Isolation Of Spermatogonial Stem Cells Research Articles

A Preliminary Technique for the Isolation and Culture of Brown Trout (Salmo trutta macrostigma, Dumeril, 1858) Spermatogonial Stem Cell

This study was aimed to find a practical technique for isolation and culture spermatogonial stem cells from male brown trout (Salmo trutta macrostigma). Twelve wild juvenile male were obtained from Kılıç Trout Fish Farm (Kahramanmaraş, Turkey). The juveniles were taken alive to the aquaria unit and stored in a 1000-liter capacity fiberglass tank. In order to identify the best size, age and testis structure of S.t. macrostigma for spermatogonial stem cell isolation and culture. Morphological and histological testis conditions were assessed. Fish were anesthetized with 0.04% 2-phenoxethanol. The surface of the fish was sterilized with 70% ethanol. Twelve fish were divided into two groups for enzyme digestion, and each group was divided into two replicates (three fish per replicate). Testis tissue of group one were digested by 0.25% trypsin- EDTA, and testis tissues of group two were digested by 0.05% trypsin-EDTA. At the end of the trial, first, the best age, size and weight of the male fish for spermatogonial stem cell isolation and culture were identified as 5+ month old, 12.13±1.5 cm, 19, 25±7.05 g respectively. Then, the highest spermatogonial stem cells were measured in the stage one and two of the testes. Finally, isolation and culture conditions were optimized for male S.t. macrostigma. Spermatogonial stem cell isolation and culture techniques were defined for fish in order to be used in surrogate reproduction technologies and gene transfer systems.

Identification of a Technique Optimized for the Isolation of Spermatogonial Stem Cells from Mouse Testes

Identification of a Technique Optimized for the Isolation of Spermatogonial Stem Cells from Mouse Testes

Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation.

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.

Advances in Isolation Methods for Spermatogonial Stem Cells.

Stem cell research has led to many remarkable achievements in recent years, but progress in the study of spermatogonial stem cells (SSCs) has been relatively slow, partly due to the slow development of techniques for spermatogonial stem cell isolation. The major accomplishments of SSC sorting and identification occurred approximately 10 years ago, and since that time, these techniques have been widely used without major improvements. In this article, we briefly introduce the biological properties of SSCs before reviewing the development of sorting techniques for SSCs in the past decades. We then summarize recent achievements in SSC sorting and finally discuss the advantages and disadvantages of SSC isolation methods, to provide new insight into techniques and research related to spermatogonial stem cells and promote the development of reproductive biology.

Expression of the chicken GDNF family receptor α-1 as a marker of spermatogonial stem cells

The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFRα1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFRα1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFRα1 proteins. Using specific polyclonal mouse anti-cGFRα1 serum, a total of 2.8% cells were recognized as cGFRα1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFRα1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFRα1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFRα1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipient's testes with transplanted cGFRα1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.

Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.

Cryopreservation of mouse testicular tissue: prospect for harvesting spermatogonial stem cells for fertility preservation

To investigate the efficacy of vitrification, rapid freezing, and slow freezing in preserving testicular tissue for subsequent isolation of spermatogonial stem cells. Experimental study. University-based laboratory. Immature mouse testicular tissue. The tunica of the testis was manipulated before cryopreservation. The tunica was either breached with a fine needle or completely removed, or the testis was sectioned longitudinally into halves. Cell viability by Trypan blue exclusion test and flow cytometry analysis of live-dead cytotoxicity test, measurement of hormonal production, enrichment of spermatogonial stem cells with use of magnetic-activated cell sorting technology. Samples with tunica minimally penetrated with a needle point gave the highest cell viability after freezing and thawing. Vitrification protocol with use of an ethylene glycol-sucrose-based vitrification solution (40% vol/vol ethylene glycol-0.6 mol/L sucrose) was able to maintain postwarming cell viability and functions similar to those of noncryopreserved controls and significantly better than both conventional slow and rapid freezing protocols. Primitive spermatogonial stem cells were enriched successfully from vitrified tissue via magnetic-activated cell sorting. Vitrification of testicular tissue is a time- and cost-efficient strategy to preserve spermatogonial stem cells for potential transplantation procedure.

Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1‐positive subpopulations in men

Isolation of spermatogonial stem cells (SSCs) could enable in vitro approaches for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. SSC isolation from adult testes is difficult due to low cell numbers and lacking cell surface markers. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha1) plays a crucial role for the maintenance of SSCs in rodents and is expressed in monkey spermatogonia. Magnetic activated cell sorting was employed for the enrichment of GFRalpha1+ spermatogonia from adult primate testes. Magnetic activated cell sorting of monkey cells enriched GFRalpha1+ cells threefold. 11.4% of GFRalpha1+ cells were recovered. 42.9% of GFRalpha1+ cells were recovered in sorted fractions of human testicular cells, representing a fivefold enrichment. Interestingly, a high degree of morphological heterogeneity among the GFRalpha1+ cells from human testes was observed. Magnetic activated cell sorting using anti-GFRalpha1 antibodies provides an enrichment strategy for spermatogonia from monkey and human testes.

Effect of Childhood Acute Lymphoblastic Leukemia Therapy on Spermatogonia Populations and Future Fertility

Isolation of spermatogonial stem cells before potentially sterilizing cancer therapy, followed by transplantation of these cells into the testis after such treatment, may be an effective approach to prevent infertility among prepubertal boys suffering from acute lymphoblastic leukemia (ALL). A key clinical consideration in this context is the timing of biopsy, if collection of spermatogonia could be delayed from diagnosis to the later phase of leukemia treatment, better patient selection could be offered. The objective of the study was to examine the routine testicular biopsy material collected to detect testicular leukemia to evaluate if treatment for leukemia affects numbers and maturation of the spermatogonia during the prepubertal period. The study involved 28 testicular biopsies from 23 prepubertal boys treated for ALL. Samples were stained immunohistochemically to evaluate the expression of the spermatogonial markers MAGE 4A, OCT4, CD9, and AP2gamma, and of the Sertoli cell marker WT-1. To relate these findings to gonadal function after sexual maturation, the surviving patients were evaluated as adults. Several MAGE 4A-, CD9-, or OCT4-positive spermatogonia were detected in testicular biopsies after standard risk therapy without cyclophosphamide, whereas their numbers were significantly reduced in six patients receiving high-risk ALL therapy involving cyclophosphamide. No significant alteration in spermatogonial numbers was associated with testicular leukemia. All patients not treated with cyclophosphamide recovered normal testicular function, with normal sperm quality and endocrine function. Treatment for childhood leukemia without high-dose cyclophosphamide seldom depletes the spermatogonial stem cell pool totally.

438. PLZF is a spermatogonia stem cell-specific marker in the sheep testis: application to enrichment of ovine spermatogonial stem cell

Identification and isolation of spermatogonial stem cells (SSCs) are prerequisite for long-term culture, genetic manipulation, and transplantation research. The promyelocytic leukemia zinc-finger (PLZF) has been identified as a spermatogonia stem cell marker in rodent and other species, however its expression in sheep testis has not been reported yet. In this study, we validated an antibody that specifically binds to spermatogonia stem cell in sheep testis, thus demonstrated that PLZF is a spermatogonia stem cell marker and can be used for its identification. Testes from 12 Merino rams were selected to represent four stages of testis development at testis weights of 3–5 g (neonatal), 30 g (peripubertal), 50 g (prepubertal) and 100 g (mature). Three testes sections from 4 different developmental stage were stained with PLZF antibody and 25 individual tubules in each section were counted. In the sections, the percentage of PLZF positive cells/per tubule was increased nearly 2-fold from neonatal (6. 4 ± 0. 4%) to peripubertal (1 2.2 ± 2.8%), and then the percentage begin to decline in prepubertal (4.6 ± 0. 7%) and mature testes (3.1 ± 0.6%). A single cell suspension of testicular cells was generated by a two step enzymic digestion (n = 4) and spermatogonia stem cells were enriched by overnight differential plating with 0.2% gelatine coated flask. The percentages of spermatogonia stem cells in the single cell suspensions were assessed by PLZF antibody staining of smears. Compared with the initial isolation (3.1 ± 0.6%), spermatogonia were enriched 11-fold in overnight differential plating (34.0 ± 5.7,%) (P < 0.05). These data provide the basis for future studies aimed at refining conditions of spermatogonial stem cell culture and manipulation before male germ stem cell transplantation in sheep.

Protein gene product 9.5 is a spermatogonia‐specific marker in the pig testis: Application to enrichment and culture of porcine spermatogonia

Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P < 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (>90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (>80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.

Advances and applications of germ cell transplantation

When male germ line stem cells are transplanted from the testis of a fertile donor animal to the testis of an infertile recipient they can establish donor-derived spermatogenesis in the recipient testis, and the resulting sperm can transmit the genotype of the donor to the offspring of the recipient. Germ cell transplantation provides a bioassay to study the biology of these stem cells, to develop systems for spermatogonial stem cell isolation and culture, to examine defects in spermatogenesis and to correct male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. A potential clinical application is restoration of fertility in patients that underwent cytotoxic treatments for cancer. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of toxins or drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in mammalian species.

Spermatogonial Stem Cell Isolation, Storage, and Transplantation

The past 30 years have been marked by unparalleled accomplishments in the medical treatment of malignancy. Prior to advances in chemotherapeutic and radiation treatment, many oncologic conditions had dismal survival rates. Today, medical interventions have success rates that approach complete remission for many malignancies. An inadvertent complication of these therapies, however, has been the high rate of infertility following treatment. Male germinal tissue, like many malignancies, is mitotically active and therefore is particularly susceptible to the toxic effects of both chemotherapy and radiotherapy (Meistrich et al, 1982; Meistrich, 1993). Consequently, posttreatment patients often develop severe oligozoospermia or azoospermia (Wallace et al, 1991). Potential infertility complications can be anticipated, and adult male patients interested in future procreation are counseled to cryopreserve semen before instituting treatment. With presentday capabilities of in vitro fertilization, particularly intracytoplasmic injection, male patients can maintain posttreatment fertility. Pretreatment sperm banking, however, is not a viable option for prepubescent males. These individuals have not yet begun spermatogenesis and thus lack viable spermatozoa. It is estimated that, by the end of the decade, 1 in 250 young men will be childhood cancer survivors (Blatt, 1999). For these patients, infertility has often been an accepted consequence of their lifesaving treatment.

371 EVALUATION OF ENRICHMENT STRATEGIES FOR PORCINE SPERMATOGONIA BY EXPRESSION OF PROTEIN GENE PRODUCT 9.5, A SPERMATOGONIA-SPECIFIC MARKER IN THE PIG TESTIS

Transplantation of genetically altered male germ cells is under investigation as a novel route to generate transgenic animal models. Identification and isolation of spermatogonial stem cells are a prerequisite for this strategy. The objectives of this study were to validate a marker for identification of undifferentiated porcine spermatogonia, and to use this marker to develop a practical enrichment strategy for spermatogonia from pig testis. We established that expression of protein gene product (PGP) 9.5 is a spermatogonia-specific marker in porcine testis through analysis of its expression pattern in testis cells, by comparison with the expression of the cell-type specific proteins GATA-4 (expressed in Sertoli cells) or PLZF (expressed in undifferentiated mouse spermatogonia) in seminiferous tubules at different ages, and by comparison of expression levels of PGP 9.5 and the germ cell-specific protein VASA in different cell fractions after differential plating. Using expression of PGP 9.5 as a marker, we characterized enrichment of porcine spermatogonia from two-week-old (2wo) and 10-week-old (10wo) pigs by immunofluorescence either after differential plating only or after velocity sedimentation at unit gravity followed by differential plating. After differential plating with overnight culture to deplete testicular somatic cells that firmly attach to culture dishes, spermatogonia (mean � SEM per 1000 cells) were 5-fold enriched (P &lt; 0.05) in cells remaining in suspension (fraction I) (2wo: 54.0 � 9.1; 10wo: 162.7 � 30.5) and in populations slightly attached to the culture plate (fraction II) (2wo: 92.7 � 8; 10wo: 159.5 � 22.5) compared to the initial samples (2wo: 12.3 � 2.7; 10wo: 27.2 � 2.9). Slightly attached spermatogonia appear to be superior for future experiments due to higher viability (&gt;90%) than spermatogonia remaining in suspension (&lt;50%). Cell populations containing up to 70% spermatogonia with good viability (&gt;80%) were achieved by velocity sedimentation isolation followed by differential plating. These results indicate that expression of PGP 9.5 is a useful marker for identification of undifferentiated porcine germ cells. Simple differential adhesion culture of testis cells harvested from pre-pubertal boars can supply cell populations enriched in spermatogonia for subsequent genetic manipulation and transplantation. This work was supported by 1 R01 RR17359-01.

A novel technique of spermatogonial stem cell isolation

Objective: Adult stem cells offer the possibility of tissue regeneration following injury. However, for most stem cells, markers are not available for cell identification and purification. Accordingly, alternative approaches for stem cell identification have been developed. Hematopoetic stem cells can be enriched by exploiting their ability to home to the bone marrow of irradiated mice. In this study, we describe the development of a similar technique to isolate and enrich a population of adult spermatogonial stem cells for germ cell transplantation. Our ultimate goal is to develop a novel technique for the purification of adult spermatogonial stem cells to be used for transplantation as a therapy for primary or secondary male infertility