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COMMENTARY

Germ-line immortality

Martin M. Matzuk* Departments of Pathology, Molecular and Cellular Biology, and Molecular and Human Genetics, and Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030

ajor advances in stem cell Table 1. Pathway of differentiation in males research have occurred over Spermatogonial Differentiated the last decades. Progress Markers ES cells ¡ PGCs ¡ Gonocytes ¡ stem cells ¡ spermatogonia has included the generation ofM lines of human and mouse embryonic Kit ϩϩϩ͞– – (low) ϩ ϩ ϩϩ stem (ES) cells and the identification and Thy-1 ? (low) – Oct4 ϩϩ ϩ ϩ – purification of stem cells for multiple Plzf ϩ (low)* ϩ* ϩϩ – independent lineages. Recent studies by GCNA1 – ϩ† ϩϩ ϩ Brinster and colleagues in this issue of TNAP ϩ (high) ϩ (high) – ϩ (low)͞–– PNAS (1) also suggest that the reproduc- RET ϩ (low)* ? ? ϩ – ␣ ϩ ϩ tive potential of an organism can be pro- GFR 1 (low)* ? ? (low) – NCAM ϩ ? ϩϩ ? longed indefinitely by using germ-line stem cells. It even appears that and Markers that are known to be expressed (ϩ) or absent (–) in many of the pathway cells are listed. GCNA1, can develop from cultured mouse nuclear antigen 1; TNAP, tissue-nonspecific ; NCAM, neural cell adhesion ES cells (2–4). Although derived molecule. in vitro have yet to prove their develop- *mRNA levels. † mental potential, these studies suggest Postmigratory PGCs only. that ES cells and germ-line stem cells share many characteristics. ent males. The process was surprisingly KitϪ Sca-IϪ ␣6-integrinϩ ␣v-integrinϪ/dim In most mammalian females, efficient, with up to 100% of the injected (9, 10). initiates during in utero development, such testes containing donor cells. that no oogenic stem cells exist in the Spermatogonial Stem Cells Keep Going postnatal ovary, thereby limiting the re- Stem Cell Markers Emerge and Going ... productive lifespan of a woman. However, With transplantation methodology in The third major advance in the spermato- the reproductive lifespan of a man is in hand, Brinster and colleagues (9, 10) gonial stem cell field is presented by Kub- theory without limits. Although this re- moved to develop methods to enrich for ota et al. (1) in this issue of PNAS. For productive potential of males has long spermatogonial stem cells. Similar to the the entire adult stem cell field, it has been been known, the further characterization relatively small populations of other adult extremely difficult to generate stable lines and identification of spermatogonial stem stem cells, spermatogonial stem cells are of stem cells that can propagate in vitro. cells is only a recent advance. rare (Ϸ1 in 3,000 adult testis cells). Fluo- Kubota et al. now show that mouse sper- Three major requirements were needed rescence-activated cell sorting (FACS) was matogonial stem cells from several strains to advance re- used to identify cell-surface markers for can be maintained continuously in culture search, including (i)anin vivo assay for the spermatogonial stem cells (9, 10). for 6 months and reconstitute normal stem cell function, (ii) knowledge of stem Through a meticulously conducted series in an infertile host. There cell markers, and (iii) a method to main- of experiments, populations separated by were three keys to their success. First, by tain the stem cells continuously in culture. FACS (and, now, magnetic-activated cell using the markers described above, stem Similar to his pivotal studies to optimize sorting) are transplanted back into mice. cells were separated from contaminating culture and transgenesis (reviewed in The first major finding was that the Kit testes cells that interfere with prolifera- refs. 5 and 6), Brinster’s group has system- tyrosine kinase positive populations of tion. Second, the stem cells were grown atically made giant leaps toward these testis cells did not have increased stem on a mitotically inactive layer of STO fi- broblasts. Third, the stem cells could be goals. cell activity (9). This finding was a sur- cultured in a well defined serum-free me- prise because ES cells, primordial germ Spermatogonial Transplantation dium; absence of serum was critical be- cells (PGCs), gonocytes, and differenti- A decade ago, Brinster and Zimmerman ϩ cause serum supports the proliferation of (7) and Brinster and Avarbock (8) pub- ated spermatogonia are Kit (Table 1). other cells, including fibroblasts, but de- lished in PNAS methods for spermatogo- This result is consistent with findings that ters growth of the stem cells. This finding nial transplantation. These studies were a Kit ligand is necessary for embryonic contrasts with the long-term culture of major breakthrough in the development germ cell formation but not for prolifera- germ-line stem cells from only one mouse of an assay for in vivo stem cell function. tion of postnatal day 2 spermatogonia or strain (DBA͞2) by Kanatsu-Shinohara et Male mice lacking spermatogenesis were germ-line stem cells in culture (11). al. (11), who used 1% bovine serum and chosen as recipients. Wild-type mice or In the search for markers strongly asso- mitotically inactive mouse embryonic fi- transgenic mice carrying a round sperma- ciated with stem cell function, Brinster’s broblasts. [Interestingly, 40 years ago, tid-expressed ␤-galactosidase (lacZ) gene group (10) showed that Thy-1, expressed Brinster demonstrated that a serum-free (or, more recently, either ROSA26 or on ES cells (12), is also present on sper- medium including BSA was necessary for actin-enhanced green fluorescent matogonial stem cells. However, whereas egg culture (reviewed in ref. 6).] Depend- transgenic mice) have served as stem cell many adult stem cells, including mammary ing on the strain of mice from which they donors. After enzyme digestion of the gland stem cells (13) and cardiac stem donor tubules, the germ cells of perinatal cells (14), express stem cell antigen 1 to 4-week-old testes (along with trypan (Sca-1), spermatogonial stem cells are See companion article on page 16489. Ϫ blue dye) were injected with a 40-␮m Sca-1 (10). Thus, spermatogonial stem *E-mail: [email protected]. Ϫ ϩ glass pipette into the tubules of the recipi- cells could be sorted as MHC-I Thy-1 © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407344101 PNAS ͉ November 23, 2004 ͉ vol. 101 ͉ no. 47 ͉ 16395–16396 Downloaded by guest on October 2, 2021 were derived, the cells grew in serum-free spermatogonial stem cells fail to self- cell-specific (22), can gene targeting cor- conditions with the addition of glial cell renew, and the mice eventually lack sper- rect an infertile man’s germ-line defect in line-derived neurotrophic factor (GDNF) matogenesis. Plzf likely functions by his own spermatogonal stem cells? or also in the presence of soluble GDNF- recruiting Polycomb family members and Clearly, a robust culture system, as de- family receptor ␣-1 (GFR␣1) and basic subsequent histone deacetylases. Thus, the scribed by Kubota et al. (1), combined fibroblast growth factor. The requirement ability of Plzf to control the epigenetic with an established transplantation tech- for GDNF and a ligand-binding soluble state of the stem cell dictates the rate of nology will provide the foundation for receptor component was not surprising spermatogonial stem cell self-renewal and gene targeting, and with extension to because Gdnf ϩ/Ϫ mice lose their sper- the timing of differentiation, both pro- other species will fuel a major advance in matogonia (15), GDNF can stimulate self- cesses likely influenced by the GDNF sig- genetic modification for farm animals, renewal of spermatogonia (15, 16), and naling pathway. endangered species, and primates, includ- the spermatogonial stem cells express Although not true of other species, ing humans. three GDNF receptors [RET, GFR␣1, spermatogonial stem cells from rat are The current study by Kubota et al. (1) and neural cell adhesion molecule (1)]. not only competent to undergo all of the also has important relevance for testicular Similar to in vivo, the cultured cells dou- steps of spermatogenesis in the seminifer- , stem cell potential, and male con- bled every Ϸ5.6 days and continued to ous tubule of a mouse but also determine traception. Germ cell tumors are the most have spermatogenic potential in Brinster’s the rate, performing the process in 52 common in young men; yet, un- transplantation assay (7, 8), indicating that days (the rate of rat spermatogenesis) as like ES cells, the transplanted spermato- the culture conditions are nearly perfect compared with 35 days (the rate of mouse gonial stem cells fail to form tumors. This for maintaining the cells in a viable, pro- spermatogenesis) (20). This finding is sim- finding indicates that germ cell cancers liferating, and self-renewing state. Unlike ilar to ovarian , where the derive from a precursor germ cell popula- ϩ a previously described Kit transformed state of the female germ cell () dic- tion [e.g., ES cells, PGCs, or gonocytes]. spermatogonial cell line (17), or eggs or tates the rate of folliculogenesis (21). The close resemblance of cultured sper- sperm from ES cells (2–4), these trans- Thus, the germ cells, but not the somatic matogonial stem cells, PGCs, and ES cells planted spermatogonial stem cells give cells (i.e., Sertoli cells in male and granu- [e.g., morphological appearance as well as rise to offspring, suggesting that their losa cells in female), control the timing of Oct4 and alkaline phosphatase activity chromosome complement is stable under spermatogenesis and folliculogenesis. (see Table 1)] raises the possibility that Kubota et al.’s (1) culture conditions. One beauty of the Kubota et al. (1) the spermatogonial stem cells might be work is that the culture conditions are Insights and Implications converted to ES cells by in vitro manipula- simple and defined. This result will allow tion and would then have multipotential It is amazing that transplanted spermato- the group to manipulate the culture con- capability, including tumorigenicity. Like- gonial stem cells can migrate from the ditions further to address unanswered wise, as we understand how to maintain lumen to the base- questions. For example, what levels of spermatogonial stem cells in a state of ment membrane, recapitulating gonocyte spermatogonial differentiation occur dur- self-renewal in culture, then we might pur- migration during development. This find- ing the cell culture? If spermatogonial sue in vivo maintenance of this state to ing might suggest that gonocytes and sper- differentiation does occur, can it be ma- prevent differentiation, thereby inducing a matogenic stem cells have the ability to nipulated to regulate the entrance of the contraceptive effect. Future experiments, ‘‘sense’’ similar migratory environmental cells into meiosis and possibly spermio- including studies to profile gene expres- cues. These cues are apparently conserved genesis? Which testis cells [i.e., Sertoli sion patterns of spermatogonial stem cells, because spermatogonal stem cells from cells (intratubule cells), myoid cells (which ES cells, PGCs, gonocytes, and germ cell dog, cow, mouse, rat, macaque, baboon, line the seminiferous tubule wall), and͞or cancers, will provide targets for health- and human, injected into the seminiferous Leydig cells (which lie between tubules)] care. Clearly, Brinster’s group will con- tubules of immunodeficient mice, can mi- and͞or which characteristics of the base- tinue to lead the technological advances grate to their niche apposed to the base- ment membrane contribute the necessary and address key biological questions in ment membrane and continue to prolifer- stimuli for migration and population of stem cell research and reproduction. ate within a microenvironment separated the spermatogonial stem cells? Although from that of their origin by 100 million Sertoli cells are necessary for spermato- I thank Drs. Robert Braun, Kathleen Burns, years of evolution. genesis in vivo, diffusible factors from and Margaret Goodell for critical review of With regard to spermatogonial stem elsewhere in the testis are likely to be crit- the Commentary and Ms. Shirley Baker for cell self-renewal, Braun and coworkers ical for spermatogonial stem cell renewal. assistance with formatting. Reproductive Bi- (18) and Pandolfi and coworkers (19) Finally, because there are Ͼ100 identified ology research in the Matzuk laboratory has showed that promyelocytic leukemia zinc genes that play key roles in mammalian been supported by the National Institutes of finger (Plzf) is essential. Without Plzf, spermatogenesis, many of which are germ Health and Wyeth.

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