Sirotkin A V, Grossmann R
Research Institute of Animal Production, Slovak Centre of Agricultural Studies, Hlohovská 2, 949 92 Nitra, Slovakia.
Comp Biochem Physiol A Mol Integr Physiol. 2007 Oct;148(2):422-9. doi: 10.1016/j.cbpa.2007.06.001. Epub 2007 Jun 7.
The aim of our in-vitro experiments was to examine, whether leptin can directly control functions of avian ovarian cells and to outline potential intracellular mediators of its effects. Granulosa cells or fragments of ovarian follicular wall were cultured with leptin (0, 1, 10 or 100 ng/mL medium). The expression of peptides involved in apoptosis (TdT, bax, its binding protein, bcl-2, ASK-1 and p53), cell cycle-related peptides (PCNA and cyclin B1), release of hormones (progesterone, testosterone, estradiol, arginine-vasotocin), as well as the expression of protein kinases (PKA, MAPK/ERK1,2 and CDK/p34) in the ovarian cells were examined by using immunocytochemistry, TUNEL, SDS-PAGE-Western immunoblotting, EIA and RIA. It was found that leptin inhibited expression of all markers of cytoplasmic apoptosis (bax, ASK-1 and p53), stimulated expression of anti-apoptotic peptide bcl-2, but did not affect nuclear DNA fragmentation (TdT). Furthermore, leptin inhibited expression of PCNA (marker of S-phase of mitosis), but not of cyclin B1 (marker of G phase of cell cycle). Moreover, it promoted release of progesterone and estradiol, suppressed release of testosterone, but did not affect arginine-vasotocin. Finally, leptin inhibited expression of MAPK/ERK1,2 and CDK/p34 and stimulated expression of PKA. The present observations demonstrate that leptin can directly control basic chicken ovarian functions - inhibit cytoplasmic apoptosis and proliferation (S-phase, but not G-phases of mitosis), regulate secretory activity (release of steroids, but not nonapeptide hormone) and expression of MAPK, PKA and CDC2, which might be potential intracellular mediators of leptin action.
我们体外实验的目的是研究瘦素是否能直接控制禽类卵巢细胞的功能，并概述其作用的潜在细胞内介质。将颗粒细胞或卵巢卵泡壁碎片与瘦素（0、1、10或100 ng/mL培养基）一起培养。通过免疫细胞化学、TUNEL、SDS-PAGE- Western免疫印迹、酶免疫分析和放射免疫分析，检测卵巢细胞中参与凋亡的肽（末端脱氧核苷酸转移酶、bax、其结合蛋白、bcl-2、凋亡信号调节激酶1和p53）、细胞周期相关肽（增殖细胞核抗原和细胞周期蛋白B1）、激素释放（孕酮、睾酮、雌二醇、精氨酸加压催产素）以及蛋白激酶（蛋白激酶A、丝裂原活化蛋白激酶/细胞外信号调节激酶1/2和周期蛋白依赖性激酶/p34）的表达。结果发现，瘦素抑制细胞质凋亡的所有标志物（bax、凋亡信号调节激酶1和p53）的表达，刺激抗凋亡肽bcl-2的表达，但不影响核DNA片段化（末端脱氧核苷酸转移酶）。此外，瘦素抑制增殖细胞核抗原（有丝分裂S期标志物）的表达，但不影响细胞周期蛋白B1（细胞周期G期标志物）的表达。而且，它促进孕酮和雌二醇的释放，抑制睾酮的释放，但不影响精氨酸加压催产素。最后，瘦素抑制丝裂原活化蛋白激酶/细胞外信号调节激酶1/2和周期蛋白依赖性激酶/p34的表达，并刺激蛋白激酶A的表达。目前的观察结果表明，瘦素可以直接控制鸡卵巢的基本功能——抑制细胞质凋亡和增殖（有丝分裂的S期，但不是G期），调节分泌活性（类固醇释放，但不是九肽激素）以及丝裂原活化蛋白激酶、蛋白激酶A和细胞周期蛋白依赖性激酶2的表达，这些可能是瘦素作用的潜在细胞内介质。
