Mary R. Avarbock

Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells

Spermatogonial stem cells (SSCs) are responsible for maintaining spermatogenesis throughout life in the male by continuous production of daughter cells that differentiate into spermatozoa. However, no unique phenotypic markers to identify SSCs have been described. In this study, the SSC surface phenotype was characterized by using flow cytometric cell sorting in conjunction with a transplantation functional assay for SSCs. Highly enriched stem cell activity was found in the MHC class I (MHC-I)–Thy-1+c-kit– cell fraction of the mouse cryptorchid testis. There was little or no stem cell activity in any other fraction. The antigenic phenotype of the MHC-I–Thy-1+c-kit– SSCs was α6-integrin+CD24+αvintegrin–Sca-1–CD34–. Subsequently, testis side population (SP) cells, which are defined by a Hoechst dye efflux assay, were identified. Their surface phenotype was found to be MHC-I+Thy-1–Sca-1+, and the transplantation assay demonstrated that the testis SP and SSCs are distinct populations. In several other tissues, the SP has been shown to contain stem cells, but we found that this characteristic does not define SSCs. The identification of a surface phenotype that allows production of a highly enriched SSC population will facilitate functional and genomic studies and enable further comparison with other stem cells.

Transgenic mice produced by retroviral transduction of male germ-line stem cells.

Male germ-line stem cells are the only cell type in postnatal mammals that have the capability to self-renew and to contribute genes to the next generation. Genetic modification of these cells would provide an opportunity to study the biology of their complex self-renewal and differentiation processes, as well as enable the generation of transgenic animals in a wide range of species. Although retroviral vectors have been used as an efficient method to introduce genes into a variety of cell types, postnatal male germ-line stem cells have seemed refractory to direct infection by these viruses. In addition, expression of genes transduced into several types of stem cells, such as embryonic or hematopoietic, is often attenuated or silenced. We demonstrate here that in vitro retroviral-mediated gene delivery into spermatogonial stem cells of both adult and immature mice results in stable integration and expression of a transgene in 2–20% of stem cells. After transplantation of the transduced stem cells into the testes of infertile recipient mice, approximately 4.5% of progeny from these males are transgenic, and the transgene is transmitted to and expressed in subsequent generations. Therefore, there is no intrinsic barrier to retroviral transduction in this stem cell, and transgene expression is not extinguished after transmission to progeny.

TRANSPLANTATION OF MALE GERM LINE STEM CELLS RESTORES FERTILITY IN INFERTILE MICE

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.

Culture Conditions and Single Growth Factors Affect Fate Determination of Mouse Spermatogonial Stem Cells

Abstract Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I− Thy-1+ α6-integrin+ αv-integrin−/dim throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.

Maintenance of Mouse Male Germ Line Stem Cells In Vitro

Abstract The proliferation and differentiation of a stem cell are regulated intrinsically by the stem cell and extrinsically by the stem cell niche. Elucidation of regulatory mechanisms of spermatogonial stem cells (SSCs), the stem cell of the postnatal male germ line, would be facilitated by in vitro studies that provide a defined microenvironment reconstituted ex vivo. We analyzed the effect of in vitro environment on the maintenance of adult and immature SSCs in a 7-day culture system. Allthough the number of adult and immature SSCs decreased in a time-dependent manner, nearly one in four stem cells (24%) could be maintained in vitro for 7 days. Stem cell maintenance was enhanced by coculture with OP9 bone marrow stroma or L fibroblast cell lines, addition of glial cell line-derived neurotrophic factor, or utilization of specific culture medium. In contrast, coculture with TM4 or SF7 Sertoli cell lines and addition of activin A or bone morphogenetic protein 4 (BMP4) reduced stem cell maintenance in vitro. Only 4% of the stem cells remained when cultured with TM4 cells or activin A, and 6% remained when cultured with SF7 cells or BMP4. These results lead to the hypothesis that suppression of germ cell differentiation improves in vitro maintenance of SSCs by interrupting the unidirectional cascade of spermatogenesis and blocking stem cell differentiation.

Culture of mouse spermatogonial stem cells

Spermatogenesis occurs within the seminiferous tubules of mammals by a complex process that is highly organized, extremely efficient and very productive. At the foundation of this process is the spermatogonial stem cell that is capable of both self-renewal and production of progeny cells, which undergo differentiation over a period of weeks to months in order to generate mature spermatozoa. It had been thought that germ cells survive only a brief period in culture, generally less than a few weeks. However, an accurate assessment of the presence of spermatogonial stem cells in any cell population has only recently become possible with development of the spermatogonial transplantation technique. Using this technique, we have demonstrated that mouse spermatogonial stem cells can be maintained in culture for approximately 4 months and will generate spermatogenesis following transplantation to the seminiferous tubules of an appropriate recipient. Extensive areas of cultured donor cell-derived spermatogenesis are generated in the host, and production of mature spermatozoa occurs. Cultivation of the testis cells on STO feeders is beneficial to stem cell survival. These results provide the first step in establishing a system that will permit spermatogonial stem cells to be cultivated and their number increased in vitro to allow for genetic modification before transplantation to a recipient testis.

Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility

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 (∼0.5 mm2). 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.

Glial Cell Line-derived Neurotrophic Factor Regulation of Genes Essential for Self-renewal of Mouse Spermatogonial Stem Cells Is Dependent on Src Family Kinase Signaling

Self-renewal and differentiation by spermatogonial stem cells (SSCs) is the foundation for continual spermatogenesis. SSC self-renewal is dependent on glial cell line-derived neurotrophic factor (GDNF); however, intracellular mechanisms stimulated by GDNF in SSCs are unknown. To investigate these mechanisms we utilized a culture system that maintains a mouse undifferentiated germ cell population enriched for self-renewing SSCs. In these cultures mRNA for the transcription factors Bcl6b, Erm, and Lhx1 are up-regulated by GDNF and decreased in its absence. The expression of all three molecules was further identified in undifferentiated spermatogonia in vivo. Using small interfering RNA to reduce expression and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were shown to be important for SSC maintenance in vitro. Next, GDNF was shown to activate both Akt and Src family kinase (SFK) signaling in SSCs, and culture of SSCs with inhibitors to Akt or SFKs followed by transplantation analysis showed significant impairment of SSC maintenance in vitro. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when Akt, but not SFK, signaling was impaired, indicating that multiple signaling pathways are responsible for SSC self-renewal and survival. Biochemical and gene expression experiments revealed that GDNF up-regulated expression of Bcl6b, Erm, and Lhx1 transcripts is dependent on SFK signaling. Overall, these data demonstrate that GDNF up-regulation of Bcl6b, Erm, and Lhx1 expression through SFK signaling is a key component of the intracellular mechanism for SSC self-renewal.

Germ cell transplantation from large domestic animals into mouse testes.

Donor‐derived spermatogenesis after spermatogonial transplantation to recipient animals could serve as a novel approach to manipulate the male germ line in species where current methods of genetic modification are still inefficient. The objective of the present study was to investigate germ cell transplantation from boars, bulls, and stallions, which are economically important domestic animals, to mouse recipients. Donor testis cells (fresh, cryopreserved, or cultured for 1 month) were transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. Recipient testes were analyzed from 1 to > 12 months after transplantation for the presence of donor germ cells by donor‐specific immunohistochemistry. Donor cells were present in most recipient testes with species‐dependent differences in pattern and extent of colonization. Porcine donor germ cells formed chains and networks of round cells connected by intercellular bridges but later stages of donor‐derived spermatogenesis were not observed. Transplanted bovine testis cells initially appeared similar but then developed predominantly into fibrous tissue within recipient seminiferous tubules. Few equine germ cells proliferated in mouse testes with no obvious difference between cells recovered from a scrotal or a cryptorchid donor testis. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh or cryopreserved germ cells from large animals can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion. Species‐specific differences in the compatibility of large animal donors and mouse recipients were detected which cannot be predicted solely on the basis of phylogenetic distance between donor and recipient species. Mol. Reprod. Dev. 57:270–279, 2000.

Effects of Aging and Niche Microenvironment on Spermatogonial Stem Cell Self‐Renewal

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.