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A physiological equilibrium exists between pro- and antioxidant factors. When the oxidant factors exceed the capacity of their removal or inactivation, oxidative stress (OS) occurs. The OS levels were assayed in plasma obtained from 2 bird species. Blood samples were collected from 20 healthy domestic chicken hens, 10 living in an intensive farming environment and 10 free-range, and from 18 healthy Eurasian magpies (Pica pica; 7 females and 11 males, with an estimated age of >1 year of age). For OS biomarker assessment, the determinable reactive oxygen metabolites (d-ROMs) were measured, and the plasmatic antioxidant test (PAT) was performed; the OS index (OSI) was then calculated (d-ROMs/PAT × 1000) as a parameter of overall oxidative stress. Moreover, lipid peroxidation was assessed by measuring plasmatic malondialdehyde (MDA) levels. A hematological evaluation was also performed on each bird with a hemocytometer, on which a blood sample was placed to obtain both a total and differential white blood cell (WBC) count. In hens, OSI and MDA levels were significantly higher (P = .04, and P = .004) in subjects from intensive farming (14.7 ± 7.1 and 27.2 ± 10.4 nmol/mL) than in those bred in rural conditions (5.6 ± 10.3 and 8.2 ± 13.3 nmol/mL). In magpies, a positive correlation between the total WBC count and OS was found, and both d-ROMs and OSI were significantly higher (P = .03) in subjects with a total WBC count greater than the median value (20.4 × 103 cells/µL) with respect to those with a total WBC count less than the median value. The results generated from this study indicate that higher OS levels occurred in hens bred in an intensive indoor farm environment compared with outdoor free-range conditions. Possibly the higher OS levels could be related to the higher stocking density and dust levels found in the indoor facility. Additionally, the correlation between OS biomarker levels in magpies and total WBC count suggests that OS level is influenced by immune response, in agreement with previous studies. Collectively, present data seem to be promising for the application of OS measurement in avian medicine for health and animal welfare monitoring.

Based on our previous study in follicles, the first aim of this work was to evaluate the effect of melatonin in the swine corpus luteum (CL). Luteal cells were exposed to 10 and 20pg mL-1 melatonin. We evaluated the effect on proliferation (bromo-deoxy-uridine uptake), steroidogenesis (progesterone) and redox status by means of Griess test (nitric oxide production), WST-1 test (superoxide anion generation) and FRAP test (non-enzymatic antioxidant power). The results showed a significant increase in antioxidant power, as well as a reduction in the other parameters analysed. These data and the expression of MT2 observed in luteal cells allow us to hypothesise a physiological role of melatonin in the regulation of CL functionality. The reproductive function is dependent on energy reserves stored in adipose tissue. Therefore, we sought to verify the effect of melatonin on adipose stromal cells (ASCs). MT2 receptor expression was detected in ASCs and the presence of gene markers (PPARγ and leptin) before and after adipogenic differentiation was verified. The differentiation was significantly inhibited by melatonin, as well as cell viability. In conclusion, present results suggest that melatonin exerts a potential inhibitory action on luteal function and adipogenesis, possibly mediated by MT2.

The World Health Organization defined leishmaniasis as one of the priority attention diseases. Aiming to clarify some aspects of its pathogenetic mechanisms, our study focused on the assessment of redox status in dogs, the main reservoir for Leishmania infantum. Forty-five dogs from an endemic area in southern Italy were divided into four different groups (from mild disease with negative to low positive antibody levels to very severe disease with medium to high positive antibody levels) according to the LeishVet group guidelines. Their plasma and/or sera were tested for reactive oxygen species (ROS), namely the superoxide anion (O2&minus, ), reactive nitrogen species (RNS), such as nitric oxide (NO) and hydroperoxides (ROOH), as well as activity of the detoxifying enzyme superoxide dismutase (SOD), and total nonenzymatic antioxidant capacity, as determined by the ferric reducing-antioxidant power (FRAP) assay. O2&minus, generation was significantly (p &lt, 0.05) reduced in leishmaniasis-affected dogs independently of the clinical stage, while NO production was stimulated (p &lt, 0.05) only in II and III stage patients. No difference could be found for the levels of hydroperoxides and SOD activity between healthy and pathological subjects. FRAP values were lower in affected dogs but only in stage II. Taken together, although we demonstrated that several redox status parameters are altered in the plasma of dog affected by leishmaniasis, the oxidative stress changes that are observed in this disease, are possibly mainly due to cellular blood components i.e., neutrophils responsible for the elimination of the parasite. Further studies are required to assess the clinical values of the collected data.

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 (P0.05) while it significantly (P0.05) stimulated cell viability, superoxide (O

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.

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 follicles5 mm. In addition, OXA influences proliferation (P0.05), steroidogenic activity (P0.05), and redox status of granulosa cells (P0.05). 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.

Simple Summary In recent years, the sensitivity and interest of veterinary practitioners and owners both for elderly pets and for those suffering from chronic or degenerative diseases, has increased considerably. As for the horse, both the progression of age, or an intense sport activity, are accompanied by the onset of chronic arthritic forms that affect the animal welfare and significantly limit its movements. To date, the available drugs cannot eradicate the causes of these diseases, whose symptoms are usually treated with anti-inflammatory drugs. These drugs, although extremely effective, due to the side effects, can only be used for short periods of time. As an alternative to these treatments, we investigated here the use of a natural product, the extract of Indian celery seeds, that in humans is employed as a supplement with anti-inflammatory properties, for long lasting treatments of chronic osteo-arthropathies. The improvements obtained and the lack of side effects for prolonged treatments, suggest that also in the equine species the Indian celery seed extract can be successfully administered to ameliorate both well-being and quality of life of subjects suffering from chronic joint diseases. Abstract The extract of the seeds from Indian celery, Apium greaveolens (CSE), tested in experimental animals (rodents), and in humans affected by chronic osteoarthritic diseases, exhibits anti-inflammatory effects that can be compared, to some degree, to those of non-steroid anti-inflammatory drugs (NSAID). In view of a potential use of CSE in the equine species, it was tested on horses affected by chronic articular pathologies. The trial was performed on 20 horses divided into three different groups, orally treated with 0 (controls), 7.0 or 30 g of CSE BID. Basic orthopedic examinations were conducted, vital signs were observed, and blood samples collected. Improvement was observed at the highest dosage tested (30 g of CSE BID), as reflected in the score values of three clinical parameters, (i) amplitude and (ii) sensitivity to passive flexion and (iii) flexion test. Since the improvement of these parameters can be correlated with a lower perception of the pain, the present data suggest that the CSE treatment can have an analgesic effect in horses affected by chronic osteoarthritic diseases.

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.

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 β 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 β 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.

The triazine herbicide simazine is a pesticide commonly detected in surface and ground waters, although banned in most European countries since 2004. Concerns for humans and animal health result from its potential endocrine disrupting action, that can lead to reproductive disorders. The present in vitro study was undertaken to study simazine effects on swine granulosa cell function, namely cell viability, proliferation, steroidogenesis and NO production. Moreover, the ability of this substance to interfere with the angiogenetic process, a crucial event in reproductive function, was taken into account. Our data document that simazine treatment, at 0.1 or 10 μM concentration levels, stimulates granulosa cell proliferation and viability and impairs steroidogenesis, increasing in particular progesterone production. In addition, the in vitro angiogenesis bioassay revealed a significant simazine stimulatory effect on immortalized porcine Aortic Endothelial Cell proliferation. Collectively, these results show that simazine can display disruptive effects on ovarian cell functional parameters, possibly resulting in reproductive dysfunction. This hypothesis is also supported by the observed pro-angiogenetic properties of this herbicide, as already suggested for different endocrine disruptors.

Granulosa cells, which belong to the somatic compartment of the ovarian follicle, are actively involved as endocrine cells in follicle growth. Recently, it has been proposed that these cells are not terminally differentiated and possess multipotency. Therefore, we cultured swine granulosa cells in specific endothelial cell culture medium (EBM-2), and phenotypic and functional characteristics of endothelial cells were assessed. The collected data suggest that these endocrine cells can also behave as endothelial cells, therefore potentially contributing to follicular angiogenesis, a crucial process in follicle growth and selection.

Oxidative stress plays an important role in the development of many horse diseases and it has been shown that housing has important implications for the psychophysical well-being of horses. The aim of this study is to determine if there are any differences between the redox status in horses in relation to housing conditions. The four housing conditions analyzed were: single box, without external access and without contact (Cat A), single box with external access and possibility of partial contact (Cat B), group housing with box and large paddock (Cat C), pasture with more than 7 horses and the possibility of green forage for the whole year (Cat D). A group of 117 healthy horses were selected in several private stables in Northern Italy. All subjects treated with any type of drug were excluded. At the end of the enrollment, the 117 selected horses were divided into the four housing categories. Stereotypies were highest in the group of horses in single box, without external access and without contact (Cat A). Oxidative stress was evaluated by testing plasma or serum samples for the following parameters: superoxide anion (WST), nitric oxide (NO), reactive oxygen species (d-ROMs), ferric reducing ability of plasma (FRAP), and the activity of superoxide dismutase (SOD). Simultaneously with the blood sampling, the owners completed a questionnaire with all the management aspects of the horse (signaling, feeding, equestrian activity, vaccinations, foot management etc.). The statistical evaluation was carried out based on the categories previously described, on the presence and absence of stereotypies and on some signaling data obtained from the questionnaire. There were no significant differences in the parameters analyzed between the categories. No significant redox status differences were detected based on the presence or absence of stereotypies. Interestingly, when the age was introduced as selection (&lt, 14 and &gt, 14 years old) parameter inside the categories, statistical significance was observed for some of the stress markers considered. Finally, independently of the housing conditions, the horses of the most two represented breeds exhibited different values of FRAP. All these aspects are commented in the discussion.

Oxidative stress is a prominent event in several acute and chronic diseases including neoplasia. Although its direct involvement in carcinogenesis still remains to be clearly defined, a deeper knowledge of oxidative stress in oncologic patients could help to monitor their clinical outcome and to develop new therapeutic approaches. Therefore, the present study was undertaken to explore redox status in blood of neoplastic dogs affected either by multicentric lymphoma or by primary cutaneous mastocytoma. Superoxide anion (O2 •−), nitric oxide (NO) and hydroperoxides (ROOH) were measured. Detoxifying enzyme superoxide dismutase (SOD) and total non-enzymatic antioxidant capacity (ferric reducing-antioxidant power (FRAP)) were assessed. The oxidative stress index (OSi) both for enzymatic (OSiE) and non-enzymatic (OSiNE) scavengers were evaluated. Both pathologies, showed a reduced NO generation, while O2 •− levels were decreased only in mastocytoma. The oxidative stress indexes showed a significant decrease in mastocytoma patients, only for OSiE.

Apart from its well-known role in regulating endothelial function, in mammals, nitric oxide (NO) is an important signaling molecule involved in many processes, regulating different biological functions. It has been demonstrated that NO plays a role in the physiology of the reproductive system, where it acts in controlling the activity of reproductive organs in both sexes. In the female of several animal species, experimental data suggest the presence of an intraovarian NO-generating system, which could be involved in the control of follicular development. The role of NO in regulating follicular atresia by apoptosis is still controversial, as a dual action depending mostly on its concentration has been documented. NO also displays positive effects on follicle development and selection related to angiogenic events and it could also play a modulatory role in steroidogenesis in ovarian cells. Both in monovulatory and poliovulatory species, the increase in PGE2production induced by NO via a stimulatory effect on COX-2 activity appears to be a common ovulatory mechanism. Considerable evidence also exists to support an involvement of the NO/NO synthase system in the control of meiotic maturation of cumulus–oocyte complexes.

Kisspeptin and its receptor KISS1R are involved in the neuroendocrine regulation of mammalian reproduction and their role on follicular development and function can be hypothesized. The present work was designed to confirm the immunopresence of kisspeptin and its receptor in the ovary of swine and to study the effects of kisspeptin 10 and its antagonist, kisspeptin 234, on main functional parameters of granulosa cells (i.e. cell proliferation, steroid production, and redox status) as well as their modulatory action on angiogenesis. The immunopresence of kisspeptin and KISS1R were detected in granulosa cells. Kisspeptin 10 stimulated progesterone in vitro production, thus indirectly suggesting that it can have a role in the luteinization process of granulosa cells. Kisspeptin 10 displayed potentiating effects on non-enzymatic scavenging activity, thus supporting its involvement in the control of the antioxidant defense system of ovarian follicles. In addition, results from the angiogenesis bioassay suggest that kisspeptin may have a role in the physiological development of new ovarian vessels. Additional studies are needed to confirm the functional significance of the kisspeptin/KISS1R system within the swine ovary.

Orexins are neuropeptides with pleiotropic functions, involved in the coordination of multiple versatile physiological processes, in particular related to food intake and several aspects of the reproductive process. Their actions are carried out through the bond with the related Orexin 1 (OXR1) and Orexin 2 (OXR2) G-protein-coupled receptors. Studies on the expression of the orexinergic system in the female genital organs are scarce and limited to preovulatory gametogenic follicles and corpora lutea isolated from the rest of the ovary. As the description of only these structures is insufficient to provide a complete picture of the organ, the present study is aimed to give a panoramic view of all the ovarian structures and cells expressing Orexin A (OXA) and its receptors in their original localization. Double labeling immunofluorescent methods, applied on frozen sections of the whole organ in both follicular and luteal phase, were used to highlight the particular distribution and colocalization of the proteins. For a better recognition of cellular morphology and a better distinction between gametogenic (healthy) and atretic follicles, also a single labeling immunolocalization of OXA on formalin fixed paraffin embedded tissues and a TUNEL staining were performed. The results indicate that OXA and its two receptors subtypes are expressed in all the different structures composing the swine ovary, albeit in different ways, in both phases of the ovarian cycle. In general, OXA and OXR2 appear diffusely distributed within "health", proliferating and steroid producing cells, while has granular appearance, being presumably associated to cytoplasmic vesicles, in degenerating cells, independently if apoptotic or not. The immunoreactivity for OXR1, instead, is often associated with the nuclear envelope but it is also detectable, to a lesser extent, diffusely distributed in the cytoplasm of growing or steroid producing cells. When cells undertake the path leading to degeneration, also OXR1 immunoreactivity assumes a granular appearance in the cytoplasm and is colocalized with OXA and OXR2. Different roles for the two receptors in the same cell and a different regulation of their expression remain to be investigated. Their comprehension could help studies of follicle development in pig, as part of in vitro oocyte maturation and fertilization programs in livestock.

During corpus luteum formation, impressive biological events take place to guarantee the transition from original follicular to luteal cells and to support required massive angiogenesis. It has been demonstrated that these phenomena resemble those essential for wound healing. After ovulation, blood vessels release their content in the antral cavity and coagulation takes place. Involvement of platelets in corpus luteum growth has been hypothesized both in human and in rat. On this basis, using platelet lysate (PL), a blood derivative with a higher platelet concentration, we aimed to assess a potential involvement of platelets in swine granulosa cell luteinization and on new blood vessel growth. Our results demonstrate, for the first time in the swine, that platelets could be directly involved in granulosa cell physiological luteinization, since the treatment with PL shifted steroid production from estradiol 17β to progesterone. Moreover, PL stimulated angiogenesis. Nitric oxide could be involved in these effects. These results are important to clarify complex intrafollicular molecular machinery. A better understanding of these mechanisms can be useful to develop more focused therapeutic strategies to contrast sow infertility. In addition, since the pig represents a model for translational studies, collected data could be of interest for human medicine because reproductive pathologies such as Polycystic Ovary Syndrome (PCOS) and endometriosis are often accompanied by platelet dysfunctions.

From an angiogenesis perspective, the ovary offers a unique opportunity to study the physiological development of blood vessels. The first purpose of this work was to set up a protocol for the isolation of pig corpus luteum endothelial cells, which were characterized by both morphologic parameters and the expression of typical molecular markers; we also verified their ability to form capillary-like structures in a 3-dimensional matrix, their response to hypoxia and their migration in the presence of vascular endothelial growth factor (VEGF). The effectiveness of our isolation protocol was confirmed by the characteristic "cobblestone shape" of isolated cells at confluence as well as their expression of all the examined endothelial markers. Our data also showed a significant cell production of VEGF and nitric oxide. Isolated endothelial cells were also responsive to hypoxia by increasing the expression and production of VEGF and decreasing that of nitric oxide. In the angiogenesis bioassay, cells displayed the ability of forming capillary-like structures and also exhibited a significant migration in the scratch test. Our data suggest that the isolation of luteal endothelial cells represents a promising tool in experiments designed to clarify the biology of the angiogenic process. Furthermore, we have demonstrated that the isolated population comprises a subset of cells with a multidifferentiative capacity toward the chondrocytic and adipocytic phenotypes. These data suggest the presence of a perivascular or adventitial cell niche in the vascular wall of the corpus luteum populated with cells showing mesenchymal stem cell-like features, as already demonstrated for the adipose tissue and endometrium.

Apart from the well established role of prolactin (PRL) in the control of mammary development and lactation, this hormone appears to possess a variety of physiological functions and evidence exists about its expression in many extra-pituitary sites. This experimental work was undertaken to gain knowledge about PRL and its receptor presence in the porcine antral follicle. In particular, we investigated the expression and local production of PRL in follicular fluid, theca and granulosa cells cultured in standard conditions and in hypoxia. Then, we also investigated its modulatory effect on several parameters mainly involved in granulosa cell function, namely redox status and steroidogenesis. In order to verify an involvement of PRL in the control of ovarian angiogenesis, a process strictly linked to follicle growth and development, we have verified possible PRL effects on granulosa cell production of Vascular Endothelial Growth Factor (VEGF) and nitric oxide as well as its modulatory role on the angiogenic activity of endothelial cells. Our data demonstrate that in the swine PRL is expressed in both components of the antral follicle, theca and granulosa layers, and it is produced by granulosa cells. Moreover, the hormone represents a relevant modulatory factor on key processes underlying follicular growth and development, such as steroidogenesis and angiogenesis.

Several studies have demonstrated that the endocrine disruptor bisphenol A (BPA) negatively affects animal and human health. An angiogenic process has been suggested among the events disrupted by this molecule, but the underlying mechanisms have not yet been clarified. The effect of BPA on angiogenesis was investigated by means of a bioassay previously validated in our laboratory. Using immortalized swine aortic endothelial cell line (AOC), the development of new blood vessels through a three-dimensional in vitro angiogenesis assay was evaluated. Subsequently, since vascular endothelial growth factor (VEGF) and nitric oxide (NO) are key players in the regulation of the angiogenic process, the effect of BPA on the production of these molecules by AOC was examined. BPA (10 μmol/L) stimulated AOC growth (p < 0.05) and VEGF production (p < 0.05), but did not modify NO levels. Our data suggest that the endocrine-disrupting effects of BPA could also be associated with the promotion of vascular growth, thus interfering with a physiologically finely tuned process resulting from a delicate balance of numerous molecular processes. The stimulatory effects of BPA on VEGF production may have negative implications, potentially switching the balance toward uncontrolled neovascularization. Moreover, since angiogenesis is involved in several pathologies, including cancer growth and progression, potential health risks of BPA exposure should be carefully monitored.

Contents 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.

We previously demonstrated the presence of 2-methoxyestradiol (2-ME) in swine follicular fluid. Present study was aimed first of all to investigate if swine granulosa cell produce 2-ME; in addition, we tried to assess a potential effect of hypoxia in modulating 2-ME output. Finally, we explored the effect of 2-ME in an angiogenesis bioassay set up in our lab. Our data show that cultured granulosa cells are able to produce 2-ME; interestingly, the secretion of the hormone appeared to be stimulated by hypoxia. Angiogenesis bioassay points out that 2-ME displays an inhibitory effect on neovascularisation. Therefore our data suggest that 2-ME could be a local effector in determining the fine tuning responsible for follicle angiogenesis. These data deserve special attention since the ovary is a valuable experimental model in angiogenesis research.

Summary Soya and soybean products used in swine feeding contain genistein, a non-steroidal phyto-oestrogen which has been demonstrated to influence endocrine functions. This observation leads us to design this study to evaluate the effect of genistein on swine granulosa cell steroidogenesis and proliferation. In the attempt to unravel the genistein signal transduction mechanisms, we verified the effect of lavendustin, a Tyrosine Kinase (TK) inhibitor, and the potential involvement of NO/cGMP pathway. Finally, as angiogenesis is essential for follicle development, we tested the effect of the phyto-oestrogen on vascular endothelial growth factor production and on granulosa cell redox status, because free-radical species modulate neovascularization. Our data provide evidence that genistein interferes with granulosa cell steroidogenesis while it does not modulate cell growth: this effect could be at least partially produced by inhibiting TK-dependent signalling systems. On the contrary, NO/cGMP pathway or vascular endothelial growth factor production can be excluded as signalling mechanism involved in phyto-oestrogen effects. Remarkably, genistein stimulates hydrogen peroxide production thus potentially inhibiting follicular angiogenesis. Collectively, these results suggest that genistein consumption could potentially negatively impact swine reproductive function.

Netrin-1 was first identified as a guidance factor in axon outgrowth during central nervous system development and was later shown to be involved in the morphogenesis of other organs. This study, thus, aimed to verify netrin-1 gene expression in swine antral follicles and to detect netrin-1 protein expression in follicular fluid. In addition, since netrin-1 is also a potential guidance factor for endothelial cells during angiogenesis, an essential event for follicular development, we attempted to verify its effects on swine aortic endothelial cells. Our results show that netrin-1 is present in follicular fluid and is physiologically expressed in both the thecal and granulosa layers from swine antral follicles. Furthermore, by means of an angiogenesis bioassay, we documented the inhibition of vascular neoformation by netrin-1. In conclusion, our results demonstrate that netrin-1 can be synthesized by swine follicular cells and secreted in the follicular fluid where it appears to exert regulatory effects on both follicular function and vascular development.

Stanniocalcin 1 is a potential physiological modulator of steroidogenesis in the swine ovarian follicle L. Baioni & G. Basini & S. Bussolati & F. Grasselli Published online: 4 July 2009 # Springer Science + Business Media B.V. 2009

Background:The identification of possible ways to block blood vessels formation has become a major scientific objective of the last decade and several phytochemicals are currently being exploited to target tumour angiogenesis. Aim:The effects of Sanguinarine (SA), an alkaloid from the root of Sanguinaria Canadensis, were evaluated in an in vitro angiogenesis model; moreover the effects on Akt phosphorylation in porcine aortic endothelial cell line (AOC) were also examined. Methods:SA (300 nM) was tested in the presence or absence of VEGF (100 ng/ml) in a three dimensional angiogenesis bioassay obtained pipetting a suspension of AOC on microcarrier beads in a fibrinogen solution before the addition of thrombine. Endothelial cell proliferation was measured at 48, 96, 144, 192 h. The phosphorylation of Akt was measured by ELISA in 2×105 AOC treated as described above. Results:The addition of SA abolished (p < 0.001) VEGF stimulatory effect on AOC growth at all the examined times. In addition, the stimulatory effect induced by VEGF on Akt phosphorylation was significantly (p < 0.001) inhibited by SA. Conclusion:SA appear to be an antiangiogenic natural product by directly suppressing the proliferative effect of VEGF on endothelial cell line: this effect could be mediated by blocking the VEGF-induced Akt activation.

2-Methoxyestradiol (2-ME) is an estradiol metabolite with antiangiogenic properties. It can be produced by granulosa cells and it is present in normal follicle at high concentrations. The identification of reactive oxygen species (ROS) role in the molecular pharmacology of 2-ME is an active area of research. The objective of this article was therefore to evaluate the effect of 2-ME on both superoxide anion (O(2)(-)) production and superoxide dismutase (SOD) activity in swine granulosa cells collected from follicles greater than 5 mm and treated for 48 h with 1 muM 2-ME. 2-ME inhibited (P < 0.001) O(2)(-) generation in swine granulosa cells, while it stimulated (P < 0.05) the SOD activity. We previously demonstrated that a stimulation of O(2)(-) generation triggers angiogenetic response in granulosa cells. Therefore, we argue that the inhibitory effect of 2-ME on O(2)(-) could be responsible for its antiangiogenetic effect.

Sanguinarine (SA) has been used in toothpastes and oral rinse products and has been recently applied to livestock nutrition. This study was undertaken to examine the reproductive-related consequences of this feeding supplementation, evaluating SA effects (10, 100 and 300 nM) on swine granulosa cell steroidogenesis, oxidative enzyme activities (peroxidase, catalase and superoxide dismutase), and proliferation. Since angiogenesis is fundamental for follicle development, we also tested the impact of SA exposure on vascular endothelial growth factor (VEGF) production. SA had no effect on proliferation and did not alter progesterone production, although it significantly reduced estradiol synthesis at the two highest concentrations tested (100 and 300 nM). SA addition to granulosa cell culture inhibited VEGF production and increased peroxidase and catalase activities at all tested concentrations while superoxide dismutase activity was increased only at 300 nM. Our data suggest that SA can negatively affect some key biochemical parameters of ovarian granulosa cells.

The use of herbs as additives in livestock nutrition as an alternative to antibiotics is becoming a new goal in animal production. It is known that green tea exerts antimicrobial activity owing to specific flavonoid compounds named catechins, primarily represented by epigallocatechin-3-gallate (EGCG). Remarkably, despite many potential benefits of green tea and EGCG consumption, it is also important to get an insight on the possible reproductive-related consequences of feeding supplementation. To this purpose, granulosa cells were harvested from follicles > 5mm and treated with 5 and 50 microg/ml of EGCG in order to evaluate the effects on the main parameters of granulosa cell function: steroidogenesis, by measuring progesterone and estradiol-17beta production, and proliferation, one of the major feature of ovarian follicular growth. Moreover, as the genesis of new vessels has been demonstrated to be fundamental for follicle development, we evaluated the effect of EGCG on the production of the main angiogenetic factor, VEGF, by swine granulosa cells. Finally, since reactive oxygen species (ROS) might be involved in the control of female reproductive activity, we studied the effect of EGCG on superoxide anion (O2-) and hydrogen peroxide (H2O2) production by swine granulosa cells and on the activity of the scavenging enzyme superoxide dismutase (SOD). EGCG significantly (p < 0.05) inhibited proliferation, steroidogenesis, VEGF and O2- production by swine granulosa cells; on the contrary, H2O2 levels and SOD activity were stimulated (p < 0.05) by the catechin. Therefore, since our data demonstrate that EGCG has a negative effect on reproductive performances in swine, feeding supplementation should be carefully considered.

The possible role played by hypoxia and vascular endothelial growth factor (VEGF) in the regulation of follicular angiogenesis was studied in a three-dimensional fibrin gel model. Granulosa cells from follicles5mm were subjected to normoxia (19% O2), partial (5% O2) or total (1% O2) hypoxia and their culture media were collected and used to stimulate porcine Aortic Endothelial Cells (AOC) included in the fibrin matrix. A suspension of AOC on microcarrier beads was pipetted in a fibrinogen solution (1 mg/ml PBS) before the addition of 1250 IU thrombine (250 microl) to catalize the gel formation. Granulosa cell conditioned media were tested in the presence or absence of VEGF Trap R1R2 (150 ng/ml), a potent VEGF inhibitor, that had its efficacy tested by adding VEGF (100 ng/ml) to AOC culture. Endothelial cell proliferation was measured at 48, 96, 144, 192 h by means of Scion Image Beta. A significant (p0.01) increase of AOC proliferation at each time of measurement was induced by culture media from granulosa cells subjected to partial (except at the end of the first 48 h) and total hypoxia compared to control and normoxia conditions, and by VEGF. VEGF Trap significantly (p0.01) inhibited the stimulatory effect of media conditioned by granulosa cells cultured in hypoxic conditions. These data suggest that hypoxia stimulates angiogenic activity of granulosa cells possibly by means of VEGF which could represent the main effector in promoting endothelial cell proliferation.

Ovarian angiogenesis, which is currently considered to be of crucial importance in controlling the growth of developing follicles, is a physiological process driven by a variety of angiogenic factors. Among these, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been recognized as key players in promoting cell growth and differentiation. Porcine granulosa cells from small (3 mm), medium (3-5 mm) and large (5 mm) follicles were seeded at different densities in DMEM:Ham's F12 (1:1) with or without different concentrations of VEGF or bFGF. After 48 h of culture, media were assayed for oestradiol (E2) 17beta, progesterone (P4), nitric oxide (NO) and VEGF levels; in addition, cell proliferation was evaluated by 3H-thymidine incorporation assay. Both bFGF and VEGF effects on E2 and P4 production by cultured granulosa cells resulted to be dependent on follicle size. The bFGF was always ineffective in modulating cell proliferation, while VEGF exerted an inhibitory effect on the proliferation in the small follicle group and a stimulatory one in the medium and large follicle groups. The bFGF consistently reduced NO levels in culture media. The VEGF appeared to be ineffective in modifying NO production in the small follicle group, while it was stimulatory in the medium follicle group and inhibitory in the large follicle group. Basal VEGF production was higher in cells from the large follicle as compared with the small and medium follicle groups, and it was unaffected by bFGF. These results suggest that VEGF plays a modulatory role in granulosa cell functional activity and it is possibly involved in the regulation of follicle growth; on the contrary, bFGF does not appear to represent a significant regulatory factor in our cellular model, except for an inhibitory action on the production of NO, whose anti-angiogenic properties need to be further substantiated.

The present investigation was undertaken to verify if the two nitric oxide synthase isoforms, eNOS and iNOS, are present in swine granulosa cells and whether the enzyme soluble guanylate cyclase is functionally active in the same cells and can account for NO effects. Using western blotting, the presence of endothelial NO synthase was demonstrated in freshly collected cells; on the contrary, iNOS expression was not observed in the same cells either before or after culture with the inflammatory cytokine hTNF-alpha. The treatment with a strong NO donor (S-Nitroso-L-acetyl penicillamine, SNAP) determined an increase of cGMP levels in culture media, which was attenuated by the combined treatment with an inhibitor of NO-sensitive soluble guanylate cyclase, 1H-[1,2,3]oxadiaziolo [4,3a]quinoxaline -1-one (ODQ). The cGMP analog, 8 bromo-cGMP, mimicked the strong inhibitory effect exerted by SNAP on estradiol 17 beta and progesterone production, while ODQ did not modify steroids concentrations in culture media. These observations demonstrate the presence of a follicular NO-generating system, which in swine granulosa cells seems to include only the endothelial NOS isoform. Furthermore, the nitric oxide/cyclic GMP system seems to be functionally active in these cells, since cGMP appears to mediate NO action, even if it cannot account completely for NO inhibitory effect on steroidogenesis.

Content Gonadotropins are required for follicular growth and differentiation, but increasing amounts of evidence indicate that intrafollicular factors modulate their effects at granulosa cell level. In order to study the effect of factors present in bovine follicular fluid, a partial purification of low molecular mass factors from fluids collected from small ( 8 mm) follicles was performed and the biological activity of these peptides on steroidogenesis of granulosa cells from small and large follicles was examined. The purification was carried out by filtration through membranes in 25 and 10 kDa molecular weight cutoffs. After filtration, samples were analysed by polyacrylamide gel electrophoresis and protein concentration was measured by spectrophotometric analysis. Granulosa cells from small and large follicles were cultured in serum-free DMEM/Ham's F12 (1 : 1) plus transferrin (5 mg/l) and selenium (5 µg/l) for 2 days. At the end of the culture period, media were renewed and follicular extracts from two different preparations (< 25 and < 10 kDa) were added at the concentrations of 1–10–100–1000 ng/ml. After 24 h the media were collected and stored until estradiol 17β (E2) and progesterone (P4) determination by validated radio-immuno-assays. Basal P4 production was 18.3 ± 1.4 (mean ± SEM)and 9.8 ± 1.8 ng/24 h per 3 × 104 cells from small and large follicles, respectively. Both < 10 and < 25 kDa extracts reduced P4 production by cells from both the types of follicles (p < 0.05). Basal E2 release was 671.8 ± 21.4 and 5500 ± 800 pg/24 h per 3 × 104 cells from small and large follicles, respectively. Both extracts reduced E2 production by either cells from small and large follicles (p < 0.05). No differences were observed in the inhibition of steroidogenesis by purifications obtained from large, medium or small follicles. Results of this study indicate that factors present in bovine follicular fluid can reduce steroidogenesis in granulosa cells in vitro.

In a previous work we evaluated the effects of Silymarin, a natural antioxidant and hepatoprotective polyphenolic compound, on laying hens performances and health status. The aim of the present work was to further confirm the previous results and to investigate the effects of Silymarin on ovarian endocrine activity and productivity of laying hens as well as on egg traits. Eighteen laying hens were randomly allotted into 3 groups and observed for 20 weeks: control hens were fed control diet, while treated groups received supplementations of 200 (S200) and 400 (S400) ppm of Silymarin. S200 group showed improved eggs laying rate (+2.91%), feed conversion rate (-4.52%) and a significant (P

Recent studies indicate that interleukin-1 beta may play a role in ovarian function. To explore this possibility, we examined the effects of a fragment of interleukin-1 beta (163-171 aa) on progesterone and estradiol-17 beta production by bovine granulosa cells from small (5 mm) and large (8 mm) follicles; in addition we investigated the distribution of monocytes/macrophages, a possible source of interleukin-1 beta in the follicles, to determine a possible relationship between production of the cytokine and its activity. Bovine follicles were divided according to the size, granulosa cells were obtained by aspiration and plated at a density of 10(5) cells/well; then different concentrations of interleukin-1 beta fragment (0, 0.5, 5, 20 ng/ml) with or without bFSH were added for 24 h. Progesterone (P4) and estradiol-17 beta (E2) concentrations in media were evaluated by validated RIAs. Interleukin-1 beta (0.5 and 5 ng/ml) stimulated (P0.05) P4 output in both small and large follicles, while FSH-induced P4 output was reduced (P0.05) in large but not in small follicles by all the concentrations of peptide. FSH-induced E2 output was reduced (P0.05) in large, but not in small follicles. Immunohisto- and cytochemistry revealed a higher number of monocytes/macrophages in large follicles. It appears from our data that: (1) interleukin-1 beta fragment may modulate steroidogenesis in bovine granulosa cells depending on the stage of development of the follicle, and (2) the presence of monocytes/macrophages is related to this modulation.

Melatonin exerts well-known reproductive effects, mainly acting on hypothalamic gonadotrophin-releasing hormone release. More recent data suggest that melatonin acts directly at the ovarian level, even if, at present, these aspects have been only partly investigated. Swine follicular fluid contains melatonin and its concentration is significantly reduced during follicular growth. Therefore, the present study was undertaken to examine the effects of melatonin, used at physiological concentrations, on cultured swine granulosa cells collected from small (5 mm) follicles on the main parameters of granulosa cell function such as proliferation and steroidogenesis, namely oestradiol 17β and progesterone (P4) production. Moreover, the effects of melatonin on superoxide anion and nitric oxide (NO) generation by swine granulosa cells were also investigated. Finally, since angiogenesis is crucial for follicle growth, the effects of melatonin on new vessel growth were studied. Collected data indicate that melatonin interferes with cultured granulosa cell proliferation and steroidogenesis, specifically in terms of P4 production and NO output. In addition, the events of physiological follicular angiogenesis were stimulated by melatonin as evidenced by angiogenesis bioassay. Therefore, we suggest that physiological melatonin concentrations could potentially be involved in local modulation of swine ovarian follicle function.

Bisphosphonates are known to be potent inhibitors of osteoclast activity and their only clinically relevant effect in the short-term is the selective inhibition of bone resorption. The purpose of this study was to compare the response to the intravenous administration of two bisphosphonates, clodronate and alendronate, of several biochemical markers of bone resorption, including tartrate-resistant acid phosphatase (TRAP) and cross-linked carboxyterminal telopeptide of collagen I (ICTP) in serum and hydroxyproline (OHP), free pyridinium cross-links (Pyr), and crosslinked N-telopeptides of collagen I (NTx) in urine. The study was carried out on 11 osteoporotic and 12 Pagetic subjects of both sexes, treated with clodronate (600 mg/day for 2 days) or alendronate (5 mg/day for 2 days), and monitored for 28 days after bisphosphonate administration. All the urinary markers of bone resorption showed a prompt decline after bisphosphonates, with maximum reductions after 7–14 days: Pyr decreased by 43%±9% and 42%±22% (mean ± SD), respectively in osteoporotic and pagetic subjects, OHP by 51%±14% and 51%±20%, and NTx by 55%±15% and 65%±26%. In the osteoporotic group, the urinary markers began to increase again at 30 days, though still remaining well below the basal level, whereas in the pagetic group, the excretion of all markers remained depressed until the end of the observation period. The reduction of NTx was significantly greater than that of Pyr and OHP in pagetic patients (P

Atrazine is one of the most widely employed herbicides. Due to its environmental persistence, it can be detected in ground and water thus becoming the subject of a serious concern because of its potential endocrine disrupting activity. In particular, several in vitro and in vivo studies point out adverse effects on reproduction. However, these data were mainly collected in the male, while studies on females are lacking. Present work was therefore set up on swine ovarian granulosa cells to investigate the effect of atrazine on steroidogenesis and proliferation. Moreover, since vessel growth is fundamental for reproductive function, we evaluated the herbicide's effect on two of the main angiogenesis signaling molecules, VEGF and NO. Our data show that atrazine markedly interferes with steroidogenesis while it does not modify cell proliferation; in addition, the herbicide has also been found to affect the production of the examined angiogenesis molecules. Collectively, these results indicate for the first time a potential negative effect of atrazine on ovarian functions in the swine species.

Follicular growth is allowed by extracellular matrix remodeling and vascular network development. Since matrix metalloproteinases (MMPs) play a crucial role in both of these processes, the aim of the present study was to evaluate the expression of gelatinases MMP2 and MMP9 in swine ovarian follicle (theca and granulosa compartments) during its development. Moreover, we measured gelatinase activities into follicular fluids (FF). Our data demonstrate for the first time that MMP2 and MMP9 are expressed in swine ovarian follicle both in theca and granulosa layers; moreover we show that the expression of both gelatinases increases in theca while decreases in granulosa during follicle growth. Additionally, MMP2 activity has been detected in FF. The spatial pattern of expression of gelatinases in swine follicle suggests a differential role during physiological ovarian events.

Stanniocalcin 1 (STC1) is a glycoprotein hormone expressed in different mammalian tissues. In previous studies, we showed STC1 expression in swine ovarian follicles and we demonstrated that STC1 may be a physiological regulator of follicular function. Since reactive oxygen species (ROS) are important signal transducers in the ovary, the present study was undertaken to investigate STC1 action on ROS generation and on the activity of the major enzymatic and non-enzymatic scavengers in swine granulosa cells. O 2 − generation, catalase activity and FRAP levels were increased by STC1, whereas H 2 O 2 generation and peroxidase activity were decreased by STC1. Taken together, our data show that STC1 modulates redox status in swine granulosa cells.

This study was aimed to improve knowledge about swine ovarian follicular function, paying attention to angiogenesis, since new vessel growth is a fundamental event in ovarian function. In particular, we investigated a potential involvement of netrin-1, a protein known as a guidance axon factor. Firstly, we studied the expression and immunolocalization of netrin-1 in swine ovarian follicle and its effect on cultured swine granulosa cell viability and steroidogenesis. Furthermore, aortic endothelial cells were employed to verify a possible netrin-1 effect on angiogenesis. Our data demonstrate the expression and the presence of netrin-1 in swine follicular fluid; in addition, it was shown that netrin-1 inhibits granulosa cell viability and estradiol 17β levels while it stimulates progesterone production. Netrin-1 also inhibits aortic endothelial cell growth in the angiogenesis bioassay. This effect appears to be mediated by inhibiting vascular endothelial growth factor and stimulating nitric oxide. Therefore, we hypothesize that netrin-1 could be important for follicular function in the swine.

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.

Because of its widespread use and potential adverse biological effects, bisphenol A (BPA) represents one of the most studied endocrine-disrupting compounds. Within the reproductive system, ovarian granulosa cells have been documented as a target of BPA action, but no consensus has been reached about functional modifications induced by BPA. On these bases, we studied the potential disrupting effects of BPA on the main granulosa cell functional activities, also taking into account a potential interference with the ovarian angiogenic process. Ovarian granulosa cells were isolated from porcine follicles and cultured in the presence or absence of BPA at different concentrations for 48h. Cell proliferation was studied by measuring adenosine triphosphate content. Progesterone (P4) and estradiol 17beta (E2) production was determined by radioimmunoassay. Vascular endothelial growth factor (VEGF) output was quantified by an enzyme-linked immunosorbent assay. Redox status was monitored by measuring superoxide anion and hydrogen peroxide, and by determining the activities of the scavenging enzymes superoxide dismutase, catalase, and peroxidase by colorimetric methods. Granulosa cell proliferation as well as redox status resulted unaffected by BPA. Concentrations of E2 were stimulated by the lower BPA concentration, whereas they were inhibited by the larger doses tested. P4 output was decreased by all BPA concentrations. To the contrary, VEGF production was stimulated. Data indicate that BPA can interfere with reproductive activity by affecting granulosa cell steroidogenesis in vitro; furthermore, BPA can exert a promoting effect on the ovarian angiogenic process by increasing VEGF output in pigs. A disruption of this finely tuned process seems particularly relevant because of the risk of uncontrolled neovascularization.

The ovarian follicle represents an outstanding model for the investigation of the angiogenic process. In fact, follicular development is associated with an extensive neovascularization, and physiological angiogenesis is necessary for folliculogenesis. However, although the major factors triggering the angiogenic events have been thoroughly investigated and are now relatively well defined, information about the molecular events involved in the modulation and/or arrest of neovascularization is still scarce. Therefore, this research focused on the potential involvement of stanniocalcin (STC), a protein whose biological function is still unclear, in the control mechanisms of follicular angiogenesis. We evaluated the effect of 5 and 50 ng/mL STC on the production of the main proangiogenic factor, vascular endothelial growth factor (VEGF), by swine granulosa cells. Moreover, STC's effect on cell viability and the modulation of caspase-3 and -7 activities in swine aortic endothelial cells were also examined. Granulosa cell VEGF production was significantly (P < 0.001) inhibited by STC. Endothelial cell viability was significantly (P < 0.001) increased, whereas caspase activities were reduced (P < 0.01) by STC. These data indicate that STC is not angiosuppressive for endothelial cells, although it could potentially modulate local angiogenesis acting at the granulosa cell level.

Experimental evidence documents that nutritional phytoestrogens may interact with reproductive functions but the exact mechanism of action is still controversial. Since quercetin is one of the main flavonoids in livestock nutrition, we evaluated its possible effects on cultured swine granulosa cell proliferation, steroidogenesis, and redox status. Moreover, since angiogenesis is essential for follicle development, the effect of the flavonoid on Vascular Endothelial Growth Factor output by granulosa cells was also taken into account. Our data evidence that quercetin does not affect granulosa cell growth while it inhibits progesterone production and modifies estradiol 1 7 𝛽 production in a dose-related manner. Additionally, the flavonoid interferes with the angiogenic process by inhibiting VEGF production as well as by altering redox status. Since steroidogenesis and angiogenesis are strictly involved in follicular development, these findings appear particularly relevant, pointing out a possible negative influence of quercetin on ovarian physiology. Therefore, the possible reproductive impact of the flavonoid should be carefully considered in animal nutrition.

The rapid, controlled, and cyclical nature of angiogenesis in the ovarian follicle suggests the potential for sex steroids to influence neovascularization. Angiogenesis is regulated by a local balance between the levels of endogenous stimulators and inhibitors. Multiple lines of evidence suggest that estrogens stimulate angiogenesis via effects on endothelial cells. However, it is of outstanding value to investigate the negative control of this process. Since the main estrogen metabolites, 2-hydroxyestradiol and 4-hydroxyestradiol (4-OHE2) have been demonstrated to function as anti-estrogen in several estrogen-dependent organs; the aim of this study was to investigate their potential involvement in the modulation of follicular angiogenesis. Hydroxyestrogens were quantified in swine follicular fluid and their effects were studied on granulosa cell vascular endothelial growth factor (VEGFA) production and tested in an angiogenesis bioassay. Our study documents that these molecules are physiologically present in swine follicular fluid and their concentrations significantly (PPP

A growing body of evidence indicates that the pro-oxidant/anti-oxidant balance inside the ovarian follicle plays an important role in folliculogenesis. Therefore, the aim of the present study was to assess the redox status of follicular fluids collected from different-sized swine follicles. We quantified the most important reactive oxygen species (ROS), namely superoxide anion (O2–), hydrogen peroxide and hydroperoxides (ROOH); in addition, we examined the activity of the detoxifying enzymes superoxide dismutase, catalase (CAT) and glutathione peroxidase and the total non-enzymatic antioxidant capacity as determined by the ferric-reducing anti-oxidant power assay. Our data demonstrate that oxidative stress does not affect follicle growth because O2– levels do not change during follicle development, whereas concentrations of H2O2 and ROOH are reduced (P < 0.05). Surprisingly, all non-enzymatic and enzymatic scavengers examined in the present study, except for CAT, demonstrated reduced activity during follicle development (P < 0.05). Taken together, these results suggest that other factors could be involved in ROS detoxification during follicle development.

Angiogenesis is the process of vascular growth by sprouting of preexisting vessels. This process impacts significantly on many important disease states including cancer, diabetic retinopathy, and arthritis. Endothelial cells receive multiple information from their environment, which leads them to progress along all stages of new vessel formation. Vascular endothelial growth factor (VEGF), in particular appears to be a master regulator of this process. This molecule interacts with cellular receptors and communicates with cell nucleus through a network of intracellular signaling, most of all by activating Akt pathway. This activation accounts for many of VEGF effects, including cell survival, migration, tube formation, and promotion of NO release. Sanguinarine (SA), an alkaloid isolated from Sanguinaria canadensis, is known for its antiangiogenetic effects by suppressing basal and VEGF-induced new vessel growth. This article was aimed to evaluate the possible effect of SA (300 nM) on Akt phosphorylation in a porcine aortic endothelial cell line. The alkaloid significantly (P < 0.001) inhibited the VEGF-induced Akt increase, thus suggesting that this mode of action could be responsible, at least partially, for the antiangiogenetic effect of SA.

Inhibition of angiogenesis is an important new approach for cancer treatment and the research on this topic deserve special attention. 2-Methoxyestradiol (2-ME), a molecule shown to possess antiangiogenic activity, is a naturally occurring derivative of estradiol which can be potentially produced in the ovarian follicle. This study was therefore aimed firstly to asses 2-ME content in swine follicular fluid. Moreover, we evaluated the effect of this substance on VEGF production, superoxide anion generation (O(2)(-)) and superoxide dismuatase activity in granulosa cells. Our data evidence that 2-ME is present in follicular fluid where it potentially acts as a physiological inhibitor of angiogenesis by reducing VEGF production by granulosa cells: this effect could be mediated by a decrease of O(2)(-) generation.