as the measurement of maternal plasma concentrations of pregnancy associated glycoproteins (PAG) are recommended in order to monitor the pregnancies. Humane termination of the pregnancies by Caesarian section or slaughtering of the affected animals is recommended when the pathology onset is diagnosed more than 2 weeks prior to term. Underlying mechanisms include abnormal placental vascularization, which is present early in SCNT placental development. Enzymatic response to oxidative stress is also modified. In the first trimester, several genes expressed in the trophoblast have been found to be differentially expressed between SCNT and control conceptuses, including placental lactogen (PL), the PAG, prolactin related protein-1 (PRP-1) and Dickkopf-1(DKK-1), to name a few. All these proteins are expressed in the Binucleate cells (BNC) of the trophoblast and thus, indicate that BNC function may be affected in SCNT from very early in gestation, thereby compromising placental development. Later in pregnancy, it has been shown that transplacental exchanges are disturbed, in particular those related to glucose metabolism. Moreover, endocrine function is altered compared to controls, with decreased estrogen secretion and modifications in PAG secretion, resulting in largely elevated maternal plasma concentrations. Gene expression patterns are affected, with most prominent functional effects involving cell cycle, cell signaling pathways, molecular transport, DNA replication, recombination and repair. Most of the affected genes are downregulated. Finally, many of the pathologies reported with SCNT pregnancies resemble abnormalities reported with either mutations or deletions of imprinted genes or dysregulation of imprinted gene expression, and the expression of several imprinted genes have been shown to be abnormal in SCNT placenta. Conclusions: In conclusion, pregnancy failure after SCNT is due to multiple factors affecting, implantation, placental development, morphology, vascularization, responses to oxidative stress and the epigenetic control of gene expression. If abnormal nuclear reprogramming may induce long term effects in bovine SCNT, these effects may also be due to fetal programming due to abnormal placental function and perturbed fetal development.

Transcriptome profiling of the tubular porcine conceptus identifies the differential regulation of growth and developmentally associated genes.

Gastrulation and trophectoderm elongation of the porcine conceptus coincide with peak conceptus estrogen secretion from gestational day 11 to day 12. The current study aim was to identify genes required for elongation by defining the transcriptome profile of this dynamic tubular stage. The gastrulation and proliferative status of ovoid, tubular, and filamentous conceptuses were also examined. Polarization of the embryonic disc and growth throughout the conceptus were evident. An unamplified and two distinct amplified serial analysis of gene expression (SAGE) libraries were generated from tubular conceptus mRNA. Comparing the three libraries at 12,000 tags/library indicated small-amplified RNA-SAGE was a reliable amplification procedure. The unamplified library was increased to 42,415 tags and statistical analyses of tag frequencies with previously generated ovoid and filamentous libraries revealed the differential expression (P < 0.05) of 483 and 364 tags between ovoid:tubular or tubular:filamentous libraries, respectively. Annotated transcripts known to be involved in development and also potentially regulated by estrogen (cytokeratins 8 and 18, stratifin, midkine, and glycolytic enzymes) were further analyzed by real-time PCR. The majority of glycolytic enzyme transcripts were constitutively expressed or downregulated at the filamentous stage. Likewise, cytokeratin mRNAs were less abundant in filamentous conceptuses, whereas stratifin and midkine were more abundant in tubular conceptuses. Analysis of protein revealed distinct expression patterns for cytokeratin 18, stratifin, and midkine. The function(s) of these factors and potential modulation by estrogen clearly needs to be elucidated to understand their physiological role in normal conceptus development.

Staging of ovine embryos and expression of the T-box genes Brachyury and Eomesodermin around gastrulation.

The high rates of embryonic mortalities which follow in vitro production of ruminant embryos have emphasized the need for increased knowledge of early development. It is likely that early failures in embryonic development and placenta formation involve abnormal differentiation of mesoderm. The aim of this study was to investigate the pattern of expression of two T-box genes known to control the gastrulation process, Brachyury and Eomesodermin, by whole-mount in situ hybridization. To allow a more precise comparison of both expression patterns between embryos, we describe a new staging of pre-implanted ovine embryos by gross morphology and histology from pre-gastrulation stages to the beginning of neurulation. In pre-streak embryos primitive mesoderm cells delaminated in between the primitive endoderm and the epiblast. At that stage, no expression of Brachyury or Eomesodermin could be detected in the embryos. Early expression of both T-genes was observed by the early-streak stages in epiblast cells located close to the presumptive posterior pole of the embryos. Later on, during gastrulation both genes followed a pattern of expression similar to the ones described in other mammals. These observations suggest that other genes, which remain to be identified, are responsible for extra-embryonic mesoderm differentiation in ruminant embryos.

Obtaiment of Pudu (Pudu pudu) deer embryos by the somatic nuclear transfer technique

RESUMEN: Los objetivos de este trabajo fueron los de producir embriones de pudú, obtenidos por la transferencia de núcleos de fibroblastos de la oreja de pudú en ovocitos de un rumiante domésticos que es el bovino. Para posteriormente en un trabajo futuro proceder a la transferencia de embriones de pudú, al útero de hembras receptoras sincronizadas de otra especie. Se obtuvieron biopsias de 1 mm aproximadamente del borde externo de la orejas de dos ciervos pudu machos del jardín zoológico Buin-Zoo, Santiago de Chile. Las líneas celulares han sido establecidas y conservadas según los protocolos utilizados para las bovinos. Los ovocitos son obtenidos por punción del complejo cúmulos-ovocito (COC).desde ovarios de vacas recuperados del matadero. Cada ovocito es enucleado y fusionado con un fibroblasto aislado insertado bajo la zona pelúcida. La fusión de membranas celulares es obtenida por choques eléctricos. En cuanto a la cronología, observamos que al segundo día se forma una etapa de dos blastómeras, al tercer día mórulas de 8 a 16 células, y desde el cuarto día se ha diferenciado como blastocisto, el cuál al séptimo día termina por eclosionar de la zona pelúcida.La obtención de blastocistos embrionarios indica que es posible obtener embriones de pudú mediante clonaje heteroespecífico, aunque, el porcentaje de éxito obtenido es relativamente bajo. Queda aun por verificar la viabilidad de los embriones así obtenidos después de la transferencia in útero.

Attempts at Applying Cloning to the Conservation of Species in Danger of Extinction / Intentos de Aplicar la Clonación en la Conservación de Especies en Riesgo de Extinción

The somatic cloning by transfer of the nuclei of differentiated adult cells to previously enucleated oocytes is a promising technique for the production of embryos of high genetic value. The better mastering of somatic cloning gives us the possibility to produce embryos from endangered species. The huemul is an Andean native deer, that has been declared an endangered species, it holds a great patrimonial value and it is a Chilean national emblem. In Chile the huemul has the status of protected animal on thirteen Parks and National Reserves managed by Corporacion Nacional Forestal (CONAF). This protection, however, is considered insufficient due to the few geographical connections between the different protected areas. Furthermore, a great proportion of these areas are not subjected to use or they do not constitute adequate habitats. Many authors have proposed that the use of biotechnological methods in reproduction and assisted procreation may help conservational programs orientated to the protection of deer species threatened by extinction. All the anterior prompted us to initiate this study concerning the production of cloned huemul embryos.

El clonaje somático por transferencia del núcleo de células diferenciadas adultas a un ovocito, al que se le ha extraído el núcleo (enucleado), es una técnica prometedora para la producción de embriones de alto valor genético. El mejor dominio del clonaje somático da la posibilidad de producir embriones de especies amenazadas de extinción. El huemul es un ciervo andino autóctono, declarado como especie en peligro de extinción. tiene un gran valor patrimonial, y es emblema de la nación chilena. En este país, el huemul se encuentra protegido en trece Parques y Reservas Nacionales, manejadas por la Corporación Nacional Forestal (CONAF). Sin embargo, su protección se considera insuficiente debido a la baja conectividad entre las áreas protegidas y además, una gran proporción de estas áreas no son utilizadas o no constituyen un hábitat adecuado. Para las especies de cérvidos en vías de extinción el uso de biotecnología reproductiva y métodos de procreación asistida, según varios autores, pueden ayudar a los programas de conservación. Las técnicas clásicas de producción de embriones basados en superovulación, inseminación artificial y transferencia embrionaria,en los cérvidos, han resultado muy dificultosa. Esto, sumado a las características del huemul, que no permite su estabulación en cautiverio, nos ha movido a iniciar un estudio tendiente a la producción de embriones clonados de esta especie.

Clonación, producción de quimeras y células pluripotenciales / Clonation, production of chimeras ans pluripotencial cells

La Biología del Desarrollo y la Biotecnología han avanzado enormemente en los últimos años. Los estudios que se están realizando actualmente con nuevas técnicas moleculares, nos conducen a una nueva era en el diagnóstico y en el tratamiento de muchas enfermedades congénitas. Actualmente, se ha logrado aplicar los conocimientos derivados de ella, con incipiente éxito, en los aspectos productivos y también en la terapia de distintas enfermedades del Hombre y de otros animales y, por último, existen importantes perspectivas en la protección de especies amenazadas. En este Review, se analizan algunos de los aspectos técnicos más interesantes de la clonación, producción de quimeras y obtención de células pluripotenciales, como también los objetivos y proyecciones en el campo veterinario y de la salud animal y humana.