Expression Of Germ Cell Markers Research Articles

Low Testosterone Concentration Improves Colonisation and Viability in the Co-Cultured Goat Spermatogonial Stem Cell With Sertoli Cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis through self-renewal and differentiation. The proliferation of SSCs in culture systems can provide a valuable source of germ cells. Several studies have investigated new reproductive technologies, including the production of transgenic animals and recombinant proteins secreted from milk in goats. While studies in other species exist, research on goat SSC culture remains limited. We investigated the impact of different testosterone concentrations on the survival and colonisation of cocultured goat SSCs with Sertoli cells. Cells were isolated from immature goats using two-step enzymatic digestion and enriched by differential exclusion method. DMEM/F12 culture medium containing 1% antibiotic and 5% FBS, supplemented with GDNF (20 ng/mL), EGF, bFGF and LIF (10 ng/mL), was used with different testosterone concentrations (0, 60, 120 and 240 μg/mL) and cultured for 10 days. SC subpopulations were confirmed using PGP9.5 immunocytochemistry, and the expression of germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was evaluated through RT-PCR. Alkaline phosphatase activity provided additional SSC presence. The survival rate of SSCs after isolation and the number and area of colonies on Days 4, 7 and 10 were measured using an inverted microscope. The presence of PGP 9.5 antigens and germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was confirmed by immunocytochemistry and RT-PCR, respectively. According to the results, the group with 60 μg/mL testosterone had the highest number and area of colonies. The number of colonies in the 60 μg/mL testosterone group was significantly higher than the control group (p < 0.05), but no significant difference was observed compared to other groups (p ≥ 0.05). This study suggests that a low testosterone concentration (60 μg/mL) is optimal for goat SSC colonisation and viability in coculture with Sertoli cells, potentially leading to advancements in goat reproductive technologies.

Prenatal nicotine exposure leads to epigenetic alterations in peripheral nervous system signaling genes in the testis of the rat

BackgroundPrenatal nicotine exposure (PNE) has been documented to cause numerous deleterious effects on fetal development. However, the epigenetic changes promoted by nicotine exposure on germ cells are still not well understood.ObjectivesIn this study, we focused on elucidating the impact of prenatal nicotine exposure on regulatory epigenetic mechanisms important for germ cell development.MethodsSprague-Dawley rats were exposed to nicotine during pregnancy and male progeny was analyzed at 11 weeks of age. Testis morphology was analyzed using frozen testis sections and expression of germ cell markers was examined by RT-qPCR; histone modifications were assessed by Western Blot (WB). DNA methylation analysis was performed by methylation-specific PCR of bisulfite converted DNA. Genome-wide DNA methylation was analyzed using Methylated DNA immunoprecipitation (MeDIP)-seq. We also carried out transcriptomics analysis of pituitary glands by RNA-seq.ResultsWe show that gestational exposure to nicotine reduces germ cell numbers, perturbs meiosis, affects the expression of germ line reprogramming responsive genes, and impacts the DNA methylation of nervous system genes in the testis. PNE also causes perturbation of gene expression in the pituitary gland of the brain.ConclusionsOur data demonstrate that PNE leads to perturbation of male spermatogenesis, and the observed effects are associated with changes of peripheral nervous system signaling pathways. Alterations in the expression of genes associated with diverse biological activities such as cell migration, cell adhesion and GABA signaling in the pituitary gland underscore the complexity of the effects of nicotine exposure during pregnancy.

SAG-dihydrochloride enhanced the expression of germ cell markers in the human bone marrow- mesenchymal stem cells (BM-MSCs) through the activation of GLI-independent hedgehog signaling pathway

Different studies indicated that the enhancing the expression of germ cell markers improved the efficiency of stem cells in the generation of germ line cells. The aim of the present study was to investigate the effect of SAG-dihydrochloride on the expression of germ cell markers in the human bone marrow-mesenchymal stem cells (BM-MSCs). For this purpose, the human BM-MSCs were cultured in the medium containing different concentrations of SAG-dihydrochloride (10, 20 and 30 µM). After RNA extraction and cDNA synthesis, the expression level of PTCH1, GLI1, PLZF, DDX4 and STRA8 genes were determined by using SYBR Green Real time PCR. The analysis of the results obtained from PTCH1 and GLI1 expression indicated that SAG-dihydrochloride had the ability to enhance the expression of germ cell markers in a Gli-independent manner. Furthermore, the significant increased expression of STRA8 was observed in the BM-MSCs treated by 10 µM SAG-dihydrochloride for 4 and 6 days (p < 0.05). There was also the up-regulation of DDX4 in the BM-MSCs following treatment with 20 µM SAG-dihydrochloride for 4 and 6 days. The obtained results suggested that treatment with SAG-dihydrochloride increased the expression of germ cell markers in the human BM-MSCs through the activation of non-canonical sonic hedgehog signaling pathway.

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.

An effective method for establishing animal models of azoospermia and oligospermia.

The current study aims to develop a validated animal model to predict successful spermatogenesis retrieval in azoospermia and oligospermia men. Thirty-two mice were equally divided into 4 groups: control, scrotal hyperthermia (15 times), scrotal hyperthermia group (10 times), scrotal hyperthermia group (5 times). In the scrotal hyperthermia groups, their scrotum exposed to water at a temperature of 43°C for 20min every other day. Then, the mice were euthanised and sperm samples were collected for sperm parameters analysis, and blood samples were obtained for hormonal assay. The testis samples were taken for histopathology experiments, immunofluorescence staining and Western blot in order to examine the protein expression together with RNA extraction in order to examine the gene expression of germ cell markers. The results of sperm analysis and histopathology of testicular tissue as well as the results of gene expression and Western blot showed that hyperthermia can significantly impair spermatogenesis. In conclusion, we have developed a novel model of azoospermia and oligospermia in mouse, which uses a high temperature to suppress spermatogenesis process through demolition of germ cells subsequent cell cycle arrest and apoptosis. The model will contribute to understanding azoospermia in human, oligospermia pathophysiology and the development of treatment.

Characterization of Bombyx mori nucleopolyhedrovirus infection in fat body-derived Bombyx mori cultured cells

Bombyx mori nucleopolyhedrovirus (BmNPV) is known to replicate in many tissues of Bombyx mori larvae. However, the cell lines used for BmNPV research are predominantly derived from B. mori ovaries or early embryos. In the present study, we examined the properties of NIAS-Bm-aff3 (aff3), a cell line that was established from B. mori larval fat body, which is one of the major tissues for BmNPV propagation. aff3 is a floating cell line, and cell adhesion was enhanced following the coating of the culture dish with poly-d-lysine. RT-qPCR assays demonstrated that the expression of germ cell markers, Vasa, Siwi, and BmAgo3, was much lower in aff3 cells as compared to the B. mori ovary-derived cell line BmN-4. Conversely, aff3 cells express an adipocyte marker, Fabp1, at higher levels, indicating that this cell line retains the characteristics of fat body cells. BmNPV infection induces unique cell fusion in aff3 cells, which was also observed following infection with Autographa californica multiple nucleopolyhedrovirus, a virus that does not cause productive infection in B. mori cells. Occlusion bodies (OBs) produced in BmNPV-infected aff3 cells exhibit large cuboidal shapes as compared to those produced in BmN-4 cells. Furthermore, extremely large OBs (~25 μm in side length) were produced in aff3 cells when infected with a cuboidal polyhedrin mutant. Taking into account these unusual properties, we conclude that aff3 could prove to be a useful resource for conducting baculovirus research.

Male germline stem cells in non-human primates

Over the past few decades, several studies have attempted to decipher the biology of mammalian germline stem cells (GSCs). These studies provide evidence that regulatory mechanisms for germ cell specification and migration are evolutionarily conserved across species. The characteristics and functions of primate GSCs are highly distinct from rodent species; therefore the findings from rodent models cannot be extrapolated to primates. Due to limited availability of human embryonic and testicular samples for research purposes, two non-human primate models (marmoset and macaque monkeys) are extensively employed to understand human germline development and differentiation. This review provides a broader introduction to the in vivo and in vitro germline stem cell terminology from primordial to differentiating germ cells. Primordial germ cells (PGCs) are the most immature germ cells colonizing the gonad prior to sex differentiation into testes or ovaries. PGC specification and migratory patterns among different primate species are compared in the review. It also reports the distinctions and similarities in expression patterns of pluripotency markers (OCT4A, NANOG, SALL4 and LIN28) during embryonic developmental stages, among marmosets, macaques and humans. This review presents a comparative summary with immunohistochemical and molecular evidence of germ cell marker expression patterns during postnatal developmental stages, among humans and non-human primates. Furthermore, it reports findings from the recent literature investigating the plasticity behavior of germ cells and stem cells in other organs of humans and monkeys. The use of non-human primate models would enable bridging the knowledge gap in primate GSC research and understanding the mechanisms involved in germline development. Reported similarities in regulatory mechanisms and germ cell expression profile in primates demonstrate the preclinical significance of monkey models for development of human fertility preservation strategies.

Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture.

Objective The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system. Materials and Methods In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco’s modified Eagle’s medium (DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry against the germ cell markers α6, β1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA).Results We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive. ConclusionA non-adherent culture system could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies.

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo.

BackgroundRecent findings have revealed that the female gonad may have regenerative activity with having germ line stem cells in juveniles and adults. Application of these germ line stem cells could be an alternative therapy for reproductive disorders in regenerative medicine.MethodsTo enhance the potency of differentiation into oocyte-like cells (OLCs) and folliculogenesis, we overexpressed Oct4 in ovarian stem/stromal cell (OvSCs) and examined the cellular properties related to stemness and self-renewal ability and finally demonstrated the ability of in vitro differentiation and folliculogenesis.ResultsOvarian cortex included putative stem cells in terms of AP activity, cell cycle status, cell proliferation, expression of mesenchymal lineage surface markers and pluripotent transcriptional markers. Further, Oct4 transfected OvSCs (Oct4-OvSCs) were enhanced their AP activity and cell proliferation compared to OvSCs. The potential on in vitro differentiation into OLCs and in vivo folliculogenesis was also evaluated in OvSCs and Oct4-OvSCs, respectively. Oct4-OvSCs possessed higher oogenesis potential in vitro than OvSCs, in terms of expression of germ cell markers by RT-PCR and the number of OLCs. When OvSCs and Oct4-OvSCs were xeno-transplanted into infertile mice ovaries, the OvSCs transplantation induced new primary follicle formation and hormonal levels of estradiol and FSH remained similar to that of normal mice. However, Oct4-OvSCs possessed higher ability for folliculogenesis based on inducing developing follicles with thecal layer and granulosa cells and more similar estradiol level to normal mice.ConclusionsThese findings demonstrated that putative stem cells were present in ovarian cortex and exhibited differentiation ability into OLCs and folliculogenesis in vivo, and Oct4-overexpression enhanced these ability, suggesting their cellular models based on gene therapy in understanding the mechanisms of oogenesis and folliculogenesis, and finally in view of reproductive cell therapy.

Expression of Intermediate Filaments and Germ Cell Markers in the Developing Bovine Ovary: An Immunohistochemical and Laser-Assisted Microdissection Study

In the present investigation, bovine ovary prenatal development was studied using immunohistochemistry and laser-assisted microdissection (LAM). A major aim of this study was to evaluate the protein expression pattern of intermediate filaments (IF) and distinguish S100 protein (S100 alpha and S100 beta protein) isoforms during prenatal follicle differentiation, subsequently correlating them with germ cell marker expression. A development-specific expression pattern of different keratins as well as vimentin was detected in the prenatal bovine ovary; K18-specific expression was found during all developmental stages (i.e. in surface epithelium, germ cell cord somatic cells, and follicle cells), and keratins 5, 7, 8, 14, and 19 and vimentin had a stage-specific expression pattern in the different cell populations of the prenatal ovaries. Additionally, our results represent new data on the expression pattern of germ cell markers during bovine ovary prenatal development. S100 alpha and beta protein was localized to oocyte cytoplasm of different follicle stages, and S100 alpha staining could be observed in granulosa cells. Furthermore, through isolation of characteristic ovary cell populations using LAM, specific confirmation of some genes of interest (KRT8, KRT18, S100 alpha, S100 beta, and OCT4, DDX4) could be obtained by RT-PCR in single cell groups of the developing bovine ovary.

Differentiation of primordial germ cells from induced pluripotent stem cells of primary ovarian insufficiency.

Can the induced pluripotent stem cells (iPSCs) derived from women with primary ovarian insufficiency (POI) differentiate into germ cells for potential disease modeling in vitro? The iPSC lines derived from POI patients with 46, X, del(X)(q26) or 46, X, del(X)(q26)9qh+ could differentiate into germ cells and expressed lower levels of genes in the deletion region of the X chromosome. iPSC technology has been envisioned as an approach for generating patient-specific stem cells for disease modeling and for developing novel therapies. It has also been confirmed that iPSCs differentiate into germ cells. We compared the differentiation ability of germ cells and the gene expression level of germ cell-related genes in the X chromosome deletion region of iPSC lines derived from POI patients (n = 2) with an iPSC line derived from normal fibroblasts (n = 1). We established three iPSC lines from two patients with partial Xq deletion-induced POI and normal fibroblasts by overexpressing four factors: octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), Nanog homeobox (NANOG), and lin-28 homolog (LIN28), using lentiviral vectors. We then generated stable-transfected fluorescent reporter cell lines under the control of the Asp-Glu-Ala-Asp box polypeptide 4 (DDX4, also called VASA) promoter, and selected clonal derived sublines. We induced subline differentiation into germ cells by adding Wnt3a (30 ng/ml) and bone morphogenetic protein 4 (100 ng/ml). After 12 days of differentiation, green fluorescent protein (GFP)-positive and GFP-negative cells were isolated via fluorescence-activated cell sorting and analyzed for endogenous VASA protein (immunostaining) and for germ cell markers and genes expressed in the deleted region of the X chromosome (quantitative RT-PCR). The POI- and normal fibroblast-derived iPSCs had typical self-renewal and pluripotency characteristics. After stable transfection with the VASA-GFP construct, the sublines POI1-iPS-V.1, POI2-iPS-V.1 and hEF-iPS-V.1 produced green fluorescent cells in the differentiated cultures, and the percentage of GFP-positive cells increased over the 12 days of differentiation to a maximum of 6.9 ± 0.33%, 5.3 ± 0.57% and 8.5 ± 0.29%, respectively, of the total cell population. Immunohistochemical analysis confirmed that endogenous VASA was enriched in the GFP-positive cells. Quantitative reverse transcription-PCR revealed significantly higher expression of germ cell markers [PR domain containing 1, with ZNF domain (PRDM1, BLIMP1), developmental pluripotency-associated 3 (DPPA3, STELLA), deleted in azoospermia-like (DAZL), and VASA (DDX4)] in GFP-positive cells than in GFP-negative cells. Moreover, the GFP-positive cells from POI-iPSCs had reduced expression of the family with sequence similarity 122C (FAM122C), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), and RNA binding motif protein, X-linked (RBMX), genes located in the deleted region of the X chromosome and that are highly expressed in differentiated germ cells, compared with cells from normal iPSCs. Gene expression profiling indicated that the germ cells differentiated from POI-iPSCs were pre-meiotic. Therefore, how the differentiated primordial germ cells could progress further to meiosis and form follicles remains to be determined in the study of POI. Our results might provide an in vitro model for studying germ cell development in patients with POI. This work was supported by grants from the Major State Basic Research Development Program of China (No. 2012CB944901), the National Science Foundation of China (No. 81222007 and 81471432), the Program for New Century Excellent Talents in University and the Fundamental Research Funds for Central Universities (No. 721500003). The authors have no competing interests to declare. Not applicable.

Differential expression of pluripotent and germ cell markers in ovarian surface epithelium according to age in female mice.

BackgroundMany studies have proposed that putative ovarian stem cells (OSCs) derived from the ovarian surface epithelium (OSE) layer of adult mammalian ovaries can produce oocytes. Few studies have reported that ovaries of aged mammalian females including mice and women possess rare premeiotic germ cells that can generate oocytes. However, no studies have reported the changes of OSCs according to the age of the female. Therefore, this study evaluated pluripotent and germ cell marker expression in the intact ovary, scraped OSE, and postcultured OSE according to age in female mice.MethodsC57BL/6 female mice of 2 age groups (6–8 and 28–31 weeks) were superovulated by injection with 5 IU equine chorionic gonadotropin (eCG). Both ovaries were removed after 48 hours and scrapped to obtain OSE. Gene expressions of pluripotent (Oct-4, Sox-2, Nanog) and germ cell markers (c-Kit, GDF-9, and VASA) were evaluated by RT-PCR. VASA and GDF-9 were immune-localized in oocyte-like structures.ResultsExpressions of germ cell markers in the intact ovary were significantly decreased in aged females, whereas expressions of pluripotent markers were not detected, regardless of age. Scraped OSE expression of all pluripotent and germ cell markers, except for c-Kit, was similar between both age groups. Three weeks postcultured OSE had significantly decreased expression of GDF-9 and VASA , but not c-Kit, in old mice, as compared to young mice; however there was no difference in the expression of other genes. The number of positively stained Oct-4 by immunohistochemistry in postcultured OSE was 2.5 times higher in young mice than aged mice. Oocyte-like structure was spontaneously produced in postcultured OSE. However, while that of young mice revealed a prominent nucleus, zona pellucida-like structure and cytoplasmic organelles, these features were not observed in old mice.ConclusionsThese results show that aged female mice have putative OSCs in OSE, but their differentiation potential, as well as the number of OSCs differs from those of young mice.

Separation of somatic and germ cells is required to establish primate spermatogonial cultures.

Can primate spermatogonial cultures be optimized by application of separation steps and well defined culture conditions? We identified the cell fraction which provides the best source for primate spermatogonia when prolonged culture is desired. Man and marmoset show similar characteristics in regard to germ cell development and function. Several protocols for isolation and culture of human testis-derived germline stem cells have been described. Subsequent analysis revealed doubts on the germline origin of these cells and characterized them as mesenchymal stem cells or fibroblasts. Studies using marmosets as preclinical model confirmed that the published isolation protocols did not lead to propagation of germline cells. Testicular cells derived from nine adult marmoset monkeys (Callithrix jacchus) were cultured for 1, 3, 6 and 11 days and consecutively analyzed for the presence of spermatogonia, differentiating germ cells and testicular somatic cells. Testicular tissue of nine adult marmoset monkeys was enzymatically dissociated and subjected to two different cell culture approaches. In the first approach all cells were kept in the same dish (non-separate culture, n = 5). In the second approach the supernatant cells were transferred into a new dish 24 h after seeding and subsequently supernatant and attached cells were cultured separately (separate culture, n = 4). Real-time quantitative PCR and immunofluorescence were used to analyze the expression of reliable germ cell and somatic markers throughout the culture period. Germ cell transplantation assays and subsequent wholemount analyses were performed to functionally evaluate the colonization of spermatogonial cells. This is the first report revealing an efficient isolation and culture of putative marmoset spermatogonial stem cells with colonization ability. Our results indicate that a separation of spermatogonia from testicular somatic cells is a crucial step during cell preparation. We identified the overgrowth of more rapidly expanding somatic cells to be a major problem when establishing spermatogonial cultures. Initiating germ cell cultures from the supernatant and maintaining germ cells in suspension cultures minimized the somatic cell contamination and provided enriched germ cell fractions which displayed after 11 days of culture a significantly higher expression of germ cell markers genes (DDX-4, MAGE A-4; P < 0.05) compared with separately cultured attached cells. Additionally, germ cell transplantation experiments demonstrated a significantly higher absolute number of cells with colonization ability (P < 0.001) in supernatant cells after 11 days of separate culture. This study presents a relevant aspect for the successful setup of spermatogonial cultures but provides limited data regarding the question of whether the long-term maintenance of spermatogonia can be achieved. Transfer of these preclinical data to man may require modifications of the protocol. Spermatogonial cultures from rodents have become important and innovative tools for basic and applied research in reproductive biology and veterinary medicine. It is expected that spermatogonia-based strategies will be transformed into clinical applications for the treatment of male infertility. Our data in the marmoset monkey may be highly relevant to establish spermatogonial cultures of human testes. Funding was provided by the DFG-Research Unit FOR 1041 Germ Cell Potential (SCHL394/11-2) and by the Graduate Program Cell Dynamics and Disease (CEDAD) together with the International Max Planck Research School - Molecular Biomedicine (IMPRS-MBM). The authors declare that there is no conflict of interest. Not applicable.

BMP4 can generate primordial germ cells from bone‐marrow‐derived pluripotent stem cells

Evidence of germ cell derivation from embryonic and somatic stem cells provides an in vitro model for the study of germ cell development, associated epigenetic modification and mammalian gametogenesis. More importantly, in vitro derived gametes also represent a potential strategy for treating infertility. In mammals, male and female gametes, oocyte and sperm, are derived from a specific cell population, PGCs (primordial germ cells) that segregate early in embryogenesis. We have isolated pluripotent SSEA-1+ (stage-specific embryonic antigen-1+) cells from mice bone marrow using a MACS (magnetic-activated cell sorting) system. SSEA-1+ cells were directly separated from the suspension of MMCs (murine mononuclear cells) harvested from bone marrow of 2-4-week-old mice. Flow-cytometry assay immediately after sorting and culturing under undifferentiated condition showed 55±7% and 87±4% purity respectively. RT-PCR (reverse transcription-PCR) analysis after differentiation of SSEA-1+ cells into derivations of three germ layers showed the pluripotency properties of isolated cells. SSEA-1+ cells were induced to differentiate along germ cell lineage by adding BMP4 (bone morphogenic factor-4) to the medium. Regarding the expression of germ cell markers (PGCs, male and female germ cell lineage), it was found that adding exogenous BMP4 to culture medium could differentiate pluripotent SSEA-1+ cells isolated from an adult tissue into gamete precursors, PGCs. Differentiated cells expressed specific molecular markers of PGCs, including Oct4, fragilis, Stella and Mvh (mouse vasa homologue). Therefore BMP4 is insufficient to induce SSEA-1+ cells derived from PGCs to develop further into late germ cells in vitro.

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells In-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.

GATA4 regulates Sertoli cell function and fertility in adult male mice

Transcription factor GATA4 is expressed in Sertoli and Leydig cells and is required for proper development of the murine fetal testis. The role of GATA4 in adult testicular function, however, has remained unclear due to prenatal lethality of mice harboring homozygous mutations in Gata4. To characterize the function of GATA4 in the adult testis, we generated mice in which Gata4 was conditionally deleted in Sertoli cells using Cre–LoxP recombination with Amhr2-Cre. Conditional knockout (cKO) mice developed age-dependent testicular atrophy and loss of fertility, which coincided with decreases in the quantity and motility of sperm. Histological analysis demonstrated Sertoli cell vacuolation, impaired spermatogenesis, and increased permeability of the blood–testis barrier. RT-PCR analysis of cKO testes showed decreased expression of germ cell markers and increased expression of testicular injury markers. Our findings support the premise that GATA4 is a key transcriptional regulator of Sertoli cell function in adult mice.

Establishment and characterization of human germ cells derived from embryonic stem cells (hESCs)

OBJECTIVE: The ability to generate germ cells from hESCs represents a potential treatment for patients with infertility as well as an essential means of studying the mechanism of germ cell differentiation. The aim of this study is to investigate the feasibility of efficient production of human primordial germ cells (PGCs) from hESCs.DESIGN: In vitro cell culture.MATERIALS AND METHODS: H9 hESCs were co-cultured with MEFs in an optimized media. Cell colonies were stained with germ cell specific antibodies against SSEA-1 and cKit to evaluate for the presence of PGCs by FACS analysis. Populations of SSEA1+/cKit+, and SSEA1-/cKit- cells were sorted and analyzed for mRNA and protein expression of germ cell markers as well as embryonic germ cell (EGC) forming potential.RESULTS: High numbers of SSEA1+/cKit+ PGCs (5-10%) were generated from hESCs after differentiation. Expression of early, migratory, and matured germ cell markers of Stella, Blimp1, Nanog, Dazl, ZP1, ZP3, Acrosin and AMH was detected by qPCR. Protein expression of the pluripotent marker OCT-4 and germline marker VASA was detected by microscopy. Entry into early meiosis was suggested by the detection of of Stra8, Scp1, and Scp3. Microscopy demonstrated nuclear expression of SCP3 in 29% of PGCs. Germ cell and meiotic markers were absent in the SSEA1-/cKit- population. Only SSEA+/cKit+ cells were able to form EGC colonies in vitro. Pluripotential of EGCs was validated by the formation of mature teratomas containing all 3 germ layers in nude mice. PGCs were derived by differentiation of EGCs (∼10%) in vitro after >10 passages at similar efficiency. cKit+/SSEA1+ cells demonstrated imprint erasure characteristic of PGCs in vivo as confirmed by bisulfite sequencing.CONCLUSION: These studies demonstrate that PGCs could be efficiently and consistently generated by differentiation of hESCs without the introduction of reporter genes. The ability to produce PGCs and EGCs is unique and represents an ideal model to study human germ cell biology. OBJECTIVE: The ability to generate germ cells from hESCs represents a potential treatment for patients with infertility as well as an essential means of studying the mechanism of germ cell differentiation. The aim of this study is to investigate the feasibility of efficient production of human primordial germ cells (PGCs) from hESCs. DESIGN: In vitro cell culture. MATERIALS AND METHODS: H9 hESCs were co-cultured with MEFs in an optimized media. Cell colonies were stained with germ cell specific antibodies against SSEA-1 and cKit to evaluate for the presence of PGCs by FACS analysis. Populations of SSEA1+/cKit+, and SSEA1-/cKit- cells were sorted and analyzed for mRNA and protein expression of germ cell markers as well as embryonic germ cell (EGC) forming potential. RESULTS: High numbers of SSEA1+/cKit+ PGCs (5-10%) were generated from hESCs after differentiation. Expression of early, migratory, and matured germ cell markers of Stella, Blimp1, Nanog, Dazl, ZP1, ZP3, Acrosin and AMH was detected by qPCR. Protein expression of the pluripotent marker OCT-4 and germline marker VASA was detected by microscopy. Entry into early meiosis was suggested by the detection of of Stra8, Scp1, and Scp3. Microscopy demonstrated nuclear expression of SCP3 in 29% of PGCs. Germ cell and meiotic markers were absent in the SSEA1-/cKit- population. Only SSEA+/cKit+ cells were able to form EGC colonies in vitro. Pluripotential of EGCs was validated by the formation of mature teratomas containing all 3 germ layers in nude mice. PGCs were derived by differentiation of EGCs (∼10%) in vitro after >10 passages at similar efficiency. cKit+/SSEA1+ cells demonstrated imprint erasure characteristic of PGCs in vivo as confirmed by bisulfite sequencing. CONCLUSION: These studies demonstrate that PGCs could be efficiently and consistently generated by differentiation of hESCs without the introduction of reporter genes. The ability to produce PGCs and EGCs is unique and represents an ideal model to study human germ cell biology.

Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder-free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round-shaped cells resembling immature oocytes. Mvh(+) cells formed clumps by co-aggregation with differentiation-supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency.

Dazl Promotes Germ Cell Differentiation from Embryonic Stem Cells

It has been demonstrated that through the formation of embryoid bodies (EBs) germ cells can be derived from embryonic stem (ES) cells. Here, we describe a transgene expression approach to derive germ cells directly from ES cells in vitro without EB formation. Through the ectopic expression of Deleted in Azoospermia-Like (Dazl), a germ cell-specific RNA-binding protein, both motile tailed-sperm and oocytes were induced from mouse ES (mES) cells in culture. Furthermore, transient overexpression of Dazl led to suppression of Nanog but induced germ cell nuclear antigen in mES cells. Dazl knockdown resulted in reduction in the expression of germ cell markers including Stella, MVH and Prdm1. Our study indicates that Dazl is a master gene controlling germ cell differentiation and that ectopic expression of Dazl promotes the dynamic differentiation of mouse ES cells into gametes in vitro.