Hec1 inhibition alters spindle morphology and chromosome alignment in porcine oocytes

Hec1inhibition alters spindle morphology and chromosome alignment in porcine oocytes
Xiaomou Wei •Chunhai Gao •Jia Luo •Wei Zhang •Shuhao Qi •Weijun Liang •Shengming Dai
Received:25March 2014/Accepted:10April 2014ÓSpringer Science+Business Media Dordrecht 2014
Abstract Aneuploidy is caused by incorrect chromosome segregation and can result in cancer or birth defects.The spindle assembly checkpoint (SAC)guarantees proper cell cycle progression.Highly Expressed in Cancer protein 1(Hec1,also called Ndc80)is the core component of the Ndc80complex and is involved in regulating both kine-tochore-microtubule interactions and the SAC during mitosis in multiple cell types.However,its involvement in pig oocyte meiotic maturation remains uncertain.Thus,we investigated Hec1expression,localization,and possible functions during porcine oocyte meiosis.Immunofluores-cent staining showed that Hec1was expressed in porcine oocytes and was associated with centromeres at both the metaphase I and metaphase II stages.Disrupting Hec1function with its inhibitor INH1resulted in polar body
extrusion defects in porcine oocytes.Moreover,inhibiting Hec1activity also resulted in severe chromosome misa-lignments and aberrant spindle morphology.Our results showed a unique localization pattern for Hec1in porcine oocytes and suggested that Hec1was required for chro-mosome alignment and spindle organization.Thus,Hec1might regulate spindle checkpoint activity during mam-malian oocyte meiosis.
Keywords Hec1ÁMeiosis ÁOocyte maturation ÁPolar body extrusion ÁAneuploidy
Introduction
During mitosis,cells have multiple mechanisms to guar-antee correct chromosome segregation,which is required for maintaining genomic integrity.During the M phase in somatic cells,one of these mechanisms is the spindle assembly checkpoint (SAC).This mechanism can detect the tension generated by microtubules or the attachments between microtubules and kinetochores on chromosomes.Errors that occur during this process can result in aneu-ploidy and cell cycle defects,which can manifest as genetic disorders or the development of cancer.
Highly Expressed in Cancer protein 1(Hec1,also called Ndc80)is the core component of the Ndc80complex,which comprises Ndc80[1],Nuf2,Spc25,and Spc24[2].Structural analysis has shown that the interaction between a kinetochore and a microtubule might rely on electrostatic interactions that are mediated through the 80amino acid disordered N-terminal tail domain of Hec1,whereas the mitotic cell cycle checkpoint function of Hec1is dependent on its CH domain [3,4].
In mammalian cells,Hec1is localized at chromosome kinetochores during M phase [5–7].Hec1is also involved
Xiaomou Wei and Chunhai Gao contributed equally to this work.X.Wei ÁW.Liang ÁS.Dai (&)
Department of Clinical Laboratory Services,Liuzhou Worker’s Hospital,Liuzhou 545005,China e-mail:shengming_dai@
C.Gao
Department of Clinical Laboratory Services,Linyi People’s Hospital,Linyi 276003,China
J.Luo
Technology Centre of Guangxi Entry-Exit Inspection and Quarantine Bureau,Nanning 531004,China W.Zhang
Second Department of Trauma Orthopedics,Linyi People’s Hospital,Linyi 276003,China
S.Qi
Department of Gastroenterology,The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530000,China
Mol Biol Rep
DOI 10.1007/s11033-014-3374-4
in microtubule-kinetochore attachments and regulating the SAC [8–10].Hec1was also shown to be necessary for cell cycle control in germ cells [11].In different cell types from H.sapiens ,evis ,C.elegans ,S.cerevisiae ,and S.pombe ,disrupting Hec1functions caused errors in chro-mosome segregation,spindle morphology maintenance,and cell cycle disorders [12–16].Hec1disturbed the cell cycle checkpoint and altered mitotic spindle formation in mice,as over-expressed Hec1induced tumor formation in vivo [17].
Several previous studies investigated the Hec1signaling pathway.Hec1might be involved in regulating microtu-bule attachment in a Aurora B-mediated manner,as Hec1was found to be phosphorylated by Aurora B [18].More-over,Hec1appeared to be necessary for SAC proteins of Mad family and Mps family members of kinetochores,although Hec1was not essential for the recruitment of Bub family members [15,19–23].Hec1also widely interacts with the KMN network [24–26].
Although Hec1involvement in mitosis have been reported for many species and cell types,its roles in oocyte meiotic maturation have not been thoroughly investigated.Thus,in the present study,we investigated possible roles for Hec1in oocyte meiotic maturation using a porcine model.We could demonstrate the expression and locali-zation patterns of Hec1during pig oocyte meiosis.In addition,our functional studies showed that Hec1regulated
spindle organization,chromosome alignment,and cell cycle progression during pig oocyte meiosis.
Results
Hec1localizes at porcine oocyte kinetochores
We first examined the expression and localization of Hec1in porcine oocytes.Oocytes were harvested after culture for 24and 44h,which are the time points by which most oocytes have reached the metaphase I (MI)and metaphase II (MII)stages,respectively.As shown in Fig.1,Hec1fluorescence signals were detectable in porcine oocytes.Then,Hec1subcellular localization at different meiotic stages was examined by immunofluorescent staining using a mouse monoclonal antibody.At both the MI and MII stages of porcine oocyte meiotic maturation,Hec1was localized at chromosome kinetochores.Hec1inhibition alters porcine oocyte meiotic maturation
Next,we investigated possible functions of Hec1using treatments with a specific Hec1inhibitor (INH1).As shown in Fig.2a,after inhibitor treatment,Hec1protein expres-sion was significantly reduced,as only minimal
Hec1
Fig.1Expression and subcellular localization of Hec1during porcine oocyte meiotic maturation.Subcellular localization of Hec1in porcine oocytes was determined by immunofluorescent staining.Hec1accumulated at chromosome kinetochores at both metaphase I and metaphase II.Green Hec1;red ,chromatin.Bar =10l m.(Color figure online)
Mol Biol Rep
signals were detected at chromosome kinetochores.We then examined the effects of this Hec1inhibitor on porcine oocyte maturation after culture for 44h.Representative images of morphology for control and inhibitor-treated oocytes are shown in Fig.2b.This showed that in the control group (no inhibitor),most oocytes extruded their first polar bodies,whereas in the inhibitor-treated group most oocytes failed to extrude their first polar bodies.To confirm this,we examined the rates of polar body extrusion (Fig.2c).With no INH1treatment,77.4±4.9%(n =113)of the oocytes extruded polar bodies after
culture for 44h,while in the treated groups,the polar body extrusion rates were significantly lower at the same time points.With 10l M INH1treatment,53.6±7.3%)(n =87,p \0.05)of the oocytes extruded polar bodies,and with 50l M INH1treatment,44.1±5.9%(n =78,p \0.05)of the oocytes extruded polar bodies.This indi-cated that inhibiting Hec1activity caused a failure of polar body extrusion.
Hec1inhibition alters porcine oocyte spindle formation maturation
We then attempted to determine the cause of these polar body extrusion defects by first examining spindle formation after Hec1inhibitor treatment.As shown in Fig.3a,in the control group,oocytes exhibited normal spindle morphol-ogies,whereas in INH1-treated oocytes at both the MI and MII stages,oocytes displayed disrupted spindles.
To confirm this,we examined the rates of abnormal spindles (Fig.4b).Without inhibitor treatment,13.5±4.2%of the oocytes showed abnormal spindle morphologies,whereas with 10l M INH1,32.7±6.5%(p \0.05)of oocytes had abnormal spindle morphologies,and 52.1.6±7.8%(p \0.05)of oocytes had abnormal spindle morphologies with 50l M INH1treatment.This indicated that inhibiting Hec1activity altered meiotic spindle formation in porcine oocytes.
Hec1inhibition alters porcine oocyte chromosome alignment
During mitosis,Hec1has been shown to regulate chro-mosome segregation.We next examined whether this Hec1function was conserved in porcine (i.e.,mammalian)oocyte meiosis.As shown in Fig.4a,control oocytes showed normal chromosome alignments.However,in most Hec1inhibitor-treated oocytes the chromosomes were severely misaligned.
To confirm this,we examined the rates of misaligned chromosomes (Fig.4b).Without inhibitor treatment,17.5±5.9%of oocytes had misaligned chromosomes,whereas the abnormal rates were significantly higher with 10l M INH1(48.6±9.2%,p \0.05)and with 50l M INH1(77.3±8.7%,p \0.05).This indicated that inhibiting Hec1activity also altered chromosome align-ments in porcine oocytes.
Discussion
In this investigation we examined the expression,locali-zation,and possible functions of Hec1during porcine oocyte meiotic maturation.We found that Hec1
was
Fig.2Hec1inhibition effects on porcine oocyte meiotic maturation.a Hec1expression in porcine oocytes after inhibitor treatment.Hec1fluorescence signals were barely detectable at chromosome kineto-chores.Green ,a -tubulin;red Hec1;blue chromatin.Bar =10l m.b Typical morphologies of porcine oocytes in the control and treatment groups after culture for 44h.c First polar body extrusion rates after porcine oocytes were treated with a Hec1inhibitor.Results are mean ±SEM’s of at least three independent experiments.*p \0.05.(Color figure online)
Mol Biol Rep
expressed in porcine (mammalian)oocytes and demon-strated that Hec1played important roles in regulating spindle formation and chromosome alignment during por-cine oocyte meiotic maturation.
Using immunofluorescent staining,we first found that Hec1accumulated at kinetochores during all metaphase stages (both MI and MII stages)of porcine oocyte meiotic maturation.This localization of Hec1in porcine oocyte was consistent with previous reports that showed that Hec1was expressed in mouse somatic cells.Recent studies that used the same antibody also showed that Hec1was local-ized at kinetochores in mitotic cells of human,monkey,pig,and other species [6,11,14].Our results indicated that Hec1localization was conserved among different species and different cell types.
To further explore possible roles for Hec1,we treated porcine oocytes with a specific inhibitor (INH1)during porcine oocyte meiotic maturation.Previous reports showed that Hec1regulated cell cycle progression by dis-turbing SAC activity during mitosis;and in the female germ cell of mouse,knock down of Ndc80,a homologue of Hec1,caused the aberrant cell cycle and disrupted spindle morphology [11].We examined whether Hec1also regu-lated the cell cycle in porcine oocytes,since porcine oocytes need longer culture time and smaller spindle,which is a better model compared with mouse.Polar body extrusion was examined after using different inhibitor concentrations.These results showed that inhibiting Hec1activity affected cell cycle progression due to a failure of oocyte meiotic maturation,as inhibitor-treated oocytes could not extrude polar bodies.This inhibitor effect was dose-dependent.Our results were consistent with previous studies on mitosis [8,9,27],which also showed conserved roles for Hec1in both meiosis and mitosis.
To determine a possible cause for porcine oocyte meiotic maturation failure,we first observed that inhibiting Hec1activity resulted in aberrant spindle formation.With differ-ent inhibitor concentrations used for treatment,most of the meiotic spindles displayed abnormal morphologies,which indicated that Hec1might regulate spindle
organization
Fig.3Hec1inhibition effects on spindle morphology.a Spindle morphology after Hec1inhibition.In the control group,most oocyte spindles exhibited a normal morphology.In the Hec1inhibition group,oocytes displayed disrupted spindles at both the MI and MII stages.Bar =20l m.b Rates of aberrant spindles in the control and treatment groups.Results are mean ±SEM’s of at least three independent experiments.*p \0.05
Mol Biol Rep
during porcine oocyte meiotic maturation.The dose-dependence confirmed that this effect was due to Hec1inhibition.We also found that inhibiting Hec1activity resulted in chromosome misalignments at the metaphase stage.We assumed that the interactions between microtu-bules and the kinetochore might have been disrupted,after which microtubules failed to capture chromosomes and directed them towards the equatorial plate.During mitosis,kinetochores have been shown to have multiple components,including a major microtubule attachment position at chro-mosomes.Errors in kinetochore–microtubule interactions may cause spindle morphology and chromosome alignment defects during mitosis [9].Together,our results indicated a role for Hec1in chromosome alignment and spindle for-mation in mammalian oocytes.In addition,these roles of Hec1were conserved among species and cell types,as our results were generally consistent with those in previous reports on mitosis [8,9,25,27].
In summary,our results indicated that Hec1might reg-ulate meiotic spindle formation and chromosome align-ment during porcine oocyte meiotic maturation.
Materials and methods Ethics statement
Animal use and care were in accordance with Animal Research Institute Committee guidelines of the Liuzhou Worker’s Hospital,China.Pig ovaries were obtained from 6-month-old Duroc gilts at a local slaughterhouse (Liuz-hou,China)and transported to our laboratory at 25°C in Dulbecco’s phosphate-buffered saline (dPBS).Our study was approved by the Committee of Animal Research,Li-uzhou Worker’s Hospital,China.Permission was granted by the slaughterhouse to use these animal parts.Antibodies
Mouse monoclonal anti-Hec1was from Abcam (Cambridge,UK).Mouse monoclonal anti-a -tubulin was from Sigma (St Louis,MO)and was a gift of Prof.Shao-Chen Sun of Nanjing Agricultural University,China.Alexa Fluor 488and 568goat anti-mouse antibodies were from Invitrogen (Carlsbad,
CA).
Fig.4Hec1inhibition effects on chromosome alignment.a Chromosome alignment after Hec1inhibition.In the control group,most oocytes exhibited well-aligned chromosomes (a).In the Hec1inhibition group,oocytes displayed misaligned chromosomes (b,c).b Rates of misaligned chromosomes in the control and treatment groups.Results are mean ±SEM’s of at least three independent experiments.*p \0.05
Mol Biol Rep
Oocyte harvest and culture
Porcine ovaries were harvested from prepubertal gilts at a local slaughterhouse and transported to our laboratory in sterile saline(0.9%NaCl)that contained500IU/mL of both penicillin and streptomycin at30–35°C within3h after death.After washing twice in sterile phosphate buf-fered saline(PBS),cumulus-oocyte complexes(COCs) were aspirated from antral follicles(2–5mm in diameter) using a12-gauge needle attached to a5mL disposable syringe.Oocytes were picked after subnatant precipitation of follicularfluid,then washed twice with HEPES-TLP-PVA and modified medium199that contained0.1%(wt/ vol)polyvinyl alcohol(PVA;Sigma-Aldrich Co.,USA), 32.5mM sodium bicarbonate,0.91mM sodium pyruvate, 3.05mM glucose,75mg/L of penicillin,and50mg/L of streptomycin.Only those oocytes that were surrounded by three or more layers of intact cumulus cells and had evenly granulated ooplasm were selected.COCs were cultured in a defined maturation medium of90%(v/v)modified M199, 10ng/mL of epidermal growth factor(mouse EGF; Sigma),0.57mM cysteine(Sigma),10IU/mL of hCG, 10IU/mL of PMSG,and10%(v/v)pig follicularfluid.To prepare matured oocytes in vitro,50–60COCs were transferred to500l L of maturation medium in a4-well dish(NUNC)under200l L of paraffin oil at38.5°C in a humidified atmosphere of5%CO2in air.
Inhibitor treatment during porcine oocyte maturation
For INH1inhibitor treatment,stock INH1(50mM in DMSO;Calbiochem,Germany)was diluted with maturation medium199tofinal concentrations of10and50l M.A control group was cultured using the same concentration of DMSO.COCs or denuded oocytes(DOs)were cultured with INH1to determine its effects on oocyte maturation and Hec1 expression.COCs were denuded of their cumulus cells by gentle pipetting with0.1%(w/v)hyaluronidase(Sigma). Oocytes with clearly extruded polar bodies were judged as matured oocytes.The occurrence offirst polar body extru-sion in oocytes was examined after removing cumulus cells.
Confocal microscopy
For single staining for Hec1or a-tubulin,oocytes were fixed in4%paraformaldehyde in PBS at room temperature for30min.These oocytes were then placed in a membrane permeabilization solution(1%Triton X-100)for4h. After1h in blocking buffer(1%BSA-supplemented PBS; BioX-Vision,Heifei,China),oocytes were incubated either at4°C overnight or at room temperature for4h with either mouse anti-Hec1(1:200)or anti-a-tubulin-FITC(1:200). After three washes in wash buffer(0.1%Tween20and 0.01%Triton X-100in PBS;BioX-Vision,Heifei,China), oocytes were labeled with Alexa Fluor488goat-anti-mouse(1:100)at room temperature for1h(for Hec1 staining).Oocytes were then stained with Hoechst33342 for5min,followed by three washes in wash buffer.
For double staining of Hec1and a-tubulin,oocytes were first stained with anti-Hec1,then labeled with anti-a-tubulin(1:200)for2h,washed three times in wash buffer (2min each wash),andfinally stained with Hoechst33342 (10l g/mL in PBS)for10min.
Stained oocytes were mounted on glass slides and examined with a confocal laser-scanning microscope (Zeiss LSM700META,Germany).At least25oocytes were examined for each experimental or control group. Statistical analysis
At least three replicates were used for each treatment. Results were expressed as mean±SEM’s.Statistical comparisons were made by analysis of variance(ANOVA). If significant differences were found,then group results were compared using Duncan’s multiple comparisons test.
A p-value of\0.05was considered significant. Acknowledgments We thank Prof.Shao-Chen Sun of Nanjing Agricultural University,China for helpful discussions.We also thank BioX-Vision,Hefei,China for technical assistance.
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