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Detvanová L.1, Kolesárová A.2, Kalhotka L.1
1Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic 2Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic ABSTRACT
The objective of this thesis was to examine the effect of metals on the expression of regulators of apoptosis and secretory activity of ovarian cells of rats in vitro. The following substances were observed: growth factor IGF-I, steroid hormones progesterone and estradiol, regulators of apoptosis: Bcl-2, Bax and caspase-3. Growth factor IGF-I and steroid hormones progesterone and estradiol were detected by RIA method and intracellular peptides were detected by Western- imunoblotting. In our study all examined substances were affected by various concentrations of [(NH4)6.Mo7O24.4H2O]. The release of growth factor IGF-I was inhibited by all concentrations of selected Mo. The highest dose of Mo (500 μg.ml-1) stimulated the most intense IGF-I secretion. In the case of the steroid hormone progesterone we did not achieve demonstrable (p ≥ 0,05) results, but the secretion of P4 had a decreasing tendency. The inhibitory effect of estradiol on the release of Mo was seen (p ≤ 0,05) at concentrations of 90 μg.ml-1, 170 μg.ml-1, 500 μg.ml-1, at a dose of 330 μg.ml-1 results were not conclusive, but nevertheless, we detected a reduction in estradiol secretion. Inhibition of apoptosis marker expression of Bcl-2 has been observed due to the lowest concentrations (90 and 170 μg.ml-1), increasing expression was observed in the experiment with the addition of 330 μg.ml-1. At concentrations of 90, 170, 330 μg.ml-1 was found to stimulate the expression of pro-apoptotic Bax peptide. Lower doses of Mo (90 and 170 μg.ml-1) had no effect on the expression of caspase-3, release was not significantly (p ≤ 0,05) stimulated concentrations of 330 and 500 μg.ml-1. Data obtained from in vitro experiment indicate that Mo has an effect on endocrine and apoptotic processes in the ovaries of rats. The results of this work suggest that the effect of Mo on ovarian processes is dose-dependent. Compounds of this metal may be potential regulators of intracellular ovary processes, hormone secretion and expression of regulators of Key words: ovaries, steroid hormones, regulators of apoptosis, molybdenum, rats
Acknowledgments: Autor would like to thank doc. Kolesárová for helping, valuable and patient
advices. This study was supported and financed by the research project VEGA 1/0790/11 and MENDELNET 2013
Molybdenum belongs to the Outline of the Periodic Table of Elements, is an essential trace element for the proper functioning of the plant and bacterial enzymes (eg nitrogenase). This element is also a cofactor for xanthine oxidase, sulfite oxidase, aldehyde oxidase, etc. in animal organisms. The fertility in females is affected by an amount of exogenous as well as endogenous factors, including the impact of heavy metals. High concentrations of these elements may negatively affect the endocrine system, which is closely linked to the system of reproduction. Ovaries, as organs rich in lipids, are places of accumulation of heavy metals in the female body. Thanks to experiments dealing with the regulation of reproduction, the area of reproductive biology and biotechnology can move forward by leaps and bounds. Among the main regulators we include hormones. MATERIAL AND METHODS
Laboratory rats have been selected as a model organism (n = 10). Rat ovaries obtained post-mortem were stored individually in a thermos with a physiological solution at room temperature and have been treated up to 6 hours after death. Ovaries were washed with sterile culture medium DMEM/F12 (1:1 BioWhittakerTM, Verviers, Belgium) and were cut into fragments of 2 mm in diameter. Fragments were then cultured in a sterile culture medium DMEM/F12 (1:1 BioWhittakerTM, Verviers, Belgium), which were supplemented with 10% fetal calf serum (BioWhittakerTM) and antibiotic - antimycotic (Sigma, St. Louis, MO, USA) without addition (control) or with the addition of ammonium molybdate tetrahydrate [( NH4)6.Mo7O24.4H2O] in the following concentrations : group A (500 μg.ml- 1 ( NH4 )6.Mo7O24.4H2O.ml- 1), group B (330 μg.ml- 1 [(NH4)6.Mo7O24.4H2O]), C (170 μg.ml-1 [(NH4)6.Mo7O24.4H2O]), group D (90 μg.ml–1 [(NH4)6.Mo7O24.4H2O]). Ovarian lysates were cultured (37 ° C, 5 % CO2) without Mo (control) / with the addition of Mo (experimental groups) in the culture plates (1 ml.culture- 1). After 18 hours of culture was taken syringe medium from the culture plates and stored at 70 ° C until analysis. Steroid hormones progesterone and estradiol were analyzed by RIA method using hormone labeled with radioactive iodine 125I. Concentrations of steroid hormones were determined by RIA in 25 to 100 ml incubation medium. These substances were attached using RIA kits (Immunotech SAS, Marseille Cedex, France) according to the manufacturer's instructions (Makarevich and Sirotkin, 1999). All of RIA kits were designed to be used with samples of culture medium. Proteins were divided by gel electrophoresis and they were transfered on polyvinyldenfluoride membrane. After transfer, proteins were coloured by Ponceau S. Membrane was blocked by 15 minutes incubation in 3 % H2O2. Membrane was incubated one hour in solution of primary antibody with phosphate buffer (TTBS) and 1 % albumine bovine serum. Primary antibody was diluted follows: Bcl-2 (1:250), caspase-3 (1:500) and Bax (1:500). Unbounded antibodies were washed in TTBS 2x10 minutes. Membrane was incubated with secondary antibody against mice imunoglobulins joined with peroxidase. After incubation membrane was washed in TTBS 3x10 minutes and it was incubated 5 minutes in detection reagent Super Signal. After detection membrane was drained and exposed with Rtg film. Film was developed by FOMA LP-T developer (diluted 1:4) and universal fixer FOMAFIX from FOMA Bohemia s.r.o. (Hradec Kralové, ČR). Analysis of substances in the incubation medium was performed in duplicate. Statistical differences between the control (no exposure (NH4)6.Mo7O24.4H2O) and treatment groups, which were exposed to (NH4)6.Mo7O24.4H2O - A, B, C, D), were evaluated One Way ANOVA test using MENDELNET 2013
statistical program Sigma Plot 11.0 (Jandel, Corte Madera, USA). Significance of differences between control and experimental groups was determined at p < 0,05. RESULT AND DISCUSSION
Effect of molybdenum on the secretion of hormones In the revision the secretion of IGF -I rat ovarian cells is expressed without addition of ammonium molybdate tetrahydrate [(NH4)6.Mo7O24.4H2O]. Ovary cells produced in the control group 4,16 ± 1,14 ng.ml-1 IGF -I compared to the experimental groups : A (2,60 ± 0,61 ng.ml-1), B (2,58 ± 0,36 ng.ml-1), C (1,79 ± 0,33 ng.ml-1), D (0,77 ± 0,43 ng.ml-1) with the addition of Mo. The release of IGF - I was significantly (p < 0,05) reduced the influence of all Mo concentrations used in our experiment. Kolesárová et al. (2009b) indicate that molybdenum may have an effect on ovarian cells gilts. After the experimental administration of ammonium molybdate in doses of 0,33 mg.ml-1, 0,5 mg.ml-1 and 1,0 mg.ml-1 release was inhibited by IGF-I . Another study, which looked at the influence of Mo on the release of IGF-I in layers, suggesting that the administration of 0,17 and 0,9 mg.ml-1 ammonium molybdate, conclusive (p < 0,05) differences between groups in the secretion IGF - I were recorded. Experimental group with the highest dose of Mo (0,33 mg.ml-1) significantly inhibited the release of IGF-I (Kolesárová et al., 2009b), which is in line with our study. Bersényi et al. (2008) found that the addition of Mo to the feed of rabbits does not affect their growth. Rabbits were fed pellets, carrots, containing 39 mg Mo.kg-1 dry foods and dietary supplements that contained 40 mg Mo.kg-1. During the 14 days conclusive changes were not found. The results of our work show the inhibitory effect of Mo on the secretion of growth factor IGF-I. In the control group rat ovarian cells 108,47 ± 1,51 ng.ml-1 hormone progesterone was detected. By comparing the experimental groups (A, B, C, D) significant differences were found in the secretion of progesterone influence of molybdenum. Experimental group A produced 78,02 ± 18,87 ng.ml-1, group B 67,62 ± 15,85 ng.ml-1, group C 50,90 ± 8,58 ng.ml-1, group D 46,99 ± 8,34 ng.ml-1. The tendency of reduced secretion of progesterone by the effects of Mo has been reported, but these results were not conclusive (p ≥ 0,05). According to Kolesarova et al. (2009a), by adding 1,0 mg.ml-1 ammonium molybdate, progesterone release in granulosa cells gilts was stimulated, which is consistent with the authors' earlier work (Kolesárová et al., 2009b), who observed a similar trend secretion of progesterone influence of Mo compounds. The hens were induced with progesterone secretion by addition of 0,17 and 0,33 mg.ml-1. Kolesárová et al. (2010) suggest that the release of progesterone ovarian cells gilts was affected by the addition of 1,0 mg. ml-1, but other doses used had no effect, which is in line with our work. This finding points to the impact of higher doses of Mo on ovarian cells in animals. On the release of P4 in the case of White Leghorn breed presence or absence of fetal calf serum in culture had no impact. Ovary cells that were cultured with or without the addition of addition of serum produced approximately equal amounts of progesterone (Sirotkin, 2010). After administration of molybdenum were found between control and experimental groups A, C, D had significant differences (p ≤ 0,05) in estradiol secretion. The control has fallen 223,85 ± 15,23 pg.ml-1, in group A 36,04 ± 8,68 pg.ml-1, B 177,69 ± 52,37 pg.ml-1, C 54,48 ± 17,71 pg.ml-1, D 47,31 ± 32,69 pg.ml-1 ammonium molybdate. MENDELNET 2013
Regarding the secretory activity of ovarian cells of rats with a focus on estradiol, we recorded inhibition by Mo. Kolesárová et al. (2012) report that the release of estradiol in rats is affected by other additions, for example: bee pollen incorporated in feed. Female rats increasingly produced estradiol because of a feed mixture containing 5 kg of pollen per 1000 kg of feed. In the experimental group, which was fed with a mixture of lower pollen addition of 3 kg of pollen per 1000 kg of feed, a change was observed in estradiol secretion. Sirotkin (2010) states that the lack of fetal calf serum has an effect the release of estradiol. The differences between the impact of additions for secretion of estradiol and progesterone indicate different nutritional regulation of these hormones. Other authors state that the release of estradiol was inhibited by the addition of tetrathiomolybdate at 1-μg.ml-1 first. The results suggest that the doses of Mo did not affect the secretion of steroid hormone progesterone, but inhibited the secretion of estradiol (Kendal et al., 2003). Expression of Bcl-2 has been demonstrated in fraction 1 with molecular weight 26 K. Effect of addition (NH4)6.Mo7O24.4H2O were recorded changes in expression anti-apoptic peptid. In ovary cells were noticed inhibition of expression Bcl-2 peptid impact of (NH4)6.Mo7O24.4H2O by using lowest dose, increasing expression were recorded in group B. Most intensive ihibition was detected in groups C and D, using by lowest concentration Mo. In ovary cells was detected change of expression pro-apoptic marker Bax, which has been demonstrated in fraction 1 with molecular weight 23 K. In this case was recorded only increasing expression of apoptic marker Bax impact of (NH4)6.Mo7O24.4H2O. Most intensive expression was noticed by using lowest concentration Mo. Expression of apoptic marker caspase-3 was detected in fraction 1 with molecular weight 34 K. Most intensive expression was noticed in groups A and B, where was used highest concentration of Mo, not modified expression was recorded in group D with lowest dose of Mo. Expression of anti-apoptic peptid Bcl-2, pro-apoptic markers Bax and caspase-3 was affected with addition DON. Expression of Bcl-2 was decreased by using DON, while excretion pro-apoptic factors was decreased all of using doses (10 ng.ml-1, 100 ng.ml-1,1000 ng.ml-1) DON (Kolesárová et al., 2011/11). Dose-depended way DON and apoptic regulators can develop apoptosis in human gastric carcinomes in cell line SGC-7901 a BGC-823 in vitro. Apoptic mechanisms can increase with production of homologic dimers Bax-Bax and can decrease with production dimers Bax-Bcl-2 (Liu et al., 2009). In previous studies was identified change of expression apoptic marker caspase-3 in granulose cell in gilts addition of Mo. Expression of caspase-3 was stimulated with addition Mo compound in doses: 0,09 a 1,0 mg.ml-1 (Kolesárová et al., 2010). CONCLUSIONS
Effect of molybdenum and mechanism of action on rat ovary cells, in conjunction with growth factor IGF-I, steroid hormones and apoptic markers are insufficiently investigated. Based on the findings of the presented work it is possible to believe that Mo affects the endocrine and apoptic processes in the rat ovary. Also, a potential regulator of folliculogenesis, steroidogenesis and apoptosis through intracellular regulators. Our knowledge indicates that the effect of Mo on ovarian process is dose-dependent. For a complete understanding of the mechanisms of action of Mo on ovarian processes will require further study and verification. Our work contributes only to clarify the mechanism of action of Mo in the female reproductive system. MENDELNET 2013
BERSÉNYI,A. – BERTA, E. – KÁDÁR, I. – GLÁVITS,R. – SZILÁGYI, M. – FEKETE, S.G. 2008. Effects of high dietary molybdenum in rabbits. In Acta Veterinaria Hungarica, vol. 56, no. 18, pp. 41-56. ISSN 1588-2705. KENDAL, N. R. et al. 2003. Expression of lysyl oxidase and effect of copper chloride and ammonium tetrathiomolybdate on bovine ovarian follicle granulose cells cultured in serum-free media. In Reproduction, vol. 125, no. 5, pp. 657-665. ISSN 1741-7899. KOLESÁROVÁ, A. et al. 2009a. Insuline-like growth factor-I and progesterone release by ovarian granulosa cells of hens after experimental lead and molybdenum administration in vitro. In International Journal of Poultry Science, vol. 8, no. 5, pp. 890-895. ISSN 1537-0437. KOLESÁROVÁ, A. et al. 2009b. The realease of insuline-like growth factor – I by ovarian granulosa cells of pregnant sows after lead and mercury administration in vitro. In Slovak Journal of Animal Science, vol. 42, pp. 35-41. ISSN 1337-9984. KOLESÁROVÁ, A. et al. 2010. In vitro study on the effect of lead and mercury on porcine ovarian granulosa cells. In Journal of Enviromental Science and Health, Part A. Toxic hazardous substances and enviromental engineering, vol. 45, no. 1, pp. 320-331. ISSN 1532-4117. KOLESÁROVÁ, A. et al. 2011/12. Deoxynivalenol-induced animal ovarian signaling:proliferation and apoptosis. In Journal of Microbiology, Biotechnology and Food Science, vol. 3, no. 1, pp. 323-332. ISBN 1439-0396. KOLESÁROVÁ, A. et al. 2012. Consumption of bee pollen affects rat ovarian functions. In Journal of animal physiology and animal nutrition, vol. 37, no. 5, pp. 201-209. ISSN 1439-0396. LIU, J. et al. 2009. Effects of deoxynivalenol on apoptosis of human gastric carcinoma cell line SGC-7901, BGC-823 in vitro. In Wei Sheng Yan Jiu, vol. 38, no. 4, pp. 408-412. ISSN 1000-8020. SIROTKIN, A. V. et al. 2010. The effect of deoxynivalenol on the secretory activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro. In Journal of Environmental Science and Health Part B, vol. 46, no. 3, pp. 213-219. ISSN 1532-4095

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