Potential physiological involvement of nesfatin-1 in regulating swine granulosa cell functions.

Nesfatin-1 has recently been indicated as a pleiotropic molecule that is primarily involved in the metabolic regulation of reproductive functions acting at hypothalamic level. The aim of this study was to explore the local action of nesfatin-1 in swine ovarian follicles. Nucleobindin 2 (NUCB2) was verified using real-time quantitative polymerase chain reaction in swine granulosa cells from different sized follicles and nesfatin-1 was localised by immunohistochemistry in sections of the whole porcine ovary. The effects of different concentrations of nesfatin-1 on cell growth, steroidogenesis and the redox status of granulosa cells were determined invitro. In addition, the effects of nesfatin-1 were evaluated in an angiogenesis bioassay because vessel growth is essential for ovarian follicle function. Immunohistochemistry revealed intense positivity for nesfatin-1 in swine granulosa cells in follicles at all developmental stages. Expression of the gene encoding the precursor protein NUCB2 was higher in granulosa cells from large rather than from medium and small follicles. Further, nesfatin-1 stimulated cell proliferation and progesterone production and interfered with redox status by modifying nitric oxide production and non-enzyme scavenging activity in granulosa cells from large follicles. Moreover, nesfatin-1 exhibited a stimulatory effect on angiogenesis. This study demonstrates, for the first time, that nesfatin-1 is physiologically present in the swine ovarian follicle, where it may impair granulosa cell functions.

Isolation, proliferation and characterization of endometrial canine stem cells.

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT-PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue-derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue-derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the "Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy" for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue-derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow-derived MSC in dog.

Are the new phthalates safe? Evaluation of Diisononilphtalate (DINP) effects in porcine ovarian cell cultures.

Phthalates are plasticizing chemicals, widely used in packaging materials and consumer products for several decades. These molecules have raised concerns because of their toxicity and their use have been restricted in several countries. Therefore, novel phthalates have been introduced. Among these, diisononilphtalate (DINP) is widely employed. However, its safety has not been properly addressed. Therefore, using a well validated granulosa cell model, collected from swine ovaries with a translational value, we studied potential DINP effects on important cellular functional parameters. In particular, we studied cell growth, steroidogenesis and redox status. Collected data showed that DINP stimulates (p < 0.05) cell growth, increases estrogen and inhibits progesterone production (p < 0.05), disrupts redox balance stimulating free radicals (p < 0.05) while reducing scavenger activities (p< 0.05). Taken together, DINP's impact on cultured swine granulosa cells provides cause for concern regarding its potential adverse effects on reproductive and endocrine functions.

Allogeneic adipose tissue-derived mesenchymal stem cells in combination with platelet rich plasma are safe and effective in the therapy of superficial digital flexor tendonitis in the horse.

Overstrain tendonitis are common pathologies in the sport horses. Therapeutic approaches to tendon healing do not always result in a satisfactory anatomical and functional repair, and healed tendon is often characterized by functional impairment and high risk of reinjury. Recently, mesenchymal stem cells (MSCs) and platelet rich plasma (PRP) have been proposed as novel therapeutic treatments to improve the tendon repair process. MSCs are multipotent, easy to culture and being originated from adult donors do not pose ethical issues. To date, autologous MSCs have been investigated mainly in the treatment of large bone defects, cardiovascular diseases, osteogenesis imperfecta and orthopaedic injuries both in human and veterinary medicine. The clinical applications in which autologous MSCs can be used are limited because patient-specific tissue collection and cell expansion require time. For clinical applications in which MSCs should be used right away, it would be more practical to use cells collected from a donor, expanded in vitro and banked to be readily available when needed. However, there are concerns over the safety and the efficacy of allogeneic MSCs. The safety and efficacy of a therapy based on the use of allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) associated to platelet rich plasma (PRP) were evaluated in 19 horses affected by acute or subacute overstrain superficial digital flexor tendonitis (SDFT). The application of allogeneic ASCs neither raised clinical sign of acute or chronic adverse tissue reactions, nor the formation of abnormal tissue in the long-term. After a follow-up of 24 months, 89.5% horses returned to their previous level of competition, while the reinjury rate was 10.5%, comparable to those recently reported for SDFT treated with autologous bone marrow derived MSCs. This study suggests that the association between allogeneic ASCs and PRP can be considered a safe and effective strategy for the treatment of SDF tendonitis in the horse.

Research Note: Oxidative stress and immune response following the administration of live attenuated Mycoplasma gallisepticum vaccination in backyard chicken.

In the present study, we investigated a possible relationship between the immune response and the oxidative stress (OS) state trend in a group of 12 chickens after intraocular administration of an attenuated Mycoplasma gallisepticum (MG) vaccine. Blood samples were collected at the vaccination time (T0), after 14 (T1) and 21 d (T2). White blood cell count (WBC), differential leucocyte count, and anti-MG antibodies titer (S/P) were studied as immune response indexes. As plasmatic OS biomarkers levels, we considered malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), reactive oxygen metabolites derived compounds (d-ROMs), the ferric reducing ability of plasma (FRAP), and superoxide anion (O2-). After antigenic stimulation, it was observed a significant decrease in monocythemia and a significant increase in thrombocythemia, S/P, MDA, and SOD. Furthermore, subjects with high d-ROMs levels at T0 tended to develop higher cellular mobilization with increases in WBC and lymphocytes accompanied by lower antibody release. It was also observed that the antioxidant components FRAP and SOD were moderately positively correlated to the entity of antibody response.

The effects of nanoplastics on adipose stromal cells from swine tissues.

Plastic is one of the main sources of marine and terrestrial pollution. This material can fragment into micro- (<-5 mm) and nanoplastics (NPs) (<100 nm) following degradation. Animals are exposed to these particles by ingesting contaminated food, respiration or filtration, and transdermally. In organisms, NPs can cross biological membranes, and cause oxidative stress, cell damage, apoptosis, and endocrine interference. We previously demonstrated that polystyrene - NPs interfered with ovarian cell functions. Since reproduction involves a high energy expenditure and a crucial role is played by adipose tissue, the aim of the present study was to evaluate the effects of NPs on primary adipose stromal cells (ASCs) isolated from swine adipose tissues. In particular, the effects on cell viability, proliferation, metabolic activity, inflammatory process mediators and oxidative stress markers were assessed. The obtained results did not reveal a significant variation in cell proliferation, metabolic activity was increased (P < 0.01) but only at the lowest concentration, while viability showed a significant decrease after prolonged exposure to NPs (P < 0.01). TNF-α was increased (P < 0.05), while PAI-1 was inhibited (P < 0.001). Redox status was significantly modified; in particular, the production of O2-, H2O2 and NO was stimulated (P < 0.05), the non-enzymatic antioxidant power was reduced (P < 0.05) while catalase activity was significantly (P < 0.01) increased.

Orexin B inhibits viability and differentiation of stromal cells from swine adipose tissue.

Adipose tissue is recognized as a fundamental endocrine organ. Nowadays, we are also aware that it contains the highest number of stromal cells (ASCs) per unit of volume. These cells can differentiate between different phenotypes among which the adipocytes. The aim of this work was to verify whether orexin B, crucial mediator of the energy balance, modifies the differentiation of cultured ASCs. We used the pig as a model. Our data demonstrate that swine ASCs express prepro-orexin. Orexin B treatment inhibits ASCs proliferation (P < 0.05) and adipogenic differentiation (P < 0.05). Data collected could be interesting both in animal production field because consumers require lean meat, and in human medicine study about obesity because pig can be considered a valuable animal model for translational studies.

The myokine irisin: localization and effects in swine late medium and large antral ovarian follicle.

Irisin is mainly synthesized by skeletal muscle tissue, where it is believed to be responsible for the benefits of exercise on metabolism and cardiovascular system. In adipose tissue, its best-known effect is the browning of white adipocytes, resulting in the increase of thermogenesis and energy expenditure. As it has been largely documented that metabolic dysfunctions can frequently be associated with reductions in fertility, the possible involvement of this molecule in the regulation of reproductive processes represents an issue to be addressed. On this basis, the first aim of this work was the evaluation of the presence of irisin in the swine ovary; then, we investigated the expression of the associated molecules FNDC5, PGC-1α, and PPAR-γ. To verify a potential modulatory role both on ovarian function and on redox status, cell growth, steroidogenesis, production of superoxide anion and nitric oxide, the nonenzymatic antioxidant scavengers, were assessed in vitro on granulosa cells treated with increasing concentrations of irisin (50, 100, and 150 ng/mL). The data collected demonstrate the presence of irisin in swine ovarian follicle. Moreover, the highest concentrations tested stimulated metabolic activity and inhibited cell proliferation (P < 0.05); the peptide exerted a biphasic effect on progesterone (P < 0.01) production and, at the highest concentrations, inhibited nitric oxide while stimulated the nonenzymatic antioxidant power (P < 0.05). Superoxide anion and estradiol 17ß were unaffected. The demonstration of the local presence of irisin at the ovarian level and the highlighted effects allow us to qualify this molecule as a potential physiological regulator of follicular function.

Nanoplastics impair in vitro swine granulosa cell functions.

Soil, water, and air pollution by plastic represents an issue of great concern since the particles produced by degradation of plastic materials can be ingested by animals and humans, with still uncertain health consequences. As a contribution on this crucial subject, the present work reports an investigation on the in vitro effects of different concentrations of polystyrene nanoplastics (5, 25, and 75 µg/mL) on swine granulosa cells, a model of endocrine reproductive cells. In particular, cell growth (BrDU incorporation and ATP production), steroidogenesis (17-ß estradiol and progesterone secretion) and redox status (superoxide and nitric oxide production, enzymatic and non-enzymatic scavenging activity) were studied. Nanoplastics, at the highest concentration, stimulated cell proliferation (P < 0.05), while cell viability resulted unaffected. Steroidogenesis was disrupted (P < 0.05). Both enzymatic and non-enzymatic scavenging activity were increased after exposure at the highest nanoplastic dose (P < 0.05, P < 0.001). Nitric oxide secretion was increased by 25 and 75 µg/mL (P < 0.05) while superoxide generation was stimulated (P < 0.001) only by the highest concentration tested. Taken together, main features of cultured swine granulosa cells resulted affected by exposure to nanoplastics. These results raise concerns since environment nanoplastic contamination can represents a serious threat to animal and human health.

Expression and localization of stanniocalcin 1 in swine ovary.

Stanniocalcin 1 (STC 1) is a glycoprotein involved in mineral homeostasis and was first identified in fish. Its mammalian homologue has been implicated in the regulation of various biological processes, including angiogenesis and steroidogenesis both of which are fundamental events in ovarian function. Interestingly, the highest level of STC 1 expression in mammals occurs in ovarian tissue but no information is available on swine species. Therefore, the present study was undertaken to investigate the expression and the immunolocalization of STC 1 in swine ovary. In addition, we evaluated whether swine granulosa cells synthesize STC 1 and its possible modulation by hypoxia, a physiological condition in ovarian follicle growth. Our data show STC 1 for the first time in swine ovary; moreover, we demonstrate STC 1 production by granulosa cells, both in basal condition and in response to oxygen deprivation. The latter is suggestive of a potential modulatory role for STC 1 in hypoxia-driven angiogenesis.

Expression and function of the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in the swine ovarian follicle.

The most characterized stromal cell-derived factor-1 (SDF-1) variants are the isoform α, which is the predominant one but undergoes rapid proteolysis, and the ß isoform, which is more resistant. Through the interaction with a specific chemokine receptor called CXCR4, SDF-1 is able to regulate different physiological processes. The aim of this study was to verify the expression and potential functional role of SDF-1 and CXCR4 in the porcine ovary. Firstly, the expression of SDF-1 and its receptor in different ovarian districts was verified for the first time. Thereafter, the effect of SDF-1 ß isoform (51-72) fragment on functional parameters, such as proliferation, metabolic activity, redox status, nitric oxide production, and steroidogenic activity, was assessed on granulosa cells collected from follicles. In addition, the potential effect of this protein in vascular events was verified through investigations on porcine aortic (AOC) endothelial cells, such as the production of nitric oxide and viability tests. The proliferation and metabolic activity were not affected by treatment with the cytokine. As regard to steroidogenesis, the peptide stimulated both estrogen (P = 0.049) and progesterone production (P = 0.039). Redox status was affected by the examined substance since superoxide anion was inhibited (P = 0.001), while antioxidant power (P = 0.034), as well as nitric oxide generation, were stimulated (P = 0.034). Tests performed on AOCs showed significant stimulation of nitric oxide production (P = 0.004) by the examined peptide, while cell viability was unaffected. Therefore, the potential role of cytokine in the mechanisms involved in the regulation of follicular function can be hypothesized.

Bisphenol S, a Bisphenol A alternative, impairs swine ovarian and adipose cell functions.

The high-volume-produced plastic monomer Bisphenol A (BPA) has been in the spotlight in the last years because of its endocrine disruptor (ED) behavior, leading to disclosure of the association between the widespread human and wildlife exposure to BPA and reproductive, metabolic, and developmental disorders and hormone-dependent cancer onset. These evidences caused restrictions and prohibitions of BPA industrial uses and prompted investigation of harmless alternative compounds. Above all, several countries have substituted the parental analogue with Bisphenol S (BPS) in baby care product manufacturing, even if its structural homology to BPA suggests similar ED properties not yet completely ruled out. In light of this consideration, the aim of this in vitro study was to investigate the effect of BPS exposure (0.1, 1, and 10 µM for 48 h) on granulosa cells that are considered the prime ovarian targets of BPA as a "reproductive toxicant". Our data document that BPS inhibited E2 production, cell proliferation, and scavenging nonenzymatic activity (P < 0.05) while it significantly (P < 0.05) stimulated cell viability, superoxide (O2-) and nitric oxide (NO) production in cultured swine granulosa cells, a previously validated endocrine cell model for BPA. Evidence also exists that BPA and its analogues, as environmental lipophilic pollutants, are involved in the disruption of adipose tissue (AT) endocrine function, resulting in metabolic effects and thus in potential reproductive disorders. On this basis, our second purpose was the assessment of BPS effects on mesenchymal stromal cells (MSCs) isolated from porcine AT, taking into account MSCs viability and adipogenic differentiation, a process actually demonstrated to be largely affected by EDs. Our results show that BPS decreased (P < 0.001) cell viability of proliferating adipose stromal cells. Taken as a whole, our data demonstrate an effective BPS ED activity at µM concentrations, suggesting that further studies are needed before considering its use in industrial application as an alternative to BPA.

Orexin A in swine corpus luteum.

Orexin A (OXA) is a hypothalamic neuropeptide which acts on 2 known G-protein-coupled receptors. It has been demonstrated that OXA is a central molecular link between food intake and reproduction. More recently, its peripheral role has been investigated, and we demonstrated its involvement in regulating ovarian follicle function. The present study was undertaken to explore a potential physiological role of orexin system in swine corpus luteum, a transient ovarian endocrine organ. Our aim was, first, to analyze the localization and eventual colocalization of OXA and its 2 receptors within the different cell types composing the corpus luteum structure. Second, we wanted to explore the effects of OXA on isolated luteal cells, and finally to verify a potential involvement of OXA in angiogenesis, a crucial event in corpus luteum development. Our data demonstrate the local expression of OXA and its receptors in swine corpus luteum. Luteal cell functions were affected by treatment with OXA. In particular, progesterone production was inhibited (P < 0.05) and nonenzymatic scavenging activity was increased (P < 0.05). Moreover, OXA inhibited (P < 0.05) new vessel growth. Our results suggest that OXA could act locally to play a role in corpus luteum demise.

Orexin system in swine ovarian follicles.

Successful reproduction is strictly linked to metabolic cues. The orexins are a family of hypothalamic neurohormones, well known for their key role in the control of food intake and the involvement in several aspects of the reproductive process. The biological actions of both orexins are carried out through binding to the related Orexin 1 (OX1R) and Orexin 2 (OX2R) G-protein-coupled receptors. The purpose of this study was to investigate the presence of orexin system components in the porcine ovaries, to contribute to expand the knowledge about their pleiotropic role. First, we investigated the localization of orexin A (OXA) and its receptors by immunochemistry in different ovarian districts. Thereafter, we evaluated the expression of the prepro-orexin (PPO) gene and OXA effects on granulosa cell functions. Immunohistochemical study revealed the presence of orexinergic system components in porcine ovarian follicles. Moreover, our data show the expression of PPO messenger RNA in swine ovarian follicles >5 mm. In addition, OXA influences proliferation (P < 0.05), steroidogenic activity (P < 0.05), and redox status of granulosa cells (P < 0.05). Therefore, we hypothesize that OXA could exert a local physiological role in swine ovarian follicles even if further studies are required to deeply define the function of this pleiotropic system.

Mammalian odorant binding proteins.

Odorant binding proteins (OBPs) pertain to one of the most abundant classes of proteins found in the olfactory apparatus. OBPs are a sub-class of lipocalins, defined by their property of reversibly binding volatile chemicals, that we call 'odorants'. Numerous sequences of OBPs are now available, derived from protein sequencing from nasal mucus material, or from DNA sequences. The structural knowledge of OBPs has been improved too in recent years, with the availability of two X-ray structures. The physiological role of OBPs remains, however, essentially hypothetical, and most probably, not linked to a function of odor transport. The present knowledge on OBP biochemistry, sequence and structure will be examined here in relation to the different functional hypotheses proposed for OBPs.

Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes.

Porcine odorant binding protein (pOBP) is a monomer of 157 amino acid residues, purified in abundance from pig nasal mucosa. In contrast to the observation on lipocalins as retinol binding protein (RBP), major urinary protein (MUP) or bovine odorant binding protein (bOBP), no naturally occurring ligand was found in the beta-barrel cavity of pOBP. Porcine OBP was therefore chosen as a simple model for structure/function studies with odorant molecules. In competition experiments with tritiated pyrazine, the affinity of pOBP towards several odorant molecules belonging to different chemical classes has been found to be of the micromolar order, with a 1:1 stoichiometry. The X-ray structures of pOBP complexed to these molecules were determined at resolution between 2.15 and 1.4 A. As expected, the electron density of the odorant molecules was observed into the hydrophobic beta-barrel of the lipocalin. Inside this cavity, very few specific interactions were established between the odorant molecule and the amino acid side-chains, which did not undergo significant conformational change. The high B-factors observed for the odorant molecules as well as the existence of alternative conformations reveal a non-specific mode of binding of the odorant molecules in the cavity.

Matrix attachment region regulates basal beta-lactoglobulin transgene expression.

Nuclear matrix attachment regions (MAR) have been implicated in the regulation of gene expression. We have identified a region within the proximal 3'-flanking sequences of the ovine beta-lactoglobulin (betalg) gene that interacts with the nuclear matrix in vitro. No equivalent region was detected in the 5' flanking region. We have investigated the role of this element in regulating betalg expression in vitro and in vivo. Removal of the MAR did not affect the frequency of betalg transgene expression at the mRNA level, but betalg transgenes that lacked the MAR were expressed at a lower level than wild-type betalg transgenes. In neither in-vitro HC11 transfection experiments nor transgenic mice was hormonal induction of betalg expression significantly affected by MAR removal. Nuclear run-on analysis demonstrated that the impaired basal expression of betalg transgene loci lacking the MAR was due to a reduced transcription rate. Thus, the single MAR enhances the basal transcriptional potential of the betalg gene.

Role of androgens in proliferation and differentiation of mouse mammary epithelial cell line HC11.

Androgens have been found in mammary epithelium and in milk throughout the cycle of the mammary gland in vivo. The aim of this study was to investigate the possible role of these substances in mammary epithelial growth and differentiation in the mouse HC11 cell line. Cells were stimulated with testosterone, dihydrotestosterone, androstenedione and 5alpha-androstane-3alpha,17beta-diol at concentrations ranging between 0.3 nM and 30 nM. Cyproterone acetate or flutamide, androgen receptor antagonists, (3 microM) were used to block specific androgen effects. Proliferative effects were measured by an MTT (tetrazolium blue) conversion test and [(3)H]thymidine uptake. HC11 cells were transfected with pbetacCAT, a chimeric rat beta-casein gene promoter-chloramphenicol acetyl transferase (CAT) gene construct and CAT ELISA was used to determine gene expression. RT-PCR was performed to detect androgen receptor expression. After 24, 48 and 72 h androgens significantly (P<0.05) increased proliferation. Androgen antagonists significantly (P<0.05) reduced the proliferative effects. Furthermore androgens potentiated the lactogenic effect of prolactin, insulin and dexamethasone (P<0.05). Finally, the androgen receptor gene was expressed in both proliferating and differentiated HC11 cells. These observations lead us to hypothesize an activity of this class of steroids in mammary physiology. In particular, androgens stimulate cell proliferation and beta-casein gene expression; this influence appears to be mediated by androgen receptors.

Single turnover mechanism of a trypsin-reactor with high enzyme concentration.

A small column containing 2 mM CH-Sepharose 4B-immobilized trypsin was connected to a flow injection device equipped for potentiometric measurements (0.01-2 mM protons) and for post-column analysis by spectrophotometry and capillary electrophoresis (CE). The device was engaged with N alpha-benzoyl-L-arginine pNO2-anilide (BAPNA), beta-lactoglobulin (beta-Lac) and peptides of V8-protease predigested beta-Lac. At a given flow rate, the reaction with BAPNA or beta-Lac (below 2 mM) produced about 1 proton per substrate molecule in each sample (linear relation to substrate amount); with peptides (below 22 mM), the reaction did not exceed 0.17 acid equivalents per substrate molecule (hyperbolic dependence). Final experiments demonstrated that the reactor gave a correct estimate of available lysine in peptides of beta-Lac modified with 5-nitrosalicylaldehyde. The data could be predicted by a kinetic model describing the reactor performance in 'single turnover' conditions. The interplay between resident time and the non-catalytic amount of trypsin prevented each enzyme molecule from recycling as well as each substrate molecule (containing one or more cleavage sites) from encountering the enzyme more than once. In conclusion, both from the experimental and the theoretical point of view, this work permitted the analysis of trypsin behaviour in some extreme working conditions and indicates how to modulate the performance of an endoprotease-based reactor. A brief discussion on potential applications in protein mapping and tagging and in the quantitative analysis of protein bioavailability by means of a biosensorial strategy is also described.

Evaluation of cathepsin B levels in fresh thighs selected for cured raw ham production.

Excessive meat tenderization in cured raw Parma ham has recently been correlated with abnormal levels of cathepsin B in freshly slaughtered thigh meat. We have developed a visual assay employing the substrate Z-Arg-Arg-NNapOMe for the quantitative detection of active cathepsin B levels in pork thigh muscle homogenates. The work was based on a kinetic characterization, in steady state condition, of pig muscle cathepsin B with several peptidyl chromophoric substrate analogs. This assay can easily and safely be performed by non-specialized personnel directly in the slaughterhouse or in the factory, for an early quality evaluation of thighs selected for Parma ham production. Our characterization has further indicated that the catalytic properties of porcine muscle cathepsin B and those of isoforms from other animal and plant species are practically identical. This is particularly evident in the commercially available bovine spleen isoform, which was employed as a model enzyme in most of the experiments.