Ribonucleic Acid Export 1 Is a Kinetochore-Associated Protein That Participates in Chromosome Alignment in Mouse Oocytes

International Journal of Molecular Sciences

Article Ribonucleic Acid Export 1 Is a Kinetochore-Associated Protein That Participates in Chromosome Alignment in Mouse Oocytes

Fan Chen 1, Xiao-Fei Jiao 1, Fei Meng 1, Yong-Sheng Wang 1, Zhi-Ming Ding 1, Yi-Liang Miao 1, Jia-Jun Xiong 1,2 and Li-Jun Huo 1,2,*

1 Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; [email protected] (F.C.); [email protected] (X.-F.J.); [email protected] (F.M.); [email protected] (Y.-S.W.); [email protected] (Z.-M.D.); [email protected] (Y.-L.M.); [email protected] (J.-J.X.) 2 Department of Hubei Province Engineering Research Center in Buffalo Breeding and Products, Wuhan 430070, China * Correspondence: [email protected]; Tel.: +86-27-87281813

Abstract: Ribonucleic acid export 1 (Rae1) is an important nucleoporin that participates in mRNA export during the interphase of higher eukaryotes and regulates the mitotic cell cycle. In this study, small RNA interference technology was used to knockdown Rae1, and immunofluorescence, immunoblotting, and chromosome spreading were used to study the role of Rae1 in mouse oocyte meiotic maturation. We found that Rae1 is a crucial regulator of meiotic maturation of mouse oocytes. After the resumption of meiosis (GVBD), Rae1 was concentrated on the kinetochore structure. The Citation: Chen, F.; Jiao, X.-F.; Meng, knockdown of Rae1 by a specific siRNA inhibited GVBD progression at 2 h, finally leading to a F.; Wang, Y.-S.; Ding, Z.-M.; Miao, decreased 14 h polar body extrusion (PBE) rate. However, a comparable 14 h PBE rate was found in Y.-L.; Xiong, J.-J.; Huo, L.-J. the control, and the Rae1 knockdown groups that had already undergone GVBD. Furthermore, we Ribonucleic Acid Export 1 Is a found elevated PBE after 9.5 h in the Rae1 knockdown oocytes. Further analysis revealed that Rae1 Kinetochore-Associated Protein That depletion significantly decreased the protein level of securin. In addition, we detected weakened Participates in Chromosome kinetochore–microtubule (K-MT) attachments, misaligned chromosomes, and an increased incidence Alignment in Mouse Oocytes. Int. J. Mol. Sci. 2021, 22, 4841. https:// of aneuploidy in the Rae1 knockdown oocytes. Collectively, we propose that Rae1 modulates securin doi.org/10.3390/ijms22094841 protein levels, which contribute to chromosome alignment, K-MT attachments, and aneuploidy in meiosis. Academic Editor: Katerina Komrskova Keywords: mouse oocyte; meiotic maturation; Rae1; chromosome alignment; aneuploidy; securin

Received: 22 March 2021 Accepted: 29 April 2021 Published: 3 May 2021 1. Introduction The nuclear pore complex (NPC) is a massive multiprotein complex that is embedded Publisher’s Note: MDPI stays neutral in the nuclear envelope and regulates the nucleocytoplasmic transport of diverse molecules with regard to jurisdictional claims in in the eukaryotic interphase. A single NPC is roughly composed of 30 different protein published maps and institutional affil- components termed nucleoporins [1]. Rae1 (homologs in Schizosaccharomyces pombe, Rae1p; iations. Saccharomyces cerevisiae, Gle2p) was initially identified in S. pombe as a nucleoporin involved in poly(A) mRNA export [2,3]. Subsequent studies have shown that in addition to mRNA export, Rae1 participates in mitotic progression [4]. Rae1 harbors a microtubule–associated activity in higher eukaryotes. In Xenopus egg extracts, the Rae1–RNA ribonucleoprotein Copyright: © 2021 by the authors. complex controls microtubule polymerization [5]. In HeLa cells, Rae1 interacts with the Licensee MDPI, Basel, Switzerland. nuclear mitotic apparatus protein (NuMA), and this interaction and balance is critically This article is an open access article required for normal bipolar spindle assembly [6]. A study in U2OS cells validated that distributed under the terms and ubiquitin-specific protease 11 (USP11) binds to and modulates the ubiquitination of Rae1, conditions of the Creative Commons thereby regulating the interaction with NuMA, which is critical for normal bipolar spin- Attribution (CC BY) license (https:// dle formation [7]. In addition, Rae1 interacts with chromosome subunit 1 (SMC1), and creativecommons.org/licenses/by/ 4.0/). disrupting this interaction can result in spindle abnormalities in HeLa cells [8,9].

Int. J. Mol. Sci. 2021, 22, 4841. https://doi.org/10.3390/ijms22094841 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4841 2 of 12

In higher eukaryotes, Rae1 shares an extensive sequence homology with the mitotic checkpoint protein Bub3 and can interact with Bub1 [10,11]. Further analysis has indicated that the haploinsufficiency of either Rae1 or Bub3 induces a similar phenotype involving mitotic checkpoint defects and chromosome mis-segregation, and the overexpression of Rae1 could correct defects of both Rae1 haploinsufficiency and Bub3 haploinsufficiency in mouse embryonic fibroblast (MEF) lines, which indicates that Rae1 may be analogous to Bub3 [12]. All of the above evidence suggests that Rae1 serves as a mitotic checkpoint regulator. As an important nucleoporin, Rae1 has been extensively studied in mRNA transport and mitotic processes. Rae1 is also critically required for male meiosis and spermatogenesis [13]; however, whether Rae1 exerts a regulatory function during oocyte meiosis is still unexplored. Compared with mitosis, oocyte meiosis is a unique kind of cell division that couples two successive chromosome divisions with only one round of DNA replication to yield a highly polarized egg. Moreover, oocytes undergo protracted arrest at the diplotene stage of prophase I during meiosis [14]. Oocyte meiosis tends to result in chromosome segregation errors, which are especially high in humans, and chromosome mis-segregation is highly correlated with the generation of aneuploidy, which is a leading factor of recur- rent spontaneous abortion and congenital defects [15]. Faithful chromosome segregation relies on stable connections between kinetochores and spindle microtubules. To avoid false chromosome separation and aneuploidy, the mammalian oocyte has a surveillance pathway to monitor the kinetochore–microtubule (K-MT) attachments, the spindle assem- ble checkpoint (SAC), which is composed of Bub and Mad family members [16,17]. The SAC can discriminate between correct and incorrect K-MT attachments and inhibit the activity of the anaphase-promoting complex or cyclosome (APC/C) E3 ubiquitin ligase activity to generate a “stop anaphase” signal until all incorrect attachments are eventually corrected [18]. In our previous study, we investigated the function of another nucleoporin, Nup35, in mouse oocytes. After the resumption of meiosis, Nup35 shows a dynamic spindle location and participates in spindle assembly [19]. However, unlike Nup35, Rae1 is concentrated on the kinetochore structure during meiosis I. Moreover, Rae1–RNAi oocytes showed defects in chromosome alignment, K-MT attachments and aneuploidy, which may be caused by decreased securin expression levels.

2. Results 2.1. Subcellular Localization and Expression Pattern of Rae1 during Mouse Oocyte Maturation We first investigated the subcellular localization of Rae1 at different meiotic maturation stages by immunofluorescence staining. As shown in Figure1A, the immunofluorescence signal of Rae1 was concentrated on the nuclear rim at the germinal vesicle (GV) stage, which is consistent with previous results [6,12]. Moreover, shortly after meiotic resumption, Rae1 was concentrated on the chromosome structure. By further chromosome spreading, we verified that Rae1 accumulated on the kinetochore structure of the chromosome and colocalized with the kinetochore complex CREST (Figure1B,C). The kinetochore localization of Rae1 indicates its specific role during oocyte meiotic maturation. Furthermore, the relative protein level of Rae1 was examined using Western blotting (Figure1D). Rae1 had comparable protein levels at the GV, GVBD (germinal vesicle breakdown), and metaphase I (MI) stages, but had the highest level at the metaphase II (MII) stage (Figure1E). Int.Int. J. J. Mol. Mol. Sci. Sci.2021 2021,,22 22,, 4841 x FOR PEER REVIEW 33 of of 12 13

FigureFigure 1.1.The The subcellularsubcellular localizationlocalization andand proteinprotein expressionexpression pattern pattern of of Rae1 Rae1 during during oocyte oocyte meiotic mei- maturation.otic maturation. (A) Representative (A) Representative images images of the subcellularof the subcellular localization localization of Rae1 of in Rae1 oocyte in at oocyte the germinal at the vesiclegerminal (GV), vesicle metaphase (GV), metaphase I (MI), and I (MI) metaphase, and metaphase II (MII) stages. II (MII) Rae1 stage iss. shown Rae1 is in shown red, α in-tubulin red, α- in green,tubulin and in green, 4’,6-diamidino-2-phenylindole and 4',6-diamidino-2-phenylindole (DAPI) in ( blue.DAPI) Scale in blue. bar Scale = 10 µbarm. = (10B) μm. Representative (B) Repre- imagessentative of Rae1images localization of Rae1 localization in chromosomes in chromosomes of oocytes at of the oocyte germinals at vesiclethe germinal breakdown vesicle (GVBD), break- MI, down (GVBD), MI, and MII stage by chromosome spreading. Rae1 is shown in red and DAPI in and MII stage by chromosome spreading. Rae1 is shown in red and DAPI in blue. Scale bar = 5 µm. blue. Scale bar = 5 μm. (C). The co-localization of Rae1 and CREST in chromosomes of the C (MIIstage). The co-localization oocyte by chromosome of Rae1 and spreading. CREST in Rae1 chromosomes is shown in of red, the MIIstageCREST in oocyte green, by and chromosome DAPI in spreading.blue. Scale Rae1bar = is 5 shownμm. (D in) The red, protein CREST inlevel green, of Rae1 and DAPIat thein GV, blue. GVBD, Scale MI bar, and = 5 µ MIIm. (stageD) Thes detected protein levelby Western of Rae1 blotting. at the GV, (E GVBD,) Relative MI, intensity and MII stagesof Rae1 detected at the GV, by Western GVBD, MI, blotting. and MII (E) Relativestages is intensity pre- ofsented, Rae1 atwhich the GV, were GVBD, calculated MI, and by Rae1/tubulin. MII stages is presented, NS means whichno significant were calculated difference. by ** Rae1/tubulin. p < 0.01. The NSdata means are presented no significant as mean difference. ± standar **dp error< 0.01. of Thethe datamean are (SEM presented) of three as independent mean ± standard experiments. error of the mean (SEM) of three independent experiments. 2.2. Rae1 Knockdown Weakens Meiotic Resumption but Precociously Promotes the First Polar 2.2.Extrusion Rae1 Knockdown Weakens Meiotic Resumption but Precociously Promotes the First Polar Extrusion For further functional analysis, Rae1 was knocked down by the microinjection of For further functional analysis, Rae1 was knocked down by the microinjection of Rae1-specific siRNA into GV stage oocytes. Western blotting results verified that the rel- Rae1-specific siRNA into GV stage oocytes. Western blotting results verified that the ative level of Rae1 at the GV stage was significantly decreased in the Rae1–siRNA mi- relative level of Rae1 at the GV stage was significantly decreased in the Rae1–siRNA croinjected oocytes (Figure 2A). The knockdown efficiency of Rae1 was also validated microinjected oocytes (Figure2A). The knockdown efficiency of Rae1 was also validated with the immunostaining signal of Rae1 in the GV stage oocytes (Figure 2B). These results with the immunostaining signal of Rae1 in the GV stage oocytes (Figure2B). These results indicated that Rae1–RNAi achieved a good knockdown efficiency worthy of further indicated that Rae1–RNAi achieved a good knockdown efficiency worthy of further study. study. We then evaluated the effect of Rae1 knockdown on oocyte meiotic maturation. We then evaluated the effect of Rae1 knockdown on oocyte meiotic maturation. The results showedThe results that show the knockdowned that the knockdown of Rae1 significantly of Rae1 significantly affected the affected GVBD ratethe GVBD compared rate withcom- thatpared in with the control that in groupthe control (87.3 group± 2.7% (87.3 vs. ± 70.4 2.7%± vs.5.0%, 70.4p ±< 5.0%, 0.05, p Figure < 0.05,2C). Figure The 2C). rate The of polarrate of body polar extrusion body extrusion (PBE) at (PBE) 14 h significantly at 14 h significantly decreased decreased in the Rae1 in the knockdown Rae1 knockdown oocytes (63.7oocy±tes2.1% (63.7 vs. ± 2.1% 45.7 ± vs.3.9%, 45.7 p±< 3.9%, 0.05, p Figure < 0.05,2D); Figure however, 2D); however, the proportion the proportion of oocytes withof oo- acytes polar with body a polar was comparablebody was comparable in the control in the and control Rae1–RNAi and Rae1 meiosis-resumed–RNAi meiosis-resumed oocytes (alreadyoocytes undergoing(already undergoing GVBD; 79.0 GVBD± 3.1%; 79.0 vs. ± 65.43.1%± vs.5.4%, 65.4p ±>0.05, 5.4%, Figurep >0.05,2E). Figure Moreover, 2E). More- the PBEover, rate the afterPBE 9rate h was after significantly 9 h was significantly advanced advanced in the Rae1 in the knockdown Rae1 knockdown group (14.3 group± 3.0% (14.3 vs.± 3.0% 25.9 vs.± 2.0%, 25.9 ±p 2.0%,< 0.05, p < Figure 0.05, 2FigureF), indicating 2F), indicating a precocious a precocious anaphase anaphase trigger. Thesetrigger. results These showedresults show that Rae1ed that participates Rae1 participates in the GVBD in the and GVBD PBE and processes. PBE processes.

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Figure 2. Rae1 knockdown affects the meiotic maturation process. (A) Efficiency of Rae1–RNAi after siRNA microinjection was verifiedFigure by2. Rae1 immunoblotting. knockdown (B) affects RNAi after the siRNAmeiotic microinjection maturation was process. verified ( byA) immunostaining.Efficiency of Rae1 Rae1–RNAi is shown in redafter and siRNA DAPI in microinjection blue. Scale bar = 20wasµm. verified (C) The by GVBD immunoblotting. rates in the control (B group) RNAi and after Rae1 siRNA knockdown microinjection group were recordedwas 2verified h after release by immunostaining. from 3-Isobutyl-1-Methylxanthine Rae1 is shown (IBMX) in red arrest. and DA * p PI< 0.05. in blue. (D). TheScale PBE bar rates = 20 in μm. the control (C) groupThe and GVBD Rae1 knockdown rates in the group control were group recorded and 14 Rae1 h after knockdown release from IBMXgroup arrest. were* recordedp < 0.05. (E 2) Theh after PBE release rates in the controlfrom 3 group-Isobutyl and Rae1-1-Methylxanthine knockdown group (IBMX were recorded) arrest. after * p 14< 0.05. h in 2( hD GVBD-resumed). The PBE rates oocytes in the after control release group from IBMXand arrest. Rae1p > knockdown 0.05. (F) The PBEgroup rates wer in thee recorded control group 14 h and after Rae1 release knockdown from groupIBMX were arrest. recorded * p < after0.05. 9 ( hE) in The 2 h GVBD-resumedPBE rates oocytesin the control after release group from and IBMX Rae1 arrest. knockdown * p < 0.05. The group data arewere presented recorded as mean after± 14SEM h ofin at2 leasth GVBD three- independentresumed experiments. oocytes after release from IBMX arrest. p > 0.05. (F) The PBE rates in the control group and Rae1 knockdown2.3. group Rae1 Interference were recorded Induces after Abnormal 9 h in Chromosome 2 h GVBD- Alignmentresumed andoocytes a High after Incidence release from IBMX arrest. *of p Aneuploidy< 0.05. The data are presented as mean ± SEM of at least three independent experiments. As Rae1 participated in the meiotic maturation process and localized to the kinetochore, we assessed the spindle morphology and chromosome alignment in the MI stage 2.3. Rae1 Interferenceoocytes. Induces The abnormal results showed Chromosome that oocytes Alignment had the typical and a meioticHigh Incidence spindle morphology of in Aneuploidy both the control and RNAi groups. The chromosomes were well aligned at the bipolar spindle plate in the control oocytes; however, dispersed chromosomes were observed in As Rae1 participatedthe Rae1–RNAi in the oocytes meiotic (Figure maturation3A). The rate process of misaligned and chromosomeslocalized to was the significantly kinetochore, we assessedelevated the spindle in the RNAi morphology group compared and withchromosome the control groupalignment (19.3 ± in3.3% the vs. MI 39.5 stage± 2.6%, oocytes. The resultsp < showed 0.01, Figure that3B). ooc Givenytes that had misaligned the typical chromosomes meiotic spindle at the MI morphology stage are a leading in factor of aneuploidy [20], we then examined the aneuploidy in MII oocytes by chromosome both the control and RNAi groups. The chromosomes were well aligned at the bipolar spreading, and the results showed that the aneuploid rate was also significantly increased spindle plate in thein thecontrol Rae1 knockdown oocytes; however, oocytes (22.3 dispersed± 2.6% vs. chromosomes 42.0 ± 3.6%, p < 0.01,were Figure observed3C,D). These in the Rae1–RNAi oocytesresults suggested (Figure that3A). Rae1 The is rate involved of misaligned in chromosome chromosomes alignment at thewas MI signifi- stage and cantly elevated infaithful the RNAi chromosome group segregationcompared atwith the transitionthe control of metaphase group (19.3 I to anaphase ± 3.3% vs. I. 39.5 ± 2.6%, p < 0.01, Figure 3B). Given that misaligned chromosomes at the MI stage are a leading factor of aneuploidy [20], we then examined the aneuploidy in MII oocytes by chromosome spreading, and the results showed that the aneuploid rate was also significantly increased in the Rae1 knockdown oocytes (22.3 ± 2.6% vs. 42.0 ± 3.6%, p < 0.01, Figure 3C,D). These results suggested that Rae1 is involved in chromosome alignment at the MI stage and faithful chromosome segregation at the transition of metaphase I to anaphase I.

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Figure 3. Rae1Figure knockdown 3. Rae1 induces knockdown abnormal induces chromosome abnormal chromosome alignment and alignment elevated and aneuploid elevated rate aneuploid in oocytes. rate (A) Rep- resentative confocalin oocytes. images (A) ofRepresentative spindle morphology confocal and images chromosome of spindle alignment morphology in theand control chromosome and Rae1 alignment knockdown MI oocytes. The whitein the line control and arrowand Rae1 indicate knockdown that in the MI control oocytes. group, The chromosomeswhite line and are arrow congressed indicate and that aligned in the con- at the spindle midzone, whiletrol in group, the Rae1-knockdown chromosomes are groups, congressed chromosomes and aligned are scatteredat the spindle and dispersed.midzone, whileα-tubulin in the is Rae1 shown- in green knockdown groups, chromosomes are scattered and dispersed. α-tubulin is shown in green and and DAPI is shown in blue. Scale bar = 10 µm. (B) The rate of misaligned chromosomes is recorded and compared in the DAPI is shown in blue. Scale bar = 10 μm. (B) The rate of misaligned chromosomes is recorded control (n = 96) and Rae1 knockdown MI oocytes (n = 108). ** p < 0.01 (C) Representative confocal image of chromosome and compared in the control (n = 96) and Rae1 knockdown MI oocytes (n = 108). ** p < 0.01 (C) spread at the MIIRepresentative stage in the confocal control and image Rae1 of knockdownchromosome group. spread Control at the MII oocytes stage with in the a normal control haploid and Rae1 complement of 20 chromosomesknockdown and Rae1 group. knockdown Control oocyte oocytes with with 21 chromosomes.a normal haploid Chromosomes complement were of 20 stained chromosomes with propidium and iodide (PI) and is shownRae1 in knockdown red. Scale bar oocyte = 5 µm. with (D )21 The chromosomes aneuploidy. rate Chromosomes is recorded andwere compared stained with in the propidium control (n =io- 31) and Rae1 knockdown oocytesdide (PI) (n =and 35). is ** shownp < 0.01. in red. The Scale data arebar presented = 5 μm. (D as) The mean aneuploidy± SEM of atrate least is recorded three independent and compared experiments. in the control (n = 31) and Rae1 knockdown oocytes (n = 35). ** p < 0.01. The data are presented as mean ± SEM of2.4. at least Rae1 three Depletion independent Compromises experiments. K-MT Attachments during Oocyte Meiotic Maturation Faithful chromosome alignment and segregation also depend on accurate and stable 2.4. Rae1 DepletionK-MT Compromises attachments [K21-MT]. Therefore, Attachments we during used cold Oocyte treatment Meiotic to Maturation assess the K-MT attachment; Faithfulfollowing chromosome low alignment temperature and treatment, segregation accurate also depend and strong on accurate K-MT and attachment stable remained K-MT attachmentsstable, [21]. whereas Therefore, erroneous we used and cold fragile treatm K-MTent attachmentto assess the was K-MT disassembled attachment; [22]. In this following lowstudy, temperature MI oocytes treatment, were collected accurate after and cold strong treatment K-MT andattachment were immunostained remained for the stable, whereaskinetochore erroneous proteins and fragile CREST K and-MTα attachment-tubulin. In was the controldisassembled group, we[22]. observed In this that CREST study, MI oocyteswas evenly were positionedcollected after on both cold sides treatment of the centerand were of the immunostained spindle, and microtubules for the mostly kinetochore capturedproteins CREST the kinetochores and α-tubulin. on the In the aligned control bivalents group, we on observed the spindle that equator. CREST Conversely, was evenly positionedwe found thaton both the CRESTsides of location the center pattern of the had spindle, some and deviations, microtubules and we mostly detected a higher captured thefrequency kinetochores of unattached on the aligned kinetochores bivalents in on Rae1 the knockdown spindle equator. oocytes Conversely, (Figure4A), indicating we found thatthat the some CREST kinetochores location pattern were weakly had some attached deviations to microtubules, and we detected after Rae1 a higher knockdown. The frequency ofrate unattached of unattached kinetochores kinetochores in Rae1 wasknockdown signiﬁcantly oocytes higher (Figure (20.3 4A),± indicating2.9% vs. 62.3 ± 5.7%, that some kinetochoresp < 0.01; Figure were4 B)weaklyin the attached Rae1 knockdown to microtubules oocytes after than Rae1 in knockdown. the control The oocytes. These rate of unattachedresults kinetochores indicate that was Rae1 significantly is crucial for higher proper (20.3 K-MT ± 2.9% attachment vs. 62.3 ± and 5.7%, accurate p < chromo- 0.01; Figure 4B)some in alignment.the Rae1 knockdown oocytes than in the control oocytes. These results indicate that Rae1 is crucial for proper K-MT attachment and accurate chromosome alignment.

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Figure 4. Rae1Figure knockdown 4. Rae1 weakens knockdown kinetochore–microtubule weakens kinetochore (K-MT)–microtubule attachment (K-MT) in attachment oocytes. (A )in Representative oocytes. (A) confocal image of K-MTRepresentative attachment in confocal the control image and of Rae1K-MT knockdown attachment MIin the oocytes control after and cold Rae1 treatment. knockdownα-tubulin MI oo- is shown in cytes after cold treatment. α-tubulin is shown in green, CREST in red, and DAPI in blue. White green, CREST in red, and DAPI in blue. White arrows indicate nonconnected kinetochores in the Rae1 knockdown oocytes. arrows indicate nonconnected kinetochores in the Rae1 knockdown oocytes. Scale bar = 5 μm. (B) Scale bar = 5 µm. (B) The rate of unattached kinetochores was recorded and compared in the control (n = 125) and Rae1 The rate of unattached kinetochores was recorded and compared in the control (n = 125) and Rae1 ± knockdown oocytesknockdown (n = 143). oocytes ** p < (n 0.01. = 143). The ** data p < 0.01. are presented The data asare mean presentedSEM as of mean at least ± SEM three of independent at least three experiments. independent experiments. 2.5. Rae1 Depletion Had No Effects on BubR1 and Mad1 Location 2.5. Rae1 DepletionSAC Had inhibits No Effects the on metaphase–anaphase BubR1 and Mad1 Location transition until the chromosomes are correctly SAC inhibitsaligned the and metaphase K-MT attachments–anaphase transition are accurately until connectedthe chromosomes [18]. Given are correctly the observed defects aligned and inK-MT chromosome attachments alignment, are accurately aneuploidy, connected and [18]. K-MT Given attachment, the observed we investigated defects the SAC in chromosomefunction alignment, in the controlaneuploidy and Rae1, and RNAiK-MT groups. attachment, The key we SACinvestigated components, the SAC including BubR1 function in theand control Mad1, were and Rae1assessed RNAi in the groups. control The and key Rae1 SAC knockdown components, oocytes including cultured for 8.5 h BubR1 and Mad1,after release were assessed from IBMX in the block. control As shownand Rae1 in Figureknockdown5A,B, theoocytes immunoﬂuorescence cultured for signals of both BubR1 and Mad1 normally persisted at the kinetochore in the control and Rae1 8.5 h after release from IBMX block. As shown in Figure 5A,B, the immunofluorescence Int. J. Mol. Sci. 2021, 22, x FOR PEER knockdownREVIEW oocytes. The results suggested that the activity of SAC was unaffected7 of 13 by signals of both BubR1 and Mad1 normally persisted at the kinetochore in the control and Rae1 knockdown. Rae1 knockdown oocytes. The results suggested that the activity of SAC was unaffected by Rae1 knockdown.

Figure 5. Cont.

Figure 5. The location of BubR1 and Mad1 was not affected in the Rae1 knockdown oocytes. After Rae1 knockdown, GV stage oocytes were cultured for 8.5 h and then collected for immunofluores- cent staining of BubR1 and Mad1. (A) Confocal image of BubR1 localization in the control and Rae1 knockdown oocytes by chromosome spreading. BubR1 is shown in green and DAPI in blue. Scale Bar = 5 μm. (B) Confocal image of Mad1 localization in the control and Rae1 knockdown oocytes by chromosome spreading. Mad1 is shown in red and DAPI in blue. Scale Bar = 5 μm.

2.6. Rae1 Depletion Decreases the Protein Level of securin but Not Cyclin B Anaphase-promoting complex or cyclosome (APC/C) directly controls the metaphase–anaphase transition by regulating the proper degradation of securin and cyclin B [21]. In this experiment, we assessed the protein levels of securin and cyclin B in the control and Rae1 knockdown oocytes at the MI stage. As shown in Figure 6, the protein level of securin was significantly decreased in the Rae1 knockdown oocytes; however, the protein levels of both cyclin B1 and cyclin B2 were similar between the control oocytes and the Rae1 knockdown oocytes, which indicated that Rae1 specifically modulates the protein level of securin.

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Figure 5. The location of BubR1 and Mad1 was not affected in the Rae1 knockdown oocytes. After Figure 5. The location of BubR1 and Mad1 was not affected in the Rae1 knockdown oocytes. After Rae1 knockdown, GV stage oocytes were cultured for 8.5 h and then collected for immunofluores- Rae1cent staining knockdown, of BubR1 GV and stage Mad1. oocytes (A) Confocal were cultured image forof BubR1 8.5 h andlocalization then collected in the control for immunoﬂuorescent and stainingRae1 knockdown of BubR1 oocyte ands Mad1.by chromosome (A) Confocal spreading. image BubR1 of BubR1is shown localization in green and in DAPI the in control blue. and Rae1 knockdownScale Bar = 5 μm. oocytes (B) Confocal by chromosome image of Mad1 spreading. localization BubR1 in the is showncontrol and in green Rae1 knockdown and DAPI in blue. Scale Baroocyte = 5s µbym. chromosome (B) Confocal spreading. image of Mad1 Mad1 is shown localization in red inand the DAPI control in blue. and Scale Rae1 Bar knockdown = 5 μm. oocytes by chromosome spreading. Mad1 is shown in red and DAPI in blue. Scale Bar = 5 µm. 2.6. Rae1 Depletion Decreases the Protein Level of securin but Not Cyclin B 2.6. Rae1Anaphase Depletion-promoting Decreases complex the Protein or cyclosome Level of securin (APC/C) but directly Not Cyclin controls B the metaphase–anaphase transition by regulating the proper degradation of securin and cyclin B Anaphase-promoting complex or cyclosome (APC/C) directly controls the metaphase– [21]. In this experiment, we assessed the protein levels of securin and cyclin B in the con- anaphase transition by regulating the proper degradation of securin and cyclin B [21]. In trol and Rae1 knockdown oocytes at the MI stage. As shown in Figure 6, the protein level thisof securin experiment, was significantly we assessed decreased the protein in the Rae1 levels knockdown of securin oocytes; and cyclin however, B in the the pro- control and Rae1tein levels knockdown of both c oocytesyclin B1 atand the cyclin MI stage. B2 were As similar shown betwee in Figuren the6, thecontrol protein oocytes level and of securin wasthe Rae1 signiﬁcantly knockdown decreased oocytes, which in the indicated Rae1 knockdown that Rae1 specifically oocytes; however, modulates the the protein pro- levels Int. J. Mol. Sci. 2021, 22, x FOR PEER ofREVIEWtein both level cyclin of securin B1. and cyclin B2 were similar between the control oocytes and8 of 13the Rae1

knockdown oocytes, which indicated that Rae1 speciﬁcally modulates the protein level of securin.

Figure 6. 6. Rae1Rae1 knockdown knockdown significantly signiﬁcantly decreases decreases the protein the protein level of levelsecurin of in securin MI stage in oocytes. MI stage oocytes. AfterAfter Rae1 Rae1 knockdown, knockdown, GV GV stage stage oocytes oocytes were werecultured cultured for 8 h for(MI 8 stage) h (MI after stage) release after from release IBMX from IBMX arrest and and were were collected collected for the for W theestern Western blotting blotting of cyclin of B1, cyclin cyclin B1, B2 cyclin, and securin. B2, and (A) securin. Three (A) Three independentindependent repeat repeat results results of s ofec securin,urin, cyclin cyclin B1, and B1, c andyclin cyclin B2 by W B2estern by Western blotting. blotting. (B) The r (ela-B) The relative tive intensity of securin, cyclin B1, and cyclin B2 were compared in the control group and Rae1 intensityknockdown of securin,group. ** cyclinp < 0.01. B1, The and data cyclin are presented B2 were compared as mean ± inSEM the of control at least group three independent and Rae1 knockdown group.experiments. ** p < 0.01. The data are presented as mean ± SEM of at least three independent experiments.

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3. Discussion Previous observations have shown that Rae1 serves as a nuclear exporter of poly(A) mRNA at interphase, and a microtubule-associated protein and mitotic checkpoint regulator at mitosis. In this study, we investigated the role of Rae1 in mouse oocytes. We found that Rae1 concentrates on the nuclear rim in the GV stage and translocates to the kinetochore structure after meiotic resumption. Rae1 knockdown induces decreased securin protein levels, accompanied by misaligned chromosomes, aberrant K-MT attachments, and a high rate of aneuploidy. A high intraoocyte level of cAMP activates protein kinase A (PKA), which in turn inhibits the activity of maturation-promoting factor (MPF), maintaining oocytes in the diplotene stage [23]. MPF is a heterodimer composed of the catalytic subunit CDK1 and regulatory subunit cyclin B [24]. In this study, we found that the GVBD rate was significantly decreased in the Rae1 knockdown oocytes; however, we did not observe significant changes in the cyclin B1 and cyclin B2 levels. Therefore, we concluded that the knockdown of Rae1 has no influence on MPF activity. The disassembly of NPCs is a decisive event during nuclear membrane breakdown (NEBD). Nup98 is the first component to dissociate from the nuclear envelope upon NEBD, and the phosphorylation of Nup98 is a key step in NPC disassembly [25]. Rae1 and Nup98 exist and function together as subcomplexes of Rae1/Nup98 during the interphase and mitotic phases [26,27]. Therefore, the knockdown of Rae1 may affect the dissociation of Nup98 from the nuclear envelope, which eventually interferes with the GVBD process. After examining the expression pattern of Rae1 during oocyte meiotic maturation, we found that the Rae1 protein level in the oocytes was lower in the MI stage, but much higher in the metaphase II (MII) stage. Rae1 knockdown significantly decreased the securin level, resulting in a precocious anaphase trigger and aneuploidy. Published data have validated that homologous chromosome separation at the transition of metaphase I to anaphase I depends on the proteolysis of securin [28]. In contrast, the MII stage is maintained by the securin protein. MII oocytes from aged mice have lower securin levels and a higher rate of aneuploidy than those from young mice [29]. Therefore, we speculate that the lower level of Rae1 in MI oocytes might be related to the transition from metaphase I to anaphase I (securin proteolysis), and that the higher level of Rae1 in MII oocytes might be related to the maintenance of MII stage arrest. In both mitosis and meiosis, SAC monitors chromosome alignment and K-MT attachments, and persists at the kinetochore to inhibit the precocious anaphase trigger until proper chromosome alignment and K-MT connections are corrected [30]. In this study, Rae1 knockdown induced precocious anaphase, misaligned chromosomes, and a high rate of aneuploidy, which conformed to previous data [12,31]. All these results showed that the activity of SAC might be impaired. However, in our results, we did not detect any different localization patterns of BubR1 and Mad1 (main component of SAC) between the control and Rae1 knockdown groups, which indicates that SAC functions normally after Rae1 knockdown, and we asssume the precocious anaphase may be caused by the decreased securin level. SAC can inhibit APC/C E3 ubiquitin ligase activity to generate a “stop anaphase” signal until incorrect attachments eventually become corrected [18]. Once the chromosomes are correctly aligned and SAC dissociates from the kinetochore, activated APC/C promotes the degradation of securin and cyclin B and triggers anaphase [32]. In this study, we observed decreased protein levels of securin but not Cyclin B in the Rae1 knockdown oocytes. Consistently, published data have also shown that Rae1/Nup98 can specifically inhibit APCCdh1-mediated ubiquitination and the degradation of securin in prometaphase [27,32]. However, during the metaphase–anaphase transition, another nucleoporin, Nup88, can se- quester Rae1/Nup98 away from APCCdh1, which promotes the degradation of securin [33]. In a word, Nup88 and Rae1/Nup98 composes of a regulatory network that regulates the activity of APCCdh1 to control the securin level, thus regulating chromosome alignment and anaphase onset. Int. J. Mol. Sci. 2021, 22, 4841 9 of 12

4. Materials and Methods 4.1. Animal Statement Kunming mice, a native breed widely used in biological research, were used for oocyte collection and experiments in this study. Four- to six-week-old female Kunming mice were purchased from the animal center of Huazhong Agricultural University. All experimental procedures and animal treatments were performed in accordance with the rules stipulated by the Animal Care and Use Committee of Huazhong Agricultural University (HZAUSW-2017-005).

4.2. Antibodies and Reagents Mouse anti-Rae1 monoclonal antibody (Cat# SC-393252), mouse anti-Mad1 monoclonal antibody (Cat# 376613), rabbit anti-Cyclin B2 antibody (Cat# sc-2776), and mouse anti-securin monoclonal antibody (Cat# SC-56207) were obtained from Santa Cruz Biotech- nology (Santa Cruz, CA, USA); mouse anti-α-tubulin-FITC antibody (Cat# F2168) was obtained from Sigma Chemical Company (St Louis, MO, USA); mouse anti-α-tubulin monoclonal antibody (66031-1-Ig) and mouse anti-β-actin monoclonal antibody (66009-1- Ig) were bought from Proteintech (Wuhan, China); rabbit anti-Cyclin B1 antibody (Cat# AF6168) was purchased from Afﬁnity (Cincinnati, OH, USA); human anti-CREST antibody (Cat# 15-234-0001) was purchased from Antibodies Incorporated (Davis, CA, USA); sheep anti-BubR1 polyclonal antibody (Cat# 28193) was obtained from Abcam (Cambridge, UK); and DyLight 549-conjugated goat anti-mouse IgG (H + L) was purchased from Abbkine Biotechnology (California, CA, USA). FITC-conjugated donkey anti-sheep IgG (H + L) was produced by Jackson ImmunoResearch Laboratory (West Grove, PA, USA), FITC- conjugated goat anti-human IgG (H + L) was purchased from Boster (Wuhan, China), and TRITC-conjugated goat anti-human IgG (H + L) was purchased from Proteintech (Wuhan, China). All other chemicals and culture media were purchased from Sigma Chemical Company (St Louis, MO, USA), unless otherwise stated.

4.3. Oocyte Retrieval and Culture Four- to six-week-old female Kunming mice were sacriﬁced by cervical dislocation 48 h after intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG; Solarbio, P9970). Fully grown GV stage oocytes were collected by ovarian puncture in a prewarmed F12 medium (Ham, YC-3034) supplemented with 50 µM IBMX (Sigma, 15879, St Louis, MO, USA), and were then used for culture or microinjection. For meiotic maturation, oocytes were washed three times to remove IBMX and then cultured in a prewarmed MII (Sigma, ◦ M7167) medium covered with parafﬁn oil (Sigma, M8410) at 37 C in a 5% CO2 atmosphere. Different stages of oocytes were collected for the following experiments.

4.4. Microinjection of Rae1-Targeted Short Interfering siRNA Eppendorf CellTram oil was used for the microinjection. After isolation from the ovary, fully grown GV stage oocytes were used for immediate microinjection in MII medium supplemented with 50 µM IBMX. The microinjection operation of 200 oocytes should be rapid and within 1 h, with no signiﬁcant effect on the subsequent developmental potential of oocytes. For Rae1 knockdown, 5–10 pl of 50 µM Rae1-targeted short interfering siRNA (Cat# sc-75826; Santa Cruz, CA, USA) was microinjected into the cytoplasm of fully grown GV stage oocytes, and the same amount of negative control siRNA (Cat# sc-37007; Santa Cruz, CA, USA) was used as a control. After microinjection, the oocytes were arrested at the GV stage in an MII medium containing 50 µM IBMX for 24 h to degrade the targeted mRNA. Then, the oocytes were washed three times to remove IBMX and were cultured in a prewarmed MII medium for an additional 14 h.

4.5. Immunoﬂuorescence and Confocal Microscopy Oocytes at different stages were collected and ﬁxed in 4% paraformaldehyde (in PBS, pH 7.4; Sigma, 158127) for 30 min and permeabilized with 0.5% Triton-X-100 (Biosharp, 0649) in PBS for 30 min at room temperature. Then, the oocytes were blocked with a Int. J. Mol. Sci. 2021, 22, 4841 10 of 12

blocking buffer (2% BSA-supplemented in PBS; Sigma, A2153) for 1 h and incubated with an anti-Rae1 antibody (1:100), anti-α-tubulin-FITC antibody (1:100), or anti-CREST antibody (1:200) at 4 ◦C overnight. After three washes in a washing solution (1% Tween 20 and 0.01% Triton-X 100 in PBS), the oocytes were incubated with DyLight 549-conjugated goat anti-mouse (1:100) or TRITC-conjugated goat anti-human (1:50) for 1 h at room temperature. After three washes in a washing solution, the oocytes were counterstained with DAPI (Invitrogen, D130610, 1:1000, Waltham, MA, USA) for 1–5 min. Finally, the oocytes were mounted and observed under a confocal laser scanning microscope (Carl Zeiss 800, Jena, Thüringen, Germany). Confocal images were processed using Zeiss LSM Image Browser software. Oocytes incubated with ﬂuorescence-labelled secondary antibodies were used as a negative control, and the primary antibody was replaced with nonimmune IgG.

4.6. Western Blotting Samples containing 200 oocytes were collected at the GV, GVBD, MI, and MII stages in a 2X SDS loading buffer and were boiled for 5 min. After separation by SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (Sigma, CCGL09025). After transfer, the membranes were briefly washed and blocked in a blocking buffer (5% skim milk in TBST) at room temperature for 1 h, followed by incubation overnight at 4 ◦C with a Rae1 antibody (1:400), securin antibody (1:400), cyclin B1 antibody (1:500), or cyclin B2 antibody (1:500) at 4 ◦C overnight. After three washes in TBST, the membranes were incubated at 37 ◦C for 1 h with HRP-conjugated secondary antibodies. Finally, the membranes were washed in TBST, and the immunoblot bands were visualized with an ECL kit (Thermo Scientific, A38555, Waltham, MA, USA). α-tubulin antibody (1:1000) and β-actin (1:1000) served as the loading controls. The relative intensity of the band was assessed using ImageJ software (NIH, Bethesda, MD, USA). A blank control incubated with an HRP-conjugated secondary antibody was used as the negative control, and the primary antibody was replaced by nonimmune IgG.

4.7. Oocyte Cold Treatment Fully grown GV stage oocytes microinjected with either the control siRNA or Rae1 siRNA were maintained in an MII medium containing 50 µM IBMX for 24 h, then the IBMX was washed off and the oocytes were cultured in a fresh MII medium. Two hours later, the GV stage oocytes were removed, and the remainder were cultured for another 6 h. The oocytes (MI stage) were then transferred to a precooled MII medium (4 ◦C) for 15 min of incubation. After cold treatment, immunoﬂuorescence staining and confocal microscopy were used to detect the immunoﬂuorescence signal of CREST and others. Oocytes with normal spindles in both the control and RNAi groups were used to compare the K-MT attachment.

4.8. Chromosome Spreading Chromosome spreading was performed as described previously [34]. Briefly, oocytes were collected in the MI stage and were exposed to Tyrode’s buffer (pH 2.5, Sigma, T1788) for approximately 30 s at room temperature to remove the zona pellucida. After recovery in an MII medium for 30 min, the oocytes were fixed in a drop of spreading solution (1% PFA, 0.15% Triton X-100, 3 mM DTT in ddH2O, pH 9.2) on a glass slide. After air drying, the slides were washed and blocked with 1% BSA in PBS, followed by incubation with anti-Rae1 antibody (1:100), anti-CREST antibody (1:200), anti-BubR1 antibody (1:100), or anti-Mad1 antibody (1:40) at 4 ◦C overnight. Next, the primary antibody was washed away, and the oocytes were incubated with DyLight 549-conjugated goat anti-mouse (1:100) antibody, FITC-conjugated goat anti-human (1:100) antibody, or FITC-conjugated donkey anti-sheep antibody (1:100) at 37 ◦C for 1 h. The chromosomes were stained with DAPI. Images of the immunofluorescence staining of oocytes were captured with confocal microscopy. Int. J. Mol. Sci. 2021, 22, 4841 11 of 12

4.9. Statistical Analysis All of the experiments were independently performed at least three times; data are presented as the mean ± SEM and were analyzed with two-tailed Student’s t test using GraphPad software (CA, USA); p < 0.05 was considered signiﬁcant.

5. Conclusions In conclusion, this study demonstrated that Rae1 concentrates on the kinetochore after the resumption of meiosis and regulates the securin protein level to regulate K-MT attachment and faithful chromosome alignment. Rae1 might not function alone, but instead interacts with other nucleoporins, including Nup88 and Nup98.

Author Contributions: Project administration, F.C. and L.-J.H.; methodology, X.-F.J. and F.M.; data curation, Y.-S.W. and Z.-M.D.; resources, Y.-L.M. and J.-J.X.; supervision, L.-J.H.; writing—original draft, F.C.; writing—review and editing, L.-J.H. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Fundamental Research Funds for the Central Universities (Program No. 2662020DKPY011) and the National Natural Science Foundation of China (31972533). All of the authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: The study was approved by the Institutional Review Board (or Ethics Committee) of the Hubei Research Center of Experimental Animal (approval ID: SCXK (Hubei) 20080005). Data Availability Statement: The data that support the ﬁndings of this study are available from the corresponding author upon reasonable request. Conﬂicts of Interest: The authors declare that no competing interests exist.

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