INVESTIGATION

Association of Maternal mRNA and PhosphorylatedEIF4EBP1 Variants With the Spindle in Mouse

Oocytes: Localized Translational Control SupportingFemale Meiosis in Mammals

Edward J. Romasko,* Dasari Amarnath,*,1 Uros Midic,* and Keith E. Latham*,†,2

*Fels Institute for Cancer Research and Molecular Biology and †Department of Biochemistry, Temple University School of Medicine,Philadelphia, Pennsylvania 19140

ABSTRACT In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes.We find by cDNA microarray analysis enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocytemetaphase II (MII) spindle. We also find that the key translational regulator, EIF4EBP1, undergoes a dynamic and complex spatiallyregulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. Thesephosphorylation variants appear at different positions along the spindle at different stages of meiosis. These results indicate that dynamicspatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation,maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindleformation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates andcouples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understandingmechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs atthe spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1control of localized mRNA translation is likely key to correct segregation of genetic material across cell types.

THE oocytes of many species, both invertebrate and ver-tebrate, contain a large collection of localized determi-

nants in the form of proteins and translationally inactivematernal mRNAs. Similar localized determinants in mamma-lian oocytes have been proposed (Ciemerych et al. 2000),but this aspect of mammalian reproduction remains contro-versial (Hiiragi et al. 2006). Indeed, early mammalian em-bryogenesis is considered to be quite plastic and regulativein nature, so that localized determinants would not be ex-pected to play essential functions. Embryo splitting can beused for twinning, and blastomere extirpation does not pre-vent elaboration of normal body plans and term develop-

ment. Additionally, much of the volume of the mammalianoocyte eventually becomes allocated to cells that do notcontribute to embryonic development, being destined in-stead to generate the placenta. Accordingly, prepatterningof the mammalian oocyte through localization of maternalmRNAs or proteins, if it occurs, appears to be dispensable formammalian embryogenesis.

One potential exception to this would relate to localiza-tion within the oocyte of maternal mRNAs that supporta vital process that is evolutionarily conserved betweenmammals and other species, namely the formation andmaintenance of the meiotic spindle. Recent studies in Xen-opus revealed enriched localization to spindle microtubulesof mRNAs encoding spindle proteins (Blower et al. 2007).The spindle is a complex structure; proteomic studies ofisolated spindles have identified .1100 spindle-associatedproteins, of which nearly 400 are specific to spindles andshared with proteomic studies that incorporated DNAsedigests to deplete DNA-associated proteins (Sauer et al.2005; Bonner et al. 2011), indicating that a large array of

Copyright © 2013 by the Genetics Society of Americadoi: 10.1534/genetics.113.154005Manuscript received June 4, 2013; accepted for publication July 9, 2013Supporting information is available online at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.154005/-/DC1.1Present address: Taconic Farms, 273 Hover Avenue, Germantown, NY 12526.2Corresponding author: Department of Animal Science, College of Agriculture,Michigan State University, 474 S. Shaw Lane, Anthony Hall, Room 1230E, EastLansing, MI 48824-1225. E-mail: lathamk1@msu.edu

Genetics, Vol. 195, 349–358 October 2013 349

proteins is needed to support spindle formation, maintenance,and function. Localized maternal mRNAs could be translatedin situ to provide a local high concentration of proteins, whileminimizing potential deficiencies related to limitations in thespeed or extent of protein accumulation from elsewhere with-in the ooplasm.

Many maternal mRNAs that undergo translational recruit-ment and degradation in the mouse oocyte encode spindle-associated proteins (Chen et al. 2011). Some recruited mRNAscontain recognizable cytoplasmic polyadenylation elements(CPEs), which participate in translational regulation, andother mRNAs contain binding motifs for DAZL (deleted inazoospermia-like), a CPEB-regulated protein that is criticalfor translational control of maternal mRNAs encoding spindleproteins (Chen et al. 2011). Many other mRNAs that arerecruited stage specifically lack recognizable CPEs, indicatingthat multiple translational regulatory mechanisms may oper-ate at different stages (Potireddy et al. 2010).

Given the complex and dynamic pattern of maternal mRNArecruitment during oocyte maturation and early embryogen-esis (Potireddy et al. 2006, 2010; Mtango et al. 2008; Chenet al. 2011) and the prevalence of spindle-encoding mRNAsamong these, we wished to test oocytes of a mammalian spe-cies for conservation of localized maternal mRNAs at thespindle. We tested whether the key translational regulator,EIF4EBP1, might likewise be enriched at the spindle as partof the overall regulatory mechanism. We find by cDNA micro-array analysis enrichment for maternal mRNAs encodingspindle proteins and other proteins on the mouse oocyteMII spindle. We also find that EIF4EBP1 undergoes a dy-namic and complex spatially regulated pattern of phos-phorylation at sites that regulate its association withEIF4E and its ability to repress translation. These phos-phorylation variants appear at different positions alongthe spindle–chromosome complex (SCC) at different timesthroughout meiotic maturation. These results indicate thatdynamic spatially restricted patterns of EIF4EBP1 may pro-mote localized translation within the mammalian oocytethat contributes to spindle formation, maintenance, andfunction, and other nearby processes. Thus, localization ofmaternal mRNAs at the spindle is evolutionarily conservedbetween mammals and other vertebrates, and spatially reg-ulated EIF4EBP1 phosphorylation may control the transla-tion of these mRNAs, providing a means for coordinatingspindle formation and maintenance with progression throughthe cell cycle.

Materials and Methods

Oocyte isolation and culture

Hybrid C57Bl/6 X DBA/2 (B6D2F1) females were obtainedfrom the National Cancer Institute (NCI) at 5–6 weeks ageand used from 6 to 10 weeks age. Mice were injected in-traperitoneally with 5 IU of equine chorionic gonadotropin(eCG) and were sacrificed by cervical dislocation 44–48 hr

later. Ovaries were dissected in 37� HEPES-buffered M2 me-dium with 0.2 mM isobutyl methyl xanthine (IBMX) (Sigma-Aldrich, St. Louis) to inhibit meiotic resumption of oocytes.Ovaries were held with forceps and punctured with a 27.5-gauge needle to release cumulus-enclosed oocytes (COCs)into the dish. All abnormal and dead COCs were excluded.COCs were cultured for 1 hr in 50 mL mineral oil-coveredmicrodrops of MEMa (Life Technologies/Invitrogen, GrandIsland, NY) supplemented with 10% fetal bovine serum(Life Technologies/Gibco) that had been preequilibratedovernight. Attached cumulus cells were removed by mouthpipetting using a narrow bore pipette with a diameterslightly larger than that of an oocyte. The appearance ofa perivitelline space (PVS) between the oocyte plasma mem-brane and the zona pellucida after the 1-hr culture provideda reliable indicator of oocyte meiotic and developmentalcompetence; only oocytes with a centrally located germinalvesicle (GV) and present PVS after 1 hr recovery were usedfor experiments (Inoue et al. 2007). To release meioticarrest, oocytes were washed six times in MEMa + 10%FBS lacking IBMX. The following times were used to isolateand fix oocytes at major meiotic cell cycle events: germi-nal vesicle breakdown (GVBD) at 2 hr after IBMX removal,metaphase I (MI) at 6 hr after IBMX removal, and MII at16 hr after IBMX removal. To obtain in vivo matured MIIoocytes, mice were injected with eCG, followed 48 hr laterby 5 IU of human chorionic gonadotropin (hCG). Sixteenhours later, cumulus cells were removed by incubationin M2 medium containing hyaluronidase (120 IU/ml,Sigma).

All studies were approved by the Temple University In-stitutional Animal Care and Use Committee, consistent withNational Institutes of Health (NIH) Guide for the Care andUse of Laboratory Animals, and with Association for Assess-ment and Accreditation of Laboratory Animal Care (AAALAC)accreditation.

Expression microarray analysis

Total RNA was isolated from cells using the PicoPure RNAisolation kit (Invitrogen). Up to 50 ng of total RNA fromeach array sample were subjected to two rounds of cDNAsynthesis using the RiboAmp HS Plus kit (Life Technologies/Arcturus/Invitrogen). Labeled cRNAwas produced using theAffymetrix GeneChip Expression 39 Amplification for IVTLabeling kit. The biotin-labeled cRNA samples were frag-mented and 10 mg hybridized to arrays. Posthybridizationwashing, staining, and scanning were performed as describedin the Affymetrix GeneChip Expression Analysis TechnicalManual. Microarray data were preprocessed and analyzedwith scripts written in R (R Development Core Team 2009),utilizing routines from Bioconductor (Gentleman 2004).Probeset expression values were summarized and normal-ized using robust multiarray analysis (RMA) (Irizarry et al.2003). The Bioconductor implementation of MicroarrayAnalysis Suite 5.0 algorithm was used to obtain probesetcalls (present, absent, and marginal). Probesets detected

350 E. J. Romasko et al.

in all SCC and intact MII samples but with absent calls forenucleated oocyte samples, and satisfying both thresholdcriteria for inclusions (see below) were retained. Probesetsdetected in all SCC and intact MII samples and satisfyingboth threshold criteria for inclusions (see below) wereretained, regardless of present/absent calls for enucleatedoocyte samples. Average intensities for three sample groupswere calculated from the normalized and filtered probesets,and compared as described in Results. Array data were de-posited with the Gene Expression Omnibus database (acces-sion no. GSE46875).

Oocyte fixation and immunofluorescence

Immunocytochemistry steps were performed in nine-wellglass dishes (Pyrex) using 200 ml drops of solution for incu-bations. Oocytes were fixed in 3.7% paraformaldehyde/PBS(Electron Microscopy) for 30 min at room temperature,washed twice in blocking buffer [PBS containing 0.1%BSA (Sigma), 0.01% Tween-20 (Bio-Rad), and 0.02% so-dium azide (Sigma)], and either stored at 4� or processedimmediately. Samples were permeabilized in PBS containing0.1% Triton X-100 (Bio-Rad) for 30 min and incubated inblocking buffer for 1 hr at room temperature. Primary anti-bodies were used at 1:50 dilution in blocking buffer andwere from Cell Signaling Technology (Danvers, MA) withthe exception of phospho-Ser111-EIF4EBP1 from Abgent(San Diego, cat. no. AP3473a) and MIS18A (also knownas FASP1) from Santa Cruz Biotechnology (Dallas, S-18,cat. no. sc-83615). Primary antibodies included: EIF4EBP1mAb (53H11 Cell Signaling cat. no. 9644S), EIF4EBP1 pAb(Cell Signaling cat. no. 9452S), phospho-Thr69-EIF4EBP1pAb (Cell Signaling cat. no. 9455S), phospho-Ser64-EIF4EBP1(Cell Signaling cat. no. 9451S), phospho-Ser235/236-S6(D57.2.2E Cell Signaling cat. no. 4858P), and phospho-Ser240/244-S6 (D68F8 Cell Signaling cat. no. 5364P).The polyclonal antibodies against EIF4EBP1, phospho-Thr69-EIF4EBP1, and phospho-Ser64-EIF4EBP1 have beenused extensively, and their specificity established in earlier

studies (Gingras et al. 2001a; Wang et al. 2003; Ohne et al.2008; Ma et al. 2009; Fonseca et al. 2011; Fuchs et al. 2011)including one report (Ellederova et al. 2006) in which SDS–PAGE/Western blotting of in vitro matured pig oocytesshowed specific reactions with bands of the appropriatesizes. After overnight incubation with primary antibody at4�, the oocytes were washed three times in blocking bufferfor 10 min each wash. Secondary antibody incubation usedgoat anti-rabbit-Alexa 594 (Life Technologies/MolecularProbes) at 1:300 dilution for 1 hr at room temperature.Oocytes were washed three more times and mounted onslides in Vectashield mounting solution containing 1.5 mg/mlDAPI (Vector Laboratories, Burlingame, CA), covered withcoverslips, and sealed with nail polish. Slides were stored incases at 4� and protected from light with aluminum foil untiluse for confocal microscopy. Confocal microscopic imageswere obtained using a Leica TCS SP5 confocal microscopewith a 340 1.25 NA oil objective. For DAPI excitation, thesample was excited with a UV laser; For Alexa 594 excitation,the sample was excited with a 561-nm laser. Sequential scan-ning was used to eliminate cross-talk between channels. Allsettings were kept constant within groups. For cytoplasmicsignal quantification, mean intensity comparisons were per-formed using ImageJ from the National Institutes of Health(Schneider et al. 2012).

Results

Enrichment of maternal mRNAs at the meiotic spindlerevealed by expression microarray analysis

If localized maternal mRNAs play a vital role in spindleformation, maintenance, and function in the oocyte, thereshould exist a significant number of maternal mRNAs thatare highly partitioned to the spindle. To test for enrichedlocalization of mRNAs at the MII spindle in mouse oocytes,we isolated three samples of .1000 SCCs each by microsur-gery. We also collected four samples of 25 cytoplasts fromwhich SCCs had been removed, and three samples of 25

Table 1 Prevalence of cellular compartments and processes among SCC-enriched mRNAs

Category $1000 % of $1000 500–999 % of 500–999 100–499 % of 100–499 Total % of all genes% of all

assignments

Number of genes in group 50 33 74 157Chromatin/nuclear 17 34 8 24 25 34 50 32 17Signaling 13 26 12 36 22 30 47 30 16Plasma membrane 7 14 12 36 19 26 38 24 13Spindle/cytoskeleton 8 16 5 15 10 14 23 15 8Other or unknown 3 6 4 12 18 24 25 16 9ER 5 10 4 12 11 15 20 13 7Vesicle/endocytosis/transport 6 12 7 21 6 8 19 12 6Protein degradation/ubiquitin 5 10 3 9 11 15 19 12 6Translation 4 8 4 12 6 8 14 9 5Golgi 6 12 4 12 3 4 13 8 4RNA binding 5 10 4 12 2 3 11 7 4Cytoplasmic sequestration 4 8 4 12 1 1 9 6 3Mitochondrial 1 2 0 0 7 9 8 5 3

Some genes can be members of more than one category, hence values in columns 3, 5, 7, and 9 are not additive to 100%.

mRNA Enrichment at Spindles in Mouse Oocytes 351

intact MII oocytes. The samples were processed for RNAextraction; the mRNA was reverse transcribed, amplified,and labeled; and the cRNA was hybridized to Affymetrixarrays. After normalization, average raw intensity valueswere compared to identify probesets that were different be-tween the three sample types based on fold changes as fol-lows: Assuming that the SCC would comprise no more than10% volume of the oocyte, we calculated that a twofold en-richment for mRNAs at the spindle would yield an expressionratio of 2.25 for SCC:cytoplast and 1.125 for intact MII:cyto-plast. Threefold enrichment would yield corresponding ratiosof 3.85 and 1.285.

We identified 50 mRNAs that satisfied both criteria fortwofold enrichment, and an additional 3 that satisfied theSCC:cytoplast ratio but not the MII:cytoplast ratio withmaximum raw intensity values of $1000 (Supporting Infor-mation, Table S1). The mRNAs with greatest levels ofenrichment included two that encode known spindle orcytoskeleton-associated proteins, anillin and MIS18A. Toevaluate the relationship of these localized mRNAs to thespindle and other cellular compartments, as well as the func-tions likely to be directed by their encoded proteins, weassigned the mRNAs to categories representing cellular com-partments or processes (Table 1). The four most prominentlyaffected categories included proteins associated with plasmamembrane, chromatin/nuclear, signaling, and, as expected,spindle/cytoskeletal functions, followed by vesicle/endocy-tosis/protein transport, Golgi and endoplasmic reticulum,ubiquitination and protein degradation, and RNA binding.

For mRNAs with maximum raw intensity values between500 and 999, 33 satisfied both criteria for enrichment and

one satisfied just the SCC:cytoplast criterion. The mostprominent cell compartment and functional categories forthese mRNAs were again plasma membrane, chromatin/nuclear, signaling, and spindle/cytoskeleton. This group hada higher representation of mRNAs related to signaling andplasma membrane functions.

An additional 74 mRNAs satisfied both criteria and hadmaximum raw intensity values of 100–499, and an addi-tional 20 fulfilled just the SCC:cytoplast criterion for inclu-sion. The same top four categories were repeated for thisgroup as the two higher signal intensity groups (with theexception of those listed as other or unknown functions),indicating that these categories are consistently seen acrossthe range of signal intensity values. Protein degradation/ubiquitination and mitochondrial associations were moreprominent for this group of mRNAs.

None of the SCC-enriched mRNAs fulfilled the criteria forthreefold or greater enrichment on the SCC. We note thatthe list of SCC-enriched mRNAs includes those encodingproteins found previously enriched on the SCC, such ascalmodulin, as well as proteins involved in endocytosis, alsopreviously found to be related to spindle formation andfunction (Miyara et al. 2006; Han et al. 2010). Several of thegenes associated with the plasma membrane are involved ininteraction of the cell surface with the cytoskeleton or spindle.

We compared our list of mRNAs enriched at the SCC witha list of polysomal mRNAs enriched more than threefoldin MII oocytes vs. one-cell embryos (Potireddy et al. 2006).Twelve of 50 (24%) SCC-enriched mRNAs were also selec-tively translated at the MII stage (Atrx, Glce, Etnk1, Lbr,Rcn2, Sypl, Cdh1, Tex12, Hectd2, Dnajc3, Slc35a1, and

Figure 1 Enrichment of MIS18A protein on the mouseoocyte MII spindle observed by confocal microscopy andimage quantification. (A–C) MII oocytes were maturedin vivo, fixed, and immunostained as described in Materi-als and Methods. The spindle region of a MII oocyte isshown with MIS18A immunoreactive signal in white. (B)DNA observed by fluorescent DAPI staining. (C) Mergedimage in which MIS18A is shown in red and DNA is shownin blue. (D) Quantitative analysis of increased intensity ofMIS18A localization to the spindle as compared to cyto-plasm for MIS18A. Box plot distribution is shown wherethe mean is represented by a black square (n = 6, P ,0.0047 using one-tailed t-test with unequal variance).MIS18A, MIS18 kinetochore protein homolog A; GV, ger-minal vesicle; GVBD, germinal vesicle breakdown; MI,metaphase I; MII, metaphase II.

352 E. J. Romasko et al.

Bmpr2). Conversely, comparing our SCC mRNA list to thelist of mRNAs enriched on one-cell stage polysomes revealedonly two mRNAs in common (Atp6v1 and Calm1). This con-firms that the SCC-associated mRNAs are selectively trans-lated in MII oocytes, as needed to contribute to spindleformation, maintenance, and function.

Enriched localization of MIS18A at the spindle

Fully grown immature mouse oocytes from large antralfollicles resume meiosis after the luteinizing hormone ovu-latory surge or spontaneously when removed from the ovary.Nuclear envelope dissolution and chromatin condensationare followed by spindle formation and migration, extrusionof the first polar body containing homologous chromosomes,and arrest at metaphase II until fertilization or spontaneousactivation. In rodents, maternal stores of maturation-promotingfactor are adequate to initiate the process, but SCC formationrequires protein synthesis (Hashimoto and Kishimoto 1988),indicating a possible role for translational control of localizedmRNAs in SCC formation.

If enrichment of maternal mRNA at the spindle supportsits formation and function, we would expect to observeenriched localization of that protein to the SCC. We testedfor enriched localization to the SCC of MIS18A (MIS18kinetochore protein homolog A), a protein that is enrichedin the mitotic spindle of HeLa cells, binds and recruitscentromere protein A (CENPA) to centromeres, and is es-sential for metaphase alignment and proper chromosomesegregation (Fujita et al. 2007). This mRNA had an expres-sion ratio of 3.55 for SCC:cytoplast and 1.54 for intact MII:cytoplast and therefore satisfied our criteria of being anmRNA with enriched localization to the SCC. Immunofluo-rescence detection of MIS18A in MII oocytes (Figure 1)revealed enriched localization of the protein at the SCC,with an average immunoreactive signal intensity that was1.5-fold higher in the SCC compared to the surroundingcytoplasm.

Ribosomal subunits indicative of active translationare present at the spindle

We reasoned that for localized mRNAs to direct the localizedsynthesis of their proteins in support of SCC formation andfunction, the translational machinery would need to bepresent at the SCC. A commonly used marker of activetranslation is phosphorylated ribosome protein S6 (RPS6).Phosphorylated RPS6 is associated with efficient formationof translation initiation complexes and entrance into poly-somes (Duncan and McConkey 1982; Thomas et al. 1982).RPS6 is present at Xenopus laevis meiotic spindles (Bloweret al. 2007). To test for RPS6 at mouse oocyte SCCs, weperformed immunofluorescence detection of two phosphor-ylated RPS6 variants (Ser235/236-P-RPS6 and Ser240/244-P-RPS6) in MII oocytes (Figure 2). As expected, controlsin which primary antibody was omitted from immuno-fluorescence showed no detectable signal. Both antibodiesdirected to phosphorylated RPS6 variants labeled the

cytoplasm of GV and GVBD oocytes. Ser235/236-P-RPS6coalesced in a ring around the condensing chromosomesin GVBD oocytes (Figure 2E). Though not enriched substan-tially above the surrounding cytoplasm, both phosphory-lated variants were present at the SCC (i.e., there was noprominent void indicating absence of RPS6), consistent withtranslational capacity being present.

EIF4EBP1 expression and phosphorylation in oocytes

We next examined expression of EIF4EBP1. EIF4EBP1 is akey factor that regulates mRNA translation. This proteinbinds to EIF4E and inhibits its interaction with EIF4G,thereby interfering with translation initiation. EIF4EBP1is an intrinsically disordered protein that undergoes dy-namic folding and stabilization of tertiary structure when itbinds to EIF4E (Fletcher and Wagner 1998). The effect ofEIF4EBP1 phosphorylation on EIF4E binding affinity is likelydue to an intrastructural modulation that prevents foldinginto a binding-compatible conformation, thereby leavingEIF4EBP1 disordered and unfolded (Tait et al. 2010). Ofthe seven serine/threonine phosphorylation sites reportedin EIF4EBP1 (Thr36, Thr45, Ser64, Thr69, Ser82, Ser100,and Ser111 for mouse; human sequence numbers are greaterby one), the first five are phylogenetically conserved amongall species of organisms. The residues Ser100 and Ser111 areunique to EIF4EBP1 and not present in EIF4EBP2 or EIF4EBP3orthologs. The other phosphorylation sites are present.

In general, hyperphosphorylated EIF4EBP1 is associatedwith EIF4E release leading to translation initiation, butcontroversy exists over the importance of particular sites incontrolling the release of EIF4E binding (Gingras et al.2001b; Harris and Lawrence 2003; Hay and Sonenberg

Figure 2 Presence of phosphorylated RPS6 variants at the mouse oocyteMII spindle by immunofluorescence and confocal microscopy. (A–C) Immu-noreactive signal produced by each antibody is shown in red and DNA isshown in blue. (A–C) Localization of Ser240/244-P-RPS6. (D–F) Localizationof Ser235/236-P-RPS6. (G–I) Negative control in which all conditions areidentical except omission of primary antibody. At least five oocytes wereimaged for each condition. Bar, 20 mm. RPS6, ribosomal protein S6.

mRNA Enrichment at Spindles in Mouse Oocytes 353

2004). In somatic cells, site-specific phosphorylation mayfollow an ordered, sequential pattern of acquisition (Gingraset al. 2001a; Ayuso et al. 2010), but this has not been ex-amined in detail for oocytes. Evidence for the role of specificphosphorylation sites in contributing to translation initiationis illustrated in Figure 3. We hypothesized that the trans-lational control of mRNAs localized at the SCC could befacilitated by stage-dependent, spatially-restricted EIF4EBP1phosphorylation. Specifically, we focused on three phos-phorylated residues for detailed examination as potentialregulatory candidates: two sites close to the EIF4E-bindingregion (Ser64 and Thr69) and one site in the C-terminalregulatory region (Ser111).

We examined EIF4EBP1 expression, phosphorylation, andlocalization in GV-intact oocytes before maturation, aftermeiotic resumption at GVBD, at the MI stage during in vitromaturation, and at the MII stage after in vitro maturation.These periods represent major nuclear and cytoplasmic mat-uration events in which the oocyte has stage-specificrequirements for protein synthesis; GVBD and chromatincondensation do not require protein synthesis, but progres-sion to MI and maintenance of MII arrest are dependent onprotein synthesis (Schultz and Wassarman 1977; Siracusaet al. 1978). We examined expression of EIF4EBP1 withphosphorylation at Ser64, Ser111, and Thr69 (Figures 4 and5). In addition, we examined the expression of EIF4EBP1(independent of phosphorylation) using both a monoclonaland a polyclonal antibody against total EIF4EBP1 (Figures 4and 5). For a summary of immunofluorescent stainingresults see Figure 6.

Staining GV-intact oocytes with the antibody for totalEIF4EBP1 revealed diffuse staining throughout the entirecytoplasm and nucleus, except for an absence in the nucleolus.(Figure 4, row 1). Antibodies against Ser64-phosphorylatedEIF4EBP1 (Ser64-P-BP1) showed localization to the GV andcytoplasm, with intense spots also visible within the nucleus(Figure 4, row 2). After GVBD, Ser64-P-BP1 showed an in-crease in cytoplasmic staining and a strong signal associatedwith the condensing chromosomes. The antibody specific forSer111-phosphorylated EIF4EBP1 (Ser111-P-BP1) (Figure 4,

row 3) produced a very similar pattern to Ser64-P-BP1,with an increase in cytoplasmic staining and chromosome-associated signals concomitant with germinal vesicle break-down. The antibody specific for Thr69-phosphorylatedEIF4EBP1 (Thr69-P-BP1) (Figure 4, row 4) showed a low-level homogeneous distribution within the nuclear and cy-toplasmic compartments at the germinal vesicle stage, inaddition to signals on cytoplasmic foci. After GVBD, over-all cytoplasmic levels for Thr69-P-BP1 did not change, butstaining was acquired in specific association with the con-densing chromosomes.

At first metaphase, staining with the antibody for totalEIF4EBP1 showed continued diffuse staining throughout thecytoplasm. Antibodies for Ser64-P-BP1 and Ser111-P-BP1showed strikingly similar immunostaining patterns, whichincluded signals at both poles of the spindle and on smallfoci throughout the cytoplasm (Figure 4, rows 2 and 3).Localization was also seen on kinetochores of MI chromosomesfor both of these phosphorylated forms. Interestingly, Thr69-P-BP1 was enriched along the polar spindle microtubules.

After separation of homologous chromosomes and extru-sion of the first polar body, oocytes bypass interphasewithout DNA replication and arrest at the second metaphaseof meiosis. Protein synthesis is required for the maintenanceof this arrest (Siracusa et al. 1978), and synthesis of cyclin Bis believed to play a major role in this process (Hashimotoand Kishimoto 1988). Staining with the antibody for to-tal EIF4EBP1 revealed diffuse cytoplasmic staining, similarto earlier stages of maturation. An �40% decrease inEIF4EBP1 cytoplasmic levels was seen in MII oocytes com-pared to GV-intact stage oocytes (Figure 5). The monoclonaland polyclonal antibodies produced similar results for allstages examined, with the exception that at MII, the mono-clonal antibody showed an enrichment on the polar spin-dle microtubules that matches the enrichment pattern ofThr69-P-BP1. Thr69-P-BP1 displayed a low level of stainingthroughout the cytoplasm that did not change significantlyduring maturation (Figure 5), but showed a striking signalalong the polar microtubules in all MII oocytes examined(Figure 4, row 4). Ser64-P-BP1 and Ser111-P-BP1 displayed

Figure 3 Model illustrating site-specific phosphorylationand regulation of EIF4EBP1. Motifs within mouse EIF4EBP1are shown below the primary structure along with theiramino acid sequences. Upstream signals (insulin, cell cycleM phase, and DNA damage) and downstream kinases(mTOR, CDK1, and ATM) impact the phosphorylation stateof EIF4EBP1 at several residues including the sites (P)Ser-64, (P)Thr-69, and (P)Ser-111. Phosphorylation sites areshown as solid sites within the structure and motifs areshown as open boxes. DOG 2.0 protein domain structureillustrator software (Tsukiyama-Kohara et al. 2001) wasused to generate the EIF4EBP1 protein structure model.mTOR, mammalian target of rapamycin; CDK1, cell divi-sion kinase 1; ATM, ataxia telangiectasia mutated.

354 E. J. Romasko et al.

intense signals on the spindle poles and in spots throughoutthe cytoplasm (Figure 4, rows 2 and 3). Ser64-P-BP1 stain-ing was also enhanced in the cortical granules of all oocytesexamined. Ser111-P-BP1 did not show cortical granulestaining (Figure 4). Ser64-P-BP1 and Ser111-P-BP1 wereagain enriched on the kinetochores of chromosomes, butthis was less intense than seen in MI oocytes. Whereas totallevels of EIF4EBP1 decreased throughout maturation, cyto-plasmic signals for both Ser64-P-BP1 and Ser111-P-BP1showed an �4.5-fold relative increase as compared withGV-intact stage oocytes (Figures 4 and 5).

Discussion

Maternal mRNA localization has been broadly observed ininvertebrate and anamniote vertebrates, but has not beenassociated with mammalian oocytes. Our results demon-strate that mRNAs encoding proteins associated with thespindle chromosome complex are spatially enriched at theSCC. Moreover, our data demonstrate the presence at the SCCof the protein translation apparatus and the developmentallyregulated phosphorylation of a key translational controlprotein, EIF4EBP1, as well as enriched expression of oneprotein, MIS18A, encoded by one of the localized mRNAs.Localization of mRNA to the spindle has also been reported forXenopus oocytes (Blower et al. 2007). Collectively, our resultsdemonstrate that mammals and amphibians share this aspectof maternal mRNA localization in oocytes. Such localization isalso seen in somatic cells (Mili and Macara 2009), indicating

that it likely plays a key role in the proper formation andfunction of both meiotic and mitotic spindles and chromosomesegregation during meiosis and during mitosis in diverse celltypes. However, differences between cell types suggest thatdistinct modes of regulation exist.

The translational control of these SCC-associated mRNAsis likely to be complex, as maternal mRNAs, including someencoding spindle proteins, are regulated by a combination ofbinding proteins (Chen et al. 2011). Our results demonstratethat the regulated phosphorylation of EIF4EBP1 may con-tribute to this regulation. An increase in EIF4EBP1 phos-phorylation was previously seen in porcine and bovineoocytes by Western blotting (Tomek et al. 2002; Ellederovaet al. 2006), and is confirmed here for mouse oocytes. Be-cause EIF4EBP1 phosphorylation releases EIF4E binding topermit initiation, the overall increase in phospho-EIF4EBP1in the ooplasm may facilitate maternal mRNA translationalrecruitment in the ooplasm.

The dynamic spatial and temporal pattern of localizationof phosphorylated EIF4EBP1 at the spindle is indicative ofa novel mechanism promoting localized protein production.We propose that, as the spindle forms, it captures mRNAsthat remain translationally repressed by the presence ofEIF4EBP1. Phosphorylation of EIF4EBP1 may allow localizedmRNA translation to sustain spindle formation and provide anongoing supply of proteins for spindle maintenance. This mayallow diverse cellular signals to be integrated to control thetiming of localized mRNA translation in support of spindleformation and meiotic progression.

Figure 4 Localization and phosphorylation ofEIF4EBP1 during major stages of meiotic matu-ration. Row 1 (A–D) shows confocal immuno-fluorescence results of oocytes stained withpolyclonal antibody against total EIF4EBP1(n = 9, 11, 5, and 12 for GV, GVBD, MI, andMII, respectively); row 2 (E–H) shows Ser64-P-BP1 (n = 7, 12, 6, and 8); row 3 (I–L) showsSer111-P-BP1 (n = 11, 10, 8, and 14); row 4(M–P) shows Thr69-P-BP1 (n = 5, 7, 7, and 8).All antibodies were used in two to four sepa-rate experiments, and separate lots were testedwhen available. The signal produced by eachantibody is shown in red and DNA is shownin blue. Bar, 20 mm.

mRNA Enrichment at Spindles in Mouse Oocytes 355

The regulation of EIF4EBP1 phosphorylaton at the spin-dle is likely to be temporally and mechanistically distinctfrom its regulation in the rest of the oocyte. We observe somelevel of EIF4EBP1 phosphorylation before and after matura-tion, but a spatially dynamic pattern with enriched localiza-tion at the spindle on a background of overall diminishmentof total EIF4EBP1 content (Figure 6). Thr69-P-BP1 is presentat a low, diffuse level at both GV and MII stages, but under-goes dramatic enrichment at the spindle. Kinases associatedwith the spindle and cell cycle progression are obvious likelycontrollers. Kinases implicated in EIF4EBP1 phosphorylationinclude mTOR, polo-like kinases, cyclin-dependent cell divi-sion kinases, and several others (Lawrence et al. 1997;Yang and Kastan 2000; Heesom et al. 2001; Shang et al.2012). For example, PLK1-mediated phosphorylation in theregion of human EIF4EBP1 residues 77–118, which includesSer112, is accompanied by localization to spindles in mitoticcells. The roles of mitotic kinases in EIF4EBP1 phosphoryla-tion suggests that EIF4EBP1 may help regulate spindle for-mation and function and cell cycle progression, includingproper oocyte maturation.

We observe significant differences between meiotic andmitotic spindles in the regulation of EIF4EBP1 phosphory-lation and localization. We observe two distinct localizationpatterns for phosphorylated variants of EIF4EBP1 on meioticspindles of mouse oocytes: Thr69-P-BP1 along the polarmicrotubules, and Ser64-P-BP1 (with Ser111-P-BP1) at thespindle poles and kinetochores. In HeLa cells, cell-cycle-dependent phosphorylation of EIF4EBP1 occurrs, and CDC2was identified as the critical kinase during mitosis at Ser65and Thr70 (Heesom et al. 2001). Identical phosphorylationpatterns at Thr70-P-BP1 and Ser65-P-BP1 in mitotic HeLacells (Heesom et al. 2001) contrasts with the distinct loca-tions during oocyte meiosis. We also observe differences

between metaphases I and II in the phosphorylation ofEIF4EBP1. These differences between MI, MII, and mitot-ic spindles point to possible functional heterogeneity forEIF4EBP1 in supporting the formation and function of spin-dles that participate in different types of cellular division.

The functional categories represented by the mRNAsenriched at the SCC bears mention. Aside from the expectedprevalence of spindle- and cytoskeleton-related proteins andsignaling proteins, many of the SCC-enriched mRNAs en-coded nuclear/chromatin-associated proteins, proteins asso-ciated with the Golgi and endoplasmic reticulum, and proteinsrelated to protein degradation and ubiquitination. The pres-ence of mRNAs encoding chromatin-associated proteins mayprovide a ready supply of proteins needed for chromatincompaction or nuclear functions after meiosis. The mRNAsencoding Golgi proteins may be important for spindlepositioning, as inhibition of Golgi-based membrane fusion bybrefeldin A treatment disrupts asymmetric spindle positioningin both MI and MII mouse oocytes (Wang et al. 2008). ThemRNAs encoding ER proteins may be related to a dynamicassociation of the ER with the spindle just prior to the firstmetaphase, when the SCC is translocated to the cell surface(FitzHarris et al. 2007). The presence of mRNAs encodingproteins associated with the plasma membrane and the cyto-skeleton may also contribute to asymmetric spindle localiza-tion in the oocyte. The presence of mRNAs encoding proteinsrelated to ubiquitination would also be consistent with a localrole for these proteins in controlling spindle formation andfunction (Mtango et al. 2012).

The decline in total EIF4EBP1 expression during matu-ration with a coincident increase in phosphorylation ofSer64 and Ser111 raises the possibility that phosphorylationregulates EIF4EBP1 stability as well as its binding to EIF4E.A dual effect of phosphorylation of EIF4EBP1 was reportedelsewhere, either reducing affinity of EIF4EBP1 for EIF4E,or promoting polyubiquitination and decreased EIF4EBP1stability (Elia et al. 2008). Our data are consistent withEIF4EBP1 degradation subsequent to phosphorylation. Over-all, the data suggest that phosphorylation and possibly ubiq-uitination regulate the availability and function of EIF4EBP1during meiosis. Regulating EIF4EBP1 expression at the spin-dle may thus comprise one aspect of the critical role forthe ubiquitin pathway previously seen in oocytes (Mtangoet al. 2012).

The 7-methylguanosine cap (m7G) and the activities of itsdirect and indirect binding proteins, including EIF4EBP1,contribute to the regulation of maternal mRNA translationin the mouse oocyte. The m7G cap is present on the majority($80%) of mRNA molecules from both unfertilized and fer-tilized mouse eggs, and nearly all mRNA extracted fromunfertilized mouse eggs are translated in vitro and sensitiveto inhibition by m7GTP (Schultz et al. 1980). In addition,mRNA decapping via maternally recruited DCP1A and DCP2is involved in the degradation of maternal transcripts duringmaturation and proper genome activation in mouse (Maet al. 2013). Thus, the cap-binding protein EIF4E and its

Figure 5 Quantification of cytoplasmic expression of EIF4EBP1 andphosphorylated variants in GV- and MII-stage oocytes. GV and MIIoocytes were isolated, matured (for MII oocytes), fixed, and immunos-tained as described in Materials and Methods. Cytoplasmic fluorescenceintensities were calculated using ImageJ software (NIH) and comparedusing a two-tailed t-test. Error bars represent SEM. Solid represents GVoocytes and shading represents MII oocytes. *P , 0.05; NS, not signif-icantly different.

356 E. J. Romasko et al.

binding partner EIF4EBP1 are important for the recruitmentand translation of maternal mRNAs during maturation andearly development.

We note that EIF4EBP1 null mice are viable and fertile,but display selective effects in tissues where the ratio ofEIF4EBP1 to other EIF4EBP orthologs is highest, as well ashypoglycemia, reduced fat deposition, and increased meta-bolic rates (Tsukiyama-Kohara et al. 2001). The potentialroles for other EIF4EBP orthologs in compensating for anabsence of EIF4EBP1 in oocytes remains to be evaluated.

Defective regulation of EIF4EBP1 may contribute to di-minished oocyte quality. Age-related increases in oocyteaneuploidy are accompanied by defects in the meiotic spindle(Chiang et al. 2012; Nagaoka et al. 2012) and aberrant reg-ulation of maternal mRNA (Pan et al. 2008). Oocytes fromdiabetic mice also display meiotic defects, including chromo-some misalignment and spindle abnormalities, which can bereversed by islet transplantation (Cheng et al. 2011). Insulinsignaling may promote the production of high-quality oocytes(Wang and Moley 2010) via mTOR-mediated phosphoryla-tion of EIF4EBP1. Future studies to determine the mecha-nisms by which insulin regulates meiosis should add to ourmechanistic understanding of how dysregulated insulin sig-naling might affect oocyte quality and developmental com-petence. Our discovery of localized maternal mRNAs andphosphorylated EIF4EBP1 at the spindle also providerenewed incentive for dissecting the mechanisms that linkmaternal age, genotype, and environmental exposures todiminished oocyte quality arising out of defective spindleformation and function.

Acknowledgments

We thank Bela Patel for her outstanding technical assistanceon this project. This work was supported in part by a grantfrom the National Institutes of Health, National Institute ofChild Health and Human Development, (RO1-HD43092 andRC1-HD063371-02) and the Office of the Director, Office ofResearch Infrastructure Programs Division of ComparativeMedicine Grants (R24 OD-012221/R24RR015253).

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358 E. J. Romasko et al.

GENETICSSupporting Information

http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.113.154005/-/DC1

Association of Maternal mRNA and PhosphorylatedEIF4EBP1 Variants With the Spindle in Mouse

Oocytes: Localized Translational Control SupportingFemale Meiosis in Mammals

Edward J. Romasko, Dasari Amarnath, Uros Midic, and Keith E. Latham

Copyright © 2013 by the Genetics Society of AmericaDOI: 10.1534/genetics.113.154005

Probe Set ID Gene Symbol Gene name SCC/EN MII/ENEN Avg.

IntensityMII Avg.

IntensitySCC Avg. Intensity Gene Function

Cytoplasmic sequestration ER

Vesicle/ endocytosis/

transportPlasma

membraneSpindle/cyto

skeletonChromatin/

nuclearRNA

binding Golgi Signaling OtherMitochon

drial Translation

Protein degradation/u

biquitin

1423256_a_at Atp6v1g1 ATPase, H+ transporting, lysosomal V1 subunit G1 3.65 1.30 536.65 697.46 1961.11 hydrogen-exporting ATPase activity 1

1439648_at Anln Anillin 3.57 1.30 677.13 878.95 2419.65 actin binding, cytokinesis 11453314_x_at Mis18a MIS18 kinetochore protein homolog A 3.55 1.54 807.05 1239.05 2864.96 mitosis 1 11460656_a_at Sft2d1 SFT2 domain containing 1 3.47 1.37 771.32 1058.16 2675.60 vesicle transport 11433537_at Atrx Alpha thalassemia/mental retardation syndrome X-

linked homolog3.30 1.18 793.15 936.99 2619.49 chromatin binding, DNA repair 1

1451154_a_at Celf2 (Cugbp2) CUGBP, Elav-like family member 2 3.26 1.22 276.54 338.23 902.71 mRNA splice site selection 11455402_at Socs7 Suppressor of cytokine signaling 7 3.13 1.27 643.04 816.07 2012.06 cytokine signaling 1 1 11428374_at Glce Glucuronyl C5-epimerase 3.12 1.30 818.18 1064.18 2549.11 glycosamino glycan synthesis, Golgi 11433514_at Etnk1 Ethanolamine kinase 1 3.10 1.36 313.79 426.38 972.68 lipid metabolism 11436900_x_at Leprot Leptin receptor overlapping transcript 3.03 1.27 635.13 808.05 1924.96 receptor 1 11422241_a_at Ndufa1 NADH dehydrogenase (ubiquinone) 1 alpha

subcomplex, 12.97 1.33 539.85 716.28 1605.93 Mitochondrial function 1

1415829_at Lbr Lamin B receptor 2.94 1.30 1155.51 1500.91 3399.25 DNA binding 11439257_x_at 2.92 1.32 455.37 599.81 1329.021456438_x_at 2.85 1.29 382.77 494.32 1091.871438943_x_at 2.77 1.27 309.50 391.77 858.461422449_s_at Rcn2 Reticulocalbin 2 2.89 1.23 398.79 491.30 1154.39 calcium ion binding, ER 11423799_at Eif1 Eukaryotic translation initiation factor 1 2.86 1.30 1122.96 1464.08 3213.92 translation initiation 11455266_at Kif5c Kinesin family member 5C 2.80 1.17 390.84 457.84 1095.94 motor activity 1 11449731_s_at Nfkbia Nuclear factor of kappa light polypeptide gene

enhancer in B cells inhibitor, alpha2.76 1.20 370.54 446.16 1024.26 cytoplasmic sequestration of transcription factor;

interacts with Chuk in NF-kappa-B signaling pathway1 1 1 1

1449039_a_at Hnrpdl Heterogeneous nuclear ribonucleoprotein D-like 2.73 1.29 308.75 397.95 841.55 mRNA metabolism, DNA binding 11428103_at Adam10 A disintegrin and metallopeptidase domain 10 2.71 1.30 623.14 809.22 1688.66 endopeptidase 11454612_at Mex3c Mex3 homolog C 2.65 1.38 406.78 562.76 1076.61 RNA binding, binds ARE 1 11452176_at Nup153 Nucleoporin 153 2.55 1.35 870.39 1171.70 2217.51 negative regulation of RNA export 1 1 11436451_a_at 2.53 1.49 2195.06 3278.57 5554.911436452_x_at 2.42 1.44 1535.91 2209.19 3722.761455968_x_at 2.37 1.43 2555.28 3644.35 6065.001422880_at Sypl Synaptophysin-like protein 2.52 1.32 1359.61 1797.84 3425.04 Transport; cytoplasmic vesicle 11423390_at Siah1a Seven in absentia 1A 2.50 1.35 390.18 525.69 974.01 ubiquitin-protein ligase activity 1 11423807_a_at Calm2 Calmodulin 2 2.49 1.33 418.69 555.71 1043.84 calcium signaling,activation of adenylate cyclase 11428272_at Eif1b Eukaryotic translation initiation factor 1B 2.47 1.18 1008.29 1189.08 2485.81 translation initiation 11448261_at Cdh1 Cadherin 1 2.46 1.32 1214.31 1598.34 2984.59 calcium ion binding, cell adhesion, actin cytoskeleton 1 1 11445684_s_at Hdac2 Histone deacetylase 2 2.45 1.40 631.12 883.27 1544.70 chromatin modification, transcription repression 11435122_x_at Dnmt1 DNA methyltransferase (cytosine-5) 1 2.43 1.24 1066.93 1319.78 2592.76 DNA methylation 11421183_at Tex12 Testis expressed gene 12 2.43 1.34 482.07 647.67 1169.62 synaptonemal complex 11422669_at Ebag9 Estrogen receptor-binding fragment-associated gene

92.42 1.42 380.20 541.24 921.66 apoptotic process, Golgi 1 1

1429043_at Smndc1 Survival motor neuron domain containing 1 2.42 1.30 318.83 413.63 772.82 mRNA processing 1 11416509_at Tm9sf3 Transmembrane 9 superfamily member 3 2.39 1.23 661.95 811.08 1579.76 membrane, binding 11433887_at Dnajc3 DnaJ (Hsp40) homolog, subfamily C, member 3 2.38 1.38 746.77 1032.60 1776.20 Protein kinase inhibitor, ER unfolded protein response 1 11433944_at Hectd2 HECT domain containing 2 2.38 1.45 410.49 595.04 976.23 E3 ubiquitin protein ligase 11433857_at Fat1 FAT tumor suppressor homolog 1 2.38 1.21 869.94 1052.98 2067.54 actin filament organizatoin, cell adhesion 1 1 11424002_at Pdcl3 Phosducin-like 3 2.36 1.29 501.23 646.18 1184.99 apoptosis inhibition 1 11455111_at Yipf6 Yip1 domain family, member 6 2.35 1.28 1169.49 1495.97 2753.37 ER to Golgi transport vesicle 1 11426776_at Wasl Wiskott-Aldrich syndrome-like 2.34 1.33 741.68 985.47 1733.93 actin cytoskeleton organization 1 11417538_at Slc35a1 Solute carrier family 35 (CMP-sialic acid transporter),

member 12.32 1.28 447.29 571.42 1039.62 nucleotide-sugar transport, Golgi 1

1422462_at Ube2t Ubiquitin-conjugating enzyme E2T (putative) 2.32 1.26 673.19 846.30 1561.25 Ubiquitin ligation, DNA repair 1 11455724_at Prrg1 Proline rich Gla (G-carboxyglutamic acid) 1 2.30 1.31 928.59 1220.04 2139.26 calcium ion binding, possible p53 target 1 1 11437313_x_at Hmgb2 High mobility group box 2 2.30 1.36 307.43 416.88 705.60 DNA bending, chromatin structure, base excision repair 1 11427221_at Slc6a20a Solute carrier family 6 (neurotransmitter transporter),

member 20A2.29 1.32 370.14 490.18 847.33 amino acid transmembrane transport 1

1434394_at N4bp2 NEDD4 binding protein 2 2.28 1.35 1514.79 2049.51 3457.59 5'-polynucleotide kinase and nicking endonuclease activity, DNA repair

1 1 1

1434901_at Zbtb2 Zinc finger and BTB domain containing 2 2.27 1.23 374.44 460.11 849.54 nucleic acid binding 11434310_at Bmpr2 Bone morphogenetic protein receptor, type II

(serine/threonine kinase)2.26 1.23 555.29 683.42 1253.29 ATP and growth factor binding 1 1

1453833_a_at Rnaseh1 Ribonuclease H1 2.26 1.33 476.64 633.69 1075.63 DNA replication 1 11434856_at Ankrd44 Ankyrin repeat domain 44 2.25 1.18 668.21 787.77 1506.51 Possible binding to protein phosphatase 6 11434030_at Gtpbp10 GTP-binding protein 10 (putative) 2.25 1.26 441.24 557.11 993.95 Ribosome biogenesis and nuclear function 1 1 1

SUM 4 5 6 7 8 17 5 6 13 3 1 4 5

1423648_at Pdia6 Protein disulfide isomerase associated 6 2.61 1.07 349.32 373.30 911.19 cell redox homeostasis, ER-Golgi intermediate compartment,protein disulfide isomerase activity

1 1

1433834_at March6 Membrane-associated ring finger (C3HC4) 6 2.52 1.11 2264.01 2512.22 5698.12 ligase activity, zinc ion binding 1 11427275_at Smc4 Structural maintenance of chromosomes 4 2.34 0.97 1346.88 1310.31 3152.97 chromosome, condensin complex 1 1

1448937_at Slc35b3 Solute carrier family 35, member B3 4.16 1.41 122.83 172.62 511.56 Translocate PAPS from cytosol/nucleus to Golgi lumen 1 11434161_at Lin52 Lin-52 homolog (C. elegans) 3.26 1.38 219.20 302.16 713.71 Component of DREAM complex; repress cell cycle-

dependent genes1 1 1

1424438_a_at Leprot Leptin receptor overlapping transcript 3.15 1.30 149.40 194.26 469.90 Regulates cell surface expression of leptin and growth hormone receptors

1 1 1

1448148_at Grn Granulin 3.04 1.24 141.20 174.66 429.00 blastocyst hatching; embryo implantation; signaling 1 11423247_at Erp44 Endoplasmic reticulum protein 44 2.93 1.26 175.89 221.82 515.41 Oxidative protein folding in ER 1 11428603_at Glcci1 Glucocorticoid induced transcript 1 2.89 1.40 284.40 397.59 822.93 Early marker for glucocorticoid-induced apoptosis 1 11436499_at Sgms1 Sphingomyelin synthase 1 2.84 1.47 171.73 251.69 487.75 Synthesize sphingomyelin, cell growth and prevention of

cell death1

1418072_at Hist1h2bc Histone cluster 1, H2bc 2.71 1.35 209.58 281.93 568.47 Nucleosome component 11438324_at 9330182L06Rik RIKEN cDNA 9330182L06 gene 2.70 1.30 142.03 184.86 383.48 BMP signaling pathway component; differentiation 1 11418327_at 1110058L19Rik RIKEN cDNA 1110058L19 gene 2.66 1.28 204.82 261.65 545.59 Unknown function 11420106_at Siah1a Seven in absentia 1A 2.66 1.30 111.72 144.81 296.81 E3 ubiquitin protein ligase 1 1 11452053_a_at Tmem33 Transmembrane protein 33 2.62 1.44 199.12 287.68 522.07 Membrane protein; possible DNA damage signaling 1 11455372_at Cpeb3 Cytoplasmic polyadenylation element binding protein

32.61 1.38 169.67 234.62 442.83 Binds CPEs in mRNA UTR to regulate translation 1 1

1438839_a_at Ywhae Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide

2.57 1.35 232.24 313.48 597.45 Phosphoserine/phosphothreonine-binding signaling protein of 14-3-3 pathway; regulates translation during mitosis

1 1 1

1423215_at Spcs2 Signal peptidase complex subunit 2 homolog (S. cerevisiae)

2.52 1.27 228.89 289.99 577.88 Remove signal peptides from nascent proteins in ER as part of microsomal complex

1

1415943_at Sdc1 Syndecan 1 2.51 1.28 209.63 269.14 525.99 Cell surface proteoglycan that connects surface to cytoskeleton

1 1

Rpn1 Ribophorin 1 protein glycosylation, ER component

Tmed2 Transmembrane emp24 domain trafficking protein 2 Golgi organization

Table S1. Probesets for mRNAs showing increased abundance in the spindle-chromosome complex

Probesets with maximum raw intensity values 1000 or greater

Probesets with maximum raw intensity values of 500-999

Probesets satisfying one of two criteria for inclusion at the level of two-fold enrichment

1 1 1

1

E. J. Romasko Ŝǘ ŀƭΦ2 SI

1453055_at Sema6d Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D

2.49 1.31 189.16 247.88 471.67 Membrane protein; changes in cell morphology 1 1

1420089_at Nfkbia Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

2.48 1.19 140.91 167.11 349.62 cytoplasmic sequestration of transcription factor; interacts with Chuk in NF-kappa-B signaling pathway

1 1 1 1

1425494_s_at Bmpr1a Bone morphogenetic protein receptor, type 1A 2.47 1.30 274.38 355.56 678.24 ATP and growth factor binding 1 11428407_at Hnrnpa0 Heterogeneous nuclear ribonucleoprotein A0 2.40 1.31 195.53 256.44 469.85 RNA binding and mRNA processing 11420132_s_at Pttg1ip Pituitary tumor-transforming 1 interacting protein 2.39 1.21 184.00 223.18 440.59 Nuclear translocation of transcription factor 1 1 11415860_at Kpna2 Karyopherin (importin) alpha 2 2.39 1.49 339.37 505.61 809.62 Nuclear protein import 1 1 11428749_at Dmxl2 Dmx-like 2 2.36 1.36 337.87 460.03 798.18 Notch signaling; vesicle scaffold protein 1 11454730_at Tapt1 Transmembrane anterior posterior transformation 1 2.35 1.33 208.47 277.95 490.11 membrane protein; axial skeletal patterning 1 11436071_at Ankrd26 Ankyrin repeat domain 26 2.33 1.32 190.66 251.32 444.84 Signaling; actin filament binding 1 11423195_at Hiat1 Hippocampus abundant gene transcript 1 2.29 1.31 215.26 281.19 492.99 Tetracycline transporter; cell surface membrane protein 1 11415826_at Atp6v1h ATPase, H+ transporting, lysosomal V1 subunit H 2.29 1.32 272.24 358.05 622.45 Regulatory component of vacuolar ATPase; formation of

endosomes1 1

1454973_at Atf7ip Activating transcription factor 7 interacting protein 2.28 1.31 226.75 296.70 517.92 Heterochromatin-associated transcriptional coactivator/corepressor

1

1448493_at Paip2 Polyadenylate-binding protein-interacting protein 2 2.28 1.51 164.17 247.47 373.49 Repressor of poly(A)-containing mRNA translation 1 11435327_at Lpgat1 Lysophosphatidylglycerol acyltransferase 1 2.27 1.33 226.83 302.05 515.10 Glycerophospholipid metabolism 1 1 11449256_a_at Rab11a RAB11a, member RAS oncogene family 2.27 1.24 386.14 480.06 875.97 intracellular membrane trafficking; component of

cleavage furrow1 1 1

1440050_at Hbs1l Hbs1-like (S. cerevisiae) 2.27 1.31 369.92 482.84 838.07 mRNA surveillance pathway; GTP-binding translation elongation factor

1 1

1433550_at Chfr Checkpoint with forkhead and ring finger domains 2.26 1.44 215.36 310.48 486.23 E3 ubiquitin protein ligase; functions in antephase checkpoint

1 1 1

SUM 4 4 7 12 5 8 4 4 12 4 0 4 3

1420138_at Slc19a1 solute  carrier  family  19  (sodium/hydrogen  exchanger),  member  1 2.62 1.20 177.72 213.43 466.14 Folate  transport 11443609_s_at Syvn1 Synovial apoptosis inhibitor 1, synoviolin 2.61 0.80 199.45 158.61 521.21 E3 ubiquitin protein ligase; ER-associated degradation of

unfolded proteins (ERAD); sequester p53 in cytoplasm and degrade

1 1 1 1

1416064_a_at Hspa5 heat shock protein 5 4.31 1.92 30.31 58.24 130.52Activation of signaling in unfolded protein response, response to glucose starvation,

1

1426254_at Tm2d1 TM2 domain containing 1 4.26 1.59 93.62 149.10 399.23 Beta-amyloid peptide-binding protein 1 11423662_at Atp6ap2 ATPase, H+ transporting, lysosomal accessory

protein 24.23 1.30 47.00 61.31 198.91 Renin and prorenin cellular receptor; interacts with

ATPases1 1

1436174_at Atad2 ATPase family, AAA domain containing 2 4.15 1.27 102.15 129.85 423.51 Estrogen-induced transcriptional coactivation and cell cycle effects; interacts with acetylated histones

1 1

1427229_at Hmgcr 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 3.64 1.36 69.61 94.81 253.18 Rate-limiting transmembrane glycoprotein enzyme in cholesterol biosysnthesis

1 1

1426798_a_at Ppp1r15b Protein phosphatase 1, regulatory (inhibitor) subunit 15b

3.55 1.54 86.34 132.72 306.90 Regulates dephosphorylation of EIF2S1; ER stress and translation

1 1 1 1

1438647_x_at Cetn2 Centrin 2 3.52 1.24 48.46 60.03 170.74 Structural component of centrosome and microtubule-organizing center (MTOC) required for correct spindle formation, cytokinesis, and genome stability; calcium ion binding

1 1

1425642_at Cep290 Centrosomal protein 290 3.46 1.27 34.18 43.33 118.24 Regulation of mitotic cell cycle; interacts with microtubule-associated protein complexes

1 1

1418244_at Naa20 N(alpha)-acetyltransferase 20, NatB catalytic subunit 3.27 1.38 53.15 73.34 173.57 Catalytic subunit of NAT complex B; Cotranslational acetylation of N-terminal methionine followed by asparagine; substrates have cell cycle functions; maintains actomyosin fiber structure

1 1 1 1

1419924_at Fnip1 Folliculin interacting protein 1 3.25 1.54 53.10 81.88 172.63 AMPK/mTOR nutrient signaling pathway; may phosphorylate RPS6KB1

1 1

1428210_s_at Chuk Conserved helix-loop-helix ubiquitous kinase 3.24 1.62 33.49 54.28 108.60 Serine/threonine protein kinase; interacts with NFKBIA in NF-kappa-B signaling pathway

1 1 1 1

1423621_a_at Slc33a1 Solute carrier family 33 (acetyl-CoA transporter), member 1

3.19 1.28 77.10 98.98 246.10 Acetyl-CoA transporter; involved in formation of O-acetylated gangliosides

1

1452954_at Ube2c Ubiquitin-conjugating enzyme E2C 3.06 1.36 25.38 34.57 77.79 E2 ubiquitin protein ligase; essential part of APC/C; regulates destruction of mitotic cyclins and spindle organization

1 1 1 1

1428161_a_at Chchd2 Coiled-coil-helix-coiled-coil-helix domain containing 2 3.02 1.73 100.87 174.80 304.70 Mitochondrial function 11425493_at Bmpr1a Bone morphogenetic protein receptor, type 1A 2.98 1.41 97.90 138.13 291.94 ATP and growth factor binding 1 11426712_at Slc6a15 Solute carrier family 6 (neurotransmitter transporter),

member 152.98 1.54 34.19 52.56 101.81 Neutral amino acid transporter 1

1433835_at Ppp3cb Protein phosphatase 3, catalytic subunit, beta isoform 2.89 1.52 34.06 51.62 98.40 Calcium-dependent, calmodulin-stimulated phosphatase; regulates mitochondrial function

1 1

1452053_a_at Tmem33 Transmembrane protein 33 2.62 1.44 199.12 287.68 522.07 Membrane protein; possible DNA damage signaling 1 11439208_at Chek1 Checkpoint kinase 1 homolog (S. pombe) 2.81 1.33 142.37 189.44 400.51 Serine/threonine protein kinase; checkpoint-mediated

cell cycle arrest and DNA repair1 1 1

1436191_at Arid4a AT rich interactive domain 4A (RBP1-like) 2.79 1.79 62.13 111.13 173.37 Interacts with retinoblastoma protein; transcriptional regulation; recruit HDACs

1

1416731_at Top2b Topoisomerase (DNA) II beta 2.79 1.35 131.40 177.36 366.03 Catalyzes breaking and rejoining of DNA strands; synaptonemal complex

1 1

1449044_at Eef1e1 Eukaryotic translation elongation factor 1 epsilon 1 2.75 1.50 89.88 134.94 247.16 Translation elongation; DNA damage response; component of aminoacyl-tRNA synthetase complex

1 1

1431255_at Calr3 Calreticulin 3 2.68 1.45 50.79 73.83 136.15 Calcium-binding chaperone; component of EIF2 complex 1 1

1429596_at Slc7a6os Solute carrier family 7, member 6 opposite strand 2.64 1.49 154.52 230.06 408.45 Directs nuclear localization of RNA polymerase II 11448460_at Acvr1 Activin A receptor, type 1 2.64 1.32 135.03 178.29 356.61 Serine/threonine protein kinase receptor of TGF-B

superfamily1 1

1434604_at Eif5b Eukaryotic translation initiation factor 5B 2.64 1.24 113.90 140.83 300.79 Conserved translation initiation factor; positioning of of initiator methionine tRNA; cap-dependent translation initiation

1 1

1423295_at Tm9sf2 Transmembrane 9 superfamily member 2 2.64 1.33 149.55 199.48 394.62 Possible small molecule transporter or channel in endosomes

1 1

1431510_s_at Cmc1 COX assembly mitochondrial protein homolog (S. cerevisiae)

2.64 1.25 39.86 49.94 105.10 Required for mitochondrial cytochrome c oxidase (COX) assembly and respiration

1

1437669_x_at 2.64 1.56 88.20 137.61 232.511437668_at 2.54 1.48 80.76 119.37 205.331435023_at Itsn2 Intersectin 2 2.62 1.36 124.39 169.27 325.73 SH3-domain containing protein; links clathrin-coated

endocytosisto actin cytoskeleton1 1 1

1424495_a_at 2.60 1.17 70.37 82.20 182.651451374_x_at 2.31 1.21 56.85 69.01 131.441439251_at Idua Iduronidase, alpha-L- 2.59 1.35 56.94 76.76 147.63 carbohydrate metabolism, cell morphogenesis, lysosome 11429723_at Spryd7 SPRY domain containing 7 2.59 1.54 46.54 71.45 120.54 chronic lymphocytic leukemia deletion region gene 6

protein; unknown function1

1428094_at Lamp2 Lysosomal-associated membrane protein 2 2.56 1.18 58.55 69.04 150.04 Glycoprotein; involved in lysosome function, inter-, and intra-cellular signaling

1 1 1

1454678_s_at Eogt EGF domain-specific O-linked N-acetylglucosamine (GlcNAc) transferase

2.54 1.31 93.91 122.82 238.89 Catalyzes transfer of N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue

1 1

Ccrl1 Chemokine (C-C motif) receptor-like 1 Receptor for C-C type chemokines

Cklf Chemokine-like factor Cytokine; secreted and membrane-bound isoforms

Probesets with maximum raw intensity values of 100-499

Probesets satisfying one of two criteria for inclusion at the level of two-fold enrichment

1 1

1 1

E. J. Romasko Ŝǘ ŀƭΦ3 SI

1450026_a_at B3gnt2 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2

2.52 1.33 116.67 154.91 294.10 Biosynthesis of poly-N-acetyllactosamine chains; glycosyl transferase

1 1

1452213_at Tex2 Testis expressed gene 2 2.50 1.35 35.25 47.45 88.02 Phospholipid binding; cell signaling 1 11424573_at Tmed5 Transmembrane emp24 protein transport domain

containing 52.49 1.40 75.31 105.24 187.25 Required for assembly and maintenance of Golgi

apparatus; ER to Golgi intermediate compartment functions

1 1 1

1433940_at Spag7 Sperm associated antigen 7 2.48 1.34 96.01 128.57 238.13 nucleic acid binding 1 11437743_at Aebp2 AE binding protein 2 2.48 1.29 72.45 93.28 179.47 Interacts with and stimulates PRC2 complex, which

methylates histone H3; DNA binding1 1

1417239_at Cetn3 Centrin 3 2.46 1.31 39.32 51.42 96.80 Structural component of centrosome and microtubule-organizing center (MTOC) required for correct spindle formation, cytokinesis, and genome stability; calcium ion binding

1 1

1436555_at Slc7a2 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 2

2.43 1.39 42.11 58.34 102.26 Cationic amino acid transporter 1

1444122_at Sycp2 Synaptonemal complex protein 2 2.42 1.28 175.96 225.96 426.16 Synaptonemal complex; chromatin organization; meiosis 1 1

1450841_at Stt3a STT3, subunit of the oligosaccharyltransferase complex, homolog A (S. cerevisiae)

2.42 1.41 140.63 198.23 340.22 Catalytic subunit of N-oligosaccharyl transferase (OST) complex which transfers oligosaccharides from lipids to nascent polypeptide chains

1 1 1

1437637_at Phtf2 Putative homeodomain transcription factor 2 2.42 1.28 140.02 179.08 338.27 DNA-binding transcriptional regulator 11424017_a_at Hint1 Histidine triad nucleotide binding protein 1 2.41 1.48 97.65 144.57 234.99 Part of E3 ubiquitin-protein ligase complex; inhibits

protein kinase C; hydrolyzes purine nucleotide phosphoramidates

1 1 1 1

1423079_a_at Tomm20 Translocase of outer mitochondrial membrane 20 homolog (yeast)

2.40 1.47 35.75 52.62 85.78 Translocation of cytosolically synthesized mitochondrial preproteins

1 1

1439836_at Asb15 Ankyrin repeat and SOCS box-containing 15 2.39 1.17 133.48 155.95 319.61 Regulatory subunit of E3ubiquitin-protein ligase complex

1

1427162_a_at Elk4 ELK4, member of ETS oncogene family 2.39 1.30 153.92 199.94 368.29 Transcriptional activation, repression, histone deacetylation, and JNK signaling

1 1

1455872_at Fam167a Family with sequence similarity 167, member A 2.37 1.44 78.65 113.22 186.75 Unknown function 11460257_a_at Mthfs 5, 10-methenyltetrahydrofolate synthetase 2.37 1.41 51.55 72.62 122.37 Tetrahydrofolate metabolism; biosynthesis of purines,

thymidine, and amino acids1

1423818_a_at Arl6ip1 ADP-ribosylation factor-like 6 interacting protein 1 2.37 1.29 142.31 183.06 337.03 Protein transport and membrane trafficking 1 11434592_at Slc16a10 Solute carrier family 16 (monocarboxylic acid

transporters), member 102.36 1.18 78.22 92.26 184.92 Aromatic amino acid transporter 1

1426941_at Muc15 Mucin 15 2.35 1.40 55.63 77.85 130.54 Cell adhesion; membrane-bound and secreted forms 1 11436175_at Atxn7 Ataxin 7 2.35 1.37 102.37 139.98 240.18 Component of STAGA acetyltransferase complex;

chromatin remodeling and transcriptional regulation; interacts with alpha tubulin

1 1

1453600_at Ccdc18 coiled-coil domain containing 18 2.34 1.27 114.54 145.40 268.58 unknown 11420478_at Nap1l1 Nucleosome assembly protein 1-like 1 2.34 1.28 136.54 174.16 319.47 DNA replication and chromatin formation 11417511_at Lyar Ly1 antibody reactive clone 2.34 1.43 79.32 113.12 185.47 Regulates cell growth; nucleolar protein 11440343_at Rps6ka5 Ribosomal protein S6 kinase, polypeptide 5 2.34 1.32 47.12 62.31 110.08 Serine/threonine protein kinase involved in mitogen and

stress response; translational regulation1 1

1421000_at Cnot4 CCR4-NOT transcription complex, subunit 4 2.34 1.43 86.54 124.17 202.07 mRNA deadenylation, E3 ubiquitin protein ligase, transcriptional reguation

1 1 1

1448543_at Slmo2 Slowmo homolog 2 (Drosophila) 2.32 1.25 100.77 125.78 233.41 Mitochondrial protein; Unknown function 1 11448443_at Serpini1 Serine (or cysteine) peptidase inhibitor, clade I,

member 12.32 1.29 81.91 105.59 189.70 Serine proteinase inhibitor; Secreted 1

1436025_at Ccdc88a Coiled coil domain containing 88A 2.30 1.23 58.32 71.95 134.11 Actin binding, cytoskeleton remodeling, activates AKT/mTOR signaling pathway

1 1 1

1458820_at Zfp560 Zinc finger protein 560 2.29 1.78 66.39 118.38 152.28 Possible transcriptional regulator 11427349_x_at Zfp931 Zfp931 zinc finger protein 931 2.29 1.50 42.37 63.68 96.97 Possible transcriptional regulator 11428945_at Uba6 Ubiquitin-like modifier activating enzyme 6 2.29 1.33 131.21 174.42 300.10 E1 ubiquitin activating enzyme 11447896_s_at Snhg8 Small nucleolar RNA host gene 8 2.28 1.23 113.91 139.98 259.37 Unknown function 11418083_at 0610009B22Rik RIKEN cDNA 0610009B22 gene 2.27 1.40 61.78 86.27 140.44 Targeting and fusion of ER to Golgi transport vesicles 1 1 1 11416288_at Dnaja1 DnaJ (Hsp40) homolog, subfamily A, member 1 2.27 1.31 109.70 144.16 248.65 Heat shock chaperone protein; Protein import into

mitochondria; unfolded protein response1 1 1

1425241_a_at Wsb1 WD repeat and SOCS box-containing 1 2.27 1.49 161.17 240.62 365.28 Regulatory subunit of E3ubiquitin-protein ligase complex

1

1428680_at Cds1 CDP-diacylglycerol synthase 1 2.26 1.20 99.32 118.77 224.64 Catalyzes conversion of phosphatidic acid to CDP-diacylglycerol

1 1

1425462_at Fbxw11 F-box and WD-40 domain protein 11 2.26 1.22 130.82 160.06 295.76 Regulatory subunit of E3 ubiquitin-protein ligase complex; oocyte meiosis; mediates ubiquitination of NFKBIA

1 1

1452870_at Apaf1 Apoptotic peptidase activating factor 1 2.26 1.48 59.01 87.28 133.13 Initiates caspase-dependent apoptosis 1 11416470_a_at Rpn1 ribophorin I 2.26 1.17 111.78 131.17 252.11 protein glycosylation 1

SUM 1 11 6 19 10 25 2 3 22 18 7 6 11

11416655_at C1galt1c1 C1GALT1-specific chaperone 1 3.88 1.48 88.11 130.10 342.21 Molecular chaperone 11429771_at 3110073H01Rik RIKEN cDNA 3110073H01 gene 3.72 1.36 38.16 51.89 142.02 unknown1417499_at Timm10 translocase of inner mitochondrial membrane 10 homolog (yeast) 3.37 1.36 64.41 87.49 217.18 Protein import into mitochondrion 11439040_at Cenpe Centromere protein E 3.22 0.89 117.92 105.34 379.47 Motor activity responsible for chromosome movement

and spindle elongation1 1

1434435_s_at Cox17 cytochrome c oxidase, subunit XVII assembly protein homolog (yeast)3.17 1.06 54.64 57.88 173.10 copper uptake to mitochondrion 11430519_a_at Cnot7 CCR4-NOT transcription complex, subunit 7 2.81 1.27 78.60 99.91 220.48 Transcription cofactor required for spermatogenesis 11420114_s_at Coprs Coordinator of PRMT5, differentiation stimulator 2.79 1.11 99.62 110.99 278.02 Histone H4 methyltransferase activity 11453734_at 2.75 1.02 40.46 41.27 111.391450051_at 2.43 0.66 73.58 48.36 179.131420948_s_at 2.36 1.09 51.30 55.95 120.91

1452224_at Morc3 microrchidia 3 2.71 1.68 33.51 56.44 90.68RNA metabolism and maintenance of nuclear structure protein localization,

1

1449849_a_at Fbxl6 F-box and leucine-rich repeat protein 6 2.70 1.20 40.12 48.24 108.18 Protein ubiquitination 11437308_s_at F2r coagulation factor II (thrombin) receptor 2.67 1.26 36.37 45.66 97.09 thrombin receptor 1 11451996_at Tm2d1 TM2 domain containing 1 2.61 1.12 30.60 34.15 79.83 G protein signaling 11436506_a_at Snhg6 Small nucleolar RNA host gene (non-protein coding)

62.57 1.11 73.78 81.81 189.83 Unknown function 1

1426834_s_at D930015E06Rik RIKEN cDNA D930015E06 gene 2.56 1.12 60.61 68.14 154.98 Transmembrane protein of unknown function 1 11416369_at Hiatl1 hippocampus abundant transcript-like 1 2.55 1.27 88.43 112.32 225.89 Transmembrane transport 11429679_at Lrrc17 Leucine rich repeat containing 17 2.44 0.95 48.49 45.97 118.38 Secreted bone differentiation regulator 11417672_at Slc4a10 solute carrier family 4, sodium bicarbonate cotransporter-like, member 102.40 1.28 61.30 78.53 147.27 Sodium bicardonate carrier 11418377_a_at Siva1 SIVA1, apoptosis-inducing factor 2.36 1.12 101.72 113.89 240.43 Apoptosis initiatior 11433772_at Hspa13 Heat shock protein 70 family, member 13 2.28 1.06 71.54 76.13 163.07 Microsomal unfolded protein response 1 1 11460599_at Ermp1 Endoplasmic reticulum metallopeptidase 1 2.27 0.95 74.23 70.51 168.62 Proteolysis; ovarian follicle development 1 1Abbreviations: SCC, spindle chromosome complex; EN "enucleated" or SCC-depleted cytoplast; MII intact MII oocyte,

Atrx Alpha thalassemia/mental retardation syndrome X-linked homolog (human)

chromatin binding, DNA repair

Probesets satisfying one of two criteria for inclusion at the level of two-fold enrichment

1

E. J. Romasko Ŝǘ ŀƭΦ4 SI