The effect of resveratrol on the developmental competence of porcine oocytes vitrified at germinal vesicle stage

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1 Received: 7 July 2017 Accepted: 26 September 2017 DOI: /rda ORIGINAL ARTICLE The effect of resveratrol on the developmental competence of porcine oocytes vitrified at germinal vesicle stage ECS Santos 1 R Appeltant 2 TQ Dang-Nguyen 2 J Noguchi 2 H Kaneko 2 K Kikuchi 2 T Somfai 1 1 Institute of Livestock and Glassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan 2 Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan Correspondence Tamás Somfai, Institute of Livestock and Glassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan. somfai@affrc.go.jp Present addresses: Ruth Appeltant, Unit of Animal Genomics, GIGA-R, University of Liège (B34) Liège, Belgium Funding information CNPq-Brasil, Grant/Award Number: /2014-8; Japan Society for the Promotion of Science; KAKENHI, Grant/ Award Number: Contents We tested the effects of resveratrol both as a pre- treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre- treatment before vitrification of oocytes for 3 hr with 2 μm resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non- vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non- vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification- related damages. 1 INTRODUCTION Oocyte cryopreservation is a desired approach for the maintenance of female germplasm in ex situ gene banks for farm animals (Woelders, Windig, & Hiemstra, 2012). In pigs, although the technologies for the in vitro maturation (IVM) and fertilization (IVF) of oocytes and in vitro production of embryos (IVP) have progressed (Grupen, 2014) and live offspring have been produced from porcine oocytes vitrified at the immature stage (Somfai et al., 2014), still only a small percentage of vitrified oocytes can develop to the blastocyst stage. Relatively high survival and maturation rates (both above 70%) of vitrified immature oocytes suggest that sublethal damages (i.e., damages which do not lead to imminent cell death) caused by vitrification hamper developmental competence which is manifested only after maturation and fertilization (oocyte activation). Nevertheless, the exact nature of these sublethal damages is still unclear. Recent investigations showed that cytoplasmic maturation and energy balance of immature porcine oocytes were not affected by vitrification (Appeltant et al., 2017). However, it has been demonstrated previously that vitrification triggers the apoptotic cascade in matured (MII stage) porcine oocytes (Vallorani et al., 2012). Apoptotic events can be triggered in oocytes by various pathways leading to DNA damage and eventually cell death (Tiwari et al., 2015). Resveratrol (3,4,5- trihydroxy- trans- stilbene), a phytoalexin with anti- apoptotic and antioxidant properties, was reported to modulate the apoptotic process and reduced the frequency of apoptosis in porcine oocytes vitrified at the MII stage (Giaretta, Spinaci, Bucci, Tamanini, & Galeati, 2013). On the other hand, there are fundamental differences in the sensitivity to cryopreservation and the Reprod Dom Anim. 2017;1 9. wileyonlinelibrary.com/journal/rda 2017 Blackwell Verlag GmbH 1

2 2 SANTOS et al. response to the vitrification procedure between mature and immature porcine oocytes (Somfai, Kikuchi, & Nagai, 2012). The aim of this study was to investigate whether resveratrol can improve the developmental competence of porcine oocytes vitrified at the immature germinal vesicle stage. We tested the effects of resveratrol both as a pre- treatment before cryopreservation and as a recovery treatment after warming during IVM on the viability, maturation, developmental competence, cytoplasmic redox status and apoptosis of porcine oocytes vitrified at the immature stage. 2 MATERIALS AND METHODS 2.1 Ethics statement Live animals raising ethical concerns were not used in this study. 2.2 Collection of cumulus oocyte complexes (COCs) Porcine ovaries of cross- bred gilts (Landrace Large White) were randomly selected from a local slaughterhouse, stored at C after washing with Dulbecco s phosphate buffered saline (PBS) and transported to the laboratory within 1 2 hr. Upon arrival, COCs were collected by scraping of 2- to 6- mm follicles with a fine surgical blade in a collection medium, which was medium 199 (M199 with Hanks salts; Sigma- Aldrich, St. Louis, MO, USA) supplemented with 5% (v/v) of foetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Life Technologies, Carlsbad, CA, USA), 20 mm of HEPES (Dojindo Laboratories, Kumamoto, Japan) and antibiotics [100 IU/ml of streptomycin sulphate (Sigma- Aldrich), 100 IU/ml penicillin G potassium (Sigma- Aldrich)]. After dissection, COCs with fully grown oocytes with at least two layers of cumulus cells were selected for the subsequent experiments. 2.3 Vitrification and warming of immature COCs Cryoprotectant treatment regimen before the vitrification of COCs was performed according to a previous report (Somfai et al., 2015). In brief, a group of COCs were incubated in 1 ml of a basic medium (BM) for 30 min, which was a modified glucose- free North Carolina State University (NCSU)- 37 medium (Petters & Wells, 1993) supplemented with 20 mm HEPES, 0.17 mm sodium pyruvate, 2.73 mm sodium lactate, 50 μm ß- mercaptoethanol and 4 mg/ ml bovine serum albumin (Fraction V, Sigma- Aldrich). The BM medium was further supplemented with 7.5 μg/ml cytochalasin B (CB; C- 6762, Sigma- Aldrich). Then the COCs were transferred into an equilibration solution (ES) comprised of BM supplemented with 7.5 μg/ml CB, 2% (v/v) ethylene glycol (EG) and 2% (v/v) propylene glycol (PG) for 5 15 min at 38.5 C. Afterwards, COCs were washed two times in 50 μl of vitrification solution (VS) at 38.0 C, loaded on Cryotop sheets (Kitazato, Biopharma, Shizuoka, Japan) in a minimum volume (<1 μl) of VS and were plunged immediately in liquid nitrogen (LN). The VS was BM supplemented with 50 mg/ml polyvinylpyrrolidone (P- 0930, Sigma- Aldrich), 0.3 M sucrose ( , Wako Pure Chemical Industries, Osaka, Japan), 17.5% (v/v) EG and 17.5% (v/v) PG. Treatment of COCs in VS medium (including washing, loading and removal of excess VS) did not exceed 40 s. Vitrified samples were stored in a LN tank until use. Warming of vitrified COCs was performed according to a previous report (Somfai et al., 2015) with slight modification. In brief, Cryotop sheets were immersed directly into 2.5 ml of warming solution (0.4 M Sucrose in BM) in a 35- mm plastic dish (Falcon , Becton Dickinson, Franklin Lakes, NJ, USA) for 1 min at 42 C. The COCs were then consecutively transferred for periods of 1 min (each) to 500- μl droplets of BM supplemented with 0.2, 0.1 and 0.05 M of sucrose at 38.0 C. The warmed COCs were washed in BM without sucrose at 38.0 C and then placed into maturation medium. 2.4 In vitro maturation (IVM) The maturation culture medium was porcine oocyte medium (POM, Yoshioka, Suzuki, & Onishi, 2008) supplemented with 10 IU/ml ecg (Serotropin; ASKA Pharmaceutical Co., Ltd., Tokyo, Japan), 10 IU/ml hcg (Puberogen, Novartis Animal Health, Tokyo, Japan) and 10 μg/ml epidermal growth factor (EGF, Sigma- Aldrich). After warming, all COCs were washed three times and cultured in 500 μl of aliquots of maturation medium supplemented with 1 mm dibutyryl camp (dbcamp; Sigma- Aldrich) in 4- well dishes (Nunc, Nunclon Delta Surface, Thermo Fisher Scientific, Roskilde, Denmark) without oil coverage, each well containing COCs for 22 hr in 5% C, 5% and 90% N 2 at 39 C. Subsequently, they were cultured for an additional hr in POM medium containing hormones and EGF but without dbcamp under the same conditions as during the first 22 hr of IVM. 2.5 Evaluation of survival and maturation of oocytes After IVM, COCs were treated with a solution of 150 IU/ml hyaluronidase (Sigma- Aldrich) in collection medium for 30 s and then transferred to collection medium in which cumulus cells were removed from the oocytes by gentle pipetting with a fine glass pipette. Then the live/dead status and maturation of the oocytes were assessed morphologically under a stereo microscope. Oocytes with a normal spherical shape and dark granulated cytoplasm surrounded by a membrane with a definite smooth surface were considered alive. Oocytes without a definite membrane surface and/or with brownish cytoplasm were considered dead. Live oocytes with the first polar body (1PB) extruded were considered matured and used in subsequent experiments. 2.6 Parthenogenetic activation (PA) of oocytes The matured oocytes were placed between a pair of electrodes of an electroporator (ECFG21, Nepa Gene Co., Ltd., Ichikawa, Chiba, Japan) in an activation medium (0.3 M mannitol, 0.1 mm CaCl 2, 0.1 mm

3 SANTOS et al. 3 MgSO 4 and 0.5 mm HEPES) and stimulated with a single direct current pulse of 1500 V/cm for 99 μs. The oocytes were then washed and cultured in 500 μl of aliquots of porcine zygote medium 3 (PZM- 3) (Yoshioka, Suzuki, Tanaka, Anas, & Iwamura, 2002) supplemented with 5 μg/ml CB, in four- well dishes without oil coverage at 39 C under 5% C, 5% and 90% N 2, for 3 hr. The day of PA was defined as Day 0. The activated oocytes were subsequently subjected to embryo culture. 2.7 In vitro embryo culture (IVC) and assessment of embryo development After PA, oocytes were washed three times and placed in 100- μl drops of PZM- 3 medium (each drop containing oocytes) in four- well dishes with oil coverage, at 38.5 C under 5% C, 5% and 90% N 2 until Day 7. On Day 2, cleavage rates were recorded according to the criteria of Dang- Nguyen et al. (2010). On Day 5, 11 μl of pre- warmed FBS was added to each culture droplet. On Day 7, blastocyst rates were recorded. Embryos without a visible perivitelline space containing more than 10 blastomeres and a blastocyst cavity were categorized as blastocysts. To verify the total cell numbers, blastocysts on Day 7 were placed in 25 μg/ml of Hoechst (H 33342, Calbiochem, San Diego, CA, USA) dissolved in 99.5% ethanol and kept at 4 C until microscopic evaluation (1 day to 3 weeks). Then, the embryos were washed in 99.5% ethanol, mounted on glass slides in glycerol droplets, flattened by coverslips and examined under UV light with an excitation wavelength of nm, using an epifluorescence microscope (BX- 51, Olympus, Tokyo, Japan). A digital image of each embryo was recorded with a digital camera (Retiga 2000R, QImaging, BC, Canada) at the focal plane where all nuclei were visible, and the total numbers of nuclei labelled by H33342 were counted with the ImageJ software (NIH, USA; imagej.nih.gov/ij/). 2.8 Measurement of hydrogen peroxide (H 2 ) levels in oocytes Matured oocytes having the first polar body were selected for analysis. The relative quantity of H 2 in oocytes after IVM was determined by the method of Karja et al. (2006) with slight modifications. In brief, the oocytes were incubated for 15 min in PZM- 3 medium containing 10 μm 2,7 - dichlorodihydrofluorescein diacetate (DCHFDA; Sigma- Aldrich). Then oocytes were washed in PBS supplemented with 3 mg/ml polyvinylpyrrolidone (Sigma- Aldrich, P- 0930) at 37 C, placed on a glass slide and covered with a coverslip. The fluorescence emissions were recorded as TIFF files using the digital camera attached to the fluorescent microscope described above, with excitation at 480 nm and emission at 510 nm using the same exposition interval at 20 s after exposure to UV light always using the same camera settings. The procedure was performed in a dark room. Mean fluorescence intensity and the surface area of each oocyte were measured in images using the ImageJ software. Fluorescent colour images were converted to 8- bit greyscale. After inverting each image, fluorescent intensity of the oocyte area was expressed by the density of grey pixels. Total fluorescence/oocyte was calculated as mean fluorescence ( mean grey value ) multiplied with oocyte surface ( area ). Relative fluorescence was calculated from total fluorescence where the control group was 100%. 2.9 Measurement of intracellular glutathione (GSH) levels in oocytes Matured oocytes having the first polar body were selected for analysis. The concentrations of GSH in vitrified oocytes after IVM were determined by dithionitrobenzoic acid glutathione disulphide (DTNB- GSSG) reductase recycling assay (Anderson, 1985). In each experimental group, a pool of 10 oocytes were placed in 5 μl of 10 mm Na2- EDTA- containing sodium phosphate buffer (0.2 M, ph 7.2) and 5 μl of 1.25 M phosphoric acid in a 1.5- ml microfuge tube and stored at 80 C until assay. Then, the samples were thawed at RT and 175 μl of sodium phosphate buffer containing 0.33 mg/ml nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma- Aldrich), 25 μl of 6 mm DTNB (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 40 μl of water were added to each sample tube. Finally, 5 μl of 125 IU/ml GSH disulphide reductase (Oriental Yeast Co., LTD, Tokyo Japan) was added to the microfuge tube to initiate the reaction. Absorbance was recorded six times by a spectrophotometer (DU7500; Beckman Coulter, Inc., CA, USA) at 30- s intervals at a wavelength of 412 nm. A GSH standard and a sample blank lacking GSH were also assayed (n = 6 in each group). Standards were prepared for each assay, and the total GSH content per sample was determined from a standard curve of GSH. The GSH concentration per oocyte was calculated by dividing the total concentration per sample by the number of oocytes (10) present in the sample Assay of apoptosis in oocytes Matured oocytes having the first polar body were selected for analysis. The onset of apoptosis was investigated in oocytes after IVM based on the annexin V binding according to the report of Vallorani et al. (2012) with a slight modification. In brief, denuded matured oocytes were washed three times with PBS and then transferred to 100 μl of a binding buffer (Alexa Fluor 488 Annexin V/ Dead Cell Apoptosis Kit, Molecular Probes, Eugene, USA) with 5 μl of Alexa Fluor Annexin V, 1 μl of propidium iodide (PI) (100 μg/ml) and 0.2 μl of 5 mg/ml Hoechst for 20 min at 38.5 C in the dark. After three consecutive washes in binding buffer, the oocytes were mounted on glass slides. The fluorescence intensity of annexin V was measured under an epifluorescence microscope (BX 51; Olympus) at an excitation wavelength of 480 nm and emission at 510 nm only in live matured oocytes characterized by PI- negative nuclei and the presence of the 1PB. A photograph of each oocytes was taken at the equatorial plane at the same magnification immediately after exposure to UV light using the same camera settings as described above. The procedure was performed in a dark room.

4 4 SANTOS et al. Relative fluorescence was assayed from digital images as described above Experimental design Experiment 1: The effects of resveratrol treatment before vitrification on viability and developmental competence of immature porcine oocytes In previous studies, treatment with 2 μm resveratrol for intervals of 2 hr and 44 hr could modulate apoptosis in porcine oocytes and subsequent embryos (Giaretta et al., 2013; Kwak et al., 2012). As extensive pre- maturation before could alter the nuclear status of oocytes and the extent of cumulus expansion, we decided to test resveratrol as a pretreatment before vitrification for 3 hr. Accordingly, COCs were collected as described above and subjected to each of the following treatments: (i) COCs were kept in pre- incubated IVM medium supplemented with or without 2 μm resveratrol (Sigma- Aldrich) for 3 hr and then vitrified, warmed and subjected to IVM as described above (Res+/Vit and Res / Vit groups, respectively); (ii) COCs were kept in pre- incubated IVM medium supplemented with or without 2 μm resveratrol (Sigma- Aldrich) for 3 hr and then subjected to IVM as described above (Res+/Cont and Res /Cont groups, respectively); and (iii) to test the effect of 3 hr prematuration before vitrification, one group of COCs were vitrified immediately after collection without pre- maturation for 3 hr, then warmed and subjected to IVM as described above (N/Vit group). After IVM, live matured oocytes were selected in all groups and subjected to PA and IVC as described above. Percentages of survival, maturation, embryonic cleavage, blastocyst development and cell numbers in blastocysts were compared amongst groups. Five replications were performed Experiment 2: The effects of resveratrol treatment during 44 hr of IVM on viability and development of vitrified immature porcine oocytes COCs were collected and vitrified immediately as described above. After warming, vitrified COCs were subjected to IVM in medium supplemented with or without 2 μm resveratrol (V/Res+ and V/ Res groups, respectively) throughout the entire IVM as described above. Other oocytes without vitrification were also subjected to IVM in media with or without 2 μm resveratrol (Cont/Res+ and Cont/ Res groups, respectively). After IVM, live matured oocytes were selected in all groups and subjected to PA and IVC as described above. Percentages of survival, maturation, embryonic cleavage, blastocyst development and cell numbers in blastocysts were compared amongst groups. Five replications were performed Experiment 3: The effects of resveratrol on H 2 and GSH levels in vitrified porcine oocytes The effects of resveratrol during IVM on redox status of vitrified and non- vitrified oocytes were assessed. The same treatment groups were set up as in Experiment 2. After IVM, H 2 and GSH levels in oocytes were compared amongst groups in three biological replications Experiment 4: The effects of resveratrol on apoptosis levels in vitrified porcine oocytes The same treatment groups were set up as in Experiment 2. After IVM, the level of apoptosis in oocytes was compared amongst the groups in four biological replications Statistical Analysis All data were analysed by one- way ANOVA followed by Tukey s multiple comparison using the KyPlot package (Ver. 2.0, KyensLab Inc., Tokyo, Japan). Percentage data were subjected to arcsine transformation before analysis. For all analysis, p <.05 was set as the significance level. Results are expressed as mean ± SEM. 3 RESULTS 3.1 The effects of resveratrol treatment before vitrification on viability and developmental competence of immature porcine oocytes (Experiment 1) The survival rates of oocytes amongst all the vitrified groups (Res / Vit, Res+/Vit and N/Vit) were statistically similar but significantly lower (p <.05) than those in the Res /Cont and Res+/Cont groups (Table 1). There was no statistical difference in the percentage of PB extrusion (nuclear maturation) amongst the groups. After PA, the mean cleavage rate in N/Vit group was significantly lower (p <.05) than that in all the other groups, and he cleavage rates in the Res / Vit, Res+/Vit groups were significantly lower than those in the Res / Cont and Res+/Cont groups. The percentage of blastocyst development calculated from all activated (matured) oocytes in the Res / Cont group was significantly higher (p <.05) than that in all vitrified groups, and the value for Res+/Cont group was significantly higher than that in the Res /Vit and N/Vit groups but did not differ significantly from that of the Res /Cont and Res+/Vit groups. The percentage of blastocyst development calculated from cleaved oocytes in the Res /Cont and Res+/Cont groups was significantly higher (p <.05) than that in the Res /Vit and N/Vit groups, whereas the value of the Res+/Vit group did not differ significantly from that of the other groups. The percentage of blastocyst development calculated from the total number of oocytes in the Res /Cont and Res+/Cont group was significantly higher than that in all the other groups, which were similar to one another. There was no significant difference in percentages of oocyte survival, maturation, cleavage and blastocyst development between the Res /Cont and Res+/Cont groups and between the Res /Vit and Res+/Vit groups. There was no significant difference in total cell numbers of blastocysts amongst the groups.

5 SANTOS et al. 5 TABLE 1 The effects of pre- treatment with 2 μm resveratrol for 3 hr on survival and development of vitrified porcine oocytes Total cells in (% cultured) (% cleaved) (% total) blastocysts Group Total oocytes Live (% total) Matured (% live) Cleaved (% cultured) Res /Cont (97.5 ± 1.8) a 137 (77.4 ± 3.0) 107 (79.3 ± 7.4) a 73 (55.2 ± 7.5) a (69.0 ± 4.8) a (41.2 ± 4.7) a 57.2 ± 4.7 Res+/Cont (99.3 ± 0.6) a 134 (77.4 ± 3.2) 110 (81.6 ± 3.0) a 58 (42.8 ± 5.6) ab (53.0 ± 8.0) a (32.4 ± 3.3) a 55.7 ± 3.1 Res /Vit (44.1 ± 7.8) b 101 (68.2 ± 11.4) 65 (65.6 ± 5.5) b 23 (21.2 ± 4.5) c (33.0 ± 6.5) b (7.0 ± 2.8) b 48.6 ± 4.1 Res+/Vit (42.1 ± 1.1) b 113 (75.8 ± 7.2) 74 (66.7 ± 4.1) b 34 (32.6 ± 10.2) bc (46.2 ± 11.8) ab (11.7 ± 4.9) b 55.2 ± 5.3 N/Vit (51.0 ± 6.5) b 110 (74.1 ± 8.5) 50 (47.5 ± 2.5) c 15 (14.2 ± 3.7) c (30.5 ± 7.9) b (4.9 ± 1.0) b 54.7 ± 4.0 Matured oocytes with the first polar body were selected under stereo microscope, parthenogenetically activated and subsequently cultured. Day 0 = the day of parthenogenetic activation. Five biological replications were performed. Data are presented as mean ± SEM. a, b and c in the same column differ significantly (p <.05). Res /Cont, non- vitrified oocytes matured after 3 hr pre- IVM without resveratrol; Res+/Cont, non- vitrified oocytes matured after 3 hr pre- IVM with resveratrol; Res /Vit, oocytes vitrified after 3 hr pre- IVM without resveratrol, then warmed and matured; Res+/Vit, oocytes vitrified after 3 hr pre- IVM with resveratrol, then warmed and matured; N/Vit, oocytes vitrified immediately after collection, then warmed and matured. 3.2 The effects of resveratrol treatment during 44 h of IVM on viability and development of vitrified immature porcine oocytes (Experiment 2) The survival rates of oocytes in both vitrified groups (Vit/Res and Vit/Res+) were statistically similar, but significantly lower (p <.05) than those in both the Cont/Res and Cont/Res+ groups (Table 2). There was no statistical difference in the percentage of PB extrusion (nuclear maturation) between the Cont/Res and Cont/Res+ groups and between the Vit/Res and Vit/Res+ groups. However, the percentage of matured oocytes in both vitrified groups was lower than that in the Cont/Res group, whereas the Cont/Res+ did not differ statistically from other groups. The mean cleavage rate after PA in the Vit/Res group was significantly lower than that of the other three groups which were statistically similar to one another. The percentage of blastocyst development calculated from activated (matured) oocytes in the Vit/Res group was significantly lower (p <.05) than that in the other 3 groups, and the value of the Vit/Res+ group was significantly lower than that of the Cont/Res+ group but did not differ significantly from that of the Cont/Res group. The percentage of blastocyst development calculated from cleaved embryos in the Vit/Res group was significantly lower (p <.05) than that in the other three groups, which on the other hand did not differ significantly from one another (Table 2). The percentage of blastocyst development calculated from the total number of oocytes in the Vit/Res group was significantly lower (p <.05) than that in the Vit/Res+ group, and the values of both vitrified groups were significantly lower than those of the Cont/Res and Cont/Res+ groups which did not differ significantly. The number of total cells in blastocysts in the Vit/Res and Vit/Res+ groups was similar (p >.05) and significantly lower than that in Cont/Res and Cont/Res+ groups. 3.3 The effects of resveratrol on H 2 and GSH levels in vitrified porcine oocytes (Experiment 3) There was no significant difference in intracellular H 2 and GSH levels in oocytes amongst treatment groups (Figure 1a,b, respectively). 3.4 The effects of resveratrol on the onset of apoptosis in vitrified porcine oocytes (Experiment 4) All the oocytes subjected to annexin V binding analysis were membrane- intact indicated by PI- negative nuclei. The level of apoptosis as measured by annexin V binding in oocytes was statistically similar amongst groups (Figure 2). 4 DISCUSSION In the present study, we tested the efficacy of treatment with 2 μm resveratrol either before or after vitrification on the survival, maturation and developmental competence of immature porcine oocytes. This concentration was chosen based on previous studies (Giaretta

6 6 SANTOS et al. TABLE 2 The effects of 2 μm resveratrol during 44 h of IVM on survival and development of vitrified porcine oocytes Total cells in (% cultured) (% cleaved) (% total) blastocysts Group Total oocytes Live (% total) Matured (% live) Cleaved (% cultured) Cont/Res (99.4 ± 0.6) a 131 (90.2 ± 1.6) a 104 (78.5 ± 6.1) a 42 (32.1 ± 4.3) ab (40.9 ± 4.6) a (28.7 ± 3.8) a 69.2 ± 5.3 a Cont/Res (100 ± 0.0) a 126 (84.2 ± 5.1) ab 102 (79.2 ± 8.5) a 46 (36.0 ± 3.4) a (46.2 ± 3.2) a (30.6 ± 3.7) a 66.6 ± 6.0 a Vit/Res (47.4 ± 7.8) b 88 (78.1 ± 3.7) b 52 (54.7 ± 4.7) b 7 (7.5 ± 1.9) c (15.0 ± 3.8) b (2.7 ± 0.7) c 48.3 ± 3.7 b Vit/Res (51.8 ± 9.1) b 91 (76.1 ± 4.6) b 66 (72.1 ± 4.2) b 21 (26.2 ± 4.3) b (37.3 ± 7.3) a (9.5 ± 1.5) b 40.7 ± 5.5 b Matured oocytes with the first polar body were selected under stereo microscope, parthenogenetically activated and subsequently cultured. Day 0 = the day of parthenogenetic activation. Five biological replications were performed. Data are presented as mean ± SEM. a and b in the same column differ significantly (p <.05). Cont/Res, non- vitrified oocytes matured in the absence of resveratrol; Cont/Res+, non- vitrified oocytes matured in the presence of resveratrol; Vit/Res, vitrified oocytes matured in the absence of resveratrol; Vit/Res+, vitrified oocytes matured in the presence of resveratrol. FIGURE 1 The effects of resveratrol treatment during IVM on hydrogen peroxide (H 2 ) (a) and glutathione (GSH) (b) levels in porcine oocytes. Cont/Res = non- vitrified oocytes matured in the absence of resveratrol; Cont/Res+ = non- vitrified oocytes matured in the presence of resveratrol; Vit/Res = vitrified oocytes matured in the absence of resveratrol; Vit/Res+ = vitrified oocytes matured in the presence of resveratrol. Data are presented as mean ± SEM. Three biological replications were performed. The total number of oocytes analysed in each group is given in parentheses FIGURE 2 The effects of resveratrol treatment during IVM on annexin V binding levels in porcine oocytes. Cont/Res = nonvitrified oocytes matured in the absence of resveratrol; Cont/ Res+ = non- vitrified oocytes matured in the presence of resveratrol; Vit/Res = vitrified oocytes matured in the absence of resveratrol; Vit/Res+ = vitrified oocytes matured in the presence of resveratrol. Data are presented as mean ± SEM. Four biological replications were performed. The total number of oocytes analysed in each group is given in parentheses

7 SANTOS et al. 7 et al., 2013; Kwak et al., 2012). The first experiment demonstrated that a pre- treatment with resveratrol for 3 hr had no effect on survival and subsequent development of oocytes after vitrification. However, in Experiment 2, when applied during IVM after vitrification and warming, resveratrol enhanced the development of surviving oocytes to the blastocyst stage although it had no effect on survival rates. Furthermore, our results indicate that the positive effect of resveratrol on embryo development was manifested both at and after the first cleavage of embryos (as cleavage rates and the development of cleaved embryos were both improved) and that this effect was exerted only on vitrified, but not on fresh (non- vitrified) oocytes. As a result, the overall efficacy of the vitrification system measured by the percentage of blastocyst development from the total number of vitrified oocytes was significantly improved. The result that pre- treatment with resveratrol before vitrification did not affect survival embryo development, but post- warming treatment with this drug improved their embryo development suggests that resveratrol did not prevent vitrification- related cellular damages, but it enhanced the recovery from sublethal damages. Regarding the mechanism of action of resveratrol on the developmental competence of porcine oocytes, our results are not in agreement with those of the study of Kwak et al. (2012), which described improved embryo development of non- vitrified porcine oocytes after maturation in the presence of 2 μm resveratrol. Another marked difference in the results between the two studies is that in the study of Kwak et al. (2012), resveratrol treatment (and hence improved embryo development) was associated with reduced H 2 and increased GSH levels in oocytes, which demonstrated the antioxidant properties of the drug. However, these effects of resveratrol on oocytes were not observed in our study. The reason for the discrepancy between the two studies might be related to the differences in the culture systems used. In their study, Kwak et al. (2012) matured oocytes in a TCM199- based medium at an atmosphere of 5% C in 95% humidified air (containing 20% ), without the synchronization of nuclear maturation using dbcamp during the first half of IVM. On the other hand, we used POM medium under a low (5%) tension and applied meiosis synchronization with 1 mm dbcamp during the first 22 hr of IVM. The concentration of glucose in the TCM199 medium is higher than that in the POM medium (5.5 mm and 4 mm, respectively). Ambient and high concentration of glucose in medium can contribute to ROS formation in cultured cells (reviewed by Guérin, El Mouatassim, & Ménézo, 2001) and could potentially cause higher ROS levels in oocytes used in the study of Kwak et al. (2012). Furthermore, meiosis synchronization with camp modulating agents such as dbcamp (used in our study) increases GSH content in oocytes, thus improving its antioxidant defence (reviewed by Gilchrist et al., 2016). Taken together, it is possible that the ROS level in IVM oocytes produced in our system was fundamentally lower than that in the oocytes produced by Kwak et al. (2012) which could veil the antioxidant effect of resveratrol in our study. In the present study, similar H 2 and GSH levels were measured in oocytes matured with and without the addition of resveratrol in IVM medium, irrespective of oocyte vitrification. It suggests that in our IVM system, oxidative stress may not be a major issue compromising the developmental competence of oocytes. Also, these results indicate that in our system, vitrification at the germinal vesicle stage does not alter the redox status (measured by both the levels of H 2 and the antioxidant GSH) in the oocytes, which confirms our previous reports (Appeltant et al., 2017; Somfai et al., 2010). To understand the mechanism of action of resveratrol upon vitrified oocytes, we also assayed the level of apoptosis based on the annexin V binding. In previous reports, apoptosis in porcine oocytes vitrified at the MII stage was detected by this approach (Giaretta et al., 2013; Vallorani et al., 2012) and found to be moderated by resveratrol (Giaretta et al., 2013). However, in the present study, significant differences in the level of annexin V binding caused by resveratrol treatment or vitrification were not detected. This result could either mean that vitrification did not induce apoptosis in immature oocytes or that it could not be effectively detected at the MII stage by the annexin V binding assay. Further research will be necessary to clarify this point. Also, it must be noted that the vitrification procedure used in the present study applies a different combination of permeating cryoprotectant agents used at much lower percentages during equilibration compared to the protocol of the above- mentioned two studies, which might also affect the onset of apoptosis in vitrified oocytes. In the studies of Vallorani et al. (2012) and Giaretta et al. (2013), porcine oocytes were vitrified after equilibration in the combination of 7.5% EG and 7.5% dimethyl sulphoxide (DMSO) for 5 15 min. Previously, we demonstrated significantly lower survival rates when immature porcine oocytes were vitrified after such equilibration regimen compared with equilibration in the combination of 2% EG and 2% PG (Somfai et al., 2015) which was used in our present study. Exposure to permeating CPA such as EG at the concentration of 10% (v/v) was reported to induce DNA fragmentation, a hallmark of apoptosis in porcine embryos (Rajaei et al., 2005). Although in the study of Vallorani et al. (2012) treatment of oocytes with CPA without cooling did not trigger apoptosis, the possibility that the treatment of oocytes with a relatively high concentration of CPA during equilibration contributed to the trigger of apoptotic cascade during the cooling/warming process cannot be ruled out. The mechanism by which resveratrol treatment improved the competence specifically of vitrified oocytes remains unclear. It is possible that resveratrol exerted its effects on cumulus cells (which were not investigated in this study) and thus affected the competence of oocytes indirectly. It is known that in pigs, high frequency of apoptotic cells in the cumulus mass is associated with reduced developmental competence of oocytes (Grupen & Armstrong, 2010), which supports this theory. Also, resveratrol has been reported to upregulate the expression of Sirtuin 1 (SIRT1) in porcine oocytes (Sato et al., 2014), which is known to prevent ageing- related loss of developmental competence (Ma, Zhang, Zhang, Han, & Rui, 2015) and even improve the competence of early antral oocytes (Itami, Shirasuna, Kuwayama, & Iwata, 2015). Nevertheless, SIRT1 has many biological functions in animal cells (Chung et al., 2010), and its possible contribution upon recovery from vitrification- related cellular damages needs further clarification. Regarding the pre- vitrification treatment of oocytes in Experiment 1, it must be noted that although treatment with resveratrol did not

8 8 SANTOS et al. affect the developmental competence of oocytes, the cleavage rates were significantly higher (p <.05) when immature oocytes were vitrified after 3 hr of pre- IVM irrespective of resveratrol supplementation compared with the group vitrified immediately after oocyte collection (N/Vit). This result suggests that a short culture after before the vitrification of immature porcine COCs is beneficial to the maintenance of oocyte competence; however, the exact reason is not clear yet. The collection procedure has been known to be stressful for mammalian oocytes often reducing their developmental competence by the activation of intracellular phosphodiesterase which causes the deactivation of the camp- dependent protein kinase A (PKA) and thus premature meiotic resumption (reviewed by Gilchrist et al., 2016). Recently, we reported that vitrification after the collection triggered precocious nuclear resumption in approximately 20% of the of immature porcine oocytes even in the presence of the camp analogue dbcamp (Appeltant et al., 2017) which raises the possibility that the stresses of oocyte collection and vitrification might cause similar cellular malfunctions such as PKA deactivation and could possibly act on the oocyte synergistically. It seems reasonable to speculate that when oocytes are vitrified immediately after collection, they suffer accumulated stresses in a relatively short time. Providing a resting period between after collection and before vitrification might be beneficial by preventing stress accumulation. In conclusion, we have demonstrated that resveratrol did not prevent the cellular damages in immature oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of surviving vitrified oocytes presumably by enhancing their recovery from sublethal damages. Further research will be necessary to clarify the mechanisms of action. ACKNOWLEDGEMENTS The authors are grateful to Ms. M. Osaki and Ms. M. Nagai for technical assistance. E.C.S. Santos was supported by CNPq- Brasil (Grant number /2014-8). This work was supported by the Japan Society for the Promotion of Science (KAKENHI, Grant Number: ). CONFLICT OF INTEREST None of the authors have any conflict of interest to declare. AUTHOR CONTRIBUTION E.C.S. SANTOS did data acquisition (experimenting), involved in experiment design and data analysis and did manuscript drafting. T. SOMFAI involved in experiment design and data analysis, supervised the project, revised the draft and funded chemicals and expendables used. R. APPELTANT, T.Q. DANG- NGUYEN, J. NOGUCHI, H. KANEKO and K. KIKUCHI assisted in experiments, revised the draft and provided equipments for experiments. ORCID T Somfai REFERENCES Anderson, M. E. (1985). Determination of glutathione and glutathione disulfide in biological samples. In A. Meister (Ed.), Glutamate, Glutathione and related compounds (pp ). New York, NY: Academic Press. Appeltant, R., Somfai, T., Santos, E. C. S., Dang-Nguyen, T. Q., Nagai, T., & Kikuchi, K. (2017). Effects of vitrification of cumulus- enclosed porcine oocytes at the germinal vesicle stage on cumulus expansion, nuclear progression and cytoplasmic maturation. Reproduction, Fertility, and Development. in press. Chung, S., Yao, H., Caito, S., Hwang, J. W., Arunachalam, G., & Rahman, I. (2010). Regulation of SIRT1 in cellular functions: Role of polyphenols. 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