ABSTRACT
In the male germline, the machinery to repress retrotransposons that threaten genomic integrity via the piRNA pathway is established in gonocytes. It has been reported that disruption of the piRNA pathway leads to activation of retrotransposons and arrests spermatogenesis before it enters the second meiosis; however, its effects on gonocytes have not been fully elucidated. In this study, we analyzed the effects of Asz1 deletion, which is a crucial component of the piRNA pathway, on the gonocyte transcriptome. In Asz1-null gonocytes, MIWI2, which is responsible for introducing DNA methylation to retrotransposons in a piRNA-dependent manner, disappeared from the nuclei of fetal gonocytes. Transcriptome analysis revealed that retrotransposons targeted by the piRNA pathway and non-annotated transcript variants were upregulated in gonocytes from neonatal Asz1-/- mice. These non-annotated transcript variants were chimeras generated by joining exons transcribed from retrotransposons and canonical genes. DNA methylation analysis showed that retrotransposons that induce the expression of aberrant chimeric transcripts are not fully methylated. This was consistent with the impaired nuclear localization of MIWI2 in Asz1-null gonocytes. Furthermore, heterogeneity of DNA methylation status in retrotransposons was observed in both gonocytes and their descendants. This suggests that the piRNA system in gonocytes can potentially prevent spermatogenic cell populations bearing aberrant chimeric transcripts from propagating later in spermatogenesis. In conclusion, Asz1 is required to repress retrotransposons and retrotransposon-driven aberrant chimeric transcripts in gonocytes through the piRNA pathway.
Subject(s)
Adaptor Proteins, Signal Transducing , Germ Cells , Spermatogenesis , Adaptor Proteins, Signal Transducing/genetics , Animals , Argonaute Proteins/genetics , Chimera , Gene Deletion , Germ Cells/metabolism , Male , Mice , RNA, Small Interfering/metabolism , Retroelements , Spermatogenesis/genetics , Testis/metabolismABSTRACT
In vitro systems capable of reconstituting the process of mouse oogenesis are now being established to help develop further understanding of the mechanisms underlying oocyte/follicle development and differentiation. These systems could also help increase the production of useful livestock or genetically modified animals, and aid in identifying the causes of infertility in humans. Recently, we revealed, using an in vitro system for recapitulating oogenesis, that the activation of the estrogen signaling pathway induces abnormal follicle formation, that blocking estrogen-induced expression of anti-Müllerian hormone is crucial for normal follicle formation, and that the production of α-fetoprotein in fetal liver tissue is involved in normal in vivo follicle formation. In mouse fetuses, follicle formation is not carried out by factors within the ovaries but is instead orchestrated by distal endocrine factors. This review outlines findings from genetics, endocrinology, and in vitro studies regarding the factors that can affect the formation of primordial follicles in mammals.
Subject(s)
Anti-Mullerian Hormone , Ovarian Follicle , Animals , Anti-Mullerian Hormone/metabolism , Anti-Mullerian Hormone/pharmacology , Female , Mammals/metabolism , Mice , Oocytes/metabolism , Oogenesis , Ovarian Follicle/metabolism , Ovary/metabolismABSTRACT
Mammalian ovaries contain a large number of immature follicles. Follicular culture can contribute to the production of fertile oocytes from latent immature follicles, providing a useful tool for exploring the developmental competencies and related factors that oocytes acquire during growth. However, the potential of oocytes produced by follicular culture is limited. Herein, the optimal follicular culture conditions for the addition of polyvinylpyrrolidone to the medium and oxygen concentration were investigated. Polyvinylpyrrolidone with a high molecular weight (≥ 360,000) and a 7% oxygen concentration were found to increase the blastocyst formation rate by more than 20% compared with conventional culture conditions. Although the developmental ability of oocytes produced by follicular culture remained inferior to that of in vivo-derived oocytes, these findings may pave the way for enhanced production of fertile oocytes in vitro and for studying the process of full developmental potency acquisition by oocytes.
Subject(s)
Culture Techniques , Oocytes/growth & development , Ovarian Follicle/drug effects , Povidone/administration & dosage , Animals , Female , MiceABSTRACT
In vitro generation of fertile oocytes has been reported in several mammalian species. However, oocyte integrity is compromised by in vitro culture. Here, we aimed to understand the factors affecting oocyte competency by evaluating mitochondrial function and transcriptome as well as lipid metabolism in in vivo-derived oocytes and in vitro grown and matured (IVGM) oocytes under atmospheric (20%) and physiological (7%) O2 concentration. We used single-cell RNA-sequencing as well as Gene Ontology and KEGG analyses to identify the molecular pathways affecting the developmental competence of oocytes. Oocytes grown under 20% O2 conditions showed a significant decrease in mitochondrial membrane potential, upregulation of ceramide synthesis pathway-associated genes, and high ceramide accumulation compared with oocytes grown under 7% O2 conditions and in vivo-grown oocytes. This suggests that excess ceramide level causes mitochondrial dysfunction and poor developmental ability of the oocytes. Mitochondrial DNA copy number was lower in IVGM oocytes irrespective of O2 concentration in culture, although there was no common abnormality in the expression of genes related to mitochondrial biosynthesis. In contrast, some oocytes produced under 7% O2 conditions showed gene expression profiles similar to those of in vivo-grown oocytes. In these oocytes, the expression of transcription factors, including Nobox, was restored. Nobox expression correlated with the expression of genes essential for oocyte development. Thus, Nobox may contribute to the establishment of oocyte competency before and after the growth phase. The comprehensive analysis of IVGM oocytes presented here provides a platform for elucidating the mechanism underlying functional oocyte production in vivo.
Subject(s)
Oocytes , Transcriptome , Animals , In Vitro Oocyte Maturation Techniques , Membrane Potential, Mitochondrial , Mice , Mitochondria/metabolism , OogenesisABSTRACT
Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.
Subject(s)
Oocytes/metabolism , Ovarian Follicle/metabolism , Animals , Female , Ovarian Follicle/cytology , ZebrafishABSTRACT
Conditional knockout technology is a powerful tool for investigating the spatiotemporal functions of target genes. However, generation of conditional knockout mice involves complicated breeding programs and considerable time. A recent study has shown that artificially designed microRNAs (amiRNAs), inserted into an intron of the constitutively expressed gene, induce knockdown of the targeted gene in mice, thus creating a simpler method to analyze the functions of target genes in oocytes. Here, to establish an oocyte-specific knockdown system, amiRNA sequences against enhanced green fluorescent protein (EGFP) were knocked into the intronic sites of the Zp3 gene. Knock-in mice were then bred with EGFP transgenic mice. Our results showed that Zp3-derived amiRNA successfully reduced EGFP fluorescence in the oocytes in a size-dependent manner. Importantly, knockdown of EGFP did not occur in somatic cells. Thus, we present our knockdown system as a tool for screening gene functions in mouse oocytes.
Subject(s)
MicroRNAs/metabolism , Oocytes/metabolism , Zona Pellucida Glycoproteins/metabolism , Animals , Gene Editing , Gene Knockdown Techniques , Introns , Mice , MicroRNAs/genetics , Zona Pellucida Glycoproteins/geneticsABSTRACT
In mammals, primordial follicles assembled in fetuses or during infancy constitute the oocyte resources for life. Exposure to 17beta-estradiol and phytogenic or endocrine-disrupting chemicals during pregnancy and/or the perinatal period leads to the failure of normal follicle formation. However, the mechanisms underlying estrogen-mediated abnormal follicle formation and physiological follicle formation in the presence of endogenous natural estrogen are not well understood. Here, we reveal that estrogen receptor 1, activated by estrogen, binds to the 5' region of the anti-Mullerian hormone (Amh) gene and upregulates its transcription before follicle formation in cultured mouse fetal ovaries. Ectopic expression of AMH protein was observed in pregranulosa cells of these explants. Furthermore, the addition of AMH to the culture medium inhibited normal follicle formation. Conversely, alpha-fetoprotein (AFP) produced in the fetal liver reportedly blocks estrogen action, although its role in follicle formation is unclear. We further demonstrated that the addition of AFP to the medium inhibited ectopic AMH expression via estrogen, leading to successful follicle formation in vitro Collectively, our in vitro experiments suggest that upon estrogen exposure, the integrity of follicle assembly in vivo is ensured by AFP.
Subject(s)
Anti-Mullerian Hormone/genetics , Estrogen Receptor alpha/genetics , Ovarian Follicle/growth & development , alpha-Fetoproteins/genetics , Animals , Endocrine Disruptors/toxicity , Estradiol/pharmacology , Estrogens/genetics , Estrogens/metabolism , Female , Humans , Mice , Oocytes/growth & development , Ovarian Follicle/metabolism , Transcription, Genetic/geneticsABSTRACT
RNA aptamers have garnered attention for diagnostic applications due to their ability to recognize diverse targets. Oligomers of 42-mer amyloid ß-protein (Aß42), whose accumulation is relevant to the pathology of Alzheimer's disease (AD), are among the most difficult molecules for aptamer recognition because they are prone to aggregate in heterogeneous forms. In addition to designing haptens for in vitro selection of aptamers, the difficulties involved in determining their effect on Aß42 oligomerization impede aptamer research. We previously developed three RNA aptamers (E22P-AbD4, -AbD31, and -AbD43) with high affinity for protofibrils (PFs) derived from a toxic Aß42 dimer. Notably, these aptamers recognized diffuse staining, which likely originated from PFs or higher-order oligomers with curvilinear structures in a knock-in AppNL-G-F/NL-G-F mouse, carrying the Arctic mutation that preferentially induced the formation of PFs, in addition to a PS2Tg2576 mouse. To determine which oligomeric sizes were mainly altered by the aptamer, ion mobility-mass spectrometry (IM-MS) was carried out. One aptamer, E22P-AbD43, formed adducts with the Aß42 monomer and dimer, leading to suppression of further oligomerization. These findings support the utility of these aptamers as diagnostics for AD.
ABSTRACT
Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3LDat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[18F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3LDat/wt mice compared to Dnmt3a2/3Lwt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3LDat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[18F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3LDat/wt mice and controls. [18F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3LDat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.
Subject(s)
Behavior, Animal , DNA (Cytosine-5-)-Methyltransferases/physiology , Dopaminergic Neurons/metabolism , Hypothalamus/metabolism , Motor Activity , Physical Conditioning, Animal , Animals , DNA Methyltransferase 3A , Female , Male , Mice , Mice, Transgenic , Signal TransductionABSTRACT
Oligomers of ß-amyloid 42 (Aß42), rather than fibrils, drive the pathogenesis of Alzheimer's disease (AD). In particular, toxic oligomeric species called protofibrils (PFs) have attracted significant attention. Herein, we report RNA aptamers with higher affinity toward PFs derived from a toxic Aß42 dimer than toward fibrils produced from WT Aß42 or from a toxic, conformationally constrained Aß42 variant, E22P-Aß42. We obtained these RNA aptamers by using the preincubated dimer model of E22P-Aß42, which dimerized via a linker located at Val-40, as the target of in vitro selection. This dimer formed PFs during incubation. Several physicochemical characteristics of an identified aptamer, E22P-AbD43, suggested that preferential affinity of this aptamer toward PFs is due to its higher affinity for the toxic dimer unit (KD = 20 ± 6.0 nm) of Aß42 than for less-toxic Aß40 aggregates. Comparison of CD data from the full-length and random regions of E22P-AbD43 suggested that the preferential binding of E22P-AbD43 toward the dimer might be related to the formation of a G-quadruplex structure. E22P-AbD43 significantly inhibited the nucleation phase of the dimer and its associated neurotoxicity in SH-SY5Y human neuroblastoma cells. Of note, E22P-AbD43 also significantly protected against the neurotoxicity of WT Aß42 and E22P-Aß42. Furthermore, in an AD mouse model, E22P-AbD43 preferentially recognized diffuse aggregates, which likely originated from PFs or higher-order oligomers with curvilinear structures, compared with senile plaques formed from fibrils. We conclude that the E22P-AbD43 aptamer is a promising research and diagnostic tool for further studies of AD etiology.
Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Aptamers, Nucleotide/metabolism , Disease Models, Animal , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Plaque, Amyloid/pathology , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Animals , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Humans , Immunohistochemistry , Mice , Plaque, Amyloid/genetics , Plaque, Amyloid/metabolismABSTRACT
DNA methylation is generally known to inactivate gene expression. The DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, catalyze somatic cell lineage-specific DNA methylation, while DNMT3A and DNMT3L catalyze germ cell lineage-specific DNA methylation. How such lineage- and gene-specific DNA methylation patterns are created remains to be elucidated. To better understand the regulatory mechanisms underlying DNA methylation, we generated transgenic mice that constitutively expressed DNMT3A and DNMT3L, and analyzed DNA methylation, gene expression, and their subsequent impact on ontogeny. All transgenic mice were born normally but died within 20 weeks accompanied with cardiac hypertrophy. Several genes were repressed in the hearts of transgenic mice compared with those in wild-type mice. CpG islands of these downregulated genes were highly methylated in the transgenic mice. This abnormal methylation occurred in the perinatal stage. Conversely, monoallelic DNA methylation at imprinted loci was faithfully maintained in all transgenic mice, except H19. Thus, the loci preferred by DNMT3A and DNMT3L differ between somatic and germ cell lineages.
Subject(s)
Cardiomegaly/enzymology , DNA (Cytosine-5-)-Methyltransferases/biosynthesis , DNA Methylation , Ectopic Gene Expression , Animals , Cardiomegaly/genetics , Cardiomegaly/pathology , CpG Islands , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , Female , Germ Cells/enzymology , Germ Cells/pathology , Male , Mice , Mice, TransgenicABSTRACT
This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take â¼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.
Subject(s)
Cell Culture Techniques/methods , Oocytes/cytology , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation/physiology , Female , Male , Mice , Mice, Inbred ICR , Oocytes/physiology , OogenesisABSTRACT
Mammalian fetal ovaries contain numerous primordial germ cells (PGCs), although few mature oocytes are obtained from females, owing to apoptosis and follicle atresia. The regulatory mechanisms underlying oogenesis/folliculogenesis remain unknown. Development of methods for obtaining mature oocytes from PGCs in fetal ovaries in vitro could contribute to clarifying these mechanisms. The failure of follicle assembly has been found to be the most challenging aspect in conventional culture conditions. Recently, we established novel culture conditions that enable successful follicle assembly, sustaining interactions between the oocyte and somatic cells, and, in turn, promoting oocyte growth and maturation. Mature oocytes were differentiated from PGCs after a 1-month culture period. A hundred mouse offspring were obtained from approximately a thousand mature oocytes, indicating that oocytes that were differentiated from PGCs in vitro acquired totipotency after fertilization. Here we provide a detailed protocol for using this in vitro system. This in vitro system will potentially provide a novel platform for studying oogenesis and preservation of female germ cells.
Subject(s)
Cell Differentiation/physiology , Embryo, Mammalian/cytology , Germ Cells , Oocytes , Animals , Female , Germ Cells/cytology , Germ Cells/physiology , Male , Mice , Mitosis , Oocytes/cytology , Oocytes/physiology , Tissue Culture TechniquesABSTRACT
Various complex molecular events in oogenesis cannot be observed in vivo. As a bioengineering technique for female reproductive tissues, in vitro culture systems for female germ cells have been used to analyze oogenesis and preserve germ cells for over 20 years. Recently, we have established a new methodological approach for the culture of primordial germ cells (PGCs) and successfully obtained offspring. Our PGC culture system will be useful to clarify unresolved mechanisms of fertility and sterility from the beginning of mammalian oogenesis, before meiosis. This review summarizes the history of culture methods for mammalian germ cells, our current in vitro system, and future prospects for the culture of germ cells.
Subject(s)
Cell Differentiation , Fertility , Infertility, Female/metabolism , Oocytes/metabolism , Oogenesis , Animals , Female , Infertility, Female/pathology , Mice , Oocytes/pathologyABSTRACT
The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.
Subject(s)
Oocytes/cytology , Oogenesis/physiology , Pluripotent Stem Cells/cytology , Animals , Cell Line , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Female , Fertilization , In Vitro Techniques , Male , Meiosis , Mice , Mouse Embryonic Stem Cells/cytology , Oocytes/metabolism , Oogenesis/genetics , Transcriptome/geneticsABSTRACT
Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells.
Subject(s)
Oogenesis/physiology , Animals , Cryopreservation , Female , Male , Meiosis , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Oocytes/physiology , Ovarian Follicle/physiologyABSTRACT
In vitro growth of immature oocytes provides opportunities to increase gametic resources and to understand the mechanisms underlying oocyte development. Many studies on the in vitro growth of oocytes have been reported thus far; however, only a few cases have been reported, which demonstrated that oocytes can support full-term development after in vitro fertilization. Our research group recently found that culture of mouse neonatal primordial follicles increased the birthrate; however, the establishment of an in vitro system that can completely mimic follicle or oocyte growth in vivo and control oogenesis remains an ongoing challenge.
Subject(s)
Oocytes/cytology , Organ Culture Techniques/methods , Ovarian Follicle/cytology , Animals , Culture Media/chemistry , Cytoplasm/metabolism , Female , Fertilization in Vitro , Germ Cells/cytology , Humans , Mice , Oogenesis , RNA Interference , Reproductive Medicine/methods , TranscriptomeABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0040481.].
ABSTRACT
Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken ß-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.
Subject(s)
Gene Transfer Techniques , Genes, Reporter , Germ Cells/metabolism , Peptide Fragments/genetics , Animals , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , Female , Gene Transfer Techniques/veterinary , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Mice , Mice, Transgenic , NIH 3T3 Cells , Oogenesis/genetics , Peptide Chain Initiation, Translational/genetics , Peptide Fragments/metabolism , Pregnancy , Promoter Regions, Genetic , Transgenes , Red Fluorescent ProteinABSTRACT
In mammals, genomic imprinting governed by DNA methyltransferase DNMT3A and its cofactor DNMT3L is essential for functional gametes. Oocyte-specific methylation imprints are established during oocyte growth concomitant with DNMT3A/DNMT3L expression, although the mechanisms of oocyte-specific imprinting are not fully understood. To determine whether the presence of DNMT3A/DNMT3L in oocytes is sufficient for acquisition of methylation imprints, we produced transgenic mice to induce DNMT3A/DNMT3L expression prematurely in oogenesis and analyzed DNA methylation imprints. The results showed that 2- to 4-fold greater expression of DNMT3A/DNMT3L was achieved in non-growing (ng) oocytes versus fully grown oocytes derived from wild-type mice, but the analyzed imprint domains were not methylated. Thus, the presence of DNMT3A/DNMT3L in ng oocytes is insufficient for methylation imprints, and imprinted regions are resistant to DNMT3A/DNMT3L in ng oocytes. In contrast, excess DNMT3A/DNMT3L accelerated imprint acquisition at Igf2r, Lit1, Zac1 and Impact but not Snrpn and Mest in growing oocytes. Therefore, DNMT3A/DNMT3L quantity is an important factor for imprint acquisition. Transcription at imprinted domains is proposed to be involved in de novo methylation; however, transcription at Lit1, Snrpn and Impact was observed in ng oocytes. Thus, transcription cannot induce DNMT3A catalysis at imprinted regions even if DNMT3A/DNMT3L is present. However, the accelerated methylation imprints in oocytes, with the exception of Igf2r, were erased during embryogenesis. In conclusion, a sufficient amount of DNMT3A/DNMT3L and a shift from the resistant to permissive state are essential to establish oocyte-specific methylation imprints and that maintenance of the acquired DNA methylation imprints is essential for functional imprinting.