Little is known of the molecular mechanisms whereby spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm 1,2 . Here we show that Zfp145, encoding the transcriptional repressor Plzf, has a crucial role in spermatogenesis. Zfp145 expression was restricted to gonocytes and undifferentiated spermatogonia and was absent in tubules of W/W v mutants that lack these cells. Mice lacking Zfp145 underwent a progressive loss of spermatogonia with age, associated with increases in apoptosis and subsequent loss of tubule structure but without overt differentiation defects or loss of the supporting Sertoli cells. Spermatogonial transplantation experiments revealed a depletion of spermatogonial stem cells in the adult. Microarray analysis of isolated spermatogonia from Zfp145-null mice before testis degeneration showed alterations in the expression profile of genes associated with spermatogenesis. These results identify Plzf as a spermatogoniaspecific transcription factor in the testis that is required to regulate self-renewal and maintenance of the stem cell pool.

This article will provide an updated review of spermatogonial stem cells and their role in maintaining the spermatogenic lineage. Experimental tools used to study spermatogonial stem cells (SSCs) will be described, along with research using these tools to enhance our understanding of stem cell biology and spermatogenesis. Increased knowledge about the biology of SSCs improves our capacity to manipulate these cells for practical application. The chapter concludes with a discussion of future directions for fundamental investigation and practical applications of SSCs.

The spermatogonial stem cell initiates and maintains spermatogenesis in the testis. To perform this role, the stem cell must self replicate as well as produce daughter cells that can expand and differentiate to form spermatozoa. Despite the central importance of the spermatogonial stem cell to male reproduction, little is known about its morphological or biochemical characteristics. This results, in part, from the fact that spermatogonial stem cells are an extremely rare cell population in the testis, and techniques for their enrichment are just beginning to be established. In this investigation, we used a multiparameter selection strategy, combining the in vivo cryptorchid testis model with in vitro fluorescence-activated cell sorting analysis. Cryptorchid testis cells were fractionated by fluorescence-activated cell sorting analysis based on light-scattering properties and expression of the cell surface molecules ␣6-integrin, ␣v-integrin, and the c-kit receptor. Two important observations emerged from these analyses. First, spermatogonial stem cells from the adult cryptorchid testis express little or no c-kit. Second, the most effective enrichment strategy, in this study, selected cells with low side scatter light-scattering properties, positive staining for ␣6-integrin, and negative or low ␣v-integrin expression, and resulted in a 166-fold enrichment of spermatogonial stem cells. Identification of these characteristics will allow further purification of these valuable cells and facilitate the investigation of molecular mechanisms governing spermatogonial stem cell self renewal and hierarchical differentiation.

Summary Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a man’s life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from 4-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.

Little is known about stem cell biology or the specialized environments or niches believed to control stem cell renewal and differentiation in self-renewing tissues of the body. Functional assays for stem cells are available only for hematopoiesis and spermatogenesis, and the microenvironment, or niche, for hematopoiesis is relatively inaccessible, making it difficult to analyze donor stem cell colonization events in recipients. In contrast, the recently developed spermatogonial stem cell assay system allows quantitation of individual colonization events, facilitating studies of stem cells and their associated microenvironment. By using this assay system, we found a 39-fold increase in male germ-line stem cells during development from birth to adult in the mouse. However, colony size or area of spermatogenesis generated by neonate and adult stem cells, 2-3 months after transplantation into adult tubules, was similar (ϳ0.5 mm 2 ). In contrast, the microenvironment in the immature pup testis was 9.4 times better than adult testis in allowing colonization events, and the area colonized per donor stem cell, whether from adult or pup, was about 4.0 times larger in recipient pups than adults. These factors facilitated the restoration of fertility by donor stem cells transplanted to infertile pups. Thus, our results demonstrate that stem cells and their niches undergo dramatic changes in the postnatal testis, and the microenvironment of the pup testis provides a more hospitable environment for transplantation of male germ-line stem cells.

Summary Human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia nor haploid spermatocytes or spermatids. Here we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages including post-meiotic, spermatid-like cells in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating pluripotent stem cells into UTF1, PLZF and CDH1-positive spermatogonia-like cells, HIWI and HILI-positive spermatocyte-like cells, and haploid cells expressing acrosin, transition protein 1 and protamine 1, proteins found uniquely in either spermatids and/or sperm. These spermatids show uniparental genomic imprints similar to human sperm on two loci: H19 and IGF2. These results demonstrate that male pluripotent stem cells have the capability to directly differentiate into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A single spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A dark and A pale spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFR␣1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/ niche interactions in nonhuman primate testes. STEM CELLS

Aging is evident in most tissues and organ systems, but the mechanisms of aging are difficult to identify and poorly understood. Here, we test the hypothesis that aging results in uncorrected defects in stem cell and/or niche function, which lead to system failure. We used the spermatogonial stem cell (SSC) transplantation assay to determine the effect of aging on testis stem cell/niche function in mice. Between 12 and 24 months of age, male mice experienced a declining level of fertility associated with decreased testis weight, level of spermatogenesis, and total stem cell content. However, when stem cells were consecutively passaged at 3-month intervals to testes of young males, these stem cells continued to produce spermatogenesis for more than 3 years. Thus, SSC self-renewal continues long past the normal life span of the animal when the stem cell is continually maintained in a young niche/microenvironment. Moreover, these data suggest that infertility in old males results from deterioration of the SSC niche and failure to support an appropriate balance between stem cell self-renewal and differentiation. STEM CELLS 2006;24:1505-1511