Human proinsulin production in the milk of transgenic cattle.

BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Characterization of histone lysine ß-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes.

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body ß-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine ß-hydroxybutyrylation (Kbhb), which is associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation, and the effects of this increase on cellular epigenomes are unknown. We searched for and identified that Kbhb is present in bovine tissues in vivo and confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. Maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation or to develop until the blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but faintly in oocytes. RNA-seq analysis in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The downregulated genes were mainly involved in glycolysis and ribosome assembly pathways, while the upregulated genes were involved in mitochondrial metabolism and oocyte development. The genes and pathways altered by BHB will provide entry points to carry out functional experiments aiming to mitigate metabolic disorders and improve fertility in cattle.

Small extracellular vesicles derived from in vivo- or in vitro-produced bovine blastocysts have different miRNAs profiles-Implications for embryo-maternal recognition.

In vivo- and in vitro-produced bovine embryos have different metabolic profiles and differences in gene transcription patterns. These embryos also have a distinct ability to establish and sustain early pregnancies. Small extracellular vesicles (sEVs) are secreted by embryos and carry bioactive molecules, such as miRNAs. We hypothesize that in vivo or in vitro-produced bovine hatched blastocysts on Day 9 and the sEVs secreted by them have different miRNA profiles. To address this hypothesis, embryos of both groups were placed in in vitro culture on Day 7. After 48 h, hatched embryos and hatched embryo-conditioned media (eCM) of both groups were collected. A total of 210 miRNAs were detected in embryos of both groups, of these 6 miRNAs were downregulated, while 7 miRNAs were upregulated in vitro group when compared to in vivo group. sEVs were isolated from eCM to determine miRNA profile. A total of 106 miRNAs were detected in both groups, including 14 miRNAs upregulated in sEVs from in vivo-eCM, and 2 miRNAs upregulated in sEVs from in vitro-eCM. These miRNAs express in embryos and sEVs secreted by them regulate early embryonic developmental and endometrial pathways, which can modify embryo-maternal communication during early pregnancy and consequently affect pregnancy establishment.

ZEB1 and ZEB2 transcription factors are potential therapeutic targets of canine mammary cancer cells.

Mammary tumours are the most frequent in female dogs as in women and half are malignant. Tumorigenicity and invasiveness are important acquired characteristics for the development and progression of cancers and could be regulated by transcription factors associated with epithelial-mesenchymal transition (EMT) as ZEB1, ZEB2, SNAI1, SLUG and STAT3. Thus, here, we evaluate the expression of EMT-associated transcription factors in canine mammary cancer (CMC) cell lines characterized for invasiveness and tumorigenicity to determine if these could be considered good targets for future development of therapies. Five CMC cell lines were characterized regarding their morphology, doubling time and expression of intermediate and actin filaments. In addition, gene expression of SLUG, STAT3, ZEB1, ZEB2 and CDH1, tumorigenicity and invasiveness were assessed. Two of these cells presented an epithelial-like morphology (E20 and E37) and three a mesenchymal-like morphology (M5, M25 and CF41.Mg). M25 and CF41.Mg presented higher invasiveness. Furthermore, only mesenchymal-like cells formed tumorspheres and CF41.Mg made more and larger tumorspheres. The mesenchymal-like cells are more malignant than the epithelial-like cells being the CF41.Mg the most malignant. This cell presented higher ZEB1 and ZEB2 and lower CDH1 gene expression. Finally, our results revealed that there is a positive correlation between ZEBs and the tumorsphere number and size. In conclusion, these findings support ZEB1 and ZEB2 as potential therapeutic targets for CMC cells, demonstrating a great potential of canine models for comparative and translational studies.

Antioxidant responses and deregulation of epigenetic writers and erasers link oxidative stress and DNA methylation in bovine blastocysts.

Early mammalian embryos derived from in vitro fertilization are exposed to conditions distinct from the native oviduct-uterine environment, including atmospheric oxygen that promotes cellular oxidative stress and alters gene expression. High oxygen partial pressure during embryo development is associated with low pregnancy rates and increased embryonic apoptosis. We investigated how bovine embryos responded to high (20%) or low (5%) oxygen partial pressure during in vitro culture, evaluating levels of reactive oxygen species (ROS) as well as changes in the expression of oxidative stress- and epigenetic-related transcripts and miRNAs in blastocysts. Additionally, we determined the global DNA methylation levels in the resulting embryos. Our data indicated that bovine blastocysts produced in vitro under high oxygen partial pressure possessed elevated ROS abundance and exhibited increased expression of CAT, GLRX2, KEAP1, NFR2, PRDX1, PRDX3, SOD1, TXN, and TXNRD1, versus reduced levels of the oxidative stress-related bta-miR-210. These stressed embryos also presented altered expression of the epigenetic-associated transcripts DNMT3A, H2AFZ, H3F3B, HDAC2, MORF4L2, REST, and PAF1. In addition, we demonstrated that embryos cultured under high oxygen partial pressure have increased global DNA methylation, suggesting that DNA hypermethylation is mediated by the deregulation of epigenetic-related enzymes due to oxidative stress.

Expression of tissue-specific imprinted gene tumor Suppressing Subtransferable Candidate 4 (TSSC4) is altered in placentae produced by nuclear transfer in cattle.

Embryonic and placental development is highly orchestrated by epigenetic processes. Disruptions in normal placental development, commonly observed in pregnancies produced by nuclear transfer, are associated with abnormal gene expression and altered epigenetic regulation of imprinted and vital placental genes. The objective of this study was to evaluate expression and epigenetic regulation of the imprinted gene TSSC4 in cotyledonary and intercotyledonary tissues from day 60 pregnancies produced by embryo transfer (ET), in vitro fertilization (IVF) and nuclear transfer (NT) in cattle. TSSC4 expression was reduced by 30% in cotyledons at 60days of gestation in the NT group. The proximal promoter region of TSSC4 showed an increase in the permissive histone mark (H3K4me2) and a reduction in the inhibitory histone mark (H3K9me2) in the cotyledons produced by NT, in relation to cotyledons produced by embryo transfer. Interestingly, H3K9me2 was also significantly reduced in cotyledons produced by IVF, compared to the ET controls. DNA methylation, in CpG-rich regions located at the proximal promoter region and the coding region of TSSC4 did not differ. These results suggest that the reduction in TSSC4 expression, observed following NT, can not be explained by the histone changes investigated in the proximal promoter region of the gene, or by changes in methylation in three regions evaluated. Also, a decrease in the levels of H3K9 dimethylation in IVF samples, indicate that in vitro culturing could corroborate with the alterations seen in the NT group.

Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development.

Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3-6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3-6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3-6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3-6 mm follicles) in embryos. Interestingly, the addition of EVs from 3-6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3-6 mm follicles during oocyte maturation and early embryo development can partially modify metabolic and developmental related genes as well as miRNA and global DNA methylation and hydroxymethylation, suggesting that EVs play an important role during oocyte maturation and early embryo development in vitro.

Nuclear transfer alters placental gene expression and associated histone modifications of the placental-specific imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2) in cattle.

Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P<0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P<0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications.

Genome-Wide Scan for Visceral Leishmaniasis in Mixed-Breed Dogs Identifies Candidate Genes Involved in T Helper Cells and Macrophage Signaling.

We conducted a genome-wide scan for visceral leishmaniasis in mixed-breed dogs from a highly endemic area in Brazil using 149,648 single nucleotide polymorphism (SNP) markers genotyped in 20 cases and 28 controls. Using a mixed model approach, we found two candidate loci on canine autosomes 1 and 2. The positional association on chromosome 2 mapped to a predicted DNAse sensitive site in CD14+ monocytes that serve as a cis-regulatory element for the expression of interleukin alpha receptors 2 (IL2RA) and 15 (IL15RA). Both interleukins were previously found to lead to protective T helper 1 cell (Th1) response against Leishmania spp. in humans and mice. The associated marker on chromosome 1 was located between two predicted transcription factor binding sites regulating the expression of the transducin-like enhancer of split 1 gene (TLE1), an important player in Notch signaling. This pathway is critical for macrophage activity and CD4+ T cell differentiation into Th1 and T helper 2. Together, these findings suggest that the human and mouse model for protective response against Leishmania spp., which involves Th1 and macrophage modulation by interleukins 2, 15, gamma interferon and Notch signaling, may also hold for the canine model.

Insights on bovine genetic engineering and cloning / Experiências em clonagem e transgenia em bovinos

Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT.

Tecnologias de modificação genética têm se tornado ferramentas essenciais para o desenvolvimento de biotecnologias animais. A clonagem animal mediante transferência nuclear de célula somática (TNCS) possibilitou a geração de animais geneticamente modificados através da utilização de linhagens celulares previamente modificadas e selecionadas como doadoras de núcleo, garantindo desta maneira a geração de rebanhos homogênoes expressando a modificação desejada. O presente estudo objetivou discutir o uso da TNCS como importante metodologia para a produção de rebanhos transgênicos, assim como experiências recentes na manipulação genética de células doadoras de núcleo e possíveis efeitos da indução gênica à pluripotência na TNCS.

Viable calves produced by somatic cell nuclear transfer using meiotic-blocked oocytes.

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Células-tronco derivadas de tecido adiposo: isolamento, cultivo in vitro e perspectivas de utilização em dermatologia / Adipose tissue derived stem cells: isolation, in vitro culture and potential uses in dermatology

É extensa a literatura existente sobre as características e potenciais terapêuticos das células-tronco mesenquimais adultas, e a possibilidade de seu isolamento a partir de tecido adiposo excedente de outros procedimentos é promissora e extremamente desejável. Os protocolos para tal utilização, porém, ainda são descritos de maneira sumarizada e não padronizada. O objetivo dessa comunicação é apresentar um protocolo simples, prático e eficaz tanto da coleta do tecido adiposo em ambiente hospitalar quanto do isolamento e cultivo, em laboratório, in vitro de células-tronco mesenquimais adultas derivadas de tecido adiposo, com a finalidade de discutir as perspectivas da utilização dessa técnica na dermatologia.

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'.

Single embryo and oocyte lipid fingerprinting by mass spectrometry.

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.