- Email: [email protected]
Wee1B depletion promotes nuclear maturation of canine oocytes
Accepted Manuscript Wee1B depletion promotes nuclear maturation of canine oocytes Yu-Gon Kim, Dong-Hoon Kim, Seok-Hwan Song, Kyeong-Lim Lee, Byoung-Chul Yang, Jeong Su Oh, Sang-Ryeul Lee, Il-Keun Kong PII:
S0093-691X(14)00564-0
DOI:
10.1016/j.theriogenology.2014.10.017
Reference:
THE 12963
To appear in:
Theriogenology
Received Date: 2 June 2014 Revised Date:
13 October 2014
Accepted Date: 18 October 2014
Please cite this article as: Kim Y-G, Kim D-H, Song S-H, Lee K-L, Yang B-C, Oh JS, Lee S-R, Kong I-K, Wee1B depletion promotes nuclear maturation of canine oocytes, Theriogenology (2014), doi: 10.1016/ j.theriogenology.2014.10.017. 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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To: THERIOGENOLOGY
“Revised highlighted”
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Wee1B depletion promotes nuclear maturation of canine oocytes
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Yu-Gon Kima,†, Dong-Hoon Kimc,†, Seok-Hwan Songa, Kyeong-Lim Leea, Byoung-Chul
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Yangc, Jeong Su Ohd, Sang-Ryeul Leee, and Il-Keun Konga,b,†
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Department of Animal Science, Division of Applied Life Science, bInstitute of Agriculture and
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Life Science, Gyeongsang National University, Jinju 660-701, Gyeongsangnam-do, Republic
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of Korea, cAnimal Biotechnology Division, National Institute of Animal Science, Suwon 441-
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706, Gyeonggi-do, Republic of Korea, dDepartment of Genetic Engineering, College of
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Biotechnology and Bioengineering, Sungkyunkwan University, Gyeonggi-do 440-746,
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Republic of Korea, eAnimal, Dairy, and Veterinary Sciences Department, Utah State University,
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Logan, UT 84322-4700, USA
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†
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†
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These authors contributed equally to this work. Corresponding author: Tel: +82-(0)55-772-1942, Email: [email protected]
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ABSTRACT
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Most mammalian oocytes are arrested at the germinal vesicle (GV) stage by
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activation of Wee1B. Meiotic resumption is regulated by inactivation of Wee1B and
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activation of cell division cycle 25B (cdc25B). The aim of this study was to determine
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whether treatment with Wee1B-targeting small interfering RNA (Wee1B-siRNA) promotes
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nuclear maturation of canine oocytes from GV stage to metaphase II (MII) stage. In
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Experiment 1, the percentage of canine oocytes that matured to MII stage was higher (P <
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0.05) among oocytes cultured in vitro for 72 h than among those cultured for 24 and 48 h
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(5.4% ± 2.5 vs. 0.0% ± 0.0 and 1.4% ± 1.0, respectively). Furthermore, the percentage of
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oocytes that matured to metaphase I (MI) stage was higher (P < 0.05) among oocytes cultured
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for 48 and 72 h than among those cultured for 24 h (14.9% ± 10.0 and 22.4% ± 8.1,
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respectively, vs. 5.7% ± 6.0). In Experiment 2, canine oocytes were intracytoplasmically
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microinjected with Wee1B-siRNA (50 µM) at various culture time points (0, 24, 48, or 72 h).
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The nuclear configuration of the exception of oocytes in the 72 h group was examined after
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84 h of culture. The percentage of oocytes that matured to the MII stage was higher (P < 0.05)
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among those injected with Wee1B-siRNA at 0 h than among control oocytes and those
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injected at 72 h (18.0% ± 1.7 vs. 2.1% ± 2.8 and 0.0% ± 0.0, respectively). Moreover, the
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percentage of oocytes that matured to the MI stage was higher (P < 0.05) among those
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injected at 0 h than among control oocytes and those injected at 24 and 72 h (45.9% ± 6.8 vs.
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22.1% ± 3.5, 22.8% ± 10.0, and 10.0% ± 4.4, respectively). In Experiment 3, oocytes were
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intracytoplasmically microinjected with Wee1B-siRNA at 0 h of IVM and cultured for 0, 24,
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48, or 72 h. Thereafter, maturation-related gene expression was analyzed by quantitative real-
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ACCEPTED MANUSCRIPT time PCR. mRNA expression of cAMP and cdc25B was lower (P < 0.05) in oocytes injected
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at 48 h than in the other groups. mRNA expression of cAMP was lower (P < 0.05) in oocytes
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injected at 0 h than in control oocytes and those injected at 72 h. mRNA expression of
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mitogen-activated protein kinase 1 and mitogen-activated protein kinase 3 was higher (P <
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0.05) in oocytes injected at 72 h than in the other groups. In conclusion, we confirmed that
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Wee1B-siRNA microinjection enhances the percentages of canine oocytes that reach the MI
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and MII stages. These data suggest that Wee1B-siRNA microinjection could be a useful
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strategy to obtain mature canine oocytes for research and assisted canine reproduction.
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Keywords: IVM, oocyte, Wee1B-siRNA, cAMP, dog
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1. Introduction
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In most mammalian species, oocytes are ovulated at metaphase II (MII) stage. By contrast, canine oocytes are ovulated at the germinal vesicle (GV) or germinal vesicle
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breakdown (GVBD) stage, and subsequently mature in the oviduct for 2–5 d [1]. Furthermore,
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IVM of canine oocytes is not well established in comparison with that of other mammalian
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oocytes [2,3]. Oh et al. [4] reported that when oocytes are collected at the follicular stage and
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cultured for 72 h, the percentage of oocytes that mature to the MII stage is higher among
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those cultured in medium supplemented with 10% canine estrous serum (14.2%) than among
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those cultured with other percentages of serum (2.2–6.3%) and control oocytes (2.2%). The
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highest rate of meiotic resumption was reported by Willingham-Rocky et al. [5], who used
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medium containing 200 ng/mL progesterone; however, this did not affect maturation to the
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MII stage. Consequently, the only reliable source of canine oocytes for research is in vivo-
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matured oocytes from bitches (with spontaneous or induced ovulation). However, due to
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prolonged intervals between cycles and the logistics of monitoring and inducing cycles,
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oocytes are inconvenient to obtain.
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Inhibitory signals transmitted through gap junctions between mammalian oocytes
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and cumulus cells (CCs) arrest oocyte nuclei in prophase I. Prior to the LH surge, there is a
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high concentration of cyclic guanosine monophosphate (cGMP) in mural granulose cells due
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to active guanylyl cyclase and inactive cGMP phosphodiesterase. Furthermore, the cAMP
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concentration is low in mural granulose cells due to inactive LH receptor, G protein, and
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adenylyl cyclase [6,7]. However, these molecules are activated by the preovulatory LH surge 4
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closes gap junctions. Thus, cAMP does not diffuse from CCs through gap junctions [6,7].
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This leads to a decreased cAMP concentration in oocytes, the initial signal for meiotic
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resumption.
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Maturation-promoting factor (MPF) is a complex of cyclin-dependent kinase 1
(CDK1, also known as cell division cycle protein 2) and cyclin B, and is the master regulator
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of meiotic resumption. MPF activity is regulated by inhibitory phosphorylation of CDK1.
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CDK1 phosphorylation is catalyzed by Wee1B, which is a protein kinase of the WEE family
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expressed only in oocytes, whereas CDK1 dephosphorylation is mediated by cell division
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cycle 25B (cdc25B). Wee1B and cdc25B are both regulated by cAMP-dependent protein
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kinase A (PKA) [8,9]. During meiotic arrest, a high concentration of cAMP activates PKA
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and thereby increases the activity of Wee1B and decreases the activity of cdc25B, which in
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turn prevents activation of MPF [10]. Once meiosis resumes owing to the LH surge, the
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cAMP concentration decreases and PKA is thereby inactivated, which increases the activity
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of MPF owing to the inactivation of Wee1B and activation of cdc25B [11,12]. Therefore,
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MPF activity for meiotic resumption is mainly regulated by the balance between Wee1B and
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cdc25B [13,14].
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Unlike other mammalian oocytes, canine oocytes are ovulated at an immature stage
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and undergo meiotic maturation in the oviduct [11,15,16]. This unique post-ovulatory
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maturation of canine oocytes compromises the efficiency of their IVM. Although numerous
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approaches have been used to improve the efficiency of canine oocyte IVM, most of these
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involve the selection of oocytes or modulation of their culture conditions. Moreover, the
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results obtained using these approaches are not satisfactory. Therefore, different approaches 5
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are required to overcome the low IVM efficiency of canine oocytes. In the present study, we
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attempted to activate MPF in canine GV-stage oocytes by injecting Wee1B-targeting small interfering RNA (Wee1B-siRNA) into the oocyte cytoplasm, thereby inhibiting the activity of
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Wee1B.
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2. Materials and methods
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2.1. Reagents
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Aldrich (St. Louis, MO, USA).
2.2. Animal care and recovery of ovaries
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Unless otherwise indicated, all chemicals and media were purchased from Sigma-
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To recover immature oocytes, ovaries were collected from bitches undergoing an
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ovariohysterectomy at a local veterinary hospital. This study was conducted in accordance
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with Gyeongsang National University guidelines (approval no.: GNU-131105-D0066).
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2.3. Collection of immature oocytes 6
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Ovaries were used without regard to the age of the dog, pubertal status, or the stage of the estrus cycle. Ovaries were transported to the laboratory in 0.9% saline at 35 ºC within 1
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h of removal. Upon arrival at the laboratory, ovaries were placed in tissue culture media
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(TCM)-199 containing 25 mM HEPES and supplemented with 0.1% BSA at 38.5 ºC. The
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ovarian cortex was immediately sliced with a razor blade, and oocytes (cumulus-oocyte
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complexes (COCs)) that were dark, spherical, had a cytoplasmic diameter of ≥ 100 µm, and
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were surrounded by compact CCs [17] were selected.
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2.4. IVM of canine COCs
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The COCs were cultured (minimum of 15 oocytes per treatment) in 4-well culture
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dishes (Nunc, Roskilde, Denmark) at 38.5 ºC in a humidified incubator containing 5% CO2 in
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air for 72 h. The media for IVM was TCM-199 supplemented with 0.6% glucose, 60 nM
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putrescine, 20 nM progesterone, 30 nM sodium selenite, 25 µg/mL insulin, 100 µg/mL apo-
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transferrin, 40 ng/mL EGF, and 1% penicillin [18].
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2.5. Denuded oocytes and assessment of nuclear maturation
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To remove CCs when culture was complete, oocytes were placed into a 4-well dish along with 750 µL of TCM-199 containing 0.1% hyaluronidase and denuded by repeated
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pipetting. Thereafter, denuded oocytes were washed (three times) in Dulbecco’s PBS
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supplemented with 0.1% polyvinylpyrrolidone, fixed with 3.7% formaldehyde, stained with
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the DNA-specific fluorescent dye Hoechst 33342 for 5 min, placed on a slide, and overlaid
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with a cover-glass. Fluorescence microscopy was used to determine nuclear maturation,
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classified as GV, GVBD, MI, or MII stage (Fig.1) [19].
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2.6. Microinjection
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Ten picoliters of 50 µM Wee1B-siRNA [20] was microinjected (Femtojet 5247 microinjection system; Eppendorf, Hamburg, Germany) into the cytoplasm of a denuded GV-
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stage oocyte using a glass micropipette mounted on a micromanipulator (Narishige, Tokyo,
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Japan). Thereafter, oocytes were cultured in IVM culture media at 38.5 ºC in a humidified
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incubator containing 5% CO2 in air beneath mineral oil for up to 72 h, with the exception of
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the 72 h group, in which oocytes were cultured for a total of 84 h.
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2.7 Gene expression analysis of Wee1B-siRNA-injected oocytes
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RNA was isolated from oocytes cultured in vitro for 0, 24, 48, or 72 h after
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cytoplasmic microinjection of Wee1B-siRNA from three biological replicates each with 20 8
ACCEPTED MANUSCRIPT oocytes using an Arcturus PicoPure RNA Isolation Kit (Life Technologies Inc., Foster City,
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CA, USA) according to the manufacturer's instructions. Thereafter, cDNA was synthesized
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using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). To
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investigate the mRNA levels of cAMP, cdc25B, mitogen-activated protein kinase 1 (MAPK1),
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and mitogen-activated protein kinase 3 (MAPK3) relative to that of the housekeeping gene
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glyceraldehyde 3-phosphate dehydrogenase (GAPDH), quantitative real-time PCR (qPCR)
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was performed on a CFX96 real-time system (Bio-Rad Laboratories) using iQ™ SYBR®
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Green Supermix (Bio-Rad Laboratories), according to the manufacturer’s instructions. The
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cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s,
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55 °C for 40 s, and 72 °C for 40 s. The primers used for the qPCR reaction are shown in
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Table 1.
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2.8. Experimental design
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In this study, all experiments were replicated at least three times.
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Experiment 1 was designed to evaluate the optimal duration of IVM. In total, 337
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COCs were matured at 38.5 ºC in a humidified incubator containing 5% CO2 in air for 24, 48,
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or 72 h. Thereafter, CCs were removed and denuded oocytes were stained to assess their
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nuclear maturation. The percentages of oocytes at each stage of meiotic resumption were
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recorded.
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Experiment 2 was designed to determine the optimal time point of Wee1B-siRNA injection into the cytoplasm of denuded canine oocytes. Oocytes were injected at 0 h
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(immediately after high-quality oocytes were recovered) or after 24, 48, or 72 h of culture.
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Thereafter, oocytes were matured in vitro for up to 72 h at 38.5 ºC in a humidified incubator
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containing 5% CO2 in air, with the exception of oocytes in the 72 h group, which were
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examined after 84 h of culture. The percentages of oocytes at each stage of meiotic
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resumption were recorded. In total, 384 injected oocytes and non-injected (control) oocytes
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were used.
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Experiment 3 was designed to investigate the mechanism via which Wee1B functions
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in the nuclear maturation of canine oocytes. Wee1B-siRNA was microinjected into the
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cytoplasm of oocytes at 0 h of IVM, and then oocytes were cultured for various amounts of
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time (0, 24, 48, or 72 h). Thereafter, total RNA was isolated and analyzed by qPCR. The
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mRNA levels of cAMP, cdc25B, MAPK1, and MAPK3 were investigated.
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2.8. Statistical analysis
The percentages of canine oocytes at various maturation stages were recorded.
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Significant differences in oocyte maturation were confirmed by calculating the least-square
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means using SAS 9.3 software (SAS Institute, Inc., Cary, NC, USA). Maturation-related gene
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expression data are expressed as the mean ± SEM and were compared using the Student’s t-
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test. All experiments were performed at least three times. P < 0.05 was considered significant.
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3. Results
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3.1. Optimal duration of IVM
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The percentage of oocytes that matured to MII stage was higher (P < 0.05) among those cultured for 72 h than among those cultured for 24 and 48 h (5.4% ± 2.5 vs. 0.0% ± 0.0
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and 1.4% ± 1.0, respectively; Table 2). Most oocytes were arrested at GV after maturation for
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24, 48, and 72 h (71.4% ± 16.5, 59.7% ± 8.8, and 44.8% ± 9.7, respectively). The percentage
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of oocytes that matured to the MI or MII stage was higher (P < 0.05) among those cultured
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for 72 h (27.8% ± 8.3) than among those cultured for 24 h (5.7% ± 6.0), but did not differ
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between oocytes cultured for 72 h and those cultured for 48 h (27.8% ± 8.3 vs. 16.4% ± 11.0;
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Table 2).
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3.2. Effect of Wee1B-siRNA microinjection into cytoplasm
The percentage of oocytes that matured to MII was higher (P < 0.05) among those
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microinjected with Wee1B-siRNA at 0 h than among control oocytes and those microinjected
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with Wee1B-siRNA at 72 h (18.0% ± 1.7 vs. 2.1% ± 2.8 and 0.0% ± 0.0, respectively), but
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did not differ among oocytes microinjected with Wee1B-siRNA at 0 h and those injected at
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24 or 48 h (18.0% ± 1.7 vs. 11.4% ± 5.0 and 14.7% ± 1.4, respectively; Table 3).
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Furthermore, the percentage of oocytes that matured to MI was higher (P < 0.05) among
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oocytes microinjected with Wee1B-siRNA at 0 h than among control oocytes and those
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microinjected with Wee1B-siRNA at 24 and 72 h (45.9% ± 6.8 vs. 22.1% ± 3.5, 22.8% ±
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10.0, and 10.0% ± 4.4, respectively), but did not differ between oocytes microinjected with
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Wee1B-siRNA at 0 h and those microinjected with Wee1B-siRNA at 48 h (45.9% ± 6.8 vs.
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26.1% ± 9.0; Table 3).
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3.3 Assessment of maturation-related genes by qPCR
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The mRNA levels of cAMP, cdc25B, MAPK1, and MAPK3 were analyzed by qPCR
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at 0, 24, 48, and 72 h after microinjection of Wee1B-siRNA into the cytoplasm of canine
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oocytes, which was performed at 0 h of IVM. Expression levels were normalized against that
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of GAPDH. The mRNA level of cAMP was lowest (P < 0.05) in oocytes cultured for 48 h
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after Wee1B-siRNA microinjection, and there were no significant differences among the
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other groups (Fig. 2A). Moreover, the mRNA level of cdc25B did not differ among control
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oocytes and those cultured for 24 or 72 h after Wee1B-siRNA microinjection; however, it
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was lower (P < 0.05) in oocytes cultured for 48 h after Wee1B-siRNA microinjection than in
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the other groups (Fig. 2B). The mRNA levels of MAPK1 and MAPK3 were higher (P < 0.05)
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in oocytes cultured for 72 h after Wee1B-siRNA microinjection than in the other groups (Fig.
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2C and 2D).
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Discussion
In the present study, the percentage of canine oocytes that matured to the MII stage
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was higher among those that underwent IVM for 72 h (P < 0.05) than among those that
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underwent IVM for 24 or 48 h. Oocytes have been previously cultured for 72 h in
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ACCEPTED MANUSCRIPT supplemented TCM-199. Canine oocytes are ovulated at prophase I and mature in the distal
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part of the oviduct for at least 48–72 h [4]. Hence, we investigated the optimal duration of
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IVM of canine oocytes. Microinjection of Wee1B-siRNA into the oocyte cytoplasm
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significantly enhanced nuclear maturation. The percentages of oocytes at MI and MII stages
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were higher (P < 0.05) among oocytes microinjected with Wee1B-siRNA at 0 h of IVM
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(45.9% ± 6.8 and 18.0% ± 1.7, respectively) than among control oocytes (22.1% ± 3.5 and
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2.1% ± 2.8, respectively). This is the first report regarding intracytoplasmic microinjection of
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Wee1B-siRNA to promote nuclear maturation of canine oocytes. In this study, the MII
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maturation rate was not higher than in previous studies, but the maturation rate from the GV
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to MI stage was high. Compared to other mammalian species, the developmental competence
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of canine oocytes (cultured in vitro) from the GV to MII stage is severely compromised.
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Consequently, there is an urgent need to develop improved systems for IVM of canine
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oocytes. The most common approach to improve the IVM of canine oocytes is to add various
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compounds to the culture media to mitigate meiotic arrest. Pretreatment with 10 mM caffeine
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for 12 h prior to culture for a further 60 h in basal culture medium enhances the development
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of oocytes to MII (16.9% ± 2.4) and MI (8.1% ± 1.2) stages [17]. The addition of 200 ng/mL
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progesterone resulted in 10.7% of oocytes reaching the MII stage and 43.3% of oocytes
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reaching the GVBD/MI stage [5]. The current study sought to explore and validate a novel
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approach that could be used to improve the efficiency of canine IVF.
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In mice, pigs, cattle, and other mammals, oocytes are arrested at prophase I prior to
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the LH surge. The MPF complex, comprising CDK1 and cyclin B, is required for meiotic
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resumption. The CDK1 kinase is inactive at the GV stage and its activation triggers GVBD
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[21-24]. Furthermore, cAMP in oocytes plays critical roles in meiotic arrest and resumption. 13
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phosphorylation of Wee1B and thereby inhibits MPF activity. There are several prerequisites
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for the activation of MPF: a low cAMP concentration, which inactivates PKA, inactive
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Wee1B, and dephosphorylation of MPF by cdc25B [9,22,25-29]. Therefore, maintenance of a
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high cAMP concentration activates Wee1B and prevents activation of MPF [30].
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Consequently, we speculated that intracytoplasmic microinjection of Wee1B-siRNA into
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canine oocytes would activate MPF. Following Wee1B-siRNA microinjection at 0 h of IVM
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and subsequent culture for 72 h, 18.0% ± 1.7 of oocytes reached MII stage and 63.9% ± 5.3
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of oocytes reached the MI or MII stage (Table 3). These maturation rates (MI or MII) were
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significantly different from those of control oocytes. The maturation rates of oocytes cultured
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for 24 or 48 h following Wee1B-siRNA microinjection were not significantly different from
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those of control oocytes, although there were fewer of the former oocytes (Table 3).
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Therefore, we analyzed the mRNA levels of genes related to the mechanism in which Wee1B
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functions by qPCR in oocytes cultured for various amounts of times following Wee1B-siRNA
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microinjection at 0 h of IVM. The mRNA level of cAMP was lower in oocytes cultured for
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48 h after Wee1B-siRNA microinjection than in control oocytes, but did not differ between
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oocytes cultured for 72 h after Wee1B-siRNA microinjection and control oocytes (Fig. 2A).
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This shows that cAMP was not influenced by Wee1B-siRNA, although it was expected to
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downregulate cAMP. The mRNA level of cdc25B was significantly lower and higher in
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oocytes cultured for 48 h and 72 h after Wee1B-siRNA microinjection, respectively, in
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comparison with that in control oocytes (Fig. 2B). Increased cdc25B expression can stimulate
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downstream genes, such as MAPK1 and MAPK3, and induce nuclear maturation. mRNA
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levels of MAPK1 and MAPK3, which are related to nuclear maturation, were increased in
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expression of factors related to nuclear maturation was increased following Wee1B-siRNA
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microinjection, even though mRNA expression of cAMP was not altered. These results are in
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accordance with the mechanism via which Wee1B functions in oocyte nuclear maturation.
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Therefore, our results suggest that Wee1B-siRNA can promote nuclear activation of canine
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oocytes.
In conclusion, microinjection of Wee1B-siRNA into the cytoplasm of canine oocytes
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significantly promoted nuclear maturation. This is the first report of Wee1B-siRNA injection.
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Although further refinement is needed to enhance the proportion of oocytes that reach MII
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stage, this study provides the proof-of-concept for this approach.
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Acknowledgments
This study was supported by the Rural Development Administration, Republic of
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Korea (grant nos. PJ008975042014 and PJ009321012014). Yu-Gon Kim, Seok-Hwan Song,
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and Kyeong-Lim Lee were supported by BK21 Plus fellowships from Gyeongsang National
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University, Republic of Korea.
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[10] Mehlmann LM. Stops and starts in mammalian oocytes: Recent advances in understanding the regulation of meiotic arrest and oocyte maturation. Reproduction 2005;130(6):791-9.
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[11] Chastant-Maillard S, Viaris de Lesegno C, Chebrout M, Thoumire S, Meylheuc T, Fontbonne A, et al. The canine oocyte: Uncommon features of in vivo and in vitro maturation. Reprod Fertil Dev 2011;23(3):391-402.
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[12] Holst PA, Phemister RD. The prenatal development of the dog: Preimplantation events. Biol Reprod 1971;5(2):194-206.
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ACCEPTED MANUSCRIPT [13] Mehlmann LM, Kalinowski RR, Ross LF, Parlow AF, Hewlett EL, Jaffe LA. Meiotic resumption in response to luteinizing hormone is independent of a gi family G protein or calcium in the mouse oocyte. Dev Biol 2006;299(2):345-55.
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[14] Kang H, Hwang SC, Park YS, Oh JS. Cdc25B phosphatase participates in maintaining metaphase II arrest in mouse oocytes. Mol Cells 2013;35(6):514-8.
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[15] Concannon PW, McCann JP, Temple M. Biology and endocrinology of ovulation, pregnancy and parturition in the dog. J Reprod Fertil Suppl 1989;39:3-25.
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[16] Wildt DE, Panko WB, Chakraborty PK, Seager SW. Relationship of serum estrone, estradiol-17beta and progesterone to LH, sexual behavior and time of ovulation in the bitch. Biol Reprod 1979;20(3):648-58.
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[17] Salavati M, Ghafari F, Zhang T, Fouladi-Nashta AA. Influence of caffeine pretreatment on biphasic in vitro maturation of dog oocytes. Theriogenology 2013;80(7):784-92.
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[18] Saikhun J, Sriussadaporn S, Thongtip N, Pinyopummin A, Kitiyanant Y. Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells. Theriogenology 2008;69(9):1104-10.
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[19] Kim MK, Fibrianto YH, Oh HJ, Jang G, Kim HJ, Lee KS, et al. Effects of estradiol17beta and progesterone supplementation on in vitro nuclear maturation of canine oocytes. Theriogenology 2005;63(5):1342-53.
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[20] Suzuki MG, Ito H, Aoki F. Effects of RNAi-mediated knockdown of histone methyltransferases on the sex-specific mRNA expression of imp in the silkworm bombyx mori. Int J Mol Sci 2014;15(4):6772-96.
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[21] Han SJ, Conti M. New pathways from PKA to the Cdc2/cyclin B complex in oocytes: Wee1B as a potential PKA substrate. Cell Cycle 2006;5(3):227-31.
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[22] Shimaoka T, Nishimura T, Kano K, Naito K. Critical effect of pigWee1B on the regulation of meiotic resumption in porcine immature oocytes. Cell Cycle 2009;8(15):2375-84.
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[23] Shimaoka T, Nishimura T, Kano K, Naito K. Analyses of the regulatory mechanism of porcine WEE1B: The phosphorylation sites of porcine WEE1B and mouse WEE1B are different. J Reprod Dev 2011;57(2):223-8.
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[24] Bilodeau-Goeseels S. Bovine oocyte meiotic inhibition before in vitro maturation and its value to in vitro embryo production: Does it improve developmental competence? Reprod Domest Anim 2012;47(4):687-93.
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ACCEPTED MANUSCRIPT [25] Nakanishi M, Ando H, Watanabe N, Kitamura K, Ito K, Okayama H, et al. Identification and characterization of human Wee1B, a new member of the Wee1 family of cdkinhibitory kinases. Genes Cells 2000;5(10):839-47.
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[26] Bornslaeger EA, Schultz RM. Regulation of mouse oocyte maturation: Effect of elevating cumulus cell cAMP on oocyte cAMP levels. Biol Reprod 1985;33(3):698-704.
379 380 381 382
[27] Tsafriri A, Chun SY, Zhang R, Hsueh AJ, Conti M. Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: Studies using selective phosphodiesterase inhibitors. Dev Biol 1996;178(2):393-402.
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[28] Han SJ, Chen R, Paronetto MP, Conti M. Wee1B is an oocyte-specific kinase involved in the control of meiotic arrest in the mouse. Curr Biol 2005;15(18):1670-6.
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[29] Hanna CB, Yao S, Patta MC, Jensen JT, Wu X. WEE2 is an oocyte-specific meiosis inhibitor in rhesus macaque monkeys. Biol Reprod 2010;82(6):1190-7.
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[30] Conti M, Hsieh M, Zamah AM, Oh JS. Novel signaling mechanisms in the ovary during oocyte maturation and ovulation. Mol Cell Endocrinol 2012;356(1-2):65-73.
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Table Legends
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Table 1
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Quantitative real-time PCR primers used in this study.
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Table 2
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Effect of the duration of IVM on the nuclear development of canine oocytes.
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Table 3
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Effect of the time point of Wee1B-targeting small interfering RNA microinjection on the nuclear developmental of canine oocytes.
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Figure Legends
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Fig. 1. Stages of nuclear maturation of canine oocytes (stained with Hoechst 33342). A:
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germinal vesicle (GV); B: germinal vesicle breakdown (GVBD); C: metaphase I (MI); D:
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metaphase II (MII). Scale bars = 100 µm.
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Fig. 2. Relative expression of genes related to the mechanism in which Wee1B functions in
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mammalian oocytes. Control: culture for 72 h without Wee1B-targeting small interfering
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RNA (Wee1B-siRNA) microinjection; 0 h group: oocytes not cultured after Wee1B-siRNA
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microinjection; 24 h group: oocytes cultured for 24 h after Wee1B-siRNA microinjection; 48
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h group: oocytes cultured for 48 h after Wee1B-siRNA microinjection; 72 h group: oocytes
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cultured for 72 h after Wee1B-siRNA microinjection. a–e Data are from three independent
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experiments. Different letters indicate a significant difference (P < 0.05).
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ACCEPTED MANUSCRIPT To: THERIOGENOLOGY
Revised
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Wee1B depletion promotes nuclear maturation of canine oocytes
Yu-Gon KimA,†, Dong-Hoon KimC,†, Seok-Hwan SongA, Han-Seul ParkA,
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Byoung-Chul YangC, Jeong-Su OhD, Il-Keun KongA,B,† A
Department of Animal Science, Division of Applied Life Science, BInstitute of Agriculture
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and Life Science, Gyeongsang National University, Jinju 660-701, Gyeongsangnam-do, Republic of Korea, CAnimal Biotechnology Division, National Institute of Animal Science, Suwon 441-706, Gyeonggi-do, Republic of Korea, DDepartment of Genetic Engineering,
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746, Republic of Korea
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College of Biotechnology and Bioengineering, Sungkyunkwan University, Gyeonggi-do 440-
ACCEPTED MANUSCRIPT Table 1 Real time PCR primers for quantification of gene expression. Gene
Product size Sequence
Accession number (bp)
F; AACAGTGACACCCACTCTTC GAPDH
110 R; CGGTTGCTGTAGCCAAATTC F; CAGGTGACAGACTTCGGTTT
cAMP
105 F; AATATCTGGGCAGTCCCATTAC
cdc25B
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R; CCTTGCCCTCTTCTTGATCTT
AB_038240
NM_001003032.1
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R; GTAGCCTTTGCTCAGGATGAT
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symbol
XM_005626604.1
F; AGAGAACCCTGAGGGAGATAAA MAPK1
90
NM_001110800
112
NM_001252035.1
R; CGATGGTTGGTGCTCGAATA
F; CAACGACCATGTTTGCTACTTC MAPK3
R; GGTGTTGATGAGCAGGTTAGA
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GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; cAMP: Cyclic adenosine monophosphate; cdc25B: cell division cycle 25; MAPK1: Mitogen-activated protein kinase 1; MAPK3: Mitogen-activated protein kinase 3.
ACCEPTED MANUSCRIPT Table 2 Effects of in vitro maturation time on nuclear development of canine oocytes. No. oocytes developed to (Mean ± SEM) & culture time (h)
Stages
24
48
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75 (71.4 ± 16.5) 24 (22.8 ± 12.3)a
40 (59.7 ± 8.8) 16 (23.8 ± 9.8)a
74 (44.8 ± 9.7)a 45 (27.2 ± 6.7)a
MI
6 (5.7 ± 6.0)b
10 (14.9 ± 10.0)a
37 (22.4 ± 8.1)a
MII
0 (0.0 ± 0.0)b
1 (1.4 ± 1.0 )b
9 (5.4 ± 2.5)a
MI or MII
6 (5.7 ± 6.0)b
11 (16.4 ± 11.0)a,b
46 (27.8 ± 8.3)a
GV GVBD
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Total no. oocytes cultured 105 67 Within a row, percentages without a common superscript differed (P < 0.05).
a,b
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metaphase II.
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Table 3
Stages
No. oocytes developed to (Mean ± SEM) & culture time (h)⃰ 48
72§
34 (48.5 ± 17.6)a,b
14 (15.9 ± 5.2)b
35 (50.0 ± 9.2)a
6 (9.8 ± 2.5)c
12 (17.1 ± 3.5)b,c
38 (43.1 ± 7.5)a
28 (40.0 ± 4.7)a,b
21 (22.1 ± 3.5)b
28 (45.9 ± 6.8)a
16 (22.8 ± 10.0)b
23 (26.1 ± 9.0)a,b
7 (10.0 ± 4.4)b
2 (2.1 ± 2.8)b,c
11 (18.0 ± 1.7)a
8 (11.4 ± 5.0)a,b
13 (14.7 ± 1.4)a
0 (0.0 ± 0.0)c
23 (24.2 ± 2.8)b,c
39 (63.9 ± 5.3)a
24 (34.2 ± 15.0)b,c
36 (40.9 ± 10.2)a,b
7 (10.0 ± 4.4)c
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70
0
24
GV
48 (50.5 ± 13.8)a,b
16 (26.2 ± 5.6)a,b
GVBD
24 (25.2 ± 11.0)a,c
MI MII
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Control
MI or MII
Total no. oocytes cultured 95 61 Within a row, percentages without a common superscript differed (P < 0.05).
a-c
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All of experimental groups were cultured on 72 hours after Wee1B-siRNA microinjection. §72 h of Wee1B-siRNA microinjection group was
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cultured for 12 hours more. GV: germinal vesicle; GVBD: germinal vesicle break-down; MI: metaphase I; MII: metaphase II.
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Effects of Wee1B-siRNA injection time on nuclear development of canine oocytes.
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Figure 1
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S0093-691X(14)00564-0
DOI:
10.1016/j.theriogenology.2014.10.017
Reference:
THE 12963
To appear in:
Theriogenology
Received Date: 2 June 2014 Revised Date:
13 October 2014
Accepted Date: 18 October 2014
Please cite this article as: Kim Y-G, Kim D-H, Song S-H, Lee K-L, Yang B-C, Oh JS, Lee S-R, Kong I-K, Wee1B depletion promotes nuclear maturation of canine oocytes, Theriogenology (2014), doi: 10.1016/ j.theriogenology.2014.10.017. 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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To: THERIOGENOLOGY
“Revised highlighted”
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Wee1B depletion promotes nuclear maturation of canine oocytes
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Yu-Gon Kima,†, Dong-Hoon Kimc,†, Seok-Hwan Songa, Kyeong-Lim Leea, Byoung-Chul
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Yangc, Jeong Su Ohd, Sang-Ryeul Leee, and Il-Keun Konga,b,†
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7 a
Department of Animal Science, Division of Applied Life Science, bInstitute of Agriculture and
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Life Science, Gyeongsang National University, Jinju 660-701, Gyeongsangnam-do, Republic
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of Korea, cAnimal Biotechnology Division, National Institute of Animal Science, Suwon 441-
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706, Gyeonggi-do, Republic of Korea, dDepartment of Genetic Engineering, College of
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Biotechnology and Bioengineering, Sungkyunkwan University, Gyeonggi-do 440-746,
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Republic of Korea, eAnimal, Dairy, and Veterinary Sciences Department, Utah State University,
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Logan, UT 84322-4700, USA
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†
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†
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These authors contributed equally to this work. Corresponding author: Tel: +82-(0)55-772-1942, Email: [email protected]
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ABSTRACT
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Most mammalian oocytes are arrested at the germinal vesicle (GV) stage by
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activation of Wee1B. Meiotic resumption is regulated by inactivation of Wee1B and
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activation of cell division cycle 25B (cdc25B). The aim of this study was to determine
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whether treatment with Wee1B-targeting small interfering RNA (Wee1B-siRNA) promotes
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nuclear maturation of canine oocytes from GV stage to metaphase II (MII) stage. In
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Experiment 1, the percentage of canine oocytes that matured to MII stage was higher (P <
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0.05) among oocytes cultured in vitro for 72 h than among those cultured for 24 and 48 h
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(5.4% ± 2.5 vs. 0.0% ± 0.0 and 1.4% ± 1.0, respectively). Furthermore, the percentage of
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oocytes that matured to metaphase I (MI) stage was higher (P < 0.05) among oocytes cultured
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for 48 and 72 h than among those cultured for 24 h (14.9% ± 10.0 and 22.4% ± 8.1,
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respectively, vs. 5.7% ± 6.0). In Experiment 2, canine oocytes were intracytoplasmically
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microinjected with Wee1B-siRNA (50 µM) at various culture time points (0, 24, 48, or 72 h).
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The nuclear configuration of the exception of oocytes in the 72 h group was examined after
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84 h of culture. The percentage of oocytes that matured to the MII stage was higher (P < 0.05)
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among those injected with Wee1B-siRNA at 0 h than among control oocytes and those
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injected at 72 h (18.0% ± 1.7 vs. 2.1% ± 2.8 and 0.0% ± 0.0, respectively). Moreover, the
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percentage of oocytes that matured to the MI stage was higher (P < 0.05) among those
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injected at 0 h than among control oocytes and those injected at 24 and 72 h (45.9% ± 6.8 vs.
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22.1% ± 3.5, 22.8% ± 10.0, and 10.0% ± 4.4, respectively). In Experiment 3, oocytes were
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intracytoplasmically microinjected with Wee1B-siRNA at 0 h of IVM and cultured for 0, 24,
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48, or 72 h. Thereafter, maturation-related gene expression was analyzed by quantitative real-
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at 48 h than in the other groups. mRNA expression of cAMP was lower (P < 0.05) in oocytes
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injected at 0 h than in control oocytes and those injected at 72 h. mRNA expression of
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mitogen-activated protein kinase 1 and mitogen-activated protein kinase 3 was higher (P <
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0.05) in oocytes injected at 72 h than in the other groups. In conclusion, we confirmed that
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Wee1B-siRNA microinjection enhances the percentages of canine oocytes that reach the MI
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and MII stages. These data suggest that Wee1B-siRNA microinjection could be a useful
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strategy to obtain mature canine oocytes for research and assisted canine reproduction.
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Keywords: IVM, oocyte, Wee1B-siRNA, cAMP, dog
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1. Introduction
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In most mammalian species, oocytes are ovulated at metaphase II (MII) stage. By contrast, canine oocytes are ovulated at the germinal vesicle (GV) or germinal vesicle
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breakdown (GVBD) stage, and subsequently mature in the oviduct for 2–5 d [1]. Furthermore,
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IVM of canine oocytes is not well established in comparison with that of other mammalian
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oocytes [2,3]. Oh et al. [4] reported that when oocytes are collected at the follicular stage and
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cultured for 72 h, the percentage of oocytes that mature to the MII stage is higher among
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those cultured in medium supplemented with 10% canine estrous serum (14.2%) than among
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those cultured with other percentages of serum (2.2–6.3%) and control oocytes (2.2%). The
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highest rate of meiotic resumption was reported by Willingham-Rocky et al. [5], who used
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medium containing 200 ng/mL progesterone; however, this did not affect maturation to the
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MII stage. Consequently, the only reliable source of canine oocytes for research is in vivo-
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matured oocytes from bitches (with spontaneous or induced ovulation). However, due to
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prolonged intervals between cycles and the logistics of monitoring and inducing cycles,
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oocytes are inconvenient to obtain.
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Inhibitory signals transmitted through gap junctions between mammalian oocytes
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and cumulus cells (CCs) arrest oocyte nuclei in prophase I. Prior to the LH surge, there is a
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high concentration of cyclic guanosine monophosphate (cGMP) in mural granulose cells due
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to active guanylyl cyclase and inactive cGMP phosphodiesterase. Furthermore, the cAMP
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concentration is low in mural granulose cells due to inactive LH receptor, G protein, and
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adenylyl cyclase [6,7]. However, these molecules are activated by the preovulatory LH surge 4
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closes gap junctions. Thus, cAMP does not diffuse from CCs through gap junctions [6,7].
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This leads to a decreased cAMP concentration in oocytes, the initial signal for meiotic
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resumption.
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Maturation-promoting factor (MPF) is a complex of cyclin-dependent kinase 1
(CDK1, also known as cell division cycle protein 2) and cyclin B, and is the master regulator
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of meiotic resumption. MPF activity is regulated by inhibitory phosphorylation of CDK1.
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CDK1 phosphorylation is catalyzed by Wee1B, which is a protein kinase of the WEE family
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expressed only in oocytes, whereas CDK1 dephosphorylation is mediated by cell division
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cycle 25B (cdc25B). Wee1B and cdc25B are both regulated by cAMP-dependent protein
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kinase A (PKA) [8,9]. During meiotic arrest, a high concentration of cAMP activates PKA
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and thereby increases the activity of Wee1B and decreases the activity of cdc25B, which in
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turn prevents activation of MPF [10]. Once meiosis resumes owing to the LH surge, the
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cAMP concentration decreases and PKA is thereby inactivated, which increases the activity
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of MPF owing to the inactivation of Wee1B and activation of cdc25B [11,12]. Therefore,
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MPF activity for meiotic resumption is mainly regulated by the balance between Wee1B and
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cdc25B [13,14].
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Unlike other mammalian oocytes, canine oocytes are ovulated at an immature stage
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and undergo meiotic maturation in the oviduct [11,15,16]. This unique post-ovulatory
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maturation of canine oocytes compromises the efficiency of their IVM. Although numerous
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approaches have been used to improve the efficiency of canine oocyte IVM, most of these
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involve the selection of oocytes or modulation of their culture conditions. Moreover, the
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results obtained using these approaches are not satisfactory. Therefore, different approaches 5
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are required to overcome the low IVM efficiency of canine oocytes. In the present study, we
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attempted to activate MPF in canine GV-stage oocytes by injecting Wee1B-targeting small interfering RNA (Wee1B-siRNA) into the oocyte cytoplasm, thereby inhibiting the activity of
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Wee1B.
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2. Materials and methods
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2.1. Reagents
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Aldrich (St. Louis, MO, USA).
2.2. Animal care and recovery of ovaries
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Unless otherwise indicated, all chemicals and media were purchased from Sigma-
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To recover immature oocytes, ovaries were collected from bitches undergoing an
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ovariohysterectomy at a local veterinary hospital. This study was conducted in accordance
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with Gyeongsang National University guidelines (approval no.: GNU-131105-D0066).
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2.3. Collection of immature oocytes 6
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Ovaries were used without regard to the age of the dog, pubertal status, or the stage of the estrus cycle. Ovaries were transported to the laboratory in 0.9% saline at 35 ºC within 1
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h of removal. Upon arrival at the laboratory, ovaries were placed in tissue culture media
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(TCM)-199 containing 25 mM HEPES and supplemented with 0.1% BSA at 38.5 ºC. The
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ovarian cortex was immediately sliced with a razor blade, and oocytes (cumulus-oocyte
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complexes (COCs)) that were dark, spherical, had a cytoplasmic diameter of ≥ 100 µm, and
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were surrounded by compact CCs [17] were selected.
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2.4. IVM of canine COCs
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The COCs were cultured (minimum of 15 oocytes per treatment) in 4-well culture
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dishes (Nunc, Roskilde, Denmark) at 38.5 ºC in a humidified incubator containing 5% CO2 in
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air for 72 h. The media for IVM was TCM-199 supplemented with 0.6% glucose, 60 nM
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putrescine, 20 nM progesterone, 30 nM sodium selenite, 25 µg/mL insulin, 100 µg/mL apo-
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transferrin, 40 ng/mL EGF, and 1% penicillin [18].
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2.5. Denuded oocytes and assessment of nuclear maturation
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To remove CCs when culture was complete, oocytes were placed into a 4-well dish along with 750 µL of TCM-199 containing 0.1% hyaluronidase and denuded by repeated
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pipetting. Thereafter, denuded oocytes were washed (three times) in Dulbecco’s PBS
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supplemented with 0.1% polyvinylpyrrolidone, fixed with 3.7% formaldehyde, stained with
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the DNA-specific fluorescent dye Hoechst 33342 for 5 min, placed on a slide, and overlaid
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with a cover-glass. Fluorescence microscopy was used to determine nuclear maturation,
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classified as GV, GVBD, MI, or MII stage (Fig.1) [19].
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2.6. Microinjection
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Ten picoliters of 50 µM Wee1B-siRNA [20] was microinjected (Femtojet 5247 microinjection system; Eppendorf, Hamburg, Germany) into the cytoplasm of a denuded GV-
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stage oocyte using a glass micropipette mounted on a micromanipulator (Narishige, Tokyo,
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Japan). Thereafter, oocytes were cultured in IVM culture media at 38.5 ºC in a humidified
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incubator containing 5% CO2 in air beneath mineral oil for up to 72 h, with the exception of
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the 72 h group, in which oocytes were cultured for a total of 84 h.
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2.7 Gene expression analysis of Wee1B-siRNA-injected oocytes
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RNA was isolated from oocytes cultured in vitro for 0, 24, 48, or 72 h after
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cytoplasmic microinjection of Wee1B-siRNA from three biological replicates each with 20 8
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CA, USA) according to the manufacturer's instructions. Thereafter, cDNA was synthesized
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using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). To
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investigate the mRNA levels of cAMP, cdc25B, mitogen-activated protein kinase 1 (MAPK1),
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and mitogen-activated protein kinase 3 (MAPK3) relative to that of the housekeeping gene
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glyceraldehyde 3-phosphate dehydrogenase (GAPDH), quantitative real-time PCR (qPCR)
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was performed on a CFX96 real-time system (Bio-Rad Laboratories) using iQ™ SYBR®
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Green Supermix (Bio-Rad Laboratories), according to the manufacturer’s instructions. The
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cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s,
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55 °C for 40 s, and 72 °C for 40 s. The primers used for the qPCR reaction are shown in
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Table 1.
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2.8. Experimental design
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In this study, all experiments were replicated at least three times.
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Experiment 1 was designed to evaluate the optimal duration of IVM. In total, 337
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COCs were matured at 38.5 ºC in a humidified incubator containing 5% CO2 in air for 24, 48,
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or 72 h. Thereafter, CCs were removed and denuded oocytes were stained to assess their
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nuclear maturation. The percentages of oocytes at each stage of meiotic resumption were
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recorded.
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Experiment 2 was designed to determine the optimal time point of Wee1B-siRNA injection into the cytoplasm of denuded canine oocytes. Oocytes were injected at 0 h
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(immediately after high-quality oocytes were recovered) or after 24, 48, or 72 h of culture.
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Thereafter, oocytes were matured in vitro for up to 72 h at 38.5 ºC in a humidified incubator
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containing 5% CO2 in air, with the exception of oocytes in the 72 h group, which were
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examined after 84 h of culture. The percentages of oocytes at each stage of meiotic
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resumption were recorded. In total, 384 injected oocytes and non-injected (control) oocytes
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were used.
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in the nuclear maturation of canine oocytes. Wee1B-siRNA was microinjected into the
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cytoplasm of oocytes at 0 h of IVM, and then oocytes were cultured for various amounts of
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time (0, 24, 48, or 72 h). Thereafter, total RNA was isolated and analyzed by qPCR. The
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mRNA levels of cAMP, cdc25B, MAPK1, and MAPK3 were investigated.
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2.8. Statistical analysis
The percentages of canine oocytes at various maturation stages were recorded.
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Significant differences in oocyte maturation were confirmed by calculating the least-square
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means using SAS 9.3 software (SAS Institute, Inc., Cary, NC, USA). Maturation-related gene
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expression data are expressed as the mean ± SEM and were compared using the Student’s t-
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test. All experiments were performed at least three times. P < 0.05 was considered significant.
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3. Results
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3.1. Optimal duration of IVM
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The percentage of oocytes that matured to MII stage was higher (P < 0.05) among those cultured for 72 h than among those cultured for 24 and 48 h (5.4% ± 2.5 vs. 0.0% ± 0.0
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and 1.4% ± 1.0, respectively; Table 2). Most oocytes were arrested at GV after maturation for
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24, 48, and 72 h (71.4% ± 16.5, 59.7% ± 8.8, and 44.8% ± 9.7, respectively). The percentage
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of oocytes that matured to the MI or MII stage was higher (P < 0.05) among those cultured
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for 72 h (27.8% ± 8.3) than among those cultured for 24 h (5.7% ± 6.0), but did not differ
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between oocytes cultured for 72 h and those cultured for 48 h (27.8% ± 8.3 vs. 16.4% ± 11.0;
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Table 2).
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3.2. Effect of Wee1B-siRNA microinjection into cytoplasm
The percentage of oocytes that matured to MII was higher (P < 0.05) among those
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microinjected with Wee1B-siRNA at 0 h than among control oocytes and those microinjected
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with Wee1B-siRNA at 72 h (18.0% ± 1.7 vs. 2.1% ± 2.8 and 0.0% ± 0.0, respectively), but
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did not differ among oocytes microinjected with Wee1B-siRNA at 0 h and those injected at
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24 or 48 h (18.0% ± 1.7 vs. 11.4% ± 5.0 and 14.7% ± 1.4, respectively; Table 3).
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Furthermore, the percentage of oocytes that matured to MI was higher (P < 0.05) among
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oocytes microinjected with Wee1B-siRNA at 0 h than among control oocytes and those
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microinjected with Wee1B-siRNA at 24 and 72 h (45.9% ± 6.8 vs. 22.1% ± 3.5, 22.8% ±
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10.0, and 10.0% ± 4.4, respectively), but did not differ between oocytes microinjected with
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Wee1B-siRNA at 0 h and those microinjected with Wee1B-siRNA at 48 h (45.9% ± 6.8 vs.
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26.1% ± 9.0; Table 3).
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3.3 Assessment of maturation-related genes by qPCR
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The mRNA levels of cAMP, cdc25B, MAPK1, and MAPK3 were analyzed by qPCR
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at 0, 24, 48, and 72 h after microinjection of Wee1B-siRNA into the cytoplasm of canine
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oocytes, which was performed at 0 h of IVM. Expression levels were normalized against that
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of GAPDH. The mRNA level of cAMP was lowest (P < 0.05) in oocytes cultured for 48 h
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after Wee1B-siRNA microinjection, and there were no significant differences among the
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other groups (Fig. 2A). Moreover, the mRNA level of cdc25B did not differ among control
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oocytes and those cultured for 24 or 72 h after Wee1B-siRNA microinjection; however, it
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was lower (P < 0.05) in oocytes cultured for 48 h after Wee1B-siRNA microinjection than in
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the other groups (Fig. 2B). The mRNA levels of MAPK1 and MAPK3 were higher (P < 0.05)
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in oocytes cultured for 72 h after Wee1B-siRNA microinjection than in the other groups (Fig.
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2C and 2D).
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Discussion
In the present study, the percentage of canine oocytes that matured to the MII stage
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was higher among those that underwent IVM for 72 h (P < 0.05) than among those that
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underwent IVM for 24 or 48 h. Oocytes have been previously cultured for 72 h in
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ACCEPTED MANUSCRIPT supplemented TCM-199. Canine oocytes are ovulated at prophase I and mature in the distal
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part of the oviduct for at least 48–72 h [4]. Hence, we investigated the optimal duration of
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IVM of canine oocytes. Microinjection of Wee1B-siRNA into the oocyte cytoplasm
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significantly enhanced nuclear maturation. The percentages of oocytes at MI and MII stages
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were higher (P < 0.05) among oocytes microinjected with Wee1B-siRNA at 0 h of IVM
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(45.9% ± 6.8 and 18.0% ± 1.7, respectively) than among control oocytes (22.1% ± 3.5 and
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2.1% ± 2.8, respectively). This is the first report regarding intracytoplasmic microinjection of
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Wee1B-siRNA to promote nuclear maturation of canine oocytes. In this study, the MII
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maturation rate was not higher than in previous studies, but the maturation rate from the GV
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to MI stage was high. Compared to other mammalian species, the developmental competence
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of canine oocytes (cultured in vitro) from the GV to MII stage is severely compromised.
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Consequently, there is an urgent need to develop improved systems for IVM of canine
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oocytes. The most common approach to improve the IVM of canine oocytes is to add various
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compounds to the culture media to mitigate meiotic arrest. Pretreatment with 10 mM caffeine
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for 12 h prior to culture for a further 60 h in basal culture medium enhances the development
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of oocytes to MII (16.9% ± 2.4) and MI (8.1% ± 1.2) stages [17]. The addition of 200 ng/mL
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progesterone resulted in 10.7% of oocytes reaching the MII stage and 43.3% of oocytes
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reaching the GVBD/MI stage [5]. The current study sought to explore and validate a novel
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approach that could be used to improve the efficiency of canine IVF.
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In mice, pigs, cattle, and other mammals, oocytes are arrested at prophase I prior to
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the LH surge. The MPF complex, comprising CDK1 and cyclin B, is required for meiotic
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resumption. The CDK1 kinase is inactive at the GV stage and its activation triggers GVBD
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[21-24]. Furthermore, cAMP in oocytes plays critical roles in meiotic arrest and resumption. 13
ACCEPTED MANUSCRIPT A high cAMP concentration in oocytes maintains the activity of PKA, which induces
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phosphorylation of Wee1B and thereby inhibits MPF activity. There are several prerequisites
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for the activation of MPF: a low cAMP concentration, which inactivates PKA, inactive
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Wee1B, and dephosphorylation of MPF by cdc25B [9,22,25-29]. Therefore, maintenance of a
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high cAMP concentration activates Wee1B and prevents activation of MPF [30].
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Consequently, we speculated that intracytoplasmic microinjection of Wee1B-siRNA into
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canine oocytes would activate MPF. Following Wee1B-siRNA microinjection at 0 h of IVM
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and subsequent culture for 72 h, 18.0% ± 1.7 of oocytes reached MII stage and 63.9% ± 5.3
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of oocytes reached the MI or MII stage (Table 3). These maturation rates (MI or MII) were
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significantly different from those of control oocytes. The maturation rates of oocytes cultured
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for 24 or 48 h following Wee1B-siRNA microinjection were not significantly different from
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those of control oocytes, although there were fewer of the former oocytes (Table 3).
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Therefore, we analyzed the mRNA levels of genes related to the mechanism in which Wee1B
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functions by qPCR in oocytes cultured for various amounts of times following Wee1B-siRNA
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microinjection at 0 h of IVM. The mRNA level of cAMP was lower in oocytes cultured for
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48 h after Wee1B-siRNA microinjection than in control oocytes, but did not differ between
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oocytes cultured for 72 h after Wee1B-siRNA microinjection and control oocytes (Fig. 2A).
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This shows that cAMP was not influenced by Wee1B-siRNA, although it was expected to
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downregulate cAMP. The mRNA level of cdc25B was significantly lower and higher in
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oocytes cultured for 48 h and 72 h after Wee1B-siRNA microinjection, respectively, in
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comparison with that in control oocytes (Fig. 2B). Increased cdc25B expression can stimulate
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downstream genes, such as MAPK1 and MAPK3, and induce nuclear maturation. mRNA
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levels of MAPK1 and MAPK3, which are related to nuclear maturation, were increased in
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expression of factors related to nuclear maturation was increased following Wee1B-siRNA
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microinjection, even though mRNA expression of cAMP was not altered. These results are in
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accordance with the mechanism via which Wee1B functions in oocyte nuclear maturation.
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Therefore, our results suggest that Wee1B-siRNA can promote nuclear activation of canine
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oocytes.
In conclusion, microinjection of Wee1B-siRNA into the cytoplasm of canine oocytes
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significantly promoted nuclear maturation. This is the first report of Wee1B-siRNA injection.
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Although further refinement is needed to enhance the proportion of oocytes that reach MII
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stage, this study provides the proof-of-concept for this approach.
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Acknowledgments
This study was supported by the Rural Development Administration, Republic of
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Korea (grant nos. PJ008975042014 and PJ009321012014). Yu-Gon Kim, Seok-Hwan Song,
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and Kyeong-Lim Lee were supported by BK21 Plus fellowships from Gyeongsang National
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University, Republic of Korea.
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[17] Salavati M, Ghafari F, Zhang T, Fouladi-Nashta AA. Influence of caffeine pretreatment on biphasic in vitro maturation of dog oocytes. Theriogenology 2013;80(7):784-92.
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[19] Kim MK, Fibrianto YH, Oh HJ, Jang G, Kim HJ, Lee KS, et al. Effects of estradiol17beta and progesterone supplementation on in vitro nuclear maturation of canine oocytes. Theriogenology 2005;63(5):1342-53.
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[24] Bilodeau-Goeseels S. Bovine oocyte meiotic inhibition before in vitro maturation and its value to in vitro embryo production: Does it improve developmental competence? Reprod Domest Anim 2012;47(4):687-93.
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ACCEPTED MANUSCRIPT [25] Nakanishi M, Ando H, Watanabe N, Kitamura K, Ito K, Okayama H, et al. Identification and characterization of human Wee1B, a new member of the Wee1 family of cdkinhibitory kinases. Genes Cells 2000;5(10):839-47.
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[26] Bornslaeger EA, Schultz RM. Regulation of mouse oocyte maturation: Effect of elevating cumulus cell cAMP on oocyte cAMP levels. Biol Reprod 1985;33(3):698-704.
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[27] Tsafriri A, Chun SY, Zhang R, Hsueh AJ, Conti M. Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: Studies using selective phosphodiesterase inhibitors. Dev Biol 1996;178(2):393-402.
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[28] Han SJ, Chen R, Paronetto MP, Conti M. Wee1B is an oocyte-specific kinase involved in the control of meiotic arrest in the mouse. Curr Biol 2005;15(18):1670-6.
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[29] Hanna CB, Yao S, Patta MC, Jensen JT, Wu X. WEE2 is an oocyte-specific meiosis inhibitor in rhesus macaque monkeys. Biol Reprod 2010;82(6):1190-7.
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[30] Conti M, Hsieh M, Zamah AM, Oh JS. Novel signaling mechanisms in the ovary during oocyte maturation and ovulation. Mol Cell Endocrinol 2012;356(1-2):65-73.
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Table Legends
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Table 1
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Quantitative real-time PCR primers used in this study.
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Table 2
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Effect of the duration of IVM on the nuclear development of canine oocytes.
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Table 3
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Effect of the time point of Wee1B-targeting small interfering RNA microinjection on the nuclear developmental of canine oocytes.
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Figure Legends
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Fig. 1. Stages of nuclear maturation of canine oocytes (stained with Hoechst 33342). A:
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germinal vesicle (GV); B: germinal vesicle breakdown (GVBD); C: metaphase I (MI); D:
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metaphase II (MII). Scale bars = 100 µm.
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Fig. 2. Relative expression of genes related to the mechanism in which Wee1B functions in
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mammalian oocytes. Control: culture for 72 h without Wee1B-targeting small interfering
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RNA (Wee1B-siRNA) microinjection; 0 h group: oocytes not cultured after Wee1B-siRNA
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microinjection; 24 h group: oocytes cultured for 24 h after Wee1B-siRNA microinjection; 48
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h group: oocytes cultured for 48 h after Wee1B-siRNA microinjection; 72 h group: oocytes
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cultured for 72 h after Wee1B-siRNA microinjection. a–e Data are from three independent
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experiments. Different letters indicate a significant difference (P < 0.05).
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ACCEPTED MANUSCRIPT To: THERIOGENOLOGY
Revised
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Wee1B depletion promotes nuclear maturation of canine oocytes
Yu-Gon KimA,†, Dong-Hoon KimC,†, Seok-Hwan SongA, Han-Seul ParkA,
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Byoung-Chul YangC, Jeong-Su OhD, Il-Keun KongA,B,† A
Department of Animal Science, Division of Applied Life Science, BInstitute of Agriculture
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and Life Science, Gyeongsang National University, Jinju 660-701, Gyeongsangnam-do, Republic of Korea, CAnimal Biotechnology Division, National Institute of Animal Science, Suwon 441-706, Gyeonggi-do, Republic of Korea, DDepartment of Genetic Engineering,
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746, Republic of Korea
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College of Biotechnology and Bioengineering, Sungkyunkwan University, Gyeonggi-do 440-
ACCEPTED MANUSCRIPT Table 1 Real time PCR primers for quantification of gene expression. Gene
Product size Sequence
Accession number (bp)
F; AACAGTGACACCCACTCTTC GAPDH
110 R; CGGTTGCTGTAGCCAAATTC F; CAGGTGACAGACTTCGGTTT
cAMP
105 F; AATATCTGGGCAGTCCCATTAC
cdc25B
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R; CCTTGCCCTCTTCTTGATCTT
AB_038240
NM_001003032.1
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R; GTAGCCTTTGCTCAGGATGAT
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symbol
XM_005626604.1
F; AGAGAACCCTGAGGGAGATAAA MAPK1
90
NM_001110800
112
NM_001252035.1
R; CGATGGTTGGTGCTCGAATA
F; CAACGACCATGTTTGCTACTTC MAPK3
R; GGTGTTGATGAGCAGGTTAGA
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GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; cAMP: Cyclic adenosine monophosphate; cdc25B: cell division cycle 25; MAPK1: Mitogen-activated protein kinase 1; MAPK3: Mitogen-activated protein kinase 3.
ACCEPTED MANUSCRIPT Table 2 Effects of in vitro maturation time on nuclear development of canine oocytes. No. oocytes developed to (Mean ± SEM) & culture time (h)
Stages
24
48
72
75 (71.4 ± 16.5) 24 (22.8 ± 12.3)a
40 (59.7 ± 8.8) 16 (23.8 ± 9.8)a
74 (44.8 ± 9.7)a 45 (27.2 ± 6.7)a
MI
6 (5.7 ± 6.0)b
10 (14.9 ± 10.0)a
37 (22.4 ± 8.1)a
MII
0 (0.0 ± 0.0)b
1 (1.4 ± 1.0 )b
9 (5.4 ± 2.5)a
MI or MII
6 (5.7 ± 6.0)b
11 (16.4 ± 11.0)a,b
46 (27.8 ± 8.3)a
GV GVBD
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Total no. oocytes cultured 105 67 Within a row, percentages without a common superscript differed (P < 0.05).
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Table 3
Stages
No. oocytes developed to (Mean ± SEM) & culture time (h)⃰ 48
72§
34 (48.5 ± 17.6)a,b
14 (15.9 ± 5.2)b
35 (50.0 ± 9.2)a
6 (9.8 ± 2.5)c
12 (17.1 ± 3.5)b,c
38 (43.1 ± 7.5)a
28 (40.0 ± 4.7)a,b
21 (22.1 ± 3.5)b
28 (45.9 ± 6.8)a
16 (22.8 ± 10.0)b
23 (26.1 ± 9.0)a,b
7 (10.0 ± 4.4)b
2 (2.1 ± 2.8)b,c
11 (18.0 ± 1.7)a
8 (11.4 ± 5.0)a,b
13 (14.7 ± 1.4)a
0 (0.0 ± 0.0)c
23 (24.2 ± 2.8)b,c
39 (63.9 ± 5.3)a
24 (34.2 ± 15.0)b,c
36 (40.9 ± 10.2)a,b
7 (10.0 ± 4.4)c
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70
0
24
GV
48 (50.5 ± 13.8)a,b
16 (26.2 ± 5.6)a,b
GVBD
24 (25.2 ± 11.0)a,c
MI MII
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MI or MII
Total no. oocytes cultured 95 61 Within a row, percentages without a common superscript differed (P < 0.05).
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All of experimental groups were cultured on 72 hours after Wee1B-siRNA microinjection. §72 h of Wee1B-siRNA microinjection group was
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Effects of Wee1B-siRNA injection time on nuclear development of canine oocytes.
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Figure 1
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Figure 2