INTRAGONADOTROPE PATHWAYS REGULATING GENE TRANSCRIPTION

描述(由申请人提供)：本提案的目标是确定原代垂体促性腺激素细胞内的促性腺激素信号转导通路，该通路介导对促性腺激素释放激素(GnRH)脉冲频率的差异转录反应。哺乳动物的生殖受黄体生成素(LH)和卵泡刺激素(FSH)的控制，它们由共同的α(A)亚基和独特的β(B)亚基组成，提供生物特异性。促性腺激素亚基和其他促性腺激素基因受GnRH脉冲模式，特别是脉冲频率的调节。促性腺激素基因也受性腺激素和多肽的调控，这些激素和多肽部分作用于脑垂体，改变基因的表达和对GnRH的反应。此外，体内的促性腺激素系统，激动素B和卵泡抑素(FS)，调节FSHB的表达。我们建议用分离的雌性大鼠垂体细胞来研究GnRH受体数目和特定的细胞内(第3)信使通路(ERK、JNK、p38、PKC、Ca/CaMK II和IV、PKA)对GnRH脉冲频率的不同反应。由于最近的数据显示，性腺类固醇可以调节其中的几个途径，我们还将评估类固醇在调节GnRH的特定第三信使反应中的作用。这一科学方法在某种程度上是独一无二的，因为正常大鼠垂体细胞将接受生理学实验范式(即搏动性促性腺激素释放激素)，并通过定量内源性基因产物(即促性腺激素初级转录本，PTS；通过RT-PCR测量)来评估对转录活性的影响。由于PT半衰期相对较短(LHb和FSHB pt的半衰期为20分钟)，该方法可以评估近实时的转录活性。 在女性中，FSH和LH的顺序刺激对周期排卵功能是必不可少的，无排卵障碍患者会出现GnRH脉搏模式和性腺类固醇反馈的异常。在下丘脑闭经和高催乳素血症中出现慢频率脉冲。相反，持续快速的GnRH脉冲频率是多囊卵巢综合征(PCOS)的一个特征。最近的数据显示，睾酮升高扰乱了正常的下丘脑-垂体对促性腺激素功能的调节，似乎在多囊卵巢综合征的发生中发挥了作用。因此，了解促性腺激素释放激素和类固醇在促性腺激素中的作用机制，对于发展恢复正常的促黄体生成素和促性腺激素分泌和治疗人类不孕症的有效方法是至关重要的。

DESCRIPTION (provided by applicant): The goal of this proposal is to define the intra-gonadotrope signal transduction pathways in primary pituitary gonadtrope cells that mediate differential transcriptional responses to gonadotropin releasing hormone (GnRH) pulse frequency. Mammalian reproduction is controlled by luteinizing hormone (LH) and follicle stimulating hormone (FSH), which consist of a common alpha (a) subunit and unique beta (b) subunits that provide biological specificity. Gonadotropin subunit and other gonadotrope genes are regulated by GnRH pulse pattern, particularly pulse frequency. Gonadotrope genes are also regulated by gonadal steroids and peptides, which act in part on the pituitary to modify gene expression and responses to GnRH. Additionally, an intra-gonadotrope system, activinbB and follistatin (FS), regulates FSHB expression. We propose to use dissociated female rat pituitary cells to delineate the roles of GnRH receptor number and specific intracellular (3rd) messenger pathways (ERK, JNK, p38, PKC, Ca/CaMK II and IV, PKA) in effecting differential gonadotrope gene (a, LHb, FSHb, GnRH receptor, activinbB, FS) responses to GnRH pulse frequency. As recent data show that gonadal steroids can modulate several of these pathways, we will also assess the actions of steroids in modulating specific 3rd messenger responses to GnRH. This scientific approach will be somewhat unique, in that normal rat pituitary cells will receive physiological experimental paradigms (i.e. pulsatile GnRH) with effects on transcriptional activity assessed by quantitation of endogenous gene products (i.e. gonadotrope primary transcripts, PTs; measured by RT-PCR). Since PT half-life is relatively short (< 20 min for LHb and FSHb PTs), this method allows assessment of near 'real-time' transcriptional activity. In women, the sequential stimulation of FSH and LH is essential for cyclic ovulatory function and abnormalities in GnRH pulse pattern and gonadal steroid feedback occur in anovulatory disorders. Slow frequency pulses are present in hypothalamic amenorrhea and hyperprolactinemia. In contrast, persistently rapid GnRH pulse frequency is a feature of polycystic ovarian syndrome (PCOS). Recent data show that elevated testosterone disrupts normal hypothalamic-pituitary regulation of gonadotrope function and appears to play a role in the genesis of PCOS. Thus, understanding the mechanisms of GnRH and steroid actions at the gonadotrope are essential to evolving effective methods to restore normal LH and FSH expression/secretion and treating human infertility.