Profiling of miRNAs in porcine Sertoli cells

Background: Sertoli cells(SCs) create a specialized environment to support and dictate spermatogenesis.MicroRNAs(miRNAs), a kind of ~ 22 nt small noncoding RNAs, have been reported to be highly abundant in mouse SCs and play critical roles in spermatogenesis. However, the miRNAs of porcine SCs remain largely unknown.Methods: We isolated porcine SCs and conducted small RNA sequencing. By comparing miRNAs in germ cells, we systematically analyzed the miRNA expression pattern of porcine SCs. We screened the highly enriched SC miRNAs and predicted their functions by Gene Ontology analysis. The dual luciferase assay was used to elucidate the regulation of tumor necrosis factor receptor(TNFR)-associated factor 3(TRAF3) by ssc-miR-149.Results: The analysis showed that 18 miRNAs were highly expressed in SCs and 15 miRNAs were highly expressed in germ cells. These miRNAs were predicted to mediate SC and germ cell functions. In addition, ssc-miR-149 played critical roles in SCs by targeting TRAF3.Conclusion: Our findings provide novel insights into the miRNA expression pattern and their regulatory roles of porcine SCs.     

Induced pluripotent stem cells from pigs and other ungulate species: an alternative to embryonic stem cells?

Robust embryonic stem cell (ESC) lines from livestock species have been difficult to derive and maintain, and unlike mouse ESC, have not contributed to our ability to understand directed differentiation in vitro. Nor have such cells yet provided a simpler means than pronuclear injection to manipulate the genomes of agriculturally important species, such as cattle, sheep and pigs. Induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells, such as fibroblasts, with a set of stemness genes, most usually but not exclusively POU5F1, SOX2, KLF4 and c-MYC, offer an alternative to ESC in these regards, as they exhibit a pluripotent phenotype resembling that of ESC, yet are readily generated in the laboratory. Accordingly, such cells, in association with cloning technologies, may be useful for introducing complex genetic changes into livestock, although this potential has yet to be demonstrated. Porcine iPSC may be especially valuable because the pig is a prime biomedical model for tissue transplantation. In general, iPSC from livestock, like those from humans, are of the epiblast type and depend upon FGF2 and activin/nodal signalling systems to maintain their pluripotency and growth. Recent experiments, in which newly reprogrammed porcine and bovine cells were selected on a LIF-based medium in presence of specific protein kinase inhibitors, have allowed iPSC cells of the na?ve type, resembling the more amenable blastocyst-derived mouse ESC and iPSC to be isolated. However, hurdles still remain if such cells are to achieve their biotechnological promise.     

Mammary stem cells： expansion and animal productivity

Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods （non-milking period between two consecutive lactation cycles）, abrupt cessation of lactation （common in water buffaloes） and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells （MaSCs）/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally.     

Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection

Vaccines to prevent Trypanosoma cruzi infection in humans or animals are not available, and in many settings, dogs are an important source of domestic infection for the insect vector. Identification of infected canines is crucial for evaluating peridomestic transmission dynamics and parasite control strategies. As immune control of T. cruzi infection is dependent on humoral and cell-mediated immune responses, we aimed to define a serodiagnostic assay and T cell phenotypic markers for identifying infected dogs and studying the canine T. cruzi-specific immune response. Plasma samples and peripheral blood mononuclear cells (PBMCs) were obtained from forty-two dogs living in a T. cruzi-endemic region. Twenty dogs were known to be seropositive and nine seronegative by conventional serologic tests two years prior to our study. To determine canine seroreactivity, we tested sera or plasma samples in a multiplex bead array against eleven recombinant T. cruzi proteins. Ninety-four percent (17/18) of dogs positive by multiplex serology were initially positive by conventional serology. The frequency of IFNγ-producing cells in PBMCs responding to T. cruzi correlated to serological status, identifying 95% of multiplex seropositive dogs. Intracellular staining identified CD4⁺ and CD8⁺ T cell populations as the sources of T. cruzi lysate-induced IFNγ. Low expression of CCR7 and CD62L on CD4⁺ and CD8⁺ T cells suggested a predominance of effector/effector memory T cells in seropositive canines. These results are the first, to our knowledge, to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs and validate these methods for identifying dogs exposed to T. cruzi.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.     

Antiproliferative effect of α-mangostin on canine osteosarcoma cells

Osteosarcoma is the most frequently diagnosed primary bone tumor in dog. Since chemotherapeutics are quite limited due to high cost and severe toxicity, therefore, the ultimate goal is to discover cost-effective therapeutics with less toxicity. We have studied the effect of α-mangostin, a xanthone derivative isolated from pericarp of mangosteen (Garcinia mangostana Linn.) in canine osteosarcoma, D-17 cells. The results showed that α-mangostin induced antiproliferation with IC(50) at 15 μg/ml. Hoechst 33342 nuclear staining and nucleosomal DNA-gel electrophoresis revealed that α-mangostin could induce nuclear condensation and fragmentation, typically seen in apoptosis. Cell cycle analysis demonstrated that α-mangostin induced sub-G1 peak. In addition, α-mangostin also induced membrane flipping of the phosphatidylserine and the loss of mitochondrial membrane potential in D-17 cells. In conclusion, α-mangostin, induced apoptotic cell death against canine osteosarcoma D-17 cells, could be a potential candidate for preventive and therapeutic application for bone cancer treatment in dogs.     

Do progenitor cells from different tissue have the same phenotype?

The purpose of this work was to validate isolation methods for sheep mesenchymal stem cells (MSC) from different sources and to explore the hypothesis that MSC exhibit markers of the same phenotype independent from tissue source. Cells derived from ovine bone marrow, synovial membrane and adipose tissue were characterized using the following markers: CD44, CD45, CD11b and MHC-I. The isolated MSC were cultivated, went through osteogenic, chondrogenic and adipogenic differentiation, and were characterized by flow cytometry using mouse anti-ovine CD44, CD45 and MHC-I monoclonal antibody (mAb), and mouse anti-bovine CD11b mAb. Ovine MSC from all three sources differentiated under chondorgenic, osteogenic and adipogenic conditions. Also, MSC from the three tissues were found to express CD44 and MHC-I but lack of CD11b and CD45. The results obtained revealed that our isolation methods for the different tissues tested are valid and that MSC from the three sources studied have same immunophenotic characteristics.     

Are there M cells in the cecal tonsil of chickens?

A unique morphological cell type, "microfold or membranous (M) cell-like cell", was detected electron-microscopically in the cecal tonsil epithelium of the chicken. M cell-like cells possessed a few short microvilli of irregular arrangement and a large number of lymphocytes and macrophages wedged into their basal surfaces. Triticum vulgaris was found to bind to M cell-like cells. With horseradish peroxidase (HRP) treatment, M cell-like cells showed an active HRP uptake just as did the neighbouring usual absorptive epithelial cells. No uptake of colloidal carbon particles from the intestinal lumen was recognized in any part of the intestinal epithelium. These results suggest that M cell-like cells of the chicken possess some M cell-characteristic morphological and histochemical features, but that their active uptake of foreign materials is not so developed as in mammalian M cells.     

Identification and phenotypic characterization of γδ T cells in rat lymph

γδ T cells represent an unconventional subset of T lymphocytes that are abundant in epithelial tissues and serve as an early immune defense against microbes. We have, for the first time, identified γδ T cells in steady-state thoracic duct lymph (TDL) from rats. The lymph contains γδ T cells expressing CD8 but not CD4, CD25, MHC-II or CD103. The percentage of TDL γδ T cells in rats does not change when the mesenteric lymph nodes (MLN) are surgically removed. Our data suggest that a proportion of γδ T cells migrate from the intestine into rat TDL, under steady-state conditions.     

PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-na?ve regulatory T cells to proliferate

In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)⁺ and N-protein⁻ moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein⁺ moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein⁻ moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3⁺ regulatory T cells present within CD4⁺ T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4⁺ T cells showed differences in regard to proliferation and frequency of Foxp3⁺ T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.     

Canine microglial cells: stereotypy in immunophenotype and specificity in function?

Microglial cells represent the endogenous immune system of the central nervous system (CNS). Upon pathological insults they reveal their immunological potential aimed at regaining homeostasis. These reactions have long been believed to follow a uniform and unspecific pattern which is irrespective to the underlying disease entity. Evidence is growing that this view seriously underrates microglial competence as the defenders of the CNS. In the present study, microglial cells of 47 dogs were examined ex vivo by means of flow cytometry. Ex vivo examination included immunophenotypic characterization using eight different surface markers and functional studies such as phagocytosis assay and the reactive oxygen species (ROS) generation test. The dogs were classified according to their histopathological diagnoses in disease categories (controls, canine distemper virus (CDV) induced demyelination, other diseases of the CNS) and results of microglial reaction profiles were compared. Immunophenotypic characterization generally revealed relative high conformity in the microglial disease response among the different groups, however the functional response was shown to be more specific. Dogs with intracranial inflammation and dogs with demyelination showed an enhanced phagocytosis, whereas a significant up-regulation of ROS generation was found in dogs with demyelination due to CDV infection. This strongly suggests a specific response of microglia to infection with CDV in the settings of our study and underlines the pivotal role of microglial ROS generation in the pathogenesis of demyelinating diseases, such as canine distemper.     

Abnormal functions of Leydig cells in crossbred cattle–yak showing infertility

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.     

Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells

Coliform mastitis may be severe in periparturient cows due to enhanced expression of pro-inflammatory cytokines that contribute to disease pathogenesis. Tumor necrosis factor (TNF)-α is implicated with the severity of coliform mastitis by provoking inflammatory responses in affected tissues. The endothelium is an integral organ in regulating inflammatory responses and loss of endothelial integrity may be fatal. Studies in humans suggest that endothelial cell apoptosis may be a consequence of TNF-α exposure and contributes to the development of sepsis, however, its impact on bovine mammary endothelial cells (BMEC) is unknown. We sought to determine the inflammatory and apoptotic responses of primary BMEC exposed to TNF-α in vitro. Stimulation of endothelial monolayers with TNF-α resulted in significant increase of toll-like receptor 4, interleukin-6 and -8, and intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 gene expression in a time-dependent manner. Caspase-8 and caspase-3 mRNA expression, as well as caspase enzyme activity, also increased significantly following TNF-α stimulation. Cell viability assessed by ATP activity and BMEC apoptosis determined by flow cytometry revealed no significant changes across time with TNF-α stimulation. Results suggest that TNF-α stimulation, at the dose used in this study, can elicit a pro-inflammatory response in BMEC, but not induce apoptosis. The impact of TNF-α on mammary vascular function and the subsequent impact on the pathophysiology of severe coliform mastitis warrant further investigation.