Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase.

The oocytes of vertebrates are typically arrested at metaphase II (mII) by the cytostatic factor Emi2 until fertilization. Regulatory mechanisms in Xenopus Emi2 (xEmi2) are understood in detail but contrastingly little is known about the corresponding mechanisms in mammals. Here, we analyze Emi2 and its regulatory neighbours at the molecular level in intact mouse oocytes. Emi2, but not xEmi2, exhibited nuclear targeting. Unlike xEmi2, separable N- and C-terminal domains of mouse Emi2 modulated metaphase establishment and maintenance, respectively, through indirect and direct mechanisms. The C-terminal activity was mapped to the potential phosphorylation target Tx(5)SxS, a destruction box (D-box), a lattice of Zn(2+)-coordinating residues and an RL domain. The minimal region of Emi2 required for its cytostatic activity was mapped to a region containing these motifs, from residue 491 to the C terminus. The cytostatic factor Mos-MAPK promoted Emi2-dependent metaphase establishment, but Mos autonomously disappeared from meiotically competent mII oocytes. The N-terminal Plx1-interacting phosphodegron of xEmi2 was apparently shifted to within a minimal fragment (residues 51-300) of mouse Emi2 that also contained a calmodulin kinase II (CaMKII) phosphorylation motif and which was efficiently degraded during mII exit. Two equimolar CaMKII gamma isoform variants were present in mII oocytes, neither of which phosphorylated Emi2 in vitro, consistent with the involvement of additional factors. No evidence was found that calcineurin is required for mouse mII exit. These data support a model in which mammalian meiotic establishment, maintenance and exit converge upon a modular Emi2 hub via evolutionarily conserved and divergent mechanisms.

Broad, ectopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice.

Mammalian metaphase II (mII) exit and embryogenesis are induced at fertilisation by a signal thought to come from the sperm protein, phospholipase C-zeta (PLCZ1). Meiotic progression can also be triggered without sperm, as in parthenogenesis, although the classic mouse in vivo parthenogenetic model, LT/Sv, fails in meiosis I owing to an unknown molecular etiology. Here, we dissect PLCZ1 specificity and function in vivo and address its ability to interfere with maternal meiotic exit. Wild-type mouse Plcz1 expression was restricted to post-pubertal testes and the brains of both sexes, with region-specifying elements mapping to a 4.1 kb Plcz1 promoter fragment. When broad ectopic PLCZ1 expression was forced in independent transgenic lines, they initially appeared healthy. Their oocytes underwent unperturbed meiotic maturation to mII but subsequently exhibited autonomous intracellular free calcium oscillations, second polar body extrusion, pronucleus formation and parthenogenetic development. Transfer of transgenic cumulus cell nuclei into wild-type oocytes induced activation and development, demonstrating a direct effect of PLCZ1 analogous to fertilisation. Whereas Plcz1 transgenic males remained largely asymptomatic, females developed abdominal swellings caused by benign ovarian teratomas that were under-represented for paternally- and placentally-expressed transcripts. Plcz1 was not overexpressed in the ovaries of LT/Sv or in human germline ovarian tumours. The narrow spectrum of PLCZ1 activity indicates that it is modulated by tissue-restricted accessory factors. This work characterises a novel model in which parthenogenesis and tumourigenesis follow full meiotic maturation and are linked to fertilisation by PLCZ1.

Viable mice with compound mutations in the Wnt/Dvl pathway antagonists nkd1 and nkd2.

Gradients of Wnt/beta-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/beta-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd(lacZ) allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/beta-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.

Epigenetic discrimination by mouse metaphase II oocytes mediates asymmetric chromatin remodeling independently of meiotic exit.

In mammalian fertilization, paternal chromatin is exhaustively remodeled, yet the maternal contribution to this process is unknown. To address this, we prevented the induction of meiotic exit by spermatozoa and examined sperm chromatin remodeling in metaphase II (mII) oocytes. Methylation of paternal H3-K4 and H3-K9 remained low, unlike maternal H3, although paternal H3-K4 methylation increased in zygotes. Thus, mII cytoplasm can sustain epigenetic asymmetry in a cell-cycle dependent manner. Paternal genomic DNA underwent oocyte-mediated cytosine demethylation and acquired maternally-derived K12-acetylated H4 (AcH4-K12) independently of microtubule assembly and maternal chromatin. AcH4-K12 persisted without typical maturation-associated deacetylation, irrespective of paternal pan-genomic cytosine methylation. Contrastingly, somatic cell nuclei underwent rapid H4 deacetylation; sperm and somatic chromatin exhibited asymmetric AcH4-K12 dynamics simultaneously within the same mII oocyte. Inhibition of somatic histone deacetylation revealed endogenous histone acetyl transferase activity. Oocytes thus specify the histone acetylation status of given nuclei by differentially targeting histone deacetylase and acetyl transferase activities. Asymmetric H4 acetylation during and immediately after fertilization was dispensable for development when both parental chromatin sets were hyperacetylated. These studies delineate non-zygotic chromatin remodeling and suggest a powerful model with which to study de novo genomic reprogramming.

A restricted role for sperm-borne microRNAs in mammalian fertilization.

Prototypical microRNAs (miRNAs) are 21 approximately 25-base-pair RNAs that regulate differentiation, carcinogenesis, and pluripotency by eliminating mRNAs or blocking their translation, in a process that is collectively termed RNA interference (RNAi). In zebrafish, RNAi mediated by miRNAs regulates early development, and in mice embryos that lack the miRNA precursor processor Dicer are nonviable. However, the roles of miRNAs in mammalian fertilization are unknown. In this report, we show using microarrays that miRNAs are present in mouse sperm structures that enter the oocyte at fertilization. The sperm contained a broad profile of miRNAs and a subset of potential mRNA targets, which were expressed in fertilizable metaphase II (mII) oocytes. Oocytes contained transcripts for the RNA-induced silencing complex (RISC) catalytic subunit, EIF2C3 (formerly AGO3). However, the levels of sperm-borne miRNA (measured by quantitative PCR) were low relative to those of unfertilized mII oocytes, and fertilization did not alter the mII oocyte miRNA repertoire that included the most abundant sperm-borne miRNAs. Coinjection of mII oocytes with sperm heads plus anti-miRNAs to suppress miRNA function did not perturb pronuclear activation or preimplantation development. In contrast, nuclear transfer by microinjection altered the miRNA profile of enucleated oocytes. These data suggest that sperm-borne prototypical miRNAs play a limited role, if any, in mammalian fertilization or early preimplantation development.

Injection of mammalian metaphase II oocytes with short interfering RNAs to dissect meiotic and early mitotic events.

The manipulation of mammalian metaphase II (mII) oocytes has illuminated the mechanisms of fertilization and early embryogenesis and is central to nuclear transfer. Although RNA interference (RNAi) would greatly facilitate this type of manipulation, its application to mature, developmentally competent mII oocytes has not been evaluated. We report efficient RNAi by the injection of short interfering RNAs (siRNAs) into mII oocytes. The levels of the target mRNA and corresponding protein were rapidly and efficiently reduced. The siRNAs were effective when injected in the subnanomolar to nanomolar range and induced concurrently RNAi of multiple targets, revealing the kinetic parameters of RNAi in mII oocytes. Coinjection of sperm with siRNA functionally abolished the transcripts in the resultant blastocysts and in cloned embryos into which siRNA was coinjected during somatic cell nuclear transfer. The RNAi method was used to dissect the early mitotic roles of meiotic regulators, which suggests that CDC20 is essential for the first mitotic division, while EMI1 and EMI2 are not essential for this process. Our results show that siRNA injection of oocytes confers temporal control of RNAi in the analysis and manipulation of key processes in mammalian meiosis and early embryogenesis.

An unconventional nuclear localization motif is crucial for function of the Drosophila Wnt/wingless antagonist Naked cuticle.

Wnt/beta-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, we first must understand how Wnt signals are normally controlled. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/beta-catenin signaling that is crucial for segmentation in the model genetic organism, the fruit fly Drosophila melanogaster. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. Here we show that nkd mutants exhibit elevated levels of the beta-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, we identify a conserved 30-amino-acid motif, separable from Dsh-binding regions, that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. Our studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20.

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.

Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix.

Mammalian sperm-borne oocyte activating factor (SOAF) induces oocyte activation from a compartment that engages the oocyte cytoplasm, but it is not known how. A SOAF-containing extract (SE) was solubilized from the submembrane perinuclear matrix, a domain that enters the egg. SE initiated activation sufficient for full development. Microinjection coupled to tandem mass spectrometry enabled functional correlation profiling of fractionated SE without a priori assumptions about its chemical nature. Phospholipase C-zeta (PLCzeta) correlated absolutely with activating ability. Immunoblotting confirmed this and showed that the perinuclear matrix is the major site of 72-kDa PLCzeta. Oocyte activation was efficiently induced by 1.25 fg of sperm PLCzeta, corresponding to a fraction of one sperm equivalent (approximately 0.03). Immunofluorescence microscopy localized sperm head PLCzeta to a post-acrosomal region that becomes rapidly exposed to the ooplasm following gamete fusion. This multifaceted approach suggests a mechanism by which PLCzeta originates from an oocyte-penetrating assembly--the sperm perinuclear matrix--to induce mammalian oocyte activation at fertilization.