Relationship between body conditions and environmental contaminants in bovine ovarian 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.

The influence of deoxynivalenol and zearalenone on steroid hormone production by porcine ovarian granulosa cells in vitro.

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.

Assessment of a potential preventive ability of amygdalin in mycotoxin-induced ovarian toxicity.

The possible effects of a natural substance amygdalin and its combination with the mycotoxin deoxynivalenol (DON) on the steroid hormone secretion (progesterone and 17-ß-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-ß-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-ß-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-ß-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-ß-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.

In vitro assessment of silver effect on porcine ovarian 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.

The effect of deoxynivalenol on the secretion activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro.

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.

In vitro assessment of molybdenum-induced secretory activity, proliferation and apoptosis of porcine ovarian granulosa cells.

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.

Cobalt-induced changes in the IGF-I and progesterone release, expression of proliferation- and apoptosis-related peptides in porcine ovarian granulosa cells in vitro.

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.