Single-Cell Expression Profiling and Proteomics of Primordial Germ Cells, Spermatogonial Stem Cells, Adult Germ Stem Cells, and Oocytes

  • Sabine Conrad
  • Hossein Azizi
  • Thomas SkutellaEmail author
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1083)


The mammalian germ cells, cell assemblies, tissues, and organs during development and maturation have been extensively studied at the tissue level. However, to investigate and understand the fundamental insights at the molecular basis of germ and stem cells, their cell fate plasticity, and determination, it is of most importance to analyze at the large scale on the single-cell level through different biological windows. Here, modern molecular techniques optimized for single-cell analysis, including single fluorescence-activated cell sorting (FACS) and single-cell RNA sequencing (scRNA-seq) or microfluidic high-throughput quantitative real-time polymerase chain reaction (qRT-PCR) for single-cell gene expression and liquid chromatography coupled to tandem mass spectrometry (LC-MSMS) for protein profiling, have been established and are still getting optimized.

This review aims on describing and discussing recent single-cell expression profiling and proteomics of different types of human germ cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), human adult germ stem cells (haGSCs), and oocytes.


Germ cells Single-cell expression profiling Single-cell proteomics 



Fluorescence-activated cell sorting


Human adult germ stem cell


Human embryonic stem cells


Human fibroblast


Human pluripotent stem cell


Human spermatogonial stem cell


Liquid chromatography mass spectrometry


Magnetic activated cell sorting


Mesenchymal stem cells


Human primordial germ cell


Quantitative real-time polymerase chain reaction


Single-cell RNA sequencing


Human spermatogonial stem cell


  1. Chikhovskaya, J. V., Jonker, M. J., Meissner, A., Breit, T. M., Repping, S., & van Pelt, A. M. (2012). Human testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mesenchymal progenitors. Human Reproduction, 27, 210–221.CrossRefPubMedGoogle Scholar
  2. Choi, I., Carey, T. S., Wilson, C. A., & Knott, J. G. (2012). Transcription factor AP-2gamma is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis. Development, 139, 4623–4632.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Conrad, S., Azizi, H., Hatami, M., Kubista, M., Bonin, M., Hennenlotter, J., Sievert, K. D., & Skutella, T. (2016). Expression of genes related to germ cell lineage and Pluripotency in single cells and colonies of human adult germ stem cells. Stem Cells International, 2016, 8582526.CrossRefPubMedGoogle Scholar
  4. Gerovska, D., & Arauzo-Bravo, M. J. (2016). Does mouse embryo primordial germ cell activation start before implantation as suggested by single-cell transcriptomics dynamics? Molecular Human Reproduction, 22, 208–225.CrossRefPubMedGoogle Scholar
  5. Giritharan, G., Li, M. W., Di Sebastiano, F., Esteban, F. J., Horcajadas, J. A., Lloyd, K. C., Donjacour, A., Maltepe, E., & Rinaudo, P. F. (2010). Effect of ICSI on gene expression and development of mouse preimplantation embryos. Human Reproduction, 25, 3012–3024.CrossRefPubMedGoogle Scholar
  6. Gkountela, S., Li, Z., Vincent, J. J., Zhang, K. X., Chen, A., Pellegrini, M., & Clark, A. T. (2013). The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nature Cell Biology, 15, 113–122.CrossRefPubMedGoogle Scholar
  7. Gonzalez, R., Griparic, L., Vargas, V., Burgee, K., Santacruz, P., Anderson, R., Schiewe, M., Silva, F., & Patel, A. (2009). A putative mesenchymal stem cells population isolated from adult human testes. Biochemical and Biophysical Research Communications, 385, 570–575.CrossRefPubMedGoogle Scholar
  8. Graf, T., & Enver, T. (2009). Forcing cells to change lineages. Nature, 462, 587–594.CrossRefPubMedGoogle Scholar
  9. Grassetti, D., Paoli, D., Gallo, M., D’Ambrosio, A., Lombardo, F., Lenzi, A., & Gandini, L. (2012). Protamine-1 and -2 polymorphisms and gene expression in male infertility: An Italian study. Journal of Endocrinological Investigation, 35, 882–888.PubMedGoogle Scholar
  10. Grindberg, R. V., Yee-Greenbaum, J. L., McConnell, M. J., Novotny, M., O’Shaughnessy, A. L., Lambert, G. M., Arauzo-Bravo, M. J., Lee, J., Fishman, M., Robbins, G. E., et al. (2013). RNA-sequencing from single nuclei. Proceedings of the National Academy of Sciences of the United States of America, 110, 19802–19807.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Guo, F., Yan, L., Guo, H., Li, L., Hu, B., Zhao, Y., Yong, J., Hu, Y., Wang, X., Wei, Y., et al. (2015). The Transcriptome and DNA Methylome landscapes of human primordial germ cells. Cell, 161, 1437–1452.CrossRefPubMedGoogle Scholar
  12. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., & Saitou, M. (2011). Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell, 146, 519–532.CrossRefPubMedGoogle Scholar
  13. Hermann, B. P., Mutoji, K. N., Velte, E. K., Ko, D., Oatley, J. M., Geyer, C. B., & McCarrey, J. R. (2015). Transcriptional and translational heterogeneity among neonatal mouse spermatogonia. Biology of Reproduction, 92, 54.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hough, S. R., Thornton, M., Mason, E., Mar, J. C., Wells, C. A., & Pera, M. F. (2014). Single-cell gene expression profiles define self-renewing, pluripotent, and lineage primed states of human pluripotent stem cells. Stem Cell Reports, 2, 881–895.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Huang, S., Ernberg, I., & Kauffman, S. (2009). Cancer attractors: A systems view of tumors from a gene network dynamics and developmental perspective. Seminars in Cell & Developmental Biology, 20, 869–876.CrossRefGoogle Scholar
  16. Huang, L., Ma, F., Chapman, A., Lu, S., & Xie, X. S. (2015). Single-cell whole-genome amplification and sequencing: Methodology and applications. Annual Review of Genomics and Human Genetics, 16, 79–102.CrossRefPubMedGoogle Scholar
  17. Hughes, C. S., Foehr, S., Garfield, D. A., Furlong, E. E., Steinmetz, L. M., & Krijgsveld, J. (2014). Ultrasensitive proteome analysis using paramagnetic bead technology. Molecular systems biology 10, 757.CrossRefPubMedGoogle Scholar
  18. Jang, S., Choubey, S., Furchtgott, L., Zou, L.N., Doyle, A., Menon, V., Loew, E.B., Krostag, A.R., Martinez, R.A., Madisen, L., et al. (2017). Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states. eLife, 6.Google Scholar
  19. Kooistra, S. M., Thummer, R. P., & Eggen, B. J. (2009). Characterization of human UTF1, a chromatin-associated protein with repressor activity expressed in pluripotent cells. Stem Cell Research, 2, 211–218.CrossRefPubMedGoogle Scholar
  20. Kristensen, D. M., Nielsen, J. E., Skakkebaek, N. E., Graem, N., Jacobsen, G. K., Rajpert-De Meyts, E., & Leffers, H. (2008). Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms. Human Reproduction, 23, 775–782.CrossRefPubMedGoogle Scholar
  21. Li, L., Dong, J., Yan, L., Yong, J., Liu, X., Hu, Y., Fan, X., Wu, X., Guo, H., Wang, X., et al. (2017). Single-cell RNA-Seq analysis maps development of human Germline cells and gonadal niche interactions. Cell Stem Cell, 20, 891–892.CrossRefPubMedGoogle Scholar
  22. Liu, Q., Li, Y., Feng, Y., Liu, C., Ma, J., Li, Y., Xiang, H., Ji, Y., Cao, Y., Tong, X., et al. (2016). Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients. Scientific Reports, 6, 39638.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Luetjens, C. M., Xu, E. Y., Rejo Pera, R. A., Kamischke, A., Nieschlag, E., & Gromoll, J. (2004). Association of meiotic arrest with lack of BOULE protein expression in infertile men. The Journal of Clinical Endocrinology and Metabolism, 89, 1926–1933.CrossRefPubMedGoogle Scholar
  24. Maekawa, M., Yamamoto, T., Kohno, M., Takeichi, M., & Nishida, E. (2007). Requirement for ERK MAP kinase in mouse preimplantation development. Development, 134, 2751–2759.CrossRefPubMedGoogle Scholar
  25. Magnusdottir, E., Dietmann, S., Murakami, K., Gunesdogan, U., Tang, F., Bao, S., Diamanti, E., Lao, K., Gottgens, B., & Azim Surani, M. (2013). A tripartite transcription factor network regulates primordial germ cell specification in mice. Nature Cell Biology, 15, 905–915.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mizrak, S. C., Chikhovskaya, J. V., Sadri-Ardekani, H., van Daalen, S., Korver, C. M., Hovingh, S. E., Roepers-Gajadien, H. L., Raya, A., Fluiter, K., de Reijke, T. M., et al. (2010). Embryonic stem cell-like cells derived from adult human testis. Human Reproduction, 25, 158–167.CrossRefPubMedGoogle Scholar
  27. Nakamura, T., Yabuta, Y., Okamoto, I., Aramaki, S., Yokobayashi, S., Kurimoto, K., Sekiguchi, K., Nakagawa, M., Yamamoto, T., & Saitou, M. (2015). SC3-seq: A method for highly parallel and quantitative measurement of single-cell gene expression. Nucleic Acids Research, 43, e60.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Neuhaus, N., Yoon, J., Terwort, N., Kliesch, S., Seggewiss, J., Huge, A., Voss, R., Schlatt, S., Grindberg, R. V., & Scholer, H. R. (2017). Single-cell gene expression analysis reveals diversity among human spermatogonia. Molecular Human Reproduction, 23, 79–90.PubMedGoogle Scholar
  29. Novershtern, N., Subramanian, A., Lawton, L. N., Mak, R. H., Haining, W. N., McConkey, M. E., Habib, N., Yosef, N., Chang, C. Y., Shay, T., et al. (2011). Densely interconnected transcriptional circuits control cell states in human hematopoiesis. Cell, 144, 296–309.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 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.CrossRefPubMedGoogle Scholar
  31. Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Nabeshima, Y., Kuro-o, M., Nabeshima, Y., Boon, K., Keaveney, M., et al. (1998). UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells. The EMBO Journal, 17, 2019–2032.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Ooi, S. K., & Bestor, T. H. (2008). The colorful history of active DNA demethylation. Cell, 133, 1145–1148.CrossRefPubMedGoogle Scholar
  33. Perrett, R. M., Turnpenny, L., Eckert, J. J., O’Shea, M., Sonne, S. B., Cameron, I. T., Wilson, D. I., Rajpert-De Meyts, E., & Hanley, N. A. (2008). The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture. Biology of Reproduction, 78, 852–858.CrossRefPubMedGoogle Scholar
  34. Robinson, L. L., Gaskell, T. L., Saunders, P. T., & Anderson, R. A. (2001). Germ cell specific expression of c-kit in the human fetal gonad. Molecular Human Reproduction, 7, 845–852.CrossRefPubMedGoogle Scholar
  35. Saliba, A. E., Westermann, A. J., Gorski, S. A., & Vogel, J. (2014). Single-cell RNA-seq: Advances and future challenges. Nucleic Acids Research, 42, 8845–8860.CrossRefPubMedPubMedCentralGoogle Scholar
  36. Shapiro, E., Biezuner, T., & Linnarsson, S. (2013). Single-cell sequencing-based technologies will revolutionize whole-organism science. Nature Reviews Genetics, 14, 618–630.CrossRefPubMedGoogle Scholar
  37. Tanay, A., & Regev, A. (2017). Scaling single-cell genomics from phenomenology to mechanism. Nature, 541, 331–338.CrossRefPubMedPubMedCentralGoogle Scholar
  38. Tang, F., Lao, K., & Surani, M. A. (2011). Development and applications of single-cell transcriptome analysis. Nature Methods, 8, S6–11.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Valli, H., Sukhwani, M., Dovey, S. L., Peters, K. A., Donohue, J., Castro, C. A., Chu, T., Marshall, G. R., & Orwig, K. E. (2014). Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells. Fertility and Sterility, 102(566–580), e567.Google Scholar
  40. Virant-Klun, I., Knez, K., Tomazevic, T., & Skutella, T. (2013). Gene expression profiling of human oocytes developed and matured in vivo or in vitro. BioMed Research International, 2013, 879489.PubMedPubMedCentralGoogle Scholar
  41. Virant-Klun, I., Leicht, S., Hughes, C., & Krijgsveld, J. (2016). Identification of maturation-specific proteins by single-cell proteomics of human oocytes. Molecular & Cellular Proteomics: MCP, 15, 2616–2627.CrossRefPubMedGoogle Scholar
  42. von Kopylow, K., Kirchhoff, C., Jezek, D., Schulze, W., Feig, C., Primig, M., Steinkraus, V., & Spiess, A. N. (2010). Screening for biomarkers of spermatogonia within the human testis: A whole genome approach. Human Reproduction, 25, 1104–1112.CrossRefGoogle Scholar
  43. von Kopylow, K., Staege, H., Schulze, W., Will, H., & Kirchhoff, C. (2012a). Fibroblast growth factor receptor 3 is highly expressed in rarely dividing human type a spermatogonia. Histochemistry and Cell Biology, 138, 759–772.CrossRefGoogle Scholar
  44. von Kopylow, K., Staege, H., Spiess, A. N., Schulze, W., Will, H., Primig, M., & Kirchhoff, C. (2012b). Differential marker protein expression specifies rarefaction zone-containing human Adark spermatogonia. Reproduction, 143, 45–57.CrossRefGoogle Scholar
  45. von Kopylow, K., Schulze, W., Salzbrunn, A., & Spiess, A. N. (2016). Isolation and gene expression analysis of single potential human spermatogonial stem cells. Molecular Human Reproduction, 22, 229–239.CrossRefGoogle Scholar
  46. Wagner, A., Regev, A., & Yosef, N. (2016). Revealing the vectors of cellular identity with single-cell genomics. Nature Biotechnology, 34, 1145–1160.CrossRefPubMedPubMedCentralGoogle Scholar
  47. Woodworth, M. B., Girskis, K. M., & Walsh, C. A. (2017). Building a lineage from single cells: Genetic techniques for cell lineage tracking. Nature Reviews Genetics, 18, 230–244.CrossRefPubMedPubMedCentralGoogle Scholar
  48. Yosef, N., & Regev, A. (2011). Impulse control: Temporal dynamics in gene transcription. Cell, 144, 886–896.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.TübingenGermany
  2. 2.Faculty of BiotechnologyAmol University of Special Modern TechnologiesAmolIran
  3. 3.Institute for Anatomy and Cell Biology III, Medical FacultyHeidelberg UniversityHeidelbergGermany

Personalised recommendations