Activation of follicle development: the primordial follicle

Investigations of primordial follicle formation and growth are fundamental to our understanding of female gamete production. In all mammalian females the full complement of oocytes is established during fetal development. This store of primordial follicles is not renewable and serves the entire reproductive life span of the adult. The correct programming of fetal ovarian development and the number of primordial follicles formed will therefore limit the fecundity of the ovary. Primordial follicles are characterized by the presence of a single oocyte surrounded by a varying number of pregranulosa cells. The relatively small size, undifferentiated status and large numbers of primordial follicles make them prime candidates for use in basic and applied research in animal production, gene transfer and cloning. Furthermore, the development of cell culture systems that use primordial follicles as a source of oocytes for in vitro growth and maturation will enable us to maximize the potential of high genetic merit females and to shorten generation intervals. Despite these possibilities, primordial follicles are the least understood of all stages of follicle development. The factor(s) responsible for maintaining the primordial pool or, conversely, for activating primordial follicle growth remain elusive.     

Formation of ovarian follicles during fetal development in sheep

The origin of follicle (i.e., pregranulosa) cells that become the somatic component of primordial follicles is obscure. In addition, information regarding the structural changes that accompany the concomitant regression of ovigerous cords and the appearance of primordial follicles is lacking. In the present study, ovine ovaries collected at frequent time intervals between Day 38 and Day 100 of fetal life were examined by light and electron microscopy. To gain new information regarding the origin of follicular cells, incorporation of 5-bromo-2'-deoxyuridine was used to identify proliferating cells at selected stages of development. Based on the location and identity of proliferating cells, apoptotic cells, and sequential changes in histoarchitecture, we hypothesize 1) that most (i.e., >95%) of the granulosal cells in newly formed primordial follicles originate from the ovarian surface epithelium; 2) that the sequential events leading to follicle formation take place entirely within ovigerous cords, with the first follicles forming at the interface of the cortex and medulla; and 3) that the loss (i.e., >75%) of germ cells, but not of somatic cells, within the ovigerous cords is a means by which each surviving oocyte gains additional pregranulosal cells before follicle formation. Conceptual models detailing the chronology of developmental events involved in the formation of primordial follicles in sheep are discussed.     

Live offspring produced by mouse oocytes derived from premeiotic fetal germ cells

Mature mouse oocytes currently can be generated in vitro from the primary oocytes of primordial follicles but not from premeiotic fetal germ cells. In this study we established a simple, efficient method that can be used to obtain mature oocytes from the premeiotic germ cells of a fetal mouse 12.5 days postcoitum (dpc). Mouse 12.5-dpc fetal ovaries were transplanted under the kidney capsule of recipient mice to initiate oocyte growth from the premeiotic germ cell stage, and they were recovered after 14 days. Subsequently, the primary and early secondary follicles generated in the ovarian grafts were isolated and cultured for 16 days in vitro. The mature oocytes ovulated from these follicles were able to fertilize in vitro to produce live offspring. We further show that the in vitro fertilization offspring were normal and able to successfully mate with both females and males, and the patterns of the methylated sites of the in vitro mature oocytes were similar to those of normal mice. This is the first report describing premeiotic fetal germ cells able to enter a second meiosis and support embryonic development to term by a combination of in vivo transplantation and in vitro culture. In addition, we have shown that the whole process of oogenesis, from premeiotic germ cells to germinal vesicle (GV)-stage oocytes, can be carried out under the kidney capsule.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.     

The capacity of primordial follicles in fetal bovine ovaries to initiate growth in vitro develops during mid-gestation and is associated with meiotic arrest of oocytes

In cattle and other species in which the pool of resting, primordial follicles is formed during fetal life, little is known about the regulation of the early stages of ovarian follicular development. We used histological morphometry and a combination of observations in vivo and experiments in vitro to study the timing and regulation of follicle formation and the acquisition of the capacity of primordial follicles to initiate growth in cattle. In vivo, primordial, primary, and secondary follicles were first observed around Days 90, 140, and 210 of gestation, respectively. The long interval between the first appearance of primordial and primary follicles suggests that primordial follicles are not capable of activating when they are first formed, or they are inhibited from activating. This hypothesis was confirmed by the finding that most primordial follicles in pieces of ovarian cortex obtained from fetal ovaries older than 140 days activated (i.e., initiated growth) after 2 days in vitro, whereas follicles in cortical pieces from 90- to 140-day-old fetal ovaries did not. We tested the hypothesis that the oocytes of newly formed primordial follicles are not in meiotic arrest and found that before Day 141, most oocytes ( approximately 73%) were in prediplotene stages of prophase I, whereas after Day 140, the majority of oocytes ( approximately 85%) had arrested at the diplotene stage. This observation was further confirmed by the finding that levels of mRNA for YBX2, a protein associated with meiotic arrest, were 2.3 times higher in ovarian cortical pieces isolated after versus before Day 141. Primordial follicles in cortical pieces from 90- to 140-day-old fetal ovaries did activate during a longer, 10-day culture, but activation could be inhibited by adding estradiol or progesterone, but not dihydrotestosterone (all at 10(-6) M). Fetal ovaries secreted estradiol in vitro, and secretion by ovaries from 83 to 140-day-old fetuses declined precipitously ( approximately 30-fold) with age, consistent with the hypothesis that estradiol inhibits activation of newly formed primordial follicles in vivo. In summary, the results show that newly formed primordial follicles do not activate in vivo or within 2 days in vitro and that capacity to activate is correlated with achievement of meiotic arrest by the oocyte and can be inhibited by estradiol, which fetal ovaries actively produce around the time of follicle formation.     

The role of neurotrophin receptors in female germ-cell survival in mouse and human

During mammalian ovary formation, the production of ovarian follicles is accompanied by an enormous loss of germ cells. It is not known how this loss is regulated. We have investigated the role of the Trk tyrosine kinase receptors, primarily TrkB, in this process. The ovaries of TrkB-/- and TrkC-/- mice with a mixed (129Sv x C57BL/6) genetic background were examined shortly after birth. Around 50% of TrkB-/- mice had grossly abnormal ovaries that contained greatly reduced numbers of follicles. No defects were found in the ovaries of TrkC-/- mice. Congenic TrkB-/- mice were generated on 129Sv and C57BL/6 backgrounds: whereas the former had a mixed ovarian phenotype similar to that of the original colony of mice, the ovaries of all offspring of the C57BL/6 congenic line contained reduced numbers of follicles. RT-PCR showed that mRNA encoding TrkB and its two ligands, neurotrophin 4 (NT4) and brain-derived neurotrophic factor (BDNF), were present throughout the period of follicle formation in the mouse. In situ hybridisation showed that TrkB was expressed primarily in the germ cells before and after follicle formation. Mouse neonatal and fetal ovaries and human fetal ovaries were cultured in the presence of K252a, a potent inhibitor of all Trk receptors. In mice, K252a inhibited the survival of germ cells in newly formed (primordial) follicles. This effect was rescued by the addition of basic fibroblast growth factor (bFGF) to the culture medium. Combined addition of both BDNF and NT4 blocking antibodies lowered germ-cell survival, indicating that these TrkB ligands are required in this process. The results indicate that signalling through TrkB is an important component of the mechanism that regulates the early survival of female germ cells.     

In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation

As cancer treatment improves, more young women of reproductive age are surviving, but they suffer from infertility as a consequence of the radiation and chemotherapy. Human ovarian tissue containing immature primordial follicles has been successfully cryopreserved. The ultimate aim of this technique is to induce ovarian function by re-plantation of ovarian tissue or, further into the future, by in vitro maturation (IVM) of the oocytes derived from the cryopreserved-thawed ovarian tissue, followed by routine in vitro fertilization. IVM of primordial follicles from young cancer survivors would avoid the risk of cancer re-transmission by the ovarian grafts. The present review discusses the current achievements in IVM of female germ cells and primordial ovarian follicles and the attempts to improve their development by adding various factors to the culture medium. The established methods for the evaluation of survival and growth in culture are also discussed: follicular counts, immunocytochemical methods, transmission electron microscopy, fluorescent viability markers and endocrine assays. Although the development of IVM systems is still in its infancy, researchers need to pursue their approach step-by-step, especially with regard to factors that might be involved in the activation of the ovarian follicles or female germ cells. The final measure of success will be the ability of the in vitro matured oocytes to fertilize and produce healthy offsprings. The availability of such treatment will probably lead to its demand not only by cancer patients but by other women as well.     

Lanosterol metabolic product(s) is involved in primordial folliculogenesis and establishment of primordial follicle pool in mouse fetal ovary

The primordial follicles present in neonatal ovary represent the fecundity of a female throughout her reproductive life. Germ cell meiosis and apoptosis are two important events during primordial folliculogenesis. In this study, through focusing on the cytochrome P450 lanosterol 14 alphademethylase (CYP51) and its lanosterol metabolic product(s), we explored the possible regulatory mechanism of the initiation of germ cell meiosis and primordial follicle formation. The expression of CYP51 could be detected in both oocytes and granulosa cells during primordial folliculogenesis by immunochemistry. RS21745, which leads to the reduction of lanosterol metabolic product(s) level, inhibited the primordial follicle formation in a dose-dependent manner, and thus postpone the establishment of the primordial follicle pool when the mouse fetal ovaries were cultured in serum-free medium. In contrast, the number of primordial follicle increased significantly with the accumulation of the lanosterol metabolic products caused by 0.025, 0.0625, and 0.125 microM AY9944-A-7 supplements. AY9944-A-7 also up-regulated the expression of meiotic diplotene stage marker gene msy2 and primordial follicle formation regulatory gene fig-alpha. Furthermore, AY9944-A-7 decreased the expression of apoptosis gene bax and significantly prevented oocyte apoptosis from 15.37 +/- 1.97% to 3.68 +/- 0.27% (P < 0.01) in neonatal ovary in vitro. In conclusion, our results indicate that lanosterol metabolic product(s) is involved in the primordial folliculogenesis by regulating the oocyte meiosis and apoptosis.     

Follistatin288 Regulates Germ Cell Cyst Breakdown and Primordial Follicle Assembly in the Mouse Ovary

In mammals, the primordial follicle pool represents the entire reproductive potential of a female. The transforming growth factor-β (TGF-β) family member activin (ACT) contributes to folliculogenesis, although the exact mechanism is not known. The role of FST288, the strongest ACT-neutralizing isoform of follistatin (FST), during cyst breakdown and primordial follicle formation in the fetal mice ovary was assessed using an in vitro culture system. FST was continuously expressed in the oocytes as well as the cuboidal granulosa cells of growing follicles in perinatal mouse ovaries. Treatment with FST288 delayed germ cell nest breakdown, particularly near the periphery of the ovary, and dramatically decreased the percentage of primordial follicles. In addition, there was a dramatic decrease in proliferation of granulosa cells and somatic cell expression of Notch signaling was impaired. In conclusion, FST288 impacts germ cell nest breakdown and primordial follicle assembly by inhibiting somatic cell proliferation.     

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage.     

Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development

The cryopreservation of ovarian tissue has been reported to affect the development of preantral follicles. However, the effect of cryopreservation of ovarian tissue on the development of primordial follicles remains to be elucidated. This study was conducted to evaluate the effect of cryopreservation on the development of frozen-thawed mouse primordial follicles. One-day-old mouse ovaries were cryopreserved by either slow-freezing or a vitrification method. The development of primordial follicles was evaluated histologically and also with markers for follicle development such as: GDF-9, inhibin-alpha subunit and ZP3 in fresh and frozen-thawed ovaries cultured for five days. The proportion of apoptotic and necrotic areas was analyzed in fresh and frozen-thawed ovaries at one and five days after culture, in order to examine the viability of ovarian cells that influence primordial follicle development. The development rate of primordial follicles was significantly lower in slow-frozen and vitrified ovaries than the fresh controls after five days of in vitro culture (P<0.05). The mRNA expression for all developmental markers was slightly decreased in the frozen-thawed ovaries; this difference was not significant. The proportion of apoptosis was significantly increased in the slow-frozen and vitrified ovaries compared to the fresh ovaries at one day (P<0.05); however, there was no difference at five days after culture. The proportion of the area of necrosis was significantly higher in slow-frozen and vitrified ovaries compared to the fresh ovaries at one and five days after culture (P<0.05). Our preliminary data suggest that ovarian tissue cryopreservation using slow-freezing and vitrification methods inhibits development of primordial follicles. This may be caused by the death of ovarian cells through apoptosis and necrosis after cryopreservation.     

Germ cell development in Meishan and White Composite gilts

This study compared dynamics of the germ cell population in two swine breeds that differ in prolifacy, White Composite (WC) and Meishan (MS), during fetal and neonatal life and in mature sows. Germ cell populations developed in a similar pattern in these two diverse breeds during fetal life. Maximal germ cell number was observed at 90 days postcoitum (dpc) in both WC and MS gilts, and substantial oogonial apoptosis was evident thereafter with approximately 30% of maximal numbers present at 25 days postpartum (dpp). Neither gilt nor sow germ cell number was correlated with maternal ovulation rate. Postnatal MS gilts had larger pools of primordial follicles and consistently greater proportions and numbers of primary and secondary follicles compared to postnatal WC gilts, indicative of enhanced follicular recruitment and primordial follicle activation. Occasional antral follicles were present in MS ovaries by 25 dpp and numerous surface follicles were observed at 56 dpp in MS but not WC ovaries, indicative of more rapid ovarian maturation and early onset of puberty. Total germ cell number is unlikely to influence or to predict subsequent ovulation rate. These observations highlight important developmental events during late fetal and early postnatal life that prepare the ovarian environment for early onset of puberty and subsequent ovulation in MS gilts.     

Consequences of fetal irradiation on follicle histogenesis and early follicle development in rat ovaries

Follicle histogenesis, in which follicles arise from fragmenting ovigerous cords, is a poorly understood mechanism that is strictly dependent upon the presence of germ cells. Our previous studies have shown that severely germ cell-depleted rat ovaries after fetal gamma-irradiation display modifications of follicular endowment and dynamics during the immature period. The primordial follicle stock was absent and the follicles with primary appearance remained quiescent longer than in control ovaries during the neonatal period. The aim of the present work was to analyze the initial steps of follicle histogenesis, and to investigate the etiology of the alterations observed in the development of irradiated ovaries. Just after birth, we observed, in addition to sterile ovigerous cords, the emergence of the first follicles which exhibited several abnormal features as compared to those of control ovaries. Most of the follicles appeared as primary follicles, as they were composed of a layer of cuboidal-shaped granulosa cells surrounding an enlarged oocyte. Interestingly, the granulosa cells of these primary-like follicles did not proliferate and did not express the genes for anti-Müllerian hormone (Amh) or bone morphogenetic protein receptor type II (Bmpr2), both of which are normally expressed from the primary stage onwards. In contrast, the oocytes strongly expressed the gene for growth and differentiation factor 9 (Gdf9), which is normally upregulated from the primary follicle stage onwards, which suggests an uncoupling of granulosa cell development from oocyte development. In addition, irradiated ovaries displayed a higher frequency of follicles that contained 2 or 3 oocytes, which are also referred to as multi-oocyte follicles (MOFs). Examination at the time of follicle histogenesis indicated that MOFs arise from incomplete ovigerous cord breakdown. Taken together, the results of this study indicate that severe perturbations of follicular histogenesis take place following irradiation and massive germ cell depletion during fetal life. In addition to the classically described sterile cords, we have pointed out the differentiation of MOFs and primary-like quiescent follicles, which finally evolve into growing follicles and participate in ovarian function. We propose that these phenotypes are closely correlated to the proportion of granulosa cells to oocytes at the time of neonatal follicle histogenesis.     

Primordial follicle activation and follicular development in the juvenile rabbit ovary

Of all the stages of mammalian folliculogenesis, the primordial to primary follicle transition is the least understood. In order to gain new insights into this process, we have conducted a comprehensive morphological, morphometric and molecular study of ovarian organisation and early follicle development in the rabbit. The structure of ovaries collected from rabbits aged from 2–12 weeks (a period encompassing primordial follicle formation, activation and the first wave of folliculogenesis in this species) has been analysed by light microscopy and the follicles present have been measured and scored for their developmental stage. To establish useful molecular markers of activation, we have further classified follicles according to their expression of the proliferative marker, proliferating cell nuclear antigen, and the zona pellucida protein, ZPB. The activation of primordial follicles is initiated immediately following their formation in the rabbit ovary and is characterised by oocyte growth, granulosa cell morphogenesis and increased granulosa cell mitosis. Enhanced ZPB protein expression at the oolemma is also associated with follicle activation and development. Few primordial follicles in the juvenile rabbit ovary are lost by atresia, as assessed by the TUNEL assay. The appearance of apoptotic granulosa cells is however coincident with the development of antral follicles. This study thus describes the temporal and spatial regulation of early follicular development in the post-natal rabbit ovary and, for the first time, shows that the primordial to primary transition in the juvenile rabbit is a highly ordered process occurring within quantifiable parameters.K.J.H. was supported by the Pest Animal Control CRC and Post Graduate scholarships from the Australian National University.     

Growth differentiation factor-9 and stem cell factor promote primordial follicle formation in the hamster: modulation by follicle-stimulating hormone

Growth differentiation factor-9 (GDF-9) and stem cell factor (SCF) influence follicle formation beyond the primary stage; however, factors influencing the formation of primordial follicles remain elusive. To determine whether GDF-9 and SCF promoted primordial follicle formation during ovarian morphogenesis in the hamster, and whether FSH had any modulatory influence, fetal ovaries were collected on Gestation Day 15 from pregnant hamsters treated with or without an FSH antiserum on Gestation Day 12 and cultured in vitro up to Day 9 with SCF, GDF-9, or FSH. The percentages and diameters of primordial, primary, and secondary follicles and their oocytes were determined by morphometric evaluation, and the expression of GDF-9 was detected by immunolocalization. SCF, GDF-9, and FSH promoted primordial and primary follicle formation, but GDF-9 was more efficient. The diameters of the follicles developed under GDF-9 or FSH, but not SCF, compared well with those developed in vivo. FSH- and GDF-9-induced folliculogenesis was attenuated by the SCF antibody. Similarly, in vitro formation of primordial follicles decreased markedly in ovaries exposed to the FSH antiserum in utero, which was reversed by SCF, GDF-9, or FSH; however, GDF-9 had a profound effect on follicular development. GDF-9 protein appeared exclusively in the oocytes on Postnatal Day 4; however, it appeared in vitro by 48 h, and the expression was upregulated by FSH. These results suggest that although SCF-induced primordial follicle formation constitutes primarily somatic cell development, GDF-9 influences both the oocyte and its companion somatic cells. FSH plays an important role in primordial folliculogenesis in the hamster via GDF-9 and SCF.     

Prostaglandin signaling and ovarian follicle development in the silkmoth, Bombyx mori

Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.     

Primordial follicle assembly was regulated by notch signaling pathway in the mice

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.     

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary

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.