Article Elevated NaCl concentration improves cryotolerance and developmental competence of porcine oocytes

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1 RBMOnline - Vol 18. No Reproductive BioMedicine Online; on web 16 January 2009 Article Elevated NaCl concentration improves cryotolerance and developmental competence of porcine oocytes Lin Lin studied Life Science in the Honours Programme at the China Agricultural University and received her Bachelor s degree in She then studied cancer genetics at the Beijing Genomic Institute Chinese Academy of Science. She is now a PhD student in Aarhus University Denmark. Her research interests focus on the effects of physicochemical stress on developmental competence and tolerance to vitrification of embryos. Ms Lin Lin Lin Lin 1347 Yutao Du 134 Ying Liu 1 Peter M Kragh 13 Juan Li 1 Stig Purup 2 Masashige Kuwayama 5 Xiuqing Zhang 4 Huanming Yang 4 Lars Bolund 3 Gábor Vajta 6 1 Population Genetics and Embryology Institute of Genetics and Biotechnology; 2 Nutrition and Production Physiology Institute of Animal Health Welfare and Nutrition Faculty of Agricultural Sciences University of Aarhus DK-8830 Tjele; 3 Institute of Human Genetics University of Aarhus DK-8000 Aarhus Denmark; 4 Beijing Genomics Institute Airport- Industrial zone B-6 Beijing China; 5 Kato Ladies Clinic Nishishinjuku Shinjuku Tokyo Japan; 6 PIVET Medical Centre Cambridge Street Leederville Perth WA 6007 Australia 7 Correspondence: Lin.Lin@agrsci.dk Abstract High hydrostatic pressure has been reported to improve the fertilizing or developmental ability of mammalian spermatozoa oocytes and embryos. This study investigated the effect of another stress temporarily increased NaCl concentration on cryotolerance and developmental competence of porcine oocytes. In Experiment 1 survival rates were compared after 1 h exposure to seven elevated NaCl concentrations and 1 h recovery time. In Experiment 2 oocytes were exposed to 593 and 1306 mosmol NaCl subsequently recovered vitrified then subjected to parthenogenetic activation. Both cleavage and blastocyst rates increased after NaCl treatment compared with untreated controls. In Experiment 3 oocytes were treated with 593 mosmol NaCl followed by 1 and 2 h recovery respectively then used as recipients for somatic cell nuclear transfer (SCNT). Cleavage rates were not different from those in untreated controls but blastocyst rates increased in both NaCltreated groups. In conclusion treatment of porcine oocytes with elevated NaCl concentrations improved their developmental competence after vitrification and parthenogenetic activation or SCNT. Further experiments are required to investigate invivo consequences and the effect on gametes and embryos of different mammalian species. Keywords: handmade cloning NaCl parthenogenetic activation somatic cell nuclear transfer vitrification Introduction 360 Owing to the similarities in size anatomy physiology nutrition metabolism and genetics the domestic pig (Sus scrofa) is regarded as one of the most valuable mammals for modeling human diseases (Schook et al. 2005; Vodicka et al. 2005; Lunney 2007). Additionally pigs have short reproductive cycles large litters and a relatively long lifespan; their breeding is inexpensive and based on 9000 years experience (Giuffra et al. 2000). On the other hand production of transgenic pigs is still a demanding challenge owing to the sensitivity of porcine oocytes and embryos to various in-vitro procedures including in-vitro embryo production cryopreservation and somatic cell nuclear transfer (SCNT). Recently considerable advances have been achieved in porcine SCNT thanks to new techniques applied for maturation (Wu et al. 2006; Schoevers et al. 2007; Song and Lee 2007) activation (Lee et al. 2004; Kragh et al. 2005) modification of zygote culture medium (Suzuki and Yoshioka 2006) application of zona free techniques for enucleation (Kragh et al. 2004; Li et al. 2006) fusion and embryo culture (Du et al. 2005; Lagutina et al. 2006) Published by Reproductive Healthcare Ltd Duck End Farm Dry Drayton Cambridge CB23 8DB UK

2 Cryopreservation of oocytes is a demanding challenge in all mammalian species. Pig oocytes are even more sensitive for various reasons including extreme sensitivity to chilling injuries due to the high cytoplasmic lipid content. Removal of the lipid by centrifugation and micromanipulation (Nagashima et al. 1995; Hara et al. 2005) or recently with a simplified procedure including partial zona digestion before centrifugation (Du et al. 2007) has resulted in improved survival after both traditional freezing and vitrification. A new approach to improve the survival fertilizing ability or developmental competence of mammalian gametes and embryos is to expose them to a severe sublethal shock high hydrostatic pressure (HHP). The positive effect of HHP pretreatment has been reported to increase cryosurvival fertilizing ability and/or developmental competence of spermatozoa oocytes and embryos of bovine porcine and murine species (Pribenszky et al. 2005abc 2006; Du et al. 2008ab). The purpose of this study was to investigate if a simple treatment with medium containing elevated NaCl concentrations increases the cryotolerance and developmental competence of in-vitro matured porcine oocytes after vitrification parthenogenetic activation or handmade cloning (HMC; Vajta et al. 2001; Kragh et al. 2004). In the first experiment morphological survival was tested after incubation with media containing various concentrations of NaCl. In the second experiment oocytes were treated with selected NaCl concentrations then vitrified parthenogenetically activated and cultured to the blastocyst stage. In the third experiment the most efficient concentration of NaCl was used with two different recovery periods after the treatment then oocytes were enucleated and used for HMC as recipients and in-vitro developmental rates were compared. Materials and methods Except where otherwise indicated all chemicals were obtained from Sigma Chemical Co. (St. Louis MO USA); all manipulations were performed on a heated stage adjusted to 39 C; and all drops used for handling oocytes were 20 μl covered with mineral oil. Oocyte collection and in-vitro maturation Cumulus oocyte complexes (COC) were aspirated from 2 6 mm follicles from slaughterhouse-derived sow ovaries and matured in groups of 50 in 400 μl in-vitro maturation (IVM) medium consisting of bicarbonate-buffered TCM- 199 (GIBCO BRL USA) supplemented with 10% (v/v) cattle serum (CS) 10% (v/v) pig follicular fluid 10 IU/ml equine chorionic gonadotrophin 5 IU/ml human chorionic gonadotrophin (Suigonan Vet; Skovlunde Denmark) in the Submarine Incubation System (SIS; Vajta et al. 1997a) for h. NaCl treatment Denuded oocytes (Experiment 1) or COC (Experiments 2 and 3) were put into 800 μl T2 (HEPES-buffered TCM- 199 containing 2% (v/v) cattle serum) supplemented with additional NaCl concentrations (see details later) in 4-well dishes and incubated for 1 h at 38.5 C in air. COC incubated in T2 without NaCl supplementation under the same conditions were used as controls. Subsequently oocytes or COC were placed in IVM medium for 1 or 2 h at 38.5 C in 5% CO 2 with maximum humidity for recovery. After recovery survival rates were checked in Experiment 1 while cumulus cells were removed with 1 mg/ml hyaluronidase in Experiments 2 and 3 and oocytes were subjected to additional treatments (see below). Vitrification of oocytes with Cryotop Cryopreservation was carried out by vitrification with Cryotop device and factory-prepared vitrification and warming solutions (Kitazato Supply Co. Fujinomiya Japan) as described previously (Kuwayama et al. 2005a b). Oocytes were transferred into equilibration solution (ES) consisting of 7.5% ethylene glycol (EG) 7.5% dimethyl sulphoxide (DMSO) and 20% synthetic serum substitute (SSS; Cat. No Irvine Scientific Santa Ana California USA) in T0 at 39 C for 5 15 min. Ten to fifteen oocytes were then transferred into a 20 μl drop of vitrification solution (VS) consisting of 15% EG 15% DMSO 0.5 M sucrose and 20% SSS dissolved in T0. After incubation for s oocytes were loaded on Cryotop and plunged into liquid nitrogen. The process from exposure in VS to plunging was completed within 1 min. Vitrified oocytes were warmed by immersing Cryotop directly into 39 C warming solution (1.0 M sucrose dissolved in T0 and 20% SSS) for 1 min then transferred to dilution solution (0.5 M sucrose dissolved in T0 and 20% SSS) for 3 min. Subsequently oocytes were incubated twice for 5 min in washing solutions (T0 supplemented with 20% SSS). Parthenogenetic activation and embryo culture Vitrified-warmed oocytes were equilibrated for s in drops of activation medium (0.3 M mannitol 0.1 mm MgSO 4 0.1mM CaCl 2 and 0.01% polyvinyl alcohol). Under a 0.12 kv/cm alternative current (AC) oocytes were aligned to the wire of a fusion chamber (Microslide 0.5 mm fusion chamber model 450; BTX San Diego CA USA). Then a single direct current (DC) pulse (1.26 kv/cm 80 µs) was applied to the oocytes for electrical activation. During this whole procedure including equilibration the oocytes were exposed to activation medium for s. After washing three times in drops of T10 activated oocytes were incubated in culture medium (PZM-3 medium supplemented with 4 mg/ml bovine serum albumin; Yoshioka et al. 2002) 5 mg/ml cytochalasin B and 10 mg/ml cycloheximide at 38.5 C in 5% CO 2 5% O 2 and 90% N 2 with maximum humidity. After 4 h treatment putative parthenogenetically activated embryos were washed three times in culture medium before culture. Embryos were then cultured in groups of in wells of 4-well dishes (Nunc Skovlunde Denmark) in 400 µl culture medium covered with 400 µl mineral oil at 38.5 C in 5% O 2 5% CO 2 and 90% N 2 with maximum humidity. Blastocyst rates were determined under a stereomicroscope on day

3 362 Handmade cloning and embryo culture Zonae pellucidae of oocytes were partially digested with 3.3 mg/ml pronase solution dissolved in T33 for 20 s then washed quickly in T2 and T20 drops. Oocytes with distended and softened zonae pellucidae were lined up in T2 drops supplemented with 2.5 μg/ml cytochalasin B. With a finely drawn and fire-polished glass pipette oocytes were rotated to locate the polar body. Oriented bisection was performed manually with ultra-sharp splitting blades (AB Technology Pullman WA USA) under a stereomicroscope. Less than half of the cytoplasm close to the polar body was removed from the remaining putative cytoplast. Suspensions of porcine fetal fibroblast cells were obtained by trypsin digestion of monolayers as described previously (Kragh et al. 2005). The cells were allowed to settle in a 20-μl drop of T2. Fusion was performed in two steps where the second one included the initiation of activation as well. For the first step 50% of the available cytoplasts were transferred into 1 mg/ml of phytohaemagglutinin (PHA; ICN Pharmaceuticals Girraween Australia) dissolved in T0 for 3 s and then each one was quickly dropped over a single fibroblast cell. After attachment cytoplast-fibroblast pairs were equilibrated in fusion medium (0.3 M mannitol and 0.01% polyvinyl alcohol; PVA) for 10 s and transferred to a fusion chamber (BTX microslide 0.5 mm fusion chamber model 450; BTX San Diego CA USA). Using AC of 0.06 kv/cm and 700 khz pairs were aligned to the wire of the fusion chamber with the somatic cells farthest from the wire then fused with a DC pulse of 2.0 kv/cm for 9 μs. After the DC pulse pairs were removed carefully from the wire transferred to T10 drops and incubated further to observe whether fusion had occurred. Approximately 1 h after the first fusion each pair was fused with another cytoplast in activation medium (0.3 M mannitol 0.1 mm MgSO mm CaCl 2 and 0.1% PVA). By using an AC of 0.06 kv/cm and 700 khz one fused pair and one cytoplast were aligned to one wire of the fusion chamber with fused pairs contacting the wire. A single DC pulse of 0.86 kv/ cm was applied for 80 μs. When fusion had been observed in T10 drops reconstructed embryos were transferred into PZM- 3 supplemented with 5 μg/ml cytochalasin B and 10 μg/ml cycloheximide. After a 4 h incubation at 38.5 C in 5% CO 2 5% O 2 and 90% N 2 with maximum humidity embryos were washed three times and cultured under conditions described earlier but in addition by using the well of the well system (Vajta et al. 2000). Blastocyst rates were determined under a stereomicroscope on day 6 after activation. Cell number counting Blastocysts were individually fixed and mounted on a glass microscopic slide in glycerol supplemented with 20 μg/ml Hoechst fluorochrome. After staining for 24 h embryos were observed under a Diaphot 200 inverted microscope with epifluorescent attachment and UV-2A filter (Nikon Tokyo Japan). Statistical analysis Statistical analysis was performed using Statistics Package for Social Sciences (SPSS) 13.0 (SPSS Chicago USA). A t-test was performed to analyse differences in survival rates after treatment cleavage rates blastocyst rates and cell numbers. A probability of P < 0.05 was considered to be statistically significant. Experimental design Experiment 1 In four replicates a total of 534 in-vitro matured and denuded metaphase II (MII)-stage oocytes were distributed into eight groups. A2 A8 were put into T2 supplemented with additional 0.25%; 0.5%; 1%; 1.5%; 2%; 3% and 4% (w/v) NaCl for 1 h with osmotic level mosmol respectively (measured with Osmomat 030 Gonotec). Oocytes in group A1 were incubated in T2 (288 mosmol) for 1 h and served as control. After 1 h recovery in IVM medium morphological survival rates of oocytes were checked under a stereomicroscope. Experiment 2 A total of 1000 oocytes were used for osmotic treatment and control vitrification and parthenogenetic activation in five replicates. After h maturation COC were divided into three groups (groups B1 B3). COC of group B2 and B3 were put into T2 with additional 1% and 4% NaCl for 1 h respectively. COC of group B1 were put into T2 for 1 h and served as control. After 1 h recovery in IVM medium these MII-stage oocytes were denuded vitrified and parthenogenetically activated. Cleavage rates of activated oocytes were checked at 36 h and blastocyst rates were checked on day 7. Experiment 3 In four replicates a total of 800 in-vitro maturated COC were divided into three groups (C1 C3). Group C2 and C3 COC were put into T2 with additional 1% NaCl for 1 h. COC of Group C1 were incubated in T2 medium for 1 h and served as control. After 1 or 2 h recovery (C2 and C3 respectively) in IVM medium these MII-stage oocytes were denuded and HMC was performed. Cleavage rates were checked at 24 h and blastocyst rates were checked on day 6. Then the day 6 blastocysts were stained with Hoechst and cell numbers were determined as described above. Results Experiment 1 As shown in Table 1 survival rates based on intact morphology were close to 100% and identical to the control (288 mosmol) in groups treated with low ( mosmol) and high ( mosmol) elevated NaCl concentrations. In contrast markedly decreased survival levels were observed after treatment with medium-level NaCl concentration (52 and 46% for 710 and 860 mosmol respectively). It also demonstrates the lack of linear correlation between the concentration of NaCl and the severity of the morphological injury.

4 Experiment 2 NaCl treatment was performed according to best survival rates in Experiment 1 (Table 2). After thawing 100% of oocytes were intact in all of these three groups. Cleavage rates after vitrification and parthenogenetic activation were higher in NaCl treated groups than in controls. The highest blastocyst rate was obtained after treatment with the 593 mosmol solution while the 1306 mosmol treatment resulted in compromised development (16 and 5% respectively). No blastocyst was obtained in the control group. Experiment 3 No difference in cleavage rates compared with controls was observed after NaCl treatment regardless of the 1 versus 2 h recovery period. (Table 3) Blastocyst rates were lower in the control group than in both treated groups (45 versus 64 and Table 1. Survival rate of porcine oocytes after osmotic treatment at eight different concentrations of NaCl (Experiment 1). Parameter Treatment group A1 (control) A2 A3 A4 A5 A6 A7 A8 Additional concentration of NaCl (%) Osmotic pressure (mosmol) Number of treated oocytes Mean percent survival 100 ± 0 a 98 ± 2 a 100 ± 0 a 99 ± 1 a 52 ± 6 b 46 ± 7 b 99 ± 1 a 98 ± 2 a after treatment ab Values with different superscript letters are statistically significantly different (P < 0.05). Table 2. Developmental competence after vitrification of oocytes following osmotic treatment and parthenogenetic activation (Experiment 2). Parameter Treatment group B1 (control) B2 B3 Additional concentration of NaCl (%) Osmotic pressure (mosmol) Number of vitrified oocytes Percentage of intact oocytes after vitrification Number of activated oocytes Cleavage rate at 36 h d 8 ± 3 a 34 ± 8 b 20 ± 3 b Blastocyst rate on day 7 d 0 ± 0 a 16 ± 4 b 5 ± 1 c abc Values within rows with different superscript letters are statistically significantly different (P < 0.05). d Values are mean percentage ± SEM of number of oocytes vitrified. Table 3. Developmental competence of porcine handmade cloning embryos derived from osmotically treated oocytes with different recovery times. Parameter Treatment group C1 (control) C2 C3 Additional concentration of NaCl (%) Osmotic pressure (mosmol) Recovery time after osmotic treatment (h) Number of reconstructed embryos Cleavage rate at 24 h c 89 ± 5 a 95 ± 3 a 92 ± 4 a Blastocyst rate on day 6 c 45 ± 2 a 64 ± 4 b 65 ± 4 b Mean cell number of day 6 blastocyst 63 ± 4 a 62 ± 3 a 50 ± 3 b ab Values within rows with different superscript letters are statistically significantly different (P < 0.05). c Values are mean percentage ± SEM of number of oocytes vitrified. 363

5 364 65% respectively) while the cell numbers of blastocysts was higher in controls and after 1 h recovery than after 2 h (63 versus 62 and 50 respectively). Discussion Porcine oocytes and embryos are generally regarded as very sensitive to in-vitro manipulations including parthenogenetic activation SCNT and cryopreservation. The causes of this sensitivity are only partially understood. One important factor seems to be the high amount of lipid droplets in their cytoplasm. The function of these lipid droplets is unclear and it has been proven that their removal by high speed centrifugation accompanied in some methods with micromanipulation (Nagashima et al ) does not impair full-term developmental competence. However these lipid droplets are extremely sensitive to chilling injuries and their irreversible damage leads to the death of the oocytes and embryos. Accordingly successes in cryopreservation have been achieved after removal of lipid droplets or by highrate cooling and warming using vitrification (Vajta et al. 1997b; Berthelot et al. 2000). Compared with embryos oocytes are even more difficult to cryopreserve and the reasons for this are unclear. The different genetic states of oocytes and embryos might be one of the reasons (Stachecki and Cohen 2004). The DNA of mature unfertilized oocytes is compacted into chromosomes that are aligned on a metaphase plate while the majority of DNA in embryos exists as decondensed chromatin at metaphase. Cryopreservation could alter the physical state of DNA and cause spindle disorganization. Compared with in-vivo maturated oocytes invitro maturated oocytes are more tolerant to cryopreservation (Stachecki et al. 2006). A new approach to improve cryosurvival of mammalian gametes and embryos has been published recently. Instead of improving cryopreservation procedures or removing cryosensitive structures high hydrostatic pressure (HHP) treatment was applied to increase the general resistance and preparedness of gametes and embryos to subsequent stresses including cryopreservation. HHP was reported to improve the cryosurvival of porcine and bovine spermatozoa (Pribenszky et al. 2005b 2006) mouse and bovine blastocysts (Pribenszky et al. 2005a c) and the developmental competence of vitrified porcine oocytes after parthenogenetic activation (Du et al. 2008ab). This technique also improved the developmental competence of porcine IVM oocytes after HMC increased the cryotolerance of produced blastocysts and allowed full-term development after transfer of fresh embryos to recipient sows (Du et al. 2008b). The mechanism of the beneficial effect of HHP is only partially understood but heat shock proteins (HSP) are supposed to play a crucial role. HSP were first reported to be produced after treatment with increased temperature (Alahiotis 1983) but further investigations proved that a number of physical or chemical effects may provoke their production including HHP cold osmotic stress (caused both by permeable substances including salts or impermeable materials such as sucrose) changes in ph and starvation (Liu et al. 2006). As one kind of HSP HSP 70 was reported to perform as an a priori activation mechanism in the process of response to stress stimulation (Geraci et al. 2006). In bovine matured oocytes HSP70 was not only evenly distributed in smaller aggregates in cytoplasm to help nascent polypeptides as a molecular chaperone but also interacted with polymerized tubulin as microtube-associated protein through a region containing a sequence related to the tubulin-binding motifs (Kawarsky and King 2001). Many types of cancer cells contain a high amount of HSP70 on their surface causing additional problems for therapy in oncology (Guzhova et al. 2005; Guzhova and Margulis 2006). Osmotic stress was reported to induce a similar effect to HHP regarding the expression pattern of HSPs (Hormann et al. 2006). In the present study an attempt was made to replace HHP treatment before cryopreservation with incubation in solutions containing elevated NaCl concentrations. Although the osmotic shock is supposed to be the main factor other mechanisms may also play a role including elevated intracellular NaCl concentration and its consequences. According to previous observations the stronger the stress (below the lethal dose) the more HSP are produced and the higher cryosurvival may occur. However other effects should also be considered. Osmotic stress for example may induce damage of the meiotic spindle with serious consequences for further developmental competence (Agca et al. 2000; Mullen et al ). The first goal of this study was therefore to find concentrations of NaCl resulting in a survival rate comparable with the controls. Experiment 1 showed that ranges of and mosmol met this requirement. The surprisingly sharp decrease in survival rates between these values has not been previously described and the underlying mechanism needs further investigation. The dangerous zone was found not only in osmotic stress for oocytes but also in cold stress for embryos (Mazur et al. 1992; Vajta et al. 1998). In Experiment 2 oocytes were exposed to 593 and 1306 mosmol before cryopreservation parthenogenetic activation and embryo culture. The best developmental rates were achieved with the 593 mosmol treatment. In Experiment 3 this dose was used for treatment of oocytes before SCNT. Two different recovery times (1 and 2 h) after NaCl treatment and HMC were applied. Blastocyst rates were significantly higher in the treated than in the control group. In order to evaluate the differences between oocytes before and after osmotic treatment according to morphology denuded oocytes were used in Experiment 1. In order to keep the oocytes in better condition for subsequent manipulation COC were used in Experiments 2 and 3 to take advantage of the protective role of cumulus cells (Tatemoto et al. 2000). As far as is known this report is the first to describe the improved developmental competence of SCNT embryos after NaCl or any other osmotic stress applied to oocytes. Different levels of tolerance to osmotic stress have been reported by different groups (van Os and Zeilmaker 1986; Oda et al. 1992; McWilliams et al. 1995; Agca et al. 2000;). It has been suggested that cellular demise from osmotic stress could be due to the type of stressor and not necessarily the ability of the cell to handle osmotic shock (Toner et al. 1993). It could be implied that besides osmotic stress some other effects may be caused by the stressor such as ionic effect. Further studies will be performed to investigate the different effects of osmotic stress by different stressors. It has also been reported that the vitrification solution containing DMSO and EG could parthenogenetically activate

6 in-vitro maturated oocytes including both ovine (Tian et al. 2007) and porcine (Somfai et al. 2007). Whether the osmotic stress caused by the cryoprotectants contributes to this effect is unknown. It is not known if the osmotic stress induced by NaCl causes the same effects. This could be part of the reason for the improvement of cryotolerance and developmental competence of porcine oocytes but further studies are needed to answer these questions. In conclusion this study showed that appropriate NaCl treatment of porcine oocytes increases the developmental rates of porcine IVM oocytes after vitrification and parthenogenetic activation. NaCl treatment of oocytes also improved developmental competence after SCNT. Embryo transfer experiments are required to investigate the in-vivo developmental competence of produced SCNT blastocysts. Additionally the mechanism of the beneficial effect of NaCl treatment requires detailed examination including molecular biological investigations. Future investigations are also needed to determine if a similar effect can be induced in gametes and embryos of different mammalian species including humans. Acknowledgements Authors would like to thank Dr John Yovich for critical reading of the manuscript and Anette M Pedersen Ruth Kristensen Janne Adamsen and Klaus Villemoes for excellent technical assistance. References Agca Y Liu J Rutledge JJ et al Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation. Molecular Reproduction and Development Alahiotis SN 1983 Heat shock proteins. A new view on the temperature compensation. Comparative Biochemistry and Physiology B Berthelot F Martinat-Botte F Locatelli A et al Piglets born after vitrification of embryos using the open pulled straw method. 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