Testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice

Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin (KISS1) do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. Using micro-dissected hypothalamic tissues to isolate RNA from our mice, we first set out to define the transcriptional differences among the wild type and knockout mice. Since the GPR54-kisspeptin axis is subject to hormonal feedback and the knockout mice are pre-pubertal, we also tested the hormonal dependence/independence of each differentially expressed transcript. To avoid variation in gene expression due to fluctuations in the levels of circulating hormones during the female estrous cycle, only male mice were used in this study. 17 samples were successfully hybridized; 4 GKO, 5 KKO, 3 WT 3 weeks old, 5 WT 6 weeks old. We compared GKO to KKO, GKO to WT, KKO to WT and all KO to all WT.携带GPR54（KISS1R）或kisspeptin（KISS1）功能丧失性突变的人类与小鼠均无法完成青春期发育，其病因是促性腺激素释放激素（Gonadotropin-Releasing Hormone, GnRH）释放障碍。 上述蛋白在下丘脑（hypothalamus）中调控的转录调控网络，目前尚未通过全基因组方法开展研究。 本研究通过显微解剖下丘脑组织以提取小鼠RNA，首先旨在明确野生型（wild type, WT）与基因敲除（knockout, KO）小鼠之间的转录水平差异。 鉴于GPR54-kisspeptin信号轴存在激素反馈调控，且基因敲除小鼠处于青春期前阶段，本研究同时检测了每一个差异表达转录本的激素依赖性与非依赖性。 为避免雌性小鼠动情周期（estrous cycle）中循环激素水平波动导致的基因表达差异，本研究仅选用雄性小鼠开展实验。 本次实验共计17份样本成功完成杂交：其中GPR54敲除（GKO）样本4份、kisspeptin敲除（KKO）样本5份、3周龄野生型（WT）样本3份、6周龄野生型（WT）样本5份。 本研究开展了GKO与KKO组、GKO与WT组、KKO与WT组以及全部敲除组与全部野生型组之间的差异比较。