Multi-omics analyses and machine learning prediction of oviductal responses in the presence of gametes and embryos

The oviduct is the site of fertilization and preimplantation embryo development in mammals. Evidence suggests that gametes alter oviductal gene expression. To delineate the adaptive interactions between the oviduct and gamete/embryo, we performed a multi-omics characterization of oviductal tissues utilizing bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and proteomics collected from distal and proximal at various stages after mating in mice. We observed robust region-specific transcriptional signatures. Specifically, the presence of sperm induces genes involved in pro-inflammatory responses in the proximal region at 0.5 days post-coitus (dpc). Genes involved in inflammatory responses were produced specifically by secretory epithelial cells in the oviduct. At 1.5 and 2.5 dpc, genes involved in pyruvate and glycolysis were enriched in the proximal region, potentially providing metabolic support for developing embryos. Abundant proteins in the oviductal fluid were differentially observed between naturally fertilized and superovulated samples. RNA-seq data were used to identify transcription factors predicted to influence protein abundance in the proteomic data via a novel machine learning model based on transformers of integrating transcriptomics and proteomics data. The transformers identified influential transcription factors and correlated predictive protein expressions in alignment with the in vivo-derived data. In conclusion, our multi-omics characterization and subsequent in vivo confirmation of proteins/RNAs indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner. Adult C57B6/J wild-type female mice were mated with either vasectomized or proven breeder males. In some cases, females were superovualted with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrpin (hCG) before mating. The oviduct tissues were collected at 0.5, 1.5, 2.5, and 3.5 days post coitus (dpc) or in some case days of pseudopregnancy (dpp). The oviducts were dissected into infundibulum+ampulla (IA) or isthmus+uterotubal junction (IU) regions. Samples were then processed for bulk RNA-sequencing or single cell RNA-sequencing.输卵管是哺乳动物体内受精及着床前胚胎发育的核心场所。已有研究表明，配子可调控输卵管的基因表达模式。为阐明输卵管与配子/胚胎之间的适应性互作机制，本研究针对交配后不同时间点的小鼠输卵管组织（分为远端与近端区域），整合批量RNA测序（bulk RNA-sequencing, RNA-seq）、单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）及蛋白质组学（proteomics）技术开展多组学表征分析。研究观察到显著的区域特异性转录特征：具体而言，在交配后0.5天（days post-coitus, dpc）的近端输卵管区域中，精子的存在可诱导促炎症反应相关基因的表达，且该类炎症反应基因由输卵管内的分泌型上皮细胞特异性表达。在1.5和2.5 dpc时，近端输卵管区域富集了丙酮酸代谢与糖酵解相关基因，可为发育中的胚胎提供代谢支持。输卵管液中的高丰度蛋白在自然受精与超数排卵样本中呈现显著差异表达。本研究基于整合转录组与蛋白质组数据的新型Transformer机器学习模型，利用RNA-seq数据筛选可调控蛋白质组数据中蛋白丰度的转录因子；该Transformer模型成功识别出关键转录因子，其预测的蛋白表达水平与体内实验获得的真实数据高度吻合。综上，本研究通过多组学表征及后续的蛋白/RNA体内验证实验证实，输卵管可通过时空特异性方式响应精子与胚胎的存在，展现出适应性调控能力。实验选用成年C57B6/J野生型雌性小鼠，与输精管结扎雄性小鼠或已证实可育的雄性小鼠进行交配。部分实验中，雌性小鼠在交配前通过孕马血清促性腺激素（pregnant mare's serum gonadotropin, PMSG）与人绒毛膜促性腺激素（human chorionic gonadotropin, hCG）进行超数排卵处理。分别在交配后0.5、1.5、2.5、3.5 dpc，或部分实验的假孕天数（days of pseudopregnancy, dpp）收集输卵管组织。将输卵管解剖为漏斗部+壶腹部（infundibulum+ampulla, IA）与峡部+子宫输卵管连接部（isthmus+uterotubal junction, IU）两个区域，随后将样本分别用于批量RNA测序或单细胞RNA测序分析。