Characteristics Of Spermatogonial Stem Cells Research Articles

Single-cell transcriptomes reveal spermatogonial stem cells and the dynamic heterogeneity of spermatogenesis in a seasonal breeding teleost.

Seasonal spermatogenesis in fish is driven by spermatogonial stem cells (SSCs), which undergo a complex cellular process to differentiate into mature sperm. In this study, we characterized spermatogenesis in the large yellow croaker (Larimichthys crocea), a marine fish of significant commercial value, based on a high-resolution single-cell RNA-sequencing atlas of testicular cells from three distinct developmental stages: juvenile, adult differentiating and regressed testes. We detailed a continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, elucidating the molecular events involved in spermatogenesis. We uncovered dynamic heterogeneity in cellular compositions throughout the annual reproductive cycle, accompanied by strong molecular signatures within specific testicular cells. Notably, we identified a distinct population of SSCs and observed a critical metabolic transition from glycolysis to oxidative phosphorylation, enhancing our understanding of the biochemical and molecular characteristics of SSCs. Additionally, we elucidated the interactions between somatic cells and spermatogonia, illuminating the mechanisms that regulate SSC development. Overall, this work enhances our understanding of spermatogenesis in seasonal breeding teleosts and provides essential insights for the further conservation and culture of SSCs.

Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions.

RETRACTED: Cellular functions of spermatogonial stem cells in relation to JAK/STAT signaling pathway

This manuscript comprehensively reviews the interrelationship between spermatogonial stem cells (SSCs) and the JAK/STAT signaling pathway. Spermatogonial stem cells in the testes of male mammals, characterized by their self-renewal and pluripotential differentiation capabilities, are essential for tissue regeneration, immunomodulation, and advancements in regenerative medicine. This review delves into the historical background and biological characteristics of SSCs, with a particular emphasis on the pivotal role of the JAK/STAT signaling pathway in their proliferation, maturation, and differentiation processes. Research indicates that the JAK/STAT pathway extensively influences various functionalities of spermatogonial stem cells, encompassing immunomodulation, tissue differentiation, homing, and adaptation to the microenvironment. Herein, we collate and dissect related studies, shedding light on the intricate dynamics between SSCs and the JAK/STAT signaling pathway, and examine the implications of these interactions on the biological attributes and functionalities of SSCs. Furthermore, the review discusses the profound implications of these findings for preclinical research and the domain of cellular engineering. It is acknowledged that, despite advancements in the research of SSCs and the JAK/STAT signaling pathway, investigations in humans and larger mammals remain inadequate, necessitating more in-depth exploration to establish a comprehensive theoretical framework. Overall, this review offers an invaluable reference for deciphering the mechanisms of the spermatogonial stem cell signaling pathways and establishes a theoretical groundwork for related preclinical research.

MiR-101–5p overexpression suppresses the proliferation of goat spermatogonial stem cells by targeting EZH2

MicroRNAs (miRNAs), as post-transcriptional gene mediators, regulate the biological characteristics of spermatogonial stem cells (SSCs), including proliferation, differentiation and apoptosis. However, the potential roles and mechanisms by which miR-101–5p affected the biological characters of goat SSCs have not been fully elucidated. Herein, we reported that miR-101–5p overexpression decreased cell viability (P < 0.01), arrested cell cycle in the G1 phase (P < 0.05), and aggravated apoptosis of goat SSCs (P < 0.01) compared with negative control (NC), as determined by CCK-8 assay and flow cytometry analysis. Additionally, PCNA protein expression was attenuated by miR-101–5p overexpression (P < 0.05). Notably, the expression of SSCs specific genes Oct4 (P < 0.05), PLZF (P < 0.01) and DAZL (P < 0.01) were decreased in miR-101–5p overexpressed SSCs. Furthermore, the dual luciferase reporter assay showed that, when co-transfected with miR-101–5p mimics, the relative luciferase activity of EZH2 wide-type (WT) was inhibited (P < 0.05) compared with the transfection of EZH2 mutant (MUT). EZH2 expression was negatively correlated with miR-101–5p expression in goat SSCs. Collectively, our data implicates that miR-101–5p overexpression aggravates cell apoptosis, and suppresses cell proliferation of goat SSCs via targeting EZH2, which may impair spermatogenesis.

Establishment of a Spermatogonial Stem Cell Line with Potential of Meiosis in a Hermaphroditic Fish, Epinephelus coioides.

Spermatogonial stem cells (SSCs) are unique adult stem cells capable of self-renewal and differentiation into sperm. Grouper is a protogynous hermaphroditic fish farmed widely in the tropical and subtropical seas. In this study, we established an SSC line derived from adult testis of orange-spotted grouper, Epinephelus coioides. In the presence of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), the cells could be maintained with proliferation and self-renewal over 20 months and 120 passages under in vitro culture conditions. The cells exhibited strong alkaline phosphatase activity and the characteristics of SSCs with the expression of germ cell markers, including Vasa, Dazl, and Plzf, as well as the stem cell markers Nanog, Oct4, and Ssea1. Furthermore, the cultured cells could be induced by 11-ketotestosterone treatment to highly express the meiotic markers Rec8, Sycp3, and Dmc1, and produce some spherical cells, and even sperm-like cells with a tail. The findings of this study suggested that the cultured grouper SSC line would serve as an excellent tool to study the molecular mechanisms behind SSCs self-renewal and differentiation, meiosis during spermatogenesis, and sex reversal in hermaphroditic vertebrates. Moreover, this SSC line has great application value in grouper fish aquaculture, such as germ cell transplantation, genetic manipulation, and disease research.

Effect of stage of puberty on culture characteristics of goat spermatogonial stem cells

The aim of experiment was to study the culture characteristics of spermatogonial stem cells (SSCs) obtained from pre-and post-pubertal goat testes. Six animals each of two age groups (3-6 months and 1.0-1.5 years) were included in the study. The testes were collected from slaughtered animals and after washing, morpho-biometric characters such as weight, length, mid circumference and volume of both the left and right testes were recorded. The isolated SSCs were enriched by double filtration through 80 and 60 μm nylon mesh filters. The filtrate was cultured overnight with SSC medium Dulbecco's modified eagle's medium / HamF-12 medium. The colonies were also characterized by alkaline phosphatase activity and demonstration of specific markers octamer binding transcription factor and protein gene product.All the morpho-biometrical parameters of left and right testes, except density, were significantly (P<0.01) different between the groups. The total number of SSCs isolated from pre-pubertal testes (6.85×106 cells/ml) was significantly (P<0.05) higher than post-pubertal testes (2.40×106 cells/ml). The colonies of SSCs inpre-pubertal culture appeared within seven days of culture whereas in post-pubertal SSCs, colonies appeared after 14 days of culture. The results demonstrated that the SSCs in prepubertal goat testes were higher in number, grew faster and formed colonies at least one week earlier compared to the SSCs of post-pubertal testes. Thus, the testes of pre-pubertal goats were a better source to study the physiological and regulatory mechanisms of SSCs.

Stage-specific embryonic antigen 4 is a membrane marker for enrichment of porcine spermatogonial stem cells.

Spermatogonial stem cells (SSCs), as tissue-specific stem cells, are capable of both self-renewal and differentiation and supporting the continual and robust spermatogenesis for male fertility. As a rare sub-fraction of undifferentiated spermatogonia, SSCs share most molecular markers with the progenitor spermatogonia. Thus, the heterogeneity of the progenitor cells often obscures the characteristics of stem cells. Distinguishing SSCs from the progenitors is of paramount importance to understand the regulatory mechanisms governing their actions. The present study was designed to reveal that SSEA4 can be a marker for putative porcine SSCs that distinguished it from the progenitors and to build a sorting program for efficient enrichment of porcine SSCs. To explore expression of SSEA4 within the undifferentiated spermatogonial population, we performed co-immunofluorescent staining for SSEA4 and common molecular markers (VASA, DBA, PLZF, c-KIT, and SOX9) in the 7-, 90-, and 150-day-old porcine testicular tissues. SSEA4-positive cells were isolated from the 90-day-old porcine testes by flow cytometry. Immunofluorescent, RNA-sequencing, and transplantation analysis were used to reveal that SSEA4-positive fraction holds the stem cell capacity. We found that SSEA4 was expressed in a rare sub-fraction of porcine undifferentiated spermatogonia, and RNA-sequencing analysis revealed that the genes for stem cell maintenance and SSC-specific markers (ID4 and PAX7) were up-regulated in the SSEA4-sorted fraction, compared with undifferentiated spermatogonia. In addition, germ cell transplantation assay demonstrated that SSEA4-positive spermatogonia colonized in the recipient testicular tubules. Sorting of the undifferentiated spermatogonia with anti-SSEA4 antibody resulted in a 2.5-fold enrichment of SSCs compared with the germ cell gate group, and 21-fold enrichment of SSCs compared with the SSEA4-negative spermatogonia group. Our findings revealed that SSEA4 is a new surface marker for porcine undifferentiated spermatogonia. This finding helps to elucidate the characteristics of porcine SSCs and facilitates the culture and manipulation of SSCs.

EZH2 expression and its role in spermatogonial stem cell self-renewal in goats

Enhancer of zeste homolog 2 (EZH2) is a histone H3 lysine 27 (H3K27) methyltransferase that plays vital roles in mouse spermatogenesis. However, the expression pattern and role of EZH2 in goat spermatogonial stem cells (SSCs) is unknown. In the present study, we investigated EZH2 expression in the testis of postpubertal goats and its effect on the biological characteristics of goat SSCs. We found that EZH2 mRNA (P < 0.01) and protein (P < 0.05) expression was increased in the testes of postpubertal goats compared to that of prepubertal goats. Moreover, EZH2 was more highly expressed in goat SSCs than in Leydig cells (P < 0.01) and Sertoli cells (P < 0.01) as determined by qPCR, Western blot, and immunofluorescence. Compared to a negative control (NC), cell proliferation (P < 0.01) and viability (P < 0.01) were decreased in SSCs in which EZH2 was knocked down, and the G2/M phase of the cell cycle was blocked (P < 0.01), as determined by Edu staining, CCK-8 assay, and flow cytometry analysis. Additionally, the expression of CASP3, CASP9, and BAX was significantly increased (P < 0.01) while BCL2 expression was decreased (P < 0.01) in EZH2 knockdown SSCs. Notably, the expression of GDNF, a SSCs marker gene, and DAZL, a spermatogenesis-related gene, were significantly decreased (P < 0.01) while GFRA1 expression was significantly up-regulated (P < 0.01) in EZH2 knockdown SSCs. Our data suggest that EZH2 plays a pivotal role in the self-renewal of goat SSCs, and knockdown of EZH2 might impair spermatogenesis in goats.

Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats.

Paternal metabolic status is an important factor in the health status of offspring. Cholestasis, as a metabolic disorder, significantly disrupts spermatogenesis. Spermatogonial stem cells (SSCs) are considered the dividing germ cells, which maintain spermatogenesis throughout the lifespan. Here, we investigated the in vivo and in vitro effect(s) of cholestasis on SSCs. Cholestasis was induced in rats by bile duct ligation. Four weeks after the cholestasis induction, testicular tissues were analysed using histopathological examinations. The expression of SSC markers, including Plzf and Thy-1, was determined using the immunofluorescent technique. Also, SSCs were isolated from animals, and their proliferation was examined in vitro. The histological examinations revealed that cholestasis caused irregularities in the structure of seminal tubes. Immunostaining showed that the total number of Thy-1- and Plzf-expressing cells was declined in the cholestasis group compared with the control group. In vitro culture of SSCs indicated that the number of SSC colonies and those expressing Plzf were significantly reduced in the culture medium of the cholestasis group. Our results indicated that cholestasis affects the functionality of SSCs and reduces the number and proliferation of them. This finding may be of interest to the effect of metabolic diseases such as cholestasis on spermatogenesis.

Human Spermatogonial Stem Cells Scrutinized under the Single-Cell Magnifying Glass.

Spermatogonial stem cells (SSCs) are essential for adult spermatogenesis. Recently, Wang etal. (2018), Guo etal. (2018), and Hermann etal. (2018) used single-cell RNA sequencing to define and molecularly characterize human testicular cell populations, including spermatogonial subsets with characteristics of human SSCs.

PD33-09 MIR-135A IS ASSOCIATED WITH CRYPTORCHIDISM INFERTILITY THROUGH SUPPRESSION OF FOXO1 IN SPERMATOGONIAL STEM CELLS

You have accessJournal of UrologyStem Cell Research: Stem Cell Research I1 Apr 2018PD33-09 MIR-135A IS ASSOCIATED WITH CRYPTORCHIDISM INFERTILITY THROUGH SUPPRESSION OF FOXO1 IN SPERMATOGONIAL STEM CELLS Yoshinobu Moritoki, Kentaro Mizuno, Hideyuki Kamisawa, Satoshi Kurokawa, Akihiro Nakane, Tetsuji Maruyama, Yutaro Hayashi, and Takahiro Yasui Yoshinobu MoritokiYoshinobu Moritoki More articles by this author , Kentaro MizunoKentaro Mizuno More articles by this author , Hideyuki KamisawaHideyuki Kamisawa More articles by this author , Satoshi KurokawaSatoshi Kurokawa More articles by this author , Akihiro NakaneAkihiro Nakane More articles by this author , Tetsuji MaruyamaTetsuji Maruyama More articles by this author , Yutaro HayashiYutaro Hayashi More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1564AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogonial stem cells (SSCs) function as a life-long spermatogenesis reserve. Self-renewal and stem maintenance are foundational characteristics of SSCs. To maintain these cellular characteristics, the regulation of genome transcription is mandatory, although the basis of this mechanism remains unknown. Recently, small mRNA, called microRNA (miRNA), consisting of 18–25 nucleotide bases have been recognized as key transcription regulators. The effect involves more than one-third of all transcription regulation. We hypothesized that miRNAs function as one of the key SSC regulators and are likely associated with SSC differentiation and thus aimed to find the miRNAs expressing in SSCs and its target mRNAs. METHODS We used a cryptorchid rat testes model of SSC disturbance. The model was generated using intra-abdominal injection of flutamide (7.5 mg/rat) into pregnant Sprague-Dawley rats. Normal testes, unilateral undescended testes (UDT), and contralateral descended testes (DT) were extirpated on postnatal day 9, when two types of SSCs (gonocytes and spermatogonia) co-existed and gonocytes began differentiation into spematogonia. A microarray examining DT and UDT focused on miR-135a, which exhibited lower expression in UDT. In situ hybridization (ISH) was used for the detection of the localization of miR-135a and quantitative polymerase chain reaction (qPCR) to determine the organ specificity of the gene. We then searched the miR-135a target genes using a computer program, followed by the luciferase assay to demonstrate the suppression effect of miR-135a in vitro. RESULTS qPCR revealed that miR-135a was 8.3–21.3 times over-expressed in the testes than in other organs; ISH showed miR-135a localized in SSCs. In silico analysis identified FOXO1 as a putative target gene of miR-135a. FOXO1 has a sequence complementary to miR-135a's seed sequence at its 3' untranslated region. FOXO1 localized in SSCs, and a luciferase assay demonstrated that miR-135a directly suppressed FOXO1 expression in mouse GC-1 cells. CONCLUSIONS We demonstrated that miR-135a is associated with the maintenance of SSCs and that its putative target gene is FOXO1. Because FOXO1 is already proven necessary for maintenance of SSCs and we proved that SSCs are reduced in UDT, we hypothesized that miR-135a was lowered to facilitate the active FOXO1 expression, leading to SSC maintenance. Thus, miR-135a is associated with SSC maintenance through regulation of FOXO1 expression. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e653-e654 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Yoshinobu Moritoki More articles by this author Kentaro Mizuno More articles by this author Hideyuki Kamisawa More articles by this author Satoshi Kurokawa More articles by this author Akihiro Nakane More articles by this author Tetsuji Maruyama More articles by this author Yutaro Hayashi More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Stem cell activity of type A spermatogonia is seasonally regulated in rainbow trout.

Spermatogonial stem cells (SSCs) support continuous production of sperm throughout the male's life. However, the biological characteristics of SSCs are poorly understood in animals exhibiting seasonal reproduction, even though most wild animals are seasonal breeders. During the spermiation season in rainbow trout, the lumen of the testes contains only spermatozoa and scattered type A spermatogonia (ASG) along the walls of the testicular lobules. These few remaining ASG, designated "residual ASG," are the only germ cells capable of supporting the next spermatogenesis, suggesting that the residual ASG are true SSCs. However, whether residual ASG can behave as SSCs in any teleost species is unknown. In this study, we attempted to clarify the biological characteristics of SSCs associated with seasonal reproduction in rainbow trout using spermatogonial transplantation. We found that the stem cell activity was clearly regulated seasonally during the annual reproductive cycle. Although the residual ASG exhibited moderate transplantability and colony-forming ability at the beginning of the spermiation season, these parameters decreased dramatically later and remained low until the next spermatogenesis was initiated. Furthermore, no clear correlations were observed between these qualitative changes and previously described morphologic characteristics of ASG or plasma sex steroid levels. Our results suggest that the biological properties of SSC populations in rainbow trout are seasonally regulated by a novel mechanism.

The applications and recovery outcome of spermatogonia stem cells in regenerative medicine

Abstract Spermatogonial stem cells are unique cells that line the basal lamina of seminiferous tubules in the testis and have the potential of self-renewal and differentiation. These cells pass on genetic information to the next generation via the process of spermatogenesis and have an important role in the maintenance of male fertility. Recent reports have shown the pluripotency characteristics of spermatogonial stem cells at the cellular and molecular level. These cells can convert into Embryonic stem like cells that exhibit similar phenotype with embryonic stem cells and the capacity to differentiate into all three germ layers: Ectoderm, endoderm and mesoderm. These properties make spermatogonial stem cells an appropriate source for use in regenerative medicine. Spermatogonial stem cells considered as an alternative and novel cell source that limits the technical and ethical problems accompanied with the other pluripotent stem cells. Patient-specific cell lineages without limitations of immunological rejection and specific auto transplantation are the beneficial’s associated with these cell population. Also, cell therapy based on spermatogonial stem cells engraftment, promises for preservation of fertility and induces the reproductive potential in infertile patients. Here, we discuss the applications of spermatogonial stem cells in regenerative medicine with focus on all important aspects, including: cell isolation, propagation, differentiation and especially cell transplantation.

Transplantation as a Quantitative Assay to Study Mammalian Male Germline Stem Cells.

In mammals, the activities of spermatogonial stem cells (SSCs) provide the foundation for continual spermatogenesis throughout a male's reproductive lifetime. At present, the defining characteristics of SSCs and mechanisms controlling their fate decisions are not well understood. Transplantation is a definitive functional measure of stem cell capacity for male germ cells that can be used as an assay to provide an unequivocal quantification of the SSC content in an experimental cell population. Here, we discuss the procedure for mice and provide protocols for preparing donor germ cell suspensions from testes directly or primary cultures of spermatogonia for transplantation, enriching for SSCs, preparing recipient males, microinjection into recipient testes, and considerations for experimental design.

Multipotent male germline stem cells (mGSCs) from neonate porcine testis

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, during which unlimited spermatozoa is produced daily derived from SSCs in the testis throughout life of the male. Germline stem (GS) cells can be isolated from spermatogonia, which shared the characteristics of SSCs and embryonic stem cells (ESCs), and can be passaged stably in vitro. The study of GS cells contributes to understanding spermatogenesis process. However, little is known about the GS cells in domestic animals. Here, we report the successful establishment of a serum- and feeder-free system for multipotent male GS cells (mGSCs) from postnatal porcine testis. These cells expressed pluripotent markers, such as Oct4, Nanog, C-myc, and germline-specific markers including Vasa, CD90, CD49f, Gfrα1, Plzf and Dazl. Then we assayed the developmental potential of these cells in vitro. The porcine multipotent male germline stem cells (pmGSCs) can form embryoid bodies (EBs) by suspension culture. Immunofluorescence analysis showed that the EBs differentiated into neuron-specific enolase (NSE, ectoderm), α-actin (mesoderm), and Pdx1 (endoderm) positive cells. These cells induced by 10-6 M retinoic acid (RA) could be differentiated into spermatid-like cells which were positive for Acrosin. The pmGSCs has been cultured over 14 passages. Thus, we have established a long-term culture system for pmGSCs. This culture system provides a platform for the study of porcine mGSCs.

Characteristics of spermatogonial stem cells derived from neonatal porcine testis.

The aim of this study was to isolate and characterise porcine spermatogonial stem cells (PSSCs). The putative porcine germline stem cells from testis were isolated successfully by an improving way of enrichment with lymphocyte separation medium (LSM). Results from RT-PCR analyses showed that PSSCs were positive for OCT4, SOX2, NANOG, PGP9.5, c-MYC, KEL4 and PRDM-14 which are multipotent stem cell markers. At the protein level, the results of immunofluorescence analyses showed that PSSCs were positive for OCT4, PGP9.5, SOX2 and CD29. We successfully differentiated these PSSCs into adipocytes and muscle cells and then defined their characteristics, including morphology, surface stem cell markers, and mechanical properties. But the experiment of teratoma formation was negative. The results indicated the PSSCs could be multipotent. Atomic force microscopy was used to characterise the morphological and mechanical properties of undifferentiated PSSCs, as well as the differentiated adipocytes and muscle cells, which could be potentially useful for distinguishing PSSCs from differentiated cells.

A simple method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells

Spermatogonial stem cells (SSCs) are expected to participate in male infertility therapy, endangered species preservation, and transgenic animal technology by their unique unipotency to differentiate into spermatozoa. The main challenges, however, remain to be addressed including the appropriate conditions to reach good number of these cells and how to derive, culture, and maintain them in vitro. In the present study, the testicular tissues were isolated from 1-d-old male chickens to establish primary cell cultures. This culture led to development of distinguished colonies which were further characterized by alkaline phosphatase (AP) activity assay and gene expression analysis. They were shown to be positive for AP activity and expressed two main transcription factors of OCT4 and STRA8 as indicated by reverse transcription-polymerase chain reaction. These were indications of carrying characteristics of SSCs by these colonies. The cultures were also exposed to different concentrations of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) growth factors to seek optimum colony-forming conditions. Colony-forming activity assay indicated that they were able to propagate in vitro with an increased self-renewal property when cultured in the presence of 15 ng/mL of GDNF, 20 ng/mL of bFGF, and 15 ng/mL of LIF. The present work provides an easy and practical method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells and introduces appropriate cell culture conditions to improve and maintain their self-renewal property based on supplying the necessary growth factors.

Mouse undifferentiated spermatogonial stem cells cultured as aggregates under simulated microgravity.

Dynamic simulated microgravity (SMG) culture systems provide environments that stimulate stem cell proliferation and differentiation. However, the effect of SMG on spermatogonial stem cells (SSCs) remains unclear. Here, we used a rotating cell culture system (RCCS) to determine its effect on mouse SSC proliferation and differentiation. SSCs were enriched from mouse pub testis and cocultured with Sertoli cell feeders pre-treated with mitomycin C on fibrin scaffolds in a rotary bioreactor for 14 days. Our results show that mouse SSCs cultured in a rotary bioreactor exhibited enhanced proliferation surpassing those cultured in static conditions, although SSC cultures in SMG underwent a growth lag at initial 3 days. After 14 days, mouse SSCs and feeders grew into cell aggregates with average diameters of 242.63 ± 16.53 μm compared with those in conventional static culture (49.51 ± 15.64 μm). Related detection revealed that proliferating SSCs in SMG remained undifferentiated, maintained clone-forming capacity and were capable of differentiation into round spermatids with flagella. The growth characteristics of mouse SSCs in RCCS suggest that the resulting aggregates are similar to native in vivo cells. Rotary bioreactors that create SMG environments may be an alternative to conventional systems for the clinical application of SSCs.

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting. The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture. One month old goats' testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated. The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively. The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.

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.