The human spermatogonial compartment is essential for daily production of millions of sperm. Despite this crucial role, the molecular signature, kinetic behavior and regulation of human spermatogonia are poorly understood. Using human testis biopsies with normal spermatogenesis and by studying marker protein expression, we have identified for the first time different subpopulations of spermatogonia. MAGE-A4 marks all spermatogonia, KIT marks all B spermatogonia and UCLH1 all Apale-dark (Ap-d) spermatogonia. We suggest that at the start of the spermatogenic lineage there are Ap-d spermatogonia that are GFRA1, likely including the spermatogonial stem cells. Next, UTF1 becomes expressed, cells become quiescent and GFRA1 expression decreases. Finally, GFRA1 expression is lost and subsequently cells differentiate into B spermatogonia, losing UTF1 and acquiring KIT expression. Strikingly, most human Ap-d spermatogonia are out of the cell cycle and even differentiating type B spermatogonial proliferation is restricted. A novel scheme for human spermatogonial development is proposed that will facilitate further research in this field, the understanding of cases of infertility and the development of methods to increase sperm output.

To date, in the human seminiferous epithelium, only six associations of cell types have been distinguished, subdividing the epithelial cycle into six stages of very different duration. This hampers comparisons between studies on human and laboratory animals in which the cycle is usually subdivided into 12 stages. We now propose a new stage classification on basis of acrosomal development made visible by immunohistochemistry (IHC) for (pro)acrosin. IHC for acrosin gives results that are comparable to periodic acid Schiff staining. In the human too, we now distinguish 12 stages that differ from each other in duration by a factor of two at most. B spermatogonia are first apparent in stage I, preleptotene spermatocytes are formed in stage V, leptonema starts in stage VII, and spermiation takes place at the end of stage VI. A similar timing was previously observed in several monkeys. Stage identification by way of IHC for acrosin appeared possible for tissue fixed in formalin, Bouin fixative, diluted Bouin fixative, Cleland fluid, and modified Davidson fixative, indicating a wide applicability. In addition, it is also possible to distinguish the 12 stages in glutaraldehyde/osmium-tetroxide fixed/plastic embedded testis material without IHC for acrosin. The new stage classification will greatly facilitate research on human spermatogenesis and enable a much better comparison with results from work on experimental animals than hitherto possible. In addition, it will enable a highly focused approach to evaluate spermatogenic impairments, such as germ cell maturation arrests or defects, and to study details of germ cell differentiation.