Germline Stem Cell Population Research Articles

Germ cells of the mammalian female: A limited or renewable resource?†.

In many non-mammalian organisms, a population of germ-line stem cells supports continuing production of gametes during post-natal life, and germ-line stem cells are also present and functional in male mammals. Traditionally, however, they have been thought not to exist in female mammals, who instead generate all their germ cells during fetal life. Over the last several years, this dogma has been challenged by several reports, while being supported by others. We describe and compare these conflicting studies with the aim of understanding how they came to opposing conclusions. We first consider studies that, by examining marker-gene expression, the fate of genetically marked cells, and consequences of depleting the oocyte population, addressed whether ovaries of post-natal females contain oogonial stem cells that give rise to new oocytes. We next discuss whether ovaries contain cells that, even if inactive under physiological conditions, nonetheless possess oogonial stem cell properties that can be revealed through cell culture. We then examine studies of whether cells harvested after long-term culture of cells obtained from ovaries can, following transplantation into ovaries of recipient females, give rise to oocytes and offspring. Finally, we note studies where somatic cells have been re-programmed to acquire a female germ-cell fate. We conclude that the weight of evidence strongly supports the traditional interpretation that germ-line stem cells do not exist post-natally in female mammals. However, the ability to generate germ cells from somatic cells in vitro establishes a method to generate new gametes from cells of post-natal mammalian females.

Autologous activated platelet-rich plasma injection into adult human ovary tissue: molecular mechanism, analysis, and discussion of reproductive response.

In clinical infertility practice, one intractable problem is low (or absent) ovarian reserve which in turn reflects the natural oocyte depletion associated with advancing maternal age. The number of available eggs has been generally thought to be finite and strictly limited, an entrenched and largely unchallenged tenet dating back more than 50 years. In the past decade, it has been suggested that renewable ovarian germline stem cells (GSCs) exist in adults, and that such cells may be utilized as an oocyte source for women seeking to extend fertility. Currently, the issue of whether mammalian females possess such a population of renewable GSCs remains unsettled. The topic is complex and even agreement on a definitive approach to verify the process of ‘ovarian rescue’ or ‘re-potentiation’ has been elusive. Similarities have been noted between wound healing and ovarian tissue repair following capsule rupture at ovulation. In addition, molecular signaling events which might be necessary to reverse the effects of reproductive ageing seem congruent with changes occurring in tissue injury responses elsewhere. Recently, clinical experience with such a technique based on autologous activated platelet-rich plasma (PRP) treatment of the adult human ovary has been reported. This review summarizes the present state of understanding of the interaction of platelet-derived growth factors with adult ovarian tissue, and the outcome of human reproductive potential following PRP treatment.

Identification of Germ-Line Stem Cells in Zebrafish.

Both male and female zebrafish have a population of germ-line stem cells that produce gametes throughout the life of the fish. These cells localize to specific regions in the gonads and can be identified because they uniquely express the nanos2 gene, which encodes a conserved regulator of translation. A method is presented here for identifying germ-line stem cells in the ovary and testis using a combined protocol for whole-mount fluorescent RNA in situ hybridization to detect nanos2 mRNA and immunofluorescence to detect the pan-germ cell marker Vasa.

Characterization and cardiac differentiation of chicken spermatogonial stem cells

Spermatogonial stem cells (SSCs), a unique population of germline stem cells in adult testis, have the capability to self-renew and produce daughter cells destined to differentiate into spermatozoa throughout the life of the bird. Chicken SSCs were successfully isolated from testicular cells and subsequent analysis was performed to identify pluripotent cells by investigation with cytochemical reagents including Periodic acid-Schiff (PAS), alkaline phosphatase (AP), and antibodies to germline cell specific (DAZL or VASA) and stage-specific embryonic antigens (Oct4, SSEA1, SSEA3, SSEA4, TRA-1-60, and TRA-1-81). Results confirmed these as germline cells with the expression of DAZL (Deleted in Azoospermia-Like) and VASA genes in isolated cells. Immunochemistry results showed that multipotent germline stem cells (mGSCs) expressed these gene markers related to embryonic stem cells (ESCs) and could spontaneously differentiate into three embryonic germ (EG) layers in vitro. The mGSC-derived cardiomyocytes expressed cardiac-specific markers such as sarcomeric alpha actinin, alpha-cardiac actinin; conexin-43, the major protein of gap junctions which are thought to have an important role in the synchronized contraction of the heart and in embryonic development; and cardiac troponin T, the tropomyosin binding subunit of the troponin complex which regulates muscle contraction. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) results indicated that the genes related to cardiac transcription factors were expressed following differentiation. Results of the present study strongly contribute to the information related to the ability of chicken mGSCs to differentiate into cells such as contraction cardiomyocytes similar to ESCs and may provide a new source of cardiomyocytes for basic research and potential therapeutic application in various cardiac degenerative diseases of birds and other animals.

Functional and molecular features of the ID4+ germline stem cell population in mouse testes

Functional and molecular features of the ID4+ germline stem cell population in mouse testes

Functional and molecular features of the Id4+ germline stem cell population in mouse testes.

The maintenance of cycling cell lineages relies on undifferentiated subpopulations consisting of stem and progenitor pools. Features that delineate these cell types are undefined for many lineages, including spermatogenesis, which is supported by an undifferentiated spermatogonial population. Here, we generated a transgenic mouse line in which spermatogonial stem cells are marked by expression of an inhibitor of differentiation 4 (Id4)-green fluorescent protein (Gfp) transgene. We found that Id4-Gfp(+) cells exist primarily as a subset of the type A(single) pool, and their frequency is greatest in neonatal development and then decreases in proportion during establishment of the spermatogenic lineage, eventually comprising ∼ 2% of the undifferentiated spermatogonial population in adulthood. RNA sequencing analysis revealed that expression of 11 and 25 genes is unique for the Id4-Gfp(+)/stem cell and Id4-Gfp(-)/progenitor fractions, respectively. Collectively, these findings provide the first definitive evidence that stem cells exist as a rare subset of the A(single) pool and reveal transcriptome features distinguishing stem cell and progenitor states within the mammalian male germline.

Nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary

Female zebrafish have a prolific reproductive capacity, suggesting that a germline stem cell (GSC) population drives oocyte production. However, a zebrafish female GSC population has yet to be identified. Adult stem cells are defined by their ability to both self-renew and differentiate, and by their localization to a stem cell niche. We show here that mitotic and early meiotic germ cells are present in the adult ovary and that the zebrafish homolog of the conserved vertebrate GSC marker, nanos2, is expressed in a subset of pre-meiotic oogonia in the adult gonad. We propose that these nanos2+ cells are GSCs. Importantly, we find that mitotic, nanos2+, and early meiotic germ cells localize to the germinal zone, thus identifying this region as the probable ovarian GSC niche in zebrafish. nanos3, which encodes a conserved RNA-binding protein, is known to be required for the continued production of oocytes in the zebrafish. Although mammalian homologs of nanos3 are expressed in early spermatogonia, no study has defined the role of nanos3 in the regulation of vertebrate GSCs. Here we demonstrate that nanos3 function is required for the maintenance of GSCs, but not for their specification, and propose that nanos2 and nanos3 are partially redundant in this role.

Of Mice and (wo)Men: Purified Oogonial Stem Cells from Mouse and Human Ovaries

When it comes to reproducing, James Brown summed up the human experience: This is a man’s world. Women produce a limited number of viable eggs (probably considerably fewer than 400) and become reproductively senescent in the forth or fifth decade of life. In contrast, men produce millions of sperm per day, with little evidence of age-related decline, and can father offspring into their seventh decade and beyond—if they can find willing mates. Universal agreement exists that a germline stem cell population in the testis makes continuous sperm production possible. The existence of a corresponding stem germ cell population in the ovary, however, has been the subject of intense debate. The latest chapter in this debate was recently published online in Nature Medicine [1]. What makes this paper especially noteworthy is that it provides the first evidence that isolation techniques used to obtain putative oogonial stem cells (OSCs) from the mouse ovary can be used to isolate similar cells from human ovaries. The findings are the result of collaborative studies between investigators at Harvard and at Saitama Medical Center in Japan. The Harvard group includes Jonathan Tilly, whose laboratory challenged the ‘‘central dogma’’ of reproductive biology—that all eggs produced by the mammalian ovary initiate oogenesis during fetal development—with a paper in 2004 reporting OSCs in the mouse ovary [2]. In males, spermatogonial stem cells (SSCs) at the basement membrane of the seminiferous tubules divide to replenish their numbers and give rise to progenitor spermatogonia that provide the steady supply of new spermatocytes required for continuous sperm production. The SSC population is thought to be small in number, making up 0.03% of the total testicular cell population in adult mice [3]. Techniques for the isolation and in vitro expansion of SSCs have been developed, and the litmus test of the putative SSC cells obtained—their capacity to regenerate continual sperm production following transplantation into a recipient testis—has been firmly established [4, 5]. Despite a flurry of research, the existence of the female germline stem cell counterpart—OSCs—has remained controversial. White et al. [1] expand our understanding of OSCs with the demonstration that a population of mitotically active cells can be isolated from both mouse and human ovarian tissue by cell sorting using an antibody that recognizes the Cterminal portion of DDX4, a germ cell-specific RNA helicase. The isolated cells express genes known to be restricted to germ cells, and like male SSCs, these appear to be a rare type (an estimated 0.014% of the ovarian cell population). Interestingly, in contrast to the male, where isolated SSCs form colonies of actively dividing cells after several days in culture [6–9], from 10 to 12 wk (mouse) and from 4 to 8 wk (human) of culture were required to obtain actively dividing OSC colonies. This smacks of in vitro transformation, raising concern about whether the population of cells that exists in the ovary are

Cytoneme-Mediated Delivery of Hedgehog Regulates the Expression of Bone Morphogenetic Proteins to Maintain Germline Stem Cells in Drosophila

Author SummaryThe Drosophila ovary contains a well-defined stem cell niche that hosts 2–3 germline stem cells (GSCs). The Hedgehog (Hh) family of signalling proteins mediates cellular homeostasis in several adult tissues, and here we decipher the detailed mechanism of action of Hh in the adult female GSC niche. We demonstrate that Hh acts in a juxtacrine manner (i.e., it requires physical contact between the cells involved) to maintain the normal pool of GSCs in the ovarian niche. Hh is produced in one type of niche support cell (the cap cells), and it is received, upon secretion, by a second, neighbouring population of niche cells (the escort cells). In the latter, we show that the Hh signalling pathway regulates the expression of the Drosophila Bone Morphogenetic Protein (BMP) homologues and essential stem cell factors decapentaplegic (dpp) and glass bottom boat (gbb). We also find that Hh distribution in the GSC niche is mediated by short cellular projections, reminiscent of wing disc cytonemes, although they grow from the (Hh) signal-producing cells towards the receiving cells. Under conditions of low levels of Hh protein and/or Hh signalling within the niche, cap cells emit up to 6-fold longer Hh-decorated cytonemes towards the signalling-deficient area of the niche. Our data reveal that stem cell niches are dynamic structures that can sense, and react to, changes in the activity of essential stem cell factors to prevent stem cell differentiation.

The quest for human ovarian stem cells

Researchers have isolated a rare population of germline stem cells from adult mouse and human ovaries that are capable of forming oocytes. The ability to harvest such cells from human ovaries could change the options available for fertility preservation and the treatment of infertility (pages 413–421).

Neo-oogenesis: Has its existence been proven?

Neo-oogenesis: Has its existence been proven?

The C. elegans adult male germline: Stem cells and sexual dimorphism

The hermaphrodite Caenorhabditis elegans germline has become a classic model for stem cell regulation, but the male C. elegans germline has been largely neglected. This work provides a cellular analysis of the adult C. elegans male germline, focusing on its predicted stem cell region in the distal gonad. The goals of this study were two-fold: to establish the C. elegans male germline as a stem cell model and to identify sex-specific traits of potential relevance to the sperm/oocyte decision. Our results support two major conclusions. First, adult males do indeed possess a population of germline stem cells (GSCs) with properties similar to those of hermaphrodite GSCs (lack of cell cycle quiescence and lack of reproducibly oriented divisions). Second, germ cells in the mitotic region, including those most distal within the niche, exhibit sex-specific behaviors (e.g. cell cycle length) and therefore have acquired sexual identity. Previous studies demonstrated that some germ cells are not committed to a sperm or oocyte cell fate, even in adults. We propose that germ cells can acquire sexual identity without being committed to a sperm or oocyte cell fate.

Evolution and spermatogenesis

Sexual reproduction depends on the production of haploid gametes, and their fusion to form diploid zygotes. Here, we discuss sperm production and function in a molecular and functional evolutionary context, drawing predominantly from studies in model organisms (mice, Drosophila, Caenorhabditis elegans). We consider the mechanisms involved in establishing and maintaining a germline stem cell population in testes, as well as the factors that regulate their contribution to the pool of differentiating cells. These processes involve considerable interaction between the germline and the soma, and we focus on regulatory signalling events in a variety of organisms. The male germline has a unique transcriptional profile, including expression of many testis-specific genes. The evolutionary pressures associated with gene duplication and acquisition of testis function are discussed in the context of genome organization and transcriptional regulation. Post-meiotic differentiation of spermatids involves very dramatic changes in cell shape and acquisition of highly specialized features. We discuss the variety of sperm motility mechanisms and how various reproductive strategies are associated with the diversity of sperm forms found in animals.

Insulin signaling promotes germline proliferation in C. elegans

Cell proliferation must be coordinated with cell fate specification during development, yet interactions among pathways that control these two critical aspects of development are not well understood. The coordination of cell fate specification and proliferation is particularly crucial during early germline development, when it impacts the establishment of stem/progenitor cell populations and ultimately the production of gametes. In C. elegans, insulin/IGF-like receptor (IIR) signaling has been implicated in fertility, but the basis for the fertility defect had not been previously characterized. We found that IIR signaling is required for robust larval germline proliferation, separate from its well-characterized role in preventing dauer entry. IIR signaling stimulates the larval germline cell cycle. This activity is distinct from Notch signaling, occurs in a predominantly germline-autonomous manner, and responds to somatic activity of ins-3 and ins-33, genes that encode putative insulin-like ligands. IIR signaling in this role acts through the canonical PI3K pathway, inhibiting DAF-16/FOXO. However, signaling from these ligands does not inhibit daf-16 in neurons nor in the intestine, two tissues previously implicated in other IIR roles. Our data are consistent with a model in which: (1) under replete reproductive conditions, the larval germline responds to insulin signaling to ensure robust germline proliferation that builds up the germline stem cell population; and (2) distinct insulin-like ligands contribute to different phenotypes by acting on IIR signaling in different tissues.

Insulin signaling promotes germline proliferation inC. elegans

Cell proliferation must be coordinated with cell fate specification during development, yet interactions among pathways that control these two critical aspects of development are not well understood. The coordination of cell fate specification and proliferation is particularly crucial during early germline development, when it impacts the establishment of stem/progenitor cell populations and ultimately the production of gametes. In C. elegans, insulin/IGF-like receptor (IIR) signaling has been implicated in fertility, but the basis for the fertility defect had not been previously characterized. We found that IIR signaling is required for robust larval germline proliferation, separate from its well-characterized role in preventing dauer entry. IIR signaling stimulates the larval germline cell cycle. This activity is distinct from Notch signaling, occurs in a predominantly germline-autonomous manner, and responds to somatic activity of ins-3 and ins-33, genes that encode putative insulin-like ligands. IIR signaling in this role acts through the canonical PI3K pathway, inhibiting DAF-16/FOXO. However, signaling from these ligands does not inhibit daf-16 in neurons nor in the intestine, two tissues previously implicated in other IIR roles. Our data are consistent with a model in which: (1) under replete reproductive conditions, the larval germline responds to insulin signaling to ensure robust germline proliferation that builds up the germline stem cell population; and (2) distinct insulin-like ligands contribute to different phenotypes by acting on IIR signaling in different tissues.

Neural differentiation from equine inner cell mass cells in vitro

Spermatogonial stem cells (SSCs), a unique population of germline stem cells in adult testis, have the capability to self-renew and produce daughter cells destined to differentiate into spermatozoa throughout the life of the bird. Chicken SSCs were successfully isolated from testicular cells and subsequent analysis was performed to identify pluripotent cells by investigation with cytochemical reagents including Periodic acid-Schiff (PAS), alkaline phosphatase (AP), and antibodies to germline cell specific (DAZL or VASA) and stage-specific embryonic antigens (Oct4, SSEA1, SSEA3, SSEA4, TRA-1-60, and TRA-1-81). Results confirmed these as germline cells with the expression of DAZL (Deleted in Azoospermia-Like) and VASA genes in isolated cells. Immunochemistry results showed that multipotent germline stem cells (mGSCs) expressed these gene markers related to embryonic stem cells (ESCs) and could spontaneously differentiate into three embryonic germ (EG) layers in vitro. The mGSC-derived cardiomyocytes expressed cardiac-specific markers such as sarcomeric alpha actinin, alpha-cardiac actinin; conexin-43, the major protein of gap junctions which are thought to have an important role in the synchronized contraction of the heart and in embryonic development; and cardiac troponin T, the tropomyosin binding subunit of the troponin complex which regulates muscle contraction. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) results indicated that the genes related to cardiac transcription factors were expressed following differentiation. Results of the present study strongly contribute to the information related to the ability of chicken mGSCs to differentiate into cells such as contraction cardiomyocytes similar to ESCs and may provide a new source of cardiomyocytes for basic research and potential therapeutic application in various cardiac degenerative diseases of birds and other animals.

Dedifferentiating Spermatogonia Outcompete Somatic Stem Cells for Niche Occupancy in the Drosophila Testis

Differentiating cells can dedifferentiate to replace stem cells in aged or damaged tissues, but the underlying mechanisms are unknown. In the Drosophila testis, a cluster of stromal cells called the hub creates a niche by locally activating Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling in adjacent germline and somatic stem cells. Here, we establish a system to study spermatogonial dedifferentiation. Ectopically expressing the differentiation factor bag-of-marbles (Bam) removes germline stem cells from the niche. However, withdrawing ectopic Bam causes interconnected spermatogonia to fragment, move into the niche, exchange positions with resident somatic stem cells, and establish contact with the hub. Concomitantly, actin-based protrusions appear on subsets of spermatogonia, suggesting acquired motility. Furthermore, global downregulation of Jak-STAT signaling inhibits dedifferentiation, indicating that normal levels of pathway activation are required to promote movement of spermatogonia into the niche during dedifferentiation, where they outcompete somatic stem cells for niche occupancy.

MILI, a PIWI-interacting RNA-binding Protein, Is Required for Germ Line Stem Cell Self-renewal and Appears to Positively Regulate Translation

The Argonaute/PIWI protein family consists of Argonaute and PIWI subfamilies. Argonautes function in RNA interference and micro-RNA pathways; whereas PIWIs bind to PIWI-interacting RNAs and regulate germ line development, stem cell maintenance, epigenetic regulation, and transposition. However, the role of PIWIs in mammalian stem cells has not been demonstrated, and molecular mechanisms mediated by PIWIs remain elusive. Here we show that MILI, a murine PIWI protein, is expressed in the cytoplasm of testicular germ line stem cells, spermatogonia, and early spermatocytes, where it is enriched in chromatoid bodies. MILI is essential for the self-renewing division and differentiation of germ line stem cells but does not affect initial establishment of the germ line stem cell population at 7 days postpartum. Furthermore, MILI forms a stable RNA-independent complex with eIF3a and associates with the eIF4E- and eIF4G-containing m7G cap-binding complex. In isolated 7 days postpartum seminiferous tubules containing mostly germ line stem cells, the mili mutation has no effect on the cellular mRNA level yet significantly reduces the rate of protein synthesis. These observations indicate that MILI may positively regulate translation and that such regulation is required for germ line stem cell self-renewal.

Identification of Candidate Genes Involved in Avian Germline Stem Cell Migration.

In vertebrates, the germ line stem cell population arises from extraembryonic regions and must actively migrate to the gonadal anlagen. In the avian embryo, the process of germ line stem cell migration proceeds through a series of active and passive migratory phases. The germ line stem cells or primordial germ cells (PGCs) located in the epiblast of the unincubated embryo translocate to the hypoblast between stages X-XIII (E-G&K). Subsequently, during gastrulation the PGCs are passively carried by the hypoblast to the germinal crescent at about stage 4 (H&H). As blood islands develop and the embryo becomes vascularized, the PGCs are passively carried through the blood stream between stages 13-16 (H&H). Subsequently, the PGCs leave the blood vessels and actively migrate to the gonadal anlagen. Recently, it has become possible to culture chicken germ line stem cells using PGC populations harevested from embryonic blood. We have established several cultures of the germ line stem cells. To examine the ability of the cultured germ cells to migrate, an in ovo assay was developed. The PGCs were loaded with a vital fluorescent dye (PKH-26) and injected into the stage X embryos. At stage 28-30 (H&H), the gonads were removed and examined for the presence of fluorescent PGCs indicating that the injected germ cells successfully migrated. Variability in the ability of cultured PGCs to migrate was found. Therefore, we compared the global gene expression profile between a line of germ line stem cells that exhibited good migration and a line with low migration. A chicken 44K Agilent microarray was used to interrogate the expression profiles of the two cell lines. Total RNA was obtained from 4 replicate cell cultures labeled by Cy3 or Cy5 with dye swap. Gene signal intensity was globally normalized by LOWESS and expressed on natural log scale. A mixed model including the fixed effects of dye, line, and random effects of slide and array was used to identify differentially expressed genes at P < 0.05. Of the 42,035 identified genes scanned, 5013 genes showed differential expression. As expected, several germ cell specific genes were not differentially expressed including VASA and DAZL. Of the differentially expressed genes 32 showed a greater than five-fold difference between the two lines. However, one gene showed an 18 fold difference, BU455352, Gga.19961 (alpha-3-catenin). This gene produces a beta-catenin-binding protein that mediates strong cell-cell adhesion and is associated with cell adhesion and migration in other cells. The results of this study confirm that cultured avian PGCs can retain their ability to migrate to the gonadal anlagen after long-term culture and suggests that alpha-3-catenin may be associated with avian germ cell migration.

Drosophila spermatogenesis: insights into testicular cancer

Geneticists have a long history of studying reproduction in the fruitfly, Drosophila melanogaster, and in recent years it has become apparent that many of the genes that regulate invertebrate reproduction have been conserved through vertebrate evolution. As with other higher eukaryotes, spermatogenesis in Drosophila is characterized by a regenerative germline stem cell population that divides asymmetrically to produce mitotic spermatogonia which will eventually differentiate into spermatocytes. Germline tumours consisting of undifferentiated germ cells have been associated with both loss-of-function mutations and ectopic gene expression. While the genesis of these tumours may not be identical to human germ cell tumours many of the genes that regulate stem cell proliferation and aberrant over-proliferation in the Drosophila testis provide candidate molecules that may underlie the genetic programmes that contribute to human testicular oncogenesis.