Cilostamide and forskolin treatment during pre-IVM improves preimplantation development of cloned embryos by influencing meiotic progression and gap junction communication in pigs

Theriogenology xxx (2016) 1–9

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Cilostamide and forskolin treatment during pre-IVM improves preimplantation development of cloned embryos by influencing meiotic progression and gap junction communication in pigs Bola Park a, Hanna Lee a, Yongjin Lee a, Fazle Elahi a, Joohyeong Lee a, Seung Tae Lee b, Choon-Keun Park b, Sang-Hwan Hyun c, Eunsong Lee a, d, * a

Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea Division of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Korea c Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea d Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 September 2015 Received in revised form 24 February 2016 Accepted 27 February 2016

This study was conducted to evaluate the effects of treatment with the cAMP modulators cilostamide and/or forskolin during pre-IVM culture on meiotic progression, gap junction communication, intraoocyte cAMP level and glutathione content, embryonic development after parthenogenesis, and somatic cell nuclear transfer in pigs. Cumulus–oocyte complexes were cultured for 24 hours in unsupplemented medium or media containing 20 mM cilostamide and/or 50 mM forskolin. After pre-IVM, oocytes were cultured for 41 to 44 hours in a standard IVM medium to induce oocyte maturation. When the nuclear status of oocytes was examined after pre-IVM for 24 hours, a higher (P < 0.01) proportion of oocytes treated with forskolin (85.5%) and cilostamide þ forskolin (92.6%) remained at the germinal vesicle stage compared with untreated (20.6%) and cilostamide-treated oocytes (54.7%). cAMP level in pre-IVM oocytes was significantly increased by combined treatment with cilostamide þ forskolin (21.38 fmol/oocyte) relative to the no pre-IVM control, no treatment, cilostamide, and forskolin groups (2.85, 1.88, 1.74, and 8.95 fmol/oocyte, respectively). Forskolin with or without cilostamide significantly maintained open-gap junction communication relative to no treatment. Blastocyst formation in parthenogenesis was significantly (P < 0.01) improved by forskolin (65.3%) relative to other treatments (28.3% to 48.1%). Supplementation of pre-IVM with dibutyryl cAMP showed similar blastocyst formation as forskolin treatment (61.1% and 61.0%, respectively). In somatic cell nuclear transfer, simultaneous treatment with cilostamide þ forskolin significantly (P < 0.05) increased embryonic development to the blastocyst stage (42.9%) relative to the no pre-IVM, control, and cilostamide groups (32.3, 28.6, and 32.8%, respectively). The glutathione contents in pre-IVM oocytes were increased by no treatment, forskolin, and cilostamide þ forskolin (1.38, 1.39, and 1.27 pixels/oocyte, respectively) compared with no pre-IVM and cilostamide (1.00 and 0.99 pixels/oocyte, respectively; P < 0.05). Our results reported that the meiotic progression of immature pig oocytes could be reversibly attenuated by cAMP, whereas treatment with cilostamide and forskolin during pre-IVM had positive effects on developmental competence of oocytes in pigs, probably by improving cytoplasmic maturation. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Cyclic AMP Forskolin Oocyte maturation Nuclear transfer

* Corresponding author. Tel.: þ82 33 250 8670; fax: þ82 33 259 5625. E-mail address: [email protected] (E. Lee). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.02.029

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1. Introduction Pigs are frequently used as a suitable animal model for human diseases or bio-organ donors because of the similarity of their physiology to that of humans [1]. Somatic cell nuclear transfer (SCNT) is an assisted reproductive technology commonly used to produce cloned animals for specific purposes. However, the efficiency of cloned animal production is currently not satisfactory [2,3]. SCNT efficiency is influenced by various factors including type of donor cells, oocyte quality, and nuclear remodeling and reprogramming regimens. Among these, quality of oocytes derived from IVM is one of the most important factors determining the success of SCNT because remodeling and reprogramming of donor nuclei are controlled by cytoplasmic factors of recipient oocytes [4,5]. Oocytes with incomplete nuclear and cytoplasmic maturation cannot support normal embryonic development after SCNT. Extensive studies were carried out to produce competent oocytes through IVM; however, their quality is still low compared with their in vivo counterparts. In vivo oocytes have been arrested at the dictyate stage of meiosis, and it takes several days to mature from antral follicles to pre-ovulatory follicles [6]. Nuclear and cytoplasmic maturation are completed by luteinizing hormone [7]. However, in vitro oocytes spontaneously start meiosis once removed artificially from follicles. This phenomenon leads to early or premature extrusion of the first polar body (PB) compared with in vivo oocytes before sufficient cytoplasmic maturation occurs, which may be because of unstable progression of meiosis [8]. Additionally, gap junction communication (GJC) status is altered depending on the stage of nuclear maturation [9,10]. Early loss of GJC probably occurs in IVM oocytes because of premature nuclear maturation and, therefore, may be needed to induce better synchronization between nuclear and cytoplasm maturation to obtain competent oocytes [11]. Delaying artificial nuclear maturation was previously reported to have a positive effect on cytoplasmic maturation and subsequent embryonic development after IVF and SCNT. Accordingly, IVM of oocytes for the first 20 hours in medium containing 1 mM dibutyryl cAMP (dbcAMP) increased developmental competence of oocytes after IVF [12]. Additionally, pre-maturation culture with 50 mM roscovitine (p34cdc2/cyclin B kinase inhibitor) for 22 hours increased intraoocyte glutathione (GSH) content and blastocyst formation after IVF [13]. Although the mechanism responsible for oocyte maturation in vitro and in vivo is not fully understood, it is known that the second messenger, cAMP, plays a critical role in maintenance of meiotic arrest in mammalian oocytes [14]. cAMP is synthesized in oocytes and additionally by granulosa and cumulus cells, after which it enters into oocytes through gap junctions [15]. The optimum level of intraoocyte cAMP has a positive effect on maintenance of meiotic arrest, whereas decreases in the level of cAMP lead to spontaneous resumption of meiosis [16]. Increases in cAMP levels activate cAMP-dependent protein kinase A, resulting in meiotic arrest through inhibition of maturationpromoting factor and mitogen-activated protein kinase [17]. Intraoocyte cAMP levels are regulated by phosphodiesterase (PDE) and adenyl cyclase (AC), which play roles in the degradation and synthesis of cAMP, respectively.

Inhibition of PDE type 3A (PDE3A), which is normally present in pig oocytes, has been found to transiently prevent oocytes from spontaneous meiotic resumption after removal of cumulus–oocyte complex (COC) from follicles by protecting against cAMP degradation [16,18]. Cilostamide inhibits the activity of PDE3A [19], and Dieci et al. [20] reported that treatment of pig oocytes with 1 mM cilostamide for 24 hours during IVM improved oocyte quality and developmental competence. High levels of intraoocyte cAMP in cattle have been reported to maintain meiotic arrest of oocytes [21]. Conversely, forskolin has been reported to regulate cAMP levels of oocytes in many species, including mice [22], rats [23], and pigs [24]. Olsiewski and Beers [25] reported that cAMP levels increased in response to forskolin treatment in denuded oocytes and intact COCs in rats. As shown in previous studies [26,27], cAMP modulators play a key role in nuclear maturation because cAMP is a fundamental factor involved in the arrest and resumption of meiosis that is also associated with further developmental competence. Despite many studies being conducted to improve the quality of IVM oocytes and SCNT efficiency, IVM oocytes and SCNT embryos derived from IVM oocytes still show lower developmental competence than their counterparts. Therefore, we investigated whether increasing the cAMP level by treatment with the cAMP modulators, cilostamide and forskolin, during pre-IVM will improve oocyte maturation and embryonic development by preventing premature nuclear maturation and early loss of GJC. To test this hypothesis, immature COCs were untreated or treated for 24 hours during pre-IVM with cilostamide and/ or forskolin. Meiotic progression, GJC status, intraoocyte cAMP level, GSH content, and developmental competence after parthenogenesis (PA) and SCNT were then examined. 2. Materials and methods 2.1. Culture media All chemicals were purchased from Sigma–Aldrich unless otherwise stated. Stock solutions of 74.0 mM cilostamide (BML-PD125; Enzo Life Science, Farmingdale, NY, USA) and 12.2 mM forskolin (BML-CN100; Enzo Life Science) were prepared in DMSO and stored in the dark. The stock solution of dbcAMP (D0627) was prepared in purified water at 101.8 mM. Cilostamide, forskolin, and dbcAMP were added to pre-IVM medium at final concentrations of 20, 50, and 1 mM, respectively, according to the experimental design. The basic medium used for IVM was medium-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (vol/vol) porcine follicular fluid, 0.6 mM cysteine, 0.91 mM pyruvate, 75 mg/mL kanamycin, and 1 mg/mL insulin. The IVC medium for embryo development was porcine zygote medium-3 containing 0.3% (wt/vol) fatty acid–free BSA [28]. 2.2. Oocyte collection and IVM Ovaries were obtained from prepubertal gilts at a local abattoir. Follicular contents were aspirated from superficial follicles (3–8 mm in diameter), pooled into 15-mL conical tubes, and allowed to settle. The sediment was then placed in HEPES-buffered Tyrode’s medium (TLH) containing 0.05%

B. Park et al. / Theriogenology xxx (2016) 1–9

(wt/vol) polyvinyl alcohol (TLH–PVA) [29] and observed under a stereomicroscope. Only COCs with more than three layers of compact cumulus cells were selected. After washing twice in TLH–PVA and once in basic medium, a group of 50 to 60 COCs was placed into each well of a four-well multidish (Nunc, Roskilde, Denmark) containing 500 mL of IVM medium with cAMP modulators. COCs were cultured for 24 hours in pre-IVM medium at 39  C in a humidified atmosphere of 5% CO2 in air. Next, pre-IVM, COCs were cultured in 500 mL of IVM medium with 10 ng/mL epidermal growth factor, 10 IU/mL hCG (Intervet International BV, Boxmeer, Holland) and 80 mg/mL FSH (Antrin R-10; Kyoritsu Seiyaku, Tokyo, Japan) for 22 hours at 39  C under 5% CO2 in air. The COCs were then washed three times in fresh hormone-free IVM medium and cultured in hormone-free IVM medium for an additional 19 hours for SCNT and 22 hours for PA.

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nutrient mixture (Invitrogen) supplemented with 15% (vol/vol) fetal bovine serum and 75 mg/mL kanamycin until a complete monolayer formed. Donor cells were synchronized at the G0/G1 stage of the cell cycle by contact inhibition for 48 to 72 hours. Cells of the same passage (passages 3–8) were used in each replicate. A single-cell suspension was prepared by trypsinization of cultured cells and resuspension in TLH containing 0.4% (wt/vol) BSA (TLH–BSA) before nuclear transfer. 2.5. SCNT, PA, and embryo culture

During pre-IVM, immature COCs were untreated (control) or treated with 20 mM cilostamide and/or 50 mM forskolin, after which nuclear maturation, GJC, and embryonic development following PA and SCNT were examined. The concentrations of forskolin and cilostamide to maintain cAMP level and prevent meiotic resumption in this study were adopted from the previous results [24,30] obtained in pigs. The duration (24 hours) of pre-IVM in this study was on the basis of the previous results determined in pigs [24] and human [31] that nuclear maturation occurred normally even after meiotic resumption was arrested for 24 hours by forskolin and cilostamide. In our preliminary study, when pre-IVM oocytes were cultured for 22 and 44 hours for IVM, 44-hour culture showed significantly higher nuclear maturation and blastocyst formation after PA than IVM culture for 22 hours (data not shown). Thus, we cultured immature oocytes for 24 and 44 hours for pre-IVM and IVM, respectively. In experiment 1, the effects of cilostamide and forskolin treatment during pre-IVM on intraoocyte GSH content, cumulus cell expansion, and embryonic development after PA were examined. Subsequently, the effects of cilostamide and forskolin treatment on nuclear status, cAMP level, and GJC status were examined in pre-IVM oocytes in experiments 2 and 3, respectively. In experiment 4, the effects of direct treatment with dbcAMP during pre-IVM on developmental competence of oocytes were compared with those of forskolin and cilostamide treatment to test our hypothesis that increased embryonic development after forskolin and cilostamide treatment would be attributed to the effect of increased cAMP level on oocyte maturation during pre-IVM. Maturation and embryonic development of oocytes derived from pre-IVM and IVM using cAMP modulators were compared with those of oocytes from a standard IVM (no pre-IVM) in this experiment. Finally, the effects of cilostamide and forskolin on embryonic development after SCNT were examined in experiment 5.

The base medium for oocyte manipulation was calciumfree TLH–BSA containing 5 mg/mL cytochalasin B. After 40 hours of maturation, cumulus cells were removed by repeated pipetting in 0.1% (wt/vol) hyaluronidase in hormone-free IVM medium. Denuded oocytes were incubated for 15 minutes in a manipulation medium that contained 5 mg/mL Hoechst 33342, washed twice in fresh medium, and then placed into a manipulation medium droplet that was overlaid with mineral oil. Metaphase II (MII) oocytes were enucleated by aspirating the first PB and MII chromosomes using a 17-mm beveled glass pipette (Humagen, Charlottesville, VA). Enucleation was confirmed under an epifluorescent microscope (TE300; Nikon, Tokyo, Japan). After enucleation, 20 to 30 donor cells were aspirated into a 17-mm beveled glass pipette, and a single cell was inserted into the perivitelline space of each oocyte [32]. COCs were then placed on a 1-mm fusion chamber overlaid with 1 mL of 280 mM mannitol that contained 0.001 mM CaCl2 and 0.05 mM MgCl2. Membrane fusion was induced by applying an alternating current field of 2 V cycling at 1 MHz for 2 seconds, followed by two direct current pulses of 175 V/mm for 30 microseconds using a cell fusion generator (LF101; NepaGene, Japan). The oocytes were then incubated for 1 hour in TLH–BSA, after which they were evaluated for fusion under a stereomicroscope. Reconstructed oocytes were activated by two pulses of a 120 V/mm direct current for 60 microseconds in 280 mM mannitol that contained 0.01 mM CaCl2 and 0.05 mM MgCl2. For PA, oocytes with PBs at 44 hours of IVM were activated using a pulse sequence identical to that used to activate SCNT oocytes. Following electrical activation, the PA and SCNT embryos were treated with 7.5 mg/mL cytochalasin B and 0.4 mg/mL demecolcine, respectively, combined with 1.9 mM 6-dimethylaminopurine in IVC medium for 4 hours. The SCNT and PA embryos were washed three times in fresh IVC medium, transferred into 30 mL IVC droplets under mineral oil, and then cultured at 39  C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 7 days. Cleavage and blastocyst formation were evaluated on Days 2 and 7, respectively, with the day of SCNT or PA designated as Day 0. Total blastocyst cells were counted under an epifluorescent microscope after Hoechst 33342 staining.

2.4. Preparation of donor cells

2.6. Measurement of intraoocyte GSH content

Pig fetal fibroblasts were seeded in a four-well plate and grown in Dulbecco’s modified Eagle’s medium with F-12

Intracellular GSH levels of oocytes were measured as previously described [33]. Briefly, Cell-Tracker Blue

2.3. Experimental design

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CMF2HC (4-chloromethyl-6.8-difluoro-7-hydroxycoum arin; Invitrogen) was used to detect intracellular GSH levels as blue fluorescence. A group of denuded oocytes at the MII stage from each group was collected 44 hours after IVM and incubated for 30 minutes in TLH–PVA that had been supplemented with 10 mM Cell-Tracker in the dark. After incubation, oocytes were washed with Dulbecco’s phosphate-buffered saline (Invitrogen) containing 0.1% (wt/vol) PVA, placed into 10-mL droplets, and observed for fluorescence under an epifluorescence microscope (TE-300; Nikon) with a UV filter (370 nm). Fluorescent images were recorded and saved as graphic files in TIFF format. The fluorescence intensity of oocytes was analyzed using the ImageJ software (version 1.45r; National Institutes of Health, Bethesda, MD, USA). 2.7. Examination of nuclear status and cumulus expansion After 24 hours of pre-IVM, oocytes were mounted onto glass slides and fixed for 5 to 10 minutes in 25% (vol/vol) acetic acid in ethanol. Fixed oocytes were then stained in a solution of 1% (wt/vol) orcein in 45% (vol/vol) acetic acid. The oocytes were assessed under a phase contrast microscope and designated as germinal vesicle (GV), metaphase I, anaphase I and telophase I, or MII according to the morphologic criteria for characterization of meiotic stages reported by Funahashi et al. [34]. After pre-IVM and IVM, cumulus cell expansion was assessed subjectively as previously described [35]. Briefly, no response was scored as 0, minimum observable response with the cells in the outermost layer of the cumulus became round and glistening as 1, the expansion of outer cumulus cell layers as 2, the expansion of all cumulus cell layers except corona radiata as 3, and the expansion of all cumulus cell layer including corona radiata as 4. 2.8. Assay of intraoocyte cAMP level The levels of cAMP in oocytes were determined using a Cyclic AMP Complete ELISA Kit (Assay Designs, Ann Arbor, MI, USA). Briefly, samples were washed in Dulbecco’s phosphate-buffered saline containing 0.1% (wt/vol) PVA. Oocytes (about 30 oocytes per group) were then transferred to 200 mL of 0.1 N HCl solution and stored at 80  C until assay. During the assay, all samples were acetylated according to the manufacturer’s protocols, after which plates were read at 405 nm using a plate reader. 2.9. Measurement of GJC Functional communication of the gap junction was assessed by lucifer yellow microinjection as previously described [36], with some modifications. A 3% (wt/vol) solution of lucifer yellow in 5 mM lithium chloride was treated into oocytes, after which the spreading of dye into the surrounding cumulus cells was monitored under an inverted epifluorescent microscope (excitation 430 nm, emission 540 nm; TE-300; Nikon). The status of GJC in each COC was classified as open, partially open, or closed as previously described [37].

2.10. Statistical analyses All statistical analyses were performed using the Statistical Analysis System (version 9.3; SAS Institute, Cary, NC, USA). Data were analyzed using a general linear model followed by the least significant difference mean separation procedure when treatments differed at P < 0.05. Percentage data were arcsine transformed before analysis to maintain homogeneity of variances. All results are expressed as the mean  SEM. 3. Results 3.1. Effects of cilostamide and forskolin treatment during preIVM on nuclear maturation, intraoocyte GSH contents, and embryonic development after PA (experiment 1) As listed in Table 1, nuclear maturation of oocytes was not altered by pre-IVM treatment using cAMP modulators. However, expansion of cumulus cells during/after pre-IVM and IVM was influenced by cAMP modulator treatment. Additionally, forskolin with and without cilostamide treatment showed more expanded cumulus cell layers after preIVM than the control and cilostamide treatment (Fig. 1 and Table 1). The intraoocyte GSH content was significantly higher in the control (1.38 pixels/oocyte), forskolin (1.39 pixels/ oocyte), and cilostamide þ forskolin (1.27 pixels/oocyte) groups than in the no pre-IVM (1.00 pixels/oocyte) and cilostamide (0.99 pixels/oocyte) groups (P < 0.05). After PA of oocytes that were treated with cilostamide and/or forskolin, embryo cleavage increased in response to forskolin (96.7%) relative to cilostamide (81.9%) and cilostamide þ forskolin (84.6%) (P < 0.05). Blastocyst formation also increased (P < 0.01) in response to forskolin treatment (65.3%) relative to the no pre-IVM (48.1%), control (40.8%), cilostamide (41.1%), and cilostamide þ forskolin (28.3%) treatments (Table 2). The mean cell number per blastocyst was higher (P < 0.05) in cilostamide þ forskolin (41.4 cells) than in the no pre-IVM (36.4 cells/blastocyst) group.

Table 1 Effects of cilostamide and forskolin treatment during pre-IVM for 24 hours on nuclear maturation, intraoocyte glutathione content, and embryonic development after parthenogenesis. Pre-IVM treatment

No. of Percentage oocytes of oocytes culturedd reaching metaphase II

No pre-IVM Control Cilostamide (C) Forskolin (F) CþF

199 151 147

95.3  1.8 90.4  4.5 93.6  3.7

1.00  0.09a 1.38  0.10b 0.99  0.11a

0  0a 0.73  0.16b 0.43  0.13b

194

89.6  2.0

1.39  0.08b

3.20  1.11c

192

93.1  1.4

1.27  0.01b

3.16  0.14c

a,b,c

Relative level Cumulus cell (pixels/oocyte) expansionf of GSH (n ¼ 60e)

Within a column, values with different superscripts are different (P < 0.05). d Three replicates. e Number of metaphase II oocytes examined for GSH contents. f Cumulus cell expansion was scored as 0 (no response), 1 (minimum observable response with the cells in the outermost layer of the cumulus become round and glistening), 2 (the expansion of outer cumulus cell layers), 3 (the expansion of all cumulus cell layers except corona radiata), and 4 (the expansion of all cumulus cell layers).

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Fig. 1. Morphology of cumulus–oocyte complexes (COCs) after pre-IVM and IVM. COCs without pre-IVM culture (A) and COCs that had been cultured for 24 hours in a pre-IVM medium containing no treatment (D), cilostamide (G), forskolin (J), and C þ F (M) were cultured for 22 hours in IVM medium with hormones (B, E, H, K, and N) and then for an additional 22 hours in hormone-free medium (C, F, I, L, and O).

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only 3.3% of COCs had closed GJC, whereas 78.6% of COCs had open GJC. Pre-IVM treatment with cilostamide þ forskolin (66.7%) revealed a significantly higher proportion of oocytes with opened GJC than the control (42.2%) (P < 0.05).

Table 2 Effects of cilostamide and forskolin treatment during pre-IVM for 24 hours on embryonic development after parthenogenesis. Pre-IVM treatment

No. of PA Percentage of embryos embryos developed to culturedf 2 Cells Blastocyst

No pre-IVM Control Cilostamide (C) Forskolin (F) CþF

175 124 110 164 119

92.0 93.0 81.9 96.7 84.6

    

2.9ab 1.9ab 4.5a 1.6b 5.3a

48.1 40.8 41.1 65.3 28.3

    

2.5c 4.9c 3.1c 2.6d 4.6e

No. of cells in blastocyst

36.4 40.0 40.0 39.3 41.4

    

3.4. Effect of cilostamide and forskolin compared with dbcAMP during pre-IVM on embryonic development after PA (experiment 4)

1.3a 2.0ab 2.1ab 1.2ab 2.6b

Following PA of oocytes that were treated with forskolin, cilostamide þ forskolin, and dbcAMP during preIVM, oocytes in the dbcAMP (96.7%) and forskolin (98.5%) groups showed higher (P < 0.01) embryo cleavage than those treated with cilostamide þ forskolin (79.5%). Blastocyst formation also increased (P < 0.01) in response to the dbcAMP (61.1%) and forskolin (61.0%) treatment relative to the standard IVM (no pre-IVM) (49.2%) and cilostamide þ forskolin treatment (38.8%) (Table 4). The mean cell number of blastocysts was not altered by the preIVM treatments (39.6–42.6 cells/blastocyst).

a and b, Within a column, values with different superscripts are different (P < 0.05); c–e, within a column, values with different superscripts are different (P < 0.01). f Three replicates.

3.2. Nuclear status and cAMP levels of oocytes treated with cilostamide and forskolin during pre-IVM (experiment 2) After pre-IVM, the nuclear status of oocytes was assessed to determine the effects of cilostamide and/or forskolin treatment during pre-IVM on meiotic progression. A higher (P < 0.01) proportion of oocytes was arrested at the GV stage by forskolin (85.5%) and cilostamide þ forskolin (92.6%) compared with the control (20.6%) and cilostamide (54.7%) groups (Table 3). More oocytes progressed to the MII stage in the control (29.9%) and cilostamide (19.6%) than in forskolin (7.5%) and cilostamide þ forskolin (5.7%) groups. The nuclear status after 22 hours of pre-IVM with cilostamide and forskolin indicated that cilostamide þ forskolin treatment effectively blocks the resumption of meiosis. In addition, the intraoocyte cAMP level increased drastically from 0 hours (2.85 fmol/oocyte) to 24 hours of pre-IVM culture (21.38 fmol/oocyte) in response to the cilostamide þ forskolin treatment, and the cAMP level was higher (P < 0.05) in other groups. Oocytes treated with forskolin (8.95 fmol/oocyte) also showed a higher (P < 0.05) cAMP level than untreated and cilostamide-treated oocytes (1.88 and 1.74 fmol/oocyte, respectively).

3.5. Effect of cilostamide and forskolin treatment during preIVM on embryonic development after SCNT (experiment 5) Early cleavage of SCNT embryos was not influenced by the tested pre-IVM treatments. However, blastocyst formation increased significantly in response to cilostamide þ forskolin treatment (42.9%) relative to the no pre-IVM (32.3%), control (28.6%), and cilostamide (32.8%) treatments (Table 5). The mean cell numbers of blastocysts were significantly higher (P < 0.05) in the cilostamide (44.6 cells) and cilostamide þ forskolin group (44.6 cells) than in the no pre-IVM group (37.1 cells/blastocyst). 4. Discussion During IVM of mammalian oocytes, various cAMP modulators, such as milrinone, cilostamide, and 3-isobutyl1-methylxanthine, show various effects on oocyte maturation and later embryonic development to the blastocyst stage depending on the concentrations and/or time of treatment [38]. cAMP levels are regulated by AC activator and/or PDE inhibitors. A high level of cAMP arrests meiotic progression or synchronizes cytoplasmic and nuclear maturation of oocytes when applied during IVM or preIVM. Previous reports [39,40] showed that cAMP

3.3. GJC between oocytes and cumulus cells after 24 hours of pre-IVM (experiment 3) To confirm the effects of cilostamide and forskolin on GJC between the oocytes and their surround cells, a scrapeloading and dye transfer assay was conducted. The status of GJC is presented in Figure 2. At the time of COC collection,

Table 3 Nuclear status, cAMP level, and cumulus cell expansion of oocytes treated for 24 hours with cilostamide and forskolin during pre-IVM. Treatment

No. of oocytes examinedh

Nuclear status (%)

No pre-IVM Control Cilostamide (C) Forskolin (F) CþF

75 87 93 81 53

100.0 20.6 54.7 85.5 92.6

GV

cAMP level (fmol/oocyte) MI

    

0.0a 5.8b 8.8c 5.9d 1.3d

0 48.7 25.7 7.0 3.6

AI/TI     

0.0a 7.0b 7.5b 5.4a 1.8a

0 0.9 0 0 0

    

MII 0.0 0.9 0.0 0.0 0.0

0 29.9 19.6 7.5 5.7

    

0.0a 7.5b 1.3b 4.9c 0.4c

2.85 1.88 1.74 8.95 21.38

    

0.45e 0.65e 0.34e 0.40f 3.59g

a–d, Within a column, values with different superscripts are different (P < 0.01); e–g, within a column, values with different superscripts are different (P < 0.05). Abbreviation: AI/TI, anaphase I/telophase I; GV, germinal vesicle; MI, metaphase I; MII, metaphase II. h Three replicates.

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Fig. 2. Gap junction communication (GJC) of oocytes treated with cilostamide and/or forskolin for 24 hours during pre-IVM. Treatment with cilostamide and forskolin significantly (P < 0.05) maintained GJC open compared with no treatment. Different letters (a–c) in the bar indicate significant differences among treatment groups.

modulators exhibited beneficial effects on the developmental competence of oocytes after IVF and SCNT in pigs when IVM media were supplemented with these modulators. However, cAMP modulators may negatively regulate oocyte meiosis if improper concentration is used and/or oocytes are treated for too long [24]. Promotion of oocyte maturation by luteinizing hormone is believed to be the result of interruption of GJC between oocytes and surrounding cells and the subsequent reduction of cAMP transfer into oocytes. In this study, oocytes were cultured for 24 hours during pre-IVM because no beneficial effects on meiotic resumption and GJC were observed when pig oocytes were cultured for longer than 24 hours in the presence of forskolin [24]. Two cAMP modulators, cilostamide and forskolin, applied during pre-IVM on the meiotic arrest of COCs effectively arrested nuclear status at the GV stage after pre-IVM for 24 hours, and the arresting effect of forskolin and cilostamide was completely reversible that nearly 90% of pre-IVM oocytes reached the MII stage after IVM. Additionally, these modulators exerted beneficial effects on the developmental competence of IVM oocytes. It is well known that low developmental competence of in vitro–produced embryos might be because of a premature nuclear maturation with insufficient cytoplasmic maturation of IVM oocytes and that transient meiotic arrest to allow oocytes for proper cytoplasmic maturation is beneficial for embryonic development [41]. cAMP is involved in cell metabolism, cumulus cell expansion, GJC,

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and steroidogenesis during oocyte maturation through protein kinase A [42,43]. Considering the role of cAMP in regulating meiotic resumption and other cell metabolism during oocyte maturation, these positive effects of cAMP modulators could be attributed to improved cytoplasmic maturation including the better synchronization of nuclear and cytoplasmic maturation by allowing oocytes additional time for cytoplasmic maturation while nuclear maturation was arrested. When the effects of cAMP modulator treatment during pre-IVM of oocytes on PA embryonic development were examined, blastocyst formation was increased by forskolin treatment relative to the other treatments. These results were inconsistent with previous findings in humans [31] that showed that combined treatment with cilostamide and forskolin during pre-IVM slightly increased blastocyst formation of oocytes after intracytoplasmic sperm injection. Differences in oocyte sources, species, and other culture conditions, such as duration of treatment and concentrations of cAMP modulators, might have resulted in inconsistent results between present and the previous study in human [31]. Intraoocyte GSH content has been reported to be an important factor indicating oocyte cytoplasmic maturation and normal embryonic development in porcine and bovine animals in vitro [33,44]. However, in this study, intraoocyte GSH contents did not correspond with the ability to develop to the stage of PA and SCNT embryos. Control oocytes showed similar GSH contents as oocytes treated with forskolin or/and cilostamide but lower blastocyst formation. Our pre-IVM system using cAMP modulators might influence other unknown cytoplasmic factors than GSH content of oocytes and later embryonic development. Exposure to cilostamide and/or forskolin during pre-IVM altered cAMP level. This effect was prominent in oocytes treated with cilostamide and forskolin, but cilostamide did not increase the cAMP level. Although the reason for the absence of an increase in cAMP level in response to cilostamide in this study was unclear, it was considered to occur because cAMP level was low in immature oocytes. Accordingly, the preventing effect of cilostamide on cAMP degradation might not have occurred when cAMP synthesis was stimulated by forskolin. GJC between oocytes and their surrounding cumulus cells is closely related to meiotic progression. When meiosis resumes, the GJC gradually decreases and ultimately stops during oocyte maturation in vitro and in vivo. A negative relationship between GVBD and GJC has been reported in pigs, with GVBD reducing GJC [36,45]. In the

Table 4 Comparison of the effects of cilostamide and forskolin to dbcAMP during pre-IVM for 24 hours on embryonic development after parthenogenesis. Pre-IVM treatment

No pre-IVM DbcAMP Forskolin (F) Cilostamide þ F

No. of oocytes culturedd

Percentage of oocytes reaching metaphase II

278 272 273 272

94.2 94.7 93.4 92.4

   

1.4 1.4 0.7 0.8

No. of PA embryos culturedd

Percentage of embryos developed to

232 218 197 163

96.6 96.7 98.5 79.5

a–c, Within a column, values with different superscripts are different (P < 0.01). Abbreviation: dbcAMP, dibutyryl cAMP. d Six replicates.

2 cells    

0.1a 1.4a 0.7a 3.4b

Blastocyst 49.2 61.1 61.0 38.8

   

1.4a 1.6b 2.1b 3.9c

No. of cells in blastocyst 39.6 42.2 42.6 42.4

   

1.4 1.2 1.6 2.0

8

B. Park et al. / Theriogenology xxx (2016) 1–9

Table 5 Effects of cilostamide and forskolin treatment during pre-IVM for 24 hours on in vitro development of SCNT pig embryos. Pre-IVM treatment

No. of SCNT embryos culturedc

Percentage of embryos developed to 2-cells

Blastocyst

No pre-IVM Control Cilostamide (C) Forskolin (F) CþF

161 175 148

89.5  3.7 87.9  4.9 89.6  2.9

32.3  3.2a 28.6  3.5a 32.8  2.9a

37.1  1.8a 40.9  2.6 ab 44.6  2.9b

170

85.7  4.5

36.9  2.7ab

40.3  2.3

124

88.6  1.8

42.9  3.3b

44.6  2.5b

No. of cells in blastocyst

ab

a and b, Within a column, values with different superscripts are different (P < 0.05). Abbreviation: SCNT, somatic cell nuclear transfer. c Six replicates.

present study, similar results were obtained, with GJC being maintained by treatment with cilostamide and/or forskolin. These findings corresponded well with nuclear arrest at the GV stage induced by the treatments. Our findings were consistent with those of previous studies that showed maintenance of GJC-enhanced cytoplasmic maturation and subsequent embryonic development after IVF in cattle [46]. Luciano et al [47] reported a relationship between GJC and chromatin remodeling in bovine oocytes. GJC is fully open at the GV0 stage but gradually declines from the GV1 to GV4 stages. The results of nuclear status examined at 24 hours after pre-IVM in this study indicate that forskolin with or without cilostamide effectively inhibited spontaneous resumption of meiosis. Nuclear arrest at the GV stage was induced more effectively by forskolin than cilostamide, indicating that the AC activator, forskolin, was more effective at arresting meiotic resumption in pigs and humans [24,31]. In the present and previous studies, meiotic arrest of oocytes at the GV stage after forskolin treatment (w88% to 92%) was higher in pigs than humans (60%). This difference may have depended on the species, collection methods of immature oocytes, and culture conditions. DbcAMP is commonly used to modulate the cAMP level in oocytes [40]. In this study, dbcAMP was used to determine if the beneficial effects of cAMP modulators were mediated by increasing cAMP levels during pre-IVM. Thus, the effects of forskolin treatment on developmental competence after PA were examined and compared with those of dbcAMP treatment during pre-IVM or/and IVM. Treatment with dbcAMP during pre-IVM, but not IVM, showed a beneficial effect on embryonic development, with blastocyst formation comparable with that of oocytes treated with forskolin. The cAMP level and effect of dbcAMP on embryonic development indicate that improved blastocyst formation of forskolin-treated oocytes may be mediated by the increase in cAMP level after forskolin treatment. However, considering the inhibited embryonic development in oocytes showing increased cAMP level after cilostamide and forskolin treatment, excessive cAMP may be detrimental to cytoplasmic maturation and subsequent embryonic development. Forskolin treatment during pre-IVM stimulated blastocyst formation of PA embryos, whereas combined treatment with cilostamide and forskolin significantly inhibited

blastocyst formation. These findings were in contrast to the SCNT results, which showed that cilostamide þ forskolin had the greatest stimulatory effect on blastocyst formation. Although the reasons for the different responses of PA and SCNT embryos to cAMP modulators observed in this study are not clear, the different nature of nuclear remodeling and reprogramming between them might have contributed to the contradictory results. Our results also reported that the meiotic progression of immature porcine oocytes could be reversibly attenuated by cilostamide and forskolin. Moreover, treatment of oocytes with cilostamide and forskolin during pre-IVM had positive effects on the developmental competence of oocytes in pigs, probably by improving cytoplasmic maturation. However, further study is needed to evaluate the effects of cilostamide and forskolin during pre-IVM on the in vivo development of IVF and SCNT embryos. Acknowledgments The authors would like to thank Gangwon Veterinary Service for the help in collecting pig ovaries used in this study. This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (grant no. 2015R1A2A2A01005490). References [1] Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, et al. Production of alpha-1, 3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002;295:1089–92. [2] Polejaeva IA, Chen S, Vaught TD, Page RL, Mullins J, Ball S, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 2000;407:86–90. [3] De Sousa PA, Dobrinsky JR, Zhu J, Archibald AL, Ainslie A, Bosma W, et al. Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy. Biol Reprod 2002;66:642–50. [4] Ikeda K, Takahashi Y. Effects of maturational age of porcine oocytes on the induction of activation and development in vitro following somatic cell nuclear transfer. J Vet Med Sci 2001;63:1003–8. [5] Kim J, You J, Hyun SH, Lee G, Lim J, Lee E. Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig. Mol Reprod Dev 2010;77:330–9. [6] Motlik J, Crozet N, Fulka J. Meiotic competence in vitro of pig oocytes isolated from early antral follicles. J Reprod Fertil 1984;72: 323–8. [7] Mattioli M, Bacci M, Galeati G, Seren E. Effects of LH and FSH on the maturation of pig oocytes in vitro. Theriogenology 1991;36:95–105. [8] Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965;208:349–51. [9] Anderson E, Albertini DF. Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J Cell Biol 1976; 71:680–6. [10] Carabatsos MJ, Sellitto C, Goodenough DA, Albertini DF. Oocyte– granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence. Dev Biol 2000;226:167–79. [11] Wu D, Cheung QC, Wen L, Li J. A growth-maturation system that enhances the meiotic and developmental competence of porcine oocytes isolated from small follicles. Biol Reprod 2006;75:547–54. [12] Funahashi H, Cantley TC, Day BN. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod 1997;57:49–53. [13] Coy P, Romar R, Ruiz S, Canovas S, Gadea J, Garcia Vazquez F, et al. Birth of piglets after transferring of in vitro-produced embryos prematured with R-roscovitine. Reproduction 2005;129:747–55. [14] Downs SM, Daniel SA, Bornslaeger EA, Hoppe PC, Eppig JJ. Maintenance of meiotic arrest in mouse oocytes by purines: modulation of

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