Roychoudhury Shubhadeep, Bulla Jozef, Sirotkin Alexander V, Kolesarova Adriana
a Department of Life Science & Bioinformatics , Assam University , Silchar , India.
J Environ Sci Health A Tox Hazard Subst Environ Eng. 2014;49(6):625-33. doi: 10.1080/10934529.2014.865404.
Objective of this in vitro study was to examine the secretion activity (progesterone and insulin-like growth factor I) of porcine ovarian granulosa cells after copper (Cu) addition and to outline a potential intracellular mediator (cyclin B1) of its effects. It also aimed at investigating the apoptotic potential of Cu on porcine ovarian granulosa cells after addition in vitro. Ovarian granulosa cells were incubated with copper sulphate (CuSO4·5H2O) at the doses 0.33, 0.40, 0.50, 1.0 and 2.0 μL mL(-1) for 18 h and compared with control group without Cu addition. Release of progesterone (P4) and insulin-like growth factor I (IGF-I) by granulosa cells was assessed by RIA, expression of cyclin B1 by immunocytochemistry and apoptosis by TUNEL assay. Observations show that P4 release by granulosa cells was inhibited while the release of IGF-I and cyclin B1 was stimulated significantly (P < 0.05) by CuSO4·5H2O addition at the dose 2.0 μL mL(-1). Also, addition of CuSO4.5H2O at the lowest dose used in the study (0.33 μL mL(-1)) significantly (P < 0.05) decreased apoptosis in granulosa cells. In conclusion, results indicate dose dependent effect of Cu on (1) secretion of steroid hormone progesterone and growth factor IGF-I, (2) expression of cyclin B1 as marker of proliferation of porcine ovarian granulosa cells, (3) apoptosis of porcine ovarian granulosa cells and, (4) that the effect of Cu on ovarian cell proliferation could be mediated by IGF-I and cyclin B1. Obtained data suggest interference of Cu in the pathways of proliferation of porcine ovarian granulosa cells through hormonal and intracellular peptide cyclin B1.
本体外研究的目的是检测添加铜（Cu）后猪卵巢颗粒细胞的分泌活性（孕酮和胰岛素样生长因子I），并概述其作用的潜在细胞内介质（细胞周期蛋白B1）。它还旨在研究体外添加Cu后其对猪卵巢颗粒细胞的凋亡潜力。将卵巢颗粒细胞与剂量为0.33、0.40、0.50、1.0和2.0 μL mL⁻¹的硫酸铜（CuSO₄·5H₂O）孵育18小时，并与未添加Cu的对照组进行比较。通过放射免疫分析法评估颗粒细胞孕酮（P4）和胰岛素样生长因子I（IGF-I）的释放，通过免疫细胞化学法检测细胞周期蛋白B1的表达，通过TUNEL法检测细胞凋亡。观察结果表明，在2.0 μL mL⁻¹剂量下添加CuSO₄·5H₂O可显著抑制颗粒细胞P4的释放，同时显著刺激IGF-I和细胞周期蛋白B1的释放（P < 0.05）。此外，在研究中使用的最低剂量（0.33 μL mL⁻¹）添加CuSO₄·5H₂O可显著（P < 0.05）降低颗粒细胞的凋亡。总之，结果表明Cu对（1）甾体激素孕酮和生长因子IGF-I的分泌、（2）作为猪卵巢颗粒细胞增殖标志物的细胞周期蛋白B1的表达、（3）猪卵巢颗粒细胞的凋亡具有剂量依赖性影响，以及（4）Cu对卵巢细胞增殖的影响可能由IGF-I和细胞周期蛋白B1介导。获得的数据表明Cu通过激素和细胞内肽细胞周期蛋白B1干扰猪卵巢颗粒细胞的增殖途径。
