Female Germline Stem Cells Research Articles

A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function.

Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila. miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.

Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells.

BackgroundGermline stem cells play an essential role in establishing the fertility of an organism. Although extensively characterized, the regulatory mechanisms that govern the fundamental properties of mammalian female germline stem cells remain poorly understood.ResultsWe generate genome-wide profiles of the histone modifications H3K4me1, H3K27ac, H3K4me3, and H3K27me3, DNA methylation, and RNA polymerase II occupancy and perform transcriptome analysis in mouse female germline stem cells. Comparison of enhancer regions between embryonic stem cells and female germline stem cells identifies the lineage-specific enhancers involved in germline stem cell features. Additionally, our results indicate that DNA methylation primarily contributes to female germline stem cell unipotency by suppressing the somatic program and is potentially involved in maintenance of sexual identity when compared with male germline stem cells. Moreover, we demonstrate down-regulation of Prmt5 triggers differentiation and thus uncover a role for Prmt5 in maintaining the undifferentiated status of female germline stem cells.ConclusionsThe genome-wide epigenetic signatures and the transcription regulators identified here provide an invaluable resource for understanding the fundamental features of mouse female germline stem cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1023-z) contains supplementary material, which is available to authorized users.

Human GV oocytes generated by mitotically active germ cells obtained from follicular aspirates.

Human female germline stem cells (FGSCs) have opened new opportunities for understanding human oogenesis, delaying menopause, treating infertility, and providing a new strategy for preserving fertility. However, the shortage of adult human ovaries tissues available impedes their future investigations and clinical applications. Here, we have established FGSC lines from scarce ovarian cortical tissues that exist in follicular aspirates (faFGSCs), which are produced and discarded in in vitro fertilization centers worldwide. The faFGSCs have characteristics of germline stem cells involved in the gene expression profile, growth characteristics, and a normal karyotype consistent with that of FGSCs obtained from ovarian cortexes surgically removed from patients (srFGSCs). Furthermore, faFGSCs have developmental potentials including spontaneous differentiation into oocytes under feeder-free conditions, communicating with granulosa cells by gap junctions and paracrine factors, entering meiosis after RA induction, as well as forming follicles after injection into human ovarian cortical tissues xenografted into adult immunodeficient female mice. Lastly, we developed a strategy guiding FGSCs differentiated into germinal vesicle (GV) stage oocytes in vitro and revealed their developmental mechanisms. Our study not only provides a new approach to obtain human FGSCs for medical treatment, but also opens several avenues to investigate human oogenesis in vitro.

Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila.

Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating.

Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin

The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. Here we present our studies of one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). We demonstrate that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. We show germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, we investigate the requirement of Ote in the male fertility. We show that ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, our studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, our data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis.

Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms.

Few animals are known to lay eggs in the absence of ovulation or copulation, as it is presumably energetically wasteful and subjected to negative selection. Characterization of Smed-boule, a member of the DAZ family of germline RNA-binding proteins, revealed that egg capsule (or capsule) production and deposition occurs independently of the presence of gametes in the planarian flatworm Schmidtea mediterranea. Reduction of Smed-boule expression by RNA-interference (RNAi) causes ablation of spermatogonial stem cells and the inability of ovarian germline stem cells to undergo oogenesis. Although animals subjected to Smed-boule RNAi lose their gametes and become sterile, they continue to lay egg capsules. Production of sterile capsules is even observed in virgin Smed-boule(RNAi) and control planarians maintained in complete isolation, demonstrating that egg production in S. mediterranea occurs independently of ovulation, fertilization, or mating. Evidence suggests that this is a conserved feature amongst Platyhelminthes, and therefore relevant to the pathology and dissemination of parasitic flatworms. These findings demonstrate that Smed-boule functions at different stages during male and female germline stem cell development, and also demonstrate that egg capsule production by planarian flatworms occurs independently of signals produced by mating or ova.

Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice.

We investigated whether DEAD-box polypeptide 4 (DDX4) positive cells from post-natal ovaries of germline lineage reporter mice can be isolated based on endogenously expressed fluorescent proteins and used to establish a cell line for producing offspring. DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be isolated and used to establish a cell line for producing offspring. In recent years, female germline stem cells (FGSCs) have been isolated from the ovaries of post-natal mice by magnetic-activated cell sorting or fluorescence-activated cell sorting (FACS) relying on an antibody against DDX4. However, whether DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be established without using an antibody, as well as a cell line established for producing offspring, remains unknown. To obtain the expected offspring (Ddx4-Cre;mT/mG mice), Ddx4-Cre mice were crossed with mT/mG mice. In the ovaries of Ddx4-Cre;mT/mG mice, germ cells were destined to express enhanced green fluorescent protein (EGFP) while somatic cells still express tandem dimer Tomato (tdTomato). Therefore, the germ cells could be clearly distinguished from somatic cells by fluorescent proteins. Then, we investigated the pattern of fluorescent cells in the ovaries of 21-day-old Ddx4-Cre;mT/mG mice under a fluorescent microscope. Germ cells were sorted by FACS without using antibody and used to establish a FGSC line. The FGSC line was analyzed by DDX4 immunostaining, Edu (5-ethynyl-2'-deoxyuridine) labeling, and RT-PCR for germ cell markers. Finally, the physiological function of the FGSC line was examined by transplanting FGSCs into the ovaries of sterilized recipients and subsequent mating. Firstly, we have successfully isolated FGSCs from the ovaries of 21-day-old Ddx4-Cre;mT/mG mice based on endogenously expressed fluorescent proteins. FACS was used to separate the cells and 2.3% of all viable cells was EGFP-positive germ cells. Subsequently, a FGSC line was established that was doubly positive for DDX4 immunostaining and Edu labeling. The mRNA expression of several germ cell markers in this cell line, such as Ddx4, Deleted in azoospermia-like (Dazl), B lymphocyte-induced maturation protein-1 (Blimp1), Stella and Fragilis, was detected. Lastly, the FGSC line was proven to be functional under physiological conditions, as offspring were produced after transplanting FGSCs into ovaries of sterilized recipients and a subsequent mating. The molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro still need to be elucidated. Our results confirm that DDX4-positive cells can be separated from post-natal mouse ovaries and used to establish cell lines that are functional in producing offspring, and provide further evidence for the existence of post-natal FGSCs in mammals. The Ddx4-Cre;mT/mG mouse strain is an ideal model for the isolation, characterization and propagation of FGSCs and is a useful tool for fully elucidating the molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro. none. This work was supported by National Basic Research Program of China (2013CB967401) and the National Nature Science Foundation of China (81370675, 81200472 and 81421061). The authors declare no competing interests.

Quantitative Evaluation of Primordial Follicles in Rat Ovaries during the Early and Delayed Terms after Different Cytostatic Exposures.

Experiments on female Wistar rats showed that cytostatic agents (farmorubicin, platidiam, carboplatin, and etoposide) induce an initial significant decrease in the number of primordial follicles. Over the next 2-3 estrous cycles after administration of farmorubicin, platidiam, and carboplatin, this index practically did not differ from the control. The number of primordial follicles in the third and fourth estrous cycles after farmorubicin administration, as well as in the second and sixth estrous cycles after etoposide administration was much higher than the follicular reserve after cytostatic treatment (first estrous cycle). The ovarian reserve was exhausted in the delayed period after the start of the experiment. This dynamics of the pool of primordial follicles suggests that the ovary of rats in the postnatal period of life contains a limited number of female germline stem cells.

Stem Cells in Mammalian Gonads.

Stem cells have great value in clinical application because of their ability to self-renew and their potential to differentiate into many different cell types. Mammalian gonads, including testes for males and ovaries for females, are composed of germline and somatic cells. In male mammals, spermatogonial stem cells maintain spermatogenesis which occurs continuously in adult testis. Likewise, a growing body of evidence demonstrated that female germline stem cells could be found in mammalian ovaries. Meanwhile, prior studies have shown that somatic stem cells exist in both testes and ovaries. In this chapter, we focus on mammalian gonad stem cells and discuss their characteristics as well as differentiation potentials.

Isolation of Mammalian Oogonial Stem Cells by Antibody-Based Fluorescence-Activated Cell Sorting.

The ability to isolate and subsequently culture mitotically active female germ cells from adult ovaries, referred to as either oogonial stem cells (OSCs) or adult female germline stem cells (aFGSCs), has provided a robust system to study female germ cell development under multiple experimental conditions, and in many species. Flow cytometry or fluorescence-activated cell sorting (FACS) is an integral part of many isolation and characterization protocols. Here, we provide methodological details for antibody-based flow cytometric isolation of OSCs using antibodies specific for external epitopes of the proteins Ddx4 or Ifitm3, alone or in combination with the use of fluorescent reporter mice. Beginning with sample preparation, we provide point-by-point instructions to guide researchers on how to isolate OSCs using flow cytometry.

The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals.

The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years.

Improvement in Isolation and Identification of Mouse Oogonial Stem Cells.

Female germline stem cells (FGSCs) or oogonial stem cells (OSCs) have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary results in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days and then were purified by magnetic-activated cell sorting (MACS). The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were also used as feeder cells to support the proliferation of OSCs. The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feeder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse.

Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries.

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.

Detection and characterization of female germline stem cells in menopausal/perimenopausal women and related clinical analysis

To investigate the existence of human germline stem cells in menopausal/perimenopausal women, and to explore the relating characteristics of patients and specimen that may predict the success of subsequent cell culture.

Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche.

Stem cell self-renewal versus differentiation is regulated by the niche, which provides localized molecules that favor self-renewal. In the Drosophila melanogaster female germline stem cell (GSC) niche, Decapentaplegic (Dpp), a fly transforming growth factor β molecule and well-established long-range morphogen, acts over one cell diameter to maintain the GSCs. Here, we show that Thickveins (Tkv; a type I receptor of Dpp) is highly expressed in stromal cells next to Dpp-producing cells and functions to remove excess Dpp outside the niche, thereby spatially restricting its activity. Interestingly, Tkv expression in these stromal cells is regulated by multiple Wnt ligands that are produced by the niche. Our data demonstrate a self-restraining mechanism by which the Drosophila ovarian GSC niche acts to define its own boundary.

Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila.

The intestinal epithelium is the most rapidly self-renewingtissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed agenome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC) lineage differentiation, and ISC-to-enteroendocrine (EE) lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo.

Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.

Isolation and enrichment of mouse female germ line stem cells.

The existence of female germ-line stem cells (FGSCs) has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS) and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH) and stage-specific embryonic antigen-1 (SSEA1) markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR) (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3), alkaline phosphatase (AP) activity test and immunocytochemistry. Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 ± 0.49% (Mean ± SDV) positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl) whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries.

Insulin-independent role of adiponectin receptor signaling in Drosophila germline stem cell maintenance

Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate.

An enhanced gene targeting toolkit for Drosophila: Golic+.

Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer "genes" in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report G: ene targeting during O: ogenesis with L: ethality I: nhibitor and C: RISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.