Attenuated retinoic acid signaling is among the early responses in mouse uterus approaching embryo attachment

The uterus is transiently receptive for embryo implantation. It remains to be understood why the uterus does not reject semi-allogeneic or allogeneic embryos for implantation. Uterine early response genes at the time approaching embryo attachment may provide insights. Uteri from C57BL/6 pseudo-pregnant (PP) mice and pregnant (P) mice approaching embryo attachment on day 3 post-coitum (D3) @22 h were analyzed for early response genes by microarray. SAM algorithm retained 21,858 unique probesets. Principal component analysis (PCA) indicated a clear separation between the PP and P groups. There were 105 upregulated and 5 downregulated protein-coding genes in the pregnant uterus (fc>1.5 & q-value<5%). Gene ontology (GO) analysis of these upregulated genes revealed that 84% of the GO terms were related to immune responses, with a dominant natural killer (NK) cell activation signature. Among the top 8 upregulated protein-coding genes, Cyp26a1 inactivates retinoic acid (RA) while Lrat promotes vitamin A storage, both of which attenuate RA bioavailability; Atp6v0d2 and Gjb2 play roles in ion transport and transmembrane transport; Gzmb, Gzmc, and Il2rb are involved in immune responses; and Tdo2 is important for kynurenine pathway. Most of these genes or their related pathways have been implicated in immune regulations. RA signaling has been implicated in tolerance and immunity and uterine NK cells have been implicated in immunotolerance at the maternal-fetal interface in the placenta. The mechanisms of immune responses approaching embryo attachment remain to be elucidated. The coordinated effects of the early response genes may hold the keys to the question of why the uterus does not reject an implanting embryo. Embryo attachment can be detected by faint blue dye reaction in mice and it normally occurs ~midnight 4 days post-coitum (D4 @0h or D3 @24h). The earliest time for faint blue dye reaction to be detectable in some C57BL/6 mice is D3 @22h (DOI: 10.1016/j.fertnstert.2011.01.160). Young virgin C57BL/6 mice were purchased from The Jackson Laboratory. They were randomly splitted into two sets. One set of mice were mated with stud males. Mating night was defined as D0 and mating was verified by a vaginal plug the next morning. The pregnant uterine tissues were collected on D3 @22h. Only the ones without blue dye reaction (to verify no embryo attachment yet) but with healthy-looking blastocysts (to verify pregnancy status) flushed from the uterine horns were included. The other set of mice were mated with vasectomized males to produce pseudopregnant mice. The pseudopregnant uterine tissues were also collected on D3 @22h. The uterine tissues were processed for total RNA isolation. Four samples each from the pregnant and pseudopregnant groups with the best RNA qualities (260/280 ratio: 1.84~1.99; RIN: 6.9~8.7) were selected from microarry.

子宫对胚胎植入具有一过性接受态。目前学界仍未阐明子宫为何不会排斥半同种异体或同种异体的植入胚胎。接近胚胎附着阶段的子宫早期应答基因或可提供相关研究思路。本研究通过微阵列技术，对交配后第3天22小时（D3 @22h）、接近胚胎附着阶段的C57BL/6假孕（PP）小鼠与妊娠（P）小鼠的子宫组织开展早期应答基因分析。经SAM（Significance Analysis of Microarrays）算法筛选，共保留21858个独特探针集。主成分分析（Principal Component Analysis, PCA）结果显示，假孕组与妊娠组的子宫组织呈现清晰分离。妊娠子宫中共有105个上调、5个下调的蛋白编码基因（折叠变化>1.5且校正后q值<5%）。对上述上调基因进行基因本体（Gene Ontology, GO）富集分析后发现，84%的GO术语与免疫应答相关，且呈现自然杀伤（Natural Killer, NK）细胞活化的核心特征。在上调排名前8的蛋白编码基因中：Cyp26a1可降解视黄酸（Retinoic Acid, RA），Lrat可促进维生素A储存，二者均会降低RA的生物利用度；Atp6v0d2与Gjb2参与离子转运与跨膜转运过程；Gzmb、Gzmc及Il2rb参与免疫应答；Tdo2则在犬尿氨酸通路中发挥重要作用。上述多数基因及其相关通路均已被证实与免疫调控相关。视黄酸信号通路已被证实与免疫耐受及免疫应答相关，而子宫NK细胞则在胎盘母胎界面的免疫耐受中发挥作用。接近胚胎附着阶段的免疫应答机制仍有待阐明。早期应答基因的协同调控效应或可揭开子宫不排斥植入胚胎这一科学问题的谜底。小鼠胚胎附着可通过微弱蓝色染料反应检测，通常发生在交配后第4天午夜左右（D4 @0h或D3 @24h）。部分C57BL/6小鼠的微弱蓝色染料反应最早可在D3 @22h被检测到（DOI: 10.1016/j.fertnstert.2011.01.160）。年轻未产C57BL/6小鼠购自杰克逊实验室（The Jackson Laboratory），随机分为两组。一组与种公鼠合笼，合笼当晚记为D0，次日清晨通过阴道栓验证交配成功。于D3 @22h收集妊娠子宫组织，仅纳入未出现蓝色染料反应（确认尚未发生胚胎附着）且从子宫角冲洗出形态健康的囊胚（确认妊娠状态）的样本。另一组与输精管结扎的公鼠合笼以构建假孕小鼠，同样于D3 @22h收集假孕子宫组织。收集子宫组织后进行总RNA提取。从微阵列实验的样本中，筛选出妊娠组与假孕组各4份RNA质量最优的样本（260/280比值：1.84~1.99；RNA完整性指数（RNA Integrity Number, RIN）：6.9~8.7）用于后续分析。