The toxic influence of silver and titanium dioxide nanoparticles on cultured ovarian granulosa cells.

The application of metal nanoparticles in modern society is growing, but there is insufficient data concerning their influence on reproductive processes and comparison of their biological activity. The present experiments aimed to compare the effects of silver and titanium dioxide nanoparticles (AgNPs and TiO2NPs) on ovarian granulosa cell functions. AgNPs and TiO2NPs were added to culture of porcine granulosa cells at doses 0, 0.01, 0.1, 1 or 10 µg/mL. The mRNAs for proliferating cell nuclear antigen (PCNA), cyclin B1, bax and caspase 3 were quantified by RT-PCR; release of progesterone was analyzed by ELISA. It was shown that both AgNPs and TiO2NPs significantly reduced all the measured parameters. ED50 of the inhibitory influence of AgNPs on the main ovarian cell parameters was higher than ED50 of TiO2NPs. The ability of AgNPs and TiO2NPs to suppress ovarian granulosa cell functions should be taken into account by their application.

Mechanisms of the direct effects of oil-related contaminants on ovarian cells.

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.

Quercetin directly inhibits basal ovarian cell functions and their response to the stimulatory action of FSH.

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.

Resveratrol directly affects ovarian cell sirtuin, proliferation, apoptosis, hormone release and response to follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I).

The objective of our study was to examine the influence of the plant polyphenol resveratrol (R) on the rapamycin signalling pathway (mammalian target of rapamycin; mTOR) and basic ovarian cell functions in mammalian targets, as well as on their response to the physiological hormonal stimulators follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I). Resveratrol was found to stimulate sirtuin 1 accumulation and apoptosis, inhibit proliferation, suppress P and promote T and E release. Alone, FSH promoted proliferation and had no effect on apoptosis, but had an inhibitory effect on these processes when combined with R. IGF-I alone stimulated proliferation and inhibited apoptosis and promoted P production but not that of T; however, in the presence of R, the addition of IGF-I switched from having an anti-apoptotic to a pro-apoptotic effect and stimulated T release, but it did not modify the effect of IGF-I on proliferation and P output. These observations: (1) demonstrate that R directly affects the basic ovarian cell functions of proliferation, apoptosis and steroidogenesis, (2) provide further evidence of the involvement of FSH and IGF-I in the regulation of these processes, (3) demonstrate the ability of R to prevent and even invert the effects of FSH and IGF-I on ovarian cells and (4) indicate that the effects of R may be mediated by the mTOR-sirtuin intracellular signalling system.

Direct effect of curcumin on porcine ovarian cell functions.

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.

Dietary supplementation of yucca (Yucca schidigera) affects ovine ovarian functions.

Yucca (Yucca schidigera) is a popular medicinal plant due to its many positive effects on animal and human physiology, including their reproductive systems. To examine the effect of supplemental yucca feeding on sheep reproduction, including ovarian functions and their hormonal regulators, ewes were fed (or not fed, control) yucca powder (1.5 g/head/day, 30 days). Macromorphometric indexes of the oviduct, ovary, and ovarian folliculogenesis were measured. Reproductive hormone levels in the blood were measured using a radioimmunoassay. Granulosa cells were aspirated from the ovary, and their proliferation and apoptosis were detected using immunocytochemistry. To assess secretory activity and its response to gonadotropin, ovarian fragments of treated and control ewes were cultured with and without follicle-stimulating hormone (FSH; 0, 0.1, 1, 10, or 100 IU/mL), and the release of reproductive hormones into the culture medium was evaluated. Finally, to examine the direct action of yucca on the ovary, ovarian fragments from control ewes were cultured with and without yucca extract (1, 10, or 100 µg/mL), and the release of reproductive hormones was measured. Yucca supplementation significantly decreased the size of small antral follicles (2 to <5 mm in diameter), increased accumulation of the apoptosis marker bax, and decreased serum progesterone (P4) and estradiol (E2) levels. It inhibited the release of P4 (but not other hormones), to prevent the stimulatory action of FSH on P4 output and promoted insulin-like growth factor I (IGF-I) release by fragments cultured with FSH. However, yucca supplementation did not affect the size of larger follicles and number of follicles, volume and weight of ovaries, length and weight of oviducts, caspase 3 accumulation, cell proliferation, testosterone (T) or IGF-I serum levels, or T or E2 release by cultured ovarian fragments and their response to FSH. Yucca addition to culture medium inhibited P4 and IGF-I, but not T or E2 release at the lowest (1 µg/mL) dose, and stimulated P4, but not T, E2, or IGF-I release at the highest (100 µg/mL) dose. These data suggest that yucca supplementation can reduce small antral ovarian follicle development possibly via the stimulation of apoptosis of their granulosa cells, suppression of ovarian P4 and E2 release, and alteration of ovarian IGF-I output and ovarian response to gonadotropin. Thus, yucca can directly affect P4 and IGF-I release by ovine ovarian cells.

Yucca schidigera extract can promote rabbit fecundity and ovarian progesterone release.

The aim of the present in vivo study was to determine the effects of yucca powder extract added to the rabbit females feed mixtures on kindling and conception rate. Rabbit does of the experimental groups were fed with the standard diet enriched with supplement of yucca dry extract at doses of 5 g/100 kg feed (E1 group) or 20 g/100 kg feed (E2 group) for 50 days. In our preliminary in vivo results, we shown that conception rate was significantly higher in both experimental E1 and E2 groups (82.4% and 100.0%, respectively) than in the control group (47.1%). The kindling rate was also significantly higher in the experimental groups (70.6% and 100.0%, respectively) than in the control group (41.2%). The differences between control and yucca-treated groups in the number of liveborn, stillborn, and weaned pups per doe were not statistically significant. To understand possible endocrine mechanisms of yucca action on fertility rate, we have examined the influence of yucca extract additions on the release of steroid hormones by isolated and cultured rabbit ovarian fragments. Yucca additions promoted release of progesterone (at dose of 1 µg/mL, but not at doses of 10 and 100 µg/mL). Yucca addition at these doses did not affect testosterone or estradiol release. Our observations show the stimulatory effect of yucca consumption on rabbit fecundity, which can be due to its direct stimulatory influence on ovarian progesterone but not on testosterone or estradiol output.

Assessment of T-2 toxin effect and its metabolite HT-2 toxin combined with insulin-like growth factor I, leptin and ghrelin on progesterone secretion by rabbit ovarian fragments.

Assessment of A-trichothecene mycotoxins (T-2 and HT-2 toxins) effect combined with growth factor IGF-I, and the metabolic hormones leptin and ghrelin on progesterone secretion by rabbit ovarian fragments was studied. Rabbit ovarian fragments were incubated without (control group) or with T-2/HT-2 toxin, or their combinations with insulin-like growth factor I (IGF-I), leptin or ghrelin at various concentrations for 24 h. Secretion of progesterone was determined by ELISA. First, T-2 toxin and HT-2 toxins at all doses used (0.01, 0.1, 1, 10, and 100 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Second, T-2 toxin but not HT-2 toxin combined with IGF-I was shown to be potential regulator of progesterone secretion in rabbit ovarian fragments. T-2 toxin at all doses used (0.01; 0.1; 1; 10; and 100 ng mL(-1)) combined with IGF-I (at dose 100 ng mL(-1)) significantly (P < 0.05) decreased progesterone secretion by rabbit ovarian fragments. Third, T-2 toxin and HT-2 toxin at all doses used in the study (0.01, 0.1, 1, 10, and 100 ng mL(-1)) combined with leptin (at dose 1000 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Furthermore, T-2 toxin and HT-2 toxin at all doses used in the study (0.01, 0.1, 1, 10, and 100 ng mL(-1)) combined with ghrelin (500 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Results in this study showed that trichothecene as T-2 toxin combined with IGF-I but not HT-2 toxin was able to decrease progesterone secretion in rabbit ovarian fragments in vitro. Experimental results of T-2 and HT-2 toxins combined with leptin and ghrelin did not confirm ability to modulate progesterone secretion by ovarian fragments in rabbits.

T-2 toxin and its metabolite HT-2 toxin combined with insulin-like growth factor-I modify progesterone secretion by porcine ovarian granulosa cells.

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.