Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice

Genes of the DAZ family play critical roles in germ cell development in mammals and other animals. In mice, Dazl mRNA is first observed at embryonic day 11.5 (E11.5), but previous studies using Dazl-deficient mice of mixed genetic background have largely emphasized postnatal spermatogenic defects. Using an inbred C57BL/6 background, we show that Dazl is required for embryonic development and survival of XY germ cells. By E14.5, expression of germ cell markers (Mvh, Oct4, Dppa3/Stella, GCNA and MVH protein) was reduced in XY Dazl-/- gonads. By E15.5, most remaining germ cells in XY Dazl-/- embryos exhibited apoptotic morphology, and XY Dazl-/- gonads contained increased numbers of TUNEL-positive cells. The rare XY Dazl-/- germ cells that persisted until birth maintained a nuclear morphology that resembled that of wildtype germ cells at E12.5-E13.5, a critical developmental period when XY germ cells lose pluripotency and commit to a spermatogonial fate. We propose that Dazl is required as early as E12.5-E13.5, shortly after its expression is first detected, and that inbred Dazl-/- mice of C57BL/6 background provide a reproducible standard for exploring Dazl's roles in embryonic germ cell development.     

Telomere DNA deficiency is associated with development of human embryonic aneuploidy

Aneuploidy represents the most prevalent form of genetic instability found in human embryos and is the leading genetic cause of miscarriage and developmental delay in newborns. Telomere DNA deficiency is associated with genomic instability in somatic cells and may play a role in development of aneuploidy commonly found in female germ cells and human embryos. To test this hypothesis, we developed a method capable of quantifying telomere DNA in parallel with 24-chromosome aneuploidy screening from the same oocyte or embryo biopsy. Aneuploid human polar bodies possessed significantly less telomere DNA than euploid polar bodies from sibling oocytes (−3.07 fold, P = 0.016). This indicates that oocytes with telomere DNA deficiency are prone to aneuploidy development during meiosis. Aneuploid embryonic cells also possessed significantly less telomere DNA than euploid embryonic cells at the cleavage stage (−2.60 fold, P = 0.002) but not at the blastocyst stage (−1.18 fold, P = 0.340). The lack of a significant difference at the blastocyst stage was found to be due to telomere DNA normalization between the cleavage and blastocyst stage of embryogenesis and not due to developmental arrest of embryos with short telomeres. Heterogeneity in telomere length within oocytes may provide an opportunity to improve the treatment of infertility through telomere-based selection of oocytes and embryos with reproductive competence.     

Expression of Fas,p53 and AFP in development of human fetal germ cells in vitro

In the present study we employed a two-step culture system to study the expression of Fas, p53 and alpha-fetoprotein (AFP) in the development in vitro of human fetal germ cells. p53 mRNA was determined by Northern blotting, and Fas content was assessed by western blotting. RT-nested polymerase chain reaction (RT-nPCR) analysis was performed to determine the expression of AFP mRNA in different stages of fetal follicular development. Follicular cell apoptosis was evaluated by DNA fragmentation analyses (DNA ladder). The results showed that by day 7 of culture approximately one-sixth of fetal germ cells grew to class C oocytes (primary oocytes) from class B oocytes (primordial oocytes) or class A oocytes. On day 45 of culture, one-third of these primary follicles doubled in size. In the meantime, there was a high proportion apoptosis of follicular cells on days 35 or 45 of culture, as evident by a clear ladder pattern of DNA fragmentation upon electrophoretic analysis. Expression of Fas antigen and p53 mRNA increased in a time-dependent manner, while AFP mRNA was expressed on days 10 to 35, and disappeared on day 45. These results indicate that human fetal germ cells can develop in a two-step culture system and AFP may play an active role in the proliferation of these germ cells. At the late stage of follicular development in vitro, a number of follicular cells became apoptotic. Moreover, apoptosis may be the mechanism responsible for fetal germ cell regression and the Fas antigen and/or p53-mediated death pathway may be central in the induction of germ cell regression.     

Analysis of factors decreasing testis weight in MRL mice

MRL/MpJ (MRL) mouse testes have several unique characteristics, including the appearance of oocytes, the occurrence of metaphase-specific apoptosis of meiotic spermatocytes, and the presence of heat-shock-resistant spermatocytes. In the present study we used chromosomal mapping to determine the genomic background associated with small testis size in MRL mice. We prepared and analyzed C57BL/6-based congenic mice carrying MRL mouse loci. Quantitative trait loci (QTL) analysis revealed susceptibility loci for small testis size at 100 cM on chromosome (Chr) 1 and at around 80 cM on Chr 2. Analysis with B6.MRLc1 and B6.MRLc2 congenic mice and double-congenic mice confirmed the QTL data and showed that low testis weight in MRL mice was caused by germ cell apoptosis. Through histological examinations we found that B6.MRLc1 and B6.MRLc2 mice showed stage-specific apoptosis in their testes, the former at metaphase stage XII and the later at pachytene stage IV. Metaphase-specific apoptosis of spermatocytes occurs due to mutation of the exonuclease 1 (Exo1) gene located at 100 cM on Chr 1. Thus, the mutation of the Exo1 gene is also responsible for low testis weight caused by metaphase-specific apoptosis. In conclusion, testis weight is reduced in MRL mice due to apoptosis of germ cells caused by mutations in loci on Chrs 1 and 2.     

Caspase-12 compensates for lack of caspase-2 and caspase-3 in female germ cells

Previously, we analyzed mice lacking either caspase-2 or caspase-3 and documented a role for caspase-2 in developmental and chemotherapy-induced apoptosis of oocytes. Those data also revealed dispensability of caspase-3, although we found this caspase critical for ovarian granulosa cell death. Because of the mutual interdependence of germ cells and granulosa cells, herein we generated caspase-2 and -3 double-mutant (DKO) mice to evaluate how these two caspases functionally relate to each other in orchestrating oocyte apoptosis. No difference was observed in the rate of spontaneous oocyte apoptosis between DKO and wildtype (WT) females. In contrast, the oocytes from DKO females were more susceptible to apoptosis induced by DNA damaging agents, compared with oocytes from WT females. This increased sensitivity to death of DKO oocytes appears to be a specific response to DNA damage, and it was associated with a compensatory upregulation of caspase-12. Interestingly, DKO oocytes were more resistant to apoptosis induced by methotrexate (MTX) than WT oocytes. These results revealed that in female germ cells, insults that directly interfere with their metabolic status (e.g. MTX) require caspase-2 and caspase-3 as obligatory executioners of the ensuing cell death cascade. However, when DNA damage is involved, and in the absence of caspase-2 and -3, caspase-12 becomes upregulated and mediates apoptosis in oocytes. Takai and Matikainen contributed equally to this work.     

Hypersensitivity of Primordial Germ Cells to Compromised Replication-Associated DNA Repair Involves ATM-p53-p21 Signaling

Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs), which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA), a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.     

Differential subcellular sequestration of proapoptotic and antiapoptotic proteins and colocalization of Bcl-x(L) with the germ plasm, in Xenopus laevis oocytes

Apoptosis is an important element of normal embryonic development and gametogenesis in invertebrate and vertebrate species. Although the components of apoptotic machinery are present in Xenopus laevis fully grown stage VI oocytes and eggs, apoptosis in the developing Xenopus ovary is limited to the somatic cells with no indication of apoptosis in the germ cells. Considering the possibility that Xenopus previtellogenic oocytes might lack the components of the apoptotic pathway, we analyzed Xenopus Stage I oocytes for the presence of the proapoptotic factors Bax and tumor suppressor p53, and antiapoptotic factors Bcl-x(L) and mitochondrial heat shock protein 60 (Hsp60). We found that pro- and antiapoptotic proteins are present in Xenopus oocytes but, surprisingly, they are located in distinct subcellular compartments with proapoptotic proteins Bax and p53 being sequestered in the oocyte nucleus and antiapoptotic protein Bcl-x(L) sequestered in the cytoplasm and highly enriched in the METRO region of the mitochondrial cloud, where it colocalized with the germ plasm, and Hsp60 colocalizing with all mitochondria. The absence of apoptosis in Xenopus early oogenesis is maybe due to differential sequestration of pro- and antiapoptotic molecules.     

Tools for the genetic analysis of germ cells

Germ cells are essential for the propagation of individual species. Studies on germ cell development in mice highlight important biological paradigms. Beginning with their first appearance around embryonic day 7 (E7), germ cells undergo specific cellular changes at different stages of their embryonic and adult development. Germ cells migrate through the hind‐regions of the embryo to eventually home into the developing gonads. Further differentiation and development of germ cells differ in males and females. The processes involved in germ cell development and their eventual differentiation into sperm and oocytes have been under extensive investigation in recent years. Studies on germ cells have shed light on the cellular and molecular processes involved in their specification, migration, proliferation, death, and differentiation. These studies have also revealed much about maintenance of stem cell populations and fertility. Here we review the genetic tools that are at present available to study germ cells in the mouse. genesis 47:617–627, 2009. © 2009 Wiley‐Liss, Inc.     

p53蛋白在周期调节蛋白A1变异引起的雄性小鼠生殖细胞凋亡中的作用

为研究p5 3蛋白在周期调节蛋白A1(cyclinA1)变异引起的雄性小鼠生殖细胞凋亡中的作用 ,以p5 3基因敲除的小鼠和周期调节蛋白A1基因敲除的小鼠杂交 ,获取同胎生单基因变异和双基因同时变异的雄性后代共 4组 12只 .比较它们的性腺和生殖细胞发育 ,并用TUNEL染色法观察和比较生殖细胞的凋亡情况 .在睾丸最大横切面上观察到 :周期调节蛋白A1变异组凋亡细胞最多 (348± 10 4个 ) ,明显高于p5 3 周期调节蛋白A1双基因变异组 (12 1± 38个 ) ,t=3 2 5 79,P =0 0 4 72 .p5 3变异组凋亡细胞最少 (45± 2 4个 ) ,配对t检验显示有非常显著性差异 ,t=8 4 0 13,P =0 0 0 35 .这一研究结果提示 ,p5 3基因可能在雄性生殖细胞的发育中起监视作用 ,并在周期调节蛋白A1变异引起发育异常时启动p5 3途径造成异常细胞的凋亡 .     

A phenotypic spectrum of sexual development in Dax1 (Nr0b1)-deficient mice: consequence of the C57BL/6J strain on sex determination

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1; hereafter referred to as Dax1) is an orphan nuclear receptor that regulates adrenal and gonadal development. Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (Dax1) mutations in the mouse are sensitive to genetic background. In this report, a spectrum of impaired gonadal differentiation was observed as a result of crossing the Dax1 knockout on the 129SvIm/J strain onto the C57BL/6J strain over two generations of breeding. Dax1-mutant XY mice of a mixed genetic background (129;B6Dax1(-/Y) [101 total]) developed gonads that were predominantly testislike (n = 61), ovarianlike (n = 27), or as intersex (n = 13). During embryonic development, Sox9 expression in the gonads of 129;B6Dax1(-/Y) mutants was distributed across a wide quantitative range, and a threshold level of Sox9 (>0.4-fold of wild-type) was associated with testis development. Germ cell fate also varied widely, with meiotic germ cells being more prevalent in the ovarianlike regions of embryonic gonads, but also observed within testicular tissue. Ptgds, a gene associated with Sox9 expression and Sertoli cell development, was markedly downregulated in Dax1(-/Y) mice. Stra8, a gene associated with germ cell meiosis, was upregulated in Dax1(-/Y) mice. In both cases, the changes in gene expression also occurred in pure 129 mice but were amplified in the B6 genetic background. Sertoli cell apoptosis was prevalent in 129;B6Dax1(-/Y) gonads. In summary, Dax1 deficiency on a partial B6 genetic background results in further modulation of gene expression changes that affect both Sertoli cell and germ cell fate, leading to a phenotypic spectrum of gonadal differentiation.     

Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro.

Members of the Bcl-2 family serve as central checkpoints for cell death regulation, and overexpression of Bcl-2 is known to inhibit apoptosis in many cell types. To determine whether targeted expression of Bcl-2 could be used to protect female germ cells from apoptosis, we generated transgenic mice expressing fully functional human Bcl-2 protein only in oocytes. Transgenic mice were produced using a previously characterized 480-bp fragment of the mouse zona pellucida protein-3 (ZP3) gene 5'-flanking region to direct oocyte-specific expression of a human bcl-2 complementary DNA. Immunohistochemical analyses using a human Bcl-2-specific antibody showed that transgene expression was restricted to growing oocytes and was not observed in the surrounding ovarian somatic cells or in any other nonovarian tissues. Histomorphometric analyses revealed that ovaries collected from transgenic female mice possessed significantly fewer atretic small preantral follicles compared with wild-type sisters, resulting in a larger population of healthy maturing follicles per ovary. However, the number of oocytes ovulated in response to exogenous gonadotropin priming and the number of pups per litter were not significantly different among wild-type vs. transgenic female mice. Nonetheless, oocytes obtained from transgenic mice and cultured in vitro were found to be resistant to spontaneous and anticancer drug-induced apoptosis. We conclude that targeted expression of Bcl-2 only in oocytes can be achieved as a means to convey resistance of the female germ line to naturally occurring and chemotherapy-induced apoptosis.     

Generation of functional oocytes and spermatids from fetal primordial germ cells after ectopic transplantation in adult mice

Primordial germ cells (PGCs) are undifferentiated germ cells in developing fetuses. As these cells give rise to definitive oocytes and spermatozoa that contribute to new life in the next generation, their development must be under strict control, regarding genetic and epigenetic aspects. However, we do not know to what extent their development depends on the specific milieu. In this study, we transplanted mouse PGCs collected from male and female gonads at 12.5 days postcoitum, together with gonadal somatic cells, under kidney capsules of adult mice. The transplanted PGC and gonadal somatic cells constructed testis-like and ovary-like tissues, respectively, under the kidney capsules within 4 wk. Normal-appearing round spermatids and fully grown germinal vesicle (GV) oocytes developed within these tissues. Ectopic spermatogenesis continued thereafter, while oogenesis consisted of only a single wave. The injection of these round spermatids directly into mature in vivo-derived oocytes led to the birth at term of normal pups. PGC-derived GV oocytes were isolated, induced to mature in vitro, and injected with normal spermatozoa. The injected oocytes were successfully fertilized and developed into normal pups. Our findings demonstrate the remarkable flexibility of PGC development, which can proceed up to the functional gamete stage under spatially and temporally noninnate conditions. This transplantation system may provide a unique technical basis for induction of the development of early germ cells of exogenous origins, such as those from embryonic stem cells.     

Developmental potential of mouse primordial germ cells

There are distinctive and characteristic genomic modifications in primordial germ cells that distinguish the germ cell lineage from somatic cells. These modifications include, genome-wide demethylation, erasure of allele-specific methylation associated with imprinted genes, and the re-activation of the X chromosome. The allele-specific differential methylation is involved in regulating the monoallelic expression, and thus the gene dosage, of imprinted genes, which underlies functional differences between parental genomes. However, when the imprints are erased in the germ line, the parental genomes acquire an equivalent epigenetic and functional state. Therefore, one of the reasons why primordial germ cells are unique is because this is the only time in mammals when the distinction between parental genomes ceases to exist. To test how the potentially imprint-free primordial germ cell nuclei affect embryonic development, we transplanted them into enucleated oocytes. Here we show that the reconstituted oocyte developed to day 9.5 of gestation, consistently as a small embryo and a characteristic abnormal placenta. The embryo proper also did not progress much further even when the inner cell mass was 'rescued' from the abnormal placenta by transfer into a tetraploid host blastocyst. We found that development of the experimental conceptus was affected, at least in part, by a lack of gametic imprints, as judged by DNA methylation and expression analysis of several imprinted genes. The evidence suggests that gametic imprints are essential for normal development, and that they can neither be initiated nor erased in mature oocytes; these properties are unique to the developing germ line.     

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.     

范可尼贫血基因在卵泡发育中的调节作用

范可尼贫血(Fanconi anemia, FA)是一种罕见的常染色体或X染色体连锁的隐性遗传病,其发生源于范可尼贫血基因(FA基因)突变。FA基因是一组在DNA交联损伤中起同源重组修复作用的基因。FA女性患者常见早发性卵巢功能衰退(premature ovarian insufficient, POI)的特征,而FA小鼠也表现出生殖细胞严重缺乏,这些结果提示FA基因在哺乳动物卵泡发育中起重要作用。研究显示FA基因在促进原始生殖细胞增生,维持正常卵母细胞减数分裂,参与卵泡发育的促性腺激素调节以及卵母细胞与颗粒细胞生长过程中的相互调节等方面调节卵泡发育。本文综述了FA基因在卵泡发育中的作用和分子机制方面的研究进展,为POI的病因学解析提供遗传基础。     

Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption

Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints.     

Deficiency of Bloom's syndrome protein causes hypersensitivity of C. elegans to ionizing radiation but not to UV radiation, and induces p53-dependent physiological apoptosis

Caenorhabditis elegans him-6 mutants, which show a high incidence of males and partial embryonic lethality, are defective in the orthologue of human Bloom's syndrome protein (BLM). When strain him-6(e1104) containing a missense him-6 mutation was irradiated with gamma-rays during germ cell development or embryogenesis, embryonic lethality was higher than in the wild type, suggesting a critical function of the wild type gene in mitotic and pachytene stage germ cells as well as in early embryos. Even in the absence of gamma-irradiation, apoptosis was elevated in the germ cells of the him-6 strain and this increase was dependent on a functional p53 homologue (CEP-1), suggesting that spontaneous DNA damage accumulates due to him-6 deficiency. However, induction of germline apoptosis by ionizing radiation was not significantly affected by the deficiency, indicating that HIM-6 has no role in the induction of apoptosis by exogenous DNA damage. We conclude that the C. elegans BLM orthologue is involved in DNA repair in promeiotic cells undergoing homologous recombination, as well as in actively dividing germline and somatic cells.     

Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.     

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.