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Serial gene co-expression network approach to mine biological meanings from integrated transcriptomes of the porcine endometrium during estrous cycle

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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.

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References

  1. 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

  2. Andrews, S (2014). FastQC: a quality control tool for high throughput sequence data. Babraham Bioinformatics. http://www.bioinformatics.babraham.ac.uk/projects/fastqc.

  3. 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

  4. 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

  5. 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

  6. Bindea G, Galon J, Mlecnik B (2013) CluePedia Cytoscape plugin: pathway insights using integrated experimental and in silico data. Bioinformatics 29:661–663

  7. 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

  8. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120

  9. 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

  10. 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

  11. 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

  12. Consortium, G.O (2001) Creating the gene ontology resource: design and implementation. Genome Res 11:1425–1433

  13. Curry TE Jr, Osteen KG (2001) Cyclic changes in the matrix metalloproteinase system in the ovary and uterus. Biol Reprod 64:1285–1296

  14. 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.

  15. 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

  16. 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

  17. Dimitriadis E, Robb L, Salamonsen L (2002) Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells. Mol Hum Reprod 8:636–643

  18. Drummond AE (2006) The role of steroids in follicular growth. Reprod Biol Endocrinol 4:16

  19. 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

  20. 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

  21. 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

  22. Edwards DP (2005) Regulation of signal transduction pathways by estrogen and progesterone. Annu Rev Physiol 67:335–376

  23. Esbenshade K, Ziecik A, Britt J (1990) Regulation and action of gonadotrophins in pigs. J Reprod Fertil Suppl 40:19–32

  24. 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

  25. 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

  26. 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

  27. 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

  28. 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

  29. 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

  30. 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

  31. Howe EA, Sinha R, Schlauch D, Quackenbush J (2011) RNA-Seq analysis in MeV. Bioinformatics 27:3209–3210

  32. Huang DW, Sherman BT, Lempicki RA (2008) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44

  33. 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

  34. Johnson GA, Burghardt RC, Bazer FW, Spencer TE (2003) Osteopontin: roles in implantation and placentation. Biol Reprod 69:1458–1471

  35. 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

  36. 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

  37. Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M (2004) The KEGG resource for deciphering the genome. Nucleic Acids Res 32:D277–D280

  38. 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

  39. 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

  40. 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

  41. Kim D, Langmead B, Salzberg SL (2015a) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357

  42. 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

  43. 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

  44. 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

  45. 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

  46. 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

  47. 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

  48. 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

  49. 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

  50. 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

  51. 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

  52. 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

  53. 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

  54. 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

  55. 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

  56. 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

  57. 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

  58. 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

  59. 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

  60. 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

  61. Sharpe RM, Franks S (2002) Environment, lifestyle and infertility—an inter-generational issue. Nat Med 8:S33

  62. 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

  63. 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

  64. 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

  65. Soede N, Langendijk P, Kemp B (2011) Reproductive cycles in pigs. Anim Reprod Sci 124:251–258

  66. 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

  67. Team RC (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2015. https://www.R-project.org.

  68. 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

  69. Tur I (2013) General reproductive properties in pigs. Turk J Vet Anim Sci 37:1–5

  70. 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

  71. 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

  72. Waclawik A (2011) Novel insights into the mechanisms of pregnancy establishment: regulation of prostaglandin synthesis and signaling in the pig. Reproduction 142:389–399

  73. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57

  74. Wickham H (2011) ggplot2. Wiley Interdiscip Rev Comput Stat 3:180–185

  75. 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

  76. 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

  77. 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.

  78. 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

  79. 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

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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

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.

Correspondence to Hakhyun Ka or Jong-Eun Park or Jun-Mo Kim.

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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

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Keywords

  • DEGs
  • Estrous cycle
  • Gene co-expression network
  • Porcine endometrium
  • RNA-seq