Prepubertal Human Spermatogonia and Mouse Gonocytes Share Conserved Gene Expression of Germline Stem Cell Regulatory Molecules

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Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules

Xin Wua, Jonathan A. Schmidta, Mary R. Avarbocka, John W. Tobiasb, Claire A. Carlsonc, Thomas F. Kolond, Jill P. Ginsbergc, and Ralph L. Brinstera,1

aDepartment of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; bPenn Bioinformatics Core, University of Pennsylvania, Philadelphia, PA 19104; and cDivision of Oncology and dDepartment of Urology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104

Contributed by Ralph L. Brinster, October 30, 2009 (sent for review October 8, 2009) In the human testis, beginning at Ϸ2 months of age, gonocytes are robust methods were developed for rodent germ cell transplan- replaced by adult dark (Ad) and pale (Ap) spermatogonia that make tation and SSC culture conditions (1, 4–8). These techniques led up the spermatogonial stem cell (SSC) pool. In mice, the SSC pool to the characterization of many aspects of SSC biology, including arises from gonocytes Ϸ6 days after birth. During puberty in both the identification of glial cell line-derived neurotrophic factor species, complete spermatogenesis is established by cells that (GDNF) as the main regulator of rodent SSC self-renewal (7, 8). differentiate from SSCs. Essentially pure populations of prepuber- GDNF binds to the c-Ret receptor tyrosine kinase (RET) in tal human spermatogonia and mouse gonocytes were selected combination with the cofactor GDNF-family receptor ␣1 from testis biopsies and validated by confirming the presence of (GFR␣1) to initiate intracellular signaling in SSCs (7, 9, 10). specific marker proteins in cells. Stem cell potential of germ cells Using SSC culture and transplantation in conjunction with was demonstrated by transplantation to mouse testes, following combinations of GDNF withdrawal, microarray analysis, small which the cells migrated to the basement membrane of the interference RNA (siRNA), and signaling molecule inhibition, seminiferous tubule and were maintained similar to SSCs. Differ- several GDNF-regulated genes involved in SSC self-renewal ential gene expression profiles generated between germ cells and were identified and studied in the mouse, including B-cell testis somatic cells demonstrated that expression of genes previ- CLL/lymphoma 6, member B (Bcl6b), Ets variant gene 5 (Etv5), ously identified as SSC and spermatogonial-specific markers (e.g., and LIM homeobox 1 (Lhx1) (10). Three additional GDNF- zinc-finger and BTB-domain containing 16, ZBTB16) was greatly regulated genes, basic helix–loop–helix family, member e 40 elevated in both human spermatogonia and mouse gonocytes (Bhlhb2), homeobox C4 (Hoxc4), and Tec protein tyrosine compared to somatic cells. Several genes were expressed at sig- kinase (Tec) were validated in rat SSCs (11). Among these six nificantly higher levels in germ cells of both species. Most impor- genes, Bcl6b and Etv5 have now been identified as important by tantly, genes known to be essential for mouse SSC self-renewal several studies and appear to play a central role in rodent SSC (e.g., Ret proto-oncogene, Ret; GDNF-family receptor ␣1, Gfr␣1; self-renewal (10–13). GDNF also regulates downstream signal- and B-cell CLL/lymphoma 6, member B, Bcl6b) were more highly ing and ultimately rodent SSC maintenance and self-renewal, expressed in both prepubertal human spermatogonia and mouse which involves phosphatidylinositol 3-kinase/serine-threonine gonocytes than in somatic cells. The results indicate remarkable kinase AKT family (PI3K/AKT), and Src family kinase (SFK) conservation of gene expression, notably for self-renewal genes, in signaling mechanisms (14–16). In contrast to rodent SSCs, little these prepubertal germline cells between two species that di- is known regarding the mechanisms regulating primate SSC verged phylogenetically Ϸ75 million years ago. function. This lack of knowledge is in part due to the absence of techniques for identification and isolation of essentially pure mouse spermatogonia ͉ spermatogenesis populations of primate SSCs for in vitro study. Several groups have attempted to characterize gene expression in human testes. permatogonial stem cells (SSCs) are the foundation for However, the lack of purified cell populations made interpre- Sspermatogenesis and are capable of both self-renewal and tation of results difficult (17, 18). production of daughter cells that differentiate into spermatozoa. To develop an understanding of SSCs and their regulation in the During embryonic development, primordial germ cells (PGCs) human germline, essentially pure populations of prepubertal hu- migrate to the genital ridge and subsequently differentiate into man spermatogonia were isolated and their gene expression profile gonocytes (1, 2). Following birth in the mouse, which has a short determined. Parallel studies were performed on mouse gonocytes (Ϸ3 weeks) prepubertal period, gonocytes undergo a transition for comparison, because extensive data exists regarding mouse to SSCs or develop directly to type A1 spermatogonia, an early SSCs and their regulation. Moreover, a similarity in self-renewal differentiation stage, by day six of life (2). However in humans, and survival mechanisms between human and mouse SSCs may which have a long (Ϸ12 years) prepubertal period, the gonocytes exist because transplantation of testis cells from nonrodent species, are gradually replaced in the first 2–3 months by adult dark (Ad) including human, into testes of immunodeficient mice allowed the and adult pale (Ap) spermatogonia that are thought to represent the reserve and active SSC pool, respectively (2, 3). Beginning at about age 5 years, these Ad and Ap spermatogonia undergo a Author contributions: X.W., J.A.S., and R.L.B. designed research; X.W. and M.R.A. performed research; C.A.C., T.F.K., and J.P.G. contributed new reagents/analytic tools; X.W., modest activation, particularly to type B spermatogonia, that J.A.S., J.W.T., and R.L.B. analyzed data; and X.W., J.A.S., and R.L.B. wrote the paper. Ϸ represent 10% of total spermatogonia by age 10. During The authors declare no conflict of interest. puberty, the SSCs in both human and mouse provide the Data deposition: The microarray data reported in this paper has been deposited in the foundation, through self-renewal and differentiation to daughter National Center for Biotechnology Information Gene Expression Omnibus (GEO) database, cells, for spermatogenesis and fertility. www.ncbi.nlm.nih.gov/geo (accession no. GSE18914). The study of rodent gonocytes and SSCs was previously 1To whom correspondence should be addressed. E-mail: [email protected]. hampered by the lack of techniques for purification and long- This article contains supporting information online at www.pnas.org/cgi/content/full/ term in vitro maintenance. However, over the past 15 years, 0912432106/DCSupplemental.

21672–21677 ͉ PNAS ͉ December 22, 2009 ͉ vol. 106 ͉ no. 51 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0912432106 Downloaded by guest on September 26, 2021 human spermatogonia and mouse gonocytes present an opportunity to potentially isolate essentially pure populations of human SSCs and mouse gonocytes, which include the immediate precursor of mouse SSCs. To confirm that the large cells located in the seminiferous tubules were indeed prepubertal human spermatogonia and mouse gonocytes, histological sections were stained for characteristic germ cell marker proteins. ZBTB16 (previously known as PLZF) (20, 21) is highly expressed in gonocytes and spermatogonia, including SSCs, but not in later differentiation stages of spermatogenesis, and the protein was found to be strongly immunostained in the large cells in the seminiferous tubules of both human and mouse testes (Fig. 1 C and D). Other characteristic spermatogonial proteins also stained in prepubertal human spermatogonia and mouse gonocytes (Fig. S1). In contrast, GATA-binding protein 4 (GATA4), which is found in Sertoli cells but not germ cells (22), was not present in the large cells (Fig. 1 E and F). These data demonstrate that the large cells in the seminiferous tubules in both species are indeed immature germ cells. Following digestion of the testis tissue to a single cell suspension, immunostaining for the presence of germ cell and Sertoli cell-specific markers confirmed germ cell markers were expressed exclusively in large round cells from both human and Fig. 1. Germ cells in prepubertal human (age 9 years) and mouse testes (age: mouse testes (Fig. S2). GATA4 was not detected in any large 3 days). Histological cross sections of human (A) and mouse (B) testis show cells, and was exclusively found in smaller cells. large cells (arrows) resting near the basement membrane of the seminiferous tubule. Immunohistochemical staining of human and mouse testes with germ Prepubertal Human Spermatogonia and Mouse Gonocytes Can Be cell markers demonstrated that the large round cells (arrows) expressed Selected and Purified from Testis Cell Suspensions. Because of the CELL BIOLOGY ZBTB16, a well-characterized marker of SSCs and spermatogonia (C, human, D, difference in size and morphological characteristics between the mouse). In contrast, the large cells did not express the somatic cell marker immature germ cells and somatic cells, micromanipulation tech- GATA-4 (E, human, F, mouse), which was present in Sertoli cells (arrow heads). niques to select these two cell types from single cell suspensions Negative control images in which gene speciﬁc antibodies were replaced with isolated from prepubertal human and mouse testes were devel- IgG control antibodies are included in each panel. (Scale bars, 20 ␮m.) oped (SI Materials and Methods). The selected populations of germ cells and somatic cells were distinctly homogenous and maintenance and limited replication of spermatogonia in the essentially pure, as demonstrated by immunofluorescence for recipient seminiferous tubules for periods of 6–12 months (1, 19). marker proteins (Fig. S3). Germ cells were significantly larger Ϯ Ϯ ␮ Thus, comparison of prepubertal human spermatogonia and mouse than somatic cells for both human (14.8 0.2 and 8.7 0.2 m, respectively, n ϭ 10, P Ͻ 0.05) and mouse (12.7 Ϯ 0.3 and 8.7 Ϯ gonocyte gene expression profiles could confirm the existence of ␮ ϭ Ͻ regulatory conservation between human and mouse in male germ- 0.3 m, respectively, n 10, P 0.05) and prepubertal human spermatogonia were significantly larger than mouse gonocytes line cells, provide details regarding species-specific gene expression Ͻ patterns, potentially allow for the extrapolation of our knowledge (P 0.05). Selected germ cells from both human and mouse testes were positive for ZBTB16, as well as for three additional about mouse SSCs to the human germline, which is difficult to spermatogonial and germ cell markers, ubiquitin carboxyl- study, and impact our understanding of human male fertility and terminal esterase L1 (UCHL1; previously known as PGP9.5), infertility. dead (Asp-Glu-Ala-Asp) box polypeptide 4 (VASA; also known Results as DDX4), and deleted in azoospermia-like (DAZL) (23–25), but were negative for GATA4 (Fig. S3). When 820 selected large Human and Mouse Germ Cell Populations Are Predicted by Morphol- cells from human and mouse testes were stained for germ cell ogy and Immunostaining. Before selection of prepubertal human markers, 811 were stained (98.9%). In contrast, selected popu- spermatogonia and mouse gonocytes for oligonucleotide mi- lations of somatic cells were negative for ZBTB16, UCHL1, croarray analysis, morphology of the germ cells was confirmed VASA, and DAZL and some are positive for GATA4 (Fig. S3). using immunostaining. In prepubertal testes from a 9-year-old When 820 selected somatic cells from human and mouse testes human (Fig. 1A) and a 3-day-old mouse (Fig. 1B) germ cells were were stained for germ cell markers, none were stained (0%). large in diameter and near the basement membrane of the Collectively, these data indicate that micromanipulator selection Ϸ seminiferous tubules. The prepubertal period in human is 12 of germ cells from prepubertal human testis and neonatal mouse Ϸ years and in mouse is 3 weeks, and the relative immature status testis cell suspensions is an efficient technique for the enrich- of both human and mouse testis is confirmed by the large size of ment of prepubertal human spermatogonia and mouse gono- the germ cells and the absence of spermatogenesis. In the mouse, cytes, which provides essentially pure populations of cells for by about day 6, the gonocytes convert to SSCs or type A1 analysis. spermatogonia, and both reside on the basement membrane, are smaller than the gonocytes and resemble more differentiated Transplantation of Selected Testis Germ Cells Demonstrates Stem Cell germ cell stages (2, 3). In human, Ad and Ap spermatogonia Potential. Germ cells from neonatal mouse testes, gonocytes, persist as large cells comprising 80–90% of the germ cells until have stem cell potential (4, 26). To prove unequivocally that puberty begins (2, 3). Then, the Ad and Ap stem cells become isolated germ cells are indeed gonocytes, populations of selected a small percentage of the total germ cell population and are putative somatic cells and gonocytes from ROSA26 transgenic difficult to distinguish from differentiating germ cells. Thus, mice that express LacZ in all cells, including germ cells, were unequivocal separation of SSCs from other germ cells once transplanted into recipient mouse testes. Two months after differentiation begins is not possible, and these prepubertal transplantation, testes were evaluated using 5-bromo-4-chloro-

Wu et al. PNAS ͉ December 22, 2009 ͉ vol. 106 ͉ no. 51 ͉ 21673 Downloaded by guest on September 26, 2021 recipient mouse would form a dense network of spermatogonia and a colony of spermatogenesis (Fig. S4). To determine whether the prepubertal human spermatogonia were capable of further differentiation in mouse testes, we made use of a modified Busulfan-treated mouse recipient in which the endogenous Sertoli cells are removed by injecting cadmium into the seminiferous tubules (27), After transplantation of a mouse testis cell suspension into such recipients, donor-derived mini- tubules and spermatogenesis are established in the recipient tubules (27). When a prepubertal human testis cell suspension from a 10-year-old boy was injected into the tubules of immunodeficient mouse recipients treated with cadmium, after 4 months, approximately seven times as many prepubertal human spermatogonia colonization sites were observed (Table S1). In addition, small colonies of three to six human germ cells could be identified, similar to initial SSC proliferation in the 2 weeks after mouse gonocyte transplantation to recipients (Fig. 2 C and Fig. 2. Colonization of recipient mouse seminiferous tubules by trans- D). No human spermatogonia differentiation or spermatogen- planted donor testis cells at posttransplantation times of approximately 4 esis beyond the small colonies were found (SI Discussion). months for human, and 2 days or 2 weeks for mouse. Whole-mount staining of transplanted human germ cells with anti-UCHL1 antibody was visualized Oligonucleotide Microarray Analysis Reveals Genes Expressed in Pre- with 3-amino-9-ethylcarbazole (AEC; red) at approximately 4 months after transplantation (A). Mouse ROSA26 transgenic donor testis cells in recipient pubertal Human Spermatogonia and Mouse Gonocytes. RNA from mouse seminiferous tubules were stained with X-gal (blue) at day 2 after selected populations of germ cells and somatic cells from human transplantation (B). Human cells transplanted to recipient mouse seminiferous and mouse testes was subjected to oligonucleotide microarray tubules previously treated with cadmium were found in small groups or analysis. This analysis showed that 10,809 and 6,076 gene probes colonies and with greater frequency (C; AEC staining; red and Table S1). These were expressed at significantly different levels between germ cells small groups of human germ cells resembled the initial germ cell expansion of and somatic cells from prepubertal human and mouse testes, mouse gonocytes transplanted to mouse recipients after approximately 2 respectively. Of these probes with significantly different expression weeks (D; X-gal staning; blue). Human germ cells (A and C) never progress levels (P Ͻ 0.05), 3909 and 2115 were expressed 2-fold or higher in beyond the initial colonization, but mouse germ cells continue to divide and form colonies of spermatogenesis (Fig. S4). (Scale bar, 20 ␮m.) prepubertal human and mouse germ cells, respectively, relative to somatic cells. In addition, 44 and 6 gene probes were expressed 100-fold or higher in human and mouse germ cells, respectively, 3-indolyl b-D--galactoside (X-gal) staining. No spermatogenic compared to somatic cells. When the top 100 genes with enriched colonies were observed in any testis transplanted with somatic expression in germ cells were compared between human (Table S2) cells (n ϭ 8 testes; Fig. S4). In contrast, colonies were readily and mouse (Table S2) samples, seven of these genes including Dazl, observed in testes transplanted with putative gonocytes (n ϭ 8 were conserved between prepubertal human spermatogonia and testes; Fig. S4), thus demonstrating that large cells selected from mouse gonocytes (Table S3). Expression and enrichment of these digested mouse neonatal testes are gonocytes with stem cell seven genes were confirmed using qRT-PCR (Table S3). The potential. specificity of this list to germ cells is validated by the presence of When adult human testis cells are transplanted to immuno- DAZL, which previously has been demonstrated to be character- deficient mice the somatic cells are lost, as above with mouse istically expressed by the germline (25). The two most differentially testis cell transplantation (19). However, the transplanted hu- expressed of the conserved genes, embryonic lethal, abnormal man SSCs remain on the recipient mouse seminiferous tubule vision, Drosophila-like 2 (Hu antigen B) (Elavl2) and serine/ threonine kinase 31(Stk31) were selected for further analysis to basement membrane for periods up to 6 months as single cells, confirm their presence in germ cells and validate the oligonucleo- with an occasional doublet or slightly higher number of germ tide microarrays. Expression of Elavl2 has previously been observed cells, representing slow division (19). Differentiation of the in testis tissue; however, this expression was not characterized (28). human SSCs to later germ cell stages does not occur in mouse ␮ Stk31 was recently identified as a novel cancer/testis antigen ex- seminiferous tubules. When 10 L of a testis cell suspension pressed in adult mouse spermatogonia (29). In human testis, Elavl2 from a prepubertal boy (age: 10 years) containing human Ϸ and Stk31 were both expressed in germ cells (Fig. S1). ELAVL2 prepubertal spermatogonia ( 150 cells) plus somatic cells protein was localized to the nucleus, whereas STK31 was localized (Ϸ850 cells) was transplanted to each testis of immunodeficient ϭ to the cytoplasm. ELALVL2 was also expressed in mouse gono- nude mice and the testes (n 6) of the recipients examined 3 to cytes; however, no antibodies exist for localizing mouse STK31. 6 months later, germ cells were found as singlets or doublets on the seminiferous tubule basement membrane (Fig. 2A). These Comparison of Gene Expression between Prepubertal Human Sper- results indicate that the spermatogonia in the testis cell suspen- matogonia and Mouse Gonocytes Reveals a High Level of Conserva- sion of the prepubertal boy migrate to the basement membrane tion. Many relevant genes that previously have been reported to of the mouse recipient seminiferous tubule and are maintained be enriched in germ cells were expressed in selected populations as germ cells in a manner similar to transplanted adult human of both prepubertal human spermatogonia and mouse gonocytes SSCs (19). The appearance of the human cells on the basement (Table 1). These genes are important for several aspects of male membrane after 3–6 months resembles that of mouse gonocytes germ cell biology. Specific extracellular surface marker genes in the first few days after transplantation (Fig. 2B) suggesting that have been used to enrich for germline stem cells were that the prepubertal human spermatogonia home to the base- conserved and expressed significantly higher in germ cells than ment membrane but are unable to differentiate. UCHL1 immu- in somatic cells including, EpCam for human and mouse (8), nostaining identifies these cells as spermatogonia(Fig. 2A and Gpr125 for human (30), and Itga6 for mouse (31). Several Fig. S4). The species origin of the cells was confirmed using an intracellular marker genes associated with germ cell function anti-baboon antibody known to stain human germ cells (Fig. S4) were also expressed higher in prepubertal human spermatogonia (19). Moreover, any residual endogenous mouse SSCs in a and mouse gonocytes. These included, Dazl, Vasa (24), Zbtb16,

21674 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0912432106 Wu et al. Downloaded by guest on September 26, 2021 Table 1. Expression of genes in prepubertal human spermatogonia and mouse gonocytes that were previously identiﬁed in mice as enriched in spermatogonial stem cells relative to testis somatic cells Human Mouse

Prepubertal Somatic Fold Somatic Fold Gene symbol* spermatogonia† cells† difference‡ P value Gonocytes† cells† difference‡ P value

Surface markers Epcam 1979 535 4 0.00 2464 52 47 0.00 Gpr125 165 17 10 0.05 361 219 2 0.13 Itga6 897 1311 1 0.50 651 170 4 0.05 Kit 621 15 41 0.00 65 6 11 0.01 Intracelluar markers Dazl 443 5 96§ 0.00 1451 19 76§ 0.00 Vasa 1767 14 124 0.00 856 87 10 0.00 Zbtb16 28 25 1§ 0.57 931 65 14§ 0.00 Uchl1 900 1224 1§ 0.59 606 34 18§ 0.02 Taf4b 1032 28 36 0.00 229 110 2 0.39 Ngn3 10 24 Ϫ2 0.06 11 16 1 0.13 Self-renewal markers Ret 190 29 7 0.02 808 45 18 0.00 Gfra1 100 12 8 0.01 388 13 30 0.00 Etv5 1228 39 31 0.00 981 515 2 0.21 Bcl6b 235 66 4 0.03 2649 56 47 0.00

*Affymetrix probe which represent the signals are shown in SI Materials and Methods. †Average of the normalized signal for expression on the three replicate microarrays (represented by probe with the highest relative expression values). ‡Fold difference between germ cells and somatic cells. §

qRT-PCR values for fold enrichment of Dazl, Zbtb16, Uchl1 were 138 and 76, 22 and 36, 70 and 32 for prepubertal human spermatogonia and mouse gonocytes, CELL BIOLOGY respectively.

TATA box-binding protein (TBP)-associated factor (Taf4b) GDNF Is Important for Prepubertal Human Spermatogonia Mainte- (32), and Uchl1 (23) but not neurogenin 3 (33). While the nance. When mouse gonocytes are placed in serum-free culture oligonucleotide microarray analysis failed to show enrichment of medium containing GDNF and GFR␣1 on STO (SIM mouse Zbtb16 and Uchl1 gene expression in prepubertal human sper- embryo derived thioguanine and ouabain resistant) feeder cells, matogonia, protein staining and qRT-PCR indicated the pres- in conditions that support mouse SSC self-renewal, they quickly ence of high levels of proteins (Fig. 1 and Figs. S1 and S2) and form germ cell clumps that contain a high percentage of cells mRNA (Table 1, legend) for these genes. with stem cell potential and the SSCs continue to multiply (7). Most important, the expression of genes previously demon- However, these conditions do not support prepubertal human strated to be essential for GDNF regulation of mouse and rat spermatogonia maintenance, and the human germ cells quickly SSC self-renewal were also conserved between prepubertal detach from the feeder cells and are lost. Serum-free medium for human spermatogonia and mouse gonocytes (Table 1). These culture of rat gonocytes and SSCs is slightly modified from the genes include the cognate receptors for GDNF, Ret and GFR␣1, mouse medium, with several components increased in concen- as well as for the GDNF regulated transcription factors Bcl6b tration (8). When this medium is used for prepubertal human and Etv5 (10, 11), which are all critical for SSC self-renewal. Etv5 spermatogonia culture and the feeder cells are changed to C166 is reported to be expressed in mouse Sertoli cells (34), and thus, (ATCC), the human germ cells can be maintained for at least 19 the signal is high in the microarray data for mouse somatic cells days but do not form germ cell clumps (Fig. S5). However, if as well as for gonocytes, resulting in an insignificant enrichment GDNF and GFR␣1 are omitted from the medium, the germ cells calculation for mouse gonocytes. Nonetheless, it has been es- quickly detach from the feeder cells and are lost (Fig. S5). By the tablished that Etv5 is highly expressed in mouse SSCs and critical end of 1 week in culture, 80% of the prepubertal human for self-renewal (10). In contrast, genes (Nanog, Pou5f1, and spermatogonia have detached in medium lacking GDNF and Sox2) known to be important for self-renewal and pluripotency GFR␣1 compared to medium with the growth factors (Fig. S5). of embryonic stem cells (ESCs) and induced pluripotent stem Clearly, the GDNF signaling pathway is important for prepu- cells (iPSCs) were not expressed at high levels or greatly enriched bertal human spermatogonia maintenance, but other mecha- in prepubertal human spermatogonia and mouse gonocytes nisms are necessary for continual self-renewal and formation of (Table S4) (35, 36). An exception appears to be lin-28 homolog germ cell clumps. B(Caenorhabditis elegans)(Lin28b) in prepubertal human spermatogonia and lin-28 homolog (C. elegans)(Lin28) in mouse Discussion gonocytes (Table S4) that are expressed in pluripotent cells and Little is known about the biology and regulation of human at relatively high levels in the germ cells from prepubertal testes, germline cells, particularly regarding maintenance and regula- which may suggest a role for these genes in the biology of these tion of SSCs, which are the foundation of spermatogenesis germ cells (SI Text). Thus, prepubertal human spermatogonia throughout adult life. The oligonucleotide microarray analyses and mouse gonocytes share a remarkable similarity in gene provided compelling data regarding the similarity of prepubertal expression for surface and intracellular markers, as well as in human spermatogonia and mouse gonocytes. Remarkably, seven gene expression for molecules known to be critical to mouse and of the 100 most highly differentially expressed genes between rat SSC survival and self-renewal, but little similarity in expres- germ cells and somatic cells were conserved in human and mice, sion of the genes critical for ESC and iPSC pluripotency and despite phylogenetic divergence of these species approximately self-renewal. 75 million years ago, attesting to the fundamental importance of

Wu et al. PNAS ͉ December 22, 2009 ͉ vol. 106 ͉ no. 51 ͉ 21675 Downloaded by guest on September 26, 2021 germ cells in species evolution (37). While indicative of a high degree of conservation, a specific role for these seven genes in germ cell biology has not been identified. DAZL is present in germ cells, but its function is unclear. More informative is the conservation of expression of genes already known to be present and often of functional importance in germ cells, particularly SSCs. The expression of genes for four surface protein markers found on SSCs was significantly enriched in the germ cell populations. In mice, EPCAM has previously been identified on neonatal male germ cells and in adult testes only on spermatogonia (38). In rat, EPCAM is an excellent, and perhaps the best, surface antigen for enrichment of rat gonocytes and SSCs of pups (8). The expression of Gpr125 is present in prepubertal human spermatogonia but not mouse gonocytes. However, GPR125 has been reported to be present on mouse spermatogonia and useful for selection (30). Expression of Itga6 is enriched in mouse gonocytes, and the antigen is known to be useful in selection (31). The absence of enriched expression in the prepubertal Fig. 3. A proposed model of human spermatogonial stem cell (SSC) self- human spermatogonia reflects not so much the absence of renewal regulation by glial cell line-derived neurotrophic factor (GDNF), expression in the germ cells, but rather its expression in somatic which has been demonstrated to have an essential role in regulating rodent SSC self-renewal. The model is similar to those suggested for mouse SSC cells as indicated by the high probe signal for human testis self-renewal (42). In this model, GDNF binds to RET and the GFR␣1 coreceptor somatic cells. KIT is a well-known characteristic surface marker with possible intracellular protein kinase signaling through SFK and PI3K/AKT of germ cells, often expressed in the fetal testis and on differ- downstream pathways to regulate the expression of speciﬁc genes, such as entiating spermatogonia (39). While a high signal for mRNA is Etv5 and Bcl6b, which are involved in SSC self-renewal. However; other genes present in the prepubertal human spermatogonia, the signal is not regulated by GDNF (e.g., Zbtb16, Taf4b, and Lin28), are likely controlled much lower in the mouse gonocyte, and the protein may be by different signals and may block differentiation but not be involved directly absent or low on the cell surface. The relatively high level of the in self-renewal. Genes for these regulatory molecules have been shown to be Kit signal in prepubertal human spermatogonia may reflect a highly expressed in prepubertal human spermatogonia, mouse gonocytes and signal for some Ad and Ap stem cells to differentiate into type mouse SSCs. The basement membrane (green), on which the SSC rests, is generated by the peritubular myoid cells (dark brown) and Sertoli cells (tan). B spermatogonia, a process known to be occurring during the prepubertal period in humans (2, 3). While the match between human and mouse surface antigen gene expression in germ cells self-renewal of the SSC (10, 11). Protein kinases SFK, P13K, and is not exact, the similarity is compelling. Moreover, the biological AKT, play a role in the intracellular self-renewal signals from importance of surface antigen similarity is confirmed, in part, by RET in mouse SSCs, and in each of these signaling pathways the ability of human SSCs (19) and prepubertal spermatogonia several molecules are expressed at high levels in prepubertal (Fig. 2 and Table S4) after transplantation to migrate to the human spermatogonia and mouse gonocytes (14) (SI Text). basement membrane of the mouse seminiferous tubule. This Thus, the data in Table 1 suggest that this regulatory pathway is migration of prepubertal human spermatogonia in a direction already present in the mouse gonocyte for maintenance and opposite to normal differentiating germ cell movement, through certainly for use immediately when the transition to SSC occurs the mouse Sertoli cell tight junctions, which separate the sem- before puberty. Moreover, these data imply that prepubertal iniferous tubule lumen from the basement membrane in the human spermatogonia and mouse gonocytes as well as mouse recipient testis, represents a dramatic conservation of the ‘‘hom- SSCs share, at least in part, critical aspects of maintenance and ing’’ mechanism in these human germ cells despite an enormous self-renewal (Fig. 3). Certainly other regulatory factors are phylogenetic divergence from the recipient. involved in these processes for prepubertal human spermatogo- The enrichment in expression of genes for intracellular markers of SSCs in prepubertal human spermatogonia and mouse gonocytes nia, because a medium containing GDNF that supports mouse is just as remarkable and important as found for cell surface gonocyte transition to SSC is not adequate to support self- proteins. On the basis of oligonucleotide microarray analyses and renewal and germ cell clump formation for prepubertal human qRT-PCR, the expression of Dazl, Vasa, Zbtb16, and Uchl1 are all spermatogonia. Nonetheless, the importance of GDNF for enriched in these prepubertal germ cells of both species as they are prepubertal human spermatogonia is attested to, not only by the in mouse SSCs (20, 21, 23–25). Rhesus monkey spermatogonia have microarray data, but also by the maintenance of prepubertal also been shown to express Dazl, Vasa, and Zbtb16 (40). While human spermatogonia in medium containing GDNF in contrast Taf4b expression showed enrichment on the microarray in prepu- to their rapid disappearance when the growth factor is absent bertal human spermatogonia but not mouse gonocytes, it is known (Fig. S5). Moreover, the maintenance of human putative SSCs in to be important for mouse SSC maintenance (32). In contrast to the mouse testes for long periods indicates important growth factors previous genes, Ngn3 has been reported to be present in mouse and gene activity are common to the two species. In a medical SSCs and early spermatogonia but not in gonocytes (33). Thus, the context, information regarding maintenance of human SSCs in intracellular markers in these germ cells mirror the similarity vitro has particular relevance for prepubertal boys undergoing between the two species in surface markers and suggest that cancer treatment (SI Discussion). intracellular regulation in prepubertal human spermatogonia and The morphology and location of the prepubertal germ cells mouse gonocytes is comparable in important aspects and similar to made identification and isolation possible, and protein markers mouse SSCs. confirmed the selection of essentially pure populations. Subse- The most striking and significant pattern of similarity between quent oligonucleotide microarray analyses demonstrated a re- prepubertal human spermatogonia and mouse gonocytes is markable similarity in these cells from human and mouse and regarding expression of genes known to be critical for self- demonstrated that they share high expression levels for the renewal of mouse SSCs. In mouse, rat, and probably hamster, receptors for GDNF, the essential self-renewal growth factor for RET and its coreceptor, GFR␣1, bind GDNF and signal, in part, mouse and rat SSCs, as well as for critical intracellular tran- through transcription factors ETV5 and BCL6B to regulate scription factors controlling SSC self-renewal. These data sug-

21676 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0912432106 Wu et al. Downloaded by guest on September 26, 2021 gest that prepubertal human spermatogonia and mouse gono- biopsies from boys aged 2–10 years. An average of 31.5 mg tissue was obtained cytes share regulatory mechanisms with mouse SSCs. The ability from human biopsies. of prepubertal human spermatogonia to migrate to the basement membrane and be maintained as germ cells, and likely stem cells, Microarray Processing and Analysis. Four hundred cells were selected for each in mouse seminiferous tubules, as well as the positive influence group of germ cells or testis somatic cells from three independent testis cell of GDNF on the human spermatogonia in vitro, lends biological preparations from both human (ages 2, 8, and 10 years) and mouse (age 3 support to the similarity of the two species in their SSCs. This days). relationship is remarkable because of the large phylogenetic Further details of procedures are described in the SI Text. separation between the two species and is important because it While this manuscript was under review, an article appeared describing opens a window of opportunity to learn about human SSCs staining of human adult germ cells for some the same antigenic proteins (41) through studies on prepubertal human spermatogonia that can be identified and isolated in essentially pure populations and ACKNOWLEDGMENTS. We thank Drs. R. Behringer, S. Goodyear, M. Kotlikoff, relating observations to the rapidly developing information base Z. Niu, and J. Oatley for critical evaluation of the manuscript; C. Freeman and R. Naroznowski for assistance with animal maintenance; J. Hayden for assis- about mouse SSCs. tance with photography and art work; and Juliana Burns for histological preparations. Financial support by an Ethel Foerderer Award (J.P.G.), National Materials and Methods Institute of Child Health and Human Development (NICHD) Grant HD061217 Cell Preparation and Selection. Mouse donor cells were isolated from 3-day-old (to J.P.G.), NICHD Grant HD044445 (to R.L.B.), NICHD Grant HD 052728 (to C57BL/6 or ROSA26 mouse pups. Human donor cells were isolated from testis R.L.B.), and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (R.L.B.).

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