Transcriptomics of the Fetal Hypothalamic Response to Brachiocephalic Occusion and Estradiol Treatment

Estradiol (E2) is a well-known modulator of fetal neuroendocrine activity, and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic occlusion or sham occlusion. Half of the fetuses received subcutaneous pellets that released E2 (5 days@~250 μg/day, increasing plasma E2 from 62±6 to 102±12 pg/mL). Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Analysis of the data by ANOVA revealed that E2 differentially regulated (DR) 561 genes, and BCO DR 894 genes compared to control and estradiol+BCO DR 1153 genes compared to BCO alone (all p<0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis, but did not significantly involve genes known to directly respond to the estrogen receptor (ie., contain ERE sequences). BCO upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain, and downregulated neuropeptide synthesis. E2 upregulated immune- and apopotosis- related pathways after BCO, and reduced kinase and epigenetic pathway responses to the BCO. We conclude that array results are consistent with the results of more limited hypotheses previously published from these experiments. Chronically catheterized fetal sheep; 4 groups: control (no treatment); estradiol treatment (250 ug/day x 5 days); brachiocephalic occlusion of fetal sheep (BCO, 10 min, no other treatment); BCO plus estradiol (combination of estradiol treatment and BCO). All fetuses had blood drawn from chronically implanted catheters at 0, 5, 10, and 30 min after start of BCO or sham-BCO period. Fetal sheep 124-128 days gestation (term=147 days) at the time of study. Fetuses euthanized and fetal hypothalami collected for gene array studies 1 hour after first blood sample drawn (and start of BCO or sham-BCO period).

雌二醇（Estradiol, E2）是公认的胎儿神经内分泌活动调节因子，此前被提出是帮助胎儿为分娩及产后生命做好准备的关键内分泌信号。为探究E2对胎儿应激反应以及胎儿脑对窒息性应激的调控作用，本研究对长期留置导管的胎羊实施暂时性（10分钟）头臂动脉闭塞（brachiocephalic occlusion, BCO）或假闭塞处理。其中半数胎羊接受皮下植入缓释颗粒，持续5天释放E2（剂量约250 μg/天，可将血浆E2水平从62±6 pg/mL提升至102±12 pg/mL）。

本研究采用安捷伦8x15k绵羊基因芯片（Agilent 8x15k ovine array，货号019921）分析下丘脑mRNA表达，实验流程及注释方法参照本实验室此前发表的方案。通过方差分析（ANOVA）对数据进行分析后结果显示：与对照组相比，E2组差异调控（differentially regulated, DR）561个基因，BCO组差异调控894个基因；而E2+BCO联合组相较于单纯BCO组，差异调控1153个基因（所有结果均满足p<0.05）。

进一步分析发现，E2可上调表观遗传通路、下调局部类固醇合成通路，但未显著影响已知可直接应答雌激素受体的基因（即含有雌激素应答元件（estrogen response element, ERE）序列的基因）。BCO可上调激酶通路以及与淋巴细胞浸润脑组织相关的基因，并下调神经肽合成。在BCO处理后，E2可上调免疫与凋亡相关通路，并减弱机体对BCO的激酶及表观遗传通路应答。

本研究结论为，本次芯片分析结果与本团队此前基于有限假设开展的同类实验发表结果一致。

本研究的实验对象与分组如下：长期留置导管的胎羊，共分为4组：1. 对照组：无任何处理；2. 雌二醇处理组：250 μg/天，持续5天；3. 头臂动脉闭塞组（brachiocephalic occlusion, BCO）：实施10分钟闭塞处理，无其他额外干预；4. BCO联合雌二醇处理组：联合雌二醇处理与BCO闭塞操作。

所有胎羊在BCO或假BCO处理开始后的0、5、10、30分钟，通过长期留置的导管采集血液样本。本研究开展时，受试胎羊的妊娠天数为124~128天（足月妊娠为147天）。在首次采集血液样本（即BCO或假BCO处理开始）1小时后，对胎羊实施安乐死并采集下丘脑组织，用于后续基因芯片分析。