Corpus luteum proximity alters molecular signature of the small extracellular vesicles and cumulus cells in the bovine ovarian follicle environment.

Progesterone (P4) is predicted to act as a negative regulatory hormone for oocyte maturation events; however, its local effects during follicular development remain poorly understood in bovine. The complex process of oocyte meiosis progression is dependent on cellular communication among follicular cells. Besides, the breakdown of this communication, mainly between cumulus cells (CC) and oocyte, through the retraction of cumulus projections connecting these cells can impact oocyte maturation. In our study, we observed that follicles from the ovary ipsilateral to the corpus luteum (CL) containing high intrafollicular P4 concentrations enhance the abundance of proteins detected in follicular-derived small extracellular vesicles (sEVs) predicted to be involved in the retraction of membrane projections based on actin filaments, such as transzonal projections (TZPs). Conversely, we found that follicles from the ovary contralateral to the CL, which contained low intrafollicular P4 concentrations, had a high detection of proteins predicted to regulate the maintenance of TZPs. We also performed RNAseq analysis which demonstrated that 177 genes were differentially expressed in CC under the different P4 environments. Bioinformatic analysis points to changes associated to cell metabolism in cells from follicles ipsilateral to the CL in comparison to genes involved in cell communication in CC from follicles contralateral to the CL. Our functional analysis experiment confirmed that supplementation of cumulus-oocyte complexes during in vitro maturation with P4 at concentration similar to ipsilateral follicles reduces the number of TZPs. In summary, our study underscores a direct association between P4 concentration and cumulus-oocyte interaction, with potential consequences for the acquisition of oocyte competence.

Corpus luteum presence in the bovine ovary increase intrafollicular progesterone concentration: consequences in follicular cells gene expression and follicular fluid small extracellular vesicles miRNA contents.

BACKGROUND: It is well described that circulating progesterone (P4) plays a key role in several reproductive events such as oocyte maturation. However, during diestrus, when circulating P4 is at the highest concentrations, little is known about its local impact on the follicular cells such as intrafollicular P4 concentration due to corpus luteum (CL) presence within the same ovary. Based on that, our hypothesis is that the CL presence in the ovary during diestrus alters intrafollicular P4 concentrations, oocyte competence acquisition, follicular cells gene expression, and small extracellular vesicles (sEVs) miRNAs contents. RESULTS: P4 hormonal analysis revealed that ipsilateral to the CL follicular fluid (iFF) presented higher P4 concentration compared to contralateral follicular fluid (cFF). Furthermore, oocyte maturation and miRNA biogenesis pathways transcripts (ADAMTS-1 and AGO2, respectively) were increased in cumulus and granulosa cells of iFF, respectively. Nevertheless, a RT-PCR screening of 382 miRNAs showed that three miRNAs were upregulated and two exclusively expressed in sEVs from iFF and are predicted to regulate cell communication pathways. Similarly, seven miRNAs were higher and two exclusively expressed from cFF sEVs and are predicted to modulate proliferation signaling pathways. CONCLUSION: In conclusion, intrafollicular P4 concentration is influenced by the presence of the CL and modulates biological processes related to follicular cell development and oocyte competence, which may influence the oocyte quality. Altogether, these results are crucial to improve our knowledge about the follicular microenvironment involved in oocyte competence acquisition.

Expression of acetylated histones H3 and H4 and histone deacetylase enzymes HDAC1, HDAC2 and HDAC6 in simple mammary carcinomas of female dogs.

Histone deacetylation is an important mechanism involved in human breast cancer tumorigenesis and recent veterinary oncology studies also demonstrate a similar relationship in some canine neoplasms. The use of HDAC inhibitors in vitro and in vivo has demonstrated antitumor action on several strains of human and animal cancers. The present study aims to correlate the expression of H3K9Ac, H4K12Ac, HDAC1, HDAC2 and HDAC6 in simple mammary carcinomas in dogs with clinicopathological parameters and overall survival time. To this end, 61 samples of simple breast carcinomas were analyzed by the immunohistochemistry technique with subsequent validation of the antibodies by the Western Blot technique. The expressions obtained via a semi-quantitative way were categorized by assigning scores and classified into high or low expressions according to the given score, except for HDAC6, when the marking percentage was considered and subdivided into high and low expressions using the median value. For statistical analysis, the chi-square test or Fisher exact test were used as univariate analysis and correspondence analysis as a multivariate test, in addition to the Kaplan-Meier survival analysis. In the studied samples, the highest frequencies were determined for the high expression proteins H4K12Ac (88.5%), HDAC2 (65.6%) and HDAC6 (56.7%) and the low expression proteins H3K9Ac (73.8%) and HDAC1 (54.1%). An association between the low expression of HDAC1 and the presence of lymph node metastasis (p = 0.035) was indicated by univariate analysis while the high expression of HDAC1 was associated with favorable prognostic factors, such as the absence of lymph node metastasis and low mitotic index by multivariate analysis. Also, by multivariate analysis, the low expression of HDAC6 was correlated with the low expression of Ki67, smaller tumors, and better prognosis factors as well. Protein expression was not correlated with patients' overall survival time (p > 0.05). The high expressions of HDAC2 and HDAC6 in mammary carcinomas in female dogs may be useful information for research involving therapeutic targets with iHDACs since their inhibition favors hyperacetylation and transcription of tumor suppressor genes.

Cattle Cloning by Somatic Cell Nuclear Transfer.

Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.

High body energy reserve influences extracellular vesicles miRNA contents within the ovarian follicle.

Aiming to evaluate the effects of increased body energy reserve (BER) in Nellore cows' reproductive efficiency, cows were fed with different nutritional plans to obtain animals with high BER (HBER; Ad libitum diet) and moderate BER (MBER: cows fed 70% of HBER group ingestion). To evaluate the BER, cows were weekly weighted and evaluated for subcutaneous fat thickness and insulin serum concentration along the experimental period. At the end of the experimental period, animals were submitted to estrous synchronization and artificial insemination. Animals were slaughtered approximately 120 h after ovulation induction and the reproductive tracts were collected for embryo recovery and samples collection. Cumulus-oocyte-complexes (COC) and follicular fluid were collected from 3-6 mm in diameter ovarian follicles to perform miRNA analysis of cumulus cells (CC) and extracellular vesicles from follicular fluid (EV FF). As expected, differences were observed among MBER and HBER groups for body weight, fat thickness, and insulin serum concentration. HBER animals showed lower ovulation and embryo recovery rates compared to MBER animals. Different miRNAs were found among CC and EV FF within groups, suggesting that the BER may influence follicular communication. This suggests that small follicles (3-6 mm diameter) are already under BER effects, which may be greater on later stages of follicular development.

Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in Bos indicus females with a high or low antral follicle count.

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.

Resiliency of equid H19 imprint to somatic cell reprogramming by oocyte nuclear transfer and genetically induced pluripotency†.

Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetically stable after reprogramming in this species. In light of this, we aimed at characterizing the equid H19 gene to determine whether imprinting is altered in somatic cell nuclear transfer (SCNT)-derived conceptuses and induced pluripotent stem cell (iPSC) lines using the mule hybrid model. A CpG-rich region containing five CTCF binding sites was identified upstream of the equine H19 gene and analyzed by bisulfite sequencing. Coupled with parent-specific and global H19 transcript analysis, we found that the imprinted H19 remains monoallelic and that on average the methylation levels of both parental differentially methylated regions in embryonic and extra-embryonic SCNT tissues and iPSC lines remained unaltered after reprogramming. Together, these results show that, compared to other species, equid somatic cells are more resilient to epigenetic alterations to the H19-imprinted locus during SCNT and iPSC reprogramming.

Neurons-derived extracellular vesicles promote neural differentiation of ADSCs: a model to prevent peripheral nerve degeneration.

Potential mechanisms involved in neural differentiation of adipocyte derived stem cells (ADSCs) are still unclear. In the present study, extracellular vesicles (EVs) were tested as a potential mechanism involved in the neuronal differentiation of stem cells. In order to address this, ADSCs and neurons (BRC) were established in primary culture and co-culture at three timepoints. Furthermore, we evaluated protein and transcript levels of differentiated ADSCs from the same timepoints, to confirm phenotype change to neuronal linage. Importantly, neuron-derived EVs cargo and EVs originated from co-culture were analyzed and tested in terms of function, such as gene expression and microRNA levels related to the adult neurogenesis process. Ideal neuron-like cells were identified and, therefore, we speculated the in vivo function of these cells in acute sciatic nerve injury. Overall, our data demonstrated that ADSCs in indirect contact with neurons differentiated into neuron-like cells. Neuron-derived EVs appear to play an important role in this process carrying SNAP25, miR-132 and miR-9. Additionally, in vivo neuron-like cells helped in microenvironment modulation probably preventing peripheral nerve injury degeneration. Consequently, our findings provide new insight of future methods of ADSC induction into neuronal linage to be applied in peripheral nerve (PN) injury.

Sperm-borne miR-216b modulates cell proliferation during early embryo development via K-RAS.

Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen-thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.

Stem cells on regenerative and reproductive science in domestic animals.

Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.

Cumulus-oocyte interactions and programmed cell death in bovine embryos produced in vitro.

This study investigated the interactions between cumulus cells (CCs) and oocytes and programmed cell death in bovine cumulus-oocyte complexes (COCs) with different morphological characteristics. DNA fragmentation was assessed in CCs at 0 and 24 h of maturation, as well as parthenogenetic developmental competence on the 9th day post-activation, blastocyst quality and BCL-2 and BAX transcript levels in matured CCs. Most immature oocytes in the COC-A group (full cumulus and several compact layers) were in the initial germinal vesicle (iGV) stage, exhibiting minimal or no DNA damage. In contrast, after follicle removal, the COCB (partial cumulus and one or two cell layers) and C (expanded cumulus) groups presented in more advanced GV stages and exhibited DNA fragmentation. After maturation, significant increases in fragmented nuclei were noted in COCC and COCB groups. Embryos resulting from the COC-A developed more rapidly and had increased competence compared to embryos resulting from groups COCB and COCC. The COCB group exhibited the highest BAX protein levels and a reduced BCL-2/BAX protein ratio. The results show a negative correlation between nuclear fragmentation and embryonic development potential in COCs with different morphologies. In addition, a low BCL-2/BAX protein ratio might be associated with an increase in nuclear fragmentation in CCs.

Distinct features of rabbit and human adipose-derived mesenchymal stem cells: implications for biotechnology and translational research.

INTRODUCTION: Owing to their similarity with humans, rabbits are useful for multiple applications in biotechnology and translational research from basic to preclinical studies. In this sense, mesenchymal stem cells (MSCs) are known for their therapeutic potential and promising future in regenerative medicine. As many studies have been using rabbit adipose-derived MSCs (ASCs) as a model of human ASCs (hASCs), it is fundamental to compare their characteristics and understand how distinct features could affect the translation to human medicine. OBJECTIVE: The aim of this study was to comparatively characterize rabbit ASCs (rASCs) and hASCs to further uses in biotechnology and translational studies. MATERIALS AND METHODS: rASCs and hASCs were isolated and characterized by their immunophenotype, differentiation potential, proliferative profile, and nuclear stability in vitro. RESULTS AND DISCUSSION: Both ASCs presented differentiation potential to osteocytes, chondrocytes, and adipocytes and shared similar immunophenotype expression to CD105+, CD34-, and CD45-, but rabbit cells expressed significantly lower CD73 and CD90 than human cells. In addition, rASCs presented greater clonogenic potential and proliferation rate than hASCs but no difference in nuclear alterations. CONCLUSION: The distinct features of rASCs and hASCs can positively or negatively affect their use for different applications in biotechnology (such as cell reprogramming) and translational studies (such as cell transplantation, tissue engineering, and pharmacokinetics). Nevertheless, the particularities between rabbit and human MSCs should not prevent rabbit use in preclinical models, but care should be taken to interpret results and properly translate animal findings to medicine.

Metabolic gene expression and epigenetic effects of the ketone body ß-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos.

The rapid decline in fertility that has been occurring to high-producing dairy cows in the past 50 years seems to be associated with metabolic disturbances such as ketosis, supporting the need for research to improve our understanding of the relations among the diet, metabolism and embryonic development. Recently, the ketone body ß-hydroxybutyrate (BOHB) was demonstrated to be a potent inhibitor of histone deacetylases (HDACs). Herein, we performed a series of experiments aiming to investigate the epigenetic effects of BOHB on histone acetylation in somatic cells, cumulus-oocyte complexes (COCs) and somatic cell nuclear transfer (SCNT) embryos. Treatment with BOHB does not increase histone acetylation in cells but stimulates genes associated with ketolysis and master regulators of metabolism. We further demonstrated that maturing COCs with high levels of BOHB does not affect their maturation rate or histone acetylation but increases the expression of PPARA in cumulus cells. Treatment of somatic cell nuclear transfer zygotes with BOHB causes hyperacetylation, which is maintained until the blastocyst stage, causing enhanced FOXO3A expression and blastocyst production. Our data shed light on the epigenetic mechanisms caused by BOHB in bovine cells and embryos and provide a better understanding of the connection between nutrition and reproduction.

TAX-mRNA-Carrying Exosomes from Human T Cell Lymphotropic Virus Type 1-Infected Cells Can Induce Interferon-Gamma Production In Vitro.

Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. The development of HAM/TSP, a chronic neuroinflammatory disease, is correlated to complex interaction between the host immune response and the infecting virus. Tax expression plays an important role in HAM/TSP pathogenesis by activating various cellular genes, including the cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Exosomes have emerged as an important factor of cell-to-cell communication contributing to diverse cellular processes, including immune modulation. Considering the potential role of exosomes in modulating the immune response and inflammation, the main objective of this study was to examine if HTLV-1-infected cells produce exosomes carrying viral proteins or inflammatory molecules, which can participate in the chronic inflammation that is observed in patients with HAM/TSP. Exosomes were isolated from HTLV-1-infected cell line, evaluated for the tax mRNA presence, and tested for the ability to activate peripheral mononuclear cells (PBMC) in inducing an inflammatory immune response. We observed that the proinflammatory cytokines, IFN-γ and TNF-α, were upregulated in T cells after treatment of the PBMC with Tax-carrying exosomes compared to the negative control. Interleukin-4, Granzyme B, and Perforin did not show alterations. Taken together, these results suggest that exosomes carrying tax-mRNA isolated from HTLV-1-infected cells might induce the production of proinflammatory cytokines and activate T helper (Th)1, and not Th2-immune response. If this finding is further confirmed, this study may have impact on investigations on the pathogenesis of HAM-TSP and the inflammatory response involved in this disease.

The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells) and cumulus-oocyte complexes (COCs) to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

Vascularization and VEGF expression altered in bovine yolk sacs from IVF and NT technologies.

Reproductive technologies are widely used in cattle, although many are associated with high-embryonic mortality, especially during early gestation, when the yolk sac undergoes macroscopic changes in structure. We hypothesized that vasculogenesis and angiogenesis are affected, thereby affecting embryonic and placental differentiation. To test this, we studied yolk sac development and gene expression of the vascular endothelial growth factor system (VEGF-A, VEGFR-1/Flt-1, VEGFR-2/KDR). Samples from Days 25 to 40/41 of pregnancy from control cattle (n = 8) and from pregnancies established with IVF, (n = 7) or somatic cell nuclear transfer/clones (n = 5) were examined by histology, immunohistochemistry, and quantitative reverse transcriptase PCR. Yolk sacs in IVF- and nuclear transfer-derived pregnancies were immature. Development of villi was sparse in IVF yolk sacs, whereas vascularization was barely formed in clones and was associated, in part, with thin or interrupted endothelium. Transcript levels of the genes characterized exceed minimum detection limits for all groups, except in the mentioned clone with interrupted endothelium. Levels of mRNA for VEGF-A and VEGFR-2 were significantly higher in IVF yolk sacs. Clones had substantial individual variation in gene expression (both upregulation and downregulation). Our data confirmed the broad range in expression of VEGF genes. Furthermore, overexpression in IVF yolk sacs may compensate for an immature yolk sac structure, whereas in clones, patchy expression may cause structural alterations of blood vessels. In conclusion, we inferred that disturbances of yolk sac vasculature contributed to increased early embryonic mortality of bovine pregnancies established with reproductive technologies.

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.

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.

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles / Desafios e perspectivas na produção in vitro de embriões bovinos: enfoque epigenético e em vesículas secretadas por células

This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.

Nesta revisão, apresentamos alguns problemas clínicos encontrados nos animais derivados de PIV, principalmente derivados de transferência de núcleo, e discutimos o possível papel da epigenética em tais processos. Além disso, uma vez que vesículas secretadas por células têm sido descritas como possíveis reguladores de processos reprodutivos fisiológicos importantes, tais como a foliculogênese e a fertilização, estas são aqui apresentadas como uma possível nova ferramenta para a manipulação do período embrionário pré-implantacional através de moléculas efetoras, contidas em tais vesículas.

Isolamento e cultivo de neurônios e neuroesferas de córtex cerebral aviar / Isolation and culture of neurons and neurospheres from chicken brain cortex

Métodos de cultivo celular são convenientes na realização de análises funcionais de alterações/interações protéicas das células neuronais, auxiliando a decifrar o interactoma de proteínas chaves na neurogênese de doenças do Sistema Nervoso Central. Por esse motivo, culturas de neurônios e neuroesferas isolados do córtex cerebral aviar representam um modelo acessível para o estudo de diversas doenças neurológicas, tal como a epilepsia. A espécie aviar apresenta peculiaridades em seu proteoma neuronal, visto a presença de uma expressão diferenciada de proteínas chaves no metabolismo energético cerebral, algumas destas (VDAC1 e VDAC2) desempenham papel importante na compreensão do mecanismo da epilepsia refratária. A metodologia estabelecida no presente estudo obteve cultivo de neuroeferas, onde as células cresceram tipicamente em aglomerados atingindo, dentro de 7 dias, o diâmetro ideal de 100-200 µm. A diferenciação celular das neuroesferas foi obtida após a aderência destas às placas tratadas com poli-D-lisina, evidenciada pela migração de fibras do interior da neuroesfera. Ao contrário das neuroesferas, os neurônios em cultivo extenderam seus neuritos após 11 dias de isolamento. Tal modelo in vitro pode ser utilizado com sucesso na identificação das variáveis neuroproteômicas, propiciando uma avaliação global das alterações dinâmicas e suas interações protéicas. Tal modelo pode ter aplicações em estudos dos efeitos de indutores da morte celular e bloqueadores de canais de membrana mitocondriais em proteínas chaves do metabolismo energético cerebral.

Cell culture methods are used for studies of protein interactions in neural cells, helping to detect the interactome of proteins linked to generation of central nervous system diseases. For this reason, neural cells and neurospheres isolated from cortical chicken brain are a current model for studies of neurological diseases, such as epilepsy. Chicken brain has key characteristics on its proteome, with a differential expression of proteins linked to energy metabolism, some of them (VDAC 1 and VDAC 2) play an important role in understanding mechanism of refractory epilepsy. Using the methods described, we found neurospheres, in which cells grow in structures with the ideal diameter of 100-200µm within seven days after isolation. Neurospheres differentiation was obtained after adhesion of these cells to surfaces coated with poly-D-Lysine, detected by migration of fibers inside them. Unlike neurospheres, neurons extended neurites after 11 days of isolation. Here we describe a method to isolate and culture neurons and neurospheres from chicken cerebral cortex. Such "in vitro" model can be utilized on studies of neuronal protein differential expression and interaction. Cultures of isolated neurons represent an accessible model on studies of apoptosis and channel blockers of key proteins linked to brain metabolism.