Rodrigo da Silva Nunes Barreto

Modulation of Maternal Immune System During Pregnancy in the Cow

There is a molecular crosstalk between the trophoblast and maternal immune cells of bovine endometrium. The uterine cells are able to secrete cytokine/chemokines to either induce a suppressive environment for establishment of the pregnancy or to recruit immune cells to the endometrium to fight infections. Despite morphological differences between women and cows, mechanisms for immune tolerance during pregnancy seem to be conserved. Mechanisms for uterine immunesuppression in the cow include: reduced expression of major histocompatability proteins by the trophoblast; recruitment of macrophages to the pregnant endometrium; and modulation of immune-related genes in response to the presence of the conceptus. Recently, an eGFP transgenic cloned embryo model developed by our group showed that there is modulation of foetal proteins expressed at the site of syncytium formation, suggesting that foetal cell can regulate not only by the secretion of specific factors such as interferon-tau, but also by regulating their own protein expression to avoid excessive maternal recognition by the local immune system. Furthermore, foetal DNA can be detected in the maternal circulation; this may reflect the occurrence of an invasion of trophoblast cells and/or their fragment beyond the uterine basement membrane in the cow. In fact, the newly description of exosome release by the trophoblast cell suggests that could be a new fashion of maternal-foetal communication at the placental barrier. Additionally, recent global transcriptome studies on bovine endometrium suggested that the immune system is aware, from an immunological point of view, of the presence of the foetus in the cow during early pregnancy.

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.

Rabbit olfactory stem cells. Isolation protocol and characterization.

PURPOSE To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Gene expression in placentation of farm animals: an overview of gene function during development.

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish - a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] - for humans, and tissue-specific genes database (TiSGeD) - for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta.

Decellularized bovine cotyledons may serve as biological scaffolds with preserved vascular arrangement

Technically produced scaffolds are common to establish transplantable tissues for regenerative medicine, but also biological ones that are closer to the natural condition become of interest. Placentas are promising, because they represented available, complete organs with rich extracellular matrix (ECM) and well‐developed vasculature that easily could build anastomoses to a hosts organ. Only placentas from larger animal models such as the bovine meet the dimensions large enough for most organs but are not adequately described yet. We here studied the nature of the ECM in 27 natural and decellularized bovine cotyledons, that is, the fetal part of the placentomes, by means of histology, immunohistochemistry, and electron microscopy. Successful decellularization was done by perfusion with 0.01%, 0.1%, and 0.5% sodium dodecyl sulfate each and subsequent immersion in 1% Triton X‐100, resulting in a removal of cells and DNA, whereas the structure of the allantochorionic surface and villi was preserved. Although some fibres disappeared, also the arrangement of the main ECM proteins was largely similar before and after decellularization: Along the larger vessels, a densely packed network of thick fibres occurred, organized in layers without cells or spaces in between. Collagen IV, fibronectin, and laminin contributed to those areas. In contrast, collagen I and III characterized the meshwork of medium‐sized and thin fibres in the mesenchyme, respectively. In conclusion, decellularized bovine cotyledons indeed had characteristics of a biological scaffold and provide an interesting alternative to develop large‐scale scaffolds with complex vascular architecture for tissue engineering purposes.

Optimization of Canine Placenta Decellularization: An Alternative Source of Biological Scaffolds for Regenerative Medicine

Due to the scarcity of tissues and organs for transplantation, the demand for bioengineered tissues is increasing with the advancement of technologies and new treatments in human and animal regenerative medicine. Thus, decellularized placental extracellular matrix (ECM) has emerged as a new tool for the production of biological scaffolds for subsequent recellularization and implantation for recovery of injured areas or even for replacement of organ and tissue fractions. To be classified as an ideal biological scaffold, the ECM must be acellular and preserve its proteins and physical features to be useful for cellular adhesion. In this context, we developed a process of decellularization of canine placentas with 35 and 40 days of gestation using dodecyl sulfate sodium under immersion and agitation in sterile conditions. Before use of this scaffold in recellularization processes, the decellularization efficiency needs to be confirmed by the absence of cellular content and an irrelevant amount of reminiscent DNA. Both vasculature architecture and ECM proteins, such as collagen types I, III, and IV, laminin, and fibronectin, were preserved with our method. In this way, we established a new biological scaffold model that could be used for recellularization in regenerative medicine of tissues.

115 ROLE OF THE OVARY AND UTERUS FOR THE PLAINS VISCACHA (LAGOSTOMUS MAXIMUS MAXIMUS, CHINCHILLIDAE) REPRODUCTION

A dogma of mammalian reproduction states that primordial germ cells in females are restricted to the intrauterine phase and only small portions of oocytes are available for ovulation during the adult life. Among the rare exceptions to this rule is the plains viscacha. It polyovulates up to 800 oocytes per cycle, from which 10 to 12 are implanted, but only 1 to 2 conceptuses survive. To better understand the main mechanisms involved in these patterns of super-ovulation, super-implantation, and embryonic loss in the viscacha, we conducted an analysis of the ovary and uterus of pregnant females and their conceptuses. Pregnant females (n=16) of ~50 and 90 days of gestation (early to mid-gestation) were selected for conceptus recovery. Hemi-ovariohysterectomy was performed following surgical and anaesthetic protocols used for laboratory animals and the conceptuses collected. Female fetuses of Day 50 (n=2) and 90 (n=1) of gestation were obtained from Estación de Cría de Animales Silvestres, Buenos Aires, Argentina. The reproductive organs were investigated by means of gross morphology, histology (hematoxilin and eosin), stereology (quantification of the volume of the ovary and number of ovary follicles), immunohistochemistry (PCNA, Oct-4, VEGF, and Caspase-3), and transmission electron microscopy. In the Day 50 fetal samples, the ovaries had an ovoid shape with smooth surface without apparent folds. First steps of subdivision were observed in the ovary of fetus of 90 days. The total volume of all fetal ovaries was of 4.8mm2 and a coefficient of variation (CV) of 0.32. The ovaries of adult individuals had remarkable invaginations with surface projections and were small, asymmetrical, and dorsoventrally flattened with a mean of 77.6mm3 (CV=0.47). Only adult females had differentiation of germ cells. Primordial follicles had a mean of 9.9×105 (CV=0.19), representing 93% of the total number of ovarian follicles. The mean of primary follicles was 3.05×104 (CV=0.36), whereas for secondary follicles it was 2.75×104 (CV=0.50), each representing 3% of all ovarian follicles. The number of antral follicles in several stages of development was 8.64×103 (CV=0.75), representing 1% of the follicles. Primordial follicles expressed pluripotency (Oct-4+) and proliferation (PCNA+) markers, as well as the primary follicles. The cells did not react for Caspase-3 as marker for apoptosis. Variations regarding to the vascularization of the different regions of the uterine horn were observed, which were more intense and efficient near to the cervix. Data showed that a specialised, highly convoluted structure of the ovarian cortex developed in the intrauterine phase as a prerequisite for massive super-ovulation, associated with the inhibition of apoptosis and continued proliferation of germ cells, as well as maintenance of several corpora lutea during the adult life. Thus, a highly complex pattern of polyovulation, polyimplantation, and controlling mechanisms has evolved in the female reproductive system of the viscacha that mainly was associated with the maternal side. After an in-depth analysis of the arterial and venous vascularization of the uterine horns and uteri, we speculate that specializations regarding the vasculature and musculature evolved first and then contributed as a compensatory or controlling mechanism for polyovulation and polyimplantation. In conclusion, polyovulation in the viscacha represents a unique enigma in reproductive biology.

Organogenesis of the Musculoskeletal System in Horse Embryos and Early Fetuses.

Musculoskeletal system development involves heterotypical inductive interactions between tendons, muscles, and cartilage and knowledge on organogenesis is required for clarification of its function. The aim of this study was to describe the organogenesis of horse musculoskeletal system between 21 and 105 days of gestation, using detailed macroscopic and histological analyses focusing on essential developmental steps. At day 21 of gestation the skin was translucid, but epithelial condensation and fibrocartilaginous tissues were observed on day 25 of pregnancy. Smooth muscle was seen in lymphatic and blood vessel walls and the beginning of cartilaginous chondrocranium was detected at day 30 of gestation. At day 45, typical chondroblasts and chondrocytes were observed and at day 55, mandibular processes expanded toward the ventral midline of the pharynx. At day 75, muscles became thicker and muscle fibers were seen developing in carpal and metacarpal joints with the beginning of the ossification process. At day 105, major muscle groups, similar to those seen in an adult equine, were observed. The caudal area of the nasal capsule and trabecular cartilages increased in size and became ossified, developing into the ethmoid bone. The presence of nasal, frontal, parietal, and occipital bones was observed. In conclusion, novel features of equine musculoskeletal system development have been described here and each process was linked with an early musculoskeletal event. Data presented herein will facilitate a better understanding of the equine muscular system organogenesis and aid in the detection of congenital deformities. Anat Rec, 299:722–729, 2016.