Identification of oogonial stem cells in chicken ovary.

OBJECTIVES: Oogonial stem cells (OSCs) are germ cells that can sustain neo-oogenesis to replenish the pool of primary follicles in adult ovaries. In lower vertebrates, fresh oocytes are produced by numerous OSCs through mitosis and meiosis during each reproduction cycle, but the OSCs in adult mammals are rare. The birds have retained many conserved features and developed unique features of ovarian physiology during evolution, and the presence of OSCs within avian species remain unknown. MATERIALS AND METHODS: In this study, we investigated the existence and function of OSCs in adult chickens. The chicken OSCs were isolated and expanded in culture. We then used cell transplantation system to evaluate their potential for migration and differentiation in vivo. RESULTS: DDX4/SSEA1-positive OSCs were identified in both the cortex and medulla of the adult chicken ovary. These putative OSCs undergo meiosis in the reproductively active ovary. Furthermore, the isolated OSCs were expanded in vitro for months and found to express germline markers similar to those of primordial germ cells. When transplanted into the bloodstream of recipient embryos, these OSCs efficiently migrated into developing gonads, initiated meiosis, and then derived oocytes in postnatal ovaries. CONCLUSIONS: This study has confirmed the presence of functional OSCs in birds for the first time. The identification of chicken OSCs has great potential for improving egg laying and preserving endangered species.

Regulation of adult female germline stem cells by nutrient-responsive signaling.

Insect oogenesis is greatly affected by nutrient availability. When nutrients are abundant, oocytes are rapidly generated, but the process is slowed to conserve energy under nutrient-deficient conditions. To properly allocate limited resources toward oogenesis, systemic factors coordinate the behavioral response of ovarian germline stem cells (GSCs) to nutritional inputs by acting on the GSC itself, GSC supporting cells (the niche), or the adipose tissue surrounding the ovary. In this review, we describe current knowledge of the Drosophila ovarian GSC-niche-adipocyte system and major nutrient sensing pathways (insulin/IGF signaling, TOR signaling, and GCN2-dependent amino acid sensing) that intrinsically or extrinsically regulate GSC responses to nutrient signals.

Traffic jam regulates the function of the ovarian germline stem cell progeny differentiation niche during pre-adult stage in Drosophila.

Stem cell self-renewal and the daughter cell differentiation are tightly regulated by the respective niches, which produce extrinsic cues to support the proper development. In Drosophila ovary, Dpp is secreted from germline stem cell (GSC) niche and activates the BMP signaling in GSCs for their self-renewal. Escort cells (ECs) in differentiation niche restrict Dpp outside the GSC niche and extend protrusions to help with proper differentiation of the GSC daughter cells. Here we provide evidence that loss of large Maf transcriptional factor Traffic jam (Tj) blocks GSC progeny differentiation. Spatio-temporal specific knockdown experiments indicate that Tj is required in pre-adult EC lineage for germline differentiation control. Further molecular and genetic analyses suggest that the defective germline differentiation caused by tj-depletion is partly attributed to the elevated dpp in the differentiation niche. Moreover, our study reveals that tj-depletion induces ectopic En expression outside the GSC niche, which contributes to the upregulated dpp expression in ECs as well as GSC progeny differentiation defect. Alternatively, loss of EC protrusions and decreased EC number elicited by tj-depletion may also partially contribute to the germline differentiation defect. Collectively, our findings suggest that Tj in ECs regulates germline differentiation by controlling the differentiation niche characteristics.

Molecular control of the female germline stem cell niche size in Drosophila.

Adult stem cells have a unique capacity to renew themselves and generate differentiated cells that are needed in the body. These cells are recruited and maintained by the surrounding microenvironment, known as the stem cell niche, during organ development. Thus, the stem cell niche is required for proper tissue homeostasis, and its dysregulation is associated with tumorigenesis and tissue degeneration. The identification of niche components and the mechanisms that regulate niche establishment and maintenance, however, are just beginning to be uncovered. Germline stem cells (GSCs) of the Drosophila ovary provide an excellent model for studying the stem cell niche in vivo because of their well-characterized cell biology and the availability of genetic tools. In this review, we introduce the ovarian GSC niche, and the key signaling pathways for niche precursor segregation, niche specification, and niche extracellular environment establishment and niche maintenance that are involved in regulating niche size during development and adulthood.

Ddx4+ Oogonial Stem Cells in Postmenopausal Women's Ovaries: A Controversial, Undefined Role.

Recent studies support the existence of oogonial stem cells (OSCs) in the ovarian cortex of different mammals, including women.These cells are characterized by small size, membrane expression of DEAD(Asp-Glu-Ala-Asp)-box polypeptide-4 (Ddx4), and stemness properties (such as self-renewal and clonal expansion) as well as the ability to differentiate in vitro into oocyte-like cells. However, the discovery of OSCs contrasts with the popular theory that there is a numerically defined oocyte pool for female fertility which undergoes exhaustion with menopause. Indeed, in the ovarian cortex of postmenopausal women OSCs have been detected that possess both viability and capability to differentiate into oocytes, which is similar to those observed in younger patients. The pathophysiological role of this cell population in aged women is still debated since OSCs, under appropriate stimuli, differentiate into somatic cells, and the occurrence of Ddx4+ cells in ovarian tumor samples also suggests their potential involvement in carcinogenesis. Although further investigation into these observations is needed to clarify OSC function in ovary physiology, clinical investigators and researchers studying female infertility are presently focusing on OSCs as a novel opportunity to restore ovarian reserve in both young women undergoing early ovarian failure and cancer survivors experiencing iatrogenic menopause.

Extracellular matrix signaling activates differentiation of adult ovary-derived oogonial stem cells in a species-specific manner.

OBJECTIVE: To test if ovarian microenvironmental cues affect oogonial stem cell (OSC) function in a species-specific manner. DESIGN: Animal and human study. SETTING: Research laboratory. PATIENT(S)/ANIMAL(S): Human ovarian cells obtained from cryopreserved ovarian cortical tissue of reproductive-age women, and ovarian cells and tissues from female C57BL/6 mice. INTERVENTION(S): Mouse ovarian tissue, mouse OSCs (mOSCs) and human OSCs (hOSCs) were analyzed for extracellular matrix (ECM) protein expression, and OSCs isolated from adult mouse and human ovaries were cultured in the absence or presence of ECM proteins without or with an integrin signaling inhibitor. MAIN OUTCOME MEASURE(S): Gene expression and in vitro derived (IVD) oocyte formation. RESULT(S): Culture of mOSCs on a collagen-based ECM significantly elevated the rate of differentiation of the cells into IVD oocytes. Mouse OSCs expressed many integrins, including Arg-Gly-Asp (RGD)-binding subunits, and ECM-mediated increases in mOSC differentiation were blocked by addition of integrin-antagonizing RGD peptides. In comparison, hOSCs expressed a different pattern of integrin subunits compared with mOSCs, and hOSCs were unresponsive to a collagen-based ECM; however, hOSCs exhibited increased differentiation into IVD oocytes when cultured on laminin. CONCLUSION(S): These data, along with in silico analysis of ECM protein profiles in human ovaries, indicate that ovarian ECM-based niche components function in a species-specific manner to control OSC differentiation.

Preservation of female genetic resources of common carp through oogonial stem cell manipulation.

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1 °C/min) and short-term storage (-80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.

Genetic studies in mice directly link oocytes produced during adulthood to ovarian function and natural fertility.

Multiple labs have reported that mammalian ovaries contain oogonial stem cells (OSCs), which can differentiate into oocytes that fertilize to produce offspring. However, the physiological relevance of these observations to adult ovarian function is unknown. Here we performed targeted and reversible ablation of premeiotic germ cells undergoing differentiation into oocytes in transgenic mice expressing the suicide gene, herpes simplex virus thymidine kinase (HSVtk), driven by the promoter of stimulated by retinoic acid gene 8 (Stra8), a germ cell-specific gene activated during meiotic commitment. Over a 21-day ablation phase induced by the HSVtk pro-drug, ganciclovir (GCV), oocyte numbers declined due to a disruption of new oocyte input. However, germ cell differentiation resumed after ceasing the ablation protocol, enabling complete regeneration of the oocyte pool. We next employed inducible lineage tracing to fate map, through Cre recombinase-mediated fluorescent reporter gene activation only in Stra8-expressing cells, newly-formed oocytes. Induction of the system during adulthood yielded a mosaic pool of unmarked (pre-existing) and marked (newly-formed) oocytes. Marked oocytes matured and fertilized to produce offspring, which grew normally to adulthood and transmitted the reporter to second-generation offspring. These findings establish that oocytes generated during adulthood contribute directly to ovarian function and natural fertility in mammals.

Oocyte stem cells: fact or fantasy?

For many decades, the dogma prevailed that female mammals had a finite pool of oocytes at birth and this was gradually exhausted during a lifetime of reproductive function. However, in 2004, a new era began in the field of female oogenesis. A study was published that appeared to detect oocyte-stem cells capable of generating new eggs within mouse ovaries. This study was highly controversial and the years since this initial finding have produced extensive research and even more extensive debate into their possibility. Unequivocal evidence testifying to the existence of oocyte-stem cells (OSCs) has yet to be produced, meanwhile the spectrum of views from both sides of the debate are wide-ranging and surprisingly passionate. Although recent studies have presented some convincing results that germ cells exist and are capable of creating new oocytes, many questions remain. Are these cells present in humans? Do they exist in physiological conditions in a dormant state? This comprehensive review first examines where and how the dogma of a finite pool was established, how this has been challenged over the years and addresses the most pertinent questions as to the current status of their existence, their role in female fertility, and perhaps most importantly, if they do exist, how can we harness these cells to improve a woman's oocyte reserve and treat conditions such as premature ovarian insufficiency (POI: also known as premature ovarian failure, POF).

FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event.