Follicular Renewal Research Articles

Follicular renewal and stemness potency of follicular cells depended of telomerase activity and TERT expression – short review

Abstract Several hypotheses have been proposed, relating to the potential genesis of follicular cells in the ovarian niche. Reports using mice as an experimental model have suggested that the ovaries may contain stem cells that are likely involved in the formation of new follicles in adult reproductive life. Over recent years, various types of ovarian cells have been identified and described to confirm or disprove the existence of ovarian adult stem cells. Most research is focused on granulosa cells (GCs), which are essential for follicular development and maturation of female germ cells (oocytes). GCs exhibit the features of stem cells, such as expression of stem cell markers: OCT-4, Sox-2, Nanog as well as certain markers of mesenchymal stem cells, including CD29, CD44, CD90, CD105, CD117, and CD166. Another discovery in favor of the potential stemness of GCs is their ability to transdifferentiate towards other cell lines and high telomerase (TERT) activity in dividing compartments of the follicle during its maturation.

ISOTRETINOIN FOR TREATING ACNE VULGARIS

Acne vulgaris (AV) occurs in more than 80% of dermatological cases in adolescents and young adults and affects the quality of life. Oral isotretinoin, a metabolite product of vitamin A, is well-known to be the most effective treatment for severe nodulocystic AV and moderate AV that does not respond to other therapeutic modalities. Thus, this literature review was conducted to explain the mechanism of action, effectiveness, contraindications, side effects, and safety of oral isotretinoin in AV, which provided essential information for dermatologists. Furthermore, isotretinoin is the only treatment modality that has implications for the entire pathogenesis of acne. It contributes to decreasing corneocytes’ adhesion, supporting cellular proliferation and follicular renewal, induction of cell apoptosis, and immune regulation. The effectiveness of AV therapy with isotretinoin has expanded as it is also indicated for moderate to severe AV that does not respond well to topical combination therapy or systemic antibiotics. However, isotretinoin is included in Category X drugs and may induce many side effects from mucocutaneous effects to teratogenicity. From this extensive literature review, it can be concluded that isotretinoin is one of the treatment modalities for acne with good effectivity due to its mechanism of actions that affect the entire acne pathogenesis. Considering the various side effects of isotretinoin, its use requires adequate clinical assessment and monitoring by a dermatologist.

Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries

It has been generally accepted for more than half a century that, in most mammalian species, oocytes cannot renew themselves in postnatal or adult life, and that the number of oocytes is already fixed in fetal or neonatal ovaries. This assumption, however, has been challenged over the past decade. In this study, we have taken an endogenous genetic approach to this question and generated a multiple fluorescent Rosa26(rbw/+);Ddx4-Cre germline reporter mouse model for in vivo and in vitro tracing of the development of female germline cell lineage. Through live cell imaging and de novo folliculogenesis experiments, we show that the Ddx4-expressing cells from postnatal mouse ovaries did not enter mitosis, nor did they contribute to oocytes during de novo folliculogenesis. Our results provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active Ddx4-expressing female germline progenitors exist in postnatal mouse ovaries.

The primordial follicle reserve is not renewed after chemical or γ-irradiation mediated depletion

Reports indicate that germ-line stem cells present in adult mice can rapidly generate new oocytes and contribute to the primordial follicle reserve following conditions of ovotoxic stress. We further investigated the hypothesis that adult mice have the capacity to generate new oocytes by monitoring primordial follicle numbers throughout postnatal life and following depletion of the primordial follicle reserve by exposure to doxorubicin (DXR), trichostatin A (TSA), or whole-body γ-irradiation. We show that primordial follicle number remains stable in adult C57BL/6 mice between the ages of 25 and 100 days. However, within 2 days of treatment with DXR or TSA, primordial follicle numbers had declined to 65 and 51% respectively (P<0.05-0.01 when compared to untreated controls), with no restoration of follicle numbers evident after 7 days for either treatment. Furthermore, ovaries from mice subjected to sterilizing doses of γ-irradiation (0.45 or 4.5 Gy) revealed complete ablation of all primordial follicles 5 days after treatment, with no indication of follicular renewal. We conclude that neo-folliculogenesis does not occur following chemical or γ-irradiation mediated depletion of the primordial follicle reserve.

Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a clinical trial.

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.

Ovarian Stem Cell Niche and Follicular Renewal in Mammals

Stem cell niche consists of perivascular compartment, which connects the stem cells to the immune and vascular systems. During embryonic period, extragonadal primordial germ cells colonize coelomic epithelium of developing gonads. Subsequently, ovarian stem cells (OSC) produce secondary germ cells under the influence of OSC niche, including immune system-related cells and hormonal signaling. The OSC in fetal and adult human ovaries serve as a source of germ and granulosa cells. Lack of either granulosa or germ cell niche will result in premature ovarian failure in spite of the presence of OSC. During perinatal period, the OSC transdifferentiate into fibroblast-like cells forming the ovarian tunica albuginea resistant to environmental threats. They represent mesenchymal precursors of epithelial OSC during adulthood. The follicular renewal during the prime reproductive period (PRP) ensures that there are fresh eggs available for a healthy progeny. End of PRP is followed by exponentially growing fetal genetic abnormalities. The OSC are present in adult, aging, and postmenopausal ovaries, and differentiate in vitro into new oocytes. During in vitro development of large isolated oocytes reaching 200 μm in diameter, an ancestral mechanism of premeiotic nurse cells, which operates during oogenesis in developing ovaries from invertebrates to mammalian species, is utilized. In vitro developed eggs could be used for autologous IVF treatment of premature ovarian failure. Such eggs are also capable to produce parthenogenetic embryos like some cultured follicular oocytes. The parthenotes produce embryonic stem cells derived from inner cell mass, and these cells can serve as autologous pluripotent stem cells.

How Can Female Germline Stem Cells Contribute to the Physiological Neo-Oogenesis in Mammals and Why Menopause Occurs?

At the beginning of the last century, reproductive biologists have discussed whether in mammalian species the fetal oocytes persist or are replaced by neo-oogenesis during adulthood. Currently the prevailing view is that neo-oogenesis is functional in lower vertebrates but not in mammalian species. However, contrary to the evolutionary rules, this suggests that females of lower vertebrates have a better opportunity to provide healthy offspring compared to mammals with oocytes subjected to environmental threats for up to several decades. During the last 15 years, a new effort has been made to determine whether the oocyte pool in adult mammals is renewed as well. Most recently, Ji Wu and colleagues reported a production of offspring from female germline stem cells derived from neonatal and adult mouse ovaries. This indicates that both neonatal and adult mouse ovaries carry stem cells capable of producing functional oocytes. However, it is unclear whether neo-oogenesis from ovarian somatic stem cells is physiologically involved in follicular renewal and why menopause occurs. Here we review observations that indicate an involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from ovarian stem cells during the prime reproductive period and propose why menopause occurs in spite of persisting ovarian stem cells.

Immune physiology and oogenesis in fetal and adult humans, ovarian infertility, and totipotency of adult ovarian stem cells

It is still widely believed that while oocytes in invertebrates and lower vertebrates are periodically renewed throughout life, oocytes in humans and higher vertebrates are formed only during the fetal/perinatal period. However, this dogma is questioned, and clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal from ovarian stem cells (OSCs) in adult human ovaries, and of the role of third-party bone marrow-derived cells (monocyte-derived tissue macrophages and T lymphocytes) could help provide a better understanding of the causes of ovarian infertility, its prevention, and potential treatment. We have reported differentiation of distinct cell types from OSC and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. OSCs are also capable of producing neural/neuronal cells in vitro after sequential stimulation with sex steroid combinations. Hence, OSC represent a unique type of totipotent adult stem cells, which could be utilized for autologous treatment of premature ovarian failure and also for autologous stem cell therapy of neurodegenerative diseases without use of allogeneic embryonic stem cells or somatic cell nuclear transfer. The in vivo application of sex steroid combinations may augment the proliferation of existing neural stem cells and their differentiation into mature neuronal cells (systemic regenerative therapy). Such treatment may also stimulate the transdifferentiation of autologous neural stem cell precursors into neural stem cells useful for topical or systemic regenerative treatment.

REVIEW ARTICLE: Immune Physiology of the Mammalian Ovary – A Review

The immune system, besides orchestrating the immune response, plays an important role in the regulation of tissue homeostasis. We refer to this later activity as ‘immune physiology.’ In human ovaries, immune system‐related cells and molecules accompany corpus luteum development and regression and cancer progression. They also accompany the origination of new rat and human germ cells by asymmetric division of ovarian surface epithelium cells, symmetric division and migration of germ cells, and follicular growth. Currently prevailing dogma on the preservation of human oocytes from the fetal period until menopause (‘storage’ doctrine) vs. oocyte renewal in invertebrates and lower vertebrates (‘continued formation’ doctrine) raises question as to the disadvantage from an evolutionary point of view of prolonged oocyte storage in humans. We attempted to reconcile these two opposing views by proposing the prime reproductive period (PRP) doctrine as follows: The ‘storage’ doctrine fits two periods of the life in human females, that between the termination of fetal oogenesis and puberty or pre‐menarcheal period (about 10–12 years), and also that period from the end of PRP (at about 38 years of age) until menopause. On the contrary, the ‘continued formation’ doctrine accounts for oocyte and follicular renewal during the PRP, and insures the availability of fresh oocytes for the development of healthy progeny. Further study on ‘immune physiology’ may help us better understand ovarian physiology and pathology in general, including infertility caused by premature ovarian failure, the pathophysiology of degenerative diseases and mechanisms of malignancy and metastasis.

Oogenesis from Human Somatic Stem Cells and a Role of Immune Adaptation in Premature Ovarian Failure

The central thesis is that, while embryonic oocytes originate from extra-ovarian sources, those generated during fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE). With the assistance of immune system-related cells, primitive granulosa and germ cells appear to originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly in order to be recognized as self and renewed later. With the onset of menarche, a periodical follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs are always available during the prime reproductive period. The periodical follicular renewal ceases between 35-40 years of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. Premature ovarian failure (POF) may result from premature termination of follicular renewal during adulthood, possibly due to the alteration of fetal follicular development during immune adaptation (idiopathic POF), or due to the alteration of the adult immune system by cytostatic chemotherapy. Factors responsible for the diminution of follicular renewal may be responsible for the aging of other tissues and the whole body in general. However, our recent research shows that OSE stem cells may produce new eggs in vitro, even when derived from ovaries lacking primary follicles. Consequently, their in vitro fertilization (IVF) and subsequent utilization of embryos for intrauterine implantation may represent a novel IVF approach for providing genetically related children to women with ovarian infertility, which is worthy of consideration and further exploration.

Potential new strategies for the treatment of ovarian infertility and degenerative diseases with autologous ovarian stem cells

The 50-year-old and currently prevailing view that all oocytes in adult human ovaries persist from the fetal period of life is controversial as it clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal in adult human ovaries, and of the role of hormonal signals and third-party cells (tissue macrophages and T cells), could all be helpful in providing better understanding of the causes of ovarian infertility, its prevention and potential therapy. In addition, the authors recently reported differentiation of distinct cell types and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. It is possible that fertilisation of such eggs will open up new opportunities for providing genetically related children to infertile women for whom conventional in vitro fertilisation has failed. As ovarian stem cells appear to represent a new type of totipotent adult stem cell, they could also be utilised for autologous stem cell therapy of degenerative diseases, without any involvement of allogeneic embryonic stem cells and somatic cell nuclear transfer.

Evidence for follicular renewal in the postnatal mouse ovary

Proliferation and maturation of germ cells in the fetal ovary establishes a finite, non-growing pool of primordial follicles (PF) at birth. Believed to be non-renewable, the supply of PF declines postnatally through folliculogenesis via atresia, or survival to ovulation. Recent studies suggest that germline stem cells replenish oocytes forming new follicles in the mouse ovary. We quantitated all healthy follicles in postnatal mouse ovaries from day 1–200 using stereology, immunolabeling of oocyte meiotic maturation (GCNA, germ cell nuclear antigen) and cell proliferation (PCNA, proliferating cell nuclear antigen) and electronmicroscopy. Day 1 ovaries had 7924 ± 1564 (SEM) PF and 1986 ± 204 on day 7. Oocytes were ejected from ovaries up to day 25 with 200–800 oocytes per ovary (confirmed by ultrastructure) exiting on day 7 and 12. Discarded and some subtunical oocytes were GCNA-positive suggesting their late meiotic maturation. Total PF (range 1976 ± 306 to 2317 ± 289) and total follicles per ovary (range 2332 ± 349 to 3077 ± 322) were not significantly different from day 7 to 100. Total volumes (x106μm3) of PF oocytes per ovary were not significantly different from day 12 (7.37 ± 0.77) to day 100 (4.35 ± 0.45). PF oocytes were PCNA-negative although occasional mitotic figures enclosed by squamous cells were noted in the ovarian cortex on day 1, 7 and 12. The data provide further evidence for germline stem cells that replenish PF, thus contributing to an unabated supply of growing follicles during puberty and early adult life. Supported by Monash University and NHMRC Australia.

Eggs forever?

A group of scientists from Harvard Medical School (Johnson et al., 2004) claims to have “established the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary,” expressing no doubts about their methods, results and conclusion. Johnson et al. based their conclusions of oocyte and follicular renewal from existing germline stem cells (GSC) in the postnatal mouse ovary on three types of observations: (1) A claimed discordance in follicle loss versus follicle atresia in the neonatal period and in the following pubertal and adult period; (2) immunohistochemical detection of proliferating GSC with meiotic capacity using combined markers for meiosis, germline, and mitosis; and (3) neo-folliculogenesis in ovarian chimeric grafting experiments with adult mice. Oogenesis is the process that transforms the proliferative oogonium into an oocyte through meiosis, followed by folliculogenesis and follicular and oocyte maturation. The most crucial part in producing a functional oocyte is firstly, initiation and completion of the first meiotic prophase, and secondly, enclosure of the resulting diplotene oocyte in a follicle. Neither of these two events has been shown to take place in Johnson et al.'s study of the postnatal mouse ovary. We hereby address the observations underpinning their hypothesis and conclude that it is premature to replace the paradigm that adult mammalian neo-oogenesis/folliculogenesis does not take place.

Can ovarian infertility be treated with bone marrow- or ovary-derived germ cells?

A year ago, reproductive biologists and general public were astonished with evidence reported by Johnson et al. in Nature 428:145 that mammalian ovaries possess persisting large germline stem cells, which allegedly enable follicular renewal in adult females. Recently, the same research group declared such view obscure, and reported that mammalian oocytes originate from putative germ cells in bone marrow and are distributed by peripheral blood to the ovaries (Cell 122:303). While neglecting available data on the germ cell origin from the ovarian surface epithelium (OSE) in adult mouse and human females and complexity of follicular renewal in humans, the authors widely extrapolated their observations on formation of allogeneic oocytes after bone marrow (or blood) transplantation in ovaries of adult mice treated with cytostatics to clinical implications in the public media. Yet, the resulting outcome that such allogeneic oocytes may enable the propagation of ovarian cycles is a poor alleviation for the women with ovarian infertility. Women lacking primary follicles, or carrying follicles with low quality eggs persisting in aging ovaries, are not concerned about the lack of menstrual cycles or ovarian steroids, but about virtually no chance of having genetically related children. Johnson et al. also reported that the germ cell formation in bone marrow disappears in ovariectomized mice. Such observation, however, raises solid doubts on the bone marrow origin of oocytes. Since germ cells developing from the OSE cells of adult human ovaries during periodical follicular renewal are known to enter blood vessels in order to enable formation of primary follicles at distant ovarian sites, they also contaminate peripheral blood and hence bone marrow. Better knowledge on the complexity of follicular renewal in humans and exploration of a potential of human OSE cells to produce new oocytes in vitro are essential for novel approaches to the autologous treatment of premature ovarian failure and age induced ovarian infertility.

Origin of Germ Cells and Follicular Renewal in Adult Human Ovaries

Origin of Germ Cells and Follicular Renewal in Adult Human Ovaries

"Waste" Follicular Aspirate from Fertility Treatment–A Potential Source of Human Germline Stem Cells?

Fertility clinics worldwide routinely produce a large volume of 'waste' follicular aspirate, which is potentially an abundant source of immature ovarian follicles. Current attempts to cultivate these further in vitro to yield viable mature oocytes for fertility treatment have not yet achieved much success. Instead, recent lines of evidence have emerged that are suggestive of a potential stem cell niche within such immature ovarian follicles. The recent discovery of follicular renewal and putative germ-line stem cells within the postnatal mammalian ovary shook the foundations of reproductive biology by challenging the established dogma that mammalian females lose the capacity for germ cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. More intriguingly, another recent study in the Drosophila model provided compelling evidence that somatic progenies (nurse cells) of germ-line stem cells had the ability to revert back to the stem-cell-like state. This introduces the exciting possibility that within the mammalian ovarian follicle, similar somatic progenies of germ-line stem cells may also possess a greater intrinsic ability to revert back into functional stem cells. If this is the case, then a favored candidate would be the cumulus/granulosa of immature ovarian follicles, since such cells are true homologues of nurse cells found within the Drosophila ovary. The successful elucidation of a human germ-line stem cell niche within immature ovarian follicles is likely to have huge ramifications in stem cell biology and regenerative medicine.

Oogenesis in Adult Mammals, Including Humans: A Review

The origin of oocytes and primary follicles in ovaries of adult mammalian females has been a matter of dispute for over 100 yr. The prevailing belief that all oocytes in adult mammalian females must persist from the fetal period of life seems to be a uniquely retrogressive reproductive mechanism requiring humans to preserve their gametes from the fetal period for several decades. The utilization of modern techniques during last 10 yr clearly demonstrates that mammalian primordial germ cells originate from somatic cell precursors. This indicates that if somatic cells are precursors of germ cells, then somatic mutations can be passed on to progeny. Mitotically active germline stem cells have been described earlier in ovaries of adult prosimian primates and recently have been reported to also be present in the ovaries of adult mice. We have earlier shown that in adult human females, mesenchymal cells in the ovarian tunica albuginea undergo a mesenchymal-epithelial transition into ovarian surface epithelium cells, which differentiate sequentially into primitive granulosa and germ cells. Recently, we have reported that these structures assemble in the deeper ovarian cortex and form new follicles to replace earlier primary follicles undergoing atresia (follicular renewal). Our current observations also indicate that follicular renewal exists in rat ovaries, and human oocytes can differentiate from ovarian surface epithelium in fetal ovaries in vivo and from adult ovaries in vitro. These reports challenge the established dogma regarding the fetal origin of eggs and primary follicles in adult mammalian ovaries. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. Yet, the follicular renewal may cease at a certain age, and this may predetermine the onset of the natural menopause or premature ovarian failure. A lack of follicular renewal in aging ovaries may cause an accumulation of spontaneously arising or environmentally induced genetic alterations of oocytes, and that may be why aging females have a much higher chance of having oocytes with more mutations in persisting primary follicles.

Germline Stem Cells and Follicular Renewal in the Postnatal Mammalian Ovary

Up to now, the accepted wisdom was that, unlike males who retain germline stem cells (GSCs) through their adult lives, oocyte production in females of most mammalian species ceases before birth. Females therefore are born with a fixed pool of GSCs, or oocytes, all of them arrested in meiosis. The ovarian follicles are oocytes enclosed by somatic cells. The oocyte reserve in human females is exhausted at approximately the fifth decade of life, inducing menopause. Directly contradicting these views, the investigators have found that juvenile and adult mouse ovaries have mitotically active germ cells that continuously replenish the pool of follicles. A study of mouse ovaries, based on analyses of healthy quiescent and early growing follicle numbers, showed that approximately one third of the peak endowment of immature follicles was lost by young adulthood. In contrast to previous studies in mammals, a parallel study of atretic (degenerating) follicles showed that atresia increased markedly by days 30 to 40, reaching a peak of more than 1200 dying follicles per ovary that continued well into reproductive life (Fig. 1). There was a clear discordance between changes in numbers of nonatretic follicles and the corresponding incidence of atresia in the postnatal mammalian ovary. Histologic studies demonstrated large ovoid cells that resembled germ cells of the fetal mouse ovary; they were found in the surface epithelial cell layer covering the ovary. Immunohistochemical studies affirmed that these cells were of a germline lineage. Immunostaining confirmed the presence of germ cells in varying stages of meiosis. The investigators identified genes whose expression is necessary for replicating germ cells to produce oocytes for follicle formation in postnatal life. In studies with busulphan, a germ-cell toxicant, exposed ovaries had fewer than 5% of the primordial follicle pool present in control animals. The rate of follicle loss was very close to that expected from previously reported data. Finally, ovarian fragments taken from wild-type mice were grafted into the ovaries of transgenic female siblings after removing approximately half of the host’s ovary. The transgenic germ cells infiltrated the grafted tissue and initiated folliculogenesis with the resident wild-type somatic cells. These observations suggest that GSCs are present in the postnatal mouse ovary. The findings have therapeutic potential for expanding the follicle reserve so as to postpone normal or premature ovarian failure.Fig. 1: Postnatal ovarian germ-cell dynamics. (A and B) Numbers of nonatretic (A) and atretic (B) primordial and total immature (primordial, primary, small preantral) follicles in mouse ovaries during postnatal development (B, total immature follicles; mean ± standard error, n = 3–4 mice per age group). (C) Incidence of primordial and primary follicle atresia in ovaries exposed to DMBA on day 25 postpartum (mean ± standard error, n = 4–5 mice per time point). (D) Comparison of nonatretic and atretic immature follicle numbers in C57BL/6, CD1, and AKR/J strains of mice (mean ± standard error, n = 3–5 mice per age group). Reproduced with permission. Nature 2004;428:145–150. Copyright © 2004, Nature Publishing Group. All rights reserved.

Germline stem cells and follicular renewal in the postnatal mammalian ovary

A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.

Targeted elimination of the follicular label-retaining cells by photo-induced cell killing caused a defect on follicular renewal on mice.

One of the most well-known ways to identify stem cells is to search for the slow-cycling cells (label-retaining cells; LRCs), by labelling their DNA. The bulge of hair follicle is assumed to identify the location of the follicular stem cells. While this assumption was supported by previous analyses of cell tracing, it is still not clear whether the LRCs are critical for follicular renewal. To solve this problem, we tried to selectively eliminate the bromodeoxyuridine (BrdU)-labelled follicular LRCs by a photo-induced cell killing method, in combination with Hoechst 33258 treatment. We labelled the slow-cycling cells in the follicular bulge by repeated administration of the BrdU, followed by a chase period. When the labelled mice skin was irradiated at the telogen phase after the Hoechst 33258 treatment, we observed apoptotic cells in the bulge area 18 h after the irradiation and a defect on the follicular renewal at the next anagen phase. The time course of appearance of hair defect suggests that only the late telogen follicle is sensitive to irradiation. This finding suggests that the LRCs contribute to the renewal of the hair follicle, and they might proliferate at a restricted point in the hair cycle to supply hair germinative cells as previously predicted.