C-Phycocyanin supplementation during in vitro maturation enhances pre-implantation developmental competence of parthenogenetic and cloned embryos in pigs

Accepted Manuscript C-Phycocyanin supplementation during in vitro maturation enhances pre-implantation developmental competence of parthenogenetic and cloned embryos in pigs Shuang Liang, Jing Guo, Yong Xun Jin, Bao Yuan, Jia-Bao Zhang, Nam-Hyung Kim PII:

S0093-691X(17)30420-X

DOI:

10.1016/j.theriogenology.2017.09.001

Reference:

THE 14248

To appear in:

Theriogenology

Received Date: 29 June 2017 Revised Date:

30 August 2017

Accepted Date: 1 September 2017

Please cite this article as: Liang S, Guo J, Jin YX, Yuan B, Zhang J-B, Kim N-H, C-Phycocyanin supplementation during in vitro maturation enhances pre-implantation developmental competence of parthenogenetic and cloned embryos in pigs, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.09.001. 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.

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Revised

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C-Phycocyanin supplementation during in vitro maturation enhances

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pre-implantation developmental competence of parthenogenetic and cloned

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embryos in pigs

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Shuang Liang a, b, Jing Guo b, Yong Xun Jin a, Bao Yuan a, Jia-Bao Zhang a* &

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Nam-Hyung Kima, b*

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a

Department of Laboratory Animal Center, College of Animal Sciences, Jilin University,

Changchun, 130062, China. b

Department of Animal Science, Chungbuk National University, Cheongju, Chungbuk,

361-763, Republic of Korea.

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*Corresponding author: Nam-Hyung Kim and Jia-Bao Zhang.

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E-mail addresses: [email protected] (N.-H. Kim) and [email protected] (J.-B. Zhang).

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ABSTRACT

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C-Phycocyanin (C-PC), a protein from green microalgae, has been suggested to possess

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various biological activities, including antioxidant and free radical scavenging properties. The

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aim of the current study was to explore the effects of C-PC on the maturation of porcine

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oocytes and subsequent developmental competence after parthenogenetic activation and

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somatic cell nuclear transfer (SCNT) as well as the underlying mechanisms. There was no

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significant improvement in nuclear maturation rates between the control and C-PC

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supplementation groups (1, 3, 5, 10 µg/mL). However, supplementation of 5 µg/mL C-PC in

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the maturation medium significantly increased blastocyst formation and hatching rates after

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parthenogenetic activation (59.6 ± 3.6% and 33.0 ± 2.6% vs. 49.8 ± 3.5% and 27.4 ± 2.4%,

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respectively). In addition, the presence of C-PC during the maturation period significantly

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improved blastocyst formation rates and total cell numbers after SCNT (24.8 ± 1.9% and 42.2

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± 3.3 vs. 21.6 ± 2.2% and 39.5 ± 3.4, respectively) compared to the control group.

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Furthermore, cellular proliferation and the expression of pluripotency-related genes (SOX2

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and NANOG) were increased in cloned blastocysts derived from the C-PC supplemented

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group. Importantly, C-PC supplementation during maturation not only improved cumulus

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expansion and increased the expression of cumulus expansion-related genes (HAS2, PTX3,

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and PTGS2), but also enhanced antioxidant capacity, improved mitochondria function, and

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decreased cathepsin B activity in porcine oocytes. These results demonstrate that C-PC may

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be useful for improving porcine oocyte quality and subsequent developmental competence in

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embryos.

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Keywords: In vitro maturation, C-phycocyanin, Porcine oocyte, Parthenogenetic activation,

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Somatic cell nuclear transfer

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1. Introduction Somatic cell nuclear transfer (SCNT) is a useful technology with applications in

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transgenic animals, preserving species, stem cell research, and regenerative medicine. During

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SCNT, the nucleus of a somatic cell is transferred into a healthy enucleated metaphase II (MII)

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stage oocyte to generate a new individual compatible with the somatic cell donor [1]. It has

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been suggested that pigs share several characteristics with humans, and generating genetic

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modifications in pigs using SCNT technology may be an excellent model for biomedical

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studies and xenotransplantation [2, 3]. Many studies have attempted to improve the success

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rate of producing porcine cloned embryos in vitro [4-6]. However, compared with embryos

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derived from oocytes matured in vivo, the developmental competence of embryos derived

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from oocytes matured in vitro remains low [7, 8].

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Many factors affect the efficiency of generating cloned pigs with SCNT technology [9,

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10]. Among these factors, oocyte quality is the most important factor affecting the

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developmental competence of cloned embryos [11, 12]. Oocyte maturation is a complex

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process that includes nuclear and cytoplasmic maturation [13]. Any errors in this process can

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lead to the failure of further embryonic development [14]. Therefore, high-quality or

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developmentally competent oocytes are vital for fertilization and embryo development. In

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general, oocyte quality and developmental competence are obtained during follicular

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development under in vivo conditions [15]. When oocytes are liberated from their follicles,

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they can undergo spontaneous meiotic resumption under in vivo condition and become

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arrested in the MII stage of meiosis [16, 17]. Previous studies showed that incomplete nuclear

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maturation and cytoplasmic maturation of in vitro matured pigs oocytes explains the lower

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efficiency of embryo development in vitro [4, 18-20]. Many stressors, such as medium

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composition or culture conditions, affect the developmental competence and quality of in

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vitro matured porcine oocytes during in vitro maturation (IVM) [21-24]. To improve the

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quality of in vitro matured porcine oocytes, which undergo complete nuclear maturation and

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cytoplasmic maturation, several studies have focused on improving the efficiency of the IVM

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system via supplementation and/or the use of chemically defined media [25-28].

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ACCEPTED MANUSCRIPT C-phycocyanin (C-PC) is a biliprotein present in blue green algae, such as Spirulina

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platensis, which shows potential biological activities [29]. An increasing number of studies

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has suggested that C-PC can be used as a dietary supplement in food because of its

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anti-microbial, anti-inflammatory, neuroprotective, and hepatoprotective effects [30-32].

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C-PC is also a potential therapeutic agent for oxidative stress-induced disease [33]. Moreover,

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C-PC can prevent early signs of osteoarthritis caused by compressive stress and attenuate

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H2O2-induced oxidative stress [34]. A recent study showed that C-PC administration to

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D-galactose-induced

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effects and prevented oocyte fragmentation and aneuploidy by maintaining cytoskeletal

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integrity [35].

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aging mice significantly increased litter size through its antioxidant

The biological activities and physiological functions of C-PC for curing diseases are

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well documented. However, information on the effects of C-PC on livestock oocyte

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maturation and their subsequent embryonic development is lacking. Therefore, the objective

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of the present study was to investigate the effect of C-PC supplementation of maturation

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medium on porcine oocyte maturation and subsequent embryonic development after

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parthenogenetic activation (PA) and SCNT. We compared blastocyst formation and quality,

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oocyte nuclear maturation, cumulus expansion degree and relative gene expression (HAS2,

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PTX3, and PTGS2), intracellular levels reactive oxygen species (ROS), and glutathione

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(GSH), total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, cathepsin B

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activity, and mitochondria function in porcine oocytes.

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2. Material and Methods

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This study was approved by the Ethics Committee of Chungbuk National University,

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Korea and conducted in accordance with institutional guidelines. All chemicals used in this

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study were purchased from Sigma Chemical Co. (Sigma, St. Louis, MO, USA) unless stated

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otherwise.

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2.1 Porcine oocyte collection and in vitro maturation (IVM)

ACCEPTED MANUSCRIPT Ovaries were obtained from slaughtered sows at a local slaughterhouse and transported

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to the laboratory in sterile saline containing 75 µg/mL penicillin G and 50 µg/mL

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streptomycin sulfate at 35–37°C within 2 h in an insulated flask. Cumulus-oocyte complexes

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(COCs) were aspirated from antral follicles and collected under a stereo microscope. The

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COCs were washed three times in Tyrode's Lactate HEPES medium. Oocytes were only

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selected for further experiments if they had a homogeneous ooplasm and were surrounded by

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a minimum of three layers of cumulus cells. The COCs were cultured in tissue culture

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medium 199 (TCM-199, Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v)

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porcine follicular fluid, 1 µg/mL insulin, 75 µg/mL kanamycin, 0.91 mM Na pyruvate, 10

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ng/mL epidermal growth factor, 0.5 µg/mL follicle stimulating hormone, and 0.5 µg/mL

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luteinizing hormone for 42 h at 38.5°C in an atmosphere containing 5% CO2 at 100%

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humidity. Oocyte maturation was performed by culturing approximately 50 COCs in 500 µL

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of maturation medium in 4-well dishes containing various concentrations of C-PC (0, 1, 3, 5,

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10 µg/mL) during the entire maturation period. Before use, C-PC was dissolved in maturation

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medium to prepare a stock solution and stored in the dark at -20°C.

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2.2 PA, SCNT, and embryo culture

For PA, matured oocytes were parthenogenetically activated by two direct-current (DC)

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pulses of 120 V/mm for 60 µs in 300 mM mannitol containing 0.1 mM CaCl2, 0.05 mM

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MgSO4, 0.01% polyvinyl alcohol (PVA) (w/v), and 0.5 mM HEPES.

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For SCNT, porcine fetal fibroblasts were used as nuclear donors and cultured as

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previously described [36]. Oocytes were enucleated by aspirating the polar body and

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metaphase chromosomes in a small amount (<15% of the oocyte volume) of cytoplasm using

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a 25-µm beveled glass pipette (Humagen, Charlottesville, VA, USA). After enucleation using

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a fine injecting pipette, a single donor cell was inserted into the perivitelline space of the

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enucleated oocyte. Membrane fusion was induced by applying an alternating current field of

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2 V cycling at 1 MHz for 2 s, followed by a DC pulse of 200 V/mm for 20 µs, using a cell

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fusion generator (LF201; Nepa Gene, Chiba, Japan). Following fusion, the reconstructed

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embryos were placed in bicarbonate-buffered porcine zygote medium 5 (PZM-5) containing

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0.4 mg/mL bovine serum albumin (BSA) for 1 h prior to activation. Activation was

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performed by applying DC pulses of 150 V/mm for 100 µs in 297 mM mannitol containing

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0.1 mM CaCl2, 0.05 mM MgSO4, 0.01% PVA (w/v), and 0.5 mM HEPES. The activated oocytes or reconstructed embryos were cultured in bicarbonate-buffered

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PZM-5 containing 4 mg/mL BSA and 7.5 µg/mL cytochalasin B for 3 h to suppress extrusion

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of the pseudo-second polar body. Next, the activated oocytes or reconstructed embryos were

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thoroughly washed and cultured in bicarbonate-buffered PZM-5 supplemented with 4 mg/mL

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BSA for 7 days at 38.5°C in an atmosphere containing 5% CO2 at 100% humidity without

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changing the medium. The development of activated oocytes or reconstructed embryos into

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blastocysts was examined at 7 days after activation. To count total cell numbers, blastocysts

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were stained with 10 µg/mL Hoechst 33342 for 15 min, mounted on glass slides, and

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examined under a fluorescence microscope (IX70, Olympus, Tokyo, Japan).

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2.3 5-Bromo-2′-deoxyuridine (BrdU) analysis

Cell proliferation was assessed by performing a BrdU assay [37]. Blastocysts were

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incubated with 100 µM BrdU in a humidified atmosphere of 5% CO2 at 38.5°C for 6 h. These

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blastocysts were washed three times with Dulbecco's phosphate-buffered saline (PBS)

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containing 0.05% Tween 20 (PBS-T), fixed in ice-cold methanol for 20 min, and

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permeabilized with 0.2% Triton X-100 for 2 min. The blastocysts were then washed with

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PBS-T and treated with 2 N HCl at room temperature for 30 min. Next, the blastocysts were

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washed and incubated with a mouse anti-BrdU monoclonal antibody (Sigma; B2531) diluted

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1:10 at 4°C overnight. After washing with 0.1% BSA prepared in PBS, the blastocysts were

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incubated with a rabbit anti-mouse IgG Alexa Fluor 568-conjugated polyclonal antibody

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(1:200; Cat: A-11061; Invitrogen) at room temperature for 1 h. After extensive washing with

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PBS-T, embryos were counterstained with 10 µg/mL Hoechst 33342 for 15 min, mounted on

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glass slides, and examined under a confocal laser scanning microscope (LSM 510 and 710

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META, Zeiss, Oberkochen, Germany). Proliferating cells in blastocysts were counted using

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NIH Image J software (National Institutes of Health, Bethesda, MD, USA).

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2.4 Real-time quantitative RT-PCR analysis Total RNA was extracted from cumulus cells from the control and C-PC groups using a

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Dynabeads mRNA Direct Kit (Invitrogen) according to the manufacturer’s instructions.

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First-strand cDNA was synthesized by reverse transcription of mRNA using the Oligo (dT)

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primer and SuperScript TM III reverse transcriptase (Invitrogen). Real-time RT-PCR was

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performed with SYBR Green, a fluorophore that binds to double-stranded DNA, in a final

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reaction volume of 20 µL using a CFX96 touch real-time RT-PCR detection system (Bio-Rad,

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Hercules, CA, USA). Finally, gene expression was quantified using the 2-△△Ct method, with

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normalization to the mRNA expression of GAPDH. The PCR primers used to amplify each

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gene are listed in Supplementary Table 1.

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2.5 Detection of intracellular ROS and GSH levels

To determine intracellular ROS levels, oocytes were incubated for 15 min in PBS containing 0.1% PVA (PBS-PVA) medium and 10

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diacetate. To detect intracellular GSH levels, the oocytes were incubated for 30 min in

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PBS-PVA medium containing 10 µM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin dye.

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Fluorescence signals were captured as TIFF files using a digital camera (DP72; Olympus)

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connected to a fluorescence microscope. The same procedures were used for all groups of

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oocytes, including incubation, rinsing, and imaging. NIH Image J software was used to

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analyze the fluorescence intensities of the oocytes.

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2.6 Measurement of TAC and SOD activity

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To measure TAC, 100 oocytes in each group were washed three times with cold PBS

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prior to lysis. Oocytes were lysed by sonication in cold PBS and centrifuged at 10,000 ×g for

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10 min at 4°C; the supernatant was retained for analysis. To measure SOD activity, 100

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oocytes in each group were placed in 500 µL cold 1X lysis buffer (10 mM Tris, PH 7.5, 150

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mM NaCl, 0.1 mM EDTA), lysed by sonication, and centrifuged at 1200 rpm for 10 min. The

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supernatant was assayed for CAT activity (STA-360-T, Cell Biolabs, San Diego, CA, USA)

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and SOD activity (STA-340-T, Cell Biolabs) using commercial assay kits according to the

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manufacturer’s instructions.

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2.7 Mitochondrial membrane potential (∆Ψm) assay To measure ∆Ψm, oocytes were washed three times with PBS-PVA and incubated in in

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5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1; Invitrogen) for 30

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min. ∆Ψm was calculated as the ratio of red florescence (corresponding to activated

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mitochondria; J-aggregates) to green fluorescence (corresponding to less active mitochondria;

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J-monomers) [38]. Fluorescence signals were captured as a TIFF file using a digital camera

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connected to a fluorescence microscope. The fluorescence intensity of the images were

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processed and measured using NIH Image J software.

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2.8 Cathepsin B activity assays

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Intracellular cathepsin B activity was detected using a commercial Magic red cathepsin

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B assay kit (Immunochemistry Technologies, LLC, Bloomington, MN, USA) according to

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the manufacturer’s protocol. Briefly, oocytes were incubated in 500 µL PBS-PVA, with 2 µL

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reaction mix for 20 min at 38.5°C in a humidified atmosphere of 5% CO2. The fluorescence

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signals were captured as a TIFF file using a digital camera connected to a fluorescence

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microscope. NIH Image J software was used to analyze the fluorescence intensities of the

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oocytes.

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2.9 Statistical analysis

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All data were subjected to arcsine transformation prior to statistical analysis and

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presented as the mean ± SEM. Comparisons of data between groups were performed with

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Student's t test. All statistical analysis was performed using the software package GraphPad

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Prism (version 6.01; GraphPad, Inc., La Jolla, CA, USA). p < 0.05 was considered

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statistically significant.

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3. Results

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3.1 Effects of C-PC supplementation during maturation period on polar body extrusion (PBE)

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and developmental competence of pig oocytes We first evaluated the effects of different concentrations (0, 1, 3, 5, 10 µg/mL) of C-PC on

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the PBE of porcine oocytes. The PBE rate was determined after 42 h of IVM and the results

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are shown in Fig. 1A and B. The rate of PBE ranged from 83.3 ± 2.3% to 87.1 ± 2.4% and did

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not differ significantly between the control group and C-PC-supplemented groups (p > 0.05).

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We further investigated the development of porcine oocyte PA after IVM. As shown in Fig.

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1C and D, there was a significant increase in blastocyst formation and the hatching rates in

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the 5 µg/mL C-PC supplementation group compared to in the control group (59.6 ± 3.6% and

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33.0 ± 2.6% vs. 49.8 ± 3.5% and 27.4 ± 2.4%, respectively; p < 0.05). No significant

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differences in blastocyst formation and hatching rates were observed among the control and 0,

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1, 3, and 10 µg/mL supplementation groups. These results revealed 5 µg/mL as the optimal

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concentration; hence, this was used in the subsequent experiments.

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3.2 C-PC supplementation during maturation period improves porcine SCNT embryo

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development

We next assayed whether C-PC supplementation during the maturation period improved

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the developmental competence of porcine SCNT embryos. The analysis of in vitro SCNT

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embryo developmental competence revealed that C-PC supplementation during the

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maturation period significantly improved the developmental potency of IVM oocytes. As

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shown in Fig. 2, the frequency of blastocyst formation and number of blastocyst cells were

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significantly increased in the C-PC group (24.8 ± 1.9% and 42.2 ± 3.3, respectively)

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compared to in the control group (21.6 ± 2.2% and 39.5 ± 3.4, respectively, p < 0.05). We

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further evaluated the cellular proliferative capacity in these SCNT embryos using the BrdU

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assay. We found that the percentage of BrdU-positive cells in blastocysts derived from mature

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COCs in the C-PC group was significantly higher than that in the control group (Fig. 3A and

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B; p < 0.05). Consistent with this result, expression of the pluripotency markers SOX2 and

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NANOG was increased significantly in blastocysts obtained from C-PC-supplemented COCs

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than those in the control group (Fig. 3C and D; p < 0.05).

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3.3 Effects of C-PC supplementation during maturation period on cumulus expansion in

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porcine COCs Cumulus expansion is necessary for oocyte maturation and is routinely employed IVM as a

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gross indicator of oocyte quality [39]. Therefore, we evaluated the effects of C-PC

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supplementation during the maturation period on cumulus expansion, followed by an analysis

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of the degree of cumulus cell expansion and expression of genes related to cumulus

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expansion genes (HAS2, PTX3, and PTGS2). As shown in Fig. 4A, C-PC supplementation

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during the maturation period significantly increased the proportion of COCs exhibiting

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complete cumulus expansion (degree 4, p < 0.05). However, other degree of cumulus cell

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expansion did not differ significantly between the C-PC-supplemented COC group and

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control group (p > 0.05). We also observed increased expression of cumulus

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expansion-related genes (HAS2, PTX3 and PTGS2) after C-PC supplementation during the

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maturation period (Fig. 4B; p < 0.05). These results suggest that C-PC supplementation

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during the maturation period improves cumulus expansion in porcine COCs.

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3.4 Effects of C-PC supplementation during maturation period on intracellular ROS and

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GSH level in porcine oocytes

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Because C-PC has antioxidant properties, we hypothesized that C-PC increases the

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resistance of embryos to oxidative stress. Therefore, we evaluated the effects of C-PC

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supplementation during the maturation period on intracellular ROS and GSH levels in

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porcine after IVM. As shown in Fig. 5A, significantly lower intracellular ROS levels were

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observed in the C-PC supplementation group during the maturation period (p < 0.05). The

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intracellular GSH level was significantly increased in the C-PC-supplemented group

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compared to in the control group (p < 0.05; Fig. 5B). In addition, we evaluated SOD activity

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and TAC in porcine after IVM. SOD activity was significantly increased in the

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C-PC-supplemented group compared to in the control group (p < 0.05; Fig. 5C). Similarly,

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TAC was significantly increased in the C-PC-supplemented group compared to in the control

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group (p < 0.05; Fig. 5D).

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3.5 Effects of C-PC supplementation during maturation period on mitochondria function in

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porcine oocytes Mitochondria in mammalian cells have a major effect on the production of cellular energy.

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To further explore the mechanism by which C-PC supplementation during the maturation

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period improves the developmental potential of porcine oocytes, ∆Ψm was evaluated.

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Representative images of mitochondrial ∆Ψm are shown in Fig. 7A. The average value of

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∆Ψm (Fig. 7B) in oocytes significantly increased upon C-PC supplementation during the

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maturation period (p < 0.05).

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3.6 Effects of C-PC supplementation during maturation period on cathepsin B activity in

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porcine oocytes

To further analyze the influence of C-PC supplementation during the maturation period on

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the quality of porcine oocytes, we evaluated cathepsin B activity in oocytes after IVM. As

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shown in Fig. 6A and B, matured oocytes derived from the supplemented group showed

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significantly decreased cathepsin B activity compared to the control group (p < 0.05).

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4. Discussion In the present study, we investigated whether supplementation of C-PC during the IVM

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period improves porcine oocyte quality and pre-implantation embryo development after PA

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and SCNT. This study revealed that C-PC enhanced the developmental potential of porcine

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oocytes when supplemented during the IVM period. Moreover, we found that C-PC

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supplementation during the IVM period improved the developmental competence of PA and

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SCNT embryos. Importantly, C-PC decreased intracellular ROS levels, increased intracellular

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GSH levels, improved mitochondria function, and decreased intracellular cathepsin B activity

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during the IVM period, suggesting that C-PC promotes porcine oocyte cytoplasm maturation.

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This is the first study showing that C-PC is correlated with porcine oocyte quality and

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subsequent embryo developmental competence after PA and SCNT.

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Oocyte quality, including nuclear and cytoplasm maturation, is an important determinant

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of embryo developmental competence [40]. Cloned embryos can be produced using in vivo or

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in vitro matured oocytes [41, 42]. However, oocytes matured in vitro frequently exhibit low

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quality, including incomplete nuclear and cytoplasmic maturation, compared to in vivo

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matured oocytes [43]. A previous study suggested that the decreased quality of porcine

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oocytes influences both pre-implantation cloned embryonic development and fetal

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development [44]. Therefore, enhancing oocyte quality is very important for the

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developmental ability of embryos produced by SCNT. In the present study, we found that the

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proportion of PA embryos undergoing blastocyst formation and hatching significantly

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increased when the IVM medium was supplemented with 5 µg/mL C-PC. However,

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unexpectedly, C-PC supplementation during the IVM period did not affect porcine oocyte

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PBE. Further analyses revealed that C-PC supplementation during the IVM period clearly

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improved SCNT embryo development and the quality of these embryos as indicated by total

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cell numbers, cellular proliferation, and pluripotency gene expression in blastocysts. These

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results highlight the positive effect of C-PC supplementation during the IVM period on the

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developmental competence of PA and SCNT embryos.

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During oocyte maturation, cumulus cell expansion is necessary for oocyte maturation

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and the acquisition of developmental competence by releasing and mediating signals to

ACCEPTED MANUSCRIPT oocytes [45, 46]. The degree of cumulus expansion is routinely evaluating during IVM as a

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gross indicator of oocyte maturation and the outcome of embryo in vitro culture [35].

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Cumulus expansion may play a role as a structural component of expanded cumuli and

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molecular signals regulating oocyte maturation [47]. The oocytes with high fertilizing

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capacity and developing competence have been attributed to expansion of cumulus cell [48].

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In the current study, supplementation with C-PC during the IVM period dramatically

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increased cumulus expansion of porcine cumulus cells and expression of expansion-related

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transcripts in cumulus cells (HAS2, PTX3, and PTGS2). Therefore, C-PC supplementation

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during the IVM period improves the developmental competence of PA and SCNT embryos in

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part by affecting porcine cumulus cell expansion.

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Oxidative stress is characterized by overproduction of free radicals, which can disrupt

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the balance of ROS and antioxidants under normal physiological conditions. ROS levels

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within an optimal range is required for normal cell metabolism, while excessive ROS

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generation may negatively influence cell membranes, protein synthesis, and lipid metabolism

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[49]. It has been demonstrated that high ROS levels in oocytes are detrimental to oocyte

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maturation and fertilization and impair embryo development [38]. The maturation status of

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oocyte cytoplasm plays an important role in successful embryonic genome activation at the

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onset of embryonic development by regulating reprogramming gene expression [50]. Porcine

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oocytes are particularly sensitive to damage caused by ROS because of the high lipid

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concentrations in the cytoplasm [51]. Therefore, suppression or removal of excess ROS using

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antioxidants may improve porcine oocyte quality and subsequently embryo development.

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Supplementation of antioxidants during the IVM period has been shown to influence oocyte

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maturation and embryo development [52]. It had been suggested that supplementation with

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antioxidants in IVM medium, such as β-mercaptoethanol and L-carnitine, improves SCNT

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embryonic development [53] [54]. This may be because of increased transcription factor

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expression, which stimulates nuclear reprogramming. Therefore, we hypothesized that C-PC

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affects porcine oocyte maturation by regulating oxidative stress-induced impairments. We

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found that IVM media supplemented with 5 µg/mL C-PC effectively decreased intracellular

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ROS levels in mature oocytes. A previous study suggested that supplementation of C-PC can

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effectively prevent H2O2-induced oxidative stress and early signs of osteoarthritis [34]. In

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addition, C-PC can protect and repair the ischemic brain via improved antioxidative,

349

neurotrophic,

350

doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes [56]. Therefore, C-PC

351

reduces ROS levels in oocytes during the IVM period, thus improving porcine oocyte quality

352

and enhancing subsequent embryo developmental competence after PA and SCNT. This

353

explains why C-PC affect porcine cumulus cell expansion may occur through decreased

354

oxygen tension in close proximity to the oocyte as a result of active cumulus cell metabolism

355

[57].

anti-inflammatory

mechanisms

[55].

C-PC

also

ameliorated

SC

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and

GSH is an important antioxidant found in mammalian cells and functions as a scavenger

357

of ROS [58]. Intracellular GSH are used to predict cytoplasmic maturation in porcine oocytes

358

[21]. In vivo matured oocytes exhibit higher GSH and developmental competence than in

359

vitro matured oocytes [59, 60]. Therefore, increasing the intracellular GSH level can protect

360

oocytes from oxidative damage and increase cytoplasmic maturation. A previous study

361

suggested that supplementation of antioxidants during IVM influences oocyte maturation and

362

improves its developmental potency by increasing intracellular GSH levels. In the present

363

study, inclusion of 5 µg/mL C-PC in the IVM media significantly improved intracellular

364

GSH levels. SOD is an effective defense enzyme that catalyzes the dismutation of superoxide

365

anions into H2O2. A previous study showed that C-PC restores SOD and GSH-Px activities in

366

CCl4-induced damaged livers and increases GSH level [61]. Furthermore, an imbalance

367

between TAC and increased ROS levels surrounding the embryo may lead to oxidative stress

368

[62, 63]. In the present study, we found that C-PC supplementation during the IVM period

369

effectively increased the TAC and activity of SOD in porcine oocytes. Therefore, C-PC

370

supplemented during the maturation period promotes cytoplasmic maturation of oocytes,

371

which may be because increased intracellular GSH levels contribute to higher embryonic

372

development following PA and SCNT.

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356

373

Mitochondria are major producers of ROS and ATP, which influence oocyte

374

developmental potential and are associated with fertilization and embryonic development

375

[64]. ∆Ψm is commonly used as an indicator of mitochondrial function in oocytes [65] and is

ACCEPTED MANUSCRIPT the driving force behind ATP production [66]. A previous study showed that C-PC restored

377

oxalate-induced decreases in ATP levels in MDCK cells [67]. In addition, C-PC can prevents

378

cisplatin-induced mitochondrial dysfunction and oxidative stress [68]. In the current study,

379

we found that supplementation of C-CP during the IVM period led to increased ∆Ψm in

380

porcine oocytes. It does not seem likely that C-CP improves porcine oocyte quality by

381

improving mitochondrial function. Cathepsin B is a lysosomal cysteine proteinase in normal

382

cells and tissues and plays an important role in intracellular proteolysis [69]. Recent studies

383

have shown that cathepsin B activity is a marker of poor-quality oocytes and embryos, which

384

is associated with low developmental competence [62, 63]. In the present study, the

385

decreased cathepsin B activity in porcine oocytes further revealed that C-CP supplementation

386

during the IVM period positively affected porcine quality.

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376

In conclusion, our results suggest that C-PC supplementation during the IVM period has

388

beneficial effects on porcine oocytes maturation, particularly cytoplasm maturation, and

389

subsequent developmental competence of embryos produced from PA and SCNT. These

390

findings will be useful for improving the efficiency of producing SCNT embryos in pig.

391

However, additional studies should examine these factors in fertilized embryos.

392

395 396 397

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Acknowledgements

This study was supported by a grant from the Next-Generation BioGreen 21 Program (PJ011126), Rural Development Administration (RDA), Republic of Korea.

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Additional Information

399

Competing Interests: The authors declare that they have no competing interests.

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Figure legends

568

Fig. 1. Effects of C-PC treatment during in vitro maturation at different concentrations on

569

porcine oocyte polar body extrusion (PBE) and subsequent in vitro developmental

570

competence of parthenogenetic embryos. (A) Representative fluorescent images of metaphase

571

II (MII) stage oocyte with an extruded polar body. Blue: DNA; Green: α-tubulin (B) PBE rate.

572

(C) Blastocyst formation rate. (D) Blastocyst hatching rate. The numbers of embryos

573

examined in each experimental group are shown in the bar.

574

differences are represented with different letters (p < 0.05). Data are expressed as the mean ±

575

SEM from at least three separate experiments. Scale bar = 20 µm.

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567

Statistically significant

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a, b

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576 577

Fig. 2. Effects of C-PC treatment during in vitro maturation on the developmental

578

competence of porcine cloned embryo. (A) Blastocyst formation was examined at day 7. (B)

579

Blastocyst rate. (C) Total cell number in each blastocyst. The numbers of embryos examined

580

in each experimental group are shown in the bar.

581

represented with different letters (p < 0.05). Data are expressed as the mean ± SEM from at

582

least three separate experiments. Scale bar = 100 µm.

Statistically significant differences are

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a, b

583

Fig. 3. Effect of C-PC treatment during in vitro maturation on expression of genes related to

585

cell proliferation, apoptosis, pluripotency, and apoptosis in porcine cloned embryo. (A)

586

Immunofluorescence staining of BrdU in porcine cloned embryo at blastocyst stage. (B)

587

Percentages

588

pluripotency-related genes in blastocyst. The numbers of embryos examined in each

589

experimental group are shown in the bar.

590

represented with different letters (p < 0.05). Data are expressed as the mean ± SEM from at

591

least three separate experiments. Scale bar = 20 µm.

BrdU-positive

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of

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584

cells

in

blastocyst.

a, b

(C)

Relative

expression

of

Statistically significant differences are

592 593

Fig. 4. Effect of C-PC treatment during in vitro maturation on cumulus cell expansion and

594

relative gene expression in porcine cumulus cell. (A) The degree of cumulus expansion in

595

porcine cumulus oocyte complexes. (B) Cumulus expansion-related genes in porcine cumulus

ACCEPTED MANUSCRIPT a, b

596

cell.

Statistically significant differences are represented with different letters (p < 0.05).

597

Data are expressed as the mean ± SEM from at least three separate experiments.

598

Fig. 5. Effect of C-PC treatment during in vitro maturation on ROS and GSH levels, total

600

antioxidant capacity (TAC), and superoxide dismutase (SOD) activity in porcine oocytes.

601

Intracellular ROS (A) and GSH (B) levels. (C) TCA. (D) SOD activity. The numbers of

602

embryos examined in each experimental group are shown in the bar.

603

significant differences are represented with different letters (p < 0.05). Data are expressed as

604

the mean ± SEM from at least three separate experiments.

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599

Statistically

SC

a, b

605

Fig. 6. Effect of C-PC treatment during in vitro maturation on mitochondrial membrane

607

potential (∆Ψm) in porcine oocytes. (A) Representative fluorescent images of 5,5′,6,

608

6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining in MII stage

609

oocytes. (B) Quantification of fluorescence intensity of JC-1. The numbers of embryos

610

examined in each experimental group are shown in the bar.

611

differences are represented with different letters (p < 0.05). Data are expressed as the mean ±

612

SEM from at least three separate experiments. Scale bar = 50 µm.

a, b

Statistically significant

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606

613

Fig. 7. Effect of C-PC treatment during in vitro maturation on cathepsin B activity in porcine

615

oocytes. (A) Representative fluorescent images showing cathepsin B activity in MII stage

616

oocytes. Red: Cathepsin B. (B) Quantification of fluorescence intensity of intracellular

617

cathepsin B activity. The numbers of embryos examined in each experimental group are

618

shown in the bar.

619

(p < 0.05). Data are expressed as the mean ± SEM from at least three separate experiments.

620

Scale bar = 50 µm.

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614

a, b

Statistically significant differences are represented with different letters

ACCEPTED MANUSCRIPT Table 1. Primer sequences used in real-time RT-PCR

Primer sequence (5’-3’) F: CCCCGCCCTATGACTTCT

POU5F1

60 R: TAGGAGCTTGGCAAATTGTTC F: CGCAGACCTACATGAACG 60 R: TCGGACTTGACCACTGAG F: CTCTCCTCTTCCTTCCTC

60

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NANONG

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SOX2

Product Size (bp)

RI PT

Gene

Annealing Temperature (°C)

269

103

139

R: CTTCTGCTTCTTGACTGG

F: AGTTTATGGGCAGCCAATGTAGTT HAS2

60

101

60

185

60

194

60

117

R: GCACTTGGACCGAGCTGTGT F: GCACTTGGACCGAGCTGTGT PTX3

TE D

R: GCTATCCTCTCCAACAAGTGA F: ACAGGGCCATGGGGTGGACT PTGS2

R: CCACGGCAAAGCGGAGGTGT F: TTCCACGGCACAGTCAAG

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GAPDH

R: ATACTCAGCACCAGCATCG

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F, forward; R, reverse.

ACCEPTED MANUSCRIPT

B

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A

Blue: DNA; Green: α-tubulin

D

AC C

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C

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A

Control

C-CP

B

AC C

EP

TE D

C

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DNA

Brdu

Merge

B

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SC

C-PC

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Control

A

AC C

EP

TE D

C

ACCEPTED MANUSCRIPT

TE D EP AC C

B

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SC

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A

B

C

SC

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A

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ACCEPTED MANUSCRIPT

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TE D

D

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Light

JC-1 green

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B

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C-CP

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Control

A

JC-1 red

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Control

C-CP

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A

Red: Cathepsin B

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B

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Highlights •

C-Phycocyanin significantly

supplementation increased

the

during

in

developmental

vitro

maturation

competence

of

period porcine

parthenogenetic and cloned embryos. C-Phycocyanin enhanced antioxidant capacity, improved mitochondria

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•

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function, and decreased cathepsin B activity in porcine oocytes.