Enhanced at Puberty-1 (Eap1) Expression Critically Regulates the Onset of Puberty Independent of Hypothalamic Kiss1 Expression
Enhanced at Puberty-1 (Eap1) Expression Critically Regulates the Onset of Puberty Independent of Hypothalamic Kiss1 Expression
复制标题
青春期增强 1 (Eap1) 表达独立于下丘脑 Kiss1 表达来关键调节青春期的开始
DOI：
10.1159/000481872
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发表时间：
2017-01-01
影响因子：
--
通讯作者：
Li, Pin
中科院分区：
文献类型：
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作者：
Li, Chenxi;Li, Pin
背景/目的：Enhance at青春期-1(Eap1)是一个非内含子基因，通过下丘脑基因网络调节青春期的开始。然而，对于EAP1调控青春期所必需的精确的机制事件还没有完全阐明。EAP1被认为通过调节下丘脑转移抑制因子Kiss1来促进青春期的启动。我们的目标是通过在体内通过RNA干扰从基因上干扰Eap1来研究这一假说。方法：我们首先设计并优化了四组针对大鼠Eap1mRNA的shRNA，以及一组阴性对照shRNA。在产生慢病毒(LV)颗粒后，我们检测了Eap1在NRK-54E细胞系中的抑制情况，以选择最有效的Eap1。连续给予21日龄大鼠脑室注射LV-Eap1-shRNA或对照组，包括LV-EGFP和生理盐水。在适宜的条件下饲养大鼠，检测幼年(出生后25天)、青春期早期(PND35天)、成年(PND42天)三个发育阶段的大鼠阴道开放时间、卵巢生理以及下丘脑青春期调控基因。结果：下丘脑抑制Eap1可延缓大鼠阴道开放的发生。苏木精-伊红(H&E)染色显示PND35时黄体(CL)明显减少，而PND42时CL水平与对照组相比正常。伴随着表型和卵巢形态的差异，GnRH在PND25和PND35的表达和转录本也降低，而它们的水平在PND42与对照相似。Kiss1mRNA和蛋白水平在三个发育阶段均无显著差异。结论：Eap1的表达与GnRH的表达一样，对青春期的发育具有重要的调控作用。然而，Eap1对青春期的调节可能不需要Kiss1/Gpr54信号。
Background/Aims: Enhance at puberty-1 (Eap1) is an intronless gene that regulates the onset of puberty through a network of hypothalamic genes. However, precise mechanistic events essential for Eap1-regulation of puberty have not been fully elucidated. Eap1 is thought to promote the initiation of puberty through regulation of the hypothalamic metastasis-suppressor KiSS1. We aim to investigate this hypothesis by genetically perturbing Eap1 through RNA interference in vivo. Methods: We first engineered and optimized four sets of shRNAs that target rat Eap1 mRNA as well as one negative control shRNA. After generating lentiviral (LV) particles, we examined the suppression of Eap1 in NRK-54E cell line to select the most efficient one. Sequencelly, LV-Eap1-shRNA or controls including LV-eGFP and saline were intraventricular microinjected into 21-day-old rats. Rats were raised in appropriate conditions and we examined the time of vaginal opening, ovary physiology as well as hypothalamic puberty-regulatory genes at three developmental stages: juvenile (postnatal day PND25), early puberty (PND35), adult (PND42). Results: Hypothalamic suppression of Eap1 delayed the onset of rat vaginal opening. Hematoxylin and eosin (H&E) staining revealed a significant reduction of corpus luteum (CL) at PND35, but at PND42 CL levels were normal relative to control. In conjunction with differences in phenotype and ovary morphology, GnRH expression and transcripts were also reduced at PND25 and PND35, while their levels were similar to control at PND42. KiSS1 mRNA and protein levels were not significantly different at all three developmental stages. Conclusion: Eap1 expression critically regulates puberty as well as GnRH expression. However, Eap1-regulation of puberty may not necessitate KiSS1/GPR54 signaling.