ACTIVITY AND CONTENT OF SUPEROXIDEDISMUTASE IZOZYMES IN GRANULOSE CELLS FROM COW OVARY FOLLICLES
The activity and content of superoxide dismutase isoforms (SOD) in granulosa cells from cow ovarian follicles were studied for research after slaughter of cows ovaries were selected, which according to the physiological state were divided into groups: with "fresh" ovulation, at the site of the ovulated follicle there is a hole, no corpus luteum or diameter up to 5 mm, red color (n = 14); with "early corpus luteum", diameter 10-20 mm, color red or brown (n = 41); with “late corpus luteum", diameter 5–15 mm, color yellow (n = 32); "follicular growth", without the corpus luteum (n = 84). The ovaries of cows with small (<4 mm), medium (4 - 7 mm) and large (> 7 mm) follicles were used. Antral fluid was obtained from the follicles, from which granulosa cells were isolated. Cells were suspended according to the volume of follicular fluid in the medium Dulbeccos modified Eagle medium (DME) with the addition of estrus serum of cows, follicular fluid, insulin and heparin. In cell culture, protein concentration, activity, and superoxide dismutase isozymes were determined.
It was found that granulosa cells are characterized by SOD activity - 12.4 ± 0.74 IU / mg protein (6.8 ± 1.72 - 19.8 ± 3.75 IU / mg protein). The activity of SOD in the culture of granulosa cells had 5–6 isoforms of the enzyme. It was found that isoforms at the site of localization are divided into cytosolic, mitochondrial and extracellular proteins of SOD. The cytosolic isoform were represented by 3 - 4, and mitochondrial and extracellular have one active protein of the enzyme. he activity of the enzyme and the content of isoforms depended on the size of the follicles from which the cells are removed and the physiological state of the ovaries. The studied indicators characterize the intensity of oxidative metabolism as a whole in cells and in their individual parts and organelles. For cultivation, it is advisable to select granulosa cells from ovarian follicles of "follicular growth" and "late corpus luteum" because they are characterized by consistently high activity of SOD, which protects intracellular components from the cytotoxic action of superoxide anion.
Beauchamp, C. & Fridovich, I. (1971). Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44. 276–287.
Chevari S.N., Andyan, T.A., Shtrenger, Ya.I. (1991). Opredelenie antioksidantnykh parametrov krovi i ikh diagnosticheskoe znachenie v pozhilom vozraste. Lab. Delo. 10. 9-13. [in Russian].
Combelles, C., Holick, E., Paolella, L., Walker, D. et al. (2010). Profiling of superoxide dismutase isoenzymes in compartments of the developing bovine antral follicles Reprod. 139. 871881.
Droge, W. (2002). Free radicals in the physiological control of cell function. Physiol. Rev. ― 2002. 82. 47–95.
Devine, P.J. & Perreault, S.D. (2012). Roles of Reactive Oxygen Species and Antioxidants in Ovarian Toxicity. Biol. of Reprod. 86. 2–27.
Friedman, C., Danforth, D., Herbosa-Encarnacion, C., Arbogast, L. et. al. (1997). Follicular fluid vascular endothelial growth factor concentrations are elevated in women of advanced reproductive age undergoing ovulation induction. Fertil. Steril. 68. 607–612.
Hensley, K., Robinson, K., Garitta, S., Salsman, S. (2000). Oxygen species, cell signalling and cell injury. Free Rad. Biol. Med. 28. 1456–1462.
Huzevatyi, O.Ye., Yasinskyi V.V., Smulka, L.V. et al. (1995). Otsinka functsionalnogo stanu ootsyt-kumulyusnykh compleksiv koriv zalezhno vid typu yayechnyca. Visnyk agrarnoi nauky. 11. 94-98. [in Ukrainian].
Kuzmina, N.V. & Ostapiv, D.D. (2008). Aktyvnist superoksyd dysmutazy i glutation peroksydazy v riznykh organakh i krovi koriv. Biologiya tvaryn. 12. 423-429. [in Ukrainian].
Kwon, H., Yang, H., Hwang, K. et al. (1999). Effects of low oxygen condition on the generation of reactive oxygen species and the development in mouse embryos cultured in vitro. Obstet. Gynaecol. Res. 25. 359 – 366.
Lenzi, A., Gandini, L., Lombardo, F., Picardo, M. et al. (2002). Polyunsaturated fatty acids of germ cell membranes, glutathione and blutathione-dependent enzyme-PHGPx: from basic to clinic Contraception. 65. 301–304.
Lowry, O.H., Rosebrough, N.J., Fair, A.L., Randall, R.J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem. 193. 1. 264–275.
Schreck, R. & Baeuerle, P.A. (1991). A role for oxygen radicals as second messenger. Trends. Cell. Biol. 1. 39–42.
Vlizlo, V.V., Fedoruk, R.S., Ratych I.B. et al. (2012). Laboratorni metody doslidzhen v biologii, tvarynnytstvi ta veterynarnij medytsyni. Lviv. Spolom. 764. [in Ukrainian].
Zeron, Y., Ocherentny, А., Kedar, O., Borochov, A. et al. (2001). Seasonal changes in bovine fertility: relation to developmental competence of oocytes, membrane properties and fatty acid composition of follicles. Reprod. 121. 447–454.
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