Placentation in cloned cattle: Structure and microvascular architecture

To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies established by somatic nucleus transfer (NT), the umbilical cord and fetal membranes were edematous. Placentomal fusion was common, resulting in increased size and a decreased number of placentomes. Extensive areas of the chorioallantoic membrane were devoid of placentomes. An increased number of functional or accessory microcotyledons (< 1 cm) were present at the maternally oriented surface of fetal membranes. Extensive areas of extravasated maternal blood were present within the placentomes and in the interplacentomal region. The crypts on the caruncular surface were dilated and accommodated complexes of more than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops and sinusoidal dilations as in normal pregnancies. At the materno-fetal interface, however, the trophoblast and uterine epithelium had normal histology. In conclusion, the NT placentas had a range of pathomorphological changes; this was likely associated with the poor clinical outcome of NT pregnancies. (c) 2007 Elsevier B.V. All rights reserved.

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

Background: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse. © 2013 Pereira et al.

Treatment of Nuclear-Donor Cells or Cloned Zygotes with Chromatin-Modifying Agents Increases Histone Acetylation But Does Not Improve Full-Term Development of Cloned Cattle

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA + TSA or HH + VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH + VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.

Developmental and Epigenetic Anomalies in Cloned Cattle

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Caracterização da fusão caruncular em gestações naturais e de conceptos bovinos clonados

Barreto R.S.N., Miglino M.A., Meirelles F.V., Visintin J.A., Silva S.M., Burioli K.C., Fonseca R., Bertan C., Assis Neto A.C. & Pereira F.T.V. 2009. [Characterization of the caruncular fusion in gestations of natural and cloned bovine conceptuses.] Caracterizacao da fusao caruncular ern gestacoes naturais e de conceptos bovinos clonados. Pesquisa Veterinaria Brasileira 29(10):779-787. Laboratorio de Morfofisiologia da Placenta e Embriao, Faculdade de Zootecnia, Universidade Estadual Paulista, SP294 Km 651, Dracena, SP 17900-000, Brazil. E-mail: fverechia@dracena.unesp.brThe objective of the study was to compare the characteristics of the caruncular fusion in gestations of non-cloned and cloned conceptuses. The non-cloned conceptuses were divided according to the gestation period: Group 1 (2 to 3 months; n=9),II (4 to 6; n=9); III (7 to 8; n=10) and IV (9 n=7). The cloned conceptuses formed the Group V: 9 months; n=4. The caruncles were observed macroscopically (number and dimensions: length, width and height), microscopically and submitted to statistical analysis (5% of significance). We observed three types of macroscopic caruncular fusions: oval (morphologically normal); two united adjacent caruncles and the lobulated type, characterized by regions with several united caruncles presenting a false fusion or deformation of the caruncular parenchyma. The length of the caruncles was 1.55 +/- 0.57; 2.45 +/- 0.55; 4.66 +/- 2.0 and 5.72 +/- 1.90cm for the groups 1, 11, 111, IV respectively. As for the height, the caruncles presented a lineal growth during the gestation: 0.40 +/- 0.15; 0.57 +/- 0.21; 1.0 +/- 0.48 and 1.80 +/- 0.91cm, for the respective groups 1, 11, 111 and IV. The width of the caruncles was similar between the groups I and 11 (0.97 +/- 0.30 e 1.42 +/- 0.71 cm) and the groups III and IV (2.68 +/- 1.22 and 3.52 +/- 1.16cm). When the group V was compared to the IV, the caruncles of the group V presented a larger length (5.72 +/- 1.90 vs. 7.88 +/-.13cm) and width (3.52 +/- 1.16 vs. 4.93 +/- 1.46cm), however they were similar in height (1.80 +/- 0.91 and 2.25 +/- 0.67cm). We verified that in gestations of cloned conceptuses the caruncles presented a larger development than in gestations of non-cloned conceptuses. The fusioned caruncles presented measurements statistically similar to the isolated ones in all the parameters and groups. Under light microscopy, we observed the formation of a stromal axis from the basis of the caruncle to the apex of the fusional fissure, with the histological constitution similar to the endometrial stroma. Three microscopic shapes were also unpublished defined: true fusion with a single axis evident below the fusional fissure; pseudofusion with a double axis in H shape and false fusion with absence of the axis. The first two formats were associated to the oval and lobulated caruncles and the last one to the false fusion with deformation of the caruncle parenchyma. The fusional axis increased in size along the gestation among the groups I, II, III and IV. The group V presented a larger length and width of the axis when compared to the group IV. Thus, in gestations of cloned conceptuses a destruction of the lateral epithelium of the caruncles is associated to an incompetence in the maternal-fetal interdigitation, that compromises the cotyledonary fusion. We suggest that, in gestations derived of cloned conceptuses, the increase of the size of the caruncular fusions is possibly associated to a compensatory mechanism for the metabolic exchanges between mother and fetus, in reason of the smallest number of isolated caruncles.

Transporte placentário via cavéola na placenta de bovinos clonados e transgênicos

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1 M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1 M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV-2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2) are part of the caveolae composition and important structures related to the molecule transfer to the fetus, nourish it through endocytosis and pinocytosis.

Apoptose em placentônios bovinos de gestações de conceptos naturais e de transgênicos clonados

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus)

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such `labour-intensive technology`.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.