怀孕的建立涉及母体免疫系统内保护和耐受性之间的微调平衡,因为女性需要接受外来抗原(半同种异体胎儿),同时仍然能够对抗来自子宫的病原体。在马,第一次子宫接触父系抗原是在交配期间,精子被引入子宫组织并排出子宫淋巴管。此外,有人认为,精浆及其中的蛋白质在为雌性肠道准备合适的免疫环境中起着至关重要的作用,但这在马中尚未得到证实。因此,本研究的目的是评估精浆授精或精浆减少授精后的子宫内膜转录组。我们假设精浆减少会改变子宫内膜转录组并影响与免疫耐受有关的转录本,抗原呈递和胚胎生长发育。要做到这一点,在四个发情周期的过程中,采用随机切换设计对六只(n=6)母马进行了授精。经直肠触诊母羊,通过超声检查检查发现排卵前卵泡(>35毫米),同时增加子宫水肿和宫颈松弛,然后用四个治疗组之一治疗,包括(1)30mL乳酸林格溶液(LRS;NegCon),(2)500×106精子与30mL精浆(SP),(3)30mL乳酸林格溶液(LRS;洗出)和(4)500×106个精子,精浆通过梯度离心减少,并重悬于30mLLRS(SP-)中。施用人绒毛膜促性腺激素(hCG)以标准化排卵时间,并在授精后7天收集子宫内膜活检。利用Trizol分离RNA,RNA-Seq由Novogene进行,总作图97.79%,读取深度4000万。p值设定为<0.05。比较SP+和SP-时,鉴定了158个差异表达基因(DEGs)。受影响的生物过程包括抗原加工和调节,胆固醇合成,和免疫/炎症反应。使用DAVIDv6.8进行的基因本体论(GO)富集分析显示,这些DEGs中的许多参与了诸如抗原呈递(HLA-DMβ链,HLA-DRB,HLA-DQA和RASGRP1),免疫细胞信号(CXCL9,CXCL1,DEFB1和MIP-2B),胚胎生长和发育(INHA,KLF2,RDH10,LAMA3和SLC34A2)和胚胎代谢(ABCA1,ABCA2,APOA1,LDL,INSR,IGFBP2和IGFBP3)。总的来说,在胚胎早期暴露于子宫环境时,从授精剂量减少的精浆会影响子宫内膜转录组。进一步的工作是合理的,以评估这些改变对胚胎成熟的影响,胎盘发育,妊娠结局和子代发育。
The establishment of pregnancy involves a fine-tuned balance between protection and tolerance within the maternal immune system, as the female needs to accept a foreign antigen (the semi-allogenic fetus) while still being able to combat pathogens from the uterus. In the horse, the first uterine exposure to paternal antigens is during mating when sperm is introduced to the tissue and draining lymphatics of the uterus. Additionally, it has been suggested that seminal plasma and its proteins within it play an essential role in preparing the female tract for a suitable immunologic environment but this has not been confirmed in the horse. Therefore, the objective of this study was to evaluate the endometrial transcriptome following insemination either with seminal plasma or with reduced seminal plasma. We hypothesised that reduced seminal plasma would alter the endometrial transcriptome and affect transcripts relating to immunotolerance, antigen presentation and embryo growth and development. To do so, six (n = 6) mares were inseminated in a randomised switch-back design over the course of four oestrous cycles. Mares were rectally palpated and scanned via ultrasonography for the detection of a pre-ovulatory follicle (>35 mm) alongside increasing uterine oedema and relaxed cervix, and then treated with one of four treatment groups including (1) 30 mL lactated Ringers solution (LRS; NegCon), (2) 500 × 106 spermatozoa in conjunction with 30 mL seminal plasma (SP+), (3) 30 mL lactated Ringers solution (LRS; wash out) and (4) 500 × 106 spermatozoa with seminal plasma reduced via gradient centrifugation and resuspended in 30 mL LRS (SP-). Human chorionic gonadotropin (hCG) was administered to standardise the time to ovulation and endometrial biopsies were collected 7 days after insemination. RNA was isolated utilising Trizol, and RNA-Seq was performed by Novogene, with 97.79% total mapping and 40 million read depth. p value was set to <0.05. When comparing SP+ to SP-, 158 differentially expressed genes (DEGs) were identified. Biological processes impacted included antigen processing and regulation, cholesterol synthesis, and immune/inflammatory response. Gene ontology (GO) enrichment analysis using DAVID v6.8 revealed that many of these DEGs were involved in biological process such as antigen presentation (HLA-DM beta chain, HLA-DRB, HLA-DQA and RASGRP1), immune cell signalling (CXCL9, CXCL1, DEFB1 and MIP-2B), embryo growth and development (INHA, KLF2, RDH10, LAMA3 and SLC34A2) and embryo metabolism (ABCA1, ABCA2, APOA1, LDL, INSR, IGFBP2 and IGFBP3). Overall, reduction of seminal plasma from the insemination dose impacted the endometrial transcriptome at the time of early embryonic exposure to the uterine environment. Further work is justified to evaluate these alterations impact on embryo maturation, placental development, pregnancy outcome and development of offspring.