Prototypical microRNAs (miRNAs) are 21~25-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.
The cytoplasmic events that immediately follow sperm-oocyte fusion in mammalian fertilization, which are collectively termed oocyte activation, are essentially posttranscriptional. Activation reduces the relatively low transcript level of oocytes even further , and broad transcriptional activity does not resume until the late 1-cell stage, which in the mouse is ~12 h later [2–4]. The transition from oocyte-like to zygote cytoplasm that occurs during this period is functionally remarkable and probably unique, since it corresponds to the emergence of a totipotent state. The genesis of totipotency presumably reflects a remodeling of the oocyte cytoplasm, although little is known about this process. One prelude to totipotency following gamete fusion is meiotic cell cycle resumption. The signaling network associated with meiotic exit exemplifies posttranscriptional regulation of protein activities during oocyte activation. For example, the levels of the recently described cytostatic factor EMI2, decline  and in Xenopus, its destruction is the result of calmodulin kinase II-mediated phosphorylation, which targets it for ubiquitination and subsequent proteolysis . A subset of oocyte proteins is thus subject to posttranscriptional regulation at the level of phosphorylation soon after fertilization.
In an analogous fashion, protein phosphorylation modulates mitotic cell cycle control  and has become a classical paradigm of mitotic posttranscriptional regulation . However, in mitotic cells, this tier of posttranscriptional regulation is supplemented with another, which involves short RNA molecules, termed microRNAs (miRNAs). This is a family of 21~25-nucleotide (nt), single-stranded RNAs with several hundred members, of the predicted order of 103 in the human genome . The miRNAs of vertebrates typically regulate mRNAs, for which they possess imperfect sequence complementarity, to inhibit translation . This functionally neutralizes the mRNA target in a reversible manner. In some cases, the miRNA forms a perfect complement with its target, thereby causing (irreversible) target destruction . Both the reversible and irreversible pathways are mediated by the RNA-induced silencing complex (RISC), which is a multi-subunit molecular machine  whose principal site of action is the cytoplasmic P-body . In addition to the degree of miRNA/mRNA complementarity, RISC composition plays a role in pathway selection; only those complexes that contain EIF2C2 (formerly AGO2) are able to cleave mRNA targets, whereas those with EIF2C1, 3 or 4 cannot cleave the targets .
It has become clear that miRNAs play a major regulatory role in diverse aspects of vertebrate differentiation and development . As examples, roles have been proposed for Mirn181a-2 in hematopoiesis , Mirn430 in brain morphogenesis , Mirn1–1 in skeletal muscle development and proliferation , and Mirn196a-1 in hind-limb formation . Some miRNAs play multiple developmental roles at distinct sites and/or timepoints. For example, in addition to its role in brain morphogenesis, zebrafish mirn430 has recently been shown to target maternally derived mRNAs immediately after fertilization, a process that results in maternal transcript purging prior to the onset of embryonic transcription (zygotic gene activation) . Zebrafish mirn430 also exemplifies the coregulation by a single miRNA of several hundred distinct mRNAs, since it has >300 mRNA targets during early development .
We reasoned that mammalian fertilization similarly involves posttranscriptional silencing of oocyte mRNAs as a prelude to embryonic development. One attractive strategy by which this regulation can occur is the delivery of sperm-borne miRNAs into the oocyte during fertilization. In the present study, we investigate this possibility in the mouse and show that if there is any miRNA contribution from spermatozoa at fertilization, it is limited.
Materials and Methods
Mice, and Preparation and Culture of Oocytes and Embryos
Oocytes were isolated from the oviducts of B6D2F1 females (8–12 weeks old) following superovulation by intraperitoneal injection of 5 IU eCG, followed by 5 IU hCG 48 h later. Oocytes were typically collected in HEPES-buffered CZB  at 12 h post-hCG injection, denuded of associated cumulus cells, washed, and placed in KSOM under mineral oil (Shire, Florence, KY) in a humidified atmosphere of 5% (v/v) CO2 in air at 37°C  until required. Embryo culture was also performed in KSOM in a humidified atmosphere of 5% (v/v) CO2 in air at 37°C. For PCR analysis of oocyte transcripts, zonae pellucidae were first removed by briefly exposing the oocytes (~30 sec) to acid Tyrode solution, and then washing them in HEPES-buffered CZB.
The mice were supplied by SLC (Shizuoka-ken, Japan) and handled according to local institutional guidelines.
Sperm Preparation for RNA Analysis
Acutely isolated, motile spermatozoa were isolated from 24–64 mouse caudae epididymides of strains B6C3F3, C3H, CBA, C57BL/6, and DBA/2 (2.0 × 108 spermatozoa, Sample #1), ICR (6.8 × 107 spermatozoa, Sample #2), ICR (7.3 × 107 spermatozoa, Sample #3), C57BL/6 (1.2 × 108 spermatozoa, Sample #4), B6D2F1 (1.8 × 108 spermatozoa, Sample #5), C57BL/6 (2.5 × 108 spermatozoa, Sample #6), and C57BL/6 (2.1 × 108 spermatozoa, Sample #7). Sperm were prepared by filtering diced caudae epididymides through a KimWipe and extracted by trituration at room temperature in a volume of 2.0–2.5 ml Nuclear Isolation Medium (125 mM KCl, 2.6 mM NaCl, 7.8 mM Na2HPO4, 1.4 mM KH2PO4, 3.0 mM EDTA.Na2 [pH 7.2]; NIM) that contained 0.1% (v/v) Triton X-100 (Sigma Ultra). Sperm were pelleted by centrifugation for 30 sec at 2°C, resuspended in a total volume of 1.5 ml ice-cold NIM, and repelleted by centrifugation for 30 sec at 2°C. The supernatant was carefully removed and the pellet was resuspended in 1 ml of ice-cold PBS, and the sperm were counted. Nonsperm cellular contamination accounted for <0.025% (Sample #1), <0.073% (Sample #2), <0.068% (Sample #3), 0.07% (Sample #4), 1.03% (Sample #5), 0.61% (Sample #6), and 0.41% (Sample #7) of each respective total. Where appropriate, samples were subjected to electron microscopic analysis at the counting stage, to confirm demembranation relative to untreated, swimming sperm. The sperm were further pelleted by centrifugation for 2 min at 2°C, resuspended in 600 μl of lysis/binding buffer (mirVana miRNA Isolation Kit; Ambion) and total RNA was extracted according to the instructions of the manufacturer. The RNA was eluted in 100 μl of elution solution and concentrated by ethanol precipitation for the microarray analysis.
Acutely isolated B6D2F1 cauda epididymidal spermatozoa collected as described above were triturated in NIM that was supplemented with 0.05% (w/v) Triton X-100 at room temperature (26°C) for 15 sec . The cells, which were subsequently held on ice or at 2°C until microinjection, were washed twice in ice-cold NIM; the preparation took ~5 min. For anti-miR coinjection (Ambion), 0.5 μl of the sperm suspension was mixed with a freshly prepared solution that contained 2.5 μl of the two anti-miRs (each at 100 μM) and 4.5 μl of 20% (w/v) PVP360 (average Mr = 360 kDa), and microinjected as described previously  within 40 min of PVP360 mixing. The injected concentration of each anti-miR was 25 μM. The anti-miR combinations were: anti-Mirn16–1 plus anti-Mirn30c-1; anti-Mirn136 plus anti-Mirn145; and anti-Mirn191 plus anti-Mirn222.
For nuclear transfer , enucleation of mII oocytes was performed in M2 medium supplemented with cytochalasin B (5 μg/ml). Enucleated oocytes were kept in KSOM and divided equally into two groups. Cumulus cell nuclei were injected into the members of one group, whilst the members of the other group did not undergo nuclear transfer. At ~30 min postinjection, both groups were collected, subjected to zona pellucida removal by exposure to acid Tyrode solution, washed in HEPES-buffered CZB, transferred in ≤1 μl to 50 or 100 μl mirVana lysis/binding solution, and maintained at −80°C until required for PCR analysis.
Bright-field images were captured with a DP-12 digital camera (Olympus). All micromanipulation was performed a piezo-actuated micropipette (Prime Tech, Japan) essentially as described previously [23, 24].
In Vitro Fertilization
RNA was prepared from oocytes soon after fertilization. Freshly collected, cumulus-denuded oocytes (12~16 h post-hCG) that generally lacked a Pb1 were placed in 1 ml of human tubal fluid (HTF) medium with 106 acutely isolated B6D2F1 cauda epididymidal spermatozoa, which had just been capacitated for 1 h in HTF medium using standard procedures. Fertilization was carried out at 37°C in a humidified atmosphere of 5% (v/v) CO2 in air. After ~2 h, most of the oocytes that had undergone second polar body (Pb2) extrusion were pooled, exposed to acid Tyrode solution for zona pellucida removal, washed extensively in HEPES-buffered CZB medium, transferred in ≤1 μl to 50 or 100 μl mirVana lysis/binding solution, and held at −80°C until required for PCR analysis. A smaller group of the fertilized oocytes was cultured and transferred to pseudopregnant recipients, to confirm that they were developmentally competent.
The RNA preparations yielded 10.4 μg (Sample #1), 4.6 μg (Sample #2), 8.2 μg (Sample #3), 4.5 μg (Sample #4), 9.5 μg (Sample #5), 14.3 μg (Sample #6), and 11.6 μg (Sample #7), and were tagged with a Cy5 dendrimer prior to hybridization overnight to mouse mParaflo superfluidic miRNA array probes, followed by a high stringency wash, which was performed blind at the laboratories of LC Sciences (http://www.lcsciences.com). The arrays were analyzed using the Axon GenePix 4000B Microarray Scanner (Axon Instruments) and the data were processed with the ArrayPro software (Media Cybernetics).
All samples, with the exception of Sample #6, were subjected to arrays that contained six repeats for each miRNA detection probe on the array; Sample #6 was probed with an array that contained four such repeats. When signals were detected for less than four of the repeats, they were considered unreliable and excluded from the sets of detected miRNAs. The array sequence contents were derived from the Sanger Institute miRBase, release 7.0 or 7.1 (http://micrororna.sanger.ac.uk/sequences). We employed a local background subtraction method and divided the data into two groups that were above and below the detection levels, as determined by two criteria: signal intensity less than three-times the background standard deviation was considered below the detection threshold, and a spot CV of >0.5 was indicative of unreliable detection, where CV is calculated as: (standard deviation)/(signal intensity). The microarray data are deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo/) with the series accession number GSE4011.
Analysis of mRNA by PCR
For mRNA analysis, 79 or 156 zona-free mII oocytes (collected 16–17 h post-hCG) were processed in Isogen (Nippon Gene, Japan), to yield 240 ng and 700 ng total RNA, respectively. The RNA was treated with DNase I and used for oligo d(T)20-primed first-strand cDNA synthesis with SuperScript III RT, followed by digestion with RNase H (Invitrogen) in a final reaction volume of 20 μl. The 0.05× or 0.1× cDNA reaction mixtures were used to program PCR reactions with GeneTaq NT DNA polymerase (Nippon Gene, Japan). The reactions were accompanied by negative, RT-minus controls. The PCR for sperm-derived cDNA involved 35 cycles of 94°C for 30 sec, 59°C for 30 sec, and 72°C for 30 sec. For zona-free oocyte-derived cDNA, the PCR involved 35 cycles of 94°C for 20 sec, 60°C for 20 sec, and 72°C for 30 sec. The PCR products were visualized on agarose gels.
The following primers were used for RT-PCR: Actb, 5′-GGCATTGTTACCAACTGGGACGAC-3′ and 5′-CCAGAGGCATACAGGGACAGCACAG-3′; Cd8b1 (AK088649), 5′-ATCCTGCTTCTGCTGGCATTCC-3′ and 5′-TCCTTGTGAGACCCCAAACCAC-3′; Ptprc (NM_011210), 5′-TTTGGGGATTCCAGAAACGC-3′ and 5′-CATTGACATAGGCAAGTAGGGACAC-3′; Cd48 (NM_007649), 5′-TCGTGTGAGGTAAAGGACCAGC-3′ and 5′-GACTCTGTGCTTCCAAGTTGCC-3′; Mtpn, 5′-CCCCTCTTCTGTCTGCTGTCTATG-3′ and 5′-ATCACATTCCCCACTGCTCCTC-3′; Lin28, 5′-GAATCCATCCGTGTCACTGGC-3′ and 5′-TTACCCCCACTTTCTCCACTCTG-3′; Smc1a, 5′-CATTGAGATTGACTACGGTGACCTG-3′ and 5′-GCCTTCTTTGCTCGCTTTCG-3′; Mapk7, 5′-CCTAATGCTTTTGATGTGGTGACC-3′ and 5′-GAGCGGCTGTGAAGAGTGAATG-3′; 2700055K07Rik, 5′-GCTGGCAACTAAGCGGTTCAAG-3′ and 5′-TTTGGCAGGTGTCACTTCTTCAC-3′; Bcl2, 5′-TTCCAGCCTGAGAGCAACCCAATG-3′ and 5′-ATCCCTGAAGAGTTCCTCCACCAC-3′; E2f1, 5′-GAGAAGTCACGCTATGAAACCTCAC-3′ and 5′-AGCCGCTTACCAATCCCCAC-3′; Kit, 5′-TGAAGTGGATGGCACCAGAGAG-3′ and 5′-GTCATACATTTCGGCAGGCG-3′; Mapk14, 5′-CCTACTGGAGAAGATGCTCGTTTTG-3′ and GGCACTTGAATGGTATTTGGAGAG-3′; Jak2, 5′-AACTCCAGCACAGCACTGAAGAGC-3′ and ATGTTCCTTGTTGCCAGGTCCC-3′; Hdac4, 5′-GCAGAGGCTGAATGTGAGCAAG-3′ and GCGATAGGCATAACCACCGTTC-3′; Lamc2, 5′-GACAAGACCCAGCAAGCAGAAAC-3′ and GGATGCCGTCCAATGTGTTG-3′; Pten, 5′-CTGAGAGACATTATGACACCGCC-3′ and TGTGAAACAGCAGTGCCACG-3′; Cdk6, 5′-GGTGACCAGCAGTGGACAGATAAAG-3′ and TGGGTTGAGCAGATTTGGAATG-3′; Tmem2, 5′-CCGAGGCTTTCAGATTTACGATG-3′ and TCCCACATAGGTGTCCTTGTATCC-3′; Eif2c1 (NM_153403), 5′-GCTGACAAGAATGAGCGGATTG-3′ and GACATAATAATGGGATGGTCGGC-3′; Eif2c2 (NM_153178), 5′-TCAAGCTGGAGAAGGACTATCAGC-3′ and 5′-TGATCTTCGTGTCCACGGTTGTG-3′; Eif2c3 (NM_153402) 5′-TCCCTGCACCAGCATATTACG-3′ and 5′-TGTGGATCTCGCCCATTGC; Eif2c4 (NM_15317), 5′-AGATAAAATGGAAAGGGTGGGG-3′ and 5′-AATGTGAAGGACGGCTGGTTC; Eif2c5 (AY135691), 5′-ATCTCTGAGAGCCAGTTCCAGCAG-3′ and 5′-GATGCCAGCATGACATGATGAAC-3′.
Analysis of miRNA by qPCR
Total RNA was isolated from zona-free mII and newly fertilized oocytes using the mirVana miRNA Isolation Kit (Ambion) according to the recommendations of the vendor. The samples corresponded to pools of 336, 370, and 482 mII oocytes or 170, 197, and 348 in vitro-fertilized eggs. Reverse transcription (RT) reactions were performed using the TaqMan MicroRNA RT Kit with RNase inhibitor and TaqMan MicroRNA Assay Human Panel Early Access Kit (Applied Biosystems) programmed with 5% of total RNA (corresponding to 8.5~24.1 cells) per miRNA being quantified. Reverse transcription was performed by sequential incubation at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min, in a final volume of 15 μl. For each RT reaction, duplicates of 7 μl were subjected to real-time, semiquantitative (q)PCR with TaqMan Universal Master Mix in a final volume of 20 μl and with the amplification parameters: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The reaction products were analyzed using the ABI Prism 7500 Sequence Detection System. Synthetic Mirn16–1 was used to construct standard curves.
Sperm-borne miRNA qPCR was performed on total RNA from demembranated cauda epididymidal spermatozoa (prepared as described above), which were enriched for miRNAs using the mirVana miRNA Isolation Kit (Ambion), yielding 37.6 ± 9.99 fg RNA/sperm (n = 3 samples). For qPCR with the mirVana qRT-PCR miRNA Detection kit (Ambion), RT was programmed with 10 ng RNA per reaction at 37°C for 30 min and 95°C for 10 min, in a volume of 10 μl. For each reaction, duplicates of 10 μl were subjected to qPCR with 0.2 μl SuperTaq (Ambion) in a volume of 25 μl with the amplification parameters of 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 35 sec. The reaction products were analyzed using the ABI Prism 7500 Sequence Detection System. Synthetic Mirn16–1 was used to construct standard curves. The expression levels are expressed in terms of ratios to Mirn16–1 or Mirn24–1. The microarray data are also normalized against the values for Mirn16–1 or Mirn24–1, to allow the comparison presented in Figure 2C.
For qPCR of embryos 8 and 24 h following coinjection of mII oocytes with sperm plus anti-miRs, three embryos per reaction were placed in 1 μl of 0.2% (w/v) sarkosyl, to give a final volume of ≤2 μl. RNA was processed using TaqMan Micro RNA RT (with RNase inhibitor) and TaqMan MicroRNA Assay ABM 000008 hsa-miR-16 kits (Applied Biosystems). Prior to the addition of Multiscribe RT, RNase inhibitor, and primer, the reactions were made up to 9.8 μl, heated to 65°C or 95°C for 5 min and quenched on ice. The RT enzyme cocktail (4.2 μl) was then added, and the reaction was performed in the following sequence: 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. Amplification of the products was performed as described above.
Mouse Spermatozoa Contain Submembrane miRNAs
Little if any sperm cytosol enters the oocyte at fertilization, although it can be expected to contain miRNAs by analogy to mitotic cells in which the major site of miRNA action is the cytoplasmic body . To exclude sperm components, which include soluble cytoplasm, that do not enter the oocyte at fertilization, and to reduce nonsperm contamination, we extracted seven independent samples (Samples #1–#7) from the mature cauda epididymidal sperm of ICR, C57BL/6, and F1–3 hybrid mouse strains with the detergent Triton X-100 prior to RNA recovery for miRNA microarray analysis. Electron microscopy (Fig. 1A) confirmed plasma membrane disruption and removal of cytosolic material (which does not enter the oocyte) but retention of nuclear and perinuclear structures (which enter the oocyte) [22, 23]. Postextraction contamination from nonspermatozoal cells was assessed microscopically as being <0.025% (Sample #1) to 1.03% (Sample #5). Thus, no contaminating cells were detected for Sample #1 (4000 spermatozoa counted) or for 1370 and 1471 spermatozoa in Samples #2 and #3, respectively. High-sensitivity transcript analysis by PCR failed to detect any mRNAs for markers of lymphocyte contamination, e.g., CD8B1, PTPCR (previously known as CD45), and CD48 (Fig. 1B).
Contaminant-free RNA samples prepared in this way were independently labeled, and the mature processed miRNA profiles were determined on a μParaFlo microfluidics platform under stringent hybridization conditions relative to a database set of 238 probes . In all, 191 distinct miRNAs were detected, with 124.7 ± 28.8 miRNAs per sample. The numbers of detected miRNAs did not correlate with the cellular contamination level (data not shown). Of the 191 detected miRNAs, a core of 54 miRNAs (28.3%) was present in all seven samples (Fig. 2, A and B).
To confirm the microarray data using an independent and sensitive method, we selected 24 of the 54 consistently identified miRNAs for qPCR analysis (Fig. 2C). The selected miRNAs represented a range of apparent abundances but included those that corresponded to the highest signals produced by the microarray probes. The data sets were normalized relative to Mirn16–1 (Fig. 2C) or Mirn24–1 (data not shown), although in only one case (Mirn200c) was a significant difference produced in both cases between the levels indicated by the microarray and qPCR analyses (P = 0.0108) (Fig. 2C). Thus, the microarray and qPCR data are in good agreement. The consistent identification (i.e., detection in all samples) of 54/238 miRNAs suggests that mouse sperm contain at least ~20% of miRNAs in the nuclear and/or perinuclear compartments that are introduced into the oocyte during fertilization.
Sperm-Borne MicroRNAs as Bystanders During Fertilization
The molecular machine responsible for miRNA-mediated RNAi is the RISC, which resides in cytoplasmic bodies . A key catalytic component of the RISC is provided by the Argonaute family of PAZ/PIWI domain endonucleases [10, 26]. We detected low levels of mRNA for Argonaute 2 (EIF2C2, formerly AGO2) in mII oocytes, and higher levels of Eif2c3 transcripts, while Eif2c1, 4, and 5 mRNAs were undetectable (Fig. 3, A and B). To address the question as to whether sperm-borne miRNAs play a role later in development, we determined the expression profiles of Argonaute family members during preimplantation development and found that whereas Eif2c2 and Eif2c3 expression was greatly reduced after the 1-cell stage, the Eif2c4 levels increased dramatically between the 4- and 8-cell stages. These data suggest that RISC activity is present in mII oocytes but that the activity is lost in the early cleavage stages, with subsequent re-establishment around the 8-cell stage.
Fertilization rapidly produces totipotent embryos, which share characteristics with embryonic stem (ES) cells. Of the 11 ES cell-enriched miRNAs, 9 (81.8%) were detected in sperm, including the ES cell-specific Mirn296 [10, 27]. This compares with a smaller overlap (19/54, 35.2%) with the miRNA profiles of other tissues (brain, spleen, lung, hematopoietic, liver, heart, and kidney), and 7/9 (77.8%) with ubiquitously expressed miRNAs . The sperm-borne miRNAs included several for which the targets have been experimentally validated in other systems ; 11/15 (73.3%) of these targets were readily detected in mII oocytes (Fig. 3C).
Although direct comparison of the levels of individual sperm-borne miRNAs with their respective levels in mII oocytes is difficult, the ratio appears to be low (data not shown). This suggests that most sperm-borne miRNAs do not alter significantly the maternal miRNA landscape during fertilization. To test this, we analyzed mII oocytes that had been fertilized in vitro, which enabled us to confirm sperm entry within ~2 h by monitoring Pb2 extrusion (Fig. 4A). This early measure of fertilization (as opposed to the later one of pronucleus formation) was adopted because we wished to address the possibility that sperm-borne miRNAs make a significant but short-lived ooplasmic contribution during fertilization.
Newly fertilized, developmentally competent eggs with nascent Pb2 were collected, stripped of their zonae pellucida (a potential source of contaminating exogenous nucleic acid), and subjected to qPCR for miRNAs that were previously detected in sperm. The corresponding miRNA profiles of the zona-free mII oocytes were determined in parallel, enabling a comparison between unfertilized (mII) and recently fertilized eggs. Data were compared either as sets of estimated copy numbers per cell (Fig. 4B) or as normalized against the level of Mirnlet7a, with no significant difference between the two (data not shown). All of the sperm-borne miRNAs were present in mII oocytes prior to fertilization (Fig. 4B). Moreover, comparison of newly fertilized eggs and control, unfertilized mII oocytes revealed superimposable miRNA profiles, with no consistently significant changes in the miRNA levels (Fig. 4B). Since the analysis included the most abundant sperm-borne submembrane miRNAs, this finding indicates a highly restricted contribution of sperm to the oocyte miRNA landscape at fertilization.
This conclusion clearly predicts that the inhibition of sperm-borne miRNAs has little or no effect on fertilization or early development. As a partial test of this notion, we coinjected membrane-challenged sperm with miRNA inhibitors (anti-miRs) corresponding to the sperm-borne miRNAs Mirn16–1, Mirn30c-1, Mirn145, Mirn191, and Mirn222. We also included anti-Mirn136 as a representative of the anti-miRs that target the miRNAs that we did not detect in sperm (Fig. 2C). These inhibitors are perfectly complementary to their miRNA targets and are stabilized by the addition of 2'-OMethyl-modified linkages at every base, rendering them poorly hydrolysable by cellular RNases [29, 30]. We validated the postinjection miRNA knockdown of Mirn16–1 and found that it was reduced to ~7% of the control levels at 8 h postinjection (Fig. 4C). Interestingly, the level of Mirn16–1 increased after 24 h. Since we included hot start protocols prior to cDNA synthesis, the apparent concentration of Mirn16–1 was unlikely to have been significantly altered by miRNA/anti-miR hybridization, and may therefore reflect de novo Mirn16–1 gene transcription or precursor processing in the period between 8 h and 24 h postinjection.
Although these findings suggest that the miRNA levels were successfully reduced following sperm/anti-miR coinjection of mII oocytes, all of the groups became activated and cleaved at rates that did not differ significantly from those of the controls (Fig. 4, D and E). The embryos developed efficiently to the morula/blastocyst stage in vitro (Fig. 4D). Thus, the presence of given miRNAs in mII oocytes and immediately postfertilization cannot be taken to imply that they perform an essential function in early preimplantation development.
One interpretation of our data is that the oocyte miRNA profile must not be augmented during fertilization if it is to support healthy development. We speculated that the high rate of preimplantation developmental failure (≥50%) of cloned embryos  might be a consequence of miRNA addition during nuclear transfer. To test this idea, we investigated whether cumulus cell nuclear transfer altered the oocyte composition with respect to a subset of ten miRNAs identified in sperm. We did not prescreen cumulus cells for the presence of these miRNAs, so the subset was essentially a random selection.
The miRNA profiles of the enucleated oocytes were superimposable upon the corresponding profiles of the nucleated mII oocytes (compare Fig. 4, B and F). This suggests that the process of enucleation does not result in a general alteration of the mII oocyte miRNA profile. In addition, the levels of most miRNAs in enucleated oocytes were not altered following cumulus cell nuclear transfer (Fig. 4F). However, nuclear transfer reproducibly augmented the level of Mirn21 to ~6-fold its level in (enucleated) mII oocytes (Fig. 4F). This provides proof of the principle that miRNA changes in the oocyte can be detected by qPCR. It also shows that nuclear transfer introduces miRNA that, in at least one case out of the ten tested (10%), causes a marked increase in the endogenous miRNA level.
We have employed microarray and qPCR technologies to investigate sperm-borne miRNA. We found that sperm contain a range of detergent-resistant miRNAs at low levels, and that these miRNAs play at most a limited role in fertilization. Current miRNA catalogs are probably incomplete, as the existence of fewer than 50% of the predicted miRNAs has been confirmed experimentally [9, 25]. Thus, it is possible that sperm contain as yet unidentified miRNAs whose tissue distribution is narrow or exclusive to sperm (because they have not been identified elsewhere) and which play a role at fertilization.
Recently, a novel class of 29~30-nt RNAs (piRNAs) has been described in the testis, where they bind the PIWI family Argonaute proteins PIWIL1 and PIWIL2 (previously MIWI and MILI) [31, 32]. However, piRNAs are apparently present at low levels, if at all, in mature spermatozoa [31, 32]. Another, possibly related class of short RNAs, termed germline small RNAs (gsRNAs) is also present in the testis, although the window of gsRNA expression occurs from the pachytene spermatocyte to early spermatid stages, which is also suggestive of one or more roles in spermatogenesis, rather than during fertilization . Consistent with this, our findings imply that many sperm submembrane miRNAs, including the most abundant, are merely carried over from spermatogenesis, after which they play no role.
Although we set out to test the hypothesis that exogenous, sperm-borne miRNAs facilitate immediate early development, we were unable to find evidence in support of this notion. However, it is also possible that the disruption of oocyte miRNA homeostasis has the opposite effect and adversely affects development. Even if such a mechanism were to operate, our data show that it would not universally affect miRNAs because the inhibition of selected miRNAs with anti-miRs did not perturb preimplantation development (although it is formally possible that later development might have been inhibited). However, perturbation of the mII miRNA landscape could account for the poor developmental potency of nuclear transfer embryos if the process of nuclear transfer introduces one or more miRNAs that critically interfere with immediate early embryonic gene expression. Our data suggest that nuclear transfer deposited a ~5-fold excess of Mirn21 into the enucleated mII oocytes; Mirn21 represented 10% of the miRNAs analyzed (Fig. 4F). If this sample is representative, it indicates that nuclear transfer could augment the levels of a functionally significant repertoire of miRNAs. This is notwithstanding the relative selectivity of nuclear transfer by microinjection; compared to cell fusion, nuclear transfer by microinjection typically introduces less somatic cell cytoplasm into the recipient oocyte. The pleiotropic nature of some miRNAs shows that they can function in different cellular environments [16, 19]. Thus, miRNAs that play a role in somatic cells may also be capable of regulating the same or different mRNAs with distinct roles in embryogenesis following their introduction to an ectopic site by nuclear transfer.
It has recently been shown that the introduction of ~103-fold molar excess (deduced from our data) of Mirn222 into the zygotes of genetic background related to the ones used in the present study efficiently induced a white tail paramutation in resultant offspring . We detected the mRNA of Kit, which is a putative Mirn222 target, in mII oocytes (Fig. 3C). However, the copy numbers of the resident Mirn222 and Kit mRNAs were not reported , and it remains unclear as to whether supplementing resident oocyte Mirn222 by injection of exogenous Mirn222 alters Kit translational regulation so as to account for the paramutation phenotype.
It is possible that the sperm preparation method we used depleted abundant miRNAs that enter the oocyte during fertilization. This is unlikely to be the general case because IVF did not alter the oocyte levels of either abundant or scarce sperm-borne miRNAs. Moreover, there is evidence that at least one of the core miRNAs (the ortholog of Mirn182) is also present in human spermatozoa , which suggests that our catalog of core miRNAs faithfully detected at least one miRNA type that is conserved in another species.
What are the implications of the findings presented here for the potential role of miRNAs in mammalian preimplantation development? We show that transcripts for the RISC catalytic components EIF2C2, EIF2C3 and EIF2C4 are developmentally regulated during preimplantation development (Fig. 3, A and B). On the principle of biological parsimony (i.e., that the energetic load of Eif2c expression is unlikely to be for nothing), this regulation implies that miRNAs indeed play a role in mouse preimplantation development. An early developmental role for miRNA-mediated regulation has been shown for zebrafish Mirn430, which functionally ablates several hundred maternally derived mRNAs by accelerating their deadenylation and clearance . Moreover, homozygously targeted Dicer1 knockout mouse embryos are nonviable and are thought to perish during preimplantation differentiation . Our unpublished preliminary observations using siRNAs against Dicer1 mRNA also hint at an early role, but one which begins well after the zygotic switch. Nevertheless, neither this finding nor the findings presented here preclude a role for maternally derived miRNAs early in preimplantation development, and such a role would explain why Eif2c3 is upregulated at the 1-cell stage (Fig. 3, A and B). Whilst the possibility of reversible early embryonic posttranscriptional regulation by miRNAs awaits detailed investigation, it seems unlikely that sperm extensively utilize this mechanism during mammalian fertilization.
We are indebted to Drs. Joe Xhou and Chris Hebel of LC Sciences for performing the microarray analyses, Dr. Naoko Yoshida for her skills with the embryo transfer pipette, and Ms. Makiko F. Uwo for electron microscopy.