Rat Spermatogonial Stem Cells Research Articles

Evaluating the effect of conditioned medium from mesenchymal stem cells on differentiation of rat spermatogonial stem cells.

In cancer patients, chemo/radio therapy may cause infertility by damaging the spermatogenesis affecting the self-renewal and differentiation of spermatogonial stem cells (SSCs). In vitro differentiation of stem cells especially mesenchymal stem cells (MSCs) into germ cells has recently been proposed as a new strategy for infertility treatment. The aim of this study was to evaluate the proliferation and differentiation of SSCs using their co-culture with Sertoli cells and conditioned medium (CM) from adipose tissue-derived MSCs (AD-MSCs). Testicular tissues were separated from 2-7 days old neonate Wistar Rats and after mechanical and enzymatic digestion, the SSCs and Sertoli cells were isolated and cultured in Dulbecco's modified eagle medium with 10% fetal bovine serum, 1X antibiotic, basic fibroblast growth factor, and glial cell line-derived neurotrophic factor. The cells were treated with the CM from AD-MSCs for 12 days and then the expression level of differentiation-related genes were measured. Also, the expression level of two major spermatogenic markers of DAZL and DDX4 was calculated. Scp3, Dazl, and Prm1 were significantly increased after treatment compared to the control group, whereas no significant difference was observed in Stra8 expression. The immunocytochemistry images showed that DAZL and DDX4 were positive in experimental group comparing with control. Also, western blotting revealed that both DAZL and DDX4 had higher expression in the treated group than the control group, however, no significant difference was observed. In this study, we concluded that the CM obtained from AD-MSCs can be considered as a suitable biological material to induce the differentiation in SSCs.

Signal regulatory protein alpha is a conserved marker for mouse and rat spermatogonial stem cells†.

Characterization of spermatogonial stem cells (SSCs) has been hampered by their low frequency and lack of features that distinguish them from committed spermatogonia. Few conserved SSC markers have been discovered. To identify a new SSC marker, we evaluated SIRPA expression in mouse and rat SSCs. SIRPA was expressed in a small population of undifferentiated spermatogonia. SIRPA, and its ligand CD47 were expressed in cultured SSCs. Expression of both SIRPA and CD47 was upregulated by supplementation of GDNF and FGF2, which promoted SSC self-renewal. Sirpa depletion by short hairpin RNA impaired the proliferation of cultured SSCs, and these cells showed decreased MAP2K1 activation and PTPN11 phosphorylation. Immunoprecipitation experiments showed that SIRPA associates with PTPN11. Ptpn11 depletion impaired SSC activity in a manner similar to Sirpa depletion. SIRPA was expressed in undifferentiated spermatogonia in rat and monkey testes. Xenogenic transplantation experiments demonstrated that SIRPA is expressed in rat SSCs. These results suggest that SIRPA is a conserved SSC marker that promotes SSC self-renewal division by activating the MAP2K1 pathway via PTPN11.

Rattus norvegicus Spermatogenesis Colony-Forming Assays.

Knowledge gaps persist on signaling pathways and metabolic states in germ cells sufficient to support spermatogenesis independent of a somatic environment. Consequently, methods to culture mammalian stem cells through spermatogenesis in defined systems have not been established. Lack of success at culturing mammalian stem cells through spermatogenesis in defined systems reflects an inability to experimentally recapitulate biochemical events that develop in germ cellswithin the testis-specificseminiferous epithelium. Complex germ and somatic cell associations that develop each seminiferous epithelial cycle support such a hypothesis, conceivably explaining why highly pure mammalian spermatogonia donot effectivelydevelopinto and through meiosis without somatic cells. Here, we outline an in vitro spermatogenesis colony-forming assay to study how differentiating spermatogonial syncytia develop from rat spermatogonial stem cell lines. Robust spermatogonial differentiation under defined culture conditions, once established, is anticipated tofacilitate molecular biology studies on pre-meiotic steps in gametogenesis by providing soma-free bioassays to systematically identify spermatogenic factors that promote meiotic progression in vitro.

Abstract 12045: Mapping the CaV1.2 Interactome in Rat Heart in vivo

Introduction: The Ca2+ channel CaV1.2 is an essential part of excitation contraction coupling, neurotransmission and vascular tone. Identifying protein partners of CaV1.2 is critical to make mechanistic insights into these fundamental processes. Prior CaV1.2 proximity proteomes used APEX labeling, which labels other amino acids, introduces oxidative stress and is catalyzed ex vivo. Hypothesis: α1C-TurboID knockin rats can generate tissue specific interactomes in vivo, in cells with intact cell and matrix contacts, which may differ substantially from interactomes made from ex vivo peroxidase catalyzed labeling. Methods: CRISPR/Cas9 homology directed repair (HDR) was applied to rat spermatogonial stem cells with a targeting vector that includes cacna1c exon 44, coding the C-terminus of CaV1.2, and birA*, which codes TurboID, cloned 3’ to the stop codon (Fig A). Figures were made with Biorender. Results: Streptavidin blotting of brain and heart lysates demonstrated robust biotinylation in knockin rats (Fig B). In paced heart myocytes, α1C-TurboID produces Ca2+ transients and responds appropriately to adrenergic stimulation (Fig C), indicating proper localization to the sarcolemma. Expression of α1C-TurboID does not alter systolic function (Fig D). Mass spectrometry of heart lysates affinity purified on streptavidin beads identified 629 proteins, of which 454 were significantly enriched in knockin rats. These include known sarcolemmal proteins and interactors of CaV1.2 (Fig E), which was confirmed by antibody detection (Fig F). 73% of these 454 proteins were not significantly enriched in the APEX-α1C proteome. Conclusion: TurboID fusion to CaV1.2 generates a reliable and substantially different proteome compared to other techniques. Successful in vivo characterization of the channel interactome confirms α1C-TurboID rats will be a powerful tool for identifying changing protein networks across developmental stages, tissues, and disease models.

Reestablishment of spermatogenesis after more than 20 years of cryopreservation of rat spermatogonial stem cells reveals an important impact in differentiation capacity.

Treatment of cancer in children is increasingly successful but leaves many prepubertal boys suffering from infertility or subfertility later in life. A current strategy to preserve fertility in these boys is to cryopreserve a testicular biopsy prior to treatment with the expectation of future technologies allowing for the reintroduction of stem cells and restoration of spermatogenesis. Spermatogonial stem cells (SSCs) form the basis of male reproduction, differentiating into all germ cell types, including mature spermatozoa and can regenerate spermatogenesis following transplantation into an infertile testis. Here, we demonstrate that rat SSCs frozen for more than 20 years can be transplanted into recipient mice and produce all differentiating germ cell types. However, compared with freshly isolated cells or those frozen for a short period of time, long-frozen cells do not colonize efficiently and showed reduced production of spermatids. Single-cell RNA sequencing revealed similar profiles of gene expression changes between short- and long-frozen cells as compared with fresh immediately after thawing. Conversely, following transplantation, long-frozen samples showed enhanced stem cell signaling in the undifferentiated spermatogonia compartment, consistent with self-renewal and a lack of differentiation. In addition, long-frozen samples showed fewer round spermatids with detectable protamine expression, suggesting a partial block of spermatogenesis after meiosis resulting in a lack of elongating spermatids. These findings strongly suggest that prolonged cryopreservation can impact the success of transplantation to produce spermatogenesis, which may not be revealed by analysis of the cells immediately after thawing. Our analysis uncovered persistent effects of long-term freezing not found in other cryopreservation studies that lacked functional regeneration of the tissue and this phenomenon must be accounted for any future therapeutic application.

Can antioxidants protect against chemotherapy in a rat spermatogonial stem cell line?

Lay summaryBoys administered chemotherapy to treat cancer are at risk of damage to their healthy testicular tissue, which can lead to infertility in adulthood. Researchers are therefore investigating treatments to protect the testis during cancer treatment. Here, cells originating from rat testicles were cultured for 4 days and exposed to chemotherapy drugs with or without antioxidants for the final 2 days. Antioxidants can reduce cellular damage by inactivating toxic compounds. Here, antioxidants such as melatonin or n-acetylcysteine were tested against chemotherapy agents cisplatin, doxorubicin, or vincristine. Cultures were repeated four times, with cell survival measured at the end of culture. The antioxidants were not damaging and partially protected against cisplatin, although not doxorubicin. Surprisingly, n-acetylcysteine enhanced vincristine-induced damage. The results suggest that using antioxidants to protect the testis could have either beneficial or harmful effects when given alongside different chemotherapy drugs: this is important, considering that patients are often treated with multiple drugs.

CD2 is a surface marker for mouse and rat spermatogonial stem cells.

The spermatogonial stem cell (SSC) population in testis is small, and the lack of SSC markers has severely handicapped research on these cells. During our attempt to identify genes involved in SSC aging, we found that CD2 is expressed in cultured SSCs. Flow cytometric analysis and spermatogonial transplantation experiments showed that CD2 is expressed in SSCs from mature adult mouse testes. Cultured SSCs transfected with short hairpin RNAs (shRNAs) against CD2 proliferated poorly and showed an increased frequency of apoptosis. Moreover, functional analysis of transfected cells revealed impairment of SSC activity. Fluorescence activated cell sorting and spermatogonial transplantation experiments showed that CD2 is expressed not only in mouse but also in rat SSCs. The results indicate that CD2 is a novel SSC surface marker conserved between mouse and rat SSCs.

Inducible dominant negative ErbB2 rat spermatogonial line for generation of transgenic rat model anddissecting ERBB2 tyrosine kinase mediated pathways.

The ERBB2receptors tyrosine kinase, also known as HER2/Neu, play an essential role in early organism development and modulation of cell behavior in varieties of tissue in an adult organism. Our aim was the generation of transgenic rat spermatogonial line capable of inducible expression of a dominant negative form of the ERBB2protein in vitro and the transgenic rat as an animal model for dissection of the ERBB2mediated signaling in vitro and in vivo. Donor derived rat spermatogonial stem cells that express green fluorescence protein and inducible ERT2CreERT2recombinase were modified with Sleeping Beauty transposon-based vector that express truncated kinase deficient form of the ERBB2receptor upon Cre mediated recombination. Clonally selected spermatogonial cell lines were extensively tested in vitro. Animals were generated via spermatogonia mediated transgenesis by transplantation of clonal cell line into testes of chemically sterilized recipients. Obtained progeny were tested for inducibility in vivo and served as donors of spermatogonia for downstream analysis. We obtained animals and clonally derived spermatogonial stem cell lines that express an inducible dominant negative form of the ERBB2protein. Isogenic nature of induced and uninduced cells allows most accurate morphological and molecular comparison of cells affected by the interruption of normal function of the ERBB2receptor and cellular dynamics in vitro and in vivo. Clonally derived spermatogonial stem cell lines that express an inducible dominant negative form of ErbB2demonstrated an obvious difference in morphological appearance and growth kinetics of induced cells comparing to uninduced ones. Western blot analysis of induced and uninduced cells revealed significant differences in presence and phosphorylation state of several tested important proteins involved in the ERBB2mediated signal transduction, such as S6ribosomal protein; AKT (the serine/threonine kinase also known as protein kinase B); and three protein kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade PRAS40, MEK, ERK1/2.

OPTIMIZATION OF FREEZING CONDITIONS FOR CRYOPRESERVATION OF RAT SPERMATOGONIAL STEM CELL

Different cell types demonstrate an individual response to the doses of DMSO in freezing media that cannot be calculated empirically. Such situation requires experimental adjustment of the dose of the main cryoprotectant for each cell type. The purpose of the work was to optimize the basic composition of the cryopreservation medium for the rat spermatogonial cell lines, to investigate the time period of the initial storage of the cells at –80° C, as well as the effect of the ratio of volume of the freezing medium to the number of cells on their subsequent survival after recovery. Our data demonstrate that the 8% DMSO is the most effective concentration of the cryoprotectant. Furthermore, we tested different time of initial storage of cryovials in a commonly used “Mr. Frosty” Freezing Container at –80ºC. Our results suggest that 12 hours period (overnight) provides enough time for primary freezing step at –80ºC. Optimization of the volume of freezing medium revealed, that 0.5 ml, compared with 1.0 ml of medium for rat spermatogonial stem cells allows more effective preservation and long-time cryostorage of low numbers of cells (3×104) with successful recovery of up to 50% of the frozen cell population. Additionally, we attempted to improve the freezing medium composition by supplementing its base formulation with sucrose and/or trehalose. The use of optimal concentration of DMSO (8%) in the medium in combination with a 200mM of trehalose resulted in an increase of spermatogonia viability after recovery by more than 12% compared to the original composition of SG (Spermatogonia Growth) medium containing 10% of DMSO.

Rattus norvegicus Spermatogenesis Colony-Forming Assays.

Knowledge gaps persist on signaling pathways and metabolic states in germ cells sufficient to support spermatogenesis independent of a somatic environment. Consequently, methods to culture mammalian stem cells through spermatogenesis in defined systems have not been established. Lack of success at culturing mammalian stem cells through spermatogenesis in defined systems reflects an inability to experimentally recapitulate biochemical events that develop in germ cells during a seminiferous epithelial cycle. Complex germ and somatic cell associations that develop each seminiferous epithelial cycle support such a hypothesis, conceivably explaining why highly pure mammalian spermatogonia have not developed into meiosis, much less through meiosis without somatic cells. Here, we outline an in vitro spermatogenesis colony-forming assay to study how differentiating spermatogonial syncytia develop from rat spermatogonial stem cell lines. Robust spermatogonial differentiation under defined culture conditions will facilitate molecular biology studies on pre-meiotic steps in gamete development, and provide a soma-free bioassay to identify spermatogenic factors that promote meiotic progression in vitro.

Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced autophagy of mouse Leydig TM3 cells in vitro.

BackgroundAs a plasticizer, plastic softener, and flame-retardant, tri-ortho–cresyl phosphate (TOCP) is and has been widely used in industry and reported to have a toxic effect on the male reproductive system in animals besides neurotoxicity and immunotoxicity. We have reported that TOCP inhibits spermatogenesis and induces autophagy of rat spermatogonial stem cells, but it is still unknown whether TOCP induces autophagy of mouse Leydig cells and its potential mechanism.MethodsCell viability was observed by MTT assay. Level of testosterone was measured by radioimmunoassay. Apoptosis was observed by AnnexinV-FITC/PI assay. The contents of LC3, Atg5-Atg12, and Beclin 1 were detected by Western blotting analysis. Autophagosomes were investigated by transmission electron microscopy. The contents of MDA and GSH and the activities of SOD, GSH-PX, total antioxidant status (TAS) and total oxidant status (TOS) were measured by oxidative stress kits.ResultsThe present study shows that TOCP markedly inhibited viability and testosterone output of mouse Leydig TM3 cells but had no effect on apoptosis. However, TOCP significantly increased both LC3-II and the ratio of LC3-II to LC3-I and the contents of autophagy proteins Atg5 and Beclin 1. Transmission electron microscopy (TEM) showed that TOCP increased autophagic vacuoles of the cytoplasm, indicating that TOCP could induce autophagy of the cells. TOCP significantly induced oxidative stress of mouse Leydig TM3 cells. H2O2 also inhibited viability and induced autophagy of the cells; however, inhibition of oxidative stress by N-acetyl-L-cysteine (NAC) could rescue the inhibition of cell viability and induction of autophagy by TOCP.ConclusionsThe results show oxidative stress might be involved in TOCP-induced autophagy of mouse Leydig TM3 cells.

Saligenin cyclic-o-tolyl phosphate (SCOTP) induces autophagy of rat spermatogonial stem cells.

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame-retardants in industry, which can be metabolized to High-toxic saligenin cyclic-o-tolyl phosphate (SCOTP). Our previous results found that TOCP could disrupt the seminiferous epithelium in the testis and induce autophagy of rat spermatogonial stem cells. Little is known about the toxic effect of SCOTP on rat spermatogonial stem cells. The present study showed that SCOTP decreased viability of rat spermatogonial stem cells in a dose-dependent manner. Both LC3-II and the ratio of LC3-II/LC3-I were significantly increased; autophagy proteins atg5 and Beclin 1 were also markedly increased after treatment with SCOTP, indicating SCOTP could induce autophagy of the cells. Ultrastructural observation under the transmission electron microscopy (TEM) indicated that there were autophagic vacuoles in the cytoplasm in the SCOTP-treated cells. However, cell cycle arrest was not observed by flow cytometry; and the mRNA levels of p21, p27, p53 and cyclin D1 in the cells were also not affected by SCOTP. Meanwhile, SCOTP didn't induce apoptosis of the cells. In summary, we showed that SCOTP could induce autophagy of rat spermatogonial stem cells, without affecting cell cycle and apoptosis.

Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells.

Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm-forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate "pure," non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetic selection of donor spermatogonia. Monoclonal enrichment of Erbb3 null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform for generating targeted germline mutations in rats and for studying spermatogenesis.

설치류에서 정소조직의 체외배양을 통한 정자형성과정에 관한 연구

Spermatogenesis is a complex developmental process of continuous cell division involving a phase of germ cell expansion, meiotic division and cytodifferentiation that ultimately generates mature gametes. The object of this study was the development of optimal culture system in order to induce spermatogenesis in vitro and for its establishment. We collected immature testicular tissues from different developmental-stage of mice and rats which were subsequently cultured on the agarose gel with serum-free culture media. After appropriate culture period, we counted multiple layered seminiferous tubules in the cultured testis tissue to find out whether any differentiation occured following spermatogenesis. Between our experimental mouse strains ICR and C57, as a result, we observed significant increased spermatogenic tubules in case of C57 mouse strain in neonatal stage. In our optimized culture condition, we detected the lectin PNA positive spermatid resulted from spermatogenesis in cultured testis tissue after 6 weeks of incubation. We also injected the lentiviral transduced rat spermatogonial stem cells (SSCs) into recipient testis to generate the transgenic gametes. The testicular fragments were then cultured based on our optimized culture condition. After culturing 8 weeks, we observed expression of GFP in differentiated germ cells including post meiotic germ cells, which suggested the induction of donor-derived spermatogenesis. Also, in order to optimize the in vitro culture system for rat testis, we have tested the additional effect of hormones and growth factors, but could not observe the significant increased effect of hormone and growth factors treated groups than the control. In conclusion, our in vitro tissue culture methods could be applicable for production of in vitro transgenic gametes in various kinds of mammalian species including domestic animal.

Tri-ortho-cresyl phosphate induces autophagy of rat spermatogonial stem cells.

Tri-ortho-cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame retardants in industry and reported to have a deleterious effect on the male reproductive system in animals besides delayed neurotoxicity. Our preliminary results found that TOCP could disrupt the seminiferous epithelium in the testis and inhibit spermatogenesis, but the precise mechanism is yet to be elucidated. This study shows that TOCP inhibited viability of rat spermatogonial stem cells in a dose-dependent manner. TOCP could not lead to cell cycle arrest in the cells; the mRNA levels of p21, p27, p53, and cyclin D1 in the cells were also not affected by TOCP. Meanwhile, TOCP did not induce apoptosis of rat spermatogonial stem cells. After treatment with TOCP, however, both LC3-II and the ratio of LC3-II/LC3-I were markedly increased; autophagy proteins ATG5 and beclin 1 were also increased after treatment with TOCP, indicating that TOCP could induce autophagy in the cells. Ultrastructural observation under the transmission electron microscopy indicated that autophagic vesicles in the cytoplasm containing extensively degraded organelles such as mitochondria and endoplasmic reticulum increased significantly after the cells were treated with TOCP. In summary, we have shown that TOCP can inhibit viability of rat spermatogonial stem cells and induce autophagy of the cells, without affecting cell cycle and apoptosis.

The induction of rat spermatogonial stem cells into neuronal-like cells and behavioral recovery following transplantation in a rat Parkinson's disease model

Parkinson's disease (PD) is a widespread age-associated neurodegenerative disorder. Current treatment is symptomatic rather than curative. However, stem cell replacement therapies may have the potential to offer curative treatment. In this study, we demonstrate that rat CD49f+ spermatogonial stem cells (rSSCs) can be induced to become functional dopaminergic neuron-like cells in vitro. Furthermore, when rSSCs were transplanted into 6-hydroxydopamine (6-OHDA)-treated PD rats, the results indicated that rSSCs expressed multiple neuron cell markers and were ameliorative to behavioral recovery in PD rats after induction both in vitro and in vivo. In addition, rSSCs demonstrated increased activity in the regeneration of dopaminergic neuron-like cells, increased migration distances and were associated with improvement in animal behavior in the PD rat model. Therefore, rSSCs could be a source of dopaminergic neuron-like cells with potential benefit in cell replacement therapy for PD.

Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months.

Genetic manipulations in the rat: progress and prospects

Several advances have been made to manipulate the rat genome in the last 2 years. This review aims to describe these advances in rat genetic manipulations, with an emphasis on their current status and their prospects and applications in the postgenomic era. Authentic rat embryonic stem cells were derived in 2008 using the 2i/3i culture system. This led to the generation of the first gene knockout rats via embryonic stem cell-based gene targeting. The development of zinc-finger nucleases (ZFNs) provided an alternative approach that avoids the necessity of germline competent embryonic stem cells. Meanwhile, improvements have been made to the well established random mutagenesis mediated by transposons or N-ethyl-N-nitrosourea (ENU). The in-vitro rat spermatogonial stem cell (SSC) system has greatly optimized these phenotype-driven approaches for future applications. The rat has long been a prime model organism in physiological, pharmacological and neurobehavioral studies. The recent advances of rat reverse genetic approaches, together with the classical ENU and transposon mutagenesis system, will contribute tremendously to the deciphering of gene functions and the creation of rat disease models.

Involvement of c-Src/STAT3 signal in EGF-induced proliferation of rat spermatogonial stem cells

Epidermal growth factor (EGF) plays a role in male germ cell development, but the precise function is yet to be defined. This study shows that EGF stimulates rat spermatogonial proliferation in a dose-dependent manner and significantly increased the protein levels of phosphated c-Src (p-c-Src) and phosphated signal transducer and activator of transcription 3 (p-STAT3). Moreover, overexpression of c-Src tagged with enhanced green fluorescence protein (EGFP) in rat spermatogonial stem cells enhances the cell viability. In contrast, knockdown or inhibition of c-Src inhibits rat spermatogonial stem cell proliferation; EGF could not abrogate the inhibitory effect. Evidently, the content of p-STAT3 protein was increased in c-Src-expressing cells and decreased in c-Src-suppressing cells. Furthermore, knockdown or inhibition of STAT3 also suppressed cell viability; neither EGF nor increased c-Src could reverse the inhibitory effect. These results are the first evidence that EGF induces proliferation of rat spermatogonial stem cells through c-Src/STAT3 signal.

Biological Characteristics of Rat Spermatogonial Stem Cells Cultured on Sertoli Cell Feeder Layer in vitro

Biological Characteristics of Rat Spermatogonial Stem Cells Cultured on Sertoli Cell Feeder Layer in vitro