Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Genome-wide DNA methylation profile of prepubertal porcine testis

Xi Chen A * , Liu-Hong Shen A * , Li-Xuan Gui B * , Fang Yang A , Jie Li A , Sui-Zhong Cao A , Zhi-Cai Zuo A , Xiao-Ping Ma A , Jun-Liang Deng A , Zhi-Hua Ren A , Zhong-Xu Chen B C and Shu-Min Yu A C
+ Author Affiliations
- Author Affiliations

A College of Veterinary Medicine, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang District, Chengdu, 611130, China.

B OnMath Science and Technology Limited Company, No. 500 Tianfu Road, Chengdu, Sichuan, 611130, China.

C Corresponding authors. Emails: czhongxu@gmail.com; yayushumin@163.com

Reproduction, Fertility and Development - https://doi.org/10.1071/RD17067
Submitted: 21 February 2017  Accepted: 17 June 2017   Published online: 21 July 2017

Abstract

The biological structure and function of the mammalian testis undergo important developmental changes during prepuberty and DNA methylation is dynamically regulated during testis development. In this study, we generated the first genome-wide DNA methylation profile of prepubertal porcine testis using methyl-DNA immunoprecipitation (MeDIP) combined with high-throughput sequencing (MeDIP-seq). Over 190 million high-quality reads were generated, containing 43 642 CpG islands. There was an overall downtrend of methylation during development, which was clear in promoter regions but less so in gene-body regions. We also identified thousands of differentially methylated regions (DMRs) among the three prepubertal time points (1 month, T1; 2 months, T2; 3 months, T3), the majority of which showed decreasing methylation levels over time. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that many genes in the DMRs were linked with cell proliferation and some important pathways in porcine testis development. Our data suggest that DNA methylation plays an important role in prepubertal development of porcine testis, with an obvious downtrend of methylation levels from T1 to T3. Overall, our study provides a foundation for future studies and gives new insights into mammalian testis development.

Additional keywords: development, epigenetics, MeDIP-seq, reproduction.


References

Anders, S., Pyl, P. T., and Huber, W. (2015). HTSeq – a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169.
HTSeq – a Python framework to work with high-throughput sequencing data.CrossRef | 1:CAS:528:DC%2BC28Xht1Sjt7vL&md5=4c20f80590246f3a2a7f8719bd5f4213CAS |

Aran, D., Toperoff, G., Rosenberg, M., and Hellman, A. (2011). Replication timing-related and gene body-specific methylation of active human genes. Hum. Mol. Genet. 20, 670–680.
Replication timing-related and gene body-specific methylation of active human genes.CrossRef | 1:CAS:528:DC%2BC3MXpsFygtw%3D%3D&md5=cafc86dfeca468fed02f25b991f83f25CAS |

Archambeault, D. R., and Yao, H. H. (2010). Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion. Proc. Natl. Acad. Sci. USA 107, 10526–10531.
Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion.CrossRef | 1:CAS:528:DC%2BC3cXnvVCiurY%3D&md5=d8b28f1899370fe4e1b97c3f12af54f8CAS |

Avelar, G. F., Oliveira, C. F., Soares, J. M., Silva, I. J., Dobrinski, I., Hess, R. A., and França, L. R. (2010). Postnatal somatic cell proliferation and seminiferous tubule maturation in pigs: a non-random event. Theriogenology 74, 11–23.
Postnatal somatic cell proliferation and seminiferous tubule maturation in pigs: a non-random event.CrossRef |

Behringer, R. R., Finegold, M. J., and Cate, R. L. (1994). Müllerian-inhibiting substance function during mammalian sexual development. Cell 79, 415–425.
Müllerian-inhibiting substance function during mammalian sexual development.CrossRef | 1:CAS:528:DyaK2cXmvFejtLs%3D&md5=556c5a452134cec720aac07944497bd5CAS |

Carrel, L., and Willard, H. F. (2005). X-inactivation profile reveals extensive variability in X-linked gene expression in females. Nature 434, 400–404.
X-inactivation profile reveals extensive variability in X-linked gene expression in females.CrossRef | 1:CAS:528:DC%2BD2MXit1yqsrY%3D&md5=12937b1198f5a68d519e5b348eb51862CAS |

Choi, M., Lee, J., Le, M. T., Nguyen, D. T., Park, S., Soundrarajan, N., Schachtschneider, K. M., Kim, J., Park, J. K., Kim, J. H., and Park, C. (2015). Genome-wide analysis of DNA methylation in pigs using reduced representation bisulfite sequencing. DNA Res. 22, 343–355.
Genome-wide analysis of DNA methylation in pigs using reduced representation bisulfite sequencing.CrossRef | 1:CAS:528:DC%2BC28Xht1CisLnM&md5=7a84a605e545111c4316d41be394826fCAS |

Chung, S. S., Wang, X., and Wolgemuth, D. J. (2016). Prolonged oral administration of a pan-retinoic acid receptor antagonist inhibits spermatogenesis in mice with a rapid recovery and changes in the expression of influx and efflux transporters. Endocrinology 157, 1601–1612.
Prolonged oral administration of a pan-retinoic acid receptor antagonist inhibits spermatogenesis in mice with a rapid recovery and changes in the expression of influx and efflux transporters.CrossRef | 1:CAS:528:DC%2BC28XhtFGgurbO&md5=ed80bc058c97c4c95ccec92c816c8833CAS |

Clark, S. J., Harrison, J., and Frommer, M. (1995). CpNpG methylation in mammalian cells. Nat. Genet. 10, 20–27.
CpNpG methylation in mammalian cells.CrossRef | 1:CAS:528:DyaK2MXlsVymsbk%3D&md5=379e0ad46dc15be6149cd3057568524cCAS |

Colaneri, A., Wang, T., Pagadala, V., Kittur, J., Jr Staffa, N. G., Peddada, S. D., Isganaitis, E., Patti, M. E., and Birnbaumer, L. (2013). A minimal set of tissue-specific hypomethylated CpGs constitute epigenetic signatures of developmental programming. PLoS One 8, e72670.
A minimal set of tissue-specific hypomethylated CpGs constitute epigenetic signatures of developmental programming.CrossRef | 1:CAS:528:DC%2BC3sXhsV2rtbbF&md5=4f35d838f9cd85a31e6c43c0effd3870CAS |

Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., and Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676.
Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.CrossRef | 1:CAS:528:DC%2BD2MXpvFGqt70%3D&md5=f0eb182d91a46c036310171c6b2a8efeCAS |

Deaton, A. M., and Bird, A. (2011). CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022.
| 1:CAS:528:DC%2BC3MXmslOgsLY%3D&md5=335f1a3e5b6215f757a37f85e5146649CAS |

Goldberg, A. D., Allis, C. D., and Bernstein, E. (2007). Epigenetics: a landscape takes shape. Cell 128, 635–638.
Epigenetics: a landscape takes shape.CrossRef | 1:CAS:528:DC%2BD2sXis12ju74%3D&md5=dbb53523d7cd85e32c26c637c503ac35CAS |

Goossens, E., De-Rycke, M., Haentjens, P., and Tournaye, H. (2009). DNA methylation patterns of spermatozoa and two generations of offspring obtained after murine spermatogonial stem cell transplantation. Hum. Reprod. 24, 2255–2263.
DNA methylation patterns of spermatozoa and two generations of offspring obtained after murine spermatogonial stem cell transplantation.CrossRef | 1:CAS:528:DC%2BD1MXhtValsbnF&md5=55aa79f2872f0c0d5ccff601030f1139CAS |

Guibert, S., Forné, T., and Weber, M. (2009). Dynamic regulation of DNA methylation during mammalian development. Epigenomics 1, 81–98.
Dynamic regulation of DNA methylation during mammalian development.CrossRef | 1:CAS:528:DC%2BD1MXhs1WhtLnK&md5=a1b62f7878ff9b07f569277db9632131CAS |

Gupta, R., Nagarajan, A., and Wajapeyee, N. (2010). Advances in genome-wide DNA methylation analysis. Biotechniques 49, iii–xi.
Advances in genome-wide DNA methylation analysis.CrossRef | 1:CAS:528:DC%2BC3MXivVansbs%3D&md5=ee8b8524ad98bf9eb942dbe4b4628b5aCAS |

Gutierrez, K., Dicks, N., Glanzner, W. G., Agellon, L. B., and Bordignon, V. (2015). Efficacy of the porcine species in biomedical research. Front. Genet. 6, 293.
Efficacy of the porcine species in biomedical research.CrossRef |

Harada, Y., Tanaka, N., Ichikawa, M., Kamijo, Y., Sugiyama, E., Gonzalez, F. J., and Aoyama, T. (2016). PPARα-dependent cholesterol/testosterone disruption in Leydig cells mediates 2,4-dichlorophenoxyacetic acid-induced testicular toxicity in mice. Arch. Toxicol. 90, 3061–3071.
PPARα-dependent cholesterol/testosterone disruption in Leydig cells mediates 2,4-dichlorophenoxyacetic acid-induced testicular toxicity in mice.CrossRef | 1:CAS:528:DC%2BC28XhvFOqu7s%3D&md5=ec5ab1ea370daa698c6e79247124b8cbCAS |

Hu, Y., Xu, H., Li, Z., Zheng, X., Jia, X., Nie, Q., and Zhang, X. (2013). Comparison of the genome-wide DNA methylation profiles between fast-growing and slow-growing broilers. PLoS One 8, e56411.
Comparison of the genome-wide DNA methylation profiles between fast-growing and slow-growing broilers.CrossRef | 1:CAS:528:DC%2BC3sXjsFSitb0%3D&md5=0cb87cb380218f4db24bda2a52b4edbbCAS |

Huang, S. Y., Lin, J. H., Teng, S. H., Sun, H. S., Chen, Y. H., Chen, H. H., Liao, J. Y., Chung, M. T., Chen, M. Y., Chuang, C. K., Lin, E. C., and Huang, M. C. (2011). Differential expression of porcine testis proteins during postnatal development. Anim. Reprod. Sci. 123, 221–233.
Differential expression of porcine testis proteins during postnatal development.CrossRef | 1:CAS:528:DC%2BC3MXit1Whs70%3D&md5=2c4e1b520ae870c0a737baa0a630392eCAS |

Illingworth, R. S., and Bird, A. P. (2009). CpG islands – ‘a rough guide’. FEBS Lett. 583, 1713–1720.
CpG islands – ‘a rough guide’.CrossRef | 1:CAS:528:DC%2BD1MXmsVCmsrc%3D&md5=ebe54c2ceda1b02c21d03906c751a2daCAS |

Illingworth, R., Kerr, A., Desousa, D., Jørgensen, H., Ellis, P., Stalker, J., Jackson, D., Clee, C., Plumb, R., Rogers, J., Humphray, S., Cox, T., Langford, C., and Bird, A. (2008). A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biol. 6, e22.
A novel CpG island set identifies tissue-specific methylation at developmental gene loci.CrossRef |

Illingworth, R. S., Gruenewald-Schneider, U., Webb, S., Kerr, A. R., James, K. D., Turner, D. J., Smith, C., Harrison, D. J., Andrews, R., and Bird, A. P. (2010). Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet. 6, e1001134.
Orphan CpG islands identify numerous conserved promoters in the mammalian genome.CrossRef |

Jacinto, F. V., Ballestar, E., and Esteller, M. (2008). Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome. BioTechniques 44, 35–43.
Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome.CrossRef | 1:CAS:528:DC%2BD1cXhsVWrt78%3D&md5=1efb4929c89907713efc56c2e3e9bf41CAS |

Jayachandran, A., Lo, P. H., Chueh, A. C., Prithviraj, P., Molania, R., Davalos-Salas, M., Anaka, M., Walkiewicz, M., Cebon, J., and Behren, A. (2016). Transketolase-like 1 ectopic expression is associated with DNA hypomethylation and induces the Warburg effect in melanoma cells. BMC Cancer 16, 134.
Transketolase-like 1 ectopic expression is associated with DNA hypomethylation and induces the Warburg effect in melanoma cells.CrossRef |

Jjingo, D., Conley, A. B., Yi, S. V., Lunyak, V. V., and Jordan, I. K. (2012). On the presence and role of human gene-body DNA methylation. Oncotarget 3, 462–474.
On the presence and role of human gene-body DNA methylation.CrossRef |

Kim, M. J., Choi, H. W., Jang, H. J., Chung, H. M., Arauzo-Bravo, M. J., Schöler, H. R., and Do, J. T. (2013). Conversion of genomic imprinting by reprogramming and redifferentiation. J. Cell Sci. 126, 2516–2524.
Conversion of genomic imprinting by reprogramming and redifferentiation.CrossRef | 1:CAS:528:DC%2BC3sXht1aqsrbJ&md5=3fa398b32d94cbe40e1e3e8827b1fc2aCAS |

Langmead, B., and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359.
Fast gapped-read alignment with Bowtie 2.CrossRef | 1:CAS:528:DC%2BC38Xjt1Oqt7c%3D&md5=a13f2ca64e62bfc24f7022f40e3e0883CAS |

Li, Q., Li, N., Hu, X., Li, J., Du, Z., Chen, L., Yin, G., Duan, J., Zhang, H., Zhao, Y., Wang, J., Wang, J., and Li, N. (2011). Genome-wide mapping of DNA methylation in chicken. PLoS One 6, e19428.
Genome-wide mapping of DNA methylation in chicken.CrossRef | 1:CAS:528:DC%2BC3MXmtFWisbw%3D&md5=97413531a6c82b97366938a595a8db8aCAS |

Liang, P., Song, F., Ghosh, S., Morien, E., Qin, M., Mahmood, S., Fujiwara, K., Igarashi, J., Nagase, H., and Held, W. A. (2011). Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development. BMC Genomics 12, 231.
Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development.CrossRef | 1:CAS:528:DC%2BC3MXmtlKlsLY%3D&md5=cf570564f12733c0346148f133a32c82CAS |

Lokk, K., Modhukur, V., Rajashekar, B., Märtens, K., Mägi, R., Kolde, R., Koltšina, M., Nilsson, T. K., Vilo, J., Salumets, A., and Tõnisson, N. (2014). DNA methylome profiling of human tissues identifies global and tissue-specific methylation patterns. Genome Biol. 15, 3248.
DNA methylome profiling of human tissues identifies global and tissue-specific methylation patterns.CrossRef |

Malyshev, I. Y. (2015). Epigenetic, post-transcriptional and metabolic mechanisms of macrophage reprogramming. Patol. Fiziol. Eksp. Ter. 3, 118–127.

Maunakea, A. K., Nagarajan, R. P., Bilenky, M., Ballinger, T. J., D’Souza, C., Fouse, S. D., Johnson, B. E., Hong, C., Nielsen, C., Zhao, Y., Turecki, G., Delaney, A., Varhol, R., Thiessen, N., Shchors, K., Heine, V. M., Rowitch, D. H., Xing, X., Fiore, C., Schillebeeckx, M., Jones, S. J. M., Haussler, D., Marra, M. A., Hirst, M., Wang, T., and Costello, J. F. (2010). Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257.
Conserved role of intragenic DNA methylation in regulating alternative promoters.CrossRef | 1:CAS:528:DC%2BC3cXosVKiu70%3D&md5=65f8a9b6c082c9acdc4fb4a66dd58a49CAS |

Ministry of Science and Technology of China (2004). The Regulations for the Administration of Affairs Concerning Experimental Animals. Laboratory Animal Science and Administration.

Moreno, S. G., Attali, M., Allemand, I., Messiaen, S., Fouchet, P., Coffigny, H., Romeo, P. H., and Habert, R. (2010). TGF-beta signaling in male germ cells regulates gonocyte quiescence and fertility in mice. Dev. Biol. 342, 74–84.
TGF-beta signaling in male germ cells regulates gonocyte quiescence and fertility in mice.CrossRef | 1:CAS:528:DC%2BC3cXlvVegu7Y%3D&md5=3d32b014f29f0a0bf2dc62b685706d49CAS |

Niederberger, C. (2012). Re: oral administration of a retinoic acid receptor antagonist reversibly inhibits spermatogenesis in mice. J. Urol. 187, 1509–1519.

Oakes, C. C., La-Salle, S., Smiraglia, D. J., Robaire, B., and Trasler, J. M. (2007). A unique configuration of genome-wide DNA methylation patterns in the testis. Proc. Natl. Acad. Sci. USA 104, 228–233.
A unique configuration of genome-wide DNA methylation patterns in the testis.CrossRef | 1:CAS:528:DC%2BD2sXjt1Oiug%3D%3D&md5=b08fee4d60c42265de4ee39d5e6bd424CAS |

Richter, A. M., Walesch, S. K., and Dammann, R. H. (2016). Aberrant promoter methylation of the tumour suppressor RASSF10 and its growth inhibitory function in breast cancer. Cancers (Basel) 8, 26.
Aberrant promoter methylation of the tumour suppressor RASSF10 and its growth inhibitory function in breast cancer.CrossRef |

Rollins, R. A., Haghighi, F., Edwards, J. R., Das, R., Zhang, M. Q., Ju, J., and Bestor, T. H. (2006). Large-scale structure of genomic methylation patterns. Genome Res. 16, 157–163.
Large-scale structure of genomic methylation patterns.CrossRef | 1:CAS:528:DC%2BD28XhsFahsLk%3D&md5=1169f77022d34a850ecac44a0e2e7a7bCAS |

Jaenisch, R., and Bird, A. (2003). Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33, 245–254.
| 1:CAS:528:DC%2BD3sXhsV2kt7s%3D&md5=e33291d4da3e94f8de6d1f3a674a4e69CAS |

Schachtschneider, K. M., Madsen, O., Park, C., Rund, L. A., Groenen, M. A., and Schook, L. B. (2015). Adult porcine genome-wide DNA methylation patterns support pigs as a biomedical model. BMC Genomics 16, 743.
Adult porcine genome-wide DNA methylation patterns support pigs as a biomedical model.CrossRef |

Shi, L., Song, R., Yao, X., and Ren, Y. (2017). Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro. Theriogenology 93, 24–32.
Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro.CrossRef | 1:CAS:528:DC%2BC2sXitFGht78%3D&md5=7e525b4a32c5fdc8d6ef978622abeb12CAS |

Sofikitis, N., Giotitsas, N., Tsounapi, P., Baltogiannis, D., Giannakis, D., and Pardalidis, N. (2008). Hormonal regulation of spermatogenesis and spermiogenesis. J. Steroid Biochem. Mol. Biol. 109, 323–330.
Hormonal regulation of spermatogenesis and spermiogenesis.CrossRef | 1:CAS:528:DC%2BD1cXmsVeitLc%3D&md5=6ab055996484bfb20446408cf886cf0dCAS |

Song, F., Mahmood, S., Ghosh, S., Liang, P., Smiraglia, D. J., Nagase, H., and Held, W. A. (2009). Tissue specific differentially methylated regions (TDMR): changes in DNA methylation during development. Genomics 93, 130–139.
Tissue specific differentially methylated regions (TDMR): changes in DNA methylation during development.CrossRef | 1:CAS:528:DC%2BD1MXmvVCmsw%3D%3D&md5=7bf0da968adbb9a5604b2957fb3fc34aCAS |

Su, J., Wang, Y., Xing, X., Liu, J., and Zhang, Y. (2014). Genome-wide analysis of DNA methylation in bovine placentas. BMC Genomics 15, 12.
Genome-wide analysis of DNA methylation in bovine placentas.CrossRef |

Suzuki, M. M., and Bird, A. (2008). DNA methylation landscapes: provocative insights from epigenomics. Nat. Rev. Genet. 9, 465–476.
DNA methylation landscapes: provocative insights from epigenomics.CrossRef | 1:CAS:528:DC%2BD1cXlvFKrtL0%3D&md5=c76f05f91b4cc2aeb39f0df2678320c2CAS |

Weber, M., Hellmann, I., Stadler, M. B., Ramos, L., Pääbo, S., Rebhan, M., and Schübeler, D. (2007). Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat. Genet. 39, 457–466.
Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.CrossRef | 1:CAS:528:DC%2BD2sXjsV2hsrk%3D&md5=d87863fb90352a0ff920a546584f4bcbCAS |

Xie, C., Mao, X., Huang, J., Ding, Y., Wu, J., Dong, S., Kong, L., Gao, G., Li, C., and Wei, L. (2011). KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39, W316–W322.
KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases.CrossRef | 1:CAS:528:DC%2BC3MXosVOmsr4%3D&md5=c479fa2be32aa9b053f234069095698dCAS |

Yang, C., Zhang, M., Niu, W., Yang, R., Zhang, Y., Qiu, Z., Sun, B., and Zhao, Z. (2011). Analysis of DNA methylation in various swine tissues. PLoS One 6, e16229.
Analysis of DNA methylation in various swine tissues.CrossRef | 1:CAS:528:DC%2BC3MXhsFWitLY%3D&md5=257909c20a71b5709526f02c93dd787dCAS |

Yao, C., Liu, Y., Sun, M., Niu, M., Yuan, Q., Hai, Y., Guo, Y., Chen, Z., Hou, J., Liu, Y., and He, Z. (2015). MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis. Reproduction 150, R25–R34.
MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis.CrossRef | 1:CAS:528:DC%2BC2MXhsV2ktL3L&md5=328561d39d9d7ab0af2dabb1bbe3722cCAS |

Ye, J., Fang, L., Zheng, H., Zhang, Y., Chen, J., Zhang, Z., Wang, J., Li, S., Li, R., Bolund, L., and Wang, J. (2006). WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34, W293–W297.
WEGO: a web tool for plotting GO annotations.CrossRef | 1:CAS:528:DC%2BD28Xps1yisLc%3D&md5=72549bd922fb6376c33a17b6895df078CAS |

Young, M. D., Wakefield, M. J., Smyth, G. K., and Oshlack, A. (2010). Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14.
Gene ontology analysis for RNA-seq: accounting for selection bias.CrossRef |

Zhou, Z. Y., Li, A., Wang, L. G., Irwin, D. M., Liu, Y. H., Xu, D., Han, X. M., Wang, L., Wu, S. F., Wang, L. X., Xie, H. B., and Zhang, Y. P. (2015). DNA methylation signatures of long intergenic non-coding RNAs in porcine adipose and muscle tissues. Sci. Rep. 5, 15435.
DNA methylation signatures of long intergenic non-coding RNAs in porcine adipose and muscle tissues.CrossRef | 1:CAS:528:DC%2BC2MXhslans73J&md5=c2b917b29f413c1ac51dd29385e96f24CAS |



Supplementary MaterialSupplementary Material (133 KB) Export Citation