INTRAGONADOTROPE PATHWAYS REGULATING GENE TRANSCRIPTION

描述（由申请人提供）：本提案的目的是确定原代垂体促性腺细胞中促性腺细胞内信号转导途径，该途径介导对促性腺激素释放激素（GnRH）脉冲频率的差异转录反应。哺乳动物的生殖受促黄体生成素（LH）和促卵泡激素（FSH）控制，它们由共同的α（a）亚基和独特的β（B）亚基组成，提供生物学特异性。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类固醇和肽的调节，其部分作用于垂体以改变基因表达和对GnRH的反应。此外，促性腺激素内系统激活素B和卵泡抑素（FS）调节FSHB表达。本实验拟用离体雌性大鼠垂体细胞研究GnRH受体数目和细胞内特异性信使途径（ERK、JNK、p38、PKC、Ca/CaMK Ⅱ和Ⅳ、PKA）在影响促性腺激素基因（a、LHb、FSHb、GnRH受体、activinbB、FS）对GnRH脉冲频率反应中的作用。最近的数据表明，性腺类固醇可以调节这些途径中的几个，我们也将评估类固醇在调节特定的第三信使反应GnRH的行动。这种科学方法将是有点独特的，因为正常大鼠垂体细胞将接受生理实验范例（即脉动GnRH），通过定量内源性基因产物（即促性腺激素初级转录物，PT;通过RT-PCR测量）评估对转录活性的影响。由于PT半衰期相对较短（LHb和FSHb PT < 20分钟），因此该方法允许评估近“实时”转录活性。 在女性中，FSH和LH的顺序刺激对于周期性排卵功能是必不可少的，并且在无排卵疾病中发生GnRH脉冲模式和性腺类固醇反馈异常。下丘脑性闭经和高催乳素血症时出现低频脉冲。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。最近的数据表明，睾酮水平升高破坏了正常的下丘脑-垂体促性腺激素功能调节，似乎在PCOS的发生中起作用。因此，了解促性腺激素释放激素和类固醇激素在促性腺激素细胞中的作用机制，对于发展恢复正常LH和FSH表达/分泌的有效方法和治疗人类不育症至关重要。

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.