Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Care and Management of Mares
2.2. RNA Isolation and Quantification
2.3. RNA Sequencing
2.4. Bioinformatic Analysis
3. Results
3.1. Sequencing Results
3.2. Transcript Assembly and Analysis
3.3. Day 9
3.4. Day 11
3.5. Day 13
3.6. Significant Transcripts on Days 9, 11 and 13
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
- Allen, W.R.; Stewart, F. Equine placentation. Reprod. Fertil. Dev. 2001, 13, 623–634. [Google Scholar] [CrossRef] [PubMed]
- Bazer, F.W.; Burghardt, R.C.; Johnson, G.A.; Spencer, T.E.; Wu, G. Interferons and progesterone for establishment and maintenance of pregnancy: Interactions among novel cell signaling pathways. Reprod. Biol. 2008, 8, 179–211. [Google Scholar] [CrossRef]
- Sharp, D.C.; Thatcher, M.J.; Salute, M.E.; Fuchs, A.R. Relationship between endometrial oxytocin receptors and oxytocin-induced prostaglandin F2 alpha release during the oestrous cycle and early pregnancy in pony mares. J. Reprod. Fertil. 1997, 109, 137–144. [Google Scholar] [CrossRef] [PubMed]
- McCracken, J.A.; Custer, E.E.; Lamsa, J.C. Luteolysis: A neuroendocrine-mediated event. Physiol. Rev. 1999, 79, 263–323. [Google Scholar] [CrossRef] [PubMed]
- Betteridge, K.J.; Eaglesome, M.D.; Mitchell, D.; Flood, P.F.; Beriault, R. Development of horse embryos up to twenty two days after ovulation: Observations on fresh specimens. J. Anat. 1982, 135, 191–209. [Google Scholar] [PubMed]
- Oriol, J.G.; Sharom, F.J.; Betteridge, K.J. Developmentally regulated changes in the glycoproteins of the equine embryonic capsule. J. Reprod. Fertil. 1993, 99, 653–664. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ginther, O.J. Mobility of the early equine conceptus. Theriogenology 1983, 19, 603–611. [Google Scholar] [CrossRef]
- McDowell, K.J.; Sharp, D.C.; Grubaugh, W.; Thatcher, W.W.; Wilcox, C.J. Restricted conceptus mobility results in failure of pregnancy maintenance in mares. Biol. Reprod. 1988, 39, 340–348. [Google Scholar] [CrossRef]
- Ginther, O.J. Internal regulation of physiological processes through local venoarterial pathways: A review. J. Anim. Sci. 1974, 39, 550–564. [Google Scholar] [CrossRef]
- Leith, G.S.; Ginther, O.J. Characterization of intrauterine mobility of the early equine conceptus. Theriogenology 1984, 22, 401–408. [Google Scholar] [CrossRef]
- Stout, T.A.; Allen, W.R. Role of prostaglandins in intrauterine migration of the equine conceptus. Reproduction 2001, 121, 771–775. [Google Scholar] [CrossRef] [PubMed]
- Baker, C.B.; Adams, M.H.; McDowell, K.J. Lack of expression of alpha or omega interferons by the horse conceptus. J. Reprod. Fertil. 1991, 44, 439–443. [Google Scholar]
- Vanderwall, D.K.; Woods, G.L.; Weber, J.A.; Lichtenwalner, A.B. Corpus luteal function in nonpregnant mares following intrauterine administration of prostaglandin E(2) or estradiol-17beta. Theriogenology 1994, 42, 1069–1083. [Google Scholar] [CrossRef]
- Wilsher, S.; Allen, W.R. Intrauterine administration of plant oils inhibits luteolysis in the mare. Equine Vet. J. 2011, 43, 99–105. [Google Scholar] [CrossRef] [PubMed]
- Diel de Amorim, M.; Nielsen, K.; Cruz, R.K.; Card, C. Progesterone levels and days to luteolysis in mares treated with intrauterine fractionated coconut oil. Theriogenology 2016, 86, 545–550. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Klohonatz, K.M.; Hess, A.M.; Hansen, T.R.; Squires, E.L.; Bouma, G.J.; Bruemmer, J.E. Equine endometrial gene expression changes during and after maternal recognition of pregnancy. J. Anim. Sci. 2015, 93, 3364–3376. [Google Scholar] [CrossRef] [PubMed]
- Klein, C.; Scoggin, K.E.; Ealy, A.D.; Troedsson, M.H. Transcriptional profiling of equine endometrium during the time of maternal recognition of pregnancy. Biol. Reprod. 2010, 83, 102–113. [Google Scholar] [CrossRef]
- Kenney, R.M. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. J. Am. Vet. Med. Assoc. 1978, 172, 241–262. [Google Scholar]
- Afgan, E.; Baker, D.; Batut, B.; van den Beek, M.; Bouvier, D.; Cech, M.; Chilton, J.; Clements, D.; Coraor, N.; Gruning, B.A.; et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018, 46, W537–W544. [Google Scholar] [CrossRef] [Green Version]
- Andrews, S. FastQC: A Quality Control Tool for High Throughput Sequence Data. 2010. Available online: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 21 May 2019).
- Ewels, P.; Magnusson, M.; Lundin, S.; Kaller, M. MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics 2016, 32, 3047–3048. [Google Scholar] [CrossRef]
- Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [PubMed]
- Kalbfleisch, T.S.; Rice, E.S.; DePriest, M.S., Jr.; Walenz, B.P.; Hestand, M.S.; Vermeesch, J.R.; BL, O.C.; Fiddes, I.T.; Vershinina, A.O.; Saremi, N.F.; et al. Improved reference genome for the domestic horse increases assembly contiguity and composition. Commun. Biol. 2018, 1, 197. [Google Scholar] [CrossRef] [PubMed]
- Kim, D.; Langmead, B.; Salzberg, S.L. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 2015, 12, 357–360. [Google Scholar] [CrossRef] [PubMed]
- Pertea, M.; Pertea, G.M.; Antonescu, C.M.; Chang, T.C.; Mendell, J.T.; Salzberg, S.L. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015, 33, 290–295. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Matlin, A.J.; Clark, F.; Smith, C.W. Understanding alternative splicing: Towards a cellular code. Nat. Rev. Mol. Cell Biol. 2005, 6, 386–398. [Google Scholar] [CrossRef] [PubMed]
- Venables, J.P. Unbalanced alternative splicing and its significance in cancer. Bioessays 2006, 28, 378–386. [Google Scholar] [CrossRef]
- Jurica, M.S.; Moore, M.J. Pre-mRNA splicing: Awash in a sea of proteins. Mol. Cell 2003, 12, 5–14. [Google Scholar] [CrossRef]
- Klohonatz, K.M.; Nulton, L.C.; Hess, A.M.; Bouma, G.J.; Bruemmer, J.E. The role of embryo contact and focal adhesions during maternal recognition of pregnancy. PLoS ONE 2019, 14, e0213322. [Google Scholar] [CrossRef] [PubMed]
- Klohonatz, K.M.; Cameron, A.D.; Hergenreder, J.R.; da Silveira, J.C.; Belk, A.D.; Veeramachaneni, D.N.; Bouma, G.J.; Bruemmer, J.E. Circulating miRNAs as Potential Alternative Cell Signaling Associated with Maternal Recognition of Pregnancy in the Mare. Biol. Reprod. 2016, 95, 124. [Google Scholar] [CrossRef]
- Vogel, V. Mechanotransduction involving multimodular proteins: Converting force into biochemical signals. Annu. Rev. Biophys. 2006, 35, 459–488. [Google Scholar] [CrossRef] [PubMed]
- Burridge, K.; Fath, K.; Kelly, T.; Nuckolls, G.; Turner, C. Focal adhesions: Transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol. 1988, 4, 487–525. [Google Scholar] [CrossRef] [PubMed]
- Hehlgans, S.; Eke, I.; Cordes, N. An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading. Int. J. Radiat. Biol. 2007, 83, 769–779. [Google Scholar] [CrossRef] [PubMed]
- Cherry, L.K.; Li, X.; Schwab, P.; Lim, B.; Klickstein, L.B. RhoH is required to maintain the integrin LFA-1 in a nonadhesive state on lymphocytes. Nat. Immunol. 2004, 5, 961–967. [Google Scholar] [CrossRef] [PubMed]
- Watanabe, T.; Tsuda, M.; Makino, Y.; Konstantinou, T.; Nishihara, H.; Majima, T.; Minami, A.; Feller, S.M.; Tanaka, S. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration. Cell Res. 2009, 19, 638–650. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rosenberger, G.; Kutsche, K. αPIX and βPIX and their role in focal adhesion formation. Eur. J. Cell Biol. 2006, 85, 265–274. [Google Scholar] [CrossRef] [PubMed]
- Frank, J.W.; Seo, H.; Burghardt, R.C.; Bayless, K.J.; Johnson, G.A. ITGAV (alpha v integrins) bind SPP1 (osteopontin) to support trophoblast cell adhesion. Reproduction 2017, 153, 695–706. [Google Scholar] [CrossRef] [PubMed]
- Tanis, K.Q.; Schwartz, M.A. Regulation of Cell Adhesion Responses by Abl Family Kinases. In Abl Family Kinases in Development and Disease; Springer: Berlin/Heidelberg, Germany, 2006; pp. 16–25. [Google Scholar]
- Han, D.C.; Guan, J.L. Association of focal adhesion kinase with Grb7 and its role in cell migration. J. Biol. Chem. 1999, 274, 24425–24430. [Google Scholar] [CrossRef] [PubMed]
- Paliashvili, K. Identification of Novel Focal Adhesion Kinase Binding Partners and Their Biological Functions in Cancer Cells. Ph.D. Thesis, Edinburgh Medical School, Edinburgh, UK, 2015. [Google Scholar]
- Sasi Kumar, K.; Ramadhas, A.; Nayak, S.C.; Kaniyappan, S.; Dayma, K.; Radha, V. C3G (RapGEF1), a regulator of actin dynamics promotes survival and myogenic differentiation of mouse mesenchymal cells. Biochim. Biophys. Acta 2015, 1853, 2629–2639. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chung, J.J.; Navarro, B.; Krapivinsky, G.; Krapivinsky, L.; Clapham, D.E. A novel gene required for male fertility and functional CATSPER channel formation in spermatozoa. Nat. Commun. 2011, 2, 153. [Google Scholar] [CrossRef] [PubMed]
- Ren, D.; Navarro, B.; Perez, G.; Jackson, A.C.; Hsu, S.; Shi, Q.; Tilly, J.L.; Clapham, D.E. A sperm ion channel required for sperm motility and male fertility. Nature 2001, 413, 603–609. [Google Scholar] [CrossRef] [PubMed]
Genes | Transcripts | Unannotated Transcripts | |
---|---|---|---|
Day 9 | 634 | 1435 | 682 |
Day 11 | 639 | 1435 | 678 |
Day 13 | 421 | 916 | 446 |
Day 9 | Day 11 | Day 13 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
All | Annotated | All | Annotated | All | Annotated | ||||||
Transcript | p-Value | Transcript | p-Value | Transcript | p-Value | Transcript | p-Value | Transcript | p-Value | Transcript | p-Value |
ZFHX3 | 4.00 × 10−76 | ZFHX3 | 4.00 × 10−76 | ACTN4 | 6.03 × 10−55 | ACTN4 | 6.03 × 10−55 | MSTRG.15333.5 | 3.09 × 10−36 | ACOX1 | 7.14 × 10−24 |
ERBB2 | 2.49 × 10−56 | ERBB2 | 2.49 × 10−56 | MSTRG.3211.2 | 6.90 × 10−45 | AGRN | 2.46 × 10−40 | ACOX1 | 7.14 × 10−24 | SORBS3 | 3.23 × 10−17 |
NF1 | 2.49 × 10−56 | NF1 | 2.49 × 10−56 | MSTRG.6009.2 | 8.48 × 10−42 | C16H3orf67 | 1.43 × 10−33 | SORBS3 | 3.23 × 10−17 | SERPINB9 | 2.70 × 10−15 |
MSTRG.27804.1 | 9.13 × 10−53 | MKLN1 | 3.03 × 10−39 | AGRN | 2.46 × 10−40 | BTF3L4 | 5.84 × 10−30 | MSTRG.22183.4 | 6.65 × 10−17 | MTMR2 | 7.01 × 10−15 |
MSTRG.15604.11 | 1.57 × 10−50 | LACTB | 1.84 × 10−37 | MSTRG.1680.8 | 6.10 × 10−35 | PITPNA | 6.19 × 10−27 | MSTRG.17471.3 | 6.54 × 10−16 | DOCK1 | 1.32 × 10−14 |
MKLN1 | 3.03 × 10−39 | EGR1 | 8.44 × 10−27 | MSTRG.26234.10 | 2.27 × 10−34 | BICRAL | 2.66 × 10−23 | MSTRG.28537.13 | 1.28 × 10−15 | FAM20B | 7.12 × 10−14 |
MSTRG.13994.1 | 1.56 × 10−38 | AKAP11 | 2.57 × 10−24 | C16H3orf67 | 1.43 × 10−33 | STX3 | 3.11 × 10−20 | MSTRG.20351.7 | 1.64 × 10−15 | TTC28 | 1.59 × 10−13 |
LACTB | 1.84 × 10−37 | AKAP11 | 1.40 × 10−23 | BTF3L4 | 5.84 × 10−30 | PATZ1 | 2.69 × 10−19 | SERPINB9 | 2.70 × 10−15 | HIP1 | 1.77 × 10−13 |
MSTRG.19114.13 | 6.66 × 10−32 | COMMD4 | 3.12 × 10−23 | PITPNA | 6.19 × 10−27 | ZNF605 | 4.10 × 10−19 | MSTRG.14125.20 | 7.01 × 10−15 | USP42 | 1.03 × 10−12 |
MSTRG.13516.12 | 2.06 × 10−30 | NXT2 | 5.60 × 10−23 | MSTRG.5600.1 | 1.31 × 10−26 | FAM104A | 1.44 × 10−18 | MTMR2 | 7.01 × 10−15 | UNK | 1.18 × 10−12 |
EGR1 | 8.44 × 10−27 | R3HDM2 | 8.69 × 10−22 | BICRAL | 2.66 × 10−23 | TNPO1 | 2.22 × 10−17 | DOCK1 | 1.32 × 10−14 | TLDC1 | 1.84 × 10−11 |
MSTRG.14475.5 | 4.73 × 10−26 | NCBP1 | 5.71 × 10−20 | MSTRG.25435.2 | 9.22 × 10−21 | MME | 8.32 × 10−17 | MSTRG.5354.16 | 2.93 × 10−14 | ZBTB37 | 2.32 × 10−11 |
MSTRG.10550.2 | 2.08 × 10−25 | C1H1orf198 | 5.81 × 10−20 | STX3 | 3.11 × 10−20 | TMED8 | 3.81 × 10−16 | MSTRG.15333.7 | 6.54 × 10−14 | C7H11orf54 | 4.06 × 10−11 |
AKAP11 | 2.57 × 10−24 | NHS | 6.50 × 10−20 | MSTRG.2169.2 | 3.76 × 10−20 | RABGAP1 | 7.47 × 10−16 | FAM20B | 7.12 × 10−14 | KDM7A | 9.32 × 10−11 |
MSTRG.10469.4 | 2.57 × 10−24 | PHF20 | 2.45 × 10−19 | PATZ1 | 2.69 × 10−19 | PLA2G2C | 1.33 × 10−15 | MSTRG.17032.9 | 9.35 × 10−14 | ATRX | 9.32 × 10−11 |
AKAP11 | 1.40 × 10−23 | C13H7orf26 | 7.43 × 10−19 | ZNF605 | 4.10 × 10−19 | LOC100064842 | 2.16 × 10−15 | TTC28 | 1.59 × 10−13 | LARS | 2.45 × 10−10 |
MSTRG.19003.10 | 1.67 × 10−23 | RPSA | 1.26 × 10−18 | FAM104A | 1.44 × 10−18 | DCAF6 | 2.73 × 10−15 | HIP1 | 1.77 × 10−13 | PHRF1 | 3.70 × 10−10 |
COMMD4 | 3.12 × 10−23 | PIGM | 1.60 × 10−18 | MSTRG.25230.44 | 4.50 × 10−18 | FBXO31 | 3.97 × 10−15 | MSTRG.26578.1 | 6.78 × 10−13 | ATP11C | 7.35 × 10−10 |
MSTRG.19003.12 | 4.22 × 10−23 | TEP1 | 3.64 × 10−18 | MSTRG.22573.1 | 7.13 × 10−18 | MYO1C | 7.40 × 10−15 | USP42 | 1.03 × 10−12 | SLC25A25 | 1.18 × 10−9 |
MSTRG.13065.7 | 5.14 × 10−23 | SSH2 | 1.22 × 10−17 | MSTRG.25564.2 | 1.99 × 10−17 | NCOA2 | 1.25 × 10−14 | MSTRG.3924.5 | 1.18 × 10−12 | CCDC181 | 1.23 × 10−9 |
Day 9 | Day 11 | Day 13 | ||||||
---|---|---|---|---|---|---|---|---|
Transcript | Log2 FC | p-Value | Transcript | Log2 FC | p-Value | Transcript | Log2 FC | p-Value |
HOOK1 | 13.8 | 2.76 × 10−2 | MBOAT2 | 13.6 | 2.93 × 10−2 | ACOX1 | −14.1 | 7.14 × 10−24 |
GOLGB1 | −13.8 | 2.79 × 10−2 | NF1 | -13.5 | 3.26 × 10−2 | MBOAT2 | 13.9 | 3.42 × 10−2 |
AKAP11 | 13.1 | 1.40 × 10−23 | PTPN4 | 13.5 | 4.45 × 10−5 | YWHAZ | 13.3 | 4.71 × 10−2 |
USF3 | −12.5 | 9.02 × 10−10 | PRKAA2 | -13.3 | 3.72 × 10−2 | ZBTB37 | 12.2 | 2.32 × 10−11 |
PFKFB3 | 11.7 | 2.22 × 10−10 | PHC3 | 13.0 | 4.64 × 10−2 | TTC28 | −11.9 | 1.59 × 10−13 |
PIGM | 11.7 | 1.60 × 10−18 | TRANK1 | 13.0 | 4.68 × 10−2 | MTMR2 | 11.9 | 7.01 × 10−15 |
SSH2 | 11.6 | 1.22 × 10−17 | NFAT5 | 12.6 | 9.18 × 10−10 | ULK2 | −11.8 | 7.92 × 10−4 |
FUK | 11.5 | 7.51 × 10−15 | STX3 | 12.5 | 3.11 × 10−20 | MSI2 | −11.7 | 1.41 × 10−5 |
TTBK2 | 11.5 | 2.00 × 10−3 | TLL1 | 12.4 | 3.35 × 10−13 | KANK2 | 11.6 | 1.38 × 10−6 |
PPP6R2 | 11.1 | 7.51 × 10−15 | TMEM181 | 12.1 | 1.57 × 10−9 | NACC2 | 11.3 | 3.92 × 10−3 |
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Klohonatz, K.M.; Coleman, S.J.; Islas-Trejo, A.D.; Medrano, J.F.; Hess, A.M.; Kalbfleisch, T.; Thomas, M.G.; Bouma, G.J.; Bruemmer, J.E. Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy. Genes 2019, 10, 749. https://doi.org/10.3390/genes10100749
Klohonatz KM, Coleman SJ, Islas-Trejo AD, Medrano JF, Hess AM, Kalbfleisch T, Thomas MG, Bouma GJ, Bruemmer JE. Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy. Genes. 2019; 10(10):749. https://doi.org/10.3390/genes10100749
Chicago/Turabian StyleKlohonatz, Kristin M., Stephen J. Coleman, Alma D. Islas-Trejo, Juan F. Medrano, Ann M. Hess, Ted Kalbfleisch, Milton G. Thomas, Gerrit J. Bouma, and Jason E. Bruemmer. 2019. "Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy" Genes 10, no. 10: 749. https://doi.org/10.3390/genes10100749