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BOR - Papers in Press, published online ahead of print August 30, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054213
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BIOLOGY OF REPRODUCTION 75, 891–898 (2006)
DOI: 10.1095/biolreprod.106.054213
© 2006 by the Society for the Study of Reproduction, Inc.

research-article

Injection of Mammalian Metaphase II Oocytes with Short Interfering RNAs to Dissect Meiotic and Early Mitotic Events1

Manami Amanai , Shisako Shoji , Naoko Yoshida , Manjula Brahmajosyula , and Anthony C.F. Perry 2

Laboratory of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

ABSTRACT

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.

early development, embryo, fertilization, gamete biology

INTRODUCTION

Fertilizable mouse oocytes are arrested at the second meiotic metaphase (mII) by one or more cytostatic factor (CSF) activities that stabilize the maturation promoting factor (MPF) by preventing ubiquitination of its cyclin component by the anaphase-promoting complex, APC [1–4]. The key net function of CSF is to prevent oocyte elimination by parthenogenetic activation. Recent work suggests that an endogenous meiotic inhibitor, EMI2 [5], contributes to mammalian CSF activity by sequestering the APC adaptor protein, CDC20 [6]. CDC20 is one of at least two APC adaptor proteins that have been demonstrated in mammalian oocytes, the other being CDH1, and although CDC20 is functionally dominant at mII, its role during the first mitotic cell cycles is unknown [6].

The removal of EMI2 from maturing or mature mII oocytes results in meiotic progression that resembles parthenogenetic activation [6], and the establishment and maintenance of meiotic metaphase arrest by EMI2 mirrors the role played in mitosis by EMI1 [4]. EMI1 is present in mII oocytes and throughout early development [6], and although it appears to be dispensable in meiosis, nothing is known about the roles of either EMI1 or EMI2 in early embryogenesis.

In addition to cell cycle changes, fertilization is associated with transcriptional flux; mII oocytes are transcriptionally subdued [7–9] but undergo further mRNA degradation [10] prior to the onset of de novo transcription (zygotic gene activation). In the mouse, this onset occurs at the late 1-cell stage [11–13], and is followed at the 4-cell stage by a second wave of enhanced transcription [14]. Throughout preimplantation development, thousands of genes are transcribed, often in a temporally regulated manner and representing a broad spectrum of potential functions, which include signaling [13, 14]. Experimental interference with meiotic and early embryonic transcripts in a stage-specific manner is a current challenge for the elucidation of the respective functions of these transcripts.

One potential solution is provided by RNA interference (RNAi), in which mRNA function is inhibited by double-stranded (ds)RNAs that exhibit complementarity to specific target mRNAs [15]. These types of dsRNAs include naturally occurring microRNA (miRNA) precursors and functionally related 21~23-base pair (bp) short interfering RNAs (siRNAs) [16–18]. miRNAs and siRNAs tend to abrogate target translation when target mRNA sequence complementarity is imperfect or to promote target mRNA destruction when complementarity is perfect or near-perfect [17, 19–21]. Both pathways involve the incorporation of the mRNA complementary strand of the siRNA/miRNA into the RNA-induced silencing complex (RISC) [22]. Microarray analyses suggest that mRNA transcripts that are not the intended target of a given siRNA must share an identical stretch of ≥11 bp with the intended target to experience off-target elimination [23, 24]. Although siRNAs induce mRNA transcript increases in some cases, these may be due to statistical scatter [25]; siRNAs are thus considered to be highly specific.

RNAi was first demonstrated in mouse zygotes and immature oocytes by injection of relatively long (550~650-bp) dsRNAs [26, 27]. However, long dsRNAs are relatively cumbersome to work with, especially for multiple targets, and they have greater potential to induce nonspecific RNAi relative to siRNAs because there is an increased chance of off-target matching and consequent hydrolysis [18, 19]. Two approaches have been employed to reduce the potential for off-target interference and increase RNAi specificity in oocytes: the introduction of shorter siRNAs, and the use of related short hairpin RNAs (shRNAs). In the first approach, siRNAs are microinjected into germinal vesicle (GV) oocytes, which are then allowed to mature in vitro, as exemplified by phenocopying c-mos null mutations generated by conventional gene targeting [28]. However, culturing in vitro from cumulus-denuded GV oocytes produces mII oocytes that lack normal spindles [6], have anomalous mitochondrial distribution [29], and yield developmentally impaired embryos after fertilization [29–31]. Therefore, the study of meiosis and embryogenesis via this method is problematic. Moreover, the injection of cumulus-enveloped oocytes is technically difficult; oocytes are difficult to visualize and the pipettes rapidly become clogged. The GV injection method can also be adapted successfully to the injection of siRNA into zygotes, and has been shown (for example) to phenocopy preimplantation embryonic phenotypes, including Pou5f1 null mutations [28].

The second method for siRNA introduction into oocytes is predicated on the ability of DNA to direct shRNA production, and consequently RNAi, in mammalian cells [32]. DNA-directed RNAi has been harnessed to generate transgenes, which when active in the mII oocytes of transgenic mice elicit a knockdown phenotype [33–35]. The drawbacks associated with transgene (tg) RNAi include: (i) the time and animal resources required, precluding their use in high-throughput screening; (ii) the absence of controls to ascertain the level and effect of tg expression in a given cell at a given time; (iii) limited cell- and tissue-specific control of expression; (iv) the delay between promoter activation and accumulation of sufficient functional RNAi-inducing molecules; and (v) the possibility of compensatory mechanisms or other adjustments that balance the loss of a particular mRNA during the relatively protracted process of ontogenesis.

Therefore, we investigated the potential of siRNAs to elicit RNAi in mII oocytes and preimplantation embryos following injection of mII oocytes. The efficiencies and kinetics of RNAi for three target mRNA transcripts following siRNA injection into mII oocytes are reported. Coinjection of siRNAs with a fertilizing sperm into mII oocytes elicited near-term and long-term preimplantation knockdown phenotypes. Efficacious targeting in nuclear transfer cloned embryos was achieved by coinjecting siRNAs with a somatic cell (donor) nucleus. We have harnessed siRNA-mediated RNAi to dissect the early mitotic roles of meiotic regulators, showing that CDC20 is essential for the first mitotic cell cycle. This establishes the utility of the approach and shows how it may facilitate the dissection and manipulation of complex genetic pathways in meiotic and early embryonic cells.

MATERIALS AND METHODS

Preparation and Culture of Oocytes and Embryos

Mature oviductal metaphase II (mII) oocytes were collected as described previously [36] from superovulated, 8–12-week-old B6D2F1 wild-type or pCAGG-eGFP transgenic female mice (B6C3 x B6C3 F0, back-crossed with C57BL/6) [37]. Oocyte maintenance and embryo culture were typically in kalium simplex optimized medium (KSOM; Specialty Media, Phillipsburg, NJ) under mineral oil (Shire, Florence, KT) in a humidified atmosphere of 5% (v/v) CO2 in air at 37–38°C. Mice were supplied by SLC (Shizuka-ken, Japan) and treated in accordance with local guidelines.

Design of siRNAs

Double-stranded siRNAs were designed according to previous recommendations [38] using top-scoring sequences identified by Target Finder (Gene Script Corp; https://www.genscript.com/ssl-bin/app/rnai) and the RNAi software tools of the Hannon laboratory (http://katahdin.cshl.org:9331/portal/scripts/main2.pl). The siRNAs and were directed against the following mRNAs: Cdc20 (NM_023223) for siCdc20#1(21+2) (nucleotide [nt] 892–912) 5'-CAGCAGCAGAAACGACUUCGA-3'; siCdc20#1 (nt 892–916) 5'-CAGCAGCAGAAACGACUUCGAAACA-3'; siCdc20#2 (nt 1037–1061) 5'-CACUGAGUGGCCAUAGCCAGGAAGU-3'; siCdc20#3 (nt 1128–1152) 5'-CGUGUGGCCUAGUGGUCCUGGAGAA-3'; siCdc20#4 (nt 915–939) 5'-CAUGACCAGUCACUCCGCUCGAGUA-3'; siCdc20#1(27+0) (nt 892–918), 5'-CAGCAGCAGAAACGACUUCGAAACAUG-3', Emi1 (NM_025995) for siEmi1#3(21+2) (nt 337–357) 5'-GAACCUUCCUGUAAUGACUGU-3'; siEmi1#1 (nt 311–331) 5'-GCUUCCUACAGUCCCGUGU-3'; siEmi1#2 (nt 1094–1114) 5'-CAGCGUGGUCAGAGAGUUUCUACCU-3'; siEmi1#3 (nt 337–361) 5'-GAACCUUCCUGUAAUGACUGUGUUA-3'; siEmi1#3(27+0) (nt 337–363) 5'-GAACCUUCCUGUAAUGACUGUGUUAGA-3'; siEmi1#4(27+0) (nt 309–335) 5'-AGAAGCUUCCUACAGUCCCGUGUGUUU-3'; eGFP (pCX-EGFP) for sieGFP#1 (nt 2322–2346) 5'-GACAACCACUACCUGAGCACCCAGU-3'; sieGFP#2 (nt 1837–1861) 5'-GCGAUGCCACCUACGGCAAGCUGAC-3'; sieGFP#3 (nt 2162–2186) 5'-CAACUACAACAGCCACAACGUCUAU-3'; sieGFP#4(27+0) (nt 2322–2346) 5'-GACAACCACUACCUGAGCACCCAGUUU-3'; and Pou5f1 (M34381) for siOct4#1 (nt 662–686) 5'-GACAACAAUGAGAACCUUCAGGAGA-3'. With the exception of the 27+0 siRNAs, the sequences included 2-bp 3'-overhangs, i.e., AG for the top strand (shown here) and AU for the complementary strand. The 27+0 siRNAs included UU at the 3'-termini of their top strands (shown above); for siEmi1#4(27+0), the UU is part of the Emi1 mRNA sequence complement, whereas it is not in the case of sieGFP#4(27+0). Modified sieGFP#1 (sieGFP#1m) contained three phosphorothioated bonds at its 5'-termini. The sieGFP#1m and 27+0 siRNAs were obtained from Hokkaido System Science (Japan); all the other siRNAs were supplied by iGENE Therapeutics (Japan).

Microinjection

All microinjections were performed using piezo-actuated micropipettes (Prime Tech, Ibaraki, Japan). The siRNAs were injected into mII oocytes (13.5 to 15 hours post-hCG administration) at the indicated final concentration, in 5~15 pl of a solution that contained 5~15% (w/v) PVP (Mr 360 000) (Wako Chemical Co., Japan) in either nuclear isolation medium (phosphate-buffered KCl) or ultrapure water. The injection volumes were estimated by observing the distance of displacement traveled by the mercury front along a length of known internal pipette diameter, and corresponded well with previous measurements. Control B6D2F1 mII oocytes were injected either with sieGFP#2 or not injected; no difference was noted between these treatments. Injected oocytes were washed and cultured in KSOM prior to analysis. Injections were completed within 1 h of sample/PVP mixing. Sperm heads extracted with Triton X-100 [36] were mixed with siRNA to give a final siRNA concentration of 25 µM in 9~10.5% (w/v) PVP, and coinjected into mII oocytes. Controls injected without siRNA or with an siRNA for which there was no intended target enabled distinction between mRNA reduction due to RNAi and fertilization. Acutely isolated cumulus cell nuclei were mixed to give a final siRNA concentration of 25 µM in 9% (w/v) PVP and injected into enucleated oocytes, essentially as described previously [39]. The data shown are from experiments that were conducted at least twice, on different days.

Polymerase Chain Reaction (PCR)

To obtain a readout of RNAi by agarose gel electrophoresis (standard RT-PCR), 1–4 cumulus-denuded oocytes or embryos were collected in 2 µl of 0.1% (w/v) sarkosyl, heated at 65°C for 5 min and subjected to oligo(dT)12–18-primed first-strand cDNA synthesis in a 25-µl reaction volume with SuperScript III RT (Invitrogen). The cDNA (0.05x or 0.1x) was used to program PCR with GeneTaq NT DNA polymerase (Nippon Gene, Japan) with the following parameters: hot start at 94°C for 4 min, followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min, and ending in an extension step at 72°C for 4 min. For each reaction, 40–50% of the product was examined by 2% (w/v) agarose gel electrophoresis. All the test reactions were accompanied by negative, RT-minus controls and were performed with at least two independent preparations. Ratiometric quantitation of mRNAs by real-time PCR (qPCR) was carried out with SYBR Green PCR Master Mix (Qiagen) and ~10% of the first-strand reverse-transcription reaction (0.1~0.4 cell equivalents) in 25 µl, with the following PCR parameters: hot start at 95°C for 15 min, followed by 45 cycles of 95°C for 10 sec, 58°C for 15 sec, and 72°C for 31 sec. The PCR primers were nondimerizing under the conditions employed and were typically intron-flanking. Product quantification was performed with the Prism 7000 Sequence Detection System (Applied Biosystems). To determine the number of cycles required to reach the threshold value for each primer set, standard curves were constructed using serial dilutions of mouse testis cDNA and/or linear cloned cDNA. The use of known amounts of cloned cDNA allowed estimation of absolute copy numbers for corresponding transcripts, assuming 100% efficiency of reverse transcription (given the low concentration of mRNA template). Normalized ratiometric values were obtained by dividing the raw value for the query sequence by that concurrently obtained from the same cDNA preparation for ß-actin in the oocytes or H2A.Z in the embryos [40]. These values were compared to those obtained in parallel for noninjected, age-matched controls or for controls that were injected with sieGFP#1 or sieGFP#2, in which the eGFP mRNA levels were not being analyzed; we found no significant differences between the noninjected and sieGFP-injected controls. Statistical analyses were performed using Student unpaired t-tests.

Standard RT-PCR was performed with the following primers: for ß -actin, 5'-TGACAGGATGCAGAAGGAGA-3' and 5'-GCTGGAAGGTGGACAGTGAG-3'; for eGFP, 5'-CTGGTCGAGCTGGACGGCGACG-3' and 5'-GGCGCGGGTCTTGTAGTTGCCG-3'; for Emi1, 5'-CTGAGCTCTCCCGCAG-3' and 5'-GCGGATCCATCACAATCTTTGTAAGTTCT-3'; and for Cdc20, 5'-GCATTCTGAGGTTTGCCG-3' and 5'-GCAGTTCGTGTTCGAGAG-3'.

Quantitative PCR was performed with the following primers: for ß-actin, 5'-TGACAGGATGCAGAAGGAGA-3' and 5'-GCTGGAAGGTGGACAGTGAG-3'; for H2A.Z (NM_016750), 5'-GCGTATCACCCCTCGTCACTTG-3' and 5'-TCTTCTGTTGTCCTTTCTTCCCG-3'; for Pou5f1 (M34381), 5'-CGTGAAGTTGGAGAAGGTGGAACC-3' and 5'-GCAGCTTGGCAAACTGTTCTAGCTC-3'; for eGFP, 5'-GCAGAAGAACGGCATCAAGGTG-3' and 5'-TGCTCAGGTAGTGGTTGTCGGG-3'; Emi1, 5'-AAATCAAGTCCTCCAGTCAGCGTG-3' and 5'-TTCGTTGTTCTTCAATGTCTTTGCC-3'; for Emi2, 5'-AGTGGTGAGCAGGTTCCAACTCTG-3' and 5'-TGTTTACTCCGTAGGTGGGTGAGG-3'; for Cdh1 (Fizzy-related 1, NM_019757), 5'-GGACTCAGTGACTTCCGTTGGC-3' and 5'-TTCTTCCCAGCAGCAGCGTC-3'; and for Cdc20, 5'-GGCACATTCGCATTTGGAACG-3' and 5'-TAGTGGGGAGACCAGAGGATGGAG-3'.

Immunoblotting

Immunoblotting was carried out with rabbit anti-mouse CDC20 polyclonal antibodies (Santa Cruz Biotechnology) or a mouse monoclonal antibody against {alpha}-tubulin (Sigma). Rabbit polyclonal antibodies raised against recombinant His-EMI1-NT [6] were supplied by Kitayama Laboratories (Nagano, Japan). Following standard SDS-PAGE, blocking (Block Ace; Yukijirusi Nyugyo, Japan), blotting, and immunodetection were performed with enhancer (Toyobo Co., Ltd., Japan) and the LumiGLO Reserve Chemiluminescent Substrate Kit (Kirkegard & Perry Laboratories, Inc.). The blots were stripped with Western blot stripping solution (Nakalai Tesque Inc., Japan) and reprobed with the anti-{alpha}-tubulin antibody (Sigma), to confirm equal loadings. Relative quantification of chemiluminescent signals following immunoblotting was carried out with the LAS-1000mini lumino-image analyzer (Fujifilm, Japan).

Image Acquisition

Oocytes and embryos were visualized under the Olympus IX-71 microscope, and the images were captured by DP-12 (Hoffman) or DP-70 (fluorescence and bright-field) cameras.

RESULTS

Reduction of mRNA Target Levels by siRNAs Injected into mII Oocytes Singly or in Combination

Cumulus-denuded oocytes that have matured in vitro are often abnormal [6, 29–31], and those with intact cumulus oophori are difficult to inject. Therefore, we considered whether mature in vivo-derived mII oocytes might support siRNA-mediated RNAi. We adopted as paradigms the cell cycle proteins EMI1 and CDC20 [6]. EMI1 regulates mitotic prometaphase by sequestering the APC activator CDC20 [41]. We also included enhanced GFP (eGFP) as an endogenous reporter in ubiquitously expressing eGFP-transgenic mice. The mII oocytes were injected with siRNA 13~15 h post-hCG, so that the resultant phenotypes could be induced within their normal fertilizable lifetime, which lasts at least 21 h after the LH surge [42], the equivalent of hCG administration.

The Emi1, Cdc20, and eGFP target mRNA levels were determined by qPCR 8 and 24 h after siRNA injection of mII oocytes and compared to those of age-matched controls. Spiked qPCR reactions that contained known amounts of cDNA gave estimations of the mRNA target copy numbers at 790 (Emi1), 1990 (Cdc20), and 3250 (eGFP) copies per noninjected B6D2F1 or in the case of eGFP, transgenic mII oocyte ~22 h post-hCG.

Initially, we employed the siRNAs of 19+2 (siEmi1#1) or 25+2 (all others) [25], each at 25 µM (Fig. 1, A–C). Of the tested siRNAs, 5 out of 9 gave target mRNA levels that were reduced by 50% or more at 8 h, and all of the siRNAs, with the exception of sieGFP#2, achieved this reduction by 24 h (average reduction = 82.5 ± 5.7%). We have previously shown that control oocytes corresponding to the 24-h timepoint typically retain their mII external, spindle, and chromatin morphologies and have high histone and myelin basic protein kinase activities [6]. These data suggest that mII oocytes are able to support rapid siRNA-mediated RNAi.


Figure 75060301
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  		FIG. 1. RNAi efficiencies of siRNAs against eGFP, Cdc20, and Emi1 mRNAs following injection into mII oocytes. A–C) qPCR (lower) and gel electrophoresis to analyze target mRNA levels when siRNAs were injected separately at the times shown (8 or 24 h postinjection), as indicated. The raw qPCR values are expressed as ratios to the value for ß-actin mRNA in the same sample, and further normalized against the values for noninjected, age-matched oocytes, set at 1.00. D) qPCR of Cdc20 and Emi1 mRNAs following injection of mII oocytes with siRNAs of different lengths, as indicated. For each target, the 5'-termini of the three siRNAs maps to the same nucleotide. E) qPCR of eGFP, Emi1, and Cdc20 mRNAs in eGFP-expressing oocytes coinjected with either sieGFP#1 siEmi1#3 and sieCdc20#1 (3x) or the siRNAs independently (1x). The data for sieGFP include those presented in A for sieGFP#1. Replicate numbers are shown in parentheses and data are plotted ± SEM.

We also investigated the effect of siRNA length on RNAi. Injection of mII oocytes with sieGFP#4(27+0) and siEmi1#4(27+0) siRNAs (each at 25 µM and with the configuration 27+0 [25]) reduced average target mRNA levels to 22.8 ± 18.3% (10 oocytes, n=4) and 108.7 ± 26.0% (18 oocytes, n=6), respectively, compared to noninjected controls; the value for eGFP after 8 h was 137.9 ± 12.6% (13 oocytes, n=5). To enable more direct comparisons between the siRNAs, we selected two efficient 25+2 siRNAs (siCdc20#1 and siEmi1#3) and designed 21+2 and 27+0 counterparts with 5'-termini that matched the nucleotides in their respective target mRNAs. The efficiency of RNAi did not significantly differ between these groups (Fig. 1D), which suggests that target site is at least as important as siRNA length.

Simultaneous RNAi for multiple targets would be advantageous for examining interacting pathway components. When we coinjected siCdc20#1, siEmi1#3, and sieGFP#1 (25+2, 25 µM each) into eGFP-expressing transgenic mII oocytes, all the target mRNA levels were reduced at 24 h postinjection (Fig. 1E). Target reduction was generally slightly lower than it was when the respective siRNAs were injected independently into oocytes of the same (transgenic) strain. The siRNAs were not equally susceptible to this effect, with siEmi1#3 being more sensitive than the other two siRNAs (Fig. 1E). Nevertheless, these data clearly demonstrate multiplex siRNA-mediated RNAi in mII oocytes.

Kinetics of RNAi by Effective siRNAs in mII Oocytes

To assess the kinetics of siRNA-mediated RNAi in greater detail, we performed qPCR assays on mII oocytes at multiple timepoints over a 24-h period postinjection of siEmi1#3 and siCdc20#1 (Fig. 2A). Reduction in the levels of the respective mRNA targets occurred within 4 h and were greater than half-maximal within 8 h (Fig. 2A). In the case of siCdc20#1, the reduction was both pronounced and rapid, eliminating 73% of the target mRNA and 60% of the CDC20 protein by 4 h (Fig. 2, A and B). Since the Cdc20 transcript level is constant throughout mII [6], this represents an average elimination of ~360 Cdc20 mRNA molecules per hour per oocyte over the 4-h period. Twenty-four hours after siRNA injection, the oocytes contained ~0% CDC20 or 22% EMI1 protein compared to age-matched controls. Oocytes injected with either siEmi1#3 or siCdc20#1 were morphologically indistinguishable from noninjected or sieGFP-injected age-matched controls at 8 or 24 h postinjection.


Figure 75060302
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  		FIG. 2. Induction of RNAi in mII oocytes as functions of time post-siRNA injection and injected siRNA concentration. A) Induction of RNAi by Cdc20#1 and siEmi1#3 as a function of time after corresponding siRNA injection. B) Protein levels at the times shown after siRNA injection of mII oocytes, as assessed by Western blotting. The quantified chemiluminescence for siCdc20#1-injected and age-matched (noninjected) controls at 4 h gives values (arbitrary units after background subtraction) of 18 480 and 47 841, respectively. C, D) The levels of target transcripts remaining at the times shown after injection of mII oocytes with siCdc20#1 (C) or siEmi1#3 (D) at the concentrations shown. The values are determined as for Figure 1, and are compared in each case to the value for age-matched oocytes at the time of injection, set to 1.0. The data are plotted ± SEM.

The effect of siRNA concentration on RNAi was evaluated using siCdc20#1 and siEmi1#3. An injected of 25 µM siRNA has previously been reported to induce RNAi in maturing oocytes [28]. However, siCdc20#1 at only 50 nM elicited a similar target reduction endpoint (94.1%, 50 nM vs. 97.0%, 25 µM), although this took longer time (24 h) (Fig. 2, C and D). Lower concentrations of injected siCdc20#1 (0.1~10 nM) produced submaximal decreases in Cdc20 mRNA. The injected volume (5~15 pl) of 100 pM siRNA contained 310~930 molecules. Given that the Cdc20 mRNA copy number (~1900/oocyte) is likely to be an underestimation, as it assumes 100% efficiency of cDNA synthesis, siCdc20#1 elicited detectable RNAi in mII oocytes when its target was present in several-fold molar excess. In analogous experiments, 50 nM siEmi1#3 induced a substantial reduction in the level of Emi1 mRNA over the time course of the experiment compared to age-matched controls (Fig. 2D). The Emi1 mRNA levels, unlike those of Cdc20, decline autonomously as the oocytes age [6]. This autonomous decline may mask siRNA-induced mRNA depletion and may explain why siEmi1#3 RNAi does not correlate absolutely with siRNA concentration (Fig. 2, C and D). These data show that siRNAs injected at nanomolar concentrations are able to cause pronounced reduction of target mRNAs in mII oocytes, with kinetics comparable to those previously reported for the 25–2 siRNA [25].

We extended siRNA-mediated RNAi by evaluating its application to preimplantation development using an eGFP reporter and the native POU transcription factor, POU5F1 (also known as OCT4).

Short Interfering RNA Delivery at Fertilization Produces Effective RNAi in Preimplantation Development

We determined whether coinjection of mII oocytes with a sperm head plus siRNA could induce RNAi in a tg model in which eGFP tg expression becomes detectable at the late morula stage [37]. As expected, injection of sperm from homozygous eGFP+/+ males with buffer alone yielded green fluorescent embryos by embryonic day (E)3.5 (Fig. 3A). In contrast, fluorescence was markedly reduced when eGFP+/+ sperm were coinjected with sieGFP#1 (Fig. 3A). It has previously been shown that siRNAs can mediate RNAi for at least 4 days [25]. As a prelude to maximizing siRNA longevity in mouse embryos, we modified the 5'-terminus of sieGFP#1 with poorly hydrolysable phosphorothioate linkages (sieGFP#1m; Fig. 3A), thereby increasing its stability [43]. However, sieGFP#1m exerted a weaker effect than its nonmodified counterpart (Fig. 3A), mirroring the in vitro results obtained previously [44]. The cause of the reduced efficiency of RNAi is unclear but may reflect poor incorporation of phosphorothioate-modified siRNAs into the RISC [22, 45].


Figure 75060303
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  		FIG. 3. Functional RNAi in embryos following coinjection of sperm or somatic cell nuclei with siRNAs. A) Bright-field (left) and fluorescence microscopy at E3.5 following coinjection of mII oocytes with eGFP+ sperm plus buffer alone (Control), sieGFP#1 or a phosphorothioate-modified derivative of sieGFP#1 (sieGFP#1m). B) Bright-field (left) and fluorescence microscopy at E3.5 following coinjection of enucleated mII oocytes with eGFP+/+ cumulus cell nuclei plus buffer alone (Control) or sieGFP#1. The data are plotted ± SEM. Bar = 50 µm.

We investigated whether siRNA delivery with sperm heads was effective for a native gene product, the POU domain transcription factor POU5F1 [46]. Coinjection of sperm plus siOct4#1 resulted in a drop of 83.8 ± 5.6% (n = 4) in the Pou5f1 mRNA levels at E3.5. After an additional day in culture, 10 (50%) embryos from the siOct4#1 group developed to the expanded blastocyst stage, compared to 15 (94%) of the control embryos derived from oocytes that were coinjected with sieGFP#1 and sperm. This finding is reminiscent of the retarded cavitation of Pou5f1 null embryos [47].

Collectively, these data show that siRNA-mediated RNAi can be used to dissect gene function from the earliest moments to the later stages of preimplantation embryogenesis. We next sought to harness this effect for the production of a different class of embryo, derived by nuclear transfer.

Efficient siRNA-Mediated RNAi in Nuclear Transfer Cloned Embryos

A major experimental utility of mII oocytes is their use as recipients in nuclear transfer. Given the efficacy of siRNAs when coinjected with a sperm head, we determined whether they induced RNAi when included in the nuclear transfer protocol. When eGFP+/+ cumulus cell nuclei were coinjected into enucleated mII oocytes with buffer (control), brightly fluorescent E3.5 embryos were produced, as expected (Fig. 3B). However, coinjection with the same volume of 25 µM sieGFP#1 produced a reduction in the level of eGFP mRNA to 7.2 ± 2.6% of the control level by E3.5. In contrast to the controls, eGFP epifluorescence was markedly ameliorated in the resultant E3.5 embryos (Fig. 3B). Nuclear transfer protocols typically include reprogramming windows of 1~6 h (10 h is permissible), during which time the donor nucleus resides within the mII ooplasm prior to artificial oocyte activation [39]. This is within the period required for siRNA-driven RNAi, which suggests that this technique will enable verification and/or screening of mII components required for reprogramming.

Short Interfering RNA Delivery During Fertilization Establishes an Immediate Early Mitotic Role for Cdc20 but Suggests that Levels of Emi1 and Emi2 Are Not Critical

The utility of siRNA-mediated RNAi lies in its ability to dissect biological processes. We used siRNAs to investigate the roles of the putative meiotic cell cycle regulators CDC20, EMI1, and EMI2 in early mitosis [6, 48]. Emi1 transcripts persist throughout preimplantation development, whereas Emi2 transcripts are detectable in zygotes but not after the 2-cell stage [6].

The levels of Cdc20 mRNA declined in 2-cell embryos to 4.0~6.6% of their zygotic levels, as judged by qPCR, and the levels remained low throughout preimplantation development, although they were higher than those of the Cdh1 transcripts, which were typically undetectable (Fig. 4A). Coinjection of mII oocytes with sperm plus siCdc20#1 (25 µM) reduced the level of Cdc20 mRNA to 10.4 ± 1.9% of the level of the age-matched control (sperm head plus sieGFP#2) at 24 h (Fig. 4B). However, whereas the controls divided efficiently (18/21 = 86%) at 24 h, cleavage was observed in only 3% (1/36) of the embryos in the siCdc20#1-injected group (Fig. 4, C and D). The injection of siCdc20#4 elicited an intermediate phenotype that correlated with its intermediate ability to induce RNAi (Fig. 4, C and D). This cleavage failure following Cdc20 depletion endured, which indicates that CDC20 is required for the first mitotic division, consistent with the requirement for APCCDC20 for the first mitotic progression.


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  		FIG. 4. siRNA-mediated RNAi suggests that CDC20, but not EMI1 or EMI2, is essential in early embryogenesis. A) Cdh1 and Cdc20 mRNA levels measured by qPCR at different stages of maturation and embryonic development. The maximal expression levels in each case are set at 1.00. GV, germinal vesicle; GVBD, GV breakdown; m, morula; b, blastocyst. qPCR (B), development to the 2-cell stage (C), and Hoffman modulation images (D) 24 hours after coinjection of mII oocytes with sperm heads plus siRNAs as indicated. Data are plotted ± SEM. Bar = 50 µm.

Analogous experiments probed the roles of the cell cycle regulators EMI1 and EMI2 in early embryogenesis. Coinjection of sperm with siEmi1#3 or siEmi2#1 reduced the levels of the target constructs by 78.4% or 98.2%, respectively, at 24 h postinjection (Fig. 4B). However, in neither case was development significantly attenuated (Fig. 4C). Since depletion of both mRNAs causes a prompt decline in protein levels and induces meiotic exit in the case of siEmi2#1 (Fig. 2B; [6]), this result suggests that the first mitotic cell cycle is not exquisitely sensitive to EMI1 or EMI2 levels.

By illuminating the involvement of oocyte proteins in the first mitosis, these experiments provide proof of the principle that the meiotic and early mitotic cell cycles can be distinguished via the timing and manner of siRNA injection.

DISCUSSION

This report describes siRNA-mediated RNAi following the injection of mII oocytes. Some of these siRNAs elicited reductions in their target mRNAs and proteins to barely detectable levels within a few hours of mII oocyte injection, which is well within the normal fertilizable lifetime [42]. Our approach was extended successfully to intracytoplasmic sperm injection (ICSI) to eliminate targeted mRNAs in the first mitotic cell cycle. The RNAi method was also demonstrated for nuclear transfer clones produced by coinjecting siRNA with somatic cell nuclei.

We utilized siRNA-mediated RNAi to dissect the roles played by the cell cycle regulators CDC20, EMI1, and EMI2 during the first mitotic cell cycle of early preimplantation development. EMI1 sequesters CDC20 during the prometaphase of the somatic cell cycle [4, 49], and the mechanism by which mammalian EMI2 sustains mII arrest in meiosis involves interactions with CDC20 [6]. These mitotic and meiotic cell cycle regulators are present in oocytes before, during, and after the transition from meiosis to mitosis, which raises the question as to whether they are functionally segregated. The experiments described here suggest that CDC20 is required both for the completion of meiosis and the first mitotic cell cycle. There is no other means by which the role of CDC20 in the first mitotic cell cycle could have been so readily demonstrated; injection of zygotes [27] might affect the cell cycle too late to prevent progression through prometaphase, whereas tgRNAi would have to be exquisitely regulated to avoid meiotic failure, since CDC20 is required for meiotic exit [6]. Thus, siRNA-mediated RNAi is a temporally controllable tool for the analysis of the key embryonic transition between meiotic exit and the onset of embryogenesis.

The dissection of mammalian meiotic and early embryonic development via siRNA injection has several advantages over gene targeting. The transience of RNAi permits the manipulation of early embryogenesis without necessarily affecting gene function at later stages. This is likely to be important for embryonic genes that are involved in subsequent development; RNAi-based screens in C. elegans suggest that a significant number of genes essential for embryogenesis subsequently play roles at much later developmental stages [50, 51]. Gene-targeted null females are often infertile [52] or sub-fertile [53], and the earliest associated phenotype may obscure the distinct roles that are played by the same protein in early development [6, 53]. MOS provides one example of this. Although the Mos-deficient maturing oocytes of c-mos gene-targeted mice often fail to arrest at mII, this does not resolve whether MOS is required only for the establishment of mII, is required later to maintain cytostatic arrest, or is required at both phases [6, 53]. This problem could be addressed by gene targeting but only if it were exquisitely tightly regulated, in this case, such that homozygous null alleles were generated within a given cell cycle. While they may be technically feasible in some cases, these types of approaches are cumbersome and time-consuming. In contrast, precisely timed siRNA injection is quick and relatively undemanding, and we have demonstrated in this study that siRNA injection into mII oocytes is efficient at mII and in preimplantation development.

Moreover, gene targeting may induce compensatory alterations in the expression of off-target genes with phenotypic consequences. Unfortunately, and in contrast to the situation with siRNA-mediated RNAi, the specificity or otherwise of gene targeting is poorly documented in terms of its possible effects on nontargeted gene transcript levels, although compensatory upregulation occurs in Septin 5 null mice [54]. Depletion of POU5F1 perturbs the expression of hundreds of genes [55]. Depending on the nature of the modified allele, these changes may require the entire lifetime of the targeted cell and its progenitors to take effect, whereas this compensation is restricted in siRNA-mediated RNAi by the milieu of the injected cell (and excludes those of its progenitors) and the relative acuteness of the procedure.

The RNAi strategy described here circumvents many of these problems by acutely and specifically generating loss-of-function. Maximal target mRNA reduction is achieved by siRNA-mediated RNAi in mII oocytes in a more rapid fashion than that previously reported for dsRNA-mediated RNAi in maturing oocytes [26], although it requires a similar number of molecules (≤5 x 105) to achieve a comparable endpoint reduction (Fig. 2C). As with RNAi in maturing oocytes, different targets appear to be differentially susceptible to siRNA-mediated RNAi in mII oocytes, although it may be possible to reduce this effect by prescreening. Although we have presented data for all the tested siRNAs without prescreening to determine which are the most effective, prescreening via surrogate assays is desirable, for example by cotransfecting a given test siRNA with a construct that encodes its target linked to a reporter and verifying loss of reporter expression [6, 38]. The efficiency of siRNA-mediated RNAi would be improved such that all siRNAs caused a rapid and complete reduction of their targets, thereby eliminating the need for screening. Recently, the 27+0 siRNAs have been found to be particularly effective in mammalian cultured cells [25], and two of the four 27+0 siRNAs we tested elicited marked target reduction in their respective mRNA target levels (Fig. 1D).

By coinjecting siRNAs with sperm or cumulus cell nuclei, we were able to interfere with embryonic development via a streamlined, one-step process. The efficiency of this modification of the nuclear transfer protocol suggests that RNAi does not require an interaction between the donor nucleus and siRNA prior to injection, and that no such interaction occurs. This contrasts with the situation in mII transgenesis, the efficiency of which is significantly enhanced by an association between the sperm head and exogenous tg DNA [37].

The starting siRNA concentration (25 µM) represented a pronounced molar excess (≥1000-fold) relative to the minimal siRNA concentrations sufficient for potent RNAi in mII oocytes after 24 h (Fig. 2, C and D). This suggests that it will be possible to reduce the effective siRNA concentrations in the study of mII oocytes and early embryos, thereby increasing specificity. Even greater reductions in concentration are promised by shRNAs [56]. Not only do shRNAs outperform siRNAs by up to ~2-fold but they are maximally effective at 1~10 nM, which is ~20-fold lower than that of maximally effective siRNAs in tissue culture [56].

In demonstrating the potency of siRNA-mediated RNAi, these studies indicate that siRNAs constitute a powerful tool for functional genomic analyses of mammalian oocytes and early embryos. Importantly, the RNAi operates from mII through preimplantation development, corroborating previous studies [26, 27]. Therefore, it can be used to modulate gene function transiently without prior knowledge of the precise stage at which the modulation is important. This could be harnessed to enhance the derivation of embryonic stem (ES) cells, such as human ES cells, in which permanent genetic alterations are undesirable.

FOOTNOTES

1Supported by grant-in-aid (no. C90-53010) from the Ministry of Education, Sports, Science, and Technology of Japan. Back

Correspondence: 2 Tony Perry, Laboratory of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047 Japan. FAX: 81 78 306 3144; e-mail: tony{at}cdb.riken.jp

Received: 27 May 2006.

First decision: 15 June 2006.

Accepted: 17 August 2006.

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