BIOLOGY OF REPRODUCTION (2015) 92(5):119, 1–4 Published online before print 25 March 2015. DOI 10.1095/biolreprod.115.129890
Commentary Diagnosing Spermatogonial Stemness1 F. Kent Hamra2 Department of Pharmacology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas Ever-Expanding Models of Spermatogonial Development
Familial health largely reflects the quality of traits transmitted by ancestral oocytes and spermatozoa. This fact of life endows gamete-producing germ cells with an intrinsic accountability for the well-being of heritable genomic architecture [1]. Consequently, it is fitting that pressure to preserve genomic integrity is reflected by lower frequencies of DNA mutations detected in germ cells than in somatic cells [2– 4]. In mammals, self-renewing germline stem cells are considered unique to male gonads during reproductive life. This is because mitotically dividing female germ cells enter meiosis to differentiate into oocytes shortly after sex determination in mammals, which occurs midway through embryogenesis [5]. Oogenic arrests, together with selective oocyte degeneration, are hypothesized to provide additional safeguards that defend the germline from genomic abnormalities, highlighting the ‘‘female-protective model’’ [6, 7]. In contrast, male germline stem cells, termed ‘‘spermatogonial stem cells,’’ sustain spermatozoan production in testes by mitotically self-renewing over relatively long periods that span both embryonic and adult life (.50 years in humans) [8]. Accordingly, whole-genome sequencing is providing evidence that sex-dependent increases in time given for a germline to replicate its DNA correlate strongly with longstanding observations of ‘‘male mutation bias’’ [9–11]. In most species, our ability to unequivocally identify spermatogonia that replicate to function as germline stem cells has yet to be firmly established, but such a hypothesis appears fundamental to understanding cellular mechanisms that buffer the accumulation of transmittable DNA mutations by germlines [12, 13]. Scientists are rapidly annotating stem and progenitor spermatogonia in rodents [14–17], and these advances are being translated to other mammalian species, including primates [18– 23]. Clinically, the ability to diagnose genomic stability in spermatogonial stem cells seems paramount in order to safely make the connection to their enormous prospective benefits for family planning and genetic medicine [24].
Existence of spermatogonial stem cells was postulated near the end of the 19th Century by radiologists studying the regenerative capacity of testicular germ cells in model organisms receiving ‘‘gonotoxic’’ doses of ionizing radiation [25]. At that time, spermatogonial developmental hierarchy was simply assigned to ‘‘type A’’ spermatogonia that selectively survived radiation exposure and, presumably, restored populations of heterochromatic ‘‘type B’’ spermatogonia representing differentiating spermatogenic progenitors destined for meiosis. A review of publications since then reveals consistent advances from decade to decade that have led to ever-evolving theories of an expanding developmental hierarchy of .10 distinct stem, progenitor, and differentiating spermatogonial types currently classified in mammals [15, 26], which mirror a diversity of proposed mechanisms by which stem spermatogonia maintain spermatogenesis [15, 27, 28]. Presently, a prevailing view in mammals is that spermatogonial stem cells reside within a population of type ‘‘A-single’’ (As) spermatogonia that maintain spermatogenesis by their dual capacity to either self-renew as As spermatogonia or to form syncytia of mitotically dividing progenitor cells termed type Apaired (Apr) and A-aligned spermatogonia (Aal) (Fig. 1) [29– 31]. Disrupting self-renewal of As spermatogonia can drive development fully toward a progenitor fate [32–37]. In rodents, progenitor spermatogonia differentiate into mitotically dividing types A1, A2, A3, and A4 and intermediate (Int) and B spermatogonia [38–40], during which time their cell numbers or syncytium often increase .100-fold prior to entering meiosis to form spermatocytes (Fig. 1). Dissecting Spermatogonial Heterogeneity In a recent issue of Biology of Reproduction, a study by Hermann et al. [41] reported a strategy to dissect the diversity of mouse spermatogonial types during a pivotal time in gametogenesis, immediately following the transition from ‘‘prespermatogenesis’’ to ‘‘spermatogenesis’’ (reviewed by J.R. McCarrey [26]) (Fig. 1). Transition from prespermatogenesis to spermatogenesis occurs in mice between Postnatal Days 3 and 6 (P3–P6) when pools of ‘‘prospermatogonia’’ give rise to functionally distinct populations of stem and progenitor spermatogonia [42–44]. The group hypothesized that subpopulations of spermatogonia with discrete mRNA signatures could be identified in neonatal mice, which potentially could unveil previously unrealized heterogeneity of germ cells at the ‘‘start’’ of spermatogenesis, shortly after their derivation from prospermatogonia. To test their hypothesis, Hermann et al. [41] performed single-cell qPCR using a Fluidigm system (San Francisco, CA) [45] to study gene expression in spermatogonia
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Supported by U.S. National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development grant 5R01HD053889. 2 Correspondence: F. Kent Hamra, Department of Pharmacology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX 75390. E-mail: [email protected] Ó 2015 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
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Germ Cells: Guardians of Heritable Traits
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Downloaded from www.biolreprod.org. FIG. 1. Spermatogonial stem cell predetermination hypothesis. A recent study by Hermann et al. [41] highlights large gaps of knowledge on the specification of spermatogonial stem cells. Based on that study and the group’s previous findings [2, 44], their current study formulates a hypothesis that spermatogonial stem cells may actually derive developmentally from a subpopulation of prospermatogonia that are selectively protected genetically by unknown factors. The illustration also demonstrates independent questions (?) that will likely be raised on how their concepts integrate with presumptive germline stem cell niches in the seminiferous epithelium postnatally, and with intrinsic mechanisms determining germ cell fate. Prosgn, prospermagonia; As, A-single spermatogonia; Apr, A-paired spermatogonia; Aal, A-aligned spermatogonial; A1-B, types A1 to B spermatogonia; PL-D, preleptotene to diplotene spermatocytes.
isolated from mice at P6). The approach previously proved successful to help classify even earlier pregonadal and prespermatogenic steps in primordial germ cell development, from embryonic precursors in vivo and in vitro [46–48]. It allowed Hermann et al. to analyze gene expression in arrays of hundreds of individual testis cells comparing three distinct isolation methods. A panel of 172 qPCR primer sets were designed to analyze the relative abundance of target transcripts with reported functions in undifferentiated spermatogonia, somatic testis cells, differentiating male and female germ cells, and pluripotent stem cells. Indeed, a battery of statistical analyses consistently predicted ;6 conspicuous clusters of amplified spermatogonial transcripts marking distinct germ cell
populations at that pivotal juncture in gametogenesis. Principal component analyses were then used to distill this heterogeneity into three prominent spermatogonial signatures, which directionally, would be consistent with subtypes of stem, progenitor, and differentiating spermatogonia present in P6 mice. Thus, their study strongly supported the concept that the P6 spermatogonial pool consists of multiple subpopulations with discrete gene expression signatures, quite possibly reflecting divergence in spermatogonial fate. Spermatogonial Stem Cell Predetermination Hypothesis The repertoire of molecular signatures in spermatogonia unearthed by Hermann et al. [41] begs new questions 2
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DIAGNOSING SPERMATOGONIAL STEMNESS
Conclusions and Future Directions The above-described hypothesis proposes that the founding population of actual spermatogonial stem cells derives from a predetermined pool of prospermatogonia, which by some unknown mechanism(s), selectively maintains their genetic integrity at more pristine levels than other prospermatogonia (Fig. 1). Identifying prospermatogonia predestined to become either stem or progenitor spermatogonia will, in essence, break the current dogma that spermatogenesis initiates after prospermatogonia migrate from the lumen of seminiferous cords to colonize the basal lamina [26]. This theory raises questions on how ‘‘prospermatogonial stem cell’’ genomes could selectively be protected. Extrinsic factors that promote protection of the germline would imply existence of specialized ‘‘prospermatogonial niches’’ to mediate this function in the embryonic gonad. For example, does a similar version of the As . Apr . Aal model occur earlier in development than generally thought, consistent with findings by Kluin and de-Rooij [42]? As presented, the spermatogonial stem cell predetermination hypothesis remains open to germline intrinsic mechanisms that, if not properly engaged, promote terminal differentiation and/or elimination of prospermatogonia. It would lead to question if DNA damage actually is a consequence of differentiation during a vulnerable developmental window or if it actually functions as a differentiation signal. In the latter case, how would mutational load ‘‘blind’’ prospermatogoniaderived progenitors to self-renewal factors (Fig. 1)? Conceivably, mutations above a relative threshold could facilitate the increased rates of apoptosis consistently observed in cohorts of first-generation spermatocytes derived more directly from prospermatogonia [55, 56]. In an analogous example, DNA double-strand breaks and chromatin abnormalities accumulate in MIWI2-deficient prospermatogonia due to persistent retrotransposon activation, but effects on germ cell fate are not manifested until induction of apoptosis post-mitotically at the
REFERENCES 1. Kirkwood TB. Evolution of ageing. Nature 1977; 270(5635):301–304. 2. Murphey P, McLean DJ, McMahan CA, Walter CA, McCarrey JR. Enhanced genetic integrity in mouse germ cells. Biol Reprod 2013; 88(1): 6. 3. Walter CA, Intano GW, McCarrey JR, McMahan CA, Walter RB. Mutation frequency declines during spermatogenesis in young mice but increases in old mice. Proc Natl Acad Sci U S A 1998; 95(17): 10015–10019. 4. Kohler SW, Provost GS, Fieck A, Kretz PL, Bullock WO, Sorge JA, Putman DL, Short JM. Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc Natl Acad Sci U S A 1991; 88(18):7958–7962. 5. Hartshorne GM, Lyrakou S, Hamoda H, Oloto E, Ghafari F. Oogenesis and cell death in human prenatal ovaries: what are the criteria for oocyte selection? Mol Hum Reprod 2009; 15(12):805–819. 6. Hunt PA, Hassold TJ. Human female meiosis: what makes a good egg go bad? Trends Genet 2008; 24(2):86–93. 7. Jacquemont S, Coe BP, Hersch M, Duyzend MH, Krumm N, Bergmann S, Beckmann JS, Rosenfeld JA, Eichler EE. A higher mutational burden in females supports a ‘‘female protective model’’ in neurodevelopmental disorders. Am J Hum Genet 2014; 94(3):415–425. 8. Amann RP. The cycle of the seminiferous epithelium in humans: a need to revisit? J Androl 2008; 29(5):469–487. 9. Venn O, Turner I, Mathieson I, de Groot N, Bontrop R, McVean G. Nonhuman genetics. Strong male bias drives germline mutation in chimpanzees. Science 2014; 344(6189):1272–1275. 10. Wilson Sayres MA, Makova KD. Genome analyses substantiate male mutation bias in many species. Bioessays. 2011; 33(12):938–945. 11. Kong A, Frigge ML, Masson G, Besenbacher S, Sulem P, Magnusson G, Gudjonsson SA, Sigurdsson A, Jonasdottir A, Jonasdottir A, Wong WS, Sigurdsson G, et al. Rate of de novo mutations and the importance of father’s age to disease risk. Nature 2012; 488(7412):471–475. 12. Aravin AA, Hannon GJ, Brennecke J. The Piwi-piRNA pathway provides
3
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zygotene-pachytene checkpoint [57]. Moreover, the abovedescribed models of prospermatogonial heterogeneity remain compatible with an ‘‘inductive’’ influence of germline stem niches postnatally as one way for predetermined stem spermatogonia to grasp their true potential (Fig. 1). A combination of these mechanisms would increase spermatogonial heterogeneity due to greater divergence in fate, which in turn, would allow a greater diversity in spermatogonial types to be captured by single-cell qPCR. Centered on their hypothesis, the study by Hermann et al. [42] highlights the potential to classify new spermatogonial types with respect to developmental origin and biological function. It will be interesting to see how such findings impact the course of biomedical research, to ultimately apply germline stem cells to human health, a field that prospectively will benefit by the ability to propagate and/or differentiate an individual’s germline [18, 24, 58]. If ‘‘spermatogonial stemness’’ is largely predetermined by mechanisms linked to protecting genomic integrity (i.e., disposable soma hypothesis), how should this impact standards for therapeutic application of donor cells produced by somatic cell reprogramming? This is an especially broad question as it relates regenerative medicine. As a consequence, practical application of spermatogonial stem cells holds enormous potential to transform numerous areas of science, medicine, industry, and conservation [58–63] with the probability of donor haplotypes possessing a more ancestral background [9, 10]. However, this untapped potential is being held at bay by limited capacity to experimentally manipulate spermatogonial proliferation and differentiation from so many species outside Rodentia. Current absence of in vitro and in vivo bioassays to unequivocally measure the sperm-forming potential of donor spermatogonia in most mammalian species means that genome-wide analytical approaches similar to those applied by Hermann et al. [41] may well prove critical to safely diagnose spermatogonial stemness, as imputed by genetic integrity.
surrounding the actual diversity of spermatogonia developmentally (Fig. 1). Answers to these questions are needed to fill key gaps of knowledge centered on understanding the long-term and short-term replicative potential of stem spermatogonia, and the fidelity at which prospermatogonia give rise to stem, progenitor and differentiating spermatogonia in mammals. Their study was motivated in part by earlier observations [2, 3] that ignited the ‘‘disposable soma hypothesis’’ posed in 1977 by Kirkwood [1]. Kirkwood’s premise suggested that, because germ cells give rise to the entire subsequent generation of individuals, it would be evolutionarily advantageous for these cells to use mechanisms to maintain an enhanced level of genetic integrity [1]. This thesis was reinforced by higher spontaneous mutational frequencies in the soma versus the germline of female and male mice harboring a LacI mutation-reporter transgene [2, 3]. Interestingly, these same studies made seminal discoveries in that spermatogonia at P6 accumulated more mutations than primary spermatocytes at P18 [2, 3]. This paradox raised questions as to why the germline harbored 5 to 10 times more DNA mutations in spermatogonia at the start of spermatogenesis than in the germ cell population it presumably gave rise to shortly after the start of spermatogenesis. To place this oxymoron in context with their current studies plus accumulating examples of molecular and functional heterogeneity within populations of As, Apr, and Aal spermatogonia [28, 29, 37, 49–54], the group formulated the following hypothesis: ‘‘Spermatogonial stem cell specification, either through a mechanism of predetermination or selection, may occur as a result of molecular divergence that first emerges among prospermatogonia.’’
HAMRA
13. 14. 15. 16. 17. 18. 19. 20. 21.
22.
24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
37. 38. 39.
40. Lok D, de Rooij DG. Spermatogonial multiplication in the Chinese hamster. I. Cell cycle properties and synchronization of differentiating spermatogonia. Cell Tissue Kinet 1983; 16(1):7–18. 41. Hermann BP, Mutoji KN, Velte EK, Ko D, Oatley JM, Geyer CB, McCarrey JR. Transcriptional and translational heterogeneity among neonatal mouse spermatogonia. Biol Reprod 2015; 92(2):54. 42. Kluin PM, de Rooij DG. A comparison between the morphology and cell kinetics of gonocytes and adult type undifferentiated spermatogonia in the mouse. Int J Androl 1981; 4(4):475–493. 43. Yoshida S, Nabeshima Y, Nakagawa T. Stem cell heterogeneity: actual and potential stem cell compartments in mouse spermatogenesis. Ann N Y Acad Sci 2007; 1120:47–58. 44. Nakagawa T, Nabeshima Y, Yoshida S. Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis. Dev Cell 2007; 12(2):195–206. 45. Wu AR, Neff NF, Kalisky T, Dalerba P, Treutlein B, Rothenberg ME, Mburu FM, Mantalas GL, Sim S, Clarke MF, Quake SR. Quantitative assessment of single-cell RNA-sequencing methods. Nat Methods 2014; 11(1):41–46. 46. Vincent JJ, Huang Y, Chen PY, Feng S, Calvopin˜a JH, Nee K, Lee SA, Le T, Yoon AJ, Faull K, Fan G, Rao A, et al. Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells. PLoS One 2011; 6(12):e28960. 47. Gkountela S, Li Z, Vincent JJ, Zhang KX, Chen A, Pellegrini M, Clark AT. The ontogeny of cKIT þ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat Cell Biol 2013; 15(1):113–122. 48. Vincent JJ, Huang Y, Chen PY, Feng S, Calvopin˜a JH, Nee K, Lee SA, Le T, Yoon AJ, Faull K, Fan G, Rao A, et al. Stage-specific roles for tet1 and tet2 in DNA demethylation in primordial germ cells. Cell Stem Cell 2013; 12(4):470–478. 49. Grisanti L, Falciatori I, Grasso M, Dovere L, Fera S, Muciaccia B, Fuso A, Berno V, Boitani C, Stefanini M, Vicini E. Identification of spermatogonial stem cell subsets by morphological analysis and prospective isolation. Stem Cells 2009; 27(12):3043–3052. 50. Zheng K, Wu X, Kaestner KH, Wang PJ. The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse. BMC Dev Biol 2009; 9: 38. 51. Chan F, Oatley MJ, Kaucher AV, Yang QE, Bieberich CJ, Shashikant CS, Oatley JM. Functional and molecular features of the Id4 þ germline stem cell population in mouse testes. Genes Dev 2014; 28(12):1351–1362. 52. Aloisio, GM, et al. ., PAX7 expression defines germline stem cells in the adult testis. J Clin Invest 2014; 124(9):3929–3944. 53. Abid SN, Richardson TE, Powell HM, Jaichander P, Chaudhary J, Chapman KM, Hamra FK. A-single spermatogonia heterogeneity and cell cycles synchronize with rat seminiferous epithelium stages VIII-IX. Biol Reprod 2014; 90(2):32. 54. Komai Y, Tanaka T, Tokuyama Y, Yanai H, Ohe S, Omachi T, Atsumi N, Yoshida N, Kumano K, Hisha H, Matsuda T, Ueno H. Bmi1 expression in long-term germ stem cells. Sci Rep 2014; 4:6175. 55. Coucouvanis EC, Sherwood SW, Carswell-Crumpton C, Spack EG, Jones PP. Evidence that the mechanism of prenatal germ cell death in the mouse is apoptosis. Exp Cell Res 1993; 209(2):238–247. 56. Jahnukainen K, Chrysis D, Hou M, Parvinen M, Eksborg S, So¨der O. Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biol Reprod 2004; 70(2):290–296. 57. Bao J, Zhang Y, Schuster AS, Ortogero N, Nilsson EE, Skinner MK, Yan W. Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice. Cell Death Differ 2014; 21(5):783–796. 58. Arregui L, Dobrinski I. Xenografting of testicular tissue pieces: 12 years of an in vivo spermatogenesis system. Reproduction 2014; 148(5):R71–R84. 59. Brinster RL. Male germline stem cells: from mice to men. Science 2007; 316(5823):404–405. 60. Kubota H, Brinster RL. Technology insight: In vitro culture of spermatogonial stem cells and their potential therapeutic uses. Nat Clin Pract Endocrinol Metab 2006; 2(2):99–108. 61. Nagano M, Brinster CJ, Orwig KE, Ryu BY, Avarbock MR, Brinster RL. Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A 2001; 98(23):13090–13095. 62. Brinster RL. Germline stem cell transplantation and transgenesis. Science 2002; 296(5576):2174–2176. 63. Arregui L, Dobrinski I, Roldan ER. Germ cell survival and differentiation after xenotransplantation of testis tissue from three endangered species: Iberian lynx (Lynx pardinus), Cuvier’s gazelle (Gazella cuvieri) and Mohor gazelle (G. dama mhorr). Reprod Fertil Dev 2014; 26:817–826.
4
Article 119
Downloaded from www.biolreprod.org.
23.
an adaptive defense in the transposon arms race. Science 2007; 318(5851): 761–764. Bao J, Yan W. Male germline control of transposable elements. Biol Reprod 2012; 86(5):162. Oatley JM, Brinster RL. The germline stem cell niche unit in mammalian testes. Physiol Rev 2012; 92(2):577–595. Phillips BT, Gassei K, Orwig KE. Spermatogonial stem cell regulation and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 2010; 365(1546): 1663–1678. Kumar TR. The quest for male germline stem cell markers: PAX7 gets ID’d. J Clin Invest 2014; 124(10):4219–4222. Hermann BP, Phillips BT, Orwig KE. The elusive spermatogonial stem cell marker? Biol Reprod 2011; 85(2):221–223. Valli H, Phillips BT, Shetty G, Byrne JA, Clark AT, Meistrich ML, Orwig KE. Germline stem cells: toward the regeneration of spermatogenesis. Fertil Steril 2014; 101(1):3–13. Plant TM. Undifferentiated primate spermatogonia and their endocrine control. Trends Endocrinol Metab 2010; 21(8):488–495. Hermann BP, Sukhwani M, Hansel MC, Orwig KE. Spermatogonial stem cells in higher primates: are there differences from those in rodents? Reproduction 2010; 139(3):479–493. Hermann BP, Sukhwani M, Winkler F, Pascarella JN, Peters KA, Sheng Y, Valli H, Rodriguez M, Ezzelarab M, Dargo G, Peterson K, Masterson K, et al. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 2012; 11(5):715–726. Ramathal C, Durruthy-Durruthy J, Sukhwani M, Arakaki JE, Turek PJ, Orwig KE. Reijo Pera RA. Fate of iPSCs derived from azoospermic and fertile men following xenotransplantation to murine seminiferous tubules. Cell Rep 2014; 7(4):1284–1297. Durruthy J, Ramathal C, Sukhwani M, Fang F, Cui J, Orwig KE, Reijo Pera RA. Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules. Hum Mol Genet. 2014; 23(12): 3071–3084. Clark AT, Phillips BT, Orwig KE. Fruitful progress to fertility: male fertility in the test tube. Nat Med 2011; 17(12):1564–5. Regaud C. Some biological aspects of the radiation therapy of cancer. Am J Roentgenol Radium Ther 1924; 12(2):97–101. McCarrey JR. Toward a more precise and informative nomenclature describing fetal and neonatal male germ cells in rodents. Biol Reprod 2013; 89(2):47. de Rooij DG, Griswold MD. Questions about spermatogonia posed and answered since 2000. J Androl 2012; 33(6):1085–1095. Nakagawa T, Sharma M, Nabeshima Y, Braun RE, Yoshida S. Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 2010; 328(5974):62–67. Huckins C. The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat Rec 1971; 169(3): 533–557. Lok D, Weenk D, De Rooij DG. Morphology, proliferation, and differentiation of undifferentiated spermatogonia in the Chinese hamster and the ram. Anat Rec 1982; 203(1):83–99. Oakberg EF. Spermatogonial stem-cell renewal in the mouse. Anat Rec 1971; 169(3):515–531. Yang QE, Gwost I, Oatley MJ, Oatley JM. Retinoblastoma protein (RB1) controls fate determination in stem cells and progenitors of the mouse male germline. Biol Reprod 2013; 89(5):113. Hu YC, de Rooij DG, Page DC. Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice. Proc Natl Acad Sci U S A 2013; 110(31):12685–12690. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig KE, Wolgemuth DJ, Pandolfi PP. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004; 36(6):653–659. Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 2004; 36(6):647–652. Meng X, Lindahl M, Hyvo¨nen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287(5457):1489–1493. Sada A, Suzuki A, Suzuki H, Saga Y. The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells. Science 2009; 325(5946):1394–1398. Monesi V. Autoradiographic study of DNA synthesis and the cell cycle in spermatogonia and spermatocytes of mouse testis using tritiated thymidine. J Cell Biol 1962; 14:1–18. Huckins C. Cell cycle properties of differentiating spermatogonia in adult Sprague-Dawley rats. Cell Tissue Kinet 1971; 4(2):139–154.
Commentary Diagnosing Spermatogonial Stemness1 F. Kent Hamra2 Department of Pharmacology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas Ever-Expanding Models of Spermatogonial Development
Familial health largely reflects the quality of traits transmitted by ancestral oocytes and spermatozoa. This fact of life endows gamete-producing germ cells with an intrinsic accountability for the well-being of heritable genomic architecture [1]. Consequently, it is fitting that pressure to preserve genomic integrity is reflected by lower frequencies of DNA mutations detected in germ cells than in somatic cells [2– 4]. In mammals, self-renewing germline stem cells are considered unique to male gonads during reproductive life. This is because mitotically dividing female germ cells enter meiosis to differentiate into oocytes shortly after sex determination in mammals, which occurs midway through embryogenesis [5]. Oogenic arrests, together with selective oocyte degeneration, are hypothesized to provide additional safeguards that defend the germline from genomic abnormalities, highlighting the ‘‘female-protective model’’ [6, 7]. In contrast, male germline stem cells, termed ‘‘spermatogonial stem cells,’’ sustain spermatozoan production in testes by mitotically self-renewing over relatively long periods that span both embryonic and adult life (.50 years in humans) [8]. Accordingly, whole-genome sequencing is providing evidence that sex-dependent increases in time given for a germline to replicate its DNA correlate strongly with longstanding observations of ‘‘male mutation bias’’ [9–11]. In most species, our ability to unequivocally identify spermatogonia that replicate to function as germline stem cells has yet to be firmly established, but such a hypothesis appears fundamental to understanding cellular mechanisms that buffer the accumulation of transmittable DNA mutations by germlines [12, 13]. Scientists are rapidly annotating stem and progenitor spermatogonia in rodents [14–17], and these advances are being translated to other mammalian species, including primates [18– 23]. Clinically, the ability to diagnose genomic stability in spermatogonial stem cells seems paramount in order to safely make the connection to their enormous prospective benefits for family planning and genetic medicine [24].
Existence of spermatogonial stem cells was postulated near the end of the 19th Century by radiologists studying the regenerative capacity of testicular germ cells in model organisms receiving ‘‘gonotoxic’’ doses of ionizing radiation [25]. At that time, spermatogonial developmental hierarchy was simply assigned to ‘‘type A’’ spermatogonia that selectively survived radiation exposure and, presumably, restored populations of heterochromatic ‘‘type B’’ spermatogonia representing differentiating spermatogenic progenitors destined for meiosis. A review of publications since then reveals consistent advances from decade to decade that have led to ever-evolving theories of an expanding developmental hierarchy of .10 distinct stem, progenitor, and differentiating spermatogonial types currently classified in mammals [15, 26], which mirror a diversity of proposed mechanisms by which stem spermatogonia maintain spermatogenesis [15, 27, 28]. Presently, a prevailing view in mammals is that spermatogonial stem cells reside within a population of type ‘‘A-single’’ (As) spermatogonia that maintain spermatogenesis by their dual capacity to either self-renew as As spermatogonia or to form syncytia of mitotically dividing progenitor cells termed type Apaired (Apr) and A-aligned spermatogonia (Aal) (Fig. 1) [29– 31]. Disrupting self-renewal of As spermatogonia can drive development fully toward a progenitor fate [32–37]. In rodents, progenitor spermatogonia differentiate into mitotically dividing types A1, A2, A3, and A4 and intermediate (Int) and B spermatogonia [38–40], during which time their cell numbers or syncytium often increase .100-fold prior to entering meiosis to form spermatocytes (Fig. 1). Dissecting Spermatogonial Heterogeneity In a recent issue of Biology of Reproduction, a study by Hermann et al. [41] reported a strategy to dissect the diversity of mouse spermatogonial types during a pivotal time in gametogenesis, immediately following the transition from ‘‘prespermatogenesis’’ to ‘‘spermatogenesis’’ (reviewed by J.R. McCarrey [26]) (Fig. 1). Transition from prespermatogenesis to spermatogenesis occurs in mice between Postnatal Days 3 and 6 (P3–P6) when pools of ‘‘prospermatogonia’’ give rise to functionally distinct populations of stem and progenitor spermatogonia [42–44]. The group hypothesized that subpopulations of spermatogonia with discrete mRNA signatures could be identified in neonatal mice, which potentially could unveil previously unrealized heterogeneity of germ cells at the ‘‘start’’ of spermatogenesis, shortly after their derivation from prospermatogonia. To test their hypothesis, Hermann et al. [41] performed single-cell qPCR using a Fluidigm system (San Francisco, CA) [45] to study gene expression in spermatogonia
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Supported by U.S. National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development grant 5R01HD053889. 2 Correspondence: F. Kent Hamra, Department of Pharmacology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX 75390. E-mail: [email protected] Ó 2015 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
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Downloaded from www.biolreprod.org. FIG. 1. Spermatogonial stem cell predetermination hypothesis. A recent study by Hermann et al. [41] highlights large gaps of knowledge on the specification of spermatogonial stem cells. Based on that study and the group’s previous findings [2, 44], their current study formulates a hypothesis that spermatogonial stem cells may actually derive developmentally from a subpopulation of prospermatogonia that are selectively protected genetically by unknown factors. The illustration also demonstrates independent questions (?) that will likely be raised on how their concepts integrate with presumptive germline stem cell niches in the seminiferous epithelium postnatally, and with intrinsic mechanisms determining germ cell fate. Prosgn, prospermagonia; As, A-single spermatogonia; Apr, A-paired spermatogonia; Aal, A-aligned spermatogonial; A1-B, types A1 to B spermatogonia; PL-D, preleptotene to diplotene spermatocytes.
isolated from mice at P6). The approach previously proved successful to help classify even earlier pregonadal and prespermatogenic steps in primordial germ cell development, from embryonic precursors in vivo and in vitro [46–48]. It allowed Hermann et al. to analyze gene expression in arrays of hundreds of individual testis cells comparing three distinct isolation methods. A panel of 172 qPCR primer sets were designed to analyze the relative abundance of target transcripts with reported functions in undifferentiated spermatogonia, somatic testis cells, differentiating male and female germ cells, and pluripotent stem cells. Indeed, a battery of statistical analyses consistently predicted ;6 conspicuous clusters of amplified spermatogonial transcripts marking distinct germ cell
populations at that pivotal juncture in gametogenesis. Principal component analyses were then used to distill this heterogeneity into three prominent spermatogonial signatures, which directionally, would be consistent with subtypes of stem, progenitor, and differentiating spermatogonia present in P6 mice. Thus, their study strongly supported the concept that the P6 spermatogonial pool consists of multiple subpopulations with discrete gene expression signatures, quite possibly reflecting divergence in spermatogonial fate. Spermatogonial Stem Cell Predetermination Hypothesis The repertoire of molecular signatures in spermatogonia unearthed by Hermann et al. [41] begs new questions 2
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DIAGNOSING SPERMATOGONIAL STEMNESS
Conclusions and Future Directions The above-described hypothesis proposes that the founding population of actual spermatogonial stem cells derives from a predetermined pool of prospermatogonia, which by some unknown mechanism(s), selectively maintains their genetic integrity at more pristine levels than other prospermatogonia (Fig. 1). Identifying prospermatogonia predestined to become either stem or progenitor spermatogonia will, in essence, break the current dogma that spermatogenesis initiates after prospermatogonia migrate from the lumen of seminiferous cords to colonize the basal lamina [26]. This theory raises questions on how ‘‘prospermatogonial stem cell’’ genomes could selectively be protected. Extrinsic factors that promote protection of the germline would imply existence of specialized ‘‘prospermatogonial niches’’ to mediate this function in the embryonic gonad. For example, does a similar version of the As . Apr . Aal model occur earlier in development than generally thought, consistent with findings by Kluin and de-Rooij [42]? As presented, the spermatogonial stem cell predetermination hypothesis remains open to germline intrinsic mechanisms that, if not properly engaged, promote terminal differentiation and/or elimination of prospermatogonia. It would lead to question if DNA damage actually is a consequence of differentiation during a vulnerable developmental window or if it actually functions as a differentiation signal. In the latter case, how would mutational load ‘‘blind’’ prospermatogoniaderived progenitors to self-renewal factors (Fig. 1)? Conceivably, mutations above a relative threshold could facilitate the increased rates of apoptosis consistently observed in cohorts of first-generation spermatocytes derived more directly from prospermatogonia [55, 56]. In an analogous example, DNA double-strand breaks and chromatin abnormalities accumulate in MIWI2-deficient prospermatogonia due to persistent retrotransposon activation, but effects on germ cell fate are not manifested until induction of apoptosis post-mitotically at the
REFERENCES 1. Kirkwood TB. Evolution of ageing. Nature 1977; 270(5635):301–304. 2. Murphey P, McLean DJ, McMahan CA, Walter CA, McCarrey JR. Enhanced genetic integrity in mouse germ cells. Biol Reprod 2013; 88(1): 6. 3. Walter CA, Intano GW, McCarrey JR, McMahan CA, Walter RB. Mutation frequency declines during spermatogenesis in young mice but increases in old mice. Proc Natl Acad Sci U S A 1998; 95(17): 10015–10019. 4. Kohler SW, Provost GS, Fieck A, Kretz PL, Bullock WO, Sorge JA, Putman DL, Short JM. Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc Natl Acad Sci U S A 1991; 88(18):7958–7962. 5. Hartshorne GM, Lyrakou S, Hamoda H, Oloto E, Ghafari F. Oogenesis and cell death in human prenatal ovaries: what are the criteria for oocyte selection? Mol Hum Reprod 2009; 15(12):805–819. 6. Hunt PA, Hassold TJ. Human female meiosis: what makes a good egg go bad? Trends Genet 2008; 24(2):86–93. 7. Jacquemont S, Coe BP, Hersch M, Duyzend MH, Krumm N, Bergmann S, Beckmann JS, Rosenfeld JA, Eichler EE. A higher mutational burden in females supports a ‘‘female protective model’’ in neurodevelopmental disorders. Am J Hum Genet 2014; 94(3):415–425. 8. Amann RP. The cycle of the seminiferous epithelium in humans: a need to revisit? J Androl 2008; 29(5):469–487. 9. Venn O, Turner I, Mathieson I, de Groot N, Bontrop R, McVean G. Nonhuman genetics. Strong male bias drives germline mutation in chimpanzees. Science 2014; 344(6189):1272–1275. 10. Wilson Sayres MA, Makova KD. Genome analyses substantiate male mutation bias in many species. Bioessays. 2011; 33(12):938–945. 11. Kong A, Frigge ML, Masson G, Besenbacher S, Sulem P, Magnusson G, Gudjonsson SA, Sigurdsson A, Jonasdottir A, Jonasdottir A, Wong WS, Sigurdsson G, et al. Rate of de novo mutations and the importance of father’s age to disease risk. Nature 2012; 488(7412):471–475. 12. Aravin AA, Hannon GJ, Brennecke J. The Piwi-piRNA pathway provides
3
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zygotene-pachytene checkpoint [57]. Moreover, the abovedescribed models of prospermatogonial heterogeneity remain compatible with an ‘‘inductive’’ influence of germline stem niches postnatally as one way for predetermined stem spermatogonia to grasp their true potential (Fig. 1). A combination of these mechanisms would increase spermatogonial heterogeneity due to greater divergence in fate, which in turn, would allow a greater diversity in spermatogonial types to be captured by single-cell qPCR. Centered on their hypothesis, the study by Hermann et al. [42] highlights the potential to classify new spermatogonial types with respect to developmental origin and biological function. It will be interesting to see how such findings impact the course of biomedical research, to ultimately apply germline stem cells to human health, a field that prospectively will benefit by the ability to propagate and/or differentiate an individual’s germline [18, 24, 58]. If ‘‘spermatogonial stemness’’ is largely predetermined by mechanisms linked to protecting genomic integrity (i.e., disposable soma hypothesis), how should this impact standards for therapeutic application of donor cells produced by somatic cell reprogramming? This is an especially broad question as it relates regenerative medicine. As a consequence, practical application of spermatogonial stem cells holds enormous potential to transform numerous areas of science, medicine, industry, and conservation [58–63] with the probability of donor haplotypes possessing a more ancestral background [9, 10]. However, this untapped potential is being held at bay by limited capacity to experimentally manipulate spermatogonial proliferation and differentiation from so many species outside Rodentia. Current absence of in vitro and in vivo bioassays to unequivocally measure the sperm-forming potential of donor spermatogonia in most mammalian species means that genome-wide analytical approaches similar to those applied by Hermann et al. [41] may well prove critical to safely diagnose spermatogonial stemness, as imputed by genetic integrity.
surrounding the actual diversity of spermatogonia developmentally (Fig. 1). Answers to these questions are needed to fill key gaps of knowledge centered on understanding the long-term and short-term replicative potential of stem spermatogonia, and the fidelity at which prospermatogonia give rise to stem, progenitor and differentiating spermatogonia in mammals. Their study was motivated in part by earlier observations [2, 3] that ignited the ‘‘disposable soma hypothesis’’ posed in 1977 by Kirkwood [1]. Kirkwood’s premise suggested that, because germ cells give rise to the entire subsequent generation of individuals, it would be evolutionarily advantageous for these cells to use mechanisms to maintain an enhanced level of genetic integrity [1]. This thesis was reinforced by higher spontaneous mutational frequencies in the soma versus the germline of female and male mice harboring a LacI mutation-reporter transgene [2, 3]. Interestingly, these same studies made seminal discoveries in that spermatogonia at P6 accumulated more mutations than primary spermatocytes at P18 [2, 3]. This paradox raised questions as to why the germline harbored 5 to 10 times more DNA mutations in spermatogonia at the start of spermatogenesis than in the germ cell population it presumably gave rise to shortly after the start of spermatogenesis. To place this oxymoron in context with their current studies plus accumulating examples of molecular and functional heterogeneity within populations of As, Apr, and Aal spermatogonia [28, 29, 37, 49–54], the group formulated the following hypothesis: ‘‘Spermatogonial stem cell specification, either through a mechanism of predetermination or selection, may occur as a result of molecular divergence that first emerges among prospermatogonia.’’
HAMRA
13. 14. 15. 16. 17. 18. 19. 20. 21.
22.
24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
37. 38. 39.
40. Lok D, de Rooij DG. Spermatogonial multiplication in the Chinese hamster. I. Cell cycle properties and synchronization of differentiating spermatogonia. Cell Tissue Kinet 1983; 16(1):7–18. 41. Hermann BP, Mutoji KN, Velte EK, Ko D, Oatley JM, Geyer CB, McCarrey JR. Transcriptional and translational heterogeneity among neonatal mouse spermatogonia. Biol Reprod 2015; 92(2):54. 42. Kluin PM, de Rooij DG. A comparison between the morphology and cell kinetics of gonocytes and adult type undifferentiated spermatogonia in the mouse. Int J Androl 1981; 4(4):475–493. 43. Yoshida S, Nabeshima Y, Nakagawa T. Stem cell heterogeneity: actual and potential stem cell compartments in mouse spermatogenesis. Ann N Y Acad Sci 2007; 1120:47–58. 44. Nakagawa T, Nabeshima Y, Yoshida S. Functional identification of the actual and potential stem cell compartments in mouse spermatogenesis. Dev Cell 2007; 12(2):195–206. 45. Wu AR, Neff NF, Kalisky T, Dalerba P, Treutlein B, Rothenberg ME, Mburu FM, Mantalas GL, Sim S, Clarke MF, Quake SR. Quantitative assessment of single-cell RNA-sequencing methods. Nat Methods 2014; 11(1):41–46. 46. Vincent JJ, Huang Y, Chen PY, Feng S, Calvopin˜a JH, Nee K, Lee SA, Le T, Yoon AJ, Faull K, Fan G, Rao A, et al. Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells. PLoS One 2011; 6(12):e28960. 47. Gkountela S, Li Z, Vincent JJ, Zhang KX, Chen A, Pellegrini M, Clark AT. The ontogeny of cKIT þ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat Cell Biol 2013; 15(1):113–122. 48. Vincent JJ, Huang Y, Chen PY, Feng S, Calvopin˜a JH, Nee K, Lee SA, Le T, Yoon AJ, Faull K, Fan G, Rao A, et al. Stage-specific roles for tet1 and tet2 in DNA demethylation in primordial germ cells. Cell Stem Cell 2013; 12(4):470–478. 49. Grisanti L, Falciatori I, Grasso M, Dovere L, Fera S, Muciaccia B, Fuso A, Berno V, Boitani C, Stefanini M, Vicini E. Identification of spermatogonial stem cell subsets by morphological analysis and prospective isolation. Stem Cells 2009; 27(12):3043–3052. 50. Zheng K, Wu X, Kaestner KH, Wang PJ. The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse. BMC Dev Biol 2009; 9: 38. 51. Chan F, Oatley MJ, Kaucher AV, Yang QE, Bieberich CJ, Shashikant CS, Oatley JM. Functional and molecular features of the Id4 þ germline stem cell population in mouse testes. Genes Dev 2014; 28(12):1351–1362. 52. Aloisio, GM, et al. ., PAX7 expression defines germline stem cells in the adult testis. J Clin Invest 2014; 124(9):3929–3944. 53. Abid SN, Richardson TE, Powell HM, Jaichander P, Chaudhary J, Chapman KM, Hamra FK. A-single spermatogonia heterogeneity and cell cycles synchronize with rat seminiferous epithelium stages VIII-IX. Biol Reprod 2014; 90(2):32. 54. Komai Y, Tanaka T, Tokuyama Y, Yanai H, Ohe S, Omachi T, Atsumi N, Yoshida N, Kumano K, Hisha H, Matsuda T, Ueno H. Bmi1 expression in long-term germ stem cells. Sci Rep 2014; 4:6175. 55. Coucouvanis EC, Sherwood SW, Carswell-Crumpton C, Spack EG, Jones PP. Evidence that the mechanism of prenatal germ cell death in the mouse is apoptosis. Exp Cell Res 1993; 209(2):238–247. 56. Jahnukainen K, Chrysis D, Hou M, Parvinen M, Eksborg S, So¨der O. Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biol Reprod 2004; 70(2):290–296. 57. Bao J, Zhang Y, Schuster AS, Ortogero N, Nilsson EE, Skinner MK, Yan W. Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice. Cell Death Differ 2014; 21(5):783–796. 58. Arregui L, Dobrinski I. Xenografting of testicular tissue pieces: 12 years of an in vivo spermatogenesis system. Reproduction 2014; 148(5):R71–R84. 59. Brinster RL. Male germline stem cells: from mice to men. Science 2007; 316(5823):404–405. 60. Kubota H, Brinster RL. Technology insight: In vitro culture of spermatogonial stem cells and their potential therapeutic uses. Nat Clin Pract Endocrinol Metab 2006; 2(2):99–108. 61. Nagano M, Brinster CJ, Orwig KE, Ryu BY, Avarbock MR, Brinster RL. Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A 2001; 98(23):13090–13095. 62. Brinster RL. Germline stem cell transplantation and transgenesis. Science 2002; 296(5576):2174–2176. 63. Arregui L, Dobrinski I, Roldan ER. Germ cell survival and differentiation after xenotransplantation of testis tissue from three endangered species: Iberian lynx (Lynx pardinus), Cuvier’s gazelle (Gazella cuvieri) and Mohor gazelle (G. dama mhorr). Reprod Fertil Dev 2014; 26:817–826.
4
Article 119
Downloaded from www.biolreprod.org.
23.
an adaptive defense in the transposon arms race. Science 2007; 318(5851): 761–764. Bao J, Yan W. Male germline control of transposable elements. Biol Reprod 2012; 86(5):162. Oatley JM, Brinster RL. The germline stem cell niche unit in mammalian testes. Physiol Rev 2012; 92(2):577–595. Phillips BT, Gassei K, Orwig KE. Spermatogonial stem cell regulation and spermatogenesis. Philos Trans R Soc Lond B Biol Sci 2010; 365(1546): 1663–1678. Kumar TR. The quest for male germline stem cell markers: PAX7 gets ID’d. J Clin Invest 2014; 124(10):4219–4222. Hermann BP, Phillips BT, Orwig KE. The elusive spermatogonial stem cell marker? Biol Reprod 2011; 85(2):221–223. Valli H, Phillips BT, Shetty G, Byrne JA, Clark AT, Meistrich ML, Orwig KE. Germline stem cells: toward the regeneration of spermatogenesis. Fertil Steril 2014; 101(1):3–13. Plant TM. Undifferentiated primate spermatogonia and their endocrine control. Trends Endocrinol Metab 2010; 21(8):488–495. Hermann BP, Sukhwani M, Hansel MC, Orwig KE. Spermatogonial stem cells in higher primates: are there differences from those in rodents? Reproduction 2010; 139(3):479–493. Hermann BP, Sukhwani M, Winkler F, Pascarella JN, Peters KA, Sheng Y, Valli H, Rodriguez M, Ezzelarab M, Dargo G, Peterson K, Masterson K, et al. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 2012; 11(5):715–726. Ramathal C, Durruthy-Durruthy J, Sukhwani M, Arakaki JE, Turek PJ, Orwig KE. Reijo Pera RA. Fate of iPSCs derived from azoospermic and fertile men following xenotransplantation to murine seminiferous tubules. Cell Rep 2014; 7(4):1284–1297. Durruthy J, Ramathal C, Sukhwani M, Fang F, Cui J, Orwig KE, Reijo Pera RA. Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules. Hum Mol Genet. 2014; 23(12): 3071–3084. Clark AT, Phillips BT, Orwig KE. Fruitful progress to fertility: male fertility in the test tube. Nat Med 2011; 17(12):1564–5. Regaud C. Some biological aspects of the radiation therapy of cancer. Am J Roentgenol Radium Ther 1924; 12(2):97–101. McCarrey JR. Toward a more precise and informative nomenclature describing fetal and neonatal male germ cells in rodents. Biol Reprod 2013; 89(2):47. de Rooij DG, Griswold MD. Questions about spermatogonia posed and answered since 2000. J Androl 2012; 33(6):1085–1095. Nakagawa T, Sharma M, Nabeshima Y, Braun RE, Yoshida S. Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 2010; 328(5974):62–67. Huckins C. The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat Rec 1971; 169(3): 533–557. Lok D, Weenk D, De Rooij DG. Morphology, proliferation, and differentiation of undifferentiated spermatogonia in the Chinese hamster and the ram. Anat Rec 1982; 203(1):83–99. Oakberg EF. Spermatogonial stem-cell renewal in the mouse. Anat Rec 1971; 169(3):515–531. Yang QE, Gwost I, Oatley MJ, Oatley JM. Retinoblastoma protein (RB1) controls fate determination in stem cells and progenitors of the mouse male germline. Biol Reprod 2013; 89(5):113. Hu YC, de Rooij DG, Page DC. Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice. Proc Natl Acad Sci U S A 2013; 110(31):12685–12690. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig KE, Wolgemuth DJ, Pandolfi PP. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004; 36(6):653–659. Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 2004; 36(6):647–652. Meng X, Lindahl M, Hyvo¨nen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287(5457):1489–1493. Sada A, Suzuki A, Suzuki H, Saga Y. The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells. Science 2009; 325(5946):1394–1398. Monesi V. Autoradiographic study of DNA synthesis and the cell cycle in spermatogonia and spermatocytes of mouse testis using tritiated thymidine. J Cell Biol 1962; 14:1–18. Huckins C. Cell cycle properties of differentiating spermatogonia in adult Sprague-Dawley rats. Cell Tissue Kinet 1971; 4(2):139–154.
