Dalkin A C, Burger L L, Aylor K W, Haisenleder D J, Workman L J, Cho S, Marshall J C
Division of Endocrinology, Department of Internal Medicine and the Center for Research in Reproduction, University of Virginia, Charlottesville, Virginia 22908, USA.
Endocrinology. 2001 Jan;142(1):139-46. doi: 10.1210/endo.142.1.7881.
GnRH regulates the synthesis and secretion of the pituitary gonadotropins LH and FSH. One of the actions of GnRH on the gonadotropin subunit genes (alpha, LHbeta, and FSHbeta) is the regulation of transcription [messenger RNA (mRNA) synthesis]. Gonadotropin subunit transcription rates increase after gonadectomy and following exogenous GnRH pulses. However, prior studies of subunit mRNA synthesis were limited by the available methodology that did not allow simultaneous measurement of gene transcription and mature mRNA concentrations. The purpose of the current studies was to: 1) develop a reliable and sensitive method for assessing transcription rates by measuring gonadotropin subunit primary transcript RNAs (PT, RNA before intron splicing); 2) investigate the PT responses to GnRH following castration or exogenous GnRH pulses; 3) characterize the half-disappearance time for the three PT species after GnRH withdrawal; and 4) correlate changes in PT concentration with steady-state gonadotropin subunit mRNA levels measured in the same pituitary RNA samples. Using oligonucleotide primers that flanked intron-exon boundaries, quantitative RT-PCR assays for each subunit PT species were developed. These assays require only ng amounts of RNA to measure each gonadotropin subunit PT and allow us to measure both PTs and steady-state mRNAs in a single pituitary RNA sample. Primary transcript concentrations in intact male rats showed a relative abundance of alpha > LHbeta congruent with FSHbeta, similar to the relationship found previously for mRNA levels. Additionally, each PT species was only 1-2% as abundant as the corresponding mRNA. One week after castration, gonadotropin subunit PT levels were increased (alpha: 3-fold, LHbeta: 6-fold, and FSHbeta: 3-fold) in a pattern similar to subunit mRNAs. Administration of GnRH antagonist to 7-day castrate male rats resulted in a rapid decline in PT concentrations with a half-disappearance time of 2.7 h for LHbeta and 0.8 h for FSHbeta, significantly faster than earlier measurements of the half-disappearance time for mature mRNA. Finally, in a GnRH-deficient male rat model, LHbeta and FSHbeta PT concentrations increased 4- to 6-fold 5 min after a GnRH pulse and then declined toward levels seen in control animals. These data indicate that the effects of GnRH on subunit gene transcription are an important determinant of gonadotropin regulation. The appearance and disappearance of PT RNA occurs more rapidly than changes in mature mRNA. Additionally, concentrations are elevated in long term castrates, and following an exogenous GnRH pulse the transcriptional burst is rapid and brief.
促性腺激素释放激素（GnRH）调节垂体促性腺激素黄体生成素（LH）和促卵泡激素（FSH）的合成与分泌。GnRH对促性腺激素亚基基因（α、LHβ和FSHβ）的作用之一是调节转录（信使核糖核酸（mRNA）合成）。性腺切除术后以及给予外源性GnRH脉冲后，促性腺激素亚基的转录速率会增加。然而，先前关于亚基mRNA合成的研究受到现有方法的限制，这些方法无法同时测量基因转录和成熟mRNA浓度。当前研究的目的是：1）通过测量促性腺激素亚基初级转录本RNA（PT，内含子剪接前的RNA）开发一种可靠且灵敏的评估转录速率的方法；2）研究阉割或给予外源性GnRH脉冲后PT对GnRH的反应；3）确定撤去GnRH后三种PT种类的半衰期；4）将PT浓度的变化与在相同垂体RNA样本中测得的促性腺激素亚基mRNA稳态水平相关联。使用位于内含子-外显子边界两侧的寡核苷酸引物，开发了针对每个亚基PT种类的定量逆转录聚合酶链反应（RT-PCR）检测方法。这些检测方法仅需纳克量的RNA来测量每个促性腺激素亚基PT，并使我们能够在单个垂体RNA样本中同时测量PT和稳态mRNA。完整雄性大鼠中的初级转录本浓度显示α> LHβ与FSHβ的相对丰度，类似于先前发现的mRNA水平之间的关系。此外，每种PT种类的丰度仅为相应mRNA的1-2%。阉割一周后，促性腺激素亚基PT水平升高（α：3倍，LHβ：6倍，FSHβ：3倍），其模式类似于亚基mRNA。给7天阉割的雄性大鼠注射GnRH拮抗剂导致PT浓度迅速下降，LHβ的半衰期为2.7小时，FSHβ的半衰期为0.8小时，明显快于先前对成熟mRNA半衰期的测量。最后，在GnRH缺乏的雄性大鼠模型中，GnRH脉冲后5分钟，LHβ和FSHβ的PT浓度增加4至6倍，然后降至对照动物中的水平。这些数据表明，GnRH对亚基基因转录的影响是促性腺激素调节的重要决定因素。PT RNA的出现和消失比成熟mRNA的变化更快。此外，长期阉割后浓度升高，并且在外源性GnRH脉冲后转录爆发迅速且短暂。
