Abstract
The estrous cycle is a complex process regulated by several hormones. To understand the dynamic changes in gene expression that takes place in the swine endometrium during the estrous cycle relative to the day of estrus onset, we performed RNA-sequencing analysis on days 0, 3, 6, 9, 12, 15, and 18, resulting in the identification of 4495 differentially expressed genes (DEGs; Q ≤ 0.05 and |log2FC| ≥ 1) at various phases in the estrous cycle. These DEGs were integrated into multiple gene co-expression networks based on different fold changes and correlation coefficient (R2) thresholds and a suitable network, which included 899 genes (|log2FC| ≥ 2 and R2 ≥ 0.99), was identified for downstream analyses based on the biological relevance of the Gene Ontology (GO) terms enriched. The genes in this network were partitioned into 6 clusters based on the expression pattern. Several GO terms including cell cycle, apoptosis, hormone signaling, and lipid biosynthetic process were found to be enriched. Furthermore, we found 15 significant KEGG pathways, including cell adhesion molecules, cytokine-cytokine receptor signaling, steroid biosynthesis, and estrogen signaling pathways. We identified several genes and GO terms to be stage-specific. Moreover, the identified genes and pathways extend our understanding of porcine endometrial regulation during estrous cycle and will serve as a good resource for future studies.
Similar content being viewed by others
References
Adams HA, Southey BR, Everts RE, Marjani SL, Tian CX, Lewin HA, Rodriguez-Zas SL (2011) Transferase activity function and system development process are critical in cattle embryo development. Funct Integr Genomics 11:139–150
Andrews, S (2014). FastQC: a quality control tool for high throughput sequence data. Babraham Bioinformatics. http://www.bioinformatics.babraham.ac.uk/projects/fastqc.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT (2000) Gene ontology: tool for the unification of biology. Nat Genet 25:25
Atli MO, Kurar E, Kayis SA, Aslan S, Semacan A, Celik S, Guzeloglu A (2010) Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy. Anim Reprod Sci 122:124–132
Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman W-H, Pagès F, Trajanoski Z, Galon J (2009) ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25:1091–1093
Bindea G, Galon J, Mlecnik B (2013) CluePedia Cytoscape plugin: pathway insights using integrated experimental and in silico data. Bioinformatics 29:661–663
Boerboom D, Brown KA, Vaillancourt D, Poitras P, Goff AK, Watanabe K, Doré M, Sirois J (2004) Expression of key prostaglandin synthases in equine endometrium during late diestrus and early pregnancy. Biol Reprod 70:391–399
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120
Burghardt RC, Johnson GA, Jaeger LA, Ka H, Garlow JE, Spencer TE, Bazer FW (2002) Integrins and extracellular matrix proteins at the maternal-fetal interface in domestic animals. Cells Tissues Organs 172:202–217
Chen X, Li A, Chen W, Wei J, Fu J, Wang A (2015) Differential gene expression in uterine endometrium during implantation in pigs. Biol Reprod 92(52):51–14
Chu Q, Zhou B, Xu F, Chen R, Shen C, Liang T, Li Y, Schinckel AP (2017) Genome-wide differential mRNA expression profiles in follicles of two breeds and at two stages of estrus cycle of gilts. Sci Rep 7:5052
Consortium, G.O (2001) Creating the gene ontology resource: design and implementation. Genome Res 11:1425–1433
Curry TE Jr, Osteen KG (2001) Cyclic changes in the matrix metalloproteinase system in the ovary and uterus. Biol Reprod 64:1285–1296
Dalman MR, Deeter A, Nimishakavi G, Duan Z-H (2012) Fold change and p-value cutoffs significantly alter microarray interpretations. BMC bioinformatics 13 Supplement 2, S11.
Davoodi S, Cooke RF, Fernandes ACDC, Cappellozza BI, Vasconcelos JLM, Cerri RLA (2016) Expression of estrus modifies the gene expression profile in reproductive tissues on day 19 of gestation in beef cows. Theriogenology 85:645–655
De Ruijter-Villani M, Van Tol H, Stout T (2015) Effect of pregnancy on endometrial expression of luteolytic pathway components in the mare. Reprod Fertil Dev 27:834–845
Dimitriadis E, Robb L, Salamonsen L (2002) Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells. Mol Hum Reprod 8:636–643
Drummond AE (2006) The role of steroids in follicular growth. Reprod Biol Endocrinol 4:16
Du ZQ, Eisley C, Onteru S, Madsen O, Groenen M, Ross J, Rothschild M (2014) Identification of species-specific novel transcripts in pig reproductive tissues using RNA-seq. Anim Genet 45:198–204
Dullo P, Vedi N (2008) Changes in serum calcium, magnesium and inorganic phosphorus levels during different phases of the menstrual cycle. J Human Reprod Sci 1:77
Ealy AD, Eroh ML, Sharp Iii DC (2010) Prostaglandin H synthase Type 2 is differentially expressed in endometrium based on pregnancy status in pony mares and responds to oxytocin and conceptus secretions in explant culture. Anim Reprod Sci 117:99–105
Edwards DP (2005) Regulation of signal transduction pathways by estrogen and progesterone. Annu Rev Physiol 67:335–376
Esbenshade K, Ziecik A, Britt J (1990) Regulation and action of gonadotrophins in pigs. J Reprod Fertil Suppl 40:19–32
Franczak A, Wojciechowicz B, Kotwica G (2013) Transcriptomic analysis of the porcine endometrium during early pregnancy and the estrous cycle. Reprod Biol 13:229–237
Franczak A, Wojciechowicz B, Kolakowska J, Zglejc K, Kotwica G (2014) Transcriptomic analysis of the myometrium during peri-implantation period and luteolysis–the study on the pig model. Funct Integr Genomics 14:673–682
Gebhardt S, Merkl M, Herbach N, Wanke R, Handler J, Bauersachs S (2012) Exploration of global gene expression changes during the estrous cycle in equine endometrium. Biol Reprod 87(136):131–113
Geisert R (1982) Establishment of pregnancy in the pig. I. Interrelationships between preimplantation development of the pig blastocyst and uterine endometrial secretions. Biol Reprod 27:925–939
Geisert RD, Thatcher WW, Michael Roberts R, Bazer FW (1982) Establishment of pregnancy in the pig: III. Endometrial secretory response to estradiol valerate administered on day 11 of the estrous cycle. Biol Reprod 27:957–965
Goff L, Trapnell C, Kelley D (2013). cummeRbund: analysis, exploration, manipulation, and visualization of Cufflinks high-throughput sequencing data. R package version 2. https://www.rdocumentation.org
Green J, Kim J, Whitworth K, Agca C, Prather R (2006) The use of microarrays to define functionally-related genes that are differentially expressed in the cycling pig uterus. Soc Reprod Fertil Suppl 62:163–176
Howe EA, Sinha R, Schlauch D, Quackenbush J (2011) RNA-Seq analysis in MeV. Bioinformatics 27:3209–3210
Huang DW, Sherman BT, Lempicki RA (2008) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44
Jang H, Choi Y, Yoo I, Han J, Hong JS, Kim YY, Ka H (2017) Vitamin D-metabolic enzymes and related molecules: expression at the maternal-conceptus interface and the role of vitamin D in endometrial gene expression in pigs. PLoS One 12:e0187221
Johnson GA, Burghardt RC, Bazer FW, Spencer TE (2003) Osteopontin: roles in implantation and placentation. Biol Reprod 69:1458–1471
Ka H, Seo H, Kim M, Choi Y, Lee CK (2009) Identification of differentially expressed genes in the uterine endometrium on day 12 of the estrous cycle and pregnancy in pigs. Mol Reprod Dev 76:75–84
Kaeoket K, Persson E, Dalin A-M (2001) The sow endometrium at different stages of the oestrous cycle: studies on morphological changes and infiltration by cells of the immune system. Anim Reprod Sci 65:95–114
Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M (2004) The KEGG resource for deciphering the genome. Nucleic Acids Res 32:D277–D280
Kawano N, Miyado K, Yoshii N, Kanai S, Saito H, Miyado M, Inagaki N, Odawara Y, Hamatani T, Umezawa A (2014) Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition. Sci Rep 4:4701
Keator CS, Mah K, Ohm L, Slayden OD (2011) Estrogen and progesterone regulate expression of the endothelins in the rhesus macaque endometrium. Hum Reprod 26:1715–1728
Kim M, Seo H, Choi Y, Shim J, Kim H, Lee C-K, Ka H (2012) Microarray analysis of gene expression in the uterine endometrium during the implantation period in pigs. Asian Australas J Anim Sci 25:1102
Kim D, Langmead B, Salzberg SL (2015a) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357
Kim M, Seo H, Choi Y, Yoo I, Seo M, Lee C-K, Kim H, Ka H (2015b) Analysis of stage-specific gene expression profiles in the uterine endometrium during pregnancy in pigs. PLoS One 10:e0143436
Kwon SG, Hwang JH, Park DH, Kim TW, Kang DG, Kang KH, Kim I-S, Park HC, Na C-S, Ha J (2016) Identification of differentially expressed genes associated with litter size in Berkshire pig placenta. PLoS One 11:e0153311
Lan B, Zhang J, Zhang P, Zhang W, Yang S, Lu D, Li W, Dai Q (2017) Metformin suppresses CRC growth by inducing apoptosis via ADORA1. Front Biosci (Landmark Edition) 22:248–257
Lin Z, Yin P, Reierstad S, O’halloran M, Pearson E, Mutlu G, Bulun S (2010) Adenosine A1 receptor, a target and regulator of estrogen receptorα action, mediates the proliferative effects of estradiol in breast cancer. Oncogene 29:1114
Lin H, Wang H, Wang Y, Liu C, Wang C, Guo J (2015) Transcriptomic analysis of the porcine endometrium during embryo implantation. Genes 6:1330–1346
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25:402–408
Martinez-Giner M, Noguera JL, Balcells I, Fernández-Rodríguez A, Pena RN (2013) Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy. PLoS One 8:e66023
Miao X, Luo Q (2013) Genome-wide transcriptome analysis between small-tail Han sheep and the Surabaya fur sheep using high-throughput RNA sequencing. Reproduction 145:587–596
Mitko K, Ulbrich SE, Wenigerkind H, Sinowatz F, Blum H, Wolf E, Bauersachs S (2008) Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle. Reproduction 135:225–240
Noguchi M, Yoshioka K, Itoh S, Suzuki C, Arai S, Wada Y, Hasegawa Y, Kaneko H (2010) Peripheral concentrations of inhibin A, ovarian steroids, and gonadotropins associated with follicular development throughout the estrous cycle of the sow. Reproduction 139:153–161
Okada S, Okada H, Sanezumi M, Nakajima T, Yasuda K, Kanzaki H (2000) Expression of interleukin-15 in human endometrium and decidua. Mol Hum Reprod 6:75–80
Okano A, Ogawa H, Takahashi H, Geshi M (2007) Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum. J Reprod Dev 53:923–930
Peltoniemi O, Easton B, Love R, Klupiec C, Evans G (1995) Effect of chronic treatment with a GnRH agonist (Goserelin) on LH secretion and early pregnancy in gilts. Anim Reprod Sci 40:121–133
Ponnampalam A, Rogers P (2006) Cyclic changes and hormonal regulation of annexin IV mRNA and protein in human endometrium. Mol Hum Reprod 12:661–669
Prunier A, Martinat-Botte F, Ravault J, Camous S (1987) Perioestrous patterns of circulating LH, FSH, prolactin and oestradiol-17β in the gilt. Anim Reprod Sci 14:205–218
Reverter A, Chan EK (2008) Combining partial correlation and an information theory approach to the reversed engineering of gene co-expression networks. Bioinformatics 24:2491–2497
Samborski A, Graf A, Krebs S, Kessler B, Reichenbach M, Reichenbach H-D, Ulbrich SE, Bauersachs S (2013) Transcriptome changes in the porcine endometrium during the preattachment phase. Biol Reprod 89(134):131–116
Seo H, Choi Y, Shim J, Yoo I, Ka H (2014) Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs. Biol Reprod 90(99):91–13
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504
Sharpe RM, Franks S (2002) Environment, lifestyle and infertility—an inter-generational issue. Nat Med 8:S33
Shehu A, Mao J, Gibori GB, Halperin J, Le J, Sangeeta Devi Y, Merrill B, Kiyokawa H, Gibori G (2008) Prolactin receptor-associated protein/17β-hydroxysteroid dehydrogenase type 7 gene (Hsd17b7) plays a crucial role in embryonic development and fetal survival. Mol Endocrinol 22:2268–2277
Shimizu T, Krebs S, Bauersachs S, Blum H, Wolf E, Miyamoto A (2010) Actions and interactions of progesterone and estrogen on transcriptome profiles of the bovine endometrium. Physiol Genomics 42:290–300
Soede N, Hazeleger W, Kemp B (1998) Follicle size and the process of ovulation in sows as studied with ultrasound. Reprod Domest Anim 33:239–244
Soede N, Langendijk P, Kemp B (2011) Reproductive cycles in pigs. Anim Reprod Sci 124:251–258
Srikanth K, Kwon A, Lee E, Chung H (2017) Characterization of genes and pathways that respond to heat stress in Holstein calves through transcriptome analysis. Cell Stress Chaperones 22:29–42
Team RC (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2015. https://www.R-project.org.
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578
Tur I (2013) General reproductive properties in pigs. Turk J Vet Anim Sci 37:1–5
Ulbrich SE, Frohlich T, Schulke K, Englberger E, Waldschmitt N, Arnold GJ, Reichenbach H-D, Reichenbach M, Wolf E, Meyer HH (2009) Evidence for estrogen-dependent uterine serpin (SERPINA14) expression during estrus in the bovine endometrial glandular epithelium and lumen. Biol Reprod 81:795–805
Van Dam S, Võsa U, Van Der Graaf A, Franke L, De Magalhães JP (2017) Gene co-expression analysis for functional classification and gene–disease predictions. Briefings in bioinformatics, 19:575–592
Waclawik A (2011) Novel insights into the mechanisms of pregnancy establishment: regulation of prostaglandin synthesis and signaling in the pig. Reproduction 142:389–399
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57
Wickham H (2011) ggplot2. Wiley Interdiscip Rev Comput Stat 3:180–185
Yamada O, Todoroki J-I, Takahashi T, Hashizume K (2002) The dynamic expression of extracellular matrix in the bovine endometrium at implantation. J Vet Med Sci 64:207–214
Yang Y, Han L, Yuan Y, Li J, Hei N, Liang H (2014) Gene co-expression network analysis reveals common system-level properties of prognostic genes across cancer types. Nat Commun 5:3231
Yang S, Zhou X, Pei Y, Wang H, He K, Zhao A (2018) Identification of differentially expressed genes in porcine ovaries at proestrus and estrus stages using RNA-Seq technique. Biomed Res Int 2018:9150723.
Yip KS, Suvorov A, Connerney J, Lodato NJ, Waxman DJ (2013) Changes in mouse uterine transcriptome in estrus and proestrus. Biol Reprod 89(13):11–12
Zhang X, Huang L, Wu T, Feng Y, Ding Y, Ye P, Yin Z (2015) Transcriptomic analysis of ovaries from pigs with high and low litter size. PLoS One 10:e0139514
Acknowledgments
The authors would like to thank the Rural Development Administration and National Institute of Animal Science for providing the funding to carry out this research. JMK was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea, grant number NRF-2019R1F1A1061542
Availability of data
The data generated in this study are deposited in NCBI SRA data base under the accession no. SRP127622.
Funding
This study was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development” (project title: Porcine epigenomic map construction and investigation of the imprinted genes, Project No. PJ011876), Rural Development Administration (RDA), Republic of Korea. KS is supported by a 2019 RDA Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korea.
Author information
Authors and Affiliations
Contributions
KS, WCP, and JMK analyzed the data. KS and JMK wrote the manuscript. JEP and JMK performed the experiment. HK and KTL designed and supervised the experiment and project. GWJ, DL, and JEP supervised the project. All authors reviewed the manuscript and approved.
Corresponding authors
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Srikanth, K., Park, W., Lim, D. et al. Serial gene co-expression network approach to mine biological meanings from integrated transcriptomes of the porcine endometrium during estrous cycle. Funct Integr Genomics 20, 117–131 (2020). https://doi.org/10.1007/s10142-019-00703-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10142-019-00703-1