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Stem cell-based strategies for ovarian regeneration and oocyte production have been proposed as future clinical therapies for treating infertility in women. However, utilization of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) to produce oocytes has had limited success in vitro. A recent report of the isolation and characterization of endogenous oocyte-producing or oogonial stem cells (OSCs) from ovaries of reproductive-age women describes the first stable and pure human female germ cell culture model in which a subset of cells appear to initiate and complete meiosis. In addition, purified human OSCs introduced into adult human ovarian cortical tissue generate oocytes that arrest at the diplotene stage of meiosis and successfully recruit granulosa cells to form new primordial follicles. This overview examines the current landscape of in vitro and in vivo gametogenesis from stem cells, with emphasis on generation of human oocytes. Future research objectives for this area of work, as well as potential clinical applications involving the use of human OSCs, are discussed.

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

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

The enrichment of female germline stem cells (FGSCs) and the establishment of cell lines are influenced by the efficiency of cell purification. A previous study using mouse vasa homolog (MVH)-magnetic bead sorting for the isolation and purification of mouse FGSCs showed a relatively low efficiency. In this study, we tested 3 further proteins with the aim of improving the efficiency of FGSC purification. Immunofluorescence assays and magnetic sorting were performed using short-type pituitary gland and brain-cadherin (Stpb-c), CD9, and interferon-inducible transmembrane protein 3 (Iftm3, Fragilis), all of which are expressed in germ cells. Although all 3 proteins were expressed in FGSCs, CD9 was unsuitable because of its lack of germline specificity, and Stpb-c was also unsuitable because of the unavailability of an appropriate primary antibody. The efficiency of FGSC purification was remarkably enhanced using the germline-specific protein Fragilis, compared with that using MVH. This new method for the purification of FGSCs may have extensive applications in stem cell studies and clinical research.

Oogonial stem cells are short-lived and endow the ovary with its lifetime store of follicles during fetal life. No compensatory mechanisms exist to replace germ cells that are lost for whatever reason after birth. Fetal germ cells and the abundant primordial follicles of immature animals can be successfully stored at low temperatures and transplanted to hosts to generate normal ovulatory cycles. Sterilized hosts are restored to fertility. Such results suggest that the abundant reserves of germ cells in the ovaries of human abortuses offer opportunities for treating patients whose sterility is due to afollicular ovaries uncomplicated by autoimmune disease. The prospects for this treatment depend largely on the vigilance of the recipient's immune system and public attitudes to a radical treatment, though one that promises to overcome sterility and hypoestrogenism in women with either premature menopause or gonadal dysgenesis.

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.

The idea that females of most mammalian species have lost the capacity for oocyte production at birth has been challenged recently by the finding that juvenile and adult mouse ovaries possess mitotically active germ cells. However, the existence of female germline stem cells (FGSCs) in postnatal mammalian ovaries still remains a controversial issue among reproductive biologists and stem cell researchers. We have now established a neonatal mouse FGSC line, with normal karyotype and high telomerase activity, by immunomagnetic isolation and culture for more than 15 months. FGSCs from adult mice were isolated and cultured for more than 6 months. These FGSCs were infected with GFP virus and transplanted into ovaries of infertile mice. Transplanted cells underwent oogenesis and the mice produced offspring that had the GFP transgene. These findings contribute to basic research into oogenesis and stem cell self-renewal and open up new possibilities for use of FGSCs in biotechnology and medicine.

There have been some proposals that stem cells exist in the ovarian surface epithelium (OSE) of the adult human ovary; however, no direct evidence of such cells has been given until now. The aim of this study was to isolate the putative ovarian stem cells (OSCs) from the OSE layer in women with no naturally present oocytes and follicles--20 postmenopausal women and five women with premature ovarian failure. Small round cells with a bubble-like structure and diameters from 2 to 4 microm were isolated from the material obtained by OSE scraping. They expressed early embryonic developmental markers such as stage-specific embryonic antigen-4 and Oct-4, Nanog, Sox-2, and c-kit transcription markers, and they displayed prominent c-kit immunohistochemical staining. These cells were separated by density gradient centrifugation and grown in vitro, where they proliferated. Some of them grew intensively and reached a diameter of approximately 20 microm after 5-7 days. In the OSE cell culture, oocyte-like cells developed, which reached a diameter of up to 95 microm and expressed Oct-4A, Oct-4B, c-kit, VASA, and ZP2 transcription markers, corresponding to early oocytes. They did not express SCP3 meiotic marker. In conclusion, the discovered cells are proposed to represent the adult OSCs with the expression of embryonic stem cell markers. The expression of germ lineage marker c-kit points toward their primordial germ cell ancestry. A new term "embryonic-like stem cells of the adult" is proposed for embryonic-like stem cells that might persist in various tissues and organs of adults. These findings could be used for further studies aimed at the autologous treatment of ovarian infertility and degenerative diseases.

Proliferation and partial meiotic maturation of germ cells in fetal ovaries is believed to establish a finite, non-renewable pool of primordial follicles at birth. The supply of primordial follicles in postnatal life should be depleted during folliculogenesis, either undergoing atresia or surviving to ovulation. Recent studies of mouse ovaries propose that intra- and extraovarian germline stem cells replenish oocytes and form new primordial follicles. We quantified all healthy follicles in C57BL/6 mouse ovaries from day 1 to 200 using unbiased stereological methods, immunolabelling of oocyte meiosis (germ cell nuclear antigen (GCNA)) and ovarian cell proliferation (proliferating cell nuclear antigen (PCNA)) and electronmicroscopy. Day 1 ovaries contained 7924±1564 (s.e.m.) oocytes or primordial follicles, declining on day 7 to 1987±203, with 200–800 oocytes ejected from individual ovaries on that day and day 12. Discarded oocytes and those subjacent to the surface epithelium were GCNA-positive indicating their incomplete meiotic maturation. From day 7 to 100 mean numbers of primordial follicles per ovary were not significantly depleted but declined at 200 days to 254±71. Mean numbers of all healthy follicles per ovary were not significantly different from day 7 to 100 (range 2332±349–3007±322). Primordial follicle oocytes were PCNA-negative. Occasional unidentified cells were PCNA-positive with mitotic figures observed in the cortex of day 1 and 12 ovaries. Although we found no evidence for ovarian germline stem cells, our data support the hypothesis of postnatal follicle renewal in postnatal and adult ovaries of C57BL/6 mice.

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).

Background The longstanding belief that women are born with a finite ovarian reserve has been debated for almost a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs). These cells, described in both mice and man, seemingly have the ability to generate oocytes when cultured in vitro under specific conditions, resulting in live offspring in the case of mice. The aims of this study were to verify independently the existence of OSCs in human ovarian tissue and to determine whether they could be isolated from bovine tissue. Methods We isolated OSCs from disaggregated bovine and human ovarian cortex using a fluorescence-activated cell sorting (FACS)-based technique, with cells being sorted for the presence of VASA, a germ-cell-specific protein. The cells were subsequently cultured in vitro without the need for a feeder layer, and mRNA expression of germline-specific markers was analysed with RT-PCR. The cells were transduced to express green fluorescent protein (GFP) by use of a lentivirus for the purposes of tracking. Findings A rare population of mitotically active VASA-positive cells was isolated from both bovine and human tissue and they have been grown in vitro for several months. Up to November, 2013, cells were retrieved from three human samples, but isolation has been replicated on five separate occasions in bovine ovary samples. RT-PCR demonstrated consistent expression of several germline markers, including IFITM3, DPPA3, PRDM1, POU5F1 , and KIT . It has been possible to cryopreserve the cells with successful re-initiation of growth on thawing, and stable expression of GFP was achieved after lentivirus transduction. Interpretation The expression of germline markers indicates that these cells have characteristics of germline (oogonial) stem cells. To our knowledge, this is the first report of such cells being isolated from bovine ovarian cortex and corroborates a previous report showing the isolation of OSCs from human ovary. These cells provide a novel model for investigating germ-cell development; future experiments will involve injection of OSCs into ovarian cortex to assess whether they have the capability to undergo follicle formation and oocyte development in an in-vitro environment. If their potential can be harnessed, OSCs might have a future role in clinical applications—eg, in fertility preservation. Funding UK Medical Research Council.

The Drosophila melanogaster ovarian tumor (otu gene, required for normal proliferation and differentiation of the female germ-line, encodes two cytoplasmic protein isoforms, 98 and 104 kDa. Mutants with defects in this gene are typically grouped into one of three phenotypic classes: quiescent (germ cells do not proliferate), oncogenic or tumorous (germ-line cells proliferate uncontrollably), and differentiated (germ-line cells initiate but do not complete differentiation). Analysis of transformants expressing only one of the otu isoforms showed that the 104-kDa isoform (otu-104) can rescue all classes of otu mutants, whereas only differentiated mutants are rescued to a significant extent by the 98-kDa isoform (otu-98). Western analysis of protein extracts prepared from ovaries of various developmental stages indicated that otu-104 predominates in predifferentiated stages, while otu-98 is prevalent in differentiated egg chambers. Immunolocalization experiments demonstrated that otu protein is present in the cytoplasm of oogonial stem cells that populate third instar larvae and in all germ-line-derived cells until late in oogenesis. In stage 10 egg chambers, otu protein shifts to the subcortical region of nurse cells. This type of analysis also showed that upon formation of a 16-cell syncytium otu-104, but not otu-98, preferentially accumulates in the developing oocyte cytoplasm. The otu11 mutant protein does not show this pattern of enhanced accumulation, nor does it occur in ovaries of egalitarian and Bicaudal-D mutants, which are defective in oocyte determination. Thus, these studies indicate that the 104-kDa isoform is required for normal proliferation of female germ-line cells and perhaps for oocyte differentiation. The 98-kDa isoform appears to be dispensable but can provide an otu function needed for the completion of oocyte maturation.

In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. We report here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morpholigical characteristics and produced mature spermatozoa. This methodology, besides opening new avenues of basic research into spermatogenesis and stem-cell self-renewal, may prove useful as a tool for biomedical science and biotechnology.

The mouse study of Johnson et al. (Nature 2004, 428: 145-150) may suggest that the prosimian primates are not the only mammals to exhibit mitotically active germ cells in adult ovaries. If the dogma is to be debunked and a new one accepted, at least in the mouse, it is sure to be challenged and tested, as should all dogma.

SummaryIt has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Here we show that adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ataxia telangiectasia-mutated gene-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes. These results identify BM as a potential source of germ cells that could sustain oocyte production in adulthood.

Although the breeding habits of hagfishes remain almost a complete mystery and we know very little about their reproductive physiology, the histology of their unpaired gonad indicates that the sexual differentiation of these animals is in a curious transitional state between gonochorism and hermaphroditism. Whether the myxinoids have a determinate life span is unknown, but they certainly produce a succession of eggs; each ovulation being followed by the renewed growth of a reserve of smaller oocytes, which are presumably replaced by the division of oogonial stem cells persisting in the ovary throughout their reproductive life. At least within the genus Eptatretus, ovulation may be restricted to a definite breeding season, but in Myxine, females containing mature eggs have been found throughout the year, although the interval between successive ovulations is not known.

It has long been accepted that the complement of follicles within the ovary is formed before birth in humans, or shortly after birth in rodents, and that no follicles are formed thereafter. This follows entry of all oogonia into meiosis in fetal life, with no remaining germ stem cells in the ovary, in contrast to the presence of spermatogonia in the testis. This has been brought back into debate in recent years, following the demonstration of isolation of cells expressing both germline and stem markers from the postnatal ovary in several species, including humans. We describe these cells as putative ovarian stem cells. Isolation of these cells is challenging, adding to the debate as to their existence, and the validity of DDX4 as the main marker used for their isolation has also to be questioned. While different groups have used varying techniques and indeed terminology to describe these cells, the body of evidence regarding their initial characterization after isolation is growing. There remain very limited data regarding their developmental potential, but the demonstration of the production of functional oocytes from induced pluripotent stem cells and the advances in ovarian follicle culture techniques provide a basis for such studies.

The existence of a population of putative stem cells with germline developmental potential (oogonial stem cells: OSCs) in the adult mammalian ovary has been marked by controversy over isolation methodology and potential for in-vitro transformation, particularly where cell sorting has been based on expression of DEAD box polypeptide 4 (DDX4). This study describes a refined tissue dissociation/fluorescence-activated cell sorting (FACS) protocol for the ovaries of adult women which results in increased cell viability and yield of putative OSCs. A FACS technique incorporating dual-detection of DDX4 with aldehyde dehydrogenase 1 (ALDH1) demonstrates the existence of two sub-populations of small DDX4-positive cells (approx. 7 µm diameter) with ALDH1 activity, distinguished by expression of differentially spliced DDX4 transcripts and of DAZL, a major regulator of germ cell differentiation. These may indicate stages of differentiation from a progenitor population and provide a likely explanation for the expression disparities reported previously. These findings provide a robust basis for the further characterisation of these cells, and exploration of their potential physiological roles and therapeutic application.

For more than a half a century, biologists have upheld the theory that in most mammalian species, oocytes are formed before or shortly after birth, but never in adulthood. This foundation of reproductive science has survived the rapid growth of new technology and knowledge and has remained virtually unchallenged until two recent papers were published by the group headed by Jonathan Tilly. The first paper claims that mouse germline stem cells (GSCs) replace ovarian follicles that have been rapidly lost through follicle death (Johnson et al., 2004).

The immune system plays an important role in the regulation of tissue homeostasis ("tissue immune physiology"). Function of distinct tissues during adulthood, including the ovary, requires (1) Renewal from stem cells, (2) Preservation of tissue-specific cells in a proper differentiated state, which differs among distinct tissues, and (3) Regulation of tissue quantity. Such morphostasis can be executed by the tissue control system, consisting of immune system-related components, vascular pericytes, and autonomic innervation. Morphostasis is established epigenetically, during morphogenetic (developmental) immune adaptation, i.e., during the critical developmental period. Subsequently, the tissues are maintained in a state of differentiation reached during the adaptation by a “stop effect” of resident and self renewing monocyte-derived cells. The later normal tissue is programmed to emerge (e.g., late emergence of ovarian granulosa cells), the earlier its function ceases. Alteration of certain tissue differentiation during the critical developmental period causes persistent alteration of that tissue function, including premature ovarian failure (POF) and primary amenorrhea. In fetal and adult human ovaries the ovarian surface epithelium cells called ovarian stem cells (OSC) are bipotent stem cells for the formation of ovarian germ and granulosa cells. Recently termed oogonial stem cells are, in reality, not stem but already germ cells which have the ability to divide. Immune system-related cells and molecules accompany asymmetric division of OSC resulting in the emergence of secondary germ cells, symmetric division, and migration of secondary germ cells, formation of new granulosa cells and fetal and adult primordial follicles (follicular renewal), and selection and growth of primary/preantral, and dominant follicles. The number of selected follicles during each ovarian cycle is determined by autonomic innervation. Morphostasis is altered with advancing age, due to degenerative changes of the immune system. This causes cessation of oocyte and follicular renewal at 38 +/-2 years of age due to the lack of formation of new granulosa cells. Oocytes in primordial follicles persisting after the end of the prime reproductive period accumulate genetic alterations resulting in an exponentially growing incidence of fetal trisomies and other genetic abnormalities with advanced maternal age. The secondary germ cells also develop in the OSC cultures derived from POF and aging ovaries. In vitro conditions are free of immune mechanisms, which prevent neo-oogenesis in vivo. Such germ cells are capable of differentiating in vitro into functional oocytes. This may provide fresh oocytes and genetically related children to women lacking the ability to produce their own follicular oocytes. Further study of "immune physiology" may help us to better understand ovarian physiology and pathology, including ovarian infertility caused by POF or by a lack of ovarian follicles with functional oocytes in aging ovaries. The observations indicating involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from OSC during the fetal and prime reproductive periods are reviewed as well as immune system and age-independent neo-oogenesis and oocyte maturation in OSC cultures, perimenopausal alteration of homeostasis causing disorders of many tissues, and the first OSC culture clinical trial.