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Beneficial effects of Sambucus nigra L. (black elder) as a traditional medicine have been associated with the phytoconstituents including polyphenols, terpenes and lectins. Various antioxidant rich natural products have also been implicated with improvement of reproductive health and fertility, however, the effect of Sambucus nigra on the ovarian cell functions has not been investigated yet. The objectives of the present study were to screen the polyphenols in the elderflower and elderberry extracts, and to examine the secretion activity of steroid hormones 17beta-estradiol and progesterone by human ovarian granulosa cells HGL5 after supplementation of the extracts at a concentration range of 12.5 to 100 microg.ml-1. Qualitative as well as quantitative screening of polyphenols by high-performance liquid chromatography with diode-array detector (HPLC-DAD) analysis revealed rutin to be the most abundant polyphenol in both elderflower and elderberry extracts. In culture, neither elderflower nor elderberry extract caused any significant impact (p>0.05) in cell viability as studied by AlamarBlue assay in comparison to control. However, a dose-dependent stimulation of 17beta-estradiol release was detected by ELISA after supplementation of elderflower (at 50 microg.ml-1; p<0.01) and elderberry (at 100 microg.ml-1; p<0.05) extracts at higher doses used in the study. On the other hand, both elderflower and elderberry extracts stimulated the secretion of progesterone by HGL5 cells at a lower dose (12.5 microg.ml-1; p<0.05), as compared to control. Therefore, elderflower and elderberry extracts may have the potential to regulate steroidogenesis in ovarian cells.

This study aimed to examine the effect of dietary flavonoid isoquercitrin on ovarian granulosa cells using the immortalized human cell line HGL5. Cell viability, survival, apoptosis, release of steroid hormones 17beta-estradiol and progesterone, and human transforming growth factor-beta2 (TGF-beta2) and TGF-beta2 receptor as well as intracellular reactive oxygen species (ROS) generation were investigated after isoquercitrin treatment at the concentration range of 5-100 microg.ml-1. It did not cause any significant change (p>0.05) in cell viability as studied by AlamarBlue assay in comparison to control. No significant change was observed (p>0.05) in the proportion of live, dead and apoptotic cells as revealed by apoptotic assay using flow cytometry. Similarly, the release of 17beta-estradiol, progesterone, TGF-beta2 and its receptor were not affected significantly (p>0.05) by isoquercitrin as detected by ELISA, in comparison to control. Except for the highest concentration of 100 microg.ml-1, which led to oxidative stress, isoquercitrin exhibited antioxidative activity at lower concentration used in the study (5, 10, 25, and 50 microg.ml-1) by hampering the production of intracellular ROS, in comparison to control, as detected by chemiluminescence assay (p<0.05). Findings of the present study indicate an existence of the antioxidative pathway that involves inhibition of intracellular ROS generation by isoquercitrin in human ovarian granulosa cells.

The effect of body condition and environmental contaminants on reproductive processes is known; however, it is not known whether basic ovarian cell functions and their response to these contaminants depend on body condition. This study aimed to understand the interrelationships between body conditions and environmental contaminants on ovarian cells. For this purpose, we compared ovarian granulosa cells isolated from cows with an emaciation tendency (body condition score, BCS2 on a scale from 1 to 5) and cows with average body condition (BCS3); proliferation, apoptosis, secretory activity and the response to environmental contaminants were all assessed in the cells. In the 1st series of experiments, ovarian granulosa cells isolated from BCS2 and BCS3 cows were cultured with and without benzene, xylene, and toluene (0.1%). The accumulation of nuclear and cytoplasmic markers of apoptosis (p53 and bax, respectively), a proliferation marker (PCNA), progesterone (P4), and insulin-like growth factor I (IGF-I) was evaluated by Western blot and radioimmunoassay (RIA) experiments. In the 2nd series of experiments, the groups of granulosa cells were cultured with and without mycotoxine deoxynivalenol (DON, 0, 10, 100, or 1000 ng/ml). The secretion of P4 and testosterone (T) was measured by RIA. In comparison to cells from BCS2 animals, ovarian cells isolated from BCS3 cows accumulated higher levels of bax and PCNA but not p53, and they secreted higher amounts of IGF-I but not P4 or T. In cells from BCS2 animals, benzene and xylene promoted p53 accumulation, and toluene reduced the accumulation. In the BCS2 group, all treatments promoted bax and PCNA expression. However, in cells from BCS3 animals, all environmental pollutants inhibited p53, toluene inhibited PCNA but not bax, and xylene did not affect the expression of proliferation or apoptosis markers. In the BCS2 group, P4 was inhibited by xylene, and IGF-I was stimulated by xylene but not by benzene or toluene. Low-dose exposure to DON (10 ng/ml) promoted P4 release from cells from both BCS2 and BCS3 animals, but high-dose exposure to DON (1000 ng/ml) reduced P4 secretion from the cells from BCS2 animals but not from the cells from BCS3 animals. The release of T was inhibited by high-dose exposure to DON (1000 ng/ml), irrespective of the BCS. An emaciation tendency reduces proliferation, apoptosis, and IGF-I release, and it induces or reverses the action of environmental contaminants on ovarian functions. Taken together, these observations demonstrate the effect of body condition and the direct influence of environmental contaminants on basic bovine ovarian functions. Furthermore, they demonstrate for the first time that the response of ovarian cells to environmental contaminants can be regulated by cow body condition., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We studied the influence of oil-related environmental contaminants (OREC) on the viability, hormone secretion, and protein expression using cultured porcine ovarian granulosa cells. Addition of benzene and xylene promoted proliferation and apoptosis and reduced ovarian cell viability whereas toluene induced apoptosis only. The release of progesterone (P4) and oxytocin (OT) was promoted by benzene and xylene, and suppressed by toluene while prostaglandin F (PGF) output was stimulated by benzene and toluene, but not xylene. The addition of FSH to the culture medium increased ovarian cell proliferation and hormone release, but did not affect apoptosis. However, this FSH's proliferative effect has been prevented in presence of benzene. On the other hand and in the presence of FSH, toluene prevented P4 release and decreased PGF release, while xylene prevented PGF release. We concluded that OREC can affect reproductive processes by directly influencing ovarian cell proliferation, apoptosis, viability, hormone release, and response to gonadotropins.

The aim of our study was to examine the direct influence of plant polyphenol resveratrol and oil-related environmental contaminant benzene on ovarian hormone release, as well as the ability of resveratrol to prevent the effect of benzene. Porcine ovarian granulosa cells were cultured with and without resveratrol (0, 1,10 or 100 ug/ml) alone or in combination with 0.1% benzene. The release of progesterone, oxytocin and prostaglandin F was measured by enzyme immunoassay (EIA). Benzene promoted the release of progesterone, oxytocin and prostaglandin F. Resveratrol, when given alone, stimulated both progesterone and prostaglandin F, but not the oxytocin output. Moreover, resveratrol prevented and even inverted the stimulatory action of benzene on all analysed hormones. These observations demonstrate the direct influence of both benzene and resveratrol on porcine ovarian hormone release, as well as the ability of resveratrol to prevent the benzene action on the ovary.

Plants, fruits, and vegetables containing the bioflavonoid quercetin are widely used in food, beverages, and medicines; however, the effects of quercetin on reproductive processes and the possible mechanisms of quercetin action require extensive investigation. The aim of our study was to examine the direct effects of quercetin on basic ovarian cell functions and their response to follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I), known hormonal stimulators of reproduction. We analyzed the effects of quercetin alone (0, 1, 10, and 100 ng/ml) on cultured porcine ovarian granulosa cells or isolated ovarian follicles; or of quercetin (10 ng/ml) in combination with FSH (0, 0.01, 0.1, or 1 IU/ml) or IGF-I (0, 1, 10, or 100 ng/ml) on cultured porcine granulosa cells. The expression of proliferative (PCNA, cyclin B1) and apoptotic (BAX) markers, as well as markers for release of progesterone (P4), testosterone (T), and leptin (L), were measured by quantitative immunocytochemistry, Western immunoblotting, RT-qPCR, and EIA/RIA. Addition of quercetin reduced the accumulation of PCNA and cyclin B1, as well as their transcript levels, promoted the accumulation of BAX, decreased the release of P4 and L, and increased the release of T in cultured granulosa cells. In ovarian follicles, quercetin reduced the levels of both P4 and T. Exposure to FSH stimulated PCNA and decreased BAX accumulation, and increased the release of P4, T, and L. Quercetin inhibited and even reversed the effects of FSH. Like FSH, IGF-I also promoted granulosa cell proliferation and suppressed apoptosis. Quercetin did not modify IGF-I effects. These data suggest that the plant molecule quercetin can directly down-regulate basal ovarian cell functions (proliferation, apoptosis, and release of ovarian steroid and peptide hormones) and their response to the stimulatory activity of the upstream hormonal stimulator FSH., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study was conducted with the aim to understand the roles of apoptosis signal-regulating kinase (ASK1) and transcription factor tumor suppressor protein TP53, as well as the possible interrelationships, in the control of healthy ovarian cell functions. Rabbit ovarian granulosa cells were transfected with constructs encoding ASK1, TP53, or TP53 + ASK1 and cultured with or without insulin-like growth factor 1 (IGF1). The accumulation of ASK1, the cytoplasmic apoptosis regulators BAX and BCL2, and proliferating cell nuclear antigen (PCNA, a cell proliferation marker), as well as progesterone release, were evaluated by quantitative immunocytochemistry and radioimmunoassay. Results indicate both ASK1 and TP53 promoted the accumulation of BAX, but suppressed that of BCL2 and PCNA. Progesterone release was inhibited by ASK1 and promoted by TP53, while TP53 also stimulated ASK1 accumulation. Additionally, IGF1 stimulated PCNA and reduced progesterone release, but did not affect ASK1. Transfection with ASK1, TP53, or TP53 + ASK1 could modify IGF1 activity, however, there was no cumulative effect with co-transfection of TP53 and ASK1. This is the first results that indicate there is ASK1 suppression of healthy ovarian granulosa cell functions, including promoting apoptosis, inhibiting proliferation, and alter progesterone release. There was also TP53 actions in rabbit ovarian granulosa cells, where it stimulated ASK1, apoptosis, and progesterone release, thus suppressing proliferation and responses to IGF1. The similarity of ASK1 and TP53 effects on apoptosis and proliferation, lack of cumulative action of these molecules, and capacity of TP53 to promote ASK1 accumulation suggest that TP53 can suppress some ovarian granulosa cell functions through ASK1 stimulation., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Quercetin is a dietary bioflavonoid used widely as a food supplement and is generally recognized as safe. The aim of this in vitro study was to examine the steroid hormone (progesterone and 17- β estradiol) release, proliferation (PCNA and cyclin B1) and apoptosis (caspase 3 and p53) of porcine ovarian granulosa cells after the addition of quercetin at concentrations 0.01, 0.1, 1, 10 and 100 μmol L -1 . Progesterone release was stimulated at the concentration 10 μmol L -1 . Quercetin neither had any impact on 17-β estradiol secretion nor on the presence of PCNA. However, a significant enhancement of the occurrence of cyclin B1 was noted except for the lowest concentration 0.01 μmol L -1 . Quercetin did not have any influence on the number of granulosa cells containing caspase 3, but at the concentration 10 μmol L -1 it inhibited p53 occurrence. Results confirm the safety of quercetin in porcine ovarian granulosa cell model and further suggest its possible concentration-dependent influence on ovarian functions through pathway that may involve progesterone, cyclin B1 and p53.

The polyphenol-rich plants rooibos and ginkgo are widely used in folk medicine and in preparation of foods and drinks, but their effect on reproduction has not been properly studied yet. The aim of our in vitro experiments was to examine the possible direct effect of rooibos and ginkgo on the basic ovarian cell functions-proliferation, apoptosis and release of hormones progesterone (P4) and leptin (L). Porcine ovarian granulosa cells were cultured in the presence of rooibos or ginkgo extract (0, 1, 10 and 100 μg/ml of medium). The accumulation of markers of proliferation (PCNA and cyclin B1) and apoptosis (bax) and their mRNAs was analysed using immunocytochemistry and by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Release of P4 and L was evaluated by radioimmunoassay. It was observed that rooibos or ginkgo addition was able to inhibit proliferation (down-regulates PCNA, cyclin B1 and their mRNAs), to promote apoptosis (accumulation of bax) and to suppress both L and P4 release by ovarian cells. These data suggest a direct inhibitory (anti-proliferative, pro-apoptotic and hormone-suppressing) effect of polyphenol-containing plants rooibos and ginkgo on ovarian functions. The potential anti-reproductive effect of these medical plants is to be taken into account by their consumption., (© 2017 Blackwell Verlag GmbH.)

Green tea is a commonly used beverage and green tea extract is a common dietary herbal supplement manufactured into different over-the-counter products. The aim of this in vitro study was to examine the steroid hormone secretion (progesterone and 17-β estradiol), proliferation and apoptosis of porcine ovarian granulosa cells after addition of green tea extract. Granulosa cells were incubated with green tea extract at five doses (0.1, 1, 10, 100 and 200 μg/ml) and the release of hormones by granulosa cells was assessed by EIA after 24 h exposure. The presence of proliferation and apoptotic markers was assessed by immunocytochemistry. Secretion of steroid hormones was not affected by green tea extract at all the doses in comparison to control. Also, markers of proliferation (PCNA and cyclin B1) were not affected by green tea extract. However, the highest dose (200 μg/ml) of green tea extract used in this study increased the accumulation of apoptotic markers caspase-3 and p53 in granulosa cells. In conclusion, our results indicate the impact of green tea extract at the highest dose used in this study on ovarian apoptosis through pathway that includes activation of caspase-3 and p53. Potential stimulation of these intracellular regulators could induce the process of apoptosis in ovarian cells., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

This study aimed at examining the secretion activity of steroid hormones progesterone and 17beta-estradiol by porcine ovarian granulosa cells after addition of green tea extract. Granulosa cells were incubated with green tea extract (at doses of 0.01, 0.1, 1, 10 and 100 microg.ml(-1). Another set of cells were incubated with green tea extract at the above doses along with additional supplementation of follicle stimulating hormone (FSH) at 10 microg.ml(-1). Release of hormones by granulosa cells was assessed by EIA after 24 h exposure. Secretion of steroid hormones was not affected either by green tea extract alone or after FSH supplementation with green tea extract. Results indicate that ovarian steroidogenesis is not affected by green tea under conditions used in the experiment.

Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) are frequently occurring in feed of pigs together. The aim of this study was to evaluate the possible in vitro effects of DON and ZEA, alone or their combination on steroid secretion of porcine ovarian granulosa cells (GCs). A species-specific model with porcine ovarian GCs was used to study the potential endocrine disrupting effects of DON and ZEA alone and in co-exposure. Progesterone (P4) and estradiol (E2) were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The results of this study demonstrate that DON alone at the higher concentrations may act to stimulate P4 (at 1,000, 2,000, 3,000 and 5,000 ng mL -1 but not 10 and 100 ng mL -1 ) and E2 (at 2,000, 3,000 and 5,000 ng mL -1 but not 10, 100 and 1000 ng mL -1 ) secretion. The effects of ZEA on P4 and E2 secretion were not confirmed. DON in combination with the other fusariotoxin ZEA may impair steroidogenesis. Results aslo demonstrate different toxicological effects of fusariotoxins on follicle stimulating hormone-induced secretion of P4 and E2. All these results taken together suggest that fusariotoxin and their interactions can impact ovarian steroidogenesis, thereby demonstrating their potential reproductive effects in pigs.

Curcuma longa Linn (L.) is a plant widely used in cooking (in curry powder a.o.) and in folk medicine, but its action on reproductive processes and its possible mechanisms of action remain to be investigated. The objective of this study was to examine the direct effects of curcumin, the major Curcuma longa L. molecule, on basic ovarian cell functions such as proliferation, apoptosis, viability and steroidogenesis. Porcine ovarian granulosa cells were cultured with and without curcumin (at doses of 0, 1, 10 and 100μg/ml of medium). Markers of proliferation (accumulation of PCNA) and apoptosis (accumulation of bax) were analyzed by immunocytochemistry. The expression of mRNA for PCNA and bax was detected by RT-PCR. Cell viability was detected by trypan blue exclusion test. Release of steroid hormones (progesterone and testosterone) was measured by enzyme immunoassay (EIA). It was observed that addition of curcumin reduced ovarian cell proliferation (expression of both PCNA and its mRNA), promoted apoptosis (accumulation of both bax and its mRNA), reduced cell viability, and stimulated both progesterone and testosterone release. These observations demonstrate the direct suppressive effect of Curcuma longa L./curcumin on female gonads via multiple mechanisms of action - suppression of ovarian cell proliferation and viability, promotion of their apoptosis (at the level of mRNA transcription and subsequent accumulation of promoters of genes regulating these activities) and release of anti-proliferative and pro-apoptotic progesterone and androgen. The potential anti-gonadal action of curcumin should be taken into account by consumers of Curcuma longa L.-containing products., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The key biological active molecule of soya is the isoflavone daidzein, which possesses phytoestrogenic activity. The direct effect of soya and daidzein on ovarian cell functions is not known. This study examined the effect of daidzein on basic porcine ovarian granulosa cell functions and the response to follicle-stimulating hormone (FSH). We studied the effects of daidzein (0, 1, 10 and 100 μm), FSH (0, 0.01, 0.1, 1 IU/ml) and combinations of FSH (0, 0.01, 0.1, 1 IU/ml) + daidzein (50 μm) on proliferation, apoptosis and hormone release from cultured porcine ovarian granulosa cells and ovarian follicles. The expression of a proliferation-related peptide (PCNA) and an apoptosis-related peptide (Bax) was analysed using immunocytochemistry. The release of progesterone (P4) and testosterone (T) was detected using EIA. Leptin output was analysed using RIA. Daidzein administration increased granulosa cell proliferation, apoptosis and T and leptin release but inhibited P4 output. Daidzein also increased T release and decreased P4 release from cultured ovarian follicles. Follicle-stimulating hormone stimulated granulosa cell proliferation, apoptosis and P4, T and leptin release. The addition of daidzein promoted FSH-stimulated apoptosis (but not proliferation) but suppressed FSH-stimulated P4, T and leptin release. Our observations of FSH action confirm previous data on the stimulatory effect of FSH on ovarian cell proliferation, apoptosis and steroidogenesis and demonstrate for the first time the involvement of FSH in the upregulation of ovarian leptin release. Our observations of daidzein effects demonstrated for the first time that this soya isoflavone affected basic ovarian cell functions (proliferation, apoptosis and hormones release) and modified the effects of FSH. Daidzein promoted FSH action on ovarian cell proliferation and apoptosis and suppressed, and even inverted, FSH action on hormone release. The direct action of daidzein on basic ovarian cell functions and the ability of these cells to respond to FSH indicate the potential influence of soya-containing diets on female reproductive processes via direct action on the ovary., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)

Ricinus communis L. has ethnopharmacological contraceptive reputation but its stem bark has unexplored mechanisms of action in female reproductive system. In the present study, the effect of methanolic and aqueous extracts from the stem bark of the plant was examined on basic porcine ovarian granulosa cell functions and its response to Luteinising hormone (LH)-the upstream hormonal regulator. Systemic treatment of methanolic and aqueous extracts stimulated cell proliferation (proliferating cell nuclear antigen, PCNA) and also promoted cell apoptosis (caspase-3). Aqueous extract has inverted the stimulatory effect of LH on PCNA but not on caspase-3. Methanolic extract stimulated as well as inhibited progesterone release and stimulated testosterone secretion. Whereas aqueous extract inhibited both steroid releases and suppressed the stimulatory effect of LH on progesterone release and promoted the inhibitory effect of LH on testosterone release. In conclusion, the present study unveils the mechanism of action of R. communis stem bark in in vitro condition. These suggest its possible contraceptive efficacy by exerting its regulatory role over LH and on basic ovarian cell functions and secretion activity.

The aim of this study was to determine the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), production of reactive oxygen species (ROS), and total antioxidant status (TAS) in porcine ovarian granulosa cells after quercetin and T-2 toxin exposure in vitro. Porcine ovarian granulosa cells were incubated with quercetin and T-2 toxin separately or in mutual combination at the doses of 1 ng/mL, 10 ng/mL, 100 ng/mL, and the control group without any additions for 24 h. In this study T-2 toxin developed stress reaction in porcine ovarian granulosa cells and increased generation of ROS. Quercetin had no effect in elimination of ROS generation induced by T-2 toxin, but was effective in maintaining and increasing of TAS, activities of SOD and GPx in porcine granulosa cells in vitro. These results contribute towards the understanding of cellular stress and its response.

The possible effects of a natural substance amygdalin and its combination with the mycotoxin deoxynivalenol (DON) on the steroid hormone secretion (progesterone and 17-β-estradiol) by porcine ovarian granulosa cells (GCs) were examined in this in vitro study. Ovarian GCs were incubated without (control group) and with amygdalin (1, 10, 100, 1,000 and 10,000 μg mL(1)), or its combination with DON (1 μg mL(1)) for 24 h. The release of steroid hormones was determined by ELISA. The progesterone secretion by porcine ovarian GCs was not affected by amygdalin in comparison to the control. However, the highest amygdalin dose (10,000 μg mL(1)) caused a significant stimulation of the 17-β-estradiol release. A combination of amygdalin with DON significantly (P < 0.05) increased the progesterone release at all concentrations. Similarly, a stimulatory effect of amygdalin co-administered with DON was detected with respect to the 17-β-estradiol secretion at the highest dose (10,000 μg mL(1)) of amygdalin and 1 μg mL(1) of DON. Noticeable differences between the effects of amygdalin alone and its combination with DON on the progesterone release were detected. In contrast, no differences between the stimulatory effects of amygdalin and its combination with DON on the 17-β-estradiol synthesis by porcine GCs were observed. Findings from this in vitro study did not confirm the expected protective effect of amygdalin on mycotoxin induced reprotoxicity. Our results indicate that the stimulatory effect of amygdalin combined with DON on the progesterone release was clearly caused by the DON addition, not by the presence amygdalin per se. On the other hand, the stimulation of 17-β-estradiol production was solely caused by the presence of amygdalin addition. These findings suggest a possible involvement of both natural substances into the processes of steroidogenesis and appear to be endocrine modulators of porcine ovaries.

Protein kinases, transcription factors and other apoptosis- and proliferation-related proteins can regulate reproduction, but their involvement in sexual maturation remains to be elucidated. The general aim of the in vivo and in vitro experiments with porcine ovarian granulosa cells was to identify possible intracellular regulators of female sexual maturation. For this purpose, proliferation (expression of proliferating cell nuclear antigen - PCNA, mitogen-activated protein kinases - ERK 1,2 related MAPK and cyclin B1), apoptosis (expression of the apoptotic protein Bax and apoptosis regulator Bcl-2 protein), expression of some protein kinases (cAMP dependent protein kinase - PKA, cGMP-dependent protein kinase - PKG, tyrosine kinase - TK) and cAMP responsive element binding protein 1 (CREB-1) was examined in granulosa cells isolated from ovaries of immature and mature gilts. Expression of PCNA, ERK1,2 related MAPK, cyclin B1, Bcl-2, Bax, PKA, CREB-1, TK and PKG in porcine granulosa cells were detected by immunocytochemistry. Sexual maturation was associated with significant increase in the expression of Bcl-2, Bax, PKA, CREB-1 and TK and with decrease in the expression of ERK1,2 related MAPK, cyclin B1 and PKG in granulosa cells. No significant difference in PCNA expression was noted. The present data obtained from in vitro study indicate that sexual maturation in females is influenced by puberty-related changes in porcine ovarian signaling substances: increase in Bcl-2, Bax, PKA, CREB-1, TK and decrease in ERK1,2 related MAPK, cyclin B1 and PKG. It suggests that these signaling molecules could be potential regulators of porcine sexual maturation.

The aim of this study was to examine the effect of A-trichothecenes T-2 and HT-2 toxins combined with insulin-like growth factor I (IGF-I) on the release of steroid hormone progesterone (P4) by porcine ovarian granulosa cells (GCs). The cells were incubated without (control) or with treatments of A-trichothecenes T-2 (100 and 1000 ng/mL)/ HT-2 (100 and 1000 ng/mL) combined with IGF-I (1, 10 and 100 ng/mL) for 24 h. Progesterone secretion was determined by RIA. The release of P4 by GCs after addition of T-2 toxin (at 100 ng/mL) combined with IGF-I (at 10 but not at 1 and 100 ng/mL) and HT-2 toxin (at 100 ng/mL) combined with IGF-I (at all doses) was significantly (P < 0.05) inhibited. On the other hand the release of P4 after addition of T-2/ HT-2 toxin (at 1000 ng/mL) combined with IGF-I (at all doses) was significantly (P < 0.05) stimulated. Alone IGF-I addition (at 10, 100 but not at 1 ng/mL) significantly (P < 0.05) stimulated P4 release by GCs. The results of our in vitro study indicate the T-2 and HT-2 toxins combined with IGF-I could modify progesterone secretion by porcine ovarian granulosa cells and potentially regulate process of steroidogenesis in the ovaries. Currently, occurrence of mycotoxins in food and feed is a worldwide problem and therefore study of these toxins as well as their interaction with different substances such as growth factors could be beneficial for better understanding of mechanism of their toxic effects in organism.

The present study was aimed at investigating the effect of nickel chloride (NiCl2) on secretion of progesterone (P), ultrastructure and apoptosis in porcine granulosa cells. NiCl2 was added to the cells to achieve a Ni(2+) concentration of 62.5, 125, 250, 500 and 1,000 μmol/L. A control group contained no NiCl2 addition. Quantification of P was performed directly from aliquots of the media from control and treated porcine granulosa cells after 48 h of culture using radioimmunoassay. Quantification of apoptotic cells was performed using terminal deoxynucleotidyl transferase dUTP nick end labelling assay, and ultrastructural changes were analyzed using transmission electron microscopy. A concentration-dependent depletion of P production was observed significantly for 1,000 μmol/L NiCl2. The percentage of apoptotic cells was increased in all experimental groups significantly only after addition of 1,000 μmol/L NiCl2. After addition of ≥250 μmol/L NiCl2, a higher incidence of euchromatin was observed. Also, lipid droplets and vacuoles in the cytoplasm increased after addition of ≥250 μmol/L NiCl2. NiCl2 induced the decrease in numbers of mitochondria and smooth endoplasmic reticulum after treatment with ≥500 μmol/L NiCl2. Our findings suggest a negative effect of NiCl2 on steroidogenesis and apoptosis as well as ultrastructure of porcine granulosa cells.

The general objective of this in vitro study was to examine the secretory activity (insulin-like growth factor I, IGF-I) of porcine ovarian granulosa cells after Ag addition and to outline the potential intracellular mediators (cyclin B1 and caspase-3) of its effects. Ovarian granulosa cells were incubated with silver nitrate (AgNO(3)) at the doses 0.09, 0.17, 0.33, 0.5 and 1.0 mg/mL for 18 h and compared to the control group without metal addition. The release of IGF-I by granulosa cells was assessed by RIA and expression of cyclin B1 and caspase-3 immunocytochemistry. Our observations show that IGF-I release by granulosa cells was significantly (P<0.05) stimulated by AgNO(3) addition at the doses (0.09-1.0 mg/mL). Similarly to IGF-I the cyclin B1 and caspase-3 expression in ovarian granulosa cells was stimulated by Ag addition (0.09-1.0 mg/mL). In conclusion, the present results indicate, a direct effect of Ag on (1) secretion of growth factor IGF-I, (2) expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) of porcine ovarian granulosa cells and (3) that the effect of Ag on ovarian cell proliferation could be mediated by IGF-I and cyclin B1. Obtained data indicate the interference of Ag in the pathways of proliferation and apoptosis of porcine ovarian granulosa cells through hormonal and intracellular peptides such as are cyclin B1 and caspase-3., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

It would be desirable to expand the existing general knowledge concerning direct action of metals on the ovary. Nevertheless, the results of testing of iron compound on porcine ovarian cells should be interpreted carefully because iron is an essential element which could also induce changes in cellular processes. The aim of this in vitro study was 1) to examine dose-dependent effects of iron on the secretory activity of porcine ovarian granulosa cells, and 2) to outline the potential intracellular mediators mediating these effects. Specifically, we evaluated the effect of iron sulphate on the release of insulin-like growth factor I (IGF-I) and progesterone, as well as the expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA, cyclin B1 and caspase-3 expression by immunocytochemistry (ICC). Our results show a significantly decreased IGF-I secretion by ovarian granulosa cells after iron sulphate addition at the doses 0.5 and 1.0 mg/ml. The iron sulphate additions at doses 0.17 and 1.0 mg/ml had no effect on progesterone secretion. In contrast, iron sulphate addition at doses 0.17-1.0 mg/ml resulted in stimulation of cyclin B1 and caspase-3 expression. In conclusion, the present results indicate a direct effect of iron on 1) secretion of growth factor IGF-I but not steroid hormone progesterone, 2) expression of markers of proliferation (cyclin B1), or 3) apoptosis (caspase-3) of porcine ovarian granulosa cells. These results support an idea that iron could play a regulatory role in porcine ovarian function: hormone release, proliferation and apoptosis.

Molybdenum (Mo) is an essential trace element and it plays an important role in cell functions. The mechanism of the action of molybdenum in connection with growth factor IGF-I, proliferation-related peptide cyclin B1 and apoptosis-related peptide caspase-3 has not been examined previously in porcine ovarian granulosa cells. The general objective of this in vitro study was to examine the secretory activity of porcine ovarian granulosa cells after experimental Mo administration and to outline the potential intracellular mediators of its effects. Ovarian granulosa cells were incubated with ammonium molybdate for 18 hours: 1.0 mg/mL; 0.5 mg/mL; 0.33 mg/mL; 0.17 mg/mL and 0.09 mg/mL, while the control group received no Mo. The secretion of IGF-I was assessed by RIA and expression of cyclin B1 and caspase-3 by immunocytochemistry. IGF-I release was decreased by Mo addition at the doses 1.0 mg/mL and 0.5 mg/mL. The expression of cyclin B1 was stimulated by Mo addition at all doses ranging from 1.0-0.09 mg/mL. Caspase-3 expression was also stimulated after experimental Mo addition at the doses 1.0 and 0.5 mg/mL. These data contribute to new insights regarding the mechanism of action of Mo on porcine ovarian functions, secretory activity, proliferation and apoptosis of granulosa cells through hormonal and intracellular substances such as are cyclin B1 and caspase-3.

The general objective of this in vitro study was to examine the effect of bee pollen on the release of insulin-like growth factor I (IGF-I) and steroid hormone progesterone, and expression of markers of proliferation (PCNA) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA method and expression of PCNA and caspase-3 by immunocytochemistry. Bee pollen addition at the dose of 10 ng/mL significantly (P<0.05) inhibited IGF-I release by porcine ovarian granulosa cells. This growth factor was not influenced by 100 and 1000 ng/mL doses of bee pollen. Progesterone release by cells was not influenced by bee pollen addition at the doses of 10, 100 and 1000 ng/mL as used in our study. Similarly expression of PCNA and caspase-3 was not affected by bee pollen addition. The present study shows dose-dependent regulation of IGF-I by experimental bee pollen addition in vitro. Progesterone release, expression of PCNA and caspase-3 in porcine ovarian granulosa cells was not induced by pollen. Our results contribute to new insights regarding the possible effect of bee pollen on IGF-I release, which is important for regulation of porcine ovarian functions.

The aim of this in vitro study was to examine the secretion activity, markers of proliferation and apoptosis in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) addition. Ovarian granulosa cells were incubated with DON for 24h: 10, 100 and 1000 ng/mL, while the control group received no DON. The secretion of insulin-like growth factor I (IGF-I) and progesterone was determined by radioimmunoassay (RIA) and expression of cyclin B1, PCNA and caspase-3 by immunocytochemistry. IGF-I release by GCs was inhibited by DON, while progesterone release and the expression of cyclin B1 was stimulated by DON (at 1000 ng/mL but not at 10 and 100 ng/mL). PCNA expression was stimulated by DON (at 100 and 1000 ng/mL but not at 10 ng/mL). Caspase-3 expression was not influenced by DON treatment (at all doses). In conclusion, our results indicate, (1) a direct effect of DON on secretion of growth factor IGF-I and steroid hormone progesterone, (2) expression of markers of proliferation (cyclin B1 and PCNA) but not on the (3) expression of marker of apoptosis (caspase-3) in porcine ovarian granulosa cells. This in vitro study suggests the dose-dependent association of DON on porcine ovarian functions.

Cobalt (Co) is an essential element. The general objective of this in vitro study was to examine dose-dependent changes in the secretory activity of porcine ovarian granulosa cells after experimental Co administration and to outline the potential intracellular mediators of its effects. Concentrations of IGF-I and progesterone were determined by RIA and expression of cyclin B1 and caspase-3 by immunocytochemistry. IGF-I release by granulosa cells was stopped by Co addition at the concentration 1 mg/mL. Progesterone release by granulosa cells was decreased at the lowest Co addition (0.09 mg/mL). In our study the changes of the expression of proliferation related peptide cyclin B1 and apoptosis related peptide caspase-3 in ovarian granulosa cells was observed after experimental Co addition. The molecular pathways stimulated by Co through the expression of cyclin B1 and caspase-3 were found. In conclusion, this study provides novel evidence that cobalt is the factor which can initiate adverse effects in ovarian granulosa cells. These results contribute towards the understanding of mechanisms relating to endocrine disruptor-induced alterations in porcine ovarian granulosa cells.

The heavy metals lead (Pb) and mercury (Hg) pose potential risks to sustainability of environment and thus to our future generations. General objective of this in vitro study was to examine the secretory activity of porcine ovarian granulosa cells after Pb and Hg administration and to outline the potential intracellular mediators of its effects. For this purpose, release of insulin-like growth factor I (IGF-I) and steroid hormone progesterone (P(4)), expression of proliferation- related (cyclin B1) and apoptosis-related (caspase-3) peptides was examined in porcine ovarian granulosa cells after heavy metals administration. Obtained data indicate Pb-induced inhibition of IGF-I release at lower doses (0.063 mg/mL and 0.046 mg/mL) by ovarian granulosa cells. However, P(4) release was not influenced by Pb addition, while the expression of cyclin B1 and caspase-3 was induced by Pb addition. These results indicate that Pb can affect the pathway of proliferation and apoptosis of porcine ovarian granulosa cells through intracellular substances such as cyclin B1 and caspase-3. On the other hand, the P(4) release by ovarian granulosa cells of pregnant gilts was stimulated by experimental Pb administration at doses of 0.25 mg/mL and 0.063 mg/mL and experimental Hg administration at doses 0.25 mg/mL and 0.083 mg/mL. P(4) release by ovarian cells of pregnant gilts was not influenced by a combinatory dose of FSH (1.0 ng/mL) + Pb (0.083 mg/mL) + Hg (0.083 mg/mL) but it was inhibited by experimental administration of FSH (10 ng/mL) + Pb (0.25 ng/mL) + Hg (0.25 ng/mL). Possible involvement of heavy metals - Pb and Hg and pituitary hormone FSH, in the regulation of P(4) release by porcine ovarian granulosa cells of pregnant gilts was noted. Data obtained from in vitro studies suggest the dose dependent association of heavy metals administration with the hormonal release by porcine ovarian granulosa cells. This association also depended on pregnancy of the gilts.

The objective of this study was to determine the activity of superoxide dismutase (SOD), total antioxidant status (TAS) and release of calcium, phosphorus, magnesium, sodium, potassium, total lipids, totals proteins, glucose, cholesterol and triglycerides by porcine ovarian granulosa cells cultured in vitro after lead acetate administration. The parameters were analyzed using semi-automated clinical chemistry analyzer Microlab 300, microprocessor-controlled analyzer EasyLite and spectrophotometer Genesys 10. Cells were cultured with lead acetate trihydrate [Pb(CH(3)COO)(2).3H(2)O] as follows: group Max (5 mg Pb(CH(3)COO)(2).3H(2)O/10 mL), group A (2.5 mg/10 mL), group B (0.83 mg/10 mL), group C (0.625 mg/10 mL), group D (0.455 mg/10 mL) and the control group without lead exposure for 18 hrs. The highest TAS was estimated in the control group without lead treatment in comparison with other groups (MAX, A, B, C, D). Statistical analyses showed significantly lower value (P < 0.05) in group B. The activity of SOD was the lowest in the control group in comparison to those exposed to in vitro lead culture. A significant decrease (P < 0.05) of calcium content in group MAX in comparison with control group was determined. Release of phosphorus by ovarian granulosa cells was significantly lower (P < 0.05; 0.01; 0.001) in all the treated groups in comparison with control group. Lead was found to stimulate the release of magnesium and potassium by granulosa cells, but the increase remained statistically insignificant. The highest concentration of glucose was noted in control group, but the differences were not significant either. No significant differences (P > 0.05) were detected in concentration of other studied parameters among observed groups, too.