Research paperThe developmental transcriptome landscape of receptive endometrium during embryo implantation in dairy goats
Introduction
Embryo implantation was a crucial step and absolutely necessary part for successful pregnancy in all mammals (Xia et al., 2014), and receptive endometrium (RE) was essential to embryo implantation what was known as endometrial receptivity (Dey et al., 2004, Chakrabarty et al., 2007). Endometrial receptivity was a result of the synchronized and integrated interaction among ovarian hormones, growth and transcription factors, lipid mediators, and cytokines with paracrine signaling (Miravet-Valenciano et al., 2015b). The formation of endometrial receptivity was a spatial and temporal process what was known as the ‘window of implantation’ (Charlet-Renard et al., 2002, Xia et al., 2010). During this period, the endometrium underwent great structural and functional changes, which made it receptive to adhesion and subsequent invasion by the activated embryo (Pan et al., 2007, Altmäe et al., 2013). Furthermore, impaired uterine receptivity was one of the major reasons for the failure of embryo transplantation in humans and other mammals during assisted reproduction with good-quality embryos (Macklon et al., 2006, Sha et al., 2011).
Transcriptome analyses of distinct physiological stages were crucial for understanding the gene regulatory network, including the establishment of endometrium receptivity in mammals. Over the last ten years, RNA-Sequencing (RNA-Seq) had become the main technology for quantifying gene expression changes in comparative transcriptome analysis (Wang et al., 2009). RNA-Seq had opened a new horizon for describing global trends in the differential expression genes (DEGs), single nucleotide polymorphisms (SNPs), insertion-deletion (InDel), alternative splicing (AS), and differential exon usage (DEU) (Anders et al., 2012, Florea et al., 2012, Brousseau et al., 2014, Sun et al., 2015), and has enabled the refinement of gene structures (Cloonan and Grimmond, 2008, Li et al., 2008, Nagalakshmi et al., 2008, Sultan et al., 2008, Wilhelm et al., 2008). In the previous research, only rRNAs were removed from total by rRNA removal kit but the other classes of RNAs, such as long non-coding RNAs, circular RNAs and even pre-mRNA, were still remained in total RNAs. Ribo-Zero RNA-Seq would help us detect additional RNA classes such as enhancer RNAs, anti-sense RNAs and novel RNA species (Derks et al., 2015). Currently, Ribo-Zero RNA-Seq was the best method to investigate the transcriptome, particularly for incompletely annotated genomes, such as goats. Moreover, studies on the endometrium have reported using RNA-Seq in pigs (Samborski et al., 2013a, Samborski et al., 2013b, Lin et al., 2015, Wang et al., 2016), cattles (Mamo et al., 2001, Palma-Vera et al., 2015), and human(Hu et al., 2014). Nevertheless, the molecular mechanisms involved in the development of the endometrium from the pre-receptive state to the receptive state remain largely unknown, and the developmental transcriptome landscape using Ribo-Zero RNA-Seq has not yet been fully elucidated in dairy goats.
In this study, the endometrium from gestational day 5 (D5, PE) and gestational day 15 (D15, RE) dairy goats were selected to systematically analyze the endometrium transcriptome using strand-specific Ribo-Zero RNA-Seq. Then, we constructed a comprehensive analysis of the endometrial transcriptional profiles at the global level to compare the genes expressed in the PE and RE and further explore DEGs, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) for DEGs. Furthermore, SNPs, InDel, AS, and DEU were also analyzed to study the molecular mechanisms underlying the development of the endometrium from the pre-receptive state to the receptive state in dairy goats. Therefore, the results of the present study provided essential information to enhance the knowledge of expression dynamics during endometrium development, extend the transcriptome information and provided good reference data for gene expression profiling of dairy goats.
Section snippets
Ethics statement
All animals in this study were maintained according to the No. 5 proclamation of the Ministry of Agriculture, P. R. China, and the animal protocols were approved by the Review Committee for the Use of Animal Subjects of Northwest A&F University.
Study design and sample collection
The first day of mating was considered to be day 0 of pregnancy, gestational days 5 (D5, PE) and 15 (D15, RE) were important time points for embryo implantation in goats (Igwebuike, 2009a). A total of 20 healthy, 24-month-old multiparous dairy goats
Overview of sequencing data
To systematically identify the transcriptome and examine the changes in expression levels between the pre-receptive and receptive endometrium in dairy goats, we purified and sequenced RNAs using the Illumina paired-end RNA-seq approach. As a result, a total of 120 million paired-end reads of 250 (± 50) bp in length were generated, and the quality control results of the sample were shown in Fig. S1-A. In addition, this yielded 18 gigabases (Gb) of sequence, representing approximately seven times
Discussion
Measuring gene expression at distinct stages is crucial for understanding the gene regulatory network, including the establishment of endometrium receptivity in mammals. Currently, Ribo-Zero RNA-Seq is the best method to investigate the gene expression, particularly for incompletely annotated genomes (Derks et al., 2015, Sun et al., 2015), such as goats. Moreover, transcriptome profiling of the endometrium was typically performed in some breeds (Mamo et al., 2001, Samborski et al., 2013a,
Conclusion
In conclusion, this RNA-Seq study markedly increased the current understanding of transcriptional events that occur during the progression of endometrial receptivity. A total of 810 DEGs were discovered between the pre-receptive endometrium and the receptive endometrium (P < 0.05), and CXCL14, IGFBP3, and LGALS15 potentially participated in the development of the endometrium. Based on GO and KEGG enrichment analyses, energy metabolism and amino acid metabolism likely played an important role in
Acknowledgments
This study was supported by Shaanxi Science and Technology Innovation Project Plan (2015KTCQ03-08), Shaanxi Science and Technology Innovation Project Plan (2013KTZBO2-02-02) and The Technology Extension Project of Northwest A&F University (TGZX2017-17). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We have received funds for covering the costs to publish as open access. The authors declare no conflict of interest.
References (115)
- et al.
Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks
Mol. Cell
(2012) - et al.
Roles of insulin-like growth factor (IGF) binding proteins in regulating IGF actions
Gen. Comp. Endocrinol.
(2005) - et al.
Tissue-specific alternative splicing remodels protein-protein interaction networks
Mol. Cell
(2012) - et al.
Dietary l-arginine supplementation during gestation in mice enhances reproductive performance and Vegfr2 transcription activity in the fetoplacental unit
J. Nutr.
(2012) - et al.
Spontaneous tandem-base mutations (TBM) show dramatic tissue, age, pattern and spectrum specificity
Mutat. Res. Fundam. Mol. Mech. Mutagen.
(2003) A review of uterine structural modifications that influence conceptus implantation and development in sheep and goats
Anim. Reprod. Sci.
(2009)- et al.
Non-linear accumulation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidized DNA damage, during aging
Mutat. Res.
(1996) - et al.
Role of energy metabolism in the pregnancy interceptive action of Ferula assafoetida and Melia azedarach extracts in rat
Contraception
(2004) - et al.
Comparing de novo and reference-based transcriptome assembly strategies by applying them to the blood-sucking bug Rhodnius prolixus
Insect Biochem. Mol. Biol.
(2016) - et al.
BRAK/CXCL14 expression suppresses tumor growth in vivo in human oral carcinoma cells
Biochem. Biophys. Res. Commun.
(2006)
MicroRNAs miR-30b, miR-30d, and miR-494 regulate human endometrial receptivity
Reprod. Sci.
Detecting differential usage of exons from RNA-seq data
Genome Res.
Gene Ontology: tool for the unification of biology
Nat. Genet.
CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth
Proc. Natl. Acad. Sci.
Uterine protein secretions: relationship to development of the conceptus
J. Anim. Sci.
Novel pathways for implantation and establishment and maintenance of pregnancy in mammals
Mol. Hum. Reprod.
Modulation of uterine function by endocrine and paracrine factors in ruminants
Anim. Reprod.
The many faces of interferon tau
Amino Acids
Sex-specific and lineage-specific alternative splicing in primates
Genome Res.
High-throughput transcriptome sequencing and preliminary functional analysis in four Neotropical tree species
BMC Genomics
Tandem-base mutations occur in mouse liver and adipose tissue preferentially as G:C to T:A transversions and accumulate with age
Environ. Mol. Mutagen.
SNP discovery in the bovine milk transcriptome using RNA-Seq technology
Mamm. Genome
MicroRNA regulation of cyclooxygenase-2 during embryo implantation
Proc. Natl. Acad. Sci.
The implantation window
J. Gynecol. Obstet. Biol. Reprod.
Alternative splicing: an important mechanism in stem cell biology
World J. Stem Cells
Label-free proteomics uncovers energy metabolism and focal adhesion regulations responsive for endometrium receptivity
J. Proteome Res.
Transcriptome content and dynamics at single-nucleotide resolution
Genome Biol.
The gene ontology (GO) database and informatics resource
Nucleic Acids Res.
Deciphering the RNA landscape by RNAome sequencing
RNA Biol.
Molecular cues to implantation
Endocr. Rev.
Biological Functions of Galectin 15 (LGALS15) in the Ovine Uterus
Trophoblast invasion: the role of intracellular cytokine signalling via signal transducer and activator of transcription 3 (STAT3)
Hum. Reprod. Update
Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues
F1000research
Transcriptomic analysis of the bovine endometrium: what is required to establish uterine receptivity to implantation in cattle?
J. Reprod. Dev.
Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant?
Biol. Reprod.
A map of local adaptation in Arabidopsis thaliana
Science
Localization of the sites of synthesis and action of insulin-like growth factor-I in the rat uterus
Mol. Endocrinol.
Analysis of RNA-Seq data using TopHat and cufflinks
Methods Mol. Biol.
Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy
Biol. Reprod.
Somatic microindels: analysis in mouse soma and comparison with the human germline
Hum. Mutat.
Endometrial glands are required for preimplantation conceptus elongation and survival
Biol. Reprod.
Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation
Reproduction
Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine Uterus1
Biol. Reprod.
Carbohydrate metabolism in the rat uterus during early pregnancy
Steroids Lipids Res.
De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-Alzheimer’s properties
BMC Genomics
Adaptation to climate across the Arabidopsis thaliana genome
Science
AMPK: a nutrient and energy sensor that maintains energy homeostasis
Nat. Rev. Mol. Cell Biol.
Characterization of transcriptional complexity during longissimus muscle development in bovines using high-throughput sequencing
PLoS One
Transcriptomic changes during the pre-receptive to receptive transition in human endometrium detected by RNA-Seq
J. Clin. Endocrinol. Metab.
Insulin-like growth factor-I stimulated DNA replication in mouse endometrial stromal cells
J. Reprod. Dev.
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These authors contributed equally to this work.