Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting

Spermatogonial stem cells (SSCs) are a documented source for adult multipotent stem cells. Thus, the isolation of SSCs is of great interest. However, the isolation of spermatogonia from mammalian testes is difficult because of their low total numbers and the lack of well-characterized cell surface markers. Glial-cell-derived neurotrophic factor family receptor alpha-1 (GFRα1) is expressed on undifferentiated mouse spermatogonia (including SSCs) and plays a crucial role, in rodents, for the maintenance of SSCs mediated by the Sertoli cell product GDNF. The present study has aimed to optimize the sorting efficiency and total cell yield of magnetic activated cell sorting (MACS) with anti-GFRα1 antibodies. Because of the technical limitations intrinsic to the magnetic columns, various sorting setups and strategies were compared. Use of Mini-MACS (MS) columns for single cell suspensions from 7-day-old rat testes resulted in a three-fold enrichment of GFRα1-positive cells in sorted fractions versus presorted fractions. However, with this method, only 1.77% of cells loaded onto the column were recovered in the sorted fraction. A sequential two-step sorting approach did not improve this poor yield. We therefore evaluated cell separation by using larger volume Midi-MACS (LS) columns. Enrichment of GFRα1-positive cells in sorted fractions was four-fold, and 14.5% of cells loaded onto the column were directed to the sorted fraction. With this method, approximately half of all GFRα1-positive cells present in the sample were found in the sorted fraction. We conclude that GFRα1 serves as a suitable surface marker for the enrichment of rat spermatogonia, and that the large-volume Midi-MACS separation system is superior to the routinely used small-volume Mini-MACS separation system. This work was financially supported by startup funds from the University Münster, NIH grant U54 HD 008610, Center grant, project 1 (to S.S.), a doctoral scholarship from the Ernst Schering Research Foundation (to K.G.), and a Young Investigator Grant from the Lance Armstrong Foundation (to J.E.).     

Isolation,in vitro study,and stem cell markers for type A spermatogonia in a Characiformes species

The objective of this study was to establish a protocol for the characterization, isolation, and culture of type A spermatogonia using specific molecular markers for these cells in fish. To this end, adult Prochilodus lineatus testes were collected and digested enzymatically and the resulting testicular suspension was separated using a discontinuous Percoll gradient, followed by differential plating. The cell cultures obtained were monitored for 15 days and analyzed using the immunofluorescence method with anti‐Vasa, anti‐GFRα1, and anti‐OCT4 antibodies. Spermatogonial enrichment was also performed using flow cytometry. Although discontinuous Percoll gradient centrifugation followed by differential plating enabled the removal of differentiated germ cells and somatic cells, enriching the pool of type A spermatogonia, the enrichment of type A spermatogonia through flow cytometry of samples without Percoll proved to be more efficient. Prominent cell agglomerates that were characterized according to different stem cell markers as type A spermatogonia were observed during the 15 days of the cell culture. The use of immunoperoxidase and western blot analysis methods confirmed the specificity of the markers for type A spermatogonia of P. lineatus. When combined with specific cell culture conditions, the positive characterization of these molecular markers clarified certain aspects of spermatogonial regulation, such as survival and proliferation. Finally, understanding the regulation of the in vitro germ cell maintenance process may contribute to the enhancement of in vivo and in vitro reproduction techniques of endangered or aquaculture fish species.     

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.     

CD9 is expressed on human male germ cells that have a long-term repopulation potential after transplantation into mouse testes

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.     

Long‐term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non‐obstructive azoospermia under exogenous feeder‐free culture conditions

Objectives: The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non‐obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology. Materials and methods: We have applied a cell‐sorting method, using collagen and magnetic activated cell separation to overcome obstacles, developing a collection system, and simple long‐term proliferation system, that yields large numbers of high‐purity SSCs from obstructive OA and NOA patients. Results: SSCs derived from OA and NOA patients proliferated and maintained their characteristics for more than 12 passages (>6 months) in vitro. Moreover, the population of cells positive for the SSC‐specific markers GFRα‐1 and integrin α6, increased to more than 80% at passage 8. Conclusion: These finding may support the idea that in vitro propagation of SSCs could be a useful tool for infertility treatment and study of reproductive biology.     

Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.     

免疫磁珠纯化小鼠精原干细胞的研究

精原干细胞（spermatogonial stem cells,SSCs）富集纯化是利用SSCs进行基因修饰新方法等研究的前提基础。采用免疫磁珠分选法,使用干细胞抗体CD90.2进行小鼠SSCs的纯化富集,并采用流式细胞分析法和定量PCR验证了磁珠分选效率。流式细胞分析结果：免疫磁珠分选后SSCs纯度为50.11%。荧光定量PCR检测结果：磁珠分选后支持细胞特异表达基因 GATA4 显著下调（6倍）、SSCs表达基因 GFRα-1 上调（6.5倍）、生殖干细胞特异表达基因 OCT4 极显著上调（5.9倍）,3个基因相对表达量的变化说明,免疫磁珠分选效率为6倍。流式细胞分析法所产生的偏差可能是受到了未解离磁珠及SSCs本身转基因荧光的影响。     

Phenotypic and functional characteristics of spermatogonial stem cells in rats

Spermatogonial stem cells (SSCs) are at the foundation of the highly productive spermatogenic process that continuously produces male gametes throughout postnatal life. However, experimental evaluation of SSCs in postnatal testes is complicated because these cells are extremely rare and few defining morphology or biochemical characteristics are known. In this study, we used the spermatogonial transplantation functional assay, combined with fluorescence-activated cell sorting (FACS) analysis to identify cellular, biochemical and surface antigenic characteristics of SSCs in rat testes during development. Our results demonstrated that forward scatter (FSc)(hi), side scatter (SSc)(hi), mitochondria membrane potential (DeltaPsim)(lo), Ep-CAM(+), Thy-1(+), beta3-integrin(+) stem cells in neonate rat testes become SSc(lo), DeltaPsim(hi), Ep-CAM(+), Thy-1(lo), beta3-integrin(-) stem cells in pup rat testes. Furthermore, prospective identification of rat testis cell populations (Ep-CAM(+)), highly enriched for SSCs (1 in 13 for neonate; 1 in 8.5 for pup) enabled us to predict the Thy-1 and beta3-integrin status of stem cells in neonate and pup testes, which was subsequently confirmed by transplantation analyses. Systematic characterization of SSCs enabled the production of testis cell populations highly enriched (up to 120-fold) for SSCs and will facilitate future investigations of functional and genomic characteristics.     

Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells

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(+) alpha 6-integrin(+) alpha v-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.     

Expression patterns of gdnf and gfrα1 in rainbow trout testis

In mice, glial cell line-derived neurotrophic factor (GDNF) is essential for normal spermatogenesis and in vitro culture of spermatogonial stem cells. In murine testes, GDNF acts as paracrine factor; Sertoli cells secrete it to a subset of spermatogonial cells expressing its receptor, GDNF family receptor α1 (GFRα1). However, in fish, it is unclear what types of cells express gdnf and gfrα1. In this study, we isolated the rainbow trout orthologues of these genes and analyzed their expression patterns during spermatogenesis. In rainbow trout testes, gdnf and gfrα1 were expressed in almost all type A spermatogonia (ASG). Noticeably, unlike in mice, the expression of gdnf was not observed in Sertoli cells in rainbow trout. During spermatogenesis, the expression levels of these genes changed synchronously; gdnf and gfrα1 showed high expression in ASG and decreased dramatically in subsequent developmental stages. These results suggested that GDNF most likely acts as an autocrine factor in rainbow trout testes.     

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.     

Functional analysis of spermatogonial stem cells in Steel and cryptorchid infertile mouse models

Spermatogenesis is a complex and productive process that originates from stem cell spermatogonia and ultimately results in formation of mature spermatozoa. The stem cell undergoes self-renewal throughout life, but study of its biological characteristics has been difficult because a very small number (2 to 3 in 10(4) cells) exist in the testis and they can only be identified by function. Although the development of the spermatogonial transplantation technique has provided an assay system for stem cells, efficient methods to enrich stem cells have not been available. Here, we examined two infertile mouse models, Steel/Steel(Dickie)(Sl/Sl(d)) and experimental cryptorchid, as a source of testis cell populations enriched in stem cells. The Sl/Sl(d) testis showed little enrichment, which raises questions about how adult stem cell number is determined and about the currently accepted belief that adult stem cells are independent of Sl factor. The cells recovered from cryptorchid testes were enriched for stem cells 25-fold (colonies) or 50-fold (area) compared to wild-type testes. The cryptorchid condition does not affect stem cell activity, but eliminates almost all differentiated cells, and about 1 in 200 cells is a stem cell. Thus, cryptorchid testes provide an important approach for purification and characterization of spermatogonial stem cells.     

The Fate of Spermatogonial Stem Cells in the Cryptorchid Testes of RXFP2 Deficient Mice

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool.     

Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation

The in vitro culture system for spermatogonial stem cells (SSCs) is a powerful tool for exploring molecular mechanisms of male gametogenesis and gene manipulation. Very little information is available for fish SSC biology. Our aim was to isolate highly pure SSCs from the testis of commercially important farmed carp, Labeo rohita. The minced testis of L. rohita was dissociated with collagenase. Dissociated cells purified by two-step Ficoll gradient centrifugation followed by magnetic activated cell sorting (MACS) using Thy1.2 (CD90.2) antibody dramatically heightened recovery rate for spermatogonial cells. The purified cells were cultured in vitro conditions for more than two months in L-15 media containing 10% fetal bovine serum (FBS), 1% carp serum, and other nutrients. The proliferative cells were dividing as validated by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay and formed colonies/clumps with the typical characteristics of SSCs A majority of enriched cell population represented a Vasa⁺, Pou5f1/pou5f1⁺, Ssea-1⁺, Tra-1-81⁺, plzf⁺, Gfrα1/gfrα1⁻, and c-Kit/c-kit⁻ as detected by immunocytochemical and/or quantitative real-time polymerase chain reaction (RT-PCR) analyses. Thus, Thy1⁺ SSCs were enriched with greater efficiency from the mixed population of testicular cells of L. rohita. A population of enriched spermatogonial cells could be cultured in an undifferentiated state. The isolated SSCs could provide avenue for undertaking research on basic and applied reproductive biology.     

Identification and IVC of spermatogonial stem cells in prepubertal buffaloes

Development of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long-term IVC conditions, and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1 as a specific marker for buffalo SSCs and revealed that it existed in two protein isoforms (large [135 kDa] and small [90 kDa] subunits) in the buffalo testis; furthermore, immunohistochemical analysis revealed that CDH1 expression was present in spermatogonia but absent in the somatic cells of 4-month-old buffalo testis. After 7 days of enrichment, expression of CDH1 was also detectable in IVC colonies (∼53% enrichment efficiency by Fluorescence-activated cell sorting (FACS)). For long-term culture of SSCs, proliferation studies with different factors showed that combination of 20 ng/mL GDNF, 10 ng/mL FGF2, and 1000 U/mL LIF could significantly promote number of colonies (∼two folds) and proliferation of buffalo SSCs (∼three folds) compared with those of control or single-treatment groups; furthermore, addition of these combination growth factors significantly upregulated the messenger RNA level of spermatogonial-specific and pluripotency-related markers (BCL6B, GFRA1, and POU5F1), whereas downregulated receptor tyrosine kinase (KIT). For confirmation of their stem cell potential, Dolichos biflorus agglutinin–stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. These findings indicate the identification of a new buffalo SSCs marker; furthermore, it may help in establishing long-term culture that would assist in genetic modification of these buffaloes.     

Sertoli cells dictate spermatogonial stem cell niches in the mouse testis

Sustained spermatogenesis in adult males relies on the activity of spermatogonial stem cells (SSCs). In general, tissue-specific stem cell populations such as SSCs are influenced by contributions of support cells that form niche microenvironments. Previous studies have provided indirect evidence that several somatic cell populations and the interstitial vasculature influence SSC functions, but an individual orchestrator of niches has not been described. In this study, functional transplantation of SSCs, in combination with experimental alteration of Sertoli cell content by polythiouracil (PTU)-induced transient hypothyroidism, was used to explore the relationship of Sertoli cells with SSCs in testes of adult mice. Transplantation of SSCs from PTU-treated donor mice into seminiferous tubules of normal recipient mice revealed a greater than 3-fold increase in SSCs compared to those from testes of non-PTU-treated donors. In addition, use of PTU-treated mice as recipients for transplantation of SSCs from normal donors revealed a greater than 3-fold increase of accessible niches compared to those of testes of non-PTU treated recipient mice with normal numbers of Sertoli cells. Importantly, the area of seminiferous tubules bordered by interstitial tissue and percentage of seminiferous tubules associated with blood vessels was found to be no different in testes of PTU-treated mice compared to controls, indicating that neither the vasculature nor interstitial support cell populations influenced the alteration of niche number. Collectively, these results provide direct evidence that Sertoli cells are the key somatic cell population dictating the number of SSCs and niches in mammalian testes.