O'Callaghan E, Sánchez J M, Rabaglino M B, McDonald M, Liu H, Spencer T E, Fair S, Kenny D A, Lonergan P
School of Agriculture and Food Science, University College Dublin, Dublin, Ireland.
Spanish National Research Council, National Institute for Agricultural and Food Research and Technology (CSIC-INIA), Madrid, Spain.
Front Cell Dev Biol. 2022 Aug 22;10:950443. doi: 10.3389/fcell.2022.950443. eCollection 2022.
The aim was to examine the effect of sire fertility status on conceptus-induced changes in the bovine endometrial transcriptome. To generate elongated conceptuses, Day 7 blastocysts produced using frozen-thawed sperm from Holstein Friesian bulls (3 High fertility, HF and 3 Low fertility, LF) were transferred in groups of 5-10 into synchronized heifers ( = 7 heifers per bull) and recovered following slaughter on Day 15. Day 15 endometrial explants recovered from the uterine horn ipsilateral to the corpus luteum were recovered from synchronized cyclic heifers ( = 4). Explants from each heifer were co-cultured for 6 h in RPMI medium alone (Control) or with 100 ng/ml ovine recombinant interferon tau (IFNT) or with a single conceptus from each HF or LF bull. After 6 h, explants were snap frozen and stored at -80°C. Extracted mRNA was subjected to RNA-seq and the resulting data were analyzed with R software. The numbers of differentially expressed genes (DEG; FDR<0.05) were: HF vs. Control: 956; LF vs. Control: 1021; IFNT vs. Control: 1301; HF vs. LF: 2. Unsurprisingly, the majority of DEG (658) were common to all comparisons and were related to IFNT-induced changes in the endometrium. Prior to applying the adjusted -value, there were 700 DEG between HF and LF, with 191 and 509 genes more expressed in HF or LF, respectively ( < 0.05). Overrepresentation analysis of KEGG pathways (FDR<0.05), revealed that DEG with higher expression in LF were involved in cell cycle and proteolysis, while those upregulated DEG by HF conceptuses were strongly associated with immune process pathways, such as TNF, NF-kappa B, cytokine-cytokine receptor interaction, and TLR signaling. These pathways were also enriched by DEG upregulated by IFNT compared to the Control. Furthermore, only the HF, and not the LF group, affected the expression of most genes in these pathways ( < 0.05) according to a negative binomial regression model. Finally, a weighted gene co-expression network analysis revealed two clusters of co-expressed genes associated with the HF conceptuses ( < 0.05), which were also enriched for the aforementioned pathways. In conclusion, HF conceptuses, similar to IFNT treatment, stimulated multiple pathways involved in immune response, which were apparently not affected by LF conceptuses.
本研究旨在探讨父本生育力状态对牛子宫内膜转录组中孕体诱导变化的影响。为了获得伸长的孕体，将利用荷斯坦弗里生公牛（3头高生育力，HF；3头低生育力，LF）的冻融精子产生的第7天囊胚，以5 - 10个为一组移植到同期发情的小母牛体内（每头公牛对应7头小母牛），并在第15天屠宰后回收。从与黄体同侧子宫角回收的第15天子宫内膜外植体，取自同期发情的周期性小母牛（n = 4）。将每头小母牛的外植体在RPMI培养基中单独培养6小时（对照），或与100 ng/ml的绵羊重组干扰素τ（IFNT）共同培养，或与来自每头HF或LF公牛的单个孕体共同培养。6小时后，将外植体速冻并储存在 - 80°C。提取的mRNA进行RNA测序，所得数据用R软件进行分析。差异表达基因（DEG；FDR<0.05）的数量分别为：HF与对照相比：956个；LF与对照相比：1021个；IFNT与对照相比：1301个；HF与LF相比：2个。不出所料，大多数差异表达基因（6个）在所有比较中都有出现，并且与IFNT诱导的子宫内膜变化有关。在应用校正后的P值之前HF和LF之间有700个差异表达基因，分别有191个和509个基因在HF或LF中表达上调（P<0.05）。KEGG通路的过表达分析（FDR<0.05）显示，在LF中高表达的差异表达基因参与细胞周期和蛋白水解，而HF孕体上调的差异表达基因则与免疫过程通路密切相关，如TNF、NF-κB、细胞因子-细胞因子受体相互作用和TLR信号传导。与对照相比，这些通路也被IFNT上调的差异表达基因所富集。此外，根据负二项回归模型，只有HF组而非LF组影响这些通路中大多数基因的表达（P<0.05）。最后，加权基因共表达网络分析揭示了与HF孕体相关的两个共表达基因簇（P<0.05），这些基因簇也富集了上述通路。总之，HF孕体与IFNT处理相似，刺激了多个参与免疫反应的通路，而LF孕体显然未对其产生影响。
