Elsevier

Theriogenology

Volume 82, Issue 6, 1 October 2014, Pages 897-909
Theriogenology

Research article
Global gene expression profiling of porcine endometria on Days 12 and 16 of the estrous cycle and pregnancy

https://doi.org/10.1016/j.theriogenology.2014.07.009Get rights and content

Abstract

The objective of the study was to investigate transcriptomic profile of pig endometrium on Days 12 and 16 of pregnancy in comparison with the respective days of the estrous cycle. Labeled complementary DNA was hybridized to Porcine Long Oligo microarray containing 13,297 oligonucleotide probes, which represented complementary DNA and expressed sequence tags. Statistical analysis revealed 110 differentially expressed genes (DEGs) on Day 12 of pregnancy and 179 DEGs on Day 16 of pregnancy. In silico analysis of gene function and functionality networks revealed links between genes implicated in cell death and survival, protein synthesis, lipid metabolism, cellular movement, tissue development, and cell-to-cell signaling. On Day 12 of pregnancy, estrogen, transforming growth factor (TGF) β1, and fibroblast growth factor (FGF) 2, and on Day 16 of pregnancy, epidermal growth factor (EGF), insulin, interleukin 11 (IL-11), and FGF family members were indicated as possible upstream regulators of several DEGs. Obtained results showed changes in global endometrial gene expression at the time of maternal recognition of pregnancy and embryo implantation. Additionally, these data revealed signaling molecules, which together with E2, may evoke molecular changes in the uterus, leading to successful pregnancy establishment.

Introduction

Embryo-maternal cross talk is based on mutual interaction between developing embryo and uterus. Reduced expression of mucin 1 [1] and progesterone (P4) receptors in the uterine epithelium [2] and increased expression of P4 receptor in the stroma after Day 12 of the estrous cycle are the main signs of attaining the state of receptivity of the endometrium. An increased number of estrogen receptors in the luminal and glandular epithelium [3] correlated with the rising concentration of estrogens released by the embryo [3] are the earliest symptoms of properly synchronized uterine environment and embryo development. Besides the biphasic estradiol (E2) secretion (Days 10–12 and Days 15–25) [4], elongating porcine embryos produce prostaglandin (PG) E2 [5], interleukin (IL) 1β [6] and transforming growth factor (TGF) β1 [7], [8]. Estrogens from the conceptuses together with the P4 from CLs regulate endometrial function and conceptus development [9]. Moreover, pig trophectoderm produces type I and type II interferons (IFNs). Together with estrogens, IFNs regulate endometrial gene expression (for review see Ref. [10]). The most important genes that change expression in the endometrium at the time of estrogen release are implicated in PG synthesis (prostaglandin-endoperoxide synthase [PTGS] 2, prostaglandin E synthase [PGES] 1), coding of transcription factors (Signal Transducer and Activator of Transcription [STAT]-1, homeobox genes [HoxA10]), growth factors (fibroblast growth factor [FGF] 7, insulin growth factor [IGF] I, TGF-β, vascular endothelial growth factor [VEGF]), or receptors (lysophosphatidic acid [LPA] receptor3, luteinizing hormone receptor [LHR], prolactin receptor [PRLR], oxytocin receptor [OXTR]). The effect of semen deposition can also have an impact on events in the female reproductive tract, leading to promotion of the conception and progression of pregnancy [11]. Interestingly, the latest studies indicate that the presence of embryo (Day 6 of pregnancy) can regulate the expression of genes implicated in immunologic tolerance even before estrogen signal occurs [12].

The number of porcine transcriptomic data arises every day. Microarrays [13], [14] and deep sequencing [15], [16] have been already used to describe endometrial gene expression on Days 12, 14, and 15 to 16 of the estrous cycle and pregnancy in the pig. Digital gene expression profiling was conducted to describe differences in gene expression of endometrial samples collected from Erhualian sows and Landrace × Large White pigs on Day 12 of pregnancy [17]. The microarrays were also performed to show blastocyst influence on the endometrial transcriptome on Day 8 [12]. To our knowledge, there is no available microarray data showing molecular changes in the Polish Landrace × Duroc pig endometrium. Moreover, our analyses were conducted to designate potential signals other than P4, E2, and IFN of trophectoderm origin, which are additionally involved in embryo-maternal communication during early pregnancy in the pig. Furthermore, the advantage of our work is that we performed the experiments on cyclic animals with natural estrus, which have not been treated with centrifuged [15] or freeze-inactivated boar semen [13]. Thus, in this study the transcriptome of endometrial tissues collected from crossbreed Polish Landrace × Duroc pigs on Days 12 and 16 of pregnancy in pairwise comparison with Days 12 and 16 of the estrous cycle was performed. Moreover, using an in silico approach, we aimed to determine the molecules that together with embryo-derived E2 and P4 from CLs may evoke molecular changes in the endometrium leading to successful embryo implantation.

Section snippets

Animals

All experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals and were approved by the Animal Research Ethics Committee, University of Warmia and Mazury in Olsztyn, Poland (approval no. 18/2009).

Animals were housed at the uniform farm conditions with feed and water ad libitum. Twenty crossbred gilts (Polish Landrace × Duroc) of similar age and genetic background from one commercial herd, which had at least one estrus, were

Microarrays' data

Pairwise comparison of samples recovered from pigs on Days 12 and 16 of the estrous cycle and Days 12 and 16 of pregnancy was performed to identify changes in the endometrial transcriptome during crucial events of early pregnancy in the pig. After background correction, statistical analyses revealed 110 DEGs (35 downregulated and 75 upregulated) on Day 12 of pregnancy (Table S1) and 179 DEGs (70 downregulated and 109 upregulated) on Day 16 of pregnancy (Table S2) in comparison with the

Discussion

In the present study, we found and confirmed the differential gene expression in the endometrium of Polish Landrace × Duroc crossbred gilts during the maternal recognition of pregnancy (Day 12) and embryo implantation (Day 16) in comparison with the estrous cycle. Moreover, we conducted in silico analysis, which determined upstream, potential regulators of genes showing differential expression. On Day 12 of the pregnancy versus Day 12 of estrous cycle, the most variables among 110 genes were

Acknowledgments

This study was supported by grant funding joint research of scientific co-operation networks OE1 from Ministry of Science and Higher Education in Poland (2006–2008). J. Kiewisz was supported by the President of Polish Academy of Sciences fellowship (No. 4/S/2007), European Union within the European Social Fund (No. 49/DrINNO/92/2008), and the Scholarship of Polish Science Foundation (48.2012). M.M. Kaczmarek was supported by a Polish Ministry of Science and Higher Education fellowship (No.

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