Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts

Accepted Manuscript Title: Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts Authors: Ya-ning Chen, Jian-jun Dai, Cai-feng Wu, Shu-shan Zhang, Ling-wei Sun, De-fu Zhang PII: DOI: Reference:

S0378-4320(18)30501-3 https://doi.org/10.1016/j.anireprosci.2018.09.012 ANIREP 5962

To appear in:

Animal Reproduction Science

Received date: Revised date: Accepted date:

5-6-2018 21-8-2018 18-9-2018

Please cite this article as: Chen Y-ning, Dai J-jun, Wu C-feng, Zhang Sshan, Sun L-wei, Zhang D-fu, Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts, Animal Reproduction Science (2018), https://doi.org/10.1016/j.anireprosci.2018.09.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts

Ya-ning Chena,b, Jian-jun Daia,b,c*, Cai-feng Wua,b,c, Shu-shan Zhanga,b,c, Ling-wei

Institute of Animal Science and Veterinary Science, Shanghai Academy of

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Suna,b,c, De-fu Zhanga,b,c*

Agricultural Sciences, Shanghai, China

Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of

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Agri-Genetics and Breeding, Shanghai, China c

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Shanghai Engineering Research Center of Breeding Pig, Shanghai, China

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Correspondence and reprint requests: Dr. Jian-jun Dai, Institute of Animal Science

and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai,

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201106, PR China. e-mail: [email protected]; Dr. De-fu Zhang, Institute of

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Animal Science and Veterinary Science, Shanghai Academy of Agricultural Sciences, Shanghai, 201106, PR China. e-mail: [email protected] Corresponding Author: Tel.: +86 021 52235475; fax: +86 021 62207858; E-mail

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address: [email protected] (Z. De-fu)

ABSTRACT This study was conducted to evaluate whether the poor developmental capacity of

pig embryos after vitrification was related to the occurrence of apoptosis. Parthenogenetic blastocysts were used as the research material. The blastocoel recovery rate, mitochondrial membrane potential (ΔΨm), amount of early apoptosis, activities of several caspases, and relative abundance of mRNA of apoptosis-related

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genes involved in mitochondria and death receptor apoptotic pathways were detected before or after vitrification. The results indicate that the blastocoel recovery rate

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(31.0%) and total cells (31.8) of vitrified blastocysts were less than those of fresh

blastocysts (100% and 38.2, P < 0.05). The ΔΨm of vitrified blastocysts was 0.46,

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which was less than that of fresh blastocysts (1.02, P < 0.05). The rate of apoptotic

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cells in vitrified blastocysts (8.1%) after TUNEL (TdT-mediated dUTP Nick-End

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Labeling) assay was markedly greater than that in fresh blastocysts (3.9%, P < 0.05).

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The pan-caspase, caspase-3, caspase-8 and caspase-9 activities of vitrified blastocysts

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(20.7, 20.6, 17.6 and 19.9) were markedly greater than those of fresh blastocysts (7.4, 6.5, 5.5 and 6.3, P < 0.05). The real-time PCR results indicated that relative

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abundance of caspase-8 and TNF-α mRNA from death receptor apoptotic pathway

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and caspase-9 for the mitochondrial apoptotic pathway genes in the vitrified group were greater than those in the fresh group P < 0.05). The relative abundance of Bcl-2 and SOD-1 mRNA for the mitochondrial pathway genes in the vitrified group was

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less than those in the fresh group (P < 0.05). In conclusion, the poor developmental capacity of vitrified parthenogenetic pig blastocysts was closely related with apoptosis. Both mitochondria and death receptor-mediated apoptotic pathways participated the occurrence of this apoptosis.

Keywords: Porcine; Parthenogenetic embryo; Vitrification; Mitochondria; Apoptosis; Apoptotic pathway

1. Introduction

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The vitrification of gametes and embryos is a feasible method for the preservation of genetic resources in farm and endangered animal species (Castillo-Martín et al.,

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2014). Although vitrification technology for embryo cryopreservation in some

animals has been greatly improved in recent decades, the efficiency of pig embryo

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cryopreservation remains low (Isachenko et al., 1998; Niu et al., 2015). Results from

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many studies indicated the poor developmental capacity of embryos was closely

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related to damage of cellular organelles, such as the cellular membrane, mitochondria,

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cytoskeleton, and chromosome (Ren et al., 2015; Inaba et al., 2016; Romao et al.,

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2016).

Apoptosis contributes to the decrease in the development of oocytes and embryos

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after vitrification. Coutinho et al. (2007) reported that freezing could induce apoptosis

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in mouse embryos. Inaba et al. (2016) found that the rate of apoptotic cells in cattle embryos increased after freezing. In the cryopreservation of pig oocytes, Vallorani et al. (2012) and Niu et al. (2016) also found that vitrification could increase apoptosis

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after thawing. There, however, are few reports on apoptosis in the vitrification of pig embryos. The present study was conducted to systematically ascertain the extent of apoptosis of parthenogenetic pig blastocysts before and after vitrification. Apoptosis is an initiative cell death process controlled by a series of genes, which

mainly include the death receptor-mediated extrinsic pathway (Guicciardi and Gores, 2009) and the mitochondrial mediated intrinsic pathway (Wang and Youle, 2009; Estaquier et al., 2012). It has been proposed that in vitrified pig oocytes (Dai et al. (2015; 2016) and Niu et al. (2016)), both the mitochondria and death receptor

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mediated-apoptotic pathways could be involved in the occurrence of apoptosis after thawing. To determine whether similar apoptotic pathways could occur in frozen

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parthenogenetic pig blastocysts, the present study was conducted to detect several

caspase activities and the gene expression in the two apoptotic pathways by in situ

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using fluorescence staining and real-time PCR.

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2. Materials and methods

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Unless otherwise indicated, all chemicals used were purchased from

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Sigma–Aldrich Chemical Company (St. Louis, MO, USA). 2.1. Collection and in vitro maturation (IVM) of oocytes

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Pig ovaries were collected from a local slaughterhouse and transported to the

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laboratory in a physiological saline solution (0.9% NaCl) containing 75 µg/mL penicillin and 50 µg/mL streptomycin at 35 to 37 °C within 2 h. Cumulus–oocyte complexes (COCs) were aspirated using a disposable syringe with an 18-gauge needle

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from follicles that were 3 to 8 mm in diameter. COCs with a homogeneous cytoplasm and at least three layers of cumulus cells were used for IVM. The IVM medium was TCM199 supplemented with 10% (v/v) follicular fluid of pigs (laboratory processed), 10% (v/v) FBS (fetal bovine serum), 10 IU/mL PMSG (pregnant mare serum

gonadotropin), 10 IU/mL hCG (human chorionic gonadotropin), 69 µg/mL L-cysteine, 10 IU/mL penicillin G sodium and 10 IU/mL streptomycin. Approximately 65 oocytes were cultured in a 500 µL IVM medium under 500 µL mineral oil and incubated for

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44 h at 39 °C in an atmosphere of 5% CO2 with saturated humidity.

2.2. In vitro production of parthenogenetic pig embryos

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After 44 h of IVM, COCs were denuded of the cumulus cells by vortexing in 0.1% (w/v) hyaluronidase for 2 min, and only those oocytes with the first polar body and

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even cytoplasm were selected for parthenogenetic activation. Matured oocytes were

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washed three times in activation medium (0.1 mg/mL PVA, 0.3 M mannitol, 0.1 mM

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CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 0.8 mM HEPES) and placed between two wires

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of a microslide 0.5 mm fusion chamber (model450; BTX, San Diego, CA, USA)

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covered with activation medium. Oocytes were stimulated with a direct current pulse of 1.2 kV/cm for 60 ms using BTX2001 (BTX, San Diego, CA, USA). Thereafter, the

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oocytes were washed three times with porcine zygote medium 3 (PZM-3)

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supplemented with 5 μg/mL cytochalasin B and transferred to the same medium for 4 h at 39 °C in an atmosphere of 5% CO2 with saturated humidity. Subsequently, 60 presumptive parthenogenetic embryos were transferred to 500 µL PZM-3 without CB

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for the next in vitro culture. The early-stage blastocysts on day 6 were collected, then they were used as fresh or vitrified blastocysts.

2.3. Vitrification and thawing of blastocysts

The OPS (open pulled straw) was used for the vitrification of pig parthenogenetic blastocysts. All procedures were performed on a 39 °C hot plate at room temperature. All solutions for vitrification and thawing were prepared using PZM-3 supplemented with 20% (v/v) fetal bovine serum as the basal medium (BM).

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For vitrification, 10 blastocysts were washed in an equilibrium solution (BM supplemented with 7.5% EG (Ethylene Glycol) and 7.5% DMSO (Dimethyl

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Sulphoxide) at 39 °C for 3 min. Then, the blastocysts were transferred to a

vitrification solution (BM supplemented with 15% EG, 15% DMSO and 0.4 M of

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sucrose) at 39 °C for 20 s. The blastocysts were subsequently loaded into OPS with

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the minimum amount of vitrification solution and immediately immersed in liquid

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nitrogen.

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For thawing, vitrified blastocysts were thawed with 0.3 M and 0.15 M sucrose in

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PZM-3 at 39 °C for 5 min, sequentially. Thawed blastocysts were washed three times with PZM-3 and then cultured with PZM-3 for the next 24 h at 39 °C. Only

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blastocysts with recovered blastocoel after culture from fresh or vitrified groups were

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used for the detection of mitochondrial function, apoptotic status and gene expression level.

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2.4. Mitochondrial membrane potential (ΔΨm) assay Mitochondrial ΔΨm was measured using 10 μM JC-1 (5,5′,6,6′-

tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) (Beyotime, Haimen, China) in PZM-3 at 39 °C for 20 min in the dark. After being washed three times with PZM-3, blastocysts were placed on slides and photographed using a laser scanning

confocal microscope (Nikon, Tokyo, Japan) with an emission wavelength of 529 nm for the green channel and 590 nm for the red channel. The signal intensities of green and red fluorescence were quantified in each image. The ratio of red to green for each

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oocyte was the value of ΔΨm.

2.5. TUNEL staining

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The amount of apoptosis in pig blastocysts was measured using the TUNEL

staining kit (Beyotime, Haimen, China). Fresh and vitrified blastocysts were fixed in 4%

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paraformaldehyde for 1 h at room temperature. After fixation, the embryos were

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washed in PZM-3 and permeabilized by incubation in 0.1% Triton X-100 for 2 min

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on ice. The embryos were then washed three times in PZM-3 and incubated with 50

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μL TUNEL staining solution (2 μL TdT enzyme and 48 μL fluorescently labeled

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liquid) in the dark for 1 h at 37 °C. After being counterstained with 5 μg/mL Hoechst 33342 for 10 min at 37 °C, embryos were washed in PZM-3, mounted with slight

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coverslip compression, and examined using a fluorescence microscope (Olympus,

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Tokyo, Japan). Blue fluorescence after Hoechst staining showed the total cells of blastocyst, and green fluorescence indicated the cells that had undergone apoptosis. The ratio of the number with green fluorescent cells to blue fluorescent cells of all

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blastocysts in different groups was the apoptotic rate of each group.

2.6. Detection of caspase activities The CaspACETM FITC-VAD-FMK in situ marker Kit (Promega, Madison, WI,

USA) and CaspGLOWTM Fluorescein Active Caspase-3, Caspase-8, and Caspase-9 Staining Kits (Beyotime, Haimen, China) were used to detect the pan-caspase, caspase-3, caspase-8, and caspase-9 activities in pig oocytes, respectively. After staining, fresh and vitrified blastocysts were observed under the fluorescence

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microscope (Olympus, Tokyo, Japan). The fluorescent intensity of each blastocyst was measured using Image-Pro plus 6.0 software (Media Cybernetics, Rockville, MD,

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USA). Background fluorescence was subtracted from the final values before analysis.

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2.7. RNA isolation and real-time PCR

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The RNA extraction and reverse transcription reactions were performed on ice and

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minimized operating time. One hundred fifty blastocysts were used for RNA

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extraction and real-time PCR analysis. Total RNA was isolated using an RNAprep

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pure Micro Kit (Tiangen, Beijing, China). Reverse transcription was performed using the Fast Quant RT Kit (Tiangen, China). Real time PCR was performed using

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SYBR®Premix Ex Taq™ II (TaKaRa, Shiga, Japan). Gene annotations were obtained

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from Gene Bank. Details of the target genes are presented in Table 1. Each reaction mixture (20 μL) consisted of 10 μL SYBR® Premix Ex Taq II, 0.8μL each of forward (10 μM) and reverse (10 μM) primers, 0.4 μL Rox Reference Dye II, 2 μL cDNA and

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6 μL dH2O. The cDNA was synthesized using the 7500 Real-time PCR system (Applied Biosystem) using the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The relative abundance of mRNA was determined by using the 2-ΔΔCt method, and GAPDH was used as a reference gene to calculate relative

abundance of mRNA.

2.8. Statistical analyses Data were analyzed by T-test (percent value) and one-way ANOVA (mean value)

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using SPSS 18.0 (SPSS Inc., Chicago, IL, USA), and P < 0.05 were considered

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statistically significant. All experiments were repeated at least three times.

3. Results

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3.1. Effect of vitrification on the in vitro developmental capacity of pig

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parthenogenetic blastocysts

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Data for the results of this study are shown in Table 2 and depicted in Figure 1. The

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blastocoel recovery rate at 24 h after thawing was much less than that in the fresh

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blastocyst group with an additional 24 h of culture (31.0% compared with 100%, respectively, P < 0.05). The total cell number per blastocyst from vitrified blastocysts

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was also much less than that from the fresh blastocyst group (31.81 ± 4.59 compared

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with 38.17 ± 5.78, respectively, P < 0.05).

3.2. Effect of vitrification on mitochondrial ΔΨm and amount of apoptosis of

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parthenogenetic blastocysts

The data for the JC-1 and TUNNEL staining are included in Table 3. Figure 2 and Figure 3 are the representative images of JC-1 staining and TUNEL staining. Compared with the fresh group (1.02), the mitochondrial ΔΨm in the vitrified group

(0.46) was less (P < 0.05). The number of apoptotic cells in vitrified blastocysts (8.1%) was much greater than that of fresh blastocysts (3.9%, P < 0.05).

3.3. Effect of vitrification on pan-caspase, caspase-8, caspase-9 and caspase-3

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activities of porcine parthenogenetic blastocysts

In situ staining was used to detect pan-caspase, caspase-8, caspase-9 and caspase-3

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activities. The data are shown in Table 4. The activities of pan-caspase, caspase-8,

caspase-9 and caspase-3 in the vitrified group (20.7, 20.6, 17.6 and 19.9, respectively)

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were markedly greater than those in the fresh group (7.4, 6.5, 5.5 and 6.3, P < 0.05).

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As depicted in Figure 4, the greater fluorescence intensity indicates greater caspase

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activity.

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in parthenogenetic blastocysts

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3.4. Effect of vitrification on the relative abundance of apoptosis-related gene mRNA

The data for relative abundance of mRNA for apoptosis-related genes in fresh and

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vitrified groups are depicted in Figure 5. In the death receptor-mediated extrinsic

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apoptotic pathway, the relative abundance of mRNA of caspase-8 and TNF-α genes was markedly greater in the vitrified group than those in the fresh group (10.0 compared with 1.0 and 9.5 compared with 1.0, respectively, P < 0.05). For the

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mitochondria-mediated intrinsic apoptotic pathway, the relative abundance of mRNA or the caspase-9 was also markedly greater in the vitrified group than that in the fresh group (10.3 compared with 1.0, respectively, P < 0.05), and the relative abundance of mRNA for the anti-apoptotic genes, Bcl-2 and SOD-1, were much less in the vitrified

group than those in the fresh group (0.3 compared with 1.0 and 0.34 compared with 1.0, respectively, P < 0.05).

4. Discussion

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In the present study, both the blastocoel recovery rate and total cells per blastocyst was markedly less in pig vitrified parthenogenetic blastocysts. These results were

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similar to those of Fujino et al. (2008) with pig embryos and Marcojiménez et al.

(2016) with rabbit embryos. The results from the present study indicate vitrification

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could affect the developmental capacity and quality of embryos.

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Mitochondria have an important role in the development of embryos, and

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mitochondrial damage was involved in the mitochondria-mediated intrinsic apoptotic

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pathway (Fulda et al., 2013; Lee et al., 2014). The mitochondrial ΔΨm could reflect

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the state of mitochondrial function. The JC-1 staining can be used to detect the mitochondrial ΔΨm in mammalian oocytes (Ou et al., 2012). In the present study JC-1

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staining was used to evaluate the mitochondrial function of vitrified parthenogenetic

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pig blastocysts and the mitochondrial ΔΨm of vitrified blastocysts was less than that of fresh blastocysts. The results of the present study are very similar to those of Dai et al. (2015) with pig oocytes and Lei et al. (2014) with mouse oocytes. Results of the

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present study indicated that vitrification could cause severe mitochondrial dysfunction of parthenogenetic pig blastocysts. Adding a mitochondrial function enhancer before and after freezing, such as MitoQ, might be an effective method to improve the embryonic development capacity after vitrification.

The DNA fragmentation is the main form of apoptosis, and as such, it can be used as an important indicator to evaluate the quality of blastocysts (Byrne et al., 1999). In previous studies it was determined that the embryonic developmental capacity is negatively correlated with the amount of apoptosis of embryos (Neuber et al., 2002;

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Knijn et al., 2003; Findikli et al., 2004; Contreras et al., 2008). As a result of cryopreservation of embryos, Park et al. (2006) found that vitrification could cause an

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increase in DNA fragmentation of frozen-thawed blastocysts of cattle. Wu et al. (2016) and Inaba et al. (2016) also found that the poor developmental capacity of pig and

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cattle embryos might be closely related with the increased amount of apoptosis after

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thawing. In the present study the rate of apoptotic cells in vitrified parthenogenetic

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blastocysts of pigs after TUNEL staining was markedly greater, which was similar to

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results of previous studies. Caspase is the inducing agent for apoptosis, reducing the

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caspase activities of frozen embryos by adding caspase inhibitor, such as Z-VAD-FMK, could be one of the methods investigated to reduce apoptosis rate and

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improve freezing efficiency of embryos. of apoptosis in

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Pan-caspase in situ staining is widely used to detect the amount

mammalian embryos (Gualtieri et al., 2009; Ebrahimi et al., 2010; Kitazumi and Tsukahara, 2011). Gualtieri et al. (2009) reported that the pan-caspase activities of

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frozen human oocytes were greater than those of fresh oocytes. Vallorani et al. (2012) also reported that vitrification could increase the amount of pan-caspase of MII stage pig oocytes. In the present study, the amount of pan-caspase of vitrified parthenogenetic pig blastocysts was greater, which was similar to the results of the

two studies previously described studies. The central process of apoptosis is induced by cysteine proteases called caspases. The mitochondria-mediated pathway and death receptor-mediated pathway are the main two pathways of cell apoptosis. Caspase-9 is an important protein in the

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mitochondria intrinsic apoptotic pathway (Basak et al., 2014), caspase-8 is an important protein that stimulates the extrinsic death receptor apoptotic pathway (Lord

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and Gunawardena, 2012), and caspase-3 is a functional protein in both of the common pathways (Kitazumi and Tsukahara, 2011). Changes in the activities of the three

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caspases can effectively reflect the main pathways of cell apoptosis caused by

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vitrification. In the present study the three caspase activities of vitrified blastocysts

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were greater than those of fresh blastocysts, which indicates that both mitochondria-

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and death receptor-mediated pathways contributed to the occurrence of apoptosis of

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parthenogenetic pig blastocysts after vitrification. This study was very similar to other studies where there was cryopreservation of pig oocytes and mesenchymal stem cells.

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Dai et al. (2015; 2016) and Niu et al. (2016) reported that both mitochondria and

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death receptor apoptotic pathways were involved in the apoptosis of vitrified pig oocytes. Bissoyi and Pramanik (2014) reported that cryopreservation induces the conversion of inactive pro-caspase-3 and pro-caspase-8 to active caspase-3 and

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caspase-8, respectively, in mesenchymal stem cells. The results of the present study provided for a greater understanding of the apoptotic process of frozen embryos, and enriched the understanding of cryobiology in these regards. The TNF-α and caspase-8 proteins are important cytokines in the death receptor

apoptotic pathway (Etewa et al., 2015). The relative abundance of TNF-α and caspase-8 mRNA was marked greater in the present study, which suggested that the death receptor apoptotic pathway was involved in the occurrence of apoptosis after vitrification. The Bcl-2 protein has the capacity to promote cell survival and

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anti-apoptosis. In studies of the vitrification of rat (Dhali et al., 2007) and human (Brenner et al., 1997) embryos, BclL-2 gene expression was decreased, which was

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consistent with the results in the present study with vitrified pig blastocysts. The

SOD-1 protein can clear the oxidative damage caused at the mitochondria and can

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cause an inhibition of apoptosis mediated by the mitochondrial pathway (Dawson et

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al., 2015); the decreased expression of the SOD-1 gene after vitrification

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demonstrated that the antioxidant and anti-apoptotic capacity in blastocysts were

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inhibited. The caspase-9 gene has a key role in the mitochondria intrinsic apoptosis

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pathway, and increase in expression of this gene indicated endogenous apoptosis increased after vitrification.

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In conclusion, the poor developmental capacity of vitrified parthenogenetic pig

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blastocysts was closely related with the decrease of mitochondrial function and increase of apoptosis. It is hypothesized that both mitochondria- and death receptor-mediated apoptotic pathways are involved in the apoptosis of vitrified pig

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blastocysts.

Declaration of Interest The authors declare that there is no conflict of interest that could be perceived as

prejudicing the impartiality of the research reported. Funding This research was funded by the Natural Science Foundation of China (31372315), the Shanghai Committee of Science and Technology (15ZR1430100) and Climbing

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plan of Shanghai Academy of Agricultural Sciences (PG171). Acknowledgements

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The authors thank all members in De-fu Zhang’s laboratory who contributed to the

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sample determination.

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Soc. Parasitol. 45, 47-60. Findikli, N., Kahraman, S., Kumtepe, Y., Donmez, E., Benkhalifa, M., Biricik, A., Sertyel, S. , Berkil, H. , Oncu, N., 2004. Assessment of DNA fragmentation and aneuploidy on poor quality human embryos. Reprod. Biomed. Online. 8, 196-206. Fujino, Y., Kojima, T., Nakamura, Y., Kobayashi, H., Kikuchi, K., Funahashi, H., 2008. Metal mesh vitrification (MMV) method for cryopreservation of porcine embryos. Theriogenology 70, 809-817. Fulda, S., 2013. Alternative cell death pathways and cell metabolism. Int. J. Cell. Biol. 8, e463637. Gualtieri, R., Iaccarino, M., Mollo, V., Prisco, M., Iaccarino, S., Talevi, R., 2009. Slow cooling of human oocytes: ultrastructural injuries and apoptotic status. Fertil. Steril. 91, 1023-1034. Guicciardi, M. E., Gores, G. J., 2009. Life and death by death receptors. FASEB. J. 23, 1625-1637. Inaba, Y., Miyashita, S., Somfai, T., Geshi, M., Matoba, S., Dochi, O., Naqai, T., 2016. Cryopreservation method affects DNA fragmentation introphectoderm and the speed of re-expansion in bovine blastocysts. Cryobiology 72, 86-92. Isachenko, V., Soler, C., Isachenko, E., Perez-Sanchez, F., Grishchenko, V., 1998. Vitrification of immature porcine oocytes: effects of lipid droplets, temperature, cytoskeleton, and addition and removal of cryoprotectant. Cryobiology 36, 250-253. Kitazumi, I., Tsukahara, M. 2011. Regulation of DNA fragmentation: the role of caspases and phosphorylation. FEBS. J. 278, 427-441. Knijn, H. M., Gjørret, J. O., Vos, P. L. A. M., Hendriksen, P. J. M., Weijden, B. C. V. D., Maddox-Hyttel, P., Dieleman, S. J., 2003. Consequences of in vivo development and subsequent culture on apoptosis, cell number, and blastocyst formation in bovine embryos. Biol. Reprod. 69, 1371-1378. Lee, S. K., Zhao, M. H., Kwon, J. W., Li, Y. H., Lin, Z. L., Jin, Y. X., Kim, N. H., Cui, X. S., 2014. The association of mitochondrial potential and copy number with pig oocytematuration and developmental potential. J. Reprod. Dev. 60, 128-135. Lei, T., Guo, N., Tan, M. H., Li, Y. F., 2014. Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution. J. Huazhong. Univ. Sci. Technolog. Med. Sci. 34, 99-102. Lord, C. E., Gunawardena, A. H., 2012. Programmed cell death in C. elegans, mammals and plants. Eur J Cell Biol. 91, 603-613. Marcojiménez, F., Jiméneztrigos, E., Almelamiralles, V., Vicente, J. S., 2016. Development of cheaper embryo vitrification device using the minimum volume method. PLoS. One. 11, e0148661. Neuber, E., Luetjens, C. M., Chan, A. W., Schatten, G. P. 2002. Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL. Theriogenology 57, 2193-202. Niu, Y., Wang, C., Xiong, Q., Yang, X., Chi, D., Li, P., Liu, H., Li, J., Huang, R., 2015. Distribution and content of lipid droplets and mitochondria in pig

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parthenogenetically activated embryos after delipation. Theriogenology 83, 131-138. Niu, Y., Dai, J., Wu, C., Chen, Y., Zhang, S., Zhang, D., 2016. The application of apoptotic inhibitor in apoptotic pathways of MII stage porcine oocytes after vitrification. Reprod. domest. anim. 51, 953-959. Ou, X. H., Li, S., Wang, Z. B., Li, M., Quan, S., Xing, F., Guo, L., Chao, S. B., Chen, Z., Liang, X. W., Hou, Y., Schatten, H., Sun, Q. Y., 2012. Maternal insulin resistance causes oxidative stress and mitochondrial dysfunction in mouse oocytes. Hum. Reprod. 27, 2130-2145. Park, S. Y., Kim, E. Y., Cui, X. S., Tae, J. C., Lee, W. D., Kim, N. H., Park, S. P., Lim, J. H., 2006. Increase in DNA fragmentation and apoptosis-related gene expression in frozen-thawed bovine blastocysts. Zygote 14, 125-131. Ren, L., Fu, B., Ma, H., Liu, D., 2015. Effects of mechanical delipation in porcine oocytes on mitochondrial distribution, ROS activity and viability after vitrification. CryoLetters 36, 30-36. Romão, R., Bettencourt, E., Pereira, R. M., Marques, C. C., Baptista, M. C., Barbas, J. P., Oliveira, E., Bettencourt, C., Sousa, M., 2016. Ultrastructural characterization of fresh and vitrified in vitro- and in vivo-produced sheep embryos. Anat. Histol. Embryol. 45, 231-239. Vallorani, C., Spinaci, M., Bucci, D., Porcu, E., Tamanini, C., Galeati, G., 2012. Pig oocyte vitrification by cryotop method and the activation of the apoptotic cascade. Anim. Reprod. Sci. 135, 68-74. Wang, C., Youle, R. J., 2009. The role of mitochondria in apoptosis. Annu. Rev. Genet. 43, 11-22. Wu, G. Q., Quan, G. B., Shao, Q. Y., Lv, C. R., Jiang, Y. T., Zhao, Z. Y., Hong, Q. H., 2016. Cryotop vitrification of porcine parthenogenetic embryos at the early developmental stages. Theriogenology 85, 434-440.

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Fig. 1. Images of fresh parthenogenetic pig blastocysts and re-expended blastocysts at 24 h after thawing. (100×) A. Fresh blastocysts; B. Re-expended blastocysts at 24 h after thawing

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Fig. 2 Fluorescence images on LSCM of blastocysts in vitrified and fresh groups （100×） Left photo is fresh blastocysts using JC-1 staining; right is vitrified blastocysts using JC-1 staining

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Fresh blastocysts

Vitrified blastocysts

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Fig. 3. TUNEL assay combined with Hoechst33342 staining for blastocysts (400×). Left photo is the blastocyst using Hoechst 33342 staining; right is the corresponding blastocyst by TUNEL staining; Positions of the arrow is TUNEL positive cells

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Pan-caspase

Caspase-8

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activity

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Caspase-9 activity

Caspase-3 activity

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Fig. 4. Comparison of fluorescence intensity of different caspase activities in fresh and vitrified blastocysts.（100×） In situ marker Kits were used to detect the pan-caspase, caspase-8, caspase-9 and caspase-3; Fluorescent intensities for the four caspases of the vitrified groups were much greater than those of the fresh groups

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Fig. 5. Effect of vitrification on the relative abundance of apoptosis-related genes in parthenogenetic blastocysts Real-time PCR was used to detect the relative abundance of mRNA of the two groups, and GAPDH was used as the housekeeping gene; After freezing, the relative abundances of caspase-8, caspase-9 and TNF-α were much greater than those of fresh blastocysts, and the relative abundances of Bcl-2 and SOD-1 were much less than those of the fresh groups; Different letters (a, b) in the same genes indicate a difference between treatments (P < 0.05)

Tables

Table 1 Details of primers used for real-time PCR

Reverse

180

56

120

56

162

55

164

56

142

56

113

54

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TTGACTGTGCCG TGGAACTT CCAGATGTCCCA GGTTGCAT CCTGTTCTCCCA GACAGTCC CATCCCAGCCTC CGTTATCC CCAAAGCCTGGA CCATTTGC GATGGTGTGGCC ACTGTGTA

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CACGATGGTGAAG GTCGGAG ATTCAGGGATGTGT GGCCTG AGGCCCTGCTGAA GGAAAATCT GAACTGGGGGAGG Bcl-2 ATTGTGG Caspa AACTTCTGCCATGA se-9 GTCGGG CuZn GATGGTGTGGCCA SOD CTGTGTA

NM_0012 06359 NM_2140 22 NM_0010 31779

AB271960 XM_0031 27618 NM_00119 0422.1

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GAP DH TNFα Caspa se-8

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Forward

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Gene

GenBank Accession numer

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Size /bp

Anneal ing Temper ature (°C)

Primer sequences (5′-3′)

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Table 2 Effect of vitrification on blastocoel recovery and total cell of parthenogenetic

Total cell number of per blastocyst a Fresh blastocysts 100.00 (60/60) 38.17±5.78a Vitrified blastocysts 31.03 (18/58)b 31.81±4.59b Data of total cell number of per blastocyst are mean values with pooled standard error of the mean (SEM); Within a row, the means without a common superscript letter (a,b) differ (P <0.05)

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Blastocoel recovery rate (%)

Table 3 Effect of vitrification on mitochondrial membrane potential (ΔΨm) and amount of apoptosis of parthenogenetic pig blastocysts

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Groups ΔΨm (n) TUNEL positive rate (n) a Fresh blastocysts 1.02 ± 0.12 (56) 3.92% (58)b Vitrified blastocysts 0.46 ± 0.04 (58)b 8.13% (59)a Data of ΔΨm are mean values with pooled standard error of the mean (SEM); Within a row, the means without a common superscript letter (a,b) differ (P <0.05) ΔΨm = mitochondrial membrane potential; n was the total blastocysts used for each experiment

Table 4 Effect of vitrification on different caspase activities of parthenogenetic pig blastocysts

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Pan-caspase Caspase-8 Caspase-9 Caspase-3 activity activity activity activity 7.41 ± 6.46 ± 5.47 ± 6.32± Fresh blastocysts b b b 2.24(63) 0.66(66) 0.69(66) 0.73(78)b Vitrified 20.65 ± 20.60± 17.58± 19.88 ± a a a blastocysts 1.26(69) 1.31(78) 1.39(69) 1.05(81)a Data are mean values with pooled standard error of the mean (SEM) Within a row, the means without a common superscript letter (a,b) differ (P <0.05) n was the total blastocysts used for each experiment

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