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Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14516

Spindle configuration and developmental competence of in vitro-matured bovine oocytes exposed to NaCl or sucrose prior to Cryotop vitrification Nu´ria Arcarons A, Roser Morato´ B, Joze´ F. W. Spricigo C, Marcia A. M. M. Ferraz D and Teresa Mogas A,E A

Department of Animal Medicine and Surgery, University Autonomous of Barcelona, Campus UAB, 08193 Cerdanyola del Valle`s, Spain. B Department of Biology, Universitat de Girona, Campus de Montilivi, 17071 Girona, Spain. C School of Agriculture and Veterinary Medicine, University of Brasilia, CEP 70910-900, Brası´lia-DF, Brazil. D Department of Farm Animal Health, University of Utrecht, 3584 CM Utrecht, The Netherlands. E Corresponding autor. Email: [email protected]

Abstract. In the present study we examined whether exposure to high concentrations of NaCl or sucrose before vitrification improves the cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, oocytes were exposed to different concentrations of NaCl (375–1517 mOsm) or sucrose (375–812 mOsm) for 1 h. On the basis of the results of this experiment, in Experiment 2 oocytes were exposed to 0.25% NaCl (375 mOsmol) or 2.77% sucrose (375 mOsmol) solution, vitrified and warmed. Microtubule and chromosome configurations were examined by immunocytochemistry. In Experiment 3, in vitro embryo development was assessed after vitrification of oocytes with or without 2.77% sucrose (375 mOsmol) pretreatment. There was a similar percentage of oocytes showing normal spindle configurations in the sucrosepretreated and control groups. Higher rates of abnormal spindles were found in groups treated with NaCl or sucrose solutions with .375 mOsmol. After vitrification and warming, a significantly higher percentage of oocytes with normal chromosome configurations was recorded for oocytes exposed to 375 mOsmol sucrose solution before vitrification compared with the control vitrified oocytes. However, these percentages were significantly lower than those recorded in untreated controls. Cleavage and blastocyst rates were higher in non-vitrified than vitrified oocytes. In conclusion, pretreatment with 375 mOsmol NaCl or sucrose solution had no adverse effects on the spindle status of vitrified–warmed cow oocytes. However, sucrose pretreatment offered no benefits for embryo development. Additional keywords: blastocyst, chromosomes, embryo development, microtubule configuration, osmotic stress.

Received 28 December 2014, accepted 26 February 2015, published online 22 April 2015

Introduction The freezing of spermatozoa and embryos is technically feasible and is a widely used practical procedure. In contrast, the cryopreservation of oocytes has proved much more difficult and has so far led to low survival rates. The long-term cryopreservation of oocytes is an important strategy to maintain genetic diversity, and is essential for research and development in the fields of biotechnology, biomedicine and assisted reproduction, including infertility treatment, cloning and gene banking. Traditionally, embryos and oocytes have been cryopreserved by slow freezing methods using low concentrations of cryoprotectants. During this procedure, osmotic shock and intracellular ice formation often cause cell damage. Today, vitrification is regarded as the best alternative to traditional slow freezing Journal compilation Ó CSIRO 2015

procedures. Vitrification is an ice-free cryopreservation method that involves high concentrations of cryoprotectants combined with very rapid cooling by direct plunging in liquid nitrogen resulting in an ice crystal-free, solid glass-like structures (Rall and Fahy 1985). In the past 2 years, successful survival rates of over 90% after vitrification and warming and live-birth rates of over 50% have been reported for human oocytes (Cobo et al. 2013). However, despite many recent advances, the ideal protocol for the cryopreservation of cow oocytes has not yet been developed. Thus, to date, the practical use of cow oocyte vitrification is limited because of the high sensitivity of these oocytes to cooling and cryopreservation and the low rates of blastocyst production that have been observed after warming compared with non-vitrified controls (Sripunya et al. 2010). www.publish.csiro.au/journals/rfd

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Reproduction, Fertility and Development

Vitrified–warmed oocytes usually exhibit several ultrastructural (Hyttel et al. 2000) and structural modifications, including abnormal distribution of chromosomes, microtubules and actin microfilaments (Rojas et al. 2004; Albarracı´n et al. 2005), disorganisation of the spindle apparatus with the consequent risk of chromosome loss and aneuploidy (Liu et al. 2003), changes to the lipid bilayer phase causing cytoplasmic membrane lysis (Arav et al. 1996) and nuclear fragmentation (Men et al. 2003a, 2003b), which is not readily repairable by the endogenous mechanisms within a haploid cell. Thus, it seems that the sensitivity to chilling of cow oocytes is determined by their complex structure. Several measures designed to optimise bovine oocyte vitrification protocols have been tested, such as the use of different cryoprotectants (CPAs; Horvath and Seidel 2008; Morato´ et al. 2008a), CPA concentrations or CPA exposure temperatures and times (Papis et al. 2000; Magnusson et al. 2008; Zhou et al. 2010; Prentice-Biensch et al. 2012), along with the addition of macromolecular supplements (Horvath and Seidel 2006; Checura and Seidel 2007). Other procedures tested have been the partial removal of cumulus cells (Zhou et al. 2010), modification of oocyte membrane constituents (Horvath and Seidel 2006, 2008), polarisation of cytoplasmic lipid droplets by centrifugation (Otoi et al. 1997) and the addition of cytoskeleton relaxants (Isachenko et al. 2001; Rho et al. 2002; Morato´ et al. 2008a) or ice blockers (Zhou et al. 2010) to vitrification solutions. The results of earlier investigations suggest that sublethal stress, properly defined and applied appropriately before manipulation, could improve the cryotolerance of oocytes through adaptation mechanisms enhancing their subsequent stress tolerance (Pribenszky et al. 2010). Thus, pretreatment with high hydrostatic pressure or osmotic, heat or oxidative stress seems to improve morphological intactness, fertilising ability and developmental competence after various in vitro and in vivo procedures, such as cryopreservation, parthenogenic activation or somatic cell nuclear transfer (Pribenszky et al. 2010, 2012). In the pig, exposure of oocytes to increased concentrations of sodium chloride, sucrose or trehalose before manipulation has been reported to improve both cryotolerance to vitrification and developmental competence (Lin et al. 2009a, 2009b). However, to the best of our knowledge, how the effect of temporarily increased NaCl or sucrose concentrations may influence cryotolerance and developmental competence of bovine oocytes has not yet been examined. In the present study, we examined whether the exposure of in vitro-matured (IVM) bovine oocytes to high concentrations of NaCl or sucrose before vitrification–warming would improve their cryotolerance. In the first experiment, microtubule and chromosome configurations were analysed after exposure of oocytes to various concentrations of NaCl and sucrose. In the second experiment, oocytes were treated with selected concentrations of NaCl or sucrose, vitrified–warmed and microtubule and chromosome configurations evaluated. Finally, and based on results obtained in the previous experiment, oocytes were exposed to 375 mOsm sucrose, vitrified–warmed, fertilised and cultured up to the blastocyst stage. Exposure of IVM bovine oocytes to concentrations of 0.25% NaCl (375 mOsmol) or

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2.77% sucrose (375 mOsmol) for 1 h had no substantial effect on spindle morphology, yet higher concentrations led to markedly reduced rates of oocytes showing normal spindle configurations. Moreover, no deleterious effects on the organisation of the meiotic spindle were observed after pretreatment of IVM bovine oocytes with 375 mOsmol NaCl or sucrose before vitrification. In addition, 375 mOsmol sucrose treatment prior to vitrification did not have any beneficial effects on embryo development of IVM bovine oocytes, in contrast with results reported in other species (Lin et al. 2009a, 2009b). Materials and methods Chemicals and supplies Unless stated otherwise, all chemicals and reagents were purchased from Sigma Chemical (St Louis, MO, USA). Bovine oocyte collection and IVM The methods used for the IVM of oocytes have been described elsewhere (Rizos et al. 2001). Briefly, ovaries from cows (.24 months old) killed at a local abattoir were transported to the laboratory in phosphate-buffered saline (PBS) at 35–378C. Cumulus–oocyte complexes (COCs) were obtained by aspirating follicles 2–10 mm in diameter. Only COCs with three or more layers of cumulus cells and a homogeneous cytoplasm were selected for maturation. After three washes in modified PBS (PBS supplemented with 36 mg mL
1 pyruvate, 50 mg mL
1 gentamicin and 0.5 mg mL
1 bovine serum albumin (BSA)), groups of up to 50 COCs were placed in 500 mL maturation medium in four-well plates and cultured for 24 h at 38.58C in a 5% CO2 humidified air atmosphere. The maturation medium (MM1) was TCM-199 supplemented with 10% (v/v) fetal calf serum (FCS), 10 ng mL
1 epidermal growth factor and 50 mg mL
1 gentamicin. NaCl and sucrose treatment After 21 h (Experiments 2 and 3) or 22 h (Experiment 1) in MM1, oocytes were randomly transferred to 500 mL MM2 (HEPES-buffered TCM-199 containing 2% (v/v) FCS) supplemented with the corresponding amount of NaCl or sucrose (see below) in four-well dishes and incubated for 1 h at 38.58C in a 5% CO2 humidified air atmosphere. As a control, one group of oocytes was incubated in MM2 alone. After recovery for 1 h in MM1, oocyte spindle configuration was evaluated in Experiment 1. Oocytes in Experiments 2 and 3 were vitrified–warmed (see below) and oocyte spindle configuration (Experiment 2) and embryo development (Experiment 3) were evaluated. Oocyte vitrification and warming Vitrification protocol Oocytes were vitrified using the Cryotop device (DibimedBiomedical Supply, S.L., Valencia, Spain) and vitrification and warming solutions described by Kuwayama et al. (2005). The holding medium (HM) for formulating all vitrification– warming solutions was HEPES-buffered TCM-199 containing 20% (v/v) FCS. Partially denuded oocytes were transferred to an equilibration solution (ES) consisting of 7.5% (v/v) ethylene

Osmotic stress on oocyte cryotolerance

glycol (EG) and 7.5% (v/v) dimethylsulfoxide (DMSO) in HM for 10 min and then transferred to the vitrification solution (VS) containing 15% (v/v) DMSO, 15% (v/v) EG and 0.5 M sucrose dissolved in HM. After incubation for 30–40 s, the oocytes were loaded onto the Cryotop device, almost all the solution was removed to leave only a thin layer covering the oocytes and the device was plunged into liquid nitrogen. The entire process from exposure in VS to plunging in liquid nitrogen was completed within 90 s. Warming protocol All warming steps were performed at 38.58C. Vitrified oocytes were warmed by directly immersing the Cryotop device into the warming solution, consisting of 1 M sucrose dissolved in HM for 1 min. The recovered oocytes were then transferred to HM solution containing 0.5 M sucrose for 3 min and then incubated in HM alone for 5 min. After a final rinse again in HM for 1 min, the oocytes were transferred to MM1 to recover for a further hour. IVF and embryo culture IVM oocytes were subjected to IVF at 38.58C in a 5% CO2 atmosphere. Briefly, oocytes were washed once in fertilisation medium before being transferred, in groups of up to 25 to fourwell plates containing 250 mL fertilisation medium per well (Tyrode’s medium supplemented with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL
1 fatty acidfree BSA and 10 mg mL
1 heparin–sodium salt; Calbiochem, Darmstadt, Germany). Frozen–thawed spermatozoa from bulls of proven fertility were used in all experimental procedures. Motile spermatozoa were obtained by centrifuging frozen– thawed sperm from Asturian bulls (ASEAVA (Asociacio´n Espan˜ola de criadores de ganado vacuno selecto de la raza Asturiana de los Valles); Llanera, Asturias, Spain) on a discontinuous gradient (Bovipure; Nidacon International, Gothenburg, Sweden) for 10 min at 100g at room temperature. Viable spermatozoa collected from the bottom were washed (Boviwash; Nidacon International) and pelleted by centrifugation at 100g for 5 min at room temperature. Spermatozoa were counted in a Neubauer chamber and diluted in an appropriate volume of fertilisation medium to give a final concentration of 2  106 spermatozoa mL
1. A 250-mL aliquot of this suspension was then added to each fertilisation well to obtain a final concentration of 1  106 spermatozoa mL
1. Plates were incubated at 38.58C in a 5% CO2 humidified air atmosphere. Approximately 22 h post-insemination (hpi), presumptive zygotes were denuded by gentle pipetting and transferred to 25-mL culture droplets of synthetic oviductal fluid (SOF; Holm et al. 1999; 1 embryo mL
1) containing FCS (5%, v/v) under mineral oil. Embryos were incubated for 6 days at 38.58C in a humidified 5% CO2/5% O2/90% N2 atmosphere. Cleavage rates were recorded at 48 hpi and the number of blastocysts determined on Day 7 after insemination. MII spindle status After IVM, oocytes were denuded of cumulus cells by gentle pipetting in PBS before immunostaining for tubulin and

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chromatin. Fresh IVM oocytes were used as controls to determine normal patterns of microtubule and chromatin organisation. Samples of oocytes from the experimental groups were fixed in 4% (w/v) formaldehyde–PBS (30 min, 38.58C), permeabilised using Triton X-100 (2.5% (v/v) in PBS) for 15 min and simultaneously immunostained for tubulin and chromatin as described previously (Boiso et al. 2002). The fixed oocytes were incubated with an anti-a-tubulin monoclonal antibody (Molecular Probes, Paisley, UK; 1 : 250 dilution) overnight at 378C, followed by incubation with the antimouse IgG antibody Alexa Fluor 488 (Molecular Probes; 1 : 5000) for 1 h at 378C. Between incubations, the oocytes were washed three times in prewarmed PBS for 5 min. Fixed and stained oocytes were mounted on poly L-lysine-treated coverslips fitted with a self-adhesive reinforcement ring in a 3-mL drop of Vectashield containing 125 ng mL
1 40 ,60 diamidino-2-phenylindole (DAPI; Vectorlabs, Burlingame, CA, USA) and flattened with a coverslip. Preparations were sealed with nail varnish and stored at 48C protected from light until observation within the following 2 days. Meiotic spindle configuration was assessed under an epifluorescence microscope (Axioscop 40FL; Carl Zeiss, Gottingen, Germany). The criteria used to classify chromosome and microtubule distributions have been described elsewhere (Albarracı´n et al. 2005). Briefly, the meiotic spindle was defined as normal when the classic symmetrical barrel shape was observed, with chromosomes aligned regularly in a compact group along the equatorial plane. In contrast, abnormal spindles showed disorganised, clumped, dispersed or unidentifiable spindle elements and chromatin was abnormally organised (showing clustering or dispersal from the spindle centre). Detailed images of these normal and abnormal patterns are shown in Fig. 1. Statistical analysis All statistical tests were performed using IBM SPSS 19 for Windows (IBM Corporation, Chicago, IL, USA). The significance of differences in meiotic spindle configuration after treatment, cleavage rates and blastocyst yield were analysed by ANOVA. All data were checked for normality using the Levene and Kolmogorov–Smirnov tests, and data that were not normally distributed were compared using the Kruskal–Wallis test. Significance was set at two-tailed P , 0.05. Experimental design Three experiments were conducted, with four (Experiments 1 and 2) or seven (Experiment 3) replicates for each experiment. Experiment 1: effects of transient exposure of IVM bovine oocytes to elevated NaCl or sucrose concentrations on meiotic spindle status To examine the effects of NaCl, 307 oocytes, matured in vitro for 22 h, were randomly allocated to on of eight groups (A1–A8). Oocytes in Groups A2–A8 were incubated for 1 h in MM2 supplemented with 0.25%, 0.5%, 1%, 1.5%, 2%, 3% and 4% (w/v) NaCl, respectively (corresponding to osmolalities of 375, 443, 602, 749, 901, 1206 and 1517 mOsmol, respectively,

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(a)

(b)

(c)

(d)

Fig. 1. Microtubule and chromosome configurations examined by immunocytochemistry of in vitromatured bovine oocytes after vitrification with or without NaCl or sucrose pretreatment. (a) Normal barrel-shaped MII spindle morphology. Scale bar ¼ 3.1 mm. (b–d) Vitrified–warmed oocytes treated with NaCl or sucrose before the vitrification procedure exhibiting abnormal spindle structures. (b) Abnormal spindle morphology showing partly disorganised chromosomes. Note that chromosomes are not aligned Scale bar ¼ 4.2 mm. (c) Completely disorganised microtubules and chromosomes. Scale bar ¼ 3.4 mm. (d ) Disrupted microtubular shape. Note the decondensation of microtubules and the less condensed appearance of chromosomes. Scale bar ¼ 2.9 mm. Red, tubulin (Alexa Fluor 594); blue, chromosomes (DAPI).

as measured using an osmometer (Osmomat 030; Gonotec GmbH, Berlin, Germany)). Oocytes in Group A1 were incubated in MM2 (294 mOsmol) for 1 h and served as the control group. To examine the effects of sucrose, 392 oocytes, matured in vitro for 22 h, were randomly allocated to one of six groups (B1–B6). Oocytes from Groups B2–B5 were incubated for 1 h in MM2 supplemented with 2.77%, 5.10%, 10.54% and 15.57% (w/v) sucrose, respectively (corresponding to osmolalities of 375, 455, 631 and 812 mOsmol, respectively, as measured using an osmometer (Osmomat 030; Gonotec)). Group B6 oocytes were treated with 0.25% NaCl (375 mOsmol) to allow comparisons with the previous experiment. Oocytes in Group B1 were incubated in MM2 (294 mOsmol) for 1 h and served as the control group. After treatment, oocytes were left to recover for 1 h in MM1. Subsequently, oocytes from the two sets of experiments were

fixed in 4% (v/v) paraformaldehyde and immunostained to examine meiotic spindle status. Experiment 2: cryotolerance IVM bovine oocytes exposed to NaCl or sucrose before vitrification After 21 h maturation in vitro, sets of 253 and 370 oocytes were randomly distributed into three groups (C1–C3 and D1– D3, respectively). Oocytes in C1 and D1 were incubated in MM2 for 2 h and served as the control groups. According to the results of Experiment 1, oocytes in Groups C2, C3, D2 and D3 were pretreated with 0% or 0.25% NaCl (375 mOsmol; Groups C2 and C3, respectively) or 0% or 2.77% sucrose (375 mOsmol; Groups D2 and D3, respectively) for 1 h, left to recover for 1 h in MM2 and then vitrified and warmed by the Cryotop method. After 1 h recovery in MM1, all oocytes were fixed in 4% (v/v) paraformaldehyde and immunostained to examine meiotic spindle status.

Osmotic stress on oocyte cryotolerance

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Table 1. Effects of previtrification treatment with increasing NaCl concentrations on the spindle configuration of in vitro-matured bovine oocytes Data are the mean  s.e.m. Within columns, values with different letters differ significantly (P , 0.05). A1, control oocytes; A2–A8, oocytes exposed to 0.25%, 0.5%, 1%, 1.5%, 2%, 3% and 4% NaCl, respectively Treatment group

n

Microtubule morphologyA (%)

Total MII (%) Normal

A1 A2 A3 A4 A5 A6 A7 A8 A

29 40 33 41 43 42 38 41

97.7  2.3 100  0.0 100  0.0 90.9  9.1 95.5  4.6 85.3  6.9 88.8  6.6 88.1  9.1

85.0  9.6 83.4  0.0ab 52.6  0.1bc 51.8  8.1bc 40.3  4.2cd 16.5  7.0de 15.5  3.6de 6.8  6.8e a

Chromosome morphologyA (%)

Dispersed

Decondensed or absent

Normal

12.5  9.5 13.5  5.6 39.3  6.0 22.5  5.3 24.8  7.0 24.2  2.8 28.4  5.8 18.7  8.4

2.5  2.5 3.1  3.1a 9.1  9.1a 25.7  6.1ab 34.9  9.7ab 59.4  6.0bc 56.2  5.3bc 74.5  12.5c

85.0  9.6 83.4  8.1a 47.2  7.0b 46.0  8.7b 37.5  4.2bc 16.5  7.0bc 9.6  3.5c 6.8  6.8c

a

a

Dispersed

Decondensed or absent

12.5  9.5 13.5  5.6 39.3  6.0 22.5  5.3 24.8  7.0 24.2  2.8 28.4  5.8 18.7  8.4

2.5  2.5a 3.1  3.1a 9.1  9.1a 25.7  6.1ab 34.9  9.7ab 59.4  6.0bc 56.2  5.3bc 74.5  12.5c

Values show the number of oocytes with given morphology as a percentage of the total number of oocytes reaching the MII stage.

Table 2. Effects of previtrification treatment with increasing sucrose concentrations on the spindle configuration of in vitro-matured bovine oocytes Data are the mean  s.e.m. Within columns, values with different letters differ significantly (P , 0.05). B1, control oocytes; B2–B5, oocytes exposed to 2.77%, 5.10%, 10.54% and 15.57% sucrose, respectively; B6, oocytes exposed to 0.25% NaCl Treatment group

n

Microtubule morphologyA (%)

Total MII (%) Normal

B1 B2 B3 B4 B5 B6 A

70 61 72 78 59 52

83.2  3.4 87.7  4.4 88.1  3.8 83.7  6.4 76.1  6.5 93.3  4.1

87.0  4.7 71.8  5.0ab 63.0  4.6ab 57.2  7.1b 31.5  6.8c 75.6  2.0ab a

Chromosome morphologyA (%)

Dispersed

Decondensed or absent

Normal

8.3  2.3 23.3  4.2ab 26.0  5.6ab 26.0  6.6ab 38.4  4.0b 20.9  0.6ab

4.7  2.9 4.9  2.2a 11.0  2.8ab 16.8  6.9ab 30.2  6.8b 3.5  2.0a

87.0  4.7 69.1  4.6ab 58.7  3.4b 55.1  7.0b 30.0  6.3c 73.9  3.5ab

a

a

a

Dispersed

Decondensed or absent

9.4  2.8 22.7  6.2ab 30.3  2.9ab 31.5  6.6ab 50.1  3.2b 22.7  2.0ab

3.6  2.5a 8.2  2.8a 11.0  2.8ab 13.5  6.4ab 19.9  4.6b 3.5  2.0a

a

Values show the number of oocytes with given morphology as a percentage of the total number of oocytes reaching the MII stage.

Experiment 3: effects of sucrose pretreatment on the developmental competence of vitrified– warmed bovine oocytes In this experiment, 747 oocytes were randomly allocated to one of four groups: E1, comprised of oocytes matured in vitro for 24 h; E2, oocytes exposed to 2.77% (375 mOsmol) sucrose for 1 h and left to recover for 1 h in MM2; E3, oocytes vitrified– warmed by the Cryotop method; and E4, oocytes exposed to 2.77% (375 mOsmol) sucrose for 1 h, left to recover for 1 h in MM2 and vitrified–warmed by the Cryotop method. Oocytes in Groups E2, E3 and E4 were allowed to mature for an additional 1 h in MM1 after treatment. After maturation, oocytes were fertilised and, at 22 hpi, presumptive zygotes were placed in culture drops. Cleavage rates were determined at 48 hpi and blastocyst rates recorded on Day 7. Results Experiment 1 Effects of transient exposure of IVM bovine oocytes to increasing NaCl concentrations on meiotic spindle status Table 1 details the effects of NaCl pretreatment on meiotic spindle configurations. As the osmolality of the NaCl solution increased, the percentage of oocytes with an abnormal spindle configuration also increased significantly. Only half the oocytes

exposed to 0.5% NaCl (433 mOsmol; Group A3) exhibited a normal spindle configuration. However, no significant differences in terms of microtubule distributions or chromosome configurations were observed in oocytes treated with 0.25% NaCl (Group A2) compared with untreated control oocytes (Group A1). Effects of transient exposure of IVM bovine oocytes to increasing sucrose concentrations on meiotic spindle status Table 2 details the effects of sucrose pretreatment on meiotic spindle configurations. No significant differences in microtubule configuration were observed when comparing oocytes in the control group (Group B1) and those exposed to 2.77% (Group B2) or 5.10% (Group B3) sucrose. However, only oocytes exposed to 2.77% sucrose (Group B2) had a similar normal spindle configuration rate to that found for non-exposed oocytes (Group B1). As seen with the NaCl treatment, increasing sucrose concentrations resulted in an increasing proportion of oocytes with decondensed or absent chromosomes. Oocytes exposed to 0.25% NaCl (Group B6) showed similar rates of normal spindles to those seen in untreated oocytes (Group B1). No significant differences were observed between oocytes pretreated with 0.25% NaCl (Group B6) and 2.77% sucrose (Group B2).

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Table 3. Cryotolerance of in vitro-matured bovine oocytes subjected to NaCl treatment before vitrification Data are the mean  s.e.m. Within columns, values with different letters differ significantly (P , 0.05). C1, in vitro-matured; C2, in vitro-matured oocytes vitrified–warmed by the Cryotop method; C3, in vitro-matured oocytes exposed to 0.25% NaCl for 1 h and then vitrified–warmed by the Cryotop method Treatment group

n

Total MII (%)

86 87 80

98.8  0.9 63.2  9.6b 71.3  3.6b

Microtubule morphologyA (%) Normal

C1 C2 C3

a

79.1  5.9 40.2  3.7b 41.9  4.3b a

Chromosome morphologyA (%)

Dispersed

Decondensed or absent

Normal

18.7  4.4 26.0  4.6 28.4  8.5

2.2  2.2 33.8  9.4b 29.7  4.2b

73.7  9.7 35.7  13.2b 41.9  4.3ab

a

a

Dispersed

Decondensed

25.2  8.7 42.6  4.9 38.7  3.8

1.1  1.1a 21.7  8.7b 19.4  2.2b

A

Values show the number of oocytes with given morphology as a percentage of the total number of oocytes reaching the MII stage.

Table 4. Cryotolerance of in vitro-matured bovine oocytes subjected to sucrose treatment before vitrification Data are the mean  s.e.m. Within columns, values with different letters differ significantly (P , 0.05). D1, in vitro-matured oocytes; D2, in vitro-matured oocytes vitrified–warmed by the Cryotop method; D3, in vitro-matured oocytes exposed to 2.77% sucrose for 1 h and then vitrified–warmed by the Cryotop method Treatment group

n

Total MII (%)

93 121 140

100  0.0 61.1  1.3b 61.3  1.3b

Microtubule morphologyA (%) Normal

D1 D2 D3

a

81.7  1.1 24.4  0.9b 32.9  1.3b a

Chromosome morphologyA (%)

Dispersed

Decondensed or absent

Normal

12.9  1.1 33.9  1.1b 38.2  1.0c

5.4  0.4 41.8  1.0b 28.9  0.8c

81.7  1.1 23.3  0.9b 34.2  1.2c

a

a

a

Dispersed

Decondensed or absent

12.9  1.1 40.5  0.6b 42.7  1.1c

5.4  0.4a 36.2  0.7b 23.1  0.7c

a

A

Values show the number of oocytes with given morphology as a percentage of the total number of oocytes reaching the MII stage.

Experiment 2 Cryotolerance of IVM bovine oocytes exposed to NaCl or sucrose before vitrification Table 3 compares the effects of vitrifying oocytes with or without NaCl (0.25%) pretreatment on meiotic spindle status. Significantly higher rates of oocytes exhibiting an MII spindle were recorded in the control group (untreated, non-vitrified oocytes) compared with the remaining vitrified oocyte groups. Pretreatment with NaCl before vitrification induced a similar normal microtubule morphology rate to that observed after vitrification, although both rates were significantly lower than that observed for fresh control oocytes, mainly due to a greater proportion of oocytes with decondensed or absent microtubules in these two groups (C2 and C3). No significant differences were observed among treatment groups when percentages of dispersed microtubules were examined. A significantly lower percentage of vitrified oocytes exhibited normal chromosomal morphology compared with nonvitrified oocytes. However, a similar percentage of oocytes exposed to 0.25% NaCl before vitrification–warming had normal chromosomal morphology to that in the non-vitrified group. Cryotolerance of IVM bovine oocytes exposed to sucrose treatment prior vitrification As indicated in Table 4, the vitrified oocyte groups had a significantly lower percentage of MII stage oocytes compared with the control non-vitrified oocytes. Regardless of the pretreatment, significantly lower normal microtubule configuration rates were observed comparing vitrified with control non-vitrified oocytes. Similarly, significantly lower normal chromosome configuration rates were seen in vitrified compared with control fresh

Table 5. Developmental competence of in vitro-matured bovine oocytes subjected to sucrose treatment before vitrification Data are the mean  s.e.m. E1: Control (non-vitrified), oocytes matured in vitro for 24 h; Control (non-vitrified), oocytes matured in vitro for 24 h; E2, in vitro-matured oocytes exposed to 2.77% sucrose for 1 h; E3, in vitromatured oocytes vitrified–warmed by the Cryotop method; E4, in vitromatured oocytes exposed to 2.77% sucrose for 1 h and then vitrified–warmed by the Cryotop method Treatment group E1 E2 E3 E4

n

Cleavage (%)

Blastocysts on Day 7 (%)

193 169 184 201

63.7  5.8 75.2  3.4a 27.7  2.4b 33.3  3.8b

18.1  2.4a 19.5  3.1a 1.1  0.9b 1.5  0.7b

a

oocytes, and a higher percentage of oocytes with dispersed, decondensed or absent chromosomes in the cytoplasm were identified in the vitrified groups. However, the percentage of oocytes with normal chromosome configurations was significantly higher when oocytes were exposed to 375 mOsmol sucrose before vitrification compared with control vitrified oocytes, mainly due to a lower proportion of decondensed or absent chromosomes. Similarly, the proportion of decondensed or absent microtubules decreased significantly after sucrose treatment. Experiment 3 Effects of sucrose pretreatment on the developmental competence of vitrified–warmed bovine oocytes As indicated in Table 5, significantly higher cleavage and blastocyst rates were recorded in the non-vitrified oocyte than vitrified oocyte groups, regardless of sucrose treatment.

Osmotic stress on oocyte cryotolerance

Discussion The aim of the present study was to examine whether pretreatment of IVM cow oocytes with elevated NaCl or sucrose concentrations would improve their cryotolerance to vitrification, in addition to their in vitro developmental competence. To define a suitable sublethal stress treatment, in an initial experiment we examined the effects of solutions with different osmolality on MII spindle structure. The cytotoxicity tests revealed a significantly higher percentage of oocytes with abnormal spindle structures, including disorganised or decondensed microtubules or chromosomes, after exposure of oocytes to the NaCl or sucrose solutions. These effects were dose dependent, because normal spindle rates decreased as the osmolality of the pretreatment solution increased. Consistent with this finding, Mullen et al. (2004) reported that osmolality was a significant predictor of the spindle morphology of human oocytes. Hyperosmotic effects of sucrose solutions at 600, 1200, and 2400 mOsmol resulted in 44%, 44%, and 100% of spindles with an abnormal structure, respectively (Mullen et al. 2004). In effect, cow oocytes have been observed to behave as a perfect osmometer (i.e. they follow the Boyle–Van’t Hoff relationship) over the range 265–800 mOsmol (Ruffing et al. 1993). Despite this, Lin et al. (2009a, 2009b) provided strong evidence that pig oocytes osmotically stressed by a transient increase in NaCl, sucrose or trehalose concentrations, had higher developmental rates following cryopreservation, parthenogenic activation or somatic cell nuclear transfer. Nevertheless, osmotic stress has been observed to be harmful to oocytes in several studies, even when similar osmotic pressures were applied for only 10 min (Agca et al. 2000; Mullen et al. 2004, 2007). In the present study, 0.25% NaCl (375 mOsmol) had no substantial effects on spindle morphology, yet higher concentrations led to a marked reduction in the number of oocytes showing normal spindle configurations. Similarly, we observed no adverse effects of 2.77% sucrose (375 mOsmol) and selected this concentration for the pretreatment of IVM oocytes before vitrification. Because the type of initial stress and its intensity are determinants of the expression of stress-related proteins that will help combat subsequent stress, this initial stress can also compromise oocyte viability (Toner et al. 1993). The difference in the percentage of oocytes with normal microtubule distributions in groups exposed to 375 and 433 mOsmol sucrose solutions (71.78% vs 62.96%, respectively) was not as great as the difference between groups exposed to 375 and 433 mOsmol NaCl solutions (83.35% vs 52.60%, respectively). This could indicate that elevated intracellular NaCl concentrations may have both osmotic effects and other consequences. In Experiment 2, the effects of exposure to NaCl or sucrose before vitrification on spindle status were examined. According to many authors, vitrification–warming procedures lead to a reduction in the number of oocytes at MII (Rojas et al. 2004; Albarracı´n et al. 2005; Succu et al. 2007; Morato´ et al. 2008a, 2008b, 2008c). Similarly, we detected a lower proportion of oocytes at the MII stage after NaCl or sucrose treatment prior to vitrification compared with the control group (non-vitrified oocytes). Moreover, there was a greater number of oocytes at

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the anaphase II stage after vitrification, regardless of whether they had been subjected to osmotic pretreatment. Some permeable cryoprotectants used in vitrification solutions, such as DMSO and EG, could parthenogenetically activate IVM oocytes from the sheep (Tian et al. 2007), pig (Somfai et al. 2007) or buffalo (Attanasio et al. 2010). Whether the osmotic stress caused by cryoprotectants contributes to this effect it is not known (Lin et al. 2009a). According to Lin et al. (2009a), is not known wether the osmotic stress induced by NaCl causes the same effects as CPAs. These authors speculated that parthenogenic activation could be the reason for the improved cryotolerance and developmental competence of pig oocytes treated with selected NaCl concentrations, then vitrified and parthenogenetically activated (Lin et al. 2009a). In the present cytotoxicity tests, no significant differences were detected in the number of oocytes at the MII and anaphase II stages between groups exposed to NaCl or sucrose solutions with different osmolalities. However, in Experiment 2, differences in the percentage of oocytes at the MII stage emerged between control and vitrified oocytes, and the vitrified group had a greater percentage of oocytes at anaphase II. This indicates that osmotic stress induced by NaCl or sucrose does not seem to cause parthenogenetic activation in IVM cow oocytes. Larman et al. (2007b) and Gook et al. (1995) reported that in addition to the vitrification solution, cooling shock can induce spontaneous activation in matured oocytes. Succu et al. (2007) showed that a large proportion of vitrified oocytes had spindle abnormalities when they were assessed immediately after warming. For this reason, we included a 60-min recovery period after warming before fixation to allow for spindle re-assembly. This postwarming interval should be sufficient for tubulin polymerisation and to restore microtubule structure. The oocyte can then resume meiosis and signal the spindles to reorganise themselves into anaphase II (Asgari et al. 2011). Similarly, after high hydrostatic pressure treatment, some authors have detected greater improvements in oocyte competence when they are left to recover for 1 or 2 h before vitrification (Du et al. 2008a, 2008b). However, results have been contradictory. According to Larman et al. (2007a) there is no reason to wait for microtubule recovery, especially if vitrification is performed at 378C. In the present study, we observed no significant effects of NaCl or sucrose pretreatment on normal microtubule configuration rates in vitrified oocytes. Nor were there any effects of NaCl on the rate of detection of normal chromosome configurations. In contrast, the rate of normally organised chromosomes was higher in oocytes treated with sucrose before than in oocytes vitrified without sucrose pretreatment. Therefore, it seems that pretreatment with 375 mOsmol NaCl or sucrose for 1 h before vitrification offers some protection against chromosome decondensation, one of the negative effects of vitrification. Exposure to 375 mOsmol sucrose solution for 1 h had no deleterious effects on cleavage or development to the blastocyst stage of IVM bovine oocytes. However, cryosurvival or developmental competence were not improved when 375 mOsmol sucrose solution was used before vitrification, in contrast with observations in pig oocytes (Lin et al. 2009b). It has been reported that precisely controlled sublethal stress treatment of cells can improve embryo and gamete performance, and

H

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consequently enhance subsequent oocyte survival, fertilisation and in vitro development (Pribenszky et al. 2012). The stress response triggers the induction of heat shock proteins (HSPs), first detected after heat stress, although later found to be involved in responses to other environmental stressors, including cold temperature, osmotic effect of salts, acid change in pH, high hydrostatic pressure and starvation (Pribenszky et al. 2010). HSPs participate in various cell functions, including protein, DNA, and chromatin stabilisation and repair, cell cycle control, redox regulation, energy metabolism, fatty acid/lipid metabolism and the elimination of damaged proteins (Csermely et al. 1998; Kultz 2003). The production of HSPs may explain the improved survival after vitrification and developmental competence of pig oocytes after osmotic stress induced by NaCl or sucrose solutions (Lin et al. 2009b). In matured cow oocytes, HSP70 (a stress response HSP; Geraci et al. 2006) has been described as acting as a molecular chaperone, helping nascent polypeptides to form properly. HSP70 also acts as a microtubule-associated protein, interacting with polymerised tubulin through a region containing a sequence related to tubulinbinding motifs (Kawarsky and King 2001). According to the structure and composition of cow oocytes, the solution we used to pretreat bovine oocytes had a lower osmolality than that used to pretreat pig oocytes, and the osmolality of this solution was perhaps insufficient to induce the production of adequate amounts of HSP to protect the oocyte from cryodamage. Thus, it could be speculated that to induce greater stress, bovine oocytes could be pretreated with higher concentrations of NaCl or sucrose but over a shorter period than 1 h. In line with previous observations, the greater the stress (below the lethal dose), the more HSPs are produced and the higher the cryosurvival (Du et al. 2008a). In the present study, we did not observe the improved developmental competence reported by Lin et al. (2009a, 2009b) in pig oocytes. A possible explanation for this could be the different amounts, composition and distribution of lipids (Silva et al. 2011). Membrane structure also differs between the two species. Thus, pig oocytes have double the total fatty acid reserve than cow oocytes, and phospholipids contain a higher proportion of polyunsaturated fatty acids than ruminant oocyte phospholipids (McEvoy et al. 2000). These difference could have marked effects on the nature of membranes and may contribute to the different responses of oocytes from the two species to cryopreservation (Leibo 2008). Cell membrane composition determines the properties of a cell. Cells with more flexible membranes are more permeable to water and intracellular cryoprotectants, and will incur less damage during cryopreservation (Seidel 2006). Ultrastructural differences between IVM pig and cow oocytes may explain their different developmental competence after treatment with NaCl or sucrose before vitrification. Because of their large intracytoplasmic lipid droplets and other specific properties that hinder successful cryopreservation (Zhou et al. 2010), pig oocytes may benefit from osmotic stress because it permits pre-adaptation of the cell to the subsequent stress of vitrification–warming (Pribenszky et al. 2010). In conclusion, treatment with 375 mOsmol NaCl or sucrose solution for 1 h before vitrification had no detrimental effects on

N. Arcarons et al.

the meiotic spindle status of IVM bovine oocytes. In particular, sucrose pretreatment was unable to improve embryo development as observed in other species. There is a clear need for further work designed to improve the cryotolerance of bovine oocytes. Acknowledgements This study was supported by the Spanish Ministry of Science and Innovation (Project AGL2013–46769) and Generalitat de Catalunya (Project No. 2014 SGR 547).

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