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In JoVE (1)
Other Publications (28)
- Biochemical and Biophysical Research Communications
- Nature Genetics
- RNA (New York, N.Y.)
- Biology of Reproduction
- Developmental Biology
- Science (New York, N.Y.)
- Science (New York, N.Y.)
- Proceedings of the National Academy of Sciences of the United States of America
- Developmental Biology
- Developmental Biology
- Nucleic Acids Research
- Biology of Reproduction
- Molecular and Cellular Biology
- Developmental Biology
- Biochemical and Biophysical Research Communications
- Genes & Development
- Human Molecular Genetics
- Nature
- Biology of Reproduction
- Fertility and Sterility
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Cold Spring Harbor Protocols
- Developmental Biology
- Methods in Enzymology
- Proceedings of the National Academy of Sciences of the United States of America
- Current Biology : CB
Articles by Paula Stein in JoVE
JoVE General
Mouse Oocyte Microinjection, Maturation and Ploidy Assessment
Department of Biology, University of Pennsylvania
Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.
Other articles by Paula Stein on PubMed
Analysis of the Role of RecQ Helicases in RNAi in Mammals
The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm, and RecQ1 genes that are related to Mut-7 and Qde3, two genes essential for RNAi in Caenorhabditis elegans and quelling in Neurospora, respectively. Our results suggest that Wrn, Blm, and RecQ1 are not involved in sequence-specific mRNA degradation in mammals in response to dsRNA, suggesting potential differences in the mammalian RNAi pathway.
Cdc25b Phosphatase is Required for Resumption of Meiosis During Oocyte Maturation
In a wide variety of animal species, oocyte maturation is arrested temporarily at prophase of meiosis I (ref. 1). Resumption of meiosis requires activation of cyclin-dependent kinase-1 (CDK1, p34cdc2), one component of maturation-promoting factor (MPF). The dual specificity phosphatases Cdc25a, Cdc25b and Cdc25c are activators of cyclin-dependent kinases; consequently, they are postulated to regulate cell-cycle progression in meiosis and mitosis as well as the DNA-damage response. We generated Cdc25b-deficient (Cdc25b-/-) mice and found that they are viable. As compared with wildtype cells, fibroblasts from Cdc25b-/- mice grew vigorously in culture and arrested normally in response to DNA damage. Female Cdc25b-/- mice were sterile, and Cdc25b-/- oocytes remained arrested at prophase with low MPF activity. Microinjection of wildtype Cdc25b mRNA into Cdc25b-/- oocytes caused activation of MPF and resumption of meiosis. Thus, Cdc25b-/- female mice are sterile because of permanent meiotic arrest resulting from the inability to activate MPF. Cdc25b is therefore essential for meiotic resumption in female mice. Mice lacking Cdc25b provide the first genetic model for studying the mechanisms regulating prophase arrest in vertebrates.
RNAi: Mammalian Oocytes Do It Without RNA-dependent RNA Polymerase
Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.
Protamine 2 Deficiency Leads to Sperm DNA Damage and Embryo Death in Mice
Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.
Transgenic RNAi in Mouse Oocytes: a Simple and Fast Approach to Study Gene Function
Double-strand RNA (dsRNA)-mediated posttranscriptional gene silencing, also known as RNA interference (RNAi), is a powerful tool to inhibit gene expression in several experimental model systems, including Arabidopsis, Caenorhabditis, and Drosophila. We previously described that the microinjection of Mos dsRNA into fully grown mouse oocytes results in the specific degradation of Mos mRNA in a time- and concentration-dependent manner. We report here a transgenic RNAi approach that is suitable to study gene function during mouse oocyte development and differentiation. The oocyte-specific Zp3 promoter was used to drive the expression of a long hairpin dsRNA ( approximately 500 bp) targeting Mos mRNA. Transgenic founder animals appeared healthy, but while males were fertile, females were not, in accordance with the known Mos null phenotype. The amount of Mos mRNA in the transgenic F(1) females was reduced by >90%, whereas there was no decrease in the nontargeted tissue plasminogen activator (Plat) mRNA. Moreover, the maturation-associated increase in mitogen-activated protein (MAP) kinase activity was not observed, and the metaphase II eggs underwent spontaneous parthenogenetic activation, thus recapitulating the Mos null phenotype. This approach provides a powerful method to study the functions of any oocyte-synthesized gene during oocyte development and early embryogenesis.
Requirement of Cks2 for the First Metaphase/anaphase Transition of Mammalian Meiosis
We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes. Sterility is due to failure of both male and female germ cells to progress past the first meiotic metaphase. The chromosomal events up through the end of prophase I are normal in both CKS2-/- males and females, suggesting that the phenotype is due directly to failure to enter anaphase and not a consequence of a checkpoint-mediated metaphase I arrest.
Transgenic RNAi Reveals Essential Function for CTCF in H19 Gene Imprinting
The imprinted regulation of H19 and Insulin-like growth factor 2 expression involves binding of the vertebrate insulator protein, CCCTC binding factor (CTCF), to the maternally hypomethylated differentially methylated domain (DMD). How this hypomethylated state is maintained during oogenesis and the role of CTCF, if any, in this process are not understood. With the use of a transgenic RNA interference (RNAi)-based approach to generate oocytes with reduced amounts of CTCF protein, we found increased methylation of the H19 DMD and decreased developmental competence of CTCF-deficient oocytes. Our results suggest that CTCF protects the H19 DMD from de novo methylation during oocyte growth and is required for normal preimplantation development.
Long-term Effects of Culture of Preimplantation Mouse Embryos on Behavior
Many procedures used in assisted reproductive technologies (ART) to treat human infertility entail culture of preimplantation embryos. Moreover, there is an increasing trend to culture embryos for longer periods of time before uterine transfer to identify the "best" embryos for transfer and to minimize multiple pregnancies. Embryo culture, however, can perturb embryo metabolism and gene expression, and the long-term consequences of culture are unknown. We have explored the behavioral consequences of embryo culture by using a 129S6/SvEvTac/C57BL/6J F(1) mouse model and find that adults derived from cultured embryos exhibit specific behavioral alterations in the elevated zero maze and Morris water maze tasks.
RNAi and Expression of Retrotransposons MuERV-L and IAP in Preimplantation Mouse Embryos
Both murine endogenous retrovirus-L (MuERV-L) and intracisternal A particle (IAP), two autonomous long terminal repeat (LTR) retrotransposons, are activated during genome activation in the preimplantation mouse embryo, and both sense and antisense transcripts are detected in 2-cell and 8-cell stage embryos. Because RNA interference (RNAi) functions in the preimplantation mouse embryo, we analyzed the relationship between RNAi and MuERV-L and IAP expression by inhibiting RNAi and measuring relative changes of the levels of these transcripts. We inhibited the initial step in the RNAi pathway by injecting 1-cell embryos with mDicer siRNA or long mDicer dsRNA and analyzed MuERV-L and IAP expression at the 8-cell stage. This approach resulted in the targeted destruction of mDicer mRNA, but not Hdac1 mRNA, inhibited the RNAi pathway, and resulted in a 50% increase in IAP and MuERV-L transcript abundance. These results suggest that RNAi constrains expression of repetitive parasitic sequences in preimplantation embryos, and thereby contributes to preserving genomic integrity at a stage of development when the organism consists of only a few cells.
Calreticulin on the Mouse Egg Surface Mediates Transmembrane Signaling Linked to Cell Cycle Resumption
Calreticulin, a protein best known as an endoplasmic reticulum chaperone, also is found on the extracellular plasma membrane surface of many cell types where it serves as a mediator of adhesion and as a regulator of the immune response. In this report, we demonstrate that calreticulin is present on the extracellular surface of the mouse egg plasma membrane and is increased in the perivitelline space after egg activation. The extracellular calreticulin appears to be secreted by vesicles in the egg cortex that are distinct from cortical granules. An anticalreticulin antibody binds to extracellular calreticulin on live eggs and inhibits sperm-egg binding but not fusion. In addition, engagement of cell surface calreticulin by incubation of mouse eggs in the presence of anticalreticulin antibodies results in alterations in the localization of cortical actin and the resumption of meiosis as indicated by alterations in chromatin configuration, decreases in cdc2/cyclin B1 and MAP kinase activities, and pronuclear formation. These events occur in the absence of any observable alterations in intercellular calcium. These data demonstrate that calreticulin functionally interacts with the egg cytoskeleton and can mediate transmembrane signaling linked to cell cycle resumption. These studies suggest a role for calreticulin as a lectin that may be involved in signal transduction events during or after sperm-egg interactions at fertilization.
Lack of Homologous Sequence-specific DNA Methylation in Response to Stable DsRNA Expression in Mouse Oocytes
Double-stranded RNA (dsRNA) induces sequence-specific mRNA degradation in most eukaryotic organisms via a conserved pathway known as RNA interference (RNAi). Post-transcriptional gene silencing by RNAi is also connected with transcriptional silencing of cognate sequences. In plants, this transcriptional silencing is associated with sequence-specific DNA methylation. To address whether this mechanism operates in mammalian cells, we used bisulfite sequencing to analyze DNA in mouse oocytes constitutively expressing long dsRNA against the Mos gene. Our data show that long dsRNA induces efficient Mos mRNA knockdown but not CpG and non-CpG DNA methylation of the endogenous Mos sequence in oocytes and early embryos. These data demonstrate that dsRNA does not directly induce DNA methylation in the trans form of this sequence in these mammalian cells.
CDC6 Requirement for Spindle Formation During Maturation of Mouse Oocytes
A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G2/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G2/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.
Mice Deficient in Oocyte-specific Oligoadenylate Synthetase-like Protein OAS1D Display Reduced Fertility
The double-stranded RNA (dsRNA)-induced interferon response is a defense mechanism against viral infection. Upon interferon activation by dsRNA, 2',5'-oligoadenylate synthetase 1 (OAS1A) is induced; it binds dsRNA and converts ATP into 2',5'-linked oligomers of adenosine (called 2-5A), which activate RNase L that in turn degrades viral and cellular RNAs. In a screen to identify oocyte-specific genes, we identified a novel murine cDNA encoding an ovary-specific 2',5'-oligoadenylate synthetase-like protein, OAS1D, which displays 59% identity with OAS1A. OAS1D is predominantly cytoplasmic and is exclusively expressed in growing oocytes and early embryos. Like OAS1A, OAS1D binds the dsRNA mimetic poly(I-C), but unlike OAS1A, it lacks 2'-5' adenosine linking activity. OAS1D interacts with OAS1A and inhibits the enzymatic activity of OAS1A. Mutant mice lacking OAS1D (Oas1d(-/-)) display reduced fertility due to defects in ovarian follicle development, decreased efficiency of ovulation, and eggs that are fertilized arrest at the one-cell stage. These effects are exacerbated after activation of the interferon/OAS1A/RNase L pathway by poly(I-C). We propose that OAS1D suppresses the interferon/OAS/RNase L-mediated cellular destruction by interacting with OAS1A during oogenesis and early embryonic development.
Absence of Non-specific Effects of RNA Interference Triggered by Long Double-stranded RNA in Mouse Oocytes
RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.
Abundant Transcripts from Retrotransposons Are Unstable in Fully Grown Mouse Oocytes
One physiological function proposed for RNA interference (RNAi) is to constrain expression of repetitive elements and thereby reduce the incidence of retrotransposition. Consistent with this model is that inhibiting the RNAi pathway results in an increase in expression of repetitive elements in preimplantation mouse embryos. Mouse oocytes are essentially transcriptionally quiescent providing a unique opportunity to assess the stability of repetitive element-derived transcripts in these cells. We compared the transcriptome of freshly isolated fully grown germinal vesicle (GV)-intact oocytes to that of oocytes in which meiotic maturation in vitro was inhibited for 48 h by milrinone. Consistent with the aforementioned function for RNAi is that the abundance of only a relatively small number of transcripts decreased in the cultured oocytes, when compared to changes that occur during maturation or following fertilization, and of those, several belonged to mobile elements.
Critical Roles for Dicer in the Female Germline
Dicer is an essential component of RNA interference (RNAi) pathways, which have broad functions in gene regulation and genome organization. Probing the consequences of tissue-restricted Dicer loss in mice indicates a critical role for Dicer during meiosis in the female germline. Mouse oocytes lacking Dicer arrest in meiosis I with multiple disorganized spindles and severe chromosome congression defects. Oogenesis and early development are times of significant post-transcriptional regulation, with controlled mRNA storage, translation, and degradation. Our results suggest that Dicer is essential for turnover of a substantial subset of maternal transcripts that are normally lost during oocyte maturation. Furthermore, we find evidence that transposon-derived sequence elements may contribute to the metabolism of maternal transcripts through a Dicer-dependent pathway. Our studies identify Dicer as central to a regulatory network that controls oocyte gene expression programs and that promotes genomic integrity in a cell type notoriously susceptible to aneuploidy.
Manipulations of Mouse Embryos Prior to Implantation Result in Aberrant Expression of Imprinted Genes on Day 9.5 of Development
In vitro culture of mouse embryos results in loss of imprinting. The aim of the present study was to examine how two of the techniques commonly used during assisted reproduction, namely embryo culture and embryo transfer, affect genomic imprinting after implantation in the mouse. F1 hybrid mouse embryos were subjected to three experimental conditions: control (unmanipulated), embryo transfer and in-vitro-culture followed by embryo transfer. Concepti were collected on d9.5 of development and allelic expression determination of ten imprinted genes (H19, Snrpn, Igf2, Kcnq1ot1, Cdkn1c, Kcnq1, Mknr3, Ascl2, Zim1, Peg3) was performed. Although control concepti had monoallelic imprinted gene expression in all tissues, both manipulated groups had aberrant expression of one or more imprinted genes in the yolk sac and placenta. Culture further exacerbated the effects of transfer by increasing the number of genes with aberrant allelic expression in extraembryonic, as well as embryonic tissues. Additionally, placentae of both groups of manipulated concepti exhibited reduced levels of Igf2 mRNA and increased levels of Ascl2 mRNA when compared with their unmanipulated counterparts. Furthermore, we show that biallelic expression of Kcnq1ot1 coincided with loss of methylation on the maternal allele of the KvDMR1 locus, a phenotype often associated with the human syndrome Beckwith-Wiedemann. In conclusion, our results show that even the most basic manipulation used during human-assisted reproduction, namely, embryo transfer, can lead to misexpression of several imprinted genes during post-implantation development. Additionally, our results serve as a cautionary tale for gene expression studies in which embryo transfer is used.
Pseudogene-derived Small Interfering RNAs Regulate Gene Expression in Mouse Oocytes
Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.
UBE2I (UBC9), a SUMO-conjugating Enzyme, Localizes to Nuclear Speckles and Stimulates Transcription in Mouse Oocytes
Sumoylation is a posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are covalently attached to their substrates. In vertebrates, developmental roles for sumoylation have been studied, but the function of sumoylation during mammalian oocyte growth and maturation is not known. As a prelude to conducting studies on the role of sumoylation during oocyte development, we analyzed the temporal and spatial pattern of expression of UBE2I, a SUMO-conjugating E2 enzyme. Immunocytochemical analysis of UBE2I revealed a punctate nuclear staining pattern, with transcriptionally quiescent, fully grown, GV-intact oocytes having larger UBE2I-containing bodies than transcriptionally active, meiotically incompetent growing oocytes. Inhibiting transcription in incompetent oocytes resulted in an increase in the size of the UBE2I-containing bodies. Overexpression of either wild-type UBE2I or catalytically inactive UBE2I resulted in an increase in the size of the UBE2I-containing bodies but also an increase in BrUTP incorporation, suggesting that transcriptional activation by UBE2I is independent of its catalytic activity. Although UBE2I-containing bodies did not completely colocalize with SUMO1 or SUMO2 and SUMO3, which were localized mainly on the nuclear membrane and in the nucleoplasm, UBE2I strikingly colocalized with SFRS2, which is a component of nuclear speckles and critical for mRNA processing. These results suggest a novel function for UBE2I and therefore sumoylation in gene expression.
The Effect of Blastomere Biopsy on Preimplantation Mouse Embryo Development and Global Gene Expression
The blastomere biopsy procedure does not affect preimplantation embryo development or global patterns of gene expression in a mouse model of Preimplantation Genetic Testing (PGT). However, zona breaching, which is inherent to the blastomere biopsy procedure, causes significant premature and sometimes abnormal hatching.
Microinjection of DsRNA into Fully-grown Mouse Oocytes
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 double-stranded RNA (dsRNA) of choice into fully-grown, germinal vesicle-intact (GV) mouse oocytes by microinjection. Microinjected oocytes can be cultured for up to 2 d or they can be matured to metaphase II. The metaphase II eggs can be fertilized in vitro and cultured up to the blastocyst stage. The efficiency of knockdown by RNAi can be assayed by quantitative reverse transcriptase (RT)-PCR (qPCR).
Microinjection of DsRNA into Mouse One-cell Embryos
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 double-stranded RNA (dsRNA) of choice into mouse one-cell embryos by microinjection. Microinjected embryos can be cultured for up to 4 d, i.e., to the blastocyst stage. The efficiency of knockdown by RNAi can be assayed by quantitative reverse transcriptase (RT)-PCR (qPCR).
Microinjection of Plasmids into Meiotically Incompetent Mouse Oocytes
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).
RNAi Experiments in Mouse Oocytes and Early Embryos
The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article provides a brief review of RNAi and discussion of the benefits and drawbacks of using long double-stranded RNA (dsRNA) in mammalian oocytes and early embryos. We also provide an introduction to protocols for RNAi experiments in mouse, including preparation and microinjection of dsRNA into mouse oocytes and early embryos, and preparation and testing of constructs for transgenic RNAi based on long hairpin RNA expression.
Recruitment of Orc6l, a Dormant Maternal MRNA in Mouse Oocytes, is Essential for DNA Replication in 1-cell Embryos
Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3' UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.
ICSI in the Mouse
Fertilization by intracytoplasmic sperm injection (ICSI) is a powerful technique that can be used to understand better the biology of fertilization, in addition to a form of assisted reproduction both in humans and in endangered species. Mouse is often the model organism of choice to study mammalian fertilization. The ability to fertilize successfully mouse eggs by sperm injection, however, has been hard to achieve. The introduction of piezo-actuated injection made possible to perform mouse ICSI in an efficient fashion. Piezo-actuated ICSI is also currently used to fertilize eggs of other mammalian species, for injection of earlier spermatogenic cells into mouse eggs, as well as for somatic cell nuclear transfer, and cloning. This chapter provides detailed experimental procedures to perform ICSI in mouse eggs.
The Gamma Isoform of CaM Kinase II Controls Mouse Egg Activation by Regulating Cell Cycle Resumption
Fertilization triggers a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the egg that initiates a series of events known as egg activation. These events include cortical granule exocytosis that establishes a block to polyspermy, resumption of meiosis, and recruitment of maternal mRNAs into polysomes for translation. Several calcium-dependent proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), have been implicated in egg activation. However, the precise role of CaMKII in mediating specific events of egg activation and the identity of the isoform(s) present in mouse eggs have not been unequivocally established. Through targeted deletion of the gamma isoform of CaMKII, we find that CaMKIIgamma is the predominant CaMKII isoform in mouse eggs and that it is essential for egg activation. Although CaMKIIgamma(-/-) eggs exhibit a normal pattern of Ca(2+) oscillations after insemination and undergo cortical granule exocytosis, they fail to resume meiosis or to recruit maternal mRNAs. Surprisingly, we find that the recruitment of maternal mRNAs does not directly depend on CaMKII, but requires elevated [Ca(2+)](i) and metaphase II exit. We conclude that CaMKIIgamma specifically controls mouse egg activation by regulating cell cycle resumption.
MicroRNA Activity is Suppressed in Mouse Oocytes
MicroRNAs (miRNAs) are small endogenous RNAs that typically imperfectly base pair with 3' untranslated regions (3'UTRs) and mediate translational repression and mRNA degradation. Dicer, which generates small RNAs in the miRNA and RNA interference (RNAi) pathways, is essential for meiotic maturation of mouse oocytes. We found that 3'UTRs of transcripts upregulated in Dicer1(-/-) oocytes are not enriched in miRNA binding sites, implicating a weak impact of miRNAs on the maternal transcriptome. Therefore, we tested the ability of endogenous miRNAs to mediate RNA-like cleavage or translational repression of reporter mRNAs. In contrast to somatic cells, endogenous miRNAs in oocytes poorly repressed translation of mRNA reporters, whereas their RNAi-like activity was much less affected. Reporter mRNA carrying let-7-binding sites failed to localize to P body-like structures in oocytes. Our data suggest that miRNA function is downregulated during oocyte development, an idea supported by normal meiotic maturation of oocytes lacking Dgcr8, which is required for the miRNA but not the RNAi pathway (Suh et al. [1], this issue of Current Biology). Suppressing miRNA function during oocyte growth is likely an early event in reprogramming gene expression during the transition of a differentiated oocyte into pluripotent blastomeres of the embryo.
