Adult Stem Cells in the Human Testis

Ellen Goossens, PhD, MSc; Herman Tournaye, MD, PhD, MSc


Semin Reprod Med. 2013;31(1):39-48. 

In This Article

Spermatogonial Stem Cell Identity

Spermatogonia are small single cells located on the basement membrane of the seminiferous tubules. They have an ovoid nucleus with the nucleoli close to the nuclear membrane. The dense cytoplasm contains a small Golgi apparatus, few mitochondria, and many free ribosomes. The SSC population is only a very small subpopulation of the spermatogonia. A commonly used method to identify SSCs by their cell surface phenotype is fluorescent-activated cell sorting (FACS) in combination with SSC transplantation. The first results of this approach were reported by Shinohara's group, who found that SSCs were enriched for β1-integrin (CD29) and α6-integrin (CD49f) but did not express αv-integrin (CD51).[27]

During subsequent years, more markers were identified. SSCs are positive for thymus cell antigen 1 (THY1 or CD90), CD9, glial cell line derived neurotropic factor (GDNF) family receptor α 1 (GFRα1), and E-cadherin,[28–31] and negative for major histocompatibility complex class I, C-Kit, and CD45.[32] Although it is possible to highly enrich cell populations for SSCs (100- to 200-fold) using combinations of positive and negative markers, a pure SSC suspension has not been obtained so far.

Apart from FACS, which is limited to the study of surface markers, other techniques are available to identify both cytoplasmic and nuclear SSC markers. Expression of green fluorescent protein (GFP) under the promoter of a candidate SSC gene is such a tool in mouse models. For example, octamer-binding transcription factor (OCT) 4-GFP mice enabled FACS-based isolation and transplantation of OCT4-expressing germ cells from a heterogeneous testis cell suspension.[21] Cells expressing OCT4 (GFP+) showed higher stem cell activity compared with the OCT4 (GFP) cells.[33] Alternative approaches to reveal SSC-specific genes or proteins are transgenic, knock-in, or knockout mice. Using these models, investigators determined the expression of GDNF and its receptor GFRα1, promyelocytic leukemia zinc-finger (PLZF), SOX3, neurogenin 3, NANOS2, and stimulated by retinoic acid (STRA) 8 in undifferentiated spermatogonia.[34–40] SOX3 expression co-localizes with neurogenin 3 and is required for spermatogonial differentiation.[37] Because transplantation experiments showed that only 11% of transplantable SSCs were neurogenin+, the idea arose that the SSC population is heterogeneous.[41] This heterogeneity among As-spermatogonia was confirmed by a study using whole-mount immunohistochemistry. Undifferentiated spermatogonia were identified by their location at the basement membrane of the seminiferous tubules, by the clone size, and by coexpression of known SSC markers. Although azoospermia-like protein (DAZL) is expressed throughout male germ cell development, its localization depends on the cell type. DAZL is found in spermatogonia in the nucleus but is transferred during meiosis to the cytoplasm.[42] Sal-like protein (SALL) 4, which is important to maintain pluripotency in mouse embryonic stem cells,[43] is specifically expressed in undifferentiated spermatogonia. Although SALL4 expression mostly overlaps with PLZF, its coexpression with GFRα1 revealed heterogeneity in the SSC population. SALL4+/GFRα1 and SALL4+/GFRα1+ populations could be detected, with GFRα1 expression more limited (clones of 1 to 4 cells) than the expression of PLZF and SALL4 (also clones of 8 and 16 cells).[44] The fact that the SSC population does not consist of cells displaying all the same phenotype was also reported by other research groups.[15,16,31,38,45] The implications of this heterogeneity on SSC function remains to be elucidated.

Very recently, Oatley et al proposed a new marker for rodent SSCs.[17] Inhibitor of DNA binding (ID) 4 has the most restricted expression pattern observed to date. However, not all single spermatogonia do express this marker, suggesting heterogeneity among the pool of single spermatogonia. Whether the SSC pool resides entirely in the population of single spermatogonia or extends to pairs and aligned spermatogonia still needs to be clarified. Although the phenotype of rodent SSCs is almost unraveled, the search for markers limited to single As spermatogonia is still ongoing.

It has to be mentioned that, in damaged testes, not only As spermatogonia have the capacity to self-renew. Apr and Aal spermatogonia were able to revert to the As state and start spermatogenesis.[41]

Human spermatogonia express many markers equivalent to those of rodent spermatogonia (e.g., α6-integrin, GFRα1, THY1). However, other markers are not shared. For example, human SSCs do not express β1-integrin[46] but are positive for testis-specific protein Y-linked (TSPY) 1, CD133, and stage-specific embryonic antigen (SSEA) 4.[47,48] Although during the last few years a lot of progress has been made concerning the characterization of human SSCs, further research is warranted.