Ultra-structural changes and developmental potential of porcine oocytes following vitrification

Animal Reproduction Science 100 (2007) 128–140

Ultra-structural changes and developmental potential of porcine oocytes following vitrification Lian-Yu Shi a , Hai-Feng Jin a , Jung-Gon Kim a , B. Mohana Kumar a , S. Balasubramanian a,b , Sang-Young Choe a , Gyu-Jin Rho a,∗ a

College of Veterinary Medicine, Gyeongsang National University, Chinju 660-701, Republic of Korea b Department of Clinics, Madras Veterinary College, Tamilnadu Veterinary and Animal Sciences University, Tamilnadu, India Received 15 November 2005; received in revised form 15 May 2006; accepted 29 June 2006 Available online 8 August 2006

Abstract This study evaluated the effects of exposure and/or vitrification of porcine metaphase II (MII) oocytes on their in vitro viability and ultra-structural changes with two experiments. Experiment 1 examined the effect of vitrified oocytes on microtubule localization, mitochondrial morphology, chromosome organization and the developmental rate in IVF control and vitrified oocytes. Oocytes matured for 44 h were subjected to IVF (IVF control). Oocytes matured for 42 h were exposed to cryoprotectants (CPA control), followed by 2 h culture, and subjected to IVF. Oocytes vitrified at 42 h post-maturation were warmed, cultured for 2 h, and subjected to IVF (vitrified). Experiment 2 evaluated the effect of oocytes freezing on development of ICSI with and without activation and parthenotes. Fresh and vitrified oocytes were subjected to ICSI with and without electrical activation. Cleavage and blastocyst rates were significantly (P < 0.05) lower in vitrified IVF, parthenote and ICSI embryos than those in fresh counterparts. Between ICSI embryos from fresh oocytes and vitrified oocytes, the rates of blastocyst were significantly higher (P < 0.05) in activated group than the group without activation. Significant differences (P < 0.05) were observed in normal spindle configuration of vitrified (43.5%) compared to control (81.0%) oocytes, but no significant difference was observed between CPA exposed and control groups. In conclusion, porcine oocytes at MII stage are very sensitive to vitrification with altered microtubule localization and mitochondrial organization thus resulting in impaired fertilization and embryo development. © 2006 Elsevier B.V. All rights reserved. Keywords: Oocyte; Vitrification; IVF; ICSI; Parthenote; Porcine

∗ Corresponding author at: Department of Obstetrics and Theriogenology, College of Veterinary Medicine, Gyeongsang National University, 900 Gazwa, Chinju 660-701, Republic of Korea. Tel.: +82 55 751 5824; fax: +82 55 751 5803. E-mail address: [email protected] (G.-J. Rho).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.06.020

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1. Introduction Although sensitivity of mammalian oocytes to cooling and freezing remains a challenge to cryobiologists, in porcine, the successful cryopreservation of oocytes would make a significant contribution as an alternative to embryo preservation for various reproductive technologies. Cryopreservation of oocytes by vitrification method seems promising according to the results from mammalian experiments (Martino et al., 1996; Vajta et al., 1998). Recently porcine has been widely used as a model for research in cloning technology, particularly for organ transplantation. Furthermore improvement in cryopreservation technology of in vitro produced porcine oocytes will further facilitate studies on pig cloning and the establishment of a gene bank for transgenic pigs. The oocytes of some mammalian species have been cryopreserved successfully through slow freezing procedures or vitrification. Intracellular lipid removal or polarization of porcine oocytes and embryos improved cryopreservation results. Nagashima et al. (1999) reported that vitrified in vivo matured porcine oocytes developed beyond the 8-cell stage after removal of cytoplasmic lipids and subzonal sperm injection. Whereas, Isachenko et al. (1998) obtained 22% maturation rate in porcine oocytes after vitrification of germinal vesicle (GV) stage oocytes without delipation. On the contrary, Dobrinsky et al. (2000) reported transfer of vitrified embryos without delipation produced viable piglets that grow normally and when mature are of excellent fecundity. Park et al. (2005) have demonstrated for the first time that removal of cytoplasmic lipids enhanced in vitro maturation of vitrified GV porcine oocytes. Furthermore, some of those matured oocytes could be successfully activated and developed to 4-cell stage in culture after ICSI with frozen-thawed spermatozoa. Recently, Hara et al. (2005) have reported a novel lipid removal method with improved cryotolerance without losing mitochondria from the cytoplasm of porcine GV stage oocytes by being centrifuged under hypertonic conditions in medium containing 0.27 M glucose. Another important aspect of cryopreserved oocytes is that, survival and developmental competency are greatly impaired, probably as a consequence of morphological and cytological damage induced by the cryopreservation process such as alterations of the cytoskeleton, mitochondria, cortical granules and plasma membrane (Vincent et al., 1990a; Massip et al., 1995; Hyttel et al., 2000; Rho et al., 2002). In fact the process of cryopreservation of oocytes including exposure to cryoprotectants resulted in extreme disorganization of meiotic spindles, leading to impairment of fertilization of such oocytes and the growth of embryos (Aman and Parks, 1994; Eroglu et al., 1998). In addition, intracellular distribution of mitochondria are known to be related to the level of cell metabolism by converting pyruvate to ATP, and the latter being required for diverse cellular processes in developing embryos, including cell division, DNA replication and genomic activation (Bavister, 1995). Earlier reports have analyzed the effect of vitrification employing open pulled straw or minimum volume cooling methods on cytology of porcine oocytes. Rojas et al. (2004) demonstrated that vitrification of porcine metaphase II (MII) oocytes using open pulled straw method resulted in partial or complete spindle disassembly and chromosome dispersal. To test the hypothesis that altered microtubule localization, mitochondrial morphology and chromosome organization following vitrification of MII stage oocytes results in impaired fertilization and embryo development we analyzed the microtubule localization, mitochondrial morphology and chromosome organization after cryoprotectant exposure and vitrification by immunoflourescence staining. Therefore, this study was conducted to assess the viability of porcine MII oocytes cryopreserved by vitrification on micro-organelles, such as microtubule

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and mitochondria, and the developmental rates of such oocytes after in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and parthenogenesis. 2. Materials and methods 2.1. Chemicals and media All chemicals used in this study were purchased from the Sigma Chemical Company (St. Louis, MO, USA) and media from Gibco (Life Technologies, Rockville, MD, USA) unless otherwise stated. For all the media, the pH was adjusted to 7.4 and osmolality to 280 mOsm/kg. 2.2. Oocytes collection and culture Ovaries were collected from an abattoir located in Chinju, Republic of Korea and transported in phosphate buffered saline (PBS) at 35–39 ◦ C within 2 h. Cumulus-oocyte-complexes (COCs) were aspirated from antral follicles (2–6 mm in diameter) with an 18 G needle fitted to a 10 mL syringe. Sets of 50 oocytes were matured in 500 ␮L TCM199 (IVM medium) containing Earle’s salts, 10 ng/mL EGF, 0.57 mM cysteine, 0.91 mM Na pyruvate, 75 ␮g/mL penicillin, 50 ␮g/mL streptomycin, 0.5 ␮g/mL FSH and 0.5 ␮g/mL LH in each well of a 4-well multidish (Nunc, Roskilde, Denmark) for 24 h at 38.5 ◦ C, 5% CO2 in air and further cultured for 20 h in IVM medium without FSH and LH in the same atmospheric conditions. At 42 h of culture, oocytes were divided into three groups, IVF, vitrification and ICSI procedures. Oocytes that possessed a polar body (PB) and even cytoplasm under a microscope (200×) were used for vitrification and ICSI procedures. 2.3. Oocytes vitrification Oocytes were cryopreserved by vitrification method as described by Isachenko et al. (1998) with minor modifications. All manipulations were performed on a stage warmer at 39 ◦ C in a room temperature (RT) at 25–27 ◦ C. COCs matured for 42 h were denuded of cumulus cells by vortexing for 30–40 s in Dulbecco’s phosphate-buffered saline (DPBS) with 0.1% hyaluronidase. Oocytes were initially equilibrated in holding medium [Ca2+ free DPBS supplemented with 20% fetal bovine serum (FBS), HM] for 5 min and transferred to IVM medium supplemented with 7.5 ␮g/mL cytochalasin B for 15 min prior to vitrification. Subsequently, oocytes were placed in 1.2 M ethylene glycol (EG) and 0.89 M dimethyl sulfoxide (DMSO) dissolved in the HM for 1 min and placed in 1.6 M EG and 1.18 M DMSO dissolved in the HM for 3 min and then transferred into the vitrification solution (2.4 M EG + 1.77 M DMSO + 0.5 M sucrose dissolved in the HM). After 40–60 s of exposure in the final vitrification solution, oocytes were loaded onto 0.25 cm3 plastic straw (IMV, France); heat sealed and directly plunged into liquid nitrogen (LN2 ). 2.3.1. Oocytes warming After being stored in LN2 for at least 1 day, warming was accomplished holding the frozen straw for 10 s in air and for 10 s in a 39 ◦ C water bath. The sealed end of the straw was cut and its contents emptied into a Petri-dish. Oocytes were sequentially rehydrated in a HM containing 0.4, 0.3, 0.2, and 0.1 M sucrose for 2 min in each, respectively, and rinsed twice in HM. The oocytes

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were then transferred back into the IVM medium and cultured for an additional 2 h before being subjected to routine IVF and ICSI with frozen-thawed spermatozoa. 2.4. Embryos production Frozen-thawed boar semen was used for ICSI and IVF procedures. After thawing the straws, semen was centrifuged in Percoll (Pharmacia, Uppsala, Sweden) density gradient (45% versus 90%) at 850 × g for 15 min in order to retrieve live and motile spermatozoa. Sperm pellet was then washed twice by centrifugation at 350 × g for 10 min in 5 mL modified Tris-buffered medium (mTBM) consisting of 113.1 mM NaCl, 3.0 mM KCl, 7.5 mM CaCl2 ·2H2 O, 20.0 mM Tris crystallized free base, 11.0 mM glucose, 5.0 mM Na pyruvate and 3 mL BSA (fatty acid free, fraction V), supplemented with 2 mM caffeine. ICSI procedure was performed by micromanipulation methods as described in bovine by Rho et al. (1998). One hour after injection, the oocytes were re-examined and those from which the spermatozoa have been expelled into the perivitelline space were removed. For activation, eggs at 1 h after ICSI were transferred in BTX electro chamber (BTX Inc., San Diego, CA) filled with 0.28 M mannitol solution containing 0.01% (w/v) BSA, 0.05 ␮M CaCl2 and 0.01 ␮M MgSO4 , and pulsed with a single 2.0 kV/cm DC for 30 ␮s using a BTX Electro-Cell Manipulator 200 and were cultured in 5.5 mM glucose free NCSU23 medium containing 7.5 ␮g/mL CCB for 3 h at 38.5 ◦ C in a humidified atmosphere of 5% CO2 in air. Parthenotes were produced with the same electric pulse as that of ICSI eggs except the procedure of sperm injection into oocytes. As IVF controls, sets of 20 cumulus free oocytes were transferred into 50 ␮L drop of mTBM supplemented with 2 mM caffeine, and inseminated with sperm at final concentration of 1 × 105 sperm/mL for 5 h at 38.5 ◦ C in a humidified atmosphere of 5% CO2 in air. In vitro culture of IVP embryos was performed by modified method of Kikuchi et al. (2002). Eggs were cultured in sets of 20 in 50 ␮L drops of 5.55 mM d-glucose free NCSU23 medium supplemented with 0.17 mM Na pyruvate, 2.73 mM Na lactate, and 0.4% BSA for 58 h and subsequently cultured in NCSU23 with 5.55 mM glucose until day 7. The rates of cleavage and subsequent embryonic development were evaluated under a microscope at 48 and 168 h, respectively, after insemination, sperm injection and electric activation. 2.5. Oocytes immunofluorescence staining Immunofluorescence staining of oocytes was performed by the method described by Rho et al. (2002) to assess the microtubule localization using mouse anti-chick ␣-tubulin monoclonal antibody and mitochondria distribution with Rhodamine-123 (R-123; Molecular Probes, Eugene, OR) staining. Briefly, to assess microtubule localization, oocytes were completely denuded of their cumulus cell by vortexing in 0.1% hyaluronidase solution. Subsequently, oocytes were fixed in 4% formaldehyde in PBS for 1 h and permeabilized using 0.3% Triton X-100. After being washed in PPB (PBS containing 0.1% PVA, 1% BSA and 1% (v/v) sodium azide), the fixed oocytes were transferred into fresh PPB supplemented with 10% goat serum at RT for 10 min, and then into PPB containing mouse anti-chick ␣-tubulin monoclonal antibody (1:100) at 4 ◦ C overnight. After washing in PBS, the oocytes were incubated in fluorescein isothiocyanate (FITC)-conjugated antimouse IgG (antibody) for 1 h in the dark at RT, and then treated with 10 ␮g/mL propidium iodide (PI) for 15–20 min at 39 ◦ C to stain the chromosomes. Microtubule localization of oocytes covered with antifade (Slow fade, Light Antifade Kit, Eugene, OR) was carried out by laser-scanning

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Fig. 1. Spindle morphology: (A) normal spindles; (B) abnormal spindles; (C) absence of spindle.

microscope (Flow view program, Olympus, Japan) equipped with a krypton–argon ion laser for the excitation of FITC for microtubules and PI for chromosomes. Microtubule changes were evaluated as normal, abnormal or absent. Acquired images were processed using Adobe Photoshop 7.0 (Adobe systems, Mountain View, CA) for documentation. Spindle morphology was classified as three categories: (1) normal spindles: barrel-shaped spindle with the chromosomes clustered as a discrete bundle at the metaphase plate and microtubules crossing the length of the spindle from pole to pole or extending from the spindle poles to chromosomes; (2) abnormal spindles: microtubules were not organized as typical spindles or some microtubules were disassembled; (3) absence of the spindle: no microtubules could be observed around the chromosome (Fig. 1). For mitochondria distribution, cumulus denuded oocytes were washed in PBS and incubated in PBS supplemented with 10 ␮g/mL R-123 at 39 ◦ C under 5% CO2 in air for 15 min. The stained oocytes were finally washed in PBS and mounted on a glass slide using antifade and examined under an epifluorescence microscope (excitation 507 nm, barrier 529 nm). Mitochondrial morphology was classified as two categories: (1) normal distribution: intact mitochondria evenly distributed in the ooplasm of MII oocytes; (2) abnormal distribution: unevenly distributed and reduced fluorescence of mitochondria (Fig. 2). 2.6. Chromosomal analysis Chromosomal organization was classified as: (1) dispersed chromosomes: these chromosomes were scattered in the cytoplasm or dispersed in a few zones in the cytoplasm; (2) decondensed

Fig. 2. Mitochondria contribution: (A) normal distribution; (B) abnormal distribution; (C) partial absence.

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chromosomes: chromosomes with an aberrant, less condensed appearance; (3) absence of chromosomes: no chromosomes were observed. 2.7. Experimental design This study comprised of two experiments. Experiment 1 was performed to examine the microtubule localization, chromosome organization, mitochondrial morphology and the developmental rates. Oocytes were divided into three groups. Group 1 (IVF control): oocytes were matured for 44 h and subjected to IVF. Group 2 (cryoprotective agent; CPA control): oocytes matured for 42 h were exposed to the cryoprotectant, followed by 2 h culture, and subjected to IVF. Group 3 (vitrified-warmed): oocytes vitrified at 42 h post-maturation were warmed, cultured for 2 h, and subjected to IVF. Experiment 2 was to investigate the cleavage and blastocyst development rates in different groups. Oocytes matured for 44 h (fresh oocytes) were subjected to ICSI without electrical activation (Treatment 1) and with electrical activation (Treatment 3). Oocytes matured for 42 h (vitrified-warmed oocytes) were cultured for 2 h and subjected to ICSI without electrical activation (Treatment 2) and with electrical activation (Treatment 4). Parthenotes derived from fresh (Treatment 5) and vitrified-warmed oocytes (Treatment 6) were produced with the same electrical activation as that of ICSI eggs. 2.8. Statistical analysis Differences among treatments in each experiment were made by one-way analysis of variance (ANOVA) by SPSS after arcsine transformation of the data of cleavage and development in vitro into blastocyst stage. Data were expressed as mean ± S.D. Comparisons of mean values among treatments were performed using Tukey–Kramer multiple comparisons test. In addition, the normality of chromosomes, microtubules and mitochondria were analyzed using the observed frequencies in Fisher’s extract test. A probability of P < 0.05 was considered to be statistically significant. 3. Results 3.1. Development rates of vitrified porcine oocytes The cleavage and development rates of IVF control and vitrified porcine oocytes are presented in Table 1. Cleavage rates in the IVF control group was similar to CPA group and both significantly higher (P < 0.05) than in vitrified one. However, in regard to blastocyst development, both Table 1 Cleavage and development rates of fresh, exposed to CPA and vitrified porcine oocytes Groups (treatment)a

1 (control) 2 (CPA control) 3 (vitrified)

Oocytes used

301 287 252

Development to (%, mean ± S.D.) Cleaved

Blastocysts

234 (78.0 ± 6.5) b 204 (71.0 ± 8.2) b 96 (38.1 ± 9.4) a

69 (23.0 ± 3.4) c 33 (11.4 ± 2.9) b 6 (2.3 ± 1.9) a

Different letters indicate significant difference within column (P < 0.05); seven replicates. a Control, IVF control; CPA control, exposed to cryoprotectant.

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Table 2 Spindle morphology and chromosome configuration of fresh, exposed to CPA and vitrified porcine oocytes Groups (treatment)a

1 (control) 2 (CPA control) 3 (vitrified)

Oocytes used

58 50 46

Spindle changes (%)

Chromosome changes (%)

Normal

Abnormal

Absent

Dispersed

Decondensed

Absent

47 (81.0) b 34 (68.0) b 20 (43.5) a

8 (13.8) a 12 (24.0) b 19 (41.3) c

3 (5.2) 4 (8.0) 7 (15.2)

8 (13.8) 10 (20.0) 15 (32.6)

0 (0) 2 (4.0) 4 (8.7)

3 (5.2) 4 (8.0) 7 (15.2)

Different letters indicate significant difference within column (P < 0.05); three replicates. a Control, IVF control; CPA control, exposed to cryoprotectant.

treatments (exposure and vitrification) led to a significant reduction (P < 0.05) when compared to control. 3.2. Changes in microtubule and chromosome of vitrified oocytes The cryopreservation of oocytes in all subsequent experiments was accomplished using vitrification method. Results of the analysis of the spindle and chromosomal organization obtained from representative samples of Experiment 1 are shown in Table 2. Significant differences (P < 0.05) were observed in normal spindle configuration in vitrified oocytes (43.5%) compared to control oocytes (81.0%), but no significant difference was observed between CPA exposed and control groups. After vitrification, the proportion of oocytes with abnormal spindles increased to 41.3% and 15.2% oocytes did not show any microtubules. Presence of abnormal chromosome configurations increased significantly after vitrification, especially most oocytes showed dispersed or decondensed chromosomes. 3.3. Mitochondria distribution of vitrified oocytes Analysis of mitochondrial distribution in representative samples obtained from Experiment 1 of control and CPA exposed groups predominantly (83.3% and 72.1%, respectively) showed an intact and even distribution in the ooplasm of MII stage porcine oocyte. On the other hand, when oocytes were vitrified, the proportion of oocytes with normal distribution decreased 47.5% and 52.5% of oocytes had uneven distribution of mitochondria (Table 3).

Table 3 Mitochondrial changes in fresh, exposed to CPA and vitrified porcine oocytes Groups (treatment)a

1 (control) 2 (CPA control) 3 (vitrified)

Oocytes used

48 43 40

Mitochondria changes (%) Normal

Reduced

40 (83.3) b 31 (72.1) b 19 (47.5) a

8 (16.7) a 12 (27.9) b 21 (52.5) c

Different letters indicate significant difference within column (P < 0.05); three replicates. a Contol, IVF control; CPA control, exposed to cryoprotectant.

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Table 4 Cleavage and development of fresh and vitrified porcine oocytes injected with sperm and with or without electric activation treatment and parthenogenetic activation Treatments

1 (ICSI/fresh-without activation) 2 (ICSI/vitrified-warmed-without activation) 3 (ICSI/fresh-with activation) 4 (ICSI/vitrified-warmed-with activation) 5 (parthenote/fresh) 6 (parthenote/vitrified-warmed)

Oocytes used

187 165 190 169 201 178

Development (%, mean ± S.D.) Cleaved

Blastocysts

122 (65.2 ± 5.7) b 68 (41.2 ± 6.2) a 133 (70.0 ± 6.8) b 75 (44.3 ± 4.4) a 154 (76.6 ± 3.6) b 74 (41.6 ± 5.7) a

15 (8.0 ± 3.2) b 3 (1.8 ± 1.2) a 55 (28.9 ± 3.1) d 9 (5.3 ± 0.6) b 40 (19.9 ± 2.7) c 3 (1.7 ± 2.0) a

Different letters indicate significant difference within column (P < 0.05); six replicates.

3.4. Development rates of embryos produced by different treatment groups The cleavage and development rates of embryos produced by different treatment groups are presented in Table 4. Cleavage rates of ICSI and parthenote embryos derived from vitrifiedwarmed oocytes were significantly lower (P < 0.05) than those derived from fresh counterparts (41.2–44.3% versus 65.2–76.6%). However, there was no significant difference in the rate of cleavage among fresh oocyte groups and among vitrified-warmed oocyte groups. The blastocyst development rates of ICSI and parthenote embryos derived from vitrifiedwarmed oocytes were significantly lower (P < 0.05) than those derived from fresh counterparts. Between ICSI embryos derived from fresh oocytes or vitrified-warmed oocytes, the rates of blastocyst were significantly higher (P < 0.05) in electrically activated group than the group without electrical activation (28.9% versus 8.0%, and 5.3% versus 1.8%, respectively). Moreover, significantly higher (P < 0.05) rates of blastocysts were observed in the ICSI-fresh oocytes with electrical activation than in parthenote-fresh group (28.9% versus 19.9%, respectively). However no significant differences on blastocyst development were observed between ICSI-fresh oocytes without electrical activation and ICSI-vitrified with electrical activation. 4. Discussion Despite of progress that has been made in the in vitro maturation and fertilization of porcine oocytes, the developmental ability of oocytes matured and fertilized in vitro is low compared with that of oocytes matured and fertilized in vivo (Prather and Day, 1998). In the present study, we evaluated the microtubule, chromosome and mitochondria organization of in vitro matured porcine oocytes following either or not exposure to CPA and vitrification; and the developmental ability of such oocytes after IVF and ICSI procedures. The cleavage and blastocyst development rates of MII oocytes following exposure to CPA and vitrification were significantly lower in vitrified oocytes than in fresh oocytes. Didion et al. (1990) examined the viability of porcine GV stage oocytes following cooling or freezing by conventional methods and found that the cumulus-intact GV stage oocytes did not survive cooling temperatures at or below 15 ◦ C. Whereas, Rojas et al. (2004) reported that porcine oocytes at different meiotic stages respond differently to cryopreservation and MII oocytes had better resistance than GV stage oocytes.

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The survivability of oocytes following cryopreservation procedures depends on several mechanism of cell injury, such as the chemical toxicity of the cryoprotectants, osmotic shock in the dehydration and rehydration procedures and freezing effects on oocytes ultra-structure. In the present study, MII oocytes exposed to CPA without vitrification showed reduced development as compared to controls. Exposure of oocytes to concentrated solution of cryoprotectants during the vitrification process could have triggered an increase in abnormal spindles and cytoskeletal disruption, thus resulting in the poor survivability and developmental capacity. The results are consistent with Rho et al. (2002) who have suggested that damage to microtubules and mitochondria during oocyte cryopreservation might be involved in the reduced viability. It has been reported that fertilization process could be compromised by the effect of cryoprotectants (Vincent et al., 1990b) and cooling (Martino et al., 1996). In our study, development of MII oocytes was significantly reduced following vitrification. Cryoprotectants disrupt the cortical microfilament network and caused depolymerization and disorganization of the spindle microtubules, which in turn results in chromosomal scattering (Vincent et al., 1990b). Disruption of the cytoskeleton might be intrinsic to changes in shape and shrinkage that accompany cryopreservation procedures, which in turn might lead to premature release of cortical granules and zona hardening (Vincent et al., 1990b) by allowing cortical granules to subjacent to the plasma membrane. Rewarming may then lead to membrane fusion and release of enzymes. Release of cortical granule enzymes and premature zona hardening may either block fertilization completely or incompletely block polyspermy, both cases resulting in a decreased cleavage rate after insemination (Lim et al., 1991). The change in the ZP appeared to overcome by micromanipulation techniques (Kazem et al., 1995; Rho et al., 2004) such as ICSI. Fujihira et al. (2004) reported for the first time in vitro development of blastocysts derived from vitrified immature porcine oocytes followed by IVM and ICSI. Further, they observed no significant difference in the cleavage and blastocyst rates between the vitrified-warmed and fresh oocytes. Our study also demonstrated no difference in the cleavage and blastocysts rates of in vitro matured vitrified-warmed oocytes between ICSI and IVF procedures. These results suggest that MII oocytes were more resistant to vitrification as demonstrated by their developmental capacity to blastocysts. A major pre-requisite for a normal embryo development is the proper activation of the oocyte. In vitro matured porcine oocytes need exogenous activation for embryo development, and electrical activation is a suitable method for producing ICSI and cloned embryos (Lai et al., 2001; Kim et al., 2005). The fusion of both gametes induces Ca2+ oscillations until the second polar body is extruded and pronuclei are formed (Day et al., 2000). Electrical stimulation to oocytes results in the formation of pores in the plasma membrane, which facilitates the uptake of extra-cellular calcium (Collas et al., 1993) leading to an increase in intracellular calcium concentration and oocyte activation. Lai et al. (2001) and Lee and Yang (2004) reported that electric activation following ICSI significantly increased porcine oocytes development to blastocyst. Similar results are observed in this study from ICSI-fresh oocyte (8.0% from without activation versus 28.9% from with activation). Blastocyst development rates of ICSI embryos without activation and parthenotes derived from vitrified-warmed oocytes were similar, but lower than ICSI embryos with activation, confirm our assumption that the reduced developmental competence is not attributable to impaired fertilization. This finding is in agreement with the observations of Martino et al. (1996) and Kubota et al. (1998). Irrespective of activation among vitrified-warmed oocytes the rates of cleavage in ICSI and parthenote are significantly lower than that of fresh oocyte groups, indicating that vitrification-warming procedures result in retarded embryonic development although the oocytes can develop up to two cell stages.

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The microtubule system of oocytes is crucial for fertilization and normal development (Pickering et al., 1990). Microtubules are dynamic components of the cell cytoskeleton; they are ubiquitously present in mammalian cells and perform diverse function such as cell shape and movement, transportation of molecules and organelles, meiosis and mitosis. The abnormality in microtubules profoundly influences formation of the spindle, and it is often associated with abnormal chromosome segregation after fertilization and reduced developmental rates of embryos as a consequence (Eroglu et al., 1998). Previous studies have shown that cryoprotectants such as DMSO and PrOH caused changes in microtubule organization in several species, including the mouse (Vincent et al., 1990b; George et al., 1996) and human (Sathananthan et al., 1988). It has been further shown in human oocytes that exposure to 1.5 M DMSO for an hour at 37 ◦ C resulted in the absence or reduction of microtubules (Johnson and Pickering, 1987). Similarly in our study, approximately 32% of oocytes exposed to CPA appeared to have abnormal or absence of spindles. However, Aman and Parks (1994) observed no changes in microtubules of bovine oocytes exposed to CPA. Dustin (1984) reported that microtubule assembly was highly temperature-dependent, with increased depolymerization rates at temperatures below 20 ◦ C and complete depolymerization at 0–4 ◦ C. Drastic changes in organization of the meiotic spindle and chromosome following freezing have been reported in mouse (Sathananthan et al., 1992) and human (Sathananthan et al., 1988). The exposure of oocytes to room temperature (∼25 ◦ C) or a lower temperature (4 ◦ C), for as short as 1 or 5 min caused depolymerization of the meiotic spindle (Aman and Parks, 1994; Wang et al., 2001). When porcine oocytes were kept for 5 min at 4 ◦ C, microtubule in the spindles of most oocytes partially or completely disassembled (Liu et al., 2003). Zhang and Nicklas (1995) reported that chromosomes enhanced spindle microtubule assembly and played an essential role in the initiation of spindle formation. The organization of meiotic spindle requires both chromosomes, which causes a local reduction in the threshold for microtubule polymerization, and the pericentriolar material to nucleate microtubule polymerization. In the present study, 32.6% of oocytes showed dispersed chromosome following vitrification. Microtubules could assemble into normal spindle structure when chromosomes and the pericentriolar material were functional. Whereas, oocytes having damaged chromosomes could assemble microtubules around the chromosomes, but would not form normal spindles. It seems that abnormal or absence of spindle observed in 56.5% of porcine oocytes in the present study indicate that the meiotic spindles in porcine MII oocytes are very sensitive to temperature and mainly resulted from vitrification procedure. The mitochondrial organization and activity are necessary features among the diverse events involved in cytoplasmic maturation and resumption of meiosis, affecting subsequent development (Van Blerkom, 1991). The primary function of mitochondria is to generate ATP, necessary for several functions including motility, maintenance of cellular homeostasis and regulating cell survival. The movement of mitochondria within different areas of the cell is mediated by cytoskeletal network of microtubules (Van Blerkom, 1991). The lack of relocation of active mitochondria to the inner part of the oocyte was related to the absence of an appropriate and timely formation of the microtubule network in the cytoplasm. Thus, the distribution of active mitochondria may be indicative of the energy or ion requirement of various key events during oocyte maturation, fertilization and early embryo development. Moreover mitochondria played an important role in Ca2+ signaling that mediated oocyte activation and development, and apoptotic cell death (Liu et al., 2001). Disruption of the plasma membrane and mitochondria has been observed in frozenthawed human (Sathananthan et al., 1988) and mouse oocytes (Eroglu et al., 1998). Disruption of mitochondria increased the cell permeability to calcium resulting in increased intracellular Ca2+

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thus activating the hydrolytic enzymes, impairing the energy production, and ultimately resulting in cell death (Berridge et al., 1998). In the present study, we investigated the role of mitochondria and microtubule damage as factors in cryopreservation failure. Among vitrified oocytes, about 52.5% showed reduced number of mitochondria compared to untreated oocytes. Normally, mitochondria are evenly dispersed through the cytoplasm before fertilization or electric activation of oocytes. Normal appearance of mitochondria in control oocytes was significantly higher (P < 0.05) than vitrified oocytes. However, the control and handled control groups showed no significant difference. Results of the present study showed that exposure of oocytes to CPA did not greatly alter the mitochondria, as that of vitrification. Contrary to the present observations, Noto et al. (1993) reported that mitochondria dislocation associated with a specific cellular phase appeared to be preserved after cryopreservation, suggesting that cellular organization and the distribution of cytoplasmic organelles remained unaffected by either exposure to 3.5 M DMSO and 0.25 M sucrose for 2.5 min or cryopreservation protocols. The reason for these variable results still remains unclear. In conclusion, the results indicate that porcine oocytes at MII stage are very sensitive to vitrification with altered microtubule localization and mitochondrial organization thus resulting in impaired fertilization and embryo development. Further, this would raise concern as chromosomal dispersion may lead to chromosomal aberrations of developing embryos. Hence, further investigation addressing this effect is necessary to clarify cellular mechanisms associated with cryoinjury. Acknowledgements The financial support of BioGreen 21 (grant no. 100052004002000) and KOSEF (grant no. R05-2004-000-10702-0) in Korea are gratefully acknowledged. References Aman, R.R., Parks, J.E., 1994. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro matured bovine oocyte. Biol. Reprod. 50, 103–110. Bavister, B., 1995. Culture of pre-implantation embryo: facts and artifacts. Hum. Reprod. 1, 91–148. Berridge, M.T., Bootman, M.D., Lipp, P., 1998. Calcium—a life and death signal. Nature 395, 645–648. Collas, P., Fissore, R., Robl, J.M., Sullivan, E.J., Barnes, F.L., 1993. Electrically induced calcium elevation, activation, and parthenogenetic development of bovine oocytes. Mol. Reprod. Dev. 34, 212–223. Day, M.L., McGuinness, O.M., Berridge, M.J., Johnson, M.H., 2000. Regulation of fertilization-induced Ca2+ spiking in the mouse zygote. Cell Calcium 28, 47–54. Didion, B.A., Pomp, D., Martin, M.J., Homanics, G.E., Markert, C.L., 1990. Observations on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage. J. Anim. Sci. 68, 2803–2810. Dobrinsky, J.R., Pursel, V.G., Long, C.R., Johnson, L.A., 2000. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol. Reprod. 62, 564–570. Dustin, P., 1984. Structure and chemistry of microtubules. In: Microtubules. Springer-Verlag, New York, pp.19-24. Eroglu, A., Toth, T.L., Toner, M., 1998. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil. Steril. 69, 944–957. Fujihira, T., Kishida, R., Fukui, Y., 2004. Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants. Cryobiology 49, 286–290. George, M.A., Pickering, S.J., Braude, P.R., Johnson, M.H., 1996. The distribution of alpha- and gamma-tubulin in fresh and aged human and mouse oocytes exposed to cryoprotectant. Mol. Hum. Reprod. 2, 445–456. Hara, K., Abe, Y., Kumada, N., Aono, N., Kobayashi, J., Matsumoto, H., Sasada, H., Sato, E., 2005. Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitrification. Cryobiology 50, 216–222.

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