Constantine The Philosopher University in Nitra, Nitra, Slovakia.
Anim Reprod Sci. 2011 Oct;128(1-4):73-84. doi: 10.1016/j.anireprosci.2011.09.005. Epub 2011 Sep 21.
The aim of these in vitro studies was to examine the involvement of transcription factor NF-κB (p50/p50, p65/p65) and FSH in control of porcine ovarian granulosa cells functions and the possible role of dimers p50/p50, p65/p65 in mediating FSH actions on these cells. Monolayer of primary granulosa cells was transfected with plasmids encoding human p50 cDNA and p65 cDNA, and cultured with or without addition of FSH (0, 1, 10 or 100 ng/ml). The accumulation of proteins p50 and p65, as well as of proliferation markers (PCNA and MAPK/ERK1,2) and marker of apoptosis (Bax) in cells was detected by using SDS-PAGE-Western immunoblotting and immunocytochemistry. DNA fragmentation was evaluated by TUNEL assay. Release of hormones insulin-like growth factor I (IGF-I), progesterone (P(4)), oxytocin (OT), prostaglandins E(2) (PGE(2)) and F(2α) (PGF(2α)) was measured by using RIA. We observed, that p50/p50 promoted the accumulation of PCNA, MAPK/ERK1,2, the release of OT, PGF(2α); inhibited the occurrence of TdT-positive cells, the release of IGF-I and P(4), and did not influence the accumulation of Bax and the release of PGE(2). p65/p65 enhanced the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, PGE(2) and PGF(2α); decreased the percentage of cell containing TdT and did not affect the release of P(4). FSH stimulated the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, P(4), PGE(2); but reduced the proportion of TdT-positive cells and the release of PGF(2α). These observations suggest (1) the involvement of NF-κB (p50/p50) in stimulation of proliferation, inhibition of apoptosis and in either stimulation (OT, PGE(2)) or inhibition (IGF-I, P(4), but not PGF(2)) of hormones release by porcine ovarian granulosa cells; (2) the involvement of NF-κB (p65/p65) in stimulation of proliferation and mitochondrial/Bax-related apoptosis, inhibition of nuclear/TdT-related apoptosis and in stimulation of ovarian hormones (IGF-I, OT, PGE(2), PGF(2α), but not P(4)) release; (3) the role of FSH in up-regulation of both ovarian cell proliferation and mitochondrial/Bax-related apoptosis, in inhibition of nuclear/TdT-related apoptosis, in promotion of IGF-I, P(4), OT, PGE(2) and suppression of PGF(2α) release by porcine ovarian cells. The majority of results demonstrates the involvement of NF-κB (p50/p50 and p65/p65) and FSH in control of basic ovarian functions (proliferation, apoptosis, and secretory activity), but not the functional interrelationships between these regulators.
这些体外研究的目的是研究转录因子 NF-κB（p50/p50、p65/p65）和 FSH 在控制猪卵巢颗粒细胞功能中的作用，以及二聚体 p50/p50、p65/p65 在介导这些细胞上 FSH 作用中的可能作用。用编码人 p50 cDNA 和 p65 cDNA 的质粒转染单层原代颗粒细胞，并在添加或不添加 FSH（0、1、10 或 100ng/ml）的情况下培养。通过 SDS-PAGE-Western 免疫印迹和免疫细胞化学检测细胞中 p50 和 p65 蛋白的积累，以及增殖标志物（PCNA 和 MAPK/ERK1,2）和细胞凋亡标志物（Bax）。通过 TUNEL 测定评估 DNA 片段化。通过 RIA 测量胰岛素样生长因子 I（IGF-I）、孕酮（P4）、催产素（OT）、前列腺素 E2（PGE2）和 F2α（PGF2α）的释放。我们观察到 p50/p50 促进 PCNA、MAPK/ERK1,2 的积累、OT、PGF2α 的释放；抑制 TdT 阳性细胞的发生、IGF-I 和 P4 的释放，并不影响 Bax 的积累和 PGE2 的释放。p65/p65 增强 PCNA、MAPK/ERK1,2 和 Bax 的积累，IGF-I、OT、PGE2 和 PGF2α 的释放；减少含 TdT 的细胞百分比，不影响 P4 的释放。FSH 刺激 PCNA、MAPK/ERK1,2 和 Bax 的积累、IGF-I、OT、P4、PGE2 的释放；但降低 TdT 阳性细胞的比例和 PGF2α 的释放。这些观察结果表明 (1) NF-κB（p50/p50）参与刺激猪卵巢颗粒细胞增殖、抑制细胞凋亡以及刺激（OT、PGE2）或抑制（IGF-I、P4，但不是 PGF2α）激素释放；(2) NF-κB（p65/p65）参与刺激增殖和线粒体/Bax 相关凋亡，抑制核/TdT 相关凋亡，以及刺激卵巢激素（IGF-I、OT、PGE2、PGF2α，但不是 P4）释放；(3) FSH 在调节猪卵巢细胞增殖和线粒体/Bax 相关凋亡、抑制核/TdT 相关凋亡、促进 IGF-I、P4、OT、PGE2 和抑制 PGF2α 释放方面的作用。大多数结果表明 NF-κB（p50/p50 和 p65/p65）和 FSH 参与控制基本的卵巢功能（增殖、凋亡和分泌活性），但这些调节剂之间没有功能相互关系。
