Cryobiology 66 (2013) 261–266

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Cryob iology

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Cytokeratin distribution and expression during the maturation of mouse germinal vesicle oocytes after vitrification

Xia Wei a,1, Fu Xiangwei a,1, Zhou Guangbin a,b, Xu Jing a, Wang Liang a, Du Ming a, Yuan Dianshuai a,Yue Mingxing a, Tian Jianhui a, Zhu Shien a,⇑a National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, PR China b Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University (Chengdu Campus), Wenjiang 611130, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 November 2012 Accepted 26 February 2013 Available online 7 March 2013

Keywords:CytokeratinOocytesVitrificationMouse

0011-2240/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.cryobiol.2013.02.062

⇑ Corresponding author.E-mail addresses: xiangweifu@126.com (F. Xian

(Z. Shien).1 These authors contributed equally to this work.

Cytokeratin (CK) is a type of the cytoskeleton that increases cell stabilization during oocyte maturation.This study was conducted to investigate the effect of vitrification on the distribution and expression of CKduring mouse oocyte maturatio n. Germinal vesicle (GV) oocytes were randomly allocated into three groups: (1) untreated (fresh), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (CPA exposure), or (3) vitrified using the open-pulled straw (OPS) method (vitrification). The oocytes were then incubated for 0 h, 6 h (metaphase I (MI) stage), and 10 h (metaphase II (MII) stag e).The CK distribution in the oocytes at the GV, MI, and MII stages was observed by immunofluorescence,and the expression at the MII stag e oocytes derived from vitrified GV oocytes was detected by Western blotting. The CK distribution in the GV oocytes (88.5%) displayed a cortical pattern in the fresh group,whereas a granular pattern was mainly found at the MI (86.7%) and MII (93.5%) stages. In the CPA expo- sure group, 90.3% of the GV oocytes were observed to display the cortical pattern, and 69.2% of the MIoocytes and 92.9% of the MII oocytes showed the granular pattern. In the vitrification group, most oocytes (GV, 88.9%; MI, 100%; MII, 93.3%) exhibited the cortical pattern. The CK fluorescence intensities of the MII stage oocytes in both the fresh (59.27) and CPA exposure (60.05) group were significantly higher than that of the vitrification (26.53) group (p < 0.05). Western blotting showed that the CK expression in the MII oocytes derived from vitrified GV oocytes was significantly lower (p < 0.05) than in the control. Inconclusion, OPS vitrification affe cts the normal CK distribution pattern during oocyte maturatio n and results in decreased CK expression in MII oocytes derived from vitrified GV oocytes.

� 2013 Elsevier Inc. All rights reserved.

Introductio n

Cryopreserv ation technology for non-human mammalian oo- cytes can be applied to human assisted reproducti ve technology and oocyte cryopreserv ation has many advantag es for infertility treatment [3]. It also provides a rationale for cryobanking oocytes of mice strains and is applied by many labs. Recently, vitrificationhas been widely used to cryopreserve oocytes from several mam- mals [4,20,25], and normal offspring have been produced from both vitrified mature [17,25,27,49] and immature oocytes [46].

Regardless of these achievements, many problems still exist.Low-temper ature stress is a major obstacle to the successful cryo- preservation of animal oocytes [31,32]. Various research indicates that vitrification results in various cytologic al changes in oocytes,

ll rights reserved.

gwei), zhushien@cau.edu.cn

including intracellular lipid droplets [42], spindle and chromosom ealteration s [6], DNA damage [29], an abnormal distribut ion ofmitochondr ia [35], structural changes in the cytoplasm and nucleolus [44], disruption of the cell membrane [18], and harden- ing of the zona pellucida [28,41]. Among these problems, cyto- plasm damage is an essential aspect in which disruption of the cytoskeleton such as intermediate filament damage contributes to oocyte death [43]. Intermed iate filaments are 10–12 nm protein filaments for which the diameter is intermediate between microfil-aments (5–8 nm) and microtubule s (25 nm). However , research re- lated to intermediate filaments remains poorly advanced relative to that of microfilaments and microtubule s. As one of the most important intermediate filaments, Cytokeratin (CK) is of particular interest.

CK was first observed as a major structural protein in epithelial cells [13,14]. It is also present in the oocytes and early embryos ofXenopus [15], mice [7], hamsters [33,34], goats [16] and humans [36] and has special functions in oocyte maturation and embryo developmen t [19]. It has been reported that CK structure is affected

262 X. Wei et al. / Cryobiology 66 (2013) 261–266

by vitrifying MII oocytes [43]. However, the effect of vitrificationon the CK distribut ion and expression in the immature oocytes remains unknown. In order to observe the dynamics of CK during oocytes maturation and the effect of vitrification on it, GV stage oocytes were chose to research in this study.

Therefore, fluorescent staining and Western blotting were used to investigate the distribution of CK during GV oocyte maturation and the expression of CK in MII oocytes derived from GV oocytes after cryopreservati on.

Materials and methods

All the reagents were obtained from Sigma Chemical Co. (St.Louis, MO, USA) unless otherwise specified.

Animals

Kunming (KM) mice (Grade II; Academy of Military Medical Sci- ences, Beijing, China) were used in the present study. They were housed under temperature -controlled (20–22 �C) and light-con- trolled conditions (lights on, 06:00–20:00) with free access to food and water. All experimental protocols concerning the handling ofmice were in accordance with the requirements of the Institutional Animal Care and Use Committee at China Agricultural University.

Preparation of open pulled straws

The middle part of a 0.25 mL straw (I.M.V., L’Aigle, France) was heat-soften ed and pulled manually. The pulled straw was cooled inair, and then cut with a bistoury at the narrowes t point, with 2.5 ± 0.5 cm left over. Straws with an outer diameter of0.23 ± 0.01 mm (measured with a micro forge; MF900, Marishige,Tokyo, Japan) and wall thickness of 0.02 mm were used.

Pretreatment and vitrification solutions

Pretreatment and vitrification solutions were prepared accord- ing to previous study [48]. The pretreatment solution (ED solution)was consist of 10% ethylene glycol (EG) + 10% dimethyl sulfoxide (DMSO) and vitrification solution (EDFS30 solution) is consist of15% EG, 15% DMSO, 18% (w/v) Ficoll (MW, 70,000) and 0.3 mol/L sucrose in PBS.

Experimenta l design

1. Fresh group . No treatment was administered.2. CPA exposure group . Oocytes were exposed to ED solution for

30 s, and EDFS30 solution for 25 s, and then rinsed in0.5 mol/L sucrose for 5 min.

3. Vitrification group . Oocytes were vitrified and warmed by OPS vitrification procedure.

Collection and maturation of GV oocytes

Female mouse, 3 weeks old, were superovulated with 10 i.u. ofpregnant mare gonadotropi n (PMSG) (Ningbo Hormone Products Co., Ningbo, Zhejiang, People’s Republic of China). Ovaries were ob- tained from females at 46 h after PMSG injection. Then they were transferred into Dulbecco’s phosphat e buffered saline containing 0.3% bovine serum albumin (DPBS-BSA). The antral follicles were punctured with a 26-gauge needle to release cumulus-oo cyte com- plexes (COCs). The COCs were then transferred into 100 lL micro- drops of a maturati on medium (M16 medium). They were cultured in an incubator (humidified atmosphere, 10% CO2, 10% O2, and 80%

N2 at 37.5 �C) for 0 h, 6 h, and 10 h, respectively, to obtain the GV,MI, and MII oocytes.

Collection of MII oocytes

Female mice were superovu lated by intraperitonea l injection of10 i.u. of PMSG and 10 i.u. of human chorionic gonadotropi n (hCG,Ningbo Hormone Products Co., Ningbo, Zhejiang, People’s Republic of China) given 48 h apart. Oocytes were collected from oviducts 14–15 h post-hCG injection. Cumulus cells were removed by the treatment with 0.1% hyaluronidas e in M2 medium (5.534 g/L NaCl,0.356 g/L KCl, 0.25 g/L CaCl 2�2H2O, 0.162 g/L KH2PO4, 0.294 g/L MgSO4�7H2O, 0.336 g/L NaHCO 3, 5.004 g/L HEPES, 23.3 mM sodium lactate, 0.022 g/L sodium pyruvate, 1 g/L Glucose, 4 g/L BSA,0.06 g/L Potassium penicillin G, 0.146 g/L glutamine) and oocytes were washed and kept in the HTF medium in an incubator before use.

Vitrification and warming of oocytes

Oocytes were vitrified using an OPS method. That is, oocytes were first equilibrated in ED solution for 30 s, then loaded into the narrow end of OPS with EDFS30 solution for 25 s. Finally, the straws containing oocytes (10 oocytes per OPS) were plunged into liquid nitrogen. For warming, oocytes were rinsed in 0.5 mol/L su- crose for 5 min, then rinsed three times in M2 medium and incu- bated in a CO2 incubator for 2 h before use. All manipulations were performed at 37 �C on a warming stage fixed on the stereomi- croscope , and the ambient atmosphere was air-conditio ned at atemperat ure of 25 ± 0.5 �C.

Evaluatio n of oocyte viability

Oocyte viability was evaluated by fluorescein diacetate (FDA)staining according to the method previously described by Mohr and Trounson [30]. Briefly, oocytes were treated with 1 lg/mLFDA in PBS supplem ented with 3 mg/mL BSA at 37 �C for 2 min in a dark room and then they were washed three times in PBS sup- plemente d with 3 mg/mL BSA and evaluated under a fluorescentmicroscop e (IX71, Olympus, Tokyo, Japan). Oocytes with high level of fluorescence and regular, spherical shape; without lysis; and not shrunken , swollen, or blackened were regarded as surviving.

Fluoresce nt staining and imaging

Totally 160 oocytes were used for fluorescence observations .Cumulus cells were removed by pipetting in calcium-free DPBS- BSA containing 0.1% hyaluronidase. Denuded oocytes were fixedin 4% paraformald ehyde for 20 min at room temperature . Fixed oo- cytes were washed three times in DPBS-BSA, and blocked in DPBS containing 3% BSA at 37 �C for 1 h. After washing, the samples were exposed to anti-cytokerati n primary antibody (1:500, Sigma C2931) at 37 �C for 1 h, and incubated with fluorescein isothiocya- nate (FITC)-conjugated secondar y antibody (1:200, Sigma F0257)at 37 �C for 1 h. To clarify the nuclear states and meiotic stages,they were then stained for DNA with Hochest 33342 (10 lg/mL)in mounting medium containing calcium-fr ee DPBS and glycerol (1:1). Then, after an extensive washing, the oocytes were mounted on slides and fluorescence was detected with a Nikon spectral con- focal scanning microscope (Nikon Corporat ion, Tokyo, Japan). Aminimum of 26 oocytes were used for each treatment.

Cytokerat in distribution pattern

In present experiment, CK distribution patterns were divided into two types according to previous report [22]. First type iscortical pattern, in which large CK aggregat es in cortical or near

Table 1The survival rate of vitrified GV and MII stage oocytes.

Oocytes stage

Number of total oocytes

Number of survival oocytes

Survival rate (%)

GV 73 32 43.8 a

MII 75 56 74.7 b

Different letters indicate significant difference.

X. Wei et al. / Cryobiology 66 (2013) 261–266 263

membrane. This distribution type is typical distribut ion pattern for GV stage oocyte. Second type is granular pattern, in which large CKaggregates disperse into multiple small spots and exhibits a homo- geneously distribut ed spotted pattern in cytoplasm. This distribu- tion type is typical for MI and MII stage oocyte.

Fluorescence intensity

Fluorescenc e intensities were quantified using EZ-C1 Free View- er software (Nikon), as described previously elsewher e [2] withsome modifications. In brief, the pixel value of fluorescence was measured within the whole region of cytoplasm in each stained oocyte.

Western blot

Samples of MII stage oocytes derived from GV oocytes after cry- operservation were collected and frozen in2 � laemmli buffer (Bio-Rad) with protease inhibitors. Prior to analysis the samples were thawed and subsequently heated to 100 �C for 5 min. The proteins were separated in 7.5% or 12% acrylamide gels containing 0.1% SDS,and then transferred onto hydrophobic PVDF membranes (Amer-sham, Piscataway, NJ). Membranes were blocked with 5% non-fat dried milk in TBS containing 0.05% (v/v) Tween 20 for overnight at 4 �C and incubated with diluted mouse monoclonal antibody against CK (Sigma C2931) (1:500) for 2 h at room temperat ure, fol- lowed by three (20 min) washes in PBST (PBS with 0.1% Tween-20).A peroxidase-con jugated secondar y antibody (Jackson Immuno Re- search, West Grove, PA) was added for 1 h prior to processing using an ECL-plus detection system (Amersham).

Data analysis

Percentages of distribution pattern and viability rate were ana- lyzed with chi-squared test. Fluorescenc e intensities and Western blotting result in the present study were statistically analyzed using Student’s t-test with SPSS 12.0 software (SPSS, Inc., Chicago,IL). p < 0.05 was considered statistically significant.

Results

Comparison of the viability between vitrified GV and MII oocytes

The comparison of viability rate between vitrified GV and MII oocytes was showed in Table 1. Viability rate of vitrified MII oo- cytes was significantly higher than that of vitrified GV ones.

Table 2CK distribution pattern in different stage oocytes during maturation after differ ent treatm

GV stage Incubatio

Cortical (%) Granular (%) Cortical (

Fresh 23/26 (88.5) 3/26 (11.5) 4/30 (13.3CPA exposure 28/31 (90.3) 3/31 (9.7) 8/26 (30.8Vitrification 24/27 (88.9) 3/27 (11.1) 26/26 (10

Different letters indicate significant difference in each column.

Effect of OPS vitrification on cytokeratin distribution

Most of the GV oocytes in all three groups (fresh, 88.5%; CPA exposure, 90.3%; and vitrification, 88.9%) showed a cortical pattern of CK expression (Table 2). Large and oval-shaped aggregat es ofnon-fibrillar CK were found in the GV oocyte ooplasm, and aless-deve loped filament network was noted in the cortical ooplasm (Fig. 1A, D, and G).

Most of the MI oocytes in both the fresh (86.7%) and CPA expo- sure groups (69.2%) showed a granular pattern (Table 2), which indicates CPA exposure affected the CK distribution at some extent,although there is no significant difference between these two groups. At this stage, the large CK aggregates began to split into small fragments. These CK fragments were occasionally broken down into several granules at the peripher al region in which nolarge cortical aggregates were observed (Fig. 1B and E). However,all of the oocytes in the vitrification group retained the cortical pat- tern (Table 2; Fig. 1H).

For the MII oocytes, 93.5% in the fresh group and 92.9% in the CPA exposure group showed a granular pattern (Table 2). The fila-ment network extended over the MII ooplasm, and numerous CKgranules were scattered across the oocyte. A fine CK network was concentrated evenly in the ooplasm (Fig. 1C and F). Similar to the MI stage, 93.3% of the oocytes in the vitrification group re- tained the cortical pattern (Table 2; Fig. 1I).

Cytokerat in fluorescence intensity

As shown in Fig. 2, the fluorescence intensity of the GV stage oo- cytes were not significantly different among the three groups (fresh, 89.65; CPA exposure, 87.73; vitrification, 93.84) (p > 0.05).For the MI oocytes, the fluorescence intensity in the vitrificationgroup (29.18) was not significantly different compared with the control (43.30). For MII oocytes, there was no significant difference of the CK fluorescence intensity between the CPA exposure (60.05)and control groups (59.27) (p > 0.05), yet the values of both were significantly higher than that in the vitrification group (26.53)(p < 0.05).

Cytokerat in expression of MII oocytes derived from GV oocytes

The CK expression in MII-stage oocytes, derived from fresh and vitrified GV-stage oocytes, were examined using the Western blot method. The CK expression of the MII oocytes derived from vitri- fied GV oocytes was significantly lower (p < 0.05) than that of the fresh oocytes (Fig. 3).

Discussion

In the present study, the CK distribut ion pattern in oocytes reor- ganized according to their nuclear configuration – from a cortical pattern at the GV stage to a granular pattern at the MII stage –and a filament network of CK extended over the ooplasm from the GV stage to the MII stage (Fig. 1). Similar CK distribution pat- terns were also observed during hamster oocyte maturation [33].Both of these results suggest that the initial cortical aggregat ion

ents.

n 6 h to MI stage Incubation 10 h to MII stage

%) Granular (%) Cortical (%) Granular (%)

)a 26/30 (86.7)a 2/31 (6.5)a 29/31 (93.5)a

)a 18/26 (69.2)a 2/28 (7.1)a 26/28 (92.9)a

0)b 0/26 (0)b 28/30 (93.3)b 2/30 (6.7)b

Fig. 1. CK distribution pattern in different stage mouse oocytes during maturation after various treatments. (A) (GV), (B) (MI), (C) (MII) stands for typical CK distribution pattern (green fluorescence) in fresh group; (D) (GV), (E) (MI), (F) (MII) stands for typical CK distribution pattern in CPA exposure group; (G) (GV), (H) (MI), (I) (MII) stands for typical CK distribution pattern in vitrification group. (A, D, G, H, and I) Should be included in ‘cortical’ pattern and (B, C, E, and F) should be included in ‘granular’ pattern. Blue fluorescence stands for oocytes nuclear stained with Hochest 33342. Bar = 20.0 lm.

A A A

AB

A

B

A A

B

Fluorescence intensity

Fig. 2. CK fluorescence intensity of oocytes at different stages. Different letters indicate significant difference. Experiments are repeated for three times.

264 X. Wei et al. / Cryobiology 66 (2013) 261–266

is a non-filamentous pool of CK in immature oocytes and that the CK network, which is called ‘meshworks’ by Plancha, gradually ap- proaches completion during oocyte maturation.

The CK distribution in the vitrified GV oocytes was disturbed and could not form a granular pattern (Table 2; Fig. 1), which is

similar to the findings found by Valojerdi and Salehnia [1,43] thatintermedi ate filaments were damaged in mouse MII oocytes after vitrification. When the GV oocytes were exposed to VS/WS and cul- tured in vitro , the CK distribut ion pattern during oocyte maturation was similar to that of the control (Table 2; Fig. 1). This might relate to our short incubation time (30 s/25 s) in the VS/WS. Many previ- ous studies in our lab also proved that CPA exposure have negative effects on the oocytes viability and function such as cortical gran- ule [39], mitochondrial [47]. While in this study results indicate itwas vitrification and not CPA exposure that is the main reason led to abnormal CK distribution , this probably due to the drastic changing between solid and liquid phase during vitrification cause CK reorganizati on. Cytoplasmic intermediate filament dynamics have been related to post-transla tional modifications, namely phospho rylation and dephosphoryla tion [11]. The phosphoryla tion of CK polypeptides that occurs in mitotic-arre sted cells appears toplay a role in the reorganiz ation of CK filaments [8,26]. The drastic changing between solid and liquid phase caused by cooling and heating during cryopreservati on might lead to a changed CK phos- phorylati on status and result in the abnormal reorganiz ation of CK.

A B

C C

A

B

(A)

(B)

(C)

Fig. 3. CK expression of MII oocytes derived from GV oocytes. One hundred MII oocytes derived from GV oocytes are tested each time using Western blot. Group 1stands for fresh oocytes and group 2 stands for vitrification oocytes. (A) The absolute expression level of Cytokeratin and Beta-actin. (B) Relative expression level of Cytokeratin/Beta-actin. (C) Typical figure of Cytokeratin and Beta-actin expression. Experiments were repeated three times as biological replication.Different letters indicate significant difference.

X. Wei et al. / Cryobiology 66 (2013) 261–266 265

Both the fluores cence inte nsit y (Fig. 2) and Wes tern blo ttin ganal yse s (Fig . 3) illu stra ted that vitr ificatio n signi ficantly dec rea sed CK expr ess ion in mouse MII oocy tes der ive d from vitri fied GV oo- cyt es. Com bin ed our resu lts and othe r refe ren ces rep orte d, we spe c-ula te how vitr ificatio n or cry opre serva tion might aff ect CK str uctu reand low er CK expr ess ion. Vit rificatio n can dis rupt the cell mem- bra ne [18 ] and ind uces Ca2+ inflow. The loc al inc rea se in Ca2+ con-cent rati on then ind uces a casc ade rea ctio n and act iva tes pro tein kina se C [12 ], whi ch might furt her ind uce CK modi fication as wel l.Aff ecte d CK los e its cons tru ctio n abi lit y and CK netw ork str uctu reappe ars loo se or eve n dis appe ars [43 ]. Then CK prot ein expr essi ontend s to be ligh ter col orin g and low er aver age opt ical (Fig s. 2 and 3).

It has been shown previousl y that intermediate filament gels in vitro exhibit stiffening under high-applied stress, suggesting that this stiffening property of IFs might be important for maintaining

cell integrity under large deformation s [21]. In Xenopus, CK mRNA and protein accumulate d in the oocyte as a function of develop- ment to assure compaction and normal gastrulation [23,24,40].Similarly , the increasing complexity of the CK filament network during oocyte maturation suggests a role in maintaining cell integ- rity under physical stress during oocyte transport in the oviduct after ovulation. GV oocytes lack an intact filament network system,and drastic temperature changes during vitrification can damage GV oocytes more easily than mature oocytes [45]. This may coin- cide with lower viability (Table 1) and developmen tal potential of vitrified GV oocytes [37,43]. Forming an intact CK filament net- work system during oocyte maturation might increase tempera- ture tolerance. However, the mechanism of the high sensitivity ofimmature oocytes to cryopreserv ation remains unknown and re- quires further research.

Taxol pretreatmen t of oocytes before vitrification could reduce the microtubule damage induced by vitrification [38]. Both cyto- chalasin B (CB) and cytochalasin D (CD) known as a cytoskeleton stabilizer was found to protect microfilament structures during cryopres ervation [10]. Also, it has been reported that alpha B-crys- tallin is a well known stabilizer of intermediate filaments (desminand vimentin), which functions as a molecular chaperone for inter- mediate filament proteins [5,9]. CK, desmin and vimentin are all belongs to intermediate filaments, therefore we can try alpha B-crystal lin to see whether it also has beneficial stabilizing role for CK after vitrification. This is one of interesting fields we intent to research about in the future in order to improve the vitrificationefficiency of GV oocytes.

In conclusion, it was not the exposure to VS/WS but the vitrifi-cation/w arming procedures that affected the CK distribution and lowered CK expression in the MII oocytes derived from vitrifiedGV oocytes.

Acknowled gments

This work was supported by the National ‘‘863’’ Project Founda- tion of China (No. 2011AA100303) and the National Natural Sci- ence Foundation Project of China (No. 30972102). We thank Nature Published Group Language Editing (NPGLE) for proof-read- ing the manuscript.

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