TGFB1 stimulates the proliferation of quiescent oogonial stem cells in chicken.

In brief: Oogonial stem cells in the adult ovary can generate oocytes, but they are usually quiescent. TGFB1 is key in stimulating the proliferation of OSC, thereby ensuring the sustained reproductive potential in poultry species. Abstract: Oogonial stem cells (OSCs) are a type of germ stem cell present in the adult ovary. They have the ability to self-renew through mitosis and differentiate into oocytes through meiosis. We have previously identified a population of OSCs in the chicken ovary, but the underlying mechanisms controlling their activation and proliferation were unclear. In this study, we observed that OSCs showed robust proliferation when cultured on a layer of chicken embryo fibroblasts (CEF), suggesting that CEF may secrete certain crucial factors that activate OSC proliferation. We further detected TGFB1 as a potent signaling molecule to promote OSC proliferation. Additionally, we revealed the signaling pathways that play important roles downstream of TGFB1-induced OSC proliferation. These findings provide insights into the mechanisms underlying OSC proliferation in chickens and offer a foundation for future research on in situ activation of OSC proliferation in ovary and improvement of egg-laying performance in chickens.

The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice.

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

Histological characterization of the maturation stages of the ovarian follicles of the goldfish Carassius auratus (Linnaeus, 1758).

The goldfish is a model organism showing great potential for research, particularly in comparative endocrinology concerning the neuroendocrine signalling and regulation of vertebrate reproduction. Furthermore, this teleost is increasingly stressed as a relevant alternative to more common fish model organisms, namely zebrafish. However, quality descriptions and illustrations of the complete goldfish gonadal histology are surprisingly scarce, but needed, to support research using this fish. Therefore, the main aim of this work is to describe in detail and adequately illustrate the goldfish oogenesis, from oogonia to late maturation, by applying routine stains (haematoxylin-eosin) and special procedures (periodic acid-Schiff and Goldner's trichrome). We hypothesized that the combined strategies would enable not only to observe the most general features but also to perceive some poorly described details of oocytes better. We describe the details of the following maturation stages: oogonia proliferation, chromatin-nucleolus, primary growth (one nucleolus step, multiple nucleoli step, perinucleolar step, cortical alveoli step) and secondary growth (early secondary growth step, late secondary growth step). Additionally, we report aspects of early and late follicular atresia. The study allowed comparisons with other species and showed that the Goldner's trichrome has the best discriminative power and should be the preferred stain, despite more time-consuming.

New insights in oocyte dynamics shed light on the complexities associated with fish reproductive strategies.

Information on temporal variations in stock reproductive potential (SRP) is essential in fisheries management. Despite this relevance, fundamental understanding of egg production variability remains largely unclear due to difficulties in tracking the underlying complex fluctuations in early oocyte recruitment that determines fecundity. We applied advanced oocyte packing density theory to get in-depth, quantitative insights across oocyte stages and seasons, selecting the commercially valuable European hake (Merluccius merluccius) as a case study. Our work evidenced sophisticated seasonal oocyte recruitment dynamics and patterns, mostly driven by a low-cost predefinition of fecundity as a function of fish body size, likely influenced also by environmental cues. Fecundity seems to be defined at a much earlier stage of oocyte development than previously thought, implying a quasi-determinate - rather than indeterminate - fecundity type in hake. These results imply a major change in the conceptual approach to reproductive strategies in teleosts. These findings not only question the current binary classification of fecundity as either determinate or indeterminate, but also suggest that current practices regarding potential fecundity estimation in fishes should be complemented with studies on primary oocyte dynamics. Accordingly, the methodology and approach adopted in this study may be profitably applied for unravelling some of the complexities associated with oocyte recruitment and thereby SRP variability.

The Mussel Mytilus galloprovincialis in the Bay of Naples: New Insights on Oogenic Cycle and Its Hormonal Control.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17ß-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3ß-HSD, 17ß-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

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.

Functions of the MRE11 complex in the development and maintenance of oocytes.

The MRE11 complex (MRE11, RAD50, and NBS1) is a central component of the DNA damage response, governing both double-strand break repair and DNA damage response signaling. To determine the functions of the MRE11 complex in the development and maintenance of oocytes, we analyzed ovarian phenotypes of mice harboring the hypomorphic Mre11 (ATLD1) allele. Mre11 (ATLD1/ATLD1) females exhibited premature oocyte elimination attributable to defects in homologous chromosome pairing and double-strand break repair during meiotic prophase. Other aspects of meiotic progression, including attachment of telomeres to the nuclear envelope and recruitment of RAD21L, a component of the meiotic cohesin complex to the synaptonemal complex, were normal. Unlike Dmc1 (-/-) and Trp13 (Gt/Gt) mice which exhibit comparable defects in double-strand break repair and oocyte depletion by 5 days post-partum, we found that oocyte attrition occurred by 12 weeks in Mre11 (ATLD1/ATLD1) . Disruption of the oocyte checkpoint pathway governed by Chk2 gene further enhanced the survival of Mre11 (ATLD1/ATLD1) follicles. Together our data suggest that the MRE11 complex influences the elimination of oocytes with unrepaired meiotic double-strand breaks post-natally, in addition to its previously described role in double-strand break repair and homologous synapsis during female meiosis.

Oogenesis in Laetacara araguaiae (Ottoni and Costa, 2009) (Labriformes: Cichlidae).

We aimed to analyze the oogenesis of adult females of the cichlid fish Laetacara araguaiae. The specimens' gonads were removed and processed for light and transmission electron microscopy. Oogenesis in L. araguaiae showed the following characteristics: a germinal epithelium with three types of oogonia (A-undifferentiated, A-differentiated and B-oogonia), oocytes at meiotic prophase stage and ovarian follicle formation. Oocytes showing primary growth with pre-vitellogenic and cortical alveolus were observed. Similar to data for other cichlids, oocytes in secondary growth or vitellogenesis were characterized by the initial deposition of yolk microgranules. The event that characterizes the maturation stage is nucleolus migration, also called the germinal vesicle, to the oocyte periphery in the direction of the micropyle. The follicular complex undergoes several changes throughout the oocyte stages. To the best of our knowledge this study is the first to describe L. araguaiae oogenesis. Moreover, this study is the first step to better understand the reproductive biology of this species, which shows great potential for use as an ornamental fish.

Programmed cell death in mouse primordial germ cells.

In a number of mammalian species, the main events of development of the primordial germ cells (PGCs), the embryonic precursors of the oocytes and spermatozoa, were described during the early twentieth century. Actually, the concept of the origin of germ cells in extragonadal sites before the formation of the gonadal anlagen, was put forward for the human embryo around the first decade of the 1900s (for a review, see De Felici, 2013). PGC development is characterized by two major cellular processes, a movement from the wall of the yolk sac, where the germline is determined, to the gonadal anlagen and an increase in number due to active proliferation. As far as we know, the notion that programmed cell death (PCD) might physiologically occur in mammalian PGCs was for the first time put forward by us in 1993 in the case of the mouse. How we arrived to such a concept and the progress made up to now in the characterization of this process in our and other laboratories mainly in the mouse are the topics of the present review.

Similar morphological and molecular signatures shared by female and male germline stem cells.

The existence of mammalian female germline stem cells (FGSCs) indicates that mammalian ovaries possess germline stem cells analogous to testis, and continue to produce gametes postnatally, which provides new insights into female fertility. In this study, we compared the morphological and molecular characteristics between FGSCs and spermatogonial stem cells (SSCs) by analysis of morphology, immunofluorescence, alkaline phosphatase activity assay, reverse transcription polymerase chain reaction (RT-PCR) and microarray hybridization. The results demonstrated that the morphology and growth patterns of FGSCs are similar to those of SSCs. Microarray analysis of global gene expression profiles of FGSCs and SSCs showed similar signatures in the transcriptome level. A list of 853 co-highly expressed genes (CEG) in female and male germline stem cells may be responsible for the morphological and molecular similarity. We constructed a continuous network of the CEG based on I2D protein-protein interaction database by breadth first search. From the network, we could observe the interactions of the CEG may be responsible for maintaining the properties of germline stem cells. This study was the first attempt to compare morphological and molecular characteristics between FGSCs and SSCs. These findings would provide some clues for further research on mammalian FGSCs.

The neonatal marmoset monkey ovary is very primitive exhibiting many oogonia.

Oogonia are characterized by diploidy and mitotic proliferation. Human and mouse oogonia express several factors such as OCT4, which are characteristic of pluripotent cells. In human, almost all oogonia enter meiosis between weeks 9 and 22 of prenatal development or undergo mitotic arrest and subsequent elimination from the ovary. As a consequence, neonatal human ovaries generally lack oogonia. The same was found in neonatal ovaries of the rhesus monkey, a representative of the old world monkeys (Catarrhini). By contrast, proliferating oogonia were found in adult prosimians (now called Strepsirrhini), which is a group of 'lower' primates. The common marmoset monkey (Callithrix jacchus) belongs to the new world monkeys (Platyrrhini) and is increasingly used in reproductive biology and stem cell research. However, ovarian development in the marmoset monkey has not been widely investigated. Herein, we show that the neonatal marmoset ovary has an extremely immature histological appearance compared with the human ovary. It contains numerous oogonia expressing the pluripotency factors OCT4A, SALL4, and LIN28A (LIN28). The pluripotency factor-positive germ cells also express the proliferation marker MKI67 (Ki-67), which has previously been shown in the human ovary to be restricted to premeiotic germ cells. Together, the data demonstrate the primitiveness of the neonatal marmoset ovary compared with human. This study may introduce the marmoset monkey as a non-human primate model to experimentally study the aspects of primate primitive gonad development, follicle assembly, and germ cell biology in vivo.

Proliferation of oogonia and folliculogenesis in the viviparous teleost Ilyodon whitei (Goodeidae).

Oogonial proliferation in fishes is an essential reproductive strategy to generate new ovarian follicles and is the basis for unlimited oogenesis. The reproductive cycle in viviparous teleosts, besides oogenesis, involves development of embryos inside the ovary, that is, intraovarian gestation. Oogonia are located in the germinal epithelium of the ovary. The germinal epithelium is the surface of ovarian lamellae and, therefore, borders the ovarian lumen. However, activity and seasonality of the germinal epithelium have not been described in any viviparous teleost species regarding oogonial proliferation and folliculogenesis. The goal of this study is to identify the histological features of oogonial proliferation and folliculogenesis during the reproductive cycle of the viviparous goodeid Ilyodon whitei. Ovaries during nongestation and early and late gestation were analyzed. Oogonial proliferation and folliculogenesis in I. whitei, where intraovarian gestation follows the maturation and fertilization of oocytes, do not correspond to the late oogenesis, as was observed in oviparous species, but correspond to late gestation. This observation offers an example of ovarian physiology correlated with viviparous reproduction and provides elements for understanding the regulation of the initiation of processes that ultimately result in the origin of the next generation. These processes include oogonia proliferation and development of the next batch of germ cells into the complex process of intraovarian gestation.

Oogenesis and ovarian histology in two populations of the viviparous lizard Sceloporus grammicus (Squamata: Phrynosomatidae) from the central Mexican Plateau.

The annual histological changes in ovarian morphology (oogenesis, follicular atresia, and corpus luteum) are described for the Mexican lizard Sceloporus grammicus, in two populations that inhabit contrasting environments (vegetation categories, climate, precipitation, and temperature) from Hidalgo State, Mexico. Two germinal beds were situated on the dorsal surface of each ovary of this species. In both the populations, oogenesis involves two major processes: previtellogenesis and vitellogenesis. The histological changes during previtellogenesis are similar to those for other reptilian sauropsids, whereas vitellogenesis differs and the features of this last process are described for the first time. In early previtellogenesis, primary oocytes have fibrillar chromosomes and the ooplasm stains slightly. The primordial follicles are surrounded by a granulosa composed of cuboidal follicular cells. During late previtellogenesis, the oocyte had an eccentric nucleus with lamp-brush chromosomes and multiple nucleoli. The granulosa becomes multilayered and polymorphic, containing three cell types: small, intermediate, and pyriform. The zona pellucida was homogeneous and clearly observed. In early vitellogenesis, the oocyte showed several small acidophilic granules distributed in the center and the periphery of the oocyte. As vitellogenesis progresses, the yolk platelets move toward the central area of the oocyte and they fuse to form acidophilic and homogeneous yolk. Lipid droplets were distributed irregularly in the ooplasm of the oocyte. In Zacualtipán, the results revealed a strong seasonal reproductive activity. Females had vitellogenic follicles from July to September, and pregnant females were founded from September to March. In Tizayuca, the results showed an unusual pattern of reproductive activity. Females with vitellogenic follicles and pregnant females were found throughout the year, indicating continuous reproduction. We suggest that the observed differences in reproductive activity from these populations indicate adaptative fine tuning in response to local environmental conditions. These results contribute to the knowledge of variation in vitellogenesis and reproductive strategies of this species and among spiny lizards overall.

Oogonial stem cells: do they exist and may they have an impact on future fertility treatment?

PURPOSE OF REVIEW: Germline stem cell research over the last decade has brought into question one of the basic tenets of reproductive biology that women are born with a finite number of oocytes without the potential for renewal. Evidence for the existence of oogonial stem cells in the postnatal ovary has gained momentum, but skepticism remains. RECENT FINDINGS: Several research studies claimed that they have identified functional oogonial stem cells in the postnatal ovary of several different species including humans. The scientific community has questioned both the methods and significance of these studies. SUMMARY: Many speculate that germline stem cells could make a significant impact on the treatment of female infertility. However, this field of research is still in its infancy. There is still much to learn about the biology of oogonial stem cells and their potential clinical application. More research is needed before oogonial stem cells can become a viable treatment modality for women with infertility.

Purification of oogonial stem cells from adult mouse and human ovaries: an assessment of the literature and a view toward the future.

Contemporary claims that mitotically active female germ line or oogonial stem cells (OSCs) exist and support oogenesis during postnatal life in mammals have been debated in the field of reproductive biology since March 2004, when a mouse study posed the first serious challenge to the dogma of a fixed pool of oocytes being endowed at birth in more than 50 years. Other studies have since been put forth that further question the validity of this dogma, including the isolation of OSCs from neonatal and adult mouse ovaries by 4 independent groups using multiple strategies. Two of these groups also reported that isolated mouse OSCs, once transplanted back into ovaries of adult female mice, differentiate into fully functional eggs that ovulate, fertilize, and produce healthy embryos and offspring. Arguably, one of the most significant advances in this emerging field was provided by a new research study published this year, which reported the successful isolation and functional characterization of OSCs from ovaries of reproductive age women. Two commentaries on this latest work, one cautiously supportive and one highly skeptical, were published soon afterward. This article evaluates the current literature regarding postnatal oogenesis in mammals and discusses important next steps for future work on OSC biology and function.

Development of the germinal ridge and ovary in the African elephant (Loxodonta africana).

The follicular reserve and its ontogeny in the elephant are of interest because elephants have the longest reproductive life of all land-based mammals. They also have the longest recorded pregnancy, which allows a protracted view of the series of significant events involved in the development of the embryonic and fetal gonads. The large elephant population of Zimbabwe provided the opportunity to collect conceptuses from elephants culled for management reasons and hunted professionally. Five embryos aged 76-96 days and the ovaries of four fetuses aged 4.8-11.2 months were fixed in 4% buffered formalin and studied by conventional histological sectioning and a stereological protocol to calculate the follicle reserve of each fetus. These observations enabled the conclusion that the migration of primordial germ cells into the indifferent gonad terminates at around 76 days of gestation while entry of oogonia into meiosis along with first follicle formation starts at around 5 months. Peak numbers of follicles are present by mid-gestation towards the end of the 6-month mitotic-meiotic transition period. It appears that the cortex of the elephant fetal ovary at mid-gestation (11 months) has already reached a developmental stage exhibited by the ovaries of many other mammals at full term.

Ovarian folliculogenesis in the smallnose fanskate sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae) / Foliculogénesis ovárica en la raya marmorada, sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae)

Chondrichthyes have become an important economic resource in the last decades, with Argentina as one of the countries that exploits more sharks and skates, even at levels that exceed de limits of many species. However, there is a scarce knowledge of the reproductive biology of this group, particularly from species inhabiting the Southern hemisphere. This work shows the most relevant facts during folliculogenesis in Sympterygia bonapartii. Results show that germinal cells are present in immature and maturing females. The most important facts that vary along de follicular development are the number of types and layers of follicular cells, the establishment of thin projections from the follicular cells and the degree of development of the thecae. Follicular cells are, at least, of two different types and both of them emit projections that break through the zona pellucida. The outer theca shows signs of synthetic activity. Atretic follicles of different sizes are present in exemplars of all the reproductive stages. These results are discussed in a physiological and adaptive context.

Los Condrictios se han convertido en un recurso económico importante en las últimas décadas, siendo Argentina uno de los países que más explota tiburones y rayas, incluso a niveles que exceden los límites de varias especies. A pesar de esto, es poco lo que se conoce sobre la biología reproductiva de este grupo, particularmente en especies del Hemisferio Sur. En este trabajo se estudian los estadios más relevantes de la foliculogénesis en Sympterygia bonapartii. Los resultados muestran que las ovogonias están presentes tanto en ejemplares inmaduros como subadultos. Las características más importantes que varían a lo largo del desarrollo folicular son el número de capas y tipos celulares que constituyen el epitelio folicular, el desarrollo de proyecciones de las células de la granulosa y el grado de desarrollo de las tecas. Las células foliculares son, al menos, de dos tipos y ambos emiten proyecciones que atraviesan la zona pelúcida. La teca externa presenta características compatibles con la actividad sintética. Folículos atrésicos de distintos tamaños están presentes en ejemplares de todos los estadios de madurez sexual. Estos resultados se discuten en un marco fisiológico y adaptativo.

Understanding follicle growth in vivo.

Ovarian reserve is determined by the number of primordial follicles in the ovary. Quiescent primordial follicles are activated for growth and pass through stages of development before they reach the antral stage. Then a cohort of antral follicles is recruited for further growth, dominance and ovulation under the cyclic stimulation of gonadotrophins. What triggers the initiation of growth in primordial follicles has remained a mystery for decades. However, recent studies on mutant mouse models have shown that primordial follicles are maintained in a dormant state by the actions of various inhibitory molecules to preserve the follicle pool, such as the transcription factor Foxo3a, PTEN (phosphotase and tensin homolog deleted on chromosome 10) and Tsc-1 (tumour suppressor tuberous schlerosis complex). Mice with deletions of these oocyte-specific genes exhibit premature activation of dormant primordial follicles, and all primordial follicles become depleted in early adulthood, causing premature ovarian failure. Other oocyte and somatic cell-derived growth factors are also involved in the early, gonadotrophin-independent phase of follicle growth via autocrine and paracrine interactions. Interestingly, some of these factors also play critical roles at later stages of follicle growth, such as the process of selecting the dominant follicle, by modifying the response of the follicles to gonadotrophins and inhibiting premature luteinization. Therefore, a thorough understanding of the molecular aspects of folliculogenesis is of paramount importance in the context of translational medicine and future clinical applications in human reproduction.

Endocrine modulation, inhibition of ovarian development and hepatic alterations in rainbow trout exposed to polluted river water.

Under laboratory conditions, female rainbow trout were exposed to graded concentrations of water from the River Lambro, a polluted tributary of the River Po, and to the effluent of a large wastewater treatment plant which flows into the River Lambro. In field exposures, trout were held in cages in the River Po upstream and downstream from the confluence of the River Lambro. After 10-day (laboratory) and 30-day (laboratory and field) exposures, trout were examined for several chemical, biochemical and histological endpoints. The results indicated that exposure to complex mixtures of chemicals, including estrogen receptor agonists, aryl-hydrocarbon receptor agonists, and probably antiandrogens, had occurred. Exposure altered the plasma levels of 17ß-estradiol and testosterone, and some treatments also enhanced the activity of hepatic ethoxyresorufin O-deethylase. Gonadal histology showed varying levels of degenerative processes characterised by oocyte atresia, haemorrhages, melano-macrophage centres (MMCs), and oogonia proliferation. Liver histology showed less severe effects.

Identification of germline stem cells in the ovary of the teleost medaka.

Germline stem cells continually produce sperm in vertebrate testes, whereas there is no direct evidence showing that germline stem cells are present in adult vertebrate ovaries. By using transgenic methods and clonal analysis, we identified germline stem cells that supported oogenesis and the production of offspring in the ovaries of adult medaka fish. Early-stage germ cells were localized in clusters along interwoven threadlike cords of sox9b-expressing somatic cells (termed germinal cradles) where the germ cells developed. Germline stem cells gave rise to germ cells that divided to produce cysts, which then underwent cell death or separated to form follicles. Our results provide insight into the germline stem cell biology of medaka and provide a model system for studying vertebrate stem cell niches.