Single-cell RNA sequencing reveals the intercellular crosstalk and the regulatory landscape of stromal cells during the whole life of the mouse ovary

Abstract

The heterogeneity of ovarian mesenchymal/stromal cells has just been revealed in both mice and humans. However, it remains unclear about the cellular development trace and the intercellular communication network in the whole life of the ovary. In the study, we integrated ours and published single-cell RNA sequencing data from E11.5 (embryonic day 11.5) until M12 (12-month-old) ovaries to show the dynamics of somatic cells along the developmental timeline. The intercellular crosstalk among somatic cell types was depicted with collagen signaling pathway as the most outgoing signals from stromal cells. We identified mesenchymal progenitor cells (CD24⁺) as the origin of stromal cells. Although their numbers decreased significantly in adults, the cells served as the major signal sender until ovarian senescence. Moreover, the ovarian injury could activate these stem cells and induce stroma remodeling in the aged ovary. Thus, mesenchymal progenitor cells may represent a new strategy to delay ovarian aging in the future.

Keywords

single-cell RNA sequencing / intercellular crosstalk / ovarian aging / stromal cell / mouse ovary

References

[1] Sarraj MA , Drummond AE . Mammalian foetal ovarian development: consequences for health and disease. Reproduction 2012; 143: 151- 63.

[2] Stevant I , Kühne F , Greenfield A , et al. Dissecting cell lineage specification and sex fate determination in gonadal somatic cells using single-cell transcriptomics. Cell Rep 2019; 26: 3272- 83.e3.

[3] Hirshfield AN . Development of follicles in the mammalian ovary. Int Rev Cytol 1991; 124: 43- 101.

[4] Jagarlamudi K , Rajkovic A . Oogenesis: transcriptional regulators and mouse models. Mol Cell Endocrinol 2012; 356: 31- 9.

[5] Hsueh EAMAJW . Initial and cyclic recruitment of ovarian follicles. Endocr Rev 2000; 21: 200- 14.

[6] Kinnear HM , Tomaszewski CE , Chang AL , et al. The ovarian stroma as a new frontier. Reproduction 2020; 160: R25- 39.

[7] Fan X , Bialecka M , Moustakas I , et al. Single-cell reconstruction of follicular remodeling in the human adult ovary. Nat Commun 2019; 10: 3164.

[8] Wang S , Zheng Y , Li J , et al. Single-cell transcriptomic atlas of primate ovarian aging. Cell 2020; 180: 585- 600.e19.

[9] Niu W , Spradling AC . Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary. Proc Natl Acad Sci U S A 2020; 117: 20015- 26.

[10] Morris ME , Meinsohn M-C , Chauvin M , et al. A single-cell atlas of the cycling murine ovary. eLife 2022; 11: e77239.

[11] Ge W , Wang J-J , Zhang R-Q , et al. Dissecting the initiation of female meiosis in the mouse at single-cell resolution. Cell Mol Life Sci 2021; 78: 695- 713.

[12] Wang JJ , Ge W , Zhai Q-Y , et al. Single-cell transcriptome landscape of ovarian cells during primordial follicle assembly in mice. PLoS Biol 2020; 18: e3001025.

[13] McInnes L , Healy J , Melville J . Umap: Uniform manifold approximation and projection for dimension reduction. arXiv preprint arXiv: 1802.03426, 2018.

[14] Xu C , Cao Y , Bao J . Building RNA-protein germ granules: insights from the multifaceted functions of DEAD-box helicase Vasa/Ddx4 in germline development. Cell Mol Life Sci 2021; 79: 4.

[15] Zhao Y , Du Y , Ge Q , et al. Identification and expression analysis of Dazl homologue in Cynops cyanurus. Zygote 2022; 30: 221- 6.

[16] Kuriyama S , Tamiya Y , Tanaka M . Spatiotemporal expression of UPK3B and its promoter activity during embryogenesis and spermatogenesis. Histochem Cell Biol 2017; 147: 17- 26.

[17] Kuony A , Michon F . Epithelial Markers aSMA, Krt14, and Krt19 unveil elements of murine lacrimal gland morphogenesis and maturation. Front Physiol 2017; 8: 739.

[18] Jones RL , Pepling ME . KIT signaling regulates primordial follicle formation in the neonatal mouse ovary. Dev Biol 2013; 382: 186- 97.

[19] Wang Z , Niu W , Wang Y , et al. Follistatin288 regulates germ cell cyst breakdown and primordial follicle assembly in the mouse ovary. PLoS One 2015; 10: e0129643.

[20] Jameson SA , Natarajan A , Cool J , et al. Temporal transcriptional profiling of somatic and germ cells reveals biased lineage priming of sexual fate in the fetal mouse gonad. PLoS Genet 2012; 8: e1002575.

[21] Kakuta H , Iguchi T , Sato T . The involvement of granulosa cells in the regulation by gonadotropins of Cyp17a1 in Theca Cells. In Vivo 2018; 32: 1387- 401.

[22] Rastetter RH , Bernard P , Palmer JS , et al. Marker genes identify three somatic cell types in the fetal mouse ovary. Dev Biol 2014; 394: 242- 52.

[23] Ku CH , Johnson PH , Batten P , et al. Collagen synthesis by mesenchymal stem cells and aortic valve interstitial cells in response to mechanical stretch. Cardiovasc Res 2006; 71: 548- 56.

[24] Choi KD , Vodyanik MA , Togarrati PP , et al. Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures. Cell Rep 2012; 2: 553- 67.

[25] Privratsky JR , Newman PJ . PECAM-1: regulator of endothelial junctional integrity. Cell Tissue Res 2014; 355: 607- 19.

[26] Michelini S , Ricci M , Amato B , et al. CDH5, a possible new candidate gene for genetic testing of lymphedema. Lymphat Res Biol 2022; 20: 496- 506.

[27] Medina-Contreras O , Geem D , Laur O , et al. CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice. J Clin Invest 2011; 121: 4787- 95.

[28] Lewis SE , Erickson RP , Barnett LB , et al. N-ethyl-N-nitrosourea-induced null mutation at the mouse Car-2 locus: an animal model for human carbonic anhydrase II deficiency syndrome. Proc Natl Acad Sci U S A 1988; 85: 1962- 6.

[29] Schwalie PC , Dong H , Zachara M , et al. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature 2018; 559: 103- 8.

[30] Xie T , Wang Y , Deng N , et al. Single-cell deconvolution of fibroblast heterogeneity in mouse pulmonary fibrosis. Cell Rep 2018; 22: 3625- 40.

[31] Rosenberg AB , Roco CM , Muscat RA , et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 2018; 360: 176- 82.

[32] Zheng W , Zhang H , Gorre N , et al. Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions. Hum Mol Genet 2014; 23: 920- 8.

[33] Zheng W , Zhang H , Liu K . The two classes of primordial follicles in the mouse ovary: their development, physiological functions and implications for future research. Mol Hum Reprod 2014; 20: 286- 92.

[34] Tesfaye D , Ghanem N , Carter F , et al. Gene expression profile of cumulus cells derived from cumulusoocyte complexes matured either in vivo or in vitro. Reprod Fertil Dev 2009; 21: 451- 61.

[35] Guan S , Xie L , Ma T , et al. Effects of melatonin on early pregnancy in mouse: involving the regulation of StAR, Cyp11a1, and Ihh expression. Int J Mol Sci 2017; 18: 1637.

[36] Liu C , Peng J , Matzuk MM , et al. Lineage specification of ovarian theca cells requires multicellular interactions via oocyte and granulosa cells. Nat Commun 2015; 6: 6934.

[37] Hatzirodos N , Hummitzsch K , Irving-Rodgers HF , et al. Transcriptome comparisons identify new cell markers for theca interna and granulosa cells from small and large antral ovarian follicles. PLoS One 2015; 10: e0119800.

[38] Lavoie HA , King SR . Transcriptional regulation of steroidogenic genes: STARD1, CYP11A1 and HSD3B. Exp Biol Med (Maywood) 2009; 234: 880- 907.

[39] Hudson QJ , Ashjaei K , Perricos A , et al. Endometriosis patients show an increased M2 response in the peritoneal CD14(+low)/CD68(+low) macrophage subpopulation coupled with an increase in the T-helper 2 and T-regulatory cells. Reprod Sci 2020; 27: 1920- 31.

[40] Ponichtera HE , Shainheit MG , Liu BC , et al. CD209a expression on dendritic cells is critical for the development of pathogenic Th17 cell responses in murine schistosomiasis. J Immunol 2014; 192: 4655- 65.

[41] Jin S , Guerrero-Juarez CF , Zhang L , et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun 2021; 12: 1088.

[42] Ikeda S , Yamada M . Midkine and cytoplasmic maturation of mammalian oocytes in the context of ovarian follicle physiology. Br J Pharmacol 2014; 171: 827- 36.

[43] Muramatsu H , Zou P , Kurosawa N , et al. Female infertility in mice deficient in midkine and pleiotrophin, which form a distinct family of growth factors. Genes to Cells 2006; 11: 1405- 17.

[44] York, JP , Ren YA , Zeng J , Bin Zhang , Wang F , Chen R , Liu J , Xia X , Zhang P . Growth Arrest Specific 2 (GAS2) is a critical mediator of germ cell cyst breakdown and folliculogenesis in mice. Sci Rep 2016; 6: 34956.

[45] Ollinger R , Reichmann J , Adams IR . Meiosis and retrotransposon silencing during germ cell development in mice. Differentiation 2010; 79: 147- 58.

[46] Xiao Y , Peng X , Peng Y , et al. Macrophage-derived extracellular vesicles regulate follicular activation and improve ovarian function in old mice by modulating local environment. Clin Transl Med 2022; 12: e1071.

[47] Briley SM , Jasti S , McCracken JM , et al. Reproductive age-associated fibrosis in the stroma of the mammalian ovary. Reproduction 2016; 152: 245- 60.

[48] Cuff CA , Puré E . A crucial role for CD44 in inflammation. Trends Mol Med 2001; 7: 213- 21.

[49] Langfelder P , Horvath S . WGCNA: an R package for weighted correlation network analysis. BMC Bioinf 2008; 9: 559.

[50] Liu R , Zhang X , Fan Z , et al. Human amniotic mesenchymal stem cells improve the follicular microenvironment to recover ovarian function in premature ovarian failure mice. Stem Cell Res Ther 2019; 10: 299.

[51] Morris ME , Meinsohn M-C , Chauvin M , et al. A single-cell atlas of the cycling murine ovary. Elife 2022; 11: e77239.

[52] Sheng X , Zhou J , Kang N , et al. Temporal and spatial dynamics mapping reveals follicle development regulated by different stromal cell populations. bioRxiv 2022: p. 2022.03.04.480328.

[53] Guahmich NL , Man L , Wang J , et al. Human theca arises from ovarian stroma and is comprised of three discrete subtypes. Commun Biol 2023; 6: 7.

[54] Liu T , Qin Q-Y , Qu J-X , et al. Where are the theca cells from: the mechanism of theca cells derivation and differentiation. Chin Med J (Engl) 2020; 133: 1711- 8.

[55] Talbott HE , Mascharak S , Griffin M , et al. Wound healing, fibroblast heterogeneity, and fibrosis. Cell Stem Cell 2022; 29: 1161- 80.

[56] Umehara T , Winstanley YE , Andreas E , et al. Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Sci Adv 2022; 8: eabn4564.

[57] Lynch MD , Watt FM . Fibroblast heterogeneity: implications for human disease. J Clin Invest 2018; 128: 26- 35.

[58] Wetzig A , Alaiya A , Al-Alwan M , et al. Differential marker expression by cultures rich in mesenchymal stem cells. BMC Cell Biol 2013; 14: 54.

[59] Romagnani P , Kalluri R . Possible mechanisms of kidney repair. Fibrogenesis Tissue Repair 2009; 2: 3.

[60] Shapira S , Ben-Amotz O , Sher O , et al. Delayed wound healing in heat stable antigen (HSA/CD24)-deficient mice.PLoS One 2015; 10: e0139787.

[61] Avivi-Arber L , Avivi D , Perez M , et al. Impaired bone healing at tooth extraction sites in CD24-deficient mice: a pilot study. PLoS One 2018; 13: e0191665.

[62] Yang W , Zhang J , Xu B , et al. HucMSC-derived exosomes mitigate the age-related retardation of fertility in female mice. Mol Ther 2020; 28: 1200- 13.

[63] Zolbin MM , Ersoy GS , Aliakbari F , et al. Basal characterization and in vitro differentiation of putative stem cells derived from the adult mouse ovary. In Vitro Cell Dev Biol Animal 2020; 56: 59- 66.

[64] Stimpfel M , Cerkovnik P , Novakovic S , et al. Putative mesenchymal stem cells isolated from adult human ovaries. J Assist Reprod Genet 2014; 31: 959- 74.

[65] He Y , Peng X , Wu T , et al. Restricting the induction of NGF in ovarian stroma engenders selective follicular activation through the mTOR signaling pathway. Cell Death Dis 2017; 8: e2817.

[66] Satija R , Farrell JA , Gennert D , et al. Spatial reconstruction of single-cell gene expression data. Nat Biotechnol 2015; 33: 495- 502.

[67] McGinnis CS , Murrow LM , Gartner ZJ . DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst 2019; 8: 329- 37.e4.

[68] Jiao X , Sherman BT , Huang DW , et al. DAVID-WS: a stateful web service to facilitate gene/protein list analysis. Bioinformatics 2012; 28: 1805- 6.

[69] Cao J , Spielmann M , Qiu X , et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 2019; 566: 496- 502.

[70] Flaws JA , Abbud R , Mann RJ , et al. Chronically elevated luteinizing hormone depletes primordial follicles in the mouse ovary. Biol Reprod 1997; 57: 1233- 7.

[71] Pedersen T . Determination of follicle growth rate in the ovary of the immature mouse. J Reprod Fertil 1970; 21: 81- 93.

RIGHTS & PERMISSIONS

2024 The Author(s). Published by Oxford University Press on behalf of Higher Education Press.