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.

Performance of photoelectron spin polarimeters with continuous and pulsed sources: from storage rings to free electron lasers.

In this work the experimental uncertainties concerning electron spin polarization (SP) under various realistic measurement conditions are theoretically derived. The accuracy of the evaluation of the SP of the photoelectron current is analysed as a function of the detector parameters and specifications, as well as of the characteristics of the photoexcitation sources. In particular, the different behaviour of single counter or twin counter detectors when the intensity fluctuations of the source are considered have been addressed, leading to a new definition of the SP detector performance. The widely used parameter called the figure of merit is shown to be inadequate for describing the efficiency of SP polarimeters, especially when they are operated with time-structured excitation sources such as free-electron lasers. Numerical simulations have been performed and yield strong implications in the choice of the detecting instruments in spin-polarization experiments, that are constrained in a limited measurement time. Our results are therefore applied to the characteristics of a wide set of state-of-the-art spectroscopy facilities all over the world, and an efficiency diagram for SP experiments is derived. These results also define new mathematical instruments for handling the correct statistics of SP measurements in the presence of source intensity fluctuations.

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.

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.

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.

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.

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.

The impact of the phyto-oestrogen genistein on swine granulosa cell function.

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.

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.

Presence and function of kisspeptin/KISS1R system in swine ovarian follicles.

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.

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.

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.

Cryopreservation of pig granulosa cells: effect of FSH addition to freezing medium.

We cryopreserved swine granulosa cells by a slow cooling rate system; FSH was added to the freezing medium to test its effectiveness in protecting the cells. After thawing, proliferative activity, viability, steroidogenesis and apoptosis were tested; moreover, we determined heat shock protein (HSP70) production, to investigate the recovery from stress and superoxide dismutase (SOD) and catalase activity to evaluate a possible impairment of the antioxidant pathway. E2 production was enhanced by cryopreservation in particular with FSH; on the contrary, P4 production was inhibited by the freezing process in particular without FSH. Only the higher FSH concentration (10 ng/ml) stimulated steroid secretion in freshly collected cells; P4 production by cells cryopreserved in the presence and in absence of FSH was increased by both 5 and 10 ng/ml while the lowest concentration was effective in stimulating E2 production only when FSH was added to freezing medium. Freezing did not modify proliferative activity, while apoptosis was higher in frozen than in fresh cells. HSP70 production was lower in cells cryopreserved in presence of FSH, whose antioxidant metabolism was also conserved: SOD and catalase activities were similar to control. In conclusion, cryopreservation does not seem to markedly affect granulosa cells, in particular if they are frozen in presence of FSH; the gonadotrophin somehow improves their performances after thawing, probably stimulating E2 production and the antioxidant metabolism.

Follicle-stimulating hormone-testosterone interaction in modulating steroidogenesis in bovine granulosa cells. I. Effect on progesterone production.

The aim of this study was to investigate the effects of testosterone on basal and follicle-stimulating hormone (FSH)-induced progesterone production by cultured bovine granulosa cells. Granulosa cells were isolated from small (< 5 mm) and large (> 8 mm) follicles and cultured for 48 h in 1 ml of Medium-199 with different concentrations of FSH (0.1, 1, 10 and 35 mg/l). In addition, the combined effects of different amounts of testosterone (1 nmol-10 mumol) and 1 mg/l FSH for 48 h on progesterone production by granulosa cells of both groups of follicles were studied; progesterone production during the subsequent 24-h incubation period was evaluated in the absence of hormones. In a third experiment, granulosa cells were treated with 500 micrograms of dibutyryl-cAMP and 10 mumol of testosterone for 48 h. At the end of each incubation period, the progesterone content in the culture media was determined by a validated radioimmunoassay. Basal progesterone release during the 48-h incubation period was higher in granulosa cells from small as compared to cells from large follicles; in both groups of cells, progesterone production was stimulated maximally by 1 mg/l FSH. The treatment with 10 mumol of testosterone induced a decrease of progesterone production in both groups of cells, while lower amounts exerted an inhibitory effect only in cells from large follicles. Furthermore, 10 mumol of testosterone inhibited FSH-induced progesterone release, while lower dosages were ineffective. Dibutyryl-cAMP stimulated significantly the progesterone output by granulosa cells of both groups and testosterone was effective in suppressing this increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiogenic activity of porcine granulosa cells co-cultured with endothelial cells in a microcarrier-based three-dimensional fibrin gel.

To verify the possible role played by pig granulosa cells in the ovarian angiogenic process, we have developed a reliable in vitro system which allows the evaluation of endothelial sprouting and capillary growth in three-dimensional matrices. Granulosa cells collected from porcine follicles of different size were co-cultured with porcine aortic endothelial cells (PAEC) in a microcarrier-based fibrin gel system; after 2 and 5 days of co-culture, we determined the number and length of all endothelial sprouts; moreover, these parameters were quantified only in capillary-like structures, which were defined as continuous multicellular sprouts at least 200 microm long. In granulosa cells- PAEC co-cultures we observed an increase of angiogenic activity as compared to controls (PAEC alone). Granulosa cells from follicles of different size regulate angiogenesis differently: cells from the small follicle group significantly enhanced endothelial sprouting, while those from the large follicle group favoured mainly capillary elongation. Our observations seem therefore to suggest that the development and growth of thecal vascular bed is controlled by paracrine factors of granulosa cell origin that may induce the formation of a primitive capillary plexus during the early phases of antral follicle growth, which will be remodelled in more advanced phases of follicular development.

Lipid hydroperoxide and cGMP are not involved in nitric oxide inhibition of steroidogenesis in bovine granulosa cells.

The present study was performed to explore two of the possible signalling mechanisms through which nitric oxide (NO) inhibits steroidogenesis in bovine granulosa cells. Because cGMP is generally known to play a pivotal role in NO signal transduction, the first aim of the present study was to verify the presence of a functional NO-cGMP signalling pathway. Because non-cGMP-dependent pathways could be involved in the inhibition of steroidogenesis by NO, we examined the formation of lipid hydroperoxides (LPOs), possibly induced by NO. Using bovine granulosa cells collected from small (< 5 mm) and large (> 8 mm) follicles, the effectiveness of the NO donor s-nitroso-N-acetylpenicillamine (SNAP; 10(-3), 10(-4) and 10(-5) M) in stimulating cGMP production and the formation of LPOs was examined. The second aim of the present study was to determine whether the effects of NO on steroidogenesis could be mimicked by treatment of cells with a cGMP analogue (8-bromo-cGMP (8-Br-cGMP); 10(-3), 10(-4) and 10(-5) M) and whether these effects could be reversed by [1H]-[1,2,3]oxadiaziolo[4,3a]quinoxaline-1-one (ODQ; 10(-5) and 10(-4) M) an inhibitor of NO-sensitive soluble guanylate cyclase. The highest dose of SNAP used induced a significant (P<0.01) increase in cGMP levels, while other concentrations tested were ineffective. Neither concentration of ODQ used significantly inhibited basal cGMP output, while both concentrations counteracted the stimulatory effect of SNAP. Treatment of cells with 8-Br-cGMP and ODQ was ineffective in modifying steroidogenesis. Treatment with SNAP, at the three concentrations tested, had no significant effect on the level of LPOs. The present results suggest that NO inhibits steroidogenesis in bovine granulosa cells without involving cGMP and LPOs.

Nitric oxide synthase expression and nitric oxide/cyclic GMP pathway in swine granulosa cells.

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.