Generation of developmentally competent oocytes and fertile mice from parthenogenetic embryonic stem cells

Abstract

Parthenogenetic embryos, created by activation and diploidization of oocytes, arrest at mid-gestation for defective paternal imprints, which impair placental development. Also, viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos, presumably attributable to their aberrant imprinting. We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring. Moreover, normal expression of imprinted genes is found in the germ cells and the mice. pESCs exhibited imprinting consistent with exclusively maternal lineage, and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background. pESCs differentiated into primordial germ cell-like cells (PGCLCs) and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function. The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs, consistent with efficient reprogramming of methylation and genomic imprinting. These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting, offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.

Key words： parthenogenetic embryonic stem cells; primordial germ cell-like cells; imprinting; meiosis; oocytes

References

1 Baker SM, Plug AW, Prolla TA, Bronner CE, Harris AC, Yao X, Christie DM, Monell C, Arnheim N, Bradley A (1996) Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nat Genet 13: 336- 342

2 Barlow DP, Bartolomei MS (2014) Genomic imprinting in mammals. Cold Spring Harb Perspect Biol. https://doi.org/10.1101/cshperspect.a018382

3 Barton SC, Surani MA, Norris ML (1984) Role of paternal and maternal genomes in mouse development. Nature 311: 374- 376

4 Blasco MA, Serrano M, Fernandez-Capetillo O (2011) Genomic instability in iPS: time for a break. EMBO J 30: 991- 993

5 Buccione R, Schroeder AC, Eppig JJ (1990) Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biol Reprod 43: 543- 547

6 Chen Z, Liu Z, Huang J, Amano T, Li C, Cao S, Wu C, Liu B, Zhou L, Carter MG (2009) Birth of parthenote mice directly from parthenogenetic embryonic stem cells. Stem Cells 27: 2136- 2145

7 Chuva de Sousa Lopes SM, Hayashi K, Shovlin TC, Mifsud W, Surani MA, McLaren A (2008) X chromosome activity in mouse XX primordial germ cells. PLoS Genet 4: e30

8 D’Antonio M, Benaglio P, Jakubosky D, Greenwald WW, Matsui H, Donovan MKR, Li H, Smith EN, D’Antonio-Chronowska A, Frazer KA (2018) Insights into the mutational burden of human induced pluripotent stem cells from an integrative multi-omics approach. Cell Rep 24: 883- 894

9 Davis TL, Yang GJ, McCarrey JR, Bartolomei MS (2000) The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development. Hum Mol Genet 9: 2885- 2894

10 De Sousa PA, Wilmut I (2007) Human parthenogenetic embryo stem cells: appreciating what you have when you have it. Cell Stem Cell 1: 243- 244

11 De Bonis ML, Ortega S, Blasco MA (2014) SIRT1 is necessary for proficient telomere elongation and genomic stability of induced pluripotent stem cells. Stem Cell Rep 2: 690- 706

12 Didie M, Christalla P, Rubart M, Muppala V, Doker S, Unsold B, ElArmouche A, Rau T, Eschenhagen T, Schwoerer AP (2013) Parthenogenetic stem cells for tissue-engineered heart repair. J Clin Investig 123: 1285- 1298

13 Edelmann W, Cohen PE, Kane M, Lau K, Morrow B, Bennett S, Umar A, Kunkel T, Cattoretti G, Chaganti R (1996) Meiotic pachytene arrest in MLH1-deficient mice. Cell 85: 1125- 1134

14 Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM III, Yanagimachi R, Jaenisch R (2001) Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci USA 98: 6209- 6214

15 Eppig JJ, O’Brien MJ, Wigglesworth K, Nicholson A, Zhang W, King BA (2009) Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring. Hum Reprod 24: 922- 928

16 Espejel S, Eckardt S, Harbell J, Roll GR, McLaughlin KJ, Willenbring H (2014) Brief report: parthenogenetic embryonic stem cells are an effective cell source for therapeutic liver repopulation. Stem Cells 32: 1983- 1988

17 Gao S, Zheng C, Chang G, Liu W, Kou X, Tan K, Tao L, Xu K, Wang H, Cai J (2015) Unique features of mutations revealed by sequentially reprogrammed induced pluripotent stem cells. Nat Commun 6: 6318

18 Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ (2004) Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427: 148- 154

19 Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E (2011) Somatic coding mutations in human induced pluripotent stem cells. Nature 471: 63- 67

20 Grive KJ, Freiman RN (2015) The developmental origins of the mammalian ovarian reserve. Development 142: 2554- 2563

21 Hackett JA, Huang Y, Gunesdogan U, Gretarsson KA, Kobayashi T, Surani MA (2018) Tracing the transitions from pluripotency to germ cell fate with CRISPR screening. Nat Commun 9: 4292

22 Hajkova P, Erhardt S, Lane N, Haaf T, El-Maarri O, Reik W, Walter J, Surani MA (2002) Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 117: 15- 23

23 Handel MA, Eppig JJ, Schimenti JC (2014) Applying “Gold Standards” to in-vitro-derived germ cells. Cell 159: 216

24 Hayashi K, Saitou M (2013) Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat Protoc 8: 1513- 1524

25 Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M (2012) Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338: 971- 975

26 Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shimamoto S, Imamura T, Nakashima K, Saitou M (2016) Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature 539: 299- 303

27 Hochberg Y, Benjamini Y (1990) More powerful procedures for multiple significance testing. Stat Med 9: 811- 818

28 Huang J, Deng K, Wu H, Liu Z, Chen Z, Cao S, Zhou L, Ye X, Keefe DL, Liu L (2008) Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation. Stem Cells 26: 1883- 1890

29 Huang K, Maruyama T, Fan G (2014) The naive state of human pluripotent stem cells: a synthesis of stem cell and preimplantation embryo transcriptome analyses. Cell Stem Cell 15: 410- 415

30 Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA (2015) SOX17 is a critical specifier of human primordial germ cell fate. Cell 160: 253- 268

31 Johannesson B, Sagi I, Gore A, Paull D, Yamada M, Golan-Lev T, Li Z, LeDuc C, Shen Y, Stern S (2014) Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors. Cell Stem Cell 15: 634- 642

32 Kagiwada S, Kurimoto K, Hirota T, Yamaji M, Saitou M (2013) Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice. EMBO J 32: 340- 353

33 Kilpinen H, Goncalves A, Leha A, Afzal V, Alasoo K, Ashford S, Bala S, Bensaddek D, Casale FP, Culley OJ (2017) Common genetic variation drives molecular heterogeneity in human iPSCs. Nature 546: 370- 375

34 Kim K, Lerou P, Yabuuchi A, Lengerke C, Ng K, West J, Kirby A, Daly MJ, Daley GQ (2007) Histocompatible embryonic stem cells by parthenogenesis. Science 315: 482- 486

35 Li C, Chen Z, Liu Z, Huang J, Zhang W, Zhou L, Keefe DL, Liu L (2009) Correlation of expression and methylation of imprinted genes with pluripotency of parthenogenetic embryonic stem cells. Hum Mol Genet 18: 2177- 2187

36 Li Z, Wan H, Feng G, Wang L, He Z, Wang Y, Wang XJ, Li W, Zhou Q, Hu B (2016) Birth of fertile bimaternal offspring following intracytoplasmic injection of parthenogenetic haploid embryonic stem cells. Cell Res 26: 135- 138

37 Lin G, OuYang Q, Zhou X, Gu Y, Yuan D, Li W, Liu G, Liu T, Lu G (2007) A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure. Cell Res 17: 999- 1007

38 Liu L, Trimarchi JR, Keefe DL (2002) Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos. Biol Reprod 66: 204- 210

39 Liu L, Franco S, Spyropoulos B, Moens PB, Blasco MA, Keefe DL (2004) Irregular telomeres impair meiotic synapsis and recombination in mice. Proc Natl Acad Sci USA 101: 6496- 6501

40 Liu Z, Hu Z, Pan X, Li M, Togun TA, Tuck D, Pelizzola M, Huang J, Ye X, Yin Y (2011) Germline competency of parthenogenetic embryonic stem cells from immature oocytes of adult mouse ovary. Hum Mol Genet 20: 1339- 1352

41 Liu M, Yin Y, Ye X, Zeng M, Zhao Q, Keefe DL, Liu L (2013) Resveratrol protects against age-associated infertility in mice. Hum Reprod 28: 707- 717

42 Lucifero D, Mertineit C, Clarke HJ, Bestor TH, Trasler JM (2002) Methylation dynamics of imprinted genes in mouse germ cells. Genomics 79: 530- 538

43 Mai Q, Yu Y, Li T, Wang L, Chen MJ, Huang SZ, Zhou C, Zhou Q (2007) Derivation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res 17: 1008- 1019

44 Miyauchi H, Ohta H, Nagaoka S, Nakaki F, Sasaki K, Hayashi K, Yabuta Y, Nakamura T, Yamamoto T, Saitou M (2017) Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice. EMBO J 36: 3100- 3119

45 Miyoshi N, Stel JM, Shioda K, Qu N, Odajima J, Mitsunaga S, Zhang X, Nagano M, Hochedlinger K, Isselbacher KJ (2016) Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells. Proc Natl Acad Sci USA 113: 9545- 9550

46 Moses RM, Kline D (1995) Calcium-independent, meiotic spindledependent metaphase-to-interphase transition in phorbol estertreated mouse eggs. Dev Biol 171: 111- 122

47 Nagaoka SI, Hassold TJ, Hunt PA (2012) Human aneuploidy:mechanisms and new insights into an age-old problem. Nat Rev Genet 13: 493- 504

48 Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC (1993) Derivation of completely cell culture-derived mice from earlypassage embryonic stem cells. Proc Natl Acad Sci USA 90: 8424- 8428

49 Ohinata Y, Ohta H, Shigeta M, Yamanaka K, Wakayama T, Saitou M (2009) A signaling principle for the specification of the germ cell lineage in mice. Cell 137: 571- 584

50 Ohta H, Kurimoto K, Okamoto I, Nakamura T, Yabuta Y, Miyauchi H, Yamamoto T, Okuno Y, Hagiwara M, Shirane K (2017) In vitro expansion of mouse primordial germ cell-like cells recapitulates an epigenetic blank slate. EMBO J 36: 1888- 1907

51 Pasque V, Tchieu J, Karnik R, Uyeda M, Sadhu Dimashkie A, Case D, Papp B, Bonora G, Patel S, Ho R (2014) X chromosome reactivation dynamics reveal stages of reprogramming to pluripotency. Cell 159: 1681- 1697

52 Picelli S, Faridani OR, Bjorklund AK, Winberg G, Sagasser S, Sandberg R (2014) Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9: 171- 181

53 Qing T, Liu H, Wei W, Ye X, Shen W, Zhang D, Song Z, Yang W, Ding M, Deng H (2008) Mature oocytes derived from purified mouse fetal germ cells. Hum Reprod 23: 54- 61

54 Reik W, Dean W, Walter J (2001) Epigenetic reprogramming in mammalian development. Science 293: 1089- 1093

55 Revazova ES, Turovets NA, Kochetkova OD, Kindarova LB, Kuzmichev LN, Janus JD, Pryzhkova MV (2007) Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 9: 432- 449

56 Sagi I, Chia G, Golan-Lev T, Peretz M, Weissbein U, Sui L, Sauer MV, Yanuka O, Egli D, Benvenisty N (2016) Derivation and differentiation of haploid human embryonic stem cells. Nature 532: 107- 111

57 Sagi I, De Pinho JC, Zuccaro MV, Atzmon C, Golan-Lev T, Yanuka O, Prosser R, Sadowy A, Perez G, Cabral T (2019) Distinct imprinting signatures and biased differentiation of human androgenetic and parthenogenetic embryonic stem cells. Cell Stem Cell 25: 419- 432.e419

58 SanMiguel JM, Bartolomei MS (2018) DNA methylation dynamics of genomic imprinting in mouse development. Biol Reprod 99: 252- 262

59 Sasaki K, Yokobayashi S, Nakamura T, Okamoto I, Yabuta Y, Kurimoto K, Ohta H, Moritoki Y, Iwatani C, Tsuchiya H (2015) Robust in vitro induction of human germ cell fate from pluripotent stem cells. Cell Stem Cell 17: 178- 194

60 Seisenberger S, Andrews S, Krueger F, Arand J, Walter J, Santos F, Popp C, Thienpont B, Dean W, Reik W (2012) The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol Cell 48: 849- 862

61 Shen W, Zhang D, Qing T, Cheng J, Bai Z, Shi Y, Ding M, Deng H (2006) Live offspring produced by mouse oocytes derived from premeiotic fetal germ cells. Biol Reprod 75: 615- 623

62 Sheng X, Tian C, Liu L, Wang L, Ye X, Li J, Zeng M, Liu L (2019) Characterization of oogonia stem cells in mice by Fragilis. Protein Cell 10: 825- 831

63 Shirane K, Kurimoto K, Yabuta Y, Yamaji M, Satoh J, Ito S, Watanabe A, Hayashi K, Saitou M, Sasaki H (2016) Global landscape and regulatory principles of DNA methylation reprogramming for germ cell specification by mouse pluripotent stem cells. Dev Cell 39: 87- 103

64 Stelzer Y, Ronen D, Bock C, Boyle P, Meissner A, Benvenisty N (2013) Identification of novel imprinted differentially methylated regions by global analysis of human-parthenogenetic-induced pluripotent stem cells. Stem Cell Rep 1: 79- 89

65 Surani MA, Barton SC, Norris ML (1984) Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308: 548- 550

66 Swann K, Ozil JP (1994) Dynamics of the calcium signal that triggers mammalian egg activation. Int Rev Cytol 152: 183- 222

67 Tam PP, Zhou SX, Tan SS (1994) X-chromosome activity of the mouse primordial germ cells revealed by the expression of an X-linked lacZ transgene. Development 120: 2925- 2932

68 Tapia N, Scholer HR (2016) Molecular obstacles to clinical translation of iPSCs. Cell Stem Cell 19: 298- 309

69 Theunissen TW, Friedli M, He Y, Planet E, O’Neil RC, Markoulaki S, Pontis J, Wang H, Iouranova A, Imbeault M (2016) Molecular criteria for defining the naive human pluripotent state. Cell Stem Cell 19: 502- 515

70 Tian C, Liu L, Ye X, Fu H, Sheng X, Wang L, Wang H, Heng D, Liu L (2019) Functional oocytes derived from granulosa cells. Cell Rep 29: 4256- 4267.e4259

71 von Meyenn F, Berrens RV, Andrews S, Santos F, Collier AJ, Krueger F, Osorno R, Dean W, Rugg-Gunn PJ, Reik W (2016) Comparative principles of DNA methylation reprogramming during human and mouse in vitro primordial germ cell specification. Dev Cell 39: 104- 115

72 Wang L, Zhang J, Duan J, Gao X, Zhu W, Lu X, Yang L, Zhang J, Li G, Ci W (2014) Programming and inheritance of parental DNA methylomes in mammals. Cell 157: 979- 991

73 Xie W, Barr CL, Kim A, Yue F, Lee AY, Eubanks J, Dempster EL, Ren B (2012) Base-resolution analyses of sequence and parent-oforigin dependent DNA methylation in the mouse genome. Cell 148: 816- 831

74 Yamashiro C, Sasaki K, Yabuta Y, Kojima Y, Nakamura T, Okamoto I, Yokobayashi S, Murase Y, Ishikura Y, Shirane K (2018) Generation of human oogonia from induced pluripotent stem cells in vitro. Science 362: 356- 360

75 Yin Y, Liu N, Ye X, Guo R, Hao J, Wang F, Liu L (2014) Telomere elongation in parthenogenetic stem cells. Protein Cell 5: 8- 11

76 Yoshihara M, Araki R, Kasama Y, Sunayama M, Abe M, Nishida K, Kawaji H, Hayashizaki Y, Murakawa Y (2017) Hotspots of de novo point mutations in induced pluripotent stem cells. Cell Rep 21: 308- 315

77 Yuan L, Liu JG, Zhao J, Brundell E, Daneholt B, Hoog C (2000) The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol Cell 5: 73- 83

78 Zhang H, Liu K (2015) Cellular and molecular regulation of the activation of mammalian primordial follicles: somatic cells initiate follicle activation in adulthood. Hum Reprod Update 21: 779- 786

79 Zhou Q, Wang M, Yuan Y, Wang X, Fu R, Wan H, Xie M, Liu M, Guo X, Zheng Y (2016) Complete meiosis from embryonic stem cell-derived germ cells in vitro. Cell Stem Cell 18: 330- 340