Growing Mouse Oocytes Research Articles

Chd9 mediates highly loosened chromatin structure in growing mouse oocytes

During oogenesis, oocytes prepare for embryonic development following fertilization. The mechanisms underlying this process are still unknown. Recently, it has been suggested that a loosened chromatin structure is involved in pluripotency and totipotency in embryonic stem (ES) cells and early preimplantation embryos, respectively. Here, we explored chromatin looseness in oocytes by fluorescence recovery after photobleaching (FRAP) using enhanced green fluorescent protein-tagged histone H2B. The results indicated that the chromatin in growing oocytes was already highly loosened to a level comparable to that in early preimplantation embryos. To elucidate the mechanism underlying the loosened chromatin structure in oocytes, we focused on chromodomain helicase DNA binding protein 9 (Chd9), which is highly expressed in growing oocytes. The oocytes from Chd9 knockout mice (Chd9−/−) generated using the CRISPR/Cas9 system exhibited a less loosened chromatin structure than that of wild-type mice, suggesting that Chd9 is involved in the loosened chromatin structure in growing oocytes. These results suggest that a loosened chromatin structure, which is mediated by Chd9, is a prerequisite for the acquisition of totipotency after fertilization.

Live, full-term mouse pups from oocytes grown and matured in vitro with serum substitutes

For in vitro growth and maturation of mouse oocytes (IVG-IVM), serum is added to media up to and including the stage of oocyte maturation; this subsequently supports oocytes through fertilization and early embryo development. However, problems may occur with sera, such as batch differences and issues of biosafety. The purpose of the present study was to determine the capacity for fertilization and pre- and post-implantation development of oocytes that underwent IVG-IVM with a serum substitute. Oocyte-granulosa cell complexes from preantral follicles were cultured in medium with either fetal bovine serum (FBS), Serum Substitute Supplement™ (SSS), or Knockout™ Serum Replacement (KSR) for 10days, and were then allowed to mature for 17 h. Subsequently, more than 90% of oocytes underwent germinal vesicle breakdown (GVBD) and more than 70% reached metaphase II, with no significant difference between the groups. A lower fertilization rate, presumably due to zona hardening, was found in the serum substitute groups. Nevertheless, more than 50% of the inseminated oocytes were fertilized and 35%–45% of them underwent first cleavage and developed to the blastocyst stage. Following embryo transfer, one and four live offspring were produced from the SSS and KSR groups, respectively. The present study demonstrated that murine IVG-IVM oocytes cultured in media with a serum substitute, achieved fertilization in vitro, pre- and post-implantation development, and the delivery of live pups, although the efficiency of the process is reduced compared to FBS supplementation.

Roles of MIWI, MILI and PLD6 in small RNA regulation in mouse growing oocytes.

The mouse PIWI-interacting RNA (piRNA) pathway produces a class of 26–30-nucleotide (nt) small RNAs and is essential for spermatogenesis and retrotransposon repression. In oocytes, however, its regulation and function are poorly understood. In the present study, we investigated the consequences of loss of piRNA-pathway components in growing oocytes. When MILI (or PIWIL2), a PIWI family member, was depleted by gene knockout, almost all piRNAs disappeared. This severe loss of piRNA was accompanied by an increase in transcripts derived from specific retrotransposons, especially IAPs. MIWI (or PIWIL1) depletion had a smaller effect. In oocytes lacking PLD6 (or ZUCCHINI or MITOPLD), a mitochondrial nuclease/phospholipase involved in piRNA biogenesis in male germ cells, the piRNA level was decreased to 50% compared to wild-type, a phenotype much milder than that in males. Since PLD6 is essential for the creation of the 5΄ ends of primary piRNAs in males, the presence of mature piRNA in PLD6-depleted oocytes suggests the presence of compensating enzymes. Furthermore, we identified novel 21–23-nt small RNAs, termed spiRNAs, possessing a 10-nt complementarity with piRNAs, which were produced dependent on MILI and independent of DICER. Our study revealed the differences in the biogenesis and function of the piRNA pathway between sexes.

Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size.

The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence. The folate receptors FOLR1 and FOLR2 as well as reduced folate carrier 1 (RFC1, SLC19A1 protein) each appeared to be present in follicular cells including granulosa cells. In growing oocytes, however, only FOLR2 immunoreactivity appeared abundant. Localization of apparent FOLR2 immunofluorescence near the plasma membrane increased with oocyte growth and peaked in oocytes as they neared full size. We assessed folate transport using the model folate leucovorin (folinic acid). Unexpectedly, there was a transient burst of folate transport activity for a brief period during oocyte growth as they neared full size, while folate transport was otherwise undetectable for the rest of oogenesis and in fully grown germinal vesicle stage oocytes. This folate transport was inhibited by dynasore, an inhibitor of endocytosis, but insensitive to the anion transport inhibitor stilbene 4-acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid, consistent with folate receptor-mediated transport but not with RFC1-mediated transport. Thus, near the end of their growth, growing oocytes may take up folates that could support the final stage of oogenesis or be stored to provide the endogenous folates needed in early embryogenesis.

Genome-wide reprogramming by DNA demethylation during mouse oocyte growth and early development

Genome-wide reprogramming by DNA demethylation during mouse oocyte growth and early development

Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth.

Recent studies of the demethylation process in murine zygotes have shown that 5-methylcytosine (5mC) is first converted into 5-hydroxymethylcytosine (5hmC) or further-oxidized cytosines in the paternal genome by the maternal ten-eleven translocation 3 (TET3) enzyme. This process is crucial for normal embryogenesis, and our aim was to elucidate the effect of Tet3 on the maternal genome during female germ-line development. Immunofluorescence analysis showed that 5hmC was clearly present in fully grown oocytes but not in nongrowing and early growth-stage oocytes. The 5hmC in the maternal genome was clearly detectable in DNA methyltransferase 3-like enzyme (Dnmt3L)-null oocytes and their fertilized zygotes, although Dnmt3L is essential for DNA methylation in oocytes. An analysis using an enzyme digestion-based method showed that 5hmC was present in LTR retrotransposons from the late growth period of oocytes. Quantitative RT-PCR analysis showed that Tet3 expression was enhanced during oocyte growth and exhibited an approximately 40-fold increase between nongrowing and fully grown oocytes. Our results show that 5hmC is generated since the oocyte growth stage, accompanied by up-regulation of Tet3; 5hmC is located mainly in LTR retrotransposons, indicating that 5hmC generated in growth-stage oocytes is responsible for genomewide demethylation after fertilization.

Efficient Translation of Dnmt1 Requires Cytoplasmic Polyadenylation and Musashi Binding Elements

Regulation of DNMT1 is critical for epigenetic control of many genes and for genome stability. Using phylogenetic analysis we characterized a block of 27 nucleotides in the 3′UTR of Dnmt1 mRNA identical between humans and Xenopus and investigated the role of the individual elements contained within it. This region contains a cytoplasmic polyadenylation element (CPE) and a Musashi binding element (MBE), with CPE binding protein 1 (CPEB1) known to bind to the former in mouse oocytes. The presence of these elements usually indicates translational control by elongation and shortening of the poly(A) tail in the cytoplasm of the oocyte and in some somatic cell types. We demonstrate for the first time cytoplasmic polyadenylation of Dnmt1 during periods of oocyte growth in mouse and during oocyte activation in Xenopus. Furthermore we show by RNA immunoprecipitation that Musashi1 (MSI1) binds to the MBE and that this element is required for polyadenylation in oocytes. As well as a role in oocytes, site-directed mutagenesis and reporter assays confirm that mutation of either the MBE or CPE reduce DNMT1 translation in somatic cells, but likely act in the same pathway: deletion of the whole conserved region has more severe effects on translation in both ES and differentiated cells. In adult cells lacking MSI1 there is a greater dependency on the CPE, with depletion of CPEB1 or CPEB4 by RNAi resulting in substantially reduced levels of endogenous DNMT1 protein and concurrent upregulation of the well characterised CPEB target mRNA cyclin B1. Our findings demonstrate that CPE- and MBE-mediated translation regulate DNMT1 expression, representing a novel mechanism of post-transcriptional control for this gene.

体外発育培養液へのポリビニルピロリドン添加がマウス卵母細胞・透明帯・卵丘細胞の接点の微細形態に及ぼす影響

体外発育培養液へのポリビニルピロリドン添加がマウス卵母細胞・透明帯・卵丘細胞の接点の微細形態に及ぼす影響

PCBP1 is required for maintenance of the transcriptionally silent state in fully grown mouse oocytes

Global transcriptional silencing in fully grown oocytes is a critical event during mammalian oogenesis. However, how this event is regulated remains elusive. Here, we provide evidence that poly(rC)-binding protein 1 (PCBP1), a protein found by us previously to be present in metaphase II (MII) mouse oocytes, participates in maintenance of the transcriptionally silent state in fully grown mouse oocytes. Knocking down Pcbp1 by microinjection of its specific siRNAs into fully grown germinal vesicle (GV) oocytes resulted in remarkable changes in their transcriptional state, including the disequilibrium between the number of oocytes with an NSN (non-surrounded nucleolus) and those with a SN (surrounded nucleolus), and obvious transcriptional reactiviation in oocytes with a SN configuration as evidenced by BrUTP incorporation assay and immunofluorescent labeling of phosphorylated RNA polymerase II CTD and trimethylated H3 lysine 4, markers for active transcription. Furthermore, in a comprehensive microarray analysis of the preovulatory oocyte transcriptome, an incredible number of nearly 4,000 transcripts were upregulated in the Pcbp1 knockdown groups. These data indicate that lack of the function of PCBP1 disrupts the quiescent status of transcription in the fully grown oocytes, and hence supporting a role of this protein in the regulation of global transcriptional silcencing in fully grown mouse oocytes.

DNA Methylation, Histone Modifications and Behaviour of AKAP95 during Mouse Oocyte Growth and Upon Nuclear Transfer of Foreign Chromatin into Fully Grown Prophase Oocytes

The poor efficiency of mammalian cloning is due to inappropriate/incomplete epigenetic reprogramming of the donor chromatin. As the success in reprogramming of the donor nucleus may require activity of similar mechanisms which reprogram the chromatin in the course of gametogenesis, we decided to follow the status of some epigenetic markers in the late phase of oogenesis in mice, i.e. in prophase oocytes during their growth and after completion of the growth phase. Our analysis reveals an increase in the level of global DNA methylation starting in oocytes with diameters around 60 microm which was further elevated until completion of oocyte growth. A similar increase was observed in respect to the acetylation of histone H4. On the other hand, the methylation of histone H4 Arg3 was constantly high until the end of oocyte growth, although it differed between fully grown oocytes depending on the type of spatial chromatin organization. We have also studied the AKAP95 protein which was abundant at earlier stages but decreased in fully grown oocytes according to changes in their chromatin organization. The nuclear transfer of different types of donor nuclei with hypomethylated DNA into fully grown prophase oocytes did not increase the global level of methylation of transferred foreign chromatin, regardless if the recipient oocyte was devoid of its own nucleus or its nucleus was left intact. This suggests a major problem in the ability of recipient oocytes to modify donor DNA methylation.

Growth of Mouse Oocytes to Maturity from Premeiotic Germ Cells In Vitro

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.

Cyclin B1 turnover and the mechanism causing insensitivity of fully grown mouse oocytes to cycloheximide inhibition of meiotic resumption

Cyclin B1 turnover and the insensitivity of fully-grown mouse oocytes to cycloheximide (CHX) inhibition of germinal vesicle breakdown (GVBD) were examined by assaying GVBD and cyclin B1 levels after treatment of oocytes with various combinations of eCG and CHX. Whereas over 95% of oocytes underwent GVBD after culture for 24 h with CHX alone, only 10% did so after culture with CHX + eCG (P < 0.05). In addition, preculture with eCG alone had no effect, but preculture with eCG + CHX prevented GVBD during a second culture with CHX alone. Therefore, we inferred that eCG delayed GVBD long enough for CHX inhibition of protein synthesis to allow cyclin B1 to decrease below a threshold where GVBD became dependent upon its de novo synthesis. However, western blot revealed no cyclin B1 synthesis, but cyclin B1 degradation, as long as GVs were maintained intact with eCG. Regarding the function of CHX in preculture without protein synthesis to block subsequent GVBD, whereas eCG delayed GVBD for only 3 h, CHX had an ongoing effect that further postponed GVBD, thus allowing cyclin B1 to decrease below the threshold. When oocytes precultured with eCG + CHX were further cultured without eCG and CHX, cyclin B1 first decreased but then, because of the ongoing effects of CHX, increased to a level sufficient to induce GVBD. The content of P34Cdc2 was not altered under any of the culture conditions (P > 0.05). We concluded that insensitivity of mouse germinal vesicle (GV) oocytes to CHX was due to the presence of sufficient cyclin B1, and that cyclin B1 level in such oocytes was maintained by an equilibrium between synthesis and degradation.

Production of Giant Mouse Oocyte Nucleoli and Assessment of Their Protein Content

Compared with advanced developmental stage embryos and somatic cells, fully grown mammalian oocytes contain specific nucleolus-like structures (NPB - nucleolus precursor bodies). It is commonly accepted that they serve as a store of material(s) from which typical nucleoli are gradually formed. Whilst nucleoli from somatic cells can be collected relatively easily for further biochemical analyses, a sufficient number of oocyte nucleoli is very difficult to obtain. We have found that isolated oocytes nucleoli fuse very efficiently when contact is established between them. Thus, well visible giant nucleoli can be obtained, relatively easily handled and then used for further biochemical analyses. With the use of colloidal gold staining, we estimated that a single fully grown mouse oocyte nucleolus contains approximately 1.6 ng of protein. We do believe that this approach will accelerate further research aiming at analyzing the composition of oocyte nucleoli in more detail.

Bisphenol A exposure modifies methylation of imprinted genes in mouse oocytes via the estrogen receptor signaling pathway

Bisphenol A (BPA), a synthetic additive used to harden polycarbonate plastics and epoxy resin, is ubiquitous in our everyday environment. Many studies have indicated detrimental effects of BPA on the mammalian reproductive abilities. This study is aimed to test the potential effects of BPA on methylation of imprinted genes during oocyte growth and meiotic maturation in CD-1 mice. Our results demonstrated that BPA exposure resulted in hypomethylation of imprinted gene Igf2r and Peg3 during oocyte growth, and enhanced estrogen receptor (ER) expression at the levels of mRNA and protein. The relationship between ER expression and imprinted gene hypomethylation was substantiated using an ER inhibitor, ICI182780. In addition, BPA promoted the primordial to primary follicle transition, thereby speeding up the depletion of the primordial follicle pool, and suppressed the meiotic maturation of oocytes because of abnormal spindle assembling in meiosis I. In conclusion, neonatal exposure to BPA inhibits methylation of imprinted genes during oogenesis via the ER signaling pathway in CD-1 mice.

Factors engaged in reactivation of DNA replication in the nuclei of growing mouse oocytes introduced into the cytoplasm of parthenogenetic one-cell embryos

Mammalian primary oocytes are arrested in the post-replicative G2 phase of the cell cycle. In contrast to other G2 nuclei, the nucleus of the growing mouse oocyte can reinitiate DNA synthesis after transfer by cell fusion under favorable cytoplasmic conditions, created by the parthenogenetic one-cell embryo. In the present study, we used the cell hybrid system to analyze the distribution of proteins involved in DNA re-replication in the oocyte nucleus. We show that this process is preceded by an extensive rearrangement of the insoluble fractions of minichromosome maintenance (MCM) proteins (Mcm2, -6 and 7). We also demonstrate that Cdc6 protein is present in primary growing mouse oocytes freshly isolated from the ovary, in a soluble and insoluble form. In contrast to MCM proteins, the insoluble fraction of Cdc6 was not rearranged in oocyte nuclei reinitiating DNA replication in hybrid cells. The rearrangement of MCM proteins and reinitiation of DNA synthesis occurred in the nuclei, in which the nuclear envelope remained intact. Reinitiation of DNA replication in the oocyte nucleus was sensitive to the inhibition of both CDK activity and polyadenylation of maternal mRNAs, indicating a role of proteins synthesized de novo by the embryo. These results allow us to understand better the mechanisms involved in the reinitiation of DNA replication in growing oocytes.

Subfertility Caused by Altered Follicular Development and Oocyte Growth in Female Mice Lacking PKBalpha/Akt11

Mammalian females are endowed with a finite number of primordial follicles at birth. Immediately following formation of the primordial follicle pool, cohorts of follicles are either culled from the ovary or are recruited to grow until the primordial follicle population is depleted. The majority of ovarian follicles, including the oocytes, undergo atresia through apoptotic cell death. As PKBalpha/Akt1 is known to regulate apoptosis, we asked whether Akt1 functioned in the regulation of folliculogenesis in the ovary. Akt1−/− females display reduced fertility and abnormal estrous cyclicity. At Postnatal Day (PND) 25, Akt1−/− ovaries possessed a reduced number of growing antral follicles, significantly larger primary and secondary oocytes, and an increase in the number of degenerate oocytes. By PND90, there was a significant decrease in the number of primordial follicles in Akt1−/− ovaries relative to Akt1+/+. In vivo granulosa cell proliferation was reduced, as were expression levels of Kitl and Bcl2l1, two factors associated with granulosa cell proliferation/survival. No compensation was observed by Akt2 or Akt3 at the mRNA/protein level. Significantly higher serum LH and trends for lower FSH and higher inhibin A and lower inhibin B relative to Akt1+/+ females were observed in Akt1−/− females. Exposure to exogenous gonadotropins resulted in an increase in the number of secondary follicles in Akt1−/− ovaries, but few mature follicles. Collectively, our results suggest that PKBalpha/Akt1 plays an instrumental role in the regulation of the growth and maturation of the ovary, and that the loss of PKBalpha/Akt1 results in premature ovarian failure.

Maternal imprinting during mouse oocyte growth in vivo and in vitro

Epigenetic regulation of gene expression is critical for oogenesis in mammals. In this study, a simple and efficient method was used to obtain the oocytes from cultured fetal mouse ovaries of 12.5 dpc. The methylation pattern of these oocytes was examined. The results showed that the establishment of imprinting of Igf2r and Peg3 in oocytes derived from cultured fetal mouse germ cells in vitro follows a slower time course than that of oocytes in vivo. However, oocytes in vitro and in vivo share similar methylation patterns. Igf2r was gradually de novo methylated, and the methylation covers 80% CpG sites in oocytes cultured for 28 days. However, only 45% of the CpG sites is methylated in Peg3 at the same stage. Furthermore, it demonstrated that the degree of DNA methylation is positively correlated with the size of oocytes in vitro and in vivo, indicating a progressive methylation process during oocyte growth.

Cytoplasmic Prep1 Interacts with 4EHP Inhibiting Hoxb4 Translation

BackgroundHomeobox genes are essential for embryonic patterning and cell fate determination. They are regulated mostly at the transcriptional level. In particular, Prep1 regulates Hox transcription in association with Pbx proteins. Despite its nuclear role as a transcription factor, Prep1 is located in the cytosol of mouse oocytes from primary to antral follicles. The homeodomain factor Bicoid (Bcd) has been shown to interact with 4EHP (eukaryotic translation initiation factor 4E homolog protein) to repress translation of Caudal mRNA and to drive Drosophila embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function.Methodology/Principal FindingsIn this paper we show by confocal microscopy and deconvolution analysis that Prep1 and 4EHP co-localize in the cytosol of growing mouse oocytes, demonstrating their interaction by co-immunoprecipitation and pull-down experiments. A functional 4EHP-binding motif present in Prep1 has been also identified by mutagenesis analysis. Moreover, Prep1 inhibits (>95%) the in vitro translation of a luciferase reporter mRNA fused to the Hoxb4 3′UTR, in the presence of 4EHP. RNA electrophoretic mobility shift assay was used to demonstrate that Prep1 binds the Hoxb4 3′UTR. Furthermore, conventional histology and immunohistochemistry has shown a dramatic oocyte growth failure in hypomorphic mouse Prep1i/i females, accompanied by an increased production of Hoxb4. Finally, Hoxb4 overexpression in mouse zygotes showed a slow in vitro development effect.ConclusionsPrep1 has a novel cytoplasmic, 4EHP-dependent, function in the regulation of translation. Mechanistically, the Prep1-4EHP interaction might bridge the 3′UTR of Hoxb4 mRNA to the 5′ cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development.

Expression of c-MYC in Nuclear Speckles During Mouse Oocyte Growth and Preimplantation Development

Myelocytomatosis oncogene (c-myc) is a major transcriptional regulator that controls various biological processes, and its deregulated expression causes carcinogenesis. To investigate the involvement of c-myc in oogenesis and preimplantation development, the expression of c-MYC during these stages was examined by immunocytochemistry. A strong c-MYC signal was detected in the nucleus of growing and fully grown oocytes as well as in preimplantation embryos before the morula stage. The signal intensity decreased slightly at the morula stage, and no signal was detected in blastocysts. Close observation of the nucleus revealed that c-MYC was localized in small granules that appeared to be nuclear speckles controlling pre-mRNA splicing. Although the number of granules decreased during oocyte growth, their size increased. After fertilization, the granules of c-MYC disappeared from the pronuclei, and c-MYC was evenly distributed in the nucleoplasm at the 1-cell stage, but the granules reappeared at the 2-cell stage. These results suggest that c-myc is involved in oocyte growth and preimplantation development and that its role changes during these stages.

Microinjection of Plasmids into Meiotically Incompetent Mouse Oocytes: Figure 1.

RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing in a number of model systems. The following protocol describes delivery of a mammalian double-stranded RNA (dsRNA) expression vector into meiotically incompetent mouse oocytes by microinjection. During mouse oocyte growth, there is a progressive decline in transcriptional activity, such that the fully-grown oocyte is transcriptionally quiescent. For this reason, the microinjection of plasmids should be performed in growing oocytes, obtained from 12- to 13-d-old females. Microinjected oocytes can be cultured for up to 2 d. The efficiency of knockdown by RNAi can be assayed by quantitative reverse transcriptase (RT)-PCR (qPCR).