RESEARCH ARTICLE Molecular Reproduction & Development 82:489–497 (2015)
Colcemid Treatment During Oocyte Maturation Improves Preimplantation Development of Cloned Pig Embryos by Influencing Meiotic Progression and Cytoplasmic Maturation JOOHYEONG LEE,1 JONG-IM PARK,2 GEUN-SHIK LEE,1,3 JUNG HOON CHOI,1,3 SEUNG TAE LEE,4 CHOON-KEUN PARK,4 DAE YOUNG KIM,5 SANG-HWAN HYUN6, AND EUNSONG LEE1,3* 1
College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea College of Veterinary Medicine, Konkuk University, Seoul, Korea 3 Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea 4 Division of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Korea 5 Department of Life Science, College of BioNano Technology, Gachon University, Incheon, Korea 6 College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea 2
SUMMARY The objective of this study was to examine the effects of colcemid treatment during oocyte in vitro maturation (IVM) and embryonic development after parthenogenetic activation (PA) and somatic-cell nucleus transfer (SCNT) in pigs. Immature oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM (designated as COL0-22, COL38-42, and COL0-22/38-42, respectively). The proportion of oocytes reaching the germinal vesicle (GV)/GV breakdown (GVBD) stage after 22 hr of IVM was higher in COL0-22 (98.4%) than in controls not exposed to colcemid (68.7%). The proportion of metaphase-II (MII) oocytes after 30 hr of IVM was higher in control (79.6%) than in COL0-22 oocytes (61.7%); overall nuclear progression to the MII stage was not influenced by colcemid treatment by the end of Corresponding author: College of Veterinary Medicine the IVM period (93.8, 86.7, 86.8, and 84.8% for control, COL0-22, COL38-42, and Kangwon National University COL0-22/38-42, respectively). COL0-22 oocytes showed higher intra-oocyte glutaChuncheon 200-701, Korea. thione content (1.7 vs. 1.01.3 pixels/oocyte) and increased blastocyst formation E-mail: [email protected] after PA (68.7% vs. 42.552.2%) and SCNT (39.4% vs. 16.328.6%) than control, Joohyeong Lee and Jong-Im Park COL38-42, and COL0-22/38-42 oocytes. Colcemid treatment for 022 and 022/38contributed equally to this work. 42 hr of IVM also stimulated the expression of cyclin-dependent kinase 1 (CDK1), Grant sponsor: Ministry of Education; Grant number: NRFproliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 2012R1A1A4A01002564 (ERK2) mRNAs. Our results thus demonstrate that the presence of colcemid during the early stage of IVM stimulates preimplantation development of PA and SCNT porcine embryos by improving the cytoplasmic microenvironment. Mol. Reprod. Dev. 82: 489497, 2015. ß 2015 Wiley Periodicals, Inc. Published online 17 May 2015 in Wiley Online Library Received 8 November 2014; Accepted 28 April 2015
(wileyonlinelibrary.com). DOI 10.1002/mrd.22498
INTRODUCTION Reproductive biotechnologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic-cell nuclear transfer (SCNT) have been routinely used in animals. The success or failure of these
ß 2015 WILEY PERIODICALS, INC.
Abbreviations: CDK1, cyclin-dependent kinase 1; ERK2, extracellular signal-regulated kinase 2; GSH, glutathione; GV(BD), germinal vesicle (breakdown); IVF, in vitro fertilization; IVM, in vitro maturation; MI/II, metaphase I or II; MPF, maturation-promoting factor; PA, parthenogenetic activation; PCNA, proliferating cell nuclear antigen; SCNT, somatic-cell nuclear transfer
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technologies is influenced by various factors, including the quality of the in vitro-produced oocytes or embryos; synchrony between estrous stages of surrogate mothers and developmental stage of in vitro-produced embryos; and the number of embryos transferred to the surrogates. A system that can produce large numbers of high-quality oocytes in vitro is necessary for the efficient production of SCNT embryos, considering that 100300 SCNT embryos are commonly transferred to the oviduct of recipient pigs to establish pregnancy, resulting in few live-birth offspring (Bang et al., 2013; Li et al., 2013). The quality of oocytes is one of the most critical factors influencing the developmental competence of in vitro-produced embryos (Suzuki et al., 2006; Mao et al., 2012). Despite extensive effort to improve the quality of oocytes by optimizing the in vitro maturation (IVM) system, oocytes matured in vitro are inferior to oocytes matured in vivo in their ability to support embryonic development after IVF and SCNT (Moor and Dai, 2001). Although it is possible to produce mammalian oocytes from the in vitro differentiation of embryonic stem cells, the oocytes used in animal reproductive technologies are still commonly obtained by IVM of immature oocytes collected from the ovaries of slaughtered animals (Akagi et al., 2008; Son et al., 2011). In pigs, there are numerous antral follicles of various sizes on the ovarian surface. Although more than 40 oocytes can be collected per ovary, only a small proportion of the oocytes are useful for IVM because oocytes from follicles less than 3 mm in diameter show a low capacity for nuclear and cytoplasmic maturation as well as reduced embryonic development after IVF and SCNT (Wu et al., 2006; Kim et al., 2010). Immature pig oocyteseven fully grown ones from medium to large (46 mm in diameter) follicles exhibit asynchronous nuclear morphology during IVM (Funahashi et al., 1997; Bagg et al., 2009). This heterogeneity in nuclear status is caused by the spontaneous maturation of oocytes, and may result in decreased developmental competence due to the dissociation between nuclear and cytoplasmic maturation rates. Immature oocytes are arrested in the germinal-vesicle (GV) stage, after which meiotic resumption and further nuclear maturation are controlled by time-dependent changes in intra-oocyte cyclic adenosine monophosphate (cAMP) (Mattioli et al., 1994; Shimada and Terada, 2002; Bagg et al., 2006). Several studies have examined the effects of meiotic arrest before IVM using exogenous inhibitors of meiotic resumptionsuch as dibutyryl cAMP, phosphodiesterase 3-inhibitors, and butyrolactone I (Wu et al., 2002; Somfai et al., 2003; Nogueira et al., 2006) and observed that the oocytes in which meiotic arrest was maintained during pre-maturation culture had enhanced meiotic potential and developmental competence. This observation suggested that inhibition of meiotic progression may provide more time for cytoplasmic maturation, leading to better coordination between the final stages of cytoplasmic and nuclear maturation, which will improve developmental competence overall. Colcemid, a cytoskeletal inhibitor, can induce mitotic arrest at the G2/M phase or meiotic arrest at the germinal
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vesicle breakdown (GVBD) stage in mammalian cells or oocytes, respectively (Alexandre et al., 1989; Moses et al., 1995). Colcemid disturbs microtubule polymerization by binding tightly to tubulin dimers, preventing the formation of spindle microtubules by depolymerization n ~ez et al., 2003) and thereby arresting cells or oocytes (Iba at a specific cell-cycle stage (Aszalos et al., 1985; Saraiva et al., 2009). This phenotype is reversible when used at the concentration of 1.08 mM for 14 hr (Song et al., 2009; Lee et al., 2010). Colcemid was first reported to inhibit meiotic progression of immature mouse oocytes by preventing meiotic spindle formation and polar body emission (Wassarman et al., 1976; Hashimoto and Kishimoto, 1988). Since then, colcemid has been used to facilitate the enucleation of bovine and pig oocytes during SCNT because it induces the protrusion of metaphase chromosomes in polar body-like structures (Li et al., 2009a,b; Saraiva et al., 2012). Post-activation treatment of reconstructed pig oocytes with colcemid is also reported to improve preimplantation development and to support normal, in vivo development of SCNT pig embryos by disrupting microtubules, and thereby facilitating the formation of a single pronucleus in an activated SCNT oocyte (Song et al., 2009). Despite the historical use of colcemid in oocytes, there are no reports on its effects on oocyte maturation and embryonic development after SCNT. Based on its mechanism of action, we hypothesized that colcemid treatment would delay meiotic progression by depolymerizing the spindle-forming microtubules, which in turn would allow for better coordination between nuclear and cytoplasmic maturation. This improved cytoplasmic microenvironments should stimulate glutathione (GSH) synthesis and accumulation of maternal transcripts, thereby enhancing the developmental competence of IVM oocytes. The developmental potential of IVM oocytes is usually tested by proxye.g., assessing embryonic development after IVF but polyspermic fertilization is common when conducting IVF with porcine oocytes; indeed, more than 50% of oocytes show polyspermy and cannot develop normally (Kohata et al., 2013; Appeltant et al., 2015). As embryonic development varies with the proportion of polyspermic oocytes, which in turn makes it difficult to clearly evaluate the quality of IVM oocytes, parthenogenesis has been employed as an alternative to IVF to examine developmental competence of IVM porcine oocytes. The objective of this study was to examine the effects of colcemid treatment during IVM on the nuclear and cytoplasmic maturation of oocytes and the subsequent embryonic developmental competence. To this end, meiotic progression of oocytes was observed at the various stages of IVM, along with molecular measurements of intra-oocyte GSH content, maturation-promoting factor (MPF) activity, and the relative transcript abundance of cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2). In addition, the embryonic development of colcemid-treated IVM oocytes was examined after parthenogenetic activation (PA) and SCNT.
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RESULTS Nuclear Status of Pig Oocytes Treated With Colcemid for the First 22 hr, After 22 and 30 hr of IVM A significantly higher proportion of oocytes (93.8 2.6%) were arrested at the GVBD stage by the colcemid treatment compared to the untreated oocytes (60.6 10.8%; Table 1). Conversely, the proportion of nuclei at the metaphase-I (MI) stage was significantly higher in untreated oocytes than in colcemid-treated oocytes (24.7 8.3% vs. 1.6 1.6%). When nuclear status was examined after 30 hr of IVM, with or without colcemid treatment for the first 22 hr, the proportion of untreated oocytes that reached the MII stage was significantly higher than colcemid-treated oocytes (79.6 2.4% vs. 61.7 6.5%; Table 2). In the colcemidtreated and untreated samples, 10.1 0.2 and 20.4 1.6% of the oocytes were at the MI stage, respectively.
Nuclear Status, Intra-Oocyte GSH Content, and CDK1 Kinase Activity of Pig Oocytes Treated With Colcemid From 0 to 22 hr and/or 38 to 42 hr of IVM, After 42 hr of IVM Immature pig oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM culture (designated as COL0-22, COL38-42, and COL0-22/ 38-42, respectively). The proportions of oocytes reaching the MII stage were 93.8 2.6, 86.7 2.4, 86.8 2.4, and 84.8 2.3% for the untreated control, COL0-22, COL38-42, and COL0-22/38-42 samples, respectively (Table 3). Meiotic progression was therefore not altered by colcemid treatment, although the nuclear progression of COL0-22/38-42 oocytes to the MII stage tended to decrease compared to untreated oocytes (P < 0.1). Intra-oocyte GSH content was significantly increased in the COL0-22 (1.7 0.1 pixels/oocyte) and COL0-22/38-42 oocytes (1.3 0.1 pixels/oocyte) compared to the
untreated control (1.0 0.1 pixels/oocyte). On the other hand, MPF activity, measured in terms of CDK1 kinase activity, was not influenced by colcemid treatment, irrespective of when colcemid was provided during IVM.
Effect of Colcemid Treatment During IVM on Embryonic Development In vitro development of PA embryos to the blastocyst stage (68.7 3.5%) was improved (P < 0.05) by colcemid treatment during the first 0-22 hr of IVM compared to the control (42.5 3.6%). Embryonic cleavage (87.3 1.3 to 92.5 3.4%) and cell numbers of the PA-derived blastocysts (36.0 2.2 to 42.5 1.8 cells/blastocyst), however, were not influenced by this treatment (Table 4). SCNT embryos derived from COL0-22 oocytes exhibited a significantly higher rate (39.4 3.4%) of blastocyst formation than the control (16.3 2.3%), COL38-42 (22.7 3.8%), and COL0-22/38-42 oocytes (28.6 3.4%; P < 0.05). The mean cell number in the blastocysts was also higher following colcemid treatment during the first 022 hr of IVM compared to no treatment (P < 0.05; Table 5).
Effect of Colcemid Treatment During IVM on the Transcript Abundance of CDK1, PCNA, and ERK2 in Matured Oocytes Treatment of oocytes with colcemid during IVM stimulated the accumulation of CDK1, PCNA, and ERK2 mRNAs in MII oocytes compared to the control (P < 0.05). The abundance of 18S rRNA, however, was not influenced by this treatment (Fig. 1).
DISCUSSION The in vitro production of high-quality, mature oocytes from immature oocytes is an essential step that can dictate the success of artificial reproductive technology with
TABLE 1. Nuclear Status of Pig Oocytes Treated With Colcemid During a 22 hr IVM Period, After 22 hr of IVM Nuclear status (%) Colcemid treatment during IVM
No. of oocytes examined
GV
GVBD
MI
AI-TI
MII
60 63
8.2 3.1 4.7 3.0
60.6 10.8a 93.8 2.6b
24.7 8.3a 1.6 1.6b
3.3 1.9 0.0 0.0
3.3 1.9 0.0 0.0
None 022 hr
Three replicates were carried out. Values in the same column with different letters (a, b) indicate statistical differences (P < 0.05). AI, anaphase I; TI, telophase I.
TABLE 2. Nuclear Status Pig Oocytes Treated With Colcemid for the First 22 hr of IVM, After 30 hr of IVM Nuclear status (%) Colcemid treatment during IVM
No. of oocytes examined
GV
GVBD
MI
79 78
0.0 0.0 1.4 1.4
5.2 2.1 5.2 2.3
10.1 0.2 20.4 1.6b
None 022 hr
a
AI-TI
MII
5.0 2.9 6.3 0.9
79.6 2.4a 61.7 6.5b
Four replicates were carried out. Values in the same column with different letters (a, b) indicate significant differences (P < 0.05). AI, anaphase I; TI, telophase I.
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TABLE 3. Nuclear Status, Intra-oocyte GSH Content, and CDK1 Kinase Activity of Pig Oocytes Treated With Colcemid From 0 to 22 and/or 38 to 42 hr During IVM, After 42 hr of IVM Nuclear status (%)b
Colcemid treatment during IVM
No. of oocytes examined
None 022 hr 3842 hr 022 þ 3842 hr
97 98 91 99
GV 0.0 1.0 1.1 1.0
MI
0.0 5.2 0.2a 1.0 11.2 0.2ab 1.1 11.0 0.2ab 1.0 14.1 1.6b
AI-TI 1.0 1.0 1.1 0.0
Relative GSH level (pixels/oocyte) (n ¼ 60*)
MII
2.9 2.9 2.9 0.0
93.8 86.7 86.8 84.8
2.6 2.4 2.4 2.3
1.0 1.7 1.2 1.3
0.1a 0.1b 0.1ac 0.1c
Relative CDK1 kinase activity (n¼120*) 1.00 0.09 0.83 0.03 0.91 0.04 0.84 0.04
Four replicates were carried out. Values in the same column with different letters (a-c) indicate significant differences (P < 0.05). AI, anaphase I; TI, telophase I. *Number of MII oocytes examined for GSH content and CDK1 kinase activity.
TABLE 4. Effects of Colcemid Treatment During IVM on the Embryonic Development of Pig Oocytes After PA % of embryos developed to Colcemid treatment during IVM None 022 hr 3842 hr 022 þ 3842 hr
No. of embryos cultured
2-cell
Blastocyst
127 114 138 132
87.6 92.5 87.7 87.3
3.0 3.4 3.0 1.3
42.5 68.7 52.2 42.9
a
3.6 3.5b 3.2a 5.8a
No. of cells in blastocyst 37.2 42.5 36.7 36.0
2.2 1.8 1.8 2.2
Four replicates were carried out. Values in the same column with different letters (a, b) indicate significant differences (P < 0.05).
mammalian species. Timely coordination between meiotic progression and cytoplasmic maturation is important for later embryonic development following IVF and SCNT. In the present study, transient arrest of meiotic progression in immature pig oocytes by colcemid treatment during IVM positively influenced the embryonic development of the treated oocytes after PA and SCNT through alteration of nuclear and/or cytoplasmic maturation. Specifically, treatment with colcemid during an early stage (022 hr) of IVM effectively, but reversibly, arrested the nuclear status of oocytes at the GV to GVBD stages. The subsequent improvement in embryonic development after PA and SCNT was likely due to the improved cytoplasmic environments, as measured by increased intra-oocyte GSH contents and maternal transcription of critical genes in IVM oocytes. Colcemid inhibits cell division by inducing the depolymerization of microtubules, arresting cells at the metaphase stage. In the present study, colcemid treatment during the first 22 hr of IVM arrested meiotic progression of pig oocytes at the GV to GVBD period, leading to delayed meiotic progression compared to untreated oocytes. This
result was consistent with a previous finding in mice (Wassarman et al., 1976), wherein colcemid arrested meiotic progression of oocytes treated during IVM while also inhibiting the cleavage of somatic cells. Although colcemid arrested porcine oocytes at GVBD, oocytes could be released from this state and progressed normally to the MII stage when further cultured in medium free of colcemid; most of the IVM pig oocytes reach the MII stage after a total of 38 hr. Treatment of bovine oocytes with colcemid for 26 hr at the final stage of IVM also improved the developmental competence of cloned embryos (Li et al., 2009a). In contrast, a 4-hr colcemid treatment starting at 38 hr of IVM, when the nuclear stage of oocytes had progressed near the MII stage, neither inhibited nuclear progression to the MII stage nor showed any beneficial effect on porcine embryonic development. The absence of an effect may have been because most of the porcine oocytes already reached the MII stage and cytoplasmic maturation was also almost complete, leaving no need for a further delay. In comparison, treating with colcemid for 69 hr starting at 22 hr of IVM, when most of the oocytes were around the MI stage, resulted in MI stage-arrested oocytes that did not easily
TABLE 5. Effects of Colcemid Treatment During IVM on the Embryonic Development of Pig Oocytes After SCNT % of embryos developed to Colcemid treatment during IVM None 022 hr 3842 hr 022 þ 3842 hr
No. of SCNT embryos cultured 139 119 134 122
2-cell 86.3 90.8 86.4 86.1
2.1 1.3 2.9 3.8
Blastocyst 16.3 39.4 22.7 28.6
2.3a 3.4b 3.8ac 3.4c
No. of cells in blastocyst 30.0 41.3 35.3 39.0
2.1a 2.4b 2.5ab 2.7b
Four replicates were carried out.Values in the same column with different letters (ac) indicate significant differences (P < 0.05).
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Figure 1. Relative transcript abundance of CDK1, PCNA, and ERK2 mRNAs in MII oocytes treated with colcemid during oocyte IVM. Means standard error are plotted. Bars with different letters (ac) for the same mRNA are significantly different (P < 0.05).
progress to the MII stage after further culture in medium free of colcemid (data not shown). Thus, the reversibility of meiotic arrest induced by colcemid depends on the nuclear stage of the oocytes at the time of treatment. Positive effects of colcemid treatment on the cytoplasmic status of oocytes were also observed. In vivomatured oocytes have higher GSH content (Brad et al., 2003) and developmental competence than IVM oocytes (Petters and Wells, 1993). GSH has a stimulating effect on in vitro development of embryos derived from IVF and SCNT by protecting them from oxidative stress induced by reactive oxygen species (Tatemoto et al., 2000; Salmen et al., 2005). For this reason, GSH has been used as an indicator of the cytoplasmic maturity of IVM oocytes (Abeydeera et al., 1998; Brad et al., 2003). Similarly, quantities of certain maternal mRNAs may be used as markers for predicting the maturation and developmental competence of oocytes as these specific transcripts correlate with the abundance of the corresponding proteins. CDK1 and ERK2, for example, regulate MPF and mitogen-activated protein kinase activity (Vigneron et al., 2004; Lee et al., 2013) while PCNA is an essential component of the DNA replication and repair machinery (Kelman, 1997). Colcemid treatment from 0 to 22 hr and from 0 to 22 along with 38 to 42 hr of IVM increased GSH content as well as the transcript abundance of CDK1, ERK2, and PCNA without influencing the expression of 18S rRNA in IVM porcine oocytes; these results together imply improved cytoplasmic maturation. The mechanism underlying the higher intra-oocyte GSH content and abundance of maternal mRNA transcripts was not determined in this study, although we speculate that the transient arrest of nuclear maturation by colcemid treatment improved the synchrony between nuclear and cytoplasmic maturation while enhancing the cytoplasmic microenvironment, thereby stimulating GSH synthesis and transcription of maternal genes that are beneficial for later embryonic development. Curiously, when the effect of colcemid on MPF activity in IVM oocytes was examined, no increase
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was observed irrespective of the stages of IVM during which treatment was carried out. This observation is in contrast to previous results in which colcemid treatment after IVM significantly increased MPF activity in porcine and bovine oocytes (Li et al., 2009a, 2014), but may be attributed to technical differences in the duration of treatment and in the nuclear stage of the oocytes at the time of colcemid treatment. Treatment of oocytes with colcemid during the first 22 hr of IVM significantly improved the developmental competence of the embryos after PA and SCNT, without decreasing nuclear maturation. This result agrees with previous observations that transient inhibition of meiotic maturation, through an increase in the cAMP level of oocytes, improved the developmental competence of IVM oocytes, likely by allowing time for cytoplasmic maturation and improving coordination between nuclear and cytoplasmic maturation (Somfai et al., 2003). Although cAMP levels were not examined in this study, the transient but reversible nuclear arrest at the GVBD stage by colcemid treatment showed beneficial effects on embryonic development whereas colcemid-induced arrest at the MI stage (when oocytes were exposed to colcemid for 69 hr starting at 22 hr of IVM) was not fully reversible and worsened the developmental competence of PA oocytes. These results together suggest that the effects of colcemid treatment on meiotic maturation of porcine oocytes could be beneficial or detrimental for later embryonic development, depending on the nuclear stage at the time of treatment; a positive effect was limited to nuclear arrest during the early phase of oocyte IVM, which likely allowed for the proper maturation of the cytoplasmic microenvironment.
MATERIALS AND METHODS Culture Media All chemicals used in this study were obtained from SigmaAldrich Chemical Company (St. Louis, MO), unless
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otherwise noted. Medium-199 (M-199, Invitrogen, Grand Island, NY) supplemented with 0.91 mM pyruvate, 0.6 mM cysteine, 10 ng/ml epidermal growth factor, 1 mg/ml insulin, and 10% (v/v) pig follicular fluid was used as the base IVM medium. Porcine zygote medium (PZM)-3 containing 0.3% (w/v) bovine serum albumin (BSA) (Yoshioka et al., 2002) plus 2.77 mM myo-inositol, 0.34 mM tri-sodium citrate, and 10 mM b-mercaptoetanol was used for the in vitro culture of embryos.
Oocyte Collection and IVM The ovaries of prepubertal gilts obtained from a local abattoir were transported to the laboratory at 378C. Cumulus-oocyte complexes (COCs) were aspirated from follicles 3 to 8-mm in diameter. COCs with multiple layers of compacted cumulus cells were selected and washed three times in HEPES-buffered Tyrode’s medium (TLH) containing 0.05% (w/v) polyvinyl alcohol (TLH-PVA). COCs were placed into each well of a four-well multi-dish (Nunc, Roskilde, Denmark). Each well contained 500 ml of IVM medium with 80 mg/ml follicle-stimulating hormone (FSH) (Antrin R-10, Kyoritsu Seiyaku, Tokyo, Japan) and 10 IU/ml human chorionic gonadotropin (hCG) (Intervet International BV). The COCs were cultured at 398C with 5% CO2 in air under maximum humidity. After 22 hr of maturation culture, the COCs were washed three times in fresh, hormone-free IVM medium. The COCs were then cultured in hormone-free IVM medium for an additional 20 hr. During IVM, COCs were treated with 0.4 mg/ml colcemid or left untreated, according to the experimental design.
Experimental Design Immature oocytes were treated with colcemid or untreated (control) during the time periods of 022 (COL 022), 3842 (COL 38-42), or 022 and 3842 hr (COL 022/38-42) of IVM. The effects of colcemid treatment on the nuclear status of oocytes at 22 and 30 hr of IVM was examined first. Nuclear maturation, intra-oocyte GSH content, and MPF (CDK1) activity of the oocytes treated with colcemid during various stages of IVM were examined next. Subsequently, the effects of colcemid treatment during IVM on embryonic development after PA and SCNT were determined under similar experimental treatment conditions. Finally, the effects of colcemid on CDK1, PCNA, and ERK2 transcript abundance in MII oocytes were examined. Each experiment was conducted with 34 replicates.
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according to a previously reported system (Hunter and Polge, 1966).
Measurement of Intra-Oocyte GSH Content and MPF (CDK1) Activity in Oocytes Oocytes at the MII stage were sampled at 42 hr after IVM to determine the intra-oocyte GSH contents, using a previously described method (Sakatani et al., 2007). Briefly, 510 oocytes from each treatment group were incubated in the dark for 30 min in TLH-PVA supplemented with 10 mM CellTracker Blue CMF2HC (4-chloromethyl6.8-difluoro-7-hydroxycoumarin) (Invitrogen). After incubation, oocytes were washed with Dulbecco’s phosphatebuffered saline (D-PBS; Invitrogen) containing 0.1% (w/v) PVA, and 10-ml droplets containing the oocytes were transferred for observation of fluorescence under a TE300 epifluorescence microscope (Nikon, Tokyo, Japan). Fluorescence images were saved as TIFF files, and the fluorescence intensities of the oocytes were analyzed by ImageJ software version 1.41o (National Institutes of Health, Bethesda, MD) and normalized to that of untreated control oocytes. MPF/CDK1 activity was measured with a MESACUP CDC2 kinase assay kit (Medical & Biological Laboratories; MBL, Nagoya, Japan), as described previously (Anas et al., 2000). Briefly, a group of 30 MII-stage oocytes was washed twice with kinase sample buffer consisting of 50 mM TrisHCl, 0.5 M NaCl, 5 mM EDTA, 2 mM EGTA, 0.01% (v/v) polyoxyethylene lauryl ether (Brij 35), 1 mM phenylmethylsulfonyl fluoride, 0.05 mg/ml leupeptin, 50 mM b-mercaptoethanol, 25 mM b-glycerophosphate, and 1 mM sodium orthovanadate. Oocytes were then transferred to microtubes containing 5 ml of kinase buffer, and lysed by repeated freezing-thawing in liquid nitrogen and warm water, resepctively. These oocyte lysates were stored at 808C until analyzed. The lysates (5 ml) were mixed with 45 ml of assay buffer containing 25 mM HEPES, 10 mM MgCl2, 10% (v/v) biotinylated MV peptide (SLTSSSPGGATC), and 0.1 mM ATP, and the mixture was then incubated for 30 min at 308C. The phosphorylation reaction was terminated by the addition of 200 ml of stop reagent (PBS containing 50 mM EGTA), after which the mixture was centrifuged for 15 sec at 14,000 rpm. The phosphorylation of biotinylated MV peptide was detected at 492 nm using a plate reader. The data were expressed relative to CDK1 kinase activity in the untreated oocytes.
Preparation of Donor Cells and Nuclear Transfer Examination of Nuclear Status of Oocytes During IVM To evaluate the nuclear status, oocytes at 22, 30, and 42 hr of IVM were denuded, mounted onto glass slides, and fixed for 48 hr in 25% (v/v) acetic acid in ethanol at 48C. The fixed oocytes were then stained with 1% (w/v) aceto-orcein. Nuclear morphology and extrusion of the second polar body were assessed under a phase-contrast microscope at 400 magnification. Nuclear stages were classified
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Skin fibroblasts from a newborn miniature piglet were seeded into four-well culture plates and cultured in Dulbecco’s modified Eagle medium (DMEM) with the nutrient mixture F-12 (Invitrogen), which was supplemented with 15% (v/v) fetal bovine serum until a complete monolayer of cells had formed. Donor cells were synchronized at the G0/ G1 stage of the cell cycle by contact inhibition for 4872 hr. A suspension of single cells was prepared by trypsinization of the cultured cells, followed by resuspension in TLH
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containing 0.4% (w/v) BSA (TLH-BSA) prior to nuclear transfer. After 42 hr of IVM, the MII stage oocytes were incubated for 15 min in manipulation medium (calcium-free TLH-BSA) containing 5 mg/ml Hoechst 33342, and then washed twice with fresh manipulation medium. Oocytes were transferred into a drop of manipulation medium containing 5 mg/ml cytochalasin B, which was overlaid with mineral oil. Oocytes were enucleated by aspirating the first polar body and MII chromosomes using a 17-mm beveled glass pipette (Humagen, Charlottesville, VA). Enucleation was confirmed under an epifluorescence microscope. After enucleation, a single cell was inserted into the perivitelline space of each oocyte. Cell-oocyte couplets were placed in a 1-mm fusion chamber overlaid with 1 ml of 280 mM mannitol solution containing 0.001 mM CaCl2 and 0.05 mM MgCl2, as previously described (Song et al., 2009). Membrane fusion was induced by applying an alternating current field of 2 V cycling at 1 MHz for 2 sec, followed by two pulses of 170 V/mm direct current (DC) for 25 msec using a LF101 cell-fusion generator (NepaGene, Chiba, Japan). Immediately after fusion, the oocytes were incubated for 1 hr in TLH-BSA, and membrane fusion was evaluated under a stereomicroscope prior to activation.
Day 0). The number of cells in the blastocysts were examined using Hoechst 33342 staining under an epifluorescence microscope.
Gene Expression Analysis by Real-Time PCR The transcript abundance of CDK1, PCNA, and ERK2 in IVM oocytes was analyzed by real-time PCR as previously described (Zhu-Ge and Yu, 2004). Total RNA was isolated from MII oocytes after IVM using AccuZolTM (Bioneer, Alameda, CA), according to the manufacturer’s instructions. Complementary DNA was synthesized from total RNA using a Reverse Transcription system (Promega, Madison, WI). The transcript abundance of specific genes in the embryos was then quantified on a Rotor-Gene 3000 system (Corbett, Mortlake, Australia) using the PrimeScript RT-PCR kit (Takara, Shiga, Japan). Melting-curve data were collected to verify PCR specificity. Primer3 software (Whitehead Institute, MIT Center for Genome Research, Cambridge, MA) was used to design the primers employed in this study (Table 6). All data were normalized to 18S rRNA. The relative mRNA level was calculated as previously described (Matusuoka et al., 1999; Pfaffl, 2001).
Statistical Analysis Oocyte Activation Reconstructed oocytes were activated with two pulses of 120 V/mm DC for 60 msec in a 280 mM mannitol solution containing 0.01 mM CaCl2 and 0.05 mM MgCl2. For PA, the oocytes that had reached the MII stage at 42 hr of IVM were electrically activated using the same pulse sequence used to activate the SCNT oocytes.
In Vitro Culture of Embryos Following activation, the SCNT and PA embryos were treated with 0.4 mg/ml demecolcine and 5 mg/ml cytochalasin B, resepectively, in in vitro culture (IVC) medium for 4 hr (Song et al., 2009). The SCNT and PA embryos were then washed three times in fresh IVC medium, placed into 30-ml droplets of IVC medium under mineral oil, and then cultured at 398C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 7 days. Embryo cleavage and blastocyst formation were evaluated on Days 2 and 7, respectively (the day of SCNT or PA was designated as
In this study, 665 oocytes were examined for the nuclear status during IVM, while 60 and 120 MII oocytes were used for the examination of GSH contents and MPF activity, respectively. In addition, 511 and 514 embryos were used to determine the developmental competence of MII oocytes after PA and SCNT, respectively. All experiments were repeated 34 times. Percentage data such as nuclear status, embryo cleavage, and blastocyst formation were analyzed using a general linear model procedure after arcsine transformation to maintain the homogeneity of the variances. Post-hoc analyses to identify between-group differences were performed using the least significant difference (LSD) test, with a model effect of treatment differences called at P < 0.05. The non-parametric Wilcoxon test with Dunn method for multiple comparisons was used to determine differences among treatments in datasets such as the cell number of blastocysts, GSH contents, and MPF activity. Results are expressed as the arithmetic mean standard error.
TABLE 6. Primers and RT-PCR Conditions Used for Gene Expression Analysis mRNA
Direction
Primer sequences
Product size (base pairs)
18S
Forward Reverse Forward Reverse Forward Reverse Forward Reverse
50 -CGGCTACCACATCCAAGGAA 50 -CTCCAATGGATCCTCGTTAAAGG 50 -TTTTCAGAGCTTTGGGCACTC 50 - TTTTCGAGAGCSGATCCAAGC 50 -CTCAAACCTTCCAACCTGCTG 50 -GTCGATGGACTTGGTGTAGCC 50 -TAATGCAGACACCTTGGCACT 50 -GCAAATTCACCAGAAGGCATC
147
CDK1 ERK2 PCNA
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153 183 153
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Molecular Reproduction & Development
ACKNOWLEDGMENTS We thank Gangwon Veterinary Service for the help in collecting pig ovaries used in this study. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012R1A1A4A01002564).
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adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod 57:4953. Hashimoto N, Kishimoto T. 1988. Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation. Dev Biol 126:242252.
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Somfai T, Kikuchi K, Onishi A, Iwamoto M, Fuchimoto D, Papp AB, Sato E, Nagai T. 2003. Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes. Zygote 11:199206. Son WY, Chung JT, Dahan M, Reinblatt S, Tan SL, Holzer H. 2011. Comparison of fertilization and embryonic development in sibling in vivo matured oocytes retrieved from different sizes follicles from in vitro maturation cycles. J Assist Reprod Genet 28:539544. Song K, Hyun SH, Shin T, Lee E. 2009. Post-activation treatment with demecolcine improves development of somatic cell nuclear transfer embryos in pigs by modifying the remodeling of donor nuclei. Mol Reprod Dev 76:611619. Suzuki M, Misumi K, Ozawa M, Noguchi J, Kaneko H, Ohnuma K, Fuchimoto D, Onishi A, Iwamoto M, Saito N, Nagai T, Kikuchi K. 2006. Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum. Theriogenology 65:374386. Tatemoto H, Sakurai N, Muto N. 2000. Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during In vitro maturation: Role of cumulus cells. Biol Reprod 63:805810. s-Tran R, Joly C, Humblot P, Vigneron C, Perreau C, Dalbie Uzbekova S, Mermillod P. 2004. Protein synthesis and mRNA storage in cattle oocytes maintained under meiotic block by roscovitine inhibition of MPF activity. Mol Reprod Dev 69:457465. Wassarman PM, Josefowicz WJ, Letourneau GE. 1976. Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression. J Cell Sci 22:531545. Wu D, Cheung QC, Wen L, Li J. 2006. A growth-maturation system that enhances the meiotic and developmental competence of porcine oocytes isolated from small follicles. Biol Reprod 75:547554. Wu GM, Sun QY, Mao J, Lai L, McCauley TC, Park KW, Prather RS, Didion BA, Day BN. 2002. High developmental competence of pig oocytes after meiotic inhibition with a specific M-phase promoting factor kinase inhibitor, butyrolactone I. Biol Reprod 67:170177. Yoshioka K, Suzuki C, Tanaka A, Anas IM, Iwamura S. 2002. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 66:112119. Zhu-Ge J, Yu YN. 2004. Enzyme activity analysis of CYP2C18 with exon 5 skipped. Acta Pharmacol Sin 25:10651069.
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Colcemid Treatment During Oocyte Maturation Improves Preimplantation Development of Cloned Pig Embryos by Influencing Meiotic Progression and Cytoplasmic Maturation JOOHYEONG LEE,1 JONG-IM PARK,2 GEUN-SHIK LEE,1,3 JUNG HOON CHOI,1,3 SEUNG TAE LEE,4 CHOON-KEUN PARK,4 DAE YOUNG KIM,5 SANG-HWAN HYUN6, AND EUNSONG LEE1,3* 1
College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea College of Veterinary Medicine, Konkuk University, Seoul, Korea 3 Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea 4 Division of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Korea 5 Department of Life Science, College of BioNano Technology, Gachon University, Incheon, Korea 6 College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea 2
SUMMARY The objective of this study was to examine the effects of colcemid treatment during oocyte in vitro maturation (IVM) and embryonic development after parthenogenetic activation (PA) and somatic-cell nucleus transfer (SCNT) in pigs. Immature oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM (designated as COL0-22, COL38-42, and COL0-22/38-42, respectively). The proportion of oocytes reaching the germinal vesicle (GV)/GV breakdown (GVBD) stage after 22 hr of IVM was higher in COL0-22 (98.4%) than in controls not exposed to colcemid (68.7%). The proportion of metaphase-II (MII) oocytes after 30 hr of IVM was higher in control (79.6%) than in COL0-22 oocytes (61.7%); overall nuclear progression to the MII stage was not influenced by colcemid treatment by the end of Corresponding author: College of Veterinary Medicine the IVM period (93.8, 86.7, 86.8, and 84.8% for control, COL0-22, COL38-42, and Kangwon National University COL0-22/38-42, respectively). COL0-22 oocytes showed higher intra-oocyte glutaChuncheon 200-701, Korea. thione content (1.7 vs. 1.01.3 pixels/oocyte) and increased blastocyst formation E-mail: [email protected] after PA (68.7% vs. 42.552.2%) and SCNT (39.4% vs. 16.328.6%) than control, Joohyeong Lee and Jong-Im Park COL38-42, and COL0-22/38-42 oocytes. Colcemid treatment for 022 and 022/38contributed equally to this work. 42 hr of IVM also stimulated the expression of cyclin-dependent kinase 1 (CDK1), Grant sponsor: Ministry of Education; Grant number: NRFproliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 2012R1A1A4A01002564 (ERK2) mRNAs. Our results thus demonstrate that the presence of colcemid during the early stage of IVM stimulates preimplantation development of PA and SCNT porcine embryos by improving the cytoplasmic microenvironment. Mol. Reprod. Dev. 82: 489497, 2015. ß 2015 Wiley Periodicals, Inc. Published online 17 May 2015 in Wiley Online Library Received 8 November 2014; Accepted 28 April 2015
(wileyonlinelibrary.com). DOI 10.1002/mrd.22498
INTRODUCTION Reproductive biotechnologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic-cell nuclear transfer (SCNT) have been routinely used in animals. The success or failure of these
ß 2015 WILEY PERIODICALS, INC.
Abbreviations: CDK1, cyclin-dependent kinase 1; ERK2, extracellular signal-regulated kinase 2; GSH, glutathione; GV(BD), germinal vesicle (breakdown); IVF, in vitro fertilization; IVM, in vitro maturation; MI/II, metaphase I or II; MPF, maturation-promoting factor; PA, parthenogenetic activation; PCNA, proliferating cell nuclear antigen; SCNT, somatic-cell nuclear transfer
Molecular Reproduction & Development
technologies is influenced by various factors, including the quality of the in vitro-produced oocytes or embryos; synchrony between estrous stages of surrogate mothers and developmental stage of in vitro-produced embryos; and the number of embryos transferred to the surrogates. A system that can produce large numbers of high-quality oocytes in vitro is necessary for the efficient production of SCNT embryos, considering that 100300 SCNT embryos are commonly transferred to the oviduct of recipient pigs to establish pregnancy, resulting in few live-birth offspring (Bang et al., 2013; Li et al., 2013). The quality of oocytes is one of the most critical factors influencing the developmental competence of in vitro-produced embryos (Suzuki et al., 2006; Mao et al., 2012). Despite extensive effort to improve the quality of oocytes by optimizing the in vitro maturation (IVM) system, oocytes matured in vitro are inferior to oocytes matured in vivo in their ability to support embryonic development after IVF and SCNT (Moor and Dai, 2001). Although it is possible to produce mammalian oocytes from the in vitro differentiation of embryonic stem cells, the oocytes used in animal reproductive technologies are still commonly obtained by IVM of immature oocytes collected from the ovaries of slaughtered animals (Akagi et al., 2008; Son et al., 2011). In pigs, there are numerous antral follicles of various sizes on the ovarian surface. Although more than 40 oocytes can be collected per ovary, only a small proportion of the oocytes are useful for IVM because oocytes from follicles less than 3 mm in diameter show a low capacity for nuclear and cytoplasmic maturation as well as reduced embryonic development after IVF and SCNT (Wu et al., 2006; Kim et al., 2010). Immature pig oocyteseven fully grown ones from medium to large (46 mm in diameter) follicles exhibit asynchronous nuclear morphology during IVM (Funahashi et al., 1997; Bagg et al., 2009). This heterogeneity in nuclear status is caused by the spontaneous maturation of oocytes, and may result in decreased developmental competence due to the dissociation between nuclear and cytoplasmic maturation rates. Immature oocytes are arrested in the germinal-vesicle (GV) stage, after which meiotic resumption and further nuclear maturation are controlled by time-dependent changes in intra-oocyte cyclic adenosine monophosphate (cAMP) (Mattioli et al., 1994; Shimada and Terada, 2002; Bagg et al., 2006). Several studies have examined the effects of meiotic arrest before IVM using exogenous inhibitors of meiotic resumptionsuch as dibutyryl cAMP, phosphodiesterase 3-inhibitors, and butyrolactone I (Wu et al., 2002; Somfai et al., 2003; Nogueira et al., 2006) and observed that the oocytes in which meiotic arrest was maintained during pre-maturation culture had enhanced meiotic potential and developmental competence. This observation suggested that inhibition of meiotic progression may provide more time for cytoplasmic maturation, leading to better coordination between the final stages of cytoplasmic and nuclear maturation, which will improve developmental competence overall. Colcemid, a cytoskeletal inhibitor, can induce mitotic arrest at the G2/M phase or meiotic arrest at the germinal
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vesicle breakdown (GVBD) stage in mammalian cells or oocytes, respectively (Alexandre et al., 1989; Moses et al., 1995). Colcemid disturbs microtubule polymerization by binding tightly to tubulin dimers, preventing the formation of spindle microtubules by depolymerization n ~ez et al., 2003) and thereby arresting cells or oocytes (Iba at a specific cell-cycle stage (Aszalos et al., 1985; Saraiva et al., 2009). This phenotype is reversible when used at the concentration of 1.08 mM for 14 hr (Song et al., 2009; Lee et al., 2010). Colcemid was first reported to inhibit meiotic progression of immature mouse oocytes by preventing meiotic spindle formation and polar body emission (Wassarman et al., 1976; Hashimoto and Kishimoto, 1988). Since then, colcemid has been used to facilitate the enucleation of bovine and pig oocytes during SCNT because it induces the protrusion of metaphase chromosomes in polar body-like structures (Li et al., 2009a,b; Saraiva et al., 2012). Post-activation treatment of reconstructed pig oocytes with colcemid is also reported to improve preimplantation development and to support normal, in vivo development of SCNT pig embryos by disrupting microtubules, and thereby facilitating the formation of a single pronucleus in an activated SCNT oocyte (Song et al., 2009). Despite the historical use of colcemid in oocytes, there are no reports on its effects on oocyte maturation and embryonic development after SCNT. Based on its mechanism of action, we hypothesized that colcemid treatment would delay meiotic progression by depolymerizing the spindle-forming microtubules, which in turn would allow for better coordination between nuclear and cytoplasmic maturation. This improved cytoplasmic microenvironments should stimulate glutathione (GSH) synthesis and accumulation of maternal transcripts, thereby enhancing the developmental competence of IVM oocytes. The developmental potential of IVM oocytes is usually tested by proxye.g., assessing embryonic development after IVF but polyspermic fertilization is common when conducting IVF with porcine oocytes; indeed, more than 50% of oocytes show polyspermy and cannot develop normally (Kohata et al., 2013; Appeltant et al., 2015). As embryonic development varies with the proportion of polyspermic oocytes, which in turn makes it difficult to clearly evaluate the quality of IVM oocytes, parthenogenesis has been employed as an alternative to IVF to examine developmental competence of IVM porcine oocytes. The objective of this study was to examine the effects of colcemid treatment during IVM on the nuclear and cytoplasmic maturation of oocytes and the subsequent embryonic developmental competence. To this end, meiotic progression of oocytes was observed at the various stages of IVM, along with molecular measurements of intra-oocyte GSH content, maturation-promoting factor (MPF) activity, and the relative transcript abundance of cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2). In addition, the embryonic development of colcemid-treated IVM oocytes was examined after parthenogenetic activation (PA) and SCNT.
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MEIOTIC ARREST, SOMATIC CELL NUCLEAR TRANSFER, GENE EXPRESSION
RESULTS Nuclear Status of Pig Oocytes Treated With Colcemid for the First 22 hr, After 22 and 30 hr of IVM A significantly higher proportion of oocytes (93.8 2.6%) were arrested at the GVBD stage by the colcemid treatment compared to the untreated oocytes (60.6 10.8%; Table 1). Conversely, the proportion of nuclei at the metaphase-I (MI) stage was significantly higher in untreated oocytes than in colcemid-treated oocytes (24.7 8.3% vs. 1.6 1.6%). When nuclear status was examined after 30 hr of IVM, with or without colcemid treatment for the first 22 hr, the proportion of untreated oocytes that reached the MII stage was significantly higher than colcemid-treated oocytes (79.6 2.4% vs. 61.7 6.5%; Table 2). In the colcemidtreated and untreated samples, 10.1 0.2 and 20.4 1.6% of the oocytes were at the MI stage, respectively.
Nuclear Status, Intra-Oocyte GSH Content, and CDK1 Kinase Activity of Pig Oocytes Treated With Colcemid From 0 to 22 hr and/or 38 to 42 hr of IVM, After 42 hr of IVM Immature pig oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM culture (designated as COL0-22, COL38-42, and COL0-22/ 38-42, respectively). The proportions of oocytes reaching the MII stage were 93.8 2.6, 86.7 2.4, 86.8 2.4, and 84.8 2.3% for the untreated control, COL0-22, COL38-42, and COL0-22/38-42 samples, respectively (Table 3). Meiotic progression was therefore not altered by colcemid treatment, although the nuclear progression of COL0-22/38-42 oocytes to the MII stage tended to decrease compared to untreated oocytes (P < 0.1). Intra-oocyte GSH content was significantly increased in the COL0-22 (1.7 0.1 pixels/oocyte) and COL0-22/38-42 oocytes (1.3 0.1 pixels/oocyte) compared to the
untreated control (1.0 0.1 pixels/oocyte). On the other hand, MPF activity, measured in terms of CDK1 kinase activity, was not influenced by colcemid treatment, irrespective of when colcemid was provided during IVM.
Effect of Colcemid Treatment During IVM on Embryonic Development In vitro development of PA embryos to the blastocyst stage (68.7 3.5%) was improved (P < 0.05) by colcemid treatment during the first 0-22 hr of IVM compared to the control (42.5 3.6%). Embryonic cleavage (87.3 1.3 to 92.5 3.4%) and cell numbers of the PA-derived blastocysts (36.0 2.2 to 42.5 1.8 cells/blastocyst), however, were not influenced by this treatment (Table 4). SCNT embryos derived from COL0-22 oocytes exhibited a significantly higher rate (39.4 3.4%) of blastocyst formation than the control (16.3 2.3%), COL38-42 (22.7 3.8%), and COL0-22/38-42 oocytes (28.6 3.4%; P < 0.05). The mean cell number in the blastocysts was also higher following colcemid treatment during the first 022 hr of IVM compared to no treatment (P < 0.05; Table 5).
Effect of Colcemid Treatment During IVM on the Transcript Abundance of CDK1, PCNA, and ERK2 in Matured Oocytes Treatment of oocytes with colcemid during IVM stimulated the accumulation of CDK1, PCNA, and ERK2 mRNAs in MII oocytes compared to the control (P < 0.05). The abundance of 18S rRNA, however, was not influenced by this treatment (Fig. 1).
DISCUSSION The in vitro production of high-quality, mature oocytes from immature oocytes is an essential step that can dictate the success of artificial reproductive technology with
TABLE 1. Nuclear Status of Pig Oocytes Treated With Colcemid During a 22 hr IVM Period, After 22 hr of IVM Nuclear status (%) Colcemid treatment during IVM
No. of oocytes examined
GV
GVBD
MI
AI-TI
MII
60 63
8.2 3.1 4.7 3.0
60.6 10.8a 93.8 2.6b
24.7 8.3a 1.6 1.6b
3.3 1.9 0.0 0.0
3.3 1.9 0.0 0.0
None 022 hr
Three replicates were carried out. Values in the same column with different letters (a, b) indicate statistical differences (P < 0.05). AI, anaphase I; TI, telophase I.
TABLE 2. Nuclear Status Pig Oocytes Treated With Colcemid for the First 22 hr of IVM, After 30 hr of IVM Nuclear status (%) Colcemid treatment during IVM
No. of oocytes examined
GV
GVBD
MI
79 78
0.0 0.0 1.4 1.4
5.2 2.1 5.2 2.3
10.1 0.2 20.4 1.6b
None 022 hr
a
AI-TI
MII
5.0 2.9 6.3 0.9
79.6 2.4a 61.7 6.5b
Four replicates were carried out. Values in the same column with different letters (a, b) indicate significant differences (P < 0.05). AI, anaphase I; TI, telophase I.
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TABLE 3. Nuclear Status, Intra-oocyte GSH Content, and CDK1 Kinase Activity of Pig Oocytes Treated With Colcemid From 0 to 22 and/or 38 to 42 hr During IVM, After 42 hr of IVM Nuclear status (%)b
Colcemid treatment during IVM
No. of oocytes examined
None 022 hr 3842 hr 022 þ 3842 hr
97 98 91 99
GV 0.0 1.0 1.1 1.0
MI
0.0 5.2 0.2a 1.0 11.2 0.2ab 1.1 11.0 0.2ab 1.0 14.1 1.6b
AI-TI 1.0 1.0 1.1 0.0
Relative GSH level (pixels/oocyte) (n ¼ 60*)
MII
2.9 2.9 2.9 0.0
93.8 86.7 86.8 84.8
2.6 2.4 2.4 2.3
1.0 1.7 1.2 1.3
0.1a 0.1b 0.1ac 0.1c
Relative CDK1 kinase activity (n¼120*) 1.00 0.09 0.83 0.03 0.91 0.04 0.84 0.04
Four replicates were carried out. Values in the same column with different letters (a-c) indicate significant differences (P < 0.05). AI, anaphase I; TI, telophase I. *Number of MII oocytes examined for GSH content and CDK1 kinase activity.
TABLE 4. Effects of Colcemid Treatment During IVM on the Embryonic Development of Pig Oocytes After PA % of embryos developed to Colcemid treatment during IVM None 022 hr 3842 hr 022 þ 3842 hr
No. of embryos cultured
2-cell
Blastocyst
127 114 138 132
87.6 92.5 87.7 87.3
3.0 3.4 3.0 1.3
42.5 68.7 52.2 42.9
a
3.6 3.5b 3.2a 5.8a
No. of cells in blastocyst 37.2 42.5 36.7 36.0
2.2 1.8 1.8 2.2
Four replicates were carried out. Values in the same column with different letters (a, b) indicate significant differences (P < 0.05).
mammalian species. Timely coordination between meiotic progression and cytoplasmic maturation is important for later embryonic development following IVF and SCNT. In the present study, transient arrest of meiotic progression in immature pig oocytes by colcemid treatment during IVM positively influenced the embryonic development of the treated oocytes after PA and SCNT through alteration of nuclear and/or cytoplasmic maturation. Specifically, treatment with colcemid during an early stage (022 hr) of IVM effectively, but reversibly, arrested the nuclear status of oocytes at the GV to GVBD stages. The subsequent improvement in embryonic development after PA and SCNT was likely due to the improved cytoplasmic environments, as measured by increased intra-oocyte GSH contents and maternal transcription of critical genes in IVM oocytes. Colcemid inhibits cell division by inducing the depolymerization of microtubules, arresting cells at the metaphase stage. In the present study, colcemid treatment during the first 22 hr of IVM arrested meiotic progression of pig oocytes at the GV to GVBD period, leading to delayed meiotic progression compared to untreated oocytes. This
result was consistent with a previous finding in mice (Wassarman et al., 1976), wherein colcemid arrested meiotic progression of oocytes treated during IVM while also inhibiting the cleavage of somatic cells. Although colcemid arrested porcine oocytes at GVBD, oocytes could be released from this state and progressed normally to the MII stage when further cultured in medium free of colcemid; most of the IVM pig oocytes reach the MII stage after a total of 38 hr. Treatment of bovine oocytes with colcemid for 26 hr at the final stage of IVM also improved the developmental competence of cloned embryos (Li et al., 2009a). In contrast, a 4-hr colcemid treatment starting at 38 hr of IVM, when the nuclear stage of oocytes had progressed near the MII stage, neither inhibited nuclear progression to the MII stage nor showed any beneficial effect on porcine embryonic development. The absence of an effect may have been because most of the porcine oocytes already reached the MII stage and cytoplasmic maturation was also almost complete, leaving no need for a further delay. In comparison, treating with colcemid for 69 hr starting at 22 hr of IVM, when most of the oocytes were around the MI stage, resulted in MI stage-arrested oocytes that did not easily
TABLE 5. Effects of Colcemid Treatment During IVM on the Embryonic Development of Pig Oocytes After SCNT % of embryos developed to Colcemid treatment during IVM None 022 hr 3842 hr 022 þ 3842 hr
No. of SCNT embryos cultured 139 119 134 122
2-cell 86.3 90.8 86.4 86.1
2.1 1.3 2.9 3.8
Blastocyst 16.3 39.4 22.7 28.6
2.3a 3.4b 3.8ac 3.4c
No. of cells in blastocyst 30.0 41.3 35.3 39.0
2.1a 2.4b 2.5ab 2.7b
Four replicates were carried out.Values in the same column with different letters (ac) indicate significant differences (P < 0.05).
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Figure 1. Relative transcript abundance of CDK1, PCNA, and ERK2 mRNAs in MII oocytes treated with colcemid during oocyte IVM. Means standard error are plotted. Bars with different letters (ac) for the same mRNA are significantly different (P < 0.05).
progress to the MII stage after further culture in medium free of colcemid (data not shown). Thus, the reversibility of meiotic arrest induced by colcemid depends on the nuclear stage of the oocytes at the time of treatment. Positive effects of colcemid treatment on the cytoplasmic status of oocytes were also observed. In vivomatured oocytes have higher GSH content (Brad et al., 2003) and developmental competence than IVM oocytes (Petters and Wells, 1993). GSH has a stimulating effect on in vitro development of embryos derived from IVF and SCNT by protecting them from oxidative stress induced by reactive oxygen species (Tatemoto et al., 2000; Salmen et al., 2005). For this reason, GSH has been used as an indicator of the cytoplasmic maturity of IVM oocytes (Abeydeera et al., 1998; Brad et al., 2003). Similarly, quantities of certain maternal mRNAs may be used as markers for predicting the maturation and developmental competence of oocytes as these specific transcripts correlate with the abundance of the corresponding proteins. CDK1 and ERK2, for example, regulate MPF and mitogen-activated protein kinase activity (Vigneron et al., 2004; Lee et al., 2013) while PCNA is an essential component of the DNA replication and repair machinery (Kelman, 1997). Colcemid treatment from 0 to 22 hr and from 0 to 22 along with 38 to 42 hr of IVM increased GSH content as well as the transcript abundance of CDK1, ERK2, and PCNA without influencing the expression of 18S rRNA in IVM porcine oocytes; these results together imply improved cytoplasmic maturation. The mechanism underlying the higher intra-oocyte GSH content and abundance of maternal mRNA transcripts was not determined in this study, although we speculate that the transient arrest of nuclear maturation by colcemid treatment improved the synchrony between nuclear and cytoplasmic maturation while enhancing the cytoplasmic microenvironment, thereby stimulating GSH synthesis and transcription of maternal genes that are beneficial for later embryonic development. Curiously, when the effect of colcemid on MPF activity in IVM oocytes was examined, no increase
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was observed irrespective of the stages of IVM during which treatment was carried out. This observation is in contrast to previous results in which colcemid treatment after IVM significantly increased MPF activity in porcine and bovine oocytes (Li et al., 2009a, 2014), but may be attributed to technical differences in the duration of treatment and in the nuclear stage of the oocytes at the time of colcemid treatment. Treatment of oocytes with colcemid during the first 22 hr of IVM significantly improved the developmental competence of the embryos after PA and SCNT, without decreasing nuclear maturation. This result agrees with previous observations that transient inhibition of meiotic maturation, through an increase in the cAMP level of oocytes, improved the developmental competence of IVM oocytes, likely by allowing time for cytoplasmic maturation and improving coordination between nuclear and cytoplasmic maturation (Somfai et al., 2003). Although cAMP levels were not examined in this study, the transient but reversible nuclear arrest at the GVBD stage by colcemid treatment showed beneficial effects on embryonic development whereas colcemid-induced arrest at the MI stage (when oocytes were exposed to colcemid for 69 hr starting at 22 hr of IVM) was not fully reversible and worsened the developmental competence of PA oocytes. These results together suggest that the effects of colcemid treatment on meiotic maturation of porcine oocytes could be beneficial or detrimental for later embryonic development, depending on the nuclear stage at the time of treatment; a positive effect was limited to nuclear arrest during the early phase of oocyte IVM, which likely allowed for the proper maturation of the cytoplasmic microenvironment.
MATERIALS AND METHODS Culture Media All chemicals used in this study were obtained from SigmaAldrich Chemical Company (St. Louis, MO), unless
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otherwise noted. Medium-199 (M-199, Invitrogen, Grand Island, NY) supplemented with 0.91 mM pyruvate, 0.6 mM cysteine, 10 ng/ml epidermal growth factor, 1 mg/ml insulin, and 10% (v/v) pig follicular fluid was used as the base IVM medium. Porcine zygote medium (PZM)-3 containing 0.3% (w/v) bovine serum albumin (BSA) (Yoshioka et al., 2002) plus 2.77 mM myo-inositol, 0.34 mM tri-sodium citrate, and 10 mM b-mercaptoetanol was used for the in vitro culture of embryos.
Oocyte Collection and IVM The ovaries of prepubertal gilts obtained from a local abattoir were transported to the laboratory at 378C. Cumulus-oocyte complexes (COCs) were aspirated from follicles 3 to 8-mm in diameter. COCs with multiple layers of compacted cumulus cells were selected and washed three times in HEPES-buffered Tyrode’s medium (TLH) containing 0.05% (w/v) polyvinyl alcohol (TLH-PVA). COCs were placed into each well of a four-well multi-dish (Nunc, Roskilde, Denmark). Each well contained 500 ml of IVM medium with 80 mg/ml follicle-stimulating hormone (FSH) (Antrin R-10, Kyoritsu Seiyaku, Tokyo, Japan) and 10 IU/ml human chorionic gonadotropin (hCG) (Intervet International BV). The COCs were cultured at 398C with 5% CO2 in air under maximum humidity. After 22 hr of maturation culture, the COCs were washed three times in fresh, hormone-free IVM medium. The COCs were then cultured in hormone-free IVM medium for an additional 20 hr. During IVM, COCs were treated with 0.4 mg/ml colcemid or left untreated, according to the experimental design.
Experimental Design Immature oocytes were treated with colcemid or untreated (control) during the time periods of 022 (COL 022), 3842 (COL 38-42), or 022 and 3842 hr (COL 022/38-42) of IVM. The effects of colcemid treatment on the nuclear status of oocytes at 22 and 30 hr of IVM was examined first. Nuclear maturation, intra-oocyte GSH content, and MPF (CDK1) activity of the oocytes treated with colcemid during various stages of IVM were examined next. Subsequently, the effects of colcemid treatment during IVM on embryonic development after PA and SCNT were determined under similar experimental treatment conditions. Finally, the effects of colcemid on CDK1, PCNA, and ERK2 transcript abundance in MII oocytes were examined. Each experiment was conducted with 34 replicates.
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according to a previously reported system (Hunter and Polge, 1966).
Measurement of Intra-Oocyte GSH Content and MPF (CDK1) Activity in Oocytes Oocytes at the MII stage were sampled at 42 hr after IVM to determine the intra-oocyte GSH contents, using a previously described method (Sakatani et al., 2007). Briefly, 510 oocytes from each treatment group were incubated in the dark for 30 min in TLH-PVA supplemented with 10 mM CellTracker Blue CMF2HC (4-chloromethyl6.8-difluoro-7-hydroxycoumarin) (Invitrogen). After incubation, oocytes were washed with Dulbecco’s phosphatebuffered saline (D-PBS; Invitrogen) containing 0.1% (w/v) PVA, and 10-ml droplets containing the oocytes were transferred for observation of fluorescence under a TE300 epifluorescence microscope (Nikon, Tokyo, Japan). Fluorescence images were saved as TIFF files, and the fluorescence intensities of the oocytes were analyzed by ImageJ software version 1.41o (National Institutes of Health, Bethesda, MD) and normalized to that of untreated control oocytes. MPF/CDK1 activity was measured with a MESACUP CDC2 kinase assay kit (Medical & Biological Laboratories; MBL, Nagoya, Japan), as described previously (Anas et al., 2000). Briefly, a group of 30 MII-stage oocytes was washed twice with kinase sample buffer consisting of 50 mM TrisHCl, 0.5 M NaCl, 5 mM EDTA, 2 mM EGTA, 0.01% (v/v) polyoxyethylene lauryl ether (Brij 35), 1 mM phenylmethylsulfonyl fluoride, 0.05 mg/ml leupeptin, 50 mM b-mercaptoethanol, 25 mM b-glycerophosphate, and 1 mM sodium orthovanadate. Oocytes were then transferred to microtubes containing 5 ml of kinase buffer, and lysed by repeated freezing-thawing in liquid nitrogen and warm water, resepctively. These oocyte lysates were stored at 808C until analyzed. The lysates (5 ml) were mixed with 45 ml of assay buffer containing 25 mM HEPES, 10 mM MgCl2, 10% (v/v) biotinylated MV peptide (SLTSSSPGGATC), and 0.1 mM ATP, and the mixture was then incubated for 30 min at 308C. The phosphorylation reaction was terminated by the addition of 200 ml of stop reagent (PBS containing 50 mM EGTA), after which the mixture was centrifuged for 15 sec at 14,000 rpm. The phosphorylation of biotinylated MV peptide was detected at 492 nm using a plate reader. The data were expressed relative to CDK1 kinase activity in the untreated oocytes.
Preparation of Donor Cells and Nuclear Transfer Examination of Nuclear Status of Oocytes During IVM To evaluate the nuclear status, oocytes at 22, 30, and 42 hr of IVM were denuded, mounted onto glass slides, and fixed for 48 hr in 25% (v/v) acetic acid in ethanol at 48C. The fixed oocytes were then stained with 1% (w/v) aceto-orcein. Nuclear morphology and extrusion of the second polar body were assessed under a phase-contrast microscope at 400 magnification. Nuclear stages were classified
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Skin fibroblasts from a newborn miniature piglet were seeded into four-well culture plates and cultured in Dulbecco’s modified Eagle medium (DMEM) with the nutrient mixture F-12 (Invitrogen), which was supplemented with 15% (v/v) fetal bovine serum until a complete monolayer of cells had formed. Donor cells were synchronized at the G0/ G1 stage of the cell cycle by contact inhibition for 4872 hr. A suspension of single cells was prepared by trypsinization of the cultured cells, followed by resuspension in TLH
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containing 0.4% (w/v) BSA (TLH-BSA) prior to nuclear transfer. After 42 hr of IVM, the MII stage oocytes were incubated for 15 min in manipulation medium (calcium-free TLH-BSA) containing 5 mg/ml Hoechst 33342, and then washed twice with fresh manipulation medium. Oocytes were transferred into a drop of manipulation medium containing 5 mg/ml cytochalasin B, which was overlaid with mineral oil. Oocytes were enucleated by aspirating the first polar body and MII chromosomes using a 17-mm beveled glass pipette (Humagen, Charlottesville, VA). Enucleation was confirmed under an epifluorescence microscope. After enucleation, a single cell was inserted into the perivitelline space of each oocyte. Cell-oocyte couplets were placed in a 1-mm fusion chamber overlaid with 1 ml of 280 mM mannitol solution containing 0.001 mM CaCl2 and 0.05 mM MgCl2, as previously described (Song et al., 2009). Membrane fusion was induced by applying an alternating current field of 2 V cycling at 1 MHz for 2 sec, followed by two pulses of 170 V/mm direct current (DC) for 25 msec using a LF101 cell-fusion generator (NepaGene, Chiba, Japan). Immediately after fusion, the oocytes were incubated for 1 hr in TLH-BSA, and membrane fusion was evaluated under a stereomicroscope prior to activation.
Day 0). The number of cells in the blastocysts were examined using Hoechst 33342 staining under an epifluorescence microscope.
Gene Expression Analysis by Real-Time PCR The transcript abundance of CDK1, PCNA, and ERK2 in IVM oocytes was analyzed by real-time PCR as previously described (Zhu-Ge and Yu, 2004). Total RNA was isolated from MII oocytes after IVM using AccuZolTM (Bioneer, Alameda, CA), according to the manufacturer’s instructions. Complementary DNA was synthesized from total RNA using a Reverse Transcription system (Promega, Madison, WI). The transcript abundance of specific genes in the embryos was then quantified on a Rotor-Gene 3000 system (Corbett, Mortlake, Australia) using the PrimeScript RT-PCR kit (Takara, Shiga, Japan). Melting-curve data were collected to verify PCR specificity. Primer3 software (Whitehead Institute, MIT Center for Genome Research, Cambridge, MA) was used to design the primers employed in this study (Table 6). All data were normalized to 18S rRNA. The relative mRNA level was calculated as previously described (Matusuoka et al., 1999; Pfaffl, 2001).
Statistical Analysis Oocyte Activation Reconstructed oocytes were activated with two pulses of 120 V/mm DC for 60 msec in a 280 mM mannitol solution containing 0.01 mM CaCl2 and 0.05 mM MgCl2. For PA, the oocytes that had reached the MII stage at 42 hr of IVM were electrically activated using the same pulse sequence used to activate the SCNT oocytes.
In Vitro Culture of Embryos Following activation, the SCNT and PA embryos were treated with 0.4 mg/ml demecolcine and 5 mg/ml cytochalasin B, resepectively, in in vitro culture (IVC) medium for 4 hr (Song et al., 2009). The SCNT and PA embryos were then washed three times in fresh IVC medium, placed into 30-ml droplets of IVC medium under mineral oil, and then cultured at 398C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 7 days. Embryo cleavage and blastocyst formation were evaluated on Days 2 and 7, respectively (the day of SCNT or PA was designated as
In this study, 665 oocytes were examined for the nuclear status during IVM, while 60 and 120 MII oocytes were used for the examination of GSH contents and MPF activity, respectively. In addition, 511 and 514 embryos were used to determine the developmental competence of MII oocytes after PA and SCNT, respectively. All experiments were repeated 34 times. Percentage data such as nuclear status, embryo cleavage, and blastocyst formation were analyzed using a general linear model procedure after arcsine transformation to maintain the homogeneity of the variances. Post-hoc analyses to identify between-group differences were performed using the least significant difference (LSD) test, with a model effect of treatment differences called at P < 0.05. The non-parametric Wilcoxon test with Dunn method for multiple comparisons was used to determine differences among treatments in datasets such as the cell number of blastocysts, GSH contents, and MPF activity. Results are expressed as the arithmetic mean standard error.
TABLE 6. Primers and RT-PCR Conditions Used for Gene Expression Analysis mRNA
Direction
Primer sequences
Product size (base pairs)
18S
Forward Reverse Forward Reverse Forward Reverse Forward Reverse
50 -CGGCTACCACATCCAAGGAA 50 -CTCCAATGGATCCTCGTTAAAGG 50 -TTTTCAGAGCTTTGGGCACTC 50 - TTTTCGAGAGCSGATCCAAGC 50 -CTCAAACCTTCCAACCTGCTG 50 -GTCGATGGACTTGGTGTAGCC 50 -TAATGCAGACACCTTGGCACT 50 -GCAAATTCACCAGAAGGCATC
147
CDK1 ERK2 PCNA
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153 183 153
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ACKNOWLEDGMENTS We thank Gangwon Veterinary Service for the help in collecting pig ovaries used in this study. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012R1A1A4A01002564).
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adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod 57:4953. Hashimoto N, Kishimoto T. 1988. Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation. Dev Biol 126:242252.
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