Peri-pubertal gonadotropin-releasing hormone agonist treatment affects sex-biased gene expression of amygdala in sheep

Background: The exact nature of the interrelationship between pubertal brain development and hormones involvement has always attracted wide interest. Brain structural changes that occur during the pubertal developmental process mainly appear in the regions most closely linked with emotion, motivation and cognitive functions. Using a sheep model, we have previously shown that peri-pubertal pharmacological blockade of gonadotropin-releasing hormone (GnRH) receptors results in increased sex-differences in cognitive executive function and emotional control, as well as in a sex- and side-specific gene expression pattern of hippocampal genes associated with synaptic plasticity and endocrine signaling. In this study, we explore the effects of this treatment regime on the amygdalar gene expression profile. Methods: The study was conducted with 30 same-sex twin lambs (14 female and 16 male), half of which were treated with the GnRH agonist (GnRHa) goserelin acetate every 4th week, beginning before puberty, until 50 weeks of age. The gene expression profiles of left and right amygdala samples from all 30 animals were measured by using ovine 8 X 15 K Agilent microarrays. Furthermore, differential expression of a selected number of genes was confirmed by qRT-PCR (Quantitative real time PCR). Networking analyses and Gene Ontology (GO) Term analyses were performed with the Ingenuity Pathway Analysis (IPA), version 7.5 DAVID (Database for Annotation, Visualization and integrated Discovery) software packages respectively. Results: GnRHa treatment was associated with significant sex- and hemisphere-specific differential expression of genes. Interestingly, genome-wide transcription showed a set of 438 (p value <0.05) genes in female treated left and 46 (p value <0.0.5) genes in females treated right amygdala were differentially expressed but not in any treatment group of male animals. Conclusion: Our results indicate that GnRH directly and/or indirectly is involved in the regulation of sex- and side-specific differential expression of genes in amygdala. Hence, this finding should be considered when long-term peri-pubertal GnRHa treatment is used in children. GnRHa-treated and untreated animals. This submission includes a total 56 samples for 8 groups (male control left and right amygdala, female control left and right amygdala, male treated left and right amygdala, and female treated left and right amygdala), with 7 biological replicates each group (n=7). Four male samples had poor RNA quality and are not included here. Dye-swaps were performed.

背景：青春期大脑发育与激素参与之间的相互作用确切本质，长期以来广受关注。青春期发育过程中发生的大脑结构变化，主要集中于与情绪、动机及认知功能紧密关联的脑区。本团队此前以绵羊为模型开展研究，发现青春期前后对促性腺激素释放激素（gonadotropin-releasing hormone, GnRH）受体进行药物阻断，可增强认知执行功能与情绪控制的性别差异，同时使与突触可塑性及内分泌信号通路相关的海马基因呈现性别与侧别特异性的基因表达模式。本研究旨在探究该给药方案对杏仁核基因表达谱的影响。
方法：本研究纳入30只同性双胞胎羔羊（雌性14只，雄性16只），其中一半于青春期前开始接受促性腺激素释放激素激动剂（gonadotropin-releasing hormone agonist, GnRHa）醋酸戈舍瑞林治疗，每4周给药1次，直至羔羊50周龄。采用绵羊8×15K安捷伦（Agilent）基因芯片，检测全部30只羔羊的左侧及右侧杏仁核样本的基因表达谱。此外，通过定量实时聚合酶链反应（quantitative real time PCR, qRT-PCR）验证了部分候选基因的差异表达情况。分别使用Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）7.5版本软件与DAVID（Database for Annotation, Visualization and Integrated Discovery，注释、可视化与集成发现数据库）软件完成了基因互作网络分析及基因本体（Gene Ontology, GO）术语富集分析。
结果：研究结果显示，GnRHa治疗与基因表达的显著性别及大脑半球特异性差异相关。值得注意的是，全基因组转录分析显示，接受治疗的雌性羔羊左侧杏仁核中有438个基因（P值<0.05）、右侧杏仁核中有46个基因（P值<0.0.5）呈现差异表达，而雄性动物的各治疗组均未出现此类差异。
结论：本研究结果表明，GnRH可直接或间接参与调控杏仁核基因表达的性别与侧别特异性差异。因此，在儿童中长期采用青春期前后GnRHa治疗时，需考虑本研究的这一发现。本次提交共包含8组56份样本，分别为雄性对照组左右杏仁核、雌性对照组左右杏仁核、雄性治疗组左右杏仁核及雌性治疗组左右杏仁核，每组含7个生物学重复（n=7）。另有4份雄性样本因RNA质量不佳未纳入本次数据集。实验采用了染料互换（dye-swaps）设计。